WO2008125816A1

WO2008125816A1 - Modulation of cellular proliferation

Info

Publication number: WO2008125816A1
Application number: PCT/GB2008/001257
Authority: WO
Inventors: Endre Kiss-Toth; David Crossman; Sheila Francis
Original assignee: University Of Sheffield
Priority date: 2007-04-16
Filing date: 2008-04-10
Publication date: 2008-10-23
Also published as: GB0707270D0

Abstract

A method of modulating cellular proliferation comprising contacting a mammalian cell with a compound that modulates the expression or activity of Trb-1.

Description

Modulation of Cellular Proliferation

The present invention relates methods of modulating cellular proliferation. More particularly, the invention relates to methods of modulating vascular smooth muscle cell proliferation and methods of treatment of cellular proliferative disorders.

BACKGROUND

Injury to the arterial wall initiates a series of changes in specialised molecular and cellular events that not only contribute to wound healing but to the pathogenesis of atherosclerosis, the presentation of acute coronary syndromes (ACS) and the complications of percutaneous coronary intervention (PCI). An important initiating event appears to be endothelial cell dysfunction or cell death caused by local abnormalities. These events result in the production and release of a number of inflammatory cytokines and chemokines. In pathogenic conditions, elevated levels of inflammatory mediators can lead to migration and proliferation of vascular smooth muscle cells (VSMC) to form a neointima.

These cellular responses are mediated via the co-ordinated action of various second messenger pathways, including activation of Mitogen Activated Protein Kinase (MAPK) cascades and IKB kinases. Activation of these systems has been reported in several pathological conditions of vessel walls (Ju, et al. (2002) J Pharmacol Exp Ther 301 , 15- 20; Surapisitchat, J.et al. (2001 ) Proc Natl Acad Sci U S A 98, 6476-6481 ; Takeishi, et al. (2001) J MoI Cell Cardiol 33, 1989-20051-3). MAPKs are involved in the regulation of development, cell activation, proliferation and vascular contraction (Bonventre and Force (1998) Curr Opin Nephrol Hype/lens 7, 425-433;5. Choukroun, et al. (1998) J Clin Invest 102, 1311-1320; Force and Bonventre. (1998) Hypertension 31 , 152-161 ; Mii, et al (1996) Am J Physiol 270, H142-50-7). Further, they are central in regulating VSMC activation. It has been demonstrated that activation of MAPK cascades occurs in response to a wide range of stimuli, including pro-inflammatory cytokines, growth factors, mechanical stimuli (stress) (Li, C. and Xu, Q. (2000) Ce// Signal 12, 435-445) and integrin-dependent cell/matrix interactions (Goldschmidt, M. E.,et al (2001) Circ Res 88, 674-680; et al. (1999) J Cell Sci 112, 435-445). MAPKs are classified into at least three distinct groups (JNK, p38 and ERK kinases) and can be activated via a variety of upstream kinases, MAPKKs. In VSMC, Jun kinases (JNK) and p38 MAPKs have been implicated in responses primarily to stress (heat, hypoxia, chemical, oxidative, etc.) and pro-inflammatory cytokines, extracellular signal regulated protein kinases (ERK) primarily respond to mitogenic stimuli such as growth factors (PDGF) (Che, et al. (2001) Circulation 104, 1399-1406), oxidised LDL (Yang, et al. (2001) Br J Pharmacol 132, 1531-1541) or Ang Il (reviewed in (Touyz, R. M. and Schiffrin, E. L. (2000) Pharmacol Rev 52, 639-672)). However, in most cases a given stimulus will activate more than one group of MAPKs. The specific contribution of each MAPK pathway to a physiological response varies from cell type to cell type. In some cases, MAPK pathways can co-operate, but they can antagonise in others (Robinson, M. J. and Cobb, M. H. (1997) Curr Opin Cell Biol 9, 180-186; Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J. and Greenberg, M. E. (1995) Science 270, 1326-1331). However, the mechanisms which are responsible for these differences in MAPK responses in VSMC are largely unknown.

A novel protein family, human tribbles (trb) (Kiss-Toth, et al (2005) Biochem Soc Trans 33, 1405-1406, Kiss-Toth, et al (2006) Cellular Signalling 18, 202-214) has been identified as regulators of MAPKK activity (Kiss-Toth, et al(2004) J Biol Chem 279, 42703-42708). trb-1 and trb-3 have been shown to bind to various MAPKKs and that their concentration regulates preferential activation of the different MAPK pathways, presumably leading to different cellular responses (Kiss-Toth, et al (2004) J Biol Chem 279, 42703-42708). Drosophila and Xenopus Tribbles have been shown to regulate cell cycle progression during embryonic development (Grosshans, J. and Wieschaus, E. (2000) Ce// 101 , 523-531; Mata, et al(2000) Ce// 101 , 511-522; Saka, Y. and Smith, J. C. (2004) Dev Biol 273, 210-225; Seher, T. C. and Leptin, M. (2000) CurrBiol 10, 623- 629). Murine trb-3 has been demonstrated to inhibit insulin-dependent activation of Akt and was suggested to play a role in the development of diabetes in a mouse model (Du.et al (2003) Science 300, 1574-1577; Koo, et al. (2004) Nat Med 10, 530-534). However, recent conflicting data suggest that this proposed role may require further clarification (lynedjian, P. B. (2004) Biochem J 386, 113-118). Further, the importance of human tribbles proteins in cell physiology has not been evaluated.

Vascular smooth muscle cell is a major component of blood vessel walls. An increase in vascular smooth muscle cells plays a crucial role in the development of atherosclerosis. Previous research has attempted to prevent atherosclerosis by inhibiting vascular smooth muscle cell proliferation. For example platelet-derived growth factor has been used as an inhibitor of VSMC (Nishio E, Watanabe Y. Br J Pharmacol. Sep 1997; 122(2): 269-274). Accordingly, there remains a need to provide methods and composition to modulate vascular smooth muscle cell proliferation.

In addition, much research has focused on increasing the ability of mammalian cells to proliferate in culture. In tissue engineering, a major rate limiting step is the availability of a large enough population of genetically identical cells. For example, in in vitro cultures of smooth muscle cells, cells senesce after around sixteen cell divisions, thereby inhibiting further cell growth. Accordingly, there remains a need to provide methods and compositions to modulate in vivo cellular proliferation.

The inventors have demonstrated that trb-1 is selectively over expressed in inflamed, atherosclerotic arteries and that trb-1 regulates vascular smooth muscle cell (VSMC) proliferation and migration via the JNK pathway. These observations define trb-1 as a novel, central regulator of VSMC function.

The identification of trb-1 as a regulator of cellular proliferation provides a means by which larger populations of cells can be obtained in culture. For example acceleration of the rate of cell division may be obtained by non-permanent manipulation of cells, by for example transient transfection with anti-trb-1 siRNA.

BRIEF SUMMARY OF THE DISCLOSURE

In a first aspect the invention provides a method of modulating cellular proliferation comprising contacting a cell with a compound that modulates the expression or activity of Trb-1.

In one embodiment the cell is a mammalian cell. Preferably the mammalian cell is a human cell. Preferably the cell is a smooth muscle cell. More preferably the cell is a vascular smooth muscle cell.

In one embodiment said modulation is an increase in cellular proliferation. Where modulation is an increase in cellular proliferation the compound is preferably a Trb-1 inhibitor. Preferably, the trb-1 inhibitor inhibits trb-1 binding to MAKK4. Preferably, the compound is an antisense Trib-1 nucleic acid molecule. Alternatively, the compound is an siRNA molecule. Alternatively, the compound is a ribozyme having specificity for trb- 1. Alternatively, the compound is an anti-Trib-1 antibody.

In one embodiment said modulation, is a decrease in cellular proliferation. Where modulation is a decrease in cellular proliferation preferably said compound up regulates

Trb-1 expression or activity. Preferably, said compound is small molecules that stimulate the MKK4 binding activity of TRB-1. Preferably said compound is an active

Trb-1 protein or a fragment thereof. More preferably, the compound is an isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide consisting of the amino acid sequence of

SEQ ID NO:1 or 3, wherein the fragment comprises at least 30 contiguous amino acids of SEQ ID NO: 1 or 3; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3, c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4; and d) a polypeptide comprising an amino acid sequence which is at least 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3. Still more preferably, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.

Alternatively, said compound is an isolated nucleic acid molecule encoding a Trb-1 protein or a fragment thereof. Preferably the isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% homologous to the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 200 nucleotides of.a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; and c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3. More preferably, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4. Still more preferably, the isolated nucleic acid molecule encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3. In a further aspect the invention provides a method of culturing . cells comprising contacting at least one mammalian cell with a Trb-1 modulator and providing conditions which promote the proliferation of said at least one mammalian cell.

In one embodiment said modulation is an increase in cellular proliferation. Where modulation is an increase in cellular proliferation the compound is preferably a Trb-1 inhibitor. Preferably, the Trb-1 inhibitor inhibits trb-1 binding to MAKK4. Preferably, the compound is an antisense Trib-1 nucleic acid molecule. Alternatively, the compound is an siRNA molecule. Alternatively, the compound is a ribozyme having specificity for trb-

1. Alternatively, the compound is an anti-Trib-1 antibody.

In a further aspect the invention provides a cell culture comprising at least one mammalian cell and a trb-1 modulator.

In a further aspect the invention provides a trb-1 modulator for use as a medicament.

In a further aspect the invention provides a trb-1 modulator for use as a modulator of cellular proliferation.

In a further aspect the invention provides a trb-1 modulator for use as a modulator of smooth muscle proliferation.

In a further aspect the invention provides a trb-1 modulator for use as a modulator of vascular smooth muscle proliferation.

In a further aspect the invention provides a trb-1 modulator for use as a modulator of JNK activity.

In a preferred embodiment the modulator up regulates Trb-1 expression or activity. More preferably, the Trb-1 modulator upregulates trb-1 binding to MAKK4. Preferably the modulator is an active Trb-1 protein or a fragment thereof. More preferably, the compound is an isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or 3, wherein the fragment comprises at least 30 contiguous amino acids of SEQ ID NO:1 or 3; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3, c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4; and d) a polypeptide comprising an amino acid sequence which is at least 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3. Still more preferably, the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.

Alternatively, said modulator is an isolated nucleic acid molecule encoding a Trb-1 protein or a fragment thereof. Preferably the isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% homologous to the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 200 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; and c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% homologous to the amino acid. sequence of SEQ ID NO:1 or 3. More preferably, the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4. Still more preferably, the isolated nucleic acid molecule encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3.

In a further aspect the invention provides use of a Trb-1 modulator in the preparation of a medicament for the treatment of a cellular proliferative disorder. Preferably, the proliferative disorder is a vascular proliferative disorder, more preferably the proliferative disorder is atherosclerosis or restenosis. In a further aspect the invention provides a method of identifying a compound capable of modulating cellular proliferation comprising assaying the ability of the compound to modulate the nucleic acid expression of a nucleic acid molecule of SEQ ID NO:2 or 4 or the polypeptide activity of a polypeptide of SEQ ID NO:1 or 3, thereby identifying a compound capable of modulating cellular proliferation.

In a further aspect the invention provides a method for identifying a compound capable of treating atherosclerosis comprising assaying the ability of the compound to modulate the nucleic acid expression of a nucleic acid molecule of SEQ ID NO:2 or 4 or the polypeptide activity of a polypeptide of SEQ ID NO:1 or 3, thereby identifying a compound capable of treating atherosclerosis.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 is a graphical representation showing trb-1 expression in cultured human Aortic Smooth Muscle Cells. hASMC were stimulated by LPS for the various time points as indicated, total RNA was prepared and qRT-PCR was performed to detect changes in (A) tribbles 1-3 mRNA expression levels. (B) IL-1β levels were also measured as positive .

8 controls in the same samples. (C) The impact of culture conditions on Trb-1 expression was assessed in control and si-trb-1 transfected hASMC cells by qRT-PCR. (D) The specificity of si-trb-1 knockdown was evaluated by qRT-PCR, comparing expression levels of trb-1 , -2 and -3 in si-trb-1 transfected cells. The values were normalised to the expression of tribbles in cells transfected with control siRNA.

Figure 2 is a graphical representation showing trb-1 function in hASMC proliferation, migration and chemotaxis. hASMC cells were transfected with a trb-1 overexpression construct (A) or si-trb1-1 siRNA (B). Proliferation rate was measured by ³H thymidine incorporation. As an independent measure of proliferation, time-lapse video microscopy was performed and the percentage of mitotic cells on each field were calculated (C). The number of cells migrated through the edge of the wound (D) and their speed of migration (E) was assessed in a wound-healing assay. (F) The effect of MAPK inhibitors on proliferation rate was measured as on panel B. (G) The number of cells migrated through the Boyden chamber in response to PDGF were compared between control and si-trb-1 transfected cells. In addition, the impact of JNK inhibitor on the migrating cells was also investigated. The impact of tribbles-1 knockdown on the expression of TNFβ mRNA (H) arid TGFβ protein levels (I) was evaluated by qRT-PCR and ELISA, respectively.

Figure 3 illustrates the overexpression and suppression of trb-1 expression modulates activation of MAPK. (A) hASMC cells were transfected with AP-1 luciferase reporter, activated by the co-expression of pFC MEKK1 (both Stratagene) in the presence and absence of overexpressed trb-1 , as indicated. (B) cells were transfected with empty vector (mock), trb-1 overexpression construct or si-trb1-1 siRNA, stimulated with LPS for 0-45 minutes, as indicated on the figure, lysed and pMAPK and β-actin levels were determined by Western blotting. Results were quantified and expressed as a relative ratio to β-actin on (C). (D) Unstimulaed cell lysates (0 time point) were used to detect the impact of altered trb-1 levels on steady state MAPK expression by Western blotting for total MAPK and β-actin. The signal intensity was quantified as above and expressed as a total MAPK/β-actin ratio. MAPK/β-actin levels in the mock transfected cells were taken as baseline (1 unit) and values measured in the si-trb-1 and overexpressed samples were plotted relative to these.

Figure 4 illustrates MKK4 - trb-1 interaction controls hASMC proliferation. (A) Expression of MKK4 and MKK7 in hASMC and the impact of trb-1 knockdown on the protein levels of these MAPKKs were assessed by Western Blotting. (B) Activation of MKK4 (30 mins, 100ng/ml LPS) was evaluated under normal arid reduced trb-1 levels. pMKK4 values were normalised to actin and expressed as a ratio. (C) The ability of MKK4 to control hASMC proliferation rate was measured as on Figure 2 (24hrs post-transfection). (D) Physical interaction between MKK4 and trb-1 in hASMC was investigated by PCA. As positive controls, EGFP expression plasmid (left upper panel) and 'zipper-PCA' (left lower panel, zip-V1 and zip-V2) constructs were used. MKK4 was fused to the N- termϊnal fragment of Venus-YFP (V1), whilst trb-1 was expressed in fusion with the C- terminal fragment of Venus-YFP (V2). Representative cells show interaction between MKK4 and trb-1 (right panels). (E) The impact of the N- and C-terminal domains of trb-1 on its ability to interact with MKK4 in live cells and the location of the trb-1/MKK4 complex was assessed by PCA. (F) The structure of trb-1 mutants and the positions of the N- and C-terminal deletions is shown. (G) To confirm the specificity of MKK4/trb-1 interaction, FACS was used to demonstrate the specific interaction between trb-1 and MKK4 in HeLa cells. Similarly to figure 5E, an increasing dose of non-fluorescent trb-1 expression plasmid was co-transfected to compete out the labelled protein from the fluorescent complex. Further, no interaction was detected between control plasmids and either MKK4-V1 or trb-1 -V2. (H) As a further control, an increasing dose of trb-1 expression plasmid (unlabelled) was co-transfected in HeLa cells with the above two constructs (left) and the average total fluorescence per cell was measured (right) by fluorescent microscopy.

Figure 5 illustrates trb-1 expression is up-regulated in IHD Total RNA was prepared from sections of human coronary arteries from explanted hearts with IHD (n=8) ("Disease") or DCM (n=6) ("Control"). qRT-PCR was performed to quantify expression levels of major inflammatory cytokines and tribbles 1-3. Expression data was normalised for GAPDH as housekeeping control. Statistical analysis was performed by PRISM, using Student's t- test. Relative expression values are presented using "box and whisker plots".

Figure 6 is a schematic representation of the role of trb-1 in VSMC biology.

Figure 7 depicts the amino acid sequence of the trb-1 polypeptide, designated SEQ ID NO:1.

Figure 8 depicts the nucleotide sequence of the nucleic acid molecule that encodes the trb-1 .polypeptide, designated SEQ ID NO:2. Figure 9 depicts the amino acid sequence of the kinase domain of trb-1 , designated SEQ ID NO:3. The kinase domain is located at amino acid residues 105 to 338 of SEQ ID NO:1.

Figure 10 depicts the depicts the nucleotide sequence of the nucleic acid molecule that encodes the trb-1 kinase domain, designated SEQ ID NO:4, and'is located at nucleic acid resideues 898 to 1597 of SEQ ID NO:2.

DETAILED DESCRIPTION

The present invention is based on the surprising finding that Tribbles-1 (Trb-1), a modulator of mitogen activated protein kinase (MAPK), controls vascular smooth muscle proliferation and chemotaxis via the Jun kinase pathway. This control is via direct interaction between Trb-1 and MKK4/SEK1 , a Jun activator kinase. In additions the inventors have demonstrated that expression of trb-1 is elevated in human atherosclerotic arteries compared to non-atherosclerotic controls, indicating a role for trb-1 in atherosclerotic disease in vivo.

The experimental evidence disclosed herein demonstrates that trb-1 expression levels are key in modulating the extent of VSMC proliferation and chemotaxis. The inventors have found that si-trb-1 treatment of VSMC leads to the spontaneous activation of JNK (Figure 3B). Using a recently developed technique (PCA), the inventors have visualised the interaction between trb-1 and MKK4 in live VSMC cells. The use of truncated trb-1 proteins in PCA demonstrates that the kinase-like domain of trb-1 is essential for its ability to interact with MAPKKs. Since this domain is similar to that of the MAPKs (MAPKK substrates), a plausible hypothesis for the molecular mechanism of trb action is that tribbles may compete for the binding site with the MAPKs, thus regulating their activation. This model may explain why evolution preserved a catalytically inactive kinase domain from unicellular organisms to mammals (Hegedus, et al. (2006) Ce// MoI Life Sci 63, 1632-1641 ; et al. (2006) Cellular Signalling in press).

The polypeptide sequence of Trb-1 is designated SEQ ID NO:1 and illustrated in figure 7. The trb-1 polypeptide sequence is encoded by an isolated nucleic acid molecule, designated SEQ ID NO:2, illustrated in figure 8. The polypeptide sequence of the trb-1 kinase-like domain is designated SEQ ID NO:3 and is illustrated in figure 9. The kinase domain is located at amino acid residues 105 to 338 of SEQ ID NO:1. The kinase domain is encoded by the nucleotide sequence of SEQ ID NO:4, illustrated in figure 10, and located at nucleic acid residues 898 to 1597 of SEQ ID NO:8.

The modulation of trb-1 nucleic acid expression or polypeptide activity can be used to modulate cellular proliferation.

As used herein, the term "modulate" refers to the alteration, i.e. the up regulation or down regulation, of gene expression, the level of RNA molecules or of activity of one or more proteins, protein fragments or protein subunits. Modulation is such that the aforementioned expression, level, or activity is greater than or less than that observed in the absence of the modulation.

Modulation can be a reduction, inhibition or down regulation of the aforementioned expression, level, or activity. Alternatively, modulation can be an increase, stimulation or up-regulation.

Modulation of cellular proliferation can be achieved by contacting a cell with or exposing a cell to a Trb-1 modulator.

As used herein, the term "Trb-1 modulator" refers to a compound or agent that has a stimulatory or inhibitory effect on, for example, expression of a Trb-1 nucleic acid molecule or activity of a Trb-1 polypeptide.

Compounds or agents that have a stimulatory effect upon expression of a Trb-1 nucleic, acid molecule or activity of a Trb-1 polypeptide include, small molecules that stimulate the MKK4 binding activity of TRB-1 , an active Trb-1 protein or a fragment thereof, or a nucleic acid molecule encoding a Trb-1 protein or a fragment thereof that has been introduced into the cell.

Compounds or agents that have inhibitory effect upon effect upon expression of a Trb-1 nucleic acid molecule or activity of a Trb-1 polypeptide (also referred to as "inhibitors") -include small molecules that inhibit Trb-1 MKK4 binding activity, antisense Trib-1 nucleic acid molecules and anti-Trib-1 antibodies.

As used herein, the term "antisense oligonucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.

It is preferred that the antisense oligonucleotide may be constructed and arranged so as to bind selectively with the target, i.e. trb-1 under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.

In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and more preferably, at least 15 consecutive bases which are complementary to the target. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.

Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions may be targeted. The 3'- untranslated regions are known to contain cis acting sequences which act as binding sites for proteins involved in stabilising mRNA molecules.

The term "antisense oligonucleotides" is to be construed as materials manufactured either in vitro using conventional oligonucleotide synthesising methods which are well known in the art or oligonucleotides synthesised recombinantly using expression vector constructs. The present invention includes pharmaceutical preparations containing natural and/or modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding proteins the modulation of which results in beneficial therapeutic effects, together with pharmaceutically acceptable carriers (eg polymers, liposomes/cationic lipids).

Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art (eg liposomes). The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.

In one embodiment, inhibition is associated with post transcriptional silencing, using a modulator such as siRNA to mediate cleavage of a target nucleic acid molecule (e.g.

RNA) or to inhibit translation via a process known as RNA interference. In a further preferred embodiment of the invention the therapeutic molecule is an inhibitory RNA

(siRNA). siRNA molecules are RNA molecules that function to bind to specific cellular target molecules, thereby inducing the specific degradation of the targeted mRNA. As a consequence, synthesis of specific proteins can be greatly diminished. This therefore allows the specific elimination of expression of certain genes. Systems for both transient and permanent expression of siRNA have been developed which may be incorporated into the said Ad or Ad vector (Brummelkamp, Bernards et al. 2002). Typically si RNA's are small double stranded RNA molecules that vary in length from between 10-100 base pairs in length although large siRNA's e.g. 100-1000 bp can be utilised. Preferably the siRNA's are about 20 base pairs in length. Preferably siRNA molecules are RNA molecules that function to bind to trb-1 molecules.

In one embodiment, inhibition is associated with pretranscriptional silencing. In one embodiment of the invention the inhibitor is a ribozyme. Ribozymes are catalytic RNA molecules having ribonuclease activity. They are capable of cleaving a single- stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (for example hammerhead ribozymes (described in Haselhbff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave Trb-1 mRNA transcripts to thereby inhibit translation of Trb-1 mRNA. A ribozyme having specificity for an Trb-1 -encoding nucleic acid can be designed based upon the nucleotide sequence of an Trb-1 encoding nucleic acid molecules disclosed herein (e.g., SEQ ID NO:2, SEQ ID NO:4).

In one embodiment of the invention the inhibitor is an antibody, or at least an effective binding part thereof, which binds to a trb-1 polypeptide according to the invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions thereof, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as trb-1. A molecule which specifically binds to trb-1 is a molecule which binds trb-1, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains trb-1. Immunoglobulins (Ig) are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (K or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non-variable or constant. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the "constant" (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the "variable" (V) region.

The H chains of Ig molecules are of several classes, α, μ, σ, α, and γ (of which there are several sub-classes). Anjassembled Ig molecule consisting of one or more units of two identical H and L chains, derives its name from the H chain that it possesses. Thus, there are five Ig isotypes: IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the differences in the H chains, i.e., IgGI , lgG2, lgG3 and lgG4). . Further detail regarding antibody structure and their various functions can be found in, Using Antibodies: A laboratory manual, Cold Spring Harbour Laboratory Press. The antibody may be a polyclonal or a monoclonal antibody that binds trb-1. As used herein, the term "monoclonal antibody" refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of trb-1. A monoclonal antibody composition thus typically displays a single binding affinity for a particular trb-1 protein with which it immunoreacts.

Preferably, the antibody is humanised. A humanised monoclonal antibody to a trb-1 polypeptide is produced as a fusion polypeptide in an expression vector suitably adapted for transfection or transformation of prokaryotic or eukaryotic cells. In a further embodiment of the invention, said antibody is humanised by recombinant methods to combine the complimentarity determining regions of said antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.

Preferably, said antibody is provided with a marker including a conventional label or tag, for example a radioactive and/or fluorescent and/or epitope label or tag.

Alternatively, said antibody is a chimeric antibody. Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V-regions. The C-regions from the human antibody are also used. The complimentarity determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V-region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.

Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not illicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is desirable when using therapeutic antibodies in the treatment of diseases. Humanised antibodies are designed to have less "foreign" antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.

The modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject).

In vitro modulation provides methods for treating cells in culture.

Preferably the cells are smooth muscle cells. More preferably the cells are vascular smooth muscle cells.

Alternatively, said cell is selected from the group consisting of: a nerve cell; a mesenchymal cell; a muscle cell (cardiomyocyte or myotube cell); a liver cell; ; a blood cell (eg erythrocyte, CD4+ lymphocyte, CD8+ lymphocyte; panceatic β cell; an endothelial cell; an epidermal keratinocyte; a fibroblast (e.g. dermal, corneal; intestinal

^• mucosa, oral mucosa, bladder, urethral, prostate, liver) an epithelial cell (e.g. corneal, dermal, corneal; intestinal mucosa, oral mucosa, bladder, urethral, prostate, liver); a neuronal glial cell or neural cell; a hepatocyte stellate cell; a mesenchymal cell; a muscle cell; a kidney cell; a blood cell (e.g. CD4+ lymphocyte, CD8+ lymphocyte; or a pancreatic β cell.

Preferably the cells are mammalian cells. Mo're preferably the cells are human.

In one embodiment, the modulatory method involves administering compound or agent that has a stimulatory effect upon expression of a Trb-1 nucleic acid molecule or activity of a Trb-1 polypeptide Alternatively, the modulatory method involves administering a compound or agent that has an inhibitory effect upon expression of a Trb-1 nucleic acid molecule or activity of a Trb-1 polypeptide.

In one embodiment the administration of a trb-1 inhibitor to a cell in culture, up regulates the proliferation of the cell.

In one aspect, in vitro modulation provides a method of culturing cells. In one embodimentthe trb-1 modulator is provided in a culture medium. In one embodiment, the trb-1 modulator is present at the start of culture. Alternatively, the trb-1 modulator is present after the start of culture. Alternatively, the trb-1 modulator is present continuously during culture.

In one embodiment, the cells are transformed or transfected with a trb-1 modulator. Preferably the cells are transiently transformed. Preferably the cultured cells are clonally homogeneous. In one embodiment there is provided a cell culture medium comprising a trb-1 modulator.

Cells can be grown or cultured in the manner with which the skilled worker is familiar, depending on the cell type.

The culturing of mammalian cells has become a routine procedure and cell culture conditions which allow cells to proliferate are well defined. Typically, cell culture of mammalian cells requires a sterile vessel, usually manufactured from plastics, defined growth medium and, in some examples, feeder cells and serum, typically calf serum.

Tissue engineering has implications with respect to many areas of clinical and cosmetic surgery and relates to the replacement and/or restoration and/or repair of damaged and/or diseased tissues to return the tissue and/or organ to a functional state. For example, and not by way of limitation, tissue engineering is useful in the provision of skin grafts to repair wounds occurring as a consequence of: contusions, or burns, or failure of tissue to heal due to venous or diabetic ulcers. Tissue engineering requires in vitro culturing of replacement tissue followed by surgical application of the tissue to a wound to be repaired.

The cell cultured in vitro in accordance with the present invention my find subsequent use in tissue engineering.

Cell-types which are typically cultured in vitro for subsequent use in tissue engineering include, by example and not by way of limitation embryonic and adult stem cells ( e.g. embryonic and germ cell stem cells ^" derived from human embryos, so called pluripotential stem cells and adult stem cells such as haemopoietic stem cells from which are derived cells which comprise blood, e.g. T- lymphocytes (helper and killer), B- lymphocytes) and adult differentiated cells which can be maintained in culture (e.g. fibroblasts, keratinocytes). In vitro modulatory methods, including cell culture may be carried out in any suitable vessel. Preferably the vessel is selected from the group consisting of: a petri-dish; cell culture bottle or flask; multiwell plate. "Vessel" is construed as any means suitable to contain a cell culture.

In vivo modulation provides methods of treating a subject having a disease or disorder, or at risk of having a disease or disorder, associated the expression or activity of a trb-1 nucleic acid or polypeptide. In addition, in vivo modulation provides methods of treating subject having a disease or disorder, or at risk of having a disease or disorder, that may be treated by modulating the expression or activity of a trb-1 nucleic acid molecule or polypeptide.

In one embodiment, the modulatory method involves administering a trb-1 modulator.

The modulatory method may be a method to stimulate trb-1 expression or activity. It is beneficial to stimulate trb-1 expression or activity of trb-1 activity in diseases or disorders in which trb.-1 is abnormally down regulated, or in diseases or disorders in which increased trb-1 activity is likely to have a beneficial effect.

The modulatory method may be a method to inhibit trb-1 expression or activity. It is beneficial to inhibit trb-1 activity in diseases or disorders in which trb-1 is abnormally up regulated or in diseases or disorders in which increased trb-1 activity is likely to have a beneficial effect. Diseases and disorders that may be treated by modulating the expression or activity of a trb-1 nucleic acid molecule or polypeptide include cellular proliferative disorders, more preferably vascular proliferative disorders. The terms "vascular proliferative disorders" and "vascular proliferative diseases" refer to conditions of the blood vessel walls, for example the arteries and veins which cause an obstruction to blood flow. Examples of vascular proliferative disorders include restenosis, macular degeneration and psoriasis. In addition, inflammatory disease and disorders may also result in vascular proliferation disorders, for example atherosclerosis.

According to a yet further aspect of the invention there is provided a method of treatment of a mammal, preferably a human, comprising administering to said mammal a modulator according to the invention. Accordingly, in vivo modulation provides both prophylactic and therapeutic methods.

The trb-1 modulators of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the trb-1 modulator and a pharmaceutically acceptable carrier. The term "pharmaceutically- acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with - which the active ingredient is combined to facilitate the application.

When administered, the pharmaceutical compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The compositions of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be topical, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal, intranasal, intracerebral or epidural.

The compositions of the invention are administered in effective amounts. An "effective amount" is the amount of a composition that alone, or together with further doses, produces the desired response.

The compositions used in the foregoing methods preferably are sterile and contain an effective amount of the active ingredient for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by measuring the physiological effects of the composition, such as decrease of disease symptoms etc. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.

The Trb-1 modulators that are nucleic acid molecules of can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054- 3057).

As used herein, the term "trb-1 nucleic acid" refers to a nucleic acid sequence encoding a Trb-1 protein.

As used herein, the terms "nucleic acid molecule" and "nucleic acid" include DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

In a preferred embodiment a trb-1 nucleic acid is the nucleic acid molecule of SEQ ID NO:2. The sequence is approximately 3630 nucleotides in length and encodes a 372 amino acid polypeptide designated Trb-1 (SEQ ID NO:1).

Alternatively the trb-1 nucleic acid is the nucleic acid molecule of SEQ ID NO:4.. The sequence is approximately 700 nucleotides in length and encodes a 234 amino acid kinase domain (SEQ ID NO:3).

In one embodiment the trb-1 nucleic acid molecule is an isolated nucleic acid molecule. With regards to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5'- and/or 3'-ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.

Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

Further trb-1 nucleic acid molecules of the present invention are described below. In one embodiment, the trb-1 nucleic acid molecule comprises a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4, or a portions or fragment thereof. In one embodiment the nucleic acid molecule comprises a fragment of the nucleic acid molecule of SEQ ID NO:2 or SEQ ID NO:4, for example a fragment of 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 consecutive nucleotides of SEQ ID NO: 2 or 4.

In one embodiment the nucleic acid molecule comprises a nucleotide sequence encoding the polypeptide of SEQ ID NO: 1 or SEQ ID NO:3. In yet another embodiment, the trb-1 nucleic acid molecule encodes fragments of SEQ ID NO:1 or 3, preferably the fragments are biologically active fragments, i.e. having MKK4 binding activity.

In another embodiment, the trb-1 nucleic acid molecule has a nucleic acid sequence that is the complement of the nucleotide sequences shown in SEQ ID NO:2 or SEQ ID NO:4, or portions or fragments thereof. In other embodiments, the trb-1 nucleic acid molecule has a nucleic acid sequence that is sufficiently complementary to the nucleotide sequence shown in of SEQ ID NO:2 or SEQ ID NO:4 such that it can hybridize to the nucleotide sequence shown in any of SEQ ID NO:2 or 4, thereby forming stable duplexes.

As used herein, the term "hybridizes under stringent conditions" describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in available references (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods are described in that reference and either can be used. A preferred example of stringent hybridization conditions are hybridization in 6χ sodium chloride/sodiurjjjcitrate (SSC) at about 45⁰C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 50⁰C. Another example of stringent hybridization conditions are hybridization in 6χ SSC at about 45⁰C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 55°C. A further example of stringent hybridization conditions are hybridization in 6χ SSC at about 45⁰C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 60⁰C. Preferably, stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in 0.2χ SSC, 0.1% (w/v) SDS at 65°C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5 molar sodium phosphate, 7% (w/v) SDS at 65°C, followed by one or more washes at 0.2χ SSC, 1% (w/v) SDS at 65°C. Preferably, a trb-1 nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO.2 or 4.

In one embodiment, the trb-1 nucleic acid molecule has a nucleic acid sequence that is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,' 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length of the nucleotide sequence shown in SEQ ID NO:2 or 4, or portions or fragments thereof.

In another embodiment, the trb-1 nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide that is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length the polypeptide of SEQ ID NO: 1 or 3, or portions or fragments thereof.

Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined,, using the Needleman et al. (1970) J. MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

In one embodiment the trb-1 nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 4. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 2 or 4, or substitution, deletion or insertion of non- critical residues in non-critical regions of the protein. Nucleic acid molecules corresponding to natural allelic variants and homologues of the Trb-1 nucleic acid molecules of the invention can be isolated based on their homology to the nucleic acid molecules of the invention using the nucleotide sequences described in SEQ ID NO:2 or 4, or a portion thereof, as a hybridization probe under stringent hybridization conditions.

As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In another embodiment any of the nucleic acid molecule described previously, comprises specific changes in the nucleotide sequence so as to optimize expression, activity or functional life of the Trb-1 polypeptides. Preferably, the nucleic acids described previously are subjected to genetic manipulation and disruption techniques. Various genetic manipulation and disruption techniques are known in the art including, but not limited to, DNA Shuffling (US 6,132,970, Punnonen J et al, Science & Medicine, 7(2): 38-47, (2000), US 6,132,970), serial mutagenesis and screening. One example of mutagenesis is error-prone PCR, whereby mutations are deliberately introduced during PCR through the use of error-prone DNA polymerases and reaction conditions as described in US 2003152944, using for example commercially available kits such as The GeneMorph^® Il kit (Stratagene^®, US). Randomized DNA sequences are cloned into expression vectors and the resulting mutant libraries screened for altered or improved protein activity.

In one embodiment the trb-1 nucleic acid molecule is a trb-1 encoding gene.

As used herein, the term "gene" refers to nucleic acid molecules which include an open reading frame encoding protein, and can further include non-coding regulatory sequences and introns.

In one embodiment a trb-1 polypeptide is the polypeptide of SEQ ID NO:1. The sequence is approximately 337 amino acid residues in length.

In one embodiment a trb-1 polypeptide is the polypeptide of SEQ ID NO:3. The sequence is approximately 233 amino acid residues in length.

In one embodiment the trb-1 polypeptide molecule is an isolated polypeptide. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the trb-1 protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of trb-1 protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinant^ produced.

Biologically active portions of an trb-1 protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the trb-1 protein (e.g., the amino acid sequence shown in SEQ ID NO:1 or 3), which include fewer amino acids than the full length trb-1 proteins, and exhibit at least one activity of an trb-1 protein. A biologically active portion of a trb-1 protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.

As used herein, a "biologically active portion" of protein includes fragment of protein that participate in an interaction between molecules and non-molecules. Biologically active portions of protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the protein, e.g., the amino acid sequences shown in SEQ ID NO: 2 or 4, which include fewer amino acids than the full length protein, and exhibit at least one activity of the encoded protein.

A biologically active portion of protein can be a polypeptide that is, for example, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acids in length of SEQ ID NO: 1 , or 3. Biologically active portions of protein can be used as targets for developing agents that modulateTrb-1 -mediated activities, e.g., biological activities described herein.

A trb-1 protein has the amino acid sequence shown of SEQ ID NO:1 or 3. Other useful trb-1 proteins are substantially identical to SEQ ID NO:1 or 3 and retain the functional activity of the protein of SEQ ID NO:1 or 3 yet differ in amino acid sequence due to natural allelic variation or mutagenesis. For example, such, trb-1 proteins and polypeptides posses at least one biological activity described herein such as, (1) the ability to bind MAKK4. Accordingly, a useful isolated trb-1 protein is a protein which includes an amino acid sequence at least about 45%, preferably 55%, 65%, 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:1 or 3 and retains the functional activity of the trb-1 proteins of SEQ ID NO;1 or 3.

In one embodiment the trb-1 protein has the amino acid sequence shown of SEQ ID NO:1 or 3, comprising conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.

In one embodiment the trb-1 protein is a chimeric or fusion protein. As used herein, a trb-1 "chimeric protein" or "fusion protein" comprises an trb-1 polypeptide operably linked to a non-trb-1 polypeptide. In a fusion protein the trb-1 polypeptide can correspond to all or a portion of a trb-1 protein, preferably at least one biologically active portion of an trb- 1 protein. "Operably linked" as used herein, refers to a combination of the polypeptide linked together in a functional relationship with one another, for example, fused in-frame to each other.

Variants of trb-1 protein which may function as either trb-1 agonists or as trb-1 antagonists can be identified by screening combinatorial libraries of mutants, of the trb-1 protein for trb-1 protein agonist or antagonist activity.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of (e.g., the sequence of SEQ ID NO:2 or 4) without removing or, more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention, e.g., those present in the conserved kinase domain of trb-1 are particularly non-amenable to alteration.

In one aspect the invention provides screening assays for identifying modulators of trb-1 nucleic acid expression or polypeptide activity. Assays can be cell free assays or cell based assays. The assays determine the ability of a test compound to modulate (stimulate or inhibit) the expression or activity of trb-1.

Examples

Plasmids, siRNA

Trb-1 overexpression plasmid has been described before (Kiss-Toth, E.et al. (2004) J Biol Chem 279, 42703-42708). siRNA SmartPool against human trb-1 was purchased from Dharmacon and used according to the manufacturer's recommendation. Plasmids for yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) (Hegedus, Z et al. (2006) Cell MoI Life Sci 63, 1632-1641 , Hegedus, Z., Czibula, A. and Kiss-Toth, E. (2006) Cellular Signalling in press) were a kind gift of Prof. S. Michnik and have been described before (Remy, I. and Michnick, S. W; (2004) Methods 32, 381-388).

Cell culture and the stimulation with LPS

VSMC were purchased from Cascade Biologies and maintained in Medium 231 with smooth muscle growth supplement (all from Cascade Biologies). VSMC were plated in 6 well plates at an initial density of 0.5x10⁵ cells/well in 2 ml of complete growth media. 24 hours post transfection ceils were stimulated by 100 ng/ml LPS, as stated in the figures. HeLa cells (ATCC) were maintained in DMEM medium (Invitrogen), containing 10% Foetal Bovine Serum (Biowhittaker), 5mM of sodium pyruvate and 100 μg/ml of penicillin and streptomycin (Sigma).

Artery biopsies

Coronary arteries from patients with a diagnosis of ischemic heart disease (IHD, n=8) and dilated cardiomyopathy (DCM, n=6) were harvested from transplantation recipients at the time of surgery. Patients diagnosed as having DCM were free of CAD before transplantation, as assessed by coronary angiography and histological analysis by a cardiac pathologist (SKS) using the American Heart Association (AHA) classification system (Stary, H. C. (2000) Arterioscler Thromb Vase Biol 20, 1177-1178; Stary, et al. (1995) Arterioscler Thromb Vase Biol 15, 1512-1531 ; Stary.et al. (1995) Circulation 92, 1355-1374; King, et al. (2003) Lab Invest 83, 1497-1508). All patients were men and white in origin. Mean ages were 55.75 ±5.11 for IHD and 48.5±10.23 for the DCM group. Mean AHA histological grades of disease were 6.42 for the IHD and 3.16 for the DCM group, respectively.

RNA isolation and Quantitative Real-time PCR analysis Total RNA was extracted from human artery biopsies and VSMC using RNeasy kit (Qiagen) according to the manufacturer's protocol. 2μg RNA was reverse transcribed into first strand cDNA and used immediately for qRT-PCR. Gene expression was analyzed by qRT-PCR using ABI prism 7900 (Applied Biosystems). Probes for human trbs, IL-1β, IL-RA, TNFα and GAPDH were synthesized by Sigma Genosys with FAM at the 5'-end and TAMRA at the 3'-end. The sequences of all primers and probes used are listed in table 1 below.

Table 1

To quantify transcripts for genes of interest, we used the GAPDH transcript as an internal control, and each sample was normalized with respect to its GAPDH transcript content. Standard curves for the 9 genes and GAPDH mRNAs were generated using serially diluted solution of plasmids incorporating each gene as a PCR template. All assays were performed in duplicate and the average values were used for analysis.

Transfections: Transfections were performed using Nucleofector (Amaxa) using program U-25 and Nucleofector solution for VSMC (Amaxa). For most experiments, 1.0 * 10^δ cells were used per nucleofectiori. Polyfect (Qiagen) was used for transfection of HeLa cells according to the manufacturer's instructions.

Proliferation assay:

Transfected VSMC were placed on 96 well culture plates and further cultured for 24 hours. Cells were then treated with [³H] thymidine (1 μCi/well) and /or MAPK inhibitors for 6, 24, 30hours. ERK MAPK inhibitor (PD98059), p38 MAPK inhibitor (SB203580) and JNK MAPK inhibitor (SP600125) were purchased from Calbiochem and used at 20μM for ERK and JNK MAPK inhibitors and 0.2μM for p38 MAPK inhibitor. After treatment, [³H] thymidine incorporation was measured by liquid scintillation counting. In Vitro wound healing assay, Time Lapse Video-Microscopy (TLVM):

Cells were transfected as described above, seeded into 35mm culture dishes and incubated overnight. Confluent cell monolayers were wounded by removing a strip of cells from the plate surface with a standard 1 ml pipette tip. Wounded monolayers were washed with PBS to remove non-adherent cells and replaced with fresh complete growth medium. TLVM (King, et al (2003) Lab Invest 83, 1497-1508; McCarthy, et al. (1997) J Cell Biol 136, 215-227) was used to film migration at the wound edges for 24 hours (1 frame every 2.5minutes).

The number of cells crossing a line marking the wound edges was counted over the 24 hour period. (Fig. 2D)

15 randomly picked cells from the wound edges were tracked over a 1 hour period. The on-screen distance travelled was measured to calculate migration speed (mm/h) irrespective of direction. (Fig. 2E)

Western blotting:

Anti-phospho-MAPK and anti-MAPK antibodies were purchased from Cell Signalling Technology and were used according to the manufacturer's recommendations. Anti-D- actin antibody was from Dako. Between probing for the various proteins, membranes were stripped by Re-Blot Plus Mild solution (Chemicon). Membranes were developed with SuperSignal West Pico Chemiluminescent Substrate and signals were quantified by Chemigenius gel-documentation system (Syngene). pMAPK and MAPK signals were expressed as a ratio to β-actin levels within the same sample.

RESULTS Tήbbles expression is regulated in response to an inflammatory stimulus in hASMC Smooth Muscle Cell proliferation is a key event in the healing response to injury, a process which is initiated by inflammatory stimuli (Morton, et al. (2005) Cardiovasc Res 68, 493-501). To identify whether tribbles expression is regulated in vascular cells under inflammatory conditions, we stimulated human aortic smooth muscle cells (hASMC, Fig. 1A) and human umbilical vein endothelial cells (HUVEC₁ not shown) with LPS and measured tribbles mRNA expression levels by using qRT-PCR. IL-1β expression was measured as positive controls in the same samples (Fig. 1 B). It has been found that trb- 1 is selectively and transiently up-regulated by LPS treatment in hASMC (Fig. 1A) but not in HUVEC. Previous reports (Wilkin et al (1997) EurJBiochem 248, 660-668) and our unpublished observations suggest that tribbles proteins may be unstable and expressed at low levels, therefore, mRNA levels are likely to correlate well with protein expression. The biological relevance of altered trb-1 levels on hASMC under inflammatory conditions has therefore been investigated.

To assess the role of trb-1 in LPS stimulated hASMC, cells were transiently transfected with a trb-1 expression plasmid or a pool of anti-trb-1 siRNA oligonucleotides and measured proliferation and migration in response to PDGF. However, first the potential impact of culture conditions on trb-1 expression (Fig. 1C) and the specificity of the siRNA pool (Fig. 1 D) used for targeting trb-1 mRNA were evaluated. We have found that whilst trb-1 siRNA had the desired activity both under standard culture conditions (10% FCS) and when cells were serum starved post-transfection (0.5%), the expression levels of this gene was significantly affected by the serum concentration. This is in line with previous work, demonstrating that expression of members of mammalian trb family is modulated through metabolic signals (Ohoka, et al. (2005) Embo J 24, 1243-1255; Schwarzer, R., Dames, S., Tondera, D., Klippel, A. and Kaufmann, J. (2005) Cell Signal). Therefore, a 10% FCS was used in all experiments in order to minimise inter- experimental variations. In addition, the siRNA pools showed high specificity towards trb- 1 and did not alter significantly trb-2 and -3 expression (Fig. 1D).

Tribbles-1 regulate specific hASMC cellular functions

To investigate the role of trb-1 in hASMC function, trb-1 levels were raised or suppressed by transient transfection of hASMC cells with trb-1 expression plasmid or siRNA and measured proliferation by ³H-thymidine incorporation (Fig. 2A-B). A modest antiproliferative effect was observed at 48 hrs post-transfection (24 hrs time point in the proliferation assay) when trb-1 was overexpressed. In contrast, depletion of trb-1 results in a significant increase in proliferation rate at the same time-point (Fig. 2B). As an independent measure of cell proliferation, time lapse video imaging was performed on control and si-trb-1 treated cells and calculated the rate of mitosis (Fig. 2C). The data obtained by both methods was in agreement. Using the same assay, VSMC migration in a "wound healing" assay was also measured. (Fig. 2D, E). These data show that neither the number of cells migrating into the wound (Fig. 2D) nor their migration speed (Fig. 2E) were affected upon depletion of trb-1.

Selective MAPK inhibitors were used to block individual pathways and investigated their involvement in the trb-1 regulated proliferation (Fig. 2F). These data demonstrate that blocking of JNK but not ERK or p38 pathways suppresses VSMC proliferation. To further investigate the impact of trb-1 levels in hASMC function, chemotaxic migration of these cells was measured in a transwell migration assay, in response to PDGF (Fig. 2G). Depletion of trb-1 led to an increase in transmigrated cells. Further, inhibition of the JNK pathway abrogated the effect of si-trb-1 treatment, suggesting that trb-1 may be a negative regulator of hASMC chemotaxis via its inhibitory activity of the JNK pathway. Activation of Vascular Smooth Muscle Cells by inflammatory signals leads to the production of a number of cytokines, including TNFα and TGFβ through MAPK mediated signalling events (Warner, S. J. and Libby, P. (1989) J Immunol 142, 100-109; Yamakawa, et al. (1999) Endocrinology 140, 3562-3572; Yue, Tet al. (1994) Biochem ₍ Biophys Res Commun 204, 1186-1192; Majesky, et al. (1991) J CHn Invest 88, 904-910 Sato, et al. (1990) J Cell Biol 111 , 757-763). We, therefore, whether depletion of trb-1 had a modulator role in the production of these inflammatory cytokines, in VSMC was therefore investigated. Our data shows no difference in the dynamics or the amplitude of cytokine expression at the mRNA (Fig. 2H) or protein levels (Fig. 2l), implying that trb-1 may be a specific regulator of VSMC proliferation and migration.

Overexpressed Trb-1 blocks AP-1 activation and Trb-1 depletion leads to constitutive JNK activation

In order to gain mechanistic insight into the regulation of VSMC proliferation via the JNK/AP-1 pathways and trb-1 , VSMC was transiently transfected with an AP-1 reporter plasmid, activated by overexpressed MEKK1 in the presence and absence of overexpressed trb-1 (Fig. 3A). The results demonstrate that activation of AP-1 can be blocked by overexpressed trb-1 in VSMC.

The activation of the various MAPKs was also assessed in control and trb-1 depleted cells. Phosphorylated MAPK (pMAPK, the activated form) and total MAPK levels were investigated in response to LPS treatment, by western blotting. pMAPK (Fig. 3B and C) and total MAPK (Fig. 3D) levels were normalised to β-actin and expressed as relative units. Alterations in trb-1 levels had a differential impact on the various MAPK pathways. However, these effects were different in VSMCs, compared to those observed in HeLa cells (Kiss-Toth, et al. (2004) J Biol Chem 279, 42703-42708). In VSMC, the amount of phospho-p38 protein but not the dynamics of activation was influenced by altered trb-1 expression. In contrast, while ERK and JNK pathways were also sensitive to altered trb-1 expression, these were no longer up-regulated by LPS stimulation. Indeed, modulation of trb-1 expression caused phosphorylation of both of MAPKs to decrease sharply once stimulated. In addition, depletion of trb-1 mRNA by the siRNA constructs led to maximal phosphorylation of JNK in the absence of any stimulus (Fig. 3B, middle graph, zero time point), suggesting that normal trb-1 levels inhibit the activation of this pathway in the non-stimulated state. Further, the expression level of JNK was somewhat altered when cells were transfected with the siRNA or overexpression constructs (Fig. 3D). However, alterations in total MAPK levels did not translate to changes in pMAPK levels suggesting that the total amount of MAPK expressed is not a rate-limiting step for the activation of these pathways.

Trb-1 VMKK4 interaction is key to the regulation of h ASMC proliferation

Details of trb/MAPKK interactions in hASMC were investigated. The generated data points to the JNK pathway as a key system in regulating proliferation and chemotaxis in these cells, in a trb-1 dependent manner. Therefore, the involvement of MKK4/SEK-1 and MKK7, the two known MAPKKs, which lead to activation of JNK, was characterised. The results show that MKK4 but not MKK7 is expressed in these cells and that this expression pattern is not influenced by sitrb-1 treatment (Figure 4A). We also found that the phosphorylation of MKK4 was not affected by depletion of trb-1 (Figure 4B), suggesting that trb-1 directly interferes with MKK4 activity, rather than with it's activation and/or expression. To confirm the direct role of MKK4 in hASMC proliferation, a [³H] thymidine incorporation assay was performed in siMKK4 treated cells (Figure 4C), as before. In line with our above model, the data demonstrates a positive role for MKK4, since depletion of this protein led to a decrease in proliferation rate.

To confirm the physical interaction between trb-1 and MKK4 in live hASMC, a yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) (Michnick, S. W. (2004) Drug Discov Today 9, 262-267; Remy, I. and Michnick, S. W. . (2004) Methods MoI Biol 2Qi, 411-426) was used. The Venus variant of YFP was used in this assay, since it provides a higher signal than EYFP. MKK4 and Trb-1 were fused to the N-terminal fragment of Venus YFP (V1) or to the C-terminal portion of Venus YFP (V2), respectively. The two expression constructs were co-transfected and the YFP signal was visualised by fluorescent microscopy (Figure 4D). These data demonstrate that the MKK4/trb-1 complex is located predominantly in the nucleus of hASMC. Truncated trb-1 forms, lacking the N-terminal, the C-terminal or both domains (Figure 4F) were expressed as V2 fusion proteins (Figure 4E). The results show that the nuclear localisation of the complex is critically dependent on the presence of the N-terminal trb-1 domain. Mutants lacking this domain still showed an interaction with MKK4, but the signal was no longer preferentially nuclear. Further, these experiments demonstrate that the central, kinase-like domain of trb-1 is sufficient to it's interaction with MKK4.

In order to confirm specificity of the observed interaction, several control experiments were performed. Co-expression of an increasing amount of "unlabelled" trb-1 led to a dose-dependent elimination of the YFP signal as detected by FACS (Figure 4G) or by fluorescent microscopy (Figure 4H). In addition, neither MKK4-Venus nor trb-1 -Venus fusion proteins interacted with their zip-Venus counterparts (these were used as positive control constructs in the system) (Figure 4G), which further supports the specific nature of this interaction.

Tribbles-1 expression in atherosclerotic arteries

Proliferation of vascular smooth muscle cells is one of the hallmarks of the development of chronic diseases of the vessel wall. In order to evaluate the potential role of tribbles in human disease, segments of whole artery wall taken from the explanted hearts of patients undergoing cardiac transplantation for ischaemic heart disease (IHD) were studied and tribbles expression characterised. Coronary arteries from patients with non- ischaemic dilated cardiomyopathy (DCM) were used as controls. In order to quantify potential differences in trb expression levels between the two groups, mRNA levels of known pro and anti-inflammatory cytokines (IL-1β and IL-1ra) and tribbles 1-3 were quantified by using qRT-PCR (Fig. 5). A "pro-inflammatory phenotype" was detected in the atherosclerotic group. Expression of trb-1 but not of trb-2 was significantly raised in the IHD group (Fig. 5), whilst trb-3 expression was not detected in these samples (not shown).

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed

Claims

1. A method of modulating cellular proliferation comprising contacting a mammalian cell with a compound that modulates the expression or activity of Trb-1.

2. A method according to claim 1 , wherein the mammalian cell is a human cell.

3. A method according to claim 1 or claim 2, wherein the cell is. a smooth muscle cell.

4. A method according to claim 3, wherein the cell is a vascular smooth muscle cell.

5. A method according to any one of the preceding claims, wherein said modulation is an increase in cellular proliferation.

6. A method according to claim 5, wherein the compound is a Trb-1 inhibitor.

7. A method according to claim 6, wherein the Trb-1 inhibitor inhibits trb-1 binding to MAKK4.

8. A method according to claim 6, wherein the compound is an antisense Trib-1 nucleic acid molecule.

9. A method according to claim 6, wherein the compound is an siRNA molecule.

10. A method according to claim 6, wherein the compound is a ribozyme having specificity for trb-1.

11. A method according to claim 6, wherein the compound is an anti-Trib-1 antibody.

12. A method according to anyone of claims 1 to 4, wherein said modulation is a decrease in cellular proliferation.

13. A method according to claim 12, wherein said compound up regulates Trb-1 expression or activity.

14. A method according to claim 14, wherein said compound is small molecules that stimulate the MKK4 binding activity of TRB-1.

15. A method according to claim 14, wherein said compound is an active Trb-1 protein or a fragment thereof.

16. A method according to claim 15 wherein the compound is an isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3, wherein the fragment comprises at least 30 contiguous amino acids of SEQ ID NO:1 or 3; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3, c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4; and d) a polypeptide comprising an amino acid sequence which is at least 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3.

17. A method according to claim 16, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.

18. A method according to claim 14, wherein said compound is an isolated nucleic acid molecule encoding a Trb-1 protein or a fragment thereof.

19. A method according to claim 18, wherein the compound is an isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% homologous to the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 200 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; and c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% homologous to the amino acid sequence of

SEQ 1D NO:1 or 3.

20. A method according to claim 19, wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4.

21. A method according to claim 19, wherein the isolated nucleic acid molecule encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3.

22. A method of culturing mammalian cells comprising contacting at least one mammalian cell with a Trb-1 modulator and providing conditions which promote the proliferation of said at least one mammalian cell.

23. A method according to claim 22, wherein the mammalian cell is a human cell.

24. A method according to claim 22 or claim 23, wherein the cell is a smooth muscle cell. ^ - ^{■ ■}

25. A method according to claim 24, wherein the cell is a vascular smooth muscle cell.

26. A method according to claim 22, wherein the trb-1 modulator is a trb-1 inhibitor.

27. A method according to claim 26, wherein the Trb-1 inhibitor inhibits trb-1 binding to MAKK4.

28. A method according to claim 26, wherein the compound is an antisense trb-1 nucleic acid molecule.

29. A method according to claim 26, wherein the compound is an siRNA molecule.

30. A method according to claim 26, wherein the compound is a ribozyme having specificity for trb-1.

31. A method according to claim 26, wherein the compound is an anti-trb-1 antibody.

32. A cell culture comprising at least one mammalian cell and a trb-1 modulator.

33. A cell culture medium comprising a trb-1 modulator.

34. A trb-1 modulator for use as a medicament.

35. A trb-1 modulator for use as a modulator of cellular proliferation.

36. A trb-1 modulator for use as a modulator of smooth muscle proliferation.

37. A trb-1 modulator for use as a modulator of vascular smooth muscle proliferation.

38. A trb-1 modulator for use as a modulator of JNK activity.

39. A trb-1 modulator according to any one of claims 34 to 38, wherein the modulator up regulates Trb-1 expression or activity.

40. A trb-1 modulator according to claim 39, wherein the Trb-1 modulator upregulates trb-1 binding to MAKK4.

41. A trb-1 modulator according to claim 39, wherein the modulator is an an active Trb-1 protein or a fragment thereof.

42. A trb-1 modulator according to claim 15 wherein the modulator is an isolated polypeptide selected from the group consisting of: a) a fragment of a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or 3, wherein the fragment comprises at least 30 contiguous amino acids of SEQ ID NO: 1 or 3; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or 3, c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 60 % identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4; and d) a polypeptide comprising an amino acid sequence which is at least 60% homologous-to the amino acid sequence of SEQ ID NO:1 or 3.

43. A trb-1 modulator according to claim 42, wherein the isolated polypeptide comprises the amino acid sequence of SEQ ID NO:1 or 3.

44. A trb-1 'modulator according to claim 39, wherein said modulator is an isolated nucleic acid molecule encoding a Trb-1 protein or a fragment thereof.

45. A trb-1 modulator according to claim 44, wherein the modulator is an isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 60% homologous to the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; b) a nucleic acid molecule comprising a fragment of at least 200 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:2 or 4, or a complement thereof; and c) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:1 or 3.

46. A trb-1 modulator according to claim 45, wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2 or 4.

47. A trb-1 modulator according to claim 19, wherein the isolated nucleic acid molecule encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3.

48. Use of a Trb-1 modulator in the preparation of a medicament for the treatment of a cellular proliferative disorder.

49. Use according to clam 48, wherein the proliferative disorder is a vascular proliferative disorder.

50. Use according to clam 48, wherein the proliferative disorder is atherosclerosis.

51. A method of identifying a compound capable of modulating cellular proliferation comprising assaying the ability of the compound to modulate the nucleic acid expression of a nucleic acid molecule of SEQ ID NO:2 or 4 or the polypeptide activity of a polypeptide of SEQ ID NO:1 or 3, thereby identifying a compound capable of modulating cellular proliferation.

52. A method for identifying a compound capable of treating atherosclerosis comprising assaying the ability of the compound to modulate the nucleic acid expression of a nucleic acid molecule of SEQ ID NO:2 or 4 or the polypeptide activity of a polypeptide of SEQ ID NO:1 or 3, thereby identifying a compound capable of treating atherosclerosis.

53. A method of modulating cellular proliferation substantially as hereinbefore described with reference to the accompanying drawings.

54. A method of culturing mammalian cells substantially as hereinbefore described with reference to the accompanying drawings.

55. A cell culture substantially as hereinbefore described with reference to the accompanying drawings.

56. A cell culture medium as hereinbefore described with reference to the accompanying drawings.