It is known that expression of the macrophage colony-stimulating factor (M-CSF) gene is induced in articular chondrocytes upon inflammation. However, the functional role of M-CSF in cartilage has been unclear. In this study, we describe possible roles of M-CSF in the protection and maintenance of the articular cartilage based on the results of experiments using human chondrocytic cells and rat primary chondrocytes. Connective tissue growth factor (CTGF/CCN2) is known to be a potent molecule to regenerate damaged cartilage by promoting the growth and differentiation of articular chondrocytes. Here, we uncovered the fact that M-CSF induced the mRNA expression of the ctgf/ccn2 gene in those cells. Enhanced production of CTGF/CCN2 protein by M-CSF was also confirmed. Furthermore, M-CSF could autoactivate the m-csf gene, forming a positive feed-back network to amplify and prolong the observed effects. Finally, promotion of proteoglycan synthesis was observed by the addition of M-CSF. These findings taken together indicate novel roles of M-CSF in articular cartilage metabolism in collaboration with CTGF/CCN2, particularly during an inflammatory response. Such roles of M-CSF were further supported by the distribution of M-CSF producing chondrocytes in experimentally induced rat osteoarthritis cartilage in vivo.

BACKGROUND

Connective Tissue Growth Factor (CTGF/CCN2) is an important mediator of kidney fibrosis. Previous observations indicated that attenuation of CCN2 expression sufficed to alleviate early kidney damage. However, little is known about the role of CCN2 in fibrosis of severely damaged and more chronically injured kidneys. Therefore, we examined the effects of CCN2 haploinsufficiency on the progression of renal scarring in long-term STZ-induced diabetic nephropathy, in a more advanced stage of obstructive nephropathy following unilateral ureteric obstruction (UUO), and in severe aristolochic acid (AA)-induced tubulotoxic nephritis.

METHODS

Wild-type (WT, CCN2(+/+)) and hemizygous CCN2(+/-) C57Bl/6 mice were studied. In the diabetes experiment, streptozotocin-injected and control mice were followed for 6 months, with regular blood pressure, glycaemia and albuminuria recordings. In the UUO experiment, the left ureter was obstructed for 14 days with the contralateral kidney serving as control. For the AA experiment, mice were followed for 25 days after 5 intraperitoneal injections with AA and compared to control mice injected with buffer alone. Organs were harvested for histology, mRNA and protein measurements. Collagen content was determined by HPLC and expressed as hydroxyproline/proline ratio.

RESULTS

CCN2 expression was significantly increased in the damaged as compared to control kidneys. In all three models, CCN2 levels in the damaged kidneys of CCN2(+/-) mice averaged about 50% of those in damaged WT kidneys. After 6 months of diabetes, albuminuria was increased 2.5-fold in WT mice, compared to 1.5-fold in CCN2(+/-) mice, mesangial matrix was expanded 5-fold in WT and 4.4-fold in CCN2(+/-) mice and the glomerular basement membrane was thickened 1.3-fold in WT and 1.5-fold in CCN2(+/-) mice (all differences between WT and CCN2(+/-) mice are NS). Tubular damage and interstitial fibrosis scores were also not different between Wt and CCN2(+/-) mice in the diabetes (1.8 vs. 1.7), UUO (2.8 vs. 2.6), and AA (1.4 vs. 1.2) models, as was the case for macrophage influx and collagen content in these three models.

CONCLUSIONS

Unlike in mild and relatively early STZ-induced diabetic nephropathy, scarring of severely and chronically damaged kidneys is not attenuated by a 50% reduction of CCN2 to (near) normal levels. This suggests that CCN2 is either redundant in severe and chronic kidney disease, or that it is a limiting factor only at subnormal concentrations requiring further reduction by available or emerging therapies to prevent fibrosis of the severely injured kidney.

BACKGROUND

Myocardial CCN2/CTGF (connective tissue growth factor) is strongly induced in heart failure (HF) and acts as a cardioprotective factor in ischemia/reperfusion injury. However, its functional role in myocardial hypertrophy remains unresolved.

RESULTS

Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) and non-transgenic littermate control (NLC) mice were subjected to chronic pressure-overload by abdominal aortic banding. After 4weeks of persistent pressure-overload, a time point at which compensatory hypertrophy of the left ventricle (LV) prevails, Tg-CTGF mice displayed diminished increase of LV mass compared with NLC. At study end-point after 12 weeks of sustained aortic constriction, the mice displayed LV dilatation and reduced cardiac function. Repeated transthoracic echocardiography during the 12 weeks of chronic pressure-overload, revealed attenuation of LV dilatation and virtually sustained systolic function in Tg-CTGF mice compared with NLC mice. Also, increase of LV mass was blunted in Tg-CTGF versus NLC mice at study end-point. Consistently, increases of myocardial ANP, BNP and skeletal α-actin mRNA levels were blunted in Tg-CTGF mice subjected to chronic pressure-overload. Furthermore, cardiac myocytes from Tg-CTGF mice displayed increased phospho-NFATc2 levels and attenuated hypertrophic response upon stimulation with α1-adrenoceptor agonist, indicating that CTGF attenuates hypertrophic signaling in cardiac myocytes. Increase of myocardial collagen contents in mice subjected to aortic banding was similar in Tg-CTGF and NLC mice, indicating that CTGF have minimal impact on myocardial collagen deposition.

CONCLUSIONS

This study provides novel evidence that CTGF attenuates cardiac hypertrophy upon chronic pressure-overload due to inhibition of signaling mechanisms that promote pathologic myocardial hypertrophy.

The lack of approved therapies for hepatic fibrosis seriously limits medical management of patients with chronic liver disease. Since extracellular vesicles (EVs) function as conduits for intercellular molecular transfer, we investigated if EVs from healthy individuals have anti-fibrotic properties. Hepatic fibrogenesis or fibrosis in carbon tetrachloride (CCl4)- or thioacetic acid-induced liver injury models in male or female mice were suppressed by serum EVs from normal mice (EVN) but not from fibrotic mice (EVF). CCl4-treated mice undergoing EVN therapy also exhibited reduced levels of hepatocyte death, inflammatory infiltration, circulating AST/ALT levels and hepatic or circulating pro-inflammatory cytokines. Hepatic histology, liver function tests or circulating proinflammatory cytokine levels were unaltered in control mice receiving EVN. As determined using PKH26-labelled EVN, principal target cells included hepatic stellate cells (HSC; a normally quiescent fibroblastic cell that undergoes injury-induced activation and produces fibrosis during chronic injury) or hepatocytes which showed increased EVN binding after, respectively, activation or exposure to CCl4. In vitro, EVN decreased proliferation and fibrosis-associated molecule expression in activated HSC, while reversing the inhibitory effects of CCl4 or ethanol on hepatocyte proliferation. In mice, microRNA-34c, -151-3p, -483-5p, -532-5p and -687 were more highly expressed in EVN than EVF and mimics of these microRNAs (miRs) individually suppressed fibrogenic gene expression in activated HSC. A role for these miRs in contributing to EVN actions was shown by the ability of their corresponding antagomirs to individually and/or collectively block the therapeutic effects of EVN on activated HSC or injured hepatocytes. Similarly, the activated phenotype of human LX-2 HSC was attenuated by serum EVs from healthy human subjects and contained higher miR-34c, -151-3p, -483-5p or -532-5p than EVs from hepatic fibrosis patients. In conclusion, serum EVs from normal healthy individuals are inherently anti-fibrogenic and anti-fibrotic, and contain microRNAs that have therapeutic actions in activated HSC or injured hepatocytes. Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCl4: carbon tetrachloride; CCN2: connective tissue growth factor; E: eosin; EGFP: enhanced green fluorescent protein; EVs: extracellular vesicles; EVF: serum EVs from mice with experimental hepatic fibrosis; EVN: serum EVs from normal mice; H: hematoxylin; HSC: hepatic stellate cell; IHC: immunohistochemistry; IL: interleukin; MCP-1: monocyte chemotactic protein-1; miR: microRNA; mRNA: messenger RNA; NTA: nanoparticle tracking analysis; PCNA: proliferating cell nuclear antigen; qRT-PCR: quantitative real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulphate - polyacrylamide gel electrophoresis; αSMA: alpha smooth muscle actin; TAA: thioacetic acid; TG: transgenic; TGF-β: transforming growth factor beta; TEM: transmission electron microscopy; TNFα: tumour necrosis factor alpha.

BACKGROUND

Carcinoid heart disease, a known complication of neuroendocrine tumors, is characterized by right heart fibrotic lesions. Carcinoid heart disease has traditionally been defined by the degree of valvular involvement. Right ventricular (RV) dysfunction due to mural involvement may also be a manifestation. Connective tissue growth factor (CCN2) is elevated in many fibrotic disorders. Its role in carcinoid heart disease is unknown. We sought to investigate the relationship between plasma CCN2 and valvular and mural involvement in carcinoid heart disease.

METHODS

Echocardiography was performed in 69 patients with neuroendocrine tumors. RV function was assessed using tissue Doppler analysis of myocardial systolic strain. Plasma CCN2 was analyzed using an enzyme-linked immunosorbent assay. Mann-Whitney U, Kruskal-Wallis, Chi-squared and Fisher's exact tests were used to compare groups where appropriate. Linear regression was used to evaluate correlation.

RESULTS

Mean strain was -21% +/- 5. Thirty-three patients had reduced RV function (strain>> -20%, mean -16% +/- 3). Of these, 8 had no or minimal tricuspid and/or pulmonary regurgitation (TR/PR). Thirty-six patients had normal or mildly reduced RV function (strain < or = -20%, mean -25% +/- 3). There was a significant inverse correlation between RV function and plasma CCN2 levels (r = 0.47, p < 0.001). Patients with reduced RV function had higher plasma CCN2 levels than those with normal or mildly reduced RV function (p < 0.001). Plasma CCN2>> or = 77 microg/L was an independent predictor of reduced RV function (odds ratio 15.36 [95% CI 4.15;56.86]) and had 88% sensitivity and 69% specificity for its detection (p < 0.001). Plasma CCN2 was elevated in patients with mild or greater TR/PR compared to those with no or minimal TR/PR (p = 0.008), with the highest levels seen in moderate to severe TR/PR (p = 0.03).

CONCLUSIONS

Elevated plasma CCN2 levels are associated with RV dysfunction and valvular regurgitation in NET patients. CCN2 may play a role in neuroendocrine tumor-related cardiac fibrosis and may serve as a marker of its earliest stages.

CCN family proteins 2 and 3 (CCN2 and CCN3) belong to the CCN family of proteins, all having a high level of structural similarity. It is widely known that CCN2 is a profibrotic molecule that mediates the development of fibrotic disorders in many different tissues and organs. In contrast, CCN3 has been recently suggested to act as an anti-fibrotic factor in several tissues. This CCN3 action was shown earlier to be exerted by the repression of the CCN2 gene expression in kidney tissue, whereas different findings were obtained for liver cells. Thus, the molecular action of CCN3 yielding its anti-fibrotic effect is still controversial. Here, using a general model of fibrosis, we evaluated the effect of CCN3 overexpression on the gene expression of all of the CCN family members, as well as on that of fibrotic marker genes. As a result, repression of CCN2 gene expression was modest, while type I collagen and α-smooth muscle actin gene expression was prominently repressed. Interestingly, not only CCN2, but also CCN4 gene expression showed a decrease upon CCN3 overexpression. These findings indicate that fibrotic gene induction is under the control of a complex molecular network conducted by CCN family members functioning together.

Fibrotic disease is a significant cause of mortality. CCN2 (connective tissue growth factor [CTGF]), a member of the CCN family of matricellular proteins, plays a significant role in driving the fibrogenic effects of cytokines such as transforming growth factor beta (TGFbeta). It has been proposed that other members of the CCN family can either promote or antagonize the action of CCN2, depending on the context. A recent elegant study published by Bruce Riser and colleagues (Am J Pathol. 174:1725-34, 2009) illustrates that CCN3 (nov) antagonizes the fibrogenic effects of CCN2. This paper, the subject of this commentary, raises the intriguing possibility that CCN3 may be used as a novel anti-fibrotic therapy.

Disruption of myelination during development has been implicated in a range of neurodevelopmental disorders including tuberous sclerosis complex (TSC). TSC patients with autism display impairments in white matter integrity. Similarly, mice lacking neuronal Tsc1 have a hypomyelination phenotype. However, the mechanisms that underlie these phenotypes remain unknown. In this study, we demonstrate that neuronal TSC1/2 orchestrates a program of oligodendrocyte maturation through the regulated secretion of connective tissue growth factor (CTGF). We characterize oligodendrocyte maturation both in vitro and in vivo. We find that neuron-specific Tsc1 deletion results in an increase in CTGF secretion that non-cell autonomously stunts oligodendrocyte development and decreases the total number of oligodendrocytes. Genetic deletion of CTGF from neurons, in turn, mitigates the TSC-dependent hypomyelination phenotype. These results show that the mechanistic target of rapamycin (mTOR) pathway in neurons regulates CTGF production and secretion, revealing a paracrine mechanism by which neuronal signaling regulates oligodendrocyte maturation and myelination in TSC. This study highlights the role of mTOR-dependent signaling between neuronal and nonneuronal cells in the regulation of myelin and identifies an additional therapeutic avenue for this disease.

Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment.

Dermal connective tissue is a supportive structure required for skin's barrier function; dysregulated dermal homeostasis results in chronic wounds and fibrotic diseases. The multifunctional cytokine transforming growth factor (TGF) β promotes connective tissue deposition, repair, and fibrosis. TGF-β acts through well-defined canonical pathways; however, the non-canonical pathways through which TGF-β selectively promotes connective tissue deposition are unclear. In dermal fibroblasts, we show that inhibition of the non-canonical TGF-β-activated kinase 1 (TAK1) selectively reduced the ability of TGF-β to induce expression of a cohort of wound healing genes, such as collagens, CCN2, TGF-β1, and IL-6. Fibroblast-specific TAK1-knockout mice showed impaired cutaneous tissue repair and decreased collagen deposition, α-smooth muscle actin and CCN2 expression, proliferating cell nuclear antigen staining, and c-Jun N-terminal kinase and p38, but not Smad3, phosphorylation. TAK1-deficient fibroblasts showed reduced cell proliferation, migration, cell attachment/spreading, and contraction of a floating collagen gel matrix. TAK1-deficient mice also showed progressively reduced skin thickness and collagen deposition. Thus, TAK1 is essential for connective tissue deposition in the dermis.

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

Matricellular proteins play a critical role in the development of tubulointerstitial fibrosis and renal disease progression. Connective tissue growth factor (CTGF/CCN2), a CCN family member of matricellular proteins, represents an important mediator during development of glomerular and tubulointerstitial fibrosis in progressive kidney disease. We have recently reported that oncostatin M (OSM) is a potent inhibitor of TGF-β1-induced CTGF expression in human proximal tubular cells (PTC). In the present study we examined the role of TGF-β1- and OSM-induced signaling mechanisms in the regulation of CTGF mRNA expression in human proximal tubular HK-2 cells. Utilizing siRNA-mediated gene silencing we found that TGF-β1-induced expression of CTGF mRNA after 2h of stimulation at least partially depends on SMAD3 but not on SMAD2. In contrast to TGF-β1, OSM seems to exert a time-dependent dual effect on CTGF mRNA expression in these cells. While OSM led to a rapid and transient induction of CTGF mRNA expression between 15 min and 1h of stimulation it markedly suppressed basal and TGF-β1-induced CTGF mRNA levels thereafter. Silencing of STAT1 or STAT3 attenuated basal CTGF mRNA levels indicating that both STAT isoforms may be involved in the regulation of basal CTGF mRNA expression. However, knockdown of STAT3 but not STAT1 prevented OSM-mediated suppression of basal and TGF-β1-induced upregulation of CTGF mRNA expression. Together these results suggest that the inhibitory effect of OSM on TGF-β1-induced CTGF mRNA expression is mainly driven by STAT3, thereby providing a signaling mechanism whereby OSM may contribute to tubulointerstitial protection.

BACKGROUND

Connective Tissue Growth Factor (CTGF/CCN2), a known matrix-associated protein, is required for the lactogenic differentiation of mouse mammary epithelial cells. An HC11 mammary epithelial cell line expressing CTGF/CCN2 was constructed to dissect the cellular responses to CTGF/CCN2 that contribute to this differentiation program.

RESULTS

Tetracycline-regulated expression of CTGF/CCN2 in HC11 cells enhanced multiple markers of lactogenic differentiation including beta-casein transcription and mammosphere formation. In a separate measure of mammary differentiation the addition of CTGF/CCN2 to cultures of MCF10A cells increased the development of acini in vitro. In HC11 cells the elevated levels of CTGF/CCN2 diminished the requirement for extracellular matrix proteins in the activation of beta-casein transcription, indicating that CTGF/CCN2 contributed to lactogenic differentiation through the regulation of matrix dependent cell adhesion. CTGF/CCN2 expression in HC11 cells increased expression of extracellular matrix proteins and integrins, enhanced the formation of focal adhesion complexes, and increased survival signaling. In addition, HC11 cells adhered to immobilized CTGF/CCN2 and this was inhibited by function-blocking antibodies to the integrins alpha6 and beta1, and to a lesser degree by antibody to beta3 integrin.

CONCLUSIONS

CTGF/CCN2 expression in HC11 cells led to an increase in multiple markers of lactogenic differentiation. The mechanisms by which CTGF/CCN2 contributed to lactogenic differentiation include direct binding of CTGF/CCN2 to integrin complexes and CTGF/CCN2-induced matrix protein expression resulting in elevated integrin functionality.

We have generated transgenic mice expressing a kinase-deficient type II transforming growth factor-beta (TGFbeta) receptor selectively on fibroblasts (TbetaRIIDeltak-fib). These mice develop dermal and pulmonary fibrosis. In the present study we explore activation of TGFbeta signaling pathways in this strain and examine the profibrotic properties of explanted transgenic fibroblasts including myofibroblast differentiation and abnormal metalloproteinase production. Gene expression profiles of littermate wild type or transgenic fibroblasts were compared using high-density gene arrays and validated by Taqman reverse transcriptase-PCR, Northern and Western blotting. Using a specific inhibitor (SD-208) we demonstrate that the abnormal phenotype of these cells is dependent upon TbetaRI kinase (ALK5) activity, and that transgenic fibroblasts show enhanced expression and activation of TGFbeta together with increased levels of wild type TbetaRII. Moreover, we confirm that transgene expression is itself regulated by TGFbeta and that expression at low levels facilitates signaling, whereas high level expression is inhibitory. For a subset of TGFbeta responsive genes basal up-regulation is normalized or suppressed by exogenous recombinant TGFbeta1 at time points coincident with increased transgene expression. These findings explain the profound refractoriness of TbetaRIIDeltak-fib fibroblasts to exogenous TGFbeta1, despite their activated phenotype. Thus, transgenic fibroblasts recapitulate many hallmark biochemical properties of fibrotic cells, including high level CTGF (CCN2) expression and type I collagen overproduction, altered MMP production, and myofibroblast differentiation. These cells also show an enhanced ability to contract collagen gel matrices. Our study demonstrates that altered high affinity TGFbeta receptor function may lead to ligand-dependent activation of downstream signaling, and provides further evidence of a pivotal role for sustained TGFbeta overactivity in fibrosis.

BACKGROUND

Connective tissue growth factor (CTGF/CCN2) is a matricellular protein that is highly expressed during bone development. Mice with global CTGF ablation (knockout, KO) have multiple skeletal dysmorphisms and perinatal lethality. A quantitative analysis of the bone phenotype has not been conducted.

RESULTS

We demonstrated skeletal site-specific changes in growth plate organization, bone microarchitecture, and shape and gene expression levels in CTGF KO compared with wild-type mice. Growth plate malformations included reduced proliferation zone and increased hypertrophic zone lengths. Appendicular skeletal sites demonstrated decreased metaphyseal trabecular bone, while having increased mid-diaphyseal bone and osteogenic expression markers. Axial skeletal analysis showed decreased bone in caudal vertebral bodies, mandibles, and parietal bones in CTGF KO mice, with decreased expression of osteogenic markers. Analysis of skull phenotypes demonstrated global and regional differences in CTGF KO skull shape resulting from allometric (size-based) and nonallometric shape changes. Localized differences in skull morphology included increased skull width and decreased skull length. Dysregulation of the transforming growth factor-β-CTGF axis coupled with unique morphologic traits provides a potential mechanistic explanation for the skull phenotype.

CONCLUSIONS

We present novel data on a skeletal phenotype in CTGF KO mice, in which ablation of CTGF causes site-specific aberrations in bone formation.

Osteocytes produce various factors that mediate the onset of bone formation and resorption and play roles in maintaining bone homeostasis and remodeling in response to mechanical stimuli. One such factor, CCN2, is thought to play a significant role in osteocyte responses to mechanical stimuli, but its function in osteocytes is not well understood. Here, we showed that CCN2 induces apoptosis in osteocytes under compressive force loading. Compressive force increased CCN2 gene expression and production, and induced apoptosis in osteocytes. Application of exogenous CCN2 protein induced apoptosis, and a neutralizing CCN2 antibody blocked loading-induced apoptosis. We further examined how CCN2 induces loaded osteocyte apoptosis. In loaded osteocytes, extracellular signal-regulated kinase 1/2 (ERK1/2) was activated, and an ERK1/2 inhibitor blocked loading-induced apoptosis. Furthermore, application of exogenous CCN2 protein caused ERK1/2 activation, and the neutralizing CCN2 antibody inhibited loading-induced ERK1/2 activation. Therefore, this study demonstrated for the first time to our knowledge that enhanced production of CCN2 in osteocytes under compressive force loading induces apoptosis through activation of ERK1/2 pathway.

Therapeutic targets of broad relevance are likely located in pathogenic pathways common to disorders of various etiologies. Screening for targets of this type revealed CCN genes to be consistently upregulated in multiple cardiomyopathies. We developed RNA interference (RNAi) to silence CCN2 and found this single-target approach to block multiple proinflammatory and profibrotic pathways in activated primary cardiac fibroblasts (PCFBs). The RNAi-strategy was developed in murine PCFBs and then investigated in "individual" human PCFBs grown from human endomyocardial biopsies (EMBs). Screening of short hairpin RNA (shRNA) sequences for high silencing efficacy and specificity yielded RNAi adenovectors silencing CCN2 in murine or human PCFBs, respectively. Comparison of RNAi with CCN2-modulating microRNA (miR) vectors expressing miR-30c or miR-133b showed higher efficacy of RNAi. In murine PCFBs, CCN2 silencing resulted in strongly reduced expression of stretch-induced chemokines (Ccl2, Ccl7, Ccl8), matrix metalloproteinases (MMP2, MMP9), extracellular matrix (Col3a1), and a cell-to-cell contact protein (Cx43), suggesting multiple signal pathways to be linked to CCN2. Immune cell chemotaxis towards CCN2-depleted PCFBs was significantly reduced. We demonstrate here that this RNAi strategy is technically applicable to "individual" human PCFBs, too, but that these display individually strikingly different responses to CCN2 depletion. Either genomically encoded factors or stable epigenetic modification may explain different responses between individual PCFBs. The new RNAi approach addresses a key regulator protein induced in cardiomyopathies. Investigation of this and other molecular therapies in individual human PCBFs may help to dissect differential pathogenic processes between otherwise similar disease entities and individuals.

Colorectal cancer (CRC) is a major health problem causing significant morbidity and mortality. Previous results from various studies indicate that CRC tumorigenicity encompasses tumor microenvironment, emphasizing the complex interacting network between cancer cells and nearby host cells, which triggers diverse signaling pathways to promote the growth and spread of cancer cells. The CCN family proteins share a uniform modular structure, mediating a variety of physiological functions, including proliferation, apoptosis, migration, adhesion, differentiation, and survival. Furthermore, CCN proteins are also involved in CRC initiation and development. Many studies have shown that CCN members, such as CCN1, CCN2, CCN3, Wnt-induced secreted protein (WISP)-1, WISP-2, and WISP-3, are dysregulated in CRC, which implies potential diagnostic markers or therapeutic targets clinically. In this review, we summarize the research findings on the role of CCN family proteins in CRC initiation, development, and progression, highlighting their potential for diagnosis, prognosis, and therapeutic application.

CCN5 is a secreted heparin- and estrogen-regulated matricellular protein that inhibits vertebrate smooth muscle cell proliferation and motility. CCN5 is expressed throughout murine embryonic development in most organs and tissues. However, after embryonic development is complete, we hypothesized that CCN5 distribution would be largely restricted to small set of tissues, including smooth muscle cells of the arteries, uterus, airway, and digestive tract. Because CCN5 inhibits proliferation of smooth muscle cells in vitro, it might function to prevent excessive growth in vivo. In contrast, another member of the CCN family, CCN2, promotes smooth muscle cell proliferation in vitro, and thus it was expected that its expression levels would be low in uninjured normal adult tissues. Frozen sections from adult tissues and organs were analyzed immunohistochemically using anti-CCN5 and anti-CCN2 antibodies. Both proteins were detected in arteries, the uterus, bronchioles, and the digestive tract as expected, and also in many other tissues including the pancreas, spleen, liver, skeletal muscle, ovary, testis, thymus, brain, olfactory epithelium, and kidney. CCN5 and CCN2 protein was found in smooth muscle, endothelial cells, epithelial cells, skeletal muscle, cells of the nervous system, and numerous other cell types. In many cells, both CCN5 and CCN2 was present in the nucleus. Rather than having opposite patterns of localization, CCN5 and CCN2 often had similar sites of expression. The wide distribution of both CCN5 and CCN2 suggests that both proteins have additional biological functions beyond those previously identified in specific cellular and pathological models.

BACKGROUND

Continuous ambulatory peritoneal dialysis (CAPD) is a major treatment modality for end-stage renal failure. The peritoneal membrane exhibits pathological changes that correlate with the duration of dialysis. These changes are due to the exposure of the peritoneum to non-physiologic peritoneal dialysis solution (PDS) with a high glucose content, and containing potentially toxic substances including glucose degradation products (GDP) and advanced glycation end products (AGE). Connective tissue growth factor (CTGF/CCN2) is one of the determinants of progressive fibrosis and peritoneal membrane dysfunction in CAPD. In this study, we examined the CCN2 expression and its regulation in peritoneal resident cells using a cell culture model.

METHODS

The expression of transforming growth factor-beta (TGF-beta), CCN2 and vascular endothelial growth factor (VEGF) in human peritoneal mesothelial cells (HPMC), human peritoneal fibroblasts (HPF) or endothelial cell line EA.hy926 (EC) cultured with various PDS and their components was examined by quantitative PCR (qPCR). The modulation of CCN2 synthesis under the crosstalk between HPMC and HPF or EC was examined using a conditioned medium transfer system in which HPMC was exposed to conditioned media obtained from HPF or EC incubated with PDS and their components. The differential effects of TGF-beta, CCN2 and VEGF in inducing the expression of transcriptional factors as well as interleukin-6 (IL-6), matrix metallopeptidase 9 (MMP-9) and collagen I were examined by electrophoretic mobility-shift assay (EMSA) and qPCR.

RESULTS

PDS and their components differentially modulated the expression of TGF-beta, CCN2 and VEGF in HPMC, HPF and EC. The expression of CCN2 by HPMC was significantly increased after cultured with a HPF-conditioned medium and an EC-conditioned medium. Neutralizing anti-TGF-beta antibodies reduced but not completely abolished the CCN2 synthesis in HPMC cultured with the HPF- or EC-conditioned medium. CCN2, TGF-beta and VEGF activated distinct transcriptional factors in HPMC, which resulted in divergent biological responses in terms of IL-6, MMP-9 and collagen I mRNA expression.

CONCLUSIONS

AGE and GDPs in PDS differentially regulate the synthesis of CCN2 by peritoneal resident cells. The CCN2 synthesis by HPMC can be further amplified by TGF-beta released from HPF or EC. The differential activation of different transcriptional factors and diverse response of HPMC towards CCN2, TGF-beta and VEGF suggest that these cytokines/growth factors have an overlapping and distinct role on HPMC.

Multiple roles have been already recognized for CCN2 in cartilage development and regeneration. However, the effects of CCN2 on bone regeneration remain to be elucidated. In this study, the utility of CCN2 on bone regeneration was examined in vitro and in vivo in combination with hydroxyapatite (HAp) as a scaffold. Human bone marrow stromal cells (hBMSCs) were isolated from human iliac bone marrow aspirates of healthy donors and expanded, and the effects of CCN2 on their proliferation and migration were examined in vitro. The proliferation of hBMSCs on a plastic or HAp plate was significantly enhanced by CCN2. Moreover, the migration of hBMSCs also dramatically increased by CCN2. Interestingly, a C-terminal signal modular fragment of CCN2 (CT-module) also enhanced the cell proliferation and migration as efficiently as the full-length CCN2. Next, in order to estimate the effect of CCN2 on the migration and survival of hBMSCs and bone formation inside the HAp scaffold in vivo, two experiments were performed. First, the porous HAp carrier was cultured with hBMSCs for a week, and the cell-scaffold hybrid was transplanted with or without CCN2 subcutaneously into immunocompromised mice. CCN2 accelerated the hBMSC-like cell migration and survival inside the porous HAp within 4 weeks after transplantation. Second, the porous HAp carrier with or without CCN2 was directly implanted into bone defects within a rabbit mandible, and bone regeneration inside was evaluated. As a result, CCN2 efficiently induced the cell invasion and bone formation inside the porous HAp scaffold. These findings suggest that CCN2 and its CT-module fragment could be useful for regeneration and reconstruction of large-scale bone defects.

OBJECTIVE

To understand the role of the androgen receptor in gingival overgrowth, the effects of flutamide on interleukin-1 beta- and nifedipine-induced gene expression of connective tissue growth factor (CTGF/CCN2) and collagen production in gingival fibroblasts were examined.

METHODS

Gingival fibroblasts from healthy subjects and patients with dihydropyridine-induced gingival overgrowth (DIGO) were used. Confluent cells were treated with nifedipine, interleukin-1 beta or both. The mRNA expression was examined using real-time polymerase chain reaction, and the concentration of total soluble collagen in conditioned media was analysed by Sircol Collagen Assay. In addition, the protein expressions of androgen receptor, CTGF/CCN2 and type I collagen in gingival tissue were determined by western blot.

RESULTS

Interleukin-1 beta was more potent than nifedipine in stimulating CTGF/CCN2 and procollagen alpha1(I) mRNA expression, and there was an additive effect of the two drugs. Healthy cells exhibited an equal or stronger response of procollagen alpha1(I) than those with DIGO, but DIGO cells displayed a stronger response in the secretion of soluble collagen in the same conditions. Flutamide, an androgen receptor antagonist, inhibited stimulation by nifedipine or interleukin-1 beta. Additionally, the protein expressions of androgen receptor and type I collagen were higher in DIGO gingival tissue than those in healthy gingival tissue.

CONCLUSIONS

The data suggest that both nifedipine and interleukin-1 beta play an important role in DIGO via androgen receptor upregulation and that gingival overgrowth is mainly due to collagen accumulation. Flutamide decreases the gene expression and protein production of collagen from dihydropyridine-induced overgrowth cells.

The CCN family of proteins consists of six high homologous matricellular proteins which act predominantly by binding to heparin sulphate proteoglycan and a variety of integrins. Interestingly, CCN proteins are regulated by ovarian steroid hormones and are able to adapt to changes in oxygen concentration, which is a necessary condition for successful implantation. CCN1 is involved in processes of angiogenesis within reproductive systems, thereby potentially contributing to diseases such as endometriosis and disturbed angiogenesis in the placenta and fetus. In the ovary, CCN2 is the key factor for follicular development, ovulation and corpora luteal luteolysis, and its deletion leads to fertility defects. CCN1, CCN2 and CCN3 seem to be regulators for human trophoblast proliferation and migration, but with CCN2 acting as a counterweight. Alterations in the expression of these three proteins could contribute to the shallow invasion properties observed in preeclampsia. Little is known about the role of CCN4-6 in the reproductive organs. The ability of CCN1, CCN2 and CCN3 to interact with numerous receptors enables them to adapt their biological function rapidly to the continuous remodelling of the reproductive organs and in the development of the placenta. The CCN proteins mediate their specific cell physiological function through the receptor type of their binding partner followed by a defined signalling cascade. Because of their partly overlapping expression patterns, they could act in a concert synergistically or in an opposite way within the reproductive organs. Imbalances in their expression levels are correlated to different human reproductive diseases, such as endometriosis and preeclampsia.