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The Connective Tissue Growth Factor/Cysteine- Rich 61/Nephroblastoma Overexpressed (CCN) Family¹

Departments of Surgery and Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210; and Department of Surgery, Children’s Hospital, Columbus, Ohio 43205

	Abstract

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 

I. Introduction

II. The Modular Structure of the Connective Tissue Growth Factor/Cysteine-Rich 61/Nephroblastoma Overexpressed (CCN) Family Members

III. Connective Tissue Growth Factor (CTGF)

A. Discovery

B. Structure of the CTGF gene and protein

C. CTGF mRNA production

D. CTGF production, secretion, and processing

E. CTGF-heparin interactions

F. Biological properties of CTGF

G. Mechanism of action of CTGF

IV. Cyr61

A. Discovery of cyr61

B. Structure of the cyr61 gene and protein

C. Cyr61 mRNA production

D. Cyr61 production and secretion

E. Biological properties of cyr61

F. Mechanism of action of cyr61

V. Nov

A. Discovery and structure of nov

B. Nov mRNA production

C. Biological properties of nov

VI. Elm1/WISP-1

VII. Heparin-Induced CCN-Like Protein (HICP)/rCop-1/CTGF-3/WISP-2

VIII. WISP-3

IX. Other CCN-Like Molecules

X. Regulation of Cellular Functions by the CCN Family

A. Cell cycle control

B. Cell adhesion and migration

C. Extracellular matrix (ECM) production

XI. Biological Processes Involving the CCN Family

A. Development and differentiation

B. Female reproductive tract function

C. Angiogenesis

D. Wound repair

E. Fibrotic disorders

F. Inflammation

G. Tumor growth

XII. Perspectives and Future Directions

	I. Introduction

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
THE LAST 5–6 yr have seen the emergence of a new gene family that currently comprises connective tissue growth factor (CTGF; also termed fisp-12), cysteine-rich 61 (cyr61), nephroblastoma overexpressed (nov), expressed low in metastasis 1 (elm1; also termed WISP-1), heparin-inducible CTGF/cyr61/nov (CCN)-like protein (HICP; also termed rCop-1, CTGF-3 or WISP-2), and WISP-3 (Fig. 1). Family members have been characterized from human, mouse, rat, pig, cow, chicken, quail, and frog and are predicted to have arisen from a common ancestral gene more than 40 million years ago (Fig. 2). Although these proteins were initially classified as immediate early gene products or growth factors, this concept has had to be modified in light of more detailed studies of their activities as well as the discovery of unique family members that exhibit quite different biological properties. Since CTGF, cyr61, and nov were the prototype members of this family, this article adopts the term "CCN family" as introduced by Bork in 1993 (1). Implicit in this usage is that reference to the CCN family applies to all paralogs, and that CTGF, cyr61, and nov are not necessarily representative of the full range of gene or protein structures and biological properties.

View larger version (96K): [in this window] [in a new window]   Figure 1. Amino acid sequence alignment of human CCN paralogs. Sequences were aligned using the J. Hein method. Shading represents residues that are identical to the consensus sequence.

View larger version (11K): [in this window] [in a new window]   Figure 2. Dendrogram of human CCN paralogs. The x-axis is millions of years.

	II. The Modular Structure of Connective Tissue Growth Factor/Cysteine Rich 61/Nephroblastoma Overexpressed (CCN) Family Members

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
Through evolution, exons with specific biological functions ("modules") have been shuffled, forming genes that encode mosaic proteins which exhibit new biological properties (2, 3, 4). CTGF, cyr61, and nov were shown to contain four distinct structural modules that exhibit homology to conserved regions in a variety of extracellular mosaic proteins (1). All four modules are represented in WISP-1 and WISP-3, and three of the modules are present in rCop-1 and its orthologs (Fig. 3). Each module is involved in protein binding and contains conserved cysteine, hydrophobic, and polar residues. Except for module 2 in WISP-3, the modules in the various CCN proteins are 38–98% conserved with the corresponding module in human CTGF (hCTGF) (Table 1). While complex intrachain disulfide bridging is likely, this is predicted to occur within, rather than between, the modules (1).

View larger version (33K): [in this window] [in a new window]   Figure 3. Modular structure of individual CCN family members. Proteins shown with black boxes are the predicted primary translational products. Proteins shown with open boxes are biologically active derivatives arising by proteolysis (CTGF) or viral DNA integration (Nov). Question marks indicate that the C termini of N-terminally truncated CTGF proteins have not been experimentally determined.

Module 1 is approximately 32% identical with the N-terminal cysteine-rich regions of the six "classic" IGF-binding proteins, IGFBP-1 to -6 (1), and contains a motif (GCGCCXXC) that is involved in binding IGF (17). ¹²⁵I-labeled IGF-I or -II specifically binds recombinant hCTGF (rhCTGF) (12), although with much lower affinity than classic IGFBPs and more comparable to mac25, a low-affinity IGFBP, also termed IGFBP-7 or IGFBP-related protein (IGFBP-rP)-1 (18). Nov was also reported to bind IGFs (19), but this result has not proven reproducible (20). Recently, the terms IGFBP-8, -9, and -10 were introduced as synonyms for CTGF, nov, and cyr61, respectively (12), although it has since been proposed to use the names IGFBP-rP-2, -3, and -4 until their relationship to the classic IGFBPs has been determined (21). Indeed, a case against classifying the CCN family as IGFBPs has recently been made (22). Whatever the terminology, it remains unclear whether the interaction of IGFs with CCN family members occurs physiologically, whether sequences within module 1 are actually responsible for the observed binding of IGF, and what consequences this interaction has on the respective half-life, bioavailability, or activity of each of the molecules involved. However, there is general agreement that direct, IGF-independent effects of CTGF on the cell cycle are likely to be more significant than its low affinity binding of IGF (12, 22).

Module 2 comprises a Von Willebrand type C domain (VWC) that occurs in Von Willebrand factor as well as various mucins, thrombospondins, and collagens (1). Many proteins that contain VWC modules participate in oligomerization, which may be preceded by a dimerization event (23). Since module 4 of CTGF is a putative dimerization domain, module 2 may mediate the formation of complexes from CTGF dimers. Unlike all other CCN proteins, module 2 in WISP-3 contains only 6 of the 10 cysteines, the functional significance of which has yet to be established (24). Module 3 is a thrombospondin type 1 (TSP1) that contains the local motif WSXCSXXCG (1) and appears to be a cell attachment motif that binds sulfated glycoconjugates (1, 25, 26, 27). While cyr61 and CTGF promote cell adhesion (15, 16), the role of module 3 in this process remains unexplored.

Module 4 is a C-terminal (CT) module that also occurs in the C termini of a variety of unrelated extracellular mosaic proteins (1). Six of the 10 cysteine residues in the CT module appear to adopt the cystine knot motif that also occurs in nerve growth factor (NGF), transforming growth factor-ß (TGF-ß), and platelet-derived growth factor (PDGF) (1). This complex structure comprises two 2-stranded ß-sheets that lie face-to-face and are linked by three interlocking disulfide bridges and has defined a new superfamily of growth regulators (28, 29) to which members of the CCN family (except orthologs of rCop-1, which lack module 4; see below) may also belong. Since some of the receptor binding properties of NGF, TGF-ß, and PDGF reside in variable regions within the cystine knot (28), the CT module likely contains both dimerization and receptor-binding domains (1). Although dimerization of CCN proteins has not been reported, bioactive forms of 10-kDa porcine CTGF (pCTGF) comprise the C-terminal 102 or 103 residues of the primary translational product (Fig. 3) (10) and thus support the proposed role of module 4 in binding cell surface receptors (1). Module 4 is absent from rCop and its orthologs (Figs. 1 and 3), suggesting that they are functionally distinct from other CCN proteins.

	III. Connective Tissue Growth Factor (CTGF)

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
A. Discovery
The first description of a CTGF ortholog occurred in 1988 when cDNA encoding "fibroblast-inducible secreted protein-12" (fisp-12) was isolated by differential screening of a cDNA library from serum-stimulated NIH 3T3 cells (30). The gene structure and predicted protein sequence of fisp-12 were subsequently reported in 1991 (5). Using a similar screening strategy, Brunner et al. (31) independently isolated the same cDNA, termed ßIG-M2, from TGF-ß2-stimulated mouse AKR-2B cells. Fisp-12/ßIG-M2 comprises 348 amino acids and contains 39 Cys residues, one of which occurs in the 25-residue N-terminal signal peptide (Fig. 1) (5, 31). The human ortholog of CTGF was discovered in 1991 due to the cross-reactivity of a PDGF antiserum with 38-kDa hCTGF secreted by cultured human vein endothelial cells (HUVECs) (13). The corresponding cDNA was isolated by screening a HUVEC cDNA expression library with anti-PDGF and shown to encode a 349-amino acid protein that is 91% homologous to fisp-12 (13) (Fig. 1). The protein was termed "CTGF" because it was both mitogenic and chemotactic for fibroblast-like cells in vitro. CTGF cDNAs have since been reported for pig, rat, cow, and frog (10, 32, 33, 34, 35).

B. Structure of the CTGF gene and protein
The fisp-12 gene comprises five exons and four introns and spans 3.1 kb (Ref. 5 ; Table 2). The organization and structure of the hCTGF gene are very similar to that of fisp-12 except that exon 1 encodes one additional amino acid in the signal peptide (13, 36, 37). There is 80% sequence identity between CTGF and fisp-12 in the 300 nucleotides that lie immediately upstream of the mRNA cap site (36), and the 5'-regions of both genes contain a variety of conventional regulatory elements as well as a unique TGF-ß response element (Fig. 4). Fisp-12 maps to the A3-B1 region of murine chromosome 10 (5) and hCTGF maps to human chromosome 6q23.1 (38).

View larger version (14K): [in this window] [in a new window]   Figure 4. Key regulatory elements in the gene promoters of hCTGF, mouse cyr61, and human nov. Note that the TGF-ß response element (TßRE) is unique to CTGF.

C. CTGF mRNA production
Among the cell types that produce CTGF mRNA are fibroblasts (5, 14, 31, 35, 39, 41, 42), endothelial cells (13, 35, 43), vascular smooth muscle cells (VSMC) (35, 44), epithelial cells (45, 46, 47), chondroctyes (48), and glioblastoma cells (37). Fisp-12/CTGF transcripts have been detected in multiple tissues of at least four mammalian species (5, 12, 32, 35, 47, 49). Most studies have reported a single CTGF transcript of 2.4 kb, although glioblastoma cells also contain 3.5-kb and 7.0-kb transcripts (37). The 2.4-kb transcript is present in unstimulated fibroblasts (35, 39) but is rapidly induced after treatment with TGF-ß or serum and is superinduced in stimulated fibroblasts in the absence of de novo protein synthesis (5, 31, 41). These studies collectively established that CTGF/fisp-12 are encoded by immediate early genes, although their kinetics of induction are more rapid and sustained than those of other immediate early genes (41). TGF-ß also increases CTGF mRNA levels in cultured human or mouse lung mesenchymal cells (34) and in human chondrocytic cells (48), the latter of which also produce CTGF mRNA in response to bone morphogenic protein-2, a member of the TGF-ß family (50). Using an enzyme-linked immunosorbent assay (ELISA), TGF-ß treatment of human fibroblasts was shown to result in increased production of the CTGF protein (51). The molecular basis for the action of TGF-ß on CTGF gene transcription has been atributed to nucleotides -157 to -145 of the hCTGF promoter (36). This region is a unique TGF-ß-inducible element (Fig. 4) that is fully conserved in the promoter of the fisp-12 gene (5) but is absent from the other parologs of the gene family (Fig. 4) as well as from other immediate early genes. In addition, inhibitors of protein kinase A, but not of tyrosine kinases or protein kinase C, block TGF-ß-stimulated CTGF transcription (52). Collectively, the TGF-ß response element and the involvement of cAMP in CTGF mRNA production represent major mechanistic differences in CTGF gene transcription as compared with other CCN family members.

Although considerable attention has been directed toward the TGF-ß inducibility of CTGF expression and the potential pathophysiological consequences thereof (53), PDGF, epidermal growth factor (EGF), and fibroblast growth factor (FGF) also stimulated hCTGF gene expression in fibroblasts (41). As compared with the effects of TGF-ß on this cell type, CTGF transcription is less robust in response to these growth factors (41) and does not result in increased CTGF protein levels (51). In contrast, pancreatic cancer cells of epithelial origin demonstrate much stronger production of CTGF mRNA by EGF or TGF- than TGF-ß, although the lack of response to TGF-ß was due, at least partly, to defects in TGF-ß signaling (54). Phenomenologically, the lack of response to TGF-ß in certain pancreatic cells is similar to that of skin epithelial cells, which (unlike fibroblasts) do not synthesize CTGF in response to local TGF-ß administration in vivo (14). Dexamethasone treatment of cultured 3T3 cells was recently shown to stimulate CTGF expression while actually down-regulating TGF-ß mRNA (55). Systemic administration of dexamethasone stimulated CTGF expression in the heart, kidney, and skin. Basal and dexamethasone-stimulated CTGF expression was strongly attenuated by tumor necrosis factor- (TNF-) (55), which was independently shown to suppress basal CTGF mRNA production in bovine aortic endothelial cells (BAECs), fibroblasts, and VSMCs (35). Finally, increased CTGF expression in renal epithelial cells in response to injury occurred without an increase in TGF-ß expression (46). These results show, not surprisingly, that mechanisms of CTGF gene regulation may be cell specific and, in addition to the action of TGF-ß, involve a variety of hormones, growth factors, and cytokines.

D. CTGF production, secretion, and processing
CTGF is present in HUVEC-conditioned medium (13) and undergoes microsomal processing (5), consistent with the presence of a signal peptide and its presumed secretion. CTGF mRNA is expressed in BAECs in which it is present at higher levels during growth than confluence (43). Similarly, expression of fisp-12 in cycling fibroblasts is relatively high (15). CTGF produced by human foreskin fibroblasts or mouse connective tissue cells under normal growth conditions appears to be cell associated, long lived, and secreted relatively inefficiently (39). On the other hand, fisp-12 was secreted efficiently from NIH 3T3 cells but was relatively unstable in the medium (5). Kireeva et al. (15) showed that fisp-12 was present in the cellular fraction, ECM, and medium and that inhibition of lysosomal acid hydrolysis increased the half-life of fisp-12 in the cellular fraction and stabilized steady-state fisp-12 levels. These data suggest that membrane-associated fisp-12 may form complexes with receptors on the surface of fisp-12-producing cells that are then internalized and degraded (15).

Various low mass (10–20 kDa) forms of pCTGF were purified from uterine secretory fluids and shown to commence between modules 2 and 3, at the beginning of module 3, or between modules 3 and 4 (10, 11) (Fig. 3). Native 38-kDa pCTGF undergoes rapid proteolytic processing by uterine fluids (11) and likely explains the presence of 38-kDa CTGF in uterine tissues (32) but not in uterine fluids (10, 11). Native low-mass CTGFs in uterine fluids are extremely stable, and their levels are strongly correlated with those of CTGF-degrading proteases (11). Although conditioned medium from serum-stimulated mouse fibroblasts did not degrade fisp-12 (15), 10- to 12-kDa CTGF, but not 38-kDa CTGF, was present in conditioned medium from cycling human or mouse fibroblasts (39). Recently, Western blot analysis demonstrated 12- to 14-kDa C-terminal forms of CTGF in mouse uterine luminal flushings (47) and 18- and 24-kDa CTGF isoforms in human serum and amniotic, follicular, peritoneal, and cerebrospinal fluids (40). The presence of CTGF in sera has been confirmed by ELISA (51). Whereas serum CTGF levels in normal human subjects were less than 28 ng/ml, those of patients with biliary atresia were up to 8-fold higher (51). These results highlight potential diagnostic or prognostic applications of measuring circulating or secreted CTGF levels.

Collectively, these data suggest that qualitative and quantitative aspects of CTGF secretion may be regulated by numerous factors including stage of cell cycle, growth factor pretreatment, cell type, species and tissue of origin, and protease activity of the pericellular environment. Moreover, the presence of CTGF in a variety of body fluids suggests that they may be important reservoirs of CTGF in vivo.

E. CTGF-heparin interactions
Heparin-affinity chromatography has been used to purify 38-kDa rhCTGF (14) and 10- to 20-kDa pCTGF (10, 11), the latter of which are eluted from heparin by 0.8 M NaCl. ³⁵S-labeled fisp-12 binds to heparin-agarose beads and is eluted by 0.4 M NaCl (15). Although module 3 is a binding motif for sulfated glycoconjugates (1), residues 247–260, 274–286, and 305–328 of hCTGF bind strongly to heparin (10, 56). Residues 247–260 contain a proposed heparin-binding consensus sequence (XBBXBX) that occurs in a variety of heparin-binding proteins (57).

As with other heparin-binding growth factors, such as FGFs, heparin-binding EGF-like growth factor, amphiregulin, keratinocyte growth factor, and vascular endothelial growth factor (58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70), the biological activity of CTGF is modulated by heparin (10, 14). The binding of fisp-12 to heparin has been proposed as the basis for the association of fisp-12 with the ECM in cultured cells (15). Heparin-like molecules on the cell surface or in the ECM may constitute a high-capacity, low-affinity binding reservoir for CTGF and thereby regulate its activity, bioavailability, or stability. Indeed, the half-life of fisp-12 in ECM (1 h) is much less than that of cyr61 (4 h) and appears to correlate with the relative affinity of each paralog for heparin (15, 71).

F. Biological properties of CTGF
Native 38-kDa hCTGF is a mitogenic and chemotactic factor for NIH 3T3 cells in vitro (13). Ten-kilodalton forms of pCTGF are mitogenic for Balb/c 3T3 cells, VSMCs, and endometrial stromal cells, but not endothelial cells (10, 56). The mitogenic activity of 38-kDa rhCTGF or 10-kDa pCTGF on fibroblasts is enhanced by EGF, PDGF, basic FGF (bFGF), or IGF-I and is either enhanced or reduced by heparin according to the CTGF-heparin ratio (10, 14). In monolayer cultures of normal rat kidney (NRK) cells, 38-kDa rhCTGF stimulated DNA synthesis and induced expression of type 1 collagen, fibronectin, and 5 integrin (14), as is characteristic of the effects of TGF-ß. These various biological effects are elicted by CTGF concentrations of about 1–20 ng/ml. In vivo, injection of TGF-ß or CTGF into the dermal/subcuticular area of the skin in neonatal mice produced nodules comprising mainly connective tissue cells and ECM (14, 72), suggesting that CTGF plays a role in TGF-ß-mediated formation of granulation tissue. Recombinant hCTGF is also mitogenic for human lung fibroblasts (73). However, recombinant 38 kDa fisp-12 did not directly stimulate DNA synthesis in HUVECs or 3T3 cells at 0.3–3 µg/ml (15), although other evidence suggests that endothelial cells do not respond mitogenically to 10-kDa CTGF, which is nonetheless mitogenic for 3T3 cells and VSMCs (10). At 0.3–3 µg/ml, the same fisp-12 protein enhanced the mitogenic activity of 10 ng/ml bFGF on HUVECs and NIH 3T3 cells and promoted attachment of HUVECs, NIH 3T3 cells, AKR2B cells, and mink lung epithelial cells at 5–20 µg/ml (15). These latter effects occurred at 100- to 1000-fold higher concentrations than were needed for the stimulation of mitosis, chemotaxis, or ECM production by CTGF in other studies (10, 13, 14). Nonetheless, a role for CTGF in cell adhesion is further supported by the localization of CTGF in ECM (15) and by the ability of antisense CTGF to suppress growth and migration of BAECs in vitro (43). While 38-kDa CTGF appears to be proteolytically cleaved into low mass derivatives (11), the relationship (if any) between processing and activity remains undefined.

Although TGF-ß induces CTGF expression and CTGF mimics some of the effects of TGF-ß in fibroblasts, CTGF-independent pathways of TGF-ß action have been identified in these and other cell types. For example, TGF-ß is a potent inhibitor of the growth of mink lung epithelial cells, whereas CTGF is not (14). In addition, while CTGF and TGF-ß both stimulate DNA synthesis in monolayer cultures of NRK cells, only TGF-ß is able to stimulate anchorage-independent growth (AIG) of the same cells (14, 74). AIG induced by TGF-ß is antagonized by CTGF antibodies or antisense CTGF and is restored by addition of CTGF to TGF-ß-stimulated cells showing that CTGF is involved in the stimulation by TGF-ß of this process (74). Since CTGF was induced by TGF-ß in these cells, it was proposed that both CTGF-dependent and -independent pathways are involved in the stimulation of AIG by TGF-ß (74). The induction of CTGF mRNA and AIG by TGF-ß in NRK cells is inhibited by elevated cAMP levels (52). This effect was reversed by addition of CTGF, but not other growth factors, and was attributed to a CTGF-specific restriction point in late G₁ of TGF-ß-activated cells (52).

G. Mechanism of action of CTGF
In addition to contributing to TGF-ß-mediated AIG (see above), CTGF interacts synergistically with EGF, PDGF, IGF-I, or bFGF (10, 14, 56), suggesting that it activates distinct receptors and/or signaling pathways to those used by other growth factors. Although 38-kDa hCTGF was suggested to bind PDGF receptors (13, 75), this possibility was not supported using a mitogenic form of 10-kDa murine CTGF (39). Binding studies with ¹²⁵I-labeled 38-kDa rhCTGF have demonstrated the presence of specific high- and low-affinity binding sites and a 280-kDa cross-linked CTGF complex (76). Although there was no clear evidence for a signal-transducing receptor, these data are nonetheless potentially very exciting. Alternatively, CTGF may function primarily as a cell adhesion molecule that regulates cell function either directly through its association with the ECM or indirectly by synergizing with other growth factors (15).

	IV. Cyr61

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
A. Discovery of cyr61
Cyr61 (originally termed 3CH61) was described in 1985 as an immediate early gene in mouse Balb/c 3T3 cells that was induced by serum or PDGF (77). It was named cyr61 when shown to encode a cysteine-rich protein that contained 10% cysteine residues (78). The same gene, named ßIG-M1, was also discovered by Brunner et al. (31) in their studies of TGF-ß-inducible immediate early genes in mouse AKR-2B cells. The gene for cef-10, the chicken ortholog of cyr61, was described in 1989 as pp60^v-src-inducible immediate early gene in chicken embryo fibroblasts (CEFs) (79). cDNA for the human ortholog of cyr61 (hcyr61) was isolated from a 6-week-old human embryonic tissue cDNA library (80).

B. Structure of the cyr61 gene and protein
The murine cyr61 gene comprises five exons and four introns that span 3.1 kb (Table 2) and contains a serum-response element (SRE) (Fig. 4) in the 5'-region that mediates serum- or PDGF-induced gene transcription (6). Induction of cyr61 is similar to that of other immediate early genes that contain SREs, consistent with its coordinated expression with c-fos during G₀ to G₁ (81), although its transcriptional suppression is relatively inefficient (6). Although cyr61 expression is induced by TGF-ß (31), its promoter lacks the novel TGF-ß response element found in the promoter of CTGF (36) (Fig. 3). Human cyr61 maps to p22.3 on chromosome 1 (80, 82). The mouse cyr61 gene encodes a 379-residue protein, which, after signal peptide cleavage, yields a 355-residue protein containing 38 conserved cysteine residues (31, 78) (Fig. 1). The cysteine-free portion of cyr61 is about twice the length of that in other CCN paralogs (Fig. 1).

C. Cyr61 mRNA production
A single cyr61 transcript of 2.4 kb has been reported in mouse AKR2B cells, 3T3 fibroblasts, and PSA-1 teratocarcinoma cells (31, 77, 81, 83) and rat H19–7 embryonic neuronal hippocampal cells (84). A 1.8-kb cef-10 transcript is present in CEFs (79), and two cyr61 transcripts of 2.5 kb and 3.5–4 kb have been described in human tissues (80, 82). Although cef-10 mRNA is expressed principally in the adult lung (79), cyr61 is expressed in multiple adult tissues of the mouse and human such as heart, uterus, skeletal muscle, and lung (49, 78, 80). Transcript levels in other tissues are variable and likely due to differences in mRNA detection techniques as well as in the stage of development or differentiation of the source material. For example, human cyr61 mRNA expression is higher in the kidney of the fetus than that of the adult (80). Cyr61 mRNA is present at high levels in mouse embryos on days 9.5–14.5, whereas placental expression of cyr61 is highest on days 17.5–18.5 (83).

Cyr61 gene expression is stimulated by diverse molecular signals, which supports its role in a variety of biological processes. In mouse cells, cyr61 mRNA is rapidly induced by serum, PDGF, FGF, TGF-ß, phorbol ester, or cholera toxin (31, 77, 78, 81). Serum stimulation of Balb/c 3T3 cells resulted in a rapid increase and sustained high levels of cyr61 mRNA, which were stabilized by cyclohexamide (78). Similarly, human cyr61 mRNA is present in proliferating, but not quiescent, skin fibroblasts (49). In CEFs, transcription of cef-10 is rapidly enhanced by pp60^v-src or serum (79). In human osteoblasts, cyr61 expression is enhanced by EGF, PDGF, and interleukins-1ß, -2, and -6, as well as by 1,25-dihydroxyvitamin D₃, a mediator of osteoblast differentiation and function (85). In the rat uterus, cyr61 is estrogen-responsive and strongly induced by tamoxifen (86). In rat H19–7 cells, cyr61 is induced within 1 h by bFGF, a stimulator of neuronal differentiation in this cell type (84). This process appears to involve both mitogen-activated protein kinase-dependent and -independent pathways (84).

D. Cyr61 production and secretion
The 41-kDa mouse cyr61 protein is present at very low amounts in quiescent Balb/c 3T3 cells but reaches peak levels within 2 h of serum stimulation (78). Cyr61 is differentially distributed in the ECM, cellular fraction, or cell surface rather than the medium (71, 78). The extracellular fate and half-life of cyr61 synthesized in response to mitogenic stimulation were shown to be dependent upon the stage of the cell cycle and presence of binding moieties in the ECM and on cell surfaces such as heparin-like molecules from which cyr61 is eluted by 0.6–0.8 M NaCl (16, 49, 71).

E. Biological properties of cyr61
Recombinant murine cyr61 promotes dose-dependent attachment of HUVECs or NIH 3T3 cells to plastic surfaces coated with 3–30 µg/ml of the protein (16). Similar concentrations of cyr61 also stimulate dose-dependent chemotaxis of NIH 3T3 cells (16) and human microvascular endothelial cells (HMVEC) (87). These concentrations of cyr61 were not mitogenic for NIH 3T3 cells or HUVECs, although they did potentiate the mitogenic activity of bFGF for these cell types (16, 49). This latter effect was abolished by cyr61 antiserum and attributed to the displacement by cyr61 of bFGF from the ECM, thus increasing the effective concentration of soluble bFGF (16, 49). In mouse limb bud micromass cultures, cyr61 promoted expression of type II collagen, incorporation of sulfate, and the production of cartilage nodules (88). Moreover, cyr61 promoted the adhesion and aggregation of limb bud mesenchymal cells. Cyr61 was also a weak stimulator of DNA synthesis and proliferation of limb mesenchymal cells (88). Cyr61 was further shown to promote chondrogenesis in cell cultures seeded at subthreshold densities that do not normally undergo chondrogenic differentiation. These various responses were elicted with 0.3–5 µg/ml cyr61. Limb bud micromass cultures showed reduced levels of chondrogenesis when incubated with neutralizing cyr61 antisera, suggesting that endogenous cyr61 plays a normal physiological role in chrondrogenic differentiation (88). Finally, cyr61 has been shown to induce neovascularization in vivo (87), consistent with its stimulation of directed migration of HMVECs in vitro (87). Stable transfection of cyr61 cDNA into RF-1 gastric adenocarcinoma cells had no effect on in vitro growth rate, yet produced larger tumors in immunocompromized mouse hosts as compared with a vector control, a phenomenon that was attributed to cyr61-stimulated tumor vascularization (87).

F. Mechanism of action of cyr61
A mechanistic framework for the biological properties of cyr61 has been provided by the finding that cyr61 binds to integrin _vß₃, which represents the first (and only) molecularly defined receptor for any member of the CCN family (89). The interaction of _vß₃ with cyr61 may account for its promotion of chemotaxis and growth factor-mediated DNA synthesis as well as cell adhesion since integrins are known to modulate cell migration and growth factor signaling in other systems (90). As well as demonstrating a direct interaction between the two molecules in binding assays in vitro (89), cyr61-mediated adhesion and migration of cultured endothelial cells were specifically inhibited by the peptide RGDS and/or antiintegrin _vß₃ (87, 89). In addition to its integrin-binding property, cyr61 appears to be localized to its site of synthesis by associating with the ECM, possibly by binding to heparin-like molecules. This interaction may limit the extent of cyr61 diffusion so that its site of action is in close proximity to its site of synthesis (15).

	V. Nov

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
A. Discovery and structure of nov
Nov (nephroblastoma overexpressed) was first recognized as an overexpressed gene in nephroblastomas induced by myeloblastosis-associated virus type 1 (MAV-1) in day-old chicks (7, 91). Subsequently, the human (novH), mouse nov (novM), quail (novQ), and Xenopus (xnov) orthologs of chicken nov (novC) were isolated by hybridization screening (8, 33, 38, 92). The nov gene demonstrates a well conserved intron/exon structure that is similar to that of other paralogs in the CCN family (Table 2). In one chicken nephroblastoma, MAV-1 envelope and long-terminal repeat sequences integrated into the second exon of the novC gene at Cys⁶³ (7); the first putative initiation codon downstream of this integration site is Met⁹⁹. The novH gene maps to chromosome 8q24.1 and appears to contain at least three transcription initiation sites in the promoter region (38). NovM maps to chromosome 15 between D15 Mit153 and D15 Mit 183 (92, 93).

Putative transciption factor-binding sites are present in the 5'-untranslated region of several nov genes (8, 33, 38, 92) (Fig. 4). However, SREs and the TGF-ß response element are absent, suggesting that transcriptional regulation of nov is distinct from that of CTGF or cyr61. The activity of the novH promoter is repressed in the presence of the Wilms tumor suppressor gene WT-1 (94). While WT-1 binding sites are present in the novH promoter (Fig. 4), WT-1-mediated repression of novH transcription was experimentally attributed to other domains in the promoter (94).

The primary translational products of nov orthologs are predicted to comprise between 343 and 357 amino acids and to contain 38 conserved cysteine residues (Fig. 1). The signal peptide in novH appears to comprise 27 residues (8). In CEFs, an immunoreactive 46-kDa novC protein was localized to the ECM (93), although the underlying mechanisms and functional significance remain unclear. NovH produced in transfected Madin Darby canine kidney cells is a glycosylated protein that is secreted about 1.5 h after synthesis and requires 0.5 M NaCl for elution from heparin (20). Secreted novH has a half-life of more than 18 h, which is more than 10 times that of intracellular novH (20). Levels of the endogenous 46-kDa novH protein are repressed in 293 kidney cells that constitutively express WT1 (94).

B. Nov mRNA production
A single 2.2-kb novC transcript is usually detected in MAV1-induced nephroblastomas (7). An exception was a tumor in which MAV1 integrated within the novC gene itself, resulting in the production of a 2.0-kb transcript (7). The 2.2-kb novC mRNA transcript was detected at high levels in the brain and heart of day 18 chick embryos, with weak signals in muscle and intestine and nondetectable levels in liver, lung, and yolk sac. In the adult, 2.2-kb novC mRNA was expressed at high levels in brain and lung, at low levels in the spleen, and was nondetectable in heart, muscle, and liver (7). Quiescent cultures of CEFs contain a 2.2-kb novC transcript that is down-regulated during proliferation, mitogenic stimulation, or activation of viral oncogenes (95). NovH is normally expressed as a 2.5-kb transcript in human glioma cell lines and Wilms tumors, although an additional novH mRNA of 1.2 kb was observed in a single Wilms tumor (8, 37). Unlike novC, this smaller transcript was not the result of rearrangement of the novH gene (8).

There is little, if any, experimental evidence supporting a role for nov as an immediate early gene. As explained above, there are no motifs in the nov gene promoter that resemble either a SRE or a TGF-ß response element. Serum stimulation of kidney-derived 293 cells or CEFs failed to increase nov mRNA levels within 90 min, even though the genes for CTGF and jun were rapidly activated (93). Moreover, in normal CEFs, nov was expressed in quiescent cells but down-regulated in proliferating cells (95). In contrast to cef-10, expression of p60^v-src in CEFs resulted in transcriptional down-regulation of the novC gene (95). NovC expression was also repressed by the viral oncogenes v-erbB, v-mil, and v-crk (95). In addition, mRNA destabilization sequences in the 3'-region are absent from most nov orthologs (except xnov), suggesting that nov RNA degradation is regulated differently from that of CTGF, cyr61, and other immediate early genes.

C. Biological properties of nov
Overexpression of full-length novC cDNA was shown to inhibit the growth of CEFs grown in soft agar (7). In contrast, transformation of CEFs occurred after overexpression of an N-terminally truncated form of novC that initiated at Met⁹⁹ and was thus comparable to that expressed after integration of MAV-1 into the novC gene (Fig. 3) (7). This truncated form of nov also induced morphological transformation of rat embryo fibroblasts (8). Within the CCN family, the ability to function as a protooncogene is thus far unique to nov. The dramatic biological differences between the normal and truncated nov genes have led to suggestions that nov is a growth suppressor gene, truncation of which results in oncogenic activation. These interpretations are consistent with the low levels of nov mRNA in mitogen-stimulated cells and the high levels of nov mRNA in resting cells (93, 95). Although the truncated nov protein lacked the signal peptide and the IGF-binding motif in module 1, it is not known how the loss of one or both of these domains contributes (if at all) to its transforming properties.

	VI. Elm1/WISP-1

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
Elm1 (expressed in low-metastatic type 1 cells) was initially identified by mRNA differential display as being expressed in low, but not in high, metastatic murine melanoma cells (96). The elm1 protein was subsequently predicted to comprise 367 amino acids and to conform to the CCN modular structure (97) (Figs. 1 and 3; Table 1). Elm1 transcripts of 1.8 and 5.0 kb were present in all mouse tissues analyzed (97). The 5.0-kb elm1 transcript was induced within 3 h of serum stimulation of quiescent Balb/c 3T3 cells, unlike cyr61 which was induced within 30 min in the same cells (97). Elm1 maps to mouse chromosome 15 between D15 Mit17 and D15 Mit3. Southern blotting demonstrated that the elm1 gene is also present in human, monkey, rat, mouse, dog, and cow (97). Transfection of elm1 into highly tumorigenic and metastatic K-1735 M-2 cells resulted in a decreased tumor growth in vivo and reduction in the frequency of lung metastatic colonies (97). In vitro, transfected cells that expressed relatively high amounts of elm1 grew somewhat slower and to lower saturation density as compared with control cells (97). Elm1 did not exhibit mitogenic activity for NIH3T3 cells, either alone or in combination with bFGF. The growth-suppressive properties of elm1 have been tentatively linked to module 3 (97). Recently, WISP-1 was identified as the human ortholog of elm1 and shown to be a component of the Wnt-signaling pathway in transformed cells (24). WISP-1 maps to human chromosome 8q24.1–8q24.3 and exhibits tissue-specific patterns of expression (24).

	VII. Heparin-Induced CCN-Like Protein (HICP)/rCop-1/CTGF-3/WISP-2

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
A novel 1.8-kb mRNA was identified by subtractive hybridization screening of heparin-treated and nontreated rat VSMCs and shown to encode a novel member of the CCN family that was termed HICP (98). Independent studies resulted in the isolation of cDNA for the same gene, termed rCop-1, when shown to be expressed in normal rat embryo fibroblasts or mouse 3T3 cells, but not in their transformed derivatives (99). The mouse ortholog, WISP-2, was recently identified as a unique gene that was up-regulated in Wnt-1-transformed mouse mammary epithelial cells in vitro (24). These molecules, together with the human ortholog, CTGF-3 (100), demonstrate an overall amino acid homology of 30% with hCTGF and conservation of all of the cysteine residues in modules 1–3 (Fig. 1). However, all of these proteins are C-terminally truncated as compared with other CCN paralogs, resulting in a total absence of module 4 (Figs. 1 and 3). While HICP/CTGF-3/WISP-2 transcripts were present in multiple tissues (24, 98, 100), rCop-1 mRNA was not detected in any of the major organs of the mouse embryo and adult rat (99). Although this discrepancy requires further investigation, expression of rCop-1 has been linked to aging and senescence (99). HICP expression patterns, similar to those of growth arrest genes, occur at high levels in quiescent or heparin-arrested VSMCs and at very low levels during proliferation (98). Somewhat different expression patterns occur for rCop-1 in cycling mouse 3T3 cells in which rCop-1 mRNA is absent during quiescence or immediately after serum stimulation and appears only during late S phase (99). Unlike normal cells, transfection of rCop-1 into transformed cells suppressed their cell growth; this was attributed to cell death rather than growth arrest (99). In transfected cells, rCop-1 is localized primarily intracellularly and to the cell surface, but not in conditioned medium or ECM. It has been speculated (99) that the unique subcellular localization and expression pattern of rCop-1/HICP is a reflection of its role in negative growth regulation, the structural basis of which may lie in the absence of module 4. Potential tumor-suppressive properties are further suggested by the finding that WISP-2 is underexpressed in human colon tumors, unlike WISP-1 and -3, which are overexpressed (24).

	VIII. WISP-3

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
Human WISP-3, a 354-residue protein containing 36 cysteine residues, was identified by screening expressed sequence tag databases with WISP-1 (24). The secreted WISP-3 protein is predicted to contain 34 of the 38 conserved cysteine residues found in most other CCN proteins since it lacks 4 of the cysteines normally present in module 2. It is possible that this structural difference results in unique properties and functions, although this aspect of WISP-3 biology has not yet been explored (24).

	IX. Other CCN-Like Molecules

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
Twisted gastrulation (tsg) and short gastrulation (sog), which are involved in dorsoventral patterning in Drosophila embryos (101, 102), contain sequences that resemble, respectively, modules 1 and 2. As with CTGF and cyr61, tsg is eluted from heparin by 0.6–0.8 M NaCl, and the binding of tsg by somatic cells may involve a heparin-like coreceptor (103). The cytoskeletal rearrangements that take place in dorsal amnioserosa cells in the presence of tsg have been likened to those in endothelial cells during CTGF-induced chemotaxis (101). Sog, like CTGF, may be susceptible to proteolytic cleavage, yielding diffusible bioactive fragments (102). The cDNA of "small CCN-like growth factor" (SCGF) was recently isolated from an 9-week human embryo library and shown to encode a 206-residue protein that had limited structural similarity to the C-terminal region of CCN family members, although cysteine residues occur at half the expected frequency and are poorly conserved (104). Overall, tsg, sog, and SCGF exhibit very weak alignment with the CCN family and are not genuine paralogs.

	X. Regulation of Cellular Functions by the CCN Family

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
A. Cell cycle control
Members of the CCN family are products either of immediate early genes (CTGF, cyr61) or of putative growth arrest/suppression genes (nov, elm1, HICP). The expression of immediate early genes represents the earliest genomic response to growth factors and is likely to initiate the program leading to cell replication. Since they encode a diverse array of regulatory molecules, the induction of immediate early genes represents a central component in the proliferative response to mitogenic stimuli (105). Since CTGF and cyr61 are transcriptionally activated by TGF-ß, PDGF, EGF, FGF, TPA, and cholera toxin, it is likely that the cellular responses to these diverse mitogenic stimuli are controlled, at least partly, by CTGF and cyr61. On the other hand, the expression of growth arrest-specific genes, DNA damage-inducible genes, and the MyD growth-arrest genes are associated with the negative regulation of cell growth (106). Growth arrest genes have been implicated in cellular differentiation, embryonic development, and apoptosis (107, 108, 109), and it is possible that these types of processes are similarly regulated by nov, elm1, and rCop-1.

In addition, each CCN family member appears to further regulate the cell cycle through their 1) direct mitogenic action (e.g., CTGF); 2) potentiation of the mitogenic activity of other growth factors (e.g., CTGF, cyr61); 3) binding of IGFs (e.g., CTGF); 4) regulation of ECM synthesis (e.g., CTGF, cyr61); 5) regulation of cell attachment and migration (e.g., CTGF, cyr61, elm1); 6) interactions with the ECM (e.g., CTGF, cyr61, nov); 7) oncogenic properties (e.g., nov); and 8) regulation of a cell cycle restriction point (e.g., CTGF). This broad spectrum of biological properties, many of which are interacting and interdependent, demonstrate the complexity by which the cell cycle is likely regulated by the CCN family.

B. Cell adhesion and migration
In vitro studies have shown that CTGF or cyr61 are chemotactic and promote cell adhesion (13, 15, 16, 87). Elm1 is expressed in low, but not high, metastatic cell lines and, when transfected into high-metastatic cell lines, causes them to exhibit slower rates of tumor growth and decreased incidence of metastasis (97). Truncated nov stimulates AIG and colony formation in CEFs (7). Collectively, these data demonstrate broad effects on cell adhesion and locomotion, which likely reflects the ability of CCN family members to regulate the composition of the ECM, the net balance between its synthesis and degradation, as well as to bind directly to ECM components such as integrins and heparan sulfate proteoglycans (see below).

C. ECM production
Tissue formation and cell proliferation and differentiation are dependent upon interactions between cells and the ECM. In processes such as tissue remodeling and malignancy, ECM components are degraded by proteases such as collagenase, plasmin, and matrix metalloproteases, and the ability of cells to interact with the ECM is altered or abolished. TGF-ß plays a central role in ECM production since it stimulates synthesis of ECM proteins such as collagen, fibronectin, laminin, elastin, glycosaminoglycans, and other glycoproteins as well as synthesis of inhibitors of ECM degradation such as plasminogen activator inhibitor and tissue inhibitors of metalloproteases (110). In view of the TGF-ß-inducibility of CTGF and cyr61 gene expression, these molecules may promote ECM deposition by stimulating synthesis of ECM components and inhibiting their degradation. While detailed studies of the regulation of ECM-degrading enzymes and their inhibitors by the CCN family have yet to be reported, initial studies have shown that 38-kDa CTGF enhances fibronectin, type IV collagen, and integrin production (14) and that cyr61 stimulates type II collagen production, albeit in a specialized cell type (88). As discussed below, numerous fibrotic disorders exhibit substantial ECM involvement and expression of CTGF. In addition, there is a striking appearance of CTGF in uterine stromal cells that are undergoing decidualization (47), a highly regulated process involving increased mitosis and synthesis and deposition of ECM molecules such as desmin, laminin, and fibronectin (111). ECM production and remodeling is also a critical component of development and differentiation during embryogenesis (112). Collectively, these data suggest that CTGF may exert diverse and important functions in normal physiology and pathological states via its net induction of ECM protein deposition and content.

	XI. Biological Processes Involving the CCN Family

Top Abstract I. Introduction II. The Modular Structure... III. Connective Tissue Growth... IV. Cyr61 V. Nov VI. Elm1/WISP-1 VII. Heparin-Induced CCN-Like... VIII. WISP-3 IX. Other CCN-Like Molecules X. Regulation of Cellular... XI. Biological Processes... XII. Perspectives and Future... References

 
A. Development and differentiation
Immunohistochemical studies showed that CTGF is present in the mouse as early as embryonic days 4.5–6.5, at which time it is most abundant in the embryonic endoderm and mesoderm (47). At later stages of gestation (days 14–18), CTGF and cyr61 are both present in various tissues and organs including the cardiovascular and pulmonary systems as well as the skin and placenta (15). Secretory structures such as kidney tubules and salivary, mucous, and sebaceous glands are positive for CTGF, but not cyr61, whereas the nervous and skeletal systems are positive for cyr61, but not CTGF (15). This differential distribution may be indicative of unique roles for each factor. A role for nov during Xenopus development is suggested by the production of its mRNA in the oocyte at stages I, III, and IV and in the embryo at all developmental stages between egg and tadpole (33). In chickens, novC is expressed in kidney, heart, and muscle of the embryo but not of the adult (7). Other organs (e.g., brain) demonstrate novC expression in both embryonic and adult life, whereas expression of novC in the lung occurs in the adult but not the embryo (7).

Evidence to date supports a role for cyr61 and CTGF in chondrogenesis. The cyr61 protein is present in cartilage and bone in day 13–18 mouse embryos (15, 88), and cyr61 mRNA is present in a variety of developing cartilaginous structures in day 8.5–14.5 embryos (83). A role for cyr61 in normal growth and development of the cartilaginous skeleton is further supported by the ability of cyr61 to stimulate collagen synthesis, sulfate incorporation, and formation of cartilage nodules in mouse limb bud micromass cultures, and adhesion, aggregation, and growth of limb bud mesenchymal cells (88). In the case of CTGF, its mRNA is present in hypertrophic chondroctyes from day 17 mouse embryos, neonatal rabbit growth cartilage tissue and cultured cells, and human chondrocytic cell lines, but not in human osteosarcoma or mouse osteocytic cell lines (48). In cultured rabbit growth cartilage cells, peak levels of CTGF occurred during the early hypertrophic stage. CTGF mRNA levels in these cells were enhanced by treatment with TGF-ß or bone morphogenic protein-2 (48). In addition to its potential role in chondrogenesis, cyr61 may also be involved in hippocampal differentiation, since it is induced in H19–7 cells by bFGF, which stimulates their differentiation into nonproliferative neuronal cells (84).

Several lines of evidence support a role for nov nephrogenesis. First, novC expression occurs in normal avian kidney cells of the embryo and neonate, yet is barely detectable in adult kidney cells (7). Second, ureteric buds, which give rise to the kidney collecting ducts, fail to demonstrate outgrowth in WT-1-null mice; as discussed above, novH promoter activity is repressed by WT-1, and a reciprocal quantitative gradient of novH and WT1 expression occurs during normal human nephrogenesis (see Ref. 94). Third, novH is localized to differentiating glomerular podocytes where it accumulates and may regulate podocyte structure or function during both pre- and postnatal life (20). Since the composition of the ECM changes during the earliest stages of metanephric induction (113) and ECM components regulate uteric bud morphogenesis, nov may also influence nephrogenesis by binding to the ECM (93).

B. Female reproductive tract function
The actions of ovarian steroids are mediated, in part, by polypeptide growth factors, which may also contribute to the uterine-embryo signaling dialogue that initiates implantation and stimulates embryonic development (114, 115, 116, 117, 118, 119, 120). Also, some growth factors are secreted into the uterine lumen or localized at the implantation site, where they may stimulate development of the embryo and placental membranes (114, 121). CTGF is present in uterine fluids of the pig and mouse, as well the uterus of the pig, mouse and human (10, 11, 13, 47, 56, 122) suggesting that it is involved in the regulation of uterine function. In uterine fluids, the levels of low mass CTGFs and CTGF proteases are highly correlated with each other and show cyclic variations during the estrous cycle (11). Moreover, there are clear differences in the levels of CTGF and CTGF protease(s) in uterine fluids at equivalent stages of the estrous cycle and early pregnancy suggesting that CTGF production or action is modified in the presence of the embryo (11, 56). A relationship between CTGF expression and TGF-ß action during the periimplantation period of the pig is supported by the presence of both molecules at this time in uterine and/or embryonic tissues (11, 123, 124, 125). In addition, a direct role of CTGF on uterine cells is indicated by its stimulation of DNA synthesis in pig stromal cells in vitro (56).

In mice and women, CTGF is localized primarily to uterine luminal and glandular epithelial cells during the estrous cycle and during the first few days of pregnancy (47, 122). On the day of implantation in mice, epithelial staining for CTGF is strongly reduced and is followed over the next 2 days by profound staining of decidualizing endometrial stromal cells, suggesting that CTGF contributes to the decidualization process or is produced as a result of it (47). Decidualization is a highly regulated differentiation process involving increased vascular permeability, DNA synthesis, and synthesis and deposition of ECM molecules (111, 126, 127, 128). The distribution of uterine CTGF during early pregnancy in the mouse does not entirely correlate with TGF-ß and its receptors, suggesting that TGF-ß-independent synthesis of CTGF may be operative (47). Alternative mechanisms may involve glucocorticoids or estrogen, which stimulate CTGF and cyr61 gene transcription, respectively (55, 86). The nonconcordance of CTGF and TGF-ß may also be related to the initial production of TGF-ß in a latent form (129). Whatever the explanation, these data support a role for uterine CTGF at the time of implantation.

C. Angiogenesis
Angiogenesis, the formation of new capillaries from preexisting blood vessels, occurs in processes such as tumor growth, diabetic retinopathy, wound healing, and placental vascularization. It is a complex process that involves degradation of the capillary basement membrane, migration and proliferation of endothelial cells, and tube formation; it is regulated by many factors (130), including cyr61, which promotes directed migration of HMVECs via _vß₃-integrin and induces neovascularization in the rat cornea (87). Expression patterns of cyr61 support its contribution to angiogenesis in the embryo, placenta, hypertrophic cartilage, wounds, and tumors (83, 87). Since cell migration is the only component of angiogenesis to be directly stimulated by cyr61, indirect effects of cyr61, such as release and action of the angiogenic factor bFGF from ECM (16, 49), may contribute to the overall process of neovascularization. These questions and the role of other CCN family members in angiogenesis will require further study.

D. Wound repair
Many studies have established that growth factors, including TGF-ß, are involved in wound healing (see Ref. 131). Collectively, CTGF and cyr61 exhibit numerous biological properties that are of potential importance in the wound healing response, including stimulation of cell proliferation, cell adhesion, chemotaxis, angioge