sFRP-1 Protein Research Articles

Sfrp5 expression and secretion in adipocytes are up‐regulated during differentiation and are negatively correlated with insulin resistance

We have examined the patterns of Sfrp5 (secreted frizzled-related protein 5) mRNA expression and protein secretion during adipocyte differentiation, and investigated the potential role of Sfrp5 in IR (insulin resistance) in adipocytes. 3T3-L1 pre-adipocytes were induced for differentiation, and RT-PCR (reverse transcription-PCR) and ELISA assays were used to determine Sfrp5 mRNA expression and protein secretion. The results showed that with the differentiation and maturity of pre-adipocytes, transcription and protein secretion of Sfrp5 gradually increased, peaking on the 9th day of differentiation. Sfrp5 mRNA expression in mature adipocytes was decreased by 20, 22 and 32 upon treatment with dexamethasone, insulin and TNF (tumour necrosis factor) respectively, whereas Sfrp5 protein secretion was decreased by 15, 17 and 30%, correspondingly. In contrast, Sfrp5 mRNA expression in mature adipose was increased by 34 and 19% upon treatment with rosiglitazone and metformin respectively, whereas Sfrp5 protein secretion was increased by 10 and 6%, correspondingly. In conclusion, Sfrp5 mRNA expression and protein secretion depend on the differentiation of adipocytes. The dysregulation of Sfrp5 expression and secretion is directly correlated with IR. Up-regulation of Sfrp5 expression and secretion in adipocytes may be one crucial mechanism by which rosiglitazone and metformin improve insulin sensitivity.

Effects of 5-Aza-2-deoxycytidine on DNA methylation of anti-oncogenes in non-small cell lung cancer cells

To observe the expression of SFRP1 gene methylation in non-small cell lung cancer (NSCLC), and study the effect of 5-Aza-2-deoxycytidine (5-Aza-CdR) on DNA methylation and expression of SFRP1, p16 and MGMT genes in the human lung cancer cell line SPC-A-1 cells. SP immunohistochemistry and methylation-specific PCR were used to detect the SFRP1 methylation in 60 NSCLC cases, and 21 cases of benign lung diseases were used as control group. SPC-A-1 cells were cultured and treated with 5-Aza-CdR. The promoter methylation status of SFRP1, p16 and MGMT genes were detected by methylation-specific polymerase (MSP) chain reaction, and mRNAs were detected by real-time PCR. The positive rate of SFRP1 gene methylation in NSCLC was significantly higher than that in normal lung tissue (58.3% vs. 14.3%; χ(2) = 12.118, P = 0.001). SFRP1 gene methylation was closely correlated with lymph node metastasis and degree of differentiation in NSCLC (P < 0.05). SFRP1 protein expression was correlated with clinical stage, degree of differentiation and lymph node metastasis in NSCLC (P < 0.05). The positive expression of SFRP1 protein in 30 cases of NSCLC tissue containing SFRP1 gene methylation was significantly higher than that in non-methylated NSCLC (68.6% vs. 24.0%; χ(2) = 9.613, P = 0.002). SFRP1 gene methylation was closely correlated with SFRP1 gene protein expression in NSCLC (P < 0.05). Negative expression of SFRP1 protein was correlated with the differentiation, clinical stage, and lymph node metastasis in NSCLC (all P < 0.05). Without 5-Aza-CdR treatment, the expressions of methylation of SFRP1, p16 and MGMT genes and their mRNA were low. After 5-Aza-CdR treatment at different concentrations, their expressions were significantly elevated (all P < 0.05). SFRP1 gene methylation is closely associated with carcinogenesis and development of NSCLC. 5-Aza-CdR may reverse the methylation of SFRP1, p16 and MGMT genes, and facilitate the re-expression of the anti-oncogenes.

Epigenetic inactivation of the canonical wnt antagonist secreted frizzled-related protein 1 in hepatocellular carcinoma cells

Secreted Frizzled-related protein 1 (sFRP1), as one of most important Wnt antagonists, is frequently silenced by promoter hypermethylation in many types of tumor, including hepatocellular carcinoma (HCC). In this study, we aimed to investigate whether restoration of sFRP1 affected HCC metastatic behavior. sFRP1 mRNA expression and promoter methylation in HCC tissues and cell lines were examined using RT-PCR and methylation-specific PCR (MS-PCR), respectively. sFRP1 protein expression was assessed by Western Blot. We generated stable HCC cell line restoration of sFRP1 in HepG2 cells, which naturally do not express detectable sFRP1 mRNA. The effects of exogenous sFRP1 on HepG2 cell invasion were investigated using trans-well assay. Also the effects of sFRP1 re-expression on the β-catenin/T-cell factor-dependent transcription activity was measured by luciferase assay.sFRP1 promoter methylation was frequently observed in HCC tissues (60%) and cell lines (75%). All samples with sFRP1 methylation showed down-regulation of sFRP1 expression in HCC cell lines. Demethylation treatment with 5-aza-20-deoxycytidine in HCC cells restored sFRP1 expression. Restoration of sFRP1 substantially impaired the invasive potentials of HepG2 cells. Moreover, exogenous sFRP1 caused significant decrease of β-catenin/T-cell factor-dependent transcription activity.These findings demonstrate that sFRP1 silencing due to promoter hypermethylation is a major event during tumorigenesis. sFRP1 is also a negative modulator of canonical Wnt signaling, which could contribute to metastasis in HCC progression, thus providing a possible therapeutic strategy against HCC.

Wnt signalling mediates the cross-talk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations

The mechanisms underlying the inverse relationship between osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) are not known in detail. We have previously established two cell lines from mouse bone marrow that are committed to either osteogenic (osteoblasts and chondrocytes) (mMSC Bone) or adipogenic (mMSC Adipo) lineage. To identify the molecular mechanism determining the lineage commitment, we compared the basal gene expression profile of mMSC Bone versus mMSC Adipo using Affymetrix GeneChip® MG430A 2.0 Array. Gene annotation analysis based on biological function revealed an over-representation of skeletal development genes in mMSC Bone while genes related to lipid metabolism and immune response were highly expressed in mMSC Adipo. In addition, there was a significant up-regulation of canonical Wnt signalling genes in mMSC Bone compared to mMSC Adipo ( p < 0.006). Dual-luciferase assay and expression analysis of genes related to Wnt signalling demonstrated significant activation of Wnt signalling pathway in mMSC Bone compared to mMSC Adipo. Reduced Wnt activity in mMSC Adipo was associated with increased expression of the Wnt inhibitor, secreted frizzled-related protein 1 (sFRP-1) at both mRNA and protein levels in mMSC Adipo. Interestingly, conditioned medium (CM) collected from mMSC Adipo (mMSC-CM Adipo) inhibited osteoblast differentiation of mMSC, while depletion of sFRP-1 protein from mMSC-CM Adipo abolished its inhibitory effect on osteoblast differentiation. Furthermore, treatment of mMSC with recombinant sFRP-1 resulted in a dose-dependent inhibition of osteoblast and stimulation of adipocyte differentiation. In conclusion, cross-talk exists between different populations of MSC in the bone marrow, and Wnt signalling functions as a molecular switch that determines the balance between osteoblastogenesis and adipogenesis.

Involvement of genetic instability in the downregulation of sFRP1 in Chinese patients with hepatocellular carcinoma

Secreted frizzled-related protein 1 (sFRP1) is a new tumor suppressor based on recent researches, but the correlation of the genetic instability of sFRP1 gene with the clinicopathologic features of the hepatocellular carcinoma (HCC) has not been studied in Chinese people. In this study, 42 pairs of paraffin-embedded HCC and adjacent non-carcinoma tissues were examined for the loss of heterozygosity (LOH) and microsatellite instability (MSI) of two microsatellite markers D8S532 and D8S1722 located in the vicinity of the sFRP1 gene. Envision immunohistochemistry was used to assess the expression of sFRP1. We found that the reduced expression of the sFRP1 protein was frequently observed in Chinese patients with HCC, which may at least partially result from the genetic instability, especially LOH. The LOH-associated sFRP1 downregulation may play an important role in the development of HCC.

Proliferation and Pluripotency of Human Embryonic Stem Cells Maintained on Type I Collagen

Human embryonic stem cells (hESC) require a balance of growth factors and signaling molecules to proliferate and retain pluripotency. Conditioned medium (CM) from a human embryonic germ-cell-derived cell culture, SDEC, was observed to support the growth of hESC on type I collagen (COL I) and on Matrigel (MAT) biomatricies. After 1 month, the population doubling of hESC grown in SDEC CM on COL I was equivalent to that of hESC grown in mouse embryonic fibroblast (MEF) CM on MAT. hESC grown in SDEC CM on COL I expressed OCT4, NANOG, SSEA-4, alkaline phosphatase (AP), and TRA-1-60; retained a normal karyotype; and were capable of forming teratomas. DNA microarray analysis was used to compare the transcriptional profiles of SDEC and the less supportive WI38 and Detroit 551 human cell lines. The mRNA level of secreted frizzled-related protein (sFRP-1), a known antagonist of the WNT/β-catenin signaling pathway, was significantly reduced in SDEC as compared with the other 2 cell lines, whereas the mRNA levels of prostaglandin-endoperoxide synthase 2 (PTGS2 or COX-2) and prostaglandin I₂ synthase (PGIS), two prostaglandin biosynthesis genes, were significantly increased in SDEC. The level of sFRP-1 protein was significantly reduced, and levels of 2 prostaglandins that are downstream products of PTGS2 and PGIS, prostaglandin E₂ and 6-keto-prostaglandin F(1α), were significantly elevated in SDEC CM compared with WI38, Detroit 551, and MEF CM. Further, addition of purified sFRP-1 to SDEC CM reduced the proliferation of hESC grown on COL I as well as MAT in a dose-dependent manner.

Abstract 4904: Epigenetic discovery screen identifies SFRP2 as a novel biomarker of high grade prostate cancer

Abstract Prostate cancer (CaP) is the most common noncutaneous malignancy and 3rd leading cause of cancer related deaths in men in the Western world. We urgently require CaP-specific biomarkers to distinguish between indolent and aggressive disease and avoid over-treatment. Hyperactivity of Wnt/ β-catenin signaling is an important mechanism of cancer progression, linked to androgen independent growth, development of bone metastases and self-renewal of CaP stem cells. Our aims were to (1) profile Wnt-related gene expression in benign and malignant prostates, (2) investigate hypermethylation of Wnt antagonists and (3) select a novel Wnt-related target for evaluation in low versus high grade CaP. TaqMan Low Density Arrays revealed 14 genes with significantly altered expression between cancer and benign tissue; 12/14 were upregulated in tumors. Only 6/19 human WNT genes were expressed in CaP (WNT3A, WNT4, WNT5B, WNT7B, WNT10A, WNT11); only WNT4 was significantly upregulated in tumors. Overall, the expression profile was highly similar between tumors of high (≥7) and low (≤6) Gleason scores. However, significant differences were observed for DACT1, BTRC, AXIN1 and DLL4. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 38 targets (≥2-fold) relative to untreated cells; 40% of these genes inhibit Wnt signalling. Pyrosequencing and QMSP evaluated hypermethylation of Wnt antagonist SFRPs (secreted frizzled related proteins), revealing frequent methylation of SFRP2 in 65% of tumors (48/74; Normalized Index of Methylation (NIM) = 116). Significantly lower frequencies and quantitative levels of methylation were found in benign tissue 9% (7/69; NIM = 0.47, P &amp;lt; 0.0001) and in preinvasive High Grade Prostatic Intraepithelial Neoplasia 30% (6/20, NIM = 7.45, P &amp;lt; 0.0001). Immunohistochemical analysis of tissue microarrays revealed moderate/strong SFRP2 protein expression in the cytoplasm of 84% (172/205) of benign epithelia; while negative/weak SFRP2 staining was noted in most tumor epithelia, particularly Gleason grades 3 &amp; 4, where only 12% (21/171) and 23% (29/124) of cases stained positive, respectively. In contrast, almost half (24/57) of Gleason grade 5 tumors showed moderate/strong SFRP2 expression (P &amp;lt; 0.0001). Microscopic evaluation of these tumors revealed different morphological patterns, corresponding with differential SFRP2 expression. Moderate/strong grade 5 tumors (designated Type A) appeared morphologically solid while negative/weak grade 5 tumors (designated Type B) appeared more diffuse. Furthermore, 80% (4/5 patients) of Type A patients experienced biochemical recurrence, compared with 0% (0/6 patients) of Type B patients (P = 0.015). This is the first report of epigenetic control of Wnt signaling antagonist SFRP2 in CaP. Our data suggest that SFRP2 may be a useful prognostic biomarker in sub-stratifying patients with high grade tumors at increased risk of recurrence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4904.

Promoter hypermethylation of the SFRP2 gene is a high-frequent alteration and tumor-specific epigenetic marker in human breast cancer

BackgroundWe have previously reported that expression of the Wnt antagonist genes SFRP1 and SFRP5 is frequently silenced by promoter hypermethylation in breast cancer. SFRP2 is a further Wnt inhibitor whose expression was recently found being downregulated in various malignancies. Here we investigated whether SFRP2 is also implicated in human breast cancer, and if so whether SFRP2 promoter methylation might serve as a potential tumor biomarker.MethodsWe analyzed SFRP2 mRNA expression and SFRP2 promoter methylation in 10 breast cell lines, 199 primary breast carcinomas, 20 matched normal breast tissues and 17 cancer-unrelated normal breast tissues using RT-PCR, realtime PCR, methylation-specific PCR and Pyrosequencing, respectively. SFRP2 protein expression was assessed by immunohistochemistry on a tissue microarray. Proliferation assays after transfection with an SFRP2 expression vector were performed with mammary MCF10A cells. Statistical evaluations were accomplished with SPSS 14.0 software.ResultsOf the cancerous breast cell lines, 7/8 (88%) lacked SFRP2 mRNA expression due to SFRP2 promoter methylation (P < 0.001). SFRP2 expression was substantially restored in most breast cell lines after treatment with 5-aza-2'-deoxycytidine and trichostatin A. In primary breast carcinomas SFRP2 protein expression was strongly reduced in 93 of 125 specimens (74%). SFRP2 promoter methylation was detected in 165/199 primary carcinomas (83%) whereas all cancer-related and unrelated normal breast tissues were not affected by SFRP2 methylation. SFRP2 methylation was not associated with clinicopathological factors or clinical patient outcome. However, loss of SFRP2 protein expression showed a weak association with unfavorable patient overall survival (P = 0.071). Forced expression of SFRP2 in mammary MCF10A cells substantially inhibited proliferation rates (P = 0.045).ConclusionThe SFRP2 gene is a high-frequent target of epigenetic inactivation in human breast cancer. Its methylation leads to abrogation of SFRP2 expression, conferring a growth advantage to epithelial mammary cells. This altogether supports a tumor suppressive function of SFRP2. Although clinical patient outcome was not associated with SFRP2 methylation, the high frequency of this epimutation and its putative specificity to neoplastic cells may qualify SFRP2 promoter methylation as a potential candidate screening marker helping to improve early breast cancer detection.

Abstract 866: Secreted Frizzled Related Protein 2 Modulates Post Myocardial Infarct Remodeling by Inhibiting Collagen Maturation through the Inhibition of Bone Morphogenetic Protein 1 Activity

Myocardial infarction and post-infarction remodeling with heart failure are the major cause of mortality and morbidity in the United States. We recently reported that intracardiac implantation of genetically engineered mesenchymal stem cell (MSC) overexpressing the Akt gene dramatically reduced the infarct size and restored cardiac functions in rodent hearts after coronary artery ligation. Further, we identified Secreted Frizzled Related Protein 2 (sfrp2) as a key factor released by Akt-MSC mediating myocardial survival and repair. However, the underlying mechanism remains elusive. Bone Morphogenetic Protein1 (BMP1)/Tolloid (TLD)-like metalloproteinases belong to a subgroup of astacin family and play key roles in the regulation of extracelluar matrix (ECM) formation and cardiac fibrosis. These proteases have procollagen C-proteinase (PCP) activities which are responsible for the cleavage of C-propeptides from procollagen precursors to produce mature collagen fibrils. In this report, we showed that three days following myocardial infarction in rats, both BMP1 protein expression and activity were upregulated in the infarcted left ventricle. Interestingly, we found recombinant sfrp2 could inhibit BMP1 activity in MI tissue samples as measured by an in vitro PCP activity assay. Furthermore, using purified recombinant proteins, we demonstrated that sfrp2, but not sfrp1 or sfrp3, inhibited BMP-1 activity in vitro. Moreover, purified sfrp2 could physically interact with BMP1 protein as shown by the co-immunoprecipitation assay. To provide further evidence that sfrp2 can interfere with collagen processing, we demonstrated that exogenously added sfrp2 interfered with procollagen processing in primary cultures of cardiac fibroblast culture medium. Similar results were obtained when these cells were transiently transfected with sfrp2 expressing plasmids. In summary, our data suggest that one of the molecular mechanisms underlying the cardioprotective and repair effects of sfrp2 protein on myocardial infarction is through the inhibition of BMP-1 activity. Therefore, sfrp2 has the potential clinical application as a novel anti-fibrotic reagent for the modulation of cardiac remodeling after acute myocardial infarction.

Regulation of Secreted Frizzled-related Protein-1 by Heparin

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin.

Androgen-Induced Wnt Signaling in Preosteoblasts Promotes the Growth of MDA-PCa-2b Human Prostate Cancer Cells

The high morbidity and mortality associated with prostate cancer (PCa) result from its tendency to metastasize to bone where it produces predominantly osteoblastic lesions. The Wnt signaling pathway plays an important role in embryogenesis, tumorigenesis, osteoblast development, and bone formation. Androgen signaling via the androgen receptor (AR) is critical in both PCa and bone cell growth. We examined the effects of androgens on cell growth and Wnt signaling in the AR-positive MDA-PCa-2b cell line and MC3T3 preosteoblasts, grown alone and in coculture. We show that the potent androgen dihydrotestosterone increases AR expression and transcriptional activity only in the preosteoblasts. Although dihydrotestosterone induced an 80% increase in PCa cell growth when the cells were grown alone, dihydrotestosterone had a more significant effect on MDA-PCa-2b cell proliferation (3.2-fold increase) when the PCa cells were cocultured with preosteoblasts. Dihydrotestosterone addition to preosteoblasts promoted Wnt-dependent transcriptional reporter activity associated with GSK3beta(S-9) phosphorylation and accumulation of nuclear beta-catenin as well as elevated Runx2 expression. In addition, the increased proliferation of PCa cells in coculture with MC3T3 cells in response to dihydrotestosterone was abrogated by the addition of either exogenous DKK-1 or sFRP-1 protein, two naturally occurring Wnt antagonists. Finally, we show that the paracrine growth-promoting effect of androgens is limited to MDA-PCa-2b cells. These data imply that Wnt signaling is involved in the androgen-regulated crosstalk between preosteoblasts and PCa cells and suggest that androgens may stimulate growth of some prostate tumor cells indirectly, via up-regulation of Wnt signaling in bone cells.

Cell‐type Specific Regulation of Recombinant Secreted Frizzled Related‐1 Production by Heparin and Fibroblast Growth Factor

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 protein synthesis in HEK293 cells but not in CHO-DUKX. The study also shows that the effect requires O-sulfation in heparin and is affected by glycosylation mutations. Further investigation reveals that the heparin effect on sFRP-1 production requires fibroblast growth factor-2 (FGF-2) and FGF receptor-1. Our data suggests that the secretion of sFRP-1 can be regulated by heparin and FGF signaling pathway.

Suppressing Wnt Signaling by the Hedgehog Pathway through sFRP-1

The hedgehog (Hh) signaling pathway is essential for embryonic development and carcinogenesis. Activation of Hh signaling has been identified in several types of gastrointestinal cancers, including esophageal, gastric, pancreatic, and liver cancers. Several recent studies suggest that Hh signaling activation can inhibit Wnt signaling. However, the molecular basis underlying this inhibition remains unclear. As transcription factors in the Hh signaling pathway, Gli molecules transform cells in culture, and their expression are associated with cancer development. Here we report that expression of a secreted frizzled-related protein-sFRP-1 in mouse embryonic fibroblasts is dependent on Gli1 and Gli2. In human gastric cancer cells, inhibition of Hh signaling reduces the level of sFRP-1 transcript, whereas ectopic expression of Gli1 increases the level of sFRP-1 transcript. Results from chromatin immunoprecipitation indicate that Gli1 is involved in transcriptional regulation of sFRP-1. In 293 cells with Gli1 expression, Wnt-1-mediated beta-catenin accumulation in the cytosol and DKK1 expression are all abrogated, which can be reversed by inhibiting sFRP-1 expression. Furthermore, while SIIA cells do not respond to Wnt-1-conditioned medium, inhibition of Hh signaling by smoothened (SMO) antagonist KAAD-cyclopamine (keto-N-aminoethylaminocaproyldihydrocinnamoylcyclopamine) leads to Wnt1-mediated beta-catenin accumulation in the cytosol. These data indicate that sFRP-1, a target gene of the hedgehog pathway, is involved in cross-talk between the hedgehog pathway and the Wnt pathway.

Comparative genomics on SFRP2 orthologs

SFRP1, SFRP2, SFRP3, SFRP4, SFRP5, DKK1, DKK2, DKK3, DKK4, and WIF1 are soluble modulators of WNT signaling. SFRP2 gene at human chromosome 4q31.3 is claimed as a tumor suppressor gene inactivated by the epigenetic CpG hypermethylation in colorectal cancer. SFRP2 methylation is a sensitive single DNA-based marker for the fecal screening of colorectal cancer. On the other hand, SFRP2 is claimed to induce cellular resistance to apoptosis in mammary tumors. Here, we identified and characterized the rat Sfrp2 gene by using bioinformatics. Rat Sfrp2 gene, consisting of three exons, was located within AC129161.3 genome sequence. Rat Sfrp2 gene was found to encode a 295-aa Sfrp2 protein, consisting of signal peptide, Frizzled domain with ten conserved Cys residues, and Netrin (NTR) domain with six conserved Cys residues. Rat Sfrp2 showed 98.6% and 97.6% total-amino-acid identity with mouse Sfrp2 and human SFRP2, respectively. Phylogenetic analyses revealed that Sfrp2 orthologs were more related to Sfrp1 and Sfrp5 orthologs than to Sfrp3 and Sfrp4 orthologs. Human SFRP2 promoter (nucleotide position 71417-71152 of AC020703.7 genome sequence) and rat Sfrp2 promoter (nucleotide position 184090-184357 of AC129161.3 genome sequence) showed 88.7% total nucleotide identity. GC content of human SFRP2 promoter was 63.9%. Binding site for POU domain transcription factors (POU2F1, POU2F2, etc.) was conserved between human SFRP2 promoter and rat Sfrp2 promoter. SFRP2 mRNA was expressed in embryonic stem (ES) cells, diffuse type gastric cancer with signet ring cell features, pancreatic cancer, colorectal cancer, retinoblastoma, and insulinoma. This is the first report on comparative integromics analyses on SFRP2 orthologs.

Secreted frizzled-related protein-1 inhibits RANKL-dependent osteoclast formation.

We determined that sFRP-1 mRNA was differentially expressed by osteoblast/stromal cell lines and that sFRP-1 neutralizing antibodies and siRNA complementary to sFRP-1 coding sequence enhanced, while recombinant sFRP-1 inhibited, osteoclast formation. In studying the mechanism of action for sFRP-1, we found that sFRP-1 could bind recombinant RANKL. These results suggest potential cross-talk between Wnt and RANKL pathways. Osteoclast formation in normal bone remodeling requires the presence of osteoblast lineage cells that express RANKL and macrophage-colony-stimulating factor (M-CSF), which interact with their cognate receptors on the osteoclast precursor. We identified secreted Frizzled-related protein-1 (sFRP-1), which is known to bind to Wnt and inhibit the Wnt signaling pathway, as an osteoblast-derived factor that impinges on osteoclast formation and activity. Differential display of mRNA from osteoblast lineage cell lines established sFRP-1 to be highly expressed in an osteoclast supporting cell line. sFRP-1 expression in bone was determined by in situ hybridization, and the effects of sFRP-1 on osteoclast formation were determined using a neutralizing antibody, siRNA, for sFRP-1 and recombinant protein. In situ hybridization revealed sFRP-1 mRNA expression in osteoblasts and chondrocytes in murine bone. sFRP-1 mRNA expression could be elevated in calvarial primary osteoblasts in response to prostaglandin E2 (PGE2) or interleukin (IL)-11, whereas many other osteotropic agents (e.g., IL-1, IL-6, calcitrol, parathyroid hormone) were without any effect. In vitro assays of osteoclast formation established sFRP-1 to be an inhibitor of osteoclast formation. Neutralizing antibodies against sFRP-1 enhanced TRACP+ mononuclear and multinuclear osteoclast formation (3- and 2-fold, respectively) in co-cultures of murine osteoblasts with spleen cells, whereas siRNA complementary to sFRP-1 coding sequence significantly enhanced osteoclast formation in co-cultures of KUSA O (osteoblast/stromal cell line) and bone marrow cells, cultured in the presence of PGE2 and 1,25(OH)2 vitamin D3. Recombinant sFRP-1 dose-dependently inhibited osteoclast formation in osteoblast/spleen co-cultures, RANKL + M-CSF-treated splenic cultures, and RANKL-treated RAW264.7 cell cultures, indicating a direct action of sFRP-1 on hematopoietic cells. Consistent with this, sFRP-1 was found to bind to RANKL in ELISAs. sFRP-1 is expressed by osteoblasts and inhibits osteoclast formation. While sFRP-1 activity might involve the blocking of endogenous Wnt signaling, our results suggest that, alternatively, it could be because of direct binding to RANKL. This study describes a new mechanism whereby osteoblasts regulate osteoclastogenesis through the expression and release of sFRP-1.

Regulated expression of sFRP-1 protein by the GeneSwitch system

The GeneSwitch system is a mifepristone-inducible expression system that provides exceptionally low uninduced and high-induced protein expression in mammalian cells. We have developed an adenovirus recombinant containing GeneSwitch protein driven by the GAL4-tk promoter, as well as recombinants containing sFRP-1 and luciferase reporter under the control of the GAL4-E1b promoter. Luciferase activity in A549 cells infected with the GeneSwitch and Luciferase viruses is very low in ethanol-treated cells, while the level of luciferase activity increases 200-fold in cells treated with mifepristone. Conditional expression of functional sFRP-1 is demonstrated in A549, human osteoblast, and CHO cell lines by either the co-infection of cells with sFRP-1 and GeneSwitch viruses or the infection of GeneSwitch expressing cell lines with sFRP-1 virus and subsequent treatment with mifepristone. The expression of sFRP-1 is seen as early as 4 h post-mifepristone treatment, reaching the highest levels at 20 h. The sFRP-1 protein is present in conditioned media, and the protein is functional based upon its ability to inhibit the Wnt-mediated activation of TCF-Luciferase reporter activity. The regulated expression of sFRP-1 utilizing adenovirus vectors provides an opportunity to address the contribution of sFRP-1 in the regulation of stem cell differentiation, maturation, and their function by modulating the Wnt signaling.