OBJECTIVE

The extrinsic coagulation cascade is involved in the fibrotic process, via thrombin-dependent induction of CCN2 (connective tissue growth factor) expression. Given the previously reported activation of the coagulation system in systemic sclerosis (SSc), we undertook the present study to investigate the involvement of cross-talk between the tissue factor (TF)-thrombin axis and endothelin 1 (ET-1) signaling in the fibrotic activity of SSc.

METHODS

Human colonic myofibroblasts (HCMFs) from 6 patients with SSc and gastrointestinal symptoms and from 6 control subjects were isolated and cultured under various conditions. Messenger RNA and protein levels of TF, CCN2, and endothelin receptor A (ET(A) ) were investigated. Collagen production and migratory activity of HCMFs were further assessed.

RESULTS

HCMFs from SSc patients demonstrated increased basal CCN2 production, collagen deposition, and migration rate, in a thrombin-dependent manner. Increased TF expression was also observed in SSc HCMFs. Subsequent activation of the extrinsic coagulation system resulted in thrombin-dependent enhancement of ET(A) expression. ET(A) overexpression led to further increases in both TF expression and fibrotic activity in HCMFs. Moreover, inhibition of ET-1 signaling by bosentan abolished the TF-mediated fibrotic capacity of HCMFs.

CONCLUSIONS

Tissue factor-thrombin signaling is involved in the increased fibrotic activity of HCMFs from patients with SSc. Moreover, the up-regulation of ET(A) expression by thrombin and the effect of ET-1 in the induction of TF expression indicate an amplification loop for enhanced collagen deposition. Therapeutic interventions targeting the extrinsic coagulation system or ET-1 signaling may provide clinical benefit by breaking this vicious circle.

Cardiac fibroblasts (CF) play a central role in the repair and remodeling of the heart following injury and are important regulators of inflammation and extracellular matrix (ECM) turnover. ECM-regulatory matricellular proteins are synthesized by several myocardial cell types including CF. We investigated the effects of pro-inflammatory cytokines on matricellular protein expression in cultured human CF. cDNA array analysis of matricellular proteins revealed that interleukin-1α (IL-1α, 10ng/ml, 6h) down-regulated connective tissue growth factor (CTGF/CCN2) mRNA by 80% and up-regulated tenascin-C (TNC) mRNA levels by 10-fold in human CF, without affecting expression of thrombospondins 1-3, osteonectin or osteopontin. Western blotting confirmed these changes at the protein level. In contrast, tumor necrosis factor α (TNFα) did not modulate CCN2 expression and had only a modest stimulatory effect on TNC levels. Signaling pathway inhibitor studies suggested an important role for the p38 MAPK pathway in suppressing CCN2 expression in response to IL-1α. In contrast, multiple signaling pathways (p38, JNK, PI3K/Akt and NFκB) contributed to IL-1α-induced TNC expression. In conclusion, IL-1α reduced CCN2 expression and increased TNC expression in human CF. These observations are of potential value for understanding how inflammation and ECM regulation are linked at the level of the CF.

The tumour microenvironment is complex and composed of many different constituents, including matricellular proteins such as connective tissue growth factor (CCN2), and is characterized by gradients in oxygen levels. In various cancers, hypoxia and CCN2 promote stem and progenitor cell properties, and regulate the proliferation, migration and phenotype of cancer cells. Our study was aimed at investigating the effects of hypoxia and CCN2 on chordoma cells, using the human U-CH1 cell line. We demonstrate that under basal conditions, U-CH1 cells express multiple CCN family members including CCN1, CCN2, CCN3 and CCN5. Culture of U-CH1 cells in either hypoxia or in the presence of recombinant CCN2 peptide promoted progenitor cell-like characteristics specific to the notochordal tissue of origin. Specifically, hypoxia induced the most robust increase in progenitor-like characteristics in U-CH1 cells, including increased expression of the notochord-associated markers T, CD24, FOXA1, ACAN and CA12, increased cell growth and tumour-sphere formation, and a decrease in the percentage of vacuolated cells present in the heterogeneous population. Interestingly, the effects of recombinant CCN2 peptide on U-CH1 cells were more pronounced under normoxia than hypoxia, promoting increased expression of CCN1, CCN2, CCN3 and CCN5, the notochord-associated markers SOX5, SOX6, T, CD24, and FOXA1 as well as increased tumour-sphere formation. Overall, this study highlights the importance of multiple factors within the tumour microenvironment and how hypoxia and CCN2 may regulate human chordoma cell behaviour.

Fibrosis may be a key factor in sensorimotor dysfunction in patients with chronic overuse-induced musculoskeletal disorders. Using a clinically relevant rodent model, in which performance of a high demand handle-pulling task induces tissue fibrosis and sensorimotor declines, we pharmacologically blocked cellular communication network factor 2 (CCN2; connective tissue growth factor) with the goal of reducing the progression of these changes. Young adult, female Sprague-Dawley rats were shaped to learn to pull at high force levels (10 min/day, 5 weeks), before performing a high repetition high force (HRHF) task for 3 weeks (2 h/day, 3 days/week). HRHF rats were untreated, or treated in task weeks 2 and 3 with a monoclonal antibody that blocks CCN2 (FG-3019), or a control immunoglobulin G (IgG). Control rats were untreated or received FG-3019, IgG, or vehicle (saline) injections. Mean task reach rate and grasp force were higher in 3-week HRHF + FG-3019 rats, compared with untreated HRHF rats. Grip strength declined while forepaw mechanical sensitivity increased in untreated HRHF rats, compared with controls; changes improved by FG-3019 treatment. The HRHF task increased collagen in multiple tissues (flexor digitorum muscles, nerves, and forepaw dermis), which was reduced with FG-3019 treatment. FG-3019 treatment also reduced HRHF-induced increases in CCN2 and transforming growth factor β in muscles. In tendons, FG-3019 reduced HRHF-induced increases in CCN2, epitendon thickening, and cell proliferation. Our findings indicate that CCN2 is critical to the progression of chronic overuse-induced multi-tissue fibrosis and functional declines. FG-3019 treatment may be a novel therapeutic strategy for overuse-induced musculoskeletal disorders. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:2004-2018, 2019.

Hepatic stem/progenitor cells (HPC) reside quiescently in normal biliary trees and are activated in the form of ductular reactions during severe liver damage when the replicative ability of hepatocytes is inhibited. HPC niches are full of profibrotic stimuli favoring scarring and hepatocarcinogenesis. The Cyr61/CTGF/NOV (CCN) protein family consists of six members, CCN1/CYR61, CCN2/CTGF, CCN3/NOV, CCN4/WISP1, CCN5/WISP2, and CCN6/WISP3, which function as extracellular signaling modulators to mediate cell-matrix interaction during angiogenesis, wound healing, fibrosis, and tumorigenesis. This study investigated expression patterns of CCN proteins in HPC and cholangiocarcinoma (CCA). Mouse HPC were induced by the biliary toxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Differential expression patterns of CCN proteins were found in HPC from DDC damaged mice and in human CCA tumors. In addition, we utilized reporter mice that carried Ccn2/Ctgf promoter driven GFP and detected strong Ccn2/Ctgf expression in epithelial cell adhesion molecule (EpCAM)+ HPC under normal conditions and in DDC-induced liver damage. Abundant CCN2/CTGF protein was also found in cytokeratin 19 (CK19)+ human HPC that were surrounded by α-smooth muscle actin (α-SMA)+ myofibroblast cells in intrahepatic CCA tumors. These results suggest that CCN proteins, particularly CCN2/CTGF, function in HPC activation and CCA development.

This research is designed to investigate the regulatory effect of miR-18a to the target gene connective tissue growth factor (CTGF, or CCN2), by participating in TGF-β1 signaling pathway and explore the pathogenic mechanism of miR-18a in pulmonary injury induced by nano-SiO2 based on our early study. miR-18a and expression of TGF-β1 in Chinese hamster lung (CHL) fibroblasts cells stimulated by supernatants of NR8383 cells exposed to 40 μg/ml nano-SiO2 for 24 h demonstrated 1.58 ± 0.22-fold and 1096.00 ± 2.60 pg/ml increase compared with blank control group analyzed by real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Expression increase of miR-18a and reduction of CCN2 mRNA expression levels and protein gray value ratio detected by Western blotting in CHL cells transfect miR-18a mimics for 48 h. The reverse of CHL cell transfection miR-18a inhibit is also true. The result of miR-18a and CCN2 binding sites tested by luciferase reporter gene assay shows that the report relative fluorescence value of miR-18a mimics wild type on CCN2 is 0.50 ± 0.02 with the control of mimics NC and mutant relative fluorescence report value 0.86 ± 0.04 (P < 0.05). Expression levels of miR-18a, CCN2 mRNA, and protein gray value ratio decreased in CHL cells treated by TGF-β1, respectively, and vice versa treated by TGF-β1corepressor. The results suggest that CCN2 is the target gene regulated by miR-18a and miR-18a participates in TGF-β1 signaling pathway by regulating the expression of CCN2 negatively through CCN2 3'UTR site, and thus may be involved in the development process of pulmonary injury.

We recently show that CCN3 is a counter-regulatory molecule for the pro-fibrotic protein CCN2, and a potentially novel fibrosis therapy. The goal of this study was to assess the role of CCN3 in fibroproliferative/fibrotic responses in human dermal fibroblasts exposed to Omniscan, one of the gadolinium-based contrast agents associated with development of nephrogenic systemic fibrosis (NSF) a rare but life-threatening disease thought to be complication of NMR diagnostics in renal impaired patients. Human dermal fibroblasts were exposed to Omniscan; or to platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) as controls. Proliferation was assessed along with matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1 and type 1 procollagen in the absence and presence of CCN3. In parallel, CCN3 production was assessed in control and Omniscan-treated cells. The results showed that PDGF stimulated fibroblast proliferation, production of Timp-1 and MMP-1 whereas exogenous CCN3 inhibited, in a dose response manner, cell proliferation (approx. 50 % max.) and production of MMP-1 (approx 35 % max.) but had little effect on TIMP-1. TGF-β stimulated type 1 procollagen production but not proliferation, Timp-1 or MMP-1 compared to non-TGF-ß treated control cells, and CCN3 treatment blocked (approx. 80 % max.) this up-regulation. Interestingly, untreated, control fibroblasts produced high constitutive levels of CCN3 and concentrations of Omniscan that induced fibroproliferative/fibrogenic changes in dermal fibroblasts correspondingly suppressed CCN3 production. The use of PDGF and TGF-β as positive controls, and the study of differential responses, including that to Omniscan itself, provide the first evidence for a role of fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes, elucidating possible mechanism(s). In conclusion, these data support our hypothesis of a role for fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes in these cells, and suggest that CCN3 may be an important regulatory molecule in NSF and a target for treatment in this and other fibrotic diseases.

Cigarette smoke has been associated with poor healing in several studies, but the precise mechanisms involving this impairment are still not elucidated. The aim of this work was to investigate cigarette smoke exposure effects on initial phases of cutaneous healing in mice, focusing mainly on gene expression of two molecules involved in wound repair (Ccn2/Ctgf and Tgfb1) and to study if these effects are strain dependent. Mice were exposed to the smoke of nine cigarettes per day, three times per day, for ten days. In the eleventh day an excisional wound was made. The control group was sham-exposed. The cigarette smoke exposure protocol was performed until euthanasia, seven days after wounding. Wound contraction was evaluated. Sections were stained with hematoxylin-eosin, Sirius red, and toluidine blue, and also immunostained for alpha-smooth muscle actin. Gene expression of Ccn2/Ctgf and Tgfb1 was evaluated by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Smoke-exposed animals presented delay in wound contraction; fibroblastic, inflammatory, and mast cell recruitment; re-epithelialization; myofibroblastic differentiation; and Ccn2/Ctgf and Tgfb1 gene expression. Those alterations were strain dependent. This work confirmed the deleterious effects of cigarette smoke exposure on mouse cutaneous healing depending on mouse strain and links these effects to an overexpression of Ccn2/Ctgf.

Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.

The syncytiotrophoblast, which regulates maternal-fetal transfer of drugs, consists of a single layer in humans, but two layers, i.e., SynI and SynII, in rodents. Polar distribution of transporters in the apical and basal plasma membranes of syncytiotrophoblast is important for placental function in terms of vectorial transport of substrates, but the mechanisms that control protein distribution in the syncytiotrophoblast remain unclear. We have previously established rat syncytiotrophoblast cell lines, TR-TBT 18d-1 and TR-TBT 18d-2, which retain characteristics of SynI and SynII, respectively. In this study, we aimed to characterize the gene expression profiles in the two layers by using these cell lines. DNA microarray analysis indicated that more than 25 mRNAs, including cytoskeleton binding proteins, ezrin and CLP36, are differentially expressed between TR-TBT 18d-1 and TR-TBT 18d-2. Quantitative real time-polymerase chain reaction (PCR) analysis indicated that mRNA expression of ezrin, CLP36, CCN1, and CCN2 is higher in TR-TBT 18d-1 and mRNA expression of elf-1a, hsc70 and flot2 is higher in TR-TBT 18d-2, compared with their counterparts. Immunohistochemical analysis indicated that ezrin is expressed in rat placental villi in vivo, and is located on the apical membranes of TR-TBT 18d-1, while CLP36 is located in the apical and basal sides of TR-TBT 18d-1. The expression of ezrin was highest at gestational days 14 and 18 and was highest among the ezrin/radixin/moesin (ERM) family members. These results may help to clarify the mechanisms controlling polarization of the syncytiotrophoblast and the significance of the double epithelial layers in rat and mouse.

Skin fibrosis occurs in a variety of human diseases but the current anti-fibrosis treatments are not sufficient. One major cause of fibrotic diseases shared across diverse organ fibrosis is uncontrolled overexpression of the connective tissue growth factor (CTGF, also known as CCN2). Here, we examine the anti-fibrotic activity of RNAi therapy utilizing siRNA against CTGF with a new drug delivery system (DDS), 'DegradaBALL', which is based on porous nanoparticles, for durable CTGF gene silencing. DegradaBALL is a modular DDS having many favorable properties for RNA delivery such as effective intracellular uptake, convenient drug loading, biocompatibility, sustained release profile and biodegradability. DegradaBALL loaded with siCTGF, named 'LEM-S401', showed highly durable and effective CTGF gene-silencing in TGF-β induced lung fibrosis and skin fibrosis model cells, A549 and HaCaT, respectively. In addition, LEM-S401 induced knockdown of collagen types I and III, which are excess extracellular matrix components in fibrotic skin in addition to CTGF in the mouse wound healing model. Most importantly, we showed that LEM-S401 effectively inhibited the formation of hypertrophic scars in wound-associated dermal fibrosis mouse models, during both the epidermis recovery and tissue remodeling process. Our findings suggest that LEM-S401 could be a highly potent therapeutic option for skin fibrotic diseases.

Transforming growth factor-β1 (TGF-β1) plays a premier role in fibrosis. To understand the molecular events underpinning TGF-β1-induced fibrogenesis, we examined the proteomic profiling of a TGF-β1-induced in vitro model of fibrosis in NRK-49F normal rat kidney fibroblasts. Mass spectrometric analysis indicated that 628 cell-lysate proteins enriched in 44 cellular component clusters, 24 biological processes and 27 molecular functions were regulated by TGF-β1. Cell-lysate proteins regulated by TGF-β1 were characterised by increased ribosomal proteins and dysregulated proteins involved in multiple metabolic pathways, including reduced Aldh3a1 and induced Enpp1 and Impdh2, which were validated by enzyme-linked immunosorbent assays (ELISA). In conditioned media, 62 proteins enriched in 20 cellular component clusters, 40 biological processes and 7 molecular functions were regulated by TGF-β1. Secretomic analysis and ELISA uncovered dysregulated collagen degradation regulators (induced PAI-1 and reduced Mmp3), collagen crosslinker (induced Plod2), signalling molecules (induced Ccn1, Ccn2 and Tsku, and reduced Ccn3) and chemokines (induced Ccl2 and Ccl7) in the TGF-β1 group. We conclude that TGF-β1-induced fibrogenesis in renal fibroblasts is an intracellular metabolic disorder and is inherently coupled with inflammation mediated by chemokines. Proteomic profiling established in this project may guide development of novel anti-fibrotic therapies in a network pharmacology approach.

Systemic sclerosis (SSc) is a rare and severe connective tissue disease combining auto-immune and vasculopathy features, ultimately leading to organ fibrosis. Impaired angiogenesis is an often silent and life-threatening complication of the disease. We hypothesize that CCN3 (NOV), a member of the CCN family of extra-cellular matrix proteins, which is an antagonist of the pro-fibrotic protein CCN2 (CTGF) as well as a pro-angiogenic factor, is implicated in SSc pathophysiology. We performed skin biopsies on 26 SSc patients, both in fibrotic and non-fibrotic areas for 17 patients, and collected 18 healthy control skin specimens, for immunohistochemistry and cell culture. Histological analysis of non-fibrotic and fibrotic SSc skin shows a systemic decrease of papillary dermis surface as well as capillaries disappearance. CCN3 expression is systematically decreased in the dermis of SSc patients compared to healthy controls, particularly in dermal blood vessels. Moreover, CCN3 is decreased in vitro in endothelial cells from SSc patients. We show that CCN3 is essential for endothelial cell migration and angiogenesis in vitro. In conclusion, CCN3 may represent a promising therapeutic target for SSc patients presenting with vascular involvement.

Urothelial bladder cancer (UBC) is the most common urinary neoplasm in China. CCN family protein 2 (CCN2), a cysteine-rich matricellular protein, is abnormally expressed in several cancer types and involved in tumor progression or chemo-resistance. However, detailed expression patterns and effects of CCN2 in UBC still remain unknown. We found that down-regulation of CCN2 suppressed proliferation, migration and invasion of UBC cells in vitro and targeting of CCN2 decelerated xenograft growth in vivo. When treated with mitomycin C (MMC), CCN2-scilencing UBC cells showed lower survival and higher apoptotic rates and these effects were probably mediated via inactivation of Akt and Erk pathways. We also demonstrated the clinical significance of CCN2 expression, which was higher in UBC tissues and associated with advanced tumor stage and high pathologic grade. Taken together, our data suggest that CCN2 is an oncogene in UBC and might serve as a matricellular target for improving chemotherapeutic efficacy.

Connective tissue growth factor (CTGF/CCN2) is a secreted protein modulating various biological processes, such as proliferation, differentiation, and survival. Tumor necrosis factor-α (TNF-α), known as a proinflammatory factor, negatively regulates osteoblast differentiation and survival. However, the potential mechanisms of CCN2 in TNF-α-induced osteoblast apoptosis are not fully understood. In the present study, we found that CCN2 was expressed in osteoblasts and downregulated after treatment with TNF-α. Overexpression of CCN2 attenuated TNF-α-induced osteoblast apoptosis. Autophagy, a pro-survival biological behavior, was triggered by TNF-α stimulation, and CCN2 overexpression enhanced this process. Inhibition of autophagy by chloroquine (CQ) affected the anti-apoptotic effect of CCN2. Moreover, the phosphorylation levels of Akt and Erk were upregulated in CCN2-over expressed cells, and LY294002 and U1026 (which inhibited the Akt and Erk signaling pathways, respectively) reversed the effect of CCN2 on autophagy and cell survival enhancement. Our data suggest that CCN2 might be a positive regulator of osteoblast survival in TNF-α stimulation by enhancing autophagy through the Akt and Erk signaling pathways.

The expression of Ccn2 (CTGF) has been linked to fibrosis in many tissues and pathologies, although its activities in fibroblastic cells and precise mechanism of action in fibrogenesis are still controversial. Here, we showed that CCN2 can induce cellular senescence in fibroblasts both in vitro and in vivo, whereupon senescent cells express an anti-fibrotic "senescence-associated secretory phenotype" (SASP) that includes upregulation of matrix metalloproteinases and downregulation of collagen. Mechanistically, CCN2 induces fibroblast senescence through integrin α6β1-mediated accumulation of reactive oxygen species, leading to activation of p53 and induction of p16INK4a. In cutaneous wound healing, Ccn2 expression is highly elevated only during the initial inflammatory phase and quickly declines thereafter to a low level during the proliferation and maturation phases of healing when myofibroblasts play a major role. Consistent with this expression kinetics, knockdown of Ccn2 has little effect on the rate of wound closure, formation of senescent cells, or collagen content of the wounds. However, application of purified CCN2 protein on cutaneous wounds leads to induction of senescent cells, expression of SASP, and reduction of collagen content. These results show that CCN2 can induce cellular senescence in fibroblasts and is capable of exerting an anti-fibrotic effect in a context-dependent manner.

CCN2, also known as connective tissue growth factor (CTGF), is one of important members of the CCN family. Generally, CTGF expresses at low levels in normal adult kidney, while increases significantly in various kidney diseases, playing an important role in the development of glomerular and tubulointerstitial fibrosis in progressive kidney diseases. CTGF is involved in cell proliferation, migration, and differentiation and can promote the progression of fibrosis directly or act as a downstream factor of transforming growth factor β (TGF-β). CTGF also regulates the expression and activity of TGF-β and bone morphogenetic protein (BMP), thereby playing an important role in the process of kidney repair. In patients with chronic kidney disease, elevated plasma CTGF is an independent risk factor for progression to end-stage renal disease and is closely related to glomerular filtration rate. Therefore, CTGF may be a potential biological marker of kidney fibrosis, but more clinical studies are needed to confirm this view. This section briefly describes the role and molecular mechanisms of CTGF in renal fibrosis and also discusses the potential value of targeting CCN2 for the treatment of renal fibrosis.

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT2Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKCε, PKCζ, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.

Although caffeine and glucocorticoids are frequently used to treat chronic lung disease in preterm neonates, potential interactions are largely unknown. While anti-inflammatory effects of glucocorticoids are well defined, their impact on airway remodeling is less characterized. Caffeine has been ascribed to positive effects on airway inflammation as well as remodeling. Connective tissue growth factor (CTGF, CCN2) plays a key role in airway remodeling and has been implicated in the pathogenesis of chronic lung diseases such as bronchopulmonary dysplasia (BPD) in preterm infants. The current study addressed the impact of glucocorticoids on the regulation of CTGF in the presence of caffeine using human lung epithelial and fibroblast cells.

The human airway epithelial cell line H441 and the fetal lung fibroblast strain IMR-90 were exposed to different glucocorticoids (dexamethasone, budesonide, betamethasone, prednisolone, hydrocortisone) and caffeine. mRNA and protein expression of CTGF, TGF-β1-3, and TNF-α were determined by means of quantitative real-time PCR and immunoblotting. H441 cells were additionally treated with cAMP, the adenylyl cyclase activator forskolin, and the selective phosphodiesterase (PDE)-4 inhibitor cilomilast to mimic caffeine-mediated PDE inhibition.

Treatment with different glucocorticoids (1 μM) significantly increased CTGF mRNA levels in H441 (p < 0.0001) and IMR-90 cells (p < 0.01). Upon simultaneous exposure to caffeine (10 mM), both glucocorticoid-induced mRNA and protein expression were significantly reduced in IMR-90 cells (p < 0.0001). Of note, 24 h exposure to caffeine alone significantly suppressed basal expression of CTGF mRNA and protein in IMR-90 cells. Caffeine-induced reduction of CTGF mRNA expression seemed to be independent of cAMP levels, adenylyl cyclase activation, or PDE-4 inhibition. While dexamethasone or caffeine treatment did not affect TGF-β1 mRNA in H441 cells, increased expression of TGF-β2 and TGF-β3 mRNA was detected upon exposure to dexamethasone or dexamethasone and caffeine, respectively. Moreover, caffeine increased TNF-α mRNA in H441 cells (6.5 ± 2.2-fold, p < 0.05) which has been described as potent inhibitor of CTGF expression.

In addition to well-known anti-inflammatory features, glucocorticoids may have adverse effects on long-term remodeling by TGF-β1-independent induction of CTGF in lung cells. Simultaneous treatment with caffeine may attenuate glucocorticoid-induced expression of CTGF, thereby promoting restoration of lung homeostasis.

OBJECTIVE

CCN family member 2/connective tissue growth factor (CCN2/CTGF) is known as an osteogenesis-related molecule and is thought to be implicated in tooth growth. Bone morphogenetic protein-1 (BMP-1) contributes to tooth development by the degradation of dentin-specific substrates as a metalloprotease. In this study, we demonstrated the correlations between CCN2/CTGF and BMP-1 in human carious teeth and the subcellular dynamics of BMP-1 in human dental pulp cells.

METHODS

Expression of CCN2/CTGF and BMP-1 in human carious teeth was analyzed by immunohistochemistry. BMP-1-induced CCN2/CTGF protein expression in primary cultures of human dental pulp cells was observed by immunoblotting. Intracellular dynamics of exogenously administered fluorescence-labeled BMP-1 were observed using confocal microscope.

RESULTS

Immunoreactivities for CCN2/CTGF and BMP-1 were increased in odontoblast-like cells and reparative dentin-subjacent dental caries. BMP-1 induced the expression of CCN2/CTGF independently of protease activity in the cells but not that of dentin sialophosphoprotein (DSPP) or dentin matrix protein-1 (DMP-1). Exogenously added BMP-1 was internalized into the cytoplasm, and the potent dynamin inhibitor dynasore clearly suppressed the BMP-1-induced CCN2/CTGF expression in the cells.

CONCLUSIONS

CCN2/CTGF and BMP-1 coexist beneath caries lesion and CCN2/CTGF expression is regulated by dynamin-related cellular uptake of BMP-1, which suggests a novel property of metalloprotease in reparative dentinogenesis.

Development of the palate is the result of an organized series of events that require exquisite spatial and temporal regulation at the cellular level. There are a myriad of growth factors, receptors and signaling pathways that have been shown to play an important role in growth, elevation and/or fusion of the palatal shelves. Altered expression or activation of a number of these factors, receptors and signaling pathways have been shown to cause cleft palate in humans or mice with varying degrees of penetrance. This review will focus on connective tissue growth factor (CTGF) or CCN2, which was recently shown to play an essential role in formation of the secondary palate. Specifically, the absence of CCN2 in KO mice results in defective cellular processes that contribute to failure of palatal shelf growth, elevation and/or fusion. CCN2 is unique in that it has been shown to interact with a number of other factors important for palate development, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), epidermal growth factor (EGF), Wnt proteins and transforming growth factor-βs (TGF-βs), thereby influencing their ability to bind to their receptors and mediate intracellular signaling. The role that these factors play in palate development and their specific interactions with CCN2 will also be reviewed. Future studies to elucidate the precise mechanisms of action for CCN2 and its interactions with other regulatory proteins during palatogenesis are expected to provide novel information with the potential for development of new pharmacologic or genetic treatment strategies for clinical intervention of cleft palate during development.

CCN proteins are extracellular and cell-associated molecules involved in several developmental processes, but their expression patterns and regulation in tooth development remain unclear. Here we first determined the expression patterns of CCN genes in mouse tooth germs. We found that at early stages CCN2 was detected in dental lamina, dental mesenchyme, and primary enamel knot, while other CCN family members were expressed broadly. By the bell stage, all members were expressed in differentiating odontoblasts and ameloblasts, but CCN1 and CCN2 transcripts were conspicuous in differentiating osteoblasts in dental follicle. Next, we asked what signalling molecules regulate CCN2 expression and what roles CCN2 may have. We found that upon surgical removal of dental epithelium CCN2 was not longer expressed in dental mesenchyme in cultured bud stage germs. Implantation of beads pre-coated with BMPs and FGFs onto E12-13 mandibular explants induced CCN2 expression in dental mesenchyme. There was a dose-dependent effect of BMP-4 on CCN2 induction; a concentration of 100 ng/μl was able to induce strong CCN2 expression while a minimum concentration of 25 ng/μl was needed to elicit appreciable expression. Importantly, Noggin treatment inhibited endogenous and BMP-induced CCN2 expression, verifying that CCN2 expression in developing tooth germs requires BMP signalling. Lastly, we found that rCCN2 stimulated proliferation in dental mesenchyme in a dose-dependent manner. Together, the data indicate that expression of CCN genes is spatio-temporally regulated in developing tooth germs. CCN2 expression appears to depend on epithelial and mesenchymal-derived signalling factors, and CCN2 can elicit strong proliferation in dental mesenchyme.