Functional role of periostin in development and wound repair: implications for connective tissue disease

Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell-matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.     

The CCN2/CTGF interactome: an approach to understanding the versatility of CCN2/CTGF molecular activities

Cellular communication network 2 (CCN2), also known as connective tissue growth factor (CTGF) regulates diverse cellular processes, some at odds with others, including adhesion, proliferation, apoptosis, and extracellular matrix (ECM) protein synthesis. Although a cause-and-effect relationship between CCN2/CTGF expression and local fibrotic reactions has initially been established, CCN2/CTGF manifests cell-, tissue-, and context-specific functions and differentially affects developmental and pathological processes ranging from progenitor cell fate decisions and angiogenesis to inflammation and tumorigenesis. CCN2/CTGF multimodular structure, binding to and activation or inhibition of multiple cell surface receptors, growth factors and ECM proteins, and susceptibility for proteolytic cleavage highlight the complexity to CCN2/CTGF biochemical attributes. CCN2/CTGF expression and dosage in the local environment affects a defined community of its interacting partners, and this results in sequestration of growth factors, interference with or potentiation of ligand-receptor binding, cellular internalization of CCN2/CTGF, inhibition or activation of proteases, and generation of CCN2/CTGF degradome products that add molecular diversity and expand the repertoire of functional modules in the cells and their microenvironment. Through these interactions, different intracellular signals and cellular responses are elicited culminating into physiological or pathological reactions. Thus, the CCN2/CTGF interactome is a defining factor of its tissue- and context-specific effects. Mapping of new CCN2/CTGF binding partners might shed light on yet unknown roles of CCN2/CTGF and provide a solid basis for tissue-specific targeting this molecule or its interacting partners in a therapeutic context.     

CCN2 expression and localization in melanoma cells

The matricellular protein connective tissue growth factor (CTGF, CCN2) is overexpressed in several forms of cancer and may represent a novel target in anti-cancer therapy. However, whether CCN2 is expressed in melanoma cells is unknown. The highly metastatic murine melanoma cell line B16(F10) was used for our studies. Real time polymerase chain reaction analysis was used to detect mRNA expression of CCN1, CCN2, CCN3 and CCN4 in Western blot and immunofluorescence analyses were used to detect CCN2 protein. Inhibitors of signal transduction cascades were used to probe the mechanism underlying CCN2 expression in B16(F10) cells. CCN2 was expressed in B16(F10) cells, and was reduced by the FAK/src inhibitor PP2 and the MEK/ERK inhibitor U0126 indicating that CCN2 acts downstream of these pathways in B16(F10) murine melanoma cells. Expression of CCN1, CCN3 and CCN4 was not reduced by PP2 or U0126; in fact, expression of CCN4 mRNA was elevated by PP2 or U0126 treatment. To our surprise, CCN2 protein was detected in the nuclei of B16(F10) cells, and was undetectable in the cytoplasm. CCN2 was expressed in B16(F10) melanoma cells, adding to the list of cancer cells in which CCN2 is expressed. Of the CCN family members tested, only CCN2 is downstream of the highly oncogenic MEK/ERK pathway. CCN2 should be further evaluated for a possible role in melanoma growth and progression.     

Cyr61 suppresses growth of human endometrial cancer cells

Cyr61 (CCN1) is a member of the CCN protein family; these secreted proteins are involved in diverse biological processes such as cell adhesion, angiogenesis, apoptosis, and either growth arrest or growth stimulation depending on the cellular context. We studied the role of Cyr61 in endometrial tumorigenesis. Levels of Cyr61 were decreased in endometrial tumors compared with normal endometrium. Knockdown of Cyr61 expression by RNA interference in a well differentiated endometrial adenocarcinoma cell line (Ishikawa) stimulated its cellular growth. Conversely, overexpression of the protein in the undifferentiated AN3CA endometrial cancer cell line decreased their growth concurrently with increased apoptosis in liquid culture. These same cells had decreased clonogenic capacity and a nearly complete loss of tumorigenicity in vivo. Furthermore, partially purified Cyr61 suppressed growth of endometrial cancer cells. The increased apoptosis in these endometrial cancer cells with forced overexpression of Cyr61 was associated with elevated expression of the pro-apoptotic proteins Bax, Bad, and TRAIL (tumor necrosis factor receptor-associated ligand). Cyr61-induced caspase-3 activation and depolarization of mitochondrial membrane. In summary, endometrial cancer cells have decreased expression of Cyr61 compared with normal endometrium, and this lowered expression may provide the transformed cells a growth advantage over their normal counterpart.     

Role of CCN,a vertebrate specific gene family,in development

The CCN family of genes constitutes six members of small secreted cysteine rich proteins, which exists only in vertebrates. The major members of CCN are CCN1 (Cyr61), CCN2 (CTGF), and CCN3 (Nov). CCN4, CCN5, and CCN6 were formerly reported to be in the Wisp family, but they are now integrated into CCN due to the resemblance of their four principal modules: insulin like growth factor binding protein, von Willebrand factor type C, thrombospondin type 1, and carboxy‐terminal domain. CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues, but most studies have focused on their principal role in osteo/chondrogenesis and vasculo/angiogenesis from the aspect of migration, growth, and differentiation of mesenchymal cells. CCN proteins simultaneously integrate and modulate the signals of integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch by direct binding. However, the priority in the use of the signals is different depending on the cell status. Even the equivalent counterparts show a difference in signal usage among species. It may be that the evolution of the CCN family continues to keep pace with vertebrate evolution itself.     

Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal

The thrombospondins are a family of secreted, oligomeric glycoproteins that interact with cell surfaces, multiple components of the extracellular matrix, growth factors and proteases. These interactions underlie complex roles in cell interactions and tissue homeostasis in animals. Thrombospondins have been grouped functionally with SPARCs, tenascins and CCN proteins as adhesion-modulating or matricellular components of the extracellular milieu. Although all these multi-domain proteins share various commonalities of domains, the grouping is not based on structural homologies. Instead, the terms emphasise the general observations that these proteins do not form large-scale ECM structures, yet act at cell surfaces and function in coordination with the structural ECM and associated extracellular proteins. The designation of adhesion-modulation thus depends on observed tissue and cell culture ECM distributions and on experimentally identified functional properties. To date, the evolutionary relationships of these proteins have not been critically compared: yet, knowledge of their evolutionary histories is clearly relevant to any consideration of functional similarities. In this article, we survey briefly the structural and functional knowledge of these protein families, consider the evolution of each family, and outline a perspective on their functional roles.     

Antiproliferative activity of CCN3: involvement of the C-terminal module and post-translational regulation

Previous work had suggested that recombinant CCN3 was partially inhibiting cell proliferation. Here we show that native CCN3 protein secreted into the conditioned medium of glioma transfected cells indeed induces a reduction in cell proliferation. Large amounts of CCN3 are shown to accumulate both cytoplasmically and extracellularly as cells reach high density, therefore highlighting new aspects on how cell growth may be regulated by CCN proteins. Evidence is presented establishing that the amount of CCN3 secreted into cell culture medium is regulated by post-translational proteolysis. As a consequence, the production of CCN3 varies throughout the cell cycle and CCN3 accumulates at the G2/M transition of the cycle. We also show that CCN3-induced inhibition of cell growth can be partially reversed by specific antibodies raised against a C-terminal peptide of CCN3. The use of several clones expressing various portions of CCN3 established that the CT module of CCN3 is sufficient to induce cell growth inhibition.     

Degradome Products of the Matricellular Protein CCN1 as Modulators of Pathological Angiogenesis in the Retina

CCN1 is a matricellular protein involved in normal vascular development and tissue repair. CCN1 exhibits cell- and context-dependent activities that are reflective of its tetramodular structure phylogenetically linked to four domains found in various matrix proteins. Here, we show that vitreal fluids from patients with proliferative diabetic retinopathy (PDR) were enriched with a two-module form of CCN1 comprising completely or partially the insulin-like growth factor-binding protein (IGFBP) and von Willebrand factor type C (vWC) domains. The two- and three-module forms comprising, in addition to IGFBP and vWC, the thrombospondin type 1 (TSP1) repeats are CCN1 degradome products by matrix metalloproteinase-2 and -14. The functional significance of CCN1 and its truncated variants was determined in the mouse model of oxygen-induced retinopathy, which simulates neovascular growth associated with PDR and assesses treatment outcomes. In this model, lentivirus-mediated expression of either CCN1 or the IGFBP-vWC-TSP1 form reduced ischemia-induced neovascularization, whereas ectopic expression of the IGFBP-vWC variant exacerbated pathological angiogenesis. The IGFBP-vWC form has potent proangiogenic properties promoting retinal endothelial cell growth, migration, and three-dimensional tubular structure formation, whereas the IGFBP-vWC-TSP1 variant suppressed cell growth and angiogenic gene expression. Both IGFBP-vWC and IGFBP-vWC-TSP1 forms exhibited predictable variations of their domain folding that enhanced their functional potential. These data provide new insights into the formation and activities of CCN1-truncated variants and raise the predictive value of the form containing completely or partially the IGFBP and vWC domains as a surrogate marker of CCN1 activity in PDR distinguishing pathological from physiological angiogenesis.     

CCN1 Secretion Induced by Cigarette Smoking Extracts Augments IL-8 Release from Bronchial Epithelial Cells

Inflammation involves in many cigarette smoke (CS) related diseases including the chronic obstructive pulmonary disease (COPD). Lung epithelial cell released IL-8 plays a crucial role in CS induced lung inflammation. CS and cigarette smoke extracts (CSE) both induce IL-8 secretion and subsequently, IL-8 recruits inflammatory cells into the lung parenchyma. However, the molecular and cellular mechanisms by which CSE triggers IL-8 release remain not completely understood. In this study, we identified a novel extracellular matrix (ECM) molecule, CCN1, which mediated CSE induced IL-8 secretion by lung epithelial cells. We first found that CS and CSE up-regulated CCN1 expression and secretion in lung epithelial cells in vivo and in vitro. CSE up-regulated CCN1 via induction of reactive oxygen spices (ROS) and endoplasmic reticulum (ER) stress. p38 MAPK and JNK activation were also found to mediate the signal pathways in CSE induced CCN1. CCN1 was secreted into ECM via Golgi and membrane channel receptor aquaporin4. After CSE exposure, elevated ECM CCN1 functioned via an autocrine or paracrine manner. Importantly, CCN1 activated Wnt pathway receptor LRP6, subsequently stimulated Wnt pathway component Dvl2 and triggered beta-catenin translocation from cell membrane to cytosol and nucleus. Treatment of Wnt pathway inhibitor suppressed CCN1 induced IL-8 secretion from lung epithelial cells. Taken together, CSE increased CCN1 expression and secretion in lung epithelial cells via induction of ROS and ER stress. Increased ECM CCN1 resulted in augmented IL-8 release through the activation of Wnt pathway.     

The role of matricellular proteins in glaucoma

Glaucoma is an optic neuropathy affecting approximately 60 million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemm's canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin.Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma.     

Cysteine-rich protein 61 (CCN1) and connective tissue growth factor (CCN2) at the crosshairs of ocular neovascular and fibrovascular disease therapy

Abstract

The vasculature forms a highly branched network investing every organ of vertebrate organisms. The retinal circulation, in particular, is supported by a central retinal artery branching into superficial arteries, which dive into the retina to form a dense network of capillaries in the deeper retinal layers. The function of the retina is highly dependent on the integrity and proper functioning of its vascular network and numerous ocular diseases including diabetic retinopathy, age-related macular degeneration and retinopathy of prematurity are caused by vascular abnormalities culminating in total and sometimes irreversible loss of vision. CCN1 and CCN2 are inducible extracellular matrix (ECM) proteins which play a major role in normal and aberrant formation of blood vessels as their expression is associated with developmental and pathological angiogenesis. Both CCN1 and CCN2 achieve disparate cell-type and context-dependent activities through modulation of the angiogenic and synthetic phenotype of vascular and mesenchymal cells respectively. At the molecular level, CCN1 and CCN2 may control capillary growth and vascular cell differentiation by altering the composition or function of the constitutive ECM proteins, potentiating or interfering with the activity of various ligands and/or their receptors, physically interfering with the ECM-cell surface interconnections, and/or reprogramming gene expression driving cells toward new phenotypes. As such, these proteins emerged as important prognostic markers and potential therapeutic targets in neovascular and fibrovascular diseases of the eye. The purpose of this review is to highlight our current knowledge and understanding of the most recent data linking CCN1 and CCN2 signaling to ocular neovascularization bolstering the potential value of targeting these proteins in a therapeutic context.     

CCN2 (Cellular Communication Network factor 2) in the bone marrow microenvironment,normal and malignant hematopoiesis

CCN2, formerly termed Connective Tissue Growth Factor, is a protein belonging to the Cellular Communication Network (CCN)-family of secreted extracellular matrix-associated proteins. As a matricellular protein it is mainly considered to be active as a modifier of signaling activity of several different signaling pathways and as an orchestrator of their cross-talk. Furthermore, CCN2 and its fragments have been implicated in the regulation of a multitude of biological processes, including cell proliferation, differentiation, adhesion, migration, cell survival, apoptosis and the production of extracellular matrix products, as well as in more complex processes such as embryonic development, angiogenesis, chondrogenesis, osteogenesis, fibrosis, mechanotransduction and inflammation. Its function is complex and context dependent, depending on cell type, state of differentiation and microenvironmental context. CCN2 plays a role in many diseases, especially those associated with fibrosis, but has also been implicated in many different forms of cancer. In the bone marrow (BM), CCN2 is highly expressed in mesenchymal stem/stromal cells (MSCs). CCN2 is important for MSC function, supporting its proliferation, migration and differentiation. In addition, stromal CCN2 supports the maintenance and longtime survival of hematopoietic stem cells, and in the presence of interleukin 7, stimulates the differentiation of pro-B lymphocytes into pre-B lymphocytes. Overexpression of CCN2 is seen in the majority of B-acute lymphoblastic leukemias, especially in certain cytogenetic subgroups associated with poor outcome. In acute myeloid leukemia, CCN2 expression is increased in MSCs, which has been associated with leukemic engraftment in vivo. In this review, the complex function of CCN2 in the BM microenvironment and in normal as well as malignant hematopoiesis is discussed. In addition, an overview is given of data on the remaining CCN family members regarding normal and malignant hematopoiesis, having many similarities and some differences in their function.     

Role of mechanical-stress inducible protein Hcs24/CTGF/CCN2 in cartilage growth and regeneration: mechanical stress induces expression of Hcs24/CTGF/CCN2 in a human chondrocytic cell line HCS-2/8, rabbit costal chondrocytes and meniscus tissue cells

Mechanical stress plays an important role in the cartilage metabolism. The aim of this study is to determine the influence of mechanical load magnitude and frequency on cartilage metabolism in terms of the expression of hypertrophic chondrocyte-specific gene product 24/connective tissue growth factor/CCN family 2 (Hcs24/CTGF/CCN2), as an essential mediator of extracellular matrix (ECM) production. When a human chondrocytic cell line, HCS-2/8 was exposed to uni-axial cyclic mechanical force (6% elongation, 10 times/min) only for 30 min, the expression level of Hcs24/CTGF/CCN2 (CCN2) increased, and c-Jun N-terminal protein kinase (JNK) was activated. These findings suggest that stretch-induced CCN2 may be mediated by the JNK pathway. When HCS-2/8 cells were subjected to cyclic tension force at 15 kPa, 30 cycles/min, which has been reported to be a degradation force for HCS-2/8 cells, the expressions of CCN2 and aggrecan were inhibited, and such expressions remained unchanged in rabbit hyaline costal cartilage cells. However, these expressions increased in rabbit meniscus tissue cells. These findings suggest that the sensitivity of mechanical stretch may be different depending on the type of cells. Furthermore, CCN2 was co-localized with aggrecan in this meniscus tissue region exposed to mechanical stress in vivo. These findings suggest that CCN2 induced by mechanical stress may therefore play some role in meniscus growth and regeneration.     

Identification and quantification of proteins differentially secreted by a pair of normal and malignant breast‐cancer cell lines

Secreted proteins play a pivotal role in cellular functions. To better understand malignant behavior, we adapted stable isotopic labeling with amino acids in cell culture technology to identify and quantify proteins differentially released into the extracellular media by a pair of normal and malignant breast‐cancer cell lines. Approximately 380 non‐redundant proteins were quantified in serum‐free media. Of the assigned proteins, 62% are classified secreted in protein databases and an additional 25% are designated secreted in the literature. A number of growth factors were found differentially regulated. Tumor necrosis factor, pigment epithelial‐differentiating factor and stem‐cell growth factor precursor showed decreased expression in breast‐cancer cell line, whereas Inhibin beta and macrophage migration inhibitory factor show increased expression. Interestingly, protease inhibitors, including plasma protease (C1) inhibitor, PZP precursor, and SerpinE2 were significantly down‐regulated in cancer cell line as were angiostatic factors from extracellular matrix (ECM) such as endorepillin. Further, the C‐terminal fragment of type XVIII collagen, endostatin, a potent angiostatic factor, was down‐regulated as well whereas extracellular collagens and osteoblast‐specific factor 2 (OSF‐2), were up‐regulated. Differential expression and secretion of SerpinE2 and OSF‐2 were confirmed using Western blotting. These results corroborate models of invasive tumors sustained by elaborate coordination of stromal cells via chemokines and growth factors, while protease inhibitors remodel the ECM to stimulate angiogenesis.     

CCN5, a secreted protein,localizes to the nucleus

CCN5, a member of the CCN family of growth factors, inhibits the proliferation and migration of smooth muscle cells in cell culture and animal models. Expressed in both embryonic and adult tissues, CCN5 exhibits a matricellular localization pattern characteristic of secreted proteins that are closely associated with the cell surface. In addition to this observed expression pattern, immunohistochemical evidence suggests the presence of nuclear CCN5 in some cells. To determine if CCN5 localizes to the nucleus we performed immunofluorescence, confocal imaging, and cell fractionation to corroborate the immunohistochemical observations. After confirming the presence of nuclear CCN5 using four independent experimental methods, we identified a single putative nuclear localization signal in the von Willebrand factor C domain of mouse and rat CCN5. Site directed mutagenesis of the three basic amino acids in the putative nuclear localization sequence did not prevent nuclear localization of CCN5 in four different cell types, suggesting that CCN5 nuclear transport is not mediated by the only canonical nuclear localization signal present in the primary amino acid sequence. Future work will address the mechanism of nuclear localization and the function of nuclear versus secreted CCN5.     

CTGF/CCN2 has a chemoattractive function but a weak adhesive property to embryonic carcinoma cells

Connective tissue growth factor (CTGF/CCN2) is a protein of the CCN family that modulates cell–ECM interactions in a variety of cell types. In this study, we investigated the chemotactic and adhesive properties of CCN2 protein in embryonic teratocarcinoma P19 cells. Initially, P19 cells were attracted to CCN2-coated agarose beads. In Boyden chamber experiments, CCN2-containing medium induced a threefold greater migration of P19 cells. CCN2 adhesion properties were studied by using optical tweezers. The specific adhesion times of P19 cells to polystyrene beads coated with laminin, fibronectin, CCN2 and bovine serum albumin were 1.8 ± 0.5s, 2.7 ± 0.4s, 10 ± 2s and 13 ± 2s, respectively, revealing an unexpectedly low adhesive capacity of CCN2 protein for P19 cells. In conclusion, our findings support the chemoattractive role of CCN2 for P19 cells, but not its adhesive role when compared to laminin or fibronectin.