WISP1 induces the expression of macrophage migration inhibitory factor in human lung fibroblasts through Src kinases and EGFR-activated signaling pathways.

Wnt1-inducible signaling protein 1 (WISP1/CCN4) is a secreted matricellular protein that is implicated in lung and airway remodeling. The macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been associated with chronic lung diseases. In this study, we aimed to investigate the WISP1 signaling pathway and its ability to induce the expression of MIF in primary cultures of fibroblasts from normal human lungs (HLFs). Our results showed that WISP1 significantly stimulated the expression of MIF in a concentration- and time-dependent fashion. In WISP1-induced expression of MIF, αvß5-integrin and chondroitin sulfate proteoglycans as well as Src tyrosine kinases, MAP kinases, phosphatidylinositol 3-kinase/Akt, PKC, and NF-κB were involved. WISP1-induced expression of MIF was attenuated in the presence of the Src kinase inhibitor PP2 or the MIF tautomerase activity inhibitor ISO-1. Moreover, WISP1 significantly increased the phosphorylation and activation of EGF receptor (EGFR) through transactivation by Src kinases. WISP1 also induced the expression of MIF receptor CD74 and coreceptor CD44, through which MIF exerts its effects on HLFs. In addition, it was found that MIF induced its own expression, as well as its receptors CD74/CD44, acting in an autocrine manner. Finally, WISP1-induced MIF promoted the expression of cyclooxygenase 2, prostaglandin E2, IL-6, and matrix metalloproteinase-2 demonstrating the regulatory role of WISP1-MIF axis in lung inflammation and remodeling involving mainly integrin αvß5, Src kinases, PKC, NF-κB, and EGFR. The specific signaling pathways involved in WISP1-induced expression of MIF may prove to be excellent candidates for novel targets to control inflammation in chronic lung diseases.NEW & NOTEWORTHY The present study demonstrates for the first time that Wnt1-inducible signaling protein 1 (WISP1) regulates migration inhibitory factor (MIF) expression and activity and identifies the main signaling pathways involved. The newly discovered WISP1-MIF axis may drive lung inflammation and could result in the design of novel targeted therapies in inflammatory lung diseases.

The TGF-ß1 target WISP1 is highly expressed in liver cirrhosis and cirrhotic HCC microenvironment and involved in pro- and anti-tumorigenic effects.

INTRODUCTION: WNT1-inducible signalling pathway protein 1 (WISP1) promotes progression of several tumor entities often correlating with worse prognosis. Here its expression regulation and role in the progression of chronic liver diseases (CLD) was investigated. METHODS: WISP1 expression was analyzed in human HCC datasets, in biopsies and serum samples and an HCC patient tissue microarray (TMA) including correlation to clinicopathological parameters. Spatial distribution of WISP1 expression was determined using RNAscope analysis. Regulation of WISP1 expression was investigated in cytokine-stimulated primary mouse hepatocytes (PMH) by array analysis and qRT-PCR. Outcome of WISP1 stimulation was analyzed by IncuCyte S3-live cell imaging, qRT-PCR, and immunoblotting in murine AML12 cells. RESULTS: In a TMA, high WISP1 expression was positively correlated with early HCC stages and male sex. Highest WISP1 expression levels were detected in patients with cirrhosis as compared to healthy individuals, patients with early fibrosis, and non-cirrhotic HCC in liver biopsies, expression datasets and serum samples. WISP1 transcripts were predominantly detected in hepatocytes of cirrhotic rather than tumorous liver tissue. High WISP1 expression was associated with better survival. In PMH, AML12 and HepaRG, WISP1 was identified as a specific TGF-ß1 target gene. Accordingly, expression levels of both cytokines positively correlated in human HCC patient samples. WISP1-stimulation induced the expression of Bcl-xL, PCNA and p21 in AML12 cells. CONCLUSIONS: WISP1 expression is induced by TGF-ß1 in hepatocytes and is associated with cirrhotic liver disease. We propose a crucial role of WISP1 in balancing pro- and anti-tumorigenic effects during premalignant stages of CLD.

An overview of CCN4 (WISP1) role in human diseases.

CCN4 (cellular communication network factor 4), a highly conserved, secreted cysteine-rich matricellular protein is emerging as a key player in the development and progression of numerous disease pathologies, including cancer, fibrosis, metabolic and inflammatory disorders. Over the past two decades, extensive research on CCN4 and its family members uncovered their diverse cellular mechanisms and biological functions, including but not limited to cell proliferation, migration, invasion, angiogenesis, wound healing, repair, and apoptosis. Recent studies have demonstrated that aberrant CCN4 expression and/or associated downstream signaling is key to a vast array of pathophysiological etiology, suggesting that CCN4 could be utilized not only as a non-invasive diagnostic or prognostic marker, but also as a promising therapeutic target. The cognate receptor of CCN4 remains elusive till date, which limits understanding of the mechanistic insights on CCN4 driven disease pathologies. However, as therapeutic agents directed against CCN4 begin to make their way into the clinic, that may start to change. Also, the pathophysiological significance of CCN4 remains underexplored, hence further research is needed to shed more light on its disease and/or tissue specific functions to better understand its clinical translational benefit. This review highlights the compelling evidence of overlapping and/or diverse functional and mechanisms regulated by CCN4, in addition to addressing the challenges, study limitations and knowledge gaps on CCN4 biology and its therapeutic potential.

Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma.

Cell Communication Network factor 4 (CCN4/WISP1) is a matricellular protein secreted by cancer cells that promotes metastasis by inducing the epithelial-mesenchymal transition. While metastasis limits survival, limited anti-tumor immunity also associates with poor patient outcomes with recent work linking these two clinical correlates. Motivated by increased CCN4 correlating with dampened anti-tumor immunity in primary melanoma, we test for a direct causal link by knocking out CCN4 (CCN4 KO) in the B16F0 and YUMM1.7 mouse melanoma models. Tumor growth is reduced when CCN4 KO melanoma cells are implanted in immunocompetent but not in immunodeficient mice. Correspondingly, CD45+ tumor-infiltrating leukocytes are significantly increased in CCN4 KO tumors, with increased natural killer and CD8+ T cells and reduced myeloid-derived suppressor cells (MDSC). Among mechanisms linked to local immunosuppression, CCN4 suppresses IFN-gamma release by CD8+ T cells and enhances tumor secretion of MDSC-attracting chemokines like CCL2 and CXCL1. Finally, CCN4 KO potentiates the anti-tumor effect of immune checkpoint blockade (ICB) therapy. Overall, our results suggest that CCN4 promotes tumor-induced immunosuppression and is a potential target for therapeutic combinations with ICB.

WISP1 and Macrophage Migration Inhibitory Factor in Respiratory Inflammation: Novel Insights and Therapeutic Potentials for Asthma and COPD.

Recent advancements highlight the intricate interplay between the extracellular matrix (ECM) and immune responses, notably in respiratory diseases such as asthma and Chronic Obstructive Pulmonary Disease (COPD). The ECM, a dynamic structural framework within tissues, orches-trates a plethora of cellular processes, including immune cell behavior and tissue repair mecha-nisms. WNT1-inducible-signaling pathway protein 1 (WISP1), a key ECM regulator, controls immune cell behavior, cytokine production, and tissue repair by modulating integrins, PI3K, Akt, ß-catenin, and mTOR signaling pathways. WISP1 also induces macrophage migration inhibitory factor (MIF) expression via Src kinases and epidermal growth factor receptor (EGFR) activation. MIF, through its wide range of activities, enhances inflammation and tissue restructuring. Rec-ognized for its versatile roles in regulating the immune system, MIF interacts with multiple immune components, such as the NLRP3 inflammasome, thereby sustaining inflammatory pro-cesses. The WISP1-MIF axis potentially unveils complex molecular mechanisms governing im-mune responses and inflammation. Understanding the intricate roles of WISP1 and MIF in the pathogenesis of chronic respiratory diseases such as asthma and COPD could lead to the identi-fication of novel targets for therapeutic intervention to alleviate disease severity and enhance patient outcomes.

HuR-dependent expression of Wisp1 is necessary for TGFß-induced cardiac myofibroblast activity.

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGFß-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGFß-induced myofibroblast activation upon HuR inhibition. We identified twenty-four mRNA transcripts that were enriched for HuR binding following TGFß treatment via photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). Eleven of these HuR-bound mRNAs also showed significant co-expression correlation with HuR, αSMA, and periostin in primary fibroblasts isolated from the ischemic-zone of infarcted mouse hearts. Of these, WNT1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 partially rescued myofibroblast-induced collagen gel contraction following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGFß. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGFß, in part through post-transcriptional regulation of Wisp1.

The tumor cell-secreted matricellular protein WISP1 drives pro-metastatic collagen linearization.

Collagen linearization is a hallmark of aggressive tumors and a key pathogenic event that promotes cancer cell invasion and metastasis. Cell-generated mechanical tension has been proposed to contribute to collagen linearization in tumors, but it is unknown whether other mechanisms play prominent roles in this process. Here, we show that the secretome of cancer cells is by itself able to induce collagen linearization independently of cell-generated mechanical forces. Among the tumor cell-secreted factors, we find a key role in this process for the matricellular protein WISP1 (CCN4). Specifically, WISP1 directly binds to type I collagen to promote its linearization in vitro (in the absence of cells) and in vivo in tumors. Consequently, WISP1-induced type I collagen linearization facilitates tumor cell invasion and promotes spontaneous breast cancer metastasis, without significantly affecting gene expression. Furthermore, higher WISP1 expression in tumors from cancer patients correlates with faster progression to metastatic disease and poor prognosis. Altogether, these findings reveal a conceptually novel mechanism whereby pro-metastatic collagen linearization critically depends on a cancer cell-secreted factor.

WISP1 Is Involved in the Pathogenesis of Kashin-Beck Disease via the Autophagy Pathway.

OBJECTIVE: Kashin-Beck disease (KBD) is a kind of endemic and chronic osteochondropathy in China. This study aims to explore the functional relevance and potential mechanism of Wnt-inducible signaling pathway protein 1 (WISP1) in the pathogenesis of KBD. DESIGN: KBD and control cartilage specimens were collected for tissue section observation and primary chondrocyte culture. Firstly, the morphological and histopathological observations were made under a light and electron microscope. Then, the expression levels of WISP1 as well as molecular markers related to the autophagy pathway and extracellular matrix (ECM) synthesis were detected in KBD and control chondrocytes by qRT-PCR, Western blot, and immunohistochemistry. Furthermore, the lentiviral transfection technique was applied to make a WISP1 knockdown cell model based on KBD chondrocytes. In vitro intervention experiments were conducted on the C28/I2 human chondrocyte cell line using human recombinant WISP1 (rWISP1). RESULTS: The results showed that the autolysosome appeared in the KBD chondrocytes. The expression of WISP1 was significantly higher in KBD chondrocytes. Additionally, T-2 toxin, a risk factor for KBD onset, could up-regulate the expression of WISP1 in C28/I2. The autophagy markers ATG4C and LC3II were upregulated after the low-concentration treatment of T-2 toxin and downregulated after the high-concentration treatment. After knocking down WISP1 expression in KBD chondrocytes, MAP1LC3B decreased while ATG4C and COL2A1 increased. Moreover, the rWISP1 protein treatment in C28/I2 chondrocytes could upregulate the expression of ATG4C and LC3II at the beginning and downregulate them then. CONCLUSIONS: Our study suggested that WISP1 might play a role in the pathogenesis of KBD through autophagy.

miRNA repertoires of cystic fibrosis ex vivo models highlight miR-181a and miR-101 that regulate WISP1 expression.

Cystic fibrosis (CF), a genetic disorder, is characterized by chronic lung disease. Small non-coding RNAs are key regulators of gene expression and participate in various processes, which are dysregulated in CF; however, they remain poorly studied. Here, we determined the complete microRNAs (miRNAs) expression pattern in three CF ex vivo models. The miRNA profiles of air-liquid interface cultures of airway epithelia (bronchi, nasal cells, and nasal polyps) samples from patients with CF and non-CF controls were obtained by deep sequencing. Compared with non-CF controls, several miRNAs were deregulated in CF samples; for instance, miR-181a-5p and the miR-449 family were upregulated. Moreover, mature miRNAs often showed variations (i.e. isomiRs) relative to their reference sequence, such as miR-101, suggesting that miRNAs consist of heterogeneous repertoires of multiple isoforms with different effects on gene expression. Analysis of miR-181a-5p and miR-101-3p roles indicated that they regulate the expression of WISP1, a key component of cell proliferation/migration programs. We showed that miR-101 and miR-181a-5p participated in aberrant recapitulation of wound healing programs by controlling WISP1 mRNA and protein level. Our miRNA expression data bring new insights into CF physiopathology and define new potential therapeutic targets in CF. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

WNT1 Inducible Signaling Pathway Protein 1 Is a Stroma-Specific Secreting Protein Inducing a Fibroblast Contraction and Carcinoma Cell Growth in the Human Prostate.

The WNT1 inducible signaling pathway protein 1 (WISP1), a member of the connective tissue growth factor family, plays a crucial role in several important cellular functions in a highly tissue-specific manner. Results of a RT-qPCR indicated that WISP1 expressed only in cells of the human prostate fibroblasts, HPrF and WPMY-1, but not the prostate carcinoma cells in vitro. Two major isoforms (WISP1v1 and WISP1v2) were identified in the HPrF cells determined by RT-PCR and immunoblot assays. The knock-down of a WISP1 blocked cell proliferation and contraction, while treating respectively with the conditioned medium from the ectopic WISP1v1- and WISPv2-overexpressed 293T cells enhanced the migration of HPrF cells. The TNFα induced WISP1 secretion and cell contraction while the knock-down of WISP1 attenuated these effects, although TNFα did not affect the proliferation of the HPrF cells. The ectopic overexpression of WISP1v1 but not WISP1v2 downregulated the N-myc downstream regulated 1 (NDRG1) while upregulating N-cadherin, slug, snail, and vimentin gene expressions which induced not only the cell proliferation and invasion in vitro but also tumor growth of prostate carcinoma cells in vivo. The results confirmed that WISP1 is a stroma-specific secreting protein, enhancing the cell migration and contraction of prostate fibroblasts, as well as the proliferation, invasion, and tumor growth of prostate carcinoma cells.

Association between WNT-1-inducible signaling pathway protein-1 (WISP1) genetic polymorphisms and the risk of gastric cancer in Guangxi Chinese.

BACKGROUND: WNT1-inducible signaling pathway protein 1 (WISP1) is a member of the CCN protein family and a downstream target of ß-catenin. Aberrant WISP1 expression may be involved in carcinogenesis. To date, no studies have investigated the association between single-nucleotide polymorphisms (SNPs) of WISP1 and gastric cancer. Therefore, we conducted this study to explore their relationship. METHODS: Polymerase chain reaction-restriction fragment length polymorphism assay was used to analyze three SNPs of WISP1 in 204 gastric cancer patients and 227 controls. RESULTS: Overall, we could not identify a significant association between WISP1 SNPs and gastric cancer risk. However, the subgroup analysis demonstrated that the presence of the rs7843546 T allele was associated with a significantly decreased risk of gastric cancer in those of Han Chinese ethnicity (CT vs. CC: OR = 0.33, 95%CI 0.14-0.78; TT vs. CC: OR = 0.29, 95%CI 0.11-0.76; CT + TT vs. CC: OR = 0.32, 95%CI 0.14-0.74). In addition, patients with the rs7843546 TT genotype display a 0.34-fold lower risk of developing stage I/II gastric cancer than those with the CC genotype Furthermore, individuals ≥ 50 years old who carried the rs10956697 AC genotype had a significantly decreased risk of gastric cancer (OR = 0.58, 95%CI 0.35-0.98). Smokers with the rs10956697 AC and AC + AA genotypes exhibited a 0.28-fold lower and 0.32-fold lower risk of gastric cancer, respectively. CONCLUSIONS: The WISP1 SNPs rs7843546 and rs10956697 were, for the first time, found to reduce susceptibility to gastric cancer in various subgroups of Guangxi Chinese.

WNT1-inducible-signaling pathway protein 1 regulates the development of kidney fibrosis through the TGF-ß1 pathway.

Fibrosis is a pathological feature of chronic kidney disease and its progression correlates with declining renal function. Kidney fibrosis is driven by multiple profibrotic factors. This project examined the regulatory function of WNT1-inducible-signaling pathway protein 1 (WISP1) in the development of kidney fibrosis. Induction of WISP1 by transforming growth factor beta 1 (TGF-ß1), and the role of WISP1 in TGF-ß1/Smad signaling and fibrotic responses, was examined in multiple kidney cells. Kidney expression of WISP1 was examined in mouse models of unilateral ureter obstruction (UUO) and streptozotocin-induced diabetic nephropathy. WISP1 antibody was administered to UUO mice during the induction of kidney injury and the impact on kidney fibrosis was examined. WISP1 expression was upregulated in both mouse models. TGF-ß1-induced expression of WISP1 and profibrotic genes in cultured kidney cells via TGF-ßR1. Recombinant WISP1-induced expression of TGF-ßR1 in kidney cells. Suppression of WISP1 by shRNA or neutralizing antibody reduced TGF-ß1-mediated activation of Smad3, fibrotic gene expression, and fibroblast proliferation. Treatment with WISP1 antibody inhibited the development of kidney fibrosis in UUO mice. WISP1 mediates the profibrotic effects of TGF-ß1 in kidney cells and in kidney disease. Pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting kidney fibrosis.

Mechanical stimulations can inhibit local and remote tumor progression by downregulating WISP1.

Mechanical stimulations can prevent bone loss, but their effects on the tumor-invaded bone or solid tumors are elusive. Here, we evaluated the effect of knee loading, dynamic loads applied to the knee, on metastasized bone and mammary tumors. In a mouse model, tumor cells were inoculated to the mammary fat pad or the proximal tibia. Daily knee loading was then applied and metabolic changes were monitored mainly through urine. Urine samples were also collected from human subjects before and after step aerobics. The result showed that knee loading inhibited tumor progression in the loaded tibia. Notably, it also reduced remotely the growth of mammary tumors. In the urine, an altered level of cholesterol was observed with an increase in calcitriol, which is synthesized from a cholesterol derivative. In urinary proteins, knee loading in mice and step aerobics in humans markedly reduced WNT1-inducible signaling pathway protein 1, WISP1, which leads to poor survival among patients with breast cancer. In the ex vivo breast cancer tissue assay, WISP1 promoted the growth of cancer fragments and upregulated tumor-promoting genes, such as Runx2, MMP9, and Snail. Collectively, the present preclinical and human study demonstrated that mechanical stimulations, such as knee loading and step aerobics, altered urinary metabolism and downregulated WISP1. The study supports the benefit of mechanical stimulations for locally and remotely suppressing tumor progression. It also indicated the role of WISP1 downregulation as a potential mechanism of loading-driven tumor suppression.

LncRNA NEAT1 Knockdown Alleviates Lipopolysaccharide-Induced Acute Lung Injury by Modulation of miR-182-5p/WISP1 Axis.

Accumulating evidence has demonstrated the vital roles of long non-coding RNAs (lncRNAs) in acute lung injury (ALI). In this study, we aimed to explore the effect of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) on ALI development. The ALI mice and cell models were constructed using lipopolysaccharide (LPS)-induced method. The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) were measured by enzyme-linked immunosorbent assay (ELISA). The levels of TNF-α mRNA, IL-6 mRNA, IL-1ß mRNA, NEAT1, miR-182-5p, and WNT-inducible secreted protein 1 (WISP1) mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. The level of lactate dehydrogenase (LDH) and the activity of caspase-3 were measured by specific kits. The interaction between miR-182-5p and NEAT1 or WISP1 was investigated by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Protein levels were measured by Western blot assay. NEAT1 level was elevated in LPS-induced ALI mice and LPS-stimulated MH-S cells. LPS treatment repressed MH-S cell viability and promoted apoptosis and inflammation, while NEAT1 silencing restored the impacts. For mechanism analysis, NEAT1 was identified as the sponge for miR-182-5p to positively regulate WISP1 expression. Moreover, NEAT1 knockdown could accelerate cell viability and inhibit cell apoptosis and inflammation in LPS-induced MH-S cells by elevating miR-182-5p and decreasing WISP1 in LPS-exposed MH-S cells. In addition, NEAT1 deficiency blocked the activation of NF-κB pathway caused by LPS in MH-S cells. NEAT1 overexpression restrained cell viability and facilitated cell apoptosis and inflammation in LPS-exposed MH-S cells through miR-182-5p/WISP1 axis.

WISP1 alleviates lipid deposition in macrophages via the PPARγ/CD36 pathway in the plaque formation of atherosclerosis.

Lipid deposition in macrophages plays an important role in atherosclerosis. The WNT1-inducible signalling pathway protein 1(WISP1) can promote proliferation and migration of smooth muscle cells. Its expression is up-regulated in obesity, which is associated with atherosclerosis, but the effect of WISP1 on atherosclerosis remains unclear. Thus, the objective of our study was to elucidate the role of WISP and its mechanism of action in atherosclerosis via in vivo and in vitro experiments. In our experiment, ApoE-/- mice were divided into 5 groups: control, high-fat diet (HFD), null lentivirus (HFD + NC), lentivirus WISP1 (HFD + IvWISP1) and WISP1-shRNA (HFD + shWISP1). Oil Red O staining, immunofluorescence and immunohistochemistry of the aortic sinuses were conducted. Macrophages (RAW264.7 cell lines and peritoneal macrophages) were stimulated with 50 µg/mL oxidized low-density lipoprotein (ox-LDL); then, the reactive oxygen species (ROS) level was measured. Oil Red O staining and Dil-ox-LDL (ox-LDL with Dil dye) uptake measurements were used to test lipid deposition of peritoneal macrophages. WISP1, CD36, SR-A and PPARγ expression levels were measured via Western blotting and ELISA. The results showed that HFD mice had increased WISP1, CD36 and SR-A levels. The plaque lesion area increased when WISP1 was down-regulated, and lipid uptake and foam cell formation were inhibited when WISP1 was up-regulated. Treatment of RAW264.7 cell lines with ox-LDL increased WISP1 expression via activation of the Wnt5a/ß-catenin pathway, whereas ROS inhibition reduced WISP1 expression. Moreover, WISP1 down-regulated CD36 and SR-A expression, and Oil Red O staining and Dil-ox-LDL uptake measurement showed that WISP1 down-regulated lipid deposition in macrophages. These results clearly demonstrate that WISP1 is activated by ox-LDL at high ROS levels and can alleviate lipid deposition in atherosclerosis through the PPARγ/CD36 pathway.

WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial-mesenchymal transition.

Besides intrinsic changes, malignant cells also release soluble signals that reshape their microenvironment. Among these signals is WNT1-inducible signaling pathway protein 1 (WISP1), a secreted matricellular protein whose expression is elevated in several cancers, including melanoma, and is associated with reduced survival of patients diagnosed with primary melanoma. Here, we found that WISP1 knockout increases cell proliferation and represses wound healing, migration, and invasion of mouse and human melanoma cells in multiple in vitro assays. Metastasis assays revealed that WISP1 knockout represses tumor metastasis of B16F10 and YUMM1.7 melanoma cells in both C57BL/6Ncrl and NOD-scid IL2Rγnull (NSG) mice. WT B16F10 cells having an invasion phenotype in a transwell assay possessed a gene expression signature similar to that observed in the epithelial-mesenchymal transition (EMT), including E-cadherin repression and fibronectin and N-cadherin induction. Upon WISP1 knockout, expression of these EMT signature genes went in the opposite direction in both mouse and human cell lines, and EMT-associated gene expression was restored upon exposure to media containing WISP1 or to recombinant WISP1 protein. In vivo, Wisp1 knockout-associated metastasis repression was reversed by the reintroduction of either WISP1 or snail family transcriptional repressor 1 (SNAI1). Experiments testing EMT gene activation and inhibition with recombinant WISP1 or kinase inhibitors in B16F10 and YUMM1.7 cells suggested that WISP1 activates AKT Ser/Thr kinase and that MEK/ERK signaling pathways shift melanoma cells from proliferation to invasion. Our results indicate that WISP1 present within the tumor microenvironment stimulates melanoma invasion and metastasis by promoting an EMT-like process.

WNT1-inducible signaling protein-1 mediates TGF-ß1-induced renal fibrosis in tubular epithelial cells and unilateral ureteral obstruction mouse models via autophagy.

Renal fibrosis is a common pathway for the progression of all chronic kidney diseases to end-stage kidney disease. Studies show that WNT1-inducible signaling pathway protein-1 (WISP-1) is involved in the fibrosis of various organs. The aim of the study was to explore the functional role and potential mechanism of WISP-1 in renal fibrosis. We observed that overexpression of WISP-1 in rat tubular epithelial cells (TECs) enhanced transforming growth factor-ß1 (TGF-ß1)-induced production of fibrotic markers, including collagen I (Col I), fibronectin (FN) and TGF-ß1, while inhibition of WISP-1 suppressed such production. In vivo, the messenger RNA and protein levels of Col I, FN, and α-smooth muscle actin were significantly inhibited after anti-WISP-1 antibody treatment for 7 days in unilateral ureteral obstruction mouse models. Moreover, blockade of WISP-1 by anti-WISP-1 antibody significantly reduced autophagy-related markers, including anti-microtubule-associated protein-1 light chain 3 (LC3) and beclin 1, while increasing sequestosome 1. In addition, overexpression of WISP-1 in TECs increased autophagy as evidenced by greater numbers of GFP-LC3 puncta and increased expression of LC3 and beclin 1 in response to TGF-ß1. In contrast, knockdown of WISP-1 by small interfering RNA decreased the number of GFP-LC3 puncta and the expression of LC3 and beclin 1 in TGF-ß1-treated TECs. Collectively, these data suggest that WISP-1, as a profibrotic protein, may mediate renal fibrosis by inducing autophagy in both obstructive nephropathy and TGF-ß1-treated TECs. WISP-1 may serve as an effective therapeutic target for the treatment of renal fibrosis.

Circ_KATNAL1 regulates prostate cancer cell growth and invasiveness through the miR-145-3p/WISP1 pathway.

Prostate cancer (PCa) is the second leading cause of death in men, and current studies have shown that circular RNAs (circRNAs) play important roles in its occurrence and development. Detection of circRNAs in PCa cells showed that circ_KATNAL1 is down-regulated, mainly located in the cytoplasm, and contains multiple binding sites of miR-145-3p, which is an anticancer miRNA. RNA immunoprecipitation with anti-AGO2 antibody, RNA pull-down assays with biotin-labeled circ_KATNAL1 probe or an miR-145-3p mimic, and dual luciferase reporter gene assays confirmed that circ_KATNAL1 binds directly to miR-145-3p in cells, and that WISP1, which is highly expressed in many types of tumors, is an important target gene of miR-145-3p. Circ_KATNAL1 and miR-145-3p promote each other's expression, and down-regulate the expression of the target gene WISP1. Both circ_KATNAL1 and miR-145-3p inhibit cell proliferation, invasiveness, and migration, down-regulate the expression of MMP-2 and MMP-9, promote cell apoptosis and the activation of caspase-3, caspase-8, caspase-9, and PARP, whereas WISP1 has the opposite effect, and the above-mentioned functions of circ_KATNAL1 were achieved through the miR-145-3p/WISP1 pathway. Therefore, circ_KATNAL1 plays an anticancer role in PCa cells through the miR-145-3p/WISP1 pathway, which could be an important target for the diagnosis and treatment of PCa.

WISP1 promotes bovine MDSC differentiation via recruitment of ANXA1 for the regulation of the TGF-ß signalling pathway.

Skeletal muscle is one of the most important tissues of the human body necessary for sporting activities. The differentiation of muscle-derived satellite cells (MDSCs) plays an important role in the development and regeneration of skeletal muscles. Similarly, the Wnt/ß-catenin signalling pathway plays an important role in the process of muscle differentiation. Wnt1-inducible signalling pathway protein-1 (WISP1), a downstream protein of the Wnt/ß-catenin signalling pathway and a member of the CCN family that also plays an important role in the differentiation process, and its expression increase during the differentiation of bovine MDSCs. However, its role in MDSC differentiation is poorly understood. Therefore, we investigated the mechanisms regulating this process via Western blot and immunofluorescence staining. Immunoprecipitation and mass spectrometry detected annexin A1 (ANXA1), a protein that interacts with WISP1. To determine whether WISP1 influences TGF-ß signalling and differentiation independently of ANXA1, the latter was knocked down, while WISP1 was activated. WISP1 expression increased significantly during bovine MDSC differentiation. However, WISP1 did not affect the TGF-ß signalling pathway protein marker when ANXA1 was inhibited. Taken together, WISP1 regulates the TGF-ß signalling pathway through ANXA1 recruitment, thereby promoting bovine MDSC differentiation, suggesting the Wnt/ß-catenin signalling pathway as another target to promote cell differentiation.

WISP1 silencing confers protection against epithelial-mesenchymal transition of renal tubular epithelial cells in rats via inactivation of the wnt/ß-catenin signaling pathway in uremia.

Uremia can affect hepatic metabolism of drugs by regulating the clearance of drugs, but it has not been clarified whether gene silencing could modulate the epithelial-mesenchymal transition (EMT) process in uremia. Hence, we investigated the effect of WISP1 gene silencing on the renal tubular EMT in uremia through the wnt/ß-catenin signaling pathway. Initially, microarray-based gene expression profiling of uremia was used to identify differentially expressed genes. Following the establishment of uremia rat model, serum creatinine, and urea nitrogen of rats were detected. Renal tubular epithelial cells (TECs) were transfected with shRNA-WISP1 lentivirus interference vectors and LiCI (the wnt/ß-catenin signaling pathway activator) to explore the regulatory mechanism of WISP1 in uremia in relation to the wnt/ß-catenin signaling pathway. Then, expression of WISP1, wnt2b, E-cadherin, α-SMA, c-myc, Cyclin D1, MMP-2, and MMP-9 was determined. Furthermore, TEC migration and invasion were evaluated. Results suggested that WISP1 and the wnt/ß-catenin signaling pathway were associated with uremia. Uremic rats exhibited increased serum creatinine and urea nitrogen levels, upregulated WISPl, and activated wnt/ß-catenin signaling pathway. Subsequently, WISP1 silencing decreased wnt2b, c-myc, Cyclin D1, α-SMA, MMP-2, and MMP-9 expression but increased E-cadherin expression, whereas LiCI treatment exhibited the opposite trends. In addition, WISP1 silencing suppressed TEC migration and invasion, whereas LiCI treatment promoted TEC migration and invasion. The findings indicate that WISP1 gene silencing suppresses the activation of the wnt/ß-catenin signaling pathway, thus reducing EMT of renal TECs in uremic rats.