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The modulation of angiogenic signaling by reactive oxygen species (ROS) is an emerging area of interest in cellular and vascular biology research. We provide evidence here that peroxynitrite, the powerful oxidizing and nitrating free radical, is critically involved in transduction of the VEGF signal. We tested the hypothesis that VEGF induces peroxynitrite formation, which causes tyrosine phosphorylation and mediates endothelial cell migration and tube formation, by studies of vascular endothelial cells in vitro and in a model of hypoxia-induced neovascularization in vivo. The specific peroxynitrite decomposition catalyst FeTPPs blocked VEGF-induced phosphorylation of VEGFR2 and c-Src and inhibited endothelial cell migration and tube formation. Furthermore, exogenous peroxynitrite mimicked VEGF activity in causing phosphorylation of VEGFR2 and stimulating endothelial cell growth and tube formation in vitro and new blood vessel growth in vivo. The selective nitration inhibitor epicatechin enhanced VEGF's angiogenic function in activating VEGFR2, c-Src, and promoting endothelial cell growth, migration, and tube formation in vitro and retinal neovascularization in vivo. Decomposing peroxynitrite with FeTPPs or blocking oxidation using the thiol donor NAC blocked VEGF's angiogenic functions in vitro and in vivo. In conclusion, peroxynitrite is critically involved in transducing VEGF's angiogenic signal via nitration-independent and oxidation-mediated tyrosine phosphorylation. [ABSTRACT FROM AUTHOR]

Background: Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein which is implicated in regulation of angiogenesis., Purpose: To characterize the changes in SPARC expression and effect of its deletion in a mouse model Oxygen Induced Retinopathy (OIR)., Materials and Methods: Wild type (wt) and SPARC-deficient mice were subjected to high oxygen (75%) for 5 days (p7-p12) before room air for additional 5 days (p12-p17). Retinas from both groups were flat mounted and retinal vessels were labeled with Isolectin-B4. Areas of Retinal Neovascularization (RNV) and vaso-obliteration were measured by Image-J and normalized to total retinal areas. SPARC expression was analyzed in both groups at p14 and p17 in retinal homogenates and sections by Western Blotting (WB) and immunofluorescence respectively. Human Retinal Endothelial Cells (HRECs) were exposed to hypoxia (1% O2) for 6 hours then SPARC was measured in cell lysate and condition medium by WB and ELISA. Moreover, HRECs were treated with VEGF or SPARC to study their mutual regulatory effect., Results: SPARC-deficient mice demonstrated significant increase in the vaso-obliteration ( p =0.03) and modest increase in RNV compared to the wt control. Retinal levels of SPARC was significantly decreased during OIR at p14 ( p =0.01) and partially restored to normal level by p17. Moreover, hypoxia significantly reduced SPARC expression and secretion in HRECs ( p =0.001). We noticed a mutual positive regulatory feedback between SPARC and VEGF., Conclusion: SPARC deletion enhances ischemic retinopathy, thus modulation of SPARC expression could be a novel therapeutic approach to prevent pathological RNV.

Epithelial ovarian cancer is the fourth leading cause of death from gynecologic malignancies in the United States. Most cases are diagnosed at late stages, with the solid tumor masses growing as peritoneal implants, or floating within the ascitic fluid (peritoneal ovarian carcinomatosis). Despite aggressive surgical "debulking," recurrence of recalcitrant disease is frequent with poor patient survival. Efforts to improve survival rates are hindered by lack of biomarkers that can detect and effectively treat ovarian cancer in its early stages. Urokinase plasminogen activator receptor (uPAR) is a multifunctional receptor involved in a myriad of tumor cell processes. However, the role of host uPAR in ovarian cancer is still elusive. To define the potential proinflammatory role of uPAR in ovarian cancer, first, using a syngeneic murine model in uPAR(-/-) mice, we found that ablation of uPAR restrained tumor take and peritoneal implants and prolonged the survival of uPAR(-/-) mice compared with their uPAR(+/+) counterparts. Ascitic fluid accumulation was significantly decreased in uPAR(-/-) mice with decreased macrophage infiltration. Second, in vitro mechanistic studies revealed that host uPAR is involved in the multiple steps of peritoneal metastatic cascade. Third, we evaluated the prognostic utility of tumor and stromal uPAR in human ovarian cancer tissue microarray. In summary, our studies indicated that uPAR plays a significant role in ovarian cancer cell-stromal crosstalk and contributes to increased vascular permeability and inflammatory ovarian cancer microenvironment. This provides a rationale for targeting the uPAR with either specific neutralizing antibodies or targeting its downstream inflammatory effectors in patients with ovarian cancer.

Medial thickening and vascular hypertrophy of resistance arteries can lead to cardiovascular complications associated with diabetes. While previous studies have established a role of type 1 diabetes in vascular remodeling, we recently extended these observations to type 2 diabetes and reported increased collagen deposition due to alterations in matrix metalloproteinase expression and activity in mesenteric resistance arteries. These studies also showed that remodeling response was mediated by endothelin-1 (ET-1) via activation of ET(A) receptors, whereas blockade of ET(B) receptors exacerbated the remodeling. However, the effectiveness of glycemic control strategies in preventing these vascular changes, including activation of the ET system still remained unclear. Also, very little is known about whether and to what extent reorganization of the extracellular matrix (ECM) affects vascular compliance and vasomotor tone. Accordingly, this study assessed structural remodeling of mesenteric microvessels, vascular compliance, and myogenic tone, as well as the role of matrix metalloproteinases (MMP) in mediating these processes. Spontaneously diabetic, non-obese Goto-Kakizaki (GK) rats, a model for type 2 diabetes, and normoglycemic Wistar rats were used for the studies. A subset of GK rats were administered metformin to achieve euglycemia. Glycemic control normalized the increased media-to-lumen ratios (M/L) and myogenic tone seen in diabetes, as well as normalizing plasma ET-1 levels and mesenteric ET(A) receptor expression. There was increased collagen synthesis in diabetes paralleled by decreased collagenase MMP-13 activity, while glycemic control attenuated the process. These findings and our previous study taken together suggest that hyperglycemia-mediated activation of ET-1 and ET(A) receptors alter vascular structure and mechanics in type 2 diabetes.

Epigenetic silencing of tumor suppressor genes is a new focus of investigation in the generation and proliferation of carcinomas. Secreted protein acidic and rich in cysteine (SPARC) is reportedly detrimental to the growth of ovarian cancer cells and has been shown to be epigenetically silenced in several cancers. We hypothesized that SPARC is downregulated in ovarian cancer through aberrant promoter hypermethylation. To that end, we analyzed SPARC expression in ovarian cancer cell lines and investigated the methylation status of the Sparc promoter using methylation-specific polymerase chain reaction. Our results show that SPARC mRNA expression is decreased in three (33%) and absent in four (44%) of the nine ovarian cancer cell lines studied, which correlated with hypermethylation of the Sparc promoter. Treatment with the demethylating agent 5-aza-2'-deoxycytidine rescued SPARC mRNA and protein expression. Addition of exogenous SPARC, as well as ectopic expression by an adenoviral vector, resulted in decreased proliferation of ovarian cancer cell lines. Investigation of primary tumors revealed that the Sparc promoter is methylated in 68% of primary ovarian tumors and that the levels of SPARC protein decrease as the disease progresses from low to high grade. Lastly, de novo methylation of Sparc promoter was shown to be mediated by DNA methyltransferase 3a. These results implicate Sparc promoter methylation as an important factor in the genesis and survival of ovarian carcinomas and provide new insights into the potential use of SPARC as a novel biomarker and/or treatment modality for this disease.

We have recently identified that the role of secreted protein acidic and rich in cysteine (SPARC) in amelioration of peritoneal ovarian carcinomatosis is mediated, at least in part, through mesothelial cell/lysophosphatidic acid-induced inflammatory response in ovarian cancer cells. The aim of this study was to elucidate the molecular mechanisms of the interactions between tumor cells and the cellular components of the ovarian cancer peritoneal microenvironment, specifically, mesothelial cells and macrophages. We found that SPARC not only significantly reduced macrophage chemoattractant protein-1 production and its macrophage chemotactic effect, but also attenuated the response of ovarian cancer cells to the mitogenic and proinvasive effects of macrophage chemo-attractant protein-1 and decreased macrophage-induced cancer cell invasiveness. Overexpression of SPARC in ovarian cancer cells significantly attenuated macrophage- and mesothelial cell-induced production and activity of interleukin-6, prostanoids (prostaglandins E2 and 8-isoprostanes) as well as matrix metalloproteinases and urokinase plasminogen activator. Moreover, the effects of SPARC overexpression in ovarian cancer cells were mediated, in part, through inhibition of nuclear factor-kappaB promoter activation. These results indicate, for the first time, that the effects of tumor SPARC as a negative regulator of ovarian cancer are mediated through decreased recruitment of macrophages and downregulation of the associated inflammation.

The matricellular protein secreted protein acidic and rich in cysteine (SPARC) modulates cell adhesion, proliferation, matrix deposition, and tissue remodeling. SPARC has been shown to regulate the expression of collagen type I and transforming growth factor-beta1 in mesangial cells and to be highly expressed during tubulointerstitial fibrosis in rat angiotensin (ANG) II infusion models. We hypothesized that SPARC is a downstream effector of ANG II and that loss of host SPARC function provides a protective effect on renal damage and fibrosis associated with ANG II hypertension. Our results revealed that cultured primary mesangial cells displayed a concentration-dependent increase in SPARC expression in response to ANG II. After a 14-day chronic infusion of ANG II, hypertensive SPARC-null mice exhibited significantly attenuated levels of urinary and renal indicators of oxidative stress and inflammation and decreased renal perivascular and tubulointerstitial fibrosis relative to wild-type hypertensive controls. Moreover, the observed renal protective changes in SPARC-null mice were found to be independent of blood pressure. These results identify SPARC as an effector of ANG II signaling and suggest an important role for SPARC in mediating ANG II-induced oxidative stress, inflammation, and fibrosis.

Malignant ascites is a major source of morbidity and mortality in ovarian cancer patients. It functions as a permissive reactive tumor-host microenvironment and provides sustenance for the floating tumor cells through a plethora of survival/metastasis-associated molecules. Using a syngeneic, immunocompetent model of peritoneal ovarian carcinomatosis in SP(-/-) mice, we investigated the molecular mechanisms implicated in the interplay between host secreted protein acidic and rich in cysteine (SPARC) and ascitic fluid prosurvival/prometastasis factors that result in the significantly augmented levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP). Ascitic fluid-enhanced ID8 invasiveness was mediated through VEGF via a positive feedback loop with MMP-2 and MMP-9 and through activation of alpha(v) and beta(1) integrins. Host SPARC down-regulated the VEGF-MMP axis at the transcriptional and posttranscriptional levels. In vitro, SPARC attenuated the basal as well as VEGF-induced integrin activation in tumor cells. SPARC inhibited the VEGF- and integrin-mediated ID8 proliferation in vitro and significantly suppressed their tumorigenicity in vivo. Relative to SP(+/+), SP(-/-) ascitic fluid contained significantly higher levels of bioactive lipids and exerted stronger chemotactic, proinvasive, and mitogenic effects on ID8 cells in vitro. SP(-/-) ascites also contained high levels of interleukin-6, macrophage chemoattractant protein-1, and 8-isoprostane (prostaglandin F(2)alpha) that were positively correlated with extensive infiltration of SP(-/-) ovarian tumors and ascites with macrophages. In summary, our findings strongly suggest that host SPARC normalizes the microenvironment of ovarian cancer malignant ascites through down-regulation of the VEGF-integrin-MMP axis, decreases the levels and activity of bioactive lipids, and ameliorates downstream inflammation.

The matricellular glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been accorded major roles in regulation of cell adhesion and proliferation, as well as tumorigenesis and metastasis. We have recently reported that in addition to its potent antiproliferative and proapoptotic functions, SPARC also abrogates ovarian carcinoma cell adhesion, a key step in peritoneal implantation. However, the underlying molecular mechanism through which SPARC ameliorates peritoneal ovarian carcinomatosis seems to be multifaceted and has yet to be delineated. Herein, we show that SPARC significantly inhibited integrin-mediated ovarian cancer cell adhesion to extracellular matrix proteins, as well as to peritoneal mesothelial cells. This counteradhesive effect of SPARC was shown to be mediated in part through significant attenuation of cell surface expression and clustering of alpha(v)-integrin subunit, alpha(v)beta(3)- and alpha(v)beta(5)-heterodimers, and beta(1)-subunit, albeit to a lesser extent, in ovarian cancer cells. Moreover, SPARC significantly suppressed both anchorage-dependent and -independent activation of AKT and mitogen-acti-vated protein kinase survival signaling pathways in ovarian cancer cells in response to serum and epidermal growth factor stimulation. In summary, we have identified a novel role of SPARC as a negative regulator of both integrin-mediated adhesion and growth factor-stimulated survival signaling pathways in ovarian cancer.

The interplay between peritoneal mesothelial cells and ovarian cancer cells is critical for the initiation and peritoneal dissemination of, and ascites formation in, ovarian cancer. The production of lysophosphatidic acid (LPA) by both peritoneal mesothelial cells and ovarian cancer cells has been shown to promote metastatic phenotype in ovarian cancer. Herein, we report that exogenous addition or ectopic overexpression of the matricellular protein SPARC (secreted protein acidic and rich in cysteine) significantly attenuated LPA-induced proliferation, chemotaxis, and invasion in both highly metastatic SKOV3 and less metastatic OVCAR3 ovarian cancer cell lines. SPARC appears to modulate these functions, at least in part, through the regulation of LPA receptor levels and the attenuation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B/AKT signaling. Moreover, our results show that SPARC not only significantly inhibited both basal and LPA-induced interleukin (IL) 6 production in both cell lines but also attenuated IL-6-induced mitogenic, chemotactic, and proinvasive effects, in part, through significant suppression of ERK1/2 and, to a lesser extent, of signal transducers and activators of transcription 3 signaling pathways. Our results strongly suggest that SPARC exerts a dual inhibitory effect on LPA-induced mesothelial-ovarian cancer cell crosstalk through the regulation of both LPA-induced IL-6 production and function. Taken together, our findings underscore the use of SPARC as a potential therapeutic candidate in peritoneal ovarian carcinomatosis.

Angiogenesis, a critical process in both health and disease, is mediated by a number of signaling pathways. Although proangiogenic stimuli, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and the phorbol ester phorbol-12 myristate-13 acetate (PMA) are known to promote blood vessel formation, their downstream targets are ill defined. We sought to investigate the signaling pathways required for vessel assembly by utilizing a three-dimensional collagen matrix in which human umbilical vein endothelial cells (HUVECs) form tubular structures. Our data show that PMA is sufficient for the induction of angiogenesis, and that protein kinase C (PKC) is necessary for this process. Evaluation of PKC isoforms alpha and sigma revealed that these proteins are uniquely regulated. Characterization of an additional PMA target, protein kinase D (PKD) demonstrated that this enzyme becomes phosphorylated in HUVECs, and may therefore be involved in proangiogenic signaling. Further examination of downstream effectors of PKC showed that extracellular signal-regulated kinase (ERK) is critical for angiogenesis, and is accordingly phosphorylated in response to PMA. Surprisingly however, phosphorylation of ERK is independent of PKC activity. In addition, we show that the PKC target sphingosine kinase (SPK) is required for vessel formation. These findings illustrate the complexities of blood vessel formation, and suggest that activators utilize multiple independent pathways to invoke a complete angiogenic response.

The matricellular glycoprotein SPARC (secreted protein acidic and rich in cysteine) possesses multifaceted roles in modulation of cell-matrix interactions, as well as tumor growth and metastasis. To investigate the influence of host-derived SPARC on peritoneal dissemination of ovarian cancer, we established a murine model that faithfully recapitulates advanced human disease by intraperitoneal injection of syngeneic ID8 ovarian cancer cells into SPARC-null and wild-type mice. Compared to wild-type mice, SPARC-null mice showed significantly shorter survival and developed extensive nodular peritoneal dissemination with hemorrhagic ascitic fluid accumulation. Ascitic fluid collected from SPARC-null mice showed significantly augmented levels and activity of vascular endothelial growth factor and gelatinases. Immunohistochemical analysis of tumor nodules from SPARC-null mice revealed higher proliferation and lower apoptosis indices with minimal staining for major extracellular matrix constituents. In vitro, SPARC significantly suppressed adhesion to and invasion of various peritoneal extracellular matrix constituents by murine and human ovarian cancer cell lines. Our findings suggest that SPARC ameliorates ovarian peritoneal carcinomatosis through abrogation of the initial steps of disease pathogenesis, namely tumor cell adhesion and invasion, inhibition of tumor cell proliferation, and induction of apoptosis. Thus, SPARC represents an important therapeutic candidate in ovarian cancer.

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-beta1) induces SPARC and collagen type I, we have recently shown that SPARC regulates the expression of TGF-beta1 and collagen type I in renal mesangial cells via a TGF-beta1-dependent pathway, and have proposed a reciprocal, autocrine regulatory feedback loop between SPARC and TGF-beta1. Herein, we sought to determine how SPARC regulates TGF-beta1-dependent signal transduction. Our data indicate that SPARC modulates the TGF-beta1-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-beta1. Furthermore, we found that SPARC increases the levels and the activity of the transcription factor c-jun. These effects of SPARC on the TGF-beta1 signaling pathway appear to be mediated through an interaction with the TGF-beta1-receptor complex, but only in the presence of TGF-beta1 bound to its cognate type II receptor. That SPARC is directly involved in the regulation of the TGF-beta1 signaling cascade is consistent with the paradigm that matricellular proteins modulate interactions among cells, growth factors, and their respective receptors., (Copyright 2004 Wiley-Liss, Inc.)

The role of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) in modulation of vascular cell proliferation is believed to be mediated, in part, by its ability to regulate the activity of certain growth factors through direct binding. In this study, we demonstrate that SPARC does not bind to basic fibroblast growth factor (bFGF/FGF-2) or interfere with complex formation between FGF-2 and its high-affinity FGF receptor-1 (FGFR1), yet both native SPARC and a peptide derived from the C-terminal high-affinity Ca(2+)-binding region of protein significantly inhibit ligand-induced autophosphorylation of FGFR1 (>80%), activation of mitogen-activated protein kinases (MAPKs) (>75%), and DNA synthesis in human microvascular endothelial cells (HMVEC) stimulated by FGF-2 (>80%). We also report that in the presence of FGF-2, a factor which otherwise stimulates myoblast proliferation and the repression of terminal differentiation, both native SPARC and the Ca(2+)-binding SPARC peptide significantly promote (>60%) the differentiation of the MM14 murine myoblast cell line that expresses FGFR1 almost exclusively. Moreover, using heparan sulfate proteoglycan (HSPG)-deficient myeloid cells and porcine aortic endothelial cells (PAECs) expressing chimeric FGFR1, we show that antagonism of FGFR1-mediated DNA synthesis and MAPK activation by SPARC does not require the presence of cell-surface, low-affinity FGF-2 receptors, but can be mediated by an intracellular mechanism that is independent of an interaction with the extracellular ligand-binding domain of FGFR1. We also report that the inhibitory effect of SPARC on DNA synthesis and MAPK activation in endothelial cells is mediated in part (>50%) by activation of protein kinase A (PKA), a known regulator of Raf-MAPK pathway. SPARC thus modulates the mitogenic effect of FGF-2 downstream from FGFR1 by selective regulation of the MAPK signaling cascade., (Copyright 2003 Wiley-Liss, Inc.)

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 beta1 (TGF-beta1) 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., (Copyright 2003 Wiley-Liss, Inc.)

Fibroblast growth factor-1 (FGF-1) is a potent angiogenic factor; its structure lacks a signal peptide for secretion. We previously reported that the overexpression of a secreted version of FGF-1 (sp-FGF-1) in microvascular endothelial cells (ECs) enhances cell migration [Partridge et al. J Cell Biochem 2000; 78(3): 487]. In the current study, we have examined the angiogenic effects of sp-FGF-1 in chicken chorioallantoic membranes (CAMs). Two methods of examining the effects of sp-FGF-1 in CAMs were used: cell-mediated transfection via bovine ECs and direct gene transfection. In the cell-mediated gene transfection, those eggs that were implanted with a gelatin sponge seeded with ECs stably transfected to over-express sp-FGF-1 protein showed a significant increase in angiogenesis inside the sponge when compared to eggs treated with vector control-transfected ECs. In the direct gene transfer, eggs received sp-FGF-1 showed a significant increase in vascularization when compared to eggs received vector alone plasmids. These CAM models are useful both for studying molecular mechanisms of angiogenesis and for developing better gene therapy strategies.

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., (Copyright 2002 Wiley-Liss, Inc.)

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.

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.

SPARC (Secreted Protein, Acidic, Rich in Cysteine/osteonectin/BM-40) is a highly conserved metal-binding extracellular matrix (ECM) glycoprotein which is first expressed by Xenopus embryos during late gastrulation/early neurulation (stage 12/13), by presumptive notochord and somitic cells. When animal cap explants of stage 9 embryos were cultured in vitro, SPARC expression was not detected until sibling embryos reached late neurula stage (stage 19). Addition of activin, a potent dorsal mesoderm inducer, to animal caps resulted in SPARC being expressed by the time sibling embryos reached stage 16. While basic fibroblast growth factor (bFGF), a ventral mesoderm inducer, had modest effects on SPARC mRNA expression, the combination of both activin and bFGF was synergistic. The appearance, however, of SPARC transcripts 11 h after the addition of activin and bFGF, indicates that unknown intermediates were likely to be involved in activating SPARC expression. In order to identify the potential intermediate regulatory factors which may activate and control SPARC expression, we examined the genomic organization of the 5' end of the Xenopus SPARC gene. No significant homology to the equivalent region that is highly conserved in the mouse, bovine and human SPARC genes was observed. Thus, while mammalian SPARC promoters lack TATA or CAAT boxes, the Xenopus gene contains a consensus TATA box. Moreover, promoter-proximal GGA-box repeats necessary for high level expression of mammalian SPARC are absent in Xenopus. When reporter constructs containing the 5' flanking region of the Xenopus gene were microinjected into two-cell embryos, 868 bp of 5' flanking DNA was sufficient to mimic the temporal and tissue-specific pattern of SPARC expression observed in whole embryos. While a bovine SPARC promoter reporter construct containing 740 bp of the 5' flanking DNA was expressed at a significant level in Xenopus embryos, significant differences in the cell-type expression of the reporter genes were obtained between the bovine and Xenopus constructs. The data indicate that zygotic activation of SPARC mRNA is mediated by regulatory factors acting downstream of major mesoderm induction events. The high DNA sequence conservation at the 5' end of mammalian SPARC genes is not conserved in Xenopus. These differences led to differences in their ability to direct tissue-specific gene expression in early Xenopus embryos.

The major regulator of lipolysis in white adipocytes and brown adipocytes is cAMP and the actions of cAMP are mediated by protein kinase A (PKA). Multiple subunits of PKA, including RII beta, R1 alpha, C alpha, and C beta 1, are expressed in fat cells but the major holoenzyme assembled under normal conditions contains RII beta and C alpha. Targeted disruption of the RII beta gene in mice revealed that both white and brown adipocytes are capable of compensating by increasing the level of RI alpha. Nevertheless, the mice display a lean phenotype, have an elevated metabolic rate due to activation and induction of uncoupling protein in brown fat, and are resistant to diet-induced obesity and insulin resistance. Although the metabolic disturbances in white and brown fat tissue may explain most of the phenotypic changes, the loss of neuronal expression of RII beta may also contribute to the alterations in energy balance. Specific neuronal defects have been characterized that prevent the normal changes in gene expression seen with drugs that act through the dopaminergic pathway. The RII beta mutant mouse provides an interesting model of obesity resistance and demonstrates that chronic changes in the PKA signaling system can lead to stable alterations in energy storage and utilization.

Cyclic AMP is an important second messenger in the coordinated regulation of cellular metabolism. Its effects are mediated by cAMP-dependent protein kinase (PKA), which is assembled from two regulatory (R) and two catalytic (C) subunits. In mice there are four R genes (encoding RI alpha, RI beta, RII alpha, and RII beta) and two C gene (encoding C alpha and C beta), expressed in tissue-specific patterns. The RII beta isoform is abundant in brown and white adipose tissue and brain, with limited expression elsewhere. To elucidate its functions, we generated RII beta knockout mice. Here we report that mutants appear healthy but have markedly diminished white adipose tissue despite normal food intake. They are protected against developing diet-induced obesity and fatty livers. Mutant brown adipose tissue exhibits a compensatory increase in RI alpha, which almost entirely replaces lost RII beta, generating an isoform switch. The holoenzyme from mutant adipose tissue binds cAMP more avidly and is more easily activated than wild-type enzyme. This causes induction of uncoupling protein and elevations of metabolic rate and body temperature, contributing to the lean phenotype. Our results demonstrate a role for the RII beta holoenzyme in regulating energy balance and adiposity.

The functional interaction between the human epsilon globin promoter and an erythroid-specific transcription enhancer, 5' HS-2, has been analyzed by transient expression assay. While stepwise deletion of DNA sequences between -852 and -122 had only small effects, removal of the CACC box at position -111 greatly decreased epsilon-globin promoter activity, as well as its response to the enhancer function of 5' HS-2 in erythroid cells. Our data demonstrated that the three ubiquitous promoter elements, the CACC, CCAAT, and TATA boxes, of the epsilon-globin-encoding gene together form a minimal promoter that would interact efficiently with 5' HS-2, and that at least the CACC box is an essential functional component of this enhancer-promoter interaction.