Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.     

Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) binds to fibrinogen fragments D and E, but not to native fibrinogen.

Secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) is a matricellular protein that functions in wound healing. Fibrinogen is a plasma protein involved in many aspects of wound healing, such as inflammation, fibrosis and thrombosis. In this study, the binding of SPARC to both native and plasmin-cleaved fibrinogen under physiological conditions was examined by the use of a surface plasmon resonance (SPR) biosensor. We show that SPARC binds to plasmin-cleaved fibrinogen, but not to native fibrinogen. SPARC binds to both fibrinogen fragments D and E fg D and fg E with similar dissociation constants (8.67 x 10(-8) M for Fg D and 1.61 x 10(-7) M for Fg E). Results from endothelial cell proliferation assays show that the binding of SPARC to Fg E suppressed the inhibition of proliferation by SPARC, whereas the binding of SPARC to Fg D did not influence the activity of SPARC on the cell cycle. The interaction of SPARC with fibrinogen fragments D and E, which are produced as a result of proteolytic activation of fibrinolysis, reveals potential storage sites in provisional extracellular matrix for SPARC during the wound healing process and indicates a regulatory role of SPARC in fibrinolysis and angiogenesis.     

Secreted protein acidic and rich in cysteine internalization and its age‐related alterations in skeletal muscle progenitor cells

Aging causes phenotypic changes in skeletal muscle progenitor cells (Skm‐PCs), such as reduced myogenesis and increased adipogenesis due to alterations in their environment or niche. Secreted protein acidic and rich in cysteine (SPARC), which is secreted into the niche of Skm‐PCs, inhibits adipogenesis and promotes myogenesis. We have previously reported that Skm‐PC responsiveness to SPARC declines with age, although the mechanism underlying this decline is unknown. In this study, we found that SPARC is internalized by Skm‐PCs and that this uptake increases with age. Internalization is dependent on integrin‐α5, a cell surface SPARC‐binding molecule, and clathrin‐mediated endocytosis. We also demonstrated that internalized SPARC is transported to Rab7‐positive endosomes. Skm‐PCs from old rats exhibited increased clathrin expression and decreased Rab7 expression exclusively in MyoD‐negative cells. In loss‐of‐function analyses, clathrin knockdown increased the anti‐adipogenic effect of SPARC, whereas Rab7 knockdown reduced it, indicating that alterations in SPARC internalization may mediate the age‐related decline in its anti‐adipogenic effect. These results provide insights into age‐related SPARC resistance in Skm‐PCs, which may lead to sarcopenia.     

Differential effects of various growth factors and cytokines on the syntheses of DNA,type I collagen,laminin, fibronectin,osteonectin/secreted protein,acidic and rich in cysteine (SPARC), and alkaline phosphatase by human pulp cells in culture

The purpose of this study is to differentiate roles of several growth factors and cytokines in proliferation and differentiation of pulp cells during development and repair. In human pulp cell cultures, laminin and type I collagen levels per cell remained almost constant during the whole culture period (22 days). On the other hand, secreted protein, acidic and rich in cysteine (SPARC/osteonectin) and alkaline phosphatase (ALPase) levels markedly increased after the cultures reached confluence. Laminin and type I collagen, as well as fibronectin, stimulated the spreading of pulp cells within 1 h. Adding transforming growth factor-β (TGF-β) decreased laminin and ALPase levels, whereas it increased SPARC and fibronectin levels 3- to 10-fold. Western and Northern blots showed that TGF-β enhanced SPARC synthesis at the protein and mRNA levels. Basic fibroblast growth factor (bFGF) decreased type I collagen, laminin, SPARC, and ALPase levels without changing the fibronectin level. Platelet-derived growth factor (PDGF) selectively decreased laminin, SPARC, and ALPase levels. Epidermal growth factor (EGF) also decreased SPARC and ALPase levels. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) decreased type I collagen and laminin levels, and abolished SPARC and ALPase syntheses. Of these peptides, bFGF and PDGF showed the greatest stimulation of [³H]thymidine incorporation into DNA. TGF-β, EGF, and TNF-α had less effect on DNA synthesis, whereas IL-1β inhibited DNA synthesis. These findings demonstrated that TGF-β, bFGF, EGF, PDGF, TNF-α, and IL-1β have characteristically different patterns of actions on DNA, laminin, type I collagen, fibronectin, ALPase, and SPARC syntheses by pulp cells. J. Cell. Physiol. 174:194–205, 1998. © 1998 Wiley-Liss, Inc.     

Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells

Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2-DE proteomic analysis of human melanoma cells following antisense-mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N-cadherin (N-CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N-CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral-mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi-transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N-CAD and CLU and increased secretion of HSP27. Transient re-expression of SPARC in SPARC-downregulated cells reverted extracellular N-CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N-CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells.     

The copper binding domain of SPARC mediates cell survival in vitro via interaction with integrin beta1 and activation of integrin-linked kinase

Secreted protein acidic and rich in cysteine (SPARC) is important for the normal growth and maintenance of the murine lens. SPARC-null animals develop cataracts associated with a derangement of the lens capsule basement membrane and alterations in lens fiber morphology. Cellular stress and disregulation of apoptotic pathways within lens epithelial cells (LEC) are linked to cataract formation. To identify molecular targets of SPARC that are linked to this disorder, we stressed wild-type (WT) and SPARC-null LEC by serum deprivation or exposure to tunicamycin. SPARC enhanced signaling by integrin-linked kinase (ILK), a serine/threonine kinase known to enhance cell survival in vitro. In response to stress, an ILK-dependent decrease in apoptosis was observed in WT relative to SPARCg-null LEC. Co-immunoprecipitation and cross-linking of cell lysates revealed enhanced levels of a SPARC-integrin beta1 complex during stress. Competition with monoclonal antibodies and peptides indicated that the copper binding domain of SPARC is required for SPARC-mediated response to stress. Inhibiting the binding and/or activity of ILK, integrin beta1, or SPARC resulted in increased apoptosis of stressed LEC. We conclude that SPARC protects cells from stress-induced apoptosis in vitro via an interaction with integrin beta1 heterodimers that enhances ILK activation and pro-survival activity.     

BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase

Activation of the mammalian mitogen-activated protein kinase known as BMK1 is required for growth factor-induced cell proliferation. To understand the mechanism by which BMK1 mediates this cellular response, this kinase was used as bait in a yeast two-hybrid-based library screening. Here, we report the identification of serum and glucocorticoid-inducible kinase (SGK) as a cellular protein that physically interacts with BMK1. During growth factor-induced cell stimulation, BMK1 activates SGK by phosphorylation at serine 78. This BMK1-mediated phosphorylation event is necessary for the activation of SGK and, more importantly, for cell proliferation induced by growth factors.     

Angiotensin II mediates cell survival through upregulation and activation of the serum and glucocorticoid inducible kinase 1

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is known to regulate a wide variety of cellular processes, including renal sodium retention and cell survival. Angiotensin II (Ang II) is one of the many signaling molecules capable of regulating SGK1 expression, and is also known to impact cell survival. Here, we examined the role of SGK1 in Ang II-mediated cell survival. We hypothesized that Ang II protects cells from apoptosis by upregulating and activating SGK1. To test this, we examined the effects of Ang II stimulation on SGK1 expression and downstream signaling. We also examined the effects of Ang II treatment and siRNA-mediated SGK1 knockdown on apoptosis after serum starvation. We found that after 2 h of Ang II treatment, SGK1 mRNA expression was increased approximately 2-fold. This induction was sensitive to reductions in intracellular calcium levels after pretreatment with BAPTA-AM, but insensitive to the L-type calcium channel blocker verapamil. SGK1 induction was also sensitive to the tyrosine kinase inhibitor genistein. Ang II treatment also caused a rapid increase in the level of phosphorylation of SGK1 at Ser422 and Thr256, and Ser422 phosphorylation was rapamycin-sensitive. We found that Ang II treatment was protective against serum starvation-induced apoptosis, and this protective effect was significantly blunted when SGK1 was silenced via siRNA. Lastly, Ang II induced FOXO3A phosphorylation in an SGK1-dependent manner, thereby reducing the pro-apoptotic actions of FOXO3A. Overall, these results indicate that Ang II upregulates and activates SGK1, leading to increased cell survival via multiple, non-redundant mechanisms.     

CCK2 (CCK(B)/gastrin) receptor mediates rapid protein kinase D (PKD) activation through a protein kinase C-dependent pathway

Addition of gastrin or cholecystokinin octapeptide (CCK-8) to cultures of Rat-1 cells stably transfected with the CCK2 (CCK(B)/gastrin) receptor induced protein kinase D (PKD) activation that was detectable within 1 min and reached a maximum ( approximately 10-fold) after 2.5 min of hormonal stimulation. Half-maximal PKD activation for both CCK-8 and gastrin was achieved at 10 nM. Treatment with various concentrations of the selective PKC inhibitors Ro 31-8220 or GF-I potently blocked PKD activation induced by subsequent addition of CCK-8 in a concentration-dependent fashion. Our results indicate that PKC-dependent PKD activation is a novel early event in the action of gastrin and CCK-8 via CCK2 receptors.     

Loss of insulin-like growth factor I receptor-dependent expression of p107 and cyclin A in cells that lack the extracellular matrix protein secreted protein acidic and rich in cysteine.

The extracellular matrix-associated glycoprotein secreted protein acidic and rich in cysteine (SPARC) has been implicated in the control of cell proliferation during tissue remodeling, wound healing, and malignant development. Here, we describe a novel mechanism through which SPARC influences cell cycle progression in embryonic fibroblasts derived from Sparc-nullizygous (-/-) mice. SPARC-deficient cells were indistinguishable from wild-type cells in their ability to initiate DNA synthesis after treatment with either fetal bovine serum or platelet-derived growth factor. In contrast, Sparc -/- cells responded poorly to activation of the insulin-like growth factor receptor (IGFI-R) by insulin. This defect was traced to reduced expression of the IGFI-R in Sparc -/- cells. Consistent with impaired cell cycle progression through S-phase, insulin-stimulated Sparc -/- cells also revealed reduced expression of two key regulators of S phase progression (cyclin A and thymidine kinase), whereas expression of the G1 phase progression regulators cmyc or cyclin D1 was unaffected. An examination of the status of retinoblastoma family pocket proteins in Sparc -/- cells revealed a selective and dramatic reduction in levels of the retinoblastoma-related protein p107. Exogenous platelet-derived growth factor restored expression of the IGFI-R and IGFI-R dependent DNA synthesis as well as induction of cyclin A, thymidine kinase, and p107 in insulin-stimulated Sparc -/- cells. These results suggest that SPARC-dependent matrix to cell interactions contribute to the regulation of p107 and cyclin A through IGFI-R dependent pathway(s).     

The immediate early 2 protein of human cytomegalovirus (HCMV) mediates the apoptotic control in HCMV retinitis through up-regulation of the cellular FLICE-inhibitory protein expression

Human CMV (HCMV) is a widespread human pathogen that causes blindness by inducing retinitis in AIDS patients. Previously, we showed that viral immediate early 2 (IE2) protein may allow HCMV to evade the immune control by killing the Fas receptor-positive T lymphocytes attracted to the infected retina with increased secretion of Fas ligand (FasL). In this study, we further demonstrate that the secreted FasL also kills uninfected Fas-rich bystander retinal cells and that IE2 simultaneously protects the infected cells from undergoing apoptotic death, in part, by activating the expression of cellular FLIP (c-FLIP), an antiapoptotic molecule that blocks the direct downstream executer caspase 8 of the FasL/Fas pathway. c-FLIP induction requires the N-terminal 98 residues of IE2 and the c-FLIP promoter region spanning nucleotides -978 to -696. In vivo association of IE2 to this region, IE2-specific c-FLIP activation, and decrease of FasL-up-regulated activities of caspases 8 and 3 were all demonstrated in HCMV-infected human retinal cells. Moreover, c-FLIP up-regulation by IE2 appeared to involve PI3K and might also render cells resistant to TRAIL-mediated death. Finally, enhanced c-FLIP signals were immunohistochemically detected in IE-positive cells in the HCMV-infected lesions of the human retina. Taken together, these data demonstrate specific activation of c-FLIP by HCMV IE2 and indicate a novel role for c-FLIP in the pathogenesis of HCMV retinitis.     

Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro.

To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse-chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCl extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCl extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time-dependent loss in sulphate from the BSP was evident. Using a 24 h pulse-labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12-day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation.     

Carbohydrate-binding protein 35 (Mac-2), a laminin-binding lectin, forms functional dimers using cysteine 186

Carbohydrate-binding protein 35 (CBP35), also known as the macrophage surface antigen Mac-2, is a lactosamine-specific lectin whose extracellular properties include the ability to agglutinate cells and to bind avidly to the basement membrane glycoprotein laminin. Although these and other properties would be facilitated by dimerization of this lectin, previous studies have argued against multimeric forms of this protein. We report here that macrophage CBP35, purified by laminin affinity chromatography, exists as several distinct species (Mr 35,000, 67,000, and 80,000) when analyzed under non-reducing conditions. This unexpected finding prompted us to study the biochemistry of multimerization using recombinant CBP35 (rCBP35). rCBP35 expressed in Escherichia coli forms disulfide-linked homodimers (Mr 67,000). The dimeric form of CBP35 binds to laminin with higher affinity than does monomer and by a lactosamine-dependent mechanism. Site-directed mutagenesis indicated that cysteine 186, the single cysteine residue in CBP35, is required for dimerization. These results raise the possibility that homo- and heterodimeric forms of CBP35 contribute to its postulated functions in cell-matrix interactions and growth regulation.