CCN Family Of Matricellular Proteins Research Articles

P147 M2 macrophage associated fibrosis in early stage systemic sclerosis: an update on plasma biomarkers

Abstract Background/Aims Systemic sclerosis is an autoimmune connective tissue disease characterised by vasculopathy and fibrosis. The CCN family of matricellular proteins have a regulatory role in inflammation and fibrosis. CD206, an M2 macrophage surface marker, is cleaved by metalloproteases to release soluble form of CD206; this acts as an active disease marker in systemic sclerosis. We investigated the profile of CCN2 and CCN3 in the presence of soluble CD206 (sCD206) in patients with early and late stage diffuse systemic sclerosis (dcSSc). Methods Patients with a diagnosis of dcSSc at the UCL Centre for Rheumatology and Connective Tissue Diseases were recruited. Informed consent was obtained from patients and healthy controls (HC). Blood samples were taken from which plasma was isolated and stored at -80 °C prior to assay. Expression of CCN2, CCN3, and sCD206 was measured in these plasma samples by ELISA. Study participants were categorised into healthy controls (n = 20), early stage dcSSc (n = 20) and late dcSSc (n = 20), with early stage defined as the blood sample taken within two years since diagnosis, and late stage defined as over five years since diagnosis. Results Plasma CCN2, CCN3 and sCD206 levels were significantly higher in early dcSSc patients, when compared to HC. CCN2 was raised in early dcSSc patients (early dcSSc mean 20,221pg/ml, SD 16,503, p < 0.05) with concentration in late disease matching that of HC (late dcSSc 12,893pg/ml, HC 11,289pg/ml). CCN3 was significantly higher in early and late disease when compared to HC (early mean 16,526pg/ml, late 12,838pg/ml, HC 4,201pg/ml, p < 0.05). sCD206 levels were higher in early dcSSc (mean 5356pg/ml, SD 3154, p < 0.05), with there being no significance between levels in late dcSSc (mean 4184pg/ml, SD 3900, p > 0.05) and HC (mean 3319pg/ml, SD 1925). CCN2 expression was higher compared to that of CCN3 in early disease, with CCN2:CCN3 ratio elevated in early dcSSc (mean 3.17, SD 8.26) compared to late dcSSc (mean 1.26, SD 0.99) and HC. Conclusion This investigation demonstrates that upregulation of CCN2 and CCN3 expression has a regulatory role in fibrosis in early stage dcSSc. These biomarkers of M2 macrophage associated fibrosis are to be correlated with their clinical phenotype and have potential for a role in targeted treatment of systemic sclerosis. Disclosure L.A. Leeves: None. Z. Kannan: None. Y. Ali: None. L. Pan: None. T. Malley: None. D. Abraham: None. A. Leask: None. B. Riser: None. B. Ahmed Abdi: None. R.J. Stratton: None.

CYR61 confers chemoresistance by upregulating survivin expression in triple-negative breast cancer.

Cysteine-rich angiogenic inducer 61 (CYR61) is a protein from the CCN family of matricellular proteins that play diverse regulatory roles in the extracellular matrix. CYR61 is involved in cell adhesion, migration, proliferation, differentiation, apoptosis, and senescence. Here, we show that CYR61 induces chemoresistance in triple-negative breast cancer (TNBC). We observed that CYR61 is overexpressed in TNBC patients, and CYR61 expression correlates negatively with the survival of patients who receive chemotherapy. CYR61 knockdown reduced cell migration, sphere formation, and the cancer stem cell (CSC) population and increased the chemosensitivity of TNBC cells. Mechanistically, CYR61 activated Wnt/β-catenin signaling and increased survivin expression, which are associated with chemoresistance, the epithelial-mesenchymal transition, and CSC-like phenotypes. Altogether, our study demonstrates a novel function of CYR61 in chemotherapy resistance in breast cancer.

A Comprehensive Study on the Association between Plasma NOV/CCN3 Levels and Insulin Resistance in Childhood Obesity

Introduction: Childhood obesity is a global health problem that is associated with various metabolic complications, such as insulin resistance, type 2 diabetes, dyslipidemia, and cardiovascular diseases. The mechanisms underlying the development of insulin resistance in childhood obesity are not fully understood. Nephroblastoma overexpressed gene (NOV), also known as CCN3, is a member of the CCN family of matricellular proteins that modulate cell proliferation, differentiation, adhesion, migration, and survival. Previous studies have shown that NOV/CCN3 is involved in glucose metabolism and insulin signaling in various tissues and cell types. However, the role of NOV/CCN3 in childhood obesity and insulin resistance remains unclear. Methods: In this study, we aimed to investigate the association between plasma NOV/CCN3 levels and insulin resistance in 58 obese and 43 non-obese children aged 6–12 years. We measured plasma NOV/CCN3 levels by enzyme-linked immunosorbent assay and assessed insulin resistance by homeostasis model assessment of insulin resistance (HOMA-IR). We also collected clinical and biochemical data, such as body mass index (BMI), waist circumference (WC), blood pressure (BP), fasting glucose (FG), fasting insulin (FI), lipid profile, and inflammatory markers. Results: We found that plasma NOV/CCN3 levels were significantly higher in obese children than in non-obese children (p r = 0.42, p r = 0.38, p r = 0.35, p r = 0.31, p r = 0.45, p r = 0.48, p r = 0.28, p r = 0.26, p r = 0.32, p p Conclusion: These results suggest that plasma NOV/CCN3 levels are elevated in childhood obesity and are associated with insulin resistance, indicating that NOV/CCN3 may play a role in the pathogenesis of metabolic disorders in obese children.

Roles for CCN proteins in Lysophosphatidic Acid and Sphingosine 1‐Phosphate Signaling in Prostate Cancer Cells

IntroductionCysteine rich angiogenic factor (CCN1/Cyr61) and connective tissue growth factor (CCN2/CTGF) are members of the CCN family of matricellular proteins. These secreted proteins interact with extracellular matrix proteins and integrins to modulate cell signaling. CCN proteins have been implicated in survival and other mitogenic responses in various cancers. CCN1 and CCN2 are the two early inducible genes in this family. Lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) are lipid growth factors known to contribute to tumor progression in prostate cancer. In previous work, our group showed that LPA stimulates proliferation and migration in prostate cancer cells via the G protein‐coupled receptor LPAR1. Free fatty acid receptor 4 (FFAR4) agonists, inhibit LPA‐induced responses in prostate cancer cells. We also demonstrated that CCN1 expression is induced by LPA in human prostate cancer cells. However, the roles of CCN1 and CCN2 in cancer cell signaling have not been fully elucidated.HypothesisWe hypothesize that CCN proteins contribute to LPA‐ and S1P‐induced adhesion and mitogenic signaling in prostate cancer cells.Methods and ResultsPC‐3 human prostate cancer cells were used in the current study. For all experiments, cells were serum starved for 24 hours and then treated with and without 10 µM LPA or S1P. Immunoblotting showed that CCN1 and CCN2 were induced by LPA after 2‐5 hours in PC‐3 cells. The response was inhibited by the LPA receptor antagonist Ki1625. TUG‐891, an FFAR4 agonist, inhibited LPA‐induced CCN1 expression in PC‐3 cells. S1P also induced CCN1 expression in PC‐3 cells. In related experiments, whole cell extracts, and extracellular matrix were subjected to immunoblotting for CCN1, and immunofluorescence microscopy was used to localize the induced CCN1. CCN1 protein levels in extracellular matrix were increased 2‐5 hours after LPA treatment, as compared to untreated controls, and CCN1 was detected outside the cells by confocal microscopy. In cell adhesion assays, LPA‐treated PC‐3 cells exhibited an increase in adhesion to fibronectin‐coated 96‐well plates that was detected by 2 hours. Erk MAPK activation in response to LPA was biphasic, with the later phase occurring after CCN1/2 induction.ConclusionThe results of this study suggest that CCN proteins may play roles in LPA‐ and S1P‐induced signaling in prostate cancer cells and are thus potential therapeutic targets.

Age-Related Downregulation of CCN2 Is Regulated by Cell Size in a YAP/TAZ-Dependent Manner in Human Dermal Fibroblasts: Impact on Dermal Aging

CCN2, a member of the CCN family of matricellular proteins, is a key mediator and biomarker of tissue fibrosis. We previously reported that CCN2 is significantly reduced in aged human dermis, which contributes to dermal aging through the downregulation of collagen production, the major structural protein in the skin. In this study, we investigated the underlying mechanisms of the age-related downregulation of CCN2 in human skin dermal fibroblasts. Dermal fibroblasts isolation and laser-capture microdissection‒coupled RT-PCR from human skin confirmed that age-related reduction of CCN2 expression is regulated by epigenetics. Mechanistic investigation revealed that age-related reduction of CCN2 is regulated by impaired dermal fibroblast spreading/cell size, which is a prominent feature of aged dermal fibroblasts in vivo. Gain-of-function and loss-of-function analysis confirmed that age-related downregulation of CCN2 is regulated by YAP/TAZ in response to reduced cell size. We further confirmed that restoration of dermal fibroblast size rapidly reversed the downregulation of CCN2 in a YAP/TAZ-dependent manner. Finally, we confirmed that reduced YAP/TAZ nuclear staining is accompanied by loss of CCN2 in aged human skin in vivo. Our data reveal a mechanism by which age-related reduction in fibroblast spreading/size drives YAP/TAZ-dependent downregulation of CCN2 expression, which in turn contributes to loss of collagen in aged human skin.

Cysteine-rich angiogenic inducer 61 (CCN1) independently predicts all-cause mortality in patients with dilated cardiomyopathy

Abstract Introduction Cysteine-rich angiogenic inducer 61 (Cyr61, CCN1) is a member of the CCN family of matricellular proteins exerting key functions in inflammation and fibrotic turnover. Recently it has been shown that CCN1 improves risk stratification for all-cause mortality in ACS patients. Purpose Since both inflammation and fibrosis are key processes involved in the pathogenesis of dilated cardiomyopathy (DCM), we aimed to investigate the prognostic value of CCN1 serum levels for survival in a large cohort of DCM patients. Methods The cohort compromised patients with a primary diagnosis of DCM, defined as a reduced left ventricular ejection fraction (LVEF <45%) and an increased left ventricular enddiastolic diameter according to the HENRY score (LVEDD according to HENRY >117%) at the time of diagnosis. Exclusion criteria were primary valvular diseases (≥ second degree), acute myocarditis, active infectious diseases, pulmonary diseases, cancer, chronic alcoholism and heart failure of other origins. CCN1 levels were determined in human serum using an enzyme-linked immunosorbent assay (R&D Systems, USA). Multivariable cox regression models for the association between CCN1 and all-cause mortality were adjusted for age, sex, disease duration, LVEF, estimated glomerular filtration rate (eGFR) measured based on the CKD-EPI formula, high-sensitivity C-reactive protein (hs-CRP) and aminoterminal-proB-type natriuretic peptide (NT-proBNP) levels. Results 306 DCM patients had available biomarker and clinico-demographic data in this single-center cohort (79.3% males) with a mean age of 55.2 years [interquartile range [IQR] 47.9, 64.8]). On average, disease duration was 0.3 years (IQR 0.9, 1.9), LVEF 31% (IQR 25, 37), LVEDD 67.7 mm (IQR 63, 72), and eGFR 91.5 ml/min (IQR 74.1, 102.4). During a median follow-up of 12.5 years (IQR 10.5, 14.1), a total of 114 (37.3%) patients died. Multivariable-adjusted cox regression models revealed an increasing all-cause mortality risk across CCN1 tertiles (p for trend = 0.03), with the highest incidence in the highest tertile (hazard ratio [HR] 1.75; 95%-CI 1.04, 2.94; P=0.034) as compared to the lowest tertile (Figure 1). Conclusion CCN1 predicts long-term survival in DCM patients independent of NT-pro-BNP and other risk determinants. Further research needs to evaluate whether this novel biomarker also plays a causal role in the pathogenesis of DCM. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Kerckhoff Foundation Figure 1

Effects of Lysophosphatidic Acid Receptor and Free Fatty Acid Receptor Agonists on CCN1/Cyr61 Expression in Prostate Cancer Cells

Expression of CCN1/Cyr61, a member of the CCN family of matricellular proteins, can be induced by growth factors in cancer cells. CCN1 participates in cell migration and other regulatory processes in various cancers. However, the role of CCN1 in prostate cancer remains poorly understood. In previous work we observed that lysophosphatidic acid (LPA), a bioactive lipid, stimulates proliferation and migration in prostate cancer cells via the G protein-coupled receptor (GPCR) LPAR1, and also induces CCN1 expression. We also showed that activation of free fatty acid receptor 4 (FFAR4), another GPCR, inhibits LPA-induced proliferation and migration. For the current study, we hypothesized that CCN1 contributes to adhesion and migration of prostate cancer cells. We first confirmed that CCN1 protein levels were increased by LPA after 2-5 hours in PC-3 human prostate cancer cells. This response was inhibited by TUG-891, an FFA4 agonist. When serum-starved PC-3 cells were treated with LPA in an extended time course from 2 minutes to 24 hours, Erk, Akt, and FAK were initially activated within 30 minutes of LPA addition, as detected by immunoblotting for the phosphorylated kinases. However, all three kinases exhibited a later phase of activation after ~6 hours. Thus, CCN1 induction is downstream of early signals initiated by LPAR1, but potentially upstream of later events. For adhesion assays, serum-starved PC-3 cells were incubated with and without 10 μM LPA ± 1 μM TUG-891. Enhanced adhesion was prominent 1 hour after LPA addition, and was sustained for at least 8 hours. For migration assays, serum-starved cells were incubated with the same agents in modified Boyden chambers. Enhanced cell migration was observed by 2 hours after LPA addition, and continued for at least 24 hours. LPA-induced adhesion and migration were blocked by TUG-891. In other experiments, extracellular matrix was subjected to immunoblotting and to immunofluorescence microscopy for CCN1. In the immunoblotting experiments, CCN1 protein was increased in the extracted extracellular matrix 2-5 hours after LPA addition. In the immunohistochemistry experiments, CCN1 was detected in the extracellular space of PC-3 cells treated with LPA for 0-5 hours. In conclusion, the results suggest a role for LPA-induced CCN1 in adhesion and migration in prostate cancer cells, making CCN1 a potential therapeutic target. In addition, the results further indicate that FFAR activation is a strategy to suppress both short- and long-term LPA responses.

Targeting CCN Proteins in Rheumatoid Arthritis and Osteoarthritis.

The CCN family of matricellular proteins (CYR61/CCN1, CTGF/CCN2, NOV/CCN3 and WISP1-2-3/CCN4-5-6) are essential players in the key pathophysiological processes of angiogenesis, wound healing and inflammation. These proteins are well recognized for their important roles in many cellular processes, including cell proliferation, adhesion, migration and differentiation, as well as the regulation of extracellular matrix differentiation. Substantial evidence implicates four of the proteins (CCN1, CCN2, CCN3 and CCN4) in the inflammatory pathologies of rheumatoid arthritis (RA) and osteoarthritis (OA). A smaller evidence base supports the involvement of CCN5 and CCN6 in the development of these diseases. This review focuses on evidence providing insights into the involvement of the CCN family in RA and OA, as well as the potential of the CCN proteins as therapeutic targets in these diseases.

The Roles of CCN1/CYR61 in Pulmonary Diseases

CCN1 (cysteine-rich 61, connective tissue growth factor, and nephroblastoma-1), previously named CYR61 (cysteine-rich angiogenic inducer 61) belongs to the CCN family of matricellular proteins. CCN1 plays critical roles in the regulation of proliferation, differentiation, apoptosis, angiogenesis, and fibrosis. Recent studies have extensively characterized the important physiological and pathological roles of CCN1 in various tissues and organs. In this review, we summarize both basic and clinical aspects of CCN1 in pulmonary diseases, including acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), lung fibrosis, pulmonary arterial hypertension (PAH), lung infection, and lung cancer. We also emphasize the important challenges for future investigations to better understand the CCN1 and its role in physiology and pathology, as well as the questions that need to be addressed for the therapeutic development of CCN1 antagonists in various lung diseases.

Et tu, CCN1….

The CCN family of matricellular proteins are recognized bona fide targets for therapeutically targeting so-called chronic inflammatory diseases, including fibrosis and cancers. The majority of the work supporting this contention has been derived from examining CCN2, formerly, and unhelpfully, termed "connective tissue growth factor." Both CCN2, and its related protein, CCN1, formerly termed "cysteine-rich protein 61", are positively regulated by not only TGFbeta, but also by the hippo/YAP/TAZ mechanotransduction pathway that appears to drive these pathologies. Indeed, increasing evidence indicates that CCN1 also contributes to these fibrosis and cancers and, consequently, targeting both CCN2 and CCN1 simultaneously could be of therapeutic value. This commentary focuses on a recent, exciting paper (Ju et al., 2020, Scientific Reports, 10, 3201) suggesting that CCN1 is a target for non-alcoholic steatohepatitis (NASH).

Identification and characterization of collagen-like glycosylation and hydroxylation of CCN1.

CCN1 is a secreted protein and belongs to the CCN family of matricellular proteins. CCN1 binds to various cell surface receptors; thus, CCN1 has important functions in cell proliferation, migration and angiogenesis through a variety of signaling pathways. We have reported that CCN1 is O-fucosylated and that this O-fucosylation regulates the secretion of CCN1 into the extracellular region. In this study, we detected collagen-like glycosylation and hydroxylation at Lys203 of recombinant CCN1 by mass spectrometry. We then examined the role of collagen-like glycosylation in the functions of CCN1. As a result, we found that a deficiency in collagen-like glycosylation decreased the secretion of CCN1 using wild-type CCN1- and collagen-like glycosylation-defective mutant CCN1-overexpressing cell lines. Further, knockout of lysyl hydroxylase3, a multifunctional protein with hydroxylase and glucosyltransferase activities, impaired the secretion and glycosylation level of recombinant CCN1. Previous studies reported that collagen glycosylation of Lys residues mediated by lysyl hydroxylase3 is glucosyl-galactosyl-hydroxylation, presuming that this collagen-like glycosylation detected at Lys203 of recombinant CCN1 in this study might be glucosyl-galactosyl-hydroxylation. Taken together, our results demonstrate the novel function of the collagen-like glycosylation of CCN1 and suggest that lysyl hydroxylase3-mediated glycosylation is important for CCN1 secretion.

CCN1 expression by fibroblasts is required for bleomycin-induced skin fibrosis

The microenvironment contributes to the excessive connective tissue deposition that characterizes fibrosis. Members of the CCN family of matricellular proteins are secreted by fibroblasts into the fibrotic microenvironment; however, the role of endogenous CCN1 in skin fibrosis is unknown. Mice harboring a fibroblast-specific deletion for CCN1 were used to assess if CCN1 contributes to dermal homeostasis, wound healing, and skin fibrosis. Mice with a fibroblast-specific CCN1 deletion showed progressive skin thinning and reduced accumulation of type I collagen; however, the overall mechanical property of skin (Young's modulus) was not significantly reduced. Real time-polymerase chain reaction analysis revealed that CCN1-deficient skin displayed reduced expression of mRNAs encoding enzymes that promote collagen stability (including prolyl-4-hydroxylase and PLOD2), although expression of COL1A1 mRNA was unaltered. CCN1-deficent skin showed reduced hydroxyproline levels. Electron microscopy revealed that collagen fibers were disorganized in CCN1-deficient skin. CCN1-deficient mice were resistant to bleomycin-induced skin fibrosis, as visualized by reduced collagen accumulation and skin thickness suggesting that deposition/accumulation of collagen is impaired in the absence of CCN1. Conversely, CCN1-deficient mice showed unaltered wound closure kinetics, suggesting de novo collagen production in response to injury did not require CCN1. In response to either wounding or bleomycin, induction of α-smooth muscle actin-positive myofibroblasts was unaffected by loss of CCN1. CCN1 protein was overexpressed by dermal fibroblasts isolated from lesional (i.e., fibrotic) areas of patients with early onset diffuse scleroderma. Thus, CCN1 expression by fibroblasts, being essential for skin fibrosis, is a viable anti-fibrotic target.

Levels of Connective Tissue Growth Factor (CTGF) Predict Development of Kidney Dysfunction in Type 2 Diabetes-The VADT Study

Connective tissue growth factor (CTGF) is a member of the CCN family of matricellular proteins that has been implicated in the inflammatory process, response to injury and wound healing. An emerging role of CTGF is that of a profibrotic factor implicated in the development of kidney disease. Our objective was to determine the role of CTGF as a predictor of macroalbuminuria (ACR ≥300µg/mg twice) and chronic kidney disease (CKD) stage 3 (GFR <60ml/min twice) and stage 4 (GFR <30ml/min once or twice) in the VADT cohort. Levels of CTGF were measured in 952 VADT patients, a median of 1.9 years after entry into the study. Participants were followed for a median of 3.8 years for renal outcomes. CTGF categories were defined as below the detectable limit (i.e., referent, 54.5%), lower half of detectable values (i.e., 22.8%) and upper half of detectable values (22.7%). Odds ratios (ORs) for kidney endpoints in relation to CTGF categories were calculated by logistic regression. Even with adjustment for baseline ACR levels, individuals in the highest compared to lowest CTGF category were at higher risk of macroalbuminuria [OR=3.21 (95% CI: 1.37, 7.53)]. In contrast, CTGF was predictive of incident stage 3 and stage 4 CKD only when baseline GFR levels were not adjusted for. Our study indicates that high levels of CTGF are associated with and predict future kidney dysfunction in patients with type 2 diabetes. Disclosure K.J. Hunt: None. M.A. Jaffa: None. S.M. Garrett: None. D.K. Luttrell: None. K.E. Lipson: Employee; Self; FibroGen, Inc.. Stock/Shareholder; Self; AbbVie Inc., Abbott, Becton, Dickinson and Company, Bristol-Myers Squibb Company, General Electric, Gilead Sciences, Inc., Medtronic, Nestlé, Novartis AG, Oracle, Pfizer Inc., Quest Diagnostics. Consultant; Spouse/Partner; Diabesen. M.F. Lopes-Virella: None. L.M. Luttrell: None. A.A. Jaffa: None.

Characterization of bone morphology in CCN5/WISP5 knockout mice.

CCN5/WISP2 is part of the CCN family of matricellular proteins, but is distinct in that it lacks the C-terminal (CT) domain. Although CCN5 has been shown to impact cell proliferation and differentiation in vitro, its role in vivo is unclear. We therefore generated mice using ES cells developed by the Knockout Mouse Project (KOMP) in which exons 2-5, which encode the all of the conserved protein coding regions, are replaced by a lacZ cassette. Ccn5 LacZ/LacZ mice were viable and apparently normal. Based on previous studies showing that CCN5 impacts osteoblast proliferation and differentiation, we performed an analysis of adult bone phenotype. LacZ expression was examined in adult bone, and was found to be strong within the periosteum, but not in trabecular bone or bone marrow. Micro-CT analysis revealed no apparent changes in bone mineral density (BMD) or bone tissue volume (BV/TV) in Ccn5 LacZ/LacZ mice. These studies indicate that CCN5 is not required for normal bone formation, but they do not rule out a role in mechanotransduction or repair processes. The availability of Ccn5 LacZ mice enables studies of CCN5 expression and function in multiple tissues.

A friend in knee: CCN3 may inhibit osteoarthritis progression

Osteoarthritis (OA) is a major clinical problem among the ageing population, yet no disease-modifying treatments currently exist. This issue arises, in part, due to the complex processes occurring in the microenvironment of articular cartilage that lead to osteoarthritic changes. Gaining a better understanding of these processes is crucial in developing a viable therapy for OA. A recent report in Journal of Bone Mineral Metabolism by Janune et al. (J Bone Miner Metab 35:582–597, 2016) suggests a novel role for CCN3 in maintaining the differentiated phenotype of articular cartilage. This report suggests that CCN3, a member of the CCN family of matricellular proteins, is important for proteoglycan accumulation, as well as expression of type II collagen, tenascin C, and lubricin in vitro. Furthermore, exogenous CCN3 increased tidemark integrity and lubricin protein expression in a rat model of OA. These results implicate the regulation of CCN3 as a potential therapeutic target in patients with OA.

Recombinant CCN1 prevents hyperoxia-induced lung injury in neonatal rats.

BackgroundCystein-rich protein 61 (Cyr61/CCN1) is a member of the CCN family of matricellular proteins that has an important role in tissue development and remodeling. However, the role of CCN1 in the pathogenesis of bronchopulmonary dysplasia (BPD) is unknown. Accordingly, we have investigated the effects of CCN1 on a hyperoxia-induced lung injury model in neonatal rats.MethodsIn experiment 1, newborn rats were randomized to room air (RA) or 85% oxygen (O2) for 7 or 14 days, and we assessed the expression of CCN1. In experiment 2, rat pups were exposed to RA or O2 and received placebo or recombinant CCN1 by daily intraperitoneal injection for 10 days. The effects of CCN1 on hyperoxia-induced lung inflammation, alveolar and vascular development, vascular remodeling, and right ventricular hypertrophy (RVH) were observed.ResultsIn experiment 1, hyperoxia downregulated CCN1 expression. In experiment 2, treatment with recombinant CCN1 significantly decreased macrophage and neutrophil infiltration, reduced inflammasome activation, increased alveolar and vascular development, and reduced vascular remodeling and RVH in the hyperoxic animals.ConclusionThese results demonstrate that hyperoxia-induced lung injury is associated with downregulated basal CCN1 expression, and treatment with CCN1 can largely reverse hyperoxic injury.

Dual roles of CCN proteins in breast cancer progression.

The tumor microenvironment has a powerful effect on the development and progression of human breast cancer, which may be used therapeutically. Despite efforts to understand the complex role of the tumor microenvironment in breast cancer development, the specific players and their contributions to tumorigenesis need further investigation. The CCN family of matricellular proteins comprises six members (CCN1-6; CYR61, CTGF, NOV, WISP1-3) with central roles in development, inflammation, and tissue repair. CCN proteins also exert functions during pathological processes including fibrosis and cancer by regulating extracellular signals in the cellular environment. Studies have demonstrated that all six CCN proteins exert functions in breast tumorigenesis. Although CCN proteins share a multimodular structure in which most cysteine residues are conserved within structural motifs, they may have opposing functions in breast cancer progression. A better understanding of the functions of each CCN member will assist in the development of specific therapeutic approaches for breast cancer.

Degradomic and yeast 2-hybrid inactive catalytic domain substrate trapping identifies new membrane-type 1 matrix metalloproteinase (MMP14) substrates: CCN3 (Nov) and CCN5 (WISP2)

Members of the CCN family of matricellular proteins are cytokines linking cells to the extracellular matrix. We report that CCN3 (Nov) and CCN5 (WISP2) are novel substrates of MMP14 (membrane-type 1-matrix metalloproteinase, MT1-MMP) that we identified using MMP14 “inactive catalytic domain capture” (ICDC) as a yeast two-hybrid protease substrate trapping platform in parallel with degradomics mass spectrometry screens for MMP14 substrates. CCN3 and CCN5, previously unknown substrates of MMPs, were biochemically validated as substrates of MMP14 and other MMPs in vitro—CCN5 was processed in the variable region by MMP14 and MMP2, as well as by MMP1, 3, 7, 8, 9 and 15. CCN1, 2 and 3 are proangiogenic factors yet we found novel opposing activity of CCN5 that was potently antiangiogenic in an aortic ring vessel outgrowth model. MMP14, a known regulator of angiogenesis, cleaved CCN5 and abrogated the angiostatic activity. CCN3 was also processed in the variable region by MMP14 and MMP2, and by MMP1, 8 and 9. In addition to the previously reported cleavages of CCN1 and CCN2 by several MMPs we found that MMPs 8, 9, and 1 process CCN1, and MMP8 and MMP9 also process CCN2. Thus, our study reveals additional and pervasive family-wide processing of CCN matricellular proteins/cytokines by MMPs. Furthermore, CCN5 cleavage by proangiogenic MMPs results in removal of an angiogenic brake held by CCN5. This highlights the importance of thorough dissection of MMP substrates that is needed to reveal higher-level control mechanisms beyond type IV collagen and other extracellular matrix protein remodelling in angiogenesis. SummaryWe find CCN family member cleavage by MMPs is more pervasive than previously reported and includes CCN3 (Nov) and CCN5 (WISP2). CCN5 is a novel antiangiogenic factor, whose function is abrogated by proangiogenic MMP cleavage. By processing CCN proteins, MMPs regulate cell responses angiogenesis in connective tissues.

CCN6: a modulator of breast cancer progression.

The expression of the CCN family of matricellular proteins is highly dysregulated in connective tissue pathologies such as fibrosis and highly metastatic cancers. Strategies targeting members of this family, especially CCN2, are under development as novel therapeutic approaches to highly metastatic cancers such as pancreatic cancer. In prior reports, the Kleer laboratory and colleagues have linked reduced expression of CCN6 (WISP3) with aggressive breast cancers. Loss of CCN6 was associated with elevated Akt phosphorylation and TAK1 activation. In a recent report, the same group reports that, by modulating Notch signaling, CCN6 can promote the maintenance of an epithelial phenotype and also reduce cancer cell migration and invasion, tumor initiation, and metastasis (Oncotarget in press DOI: 10.18632/oncotarget.7734 ). These results are consistent with the hypothesis that addition of CCN6 peptides may represent a novel, viable therapeutic approach to blocking aggressive breast cancers.

Deletion of WISP1 leads to higher sensitivity to carbon tetrachloride-induced liver damage

WISP1 (Wnt-induced secreted protein-1) is a member of the CCN family of matricellular proteins. These highly conserved and secreted proteins can interact with integrins and modulate many biological processes like migration, proliferation, differentiation and apoptosis. CCN proteins have been show to play divergent roles in liver pathophysiology, in spite of having very similar structural domains. Importantly, little is known about WISP1 in this context. Gene array analysis of mouse liver after acute injury by intraperitoneal injection of CCl4 in C56BL/6N mice showed WISP1 mRNA upregulated during early injury phase, between 2h and 24h after CCl4 injection. These results were confirmed by real time PCR, showing a maximal induction of 40 fold over healthy liver at 24h. To establish the role of WISP1 in acute liver damage we used a knockout mouse model, in which WISP1 expression is disrupted by replacing Exon 2 with a resistance cassette and a stop codon. Heterozygous mice were bred to establish homozygous knockout and wild type lines which were validated by genotyping. Real time PCR analysis confirmed the absence of WISP1 mRNA expression in KO mice challenged with CCl4. Remarkably, deletion of WISP1 led to a significant increase of necrotic area at 24h. This was show by hematoxylin & eosin staining reaching 49% in KO vs. 27% in WT mice (p < 0.0001; n = 13 wild type, n = 11 KO) and serum transaminase activity (GPT: 8230 U/l in WT, 12300 U/l in KO). Importantly, immunofluorescence and real time PCR showed that WT and WISP1 KO mice have no significant differences in CYP2E1 expression, suggesting that the difference in tissue injury does not depend on alterations of CCl4 metabolism in KO mice. To further characterize this different phenotype, we performed a time course after CCl4 intoxication (460 mg/kg, i.p.) by collecting liver tissue at 30 min, 2h, 8h, 18h, and 24h. Interestingly, already at 18h KO mice showed a clear necrotic area around the central veins whereas WT mice showed ballooned hepatocytes with cytoplasmic translocation of high-mobility group protein B1 (HGMB1), indicating that WISP1 KO mice had a more advanced stage of liver injury at this time point than the WT counterparts. This phenotype was also observed upon intoxication with lower doses of CCl4 (132.4 mg/kg and 76 mg/kg). Analysis of cytokine expression by real time PCR showed a stronger induction of TNFα in WISP1 KO mice (7-fold v/s 10-fold at 18h, and 3-fold vs. 6-fold at 24h in WT vs. WISP1 KO respectively), and IL6 (5-fold vs. 10-fold at 18h, and no induction vs. 3-fold at 24h in WT v/s WISP1 KO respectively). Furthermore, western blot analysis showed marked alterations in signal transduction pathways in liver tissue of WISP1 KO mice, with stronger activations of p-JNK at 2h and p-STAT3 at 18h and 24h. Currently we are investigating the mechanism through which WISP1 exerts a protective role upon CCl4 stimulation in liver damage and in the subsequent regeneration process. In conclusion we have strong evidence that WISP1 plays an important role in acute liver damage.