Secreted Protein, Acidic, Cysteine-rich Research Articles

Differential impact of mechanical unloading on structural and nonstructural components of the extracellular matrix in advanced human heart failure

Adverse remodeling of the extracellular matrix (ECM) is a significant characteristic of heart failure. Reverse remodeling of the fibrillar ECM secondary to mechanical unloading of the left ventricle (LV) by left ventricular assist device (LVAD) has been subject of intense investigation; however, little is known about the impacts on nonfibrillar ECM and matricellular proteins that also contribute to disease progression. Explanted failing hearts were procured from patients with nonischemic dilated cardiomyopathy (DCM) with or without LVAD support, and compared to nonfailing control hearts. LVfree wall specimens were formalin-fixed, flash-frozen or optimum cutting temperature-mount frozen. Histologic and biochemical assessment of fibrillar ECM showed that LVAD support was associated with lower levels of insoluble collagen, collagen type I mRNA, and collagen I/III ratio compared with no-LVAD hearts. Adisintegrin and Metalloproteinase with Thrombospondin Motifs-2 (ADAM-TS2), aprocollagen endopeptidase, was reduced in no-LVAD but not in LVAD hearts. The rise in ECM proteolytic activities was significantly lower in LVAD hearts. Matrix metalloproteinases (MMP1, MMP2, MMP8, MMP13, and MT1-MMP/MMP14) were comparable between DCM hearts. Tissue inhibitor of metalloproteinase (TIMP)3 and TIMP4 messenger RNA and protein showed the greatest reduction in no-LVAD hearts. Basement membrane proteins exhibited less severe disarray of laminin and fibronectin-1 in LVAD-supported hearts. The rise in matricellular protein, osteopontin, was suppressed in LVAD hearts, whereas secreted protein, acidic, cysteine-rich (SPARC) levels was unaffected by LVAD. Mechanical unloading of the failing DCM hearts can restore the fibrillar ECM and the basement membrane, contributing toward improved clinical outcomes. However, persistent elevation of matricellular proteins such as SPARC could contribute to the relapse of failing hearts on removal of LVAD support.

Extracellular calcium-binding peptide-modified ceramics stimulate regeneration of calvarial bone defects.

Secreted protein, acidic, cysteine-rich (SPARC)-related modular calcium binding 1 (SMOC1) has been implicated in the regulation of osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs). In this study, we found that a peptide (16 amino acids in length), which is located in the extracellular calcium (EC) binding domain of SMOC1, stimulated osteogenic differentiation of human BMSCs in vitro and calvarial bone regeneration in vivo. Treatment of BMSCs with SMOC1-EC peptide significantly stimulated their mineralization in a dose-dependent manner without changing their rate of proliferation. The expression of osteogenic differentiation marker genes, including type 1 collagen and osteocalcin, also increased in a dose-dependent manner. To examine the effect of the SMOC1-EC peptide on bone formation in vivo, the peptide was covalently immobilized onto hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) particles. X-ray photoelectron spectroscopy analysis showed that the peptide was successfully immobilized onto the surface of HA/β-TCP. Implantation of the SMOC1-EC peptide-immobilized HA/β-TCP particles into mouse calvarial defects and subsequent analyses using microcomputed tomography and histology showed significant bone regeneration compared with that of calvarial defects implanted with unmodified HA/β-TCP particles. Collectively, our data suggest that a peptide derived from the EC domain of SMOC1 induces osteogenic differentiation of human BMSCs in vitro and efficiently enhances bone regeneration in vivo.

Plakophilin 1-deficient cells upregulate SPOCK1: implications for prostate cancer progression.

Plakophilin (PKP) 1 is frequently downregulated in prostate cancer and therefore may play a tumor-suppressive role. In the present study, we stably knocked down PKP1 in the non-neoplastic, prostatic BPH-1 cell line. In the PKP1-deficient cells, the expression of keratin 14 was lost, and the apoptosis rate was significantly reduced indicating that the cells acquired new biological capabilities. Moreover, we analyzed the gene expression profile of the PKP1-deficient BPH-1 cells. Among the genes that were significantly altered upon PKP1 knockdown, we noticed several extracellular matrix (ECM)-related genes and identified sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1/testican-1) as a gene of interest. SPOCK1 is a component of the ECM and belongs to a matricellular protein family named secreted protein, acidic, cysteine-rich (SPARC). The role of SPOCK1 in prostate cancer has not been clearly elucidated. We analyzed SPOCK1 mRNA expression levels in different cancer databases and characterized its expression in 136 prostatic adenocarcinomas by immunohistochemistry and western blot. SPOCK1 revealed a cytoplasmic localization in the glandular epithelium of the prostate and showed a significant upregulation of mRNA and protein in prostate tumor samples. Our findings support the hypothesis that PKP1 may have a tumor-suppressive function and suggest an important role of SPOCK1 in prostate tumor progression. Collectively, altered expression of PKP1 and SPOCK1 appears to be a frequent and critical event in prostate cancer.

Recessive Osteogenesis Imperfecta Caused by Missense Mutations in SPARC

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.

Secreted protein acidic cysteine-rich (SPARC)

Secreted protein acidic cysteine-rich (SPARC)

Multicenter cohort molecular evidence of the presence of endometriosis-inducing factor (mir-130a) as a potent regulator of gene expression in endometriosis

Objective To study the role of endometriosis-inducing factor on the differentiation of cultured stem cells into endometrial cells. Methods This was a multicenter prospective cohort case-control study where 323 women were examined, but only 134 infertile women were recruited and compiled with the inclusion criteria. The study group comprised 64 women in whom laparoscopy showed endometriotic lesions, and the control group included 70 women free of endometriotic implants. The women’s sera were cocultured with mesenchymal stem cells, which were followed up weekly by quantitative real-time PCR to examine the expression of SPARC (secreted protein, acidic, cysteine-rich) and MYC (myelocytomatosis) genes using the method. Results By coculturing the serum of women with endometriosis and the control group with stem cells, none of the cultures of the sera of women within the control group showed any changes in the normal expression of either SPARC or MYC gene levels. Furthermore, the stem cells were normally multiplying in the same cell line during the entire duration of the study. Nevertheless, stem cells that were cocultured with sera from women with endometriosis showed upregulation of the SPARC gene mRNA with mean respiratory quotient of 3.534±1.129, whereas the MYC gene mRNA was downregulated with a mean respiratory quotient of 0.488±0.104. Conclusion The sera of women with endometriosis were able to induce transformation of mesenchymal stem cells into endometrial-like cells on a molecular basis. This evidence supports the endometriosis-inducing factor theory of endometriosis and may have tremendous effect on the therapeutic implications of this debilitating condition.

Clinical utility of measuring expression levels of KAP1, TIMP1 and STC2 in peripheral blood of patients with gastric cancer

BackgroundWe examined preoperative kinesin II-associated protein (KAP1), TIMP metallopeptidase inhibitor 1 (TIMP1) and stanniocalcin 2 (STC2) expression levels in patients with gastric cancers to assess their clinical application for diagnosing and monitoring diseases.MethodsReal-time reverse transcription-polymerase chain reaction was used to detect the expression levels of KAP1, TIMP1, STC2, talin 2 (TLN2), sushi-repeat-containing protein, X-linked 2 (SRPX2) and secreted protein, acidic, cysteine-rich (SPARC) in the patients’ peripheral blood karyocytes. The data were analyzed with receiver operating characteristics (ROC) curves.ResultsA total of 112 patients with gastric cancer, 42 patients with recurrence and 107 healthy volunteers were recruited. There were significant correlations between KAP1, TIMP1 and STC2 levels, and TNM tumor stages and distant metastases. The area under the ROC curves (AUC) of KAP1 was 0.803 ± 0.040 (P = 0.0001), the AUC of TIMP1 was 0.767 ± 0.043 (P = 0.0001) and the AUC of STC2 was 0.769 ± 0.045 (P = 0.0001), thus differentiating preoperative gastric cancer patients from healthy volunteers by ROC curve analysis. The AUC of STC2 was 0.739 ± 0.070 (P = 0.004) and the AUC of KAP1 was 0.418 ± 0.088 (P = 0.319), thus differentiating recurrence of gastric cancer from healthy volunteers by ROC curve analysis. High TIMP1 and STC2 expression levels were suspected to be poor prognostic factors of disease recurrence in patients with gastric cancer.ConclusionsKAP1, TIMP1 and STC2 expression levels may be potential biomarkers for the screening, diagnosis, prognosis and surveillance of gastric cancer.

The expression, regulation and function of secreted protein, acidic, cysteine-rich in the follicle–luteal transition

The role of the tissue remodelling protein, secreted protein, acidic, cysteine-rich (SPARC), in key processes (e.g. cell reorganisation and angiogenesis) that occur during the follicle-luteal transition is unknown. Hence, we investigated the regulation of SPARC in luteinsing follicular cells and potential roles of SPARC peptide 2.3 in a physiologically relevant luteal angiogenesis culture system. SPARC protein was detected mainly in the theca layer of bovine pre-ovulatory follicles, but its expression was considerably greater in the corpus haemorrhagicum. Similarly, SPARC protein (western blotting) was up-regulated in luteinising granulosa but not in theca cells during a 6-day culture period. Potential regulatory candidates were investigated in luteinising granulosa cells: LH did not affect SPARC (P>0.05); transforming growth factor (TGF) B1 (P<0.001) dose dependently induced the precocious expression of SPARC and increased final levels: this effect was blocked (P<0.001) by SB505124 (TGFB receptor 1 inhibitor). Additionally, fibronectin, which is deposited during luteal development, increased SPARC (P<0.01). In luteal cells, fibroblast growth factor 2 decreased SPARC (P<0.001) during the first 5 days of culture, while vascular endothelial growth factor A increased its expression (P<0.001). Functionally, KGHK peptide, a SPARC proteolytic fragment, stimulated the formation of endothelial cell networks in a luteal cell culture system (P<0.05) and increased progesterone production (P<0.05). Collectively, these findings indicate that SPARC is intricately regulated by pro-angiogenic and other growth factors together with components of the extracellular matrix during the follicle-luteal transition. Thus, it is possible that SPARC plays an important modulatory role in regulating angiogenesis and progesterone production during luteal development.

Two families of Xenopus tropicalis skeletal genes display well‐conserved expression patterns with mammals in spite of their highly divergent regulatory regions

The origin of bone and cartilage, and their subsequent diversification in specific vertebrate lineages, is intimately linked to the precise transcriptional control of genes involved in matrix mineralization. It is not yet clear, however, to which extent the osteoblasts, osteocytes, and chondrocytes of each of the major vertebrate groups express similar sets of genes. In this study we have focused on the evolution of two independent families of genes that code for extracellular matrix components of the skeleton and that include secreted protein, acidic, cysteine-rich (SPARC), bone sialoprotein (BSP) and dentin matrix protein 1 (DMP1) paralogues, and the osteocalcin (OC) and matrix gla protein (MGP) paralogues. Analyzing developing Xenopus tropicalis skeletal elements, we show that the expression patterns of these genes are well conserved with mammals. The fact that only a few osteoblasts express DMP1, while only some osteocytes express SPARC and BSP, reveals a significant degree of molecular heterogeneity for these two populations of X. tropicalis cells, similarly to what has been described in mouse. Although the cis-regulatory modules (CRM) of the mammalian OC, DMP1, and BSP orthologs have been functionally characterized, we found no evidence of sequence similarity between these regions and the X. tropicalis genome. Furthermore, these regulatory elements evolve rapidly, as they are only poorly conserved between human and rodents. Therefore, the SPARC/DMP1/BSP and the OC/MGP families provide a good paradigm to study how transcriptional output can be maintained in skeletal cells despite extensive sequence divergence of CRM.

Progesterone and interferon tau-regulated genes in the ovine uterine endometrium: identification of periostin as a potential mediator of conceptus elongation

During early pregnancy in ruminants, progesterone (P(4)) and interferon tau (IFNT) act on the endometrium to regulate genes hypothesized to be important for conceptus development and implantation. The present study was conducted to verify several candidate genes (actin alpha-2, smooth muscle, aorta (ACTA2), collagen, type III, alpha-1 (COL3A1), periostin (POSTN), secreted protein acidic cysteine-rich (SPARC), S100 calcium-binding protein A2 (S100A2), STAT5A and transgelin (TAGLN)) regulated by pregnancy, P(4), and/or IFNT in the endometrium determined using a custom ovine cDNA array. S100A2 mRNA was detected primarily in endometrial epithelia and conceptuses. S100A2 mRNA increased in endometrial epithelia from days 10 to 16 in cyclic ewes and from days 10 to 14 in pregnant ewes and declined thereafter. The abundance of S100A2 mRNA was less in endometrial luminal epithelium of IFNT-infused ewes receiving P(4). Expression of COL3A1, SPARC, ACTA2, and TAGLN was independent of pregnancy, P(4), or IFNT. POSTN mRNA was detected primarily in compact stroma of intercaruncular and caruncular endometria, but not in the conceptus. Endometrial POSTN mRNA increased between days 12 and 14 in pregnant but not cyclic ewes, and POSTN mRNA was more abundant in uterine stroma of ewes treated with P(4). POSTN protein was detected in uterine flushings of pregnant ewes and found to mediate attachment and stimulate migration of ovine trophectoderm cells in vitro. These results support the ideas that POSTN and S100A2 are regulated by P(4) and IFNT respectively, and that POSTN is involved in conceptus elongation during early pregnancy.

Association of an Extracellular Matrix Gene Cluster with Breast Cancer Prognosis and Endocrine Therapy Response

We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P<0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of approximately 40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P=0.001), and (b) no clinical benefit (odds ratio, 0.81; P=0.035) and shorter progression-free survival (hazard ratio, 1.19; P=0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

Transcription factor SOX‐5 enhances nasopharyngeal carcinoma progression by down‐regulating SPARC gene expression

Nasopharyngeal carcinoma (NPC) is prevalent in south-eastern Asia, and its tumourigenesis is rather complex. The purpose of this research was to identify the pivotal genes that may be altered during the early stage of NPC progression. Eleven genes were selected by comparative microarray analysis of NPC versus normal nasomucosal cells. The expression of SPARC (secreted protein, acidic, cysteine-rich) was statistically significantly down-regulated in NPC cells. In exploring the mechanism underlying the decreased transcription of SPARC in NPC cells, we found that the transcription factor SRY (sex-determining region Y)-box 5 (SOX-5) is up-regulated in NPC cells. RNA interference of SOX-5 by short hairpin RNA (shRNA) in NPC cells caused a dramatic increase in SPARC and chromosome immunoprecipitation assay showed that SOX-5 can bind directly to the SPARC promoter, suggesting that SOX-5 acts as a key transcriptional repressor of SPARC. We further demonstrated that shRNA knockdown of SOX-5 suppressed the proliferation of NPC cells, as well as their migratory ability, which was also observed when SPARC was over-expressed in NPC cells. Alternatively, blocking SPARC with an antagonistic antibody reversed the effects of SOX-5 knockdown. In 66 NPC patients, over-expression of SOX-5 in tumour cells correlated clinically with poor survival. Our study suggests that SOX-5 transcriptionally down-regulates SPARC expression and plays an important role in the regulation of NPC progression. SOX-5 is a potential tumour marker for poor NPC prognosis.

Molecular cytogenetic delineation of the critical deleted region in the 5q- syndrome.

The 5q- syndrome is a distinct type of myelodysplastic syndrome (MDS) characterised by refractory anaemia, morphological abnormalities of megakaryocytes, and del(5q) as the sole cytogenetic abnormality. In contrast to patients with therapy-related MDS with 5q deletions, 5q- syndrome patients have a favourable prognosis and a low rate of transformation to acute leukaemia. We have previously delineated a common deleted region of 5.6 Mb between the gene for fibroblast growth factor acidic (FGF1) and the subunit of interleukin 12 (IL12B) in two patients with 5q- syndrome and small deletions, del(5)(q31q33). The present study used fluorescence in situ hybridisation (FISH) analysis of these and a third 5q- syndrome patient with a small deletion, del(5)(q33q34), to refine further the critical deleted region. This resulted in the narrowing of the common deleted region within 5q31.3-5q33 to approximately 3 Mb, flanked by the adrenergic receptor beta 2 (ADRB2) and IL/2B genes. The common region of loss in these three 5q- syndrome patients includes the macrophage colony-stimulating factor-1 receptor (CSF1R), secreted protein, acidic, cysteine-rich (SPARC), and glutamate receptor (GR1A1) genes. This 5q- syndrome critical region is telomeric to and distinct from the other critical regions on 5q associated with MDS and acute myeloid leukaemia.