Title: 150 KDA TGF-B1 accessory
receptor acts a negative modulator of TGF-B signaling
United States Patent: 7,173,002
Issued: February 6, 2007
Inventors: Philip; Anie
(Montreal, CA), Tam; Betty (Ville St. Laurent, CA)
Assignee: McGill University
Appl. No.: 10/475,711
Filed: April 24, 2002
PCT Filed: April 24, 2002
PCT No.: PCT/CA02/00560
371(c)(1),(2),(4) Date: May
PCT Pub. No.: WO02/085942
PCT Pub. Date: October 31,
The present invention relates to a TGF-.beta.1
binding protein called r150. This protein has a GPI-anchor contained in
r150 itself and not on a tightly associated protein and that it binds TGF-.beta.1
with an affinity comparable to those of the signaling receptors.
Furthermore, the released (soluble) form of this protein binds TGF-.beta.1
independent of the types I and II receptors. Also, the soluble form
inhibits the binding of TGF-.beta. to its receptor. In addition, evidence
that r150 is released from the cell surface by an endogenous phospholipase
C is provided. Also, the creation of a mutant human keratinocyte cell line
with a defect in GPI synthesis which displays reduced expression of r150
is described. Our results using these mutant keratinocytes suggest that
the membrane anchored form of r150 is a negative modulator of TGF-beta
responses. These findings, taken together with the observation that r150
forms a heteromeric complex with the signaling receptors, suggest that
this accessory receptor in either its membrane anchored or soluble form
may antagonize TGF-.beta. responses in human keratinocytes. Experiments
with mutants confirmed that TGF.beta.1 activity can be modulated when the
expression of the accessory receptor r150 is silenced. The complete
nucleic acid and deduced amino acid sequences are now provided. The r150
cloned nucleic acid was used to study overexpression of r150. When r150
gene is overexpressed, TGF.beta. responses are increased. r150 and its
derivatives or precursors (fragments, variants and nucleic acids encoding
the same) will find a broad clinical utility, knowing that TGF.beta.1 is
an important cytokine.
Description of the Invention
BACKGROUND OF THE
Transforming growth factor-.beta. (TGF-.beta.)
is a 25 kDa multi functional growth factor which plays a central role in
the wound healing process (Roberts and Sporn, 1990; O'Kane and Ferguson,
1997). It is an important regulator of the immune response (Letterio and
Roberts, 1998), angiogenesis, reepithelialization (Roberts and Sporn,
1990), extracallular matrix protein synthesis and remodeling (Peltonen et
al, 1991; Yamamoto et al, 1994). During wound healing, re-epithelialization
initiates the repair process which is characterized by recruitment of
epidermal stem cells, keratinocyte proliferation and the formation of an
epithelial tongue of migrating keratinocytes at the wound edge (Clark,
1996). TGF-.beta. is chemotacfic to keratinocytes and induces the
expression of integrins on the migrating epithelium (Helbda, 1988;
Zambruno et al, 1995). In spite of its promigratory effect on
keratinocytes, TGF-.beta. is a potent inhibitor to epithelial cell
proliferation in vitro (Pietenpol et at, 1990) and in vivo (Glick et al,
1993). Targeted deletion of the TGF-.beta.1 gene in keratinocytes causes
rapid progression to squamous cell carcinoma (Glick et al, 1994). In
addition, the epidermis of transgenic mice expressing a dominant negative
TGF-.beta. receptor exhibits a hyperplastic and hyperkeratotic phenotype
(Wang et al, 1997). These results support the importance of proper
expression of TGF-.beta. and regulation of its function in epidermal
development and maintenance of epidermal homeostasis.
TGF-.beta. is a member of the TGF-.beta. superfamily which also include
activins, inhibins, bone morphogenic proteins, growth differentiation
factor 1 (GDF-1) and glial-derived neurotropic growth factor (GDNF)
There are three widely distributed TGF-.beta. receptors, type I, type II
and type III, all of which have been cloned (Roberts and Sporn, 1990;
Massague, 1998). The types I and II receptors are both transmembrane
serine/threonine kinases that are essential for TGF-.beta. signal
transduction. The type III receptor, also known as betaglycan, is a high
molecular weight proteoglycan that is not required for signaling, but is
believed to play a role in presenting the ligand to the type II receptor
(Lopez-Casillas et al, 1993). Endoglin, is another TGF-.beta. receptor
predominantly expressed on endothelial cells (Gougos and Letarte, 1990).
According to the present model of TGF-.beta. signal transduction, binding
of TGF-.beta. to the type II receptor which is a constitutively active
kinase, leads to the recruitment and phosphorylation of the type I
receptor (Wrana et al, 1994). The activated type I kinase phosphorylates
the central intracellular mediators of TGF-.beta. signalling known as the
Smad proteins (Heldin et al, 1997). Smad proteins can be classified into
three groups: the pathway restricted Smads include the Smad2 and Smad3
which are phosphorylated by the type I receptor of TGF-.beta. or activin,
while the Smads 1, 5 and 8 are implicated in BMP signalling. The
phosphorylation of the pathway restricted Smads permits their interaction
with the common Smad or Smad4 and this heteromeric complex then
translocates into the nucleus where it regulates expression of target
genes. Finally, there inhibitory Smads which include the Smad 7 and Smad 6
prevent the phosphorlyation of the R-Smads by the type I kinase. (Heldin
et al, 1997, Massague, 1998; Wrana and Attisano, 2000)
In blood circulation, TGF-.beta.1 is found bound to the carrier
.alpha..sub.2 macroglobulin (.alpha..sub.2M; Webb et al. 1998).
.alpha..sub.2M binds many other cytokines and therefore lacks selectivity
for TGF-.beta.1. .alpha..sub.2M polymorphism has been associated with
Alzheimer's disease, which polymorphism is observed as a deletion in "the
bait region" overlapping with TGF-.beta.1 binding domina (Gonias et al.
2000 and Blacker et al 1998).
Although the types I and II receptors are central to TGF-.beta. signaling,
it is possible that accessory receptors interacting with the signaling
receptors modify TGF-.beta. responses. For example, both endoglin and type
III receptor which form heteromeric complexes with the type II receptor
have been reported to modulate TGF-.beta. function. When overexpressed in
myoblasts, endoglin inhibited while type III receptor enhanced TGF-.beta.
responses (Letamendia et al, 1998). In addition, endoglin was shown to
antagonize TGF-.beta. mediated growth inhibition of human vascular
endothelial cells (Li et al, 2000). Similarly, the newly identified type
I-like receptor BAMBI which associates with TGF-.beta. family receptors
can inhibit signaling (Onichtchouk et al, 1999).
There are also a number of molecules that can impact TGF-.beta. signal
transduction by interacting with one or both of the TGF-.beta. signaling
receptors. However, the exact physiological significance of many of these
interactions are not clearly defined (for review, Massague, 1998). Three
of these interacting proteins: the type II TGF-.beta. receptor interacting
protein (TRIP-1) (Chen et al, 1995), B.alpha. (.alpha. subunit of protein
phosphatase A) (Griswold-Prenner, 1998), and serine-threonine kinase
receptor associated protein (STRAP) (Datta et al, 1998) all contain the
highly conserved tryptophan-aspartic acid (WD) repeats. WD domains are
important in protein-protein interactions and cellular functions such as
cell cycle progression and transmembrane signaling (Neer et al, 1994).
TRIP-1 is phosphorylated through its interaction with the type II receptor
kinase and exerts an inhibitory effect on TGF-.beta. induced PAI-1 gene
transcription, but has no effect on TGF-.beta. mediated growth inhibition
(Choy and Derynck, 1998). On the other hand, B.alpha. associates with the
type I receptor and positively modulates TGF-.beta. action. Finally, STRAP
can interact with both the type I and II receptors and when overexpressed,
it exerts an inhibitory effect on TGF-.beta. mediated transcriptional
activation. In addition, STRAP can also interact with the inhibitory
Smad7, but not Smad6. STRAP's interaction with Smad7 exerts a stabilizing
effect on Smad7's association with the activated type I kinase receptor
which prevents Smad2/3's association and subsequent phosphorylation (Datta
and Moses, 2000).
The immunophilin, FKBP12, interacts with the TGF-.beta. type I receptor
and acts as a negative modulator of TGF-.beta. function (Wang et al,
1996). It can interact with unactivated type I receptor and functions to
stabilize the quiescent receptor state by protecting phosphorylation sites
in the GS domain. Upon ligand stimulation, heteromerization and subsequent
phosphorylation of the GS domain by the TGF-.beta. type II kinase results
in the release of FKBP12 (Chen et al, 1997; Huse et al, 1999). In
contrast, the TGF-.beta. type I receptor associated protein-1 (TRAP-1)
interacts only with the activated type I receptor kinase (Charng et al,
1998). TRAP-1 is not phosphorylated by the type I kinase and TRAP-1's
interaction is reported to have an inhibitory effect on TGF-.beta.
signaling. However, a recent report describes a different function for
TRAP-1 (Wurthner et al, 2001). In this study, TRAP-1 was found to
associate with inactive TGF-.beta. and activin receptor complexes and upon
ligand stimulation, TRAP-1 is released. The conformationally altered
TRAP-1 is then believed to associate and subsequently chaperone Smad4 to
the activated Smad2. The .alpha. subunit of ras farnesyl protein
transferase (FNTA) preferentially interacts with the activated type I
receptor and is considered a substrate because it is phosphorylated by the
type I kinase and released thereafter (Kawabata et al, 1995). However the
functional significance of this phenomenon remains unexplained. The
accessory receptors, endoglin and type III receptor which form heteromeric
complexes with the type II receptor have also been reported to modulate
TGF-.beta. function. When overexpressed in myoblasts, endoglin inhibited
while type III receptor enhanced TGF-.beta. responses (Letamendia et al,
1998). Glycosylphosphatidyl inositol (GPI)-anchored proteins which lack
transmembrane and cytoplasmic domains have also been shown to bind TGF-.beta..
These proteins have been identified on certain cell lines (Cheifetz and
Massague, 1991), but the identity of these GPI-anchored proteins and the
role they may play in TGF-.beta. signaling remain unknown. Recently, the
present inventors reported the presence of GPI-anchored TGF-.beta. binding
proteins on early passage human endometrial stromal cells (Dumont et al,
1995), human skin fibroblasts (Tam and Philip, 1998) and keratinocytes
(Tam et al, 1998). On human keratinocytes, they identified a 150 kDa
GPI-anchored TGF-.beta.1 binding protein designated as r150 that can form
a heteromeric complex with the types I and II TGF-.beta. receptors (Tam et
al, 1998). In addition, they demonstrated that upon hydrolysis fom the
cell surface by phosphatidylinositol phospholipase C (PIPLC), the soluble
form of r150, retains its ability to bind TGF-.beta.1 in the absence of
the types I and II receptors. In addition, it was demonstrated that the
GPI anchor is contained in a protein with a molecular weight of 150 kDa
(Tam et al, 2001). This novel GPI-anchored TGF-.beta.1 binding protein,
r150, has the potential to antagonize or potentiate TGF-.beta. action in
keratinocytes. In the absence of the cDNA encoding r150, one way to
examine the effect of r150's loss in TGF-.beta. signaling is to
enzymatically release the binding protein by PIPLC treatment prior to
testing for alterations in TGF-.beta. induced responses. However, the
efficacy of exogenously added PIPLC is subject to variability, being
affected by pH, temperature, and acylation of GPI-anchored proteins (Shukla,
1982; Chen et al, 1998), thus results obtained may be difficult to
interpret. In addition, GPI-anchored proteins that are released may get
re-synthesized and re-inserted in the plasma membrane soon after PIPLC
hydrolysis. Hence, as an alternative, was have created and isolated a
keratinocyte cell line that is mutated in GPI anchor biosynthesis. These
cells display a significant loss of r150 from their cell surface, thus
allowing a comparative examination of TGF-.beta. mediated cellular
responses in the GPI anchor deficient cell line versus the parental HaCat
cells under stable experimental conditions
GPI-anchored proteins lack transmembrane and cytoplasmic domains, and are
attached to the cell membrane via a lipid anchor in which the protein is
covalently linked to a glycosyl phosphatidylinositol moiety. GPI-anchored
proteins have been reported to have roles in intracellular sorting
(Rodriguez-Boulan and Powell, 1992), in transmembrane signaling (Brown,
1993) and to associate with cholesterol and glycosphingolipid-rich
membrane microdomains (Brown and London, 1998; Hooper, 1999). Also, the
GPI anchor enables a protein to be selectively released from the membrane
by phospholipases (Metz et al, 1994; Movahedi and Hooper, 1997). r150 was
characterized as GPI-anchored, based on its sensitivity to
phosphatidylinositol phospholipase C (PIPLC). However, it is important to
rule out other possibilities, namely, (i) r150 is not itself GPI-anchored,
but is tightly associated with a protein that is GPI-anchored, and
therefore is susceptible to release by PIPLC; (ii) r150 is a complex of
two lower molecular weight proteins which became inadvertently
cross-linked during the affinity labeling procedure.
It is now demonstrated that the GPI-anchor is contained in r150 itself and
not on a tightly associated protein and that it binds TGF-.beta.1 with an
affinity comparable to those of the signaling receptors. Furthermore, the
released (soluble) form of this protein binds TGF-.beta. independent of
the types I and II receptors. Also, the soluble form inhibits the binding
of TGF-.beta. to its receptor. In addititon, we provide evidence that r150
is released from the cell surface by an endogenous phospholipase C. Also,
a mutant human keratinocyte cell line with a defect in GPI synthesis was
created, which display reduced expression of r150. The results using these
mutant keratinocytes suggest that the membrane anchored form of r150 is a
negative modulator of TGF-beta responses. These findings, taken together
with the observation that r150 forms a heteromeric complex with the
signaling receptors, suggest that this accessory receptor in either its
membrane anchored or soluble form and its down- or up-regulation may
potentiate or antagonize TGF-.beta. responses in human keratinocytes,
The complete amino acid of a molecule named CD109 was recently disclosed
as well as the nucleic acids encoding same (Lin et al. 2002). Sequences
comparisons with those of r150 suggest that CD109 is a r150 variant. No
definite role has been assigned to CD109 by Lin et al.
SUMMARY OF THE
This invention provides a molecule that
binds TGF-.beta.1 with a high level of selectivity. This molecule named
r150 can be retrieved in a membrane anchored form or as a released free
soluble form. Variants and parts of r150 which have the property to bind
TGF-.beta.1 are grouped under the name r150-like proteins or peptides.
They include those defined in SEQ ID Nos: 2, 4, 8, 10 and 12. Their
corresponding coding nucleic acids respectively defined in SEQ ID NOs: 1,
3, 5, 7, 9 and 11.
This invention provides for the use of a protein comprising any one of the
following sequences in the making of a medication for inhibiting TGF-.beta.1
activity in a biological tissue SEQ ID Nos: 2, 4, 6, 8, 10 and 12.
Also provided is the use of an antagonist to a protein comprising any one
of the following sequences in the making of a medication for increasing
TGF-.beta.1 activity in a biological tissue: SEQ ID Nos: 2, 4, 6, 8, 10
Also provided is the use of a nucleic acid encoding a protein comprising
any one of the following sequences in the making of medication for
inhibiting TGF-.beta.1 activity in a biological tissue: SEQ ID Nos: 1, 3,
5, 7, 9 and 11.
Also provided is the use of a molecule which silences the expression of a
nucleic encoding a protein comprising any one of the following sequences
in the making of medication for increasing TGF-.beta.1 activity in a
biological tissue: SEQ ID Nos: 1, 3, 5, 7, 9 and 11. Particularly, the
silencing molecule is an antisense nucleic acid.
The present inventors being the first to elucidate the complete nucleic
acid sequence of r150 and of its deduced amino acid sequence, this
invention provides an isolated nucleic acid encoding a protein comprising
any one of the following sequences: SEQ ID Nos: 2, 4, 6, 8, 10 and 12.
In a specific embodiment, the nucleic acid comprises any one of the
following nucleotide sequences: SEQ ID No: 1, 3, 5, 7, 9 and 11.
The nucleic acid is particularly one comprising the nucleotide sequence
defined in SEQ ID No: 1.
The above nucleic acids may include crude nucleic acids or recombinant
vectors; namely expression vectors capable of governing transcription and
translation of the crude nucleic acids inserted downstream to a promotor,
are preferred tools for producing r150-like proteins.
Recombinant host cells which comprise the nucleic acids or the recombinant
vector are other tools. The vectors are normally selected to comprise
sequences compatible with the host's machinery. Intervening sequences
located 5' and 3' with regard to the crude nucleic acids are adapted or
selected by the skilled artisan desirous to produce a particular type of
host cells. The signal peptide may be charged also for another one more
appropriate for a given cell type.
There host cells may be domesticated and used in a method of producing a
r150-like protein. Such a method comprises the steps of: growing a
recombinant host cell in a culture medium supporting cell growth and
expression of said nucleic acid: recovering the protein from the culture
medium or from the cell.
The nucleic acids may be antisense nucleic acids. They may be inserted in
a recombinant vector, namely an expression vector and recombinant host
cells which comprises such antisense nucleic acids can also be made.
It is further an object of this invention to provide a TGF-.beta.1 binding
reagent, which comprises a protein comprising any one of the following
sequences: SEQ ID Nos 2, 4, 6, 8, 10 and 12.
Compositions of matter which comprise these reagents and a carrier are
other objects of this invention.
The carrier may be a pharmaceutical carrier. Otherwise, it may be a solid
support to which r150 is bound to immobilize TGF-.beta.1.
DESCRIPTION OF THE
r150 is a TGF-.beta.1 binding molecule.
Its complete amino acid sequence as well as the nucleic acid sequence
encoding same appear to have been first elucidated by the present
inventors. Another group (Lin et al. 2002), using a very different
approach (affinity binding to monoclonal antibodies) has found a blood
cell surface antigen, which they called CD109. Sequence comparisons show
that CD109 (SEQ ID Nos 5 and 6) comprises a 17 amino acid insertion at
position 1218 1234 (51 nts). This addition accounts for the difference in
amino acids number (1445 for CD109 versus 1428 for r150). Besides that,
substitutions of nucleotides are noted: r150 amino acid thr.sup.1224 is
changed for a methionine (CD109 amino acid 1241). CD109 shows polymorphism
at residue 703 (Schuh et al. 2002). A tyrosine or a serine represent
different alleles of CD109. Such polymorphism would presumably exist for
r150. It is possible that CD109 or r150 are responsible for the building
of an immune response since allo antibodies are retrieved upon
administration of CD109 isoforms. It may therefore be implied that an
isoform compatible with the recipient subject's tissue may have to be
selected as an administrable r150 active ingredient.
r150 binds or sequesters TGF-.beta.1, in its membrane anchored form as
well as in its free soluble form (SEQ ID Nos: 4 and 8). As a result, TGF-.beta.1
availability is reduced. The effects induced by TGF-.beta.1 are therefore
negatively modulated (or inhibited). Such inhibition may be desirable in
conditions where overproduction of TGF-.beta.1 leads to pathological
states (cancer is a specific example of such pathology). On the contrary,
in other occasions, increasing TGF-.beta.1 activity may be sought. For
example, TGF-.beta.1 encourages tissue or organ graft success. Therefore
silencing r150 would have for effect to increase TGF-.beta.1 availability
and increase graft success.
r150 further appears to be related to .alpha..sub.2 macroglobulin
(.alpha..sub.2M) and since the TGF-.beta. binding domain has been
determined for .alpha..sub.2M by Webb et al. (1998), the corresponding
domain in r150 is presumed to be located a region corresponding to
.alpha..sub.2 macroglobulin amino acids 666 706. These corresponds to r150
amino acids 651 683 (SEQ ID No: 10). Therefore, the r150 peptide having
the sequence defined in SEQ ID NO: 10 is also contemplated as TGF-.beta.1
binding peptide within the scope of the invention; the nucleic acid
encoding this peptide is as well.
Webb et al. (2000) even found the minimal .alpha..sub.2M TGF-.beta.1
binding sequence which appears to be delineated by amino acid 717 and 733.
The corresponding strectch in r150 is found between amino acid residues
694 and 712 (SEQ ID No.12).
Gomas et al. (2000) reported that .alpha..sub.2M gene polymorphism has
been associated with Alzheimer's disease, which polymorphism is observed
as a deletion in "the bait region" overlapping with TGF-.beta.1 binding
domain. It is envisageable that r150 could be useful to sequester and
neutralize TGF-.beta.1 especially in diseases wherein .alpha..sub.2M is
deficient. Any portion of r150 or variants thereof that is capable of
binding TGF-.beta.1 activity is intended to be used in the making of a
medication or a method or composition for inhibiting TGF-.beta.1 activity.
This includes proteins or peptides comprising sequences defined in SEQ ID
NOs. 2, 4, 6, 8, 10 and 12. These r150-like proteins or peptides would
include any molecule having at least 50% homology with the above
sequences. On the opposite, any molecule having an antagonistic activity
to the above r150-like proteins or peptides would find a use in the making
of a medication or a method or a composition for increasing TGF-.beta.1
Nucleic acids encoding the above r150-like proteins or peptides represent
an alternative to the direct administration of proteins or peptides.
Antisense nucleics would on the opposite silence the expression of
r150-like proteins or peptides. All these nucleic acids include
recombinants vectors, namely expression vectors, which are available and
well known to the skilled artisan.
A very large body of literature describes diseases or disease models
involving up and down regulation of TGF-.beta.1 activity. Nowadays, TGF-.beta.1
binding proteins decorin and an anti-TGF antibody are currently under
clinical trials. The present r150-like proteins or peptides could
represent a valuable and advantageous alternative to these molecules,
because of their selectivity for TGF-.beta.1 isoform, combined to their
Here is a non-exhaustive list of disease models where alteration of TGF-.beta.
action has been shown to be of therapeutic benefit:
Note: TGF-.beta. has biphasic effects during tumorigenesis, acting early
as a tumor suppressor, but later stimulating cancer progression.
(i) Suppression of tumor progression by TGF-.beta.
Akhurst R. J. and Derynck R. (2001). TGF-.beta. signaling in cancer a
double-edged sward. TRENDS in Cell Biology 11: S44 S51.
Welch, Dr. et al (1990). Transforming growth factor-.beta. stimulates
mammary adenocarcinoma cell invasion and metastatic potential. Proc. Natl.
Acd. Sci. USA 87: 7678 7682
Markowitz, S. et al. (1995) Inactivation of the type II TGF-.beta.
receptor in colon cancer cells with microsatellite instability. Science
Massague .J. et al. (2000) TGF-.beta. signaling in growth control, cancer,
and heritable disorders. Cell 103, 295 309.
(ii) Stimulation of tumor progression by TGF-.beta.
Hojo, M. et al. (1999) Cyclosporine induces cancer progression by a
cell-autonomous mechanism. Nature 397, 530 534.
Yin, J. J. et al. (1999) TGF-.beta. signaling blockade inhibits PTHrP
secretion by breast cancer cells and bone metastases development. J. Clin.
Invest. 103, 197 206.
Exogenous TGF-.beta.1 promotes wound healing where as inhibiting TGF-.beta.1
activity or enhancing TGF-.beta.3 activity reduces scarring in animal
Roberts, A. B., and Sporn, M. B. (1996). Transforming growth factor R. A.
F. (ed), Plenum Press, New York, p275 308.
Mustoe, T. A., Pierce, G. F., Thomason, A., Gramates, P., Sporn, M. B.,
and Deuel, T. F. (1987). Accelerated healing of incisional wounds in rats
induced by transforming growth factor-.beta.. Science 237: 1333 1336.
Quaglino, D., Nanney, L. B., Ditesheim, J. A., and Davidson, J. M. (1991).
Transforming growth factor-.beta. stimulates wound healing and modulates
extracellular matrix gene expression in pig skin: incisional wound model.
J. Invest. Dermatol. 97: 34 42.
O'Kane, S., and Ferguson, M. W. J. (1997). Transforming growth factor-.beta.s
and wound healing. Int. J. Biochem. Cell. Biol. 29:63 78.
Shah, M., Foreman, D. M., and Ferguson, M. W. J. (1995). Neutralization of
TGF-.beta.1 and TGF-.beta.2 or exogenous addition of TGF-.beta.3 to
cutaneous rat wounds reduces scarring. J. Cell Science 108: 985 1002.
Choi, B-M., Kwak, H-J., Jun, C-D., Park, S-D., Kim, K-Y., Kim, H-R., and
Chung, H-T. (1996). Control of scarring in adult wounds using antisense
transforming growth factor-.beta.1 oligodeoxynucleotides. Immunol. Cell
Biol. 74:144 150.
Blocking TGF-.beta.1 overproduction reduce tissue fibrosis (pulmonary
fibrosis, liver cirhosis, glomerulonephritis, scleroderma and
Border et al, (1990). Suppression of experimental glomerulonephritis by
antiserum against transforming growth factor beta 1. Nature 346 (6282):
Isaka Y., Brees D. K., lkegaya K., Kaneda Y., Imai E., Noble N. A., Border
W. A. (1998). Gene therapy by skeletal muscle expression of decorin
prevents fibrotic disease in rat kidney. Nat. Med. 2: 418 423.
Isaka Y., Akagi Y., Ando Y., Tsujie M., Sudo T., Ohno N., Border W. A.,
Noble N. A., Kaneda Y., Hori M., and Imai E. (1999). Gene therapy by
transforming growth factor-beta receptor-IgG Fc chimera suppressed
extracellular matrix accumulation in experimental glomerulonephritis [see
comments]. Kidney Int. 55:465 475.
Khalil, N. and Greenberg A H (1991). The role of TGF-.beta. in pulmonary
fibrosis. Ciba Found Symp. 157: 194 207.
Yamamoto, T., Takagawa S., Katayama I., and Nishioka K. (1999).
Anti-Sclerotic effect of transforming growth factor-beta antibody in a
mouse model of bleomycin-induced scleroderma. Clin Immunol, 92(1):6 13.
Gressner, A. M., Weiskirchen, R., Breitkopf K., and Dooley S. (2002).
Roles of TGF-beta in hepatic fibrosis. Front Biosci (7): d793 807.
Sheppard D. (2001). Integrin-mediated activation of transforming growth
factor-beta(1) in pulmonary fibrosis. Chest 120(1 Suppl):49S-53S.
McCaffrey, T. A. (2000). TGF-.beta.s and TGF-.beta. receptors in
atherosclerosis. Cytokine. Growth Factor Rev. 11: 103 114.
TGF-beta has tissue protective effects (against ischemia reperfusion
injury) in the heart, brain and kidney.
Lefer A M., Ma X-L, Weyrich A S, Scalia R. (1993). Mechanism of the
cardioprotective effect of TGF-.beta.1 in feline myocardial ischemia and
reperfusion. Proc. Natl. Acad. Sci. USA 90: 1018 1022.
Lefer A M, Tsao P, Aoki N, Palladino M A. (1990). Mediation of
cardioprotection by transforming growth factor-.beta.. Science 249: 61 64.
McNeill H. Williams C, Guan J, Dragunow M, Lawlor P, Sirimanne E, Nikolics
Gluckman P. (1994). Neuronal rescue with transforming growth factor-beta 1
after hypoxic-ischaemio brain injury. Neuroreport 5: 901 904.
Mehta J L, Yang B C, Strates B S, Mehta P. (1999). Role of TGF-beta1 in
platelet-mediated cardioprotection during ischemia-reperfusion in isolated
rat hearts. Growth Factors 16: 179 190.
Recombinant hosts comprising the above nucleic acids or recombinant
expression vectors can be used as a biological machinery in the production
of the r150-like proteins or peptides. The elucidation of the nucleic acid
sequence of r150 therefore leads to a method of producing these proteins
or peptides by recombinant technology.
A variety of TGF-.beta.1 binding reagents and compositions may be derived
from the present invention.
First, peptides such as those defined in SEQ ID. Nos: 10 and 12 may be
used as such as a TGF-.beta.1 binding reagent. Larger molecules like those
defined in SEQ ID Nos 2, 4, 6 and 8 could be conjugated (through their
anchoring region) to a carrier. The carrier may take the form, for
example, of a solid or semi-solid medium (beads, chromatography columns,
plates, etc.), to immobilize TGF-.beta.1. Pharmaceutical compositions
would take any suitable form, depending on the selected route of
administration. A r150-like protein or peptide (SEQ ID Nos: 2, 4, 6, 8, 10
and 12) would be formulated with a pharmaceutically acceptable carrier.
Doses equivalents those used by intravenous route for decorin and/or the
TGF-antibody can be produced.
Claim 1 of 2 Claims
1. A method for inhibiting TGF-.beta.1
activity in a biological tissue of an animal comprising an administration
thereto of an effective amount of a protein comprising a sequence selected
from the group consisting of: a) SEQ ID NO:2 b) amino acids 694 712 of SEQ
ID NO:2; c) amino acids 651 683 of SEQ ID NO:2 d) a protein sequence
having a tyrosine at position 703 of SEQ ID NO:2; e) a protein sequence
having amino acids 21 to 1428 of SEQ ID NO:2 f) a protein sequence having
amino acids 21 to 1404 of SEQ ID NO:2; and g) a protein sequence having a
methionine instead of threonine at position 1224 of SEQ ID NO:2 thereby
inhibiting TGF-.beta.l activity in a biological tissue of an animal.
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