PKC inhibition increases gap junction intercellular communication and cell adhesion in human neuroblastoma.

Gap junction intercellular communication and cell-cell adhesion are essential for maintaining a normal cellular phenotype, including the control of growth and proliferation. Loss of either cell-cell adhesion or communication is common in cancers, while restoration of function is associated with tumor suppression. Protein kinase C (PKC) isozymes regulate a broad spectrum of cellular functions including growth and proliferation, and their overexpression has been correlated with carcinogenesis. Consequently, PKC inhibitors are currently undergoing clinical trials as an anti-cancer agents although the precise cellular alterations induced by PKC inhibitors remain to be elucidated. In the current study, the effects of PKC inhibitors on cell interactions were investigated using human neuroblastoma (IMR32, SKNMC, and SHSY-5Y) cell lines. An analysis of intercellular communication revealed an increase in gap junctional coupling with PKC inhibition. The observed increase in coupling was not associated with a change in Connexin 43 distribution or an alteration of phosphorylation status of the protein. There was also an increase in cell-cell adhesion with PKC inhibitor treatment as indicated by a cell aggregation assay. Therefore, the growth suppressive abilities of PKC inhibition on tumors may be due to the cancer suppressive effects of increased gap junction intercellular communication and cell-cell adhesion.

Proteomic analysis of the LPS-induced stress response in rat chondrocytes reveals induction of innate immune response components in articular cartilage.

Activation of toll-like receptors (TLR) in articular chondrocytes has been reported to increase the catabolic compartment, leading to matrix degradation, while the main consequence of TLR activation in monocytic cells is the expression and secretion of components of the innate immune response, particularly that of inflammatory cytokines. The objective of the work reported here was to obtain a more complete picture of the response repertoire of articular chondrocytes to TLR activation. Mass spectrometry was used to analyse the secretome of stimulated and unstimulated cells. Characterization of TLR expression in rat articular chondrocytes by RT/PCR indicated that TLR4 was the major receptor form. Exposure of these cells to lipopolysaccharide (LPS), the well-characterized TLR4 ligand, induced production not only of the matrix metalloproteinases MMP3 and 13, but also of components traditionally associated with the innate immune response, such as the complement components C1r, C3 and complement factor B, long pentraxin-3 and osteoglycin. Neither TNF-alpha nor IL-1 was detectable in culture media following exposure to LPS. One of the most prominently-induced proteins was the chitinase-like protein, Chi3L1, linking its expression to the innate immune response repertoire of articular chondrocytes. In intact femoral heads, LPS induced expression of Chi3L1 in chondrocytes close to the articular surface, suggesting that only these cells mount a stress response to LPS. Thus articular chondrocytes have a capacity to respond to TLR activation, which results in the expression of matrix metalloproteases as well as subsets of components of the innate immune response without significant increases in the production of inflammatory cytokines. This could influence the erosive processes leading to cartilage degeneration as well as the repair of damaged matrix.

The complex of ciliary neurotrophic factor-ciliary neurotrophic factor receptor alpha up-regulates connexin43 and intercellular coupling in astrocytes via the Janus tyrosine kinase/signal transducer and activator of transcription pathway.

Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor alpha (CNTFRalpha) (200 ng/ml), or CNTF-CNTFRalpha. Although CNTF and CNTFRalpha alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFRalpha significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFRalpha treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between -838 to -1693 was deemed necessary for CNTF-CNTFRalpha to induce heightened expression. CNTF-CNTFRalpha did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFRalpha for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling.

Bioactive glass/polymer composite materials with mechanical properties matching those of cortical bone.

Stress shielding resulting from mismatch in dynamic mechanical properties contributes to the reduced stability of osseous implants. Our objective was to develop biocompatible composites having mechanical properties similar to those of cortical bone. Polymers of urethane dimethacrylate (UDMA) and 2-hydroxyethyl methacrylate (HEMA, 0-20%) and composites containing bioactive glass particles (70% SiO(2), 25% CaO, and 5% P(2)O(5)), with or without silane treatment were prepared. Young's moduli of composites containing silane-treated glass (16 GPa) were significantly greater than those of composites containing untreated glass (12-13 GPa) or of unfilled polymers (5-6 GPa). Bioactive glass reduced water sorption by the composites and incorporation of silane-treated glass prevented HEMA-induced increases in water sorption. Osteoblast-like cells attached equally well to UDMA polymer and composite containing silane-treated bioactive glass. Thus, silane treatment improved the mechanical properties of bioactive glass composites without compromising biocompatibility. This material has a Young's modulus comparable to that of cortical bone. Therefore, silane-treated bioactive glass composites, when used as implant or cement materials, would reduce stress shielding and improve implant stability.

Functional characterization of oculodentodigital dysplasia-associated Cx43 mutants.

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.

LF15-0195 generates tolerogenic dendritic cells by suppression of NF-kappaB signaling through inhibition of IKK activity.

LF15-0195 (LF) is a potent, less toxic analog of the immunosuppressant 15-deoxyspergualine, which we previously reported to prevent graft rejection and to induce permanent tolerance in a murine cardiac transplantation model. However, the underlying mechanism of action of LF required elucidation. In this study, dendritic cells (DC) treated with LF before activation with tumor necrosis factor alpha (TNF-alpha)/lipopolysaccharide (LPS) failed to express maturation markers (major histocompatibility complex II, CD40, CD86) and interleukin-12. LF prevented, in a concentration-dependent manner, the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB) in DC following addition of TNF-alpha/LPS. Yet-activated and active IkappaB kinases (IKKs) were inhibited in cells pretreated with LF, thereby preventing the phosphorylation of IkappaB and release of NF-kappaB, a key regulator of genes associated with the maturation of DC. LF-induced inhibition of IKK activity was reversed in a dose-dependent manner by the overexpression of IKK. The T helper cell type 2 (Th2) differentiation of naïve T cells promoted by LF-treated DC in vitro correlates with Th2 polarization observed in transplant recipients made tolerant by LF. These data demonstrated that LF-induced blockade of NF-kappaB signaling at the level of IKK promoted the generation of tolerogenic DC that inhibited Th1 polarization and increased Th2 polarization in vitro and in vivo.

Activity restriction increases deoxypyridinoline excretion in hospitalized high-risk pregnant women.

PURPOSE: Activity restriction (AR), one of the most common interventions used in high-risk pregnancies, may exacerbate loss of bone mass. The purpose of this study was to determine changes over time in bone resorption in hospitalized AR women during late pregnancy. METHODS: This was a short-term prospective study conducted in two tertiary-care obstetric hospitals. We measured urinary deoxypyridinoline (Dpd) excretion, a marker of bone resorption, once per week in a convenience sample of 14 hospitalized AR women in the third trimester and compared values at 28-31 and 34-36 weeks' gestation to those of 11 ambulatory control women. Both groups completed a bone-loading questionnaire, 3-day food intake record, and pedometer step counts at the same gestational age. RESULTS: Urinary Dpd excretion increased from Days 1-7 (2.60 ± 0.32 nmol/mmol creatinine) to Days 22-28 (5.36 ± 0.83 nmol/mmol creatinine; p ≤ .05). Dpd excretion was higher in AR women (4.51 ± 0.31 nmol/mmol creatinine) than ambulatory women (2.72 ± 0.39 nmol/mmol creatinine) at 34-36 weeks' gestation (p ≤ .05). Energy intake between ambulatory and AR women was not different (p ≥ .05). All women met the daily requirements for calcium and vitamin D intake during pregnancy. Average daily pedometer steps for the AR women were significantly less compared to controls (1,329 ± 936 and 8,024 ± 1,890 steps/day, respectively; p ≤ .05). CONCLUSIONS: AR leads to increased bone resorption in hospitalized pregnant women, which may impact future risk of developing osteopenia and osteoporosis.

Egr-1 inhibits the expression of extracellular matrix genes in chondrocytes by TNFalpha-induced MEK/ERK signalling.

INTRODUCTION: TNFalpha is increased in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. TNFalpha activates mitogen-activated kinase kinase (MEK)/extracellular regulated kinase (ERK) in chondrocytes; however, the overall functional relevance of MEK/ERK to TNFalpha-regulated gene expression in chondrocytes is unknown. METHODS: Chondrocytes were treated with TNFalpha with or without the MEK1/2 inhibitor U0126 for 24 hours. Microarray analysis and real-time PCR analyses were used to identify genes regulated by TNFalpha in a MEK1/2-dependent fashion. Promoter/reporter, immunoblot, and electrophoretic mobility shift assays were used to identify transcription factors whose activity in response to TNFalpha was MEK1/2 dependent. Decoy oligodeoxynucleotides bearing consensus transcription factor binding sites were introduced into chondrocytes to determine the functionality of our results. RESULTS: Approximately 20% of the genes regulated by TNFalpha in chondrocytes were sensitive to U0126. Transcript regulation of the cartilage-selective matrix genes Col2a1, Agc1 and Hapln1, and of the matrix metalloproteinase genes Mmp-12 and Mmp-9, were U0126 sensitive--whereas regulation of the inflammatory gene macrophage Csf-1 was U0126 insensitive. TNFalpha-induced regulation of Sox9 and NFkappaB activity was also U0126 insensitive. Conversely, TNFalpha-increased early growth response 1 (Egr-1) DNA binding was U0126 sensitive. Transfection of chondrocytes with cognate Egr-1 oligodeoxynucleotides attenuated the ability of TNFalpha to suppress Col2a1, Agc1 or Hapln1 mRNA expression. CONCLUSIONS: Our results suggest that MEK/ERK and Egr1 are required for TNFalpha-regulated catabolic and anabolic genes of the cartilage extracellular matrix, and hence may represent potential targets for drug intervention in osteoarthritis or rheumatoid arthritis.

Cyclic AMP regulates extracellular matrix gene expression and metabolism in cultured primary rat chondrocytes.

In osteo- and rheumatoid arthritis, the synovial fluid surrounding chondrocytes contains increased levels of prostaglandin E(2) (PGE(2)), an agent known to elevate intracellular cyclic AMP (cAMP). However, the effect of PGE(2)/cAMP on mRNA expression in chondrocytes is largely unknown. In this report, we assess the effect of the cell-permeable cAMP analog adenosine 8-(4-chloro-phenylthio)-3',5'-cyclic monophosphate (CPT-cAMP) and PGE(2) on mRNA expression in primary neonatal rat chondrocytes. CPT-cAMP decreased type II collagen, link protein, parathyroid hormone/parathyroid hormone-related peptide receptor and alkaline phosphatase, increased glyceraldehyde-3-phosphate dehydrogenase mRNA and lactate efflux, but did not alter type X collagen or aggrecan mRNA. The effect of CPT-cAMP on type II collagen and link protein mRNAs and chondrocyte metabolism were attenuated by the transcriptional inhibitor actinomycin D, indicating that the ability of CPT-cAMP to suppress mRNA expression was not due to alterations in mRNA stability, but were instead likely due to transcriptional mechanisms. CPT-cAMP-treatment induced GSK3 beta phosphorylation and beta-catenin-mediated transcriptional activity. Pharmacological inhibition of GSK3 beta paralleled the effects of CPT-cAMP on type II collagen, link protein and chondrocyte metabolism, suggesting that the effect of CPT-cAMP on chondrocytes may be GSK3 beta/beta-catenin-dependent. The effects of CPT-cAMP on beta-catenin-mediated transcription, cell metabolism and mRNA expression were mimicked by the cAMP-elevating agent PGE(2), providing a physiologically relevant context for our studies. Collectively, these results suggest that agents that elevate cAMP signaling may impair chondrocyte function in conditions such as arthritis.

Regulation of Sox9 activity by crosstalk with nuclear factor-kappaB and retinoic acid receptors.

INTRODUCTION: Sox9 and p300 cooperate to induce expression of cartilage-specific matrix proteins, including type II collagen, aggrecan and link protein. Tumour necrosis factor (TNF)-alpha, found in arthritic joints, activates nuclear factor-kappaB (NF-kappaB), whereas retinoic acid receptors (RARs) are activated by retinoid agonists, including all-trans retinoic acid (atRA). Like Sox9, the activity of NF-kappaB and RARs depends upon their association with p300. Separately, both TNF-alpha and atRA suppress cartilage matrix gene expression. We investigated how TNF-alpha and atRA alter the expression of cartilage matrix genes. METHODS: Primary cultures of rat chondrocytes were treated with TNF-alpha and/or atRA for 24 hours. Levels of transcripts encoding cartilage matrix proteins were determined by Northern blot analyses and quantitative real-time PCR. Nuclear protein levels, DNA binding and functional activity of transcription factors were assessed by immunoblotting, electrophoretic mobility shift assays and reporter assays, respectively. RESULTS: Together, TNF-alpha and atRA diminished transcript levels of cartilage matrix proteins and Sox9 activity more than each factor alone. However, neither agent altered nuclear levels of Sox9, and TNF-alpha did not affect protein binding to the Col2a1 48-base-pair minimal enhancer sequence. The effect of TNF-alpha, but not that of atRA, on Sox9 activity was dependent on NF-kappaB activation. Furthermore, atRA reduced NF-kappaB activity and DNA binding. To address the role of p300, we over-expressed constitutively active mitogen-activated protein kinase kinase kinase (caMEKK)1 to increase p300 acetylase activity. caMEKK1 enhanced basal NF-kappaB activity and atRA-induced RAR activity. Over-expression of caMEKK1 also enhanced basal Sox9 activity and suppressed the inhibitory effects of TNF-alpha and atRA on Sox9 function. In addition, over-expression of p300 restored Sox9 activity suppressed by TNF-alpha and atRA to normal levels. CONCLUSION: NF-kappaB and RARs converge to reduce Sox9 activity and cartilage matrix gene expression, probably by limiting the availability of p300. This process may be critical for the loss of cartilage matrix synthesis in inflammatory joint diseases. Therefore, agents that increase p300 levels or activity in chondrocytes may be useful therapeutically.

ODDD-linked Cx43 mutants reduce endogenous Cx43 expression and function in osteoblasts and inhibit late stage differentiation.

INTRODUCTION: Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD. MATERIALS AND METHODS: We expressed human and mouse ODDD-linked Cx43 mutants in MC3T3-E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin. RESULTS: We showed that ODDD-linked Cx43 mutants are loss-of-function and dominant-negative to co-expressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation. CONCLUSIONS: Expression of human and mouse ODDD-linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43-based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.

Transforming growth factor alpha suppression of articular chondrocyte phenotype and Sox9 expression in a rat model of osteoarthritis.

OBJECTIVE: To define the roles of transforming growth factor alpha (TGFalpha) in cartilage degradation. METHODS: Primary rat articular chondrocytes and articular osteochondral explants were cultured with TGFalpha to assess the effects of TGFalpha on chondrocyte physiology and phenotype. RESULTS: TGFalpha altered chondrocyte morphology through reorganization of the actin cytoskeleton and formation of stress fibers. Expression of anabolic genes, including aggrecan, type II collagen, and cartilage link protein, was reduced in response to TGFalpha. Proliferation of chondrocytes and formation of articular chondrocyte clusters was stimulated by TGFalpha. Expression of matrix metalloproteinase 13 and cathepsin C was increased by TGFalpha. We demonstrated the down-regulation of Sox9 messenger RNA and protein levels by TGFalpha. This was associated with reduced levels of phosphorylated and total SOX9 in cartilage explants upon TGFalpha treatment. In contrast, another growth factor identified in our microarrays, Kitl, had no effects on the chondrocyte parameters tested. To examine correlations between the increased levels of TGFalpha in experimental knee osteoarthritis (OA) with the levels of TGFalpha in humans with knee OA, a microarray analysis of mRNA from 13 normal and 12 late-stage OA cartilage samples was performed. Seven OA samples showed TGFA mRNA levels similar to those in the normal controls, but expression was markedly increased in the other 5 OA samples. These data confirm that TGFA transcript levels are increased in a subset of patients with OA. CONCLUSION: This study adds TGFalpha to the list of dysregulated cytokines present in degrading cartilage in OA. Since TGFalpha inhibits articular chondrocyte anabolic capacity, increases catabolic factors, and contributes to the development of chondrocyte clusters, TGFalpha may be a potential target for therapeutic strategies in the treatment of OA.

P2Y nucleotide receptor signaling through MAPK/ERK is regulated by extracellular matrix: involvement of beta3 integrins.

Extracellular matrix influences cell behavior through receptors such as integrins and through transmission of mechanical forces. Nucleotides are released in response to mechanical stimuli and bind to P2 nucleotide receptors. As chondrocytes are subjected to frequent mechanical stimulation within a rich extracellular matrix, they are an excellent model for studying integration of signals induced by matrix and nucleotides. We investigated signaling of G protein-coupled P2Y receptors to MAPK/ERK and how this is influenced by matrix. Rat articular chondrocytes expressed transcripts for P2Y1, P2Y2, P2Y4, and P2Y6 receptors and responded to extracellular nucleotides by transient elevation of cytosolic calcium and MAPK/ERK phosphorylation. ERK1/2 activation was suppressed by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and rottlerin, and by the phospholipase D inhibitor 1-butanol. Thus, nucleotides stimulate P2Y receptors to activate ERK1/2 through a mechanism dependent on PKC and phospholipase D. We next examined the involvement of integrins. Both an RGD-containing pentapeptide and a beta3 integrin blocking antibody, but not a beta1 integrin blocking antibody, abolished nucleotide-induced ERK1/2 phosphorylation. Moreover, chondrocytes adhering to fibronectin (which binds to beta1 and beta3 containing integrins in an RGD-dependent manner) displayed prolonged ERK1/2 signaling compared to cells grown on type I or II collagen (which bind to beta1-containing integrins in an RGD-independent manner). In conclusion, P2Y receptor signaling through ERK1/2 is gated selectively by matrix proteins. Thus, nucleotides released in response to mechanical stimulation will have differing effects on cell function due to changes in the composition of the extracellular matrix during development and disease.

Forced mobilization accelerates pathogenesis: characterization of a preclinical surgical model of osteoarthritis.

Preclinical osteoarthritis (OA) models are often employed in studies investigating disease-modifying OA drugs (DMOADs). In this study we present a comprehensive, longitudinal evaluation of OA pathogenesis in a rat model of OA, including histologic and biochemical analyses of articular cartilage degradation and assessment of subchondral bone sclerosis. Male Sprague-Dawley rats underwent joint destabilization surgery by anterior cruciate ligament transection and partial medial meniscectomy. The contralateral joint was evaluated as a secondary treatment, and sham surgery was performed in a separate group of animals (controls). Furthermore, the effects of walking on a rotating cylinder (to force mobilization of the joint) on OA pathogenesis were assessed. Destabilization-induced OA was investigated at several time points up to 20 weeks after surgery using Osteoarthritis Research Society International histopathology scores, in vivo micro-computed tomography (CT) volumetric bone mineral density analysis, and biochemical analysis of type II collagen breakdown using the CTX II biomarker. Expression of hypertrophic chondrocyte markers was also assessed in articular cartilage. Cartilage degradation, subchondral changes, and subchondral bone loss were observed as early as 2 weeks after surgery, with considerable correlation to that seen in human OA. We found excellent correlation between histologic changes and micro-CT analysis of underlying bone, which reflected properties of human OA, and identified additional molecular changes that enhance our understanding of OA pathogenesis. Interestingly, forced mobilization exercise accelerated OA progression. Minor OA activity was also observed in the contralateral joint, including proteoglycan loss. Finally, we observed increased chondrocyte hypertrophy during pathogenesis. We conclude that forced mobilization accelerates OA damage in the destabilized joint. This surgical model of OA with forced mobilization is suitable for longitudinal preclinical studies, and it is well adapted for investigation of both early and late stages of OA. The time course of OA progression can be modulated through the use of forced mobilization.

The induction of CCN2 by TGFbeta1 involves Ets-1.

CCN2 is encoded by an immediate-early gene induced in mesenchymal cells during the formation of blood vessels, bone and connective tissue. It plays key roles in cell adhesion and migration, as well as matrix remodeling. CCN2 is overexpressed in fibrosis, arthritis and cancer; thus, an understanding of how to control CCN2 expression is likely to have importance in developing therapies to combat these pathologies. Previously, we found that the promoter sequence GAGGAATG is important for Ccn2 gene regulation in NIH 3T3 fibroblasts. In this report, we show that this sequence mediates activation of the CCN2 promoter by the ETS family of transcription factors. Endogenous Ets-1 binds this element of the CCN2 promoter, and dominant negative Ets-1 and specific Ets-1 small interfering RNA block induction of CCN2 expression by TGFbeta. In the absence of added TGFbeta1, Ets-1, but not the related fli-1, synergizes with Smad 3 to activate the CCN2 promoter. Whereas the ability of transfected Ets-1 to activate the CCN2 promoter is dependent on protein kinase C (PKC), Ets-1 in the presence of co-transfected Smad3 does not require PKC, suggesting that the presence of Smad3 bypasses the requirement of Ets-1 for PKC to activate target promoter activity. Our results are consistent with the notion that Smad3 and Ets-1 cooperate in the induction of the CCN2 promoter by TGFbeta1. Antagonizing Ets-1 might be of benefit in attenuating CCN2 expression in fibrosis, arthritis and cancer, and may be useful in modulating the outcome of these disorders.

Tumor necrosis factor alpha and epidermal growth factor act additively to inhibit matrix gene expression by chondrocyte.

The failure of chondrocytes to replace the lost extracellular matrix contributes to the progression of degenerative disorders of cartilage. Inflammatory mediators present in the joint regulate the breakdown of the established matrix and the synthesis of new extracellular matrix molecules. In the present study, we investigated the effects of tumor necrosis factor alpha (TNF-alpha) and epidermal growth factor (EGF) on chondrocyte morphology and matrix gene expression. Chondrocytes were isolated from distal femoral condyles of neonatal rats. Cells in primary culture displayed a cobblestone appearance. EGF, but not TNF-alpha, increased the number of cells exhibiting an elongated morphology. TNF-alpha potentiated the effect of EGF on chondrocyte morphology. Individually, TNF-alpha and EGF diminished levels of aggrecan and type II collagen mRNA. In combination, the effects of TNF-alpha and EGF were additive, indicating the involvement of discrete signaling pathways. Cell viability was not compromised by TNF-alpha or by EGF, alone or in combination. EGF alone did not activate NF-kappaB or alter NF-kappaB activation by TNF-alpha. Pharmacologic studies indicated that the effects of TNF-alpha and EGF alone or in combination were independent of protein kinase C signaling, but were dependent on MEK1/2 activity. Finally, we analyzed the involvement of Sox-9 using a reporter construct of the 48 base pair minimal enhancer of type II collagen. TNF-alpha attenuated enhancer activity as expected; in contrast, EGF did not alter either the effect of TNF-alpha or basal activity. TNF-alpha and EGF, acting through distinct signaling pathways, thus have additive adverse effects on chondrocyte function. These findings provide critical insights into the control of chondrocytes through the integration of multiple extracellular signals.

Inflammatory cytokines induce production of CHI3L1 by articular chondrocytes.

Elevated levels of CHI3L1 (chitinase-3-like protein 1) are associated with disorders exhibiting increased connective tissue turnover, such as rheumatoid arthritis, osteoarthritis, scleroderma, and cirrhosis of the liver. This secreted protein is not synthesized in young healthy cartilage, but is produced in cartilage from old donors or patients with osteoarthritis. The molecular processes governing the induction of CHI3L1 are currently unknown. To elucidate the molecular events involved in CHI3L1 synthesis, we investigated two models of articular chondrocytes: neonatal rat chondrocytes, which do not express CHI3L1, and human chondrocytes, which express CHI3L1 constitutively. In neonatal rat chondrocytes, the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 potently induced steady-state levels of CHI3L1 mRNA and protein secretion. Treatment of chondrocytes with TNF-alpha for as little as 1 h was sufficient for sustained induction up to 72 h afterward. Using inhibitors selective for the major signaling pathways implicated in mediating the effects of TNF-alpha and interleukin-1, only inhibition of NF-kappaB activation was effective in curtailing cytokine-induced expression, including after removal of the cytokine, indicating that induction and continued production of CHI3L1 are controlled mainly by this transcription factor. Inhibition of NF-kappaB signaling also abolished constitutive expression by human chondrocytes. Thus, induction and continued secretion of CHI3L1 in chondrocytes require sustained activation of NF-kappaB. Selective induction of CHI3L1 by cytokines acting through NF-kappaB coupled with the known restriction of the catabolic responses by CHI3L1 in response to these inflammatory cytokines represents a key regulatory feedback process in controlling connective tissue turnover.

TNFalpha suppresses link protein and type II collagen expression in chondrocytes: Role of MEK1/2 and NF-kappaB signaling pathways.

Tumor necrosis factor alpha (TNFalpha) inhibits matrix synthesis by chondrocytes in rheumatoid arthritis and osteoarthritis; however, the underlying signaling pathways are poorly characterized. This study investigated the TNFalpha-activated pathways regulating expression of two key components of the cartilage matrix-link protein and type II collagen. In rat articular chondrocytes, TNFalpha decreased link protein and type II collagen mRNA to undetectable levels within 48 h. Levels of link protein mRNA recovered more readily than type II collagen mRNA following removal of the cytokine. TNFalpha-mediated reduction in mRNA of both matrix molecules occurred at the level of transcription and, for link protein, mRNA stability. Turnover of type II collagen and link protein mRNA was dependent on new protein synthesis. In both prechondrocytes and articular chondrocytes, TNFalpha induced concentration-dependent activation of MEK1/2 and NF-kappaB, but not p38 or JNK. Sustained activation of NF-kappaB was observed for up to 72 h following continuous or transient exposure to TNFalpha. Using pharmacological and molecular approaches, the MEK1/2 and NF-kappaB pathways were found to mediate inhibition of type II collagen and link protein gene expression by TNFalpha. Both prechondrocytes and articular chondrocytes are targets of TNFalpha. This study identifies pathways through which TNFalpha perturbs the synthesis and organization of articular cartilage matrix during inflammation.

Epidermal growth factor stimulates proton efflux from chondrocytic cells.

Proton efflux from chondrocytes alters the extracellular pH and ionic composition of cartilage, and influences the synthesis and degradation of extracellular matrix. Epidermal growth factor (EGF) promotes chondrocyte proliferation during skeletal development and accumulates in the synovial fluid in rheumatoid arthritis. The purpose of this study was to investigate the effect of EGF on proton efflux from chondrocytes. When monitored using a Cytosensor microphysiometer, EGF was found to rapidly activate proton efflux from CFK2 chondrocytic cells and rat articular chondrocytes. The actions of EGF were concentration-dependent with half-maximal effects at 0.3-0.7 ng/ml. Partial desensitization and time-dependent recovery of the response were observed following repeated exposures to EGF. EGF-induced proton efflux was dependent on extracellular glucose, and inhibitors of Na(+)/H(+) exchange (NHE) markedly attenuated the initial increase in proton efflux. The response was diminished by inhibitors of phosphatidylinositol 3-kinase and phospholipase C, but not by inhibitors of MEK (MAPK/ERK kinase) or protein kinase A or C. Thus, EGF-induced proton efflux involves glucose metabolism and NHE, and is regulated by a discrete subset of EGF-activated signaling pathways. In vivo, proton efflux induced by EGF may lead to an acidic environment, enhancing turnover of cartilage matrix during development and in rheumatoid arthritis.

N-butyryl glucosamine increases matrix gene expression by chondrocytes.

Proteoglycan synthesis is dependent on N-acetyl glucosamine (GlcNAc) produced by the hexosamine biosynthetic pathway or obtained exogenously. Although used therapeutically to relieve symptoms of osteoarthritis, the actions of glucosamine and its analogs on cartilage are poorly understood. The purpose of this study was to determine the effects on chondrocytes of N-acylated-glucosamine analogs bearing alkyl chains of different lengths. Chondrocytes isolated from neonatal rat femoral condyles were cultured in the presence of glucosamine analogs. GlcNAc, N-proprionyl glucosamine (GlcNPro), or N-butyryl glucosamine (GlcNBu) did not alter cell number, lactate dehydrogenase release, or metabolic acid production, consistent with lack of cytotoxicity. Treatment of chondrocyte cultures with GlcNBu for 6 days significantly increased levels of type II collagen and aggrecan mRNA as determined by Northern blot analysis. In contrast, GlcNAc and GlcNPro had no significant effect. A significant increase in type II collagen mRNA was induced by GlcNBu within 3 days. GlcNBu did not alter stability of type II collagen mRNA, suggesting it acts on gene transcription. We have previously shown that tumor necrosis factor-alpha (TNFalpha) decreases levels of type II collagen mRNA. However, chondrocytes pretreated with GlcNBu maintained type II collagen mRNA at control levels in the presence of TNFalpha. These results establish that the N-butyrylated analog of glucosamine but not GlcNAc promotes matrix gene expression by chondrocytes. Thus, GlcNBu has the potential for use as a chondro-protective agent in osteoarthritis.