Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 15 to 23 amino acid residues in defined sequence
Reexamination Certificate
1997-06-10
2002-05-07
Gambel, Phillip (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
15 to 23 amino acid residues in defined sequence
C530S328000, C530S329000, C530S330000, C530S331000, C530S402000, C530S403000
Reexamination Certificate
active
06384189
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of regulating TGF-&bgr; activity. In particular, the present invention relates to a method of stimulating or inhibiting TGF-&bgr; activity by the application of specific peptides.
2. Background Art
Transforming growth factor-&bgr; (TGF-&bgr;) is a member of a family of growth, differentiation, and morphogenesis autocrine and paracrine factors (3,26). TGF-&bgr; can affect diverse cellular functions in virtually all cell types. Depending on the cell type and its extracellular environment, these effects can be either positive or negative. TGF-&bgr; inhibits the proliferation of endothelial cells in vitro (31), but stimulates angiogenesis in vivo (39). TGF-&bgr; has also been shown to enhance or inhibit the proliferation of fibroblasts depending on the nature of the substrate and the mitogens present (3). Myoblast differentiation can also be induced or blocked by TGF-&bgr; depending on the availability of mitogens (25, 45).
TGF-&bgr;1 is a disulfide-linked homodimer that is synthesized as part of a latent precursor molecule (26). The latent precursor molecule is 390 amino acids in length and consists of an N-terminal 278 amino acid latency associated peptide (LAP) and a C-terminal 112 amino acid active domain (15-17). The proregion of TGF-&bgr; is unique in that it remains non-covalently attached to the active region after intracellular proteolytic processing and secretion (15). Association of the LAP with the mature peptide region confers latency: the LAP-associated growth factor is unable to interact with its cellular receptors. The LAP contains three N-linked glycosylation sites, two of which have mannose-6-phosphate residues (8,28,38). These carbohydrate structures may be important for latency since endoglycosidase F treatment leads to activation of TGF-&bgr; (28). The disulfide-bonded dimeric structure of LAP is critical for latency, since site-directed mutagenesis of critical cysteine residues (cys 223, 225) in the LAP abolishes the latency function (9). The active domain contains nine conserved cysteine residues that participate in inter-and intrachain disulfide bonding (27).
TGF-&bgr; is secreted by most cell types as a latent complex (27,37). Since TGF-&bgr; synthesis and TGF-&bgr; receptor expression are not highly regulated, primary regulation of TGF-&bgr; activity occurs by controlling conversion of the latent TGF-&bgr; complex to the active molecule. Physiochemical activation can occur by extremes of pH, heat, chaotropic agents, and deglycosylation (6,27,28,37). Activation in vivo is more complex and not well understood. There is evidence from cell culture models that activation may occur through binding of the latent molecule to mannose 6-phosphate receptors (12,21), by plasmin-mediated proteolytic processing (4,23,40,41), and/or by processing in acidic cellular microenvironments (20). In some systems, activation of latent TGF-&bgr; by plasmin is relatively inefficient (41). In addition, there are reports of TGF-&bgr; activation occurring independently of these mechanisms (19). These results suggest that additional mechanisms of latent TGF-&bgr; activation may exist.
TGF-&bgr; has been demonstrated, through numerous studies, to play a significant role in wound healing and fibrosis. The three phases of inflammation, granulation tissue formation and biosynthesis of the extracellular matrix, are identical in both the wound healing process and the development of fibrosis. A fine balance in the biosynthetic and degradative pathways involved in extracellular matrix biosynthesis appears to be determinative of whether proper wound healing or fibrosis results. Due to its function of regulating genes critically involved in extracellular matrix formation, TGF-&bgr; significantly influences this phase of tissue regeneration, the final outcome of which is either wound healing or fibrosis. (46). Thus, sensitive regulation of TGF-&bgr; activity in this process will permit control of the wound healing and fibrotic processes.
The thrombospondins (TSP) are a growing family of multidomain glycoproteins (47,48,5,49). TSP1 is the best characterized and serves as the prototypical TSP molecule. TSP is secreted and incorporated into the extracellular matrix of a number of cells in culture (1-5). TSP, like TGF-&bgr;, has diverse effects on cellular functions that vary with cell type. TSP can inhibit endothelial proliferation and migration (2,42,34,51), but stimulates the growth of smooth muscle cells and dermal fibroblasts (52,36). TSP may also serve as both an attachment protein and an anti-adhesive molecule as shown by its ability to cause disassembly of focal adhesions in endothelial cells (33). TSP also plays a role in angiogenesis, fibrinolysis, platelet aggregation, and inflammation (1-5).
TSP is present transiently in wound environments and its synthesis is rapidly induced by growth factors, including TGF-&bgr; (50). TSP is detectable in incisional wound margins for 2-7 days, after which it localizes around vascular channels near the wound. Although the role of TSP is not yet clearly understood, it has been speculated that TSP may facilitate cell migration into the wound site or possibly act as a localized growth promoting agent (49).
There are three sequences in TSP known as type 1 repeats. Each repeat consists of approximately 60 amino acids, has six conserved cysteine residues and has approximately 47% sequence homology to similar repeats found in the human complement component, properdin. Within the type 1 repeats of TSP, there are two well defined consensus sequences, CSVTCG (SEQ ID NO:1) and WSXW (SEQ ID NO:2). CSVTCG (SEQ ID NO:1) inhibits metastasis of melanoma cells in a murine lung colonization assay (Tuszynski et al., 1992), and promotes cell adhesion possibly by mediating interactions of TSP with sulfated glycoconjugates (53,54). The anti-angiogenic activity of TSP might be, in part, due to CSVTCG (SEQ ID NO:1). Tolsma et al. showed that CSVTCG (SEQ ID NO:1) inhibits angiogenesis in vivo using a corneal neovascularization assay (55).
The sequence WSXW (SEQ ID NO:2) binds specifically to sulfated glycoconjugates and promotes cell adhesion and chemotaxis (56). The binding of TSP to the gelatin-binding domain of fibronectin can be blocked using the peptide GGWSHW (SEQ ID NO:3), suggesting WSHW (SEQ ID NO:4) may also promote matrix protein interactions (57). This sequence is also conserved within members of the TGF-&bgr; and cytokine receptor superfamilies (58,59).
This invention provides TSP peptides which activate latent TGF-&bgr; and TSP peptides which inhibit activation of latent TGF-&bgr;.
TSP is a potential physiological regulator of TGF-&bgr; activity. These peptides from TSP can both positively and negatively modulate TGF-&bgr; levels at nanomolar to micromolar concentrations, and, therefore, can be used as therapeutic agents in vivo for the promotion of wound healing and inhibition of fibrosis.
SUMMARY OF THE INVENTION
The invention provides a method of stimulating TGF-&bgr; activity, comprising contacting latent TGF-&bgr; with an amount of an activating peptide effective to convert latent TGF-&bgr; to active TGF-&bgr;. Also provided is a method of inhibiting the stimulation of TGF-&bgr; activity, comprising contacting latent TGF-&bgr; with an amount of an inhibiting peptide effective to inhibit the conversion of latent TGF-&bgr; to active TGF-&bgr;.
The invention also provides a method of enhancing wound healing, comprising administering to the wound site an amount of an activating peptide effective to convert latent TGF-&bgr; to active TGF-&bgr;, the activation of TGF-&bgr; resulting in enhanced wound healing.
A method of preventing fibrosis stimulated by TGF-&bgr; in pathology also provided. The method comprises administering to the site of potential fibrosis an amount of inhibiting peptide effective to inhibit conversion of latent TGF-&bgr; to active TGF-&bgr;, resulting in reduced fibrosis.
The invention also provides a method of blocking TGF-&bgr;-mediated inhibition
Krutzsch Henry C.
Murphy-Ullrich Joanne E.
Roberts David D.
Schultz-Cherry Stacey
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