U.S. patent application number 11/251061 was filed with the patent office on 2007-05-24 for 'signalin' family of tgfbeta signal transduction proteins and uses related thereto.
This patent application is currently assigned to Curis, Inc.. Invention is credited to Jonathan M. Graff, Ping Jin, Douglas A. Melton, Tod M. Woolf.
Application Number | 20070117098 11/251061 |
Document ID | / |
Family ID | 24319373 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070117098 |
Kind Code |
A1 |
Graff; Jonathan M. ; et
al. |
May 24, 2007 |
'signalin' family of TGFbeta signal transduction proteins and uses
related thereto
Abstract
The present invention concerns the discovery that proteins
encoded by a family of vertebrate genes, termed here
signalin-related genes, which are involved in a signal transduction
induced by members of the TGF.beta. superfamily. The present
invention makes available compositions and methods that can be
utilized, for example to generate and/or maintain an array of
different vertebrate tissue both in vitro and in vivo.
Inventors: |
Graff; Jonathan M.; (Newton,
MA) ; Woolf; Tod M.; (Natick, MA) ; Jin;
Ping; (Boston, MA) ; Melton; Douglas A.;
(Lexington, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Curis, Inc.
Cambridge
MA
02138
President and Fellows of Harvard College
Cambridge
MA
02138
|
Family ID: |
24319373 |
Appl. No.: |
11/251061 |
Filed: |
October 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10095492 |
Mar 12, 2002 |
7034114 |
|
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11251061 |
Oct 14, 2005 |
|
|
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08580031 |
Dec 20, 1995 |
6428977 |
|
|
10095492 |
Mar 12, 2002 |
|
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|
Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 506/14; 530/350; 536/23.5 |
Current CPC
Class: |
C12Q 1/6886 20130101;
A61P 43/00 20180101; A61K 48/00 20130101; C12Q 2600/136 20130101;
C07K 14/4702 20130101 |
Class at
Publication: |
435/006 ;
530/350; 435/069.1; 435/320.1; 435/325; 536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 14/705 20060101 C07K014/705 |
Goverment Interests
FUNDING
[0002] Work described herein was supported by funding from the
National Institutes of Health. The United States Government has
certain rights in the invention.
Claims
1-71. (canceled)
72. A method for modulating one or more of growth, differentiation,
or survival of a mammalian cell responsive to signalin-mediated
induction, comprising treating the cell with an effective amount of
an agent which modulates the signal transduction activity of a
signalin polypeptide thereby altering, relative to the cell in the
absence of the agent, at least one of (i) rate of growth, (ii)
differentiation, or (iii) survival of the cell.
73. The method of claim 72, wherein said agent mimics the effects
of a naturally-occurring signalin protein on said cell.
74. The method of claim 72, wherein said agent antagonizes the
effects of a naturally-occurring signalin protein on said cell.
75. The method of claim 72, wherein the cell is a testicular cell
and the agent modulates spermatogenesis.
76. The method of claim 72, wherein the cell is an osteogenic cell,
and the agent modulates osteogenesis.
77. The method of claim 72, wherein the cell is a chondrogenic
cell, and the agent modulates chondrogenesis.
78. The method of claim 72, wherein the agent modulates the
differentiation of neuronal cells.
79-88. (canceled)
89. A diagnostic assay for identifying a cell or cells at risk for
a disorder characterized by unwanted cell proliferation or
differentiation, comprising detecting, in a cell sample, the
presence or absence of a genetic lesion characterized by at least
one of (i) aberrant modification or mutation of a gene encoding a
signalin protein, and (ii) mis-expression of said gene; wherein a
wild-type form of said gene encodes a signalin protein
characterized by an ability to modulate the signal transduction
activity of a TGF.beta. receptor.
90. The assay of claim 89, wherein detecting said lesion includes:
i. providing a diagonistic probe comprising a nucleic acid
including a region of nucleotide sequence which hybridizes to a
sense or antisense sequence of said gene, or naturally occurring
mutants thereof, or 5' or 3' flanking sequences aurally associated
with said gene; ii. combining said probe with nucleic acid of said
cell sample; and ii. detecting, by hybridization of said probe to
said cellular nucleic acid, the existence of at least one of a
deletion of one or more nucleotides from said gene, an addition of
one or more nucleotides to said gene, a substitution of one or more
nucleotides of said gene, a gross chromosomal rearrangement of all
or a potion of said gene, a gross alteration in the level of an
mRNA transcript of said gene, or a non-wild type splicing pattern
of an mRNA transcript of said gene.
91. The assay of claim 90, wherein hybridization of said probe
further comprises subjecting the probe and cellular nucleic acid to
a polymerase chain reaction (PCR) and detecting abnormalities in an
amplified product.
92. The assay of claim 90, wherein hybridization of said probe
further comprises subjecting the probe and cellular nucleic acid to
a ligation chain notion (LCR) and detecting abnormalities in an
amplified product.
93. The assay of claim 90, wherein said probe hybridizes under
stringent conditions to a nucleic acid designated by one or more of
SEQ ID Nos. 1-13.
94. A method for screening for compounds that modulate an activity
of a signalin polypeptide, comprising providing a signalin
polypeptide and a molecule; contacting said signalin polypeptide
and said molecule with a compound; and assaying binding of said
signalin polypeptide to said molecule in the presence versus the
absence of said compound, thereby identifying a compound that
modulates the activity of a signalin polypeptide.
95. The method of claim 94, wherein the activity of a signalin
polypeptide comprises modulating signaling via the TGF.beta.
signaling pathway.
96. The method of claim 95, wherein the compound is an antagonist
that inhibits signaling via the TGF.beta. signaling pathway.
97. The method of claim 95, wherein the compound is an agonist that
promotes signaling via the TGF.beta. signaling pathway.
98. The method of claim 94, wherein the molecule is a protein.
99. The method of claim 94, wherein the molecule is a nucleic
acid.
100. The method of claim 94, wherein the compound promotes binding
of the signalin polypeptide to the molecule.
101. The method of claim 94, wherein the compound inhibits binding
of the signalin polypeptide to the molecule.
102. A method for screening for compounds that modulate an activity
of a signalin polypeptide, comprising providing a cell that
expresses a signalin polypeptide, which cell is responsive to
TGF.beta. signaling; contacting said cell with a compound; and
assaying signalin inductive responses in the cell in the presence
versus the absence of said compound, thereby identifying a compound
that modulates the activity of a signalin polypeptide.
103. The method of claim 102, wherein assaying signalin inductive
responses in the cell comprises assaying cell proliferation or cell
differentiation.
104. The method of claim 102, wherein assaying signalin inductive
responses comprises assaying signaling via the TGF.beta. signaling
pathway.
105. The method of claim 102, wherein assaying signalin inductive
responses comprises measuring expression of genes which are
regulated by the TGF.beta. signaling pathway.
106. The method of claim 102, wherein the activity of a signalin
polypeptide comprises modulating signaling via the TGF.beta.
signaling pathway.
107. The method of claim 106, wherein the compound is an antagonist
that inhibits signaling via the TGF.beta. signaling pathway.
108. The method of claim 106, wherein the compound is an agonist
that promotes signaling via the TGF.beta. signaling pathway.
109. The method of claim 102, wherein the cell expresses a
recombinant signalin polypeptide.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/095,492, filed Mar. 12, 2002, which is a continuation of
U.S. application Ser. No. 08/580,031, filed Dec. 20, 1995 (U.S.
Pat. No. 6,428,977). The foregoing applications are hereby
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Pattern formation is the activity by which embryonic cells
form ordered spatial arrangements of differentiated tissues. The
physical complexity of higher organisms arises during embryogenesis
through the interplay of cell-intrinsic lineage and cell-extrinsic
signaling. Inductive interactions are essential to embryonic
patterning in vertebrate development from the earliest
establishment of the body plan, to the patterning of the organ
systems, to the generation of diverse cell types during tissue
differentiation (Davidson, E., (1990) Development 108:365-389;
Gurdon, J. B., (1992) Cell 68:185-199; Jessell, T. M. et al.,
(1992) Cell 68:257-270). The effects of developmental cell
interactions are varied. Typically, responding cells are diverted
from one route of cell differentiation to another by inducing cells
that differ from both the uninduced and induced states of the
responding cells (inductions). Sometimes cells induce their
neighbors to differentiate like themselves (homoiogenetic
induction); in other cases a cell inhibits its neighbors from
differentiating like itself. Cell interactions in early development
may be sequential, such that an initial induction between two cell
types leads to a progressive amplification of diversity. Moreover,
inductive interactions occur not only in embryos, but in adult
cells as well, and can act to establish and maintain morphogenetic
patterns as well as induce differentiation (J. B. Gurdon (1992)
Cell 68:185-199).
[0004] Several classes of secreted polypeptides are known to
mediate the cell-cell signaling that determines tissue fate during
development. An important group of these signaling proteins are the
TGF.beta. superfamily of molecules, which have wide range of
functions in many different species. Members of the family are
initially synthesized as larger precursor molecules with an
amino-terminal signal sequence and a pro-domain of varying size
(Kingsley, D. M. (1994) Genes Dev. 8:133-146). The precursor is
then cleaved to release a mature carboxy-terminal segment of
110-140 amino acids. The active signaling moiety is comprised of
hetero- or homodimers of the carboxy-terminal segment (Massague, J.
(1990) Annu. Rev. Cell Biol. 6:597-641). The active form of the
molecule then interacts with its receptor, which for this family of
molecules is composed of two distantly related transmembrane
serine/threonine kinases called type I and type II receptors
(Massague, J. et al. (1992) Cell 69:1067-1070; Miyazono, K. A. et
al. EMBO J. 10:1091-1101). TGF.beta. binds directly to the type II
receptor, which then recruits the type I receptor and modifies it
by phosphorylation. The type I receptor then transduces the signal
to downstream components, which are as yet unidentified (Wrana et
al, (1994) Nature 370:341-347).
[0005] Several members of the TGF.beta. superfamily have been
identified which play salient roles during vertebrate development.
Dorsalin is expressed preferentially in the dorsal side of the
developing chick neural tube (Basler et al. (1993) Cell
73:687-702). It promotes the outgrowth of neural crest cells and
inhibits the formation of motor neuron cells in vitro, suggesting
that it plays an important role in neural patterning along the
dorsoventral axis. Certain of the bone morphogenetic proteins
(BMPs) can induce the formation of ectopic bone and cartilage when
implanted under the skin or into muscles (Wozney, J. M. et al.
(1988) Science 242:1528-1534). In mice, mutations in BMP5 have been
found to result in effects on many different skeletal elements,
including reduced external ear size and decreased repair of bone
fractures in adults (Kingsley (1994) Genes Dev. 8:133-146). Besides
these effects on bone tissue, BMPs play other roles during normal
development. For example, they are expressed in non skeletal
tissues (Lyons et al. (1990) Development 109:833-844), and
injections of BMP4 into developing Xenopus embryos promote the
formation of ventral/posterior mesoderm (Dale et al (1992)
Development 115:573-585). Furthermore, mice with mutations in BMP5
have an increased frequency of different soft tissue abnormalities
in addition to the skeletal abnormalities described above (Green,
M. C. (1958) J. Exp. Zool. 137:75-88).
[0006] Members of the activin subfamily have been found to be
important in mesoderm induction during Xenopus development (Green
and Smith (1990) Nature 47:391-394; Thomsen et al. (1990) Cell
63:485-493) and inhibins were initially described as gonadal
inhibitors of follicle-stimulating hormone from pituitary cells. In
addition, antagonists of this signaling pathway can be used to
convert embryonic tissue into ectoderm, the default pathway of
development in the absence of TGF.beta.-mediated signals. BMP-4 and
activin have been found to be potent inhibitors of neuralization
(Wilson, P. A. and Hemmati-Brivanlou, A (1995) Nature
376:331-333).
[0007] Further evidence for the importance of a TGF.beta. family
member in early vertebrate development comes from a retroviral
insertion in the mouse nodal gene. This insertion leads to a
failure to form the primitive streak in early embryogenesis, a lack
of axial mesoderm tissue, and an overproduction of ectoderm and
extraembryonic ectoderm (Conlon et al. (1991) Development
111:969-981; Iannaccone et al (1992) Dev. Dynamics 194:198-208).
The predicted nodal gene product is consistent with previous
studies showing that nodal is related to activins and BMPs (Zhou et
al. (1993) Nature 361:543-547). A role for TGF.beta. family members
in the development of sex organs has also been described; Mullerian
inhibitory substance functions during vertebrate male sexual
development to cause regression of the embryonic duct system that
develops into oviducts and uterus (Lee and Donahoe (1993)
Endocrinol. Rev. 14:152-164).
[0008] Members of this family of signaling molecules also continue
to function post-development. TGF.beta. has antiproliferative
effects on many cell types including epithelial cells, endothelial
cells, smooth muscle cells, fetal hepatocytes, and myeloid,
erythroid, and lymphoid cells. Animals which cannot produce
TGF.beta.1 (homozygous for null mutations in the TGF.beta.1 gene)
have been found to survive until birth with no apparent
morphological abnormalities (Shull et al. (1992) Nature
359:693-699; Kulkami et al. (1993) Proc. Natl. Acac. Sci.
90:770-774). The animals do die around weaning age, however, owing
to massive immune infiltration in may different organs. These data
are consistent with the inhibitory effects of TGF.beta. on
lymphocyte growth (Tada et al. (1991) J. Immunol 146:1077-1082). In
another system, the expression of a TGF.beta. transgene in the
mammary tissue of mice has been shown to inhibit the development
and secretory function of mammary tissue during sexual maturation
and pregnancy (Jhappan, C. et al. (1993) EMBO J. 12:1835-1845;
Pierce, D. F. et al. (1993) Genes Dev. 7:2308-2317). In addition to
these inhibitory effects, TGF.beta. can also promote the growth of
other cell types as evidenced by its role in neovascularization and
the proliferation of connective tissue cells. Because of these
activities, it plays a key role in wound healing (Kovacs, E. J.
(1991) Immunol Today 12:17-23)
SUMMARY OF THE INVENTION
[0009] The present invention relates to the discovery of a novel
family of genes, and gene products, expressed in vertebrate
organisms, which genes referred to hereinafter as the "signalin"
gene family, the products of which are referred to as signalin
proteins. The products of the signalin gene have apparent broad
involvement in the formation and maintenance of ordered spatial
arrangements of differentiated tissues in vertebrates, and can be
used or manipulated to generate and/or maintain an array of
different vertebrate tissue both in vitro and in vivo.
[0010] In general, the invention features isolated vertebrate
signalin polypeptides, preferably substantially pure preparations
of one or more of the subject signalin polypeptides. The invention
also provides recombinantly produced signalin polypeptides. In
preferred embodiments the polypeptide has a biological activity
including: an ability to modulate proliferation, survival and/or
differentiation of mesodermally-derived tissue, such as tissue
derived from dorsal mesoderm; the ability to modulate
proliferation, survival and/or differentiation of
ectodermally-derived tissue, such as tissue derived from the neural
tube, neural crest, or head mesenchyme; the ability to modulate
proliferation, survival and/or differentiation of
endodermally-derived tissue, such as tissue derived from the
primitive gut. Moreover, in preferred embodiments, the subject
signalin proteins have the ability to modulate intracellular signal
transduction pathways mediated by receptors for members of the
Transforming Growth Factor .beta. superfamily of molecules.
[0011] In one embodiment, the polypeptide is identical with or
homologous to a signalin protein. Exemplary signalin proteins are
represented by SEQ ID No. 14, SEQ ID No:15, SEQ ID No:16, SEQ ID
No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ
ID No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26.
Related members of the vertebrate signalin family are also
contemplated, for instance, a signalin polypeptide preferably has
an amino acid sequence at least 60% homologous to a polypeptide
represented by any of SEQ ID Nos:14-26, though polypeptides with
higher sequence homologies of, for example, 70, 80%, 90% or are
also contemplated. The signalin polypeptide can comprise a full
length protein, such as represented in the sequence listings, or it
can comprise a fragment corresponding to particular motifs/domains,
or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150 or
200 amino acids in length. In preferred embodiments, the
polypeptide, or fragment thereof, specifically modulates, by acting
as either an agonist or antagonist, the signal transduction
activity of a receptor for a transforming growth factor .beta..
[0012] In certain preferred embodiments, the invention features a
purified or recombinant signalin polypeptide having a molecular
weight in the range of 45 kd to 70 kd. For instance, preferred
signalin polypeptide chains of the .alpha. and .beta. subfamilies,
described infra, have molecular weights in the range of 45 kd to
about 55 kd, even more preferably, in the range of 50-55 kd. In
another illustrative example, preferred signalin polypeptide chains
of the .gamma. subfamily have molecular weights in the range of 60
kd to about 70 kd, even more preferably in the range of 63-68 kd.
It will be understood that certain post-translational
modifications, e.g., phosphorylation and the like, can increase the
apparent molecular weight of the signalin protein relative to the
unmodified polypeptide chain.
[0013] In another embodiment, the signalin polypeptide comprises a
signalin motif represented in the general formula shown in SEQ ID
No:28. In a preferred embodiment the signalin motif corresponds to
a signalin motif represented in one of SEQ ID Nos:14-26. In another
embodiment, the signalin polypeptide of the invention comprises a
.nu. domain represented in the general formula SEQ ID No:27. In a
preferred embodiment the .nu. region corresponds to a .nu. domain
represented in one of SEQ ID Nos:14-26. In another preferred
embodiment, the signalin polypeptide of the invention comprises a
.chi. domain represented in the general formula SEQ ID No:29. In a
further preferred embodiment the .chi. region corresponds to a
.chi. domain represented in one of SEQ ID Nos:14-26. In another
preferred embodiment, the signalin polypeptide can modulate, either
stimulate or antagonize, intracellular pathways mediated by a
receptor for a TGF.beta.. In still another embodiment, the
polypeptide comprises an amino acid sequence represented in the
general formula:
LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV. In still a
further embodiment, the signalin polypeptide of the present
invention comprises an amino acid sequence represented by the
general formula: LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV.
In an additional embodiment, the signalin polypeptide of the
present invention comprises an amino acid sequence represented by
the general formula:
LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
[0014] In another preferred embodiment, the invention features a
purified or recombinant polypeptide fragment of a signalin protein,
which polypeptide has the ability to modulate, e.g., mimic or
antagonize, a the activity of a wild-type signalin protein.
Preferably, the polypeptide fragment comprises a signalin
motif.
[0015] Moreover, as described below, the preferred signalin
polypeptide can be either an agonist (e.g. mimics), or
alternatively, an antagonist of a biological activity of a
naturally occurring form of the protein, e.g., the polypeptide is
able to modulate differentiation and/or growth and/or survival of a
cell responsive to authentic signalin proteins. Homologs of the
subject signalin proteins include versions of the protein which are
resistant to post-translation modification, as for example, due to
mutations which alter modification sites (such as tyrosine,
threonine, serine or aspargine residues), or which inactivate an
enzymatic activity associated with the protein.
[0016] The subject proteins can also be provided as chimeric
molecules, such as in the form of fusion proteins. For instance,
the signalin protein can be provided as a recombinant fusion
protein which includes a second polypeptide portion, e.g., a second
polypeptide having an amino acid sequence unrelated (heterologous)
to the signalin polypeptide, e.g. the second polypeptide portion is
glutathione-S-transferase, e.g. the second polypeptide portion is
an enzymatic activity such as alkaline phosphatase, e.g. the second
polypeptide portion is an epitope tag.
[0017] In a preferred embodiment the signalin polypeptide of the
present invention modulates signal transduction from a TGF.beta.
receptor. For example, the signalin polypeptide may modulate the
transduction of a TGF.beta. receptor for a member of the dpp
family, e.g., dpp, BMP2, or BMP4. In another preferred embodiment,
the signalin polypeptide modulates the signaling of a TGF.beta.
other than a dpp family member. For instance, the signalin
polypeptide may be involved in signalling from one or more of BMP5,
BMP6 BMP7, BMP8, 60A, GDF5, GDF6, GDF7, GDF1, Vg1, dorsalin, BMP3,
GDF10, nodal, inhibins, activins, TGF.beta.1, TGF.beta.2,
TGF.beta.3, MIS, GDF9 or GDNE.
[0018] In yet another embodiment, the invention features a nucleic
acid encoding a signalin polypeptide, or polypeptide homologous
thereto, which polypeptide has the ability to modulate, e.g.,
either mimic or antagonize, at least a portion of the activity of a
wild-type signalin polypeptide. Exemplary signalin polypeptides are
represented by SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID
No:17, SEQ ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, SEQ
ID No:22, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26.
In another embodiment the nucleic acid of the present invention
hybridizes under stringent conditions with one or more of the
nucleic acid sequences in SEQ ID No:1-13. In preferred embodiments,
the nucleic acid encodes a polypeptide which specifically
modulates, by acting as either an agonist or antagonist, the signal
transduction activity of a receptor for a transforming growth
factor .beta..
[0019] In another embodiment, the nucleic acid encodes an amino
acid sequence which comprises a signalin motif represented in the
general formula shown in SEQ ID No:28. In preferred embodiment the
signalin motif corresponds to a signalin motif represented in one
of SEQ ID Nos:14-26. In another embodiment, the nucleic acid of the
invention encodes an amino acid sequence which comprises a .nu.
domain represented in the general formula SEQ ID No:27. In a
preferred embodiment the encoded .nu. region corresponds to a .nu.
domain represented in one of SEQ ID Nos:14-26. In another
embodiment, the nucleic acid encodes a signalin polypeptide of the
invention which comprises a .chi. domain represented in the general
formula SEQ ID No:29. In a preferred embodiment the encoded .chi.
region corresponds to a .chi. domain represented in one of SEQ ID
Nos:14-26. In still a another embodiment, the nucleic acid sequence
encodes a polypeptide which comprises an amino acid sequence
represented in the general formula:
LDGRLQVSHRKGLPHVIYCRVWRWPDLQSHHELKPXECCEXPFXSKQKXV. In another
embodiment, the nucleic acid of the present invention encodes a
polypeptide which comprises an amino acid sequence represented by
the general formula,
LDGRLQVAGRKGFPHVIYARLWXWPDLHKNELKHVKFCQXAFDLKYDXV. In an still
another embodiment, the nucleic acid encodes a polypeptide which
comprises an amino acid sequence represented by the general
formula, LDGRLQVXHRKGLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEV.
[0020] Another aspect of the present invention provides an isolated
nucleic acid having a nucleotide sequence which encodes a signalin
polypeptide. In preferred embodiments, the encoded polypeptide
specifically mimics or antagonizes inductive events mediated by
wildtype signalin proteins. The coding sequence of the nucleic acid
can comprise a sequence which is identical to a coding sequence
represented in one of SEQ ID Nos:1-13, or it can merely be
homologous to one or more of those sequences.
[0021] Furthermore, in certain preferred embodiments, the subject
signalin nucleic acid will include a transcriptional regulatory
sequence, e.g. at least one of a transcriptional promoter or
transcriptional enhancer sequence, which regulatory sequence is
operably linked to the signalin gene sequence. Such regulatory
sequences can be used in to render the signalin gene sequence
suitable for use as an expression vector. This invention also
contemplates the cells transfected with said expression vector
whether prokaryotic or eukaryotic and a method for producing
signalin proteins by employing said expression vectors.
[0022] In yet another embodiment, the nucleic acid hybridizes under
stringent conditions to a nucleic acid probe corresponding to at
least 12 consecutive nucleotides of either sense or antisense
sequence of one or more of SEQ ID Nos:1-13; though preferably to at
least 25 consecutive nucleotides; and more preferably to at least
40, 50 or 75 consecutive nucleotides of either sense or antisense
sequence of one or more of SEQ ID Nos:1-13.
[0023] Yet another aspect of the present invention concerns an
immunogen comprising a signalin polypeptide in an immunogenic
preparation, the immunogen being capable of eliciting an immune
response specific for a signalin polypeptide; e.g. a humoral
response, e.g. an antibody response; e.g. a cellular response. In
preferred embodiments, the immunogen comprising an antigenic
determinant, e.g. a unique determinant, from a protein represented
by one of SEQ ID Nos. 14-26.
[0024] A still further aspect of the present invention features
antibodies and antibody preparations specifically reactive with an
epitope of the signalin immunogen.
[0025] The invention also features transgenic non-human animals,
e.g. mice, rats, rabbits, chickens, frogs or pigs, having a
transgene, e.g., animals which include (and preferably express) a
heterologous form of a signalin gene described herein, or which
misexpress an endogenous signalin gene, e.g., an animal in which
expression of one or more of the subject signalin proteins is
disrupted. Such a transgenic animal can serve as an animal model
for studying cellular and tissue disorders comprising mutated or
mis-expressed signalin alleles or for use in drug screening.
[0026] The invention also provides a probe/primer comprising a
substantially purified oligonucleotide, wherein the oligonucleotide
comprises a region of nucleotide sequence which hybridizes under
stringent conditions to at least 12 consecutive nucleotides of
sense or antisense sequence of SEQ ID No:1-13, or naturally
occurring mutants thereof. Nucleic acid probes which are specific
for each of the classes of vertebrate signalin proteins are
contemplated by the present invention, e.g. probes which can
discern between nucleic acid encoding an .alpha., .beta., or
.gamma. signalin. In preferred embodiments, the probe/primer
further includes a label group attached thereto and able to be
detected. The label group can be selected, e.g., from a group
consisting of radioisotopes, fluorescent compounds, enzymes, and
enzyme co-factors. Probes of the invention can be used as a part of
a diagnostic test kit for identifying dysfunctions associated with
mis-expression of a signalin protein, such as for detecting in a
sample of cells isolated from a patient, a level of a nucleic acid
encoding a subject signalin protein; e.g. measuring a signalin mRNA
level in a cell, or determining whether a genomic signalin gene has
been mutated or deleted. These so called "probes/primers" of the
invention can also be used as a part of "antisense" therapy which
refers to administration or in situ generation of oligonucleotide
probes or their derivatives which specifically hybridize (e.g.
bind) under cellular conditions, with the cellular mRNA and/or
genomic DNA encoding one or more of the subject signalin proteins
so as to inhibit expression of that protein, e.g. by inhibiting
transcription and/or translation. Preferably, the oligonucleotide
is at least 12 nucleotides in length, though primers of 25, 40, 50,
or 75 nucleotides in length are also contemplated.
[0027] In yet another aspect, the invention provides an assay for
screening test compounds for inhibitors, or alternatively,
potentiators, of an interaction between a signalin protein and a
signalin binding protein or nucleic acid sequence. An exemplary
method includes the steps of (i) combining a signalin polypeptide
or fragment thereof, a signalin binding element, and a test
compound, e.g., under conditions wherein, but for the test
compound, the signalin protein and binding element are able to
interact; and (ii) detecting the formation of a complex which
includes the signalin protein and the binding element either by
directly quantitating the complex or by measuring inductive effects
of the signalin protein. A statistically significant change, such
as a decrease, in the formation of the complex in the presence of a
test compound (relative to what is seen in the absence of the test
compound) is indicative of a modulation, e.g., inhibition, of the
interaction between the signalin protein and its binding
element.
[0028] Yet another aspect of the present invention concerns a
method for modulating one or more of growth, differentiation, or
survival of a mammalian cell responsive to signalin induction. In
general, whether carries out in vivo, in vitro, or in situ, the
method comprises treating the cell with an effective amount of a
signalin polypeptide so as to alter, relative to the cell in the
absence of signalin treatment, at least one of (i) rate of growth,
(ii) differentiation, or (iii) survival of the cell. Accordingly,
the method can be carried out with polypeptides mimics the effects
of a naturally-occurring signalin protein on the cell, as well as
with polypeptides which antagonize the effects of a
naturally-occurring signalin protein on said cell. In preferred
embodiments, the signalin polypeptide provided in the subject
method are derived from vertebrate sources, e.g., are vertebrate
signalin polypeptides. For instance, preferred polypeptides
includes an amino acid sequence identical or homologous to an amino
acid sequence (e.g., including bioactive fragments) designated in
one of SEQ ID No:14, SEQ ID No:15, SEQ ID No:16, SEQ ID No:17, SEQ
ID No:18, SEQ ID No:19, SEQ ID No:20, SEQ ID No:21, or SEQ ID
No:12, SEQ ID No:23, SEQ ID No:24, SEQ ID No:25, SEQ ID No:26.
Furthermore, the present invention contemplates the use of other
metazoan (e.g., invertebrate) homologs of the signalin polypeptides
or bioactive fragments thereof equivalent to the subject vertebrate
fragments.
[0029] In one embodiment, the subject method includes the treatment
of testicular cells, so as modulate spermatogenesis. In another
embodiment, the subject method is used to modulate osteogenesis,
comprising the treatment of osteogenic cells with a signalin
polypeptide. Likewise, where the treated cell is a chondrogenic
cell, the present method is used to modulate chondrogenesis. In
still another embodiment, signalin polypeptides can be used to
modulate the differentiation of neural cells, e.g., the method can
be used to cause differentiation of a neuronal cell, to maintain a
neuronal cell in a differentiated state, and/or to enhance the
survival of a neuronal cell, e.g., to prevent apoptosis or other
forms of cell death. For instance, the present method can be used
to affect the differentiation of such neuronal cells as motor
neurons, cholinergic neurons, dopanergic neurons, serotenergic
neurons, and peptidergic neurons.
[0030] The present method is applicable, for example, to cell
culture technique, such as in the culturing of neural and other
cells whose survival or differentiative state is dependent on
signalin function. Moreover, signalin agonists and antagonists can
be used for therapeutic intervention, such as to enhance survival
and maintenance of neurons and other neural cells in both the
central nervous system and the peripheral nervous system, as well
as to influence other vertebrate organogenic pathways, such as
other ectodermal patterning, as well as certain mesodermal and
endodermal differentiation processes. In an exemplary embodiment,
the method is practiced for modulating, in an animal, cell growth,
cell differentiation or cell survival, and comprises administering
a therapeutically effective amount of a signalin polypeptide to
alter, relative the absence of signalin treatment, at least one of
(i) rate of growth, (ii) differentiation, or (iii) survival of one
or more cell-types in the animal.
[0031] Another aspect of the present invention provides a method of
determining if a subject, e.g. a human patient, is at risk for a
disorder characterized by unwanted cell proliferation or aberrant
control of differentiation. The method includes detecting, in a
tissue of the subject, the presence or absence of a genetic lesion
characterized by at least one of (i) a mutation of a gene encoding
a signalin protein, e.g. represented in one of SEQ ID Nos:14-26, or
a homolog thereof; or (ii) the mis-expression of a signalin gene.
In preferred embodiments, detecting the genetic lesion includes
ascertaining the existence of at least one of: a deletion of one or
more nucleotides from a signalin gene; an addition of one or more
nucleotides to the gene, a substitution of one or more nucleotides
of the gene, a gross chromosomal rearrangement of the gene; an
alteration in the level of a messenger RNA transcript of the gene;
the presence of a non-wild type splicing pattern of a messenger RNA
transcript of the gene; or a non-wild type level of the
protein.
[0032] For example, detecting the genetic lesion can include (i)
providing a probe/primer including an oligonucleotide containing a
region of nucleotide sequence which hybridizes to a sense or
antisense sequence of a signalin gene, e.g. a nucleic acid
represented in one of SEQ ID Nos: 1-13, or naturally occurring
mutants thereof, or 5' or 3' flanking sequences naturally
associated with the signalin gene; (ii) exposing the probe/primer
to nucleic acid of the tissue; and (iii) detecting, by
hybridization of the probe/primer to the nucleic acid, the presence
or absence of the genetic lesion; e.g. wherein detecting the lesion
comprises utilizing the probe/primer to determine the nucleotide
sequence of the signalin gene and, optionally, of the flanking
nucleic acid sequences. For instance, the probe/primer can be
employed in a polymerase chain reaction (PCR) or in a ligation
chain reaction (LCR). In alternate embodiments, the level of a
signalin protein is detected in an immunoassay using an antibody
which is specifically immunoreactive with the signalin protein.
[0033] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
[0034] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 is an illustration of the model system used to test
the biological activities of the signalin proteins described in the
present invention.
[0036] FIG. 2 shows the morphology of animal cap explants from
control, signalin1 injected, or signalin2 injected embryos.
[0037] FIG. 3 illustrate the histologic analysis of animal cap
explants from control, signalin1 injected, or signalin2 injected
embryos.
[0038] FIG. 4 is an autoradiogram which shows the expression of
various marker RNAs in the injected embryos as detected by
polymerase chain reaction. Brachyury is a general mesodermal
marker; Goosecoid is a marker of dorsal mesoderm; Xwnt-8 is a
marker of ventral-lateral mesoderm; globin is a marker of ventral
mesoderm; actin is a marker of dorsal mesoderm; NCAM is a marker of
neural tissue; and EF-1.alpha. is ubiquitously expressed and serves
as a control for the amount of RNA included in each reaction. The
lane marked "E" contains total RNA harvested from whole embryos and
is a positive control. The lane marked "-RT" is identical to the
positive control lane, except that reverse transcriptase was not
included and serves as a negative control. The lanes designated
"S1" and "S2" correspond to samples from embryos injected with
xe-signalin1 and xe-signalin 2, respectively.
[0039] FIG. 5 is a matrix illustrating a possible grouping of the
signalin family into at least three different sub-families.
Blacked-out boxes represent >10 mismatches over the signalin
motif.
[0040] FIG. 6 depicts an alignment of a portion of the signalins
identified in both humans and Xenopus.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Of particular importance in the development and maintenance
of tissue in vertebrate animals is a type of extracellular
communication called induction, which occurs between neighboring
cell layers and tissues (Saxen et al. (1989) Int J Dev Biol 33:
21-48; and Gurdon et al. (1987) Development 99:285-306). In
inductive interactions, chemical signals secreted by one cell
population influence the developmental fate of a second cell
population. Typically, cells responding to the inductive signals
are diverted from one cell fate to another, neither of which is the
same as the fate of the signaling cells. Inductive signals are
transmitted by key regulatory proteins that function during
development to determine tissue patterning. For example, signals
mediated by the TGF.beta. superfamily have been shown to play a
variety of roles, including participating in vertebrate tissue
induction.
[0042] The present invention concerns the discovery of a family of
vertebrate genes, referred to herein as "signalins", which function
in intracellular signal transduction pathways initiated by members
of the TG.beta.-superfamily, and have a role in determining tissue
fate and maintenance. For instance, the results provided below
indicate that proteins encoded by the vertebrate signalin genes may
participate in the control of development and maintenance of a
variety of embryonic and adult tissues. For example, during
embryonic induction, certain of the signalins are implicated in the
differentiation and patterning of both dorsal and ventral
mesoderm.
[0043] The family of vertebrate signalin genes or gene products
provided by the present invention apparently consists of at least
seven different members which can be grouped into at least three
different subclasses within the signalin family. The vertebrate
signalins are related, apparently both in sequence and function, to
the drosophila and C. elegans MAD genes (Sekelsky et al. (1995)
Genetics 139:1347). The cDNAs corresponding to vertebrate signalin
gene transcripts were initially cloned from Xenopus and are,
arbitrarily, designed as Xe-signalin 1-4. As described in the
appended examples, degenerate primers from the cloning of the
Xenopus signalins were also used to clone human homologs of this
gene family. As a result, cDNA's for at least seven different human
signalin transcripts have been identified, and are designated
herein, again arbitrarily, as Hu-signalin 1-7. Provided in Table 1
below is a guide to the designated SEQ ID numbers for the
nucleotide and amino acid sequences for each signalin clone.
TABLE-US-00001 TABLE 1 Guide to signalin sequences in Sequence
Listing Nucleotide Amino Acid Xe-signalin 1 SEQ ID No. 1 SEQ ID No.
14 Xe-signalin 2 SEQ ID No. 2 SEQ ID No. 15 Xe-signalin 3 SEQ ID
No. 3 SEQ ID No. 16 Xe-signalin 4 SEQ ID No. 4 SEQ ID No. 17
Hu-signalin 1 SEQ ID No. 5 SEQ ID No. 18 Hu-signalin 2 SEQ ID No. 6
SEQ ID No. 19 Hu-signalin 3 SEQ ID No. 7 SEQ ID No. 20 Hu-signalin
4 SEQ ID No. 8 SEQ ID No. 21 Hu-signalin 5 SEQ ID No. 9 SEQ ID No.
22 Hu-signalin 6 SEQ ID No. 10 SEQ ID No. 23 Hu-signalin 7 SEQ ID
No. 11 SEQ ID No. 24
[0044] From the apparent molecular weights, the family of
vertebrate signalin proteins apparently ranges in size from about
45 kd to about 70 kd for the unmodified polypeptide chain. For
instance, Xe-signalin 1 and 3 have apparent molecular weights of
about 52.2 kd, Xe-signalin 2 has an apparent molecular weight of
about 52.4 kd, and Xe-signalin 4 has an apparent molecular weight
of about 64.9 kd.
[0045] Analysis of the vertebrate signalin sequences revealed no
obvious similarities with any previously identified domains or
motifs. However, the fact that each full-length clone lacks a
signal sequence, along with the observation that signalin proteins
can be detected in both the nucleus and the cytoplasm, indicates
that the vertebrate signalin genes encode intracellular
proteins.
[0046] The above notwithstanding, careful inspection of the clones
suggests at least two novel domains, one or both of which may be
characteristic of the vertebrate signalin family. The first
apparently conserved structural element of the signalin family
occurs in the N-terminal portion of the molecule, and is designated
herein as the ".nu. domain". With reference to xe-signalin-1, the
.nu. domain corresponds to amino acid residues Leu37-Val130. By
alignment of the vertebrate signalin clones, the element is
represented by the consensus sequence:
LVKKLK-X(1)-CVTI-X(2)-RXLDGRLQVXXRKGXPHVIYXRWXWPDL-X(3)-VCXNPYHYXRV
(SEQ ID No. 27), wherein X(1) represents from about 17-25 residues,
X(2) represents from about 1-35 residues, and X(3) represents about
20-25 residues, and each of the other X's represent any single
amino acid, though more preferably represent an amino acid residue
in the corresponding vertebrate signalin sequences of the appended
sequence listing.
[0047] Within the .nu. domain, there is a motif which is highly
conserved not only amongst the vertebrate signaling, but also
amongst the related drosophila and C. elegans MAD polypeptides. In
particular, this motif (referred to herein as a "signalin-motif")
includes the consensus sequence LDGRLQVXXRKGXPHVIYXRWXWPDL (SEQ ID
No. 28). Again, each occurrence of X independently represent any
single amino acid, though more preferably represent an amino acid
residue in the corresponding vertebrate signalin sequences of the
appended sequence listing.
[0048] Another apparent motif occurs in the C-terminal portion of
the signalin family. Referred to herein as the ".chi. motif", it
corresponds to amino acid residues Leu405-Leu450 of xe-signalin-1.
Again, by alignment of the vertebrate clones presently sequenced,
the .chi. motif can be represented by the consensus sequence
LXXXCXXRXSFVKGWGXXXXRQXXXX-TPCWIEXHLXXXLQXLDXVL (SEQ ID No. 29),
wherein each occurrence of X independently represent any single
amino acid, though more preferably represent an amino acid residue
in the corresponding vertebrate signalin sequences of the appended
sequence listing.
[0049] Not wishing to be bound by any particular theory, analysis
of one of the apparently conserved motifs (the signalin motif)
suggests that the signalin protein family can be grouped into at
least three different sub-families. As FIGS. 5 and 6 illustrate,
xe-signalins 1 and 3 and hu-signalins 1, 3 and 7 apparently form
one sub-family of signalins (the ".alpha.-subfamily" or
".alpha.-signalins"). Likewise, xe-signalin 4 and hu-signalins 4
and 2 form a second apparent sub-family (the .beta.-subfamily" or
".beta.-signalins"), and xe-signalin 2 and hu-signalins 5 and 6
form a third sub-family (the ".gamma.-subfamily" or
".gamma.-signalins"). Comparison of the amino acid sequence around
the signalin motif amongst members of the .alpha.-subfamily
demonstrates a consensus sequence for a signalin motif represented
by
Sequence CWU 1
1
* * * * *