U.S. patent application number 10/159749 was filed with the patent office on 2002-12-19 for morphogenic proteins.
This patent application is currently assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Harland, Richard, Hsu, David.
Application Number | 20020192219 10/159749 |
Document ID | / |
Family ID | 25165676 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020192219 |
Kind Code |
A1 |
Harland, Richard ; et
al. |
December 19, 2002 |
Morphogenic proteins
Abstract
DAN (Differential-screening-selected gene Aberrative in
Neuroblastoma) and gremlin proteins and related nucleic acids are
provided. Included are natural DAN and gremlin homologs from
several species and proteins comprising a DAN or gremlin domain
having specific activity, particularly the ability to antagonize a
bone morphogenic protein. The proteins may be produced
recombinantly from transformed host cells with the subject nucleic
acids. Also provided are isolated hybridization probes and primers
capable of specifically hybridizing with the disclosed genes,
specific binding agents and methods of making and using the subject
compositions.
Inventors: |
Harland, Richard; (Berkeley,
CA) ; Hsu, David; (Berkeley, CA) |
Correspondence
Address: |
RICHARD ARON OSMAN
SCIENCE AND TECHNOLOGY LAW GROUP
75 DENISE DRIVE
HILLSBOROUGH
CA
94010
|
Assignee: |
THE REGENTS OF THE UNIVERSITY OF
CALIFORNIA
|
Family ID: |
25165676 |
Appl. No.: |
10/159749 |
Filed: |
May 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10159749 |
May 29, 2002 |
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09040229 |
Mar 13, 1998 |
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6432410 |
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09040229 |
Mar 13, 1998 |
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08795501 |
Feb 5, 1997 |
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Current U.S.
Class: |
424/146.1 ;
435/6.13 |
Current CPC
Class: |
C07K 14/475
20130101 |
Class at
Publication: |
424/146.1 ;
435/6 |
International
Class: |
A61K 039/395; C12Q
001/68 |
Goverment Interests
[0002] The research carried out in the subject application was
supported in part by grants from the National Institutes of Health.
The government may have rights in any patent issuing on this
application.
Claims
What is claimed is:
1. A method for reducing morphogen-dependent bone formation of a
cell comprising an extracellular surface in contact with a medium,
said method comprising the step of: contacting said medium with an
antagonist of a bone morphogenie protein (BMP) selected from the
group consisting of human BMP2 and human BMP4, said antagonist
produced exogenously from said cell and comprising SEQ ID NO:2, 4,
6, 8 or 9 or a deletion mutant of a sequence selected from the
group consisting of SEQ ID NO:2 or 8 wherein said deletion mutant
encodes a polypeptide that retains at least 8 contiguous amino
acids of said SEQ ID NO:2 or 8 and is sufficient to specifically
bind and antagonize said BMP, under conditions whereby said
morphogen-dependent bone formation is reduced; and measuring a
resultant reduction of said morphogen-dependent bone formation.
2. A method for reducing morphogen-dependent bone formation of a
cell comprising an extracellular surface in contact with a medium,
said method comprising the step of: contacting said medium with an
antagonist of a bone morphogenic protein (BMP) selected from the
group consisting of human BMP2 and human BMP4, said antagonist
produced exogenously from said cell and comprising SEQ ID NO:2, or
a deletion mutant of SEQ ID NO:2 wherein said deletion mutant
encodes a polypeptide that retains at least 8 contiguous amino
acids of SEQ ID NO:2 and is sufficient to specifically bind and
antagonize said BMP, under conditions whereby said
morphogen-dependent bone formation is reduced; and measuring a
resultant reduction of said morphogen-dependent bone formation.
3. A method for reducing morphogen-dependent bone formation of a
cell comprising an extracellular surface in contact with a medium,
said method comprising the step of: contacting said medium with an
antagonist of a bone morphogenic protein (BMP) selected from the
group consisting of human BMP2 and human BMP4, said antagonist
produced exogenously from said cell and comprising SEQ ID NO:8, or
a deletion mutant of SEQ ID NO:8 wherein said deletion mutant
encodes a polypeptide that retains at least 8 contiguous amino
acids of SEQ ID NO:8 and is sufficient to specifically bind and
antagonize said BMP, under conditions whereby said
morphogen-dependent bone formation is reduced; and measuring a
resultant reduction of said morphogen-dependent bone formation.
4. A method according to claim 1, wherein said cell is in situ and
said medium is a physiological fluid comprising said BMP.
5. A method according to claim 2, wherein said cell is in situ and
said medium is a physiological fluid comprising said BMP.
6. A method according to claim 3, wherein said cell is in situ and
said medium is a physiological fluid comprising said BMP.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuing application under 35USC120
of U.S. Ser. No. 09/040,229, filed Mar. 13, 1998, which is a
continuing application under 35USC120 of U.S. Ser. No. 08/795,501
filed Feb. 5, 1997, the specifications of which are incorporated by
reference.
INTRODUCTION
[0003] 1. Field of the Invention
[0004] The field of this invention is proteins which regulate cell
function, and in particular, antagonize bone morphogenic
proteins.
[0005] 2. Background
[0006] Natural regulators of cellular growth, differentiation and
function have provided important pharmaceuticals, clinical and
laboratory tools, and targets for therapeutic intervention. A
variety of such regulators have been shown to have profound effects
on basic cellular differentiation and developmental pathways. For
example, the recently cloned cerberus protein induces the formation
of head structures in anterior endoderm of vertebrate embryos.
Similarly, the noggin protein induces head structures in vertebrate
embryos, and can redirect mesodermal fates from ventral fates, such
as blood and mesenchyme, to dorsal fates such as muscle and
notochord and can redirect epidermal fates to anterior neural
fates. The activities of chordin are similar to those of noggin,
reflecting a common mechanism of action; namely antagonizing bone
morphogenic proteins (BMP) and thereby preventing their function.
BMPs have diverse biological activities in different biological
contexts, including the induction of cartilage and bone, connective
tissue, and roles in kidney, tooth, gut skin and hair
development.
[0007] Different members of the TGF.beta. superfamily can instruct
cells to follow different fates, for example TGF.beta. induces
neural crest to form smooth muscle, while BMP2 induces the same
cells to become neurons. In Xenopus experiments, dissociated animal
cap cells (prospective ectoderm) become epidermis in response to
BMP4 but become mesoderm in response to activin. Since the identity
between activin and BMP4 is low, it is not surprising that they
induce different fates. It is more surprising that members of the
BMP subfamily, which are quite closely related in sequence, can
induce distinct fates. A striking example results from implantation
of a matrix impregnated with a BMP into muscle; when the effects
are monitored histologically, BMP2, 4 and 7 induce endochondral
bone formation, whereas a related molecule BMP12/GDF7 induces
connective tissue similar to tendon. Similarly, BMP4 can induce
cell death in the hindbrain neural crest, while the related protein
dorsalin does not.
[0008] Since different BMP family members can induce different
fates, then BMP antagonists that have specificity in blocking
subsets of BMPs could change the balance of BMPs that are presented
to a cell, thus altering cell fate. In view of the importance of
relative BMP expression in human health and disease, regulators of
cellular function and BMP function in particular, such as noggin
and cerberus, provide valuable reagents with a host of clinical and
biotechnological applications.
[0009] The present invention relates to a new family of regulators
of cellular function; in particular, we have identified a new
dorsalizing factor Gremlin. Gremlin 's activities are similar to
those of Noggin and Chordin, however, it has no sequence similarity
to them, and it is not expressed during gastrulation. Instead,
zygotic Gremlin expression begins in the migrating neural crest,
highlighting a potential role for organizer-like activities in the
development of this cell lineage. Furthermore, we have identified a
structurally and functionally-related family of proteins that
includes Gremlin, the head-inducing factor Cerberus and the tumor
suppressor DAN (Ozaki and Sakiyama, 1993; Bouwmeester et al.,
1996). We have named this family the DAN family for the first
member identified. Cerberus is expressed in the anterior
endomesoderm of the gastrula organizer and has been proposed to
participate in head induction (Bouwmeester et al., 1996). DAN was
isolated as a putative zinc-finger protein downregulated in
transformed cells and was subsequently shown to have tumor
suppressor activity (Ozaki and Sakiyama, 1993; Ozaki and Sakiyama,
1994). Similarly, the rat homolog of Gremlin, drm, has also been
proposed to have a role in controlling cell growth and
differentiation (Topol et al., 1997). We demonstrate that all
members of the DAN family act as BMP antagonists, while Cerberus
alone blocks the activity of Activin and Nodal-like activities. We
also show that DAN-family members are secreted proteins that bind
BMP-2. Together, our data reveal that individual DAN-family members
block the activity of specific TGF-.beta. ligands by binding them
and preventing them from interacting with their cell surface
receptors.
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SUMMARY OF THE INVENTION
[0067] The invention provides methods and compositions relating to
DAN (Differential-screening-selected gene Aberrative in
Neuroblastoma) and gremlin proteins and related nucleic acids.
Included are natural DAN and gremlin homologs from different
species and DAN and gremlin proteins comprising a DAN or gremlin
domain and having DAN or gremlin-specific activity, particularly
the ability to antagonize a bone morphogenic protein such as BMP2
or BMP4. The proteins may be produced recombinantly from
transformed host cells with the subject nucleic acids. The
invention provides isolated hybridization probes and primers
capable of specifically hybridizing with the disclosed genes,
specific binding agents such as specific antibodies, and methods of
making and using the subject compositions in diagnosis (e.g.
genetic hybridization screens for gremlin transcripts), therapy
(e.g. gene therapy to modulate gremlin gene expression) and in the
biopharmaceutical industry (e.g. reagents for screening chemical
libraries for lead pharmacological agents).
[0068] Preferred applications of the subject DAN and gremlin
proteins include modifying the physiology of a cell comprising an
extracellular surface by contacting the cell or medium surrounding
the cell with an exogenous DAN or gremlin protein under conditions
whereby the added protein specifically interacts with a component
of the medium and/or the extracellular surface to effect a change
in the physiology of the cell. Also preferred are methods for
screening for biologically active agents, which methods involve
incubating a DAN or gremlin protein in the presence of an
extracellular DAN or gremlin protein-specific binding target and a
candidate agent, under conditions whereby, but for the presence of
the agent, the protein specifically binds the binding target at a
reference affinity; detecting the binding affinity of the protein
to the binding target to determine an agent-biased affinity,
wherein a difference between the agent-biased affinity and the
reference affinity indicates that the agent modulates the binding
of the protein to the binding target.
BRIEF DESCRIPTION OF THE FIGURE
[0069] FIG. 1(A) shows a sequence alignment of Gremlin sequences
identified in Xenopus(x)(SEQ ID NO:04), chick(c)(SEQ ID NO:06),
mouse(m)(SEQ ID NO:09), and human(h)(SEQ ID NO:02), showing that
they are >80% identical. The predicted signal sequence is in
bold. A single N-linked glycosylation site is underlined. The nine
cysteines in the carboxy-terminal domain are marked with asterisks.
FIG. 1(B) shows a sequence alignment of the cysteine-rich domain
shared by Xenopus Gremlin (xGremlin)(SEQ ID NO:04),Xenopus Cerberus
(xCerberus)(SEQ ID NO:12), mouse DAN (mDAN)(SEQ ID NO:10), Xenopus
DAN (xDAN)(SEQ ID NO: 1), and C. elegans Gremlin-related-1 (ceGR-1)
(SEQ ID NO: 13). Conserved cysteines are marked with asterisks.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The nucleotide sequence of a natural cDNA encoding human,
xenopus and chick gremlin polypeptides are shown as SEQ ID NOS:1,
3, and 5, respectively, and the corresponding full conceptual
translates are shown as SEQ ID NOS:2, 4 and 6. The nucleotide
sequence of a natural cDNA encoding a human DAN polypeptide is
shown as SEQ ID NO:7, and the corresponding full conceptual
translate is shown as SEQ ID NO:8. The gremlin and DAN polypeptides
of the invention include one or more functional domains comprising
at least 8, preferably at least 12, more preferably at least 18,
more preferably at least 36 contiguous residues of the recited SEQ
ID NOS and have an assay-discernable DAN or gremlin-specific
activity, functionally distinct from each other and from cerberus
and noggin homologs. For example, gremlin specific polynucleotides
and polypeptides having human gremlin-specific sequences are
readily discernable from alignments of the sequences. Preferred
gremlin polypeptides have one or more human gremlin-specific
activities, such as cell surface receptor binding and/or binding
inhibitory activity and gremlin-specific immunogenicity and/or
antigenicity. In a particular embodiment, the DAN or gremlin
polypeptides retain the nine conserved cysteine residues, and
preferably all contiguous sequence contained within the native nine
conserved cysteine residues of the family as discemable by
alignment (FIG. 1A, 1B). FIG. 1(A) shows that gremlin is highly
conserved in vertebrates.
[0071] DAN or gremlin-specific activity or function may be
determined by convenient in vitro, cell-based, or in vivo assays:
e.g. in vitro binding assays, cell culture assays, in animals (e.g.
gene therapy, transgenics, etc.), etc. Binding assays encompass any
assay where the molecular interaction of a DAN or gremlin
polypeptide with a binding target is evaluated. The binding target
may be a natural extracellular binding target such as a TGF.beta.
protein, a morphogenic protein, preferably a bone morphogenic
protein such as BMP2 or BMP4; or non-natural binding target such as
a specific immune protein such as an antibody, or a DAN or gremlin
specific agent such as those identified in screening assays such as
described below. DAN or gremlin-binding specificity may be assayed
by binding equilibrium constants (usually at least about
10.sup.7M.sup.-1, preferably at least about 10.sup.8M.sup.-1, more
preferably at least about 10.sup.9 M.sup.-1), by growth cone
collapse assays, by the ability to elicit gremlin specific antibody
in a heterologous host (e.g a rodent or rabbit), etc.
[0072] For example, deletion mutagenesis is used to define
functional DAN and gremlin domains which specifically bind BMPs in
in vitro and cell-based assays described below.
1TABLE 1 Exemplary DAN and gremlin deletion mutants defining
functional domains. BMP Mutant Sequence Binding G.DELTA.N1 SEQ ID
NO: 2, residues 2-184 + G.DELTA.N2 SEQ ID NO: 2, residues 12-184 +
G.DELTA.N3 SEQ ID NO: 2, residues 24-184 + G.DELTA.N4 SEQ ID NO: 2,
residues 48-184 + G.DELTA.N5 SEQ ID NO: 2, residues 64-184 +
G.DELTA.C1 SEQ ID NO: 2, residues 1-183 + G.DELTA.C2 SEQ ID NO: 2,
residues 1-182 + G.DELTA.C3 SEQ lID NO: 2, residues 1-181 +
G.DELTA.C4 SEQ ID NO: 2, residues 1-180 + G.DELTA.C5 SEQ ID NO: 2,
residues 1-179 + G.DELTA.NC1 SEQ liD NO: 2, residues 21-181 +
G.DELTA.NC2 SEQ ID NO: 2, residues 34-182 + G.DELTA.NC3 SEQ ID NO:
2, residues 57-183 + G.DELTA.NC4 SEQ ID NO: 2, residues 13-183 +
G.DELTA.NC5 SEQ ID NO: 2, residues 45-181 + D.DELTA.N1 SEQ ID NO:
8, residues 3-181 + D.DELTA.N2 SEQ ID NO: 8, residues 8-181 +
D.DELTA.N3 SEQ ID NO: 8, residues 18-181 + D.DELTA.N4 SEQ ID NO: 8,
residues 23-181 + D.DELTA.N5 SEQ lID NO: 8, residues 28-181 +
D.DELTA.C1 SEQ ID NO: 8, residues 1-180 + D.DELTA.C2 SEQ ID NO: 8,
residues 1-179 + D.DELTA.C3 SEQ lID NO: 8, residues 1-178 +
D.DELTA.C4 SEQ ID NO: 8, residues 1-177 + D.DELTA.C5 SEQ ID NO: 8,
residues 1-176 + D.DELTA.NC1 SEQ ID NO: 8, residues 3-180 +
D.DELTA.NC2 SEQ ID NO: 8, residues 2-177 + D.DELTA.NC3 SEQ ID NO:
8, residues 7-179 + D.DELTA.NC4 SEQ ID NO: 8, residues 16-180 +
D.DELTA.NC5 SEQ ID NO: 8, residues 2 1-178 +
[0073] In a particular embodiment, the subject domains provide DAN
or gremlin-specific antigens and/or immunogens, especially when
coupled to carrier proteins. For example, peptides corresponding to
gremlin- and human gremlin-specific domains are covalently coupled
to keyhole limpet antigen (KLH) and the conjugate is emulsified in
Freunds complete adjuvant. Laboratory rabbits are immunized
according to conventional protocol and bled. The presence of DAN or
gremlin-specific antibodies is assayed by solid phase immunosorbant
assays using immobilized DAN or gremlin polypeptides, see, e.g.
Table 2.
2TABLE 2 Immunogenic human gremlin polypeptides eliciting
gremlin-specific rabbit polyclonal antibody: gremlin
polypeptide-KLH conjugates immunized per protocol described above.
Gremlin Polypeptide Sequence Immunogenicity SEQ ID NO: 2, residues
1-8 +++ SEQ ID NO: 2, residues 2-11 +++ SEQ ID NO: 2, residues 12-2
1 +++ SEQ ID NO: 2, residues 15-23 +++ SEQ ID NO: 2, residues 22-30
+++ SEQ ID NO: 2, residues 32-39 +++ SEQ ID NO: 2, residues 42-51
+++ SEQ ID NO: 2, residues 45-52 +++ SEQ ID NO: 2, residues 52-65
+++ SEQ ID NO: 2, residues 68-79 +++ SEQ ID NO: 2, residues 70-80
+++ SEQ ID NO: 2, residues 75-82 +++ SEQ ID NO: 2, residues 86-92
+++ SEQ ID NO: 2, residues 92-98 +++ SEQ ID NO: 2, residues 95-103
+++ SEQ ID NO: 2, residues 99-108 +++ SEQ ID NO: 2, residues
102-112 +++ SEQ ID NO: 2, residues 109-115 +++ SEQ ID NO: 2,
residues 116-123 +++ SEQ ID NO: 2, residues 119-126 +++ SEQ ID NO:
2, residues 222-130 +++ SEQ ID NO: 2, residues 124-135 +++ SEQ ID
NO: 2, residues 127-140 +++ SEQ ID NO: 2, residues 138-147 +++ SEQ
ID NO: 2, residues 145-155 +++ SEQ ID NO: 2, residues 149-151 +++
SEQ ID NO: 2, residues 157-165 +++ SEQ ID NO: 2, residues 160-168
+++ SEQ ID NO: 2, residues 167-181 +++
[0074] The claimed gremlin polypeptides are isolated or pure: an
"isolated" polypeptide is unaccompanied by at least some of the
material with which it is associated in its natural state,
preferably constituting at least about 0.5%, and more preferably at
least about 5% by weight of the total polypeptide in a given sample
and a pure polypeptide constitutes at least about 90%, and
preferably at least about 99% by weight of the total polypeptide in
a given sample. The gremlin polypeptides and polypeptide domains
may be synthesized, produced by recombinant technology, or purified
from mammalian, preferably human cells. A wide variety of molecular
and biochemical methods are available for biochemical synthesis,
molecular expression and purification of the subject compositions,
see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al.
Cold Spring Harbor Laboratory), Current Protocols in Molecular
Biology (Eds. Ausubel, et al, Greene Publ. Assoc.,
Wiley-Interscience, N.Y.) or that are otherwise known in the art.
An exemplary method for isolating natural DAN and gremlin proteins
involves expressing a cDNA library (e.g. derived from xenopus
ovarian cells) and assaying expression products for embryonic axis
formation. This method and other suitable bioassays amenable to
detecting DAN and gremlin proteins have been described by Lemaire
P; et al. (supra); Smith, W. C., and Harland, R. M. (1992 and 1991,
supra); Piccolo, S., et al. (1996, supra); and Zimmerman, L. B., et
al. (1996, supra).
[0075] The invention provides binding agents specific to DAN or
gremlin polypeptides, preferably the claimed human gremlin
polypeptides, including agonists, antagonists, natural cell surface
receptor binding targets, etc., methods of identifying and making
such agents, and their use in diagnosis, therapy and pharmaceutical
development. For example, specific binding agents are useful in a
variety of diagnostic and therapeutic applications, especially
where disease or disease prognosis is associated with improper
utilization of a pathway involving the subject proteins. Novel DAN
or gremlin-specific binding agents include DAN or gremlin-specific
receptors, such as somatically recombined polypeptide receptors
like specific antibodies or T-cell antigen receptors (see, e.g
Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory) and other natural binding agents such as Gremlin
cell surface receptors, non-natural intracellular binding agents
identified in screens of chemical libraries such as described
below, etc. Agents of particular interest modulate DAN or gremlin
function, e.g. gremlin-modulatable cellular physiology.
[0076] The subject proteins find a wide variety of uses including
use as immunogens, targets in screening assays, bioactive reagents
for modulating cell growth, differentiation and/or function, etc.
For example, the invention provides methods for modifying the
physiology of a cell comprising an extracellular surface by
contacting the cell or medium surrounding the cell with an
exogenous DAN or gremlin protein under conditions whereby the added
protein specifically interacts with a component of the medium
and/or the extracellular surface to effect a change in the
physiology of the cell. According to these methods, the
extracellular surface includes plasma membrane-associated
receptors; the exogenous DAN or gremlin refers to a protein not
made by the cell or, if so, expressed at non natural levels, times
or physiologic locales; and suitable media include in vitro culture
media and physiological fluids such as blood, synovial fluid, etc.
Effective administrations of subject proteins can be used to reduce
undesirable (e.g. ectopic) bone formation, inhibit the growth of
cells that require a morphogenic protein (e.g. BMP-dependent
neuroblastomas and gliomas), alter morphogen-dependent cell
fate/differentiation in culture, such as with cells for
transplantation or infusion, etc. The proteins may be may be
introduced, expressed, or repressed in specific populations of
cells by any convenient way such as microinjection,
promoter-specific expression of recombinant enzyme, targeted
delivery of lipid vesicles, etc.
[0077] In one embodiment, the invention provides methods for
modulating cell function comprising the step of modulating DAN or
gremlin activity, e.g. by contacting the cell with a DAN or gremlin
polypeptide. In preferred embodiments, the modulator effects
gremlin or DAN mediated decrease in resident BMP activity. The
target cell may reside in culture or in situ, i.e. within the
natural host. The modulator may be provided in any convenient way,
including by (i) intracellular expression from a recombinant
nucleic acid or (ii) exogenous contacting of the cell. For many in
situ applications, the compositions are added to a retained
physiological fluid such as blood or synovial fluid. DAN and
gremlin polypeptides or polypeptide modulators may also be amenable
to direct injection or infusion, topical, intratracheal/nasal
administration e.g. through aerosol, intraocularly, or within/on
implants e.g. fibers e.g. collagen, osmotic pumps, grafts
comprising appropriately transformed cells, etc. See e.g. Tracy M A
(1998) Biotechnol Prog 14(1):108-115; Putney S D, Burke P A (1998)
Nat Biotechnol 16(2):153-157 for encapsulation delivery methods.
Generally, the amount administered will be empirically determined,
typically in the range of about 10 to 1000 .mu.g/kg of the
recipient and the concentration will generally be in the range of
about 50 to 500 .mu.g/ml in the dose administered, see e.g. Lucidi
P, et al. (1998) J Clin Endocrinol Metab 83(2):353-357; Houdijk EC,
et al. (1997) Acta Paediatr 86(12):1301-1307 for suitable methods
of administration. Other additives may be included, such as
stabilizers, bactericides, etc. will be present in conventional
amounts. For diagnostic uses, the modulators or other gremlin
binding agents are frequently labeled, such as with fluorescent,
radioactive, chemiluminescent, or other easily detectable
molecules, either conjugated directly to the binding agent or
conjugated to a probe specific for the binding agent.
[0078] For example, rats implanted with BMP-4 collagen matrices are
used to demonstrate in vivo efficacy of gremlin and DAN
polypeptides. In these experiments, rats are implanted with BMP-4
collagen matrices substantially as described in Wozney et al.
(1988) Science 242:1528-1534 and treated with three gremlin
polypeptides (SEQ ID NO:2, SEQ ID NO:2, residues 24-182, and SEQ ID
NO:2, residues 54-180 or two DAN polypeptides (SEQ ID NO:8, SEQ ID
NO:8, residues 12-181. All five treatment groups demonstrate
significant reductions in ectopic (matrix) bone formation. Gremlin
and DAN polypeptides are also demonstrated to antagonize
recombinant human BMP-2- mediated bone formation in rabbits,
essentially as described in Zegzula HD, et al. (1997) J Bone Joint
Surg Am 79(12):1778-1790. Briefly, A unilateral segmental defect,
twenty millimeters long, is created in the radius in ninety-six
skeletally mature New Zealand White rabbits. Forty-eight rabbits
are evaluated at four weeks and forty-eight, at eight weeks. Eight
groups are studied at each time-period. The defect is filled with a
porous poly(DL-lactic acid) implant containing zero, 17, 35, or 70
micrograms of rhBMP-2 with and without either zero, 20, 100, or 500
micrograms gremlin polypeptide (SEQ ID NO:2, residues 24-184) or
100 or 250 micrograms DAN polypeptide (SEQ ID NO:8, residues
12-181). Radiographs of the defects are made every two weeks. The
percentage of the total area of the defect that is radiopaque is
determined with use of computerized radiomorphometry, and this
percentage used as a quantitative measure of the extent of new-bone
formation in the defect. Time and dose-dependent responses to
rhBMP-2 are found for as long as four weeks; thereafter, the
effects of seventeen, thirty-five, and seventy micrograms of
rhBMP-2 are independent of dose and time (p<or =0.05). The
defects treated with either thirty-five or seventy micrograms of
rhBMP-2 have a significantly greater (p <or =0.05) area of
radiopacity than the defects treated with either zero or seventeen
micrograms of rhBMP-2. No significant difference is found between
the defects treated with thirty-five or seventy micrograms of
rhBMP-2 and the defects filled with an autogenous graft. All
defects concomitantly treated with all three doses of gremlin and
both doses of DAN reveal significantly less (p <or =0.05) area
of radiopacity than the corresponding rhBMP-2 only treated groups.
The inhibition of BMP-2 mediated bone formation is confirmed by
histological and histomorphometric examinations.
[0079] The amino acid sequences of the disclosed DAN and gremlin
polypeptides are used to back-translate gremlin
polypeptide-encoding nucleic acids optimized for selected
expression systems (Holler et al. (1993) Gene 136, 323-328; Martin
et al. (1995) Gene 154, 150-166) or used to generate degenerate
oligonucleotide primers and probes for use in the isolation of
natural gremlin-encoding nucleic acid sequences ("GCG" software,
Genetics Computer Group, Inc, Madison Wis.). DAN and
gremlin-encoding nucleic acids used in DAN and gremlin-expression
vectors and incorporated into recombinant host cells, e.g. for
expression and screening, transgenic animals, e.g. for functional
studies such as the efficacy of candidate drugs for disease
associated with gremlin-modulated cell function, etc.
[0080] The invention also provides nucleic acid hybridization
probes and replication/amplification primers having a DAN or
gremlin cDNA specific sequence comprising a strand of least one of
SEQ ID NO:1 or 7, or at least 18, preferably at least 36, more
preferably at least 72 contiguous nucleotides of SEQ ID NO: 1 or 7
and sufficient to specifically hybridize with a second nucleic acid
comprising the complementary strand of the corresponding SEQ ID
NO:1 or 7. Demonstrating specific hybridization generally requires
stringent conditions, for example, hybridizing in a buffer
comprising 30% formamide in 5.times. SSPE (0.18 M NaCl, 0.01 M
NaPO.sub.4, pH 7.7, 0.001 M EDTA) buffer at a temperature of
42.degree. C. and remaining bound when subject to washing at
42.degree. C. with 0.2.times. SSPE; preferably hybridizing in a
buffer comprising 50% formamide in 5.times. SSPE buffer at a
temperature of 42.degree. C. and remaining bound when subject to
washing at 42.degree. C. with 0.2.times. SSPE buffer at 42.degree.
C.
3TABLE 3 Exemplary gremlin nucleic acids which hybridize with a
strand of SEQ ID NO: 1 under Conditions I and/or II. gremlin
Nucleic Acids Hybridization SEQ ID NO: 1, nucleotides 1-36 + SEQ ID
NO: 1, nucleotides 68-98 + SEQ ID NO: 1, nucleotides 95-130 + SEQ
ID NO: 1, nucleotides 175-220 + SEQ ID NO: 1, nucleotides 261-299 +
SEQ ID NO: 1, nucleotides 274-3 10 + SEQ ID NO: 1, nucleotides 33
1-369 + SEQ ID NO: 1, nucleotides 430-470 + SEQ ID NO: 1,
nucleotides 584-616 +
[0081] The subject nucleic acids are of synthetic/non-natural
sequences and/or are isolated, i.e. unaccompanied by at least some
of the material with which it is associated in its natural state,
preferably constituting at least about 0.5%, preferably at least
about 5% by weight of total nucleic acid present in a given
fraction, and usually recombinant, meaning they comprise a
non-natural sequence or a natural sequence joined to nucleotide(s)
other than that which it is joined to on a natural chromosome.
Recombinant nucleic acids comprising the nucleotide sequence of SEQ
ID NO:1, or requisite fragments thereof, contain such sequence or
fragment at a terminus, immediately flanked by (i.e. contiguous
with) a sequence other than that which it is joined to on a natural
chromosome, or flanked by a native flanking region fewer than 10
kb, preferably fewer than 2 kb, which is at a terminus or is
immediately flanked by a sequence other than that which it is
joined to on a natural chromosome. While the nucleic acids are
usually RNA or DNA, it is often advantageous to use nucleic acids
comprising other bases or nucleotide analogs to provide modified
stability, etc.
[0082] The subject nucleic acids find a wide variety of
applications including use as translatable transcripts,
hybridization probes, PCR primers, diagnostic nucleic acids, etc.;
use in detecting the presence of gremlin genes and gene transcripts
and in detecting or amplifying nucleic acids encoding additional
gremlin homologs and structural analogs. In diagnosis, gremlin
hybridization probes find use in identifying wild-type and mutant
gremlin alleles in clinical and laboratory samples. Mutant alleles
are used to generate allele-specific oligonucleotide (ASO) probes
for high-throughput clinical diagnoses. DAN and gremlin nucleic
acids are also used to modulate cellular expression or
intracellular concentration or availability of active DAN or
gremlin. DAN and gremlin inhibitory nucleic acids are typically
antisense: single-stranded sequences comprising complements of the
disclosed natural gremlin coding sequences. Antisense modulation of
the expression of a given DAN or gremlin protein may employ
antisense nucleic acids operably linked to gene regulatory
sequences. Cell are transfected with a vector comprising a DAN or
gremlin sequence with a promoter sequence oriented such that
transcription of the gene yields an antisense transcript capable of
binding to endogenous DAN/gremlin encoding mRNA. Transcription of
the antisense nucleic acid may be constitutive or inducible and the
vector may provide for stable extrachromosomal maintenance or
integration. Alternatively, single-stranded antisense nucleic acids
that bind to genomic DNA or mRNA encoding a given DAN or gremlin
protein may be administered to the target cell, in or temporarily
isolated from a host, at a concentration that results in a
substantial reduction in expression of the targeted protein. An
enhancement in DAN or gremlin expression is effected by introducing
into the targeted cell type DAN or gremlin nucleic acids which
increase the functional expression of the corresponding gene
products. Such nucleic acids may be DAN or gremlin expression
vectors, vectors which upregulate the functional expression of an
endogenous allele, or replacement vectors for targeted correction
of mutant alleles. Techniques for introducing the nucleic acids
into viable cells are known in the art and include retroviral-based
transfection, viral coat protein-liposome mediated transfection,
etc.
[0083] The invention provides efficient methods of identifying
agents, compounds or lead compounds for agents active at the level
of a gremlin modulatable cellular function. Generally, these
screening methods involve assaying for compounds which modulate
gremlin interaction with a natural gremlin binding target. A wide
variety of assays for binding agents are provided including labeled
in vitro protein-protein binding assays, immunoassays, cell based
assays, etc. The methods are amenable to automated, cost-effective
high throughput screening of chemical libraries for lead compounds.
Identified reagents find use in the pharmaceutical industries for
animal and human trials; for example, the reagents may be
derivatized and rescreened in in vitro and in vivo assays to
optimize activity and minimize toxicity for pharmaceutical
development.
[0084] In vitro binding assays employ a mixture of components
including a DAN or gremlin protein, which may be part of a fusion
product with another peptide or polypeptide, e.g. a tag for
detection or anchoring, etc. The assay mixtures comprise a natural
DAN/gremlin binding target, e.g. a TGF.beta. protein such as a BMP.
While native binding targets may be used, it is frequently
preferred to use portions thereof so long as the portion provides
binding affinity and avidity to the subject DAN/gremlin
conveniently measurable in the assay. The assay mixture also
comprises a candidate pharmacological agent. Candidate agents
encompass numerous chemical classes, though typically they are
organic compounds; preferably small organic compounds and are
obtained from a wide variety of sources including libraries of
synthetic or natural compounds. A variety of other reagents such as
salts,-buffers, neutral proteins, e.g. albumin, detergents,
protease inhibitors, nuclease inhibitors, antimicrobial agents,
etc. may also be included. The mixture components can be added in
any order that provides for the requisite bindings and incubations
may be performed at any temperature which facilitates optimal
binding. The mixture is incubated under conditions whereby, but for
the presence of the candidate pharmacological agent, the DAN or
gremlin specifically binds the cellular binding target, portion or
analog with a reference binding affinity. Incubation periods are
chosen for optimal binding but also minimized to facilitate rapid,
high-throughput screening.
[0085] After incubation, the agent-biased binding between the DAN
or gremlin and one or more binding targets is detected by any
convenient way. For cell-free binding type assays, a separation
step is often used to separate bound from unbound components.
Separation may be effected by precipitation, immobilization, etc.,
followed by washing by, for examples, membrane filtration, gel
chromatography. For cell-free binding assays, one of the components
usually comprises or is coupled to a label. The label may provide
for direct detection as radioactivity, luminescence, optical or
electron density, etc. or indirect detection such as an epitope
tag, an enzyme, etc. A variety of methods may be used to detect the
label depending on the nature of the label and other assay
components, e.g. through optical or electron density, radiative
emissions, nonradiative energy transfers, etc. or indirectly
detected with antibody conjugates, etc. A difference in the binding
affinity of the DAN/gremlin protein to the target in the absence of
the agent as compared with the binding affinity in the presence of
the agent indicates that the agent modulates the binding of the
DAN/gremlin protein to the corresponding binding target. A
difference, as used herein, is statistically significant and
preferably represents at least a 50%, more preferably at least a
90% difference.
[0086] The following examples are offered by way of illustration
and not by way of limitation.
EXAMPLES
[0087] Expression Cloning of Gremlin. To identify new axial
patterning activities in the Xenopus embyro, we screened for
maternally-supplied activities whose ventral expression induced a
secondary axis in otherwise normal embryos (Lemaire et al., 1995).
1 ng of synthetic mRNA from each pool of a Xenopus ovary cDNA
library was injected into a ventral blastomere of a 4-cell embryo.
Injected embryos were subsequently screened at the tailbud stage
for presence of a secondary axis. Using this procedure, two pools
with axis-inducing activity were identified. One pool contained
Activin-.beta.B (Thomsen et al., 1990), and was eliminated from
further consideration. Fractionation of the second pool identified
a single clone with potent activity. mRNA doses as low as 50 pg
induced a secondary axis or hyperdorsalization in 93% of
ventrally-injected embryos. In contrast to the complete secondary
axes that can be induced by activation of the Wnt signaling
pathway, higher doses of this clone induced rostral structures such
as eyes in fewer than 5% of injected embryos. The sequence of the
1.8 kb cDNA encodes a predicted protein that we have named
Gremlin.
[0088] To characterize Gremlin's axis-inducing activity, we traced
the fate of Gremlin-expressing cells in twinned embryos. 50 pg of
Gremlin mRNA and 80 pg of mRNA encoding a .beta.-Galactosidase
lineage tracer were injected equatorially into ventral blastomeres
of 4-cell embryos. At tailbud stage, embryos were fixed and stained
with X-Gal. Not all the cells in the secondary axis were stained,
indicating that Gremlin-expressing cells are able to recruit
neighboring cells into the induced axis. While Gremlin induced
secondary axes efficiently when injected into the marginal zone, it
did not when injected into a ventral-vegetal blastomere. Thus,
despite its maternal origin, Gremlin is not the vegetally-localized
dorsalizing signal emanating from the Nieuwkoop center (Gimlich and
Gerhart, 1984; Smith and Harland, 1991).
[0089] The composition and patterning of Gremlin-induced secondary
axes was examined by immunostaining injected embryos with
monoclonal antibodies that detect muscle, notochord, or neural
tissue. We found that Gremlin-induced secondary axes frequently
contain all three tissue types, and that each tissue is organized
in a relatively normal fashion. Muscle staining is organized in
regularly repeated somitic segments, while neural and notochord
tissue stain in patterns similar to those found in the primary
axis. Thus, the composition and pattern of Gremlin-induced axes
resembles that of normal axes.
[0090] Gremlin is Expressed in the Neural Crest. We used in situ
hybridization to determine Gremlin 's temporal and spatial
expression patterns (Lamb et al., 1993). Maternal Gremlin
transcripts are present, but not localized in early-stage oocytes,
and they are undetectable in mature oocytes and early stage embryos
(not shown). Despite its axial patterning activities, Gremlin is
not expressed during gastrulation. Instead, zygotic Gremlin
expression begins at tailbud stages, where it is largely correlated
with neural crest lineages. At stage 27, Gremlin staining appears
in the pronephric duct, with additional faint staining in the trunk
and tail bud. Staining in the pronephric duct may identify neural
crest cells known to contribute to this structure (Collazo et al.,
1993). In stage 30-40 embryos, Gremlin expression extends rostrally
and caudally to include neural crest cells at all axial levels. In
the head, staining is present in the mandibular segment of the
neural crest (Sadaghiani and Thiebaud, 1987). Caudally, staining
illuminates the dorsal migration of neural crest cells into the
fin, as well as the chevron-shaped pathways of their ventral
migration (Sadaghiani and Thiebaud, 1987; Krotoski et al., 1988;
Collazo et al., 1993). In addition, expression continues in the
pronephric duct as it extends caudally (Vize et al., 1995). These
observations were confirmed by examination of transverse sections.
Taken together, Gremlin's expression pattern indicates role in
neural crest induction and patterning.
[0091] Gremlin Identifies a Family of Secreted Proteins With Axial
Patterning Activity. The DNA sequence of the 1.8 kb Gremlin cDNA
predicts a 182 amino acid protein with an N-terminal hydrophobic
sequence, a site for N-linked glycosylation, and a C-terminal
cysteine-rich domain (FIG. 1A). For comparative studies, we
identified chick, mouse, and human Gremlin homologs, and found that
they share over 80% amino acid identity with Xenopus Gremlin (FIG.
1A). Recently, drm, a rat Gremlin homolog with over 80% similarity
to the Gremlin family was identified as a cDNA downregulated in
v-mos transformed cells (Topol et al., 1997).
[0092] BLAST searches revealed that Gremlin belongs to a new family
of proteins that includes the previously-identified proteins,
Cerberus and DAN (Topol et al., 1997)(FIG. 1B). Cerberus is a
secreted factor expressed in the Xenopus gastrula that acts as a
potent inducer of head structures (Bouwmeester et al., 1996; Belo
et al., 1997). DAN encodes a tumor suppressor originally identified
in rat as a downregulated transcript in transformed cells (Ozaki
and Sakiyama, 1993). We also isolated a partial cDNA encoding a
nematode Gremlin homolog that has been designated C. elegans
Gremlin-Related -1 (ceGR-1). We name this family the DAN family for
the first member to be reported. Sequence alignments show that each
protein has a domain with the consensus sequence
CX.sub.6QX.sub.6CX.sub.6NX.sub.2CXGXCXSX.sub.3PX(.sub.8-13)CX.sub.2CXPX.s-
ub.8TLXCX.sub.(15-18)CXC (FIG. 1B). Outside this domain, DAN-family
members show little similarity.
[0093] The protein sequences of Gremlin, Cerberus, and DAN all
contain a likely signal sequence. However, while Cerberus has been
reported as a secreted protein, DAN was initially identified as a
putative zinc-finger protein. To determine whether all DAN-family
members are secreted, synthetic mRNA encoding Gremlin, DAN, or
Cerberus was injected into oocytes that were then cultured in the
presence of .sup.35S methionine. We found that supernatants of
Gremlin and DAN-injected oocytes contained proteins of
approximately 28 kd and 27 kd, respectively. Cerberus-injected
oocytes secreted a heterogeneous product centered around 40 kd.
Thus, all DAN-family members are secreted proteins. The predicted
molecular weights for Gremlin (25 kd), DAN (19 kd), and Cerberus
(31 kd) are lower than those observed, indicating that all three
proteins undergo post-translational modification. Consistently,
Gremlin, Cerberus, and DAN all have sites for N-linked
glycosylation (Ozaki and Sakiyama, 1993; Bouwmeester et al.,
1996).
[0094] DAN Has Dorsalizing Activity in Xenopus Embryos. While axial
patterning activities have been shown for Gremlin and Cerberus,
none have been shown for DAN. However, the similarity between the
three proteins led us to test DAN for embryonic patterning
activity. As a preliminary assay, we injected 100 pg synthetic DAN
mRNA into the marginal zone of both ventral blastomeres of 4-cell
embryos. At this dose, DAN-injected embryos were mildly
hyperdorsalized (DAI 6-7) (Kao and Elinson, 1988) with slightly
enlarged heads, bloated trunks, and a reduction of tail structures.
Occasionally, injected embryos had a secondary axis (less than
10%). Immunostaining of DAN-injected embryos using the muscle
specific antibody 12/101 reveals that muscle is present, but that
it is poorly organized. Xenopus DAN had similar activity. DAN's
activity in embryos and its ability to dorsalize ventral mesoderm
and neuralize ectoderm (described below) establishes that all
DAN-family members have axial patterning activity.
[0095] DAN-Family Members Act Like BMP Antagonists in Embryonic
Explants. To further examine the biological activities of
DAN-family members, we tested their ability to pattern mesoderm
using the ventral marginal zone (VMZ) assay (Smith et al., 1993).
Consistent with their effects when expressed ventrally in embryos,
expression of 100 pg DAN or Gremlin MRNA induced the dorsal
mesodermal marker cardiac muscle actin (M. actin) in VMZ explants.
As previously reported, the same dose of Cerberus mRNA failed to
induce M. actin (Bouwmeester et al., 1996). Since Cerberus' failure
to dorsalize the VMZ is probably due to its ability to provide an
early block to mesoderm induction (described below), we repeated
the VMZ assay with a Cerberus DNA construct that is not
transcriptionally active until blastula stages. When its expression
was delayed, Cerberus also induced M. actin. In Xenopus, an
accumulated body of evidence has shown that dorsalization of
mesoderm can result from blocking ventralizing signals provided by
members of the BMP family (Reviewed in Harland and Gerhart, 1997).
In light of these observations, the dorsalizing activity of
Cerberus, DAN, and Gremlin indicates that they act by antagonizing
BMP signals.
[0096] We also tested the activities of DAN-family members in
animal cap explants. These experiments show that Gremlin, Cerberus,
and DAN induce neural tissue from animal cap ectoderm. Injection of
100 pg Gremlin, 1 ng Cerberus, or 10 ng DAN into the animal pole of
a 1-cell embryo induced neural differentiation in explants, as
evidenced by expression of the pan-neural marker NCAM (Kintner and
Melton, 1987; Bouwmeester et al., 1996). The absence of M. actin
induction indicates that neuralization occurred directly, without a
mesodermal intermediary.
[0097] In Xenopus, organizer-specific neuralizing signals act by
antagonizing signaling by BMPs that normally cause ectoderm to
adopt an epidermal fate (Reviewed in Harland and Gerhart, 1997;
Sasai and De Robertis, 1997). To test whether DAN-family members
neuralize through a similar mechanism, we co-expressed each one
with an activated form of a type I BMP-4 receptor. ALK-3(Q233D)
contains a Q to D mutation at position 233 that activates the
receptor kinase, leading to constitutive signaling in the BMP
signal transduction pathway (Wieser et al., 1995; Holley et al.,
1996). We found that expression of ALK-3(Q233D) blocks neural
induction by Gremlin, Cerberus, or DAN, indicating that they block
BMP signaling upstream of the receptor.
[0098] Cerberus expression in animal cap explants also induced
modest levels of the pan-endodermal marker endodermin (Edd), and
the cardiac marker Nkx 2.5 (Bouwmeester et al., 1996). We find that
Gremlin and DAN share these activities. 100 pg of Cerberus,
Gremlin, or DAN mRNA induced Edd and Nkx 2.5 at low to moderate
levels. We also found that induction of these markers occurs in the
presence or absence of ALK-3(Q233D), indicating that induction of
these genes is normally not suppressed by BMP signaling through
ALK-3. These findings indicate that in addition to BMP antagonizing
activities, DAN-family members can block the activities of
additional TGF-.beta. signals.
[0099] Gremlin Blocks the Activity of Purified BMP-2. Gremlin's
dorsalizing and neuralizing activities provide a strong indication
that it acts by antagonizing signaling by members of the BMP
family. As a more direct test of Gremlin's activity, we examined
its ability to antagonize the activity of purified BMP-2 in a
cytokine assay. The murine bone marrow stromal cell line W-20-17
provides a direct, quantitative bioassay for BMP activity by
induction of alkaline phosphatase in response to BMP treatment
(Thies et al., 1992). Preincubation of purified BMP-2 with a
Gremlin COS supernatant at a final concentration of .about.83 nM
Gremlin completely blocked BMP-2 activity at doses from 78 pM to 5
nM. At .about.21 nM Gremlin, BMP-2 activity was reduced, but not
eliminated. In contrast, a mock-transfected COS supernatant had no
effect. Similar results were obtained with BMP-4. Thus, like Noggin
and Chordin, Gremlin blocks BMP activity in multiple assays.
[0100] DAN-Family Members Bind BMP-2. Biochemical studies of the
BMP antagonists Noggin and Chordin have shown that they bind BMP-4
with high affinity, preventing it from interacting with its cell
surface receptors (Piccolo et al., 1996; Zimmerman et al., 1996).
To test whether DAN-family members antagonize BMP signaling through
a similar mechanism, we examined their ability to associate with
BMP-2, which has activities virtually indistinguishable from those
of BMP-4. Gremlin, DAN, or Cerberus COS-conditioned supernatants
were incubated with anti BMP-2 protein G sepharose beads in the
presence or absence of 1 .mu.g BMP-2. After binding, beads were
processed and analyzed by Western blot. We found that Cerberus,
DAN, and Gremlin are precipitated by anti-BMP-2 beads in a
BMP-2-dependent fashion. Reciprocal binding experiments confirm
that DAN-family members associate with BMP-2. When Cerberus was
precipitated using an anti-Cer polyclonal antibody, BMP-2
coprecipitated. A similar result was obtained when DAN or Gremlin
were precipitated with a monoclonal antibody directed against their
C-terminal MYC tags. Taken together, these results demonstrate a
direct physical interaction between DAN-family members and
BMP-2.
[0101] To further characterize the interaction between BMPs and
their antagonists, we determined whether Noggin could compete with
each family member for binding to BMP-2. We found that the addition
of 2 or 10 .mu.g purified Noggin blocks the interaction between
Cerberus, Gremlin, or DAN and BMP-2. Noggin's ability to block the
association between DAN-family members and BMP-2 adds to the
evidence that these interactions are specific, and further
indicates that DAN-family members and Noggin all bind a similar
domain on BMP-2.
[0102] Cerberus Blocks Signaling by Activin and Xnr-2. The sequence
divergence within the DAN family is reminiscent of the divergence
within the TGF-.beta. family, and suggested that individual
DAN-family members might antagonize different subsets of TGF-.beta.
ligands. Since radial expression of Cerberus suppresses formation
of trunk-tail mesoderm (Bouwmeester et al., 1996), we tested the
possibility that it might block the action of mesoderm-inducing
TGF-.beta. ligands like Activin, B Vg-1, and Xnr-2 (Smith et al.,
1990; Thomsen et al., 1990; Thomsen and Melton, 1993; Jones et al.,
1995). Animal caps were isolated from embryos coinjected with
Activin (1 pg), B Vg-1 (100 pg), or Xnr-2 (100 pg) mRNA, and 1 ng
of Gremlin, Cerberus, or DAN rnRNA, and were then analyzed for
induction of the pan-mesodermal marker Xbra (Smith et al., 1991).
We found that Cerberus expression significantly reduced or
completely eliminated Xbra induction by Activin or Xnr-2. In
contrast, neither Gremlin nor DAN blocked Xbra induction despite
their doses being well above those required for biological activity
in other assays. The lack of mesoderm induction in
Cerberus-injected caps is not due to their inability to become
mesodermalized since Xbra was induced in explants coinjected with
Smad-2(C), a Smad-2-.beta.-Galactosidase fusion protein that
induces mesoderm in a ligand-independent fashion (Baker and
Harland, 1996). Thus, unlike other DAN-family members, Cerberus
antagonizes the activity of both BMPs and Activin-like ligands.
[0103] The Gremlin cDNA encodes a secreted protein whose activities
are similar to those of the previously-identified BMP antagonists
Noggin, Chordin and Follistatin (Reviewed in Harland and Gerhart,
1997; Sasai and De Robertis, 1997). Like these factors, Gremlin
antagonizes BMP activity in multiple assays, and it is able to
physically associate with BMP-2. Despite these functional
similarities, Gremlin has no sequence similarity to
previously-identified BMP antagonists, and it is not expressed in
the organizer. Instead, zygotic Gremlin transcripts are largely
limited to the neural crest and its derivatives. This expression
pattern shows that organizer-like activities are not restricted to
the gastrulating embryo, and indicates that these inductive signals
are used broadly in development.
[0104] Gremlin's acts as an essential modulator of BMP signaling in
neural crest cells, preventing premature or excessive exposure to
BMP signals during their birth and subsequent migration.
Consistently, BMP-2, BMP-4, and Gremlin are expressed at similar
times and places during embryonic development. At tailbud stages,
BMP-2 and BMP-4 are expressed in a coincident pattern in the fin
neural crest (Clement et al., 1995; Hemmati-Brivanlou and Thomsen,
1995; Schmidt et al., 1995). BMP-4 is also expressed in the roof
plate of the spinal cord (Fainsod et al., 1994). By modulating
these signals, Gremlin has an important role in determining the
timing of neural crest cell differentiation and/or the choice of
differentiated fates.
[0105] While members of the DAN family have no sequence similarity
to Noggin or Chordin, their activities indicate that they
antagonize BMP signaling using the same mechanism. Support for this
model comes from the finding that Gremlin, Cerberus and DAN all
bind BMP-2. Furthermore, Noggin effectively competes with
DAN-family members for binding to BMP-2, indicating that the
interaction between a BMP and its antagonists occurs through a
similar BMP domain. Thus, despite the lack of sequence similarity,
Noggin, Gremlin, and by extension, all BMP antagonists, share
structural similarities that allow them to interact with similar
domains on their cognate BMPs.
[0106] The scope of TGF-.beta. antagonizing activities of the DAN
family is broadened by the finding that Cerberus also blocks
signaling by Activin and Nodal-like ligands. In their original
study, Bouwmeester et al. (1996) found that radial expression of
Cerberus blocked formation of trunk/tail mesoderm and prechordal
plate mesoderm. We extended this finding by showing that among
DAN-family members, only Cerberus antagonizes the mesoderm-inducing
activity of Xnr-2 and Activin. Thus, despite their structural
similarities, DAN-family members have specificity for different
TGF-.beta. signals. We conclude that DAN-family members act by
binding overlapping subsets of TGF-.beta. ligands, preventing them
from interacting with their cell surface receptors. The diverse
activities of DAN-family members result from their ability to
antagonize specific TGF-.beta. signaling pathways required for
embryonic inductions and cell proliferation. The different mRNA
doses required for Gremlin, Cerberus, and DAN to neuralize animal
cap ectoderm is consistent with the idea that they differ in their
affinity for BMP-4. In addition, we have shown in additional
binding studies that Gremlin and DAN have substantially different
affinities for BMP-2.
[0107] Embryo Manipulations and RT-PCR. Xenopus embryos were
generated and staged as described (Nieuwkoop and Faber, 1967;
Condie and Harland, 1987). Ventral marginal zone (VMZ) assays were
done by injecting the marginal zone of each blastomere of 4-cell
embryos and then explanting, culturing and harvesting VMZs as
described (Zimmerman et al., 1996). For animal cap assays, 1-cell
embryos were injected in the animal pole. Animal caps were
explanted at stage 8-9, cultured in 75% NAM (Peng, 1991) and
harvested at stage 10.5-11, stage 22, or stage 27.
[0108] RT-PCR analysis was done as described (Wilson and Melton,
1994). Primer sets and PCR conditions for Xbra, Ef1-.alpha.,_Muscle
actin, and NCAM are described in Wilson and Melton (1994), and
those for Edd, and Nkx 2.5 are described in Bouwmeester et al.
(1996).
[0109] Construction of a Xenopus Ovary cDNA Library. Poly(A)+RNA
was isolated from Xenopus ovaries by oligo(dT) cellulose selection
from a proteinase K/SDS lysate (Badley et al., 1988). This mRNA was
used synthesize a cDNA library containing .about.650,000
independent clones using the SuperScript cDNA cloning system (Life
Technologies). To maximize expression of inserts, we established
the library in CS105, a derivative of CS2+ (Turner and Weintraub,
1994) that produces synthetic mRNAs with high activity. cDNA pools
were made from 121 plates containing .about.1000 colonies each.
[0110] Expression Cloning. To make transcription templates, cDNA
pools were digested with Asc-1, followed by digestion with 0.2
mg/ml proteinase K, 0.5% SDS at 50.degree. for 1 hr,
phenol/chloroform extraction, and precipitation. Synthetic mRNA was
synthesized with the mMessage mMachine SP6 kit (Ambion). 1 ng of
each mRNA pool was injected into the marginal zone of one ventral
blastomere of a 4-cell embryo. Tadpole-stage embryos were scored
for presence of a secondary axis. The Gremlin cDNA was isolated
from an active pool by sib-selection (Smith and Harland, 1991).
[0111] In Situ Hybridization, Immunostaining, and Histology.
Whole-mount in situ hybridization used standard methods (Lamb et
al., 1993). Sense and antisense probes were generated from the full
length Gremlin cDNA. For histological analysis, stained embryos
were embedded in paraplast, and 10-20 .mu.m sections were cut.
[0112] The presence of neural tissue, muscle, and notochord in
embryos was determined by immunostaining with the antibodies 6F11
(Lamb et al., 1993), 12/101 (Kintner and Brockes, 1984), and Tor70
(Bolce et al., 1992), respectively.
[0113] Identification of Gremlin Homologs. A chick Gremlin cDNA was
isolated by probing a stage 12-15 chick embryonic cDNA library,
with the Xenopus Gremlin cDNA under moderately stringent
hybridization conditions (0.5 M Na.sub.2PO.sub.4 pH 7.2, 15%
Formamide, 7% SDS, 1 mM EDTA, and 1% BSA, at 42.degree.). A mouse
genomic Gremlin clone was isolated from a 129SVJ genomic DNA
library (Stratagene) in a similar fashion. A human Gremlin cDNA was
identified in a BLAST search of the GenBank EST database
(I.M.A.G.E. consortium clone ID 272074, GenBank accession N35377)
(Altschul et al., 1990; Lennon et al., 1996).
[0114] The mouse DAN clone was obtained as an EST (GenBank
accession number AA008891). Xenopus Cerberus and DAN cDNAs were
isolated from a stage 11 LiCl-treated gastrula library. ceGR-1 was
identified in a BLAST search of C. elegans genomic DNA sequences,
and a ceGR-1 cDNA fragment was then cloned from a him-8(e1489)
embryonic cDNA library. The identity of all clones used in these
experiments was confirmed by DNA sequence analysis.
[0115] Preparation of Secreted Proteins in Xenopus Oocytes. Oocytes
were isolated from ovaries by treatment with 0.2% collagenase
(Boehringer Mannheim), or by manual defolliculation. They were
injected with 50 ng synthetic mRNA and cultured at 20.degree. in
groups of 5 in 100 .mu.L OR2 (Peng, 1991) supplemented with 0.5
mg/ml BSA, 100 .mu.g/ml penicillin, 100 .mu.g/ml streptomycin
sulfate, and 30 .mu.Ci .sup.35S-methionine. After 1 day, 10 .mu.l
of each supernatant was analyzed by SDS-PAGE followed by
fluorography.
[0116] Production of DAN-family COS Cell Supernatants. Conditioned
COS supernatants were made by transfecting COS7 cells
(3.5.times.10.sup.6/150 mm plate initial seeding density) with
plasmids encoding Xenopus or human Gremlin, mouse DAN, or human
Cerberus using Lipofectamine (Life Technologies). Control media was
made by transfecting the expression vector pMT21. For detection in
Western blots, Gremlins and mDAN were fused to a C-terminal
triple-MYC epitope tag. human Cerberus was fused to a C-terminal
FLAG epitope tag. xGremlin supernatants were quantified by
comparison with known amounts of bacterially-expressed His-tagged
Gremlin purified on a Ni-NTA-agarose column (Qiagen). In embryo
assays, hCerberus-FLAG mRNA has comparable activities to Xenopus
Cerberus mRNA. xGremlin-MYC.sub.3 mRNA behaves like its wild-type
counterpart.
[0117] Immunoprecipitation and Immunoblotting. Initially, 250-1000
.mu.l of a hCerberus-FLAG, mDAN-MYC.sub.3, or xGremlin-MYC.sub.3
COS supernatant was incubated with 1 .mu.g BMP-2 in the presence of
protein G sepharose beads bound to the BMP-2,4-specific monoclonal
antibody h3b2 in 1 ml of binding buffer (25 mM HEPES pH 7.7, 0.2 M
KCl, 12.5 mM MgCl.sub.2, 0.1 mM EDTA, 0.1% BSA, 0.1% NP40, 0.2 mM
PMSF) overnight at 4.degree.. Beads were washed twice in binding
buffer, boiled in loading dye, and analyzed by Western blot. Tagged
proteins were detected using monoclonal antibodies directed against
the FLAG epitope (M2, Kodak), or against the MYC epitope (9E10),
and a peroxidase-conjugated secondary antibody (Pierce).
Competition experiments were done by adding 2 or 10 .mu.g purified
Xenopus Noggin to the binding reaction.
[0118] In reciprocal binding experiments, 1 .mu.g hCerberus or
hGremlin-MYC.sub.3, or 1 ml of a mDAN-MYC.sub.3 COS supernatant was
incubated with 0.5-1 .mu.g BMP-2 in the presence of 0.1% Tween-20,
1 M NaCl, or 0.1% NP-40+0.2 M KCl, respectively. hCerberus was
immunoprecipitated using protein G sepharose beads bound to
anti-hCerberus polyclonal antibodies. hGremlin-MYC.sub.3 and
mDAN-MYC.sub.3 were immunoprecipitated using 9E10 beads.
Competition experiments were done by adding 2 or 10 .mu.g human
Noggin. The presence of coprecipitating BMP-2 was assessed on
Western blots using a polyclonal antibody directed against
BMP-2.
[0119] Protocol for high throughput human gremlin-BMP binding
assay.
[0120] A. Reagents:
[0121] Neutralite Avidin: 20 .mu.g/ml in PBS.
[0122] Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hour at
room temperature.
[0123] Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 1 mM
MgCl.sub.2, 1% glycerol, 0.5% NP-40, 50 mM .beta.-mercaptoethanol,
1 mg/ml BSA, cocktail of protease inhibitors.
[0124] .sup.33P human gremlin protein 10.times. stock:
10.sup.-8-10.sup.-6M "cold" human gremlin supplemented with
200,000-250,000 cpm of labeled human gremlin (Beckman counter).
Place in the 4.degree. C. microfridge during screening.
[0125] Protease inhibitor cocktail (1000.times.): 10 mg Trypsin
Inhibitor (BMB # 109894), 10 mg Aprotinin (BMB # 236624), 25 mg
Benzamidine (Sigma # B-6506), 25 mg Leupeptin (BMB # 1017128), 10
mg APMSF (BMB # 917575), and 2 mM NaVo.sub.3 (Sigma # S-6508) in 10
ml of PBS.
[0126] BMP: 10.sup.-7-10.sup.-4M biotinylated BMP in PBS.
[0127] B. Preparation of assay plates:
[0128] Coat with 120 .mu.l of stock N-Avidin per well overnight at
4.degree. C.
[0129] Wash 2 times with 200 .mu.l PBS.
[0130] Block with 150 .mu.l of blocking buffer.
[0131] Wash 2 times with 200 .mu.l PBS.
[0132] C. Assay:
[0133] Add 40 .mu.l assay buffer/well.
[0134] Add 10 .mu.l compound or extract.
[0135] Add 10 .mu.l .sup.33P gremlin protein (20-25,000 cpm/0.1-10
pmoles/well=10.sup.-9-10.sup.-7 M fin cone).
[0136] Shake at 25.degree. C. for 15 minutes.
[0137] Incubate additional 45 minutes at 25.degree. C.
[0138] Add 40 .mu.l biotinylated BMP (0.1-10 pmoles/40 ul in assay
buffer)
[0139] Incubate 1 hour at room temperature.
[0140] Stop the reaction by washing 4 times with 200 .mu.l PBS.
[0141] Add 150 .mu.l scintillation cocktail.
[0142] Count in Topcount.
[0143] D. Controls for all assays (located on each plate):
[0144] a. Non-specific binding
[0145] b. Soluble (non-biotinylated gremlin) at 80% inhibition.
[0146] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Sequence CWU 1
1
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