Human CYR61-mediated enhancement of bFGF-induced DNA synthesis in human umbilical vein endothelial cells.

CYR61 is a member of an emerging family of growth regulators that includes the human connective tissue growth factor and the chicken protooncoprotein Nov. Encoded by a growth factor-inducible immediate-early gene, CYR61 is a secreted, cysteine-rich heparin-binding protein that associates with the extracellular matrix and cell surface. In this report, we describe the cloning of the human hCYR61 cDNA and expression and purification of the hCYR61 protein. The hCYR61 mRNA is abundant in the heart, lung, pancreas and placenta, is present at a low level in skeletal muscle and brain and is not detectable in liver and kidney. Purified recombinant hCYR61 protein does not induce thymidine incorporation in human umbilical vein endothelial cells by itself, but enhances bFGF-induced DNA synthesis. We show that hCYR61 acts in this cell system in part by displacement of bFGF bound to the extracellular matrix, thus increasing the effective concentration of bFGF. Possible biological implications of these observations are discussed.

Beta1 integrins show specific association with CD98 protein in low density membranes.

BACKGROUND: The CD98 (4F2, FRP-1) is a widely expressed cell surface protein heterodimer composed of a glycosylated heavy chain and a non-glycosylated light chain. Originally described as a T cell activation antigen, it was later shown to function in amino acid transport, cell fusion and homotypic cell aggregation. Several lines of evidence suggest its functional interaction with integrins but the biochemical basis for this interaction has been unclear. RESULTS: We demonstrate that CD98 constitutively and specifically associates with beta1 integrins (alpha2beta1,alpha3beta1, alpha5beta1 and alpha6beta1), but minimally with alpha4beta1. Integrin-CD98 association was established by reciprocal immunoprecipitation experiments, and confirmed by CD98-induced clustering of alpha3beta1 but not alpha4beta1 on the surface of rhabdomyosarcoma cells. Integrin-CD98 association is independent of the alpha subunit cytoplasmic tail, is maintained in alpha3beta1 ligand-interaction deficient mutants, and is not inhibited by EDTA. Within the CD98 heavy chain, a C109S mutation (but not a C330S mutation) caused a loss of beta1 integrin association. The same C109S mutation also caused a loss of CD98 light chain association. Importantly, CD98 associated selectively with beta1 integrins present in low density "light membrane" fractions on a sucrose gradient. CD98 was not present in dense fractions that contained the majority of beta1 integrins. Notably, the C109S mutant of CD98, that did not associate with beta1 integrins, showed also a reduced localization into light membrane fractions. CONCLUSIONS: We demonstrate that CD98 association with beta1 integrins is specific, occurs in the context of low density membranes, and may require the CD98 light chain.

[Physical factors in the pathogenetic therapy of children with scleroderma]. / Fizicheskie faktory v patogeneticheskoi terapii detei, bol'nykh sklerodermiei.

Telecapillaroscopy, biomicroscopy of the bulbar conjunctiva, rheovasography, cold vascular test, ohyprolin and glycosaminoglycans excretion measurements have been performed in 337 2-14-year-olds with systemic and localized scleroderma to study peripheral circulation and connective tissue metabolism. The results contributed to development of differentiated balneo- and pelotherapy schedules affecting the principal components of the disease pathogenesis.

EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.

A novel Ig superfamily protein, EWI-2, was co-purified with tetraspanin protein CD81 under relatively stringent Brij 96 detergent conditions and identified by mass spectrometric protein sequencing. EWI-2 associated specifically with CD9 and CD81 but not with other tetraspanins or with integrins. Immunodepletion experiments indicated that EWI-2-CD9/CD81 interactions are highly stoichiometric, with approximately 70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line. The EWI-2 molecule was covalently cross-linked (in separate complexes) to both CD81 and CD9, suggesting that association is direct. EWI-2 is part of a novel Ig subfamily that includes EWI-F (F2alpha receptor regulatory protein (FPRP), CD9P-1), EWI-3 (IgSF3), and EWI-101 (CD101). All four members of this Ig subfamily contain a Glu-Trp-Ile (EWI) motif not seen in other Ig proteins. As shown previously, the EWI-F molecule likewise forms highly proximal, specific, and stoichiometric complexes with CD9 and CD81. Human and murine EWI-2 protein sequences are 91% identical, and transcripts in the two species are expressed in virtually every tissue tested. Thus, EWI-2 potentially contributes to a variety of CD9 and CD81 functions seen in different cell and tissue types.

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells.

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton.

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth.

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate-early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin alphaVbeta3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an alphaV beta3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

The light chain of CD98 is identified as E16/TA1 protein.

The 80/40-kDa CD98 protein complex was purified using an anti-CD98 heavy chain monoclonal antibody coupled to Sepharose beads. Eluted proteins were subjected to preparative SDS-polyacrylamide gel electrophoresis, and protein corresponding to the 40-kDa CD98 light chain was excised. Following proteolysis with trypsin, a peptide fragment was sequenced by mass spectrometry. The nine residues obtained were identical to established C-terminal sequences of the human E16 and rat TA1 proteins, suggesting that TA1/E16 protein is the CD98 light chain. Consistent with this, anti-TA1/E16 antibodies specifically immunoblotted the approximately 35-40-kDa light chain present upon immunoprecipitation of the human CD98 complex. Furthermore, anti-CD98 heavy chain antibody specifically co-immunoprecipitated hemagglutinin-tagged light chain from cells transfected with hemagglutinin-tagged E16 cDNA. In conclusion, the CD98 light chain is identical to the TA1/E16 protein, based on partial amino acid sequence identity, antibody cross-reactivity, genetic reconstitution evidence, similar molecular size, and comparable cell distribution.

Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development.

cyr61 and fisp12 are homologous immediate-early genes that are transcriptionally activated upon growth factor stimulation in fibroblasts. Their gene products belong to an emerging family of secreted proteins with a high degree of sequence homology, including conservation of all 38 cysteine residues in their secreted portions. We have recently shown that Cyr61 is an extracellular matrix (ECM) signaling molecule that promotes cell proliferation, migration, and adhesion. We describe herein the first purification of the Fisp12 protein and we compare the activities of purified Cyr61 and Fisp12, their metabolism, targeting, and their localization during development. Although Fisp12 is the mouse homolog of the human connective tissue growth factor (CTGF), it has no detectable mitogenic activity by itself. Rather, Fisp12 enhances fibroblast growth factor-induced DNA synthesis. The activities of Fisp12 and Cyr61 are nearly indistinguishable in three cell types tested: fibroblasts, endothelial, and epithelial cells. Both proteins are found in the ECM, although Cyr61 associates with the ECM more strongly and binds heparin with higher affinity. Fisp12, but not Cyr61, is also found in the culture medium, suggesting that Fisp12 might be able to act at a distance from its site of secretion, whereas Cyr61 might act more locally. Both secreted proteins are internalized and degraded through the lysosomal pathway, suggesting interaction with cell surface receptors. Both Cyr61 and Fisp12 are found in the placenta and the circulatory system as detected by immunohistochemistry, whereas Cyr61, but not Fisp12, is found in the skeletal and nervous systems. Fisp12, but not Cyr61, is found in secretory organs. Taken together, we propose that Cyr61 and Fisp12 are both signaling cell adhesion molecules that have similar or overlapping activities, and their differential sites of localization and targeting may dictate specificity in their biological roles.