Wnt-1 Induced Secreted Protein 1 Research Articles

WNT1-induced Secreted Protein-1 (WISP1), a Novel Regulator of Bone Turnover and Wnt Signaling

WISP1/CCN4 (hereafter referred to as WISP1), a member of the CCN family, is found in mineralized tissues and is produced by osteoblasts and their precursors. In this study, Wisp1-deficient (Wisp1(-/-)) mice were generated. Using dual-energy x-ray absorptiometry, we showed that by 3 months, the total bone mineral density of Wisp1(-/-) mice was significantly lower than that of WT mice. Further investigation by micro-computed tomography showed that female Wisp1(-/-) mice had decreased trabecular bone volume/total volume and that both male and female Wisp1(-/-) mice had decreased cortical bone thickness accompanied by diminished biomechanical strength. The molecular basis for decreased bone mass in Wisp1(-/-) mice arises from reduced bone formation likely caused by osteogenic progenitors that differentiate poorly compared with WT cells. Osteoclast precursors from Wisp1(-/-) mice developed more tartrate-resistant acid phosphatase-positive cells in vitro and in transplants, suggesting that WISP1 is also a negative regulator of osteoclast differentiation. When bone turnover (formation and resorption) was induced by ovariectomy, Wisp1(-/-) mice had lower bone mineral density compared WT mice, confirming the potential for multiple roles for WISP1 in controlling bone homeostasis. Wisp1(-/-) bone marrow stromal cells had reduced expression of β-catenin and its target genes, potentially caused by WISP1 inhibition of SOST binding to LRP6. Taken together, our data suggest that the decreased bone mass found in Wisp1(-/-) mice could potentially be caused by an insufficiency in the osteodifferentiation capacity of bone marrow stromal cells arising from diminished Wnt signaling, ultimately leading to altered bone turnover and weaker biomechanically compromised bones.

Abstract 4: WISP-1 increases angiogenesis and VEGF-A expression in human oral squamous cell carcinomas

Abstract Oral squamous cell carcinomas (OSCC) is most common metastasis to cervical lymph nodes by angiogenesis. Wnt-1 induced secreted protein 1 (WISP-1) is a cysteine-rich protein that belongs to the CNN family (CTGF / CYR61 / NOV). However, the effects of WISP-1 on angiogenesis and Vascular endothelial growth factor- A (VEGF-A) expression in human OSCC are not clear. First, we found that the expression of WISP-1 in human OSCC tissues were higher than in normal oral tissues. We also demonstrated that WISP-1 enhanced angiogenesis and VEGF-A up-regulation in human OSCC. WISP-1 induced VEGF-A expressio and angiogenesis through αvβ3 integrin, FAK, c-Src, EGFR, and ERK pathway. Knockdown WISP-1 decreased VEGF-A expression and also abolished human OSCC condition medium-mediated angiogenesis in vitro as well as angiogenesis effects in the chick chorioallantoic membrane (CAM) and matrix gel plug nude mice model in vivo. In addition, in the xenograft tumor angiogenesis model, knockdown of WISP-1 significantly reduced tumor growth and tumor-associated angiogenesis. We characterized an important role for WISP-1 which regulates angiogenesis by increasing VEGF-A expression in human OSCC via the αvβ3 integrin, FAK, c-Src, EGFR, ERK, and HIF1-α signaling pathway. Citation Format: Ching-Wen Tsao, Chih-Hsin Tang, Jing-Yuan Chuang. WISP-1 increases angiogenesis and VEGF-A expression in human oral squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4. doi:10.1158/1538-7445.AM2014-4

Abstract 5010: Osteoblasts-derived WISP-1 increases prostate cancer metastasis through VCAM-1 upregulation

Abstract The skeleton is the most common metastatic site for prostate cancer, and increases the risk of intractable bone pain. Therefore, study of the interactions between prostate cancer and the bone microenvironment is very important. Wnt-1 induced secreted protein 1 (WISP-1) belongs to the CCN family (CTGF/CYR61/NOV), which plays an important role in bone formation and many cellular processes. However, the effects of WISP-1 on the prostate cancer metastasis are not clearly understood. First, we found that WISP-1 presents in osteoblast-condition medium (OB-CM), which increases prostate cancer cell migration and invasion. In addition, OBs were infected with lentivirus containing human WISP-1 shRNA which reduced OB-CM-mediated prostate cancer metastasis. We also demonstrated that OB-derived WISP-1 enhanced cell migration via vascular cell adhesion molecule 1(VCAM-1) up-regulation in prostate cancer. Transfection of cells with VCAM-1 siRNA reduced OB-CM and WISP-1-mediated cell migration. The incresing of prostate cancer cell migration by OB-derived WISP-1 is associated with FAK and p38 activation, as well as mir-126 downregulation. Pretreatment of cells with FAK and p38 inhibitors reduced WISP-1-mediated cell migration. In contrast, WISP-1-mediated migration activity was inhibited by mir-126 mimic. Over all, we characterized an important role for OB-derived WISP-1 which regulates cell migration and invasion by increasing VCAM-1 expression in human prostate cancer via the activation of FAK, p38 and downregulation of mir-126 signaling pathway. Citation Format: An-Chen Chang, Shih-Wei Wang, Chih-Hsin Tang. Osteoblasts-derived WISP-1 increases prostate cancer metastasis through VCAM-1 upregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2014-5010

Osteoblast-derived WISP-1 increases VCAM-1 expression and enhances prostate cancer metastasis by down-regulating miR-126

Bone metastases of prostate cancer (PCa) may cause intractable pain. Wnt-1-induced secreted protein 1 (WISP-1) belongs to the CCN family (CTGF/CYR61/NOV) that plays a key role in bone formation. We found that osteoblast-conditioned medium (OBCM) stimulates migration and vascular adhesion molecule-1 (VCAM)-1 expression in human PCa (PC3 and DU145) cells. Osteoblast transfection with WISP-1 shRNA reduced OBCM-mediated PCa migration and VCAM-1 expression. Stimulation of PCa with OBCM or WISP-1 elevated focal adhesion kinase (FAK) and p38 phosphorylation. Either FAK and p38 inhibitors or siRNA abolished osteoblast-derived WISP-1-induced migration and VCAM-1 expression. Osteoblast-derived WISP-1 inhibited miR-126 expression. Moreover, miR-216 mimic reversed the WISP-1-enhanced migration and VCAM-1 expression. This study suggests that osteoblast-derived WISP-1 promotes migration and VCAM-1 expression in human PCa cells by down-regulating miR-126 expression via αvβ1 integrin, FAK, and p38 signaling pathways. Thus, WISP-1 may be a new molecular therapeutic target in PCa bone metastasis.

Abnormal gene expression and gene fusion in lung adenocarcinoma with high-throughput RNA sequencing

To explore the universal law of the abnormal gene expression and the structural variation of genes related to lung adenocarcinoma, the gene expression profile of GSE37765 were downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) were analyzed with t-test and NOISeq tool, and the core DEGs were screened out by combining with another RNA-seq data containing totally 77 pairs of samples in 77 patients with lung adenocarcinoma. Moreover, the functional annotation of the core DEGs was performed by using the Database for Annotation Visualization and Integrated Discovery following selection of oncogene and tumor suppressor by combining with tumor suppressor genes and Cancer Genes database, and motif-finding of core DEGs was performed with motif-finding algorithm Seqpos. We also used Tophat-fusion tool to further explore the fusion genes. In total, 850 downregulated DEGs and 206 upregulated DEGs were screened out in lung adenocarcinoma tissues. Next, we selected 543 core DEGs, including 401 downregulated and 142 upregulated genes, and vasculature development (P=1.89E-06) was significantly enriched among downregulated core genes, as well as mitosis (P=6.26E-04) enriched among upregulated core genes. On the basis of the cellular localization analysis of core genes, wnt-1-induced secreted protein 1 (WISP1) and receptor (G protein-coupled) activity modifying protein 1 (RAMP1) identified mainly located in extracellular region and extracellular space. We also screened one oncogene, v-myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2). Moreover, transcription factor GATA2 was mined by motif-finding analysis. Finally, four fusion genes belonged to the human leukocyte antigen (HLA) family. WISP1, RAMP1, MYBL2 and GATA2 could be potential targets of treatment for lung adenocarcinoma and the fusion of HLA family genes might have important roles in lung adenocarcinoma.

Abstract 3792: WISP-1 increases cell migration and ICAM-1 expression in human oral squamous cell carcinomas.

Abstract Oral squamous cell carcinomas (OSCC) have a striking tendency to migration and metastasis to cervical lymph nodes. Wnt-1 induced secreted protein 1 (WISP-1) belongs to the CCN family (CTGF/CYR61/NOV), which is secreted cysteine-rich proteins. However, the effects of WISP-1 on migration and Intercellular adhesion molecule-1 (ICAM-1) expression in human OSCC are largely unknown. First, we found that the expression of WISP-1 in human OSCC tissues was higher than in normal oral tissues. We also demonstrated that WISP-1 enhanced cell migration and ICAM-1 up-regulation in human OSCC. Transfection of cells with ICAM-1 siRNA reduced WISP-1-mediated cell migration. WISP-1 activated αvβ3 integrin, ASK1, JNK/p38, c-Jun, and AP-1 signal transduction pathway, and WISP-1-induced expression of ICAM-1 and migration activity was inhibited by the αvβ3 antibody or inhibitor of ASK1, p38, JNK, and c-Jun. In addition, human OSCC were infected with lentivirus containing human WISP-1 shRNA which reduced the migratory ability and ICAM-1 expression. Over all, we characterized an important role for WISP-1 which regulates cell migratoin by increasing ICAM-1 expression in human OSCC via the αvβ3 integrin, ASK1, JNK/p38, c-Jun, and AP-1 signaling pathway. Citation Format: An-Chen Chang. WISP-1 increases cell migration and ICAM-1 expression in human oral squamous cell carcinomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3792. doi:10.1158/1538-7445.AM2013-3792

Abstract 1058: Novel Regulation of Wnt-Inducible Secreted Protein 1 by TNF-alpha in Primary Human Cardiac Fibroblasts

Wnt1-induced secreted protein-1 (WISP-1) is a member of the CCN family of growth factors known to play a role in cellular growth, transformation, and survival. We previously demonstrated that WISP-1 is upregulated in the heart post-infarct, stimulates fibroblast proliferation and collagen expression, and is induced by the proinflammatory cytokine TNF-a. The present study was designed to investigate the molecular mechanisms involved in TNF-a-mediated WISP-1 induction in primary human cardiac fibroblasts (hCF). hCF were treated with rhTNF-a. WISP-1 expression was analyzed by Northern, Western and promoter-reporter assays. ERK1/2 and JNK activations by Western blotting using activation-specific antibodies and imunecomplex kinase assays. CREB activation by Western blotting, EMSA and reporter assay. Results demonstrate that TNF-a potently induces WISP-1 mRNA and protein expression in a time- and dose-dependent manner, effects that were blocked by anti-TNFR2, but not anti-TNFR1, neutralizing antibodies. Further investigations revealed that TNF-a stimulates WISP-1 promoter-reporter activity, an effect that was blunted when the core CREB DNA binding sequence was mutated or following the overexpression of dnCREB. TNF-a treatment induced CREB phosphorylation, in vitro and in vivo DNA binding, and reporter gene activities. TNF-a also induced ERK1/2 phosphorylation and activity, effects that were blocked by the MEK1 inhibitor U0126 and the ERK1/2 inhibitor PD98059. Furthermore, inhibition of ERK1/2 blunted CREB activation while inhibition of ERK1/2 and overexpression of dnCREB attenuated TNF-a-mediated WISP-1 promoter-reporter activity and mRNA expression. However, inhibition of JNK by SP600125 failed to modulate TNF-a-mediated WISP-1 induction. Most importantly, siRNA-mediated WISP-1 knockdown attenuated TNF-a-mediated hCF proliferation. Thus, our results demonstrate for the first time that TNF-a is a potent inducer of WISP1 expression in hCF, and TNF-a induces WISP-1 expression via TNFR2, MEK1, ERK, and CREB. Since TNF-a and WISP-1 are upregulated post-infarct, our results indicate that TNF-a/WISP-1 signaling may play a critical role in post-infarct myocardial remodeling.

Wnt-induced secreted protein-1 is a prohypertrophic and profibrotic growth factor

Wnt1-induced secreted protein-1 (WISP-1) is a member of the cysteine-rich 61, connective tissue growth factor, and nephroblastoma overexpressed (CCN) family of growth factors and is expressed in the heart at low basal levels. The purpose of this study was to investigate whether WISP-1 is upregulated in postinfarct myocardium and whether WISP-1 exerts prohypertrophic and mitogenic effects stimulating myocyte hypertrophy, cardiac fibroblast (CF) proliferation, and collagen expression. Male C57Bl/6 (25 g) mice underwent permanent occlusion of the left anterior descending coronary artery. mRNA and protein levels were analyzed by Northern and Western blot analyses. Cardiomyocyte hypertrophy was quantified by protein and DNA synthesis. CF proliferation was quantified by CyQuant assay, and soluble collagen release by Sircol assay. A time-dependent increase in WISP-1 expression was detected in vivo in the noninfarct zone of the left ventricle, which peaked at 24 h (3.1-fold, P < 0.01). Similarly, biglycan expression was increased by 3.71-fold (P < 0.01). IL-1beta and TNF-alpha expression preceded WISP-1 expression in vivo and stimulated WISP-1 expression in neonatal rat ventricular myocytes in vitro. WISP-1-induced cardiomyocyte hypertrophy was evidenced by increased protein (2.78-fold), but not DNA synthesis, and enhanced Akt phosphorylation and activity. Treatment of primary CF with WISP-1 significantly stimulated proliferation at 48 h (6,966 +/- 264 vs. 5,476 +/- 307 cells/well, P < 0.01) and enhanced collagen release by 72 h (18.4 +/- 3.1 vs. 8.4 +/- 1.0 ng/cell, P < 0.01). Our results demonstrate for the first time that WISP-1 and biglycan are upregulated in the noninfarcted myocardium in vivo, suggesting a positive amplification of WISP-1 signaling. WISP-1 stimulates cardiomyocyte hypertrophy, fibroblast proliferation, and ECM expression in vitro. These results suggest that WISP-1 may play a critical role in post-myocardial infarction remodeling.

Association of a single nucleotide polymorphism in the WISP1 gene with spinal osteoarthritis in postmenopausal Japanese women

The Wnt-beta-catenin signaling pathway that regulates bone density is also involved in cartilage development and homeostasis in vivo. Here, we assumed that genetic variation in Wnt-beta-catenin signaling genes can affect the pathogenesis of cartilage related diseases, such as osteoarthritis. Wnt-1-induced secreted protein 1 (WISP1) is a target of the Wnt pathway and directly regulated by beta-catenin. In the present study, we analyzed the association of a single nucleotide polymorphism (SNP) in the WISP1 3'-UTR region with the development of radiographically observable osteoarthritis of the spine. For this purpose, we evaluated the presence of osteophytes, endplate sclerosis, and narrowing of disc spaces in 304 postmenopausal Japanese women. We compared those who carried the G allele (GG or GA, n = 184) with those who did not (AA, n = 120). We found that the subjects without the G allele (AA) were significantly over-represented in the subjects having higher endplate sclerosis score (P = 0.0069; odds ratio, 2.91; 95% confidence interval, 1.34-6.30 by logistic regression analysis). On the other hand, the occurrence of disc narrowing and osteophyte formation did not significantly differ between those with and without at least one G allele. Thus, we suggest that a genetic variation in the WISP1 gene locus is associated with spinal osteoarthritis, in line with the involvement of the Wnt-beta-catenin-regulated gene in bone and cartilage metabolism.

Structural Basis and Therapeutic Implication of the Interaction of CCN Proteins with Glycoconjugates

The CCN family of growth factors is composed of six structurally related proteins including the cysteine-rich 61 (Cyr61), connective tissue growth factor (CTGF), nephroblastoma overexpressed (NOV), Wnt-1 induced secreted protein-1 (WISP-1), WISP-2 and WISP-3. Each family member consists of four conserved cysteine rich modular domains with sequence similarity to the insulin like growth factor binding proteins, von Willebrand factor, thrombospondin repeat and the growth factors cysteine knot. The CCN proteins demonstrate a wide variety of biological activities regulating cell adhesion, proliferation, survival, migration, invasion in vitro and tumorigenesis and angiogenesis in vivo. Both cancer promoting and inhibiting roles were proposed for several CCN proteins suggesting that contextual factors could regulate their activities. Consistent with this hypothesis, structural and experimental evidence indicate that the function of these proteins is modulated by their interaction with sulfated glycosaminoglycans. Because the CCN proteins are implicated in the tumorigenic process, they are potential targets for the development of cancer therapeutics. Modulation of their glycosaminoglycan interaction by exogenous, highly sulfated polysaccharides, oligosaccharides or glycosaminoglycan mimetics could prevent their participation in cancer progression. Understanding the structural requirements for their polysaccharide interaction should provide important information to generate glycosaminoglycan-based cancer therapeutics targeting the CCN family of proteins.

WISP-1 Is an Osteoblastic Regulator Expressed During Skeletal Development and Fracture Repair

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-1 in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During embryonic development, WISP-1 expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-1 expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic differentiation. Using in situ and cell binding analysis, we demonstrated WISP-1 interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-1 on chondrocytes by generating stably transfected mouse chondrocytic cell lines. In these cells, WISP-1 increased proliferation and saturation density but repressed chondrocytic differentiation. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-1 spatiotemporal expression in a fracture repair model. We found that WISP-1 expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-1 is an osteogenic potentiating factor promoting mesenchymal cell proliferation and osteoblastic differentiation while repressing chondrocytic differentiation. Therefore, we propose that WISP-1 plays an important regulatory role during bone development and fracture repair.

MRP-1/CD9 gene transduction downregulates Wnt signal pathways.

Motility-related protein-1 (MRP-1/CD9) is a transmembrane glycoprotein that has been implicated in cell adhesion, motility, proliferation, and differentiation. It has a functional role as a tumor metastatic suppressor. During tumor progression, a reduction of MRP-1/CD9 gene expression results in tumor cells with a high metastatic potential. However, the mechanism of action of MRP-1/CD9 is still unclear. We studied changes of gene expression in relation to MRP-1/CD9 gene transduction into tumor cell lines, HT1080 and A549, using microarray assays and real-time PCR. Consequently, we have demonstrated that MRP-1/CD9 gene transduction can downregulate expression of several Wnt family genes, such as Wnt1, Wnt2b1 and Wnt5a, and their target genes, including WISP-1 (Wnt-1 induced secreted protein 1), WISP-3, c-Myc, vascular endothelial growth factor-A, and matrix metalloproteinase-26. Western blot analyses also showed that MRP-1/CD9 gene transduction downregulated expression of Wnt1 protein and its target proteins. In addition, a neutralizing anti-MRP-1/CD9 monoclonal antibody inhibited the downregulation of Wnt signal pathways in MRP-1/CD9-transfected cells. The present study has revealed that the MRP-1/CD9 signal is located upstream of the Wnt signal pathways. Therefore, MRP-1/CD9 could suppress cell transformation including epithelial to mesenchymal transition through downregulation of Wnt1, and might suppress tumor metastasis through downregulation of Wnt5a.

Overexpression of a set of genes, including WISP-1, common to pulmonary metastases of both mouse D122 Lewis lung carcinoma and B16-F10.9 melanoma cell lines.

Despite advances in the management of solid tumours, the development of metastases continues to be the most significant problem and cause of death for cancer patients. To define genetic determinants of pulmonary metastases, we have applied oligonucleotide microarrays to established murine models of highly metastatic D122 Lewis lung carcinoma and B16-F10.9 melanoma cell lines. These models are characterised by primary subcutaneous growth in C57BL/6J mice, a period of minimal residual disease and spontaneous pulmonary metastases. Microarray analysis defined seven genes, namely – arginase, brain natriuretic peptide (BNP), interleukin-1 alpha (IL-1 alpha), plasminogen activator inhibitor-2 (PAI-2), surfactant protein C (SP-C), uteroglobin (UG) and wnt-1-induced secreted protein-1 (WISP-1), which were consistently elevated in pulmonary metastases compared to the primary tumour of both D122 and B16-F10.9 models. Previous studies demonstrated that two of these seven genes, IL-1 alpha and PAI-2, are involved in the metastatic process. The results obtained by the microarrays were confirmed by real-time quantitative PCR, for three chosen genes – PAI-2, WISP-1 and UG. Our approach aimed to identify genes essential for the metastatic process in general and for pulmonary metastases specifically. Further research should address the precise role of these genes in the metastasising process to the lungs and test if they could be used as targets for future therapies.

WISP-1 Binds to Decorin and Biglycan

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Structural and experimental evidence suggests that CCN family member activities are modulated by their interaction with sulfated glycoconjugates. To elucidate the mechanism of action for WISP-1, we characterized the specificity of its tissue and cellular interaction and identified binding factors. WISP-1 binding was restricted to the stroma of colon tumors and to cells with a fibroblastic phenotype. By using a solid phase assay, we showed that human skin fibroblast conditioned media contained WISP-1 binding factors. Competitive inhibition with different glycosaminoglycans and treatment with glycosaminoglycan lyases and proteases demonstrated that binding to the conditioned media was mediated by dermatan sulfate proteoglycans. Mass spectrometric analysis identified the isolated binding factors as decorin and biglycan. Decorin and biglycan interacted directly with WISP-1 and inhibited its binding to components in the conditioned media. Similarly, WISP-1 interaction with human skin fibroblasts was inhibited by dermatan sulfate, decorin, and biglycan or by treatment of the cell surface with dermatan sulfate-specific lyases. Together these results demonstrate that decorin and biglycan are WISP-1 binding factors that can mediate and modulate its interaction with the surface of fibroblasts. We propose that this specific interaction plays a role in the regulation of WISP-1 function.

WISP-1 is a Wnt-1- and β-catenin-responsive oncogene

WISP-1 (Wnt-1 induced secreted protein 1) is a member of the CCN family of growth factors. This study identifies WISP-1 as a beta-catenin-regulated gene that can contribute to tumorigenesis. The promoter of WISP-1 was cloned and shown to be activated by both Wnt-1 and beta-catenin expression. TCF/LEF sites played a minor role, whereas the CREB site played an important role in this transcriptional activation. WISP-1 demonstrated oncogenic activities; overexpression of WISP-1 in normal rat kidney fibroblast cells (NRK-49F) induced morphological transformation, accelerated cell growth, and enhanced saturation density. Although these cells did not acquire anchorage-independent growth in soft agar, they readily formed tumors in nude mice, suggesting that appropriate cellular attachment is important for signaling oncogenic events downstream of WISP-1.