Kouros Motamed

SPARC (BM-40, Osteonectin) Inhibits the Mitogenic Effect of Vascular Endothelial Growth Factor on Microvascular Endothelial Cells

SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein that modulates cell adhesion and proliferation and is thought to function in tissue remodeling and angiogenesis. In this study, we demonstrate that SPARC inhibits DNA synthesis by >90% in human microvascular endothelial cells (HMEC) stimulated by the endothelial cell mitogen vascular endothelial growth factor (VEGF). Peptides derived from SPARC domain IV, which contains a disulfide-bonded EF-hand sequence and binds to endothelial cells, mimicked the effect of native SPARC. The inhibition was also observed with a peptide from the follistatin-like domain II, whereas peptides from SPARC domains I and III had no effect on VEGF-stimulated DNA synthesis. The inhibition of HMEC proliferation was mediated in part by the binding of VEGF to SPARC. The binding of125I-VEGF to HMEC was reduced by SPARC and SPARC peptides from domain IV in a concentration-dependent manner. In a radioimmune precipitation assay, peptides from SPARC domains II and IV each competed with native SPARC for its binding to VEGF. It has been reported that VEGF stimulates the tyrosine phosphorylation and activation of mitogen-activated protein kinases Erk1 and Erk2. We now show that SPARC reduces this phosphorylation in VEGF-stimulated HMEC to levels of unstimulated controls. SPARC thus modulates the mitogenic activity of VEGF through a direct binding interaction and reduces the association of VEGF with its cell-surface receptors. Moreover, an additional diminution of VEGF activity by SPARC is accomplished through a reduction in the tyrosine phosphorylation of mitogen-activated protein kinases.

Compensatory Regulation of RIα Protein Levels in Protein Kinase A Mutant Mice

The cAMP-dependent protein kinase holoenzyme is assembled from regulatory (R) and catalytic (C) subunits that are expressed in tissue-specific patterns. Despite the dispersion of the R and C subunit genes to different chromosomal loci, mechanisms exist that coordinately regulate the intracellular levels of R and C protein such that cAMP-dependent regulation is preserved. We have created null mutations in the RIβ and RIIβ regulatory subunit genes in mice, and find that both result in an increase in the level of RIα protein in tissues that normally express the β isoforms. Examination of RIα mRNA levels and the rates of RIα protein synthesis in wild type and RIIβ mutant mice reveals that the mechanism of this biochemical compensation by RIα does not involve transcriptional or translational control. These in vivo findings are consistent with observations made in cell culture, where we demonstrate that the overexpression of Cα in NIH 3T3 cells results in increased RIα protein without increases in the rate of RIα synthesis or the level of RIα mRNA. Pulse-chase experiments reveal a 4-5-fold increase in the half-life of RIα protein as it becomes incorporated into the holoenzyme. Compensation by RIα stabilization may represent an important biological mechanism that safeguards cells from unregulated catalytic subunit activity.

SPARC (osteonectin/BM-40)☆

SPARC (Secreted ProteinAcidic and Rich in Cysteine) is a prototype of a family of biologically active glycoproteins that bind to cells and to extracellular matrix (ECM) components. It is expressed spatially and temporally during embryogenesis, tissue remodeling and repair. SPARC is a modular protein (34 kDa) comprised of three structural domains, one or more of which are implicated in the regulation of cell adhesion, proliferation, matrix synthesis/turnover. Rapid proteolysis of SPARC by extracellular proteases accounts for its transient detection in the extracellular environment. The proposed roles of SPARC in the development of cataracts and the regulation of angiogenesis during wound healing and tumor growth account for the recent attention it has received from the biomedical community.

SPARC regulates TGF‐beta1‐dependent signaling in primary glomerular mesangial cells

Secreted protein acidic and rich in cysteine (SPARC), a member of the family of matricellular proteins, regulates the interaction of cells with pleiotropic factors and proteins of the extracellular matrix (ECM). Although it has been appreciated that transforming growth factor beta 1 (TGF‐β1) induces SPARC and collagen type I, we have recently shown that SPARC regulates the expression of TGF‐β1 and collagen type I in renal mesangial cells via a TGF‐β1‐dependent pathway, and have proposed a reciprocal, autocrine regulatory feedback loop between SPARC and TGF‐β1. Herein, we sought to determine how SPARC regulates TGF‐β1‐dependent signal transduction. Our data indicate that SPARC modulates the TGF‐β1‐dependent phosphorylation of Smad‐2 in primary mesangial cells derived from wild‐type and SPARC‐null mice. We also show that SPARC regulates the levels and activation of the stress‐activated c‐jun‐N‐terminal kinase (JNK) in mesangial cells by augmentation of the stimulatory effects of TGF‐β1. Furthermore, we found that SPARC increases the levels and the activity of the transcription factor c‐jun. These effects of SPARC on the TGF‐β1 signaling pathway appear to be mediated through an interaction with the TGF‐β1‐receptor complex, but only in the presence of TGF‐β1 bound to its cognate type II receptor. That SPARC is directly involved in the regulation of the TGF‐β1 signaling cascade is consistent with the paradigm that matricellular proteins modulate interactions among cells, growth factors, and their respective receptors.

SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation-dependent pathway

SPARC, a counteradhesive matricellular protein, inhibits endothelial cell adhesion and proliferation, but the pathways through which these activities are blocked are not known. In this study, we used inhibitors of major signaling proteins to identify mediators through which SPARC exerts its counteradhesive and antiproliferative functions. Pretreatments with the general protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, protected against the inhibitory effect of SPARC on bovine aortic endothelial (BAE) cell spreading by more than 60 %. Similar pretreatments with PTK inhibitors significantly blocked the diminishment of focal adhesions by SPARC in confluent BAE cell monolayers, as determined by the formation of actin stress‐fibers and the distribution of vinculin in focal adhesion plaques. Inhibition of endothelial cell cycle progression by SPARC and a calcium‐binding SPARC peptide, however, was not affected by PTK inhibitors. Inhibition of DNA synthesis by SPARC was not reversed by inhibitors of the activity of protein kinase C (PKC), or of cAMP‐dependent protein kinase (PKA), but was sensitive to pertussis (and to a lesser extent, cholera) toxin. The counteradhesive effect of SPARC on endothelial cells is, therefore, mediated through a tyrosine phosphorylation‐dependent pathway, whereas its antiproliferative function is dependent, in part, on signal transduction via a G protein‐coupled receptor. J. Cell. Biochem. 70:543–552, 1998.

Inhibition of PDGF-stimulated and matrix-mediated proliferation of human vascular smooth muscle cells by SPARC is independent of changes in cell shape or cyclin-dependent kinase inhibitors

Interactions among growth factors, cells, and extracellular matrix regulate proliferation during normal development and in pathologies such as atherosclerosis. SPARC (secreted protein, acidic, and rich in cysteine) is a matrix‐associated glycoprotein that modulates the adhesion and proliferation of vascular cells. In this study, we demonstrate that SPARC inhibits human arterial smooth muscle cell proliferation stimulated by platelet‐derived growth factor or by adhesion to monomeric type I collagen. Binding studies with SPARC and SPARC peptides indicate specific and saturable interaction with smooth muscle cells that involves the C‐terminal Ca2+‐binding region of the protein. We also report that SPARC arrests monomeric collagen‐supported smooth muscle cell proliferation in the late G1‐phase of the cell cycle in the absence of an effect on cell shape or on levels of cyclin‐dependent kinase inhibitors. Cyclin‐dependent kinase‐2 activity, p107 and cyclin A levels, and retinoblastoma protein phosphorylation are markedly reduced in response to the addition of exogenous SPARC and/or peptides derived from specific domains of SPARC. Thus, SPARC, previously characterized as an inhibitor of platelet‐derived growth factor binding to its receptor, also antagonizes smooth muscle cell proliferation mediated by monomeric collagen at the level of cyclin‐dependent kinase‐2 activity. J. Cell. Biochem. 84: 759–771, 2002.

SPARC regulates cell cycle progression in mesangial cells via its inhibition of IGF‐dependent signaling

Glomerular mesangial cells both synthesize and respond to insulin‐like growth factor‐1 (IGF‐1). Increased activity of the IGF signaling pathway has been implicated as a major contributor to renal enlargement and subsequent development of diabetic nephropathy. Secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, has been shown to modulate the interaction of cells with growth factors and extracellular matrix. We have reported that primary glomerular mesangial cells derived from SPARC‐null mice exhibit an accelerated rate of proliferation and produce substantially decreased levels of transforming growth factor β1 (TGF‐β1) in comparison to their wild‐type counterparts (Francki et al. [ 1999 ] J. Biol. Chem. 274: 32145–32152). Herein we present evidence that SPARC modulates IGF‐dependent signaling in glomerular mesangial cells. SPARC‐null mesangial cells produce increased amounts of IGF‐1 and ‐2, as well as IGF‐1 receptor (IGF‐1R) in comparison to wild‐type cells. Addition of recombinant SPARC to SPARC‐null cells inhibited IGF‐1‐stimulated mitogen activated protein kinase (MAPK) activation and DNA synthesis. We also show that the observed accelerated rate of basal and IGF‐1‐stimulated proliferation in mesangial cells derived from SPARC‐null animals is due, at least in part, to markedly diminished levels of cyclin D1 and the cyclin‐dependent kinase (cdk) inhibitors p21 and p27. Since expression of SPARC in the glomerulus is especially prominent during renal injury, our findings substantiate previous claims that SPARC is involved in glomerular remodeling and repair, a process commonly associated with mesangioproliferative glomerulonephritis and diabetic nephropathy.

Renaturation of SPARC expressed in Escherichia coli requires isomerization of disulfide bonds for recovery of biological activity

SPARC (secreted protein acidic and rich in cysteine, also known as osteonectin and BM-40) belongs to a group of secreted macromolecules that modulate cellular interactions with the extracellular matrix. During vertebrate embryogenesis, as well as in tissues undergoing remodeling and repair, the expression pattern of SPARC is consistent with a fundamental role for this protein in tissue morphogenesis and cellular differentiation. Human SPARC was cloned by the polymerase chain reaction from an endothelial cell cDNA library and was expressed in Escherichia coli as a biologically active protein. Two forms of recombinant SPARC (rSPARC) were recovered from BL21(DE3) cells after transformation with the plasmid pSPARCwt: a soluble, monomeric form that is biologically active (Bassuk et al., 1996, Archiv. Biochem. Biophys. 325, 8-19), and an insoluble form sequestered in inclusion bodies. Aggregated rSPARC was unfolded by urea treatment, purified by nickel-chelate affinity chromatography, and renatured by gradual removal of the denaturant. Proper isomerization of the disulfide bonds was achieved in the presence of a glutathione redox couple. After final purification by high resolution gel filtration chromatography, a monomeric form of rSPARC displaying biological activity was obtained. The recombinant protein inhibited the spreading and synthesis of DNA by endothelial cells, two properties characteristic of the native protein. We conclude that the information for the correct folding of rSPARC resides in the primary structure of the protein, and suggest that post-translational modifications are required neither for folding nor for biological activity.

Targeted Disruption of the Protein Kinase A System in Mice

The holoenzyme of cAMP-dependent protein kinase (PKA) is composed of two regulatory (R) and two catalytic (C) subunits and forms the major mediator of cAMP action in animal cells. Additional responses to cAMP are also elicited by the cyclic nucleotide-gated ion channels such as those expressed in the olfactory epithelium and recently found in heart, kidney, testis, and brain (Biel et al. 1994). Four R subunit genes (RIα, RIβ, RIIα, and RIIβ) have been cloned in mouse and are highly conserved in all mammals, whereas only two C subunit genes (Cα and Cβ) have been reported in mouse (McKnight 1991) and a third Cγ subunit has been found only in humans (Beebe et al. 1990). We have discovered a processed pseudogene (no introns) that is apparently derived from Cα and since the pseudogene is located on the mouse X chromosome, we have named it Cx. We find no transcripts from the Cx pseudogene and the sequence has mutated such that it would not give rise to a functional C subunit (Cummings et al. 1994). The human Cγ subunit is also most closely related to Cα, and although it is transcribed, no protein has yet been detected in vivo. Like Cx, the Cγ gene is intronless (T. Jahnsen, personal communication), suggesting that Cγ and Cx both evolved from a retroposon derived from the Cα gene early in the mammalian lineage and may have retained functionality only in humans.