Cooperation is the key: the CCN biological system as a gate to high complex protein superfamilies' signaling.

Cellular signaling is generally understood as the support of communication between contiguous cells belonging to the same tissue or cells being far apart of each other, at a molecular scale, when the message emitted by the transmitters is traveling in liquid or solid matter to reach recipient targets. Subcellular signaling is also important to ensure the proper cell constitution and functioning. However cell signaling is mostly used in the first understanding, to describe how the message sent from one point to another one, will reach a target where it will be interpreted. The Cellular Communication Network (CCN) factors (Perbal et al. 2018) constitute a family of biological regulators thought to be responsible for signaling pathways coordination (Perbal 2018). Indeed, these proteins interact with a diverse group of cell receptors, such as integrins, low density lipoprotein receptors, heparan sulfate proteoglycan receptors (HSPG), and the immunoglobulin superfamily expressed exclusively in the nervous system, or with soluble factors such as bone morphogenetic proteins (BMPS) and other growth factors such as vascular endothelial growth factor, fibroblastic growth factor, and transforming growth factor (TGFbeta). Starting from the recapitulation of basic concepts in enzymology and protein-ligands interactions, we consider, in this manuscript, interpretations of the mechanistic interactions that have been put forward to explain the diversity of CCN proteins biological activities. We suggest that the cross-talks between superfamilies of proteins under the control of CCNs might play a central role in the coordination of developmental signaling pathways.

The regulation and functions of the matricellular CCN proteins induced by shear stress.

Shear stress is a frictional drag generated by the flow of fluid, such as blood or interstitial fluid, and plays a critical role in regulating cellular gene expression and functional phenotype. The matricellular CCN family proteins are dynamically regulated by shear stress of different flow patterns, and their expression significantly alters the microenvironment of cells. Secreted CCN proteins mainly bind to several cell surface integrin receptors to mediate their diverse functions in regulating cell survival, function, and behavior. Gene-knockout studies indicate major functions of CCN proteins in the cardiovascular and skeletal systems, the two primary systems in which CCN expressions are regulated by shear stress. In the cardiovascular system, the endothelium is directly exposed to vascular shear stress. Unidirectional laminar blood flow generates laminar shear stress, which promotes a mature endothelial phenotype and upregulates anti-inflammatory CCN3 expression. In contrast, disturbed flow generates oscillatory shear stress, which induces endothelial dysfunction through the induction of CCN1 and CCN2. Shear-induced CCN1 binds to integrin α6ß1 and promotes superoxide production, NF-κB activation, and inflammatory gene expression in endothelial cells. Although the interaction between shear stress and CCN4-6 is not clear, CCN 4 exhibits a proinflammatory property and CCN5 inhibits vascular cell growth and migration. The crucial roles of CCN proteins in cardiovascular development, homeostasis, and disease are evident but not fully understood. In the skeletal system, mechanical loading on bone generates shear stress from interstitial fluid in the lacuna-canalicular system and promotes osteoblast differentiation and bone formation. CCN1 and CCN2 are induced and potentially mediate fluid shear stress mechanosensing in osteocytes. However, the exact roles of interstitial shear stress-induced CCN1 and CCN2 in bone are still not clear. In contrast to other CCN family proteins, CCN3 inhibits osteoblast differentiation, although its regulation by interstitial shear stress in osteocytes has not been reported. The induction of CCN proteins by shear stress in bone and their functions remain largely unknown and merit further investigation. This review discusses the expression and functions of CCN proteins regulated by shear stress in physiological conditions, diseases, and cell culture models. The roles between CCN family proteins can be compensatory or counteractive in tissue remodeling and homeostasis.

CCN proteins: opportunities for clinical studies-a personal perspective.

The diverse members of the CCN family now designated as CCN1(CYR61), CCN2 (CTGF), CCN3(NOV), CCN4(WISP1), CCN5(WISP2), CCN6(WISP3) are a conserved matricellular family of proteins exhibiting a spectrum of functional properties throughout all organs in the body. Interaction with cell membrane receptors such as integrins trigger intracellular signaling pathways. Proteolytically cleaved fragments (constituting the active domains) can be transported to the nucleus and perform transcriptional relevant functional activities. Notably, as also found in other protein families some members act opposite to others creating a system of functionally relevant checks and balances. It has become apparent that these proteins are secreted into the circulation, are quantifiable, and can serve as disease biomarkers. How they might also serve as homeostatic regulators is just becoming appreciated. In this review I have attempted to highlight the most recent evidence under the subcategories of cancer and non-cancer relevant that could lead to potential therapeutic approaches or ideas that can be factored into clinical advances. I have added my own personal perspective on feasibility.

Temperal and spatial expression of CCN1, CCN3, CCN4, CCN5 and CCN6 proteins in the developing postnatal teeth.

CCN proteins are matricellular proteins and are important modulators of development and function of adult organs. However, there is no literature reporting the localization of CCN proteins during postnatal tooth development and the formation of periodontium. Therefore, the aim of our study was to investigate the expression of CCN1, CCN3, CCN4, CCN5 and CCN6 in the developing postnatal teeth. Wistar rats were used at postnatal (PN) 3.5, 7, 16 and 21 days and maxillas were processed for immunohistochemistry. At PN3.5 and PN7, preameloblasts (PA), secretory ameloblasts (SA), odontoblasts (OD) and dental pulp (DP) showed moderate to strong staining for CCN1, CCN4 and CCN6 respectively. CCN5 was intensely expressed in predentin, whereas CCN5 was undetectable in PA, SA, OD and DP. At PN16 and PN21, moderate to strong reaction with CCN1, CCN4 and CCN6 was evident in OD, DP, reduced enamel epithelium (REE), osteoblasts (OB) and periodontal ligament (PDL) respectively, while CCN5 was negative to weakly expressed in REE, OD, DP, OB, PDL and osteocytes (OC). Interestingly, the expression of CCN1, CCN4 and CCN6 was initially negative at PN16 but strong at PN21 in OC. Furthermore, there was no staining for CCN3 in the tissues studied. These results demonstrated that the expression pattern of CCN1, CCN4 and CCN6 is similar and inversely correlated with that of CCN3. CCN5 exhibits a unique distribution pattern. These data indicate that CCN proteins might play regulatory roles in amelogenesis, dentinogenesis, osteogenesis and PDL homeostasis.

Time has come to address the spatiotemporal combinatorial model for CCN proteins biological activitites by spatial transcriptomics and genome wide association studies.

It is a renewed pleasure to wish our authors, editorial board members, and readership an excellent new year, full of professional and personal satisfactions. According to the Chinese Horoscope, 2023, the Year of Water Rabbit, is predicted to be quiet; a year to step back, assess the situation and make plans. It will be the time to carefully appraise, with the patience of the Water Rabbit, the future and scientific wealth of our Journal. Based on a few aspects of the CCN3 biology status that remain open questions, I am presenting below a short summary of a few CCN research directions that in my eyes, become necessary to undertake through wide-angle collaborative approaches.

Report on the 11th international workshop on the CCN family of genes, Nice, October 20-24, 2022.

In celebration of the twentieth anniversary of the inception of the CCN society, and of the first post-Covid-19 live meeting, the executive board of the ICCNS had chosen Nice as the venue for the 11th International workshop on the CCN family of genes. On this occasion participation in the meeting was extended to colleagues from other cell signaling fields who were invited to present both an overview of their work and the future directions of their laboratory. Also, for the first time, the members of the JCCS Editorial Board were invited to participate in a JCCS special session during which all aspects of the journal « life ¼ were addressed and opened to free critical discussion. The scientific presentations and the discussions that followed showed once more that an expansion of the session topics was beneficial to the quality of the meeting and confirmed that the ARBIOCOM project discussed last April in Nice was now on track to be launched in 2023. The participants unanimously welcomed Professor Attramadal's proposition to organize the 2024, 12th International CCN workshop in Oslo, Norway.

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.

Using the Bleomycin-Induced Model of Fibrosis to Study the Contribution of CCN Proteins to Scleroderma Fibrosis.

Approximately 45% of the deaths in the developed world result from conditions with a fibrotic component. Although no specific, focused anti-fibrotic therapies have been approved for clinical use, a long-standing concept is that targeting CCN proteins may be useful to treat fibrosis. Herein, we summarize current data supporting the concept that targeting CCN2 may be a viable anti-fibrotic approach to treat scleroderma. Testing this hypothesis has been made possible by using a mouse model of inflammation-driven skin and lung fibrosis.

Mouse Models of Tumor Bone Metastasis and Invasion for Studying CCN Proteins.

Bone metastasis and bone destruction are common occurrences in human malignancies, including breast, prostate, and lung cancer, and are associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis and bone destruction are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the left cardiac ventricle. In addition, we introduce mouse model of the bone destruction by injection of SAS oral squamous cell carcinoma cells into the bone marrow space of the right tibial metaphysis. These assays can be applied to studies on roles of cellular communication network factor/connective tissue growth factor (CTGF/CCN2) protein in tumor metastasis and development of treatment strategies targeting CCN proteins.

Potential Role of CCN Proteins in Breast Cancer: Therapeutic Advances and Perspectives.

CCNs are a specific type of matricellular protein, which are essential signaling molecules, and play multiple roles in multicellular eukaryotes. This family of proteins consists of six separate members, which exist only in vertebrates. The architecture of CCN proteins is multi-modular comprising four distinct modules. CCN Proteins achieve their primary functional activities by binding with several integrin7 receptors. The CCN family has been linked to cell adhesion, chemotaxis and migration, mitogenesis, cell survival, angiogenesis, differentiation, tumorigenesis, chondrogenesis, and wound healing, among other biological interactions. Breast cancer is the most commonly diagnosed cancer worldwide and CCN regulated breast cancer stands at the top. A favorable or unfavorable association between various CCNs has been reported in patients with breast carcinomas. The pro-tumorigenic CCN1, CCN2, CCN3, and CCN4 may lead to human breast cancer, although the anti-tumorigenic actions of CCN5 and CCN6 are also present. Several studies have been conducted on CCN proteins and cancer in recent years. CCN1 and CCN3 have been shown to exhibit a dual nature of tumor inhibition and tumor suppression to some extent in quiet recent time. Pharmacological advances in treating breast cancer by targeting CCN proteins are also reported. In our study, we intend to provide an overview of these research works while keeping breast cancer in focus. This information may facilitate early diagnosis, early prognosis and the development of new therapeutic strategies.

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.

CCN Family Proteins in Cancer: Insight Into Their Structures and Coordination Role in Tumor Microenvironment.

The crosstalk between tumor cells and the tumor microenvironment (TME), triggers a variety of critical signaling pathways and promotes the malignant progression of cancer. The success rate of cancer therapy through targeting single molecule of this crosstalk may be extremely low, whereas co-targeting multiple components could be complicated design and likely to have more side effects. The six members of cellular communication network (CCN) family proteins are scaffolding proteins that may govern the TME, and several studies have shown targeted therapy of CCN family proteins may be effective for the treatment of cancer. CCN protein family shares similar structures, and they mutually reinforce and neutralize each other to serve various roles that are tightly regulated in a spatiotemporal manner by the TME. Here, we review the current knowledge on the structures and roles of CCN proteins in different types of cancer. We also analyze CCN mRNA expression, and reasons for its diverse relationship to prognosis in different cancers. In this review, we conclude that the discrepant functions of CCN proteins in different types of cancer are attributed to diverse TME and CCN truncated isoforms, and speculate that targeting CCN proteins to rebalance the TME could be a potent anti-cancer strategy.

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.

Conjunction junction, what's the function? CCN proteins as targets in fibrosis and cancers.

Cellular communication network (CCN) proteins are matricellular proteins that coordinate signaling among extracellular matrix, secreted proteins, and cell surface receptors. Their specific in vivo function is context-dependent, but they play profound roles in pathological conditions, such as fibrosis and cancers. Anti-CCN therapies are in clinical consideration. Only recently, however, has the function of these complex molecules begun to emerge. This review summarizes and interprets our current knowledge regarding these fascinating molecules and provides experimental evidence for their utility as therapeutic targets.

CCN proteins as potential actionable targets in scleroderma.

Systemic sclerosis (SSc) is a complex autoimmune connective tissue disease combining inflammatory, vasculopathic and fibrotic manifestations. Skin features, which give their name to the disease and are considered as diagnostic as well as prognostic markers, have not been thoroughly investigated in terms of therapeutic targets. CCN proteins (CYR61/CCN1, CTGF/CCN2, NOV/CCN3 and WISP1-2-3 as CCN4-5-6) are a family of secreted matricellular proteins implicated in major cellular processes such as cell growth, migration, differentiation. They have already been implicated in key pathophysiological processes of SSc, namely fibrosis, vasculopathy and inflammation. In this review, we discuss the possible implication of CCN proteins in SSc pathogenesis, with a special focus on skin features, and identify the potential actionable CCN targets.

The official unified nomenclature adopted by the HGNC calls for the use of the acronyms, CCN1-6, and discontinuation in the use of CYR61, CTGF, NOV and WISP 1-3 respectively.

An examination of the confusion generated around the use of different acronyms for CCN proteins has been performed by the editors of the HUGO Gene Nomenclature Committee upon the request of the International CCN Society Scientific Committee. After careful consideration of the various arguments, and after polling the community of researchers who have published in the field over the past ten years, the HGNC have decided to adopt and approve the CCN nomenclature for all 6 genes. Effective October 2018, the genes referred to as CYR61, CTGF, NOV and WISP1-3 will be respectively designated by the gene symbols CCN1-6 with corresponding gene names « cellular communication Q2 network factor 1-6 ¼. We believe that this decision will be a step towards better communication between researchers working in the field, and will set the stage for fruitful collaborative projects. Accordingly, the Journal of Cell Communication and Signaling, the official journal of the International CCN Society, available both in print and online, constitutes a unique window into the CCN field. This official nomenclature will benefit the international scientific community that is supported by the established and renowned professionalism of the Springer-Nature publishing group.

Portal myofibroblasts are sensitive to CCN-mediated endoplasmic reticulum stress-related apoptosis with potential to attenuate biliary fibrogenesis.

Portal fibroblasts are mesenchyme-derived fibroblasts surrounding the bile ducts, and activated into portal myofibroblasts (pMF) during cholestatic liver injury. pMF express α-smooth muscle actin (α-SMA) and produce the fibrogenic extracellular matrix (ECM) collagen type I and fibronectin, playing important roles in portal fibrosis. A cholestatic bile duct-ligated (BDL) model is characterized by impaired hepatobiliary excretion of bile, leading to increased bile acid accumulation. Accumulation of bile acids is known to induce endoplasmic reticulum (ER) stress leading to liver damage and cell death. Additionally, a BDL fibrotic model is also associated with upregulation of CCN (CYR61, CTGF and NOV) matricellular proteins and reported to induce ER stress both in vitro and in vivo. To explore the effects of CCN proteins, we used adenovirus-mediated CCN1-4 (Ad-CCN1-4) gene transfers into cultured pMF. Overexpression of CCN proteins leads to protein accumulation in the ER lumen, causing ER stress and unfolded protein response (UPR). We further found ER stress and UPR to mitigate fibrogenesis in pMF by decreased cellular production of fibronectin, collagen type 1 and α-SMA. In this scenario, Tauroursodeoxycholic acid, a pharmaceutical chaperone and ER stress inhibitor, attenuated Ad-CCN1-4 induced pMF apoptosis and restored collagen and fibronectin levels. Since hepatic fibrogenesis is accompanied by ER stress and upregulation of CCN proteins in a BDL, we further evaluated ER stress responses after Ad-CCN1 gene transfer in such a model and found overexpressed CCN1 to enhance the ER stress-associated proteins BiP and CHOP with positive cleaved caspase 3 and 9 staining in periportal nonparenchymal cells. This indicates that these nonparenchymal cells, most likely pMF, have the tendency to undergo apoptosis during later stages of BDL. Ad-CCN1 transduction furthermore sensitized pMF for ER stress and apoptosis. We suggest that CCN proteins are key factors in the fibrotic microenvironment impacting pMF survival during fibrogenesis and pMF apoptosis during fibrosis resolution.

Activation of cancer-associated fibroblasts is required for tumor neovascularization in a murine model of melanoma.

Metastatic melanoma is highly fatal. Within the tumor microenvironment, the role of cancer-associated fibroblasts (CAFs) in melanoma metastasis and progression is relatively understudied. The matricellular protein CCN2 (formerly termed connective tissue growth factor, CTGF) is overexpressed, in a fashion independent of BRAF mutational status, by CAFs in melanoma. Herein, we find, in human melanoma patients, that CCN2 expression negatively correlates with survival and positively correlates with expression of neovascularization markers. To assess the role of CAFs in melanoma progression, we used C57BL/6 mice expressing a tamoxifen-dependent cre recombinase expressed under the control of a fibroblast-specific promoter/enhancer (COL1A2) to delete CCN2 postnatally in fibroblasts. Mice deleted or not for CCN2 in fibroblasts were injected subcutaneously with B16-F10 melanoma cells. Loss of CCN2 in CAFs resulted in reduced CAF activation, as detected by staining with anti-α-smooth muscle actin antibodies, and reduced tumor-induced neovascularization, as detected by micro-computed tomography (micro-CT) and staining with anti-CD31 antibodies. CCN2-deficient B16(F10) cells were defective in a tubule formation/vasculogenic mimicry assay in vitro. Mice deleted for CCN2 in CAFs also showed impaired vasculogenic mimicry of subcutaneously-injected B16-F10 cells in vivo. Our results provide new insights into the cross-talk among different cell types in the tumor microenvironment and suggest CAFs play a heretofore unappreciated role by being essential for tumor neovascularization via the production of CCN2. Our data are consistent with the hypothesis that activated CAFs are essential for melanoma metastasis and that, due to its role in this process, CCN2 is a therapeutic target for melanoma.