Netrin-1 Secreted by Human Osteoarthritic Articular Chondrocytes Promotes Angiogenesis in Vitro.

OBJECTIVE: Netrin-1 expression in articular cartilage is correlated with osteoarthritic changes. We aimed to investigate the contribution of Netrin-1 secreted by human osteoarthritic articular chondrocytes to angiogenesis process in vitro. DESIGN: Human articular chondrocytes were extracted from non-osteoarthritic (n = 10) and osteoarthritic (n = 22) joints obtained from surgical specimens and incubated for 24 hours. Medium conditioned by non-osteoarthritic and osteoarthritic articular chondrocytes were collected. Human umbilical vein endothelial cells (HUVEC) were treated with control and conditioned medium and assessed using assays for cell adherence, migration, and tube formation. Netrin-1 expression and secretion was compared between non-osteoarthritic and osteoarthritic chondrocytes by qPCR, Western blot, and ELISA. The role of chondrocyte-secreted Netrin-1 on HUVEC functions was assessed by immunological neutralization using an anti-Netrin-1 monoclonal antibody. RESULTS: As compared with medium conditioned by non-osteoarthritic chondrocytes, medium conditioned by osteoarthritic chondrocytes permitted tube formation by HUVEC. Both non-osteoarthritic and osteoarthritic chondrocytes expressed Netrin-1 at the RNA and protein levels. At the RNA level, Netrin-1 expression did not differ between non-osteoarthritic and osteoarthritic chondrocytes. At the protein level, Netrin-1 appeared as a full protein of 64 kDa in non-osteoarthritic chondrocytes and as two cleaved proteins of 55 kDa and 64 kDa in osteoarthritic chondrocytes. Immunological neutralization of endogenous Netrin-1 reduced the pro-angiogenic and pro-inflammatory transcriptional profile of HUVEC treated with the medium conditioned by osteoarthritic chondrocytes, as well as their capacities to form tubes. CONCLUSIONS: Medium conditioned by osteoarthritic chondrocytes permits tube formation by HUVEC in vitro. This permissive effect is mediated by Netrin-1.

Aging Cartilage in Wild-Type Mice: An Observational Study.

OBJECTIVE: To describe the spontaneous evolution of age-related changes affecting knee joint articular cartilage, walking speed and a serum biomarker of cartilage remodeling in C57BL/6-JRj wild-type male mice. DESIGN: Histological changes were assessed by the Osteoarthritis Research Society International (OARSI) score (0=normal, 6=vertical clefts/erosion to the calcified cartilage extending >75% of the articular surface) in newborn, 1-week- and 1-, 3-, 6-, 9- and 12-month-old C57BL/6-JRj wild-type male mice, walking speed by the Locotronic system, and serum C-terminal telopeptide of type II collagen (CTX-II) content by ELISA in 1-, 3-, 6-, and 9-month-old C57BL/6-JRj wild-type male mice. RESULTS: Mean (SD) OARSI score significantly increased from 0.2 (0.3) to 1.3 (0.6) (p=0.03) between 1 and 3 months of age and from 1.3 (0.6) to 3.3 (0.6) (p=0.04) between 3 and 6 months of age. Mean walking speed was stable between 1 and 6 months of age but significantly decreased from 11.4 (1.8) to 3.2 (0.8) cm.s-1 (p=0.03) between 6 and 9 months of age. Serum CTX-II content was maximal at 1 month of age, then decreased from 12.2 (8.5) to 2.4 (8.4) pg/ml (p=0.02) between 1 and 3 months of age, remaining low and stable thereafter. CONCLUSIONS: C57BL/6-JRj wild-type male mice showed continuously increasing osteoarthritic changes but delayed decreasing walking speed with age. These variations were maximal between 3 and 9 months of age. Maximal serum CTX-II content preceded these changes.

Early cognitive decline after bilateral subthalamic deep brain stimulation in Parkinson's disease patients with GBA mutations.

BACKGROUND: Subthalamic nucleus deep brain stimulation (STN-DBS) has demonstrated its efficacy on motor complications in advanced Parkinson's disease (PD) but does not modify disease progression. Genetic forms of PD have been associated with different cognitive progression profiles. OBJECTIVE: To assess the effect of PD-related genetic mutations on cognitive outcome after STN-DBS. METHODS: Patients with STN-DBS were screened for LRRK2, GBA, and PRKN mutations at the Pitié-Salpêtrière Hospital between 1997 and 2009. Patients with known monogenetic forms of PD from six other centers were also included. The Mattis Dementia Rating Scale (MDRS) was used to evaluate cognition at baseline and one-year post-surgery. The standardized Unified PD Rating Scale (UPDRS) evaluation On and Off medication/DBS was also administered. A generalized linear model adjusted for sex, ethnicity, age at onset, and disease duration was used to evaluate the effect of genetic factors on MDRS changes. RESULTS: We analyzed 208 patients (131 males, 77 females, 54.3 ± 8.8 years) including 25 GBA, 18 LRRK2, 22 PRKN, and 143 PD patients without mutations. PRKN patients were younger and had a longer disease duration at baseline. A GBA mutation was the only significant genetic factor associated with MDRS change (ß = -2.51, p = 0.009). GBA mutation carriers had a more pronounced post-operative MDRS decline (3.2 ± 5.1) than patients with LRRK2 (0.9 ± 4.8), PRKN (0.5 ± 2.7) or controls (1.4 ± 4.4). The motor response to DBS was similar between groups. CONCLUSION: GBA mutations are associated with early cognitive decline following STN-DBS. Neuropsychological assessment and discussions on the benefit/risk ratio of DBS are particularly important for this population.

SUMOylation by SUMO2 is implicated in the degradation of misfolded ataxin-7 via RNF4 in SCA7 models.

Perturbation of protein homeostasis and aggregation of misfolded proteins is a major cause of many human diseases. A hallmark of the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7) is the intranuclear accumulation of mutant, misfolded ataxin-7 (polyQ-ATXN7). Here, we show that endogenous ATXN7 is modified by SUMO proteins, thus also suggesting a physiological role for this modification under conditions of proteotoxic stress caused by the accumulation of polyQ-ATXN7. Co-immunoprecipitation experiments, immunofluorescence microscopy and proximity ligation assays confirmed the colocalization and interaction of polyQ-ATXN7 with SUMO2 in cells. Moreover, upon inhibition of the proteasome, both endogenous SUMO2/3 and the RNF4 ubiquitin ligase surround large polyQ-ATXN7 intranuclear inclusions. Overexpression of RNF4 and/or SUMO2 significantly decreased levels of polyQ-ATXN7 and, upon proteasomal inhibition, led to a marked increase in the polyubiquitination of polyQ-ATXN7. This provides a mechanism for the clearance of polyQ-ATXN7 from affected cells that involves the recruitment of RNF4 by SUMO2/3-modified polyQ-ATXN7, thus leading to its ubiquitination and proteasomal degradation. In a SCA7 knock-in mouse model, we similarly observed colocalization of SUMO2/3 with polyQ-ATXN7 inclusions in the cerebellum and retina. Furthermore, we detected accumulation of SUMO2/3 high-molecular-mass species in the cerebellum of SCA7 knock-in mice, compared with their wild-type littermates, and changes in SUMO-related transcripts. Immunohistochemical analysis showed the accumulation of SUMO proteins and RNF4 in the cerebellum of SCA7 patients. Taken together, our results show that the SUMO pathway contributes to the clearance of aggregated ATXN7 and suggest that its deregulation might be associated with SCA7 disease progression.

Transcriptome Analysis of Peripheral Blood in Chronic Inflammatory Demyelinating Polyradiculoneuropathy Patients Identifies TNFR1 and TLR Pathways in the IVIg Response.

We have studied the response to intravenous immunoglobulins (IVIg) by a transcriptomic approach in 11 chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) patients (CIDP duration = 6 [0.83-6.5] years). RNA was extracted from cells in whole blood collected before and 3 weeks after IVIg treatment, and hybridized on Illumina chips. After RNA quality controls, gene expression was analyzed using statistical tests fitted for microarrays (R software, limma package), and a pathway analysis was performed using DAVID software. We identified 52 genes with expression that varied significantly after IVIg (fold change [FC] > 1.2, P < 0.001, false discovery rate [FDR] <0.05). Among these 52 genes, 7 were related to immunity, 3 were related to the tumor necrosis factor (TNF)-α receptor 1 (TNFR1) pathway (inhibitor of caspase-activated DNase (ICAD): FC = 1.8, P = 1.7E-7, FDR = 0.004; p21 protein-activated kinase 2 [PAK2]: FC = 1.66, P = 2.6E-5, FDR = 0.03; TNF-α-induced protein 8-like protein 1 [TNFAIP8L1]: P = 1.00E-05, FDR = 0.026), and 2 were related to Toll-like receptors (TLRs), especially TLRs 7 and 9, and were implicated in autoimmunity. These genes were UNC93B1 (FC = 1.6, P = 2E-5, FDR = 0.03), which transports TLRs 7 and 9 to the endolysosomes, and RNF216 (FC = 1.5, P = 1E-05, FDR = 0.03), which promotes TLR 9 degradation. Pathway analysis showed that the TNFR1 pathway was significantly lessened by IVIg (enrichment score = 24, Fischer exact test = 0.003). TNF-α gene expression was higher in responder patients than in nonresponders; however, it decreased after IVIg in responders (P = 0.04), but remained stable in nonresponders. Our data suggest the actions of IVIg on the TNFR1 pathway and an original mechanism involving innate immunity through TLRs in CIDP pathophysiology and the response to IVIg. We conclude that responder patients have stronger inflammatory activity that is lessened by IVIg.

Dopaminergic denervation severity depends on COMT Val158Met polymorphism in Parkinson's disease.

BACKGROUND: Catecholamine-O-methyl-tranferase (COMT) initiates dopamine degradation. Its activity is mainly determined by a single nucleotide polymorphism in the COMT gene (Val158Met, rs4680) separating high (Val/Val, COMT(HH)), intermediate (Val/Met, COMT(HL)) and low metabolizers (Met/Met, COMT(LL)). We investigated dopaminergic denervation in the striatum in PD patients according to COMT rs4680 genotype. METHODS: Patients with idiopathic PD were assessed for motor severity (UPDRS-III rating scale in OFF-state), dopaminergic denervation using [123I]-FP-CIT SPECT imaging, and genotyped for the COMT rs4680 enzyme. [123I]-FP-CIT binding potential (BP) for each voxel was defined by the ratio of tracer-binding in the region of interest (striatum, caudate nucleus and putamen) to that in a region of non-specific activity. Genotyping was performed using TaqMan(®) SNP genotyping assay. We used a regression model to evaluate the effect of COMT genotype on the BP in the striatum and its sub-regions. RESULTS: Genotype distribution was: 11 (27.5%) COMT(HH), 26 (65%) COMT(HL) and 3 (7.5%) COMT(LL). There were no significant differences in disease severity, treatments, or motor scores between genotypes. When adjusted to clinical severity, gender and age, low and intermediate metabolizers showed significantly higher rates of striatal denervation (COMT(HL+LL) BP = 1.32 ± 0.04) than high metabolizers (COMT(HH), BP = 1.6 ± 0.08; F(1.34) = 9.0, p = 0.005). Striatal sub-regions showed similar results. BP and UPDRS-III motor scores (r = 0.44, p = 0.04) (p < 0.001) were highly correlated. There was a gender effect, but no gender-genotype interaction. CONCLUSIONS: Striatal denervation differs according to COMT-Val158Met polymorphism. COMT activity may play a role as a compensatory mechanism in PD motor symptoms.

Gene expression analyses identify Narp contribution in the development of L-DOPA-induced dyskinesia.

In Parkinson's disease, long-term dopamine replacement therapy is complicated by the appearance of L-DOPA-induced dyskinesia (LID). One major hypothesis is that LID results from an aberrant transcriptional program in striatal neurons induced by L-DOPA and triggered by the activation of ERK. To identify these genes, we performed transcriptome analyses in the striatum in 6-hydroxydopamine-lesioned mice. A time course analysis (0-6 h after treatment with L-DOPA) identified an acute signature of 709 genes, among which genes involved in protein phosphatase activity were overrepresented, suggesting a negative feedback on ERK activation by l-DOPA. l-DOPA-dependent deregulation of 28 genes was blocked by pretreatment with SL327, an inhibitor of ERK activation, and 26 genes were found differentially expressed between highly and weakly dyskinetic animals after treatment with L-DOPA. The intersection list identified five genes: FosB, Th, Nptx2, Nedd4l, and Ccrn4l. Nptx2 encodes neuronal pentraxin II (or neuronal activity-regulated pentraxin, Narp), which is involved in the clustering of glutamate receptors. We confirmed increased Nptx2 expression after L-DOPA and its blockade by SL327 using quantitative RT-PCR in independent experiments. Using an escalating L-DOPA dose protocol, LID severity was decreased in Narp knock-out mice compared with their wild-type littermates or after overexpression of a dominant-negative form of Narp in the striatum. In conclusion, we have identified a molecular signature induced by L-DOPA in the dopamine-denervated striatum that is dependent on ERK and associated with LID. Here, we demonstrate the implication of one of these genes, Nptx2, in the development of LID.

The autophagy/lysosome pathway is impaired in SCA7 patients and SCA7 knock-in mice.

There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contributes to the pathogenesis of spinocerebellar ataxia type 7 (SCA7), caused by expansion of a polyglutamine tract in the ataxin-7 protein, we looked for biochemical, histological and transcriptomic abnormalities in components of the autophagy/lysosome pathway in a knock-in mouse model of the disease, postmortem brain and peripheral blood mononuclear cells (PBMC) from patients. In the mouse model, mutant ataxin-7 accumulated in inclusions immunoreactive for the autophagy-associated proteins mTOR, beclin-1, p62 and ubiquitin. Atypical accumulations of the autophagosome/lysosome markers LC3, LAMP-1, LAMP2 and cathepsin-D were also found in the cerebellum of the SCA7 knock-in mice. In patients, abnormal accumulations of autophagy markers were detected in the cerebellum and cerebral cortex of patients, but not in the striatum that is spared in SCA7, suggesting that autophagy might be impaired by the selective accumulation of mutant ataxin-7. In vitro studies demonstrated that the autophagic flux was impaired in cells overexpressing full-length mutant ataxin-7. Interestingly, the expression of the early autophagy-associated gene ATG12 was increased in PBMC from SCA7 patients in correlation with disease severity. These results provide evidence that the autophagy/lysosome pathway is impaired in neurons undergoing degeneration in SCA7. Autophagy/lysosome-associated molecules might, therefore, be useful markers for monitoring the effects of potential therapeutic approaches using modulators of autophagy in SCA7 and other autophagy/lysosome-associated neurodegenerative disorders.

Dopa-decarboxylase gene polymorphisms affect the motor response to L-dopa in Parkinson's disease.

BACKGROUND: In Parkinson's disease (PD), the response to L-dopa is highly variable and unpredictable. The major pathway for dopamine synthesis from L-dopa is decarboxylation by aromatic L-amino acid decarboxylase (AAAD, encoded by the DDC gene). OBJECTIVE: To determine the motor response to L-dopa in PD patients as a function of the DDC gene promoter polymorphisms (rs921451 T > C polymorphism (DDC(T/C)) and rs3837091 AGAG del (DDC(AGAG/-))). METHODS: Thirty-three Caucasian PD patients underwent an acute l-dopa challenge together with the peripheral AAAD inhibitor benserazide and were genotyped for rs921451 and rs3837091. The primary efficacy criterion was the motor response to L-dopa, as estimated by the area under the curve for the change in the Unified Parkinson's Disease Rating Scale part III (UPDRS) score relative to baseline (AUCΔUPDRS) in the 4 h following L-dopa administration. Secondary endpoints were pharmacokinetic parameters for plasma levels of L-dopa and dopamine. Investigators and patients were blinded to genotypes data throughout the study. RESULTS: When adjusted for the L-dopa dose, the AUCΔUPDRS was significantly lower in DDC(CC/CT) patients (n = 14) than in DDC(TT) patients (n = 19) and significantly lower in DDC(-/- or AGAG/-) patients (n = 8) than in DDC(AGAG/AGAG) patients (n = 25). There were no significant intergroup differences in plasma pharmacokinetic parameters for L-dopa and dopamine. DISCUSSION: The rs921451 and rs3837091 polymorphisms of the DDC gene promoter influence the motor response to L-dopa but do not significantly change peripheral pharmacokinetic parameters for L-dopa and dopamine. Our results suggest that DDC may be a genetic modifier of the l-dopa response in Parkinson's disease.

The Val158Met COMT polymorphism is a modifier of the age at onset in Parkinson's disease with a sexual dimorphism.

The catechol-O-methyltranferase (COMT) is one of the main enzymes that metabolise dopamine in the brain. The Val158Met polymorphism in the COMT gene (rs4680) causes a trimodal distribution of high (Val/Val), intermediate (Val/Met) and low (Met/Met) enzyme activity. We tested whether the Val158Met polymorphism is a modifier of the age at onset (AAO) in Parkinson's disease (PD). The rs4680 was genotyped in a total of 16 609 subjects from five independent cohorts of European and North American origin (5886 patients with PD and 10 723 healthy controls). The multivariate analysis for comparing PD and control groups was based on a stepwise logistic regression, with gender, age and cohort origin included in the initial model. The multivariate analysis of the AAO was a mixed linear model, with COMT genotype and gender considered as fixed effects and cohort and cohort-gender interaction as random effects. COMT genotype was coded as a quantitative variable, assuming a codominant genetic effect. The distribution of the COMT polymorphism was not significantly different in patients and controls (p=0.22). The Val allele had a significant effect on the AAO with a younger AAO in patients with the Val/Val (57.1±13.9, p=0.03) than the Val/Met (57.4±13.9) and the Met/Met genotypes (58.3±13.5). The difference was greater in men (1.9 years between Val/Val and Met/Met, p=0.007) than in women (0.2 years, p=0.81). Thus, the Val158Met COMT polymorphism is not associated with PD in the Caucasian population but acts as a modifier of the AAO in PD with a sexual dimorphism: the Val allele is associated with a younger AAO in men with idiopathic PD.

Hes1, a new target for interleukin 1beta in chondrocytes.

OBJECTIVES: To investigate the effects of interleukin 1beta (IL1beta) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro. METHODS: Mouse articular chondrocytes in primary culture were challenged with IL1beta, alone or combined with Notch1 and IL1beta pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q)PCR. IL1beta-responsive genes were assessed by real-time qPCR and a specific siRNA against Hes1 was used to identify Hes1 target genes. RESULTS: Notch1 labelling remained nuclear and stable in intensity irrespective of treatment, suggesting a steady state activation of this pathway in our model. IL1beta transiently increased Hes1 mRNA (2.5-fold) and protein expression in treated versus naive chondrocytes. Hes1 mRNA level then decreased below control and its cyclic pattern of expression was lost. This was associated with nuclear translocation of the cytoplasmic Hes1 protein. IL1beta induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)kappaB activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the gamma-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), which suggested its independence from novel Notch1 activation. Hes1 expression was efficiently silenced by a specific siRNA. This experiment revealed that Hes1 did not mediate IL1beta-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1beta induction of matrix metalloproteinase (MMP)13 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). The Hes1-related repressor Hey1 was expressed at a very low level and was not inducible by IL1beta. CONCLUSION: Hes1 is a novel IL1beta target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation.

Contraceptive steroids from pharmaceutical waste perturbate junctional communication in Sertoli cells.

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERalpha and only express the cytoplasmic beta isoform. ERbeta localization was not modified by either steroid. The threshold level was surprisingly low, around 10(-16) M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.

[Cell therapy in cartilage repair: cellular and molecular bases]. / La thérapie cellulaire du cartilage : bases cellulaires et moléculaires.

The destruction of articular cartilage represents the outcome of most inflammatory and degenerative rheumatic diseases and leads to severe disability. Articular cartilage being unable to repair spontaneously, alterations of the joint surface often results in end-stage osteoarthritis, requiring surgical intervention and total joint replacement. This makes damaged tissues repair a major challenge in our aging society. Cartilage harbors only one cell type, the chondrocyte, which synthesizes and secretes specific matrix proteins such as type II collagen and high molecular weight proteoglycans. Matrix proteins are responsible for the conservation of the chondrocyte phenotype and the maintenance of the mechanical functions of cartilage. Development of therapeutic strategies for cartilage repair should thus comprise not only the replacement of lost cartilage cells but also that of extracellular matrix with cartilage-like properties. Different protocols are under investigation. The most commonly employed materials include transplantation of autologous osteochondral tissue. More recently, cell-based therapies using autologous mature chondrocytes or pre-chondrogenic stem cells have drawn particular attention. Tissue-engineering procedures represent the actual trend in cartilage repair. This approach combines biodegradable polymeric three-dimensional matrixes and isolated prechondrogenic stem cells. The cells are seeded within the biocompatible matrix and then implanted into the joint. Numerous non-degradable and degradable polymers, which efficiently "mimic" the natural surroundings of cartilage cells, are currently under investigation.

Crucial role of visfatin/pre-B cell colony-enhancing factor in matrix degradation and prostaglandin E2 synthesis in chondrocytes: possible influence on osteoarthritis.

OBJECTIVE: Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in osteoarthritis (OA), and metalloproteinases (MMPs) are crucial for cartilage degradation. PGE2 synthesis under inflammatory conditions is catalyzed by cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1), whereas NAD+-dependent 15-hydroxy-PG dehydrogenase (15-PGDH) is the key enzyme implicated in PGE2 catabolism. The present study was undertaken to investigate the contribution of visfatin, an adipose tissue-derived hormone, to the pathophysiology of OA, by examining its role in PGE2 synthesis and matrix degradation. METHODS: The synthesis of visfatin by human chondrocytes from OA patients, with and without stimulation with interleukin-1beta (IL-1beta) and the role of visfatin in PGE2 synthesis were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting. The effects of visfatin (1-10 microg/ml) on mPGES-1 and 15-PGDH synthesis, on the subsequent release of PGE2, and on MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and PG synthesis by primary immature mouse articular chondrocytes were examined by quantitative RT-PCR, immunoblotting, and enzyme-linked immunosorbent assay. Finally, small interfering RNA (siRNA) was used to assess the influence of visfatin on IL-1beta-induced release of PGE2 in immature mouse articular chondrocytes. RESULTS: Human OA chondrocytes produced visfatin, and visfatin synthesis was increased by IL-1beta treatment. Visfatin, like IL-1beta, triggered excessive release of PGE2, due to increased mPGES-1 synthesis and decreased 15-PGDH synthesis. Visfatin knockout with siRNA reduced IL-1beta-induced PGE2 overrelease. Visfatin triggered ADAMTS-4 and ADAMTS-5 expression and MMP-3 and MMP-13 synthesis and release, and reduced synthesis of high molecular weight PG by immature mouse articular chondrocytes. CONCLUSION: The findings of this study indicate that visfatin has a catabolic function in cartilage and may have an important role in the pathophysiology of OA.

Oestrogens inhibit interleukin 1beta-mediated nitric oxide synthase expression in articular chondrocytes through nuclear factor-kappa B impairment.

OBJECTIVES: To investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression. METHODS: The presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry. RESULTS: ERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2. CONCLUSIONS: Our data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

Activation of the peroxisome proliferator-activated receptor alpha pathway potentiates interleukin-1 receptor antagonist production in cytokine-treated chondrocytes.

OBJECTIVE: To determine whether peroxisome proliferator-activated receptor alpha (PPARalpha) agonists protect chondrocytes against the effects of interleukin-1beta (IL-1beta). METHODS: PPARalpha expression and function in cultured rabbit articular chondrocytes were studied by Northern blotting, electrophoretic mobility shift assay, and transient expression of a luciferase reporter construct bearing the human IL-1 receptor antagonist (Il-1Ra) gene promoter. Chondrocytes were incubated in vitro with IL-1beta alone or in combination with CloFibrate (CloF) or other PPAR ligands. Proteoglycans were evaluated by 35S-sulfate incorporation, matrix metalloproteinase (MMP) levels were assessed by zymography and enzyme-linked immunosorbent assay (ELISA), and MMP messenger RNA (mRNA) levels were measured by Northern blotting and real-time reverse transcriptase-polymerase chain reaction. IL-1beta and IL-1Ra soluble contents were measured by ELISA. RESULTS: CloF counteracted IL-1beta-induced 35S-proteoglycan degradation, gelatinolytic activity, and MMP-1, -3, and -13 mRNA expression. CloF also maximized IL-1beta-induced endogenous production of soluble IL-1Ra (sIL-1Ra). This stimulating effect on IL-1Ra gene expression was shown, by transient expression assay, to be transcriptional. Inhibition of sIL-1Ra expression by a specific small interfering RNA suppressed the effect of CloF on IL-1beta-induced MMP expression. The stimulatory effect of CloF was enhanced by cotransfection with wild-type PPARalpha and abolished by a dominant-negative PPARalpha mutant. Fenofibrate and WY-14643 displayed a similar stimulating effect on the IL-1Ra promoter, while rosiglitazone did not. Two PPAR response elements, an NF-kappaB-binding site, and a CCAAT/enhancer binding protein-binding site were identified in the IL-1Ra promoter. All 4 sites were necessary for mediation of the effects of CloF. CONCLUSION: Our findings support the notion that there is a PPARalpha-dependent mechanism that inhibits IL-1beta function in chondrocytes, which operates via an increase in sIL-1Ra production.

Basic fibroblast growth factor as a selective inducer of matrix Gla protein gene expression in proliferative chondrocytes.

Matrix Gla protein (MGP) is a member of the vitamin K-dependent gamma carboxylase protein family expressed in cartilage. Insulin-like growth factor I (IGF1) stimulates chondrocyte differentiation, whereas basic fibroblast growth factor (FGF2) acts in an opposite manner. We explored the differential expression and regulation by IGF1 and FGF2 of the MGP gene during chondrocyte differentiation. We used a primary culture system of rabbit epiphyseal chondrocytes to show that MGP mRNA is mainly expressed during serum-induced proliferation. Much lower MGP mRNA content is observed in post-mitotic chondrocytes, which newly express alpha 1X procollagen mRNA, a marker of late-differentiated cells. From studies of a series of growth factors, it was shown that IGF1 decreased chondrocyte MGP transcripts, whereas FGF2 had the opposite effect. FGF2 stimulated chondrocyte MGP production in a dose- and time-dependent manner at the mRNA and protein levels. FGF2 acted in a dose- and time-dependent manner, reaching a maximum at 10 ng/ml at 20 h. The protein synthesis inhibitor cycloheximide did not modify FGF2 action, in agreement with a direct effect. Actinomycin D abolished FGF2-induced stimulation, strongly suggesting that FGF2 modulated MGP gene transcription. We transiently transfected chondrocytes with a construct containing the mouse MGP promoter from -5000 to -168 base pairs, relative to the transcription start site of the gene linked to the luciferase gene (MGP-Luc). In transfected cells, FGF2 stimulated luciferase activity up to sevenfold while IGF1 had no effect. Hence, FGF2 induces transcription of the MGP gene via the 5'-flanking region of the gene. Using a series of deleted MGP-Luc constructs, we identified a sequence of 748 base pairs which was sufficient for transcriptional activation by FGF2. These results led us to postulate that the inhibitory chondrogenic action of FGF2 involves a mechanism whereby MGP gene transcription and protein are induced.