The matricellular protein CCN5 prevents anti-VEGF drug-induced epithelial-mesenchymal transition of retinal pigment epithelium.

Age-related macular degeneration (AMD) is one of the major causes of blindness in the elderly worldwide. Anti-vascular endothelial growth factor (VEGF) drugs have been widely used to treat the neovascular type of AMD (nAMD). However, VEGF acts not only as a pro-angiogenic factor but also as an anti-apoptotic factor in the eyes. In this study, we found that anti-VEGF drugs, including bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), induced epithelial-mesenchymal transition (EMT) in ARPE-19 cells in vitro, accompanied by the induction of CCN2, a potent pro-fibrotic factor. Similarly, intravitreal injection of Afl into mouse eyes resulted in EMT in the retinal pigmented epithelium (RPE). Co-treatment with CCN5, an anti-fibrotic factor that down-regulates CCN2 expression, significantly attenuated the adverse effects of the anti-VEGF drugs both in vitro and in vivo. Inhibition of the VEGF signaling pathway with antagonists of VEGF receptors, SU5416 and ZM323881, induced EMT and up-regulated CCN2 in ARPE-19 cells. Additionally, knock-down of CCN2 with siRNA abolished the adverse effects of the anti-VEGF drugs in ARPE-19 cells. Collectively, these results suggest that anti-VEGF drugs induce EMT in RPE through the induction of CCN2 and that co-treatment with CCN5 attenuates the adverse effects of anti-VEGF drugs in mouse eyes.

WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts' Activation and Collagen Processing.

Hypertension induces cardiac fibrotic remodelling characterised by the phenotypic switching of cardiac fibroblasts (CFs) and collagen deposition. We tested the hypothesis that Wnt1-inducible signalling pathway protein-1 (WISP-1) promotes CFs' phenotypic switch, type I collagen synthesis, and in vivo fibrotic remodelling. The treatment of human CFs (HCFs, n = 16) with WISP-1 (500 ng/mL) induced a phenotypic switch (α-smooth muscle actin-positive) and type I procollagen cleavage to an intermediate form of collagen (pC-collagen) in conditioned media after 24h, facilitating collagen maturation. WISP-1-induced collagen processing was mediated by Akt phosphorylation via integrin ß1, and disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2). WISP-1 wild-type (WISP-1+/+) mice and WISP-1 knockout (WISP-1-/-) mice (n = 5-7) were subcutaneously infused with angiotensin II (AngII, 1000 ng/kg/min) for 28 days. Immunohistochemistry revealed the deletion of WISP-1 attenuated type I collagen deposition in the coronary artery perivascular area compared to WISP-1+/+ mice after a 28-day AngII infusion, and therefore, the deletion of WISP-1 attenuated AngII-induced cardiac fibrosis in vivo. Collectively, our findings demonstrated WISP-1 is a critical mediator in cardiac fibrotic remodelling, by promoting CFs' activation via the integrin ß1-Akt signalling pathway, and induced collagen processing and maturation via ADAMTS-2. Thereby, the modulation of WISP-1 levels could provide potential therapeutic targets in clinical treatment.

Modified mRNA-Mediated CCN5 Gene Transfer Ameliorates Cardiac Dysfunction and Fibrosis without Adverse Structural Remodeling.

Modified mRNAs (modRNAs) are an emerging delivery method for gene therapy. The success of modRNA-based COVID-19 vaccines has demonstrated that modRNA is a safe and effective therapeutic tool. Moreover, modRNA has the potential to treat various human diseases, including cardiac dysfunction. Acute myocardial infarction (MI) is a major cardiac disorder that currently lacks curative treatment options, and MI is commonly accompanied by fibrosis and impaired cardiac function. Our group previously demonstrated that the matricellular protein CCN5 inhibits cardiac fibrosis (CF) and mitigates cardiac dysfunction. However, it remains unclear whether early intervention of CF under stress conditions is beneficial or more detrimental due to potential adverse effects such as left ventricular (LV) rupture. We hypothesized that CCN5 would alleviate the adverse effects of myocardial infarction (MI) through its anti-fibrotic properties under stress conditions. To induce the rapid expression of CCN5, ModRNA-CCN5 was synthesized and administrated directly into the myocardium in a mouse MI model. To evaluate CCN5 activity, we established two independent experimental schemes: (1) preventive intervention and (2) therapeutic intervention. Functional analyses, including echocardiography and magnetic resonance imaging (MRI), along with molecular assays, demonstrated that modRNA-mediated CCN5 gene transfer significantly attenuated cardiac fibrosis and improved cardiac function in both preventive and therapeutic models, without causing left ventricular rupture or any adverse cardiac remodeling. In conclusion, early intervention in CF by ModRNA-CCN5 gene transfer is an efficient and safe therapeutic modality for treating MI-induced heart failure.

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.

Silencing of METTL3 prevents the proliferation, migration, epithelial-mesenchymal transition, and renal fibrosis of high glucose-induced HK2 cells by mediating WISP1 in m6A-dependent manner.

Diabetic nephropathy (DN) is one of the most serious complications in diabetic patients. And m6A modifications mediated by METTL3 are involved multiple biological processes. However, the specific function and mechanism of METTL3 in DN remains unclear. DN model mice were first established with streptozotocin, and WISP1 expression was confirmed by qRT-PCR. Then the influences of WISP1 or/and METTL3 on the proliferation, migration, and epithelial-mesenchymal transition (EMT) and fibrosis-related proteins of high glucose (HG)-induced HK2 cells or HK2 cells were tested through CCK-8, wound healing, and western blot. We first revealed that WISP1 was highly expressed in renal tissues of DN model mice and HG-induced HK2 cells. Functionally, WISP1 or METTL3 silencing could weaken the proliferation, migration, EMT, and fibrosis of HG-treated HK2 cells, and WISP1 or METTL3 overexpression could induce the proliferation, migration, EMT, and fibrosis of HK2 cells. Additionally, METTL3 silencing could decrease WISP1 m6A modification, and silencing of METTL3 also could notably suppress the biological functions of HG-induced HK2 cells by downregulating WISP1. Silencing of METTL3 prevents DN development process by decreasing WISP1 with m6A modification pattern. Therefore, we suggest that METTL3/WISP1 axis might be a novel therapeutic target for DN.

OTUB1 inhibits breast cancer by non-canonically stabilizing CCN6.

BACKGROUND: CCN6 is a matricellular protein that critically regulates the tumourigenesis and progression of breast cancer. Although the tumour-suppressive function of CCN6 has been extensively studied, molecular mechanisms regulating protein levels of CCN6 remain largely unclear. This study aims to investigate the regulation of CCN6 by ubiquitination and deubiquitinating enzymes (DUBs) in breast cancer. METHODS: A screening assay was performed to identify OTUB1 as the DUB for CCN6. Various biochemical methods were applied to elucidate the molecular mechanism of OTUB1 in the regulation of CCN6. The role of OTUB1-CCN6 interaction in breast cancer was studied with cell experiments and the allograft model. The correlation of OTUB1 and CCN6 in human breast cancer was determined by immunohistochemistry and Western blot. RESULTS: We found that CCN6 protein levels were controlled by the ubiquitin-proteasome system. The K48 ubiquitination and degradation of CCN6 was inhibited by OTUB1, which directly interacted with CCN6 through its linker domain. Furthermore, OTUB1 inhibited the ubiquitination of CCN6 in a non-canonical manner. Deletion of OTUB1, concomitant with reduced CCN6 abundance, increased the migration, proliferation and viability of breast cancer cells. Supplementation of CCN6 abolished the effect of OTUB1 deletion on breast cancer. Importantly, OTUB1 expression was downregulated in human breast cancer and positively correlated with CCN6 levels. CONCLUSION: This study identified OTUB1 as a novel regulator of CCN6 in breast cancer.

The matricellular protein Drosophila Cellular Communication Network Factor is required for synaptic transmission and female fertility.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member. Drosophila cellular communication network factor is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. Drosophila cellular communication network factor-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of Drosophila cellular communication network factor results in reduced female fertility. In addition, Drosophila cellular communication network factor accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila cellular communication network factor family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity.

The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

CCN Proteins (Cellular Communication Network Factors): Expanding Their Repertoire Toward a New Concept.

I herein report the general structures and functions of CCN proteins and possible molecular mechanisms involved in the unique biological actions of this family of intercellular signaling regulators, which are considered matricellular proteins and were once referred to as "signal conductors" but have recently been renamed "Cellular Communication Network Factors." Their repertoire of functions beyond their role as matricellular proteins is also described to aid in future studies. Advanced research concerning their relevance to pathology is briefly introduced as well. The information provided in this chapter is expected to be useful for readers of subsequent chapters.

Optimal CCN4 Immunofluorescence for Tissue Microarray.

CCN4 (also known as WNT1-Inducible Signaling Pathway Protein 1 or WISP1) is a 367 amino acid, 40 kDa protein located on chromosome 8q24.1-8q24.3. Prior studies have provided support for a pro-inflammatory role for CCN4. We have shown recently that CCN4 expression is associated with advanced disease, epithelial-mesenchymal transition, and an inflamed tumor microenvironment in multiple solid tumors. We detail here the CCN4 tissue microarray immunofluorescence protocol related to these findings.

Cellular Fluorescence Imaging for the Evaluation of Bioactivity of CCN Family Proteins.

The method of labeling proteins of interest with fluorescent dyes that can specifically stain organelles in living cells provides a tool for investigating various cellular processes under a microscope. Visualization (imaging) of the cells using fluorescence has many advantages, including the ability to stain multiple cell organelles and intracellular proteins simultaneously and discriminately, and is used in many research fields. In this chapter, we describe the observation of cell organelles using fluorescence staining to analyze the functions of CCN family proteins involved in various cellular events.

Western Blot Protocols for Analysis of CCN Proteins and Fragments in Exosomes, Vesicle-Free Fractions, and Cells.

Cellular Communication Network (CCN) proteins are growth factors that play key roles in many pathophysiological events, including bone formation, wound healing, and cancer. CCN factors and fragments generated by metalloproteinases-dependent cleavage are often associated with extracellular matrix (ECM) or small extracellular vesicles (sEVs) such as exosomes or matrix-coated vesicles. We provide reliable methods and protocols for Western blotting to analyze CCN factors and fragments in cells, sEVs, and vesicle-free fractions.

Protocols for Screening Peptides Binding to CCN Family Proteins and Their Extended Utility.

The function of CCN family proteins is determined by their interactions with multiple cofactors that are present in the microenvironment. Therefore, determining these cofactors is critically important in understanding the molecular function of CCN family members. For this objective, a bacteriophage random peptide display library is a suitable tool. In this library, each filamentous bacteriophage is designed to display an oligopeptide of 7-20 random amino acid residues on its surface. Bacteriophage clones that possess peptides that bind to a CCN family protein are selected through several cycles of a process called biopanning or affinity selection. By determining the nucleotide sequence of the DNA that encodes the displayed peptide, the oligopeptides that specifically bind to the CCN family member can be specified. The obtained peptide sequences can be utilized to design peptide aptamers for CCN family proteins, or as a key sequence to determine new CCN family cofactor candidates in silico. Instead of a random peptide cDNA library, an antibody cDNA library from naïve lymphocytes or from B cells immunized by a CCN family protein can be used in order to obtain a highly specific CCN family detection or functional modulation tool.

Analysis of CCN4/WISP1 Effects on Joint Tissues Using Gain- and Loss-of-Function Approaches.

The matricellular protein Wnt-induced secreted protein 1 (WISP1) is the fourth member of the CCN family of proteins, which has been shown to affect tissues of the musculoskeletal system. In the context of the musculoskeletal disorder osteoarthritis, our lab studied the function of CCN4/WISP1 in joint tissues, including synovium and cartilage, using both gain- and loss-of-function approaches. In mice, this was done by genetic engineering and recombination to generate mice deficient in CCN4/WISP1 protein. Various experimental models of osteoarthritis with different characteristics were induced in these mice. Moreover, CCN4/WISP1 levels in joints were experimentally increased by adenoviral transfections. Osteoarthritis pathology was determined using histology, and the effect of different CCN4/WISP1 levels on gene expression was evaluated in individual tissues. Effects of high levels of CCN4/WISP1 on chondrocytes were studied with an in vitro chondrocyte pellet model. In this chapter, we describe the procedures to conduct these experiments.

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.

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.

The matricellular protein CCN5 induces apoptosis in myofibroblasts through SMAD7-mediated inhibition of NFκB.

We previously showed that the matricellular protein CCN5 reverses established cardiac fibrosis (CF) through inducing apoptosis in myofibroblasts (MyoFBs) but not in cardiomyocytes or fibroblasts (FBs). In this study, we set out to elucidate the molecular mechanisms underlying CCN5-mediated selective apoptosis of MyoFBs. We first observed that the apoptotic protein p53 and the anti-apoptotic protein NFκB are simultaneously induced in MyoFBs. When the expression level of p53 was suppressed using a siRNA, CCN5 did not induce apoptosis in MyoFBs. By contrast, when NFκB signaling was inhibited using IKK VII, an IκB inhibitor, MyoFBs underwent apoptosis even in the absence of CCN5. SMAD7 is one of the downstream targets of CCN5 and it was previously shown to potentiate apoptosis in epithelial cells through inhibition of NFκB. In accordance with these reports, when the expression of SMAD7 was suppressed using a siRNA, NFκB signaling was enhanced, and CCN5 did not induce apoptosis. Lastly, we used a luciferase reporter construct to show that CCN5 positively regulated SMAD7 expression at the transcriptional level. Collectively, our data suggest that a delicate balance between the two mutually antagonistic proteins p53 and NFκB is maintained for MyoFBs to survive, and CCN5 tips the balance in favor of the apoptotic protein p53. This study provides insight into the anti-fibrotic activity of CCN5 during the regression of CF.

WISP1 induces ovarian cancer via the IGF1/αvß3/Wnt axis.

BACKGROUND: This study intended to clarify the mechanisms by which WISP1-mediated IGF1/αvß3/Wnt axis might affect the progression of ovarian cancer. METHODS: Bioinformatics analysis was implemented for pinpointing expression of IGF1 and WISP1 which was verified through expression determination in clinical tissue samples and cells. Next, gain- or loss-of-function experimentations were implemented for testing CAOV4 and SKOV3 cell biological processes. The interaction between WISP1 and IGF1 was verified by co-immunoprecipitation and the molecular mechanism was analyzed. Finally, ovarian cancer nude mouse models were prepared to unveil the in vivo effects of WISP1/IGF1. RESULTS: IGF1 and WISP1 expression was elevated in ovarian cancer tissues and cells, which shared correlation with poor prognosis of ovarian cancer sufferers. Elevated IGF1 induced malignant properties of ovarian cancer cells through activation of PI3K-Akt and Wnt signaling pathway. WISP1 was positively correlated with IGF1. WISP1 could enhance the interaction between IGF1 and αvß3 to induce epithelial-mesenchymal transition. In vivo experiments also confirmed that upregulated WISP1/IGF1 induced tumorigenesis and metastasis of ovarian cancer cells. CONCLUSION: In conclusion, WISP1 can facilitate ovarian cancer by activating Wnt via the interaction between IGF1 and αvß3.

WNT1-inducible signalling pathway protein 1 stabilizes atherosclerotic plaques in apolipoprotein-E-deficient mice via the focal adhesion kinase/mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase pathway.

BACKGROUND: The migration, proliferation and apoptosis of vascular smooth muscle cells (VSMCs) are critical for plaque stability. WNT-inducible signalling pathway protein-1 (WISP1), a member of the CCN family of extracellular matrix proteins, can expedite the migration and proliferation of VSMCs. However, its underlying mechanism and relationship with atherosclerosis remain elusive. The relationship between WISP1 and apoptosis of VSMCs has not been determined previously. METHOD: In the study, we aimed to investigate the relationship between WISP1 and plaque stability and its related mechanism.ApoE-/- mice were divided following groups: the null lentivirus (NC), lentivirus WISP1 (IvWISP1) and WISP1-shRNA (shWISP1) groups. Immunofluorescence, Oil Red O and Masson's staining of the carotid arteries were performed. Transwell wound healing assay, CCK8 assay, and TdT-mediated dUTP nick-end labeling (TUNEL) staining were performed using VSMCs. The levels of WISP1, P38, C-Jun N-terminal kinase, extracellular signal-regulated kinase (ERK), mitogen-activated extracellular signal-regulated kinase (MEK), focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt (also known as PKB, protein kinase B), mammalian target of rapamycin (mTOR), cleaved caspase3, Bcl2 and Bax were detected by western blotting. RESULTS: The relative area of lipids and monocytes/macrophages in the shWISP1 group increased compared with that of the NC group. However, the relative area of smooth muscle cell and collagen in the IvWISP1 group increased compared with that in the NC group. Therefore, WISP1 could stabilize atherosclerotic plaques. Besides, WISP1 accelerate the migration and proliferation of VSMCs via integrin α5ß1 and FAK/MEK/ERK signalling pathways. In addition, WISP1 can inhibit the apoptosis of VSMCs via the PI3K/Akt/mTOR pathway. CONCLUSION: WISP1 not only inhibits the apoptosis of VSMCs via the PI3K/Akt/mTOR pathway but also enhances the migration and proliferation of VSMCs via the integrin α5ß1 and FAK/MEK/ERK pathways. Therefore, WISP1 could enhance the stability of atherosclerotic plaques.

WNT1 expression influences the development of dysplasia of the hip via regulating RBPMS2/NOG-BMP2/4-GDF5- WISP2 pathway.

OBJECTIVE: To explore the role of WNT family member 1 (WNT1) in the development of dysplasia of the hip (DDH) and the molecular mechanism involved in this process. Methods: Si-WNT1, pcDNA3.1-WNT1 or corresponding negative controls were transfected into human osteoblast hFOB1.19 and human chondrocyte C28/I2, respectively. The proliferation of cells was measured by EdU assay. The relative expressions of human noggin gene (NOG), growth differentiating factor 5 (GDF5), WNT1, and WNT1-inducible-signaling pathway protein 2 (WISP2) were determined by immunofluorescence analysis. The protein expressions of RNA-binding protein of multiple splice forms 2 (RBPMS2), NOG, bone morphogenetic protein 2 (BMP2), BMP4, WNT1 and WISP2 were determined by western blot. Animal experiment was also performed and the morphological development of hip joint was observed. Results: Overexpression of WNT1 promoted osteoblast proliferation and inhibited chondrocyte proliferation, while knockdown of WNT1 inhibited osteoblast proliferation. In chondrocytes, knockdown of WNT1 upregulated NOG expression, while overexpression of WNT1 downregulated its expression. In osteoblasts and chondrocytes, overexpression of WNT1 increased BMP2, BMP4, WNT1, and WISP2 expression. RBPMS2 and NOG were slightly expressed in each group. Conclusion: Overexpression of WNT1 promoted osteoblast proliferation, inhibited chondrocyte proliferation, and increased the expressions of BMP2, BMP4, WNT1, and WISP2. Therefore, WNT1 may be a new therapeutic target for DDH.