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 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.

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.

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.

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.

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.

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.

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.

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.

WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway.

Inflammation is a pathological feature of kidney injury and its progression correlates with the development of kidney fibrosis which can lead to kidney function impairment. This project investigated the regulatory function of WNT1-inducible signaling pathway protein 1 (WISP1) in kidney inflammation. Administration of recombinant WISP1 protein to healthy mice induced kidney inflammation (macrophage accrual and production of tumor necrosis factor α (TNF-α), CCL2 and IL-6), which could be prevented by inhibition of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). Furthermore, inhibition of WISP1, by gene knockdown or neutralising antibody, could inhibit cultured macrophages producing inflammatory cytokines following stimulation with lipopolysaccharides (LPSs) and kidney fibroblasts proliferating in response to TNFα, which both involved NF-κB signaling. Kidney expression of WISP1 was found to be increased in mouse models of progressive kidney inflammation-unilateral ureter obstruction (UUO) and streptozotocin (STZ)-induced diabetic nephropathy (DN). Treatment of UUO mice with WISP1 antibody reduced the kidney inflammation in these mice. Therefore, pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting inflammation in kidney disease.

The small-secreted cysteine-rich protein CyrA is a virulence factor participating in the attack of Caenorhabditis elegans by Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a diverse and intriguing group of fungi that live saprotrophically but can switch to a predatory lifestyle when starving and in the presence of nematodes. NTF like Arthrobotrys oligospora or Duddingtonia flagrans produce adhesive trapping networks to catch and immobilize nematodes. After penetration of the cuticle, hyphae grow and develop inside the worm and secrete large amounts of hydrolytic enzymes for digestion. In many microbial pathogenic interactions small-secreted proteins (SSPs) are used to manipulate the host. The genome of D. flagrans encodes more than 100 of such putative SSPs one of which is the cysteine-rich protein CyrA. We have chosen this gene for further analysis because it is only found in NTF and appeared to be upregulated during the interaction. We show that the cyrA gene was transcriptionally induced in trap cells, and the protein accumulated at the inner rim of the hyphal ring before Caenorhabditis elegans capture. After worm penetration, the protein appeared at the fungal infection bulb, where it is likely to be secreted with the help of the exocyst complex. A cyrA-deletion strain was less virulent, and the time from worm capture to paralysis was extended. Heterologous expression of CyrA in C. elegans reduced its lifespan. CyrA accumulated in C. elegans in coelomocytes where the protein possibly is inactivated. This is the first example that SSPs may be important in predatory microbial interactions.

Activation of Frizzled-7 attenuates blood-brain barrier disruption through Dvl/ß-catenin/WISP1 signaling pathway after intracerebral hemorrhage in mice.

BACKGROUND: Destruction of blood-brain barrier (BBB) ​​is one of the main mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Frizzled-7 is a key protein expressed on the surface of endothelial cells that controls vascular permeability through the Wnt-canonical pathway involving WNT1-inducible signaling pathway protein 1 (WISPI). This study aimed to investigate the role of Frizzled-7 signaling in BBB preservation after ICH in mice. METHODS: Adult CD1 mice were subjected to sham surgery or collagenase-induced ICH. Frizzled-7 activation or knockdown was performed by administration of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) by intracerebroventricular injection at 48 h before ICH induction. WISP1 activation or WISP1 knockdown was performed to evaluate the underlying signaling pathway. Post-ICH assessments included neurobehavior, brain edema, BBB permeability, hemoglobin level, western blot and immunofluorescence. RESULTS: The brain expressions of Frizzled-7 and WISP1 significantly increased post-ICH. Frizzled-7 was expressed in endothelial cells, astrocytes, and neurons after ICH. Activation of Frizzled-7 significantly improved neurological function, reduced brain water content and attenuated BBB permeability to large molecular weight substances after ICH. Whereas, knockdown of Frizzled-7 worsened neurological function and brain edema after ICH. Activation of Frizzled-7 significantly increased the expressions of Dvl, ß-Catenin, WISP1, VE-Cadherin, Claudin-5, ZO-1 and reduced the expression of phospho-ß-Catenin. WISP1 knockdown abolished the effects of Frizzled-7 activation on the expressions of VE-Cadherin, Claudin-5 and ZO-1 at 24 h after ICH. CONCLUSIONS: Frizzled-7 activation potentially attenuated BBB permeability and improved neurological deficits after ICH through Dvl​​/ß-Catenin/WISP1 pathway. Frizzled-7 may be a potential target for the development of ICH therapeutic drugs.

Dual roles of WISP2 in the progression of hepatocellular carcinoma: implications of the fibroblast infiltration into the tumor microenvironment.

The dismal outcome of hepatocellular carcinoma (HCC) patients is attributable to high frequency of metastasis and. Identification of effective biomarkers is a key strategy to inform prognosis and improve survival. Previous studies reported inconsistent roles of WISP2 in carcinogenesis, while the role of WISP2 in HCC progression also remains unclear. In this study, we confirmed that WISP2 was downregulated in HCC tissues, and WISP2 was acting as a protective factor, especially in patients without alcohol intake using multiple online datasets. In addition, we reported that upregulation of WISP2 in HCC was related to inhibition of the malignant phenotype in vitro, but these alterations were not observed in vivo. WISP2 also negatively correlated with tumour purity, and increased infiltration of fibroblasts promoted malignant progression in HCC tissues. The enhanced infiltration ability of fibroblasts was related to upregulated HMGB1 after overexpression of WISP2 in HCC. The findings shed light on the anticancer role of WISP2, and HMGB1 is one of the key factors involved in the inhibition of the efficiency of WISP2 through reducing the tumour purity with fibroblast infiltration.

Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis.

Collagen remodeling contributes to many physiologic and pathologic processes. In primary tumors, the linearization of collagen fibers promotes cancer cell invasion and metastasis and is indicative of poor prognosis. However, it remains unknown whether there are endogenous inhibitors of collagen linearization that could be exploited therapeutically. Here, we show that collagen linearization is controlled by two secreted matricellular proteins with antagonistic functions. Specifically, WISP1 was secreted by cancer cells, bound to type I collagen (Col I), and linearized Col I via its cysteine-rich C-terminal (CT) domain. In contrast, WISP2, which lacks a CT domain, inhibited Col I linearization by preventing WISP1-Col I binding. Analysis of patient data revealed that WISP2 expression is lower in most solid tumors, in comparison with normal tissues. Consequently, genetic or pharmacologic restoration of higher WISP2 levels impaired collagen linearization and prevented tumor cell invasion and metastasis in vivo in models of human and murine breast cancer. Thus, this study uncovers WISP2 as the first inhibitor of collagen linearization ever identified and reveals that collagen architecture can be normalized and metastasis inhibited by therapeutically restoring a high WISP2:WISP1 ratio. SIGNIFICANCE: Two secreted factors, WISP1 and WISP2, antagonistically regulate collagen linearization, and therapeutically increasing the WISP2:WISP1 ratio in tumors limits collagen linearization and inhibits metastasis.See related commentary by Barcus and Longmore, p. 5611.

Hepatic Wnt1 Inducible Signaling Pathway Protein 1 (WISP-1/CCN4) Associates with Markers of Liver Fibrosis in Severe Obesity.

Liver fibrosis is a critical complication of obesity-induced fatty liver disease. Wnt1 inducible signaling pathway protein 1 (WISP1/CCN4), a novel adipokine associated with visceral obesity and insulin resistance, also contributes to lung and kidney fibrosis. The aim of the present study was to investigate the role of CCN4 in liver fibrosis in severe obesity. For this, human liver biopsies were collected from 35 severely obese humans (BMI 42.5 ± 0.7 kg/m2, age 46.7 ± 1.8 y, 25.7% males) during bariatric surgery and examined for the expression of CCN4, fibrosis, and inflammation markers. Hepatic stellate LX-2 cells were treated with human recombinant CCN4 alone or in combination with LPS or transforming growth factor beta (TGF-ß) and examined for fibrosis and inflammation markers. CCN4 mRNA expression in the liver positively correlated with BMI and expression of fibrosis markers COL1A1, COL3A1, COL6A1, αSMA, TGFB1, extracellular matrix turnover enzymes TIMP1 and MMP9, and the inflammatory marker ITGAX/CD11c. In LX-2 cells, the exposure to recombinant CCN4 caused dose-dependent induction of MMP9 and MCP1. CCN4 potentiated the TGF-ß-mediated induction of COL3A1, TIMP1, and MCP1 but showed no interaction with LPS treatment. Our results suggest a potential contribution of CCN4 to the early pathogenesis of obesity-associated liver fibrosis.

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung.

BACKGROUND: Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. METHODS: We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. RESULTS: We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. CONCLUSION: Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

CCN5 activation by free or encapsulated EGCG is required to render triple-negative breast cancer cell viability and tumor progression.

Epigallocatechin-3-gallate (EGCG) has been considered an anticancer agent despite conflicting and discrepant bioavailability views. EGCG impairs the viability and self-renewal capacity of triple-negative breast cancer (TNBC) cells and makes them sensitive to estrogen via activating ER-α. Surprisingly, the mechanism of EGCG's action on TNBC cells remains unclear. CCN5/WISP-2 is a gatekeeper gene that regulates viability, ER-α, and stemness in TNBC and other types of cancers. This study aimed to investigate whether EGCG (free or encapsulated in nanoparticles) interacts with the CCN5 protein by emphasizing its bioavailability and enhancing its anticancer effect. We demonstrate that EGCG activates CCN5 to inhibit in vitro cell viability through apoptosis, the sphere-forming ability via reversing TNBC cells' stemness, and suppressing tumor growth in vivo. Moreover, we found EGCG-loaded nanoparticles to be functionally more active and superior in their tumor-suppressing ability than free-EGCG. Together, these studies identify EGCG (free or encapsulated) as a novel activator of CCN5 in TNBC cells and hold promise as a future therapeutic option for TNBC with upregulated CCN5 expression.