Core body temperature varies according to the time of exercise without affecting orexin-A production in the dorsal hypothalamus in male rats.

Physical exercise differentially increases body temperature according to the time of day, which shows the importance of circadian rhythm in thermal regulation. Given its contribution in central pathways involved in thermoregulation, orexin A could play a role in the regulation of core body temperature during and after exercise. To test this hypothesis, we assessed the effect of exercise, performed at two times of day, on core temperature and on the amount of orexin A in the production zone, i.e., the dorsal hypothalamus. Forty-nine male Wistar rats underwent forced treadmill exercise during the HG phase and HL phase of core temperature. Basal core temperature was recorded continuously for 48 h by implanted telemetric sensors in 11 rats. Regulation of core temperature during exercise (20 min) and after each exercise (60 min) was modeled with a modified logistic-type function. During HG exercise, core temperature curve reached a significantly higher maximum (asymptote: +0.70 ± 0.10 °C) and took longer to attain the strongest inclination of the core temperature regulation curve (Xmid: 3.46 ± 0.72 min). After HG exercise, time of recovery was significantly longer than after HL exercise. In male rats, thermoregulatory response to acute physical exercise was influenced by the time of day. There was no effect of either physical activity or time of day on the level of orexin A in the dorsal hypothalamus. Our results suggest that orexin A in the dorsal hypothalamus is not involved in the effects of physical exercise on thermoregulation.

Interleukin-1beta up-regulation of Smad7 via NF-kappaB activation in human chondrocytes.

OBJECTIVE: We have previously shown that interleukin-1beta (IL-1beta) impairs transforming growth factor beta (TGFbeta) signaling through TGFbeta receptor type II (TGFbetaRII) down-regulation and Smad7 up-regulation. This mechanism could account for the reduced responsiveness of osteoarthritic chondrocytes to TGFbeta and the cartilage breakdown linked to this disease. The aim of this study was to investigate the molecular mechanism underlying the IL-1beta-induced stimulation of Smad7 in human articular chondrocytes. METHODS: Human articular chondrocytes were treated with IL-1beta in the presence of TGFbeta1, pyrrolidine dithiocarbamate (a repressor of the NF-kappaB pathway), or cycloheximide. Then, steady-state messenger RNA and protein levels were estimated by real-time reverse transcription-polymerase chain reaction and immunocytology. In addition, transient transfections of p65 expression vector or p65-targeted short hairpin RNA were performed to define the effect of NF-kappaB on Smad7 expression. RESULTS: TGFbetaRII overexpression restored the TGFbeta response of human articular chondrocytes. However, this effect was transient, implying that a secondary mechanism was responsible for the alteration of the TGFbeta response with long-term exposure to IL-1beta. Moreover, IL-1beta caused a late induction of the inhibitory Smad7. This effect was direct, since it did not require de novo synthesis. In addition, we established, by experiments with gain/loss of function, that the up-regulation of Smad7 by IL-1beta is mediated through the NF-kappaB pathway, especially the p65 subunit. CONCLUSION: These findings clarify the regulatory process of IL-1beta on Smad7 expression. Understanding the molecular basis of IL-1beta induction of Smad7 and the reduction of chondrocyte responsiveness to TGFbeta provides new insights into the molecular mechanisms of osteoarthritis and may facilitate the identification of novel approaches for its treatment.

Chondroitin sulfate modulation of matrix and inflammatory gene expression in IL-1beta-stimulated chondrocytes--study in hypoxic alginate bead cultures.

OBJECTIVE: To determine the effect of avian chondroitin sulfate (CS) on interleukin-1beta (IL-1beta)-induced expression of genes related to catabolic, anabolic and inflammatory aspects in chondrocytes cultured in hypoxic alginate beads. DESIGN: Articular chondrocytes from bovine metacarpal joint were isolated and cultured in alginate beads, using low oxygen atmosphere (5% O2). After 1-week exposure to CS (1, 10 and 100microg/ml), they were treated by recIL-1beta (10ng/ml) for 24 or 48h, in the presence of CS. RNA was extracted and used to determine, by quantitative reverse transcription-polymerase chain reaction, the steady-state levels of mRNAs encoding several genes related to anabolic, catabolic and inflammatory aspects. Glycosaminoglycan (GAG) synthesis was also assayed by 35S-sulfate incorporation. RESULTS: CS decreased IL-1beta-induced expression of matrix metalloproteases-1, -3 and -13 and aggrecanases-1 and -2. It slightly enhanced the aggrecan core protein mRNA and the GAG synthesis. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA levels were found to be reduced by CS treatment. However, no CS-induced decrease of NO was observed in IL-1beta-treated chondrocytes, whereas prostaglandin E2 production was diminished in correlation with the COX-2 mRNA amounts. Furthermore, CS was capable of counteracting IL-1beta-depressed expression of transforming growth factor-beta (TGF-beta) receptors. CONCLUSIONS: CS can repress expression of genes encoding proteolytic enzymes involved in cartilage degradation. It also inhibits IL-1beta-induced expression of the pro-inflammatory genes iNOS and COX-2 and restores TGF-beta receptors I and II (TGF-betaRI and RII) mRNA levels. These data suggest that CS may exert both chondroprotective and anti-inflammatory limited effects on articular cartilage that could have long-term beneficial action on the osteoarthritic process.

NFkappaB mediates IL-1beta-induced down-regulation of TbetaRII through the modulation of Sp3 expression.

We previously showed that interleukin-1beta (IL-1beta) down-regulation of type II TGFbeta receptor (TbetaRII) involves NFkappaB pathway and requires de novo synthesis of a yet unknown protein. Here, we demonstrate that this effect is mediated through Sp1 site located at position -25 of human TbetaRII promoter. Inhibition of transcription factors binding (decoy oligonucleotides or mithramycin) abolished IL-1beta effect. EMSA and ChIP revealed that this treatment induced Sp3 binding to cis-sequence whereby IL-1beta exerts its transcriptional effects whereas it decreased that of Sp1. Moreover, although the cytokine did not modulate Sp1 expression, it increased that of Sp3 via NFkappaB pathway. Experiments of gain and loss of function clearly showed that Sp3 inhibited TbetaRII expression whereas its silencing abolished IL-1beta effect. In addition, both Sp1 and Sp3 were found to interact with NFkappaB, which therefore may indirectly interact with TbetaRII pro moter. Altogether, these data suggest that IL-1beta decreases TbetaRII expression by inducing Sp3 via NFkappaB and its binding on core promote at the expense of Sp1, which could explain the loss of cell responsiveness in certain conditions. These findings bring new insights in the knowledge of the interference between two antagonistic transduction pathways involved in multiple physiopathological processes.

Interleukin-1beta impairment of transforming growth factor beta1 signaling by down-regulation of transforming growth factor beta receptor type II and up-regulation of Smad7 in human articular chondrocytes.

OBJECTIVE: Extracellular matrix deposition is tightly controlled by a network of regulatory cytokines. Among them, interleukin-1beta (IL-1beta) and transforming growth factor beta1 (TGFbeta1) have been shown to play antagonistic roles in tissue homeostasis. The purpose of this study was to determine the influence of IL-1beta on TGFbeta receptor type II (TGFbetaRII) regulation and TGFbeta1 responsiveness in human articular chondrocytes. METHODS: TGFbeta1-induced gene expression was analyzed through plasminogen activator inhibitor 1 and p3TP-Lux induction. Receptor-activated Smad (R-Smad) phosphorylation, TGFbeta receptors, and Smad expression were determined by Western blotting and real-time reverse transcription-polymerase chain reaction techniques. Signaling pathways were investigated using specific inhibitors, messenger RNA (mRNA) silencing, and expression vectors. RESULTS: IL-1beta down-regulated TGFbetaRII expression at both the protein and mRNA levels and led to inhibition of the TGFbeta1-induced gene expression and Smad2/3 phosphorylation. Moreover, IL-1beta strongly stimulated the expression of inhibitory Smad7. TGFbetaRII overexpression abolished the loss of TGFbeta1 responsiveness induced by IL-1beta. The decrease in TGFbetaRII required de novo protein synthesis and involved both the NF-kappaB and JNK pathways. CONCLUSION: We demonstrate that IL-1beta impairs TGFbeta1 signaling through down-regulation of TGFbetaRII, which is mediated by the p65/NF-kappaB and activator protein 1/JNK pathways, and secondarily through the up-regulation of Smad7. These findings show that there is cross-talk in the signaling of 2 regulatory cytokines involved in inflammation.