The role of SPARC in the TRAMP model of prostate carcinogenesis and progression.

SPARC (Secreted Protein Acidic and Rich in Cysteine), is a matricellular glycoprotein that is produced by tumor and/or neighboring stroma. In human prostate cancer, SPARC immunoreactivity is highest in metastatic lesions but distinct contributions of tumoral and stromal SPARC to tumorigenesis and progression are unclear. To determine the role of SPARC in primary prostate tumorigenesis, we crossed SPARC-null (SP(-/-)) with TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. TRAMP(+)/SP(-/-) mice exhibited accelerated cancer development and progression. Compared to their TRAMP(+)/SP(-/-) counterparts, TRAMP(+)/SP(+/+) tumors had fewer proliferating cells, and decreased cyclins A and D1 with increased p21(Cip) and p27(Kip). Similar effects on proliferation and cell-cycle regulators were observed in human prostate cancer cell lines, transiently transfected with pSPARC. TRAMP(+)/SP(-/-) tumors exhibited decreased stromal collagen, enhanced matrix metalloproteinase activity and increased vascular endothelial growth factor, proinflammatory cytokines. To determine the contribution of stromal SPARC, we evaluated subcutaneous tumor growth of TRAMP cell lines in syngeneic SP(+/+) and SP(-/-) mice. Enhanced growth, decreased stromal collagen and increased proteolysis were noted in SP(-/-) mice. Our findings demonstrate that both tumor and stromal SPARC are limiting for primary prostate tumorigenesis and progression, through effects on the cell cycle and the creation of a less favorable tumor microenvironment.

IL6 suppression provides renal protection independent of blood pressure in a murine model of salt-sensitive hypertension.

Impaired cytochrome P450 epoxygenase enzyme (Cyp2c) regulation contributes to renal damage in angiotensin salt-sensitive hypertension (ANG/HS). We hypothesized that interleukin-6 null mice (IL6-/-) would improve Cyp2c regulation and reduce renal damage in hypertensive mice fed a high salt diet. Systolic blood pressure increased to a greater extent in ANG/HS hypertension as compared to angiotensin (ANG) hypertension but blood pressure did not differ between WT and IL6-/- hypertensive groups. Albuminuria, a marker for renal injury, increased significantly in ANG/HS hypertension in WT mice (5,113 +/- 1,050 mug/day) and was attenuated in the ANG/HS IL6-/- group (1,306 +/- 385 mug/day). Renal Cyp2c protein expression significantly decreased with ANG/HS hypertension in WT mice as compared to high salt alone. However, the ability to upregulate Cyp2c expression in response to a high salt diet was restored in the ANG/HS IL6 deficient hypertensive mice. Renal expression of soluble epoxide hydrolase, which inactivates protective epoxygenase metabolites, was significantly reduced in ANG/HS IL6-/- hypertensive mice compared to the ANG/HS WT group. These data suggest that IL6, while having no effect on blood pressure, impairs regulation of epoxygenase producing Cyp2c, which could contribute to the development of renal injury in angiotensin salt-sensitive hypertension.

Peroxynitrite mediates VEGF's angiogenic signal and function via a nitration-independent mechanism in endothelial cells.

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.

SPARC-null mice exhibit increased adiposity without significant differences in overall body weight.

Secreted protein acidic and rich in cysteine/osteonectin/BM-40 (SPARC) is a matrix-associated protein that elicits changes in cell shape, inhibits cell-cycle progression, and influences the synthesis of extracellular matrix (ECM). The absence of SPARC in mice gives rise to aberrations in the structure and composition of the ECM that result in generation of cataracts, development of severe osteopenia, and accelerated closure of dermal wounds. In this report we show that SPARC-null mice have greater deposits of s.c. fat and larger epididymal fat pads in comparison with wild-type mice. Similar to earlier studies of SPARC-null dermis, we observed a reduction in collagen I in SPARC-null fat pads in comparison with wild-type. Although elevated levels of serum leptin were observed in SPARC-null mice, their overall body weights were not significantly different from those of wild-type counterparts. The diameters of adipocytes from SPARC-null versus wild-type epididymal fat pads were 252 +/- 61 and 161 +/- 33 microm (means +/- SD), respectively, and there was an increase in adipocyte number within SPARC-null fat pads in comparison with wild-type pads. Thus the absence of SPARC appears to result in an increase in the size of individual adipocytes as well as an increase in the number of adipocytes per fat pad. In fat pads isolated from wild-type mice, SPARC mRNA was associated with both the stromal/vascular and adipocyte fractions. We propose that SPARC limits the accumulation of adipose tissue in mice in part through its demonstrated effects on the regulation of cell shape and production of ECM.

Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation.

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 (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 (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 of 125I-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.

Cyclic AMP, PKA, and the physiological regulation of adiposity.

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.

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.

Regulation of SPARC expression during early Xenopus development: evolutionary divergence and conservation of DNA regulatory elements between amphibians and mammals.

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.

Compensatory regulation of RIalpha 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 RIbeta and RIIbeta regulatory subunit genes in mice, and find that both result in an increase in the level of RIalpha protein in tissues that normally express the beta isoforms. Examination of RIalpha mRNA levels and the rates of RIalpha protein synthesis in wild type and RIIbeta mutant mice reveals that the mechanism of this biochemical compensation by RIalpha 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 Calpha in NIH 3T3 cells results in increased RIalpha protein without increases in the rate of RIalpha synthesis or the level of RIalpha mRNA. Pulse-chase experiments reveal a 4-5-fold increase in the half-life of RIalpha protein as it becomes incorporated into the holoenzyme. Compensation by RIalpha stabilization may represent an important biological mechanism that safeguards cells from unregulated catalytic subunit activity.

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.

Genetically lean mice result from targeted disruption of the RII beta subunit of protein kinase A.

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.

CACC box and enhancer response of the human embryonic epsilon globin promoter.

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.

The CACC box upstream of human embryonic epsilon globin gene binds Sp1 and is a functional promoter element in vitro and in vivo.

DNA sequences 179 base pairs upstream and 23 base pairs downstream of the cap site of human embryonic epsilon globin gene exhibit promoter activity in transfected cell cultures. The nuclear factor binding in vitro of this epsilon promoter region was studied by DNase I foot-printing, methylation interference, and gel mobility shift assay. Four major nuclear factor-binding sites are detected in complexes formed with unfractionated nuclear extracts: NF-E1 at -163, epsilon F1 at -143, CACC box at -111, and CBF at -81, respectively. Of these, NF-E1 is an erythroid-specific factor. epsilon F1 is probably a ubiquitous factor because it is present in both erythroid K562 cells and nonerythroid HeLa cells. The epsilon F1-binding site exhibits sequence similarity to that of the cAMP response element-binding protein family of transcription factors. Finally, the CCAAT box-binding protein (CBF)-binding site centers around the CCAAT promoter box. Comparative binding studies with unfractionated nuclear extracts and affinity purified HeLa Sp1 demonstrated that the epsilon-globin CACC box at -111 is a binding site of Sp1. The spatial arrangements of the NF-E1-binding site, the CACC box, and CCAAT box, with respect to their mutual separations by approximately integral numbers of helical turns, is well conserved in all mammalian embryonic epsilon globin promoters. Transient expression assay, using human growth hormone gene as the receptor, demonstrated that similar to the other two human beta-like globin genes (beta and gamma), the CACC box of epsilon globin gene is an essential promoter element for epsilon globin gene expression in vivo in K562 cells. This CACC promoter box also functions in vitro in nuclear extracts prepared from K562 cells. These data, together with Sp1-binding studies of the human beta and gamma globin CACC boxes, suggest that the general transcription factor Sp1, through its differential interactions with different forms of CACC promoter boxes, is an essential component of the machinery that controls the developmental program of mammalian globin gene regulation.