U.S. patent application number 09/935216 was filed with the patent office on 2003-02-13 for inhibitors of cell regulatory factors and methods for preventing or reducing scarring.
This patent application is currently assigned to The Burnham Institute. Invention is credited to Border, Wayne A., Harper, John R., Longaker, Michael T., Pierschbacher, Michael D., Ruoslahti, Erkki I., Whitby, David J..
Application Number | 20030032591 09/935216 |
Document ID | / |
Family ID | 27539658 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032591 |
Kind Code |
A1 |
Ruoslahti, Erkki I. ; et
al. |
February 13, 2003 |
Inhibitors of cell regulatory factors and methods for preventing or
reducing scarring
Abstract
The present invention provides a method of inhibiting an
activity of a cell regulatory factor comprising contacting the cell
regulatory factor with a purified polypeptide, wherein the
polypeptide comprises the cell regulatory factor binding domain of
a protein and wherein the protein is characterized by a
leucine-rich repeat of about 24 amino acids. In a specific
embodiment, the present invention relates to the ability of
decorin, a 40,000 dalton protein that usually carries a
glycosaminoglycan chain, to bind TGF-.beta.. The invention also
provides a novel cell regulatory factor designated MRF. Also
provided are methods of identifying, detecting and purifying cell
regulatory factors and proteins which bind and affect the activity
of cell regulatory factors. The present invention further relates
to methods for the prevention or reduction of scarring by
administering decorin or a functional equivalent of decorin to a
wound. The methods are particularly useful for dermal wounds
resulting from burns, injuries or surgery. In addition, the present
invention includes pharmaceutical compositions containing decorin
or its functional equivalent and a pharmaceutically acceptable
carrier useful in such methods. Finally, methods for preventing or
inhibiting pathological conditions by administering decorin are
also provided.
Inventors: |
Ruoslahti, Erkki I.; (Rancho
Santa Fe, CA) ; Longaker, Michael T.; (San Francisco,
CA) ; Whitby, David J.; (Adel, GB) ; Harper,
John R.; (Carlsbad, CA) ; Pierschbacher, Michael
D.; (San Diego, CA) ; Border, Wayne A.; (Salt
Lake City, UT) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Assignee: |
The Burnham Institute
|
Family ID: |
27539658 |
Appl. No.: |
09/935216 |
Filed: |
August 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09935216 |
Aug 21, 2001 |
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08458834 |
Jun 2, 1995 |
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6277812 |
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08458834 |
Jun 2, 1995 |
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08303238 |
Sep 8, 1994 |
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5654270 |
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08303238 |
Sep 8, 1994 |
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07978931 |
Nov 17, 1992 |
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07978931 |
Nov 17, 1992 |
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07882345 |
May 13, 1992 |
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07882345 |
May 13, 1992 |
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07792192 |
Nov 14, 1991 |
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07792192 |
Nov 14, 1991 |
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07467888 |
Jan 22, 1990 |
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07467888 |
Jan 22, 1990 |
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07212702 |
Jun 28, 1988 |
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Current U.S.
Class: |
514/112 ;
514/1.5; 514/15.4; 514/16.4; 514/18.6; 514/19.3; 514/54;
514/8.9 |
Current CPC
Class: |
C07K 14/4725 20130101;
A61K 38/00 20130101; C07K 2319/00 20130101; C07K 14/495 20130101;
C07K 14/4703 20130101; C07K 14/475 20130101 |
Class at
Publication: |
514/12 ;
514/54 |
International
Class: |
A61K 038/17; A61K
031/728 |
Goverment Interests
[0002] This invention was made with support of government grants CA
30199, CA 42507 and CA 28896 from the National Cancer Institute.
Therefore, the United States government may have rights in the
invention.
Claims
We claim:
1. A method for the prevention or reduction of scarring comprising
administering decorin or a functional equivalent of decorin to a
wound.
2. The method of claim 1, wherein said functional equivalent is
biglycan.
3. The method of claim 1, wherein said functional equivalent is
fibromodulin.
4. The method of claim 1, wherein said scarring is dermal
scarring.
5. A pharmaceutical composition comprising decorin or its
functional equivalent and a pharmaceutically acceptable
carrier.
6. The pharmaceutical composition of claim 5, wherein said
functional equivalent is biglycan.
7. The pharmaceutical composition of claim 5, wherein said
functional equivalent is fibromodulin.
8. The pharmaceutical composition of claim 5, wherein said
pharmaceutically acceptable carrier is hyaluronic acid.
9. The pharmaceutical composition of claim 5, wherein said
composition further comprises an RGD-containing polypeptide
attached to a biodegradable polymer.
10. A method of treating a pathology caused by a TGF-.beta.
regulated activity comprising contacting the TGF-.beta. with a
purified polypeptide, wherein the polypeptide comprises a
TGF-.beta. binding domain of a protein and wherein the protein is
characterized by a leucine-rich repeat of about 24 amino acids,
whereby the pathology causing activity is prevented or reduced.
11. The method of claim 10, wherein said protein is decorin.
12. The method of claim 10, wherein said protein is biglycan.
13. The method of claim 10, wherein said protein is
fibromodulin.
14. The method of claim 10, wherein said pathology is rheumatoid
arthritis, glomerulonephritis, arteriosclerosis, adult respiratory
distress syndrome, cirrhosis of the liver, fibrotic cancer,
fibrosis of the lungs, post-myocardial infarction, cardiac
fibrosis, post-angioplasty restenosis, renal interstitial fibrosis
or dermal fibrotic conditions.
15. The method of claim 14, wherein said pathology is
glomerulonephritis.
Description
[0001] This invention is a continuation-in-part of U.S. Ser. No.
07/882,245, filed May 13, 1992, which is a continuation of U.S.
Ser. No. 07/792,192, filed Nov. 14, 1991, which is a
continuation-in-part of U.S. Ser. No. 07/467,388, filed Jan. 22,
1990, which is a continuation-in-part of U.S. Ser. No. 07/212,702,
filed Jun. 28, 1988, now abandoned, the contents of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] This invention relates to cell biology and more specifically
to the control of cell proliferation. Proteoglycans are proteins
that carry one or more glycosaminoglycan chains. The known
proteoglycans carry out a wide variety of functions and are found
in a variety of cellular locations. Many proteoglycans are
components of extracellular matrix, where they participate in the
assembly of cells and effect the attachment of cells to the
matrix.
[0004] One of the key functions of the extracellular matrix is the
storage and presentation of growth factors to cells. Proteoglycans
are important mediators of growth factor binding, and they have
been shown to modulate the biological activities of a variety of
growth factors through interaction via their glycosaminoglycan
moieties as well as their core proteins (Ruoslahti, 1989; Ruoslahti
and Yamaguchi, 1991).
[0005] Growth factors that hind to glycosaminoglycans include
acidic and basic FGF (see Burgess and Maciag, 1989), GM-CSF,
interleukin-3 (Roberts et al., 1988), pleiotrophin (Li et al.,
1990), amphiregulin (Shoyab et al., 1988), HB-EGF (Higashiyama et
al., 1991) and platelet factor 4 (Huang et al., 1982), each of
which binds avidly to heparin and heparan sulfate. The binding of
FGFs to heparin or to heparan sulfate proteoglycans protects the
growth factors from proteolytic degradation and is thought to
create a matrix-bound growth factor reservoir (Saksela et al.,
1988; Gospodarowicz et al., 1990) from which the growth factor can
be released in an active form by partial proteolysis of the
proteoglycan core protein or through degradation of the heparan
sulfate moiety of the proteoglycans (Saksela and Rifkin, 1990;
Ishai-Michaeli et al., 1990). Basic FGF has to be bound to
glycosaminoglycan to be able to interact with its signal
transduction receptor (Yayon et al., 1991; Rapraeger et al.,
1991).
[0006] The binding of TGF-.beta. to proteoglycans represents a
different type of growth factor-proteoglycan interaction.
TGF-.beta. has been demonstrated to bind to the core proteins of at
least two proteoglycans. One of these proteoglycans is is decorin,
a small interstitial extracellular matrix proteoglycan that can
interact with TGF-.beta. via its core protein (Yamaguchi et al.
1990). Decorin, also known as PG-II or PG-40, is a small
proteoglycan produced by fibroblasts. Its core protein has a
molecular weight of about 40,000 daltons. The core has been
sequenced (Krusius and Ruoslahti, Proc. Natl. Acad. Sci. USA
83:7683 (1986); Day et al. Biochem. J. 248:801 (1987), both of
which are incorporated herein by reference) and it is known to
carry a single glycosaminoglycan chain of a chondroitin
sulfate/dermatan sulfate type (Pearson, et al., J. Biol. Chem.
258:15101 (1983), which is incorporated herein by reference). The
only previously known function for decorin is binding to type I and
type II collagen and its effect on the fibril formation by these
collagens (Vogel, et al., Biochem. J. 223:587 (1984); Schmidt et
al., J. Cell Biol. 104:1683, (1987)). Decorin (Krusius and
Ruoslahti, 1986) is the prototype of a group of proteoglycans
characterized by core proteins of .about.40 kDa that consist mainly
of leucine-rich repeats of 20 to 24 amino acids (Patthy, 1987). So
far, four members of this group of proteoglycans have been cloned;
in addition to decorin, these are biglycan (Fisher et al., 1989),
fibromodulin (Oldberg et al., 1989) and lumican (Blochberger et
al., 1992). Decorin and biglycan are ubiquitous, although they show
a quite divergent localization within tissues, with decorin found
more in the extracellular matrix of tissues where it is bound to
type I collagen (Vogel et al., 1984; Scott, 1986; Brown and Vogel,
1989) and biglycan localized more closely around cells (Bianco et
al., 1990). Fibromodulin has a somewhat more restricted
distribution with high concentrations in cartilage, tendon and
sclera, while low in skin and mineralized bone (Heinecard et al.,
1986). Lumican is found mainly in the cornea (Blochberger et al.,
1992). Together, these proteins form a superfamily of proteins
(Ruoslahti, Ann. Rev. Cell Biol. 4:229, (1988); McFarland et al.,
Science 245:494 (1989)).
[0007] The second type of TGF-.beta.-binding proteoglycan is the
type III TGF-.beta. receptor, betaglycan (Segarini and Seyedin et
al., 1988; Andres et al., 1989). Betaglycan is a cell membrane
proteoglycan (Lpez-Casillas et al., 1991; Wang et al., 1991) that
apparently is not involved in the TGF-.beta. signal transduction
pathway but may function as a cell-surface TGF-.beta. reservoir
presenting TGF-.beta. to its signal transduction receptors.
[0008] Transforming growth factor .beta.s (TGF-.beta.) are a family
of multi-functional cell regulatory factors produced in various
forms by many types of cells (for review see Sporn et al., J. Cell
Biol. 105:1039, (1987)). Five different TGF-.beta.'s are known, but
the functions of only two, TGF-.beta.1 and TGF-.beta.2, have been
characterized in any detail. TGF-.beta.'s are the subject of U.S.
Pat. Nos. 4,863,899; 4,816,561; and 4,742,00 which are incorporated
by reference. TGF-.beta.1 and TGF-.beta.2 are publicly available
through many commercial sources (e.g. R & D Systems, Inc.,
Minneapolis, Minn.). In some cells, TGF-.beta. promotes cell
proliferation, in others it suppresses proliferation. A marked
effect of TGF-.beta. is that it promotes the production of
extracellular matrix proteins and their receptors by cells (for
review see Keski-Oja et al., J. Cell Biochem 33:95 (1987);
Massague, Cell 49:437 (1987); Roberts and Sporn in "Peptides Growth
Factors and Their Receptors" [Springer-Verlag, Heidelberg]
(1989)).
[0009] While TGF-.beta. has many essential cell regulatory
functions, improper TGF-.beta. activity can be detrimental to an
organism. Since the growth of mesenchyme and proliferation of
mesenchymal cells is stimulated by TGF some tumor cells may use
TGF-.beta. as an autocrine growth factor. Therefore, if the growth
factor activity of TGF-.beta. could be prevented, tumor growth
could be controlled. In other cases the inhibition of cell
proliferation by TGF-.beta. may be detrimental, in that it may
prevent healing of injured tissues. The stimulation of
extracellular matrix production by TGF-.beta. is important in
situations such as wound healing. However, in some cases the body
takes this response too far and an excessive accumulation of
extracellular matrix ensues. An example of excessive accumulation
of extracellular matrix is glomerulonephritis, a disease with a
detrimental involvement of TGF-.beta..
[0010] Thus, there exists a critical need to develop compounds that
can modulate the effects of cell regulatory factors such as
TGF-.beta.. The present invention satisfies this need and provides
related advantages.
SUMMARY OF THE INVENTION
[0011] The present invention provides a method of inhibiting an
activity of a cell regulatory factor comprising contacting the cell
regulatory factor with a purified polypeptide, wherein the
polypeptide comprises a cell regulatory factor binding domain of a
protein and wherein the protein is characterized by a leucine-rich
repeat of about 24 amino acids. In a specific embodiment, the
present invention relates to the ability of decorin, a 40,000
dalton protein that usually carries a glycosaminoglycan chain, to
bind TGF-.beta.. The invention also provides a novel cell
regulatory factor designated Morphology Restoring Factor, (MRF).
Also provided are methods of identifying, detecting and purifying
call regulatory factors and proteins which bind and affect the
activity of cell regulatory factors.
[0012] The present invention further relates to methods for the
prevention or reduction of scarring by administering decorin or a
functional equivalent of decorin to a wound. The methods are
particularly useful for dermal wounds resulting from burns,
injuries or surgery. In addition, the present invention includes
pharmaceutical compositions containing decorin or its functional
equivalent and a pharmaceutically acceptable carrier useful in such
methods. Finally, methods for preventing or inhibiting pathological
conditions by administering decorin are also provided.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows expression of decorin cDNA containing a
mutation of the serine acceptor site to alanine. COS-1 cultures
were transfected with cDNA coding for wild-type decorin (lane 1),
decorin in which the serine-4 residue was replaced by an alanine
(lane 2), or decorin in which the serine-4 residue was replaced by
a threonine (lane 3). Immunoprecipitations were performed with an
anti-decorin antibody and medium which was labeled with
.sup.32S-sulfate (A) or .sup.1H-leucine B). Lane 4 shows an
immunoprecipitate from mock transfected COS-1 cultures. Arrow
indicates top of gel. The numbers indicate M.sub.r.times.10.sup.-3
for molecular weight standards.
[0014] FIG. 2 shows binding of [.sup.125I]-TGF-.beta. to
decorin-Sepharose. FIG. 2A shows fractionation of
[.sup.125I]-TGF-.beta.1 by decorin-Sepharose affinity
chromatography. [.sup.125I]-TGF-.beta.1 (5.times.10.sup.5 cpm) was
incubated in BSA-coated polypropylene tubes with 0.2 ml of packed
decorin-Sepharose (.oval-solid.) or gelatin-Sepharose
(.smallcircle.) in 2 ml of PBS pH 7.4, containing 1 M NaCl and
0.05% Tween 20. After overnight incubation, the affinity matrices
were transferred into BSA-coated disposable columns (Bio Rad) and
washed with the binding buffer. Elution was effected first with 3 M
NaCl in the binding buffer and then with 8 M urea in the same
buffer. FIG. 2B shows the analysis of eluents of decorin-Sepharose
affinity chromatography by SDS-polyacrylamide gel under nonreducing
conditions. Lane 1: the original [.sup.125I]-labeled TGF-.beta.1
sample; lanes 2-7: flow through and wash fractions; lanes 8-10: 3 M
NaCl fractions; lanes 11-14: 8 M urea fractions. Arrows indicate
the top and bottom of the 12% separating gel.
[0015] FIG. 3 shows the inhibition of binding of
[.sup.125I]-TGF-.beta.1 to decorin by proteoglycans and their core
proteins. FIG. 3A shows the competition of [.sup.125]-TGF-.beta.1
binding to decorin-coated microtiter wells by recombinant decorin
(.oval-solid.), decorin isolated from bovine skin (PGII)
(.box-solid.), biglycan isolated from bovine articular cartilage
(PGI) (.tangle-solidup.), chicken cartilage proteoglycan
(.smallcircle.), and BSA (.quadrature.). Each point represents the
mean of duplicate determinants. FIG. 3B shows the competition of
[.sup.125I]-TGF-.beta.1 binding with chondroitinase ABC-treated
proteoglycans and BSA. The concentrations of competitors were
expressed as intact proteoglycan. The symbols are the same as in
FIG. 3A.
[0016] FIG. 4 shows neutralization of the growth regulating
activity of TGF-.beta.1 by decorin. FIG. 4A shows inhibition of
TGF-.beta.1-induced proliferation of CHO cells by decorin. The
[.sup.3H]Thymidine incorporation assay was performed as described
in he legend of FIG. 1 in the presence of 5 ng/ml of TGF-.beta.1
and the indicated concentrations of purified decorin (.oval-solid.)
or BSA (.smallcircle.). At the concentration used, TGF-.beta.1
induced a 50% increase of [.sup.3H]thymidine incorporation in the
CHO cells. The data represent percent neutralization of this growth
stimulation; i.e. [.sup.3H]thymidine incorporation in the absence
of either TGF-.beta.1 or decorin=0%, incorporation in the presence
of TGF-.beta. but not decorin=100%. Each point shows the
mean.+-.standard deviation of triplicate samples. FIG. 4B shows
neutralization of TGF-.beta.1-induced growth inhibition in MvLu
cells by decorin. Assay was performed as in A except that
TGF-.beta.1 was added at 0.5 ng/ml. This concentration of
TGF-.beta.1 induces 50% reduction of [.sup.3H]thymidine
incorporation in the MvLu cells. The data represent neutralization
of TGF-.beta.-induced growth inhibition; i.e. [.sup.3H]thymidine
incorporation in the presence of neither TGF-.beta. or
decorin=100%; incorporation in the presence of TGF-.beta. but not
decorin=0%.
[0017] FIG. 5A shows separation of growth inhibitory activity from
decorin-expressing CHO cells by gel filtration. Serum-free
conditioned medium of decorin overexpressor cells was fractionated
by DEAE-Sepharose chromatography in a neutral Tris-HCl buffer and
fractions containing growth inhibitory activity were pooled, made
4M with guanidine-HCl and fractionated on a Sepharose CL-6B column
equilibrated with the same guanidine-HCl solution. The fractions
were analyzed for protein content, decorin content, and growth
regulatory activities. Elution positions of marker proteins are
indicated by arrows. BSA: bovine seru albumin (Mr=66,000); CA:
carbonic anhydrase (Mr=29,000); Cy: cytochrome c (Mr=12,400); Ap:
aprotinin (Mr=6,500); TGF: [.sup.125I]-TGF-.beta.1 (Mr=25,000)
[0018] FIG. 5B shows identification of the growth stimulatory
material from gel filtration as TGF-.beta.1. The growth stimulatory
activity from the late fractions from Sepharose 6B (bar in panel A)
was identified by inhibiting the activity with protein A-purified
IgG from an anti-TGF-.beta. antiserum. Data represent percent
inhibition of growth stimulatory activity in a [.sup.3H]thymidine
incorporation assay. Each point shows the mean.+-.standard
deviation of triplicate determinations. Anti-TGF-.beta.1
(.oval-solid.), normal rabbit TgG (.smallcircle.).
[0019] FIG. 6 shows micrographs demonstrating a decorin-binding
cell regulatory activity that is not suppressed by antibodies to
TGF-.beta.1.
[0020] FIG. 7 shows that decorin inhibits the binding of
[.sup.125I]-TGF-.beta. to Type III TGF-.beta. receptor (.beta.
glycan) on HepG2 cells. FIG. 7a shows the non-reduced lysate of
HepG2 cells resolved on 4-12% SDS-PAGE. FIG. 7b shows the reduced
lysate resolved on 4-12% SDS-PAGE. The reduction of intensity of 3
glycan band (approximately 300 kDa) and uncross-linked hand (free
TGF-.beta., 25 kDa) in the presence of decorin (10,000.times.molar
excess) is shown.
[0021] FIG. 8 shows that decorin inhibits the binding of
[.sup.125I]-TGF-.beta. to Type III TGF-.beta. receptor an MG-63
cells. FIG. 8a shows the resolution of the lysate on 4-12% SDS-PAGE
under non-reduced conditions, while FIG. 8b shows the results under
reduced conditions.
[0022] FIG. 9 shows that decorin (DC-9, DC-12) and biglycan inhibit
the binding of [.sup.125I]-TGF-.beta. to immobilized decorin.
[0023] FIG. 10 shows the concentration dependence of decorin
inhibition of [.sup.125I]-TGF-.beta. binding to HepG2 cells.
[0024] FIG. 11 shows the amino acid sequence of human fibromodulin
deduced from cDNA. The human fibromodulin sequence is shown aligned
with the amino acid sequences of bovine fibromodulin (Oldberg et
al., 1989), human decorin (Krusius and Ruoslahti, 1986) and human
biglycan (Fisher et al., 1989). A star marks the sequence position
where the NH.sub.2-termini of the proteoglycan core proteins
lacking their predicted signal sequences were fused to E.
coli-maltose binding protein (MBP) with two additional amino acids,
glycine and serine, added at the linkage site. Identical amino
acids are boxed.
[0025] FIG. 12 shows the construction of prokaryotic expression
vector for proteoglycan fusion proteins. The parent vector pQE-8
was modified by insertion of a BglII/BamHI-MBP fragment. This
fragment also included a factor Xa protease cleavage site and
provided a unique BamHI cloning site for introduction of the
proteoglycan core protein inserts. RBS=ribosomal binding site;
6xHis=coding sequence for consecutive six histidines; MBP=coding
sequence for E. coli-maltose binding protein; to=transcriptional
terminator `to` of phage lambda (Schwarz et al., 1987);
cat=promotor-free gene for chloramphenicol acetyltransferase,
Ti=transcriptional terminator T1 of the E. coli rrnB operon
(Brosius et al., 1981).
[0026] FIG. 13 shows the analyses by gel electrophoresis of
purified recombinant proteoglycan core fusion proteins. Each
purified protein (1 .mu.g/well) was loaded on a 4-20%
NaDodSO.sub.4-polyacrylamide gel. After electrophoresis under
non-reducing conditions, the gel was stained with Coomassie blue
R-250. A=maltose binding protein; B=mBP-biglycan; C=MBP-decorin;
D=MBP-fibromodulin. The sizes (kDa) of molecular weight marker
proteins are indicated.
[0027] FIG. 14 shows the binding of radiolabeled proteoglycan
fusion proteins and MBP to microtiter wells coated with
TGF-.beta.1. TGF-.beta.1 was used in the indicated concentrations
(75 .mu.l/well) to coat microtiter wells. The wells were incubated
with .sup.125I-labeled MBP-biglycan (.box-solid.), MBP-decorin
(.circle-solid.), MBP-fibromodulin (.tangle-solidup.) or IBP
(.diamond-solid.). Constant amounts (.about.50,000 cpm/well,
specific activities 2300-2800 Cl/mmol) of the labeled proteins were
added to the TGF-.beta.1-coated wells (total volume 100 .mu.l).
After incubation for 6 hours at 37.degree. C., the wells were
washed four times. TGF-.beta.1-binding was determined by counting
the entire wells in a gamma counter and is expressed (.+-.S.D.) as
percent of the total amount of labeled proteins added to the
wells.
[0028] FIG. 15 shows the specificity of proteoglycan core protein
binding to TGF-.beta.1. Microtiter wells were coated with the
indicated proteins (75 .mu.l/well, 3 .mu.g/ml).
.sup.125I-MBP-biglycan (hatched bars), .sup.125I-MBP-decorin (solid
bars) or I.sup.125I-MBP-fibromodulin (cross-hatched bars) were
added to the wells (total volume 100 .mu.l). After incubation for 6
hours at 37.degree. C., the wells were washed three times and
counted in a gamma counter. Binding (.+-.S.D.) is expressed as
percent of the total amount of labeled proteins added to the
wells.
[0029] FIG. 16 shows the time-course of MBP-biglycan binding to
TGF-.beta.1. .sup.125I-MBP-biglycan was added to TGF-.beta.1 coated
wells (75 .mu.l, 1 .mu.g/ml) at 4.degree. C. (.circle-solid.) or
37.degree. C. (.box-solid.), respectively. After the indicated
time-periods, the wells were washed three times and counted in a
gamma counter. Binding (.+-.S.D.) is expressed as percent of the
total amount of .sup.125I-MBP-biglycan added.
[0030] FIG. 17 shows the inhibition of the binding of biglycan
fusion protein to TGF-.beta.1 by proteoglycan fusion proteins and
intact proteoglycans. Binding of .sup.125I-MBP-biglycan to
TGF-.beta.1 was measured in the presence of the indicated
concentrations of (A) unlabeled MBP-BG (.box-solid.), MBP-DEC
(.circle-solid.), MBP-FM (.tangle-solidup.) or MBP
(.diamond-solid.) or (B) purified biglycan (.box-solid.), decorin
(.circle-solid.) or fibromodulin (.tangle-solidup.). After
incubation of 6 hours at 37.degree. C., the wells were washed three
times and counted in a gamma counter. Binding (.+-.S.D.) is
expressed as percent of radiolabel bound in the absence of
competitor.
[0031] FIG. 18 shows the competition for the binding of
radiolabeled TGF-.beta.1, -.beta.2 and -.beta.3 to microtiter wells
coated with biglycan fusion protein. The binding of
.sup.125I-labeled TGF-.beta.1 (solid bars), TGF-.beta.2 (hatched
bars) or TGF-.beta.3 (open bars) (50,000 cpm/well, specific
activities 5,000 to 7,000 Cl/mmol) to surface-bound MBP-biglycan
(coating concentration 10 .mu.g/ml. 75 .mu.l/well) was studied in
the absence (control) or presence of unlabeled MBP-BG, MB-DEC,
MBP-FM, MBP, biglycan, decorin or fibromodulin (1 .mu.M). Binding
was corrected for nonspecific binding as is expressed as percent
(.+-.S.D.) of the total amount of labeled TGF-.beta.1, 2 or 3 that
was added to the wells.
[0032] FIG. 19 shows the competition for the binding of labeled
TGF-.beta.1 to MvLu cells by proteoglycan fusion proteins. (A)
Subconfluent cultures of MvLu mink lung cells cultures in 48-well
plates were incubated with .sup.125I-TGF-.beta.1 (100 pM) in the
presence (n.s.) or absence (B.sub.o) of unlabeled TGF-.beta.1 (20
nM) or the indicated concentrations of proteoglycan fusion proteins
in a total volume of 100 .mu.l. After incubation for 4 hours at
4.degree. C., the cells were washed four times. The cells were then
solubilized for 40 min in 1% Triton-X 100 and assayed for
radioactivity in a gamma counter. Binding (.+-.S.D., n=3) is
expressed as percent of the total amount of .sup.125I-TGF-.beta.1
that was added. (B) Mink lung cells were incubated with
.sup.125I-TGF-.beta.1 (100 pM) in the absence or presence of
unlabeled TGF-.beta. (20 nM) or MBP-fusion proteins (3 .mu.M) in
24-well plates. After incubation for 4 hours at 4.degree. C., the
cells were treated with the cross-linker disuccinimidyl suberate
and analyzed by NaDodSO.sub.4-PAGE and autoradiography. Binding in
the absence of competitor (a), with TGF-.beta.1 (b), MBP-BG (c),
MBP-DEC (d), MBP-FM (e) or MBP (f). The positions of pre-stained
marker proteins are indicated. The positions of the TGF-.beta. type
I and type II receptors and of betaglycan (.beta.-G) are indicated.
Arrows point to the receptors and betaglycan (.beta.-G).
DETAILED DESCRIPTION OF THE INVENTION
[0033] Increased TGF-.beta. production has been found to be an
important element in a number of fibrotic diseases that are
characterized by an accumulation of extracellular matrix components
Border and Ruoslahti, 1992). Besides fibronectin, collagens, and
tenascin (Ignotz and Massague, 1986; Varga et al., 1987; Pearson et
al., 1988), TGF-.beta. also upregulates the expression of
proteoglycans (Bassols and Massagure, 1988). In mesangial cells
both decorin and biglycan can increase as much as 50-fold after
induction by TGF-.beta. (Border et al., 1990a), whereas in
fibroblasts only biglycan seems to be elevated (Romaris et al.,
1992; Kahari et al., 1991). Fibromodulin has not been studied in
this regard. TGF-.beta. plays a pivotal role in the pathogenesis of
experimentally induced glomerulonephritis, the most critical
manifestation of which is the accumulation of extracellular matrix
in the glomeruli (Border et al., 1990). A recent study shows that
injection of recombinant decorin into glomerulonephritic rats can
suppress the matrix accumulation (Border et al., 1992). The present
invention indicates that fibromodulin can be even more effective in
that situation. The TGF-.beta. neutralizing activities of the
decorin-type proteoglycans indicates that new types of therapeutics
can be developed based on these molecules.
[0034] The invention provides a method of inhibiting an activity of
a cell regulatory factor comprising contacting the cell regulatory
factor with a purified polypeptide, wherein the polypeptide
comprises the cell regulatory actor binding domain of a protein and
wherein the protein is characterized by a leucine-rich repeat of
about 24 amino acids. Since diseases such as cancer result from
uncontrolled cell proliferation, the invention can be used to treat
such diseases.
[0035] By "cell regulatory factor" is meant a molecule which an
regulate an activity of a cell. The cell regulatory factors are
generally proteins which bind cell surface receptors and include
growth factors. Examples of cell regulatory factors include the
five TGF-.beta.'s, platelet-derived growth factor, epidermal growth
factor, insulin like growth factor I and II, fibroblast growth
factor, interleukin-2, nerve growth factor, hemopoietic cell growth
factors (IL-3, GM-CSF, M-CSF, G-CSF, erythropoietin) and the newly
discovered Morphology Restoring Factor, hereinafter "MRF".
Different regulatory factors can be bound by different proteins
which can affect the regulatory factor's activity. For example,
TGF-.beta.1 is bound by decorin, biglycan and fibromodulin, and MRF
is bound by decorin.
[0036] By "cell regulatory factor binding domain" is meant the
fragment of a protein which binds to the call regulatory factor.
While the specific examples set forth herein utilize proteins, it
is understood that a protein fragment which retains the binding
activity is included within the scone of the invention. Fragments
which retain such activity can be recognized by their ability to
competitively inhibit the binding of, for example, decorin to
TGF-.beta., or of other polypeptides containing leucine-rich
repeats to their cognate growth factors. As an example, fragments
can be obtained by digestion of the native polypeptide or by
synthesis of fragments based on the known amino acid sequence. Such
fragments can then be used in a competitive assay to determine
whether they retain binding activity. For example, decorin can be
attached to an affinity matrix, as by the method of Example II.
Labelled TGF-.beta., and the fragment in question can then be
contacted with the affinity matrix and the amount of TGF-.beta.
bound thereto determined.
[0037] As used herein, "decorin" refers to a proteoglycan having
substantially the structural characteristics attributed to it in
Krusius and Ruoslahti, supra. Human fibroblast decorin has
substantially the amino acid sequence presented in Krusius and
Ruoslahti, supra. "Decorin" refers both to the native composition
and to modifications thereof which substantially retain the
functional characteristics. Decorin core protein refers to decorin
that no longer is substantially substituted with glycosaminoglycan
and is included in the definition of decorin. Decorin can be
rendered glycosaminoglycan-free by mutation or other means, such as
by producing recombinant decorin in cells incapable of attaching
glycosaminoglycan chains to a core protein.
[0038] Functional equivalents of decorin include modifications of
decorin that retain its functional characteristics and molecules
that are homologous to decorin, such as biglycan and fibromodulin,
for example, that have the similar functional activity of decorin.
Modifications can include, for example, the addition of one or more
side chains that do not interfere with the functional activity of
the decorin core protein.
[0039] Since the regulatory factor binding proteins each contain
leucine-rich repeats of about 24 amino acids which can constitute
80% of the protein, it is likely that the fragments which retain
the binding activity occur in the leucine-rich repeats. However, it
is possible the binding activity resides in the carboxy-terminal
amino acids or the junction of the repeats and the carboxy terminal
amino acids.
[0040] The invention teaches a general method whereby one skilled
in the art can identify proteins which can bind to cell regulatory
factors or identify cell regulatory factors which bind to a certain
family of proteins. The invention also teaches a general method
whereby these novel proteins or known existing proteins can be
assayed to determine if they affect an activity of a cell
regulatory factor. Specifically, the invention teaches the
discovery that decorin and biglycan bind TGF-is and MRF and that
such binding can inhibit the cell regulatory functions of
TGF-.beta.s. Further, both decorin and biglycan are about 80%
homologous and contain a leucine-rich repeat of about 24 amino
acids in which the arrangement of the leucine residues is
conserved. As defined each repeat generally contains at least two
leucine residues and can contain five or more. These proteoglycans
are thus considered members of the same protein family. See
Ruoslahti, supra, Fisher et al., J. Biol. Chem., 264:4571-4576
(1989) and Patthy, J. Mol. Biol., 198:567-577 (1987), all of which
are incorporated by reference. Other known or later discovered
proteins having this leucine-rich repeat, i.e., fibromodulin, would
be expected to have a similar cell regulatory activity. The ability
of such proteins to bind cell regulatory factors could easily be
tested, for example by affinity chromatography or microtiter assay
as set forth in Example II, using known cell regulatory factors,
such as TGF-.beta.s. Alternatively, any later discovered cell
regulatory factor could be tested, for example by affinity
chromatography using one or more regulatory factor binding
proteins. Once it is determined that such binding occurs, the
effect of the binding on the activity of all regulatory factors can
be determined by methods such as growth assays as set forth in
Example III. Moreover, one skilled in the art could simply
substitute a novel cell regulatory factor for a TGF-.beta. or a
novel leucine-rich repeat protein for decorin or biglycan in the
Examples to determine their activities. Thus, the invention
provides general methods to identify and test novel cell regulatory
factors and proteins which affect the activity of these
factors.
[0041] The invention also provides a novel purified compound
comprising a cell regulatory factor attached to a purified
polypeptide wherein the polypeptide comprises the cell regulatory
factor binding domain of a protein and the protein is characterized
by a leucine-rich repeat of about 24 amino acids.
[0042] The invention further provides a novel purified protein,
designated MRF, having a molecular weight of about 20 kd, which can
be isolated from CHO cells, copurifies with decorin under
nondissociating conditions, separates from decorin under
dissociating conditions, changes the morphology of transformed 3T3
cells, and has an activity which is not inhibited with
anti-TGF-.beta.1 antibody. Additionally, MRF separates from
TGF-.beta.1 in HPLC.
[0043] The invention still further provides a method of purifying a
cell regulatory factor comprising contacting the regulatory factor
with a protein which binds the cell regulatory factor and has a
leucine-rich repeat of about 24 amino acids and to purify the
regulatory factor which becomes bound to the protein. The method
can be used, for example, to purify TGF-.beta.1 by using
decorin.
[0044] The invention additionally provides a method of treating a
pathology caused by a TGF-.beta.-regulated activity comprising
contacting the TGF-.beta. with a purified polypeptide, wherein the
polypeptide comprises the TGF-.beta. binding domain of a protein
and wherein the protein is characterized by a leucine-rich repeat
of about 24 amino acids, whereby the pathology-causing activity is
prevented or reduced. While the method is generally applicable,
specific examples of pathologies which can se treated include
cancer, a fibrotic disease, and glomerulonephritis. In fibrotic
cancer, for example, decorin can be used to bind TGF-.beta.,
destroying TGF-.beta.'s growth stimulating activity on the cancer
cell. Other proliferative pathologies include rheumatoid arthritis,
arteriosclerosis, adult respiratory distress syndrome, cirrhosis of
the liver, fibrosis of the lungs, post-myocardial infarction,
cardiac fibrosis, post-angioplasty restenosis, renal interstitial
fibrosis and certain dermal fibrotic conditions such as keloids and
scarring.
[0045] The present invention also provides a method of preventing
the inhibition of a cell regulatory factor. The method comprises
contacting a protein which inhibits an activity of a cell regulator
factor with a molecule which inhibits the activity of the protein.
For example, decorin could be bound by a molecule, such as an
antibody, which prevents decorin from binding TGF-.beta.s, thus
preventing decorin from inhibiting the TGF-.beta.s' activity. Thus,
the TGF-.beta.s wound healing activity could be promoted by binding
TGF-.beta.1 inhibitors.
[0046] In addition, decorin has been found to inhibit the binding
of TGF-.beta.s to their receptors. FIGS. 7, 8 and 10 show the
results of these studies in which cells bearing TGF-.beta.
receptors (betaglycan) were incubated with TGF-.beta. in the
presence and absence of decorin.
[0047] The present invention further relates to methods for the
prevention or reduction of scarring by administering decorin or a
functional equivalent of decorin to a wound. Dermal scarring is a
process, following a variety of dermal injuries, that results in
the excessive accumulation of fibrous tissue comprising collagen,
fibronectin, and proteoglycans. The induction of fibrous matrix
accumulation is a result of growth factor release at the wound site
by platelets and inflammatory cells. The original growth factor
believed to induce the deposition of fibrous scar tissue is
transforming growth factor-.beta. (TGF-.beta.). Decorin binds and
neutralizes a variety of biological functions of TGF-.beta.,
including the induction of extracellular matrix. Due to the lack of
elastic property of this fibrous extracellular matrix, the scar
tissue resulting from a severe dermal injury often impairs
essential tissue function and can result in an unsightly scar.
[0048] The advantage of using decorin or a functional equivalent,
such as biglycan or fibromodulin, in the methods of the present
invention is that it is a normal human protein and is believed to
be involved in the natural TGF-.beta. regulatory pathway. Thus,
decorin can be used to prevent or reduce dermal scarring resulting
from burn injuries, other invasive skin injuries, and cosmetic or
reconstructive surgery.
[0049] Decorin-treated wounds have been found to exhibit
essentially no detectable scarring compared to control wounds not
treated with decorin. The TGF-.beta.-induced scarring process has
been shown to be unique to adults and third trimester human
fetuses, but is essentially absent in fetuses during the first two
trimesters. The absence of scarring in fetal wounds has been
correlated with the absence of TGF-.beta. in the wound bed. In
contrast, the wound bed of adult tissue is heavily deposited with
TGF-.beta. and the fully healed wound is replaced by a reddened,
furrowed scar containing extensively fibrous, collagenous matrix.
The decorin-treated wounds were histologically normal and resembled
fetal wounds in the first two trimesters.
[0050] In addition, the present invention further relates to a
pharmaceutical composition containing decorin or its functional
equivalent, such as biglycan or fibromodulin, and a
pharmaceutically acceptable carrier useful in the above methods.
Pharmaceutically acceptable carriers include, for example,
hyaluronic acid, and aqueous solutions such as bicarbonate buffers,
phosphate buffers, Ringer's solution and physiological saline
supplemented with 5% dextrose or human serum albumin, if desired.
The pharmaceutical compositions can also include other agents that
promote wound healing known to those skilled in the art. Such
agents can include, for example, biologically active chemicals and
polypeptides, including RGD-containing polypeptides attached to a
biodegradable polymer as described in PCT WO 90/06767 published on
Jun. 28, 1990, and incorporated herein by reference. Such
polypeptides can be attached to polymers by any means known in the
art, including covalent or ionic binding, for example.
[0051] It is understood that modifications which do not
substantially affect the activity of the various molecules of this
invention including TGF-.beta., MRF, decorin, biglycan and
fibromodulin are also included within the definition of those
molecules. It is also understood that the core proteins of decorin,
biglycan and fibromodulin are also included within the definition
of those molecules.
[0052] The following examples are intended to illustrate but not
limit the invention.
EXAMPLE I
Expression and Purification of Recombinant Decorin and Decorin Core
Protein
[0053] Expression System
[0054] The 1.8 kb full-length decorin cDNA described in Krusius and
Ruoslahti, Proc. Natl. Acad. Sci. USA 83:7683 (1986), which is
incorporated herein by reference, was used for the construction of
decorin expression vectors. For the expression of decorin core
protein, cDNA was mutagenized so the fourth codon, TCT, coding for
serine, was changed to ACT coding for threonine, or GCT coding for
alanine. This was engineered by site-directed mutagenesis according
to the method of Kunkel, Proc. Natl. Acad. Sci USA 32:488 1985),
which is incorporated herein by reference. The presence of the
appropriate mutation was verified by DNA sequencing.
[0055] The mammalian expression vectors pSV2-decorin and
pSV2-decorin/CP-thr4 core protein were constructed by ligating the
decorin cDNA or the mutagenized decorin cDNA into 3.4 kb
HindIII-Bam HI fragment of pSV2 (Mulligan and Berg, Science
209:1423 (1980), which is incorporated herein by reference).
[0056] Dihydrofolate reductase (dhfr)-negative CHO cells (CHO-DG44)
were cotransfected with pSV2-decorin or pSV2-decorin/CP and
pSV2dhfr by the calcium phosphate coprecipitation method. The
CHO-DG44 cells transfected with pSV2-decorin are deposited with the
American Type Culture Collection under Accession Number ATCC No.
CRL 10332. The transfected cells were cultured in nucleoside-minus
alpha-modified minimal essential medium (.alpha.-MEM), (GIBCO, Long
island) supplemented with 9% dialyzed fetal calf serum, 2 mM
glutamine, 100 units/ml penicillin and 100 .mu.g/ml streptomycin.
Colonies arising from transfected cells were picked using cloning
cylinders, expanded and checked for the expression of decorin by
immunoprecipitation from .sup.35SO.sub.4-labeled culture
supernatants. Clones expressing a substantial amount of decorin
were then subjected to gene amplification by stepwise increasing
concentration of methotrexate (MTX) up to 0.64 .mu.M (Kaufman and
Sharp, J. Mol. Biol. 159:601 (1982), which is incorporated herein
by reference). All the amplified cell lines were cloned either by
limiting dilution or by picking single MTX resistant colonies.
Stock cultures of these established cell lines were kept in
MTX-containing medium. Before use in protein production, cells were
subcultured in MTX-minus medium as from stock cultures and passed
at least once in this medium to eliminate the possible MTX
effects.
[0057] Alternatively, the core protein was expressed in COS-1 cells
as described in Adams and Rose, Cell 41:1007, (1985), which is
incorporated herein by reference. Briefly, 6-well multiwell plates
were seeded with 3-5.times.10.sup.5 cells per 9.6 cm.sup.2 growth
area and allowed to attach and grow for 24 hours. Cultures were
transfected with plasmid DNA when they were 50-70% confluent. Cell
layers were washed briefly with Tris buffered saline (TBS)
containing 50 mM Tris, 150 mM NaCl pH 7.2, supplemented with 1 nM
CaCl.sub.2 and 0.5 mM MgCl.sub.2 at 37.degree. C. to prevent
detachment. The wells were incubated for 30 minutes at 37.degree.
C. with 1 ml of the above solution containing 2 .mu.g of closed
circular plasmid DNA and 0.5 mg/ml DEAE-Dextran (Sigma) of average
molecular mass of 500,000. As a control, cultures were transfected
with the pSV2 expression plasmid lacking any decorin insert or mock
transfected with no DNA. Cultures were then incubated for 3 hours
at 37.degree. C. with Dulbeccos Modified Eagle's medium (Irvine
Scientific) containing 10% fetal calf serum and 100 .mu.M
chloroquine (Sigma), after removing the DNA/TBS/DEAE-Dextran
solution and rinsing the wells with TBS. The cell Layers were then
rinsed twice and cultured in the above medium, lacking any
chloroquine, for approximately 36 hours. WI38 human embryonic lung
fibroblasts were routinely cultured in the same medium.
[0058] COS-1 cultures were radiolabeled 36-48 hours after
transfection with the plasmid DNAs. All radiolabeled metabolic
precursors were purchased from New England Nuclear (Boston, Mass.).
The isotopes used were .sup.35S-sulfate (460 mCi/ml),
L-[3,4,5-.sup.3H(N)]-leucine (140 Ci/mi) and L-[.sup.14C(U)]-amino
acid mixture (product number 445E). Cultures were labeled for 24
hours in Ham's F-12 medium (GIBCO Labs), supplemented with 10%
dialyzed fetal calf serum, 2 .mu.M glutamine and 1 mM pyruvic acid,
and containing 200 .mu.Ci/ml .sup.35S-sulfate or .sup.3H-leucine,
or 10 .mu.Ci/ml of the .sup.14C-amino acid mixture. The medium was
collected, supplemented with 5 mm EDTA, 0.5 mM
phenylmethylsulfonylfluoride, 0.04 mg/ml aprotinin and 1 .mu.g/ml
pepstatin to inhibit protease activity, freed of cellular debris by
centrifugation for 20 minutes at 2,000.times.G and stored at
-20.degree. C. Cell extracts were prepared by rinsing the cell
layers with TBS and then scraping with a rubber policeman into 1
ml/well of ice cold cell lysis buffer: 0.05 M Tris-HCl, 0.5 M NaCl,
0.1% BSA, 1% NP-40, 0.5% Triton X-100, 0.1% SDS, pH 8.3. The cell
extracts were clarified by centrifugation for 1.5 hours at
13,000.times.G at 4.degree. C.
[0059] Rabbit antiserum was prepared against a synthetic peptide
based on the first 15 residues of the mature for of the human
decorin core protein
(Asp-Glu-Ala-Ser-Gly-Ile-Gly-Pro-Glu-Val-Pro-Asp-Asp-Arg-Asp). The
synthetic peptide and the antiserum against it have been described
elsewhere (Krusius and Ruoslahti, 1986 supra.) Briefly, the peptide
was synthesized with a solid phase peptide synthesizer (Applied
Biosystems, Foster City, Calif.) by using the chemistry suggested
by the manufacturer. The peptide was coupled to keyhole limpet
hemocyanin by using U-succinmidyl 3-(2-pyridyldithio) propionate
(Pharmacia Fine Chemicals, Piscataway, N.J.) according to the
manufacturer's instructions. The resulting conjugates were
emulsified in Freund's complete adjuvant and injected into rabbits.
Further injections of conjugate in Freund's incomplete adjuvant
were given after one, two and three months. The dose of each
injection was equivalent to 0.6 mg of peptide. Blood was collected
10 days after the third and fourth injection. The antisera were
tested against the glutaraldehyde-cross linked peptides and
isolated decorin in ELISA (Engvall, Meth. Enzymol. 70:419-439
(1980)), in immunoprecipitation and immunoclotting, and by staining
cells in immunofluorescence, as is well known in the art.
[0060] Immunoprecipitations were performed by adding 20 .mu.l of
antiserum to the conditioned medium or cell extract collected from
duplicate wells and then mixing overnight at 4.degree. C.
Immunocomplexes were isolated by incubations for 2 hours at
4.degree. C. with 20 .mu.l of packed Protein A-agarose (Sigma). The
beads were washed with the cell lysis buffer, with three tube
changes, and then washed twice with phosphate-buffered saline prior
to boiling in gel electrophoresis sample buffer containing 10%
mercaptoethanol. Immunoprecipitated proteins were separated by
SDS-PAGE in 7.5-20% gradient gels or 7.5% non-gradient gels as is
well known in the art. Fluorography was performed by using
Enlightning (New England Nuclear) with intensification screens.
Typical exposure times were for 7-10 days at -70.degree. C.
Autoradiographs were scanned with an LXB Ultroscan XL Enhanced
Laser Densitometer to compare the relative intensities and
mobilities of the proteoglycan bands.
[0061] SDS-PAGE analysis of cell extracts and culture medium from
COS-1 cells transfected with the decorin-pSV2 construct and
metabolically radiolabeled with .sup.35S-sulfate revealed a
sulfated band that was not present in mock-transfected cells.
Immunoprecipitation with the antiserum raised against a synthetic
peptide derived from the decorin core protein showed that the new
band was decorin.
[0062] Expression of the construct mutated such that the serine
residue which is normally substituted with a glycosaminoglycan
(serine-4) was replaced by a threonine residue by SDS-PAGE revealed
only about 10% of the level of proteoglycan obtained with the
wild-type construct. The rest of the immunoreactive material
migrated at the position of free core protein.
[0063] The alanine-mutated cDNA construct when expressed and
analyzed in a similar manner yielded only core protein and no
proteoglycan form of decorin. FIG. 1 shows the expression of
decorin (lanes 1) and its threonine-4 (lanes 3) and alanine-4
(lanes 2) mutated core proteins expressed in COS cell
transfectants. .sup.35SO.sub.4-labeled (A) and .sup.3H-leucine
labeled (B) culture supernatants were immunoprecipitated with
rabbit antipeptide antiserum prepared against the NE.sub.2-terminus
of human decorin.
[0064] Purification of Decorin and Decorin Core Protein from Spent
Culture Media
[0065] Cells transfected with pSV2-decorin vector and amplified as
described above and in Yamaguchi and Ruoslahti, Nature 36:244-246
(1988), which is incorporated herein by reference, were grown to
90% confluence in 8 175 cm.sup.2 culture flasks in nucleoside minus
.alpha.-MEM supplemented with 9% dialyzed fetal calf serum, 2 mM
glutamine, 100 units/ml penicillin and 100 .mu.g/ml streptomycin.
At 90% confluence culture media was changed to 25 ml per flask of
nucleoside-free .alpha.-MEM supplemented with 6% dialyzed fetal
calf serum which had been passed through a DEAE Sepharose Fast Flow
column (Pharmacia) equilibrated with 0.25 M NaCl in 0.05 M
phosphate buffer, pH 7.4. Cells were cultured for 3 days, spent
media was collected and immediately made to 0.5 mM
phenylmethylsulfonyl fluoride, 1 .mu.g/ml pepstatin, 0.04 mg/ml
aprotinin and 5 mM EDTA.
[0066] Four hundred milliliters of the spent media were first
passed through gelatin-Sepharose to remove fibronectin and
materials which would bind to Sepharose. The flow-through fraction
was then mixed with DEAE-Sepharose pre-equilibrated in 50 mM
Tris/HCl, pH 7.4, .mu.plus 0.2 M NaCl and batch absorbed overnight
at 4.degree. C. with gentle mixing. The slurry was poured into a
1.6.times.24 cm column, washed extensively with 50 mM Tris/HCl, pH
7.4, containing 0.2 M NaCl and eluted with 0.2 M-0.8 M linear
gradient of NaCl in 50 mM Tris/HCl, pH 7.4. Decorin concentration
was determined by competitive ELISA as described in Yamaguchi and
Ruoslahti, supra. The fractions containing decorin were pooled and
further fractionated on a Sephadex gel filtration column
equilibrated with 8 M urea in the Tris-HCl buffer. Fractions
containing decorin were collected.
[0067] The core protein is purified from cloned cell lines
transfected with the pSV2-decorin/CP vector or the vector
containing the alanine-mutated cDNA and amplified as described
above. These cells are grown to confluency as described above. At
confluency the cell monolayer is washed four times with serum-free
medium and incubated in .alpha. MEM supplemented with 2 mM
glutamine for 2 hours. This spent medium is discarded. Cells are
then incubated with .alpha. MEM supplemented with 2 mM glutamine
for 24 hours and the spent media are collected and immediately made
to 0.5 mM phenylmethylsulfonyl fluoride, 1 .mu.g/ml pepstatin, 0.04
mg/ml aprotinin and 5 mM EDTA as serum-free spent media. The spent
media are first passed through gelatin-sepharose and the
flow-through fraction is then batch-absorbed to CM-Sepharose Fast
Flow (Pharmacia Fine Chemicals, Piscataway, N.J.) pre-equilibrated
in 50 mM Tris/HCl, pH 7.4 containing 0.1 M NaCl. After overnight
incubation at 4.degree. C., the slurry is poured into a column,
washed extensively with the pre-equilibration buffer and eluted
with 0.1M-1M linear gradient of NaCl in 50 mM Tris/H Ci, pH 7.4.
The fractions containing decorin are pooled, dialyzed against 50 mL
NH.sub.4HCO.sub.3 and lyophilized. The lyophilized material is
dissolved in 50 mM Tris, pH 7.4, containing 8M urea and applied to
a Sephacryl S-200 column (1.5.times.110 cm). Fractions containing
decorin core proteins as revealed by SDS-polyacrylamide
electrophoresis are collected and represent purified decorin core
protein.
EXAMPLE II
Binding of TGF-.beta. to Decorin
[0068] a. Affinity Chromatography of TGF-.beta. on
Decorin-Sepharose
[0069] Decorin and gelatin were coupled to cyanogen
bromide-activated Sepharose (Sigma) by using 1 mg of protein per ml
of Sepharose matrix according to the manufacturer's instructions.
Commercially obtained TGF-.beta.1 (Calbiochem, La Jolla, Calif.)
was .sup.125I-labelled by the chloramine T method (Frolik et al.,
J. Biol. Chem. 259:10995-11000 (1984)) which is incorporated herein
by reference and the labeled TGF-.beta. was separated from the
unreacted iodine by gel filtration on Sephadex G-25, equilibrated
with phosphate buffered saline (PBS) containing 0.1% bovine serum
albumin (BSA) (FIG. 2). [.sup.125I]-TGF-.beta.1 (5.times.10.sup.5
cpm) was incubated in BSA-coated polypropylene tubes with 0.2 ml of
packed decorin-Sepharose (.oval-solid.) or gelatin-Sepharose
(.smallcircle.) in 2 ml of PBS pH 7.4, containing 1 M NaCl and
0.05% Tween 20. After overnight incubation, the affinity matrices
were transferred into BSA-coated disposable columns (Bio Rad) and
washed with the binding buffer. Elution was effected first with 3 M
NaCl in the binding buffer and then with 8 M urea in the same
buffer. Fractions were collected, counted for radioactivity in a
gamma counter and analyzed by SDS-PAGE under nonreducing condition
using 12% gels.
[0070] FIG. 2A shows the radioactivity profile from the two columns
and the SDS-PAGE analysis of the fractions is shown in FIG. 2B. The
TGF-.beta.1 starting material contains a major band at 25 kd. This
band represents the native TGF-.beta.1 dimer. In addition, there
are numerous minor bands in the preparation. About 20-30% of the
radioactivity binds to the decorin column and elutes with 8 M urea,
whereas only about 2% of the radioactivity is present in the
urea-eluted fraction in the control fractionation performed on
gelatin-Sepharose (FIG. 2A). The decorin-Sepharose nonbound
fraction contains all of the minor components and some of the 25 kd
TGF-.beta.1, whereas the bound, urea-eluted fraction contains only
TGF-.beta.1 (FIG. 2B). These results show that TGF-.beta.1 binds
specifically to decorin, since among the various components present
in the original TGF-.beta.1 preparation, only TGF-.beta.1 bound to
the decorin-Sepharose affinity matrix and since there was very
little binding to the control gelatin-Sepharose affinity matrix.
The TGF-.beta.1 that did not bind to the decorin-Sepharose column
may have been denatured by the iodination. Evidence for this
possibility was provided by affinity chromatography of unlabeled
TGF-.beta.1 as described below.
[0071] In a second experiment, unlabeled TGF-.beta.1 180 ng was
fractionated an decorin-Sepharose as described above for
.sup.125I-TGF-.beta..
[0072] TGF-.beta.1 (180 ng) was incubated with decorin-Sepharose or
BSA-agarose (0.2 ml packed volume) in PBS (pH 7.4) containing 1%
BSA. After overnight incubation at 4.degree. C., the resins were
washed with 15 ml of the buffer and eluted first with 5 ml of 3 M
NaCl in PBS then with 5 ml of PBS containing 8 M urea. Aliquots of
each pool were dialyzed against culture medium without serum and
assayed for the inhibition of [.sup.3H]thymidine incorporation in
MvLu cells (Example III) The amounts of TGF-.beta.1 in each pool
were calculated from the standard curve of [.sup.3H]thymidine
incorporation obtained from a parallel experiment with known
concentration of TGF-.beta.1. The results show that the TGF-.beta.1
bound essentially quantitatively to the decorin column, whereas
there was little binding to the control column (Table 1). The
partial recovery of the TGF-.beta.1 activity may be due to loss of
TGF-.beta.1 in the dialyses.
1TABLE I Decorin-Sepharose affinity chromatography of nonlabeled
TGF-.beta.1 monitored by growth inhibition assay in MvLu cells.
TGF-.beta.1 (ng) Elution Decorin-Sepharose BSA-Sepharose Flow
through & wash 2.7 (2.3%) 82.0 (93.9%) 3 M NaCl 2.2 (1.8%) 1.3
(1.5%) 8 M Urea 116.0 (95.9%) 4.0 (4.6%)
[0073] b. Binding of TGF-.beta.1 to Decorin in a Microtiter Assay:
Inhibition by Core Protein and Biglycan
[0074] The binding of TGF-.beta.1 to decorin was also examined in a
microtiter binding assay. To perform the assay, the wells of a
96-well microtiter plate were coated overnight with 2 .mu.g/ml of
recombinant decorin in 0.1 M sodium carbonate buffer, pH 9.5. The
wells were washed with PBS containing 0.05% Tween (PBS/Tween) and
samples containing 5.times.10.sup.4 cpm of [.sup.125I]-TGF-.beta.1
and various concentrations of competitors in PBS/Tween were added
to each well. The plates were then incubated at 37.degree. C. for 4
hours (at 4.degree. C. overnight in experiments with chondroitinase
ABC-digested proteoglycans), washed with PBS/Tween and the bound
radioactivity was solubilized with 1% SDS in 0.2 M NaOH. Total
binding without competitors was about 4% under the conditions used.
Nonspecific binding, determined by adding 100-fold molar excess of
unlabeled TGF-.beta.1 over the labeled TGF-.beta.1 to the
incubation mixture, was about 13% of total binding. This assay was
also used to study the ability of other decorin preparations and
related proteins to compete with the interaction.
[0075] Completion of the decorin binding was examined with the
following proteins (FIG. 3; symbols are indicated in the section of
BRIEF DESCRIPTION OF THE FIGURES): (1) Decorin isolated from bovine
skin (PGII), (2) biglycan isolated from bovine articular cartilage
(PGI) (both PGI and PGII were obtained from Dr. Lawrence Rosenberg,
Monteflore Medical Center, N.Y.; and described in Rosenberg et al.,
J. Biol. Chem. 250:6304-6313, (1985), incorporated by reference
herein), and (3) chicken cartilage proteoglycan (provided by Dr.
Paul Goetinck, La Jolla Cancer Research Foundation, La Jolla,
Calif., and described in Goetinck, P. F., in The Glycoconjugates,
Vol. III, Horwitz, M. I., Editor, pp. 197-217, Academic Press,
NY).
[0076] For the preparation of core proteins, proteoglycans were
digested with chondroitinase ABC (Seikagaku, Tokyo, Japan) by
incubating 500 .mu.g of proteoglycan with 0.8 units of
chondroitinase ABC in 250 .mu.l of 0.1 M Tris/Cl, pH 8.0, 30 mM
sodium acetate, 2 mM PMSF, 10 mM N-ethylmalelmide, 10 mM EDTA, and
0.36 mL pepstatin for 1 hour at 37.degree. C. Recombinant decorin
and decorin isolated from bovine skin (PGII) inhibited the binding
of [.sup.125I]-TGF-.mu.1, as expected (FIG. 3A). Biglycan isolated
from bovine articular cartilage was as effective an inhibitor as
decorin. Since chicken cartilage proteoglycan, which carries many
chondroitin sulfate chains, did not show any inhibition, the effect
of decorin and biglycan is unlikely to be due to
glycosaminoglycans. Bovine serum albumin did not shown any
inhibition. This notion was further supported by competition
experiments with the mutated decorin core protein (not shown) and
chondroitinase ABC-digested decorin and biglycan (FIG. 3B). Each of
these proteins was inhibitory, whereas cartilage proteoglycan core
protein was not. The decorin and biglycan core proteins were
somewhat more active than the intact proteoglycans. Bovine serum
albumin treated with chondroitinase ABC did not shown any
inhibition. Additional binding experiments showed that
[.sup.125-I]-TGF-.beta.1 bound to microtiter wells coated with
biglycan or its chondroitinase-treated core protein. These results
show that TGF-.beta.1 binds to the core protein of decorin and
biglycan and implicates the leucine-rich repeats these proteins
share as the potential binding sites.
EXAMPLE III
Analysis of the Effect of Decorin on Cell Proliferation Stimulated
or Inhibited by TGF-.beta.1
[0077] The ability of decorin to modulate the activity of
TGF-.beta.1 was examined in [.sup.3H]thymidine incorporation
assays. In one assay, an unamplified CHO cell line transfected only
with pSV2dhfr (control cell line A in reference 1, called CHO cells
here) was used. The cells were maintained in nucleoside-free
alpha-modified minimal essential medium (.alpha.-MEM, GIBCO, Long
Island, N.Y.) supplemented with 9% dialyzed fetal calf serum (dFCS)
and [.sup.3H]thymidine incorporation was assayed as described
(Cheifetz et al., Cell 48:409-415 (1987)). TGF-.beta.1 was added to
the CHO cell cultures at 5 ng/ml. At this concentration, it induced
a 50% increase of [.sup.3H]thymidine incorporation in these cells.
Decorin or BSA was added to the medium at different concentrations.
The results are shown in FIG. 4A. The data represent percent
neutralization of the TGF-.beta.1-induced growth stimulation, i.e.,
[.sup.3H]thymidine incorporation, in the absence of either
TGF-.beta.1 or decorin=0%, incorporation in the presence of
TGF-.beta.1 but not decorin=100%. Each point shows the
mean.+-.standard deviation of triplicate samples. Decorin
(.oval-solid.) BSA (.smallcircle.).
[0078] Decorin neutralized the growth stimulatory activity of
TGF-.beta.1 with a half maximal activity at about 5 .mu.g/ml.
Moreover, additional decorin suppressed the [.sup.3H]-thymidine
incorporation below the level observed without any added
TGF-.beta.1, demonstrating that decorin also inhibited TGF-.beta.
made by the CHO cells themselves. Both the decorin-expressor and
control CHO cells produced an apparently active TGF-.beta.
concentration of about 0.25 ng/ml concentration into their
conditioned media as determined by the inhibition of growth of the
mink lung epithelial cells. (The assay could be performed without
interference from the decorin in are culture media because, as
shown below, the effect of TGF-.beta. on the mink cells was not
substantially inhibited at the decorin concentrations present in
the decorin-producer media.)
[0079] Experiments in MvLu mink lung epithelial cells (American
Type Culture Collection CCL 64) also revealed an effect by decorin
on the activity of TGF-.beta.1. FIG. 4B shows that in these cells,
the growth of which is measured by thymidine incorporation, had
been suppressed by TGF-.beta.1. Assay was performed as in FIG. 4A,
except that TGF-.beta.1 was added at 0.5 ng/ml. This concentration
of TGF-.beta. induces 50% reduction of [.sup.3H]-thymidine
incorporation in the MvLu cells. The data represent neutralization
of TGF-.beta.-induced growth inhibition; i.e., [.sup.3H]-thymidine
incorporation in the presence of neither TGF-.beta. or
decorin=100%; incorporation in the presence of TGF-.beta. but not
decorin=0%.
EXAMPLE IV
New Decorin-Binding Factor That Controls Cell Spreading and
Saturation Density
[0080] Analysis of the decorin contained in the overexpressor
culture media not only uncovered the activities of decorin
described above, but also revealed the presence of other
decorin-associated growth regulatory activities. The overexpressor
media were found to contain a TGF-.beta.-like growth inhibitory
activity. This was shown by gel filtration of the DEAE-isolated
decorin under dissociating conditions. Serum-free conditioned
medium of decorin overexpressor CHO-DG44 cells transfected with
decorin cDNA was fractionated by DEAE-Sepharose chromatography in a
neutral Tris-HCl buffer and fractions containing growth inhibitory
activity dialyzed against 50 mM NH.sub.4HCO.sub.3, lyophilized and
dissolved in 4 M with guanidine-HCl in a sodium acetate buffer, pH
5.9. The dissolved material was fractionates on a 1.5.times.70 cm
Sepharose CL-6B column equilibrated with the same guanidine-HCl
solution. The fractions were analyzed by SDS-PAGE, decorin ELISA
and cell growth assays, all described above. Three protein peaks
were obtained. One contained high molecular weight proteins such as
fibronectin (m.w. 500,000) and no detectable growth regulatory
activities, the second was decorin with the activities described
under Example III and the third was a low molecular weight
(10,000-30,000-dalton) fraction that had a growth inhibitory
activity in the mink cell assay and stimulated the growth of the
CHO cells. FIG. 5 summarizes these results. Shown are the ability
of the gel filtration fractions to affect [.sup.3H]-thymidine
incorporation by the CHO cells and the concentration of decorin as
determined by enzyme immunoassay. Shown also (arrows) are the
elution positions of molecular size markers: BSA, bovine serum
albumin (Mr=66,000); CA, carbonic anhydrase (Mr=29,000); Cy,
cytochrome c (Mr=12,400); AP, aprotinin (Mr=6,500), TGF,
[.sup.125I]-TGF-.beta.1 (Mr=25,000).
[0081] The nature of the growth regulatory activity detected in the
low molecular weight fraction was examined with an anti-TGF-.beta.1
antiserum. The antiserum was prepared against a synthetic peptide
from residues 78-109 of the human mature TGF-.beta.1. Antisera
raised by others against a cyclic form of the same peptide, the
terminal cysteine residues of which were disulfide-linked, have
previously been shown to inhibit the binding of TGF-.beta.1 to its
receptors (Flanders et al., Biochemistry 27:739-746 (1988),
incorporated by reference herein). The peptide was synthesized in
an Applied Biosystems solid phase peptide synthesizer and purified
by HPLC. A rabbit was immunized subcutaneously with 2 mg per
injection of the peptide which was mixed with 0.5 mg of methylated
BSA (Sigma, St. Louis, Mo.) and emulsified in Freund's complete
adjuvant. The injections were generally given four weeks apart and
the rabbit was bled approximately one week after the second and
every successive injection. The antisera used in this work has a
titer (50% binding) of 1:6,000 in radioimmunoassay, bound to
TGF-.beta.1 in immunoblots.
[0082] This antiserum was capable of inhibiting the activity of
purified TGF-.beta.1 on the CHO cells. Moreover, as shown in FIG.
5, the antiserum also inhibited the growth stimulatory activity of
the low molecular weight fraction as determined by the
[.sup.3H]-thymidine incorporation assay on the CHO cells.
Increasing concentrations of an IgG fraction prepared from the
anti-TGF-.beta.1 antiserum suppressed the stimulatory effect of the
low molecular weight fraction in a concentration-dependent manner
(.oval-solid.). IgG from a normal rabbit serum had no effect in the
assay (.smallcircle.).
[0083] The above result identified the stimulatory factor in the
low molecular weight fraction as TGF-.beta.1. However, TGF-.beta.1
is not the only active compound in that fraction. Despite the
restoration of thymidine incorporation by the anti-TGF-.beta.1
antibody shown in FIG. 5, the cells treated with the low molecular
weight fraction were morphologically different from the cells
treated with the control IgG or cells treated with antibody alone.
This effect was particularly clear when the antibody-treated, low
molecular weight fraction was added to cultures of H-ras
transformed NIH 3T3 cells (Der et al., Proc. Natl. Acad. Sci. USA
79:3637-3640 (1982)). As shown in FIG. 6, cells treated with the
low molecular weight fraction and antibody (micrograph in panel B)
appeared more spread and contact inhibited than the control cells
(micrograph in panel A). This result shows that the CHO
cell-derived recombinant decorin is associated with a cell
regulatory factor, MRF, distinct from the well characterized
TGF-.beta.'s.
[0084] Additional evidence that the new factor is distinct from
TGF-.beta.1 came from HPLC experiments. Further separations of the
low molecular weight from the Sepharose CL-6B column was done on a
Vydac C4 reverse phase column (1.times..times.25 cm, 5 .mu.m
particle size, the Separations Group, Hesperia, Calif.); 0.1%
trifluoroacetic acid. Bound proteins were eluted with a gradient of
acetonitrite (22-40%) and the factions were assayed for
growth-inhibitory activity in the mink lung epithelial cells and
MRF activity in H-ras 3T3 cells. The result showed that the
TGF-.beta.1 activity eluted at the beginning of the gradient,
whereas the MRF activity eluted toward the end of the gradient.
EXAMPLE V
Inhibition of TGF-.beta. Binding
[0085] A. Cross Linking of [.sup.125I]-TGF-.beta. to HepG2
Cells
[0086] About 2.5.times.10.sup.4 HepG2 cells (human hepatocellular
carcinoma, ATCC No. HB 8065) were incubated with 100 pM
.sup.125I-TGF-.beta. in the presence of recombinant decorin,
TGF-.beta., or .alpha.-TGF-.beta. antibody for 2 hours at room
temperature. Cells were washed four times prior to suspension in
binding buffer (128 mM NaCl, 5 mM KCl, 5 mM Mg.sub.2SO.sub.4, 1.2
mM CaCl.sub.2, 50 mM HEPES, 2 mg/ml BSA, pH 7.5) containing 0.25 mM
disuccinimidyl suberate (DSS) for 15 minutes. Cells were
subsequently washed in washing buffer (binding buffer without BSA)
containing 150 mM sucrose and lysed before suspension in Laemmli
sample buffer, which is known to those skilled in the art,
containing SDS. The lysates were resolved on 4-12% SDS-PAGE under
reducing and non-reducing conditions. Cross-linked TGF-.beta. was
visualized by autoradiography.
[0087] FIG. 7 shows the results of the studies. Decorin inhibits
the binding of TGF-.beta. to .beta. glycan, a TGF-.beta. receptor
found on HepG2 cells.
[0088] B. Cross Linking of [.sup.125I]-TGF-.beta. to MG-63
Cells
[0089] About 10.sup.5 MG-63 cells (male osteosarcoma, ATCC No. CRL
1427) were incubated with 150 mm [.sup.125I]-TGF-.beta. in the
presence of a recombinant decorin preparation (designated as DC-13)
or TGF-.beta. for 2 hours at room temperature. Cells were washed
four times in ice cold binding buffer of Example V(A) prior to
suspension in binding buffer containing 0.25 mM DSS for 15 minutes.
Cells were washed in 250 mM sucrose buffer before lysis in 1%
Triton X-100 buffer, containing protease inhibitors. Lysed cells
were centrifuged at 12,000.times.g to remove nuclei. Equivalent
volumes of Laemmli SDS sample buffer were added to each supernatant
prior to electrophoresis through 4-12% tris-glycine gels. The
cross-linked TGF-225 was visualized by autoradiography.
[0090] FIG. 8 shows the results of the studies. Similar to the
above studies with HepG2 cells, decorin also inhibits TGF-.beta.
binding to its receptors on the MG-63 cells.
[0091] C. Binding Studies of .sup.125I-TGF-.beta. to Immobilized
Decorin
[0092] A 96-well Linbro microtiter plate was coated with 0.5
.mu.g/ml recombinant decorin at 50 .mu.l/well. The plate was placed
in a 37.degree. C. incubator overnight and thereafter washed 3
times with 200 .mu.l PBS (0.15 M NaCl) per well to remove unbound
decorin. TGF-.beta. labeled with .sup.125I (400 pM, New England
Nuclear, Bolton-Hunter Labeled) was pre-incubated with or without
competitors in 200 .mu.l PBS/0.05% Tween-20 for 1 hour, 45 minutes
at room temperature. Competitors included recombinant human decorin
preparations (DC-9 and DC-12) and biglycan, with MBP as a negative
control. DC-9 and DC-12 are different preparations of recombinant
human decorin; PT-71 or MBP (maltose-binding protein) is a negative
control; and biglycan is recombinant human biglycan.
[0093] Fifty .mu.l/well of the pre-incubated TGF-.beta. mixture or
control were added and incubated overnight at 0.degree. C.
Following the incubation, 50 .mu.l of the free TGF-.beta.
supernatants were transferred to labeled tubes. The plate was
washed 3 times with 0.05% Tween-20 in PBS (200 .mu.l/well).
Reducing sample suffer (2.times.Laemmli sample buffer) was added at
100 .mu.l/well and incubated at 37.degree. C. for 30 minutes. While
gently pulsing the solution, 100 .mu.l of bound
.sup.125I-TGF-.beta. was removed from each well and transferred
into tubes for counting in a gamma counter. The 50 .mu.l free
TGF-.beta. samples were counted in parallel to the 100 .mu.l bound
TGF-.beta. samples to obtain the bound:free ratio. The results of
the binding studies with immobilized decorin are summarized in FIG.
9.
[0094] D. Binding of .sup.125I-TGF-.beta. to HepG2 Cells
[0095] About 2.5.times.10.sup.4 HepG2 cells were incubated with 250
pM [.sup.125I]-TGF-.beta., in the presence of recombinant human
decorin (DC-12) or PT-71 (MBP) for 2 hours at room temperature.
Cells were washed with the washing buffer of Example V(A) four
times before determination of bound CPM.
[0096] The results are summarized in FIG. 10. Table II provides
numerical data for decorin (DC-12) inhibition of TGF-.beta. binding
to HepG2 cells. The "% Change" represents the difference in the
mean cpm of the test samples compared the cpm of the medium
(negative control). The .alpha.-TGF-.beta. antibody inhibits the
binding of labeled TGF-.beta. to cells bearing TGF-.beta. receptors
and serves as a positive control.
2TABLE II BINDING OF 125I-TGF-.beta.1 TO HEPG2 CELLS CPM Treatment
Concentration Bound Mean % Change Medium* -- 13,899** 12,872 .+-.
856 -- 13,898 12,529 11,764 12,694 12,448 TGF-.beta.1 2.5 .times.
10E-8M 3,092 2,812 .+-. 275 -78 2,543 2,800 Anti-TGF-.beta.1 2.5
.times. 10E-7M 6,191 4,959 .+-. 1,180 -61 (R&D) 4,848 3,839
Decorin 2.5 .times. 10E-6M 2,745 2,511 .+-. 493 -80 (DC-12) 2,844
1,945 2.5 .times. 10E-7M 4,258 4,741 .+-. 1,021 -63 5,914 4,052 2.5
.times. 10E-8M 13,596 12,664 .+-. 1,005 -2 12,798 11,599 PT-71 2.5
.times. 10E-6M 11,859 12,449 .+-. 636 -3 13,129 12,348 2.5 .times.
10E-7M 11,259 10,541 .+-. 1,045 -18 11,022 9,343 2.5 .times. 10E-8M
10,886 10,589 .+-. 424 -18 10,778 10,104 *25,000 HepG2 cells
obtained from subconfluent cultures were incubated with 250 pM
125I-TGF-.beta.1 and TGF-.beta., anti TGF-.beta., decorin, or
decorin fragments for 2 hours at room temperature. **Unbound
125I-TGF-.beta.1 was separated from bound by washing cells
4.times..
EXAMPLE VI
Scarring Studies
[0097] Adult mice were incised with paired longitudinal wounds on
the shaved dorsal skin. Care was taken to cut through the
panniculus down to the skeletal musculature of the dorsal skin. The
incisions were treated with a 250 .mu.l single dose of either 10
mg/ml hyaluronic acid (control), or a decorin (0.5
mg/ml)/hyaluronic acid (10 mg/ml) mixture in TBS. To form the
mixture, 0.5 mg/ml of recombinant decorin was mixed with 10 mg/ml
of hyaluronic acid. Each mouse had a blinded control and treated
incision. The wounds were sutured closed. Following 14 days, the
incisions were monitored grossly and harvested for histology.
Frozen sections of the control and treated incision sites (4
microns) were analyzed using standard histological procedures with
Masson's trichrome to visualize the staining.
[0098] The hyaluronic acid control exhibited a typical dermal scar
as is seen in normal adult animals, whereas the decorin-treated
wounds exhibited no detectable scar and were essentially normal
histologically. The decorin-treated wounds resembled fetal wounds
in the first two trimesters.
EXAMPLE VII
Cloning of Human Biglycan and Fibromodulin cDNAs
[0099] Total cellular RNA was extracted by using guanidinium
isothiocyanate (Sambrook et al., 1989) from subconfluent cultures
of WI-38 human lung fibroblasts (ATCC Accession No. CCL 75;
Rockville, Md.) that had been exposed to TGF-.beta.1 (3 ng/mi) for
12 hours. Total cellular RNA (1 .mu.g) was reverse transcribed with
MoMuLV reverse transcriptase using random hexanucleotides for cDNA
priming (Kawasaki, 1989). Double-stranded cDNAs encoding the
full-length coding sequences of human biglycan or human
fibromodulin were generated by amplification of the reverse
transcribed WI-38 RNAs using amplimers based on the published
sequences of human biglycan (Fisher et al., 1989) or bovine
fibromodulin (Oldberg et al., 1989). For decorin, a previously
described decorin cDNA was used as a template (Krusius and
Ruoslahti, 1986). The PCR products were subcloned into pBluescript
(Stratagene, La Jolla, Calif.). The identities of the resulting PCR
products were verified by DNA sequencing (Sanger, et al., 1977).
The PCR generated biglycan clone differs from the published
biglycan sequence in five bases. Two sequence differences could be
reconciled by re-sequencing of clone p16 of Fisher et al. (1989),
kindly provided by Dr. L Fisher. The remaining differences resulted
in one amino acid exchange (Lys.sub.176 to Asn.sub.176).
[0100] Human fibromodulin was found to be highly homologous to its
bovine equivalent. DNA sequencing analysis of the 1.2 kb PCR
product revealed a 1128 bp open reading frame that codes for a 376
amino acid protein. The deduced protein sequence shares 92%
sequence identity with the previously published bovine fibromodulin
sequence. FIG. 11 shows the human fibromodulin amino acid sequence
aligned with the bovine fibromodulin sequence and the sequences of
the other two proteoglycans used in this study.
EXAMPLE VIII
Expression Vector Construction and Fusion Protein Purification
[0101] A modified MBP sequence with a COCH-terminal factor
Xa-cleavage site was introduced into a vector, pQE-8 (Quiagen;
Chatsworth, Calif.), that also encoded an affinity tag consisting
of a cassette of six histidines. The pQE-8/MBP expression vector
was generated by subcloning the BglII/BamHI DNA fragment coding for
MBP and a factor Xa protease cleavage site into the BamHI site of
pQE-8 (FIG. 12). BamHI fragments coding for the core proteins of
human biglycan, decorin or fibromodulin were subsequently cloned
into the resulting single BamHI site 1 pQE-8/MBP (FIG. 12).
[0102] The histidine affinity tag allowed the purification of the
fusion proteins to >95% purity in a single purification step
that employs a Ni-metal-chelate affinity column (Hochuli et al.,
1987).
[0103] Biglycan, decorin, and fibromodulin were prepared according
to the instructions of Qiagen (Chatsworth, Calif.). Briefly,
recombinant bacteria were grown in LB medium containing ampicillin
(100 .mu.g/ml) and kanamycin (25 .mu.g/ml) at 37.degree. C. to a
density of OD.sub.600-0.6 to 0.8. IPTG was then added to a final
concentration of 2 mM and protein expression was allowed to proceed
for 3 h. The bacteria were then collected by centrifugation
(5000.times.g, 15 min) and lysed for 45 to 60 minutes in buffer A
(0.1 M NaHPO.sub.4, 0.01 M Tris, 6 M guanidinium-HCl, pH 8.0). The
lysates were centrifuged for 20 min at 20,000.times.g. Imidazole
was added to the supernatants to a final concentration of 10 mM and
the mixtures were loaded on Ni-NTA columns. The columns were washed
with several column volumes of buffer B (0.1 M NaHPO,, 0.01 M Tris,
8 M urea, pH 8.0) and was eluted with buffer C (buffer B adjusted
to pH 5.9). Protein-containing fractions were adjusted to pH 8 by
adding 1 M Tris-HCl in 8 M urea. After reduction of the proteins
with dithiothreitol and carboxymethylation with iodoacetamide
(Charbonneau, 1989), the proteins were separated from the reagents,
and buffers exchanged for the respective binding buffers by gel
filtration using PD-10 columns (Pharmacia LKB Biotechnology).
[0104] The purified MBP-fusion proteins displayed electrophoretic
mobilities in SDS-PAGE compatible with the predicted amino acid
sequences (FIG. 13). These proteins were relatively soluble in
physiological buffers, although some precipitation occurred during
prolonged storage at 4.degree. C.
EXAMPLE IX
Protein Iodination
[0105] The bacterially expressed fusion proteins were iodinated
using IODO-GEN according to the manufacturer's instructions (Pierce
Chemical Co.). Briefly, 50 .mu.l of an IODO-GEN solution (1 mg/25
ml CHCl.sub.3) were dried to the bottom of a borosilicate glass
tube. Protein (10-20 .mu.g) dissolved in iodination buffer (0.1 M
NaP, 1 mM EGTA, 150 mM NaCl, pH 7.4) and carrier-free Na.sup.125I
were added to the tube. After incubation for 12 minutes at room
temperature, 200 .mu.l of iodination buffer and the mixture were
loaded into a PD-10 column for radiochemical purification of the
labeled protein. The specific activities and radiochemical purities
of the labeled fusion proteins were calculated by determination of
the picric acid precipitable radioactive protein fraction in the
labeling mixture before and after the purification step. The
specific activities ranged from 2300 to 2800 Ci/mmol, with
radiochemical purities greater than 95%. TGF-.beta.1, 2 AND 3 (1-5
.mu.g) were labeled as described above using 0.25 M NaP., 2 M urea,
pH 7.4, as iodination buffer.
EXAMPLE X
Equilibrium Binding Experiments
[0106] Solid-phase binding assays were performed incubating
radiolabeled MBP-proteoglycan fusion proteins in microtiter wells
coated with increasing amounts of TGF-.beta.1.
[0107] Immulon-2 microtiter wells (Dynatech; Chantilly, Va.) were
coated with TGF-.beta.1 (75 .mu.l, 1 .mu.g/ml) or other proteins
dissolved in 0.1 M bicarbonate buffer, pH 9.5, overnight at
4.degree. C. The coated wells were then flicked dry and incubated
with 200 .mu.l of binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 2% BSA, 0.05% Tween-20, 0.01% NaN.sub.2) for 3 hours at
37.degree. C. to block nonspecific binding sites. Plates were
either used immediately or stored for future use at -20.degree. C.
for up to 2 weeks. For binding assays, the blocked wells were
washed once then labeled and unlabeled proteins were added in a
total volume of 100 .mu.l binding buffer and incubated for 6 hours
at 37.degree. C. if not indicated otherwise. After that, the well
contents were removed by aspiration and the wells were washed three
times with ice-cold binding buffer. Binding to the
surface-immobilized proteins was determined by counting the entire
wells in a gamma counter.
[0108] Non-specific binding to the wells was less than 5% of the
total radioactivity added. The coating efficiency for TGF-.beta.1
was 58.7.+-.0.5% (n=3), giving approximately 44 ng of TGF-.beta.1
per well. The coating efficiency was calculated by adding a small
amount of .sup.125I-labeled TGF-.beta.1 to the coating solution and
determining the surface-associated radioactivity after the
overnight incubation period and the subsequent washing step. All
experiments were performed in duplicate or triplicate.
[0109] Radiolabeled MBP-biglycan (MBP-BG), MBP-decorin (MBP-DEC)
and MBP-fibromodulin (MBP-FM) bound to TGF-.beta.1-coated wells in
a concentration-dependent manner, displaying maximum binding of
50%, 20%, and 55%, respectively (FIG. 14). Radiolabeled MBP alone
did not bind to the TGF-.beta.1-coated wells.
[0110] The binding of the radiolabeled fusion proteins was specific
for TGF-.beta., since little to no binding was observed to
immobilized NGF, EGF, insulin or platelet factor 4. MBP-FM bound
slightly to immobilized TGF-.beta.1 precursor protein, but MBP-BG
and MBP-DEC did not (FIG. 15).
[0111] Since the biglycan fusion protein, MBP-biglycan, showed high
binding activity toward TGF-.beta.1, it was used to characterize
further the proteoglycan-TGF-.beta.1 interactions. The binding of
MBP-BG to TGF-.alpha.1 was time- and temperature-dependent (FIG.
16). Binding increased rapidly at 37.degree. C. but very slowly at
4.degree. C., reaching at 4.degree. C. only about 20% of the
maximum binding seen at 37.degree. C.
[0112] Unlabeled MBP-BG competed for the binding of 125I-labeled
MBP-BG to TGF-.beta.1 in a concentration-dependent manner (FIG.
17A). MBP-DEC and MBP-FM competed for the binding of labeled MBP-BG
to TGF-.beta.1. They were about equally effective competitors as
MBP-BG, yielding half-maximal inhibitory concentrations of about 30
to 40 nM. MBP was inactive. When purified bovine proteoglycans were
used as competitors, biglycan and decorin were found to be less
potent than fibromodulin; the half-maximal inhibitory
concentrations were 150, 200 and 10 nM, respectively.
[0113] Data from the assays shown in FIG. 17A were analyzed using
the LIGAND computer program (Munson and Rodbard, 1980) to calculate
dissociation constants (K.sub.d) and maximal binding site
concentrations (B.sub.max) for the interaction of the proteoglycan
fusion proteins with TGF-.beta.1. Best results were obtained for
two-site binding models with K.sub.d values ranging from 1 to 17 nM
for high affinity binding sites and 47 to 200 nM for low affinity
binding sites, respectively. The B.sub.max values were in the range
of 10.sup.-13 moles per well for the high affinity binding sites
and 1.6.times.10.sup.-12 moles per well for the low affinity
binding sites, respectively. Given a TGF-.beta.1 coating
concentration of 1 .mu.g/ml, a coating volume of 75 .mu.l and a
coated efficiency of about 60%, these values indicate a molar ratio
between proteoglycan fusion protein and TGF-.beta.1 of one to ten
for the high affinity binding site and one to one for the low
affinity binding site, respectively.
[0114] Proteoglycans were also tested for their ability to bind
TGF-.beta.2 and TGF-.beta.3, the other known mammalian isoforms of
TGF-.beta.. Binding of TGF-.beta.1, 2, and 3 to immobilized
MBP-biglycan was inhibited by all three fusion proteins and all
three intact proteoglycans (FIG. 18). TGF-.beta.3 binding to MBP-BG
was more effectively inhibited by decorin and biglycan than
fibromodulin.
[0115] Solid-phase radioligand binding studies showed that
recombinant fusion proteins containing the core protein sequences
of human biglycan, decorin and fibromodulin compete for binding of
labeled MBP-biglycan to TGF-.beta.1 with similar affinities,
indicating that functionally highly conserved regions of the core
proteins are involved in the binding to TGF-.beta.. The fact that
bacterially produced recombinant proteoglycan core proteins had
similar activities to recombinant decorin produced by mammalian
cells (Yamaguchi et al., 1990) and tissue-derived proteoglycans
definitively established the presence of the TGF-.beta. binding
activity in the core proteins of these proteoglycans.
EXAMPLE XI
[0116] Cell Binding Experiments
[0117] The ability of the proteoglycan fusion proteins to compete
for TGF-.beta.1 binding to cells was tested in cell-binding
experiments. Cell binding experiments were performed according to
Massague (1987). Briefly, subconfluent monolayers of MvLu or HepG2
cells (ATCC Accession Nos. CCL 64 and HB 8065, respectively;
Rockville, Md.) in 48- or 24-well cell culture dishes (Costar;
Cambridge, Mass.) maintained in DMEM containing 10% FCS were used
in binding experiments. Cells were incubated with labeled
TGF-.beta.1 in the presence or absence of unlabeled TGF-.beta.1 or
proteoglycan fusion proteins. The cells were washed twice with
ice-cold binding buffer (128 mM NaCl, 5 mM KCl, 5 mM MgSO.sub.4,
1.2 mM CaCl.sub.2, 50 mM HEPES, pH 7.5, 2 mg/ml BSA) and then
incubated with cell binding buffer for 30 minutes at 4.degree. C.
to remove endogenous receptor-associated TGF-.beta.. Samples
containing labeled and unlabeled proteins were added to the wells
in a total volume of 100 .mu.l for 48-well or 200 .mu.l for 24-well
dishes and were incubated at 4.degree. C. with gentle agitation an
a rotary platform. After incubation for 4 hours, the cells were
washed three times with binding buffer. One-hundred microliters
(200 .mu.l for 24-well dishes) of solubilization buffer (25 mM
HEPES, pH 7.5, 10% glycerol, 1% Triton X-100, 1 mg/ml BSA) were
added to each well and incubated for 30 minutes at 4.degree. C.
Cell-associated radioactivity from triplicate samples was
determined by counting a portion of the solubilized cells in a
gamma counter.
[0118] Whereas unlabeled TGF-.beta.1 competed effectively for the
binding of labeled TGF-.beta.1 to all three types of TGF-.beta.
receptors, much higher concentrations of the proteoglycan fusion
proteins were needed to compete for TGF-.beta.1 binding to these
cells (FIG. 19A). Half-maximal competition was achieved by fusion
protein concentrations averaging about 3 .mu.M.
EXAMPLE XII
Cross-Linking of TGF-.beta. to Receptors
[0119] For receptor cross-linking, cells were grown in 24-well
culture dishes and were processed as described above. After
incubation with labeled and unlabeled ligands, the cells were
washed once with binding buffer and three times with binding buffer
without BSA. Then 100 .mu.l of binding buffer (without BSA)
containing disuccinimidyl suberate (final concentration 0.2 mM)
were added, and the cells were incubated for 15 min at 4.degree. C.
After the cross-linking reaction, the cells were lysed in 100 .mu.l
of solubilization buffer (125 mM NaCl, 10 mM Tris, pH 7, 1 mM EDTA,
1% Triton X-100, 10 .mu.g/ml leupeptin, 10 .mu.g/ml antipain, 50
mg/ml aprotinin, 100 .mu.g/ml benzamidine-HCI, 10 .mu.g/ml
pepstatin). The lysates were mixed with sample buffer and analyzed
by SDS-PAGE under reducing conditions using precast 4-12% Novex
gels. After electrophoresis the gels were dried and exposed to
XAR-100 x-ray film for several days at -70.degree. C.
[0120] Cross-linking experiments revealed that TGF-.beta.1 binding
to the type I and type III receptors was affected more by all three
proteoglycan fusion proteins than binding to the type II receptors,
which was essentially unchanged (FIG. 19B). Laser densitometry
analyses of the respective autoradiograms showed that the binding
of labeled TGF-.beta.1 to type I and III receptors was decreased by
approximately 25% or 50%, respectively, at the proteoglycan
concentration used.
[0121] Competition was most effective for TGF-.beta. binding to the
type I and type III TGF-.beta. receptors, perhaps because these
receptors have a lower affinity for TGF-.beta. than does the type
II receptor. While the affinities of the proteoglycans for the
TGF-.beta. are much lower than those of any of the receptors, all
of the proteoglycans are abundant in tissues potentially making up
in concentration what they may lack in affinity. Moreover, the
experimental conditions used in the cell binding experiments do not
favor the binding of TGF-.beta. to proteoglycans.
[0122] Although the invention has been described with reference to
the presently-preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Accordingly, the invention is limited only by the
following claims.
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