The culture of chondrocytes is one of the most powerful tools for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate into fibroblasts when they are subcultured, which is a major problem. This protocol, involving primary cultures to limit dedifferentiation, describes two different methods for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes, both of which represent hyaline cartilage) from mice. Mice are of particular interest for cellular and molecular studies, as many tools suitable for use in mice are available. In addition, rapid development of transgenic and gene-targeted mice provides powerful instruments for biological studies. The protocol can be divided into four stages: isolation of cartilage (15 min per animal), isolation of chondrocytes (2 h extended overnight), seeding of chondrocytes (1 h 30 min) and growth in culture (6 d). To obtain confluency of chondrocytes using this protocol takes 7 d. Methods for phenotyping chondrocytes are also provided.

Our data agree with the hypothesis of a gene-nutrient interaction between MTHFR 677C-->T polymorphism and folate status that may confer a selective advantage of TT-homozygous genotype when dietary intake of folate is adequate, at least in the areas studied.

The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura‐2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays. ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1‐100 μM range (EC50= 4·8 μM). This effect was observed in only ≈40 % of the terminals. Interestingly, ATP, in the same range (EC50= 8·6 μM), evoked AVP, but no significant OT, release from these terminals. Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP‐induced responses. Pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP‐induced [Ca2+]i response. To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2‐methylthio‐ATP > ATP‐γ‐S > α,β‐methylene‐ATP > ADP. The compounds adenosine, AMP, β,γ‐methylene‐ATP and UTP were ineffective. The ATP‐dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence‐quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP‐induced [Ca2+]i increase and AVP release were independent of and additive to a K+‐induced response, in addition to being insensitive to Cd2+. The ATP‐induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage‐dependent Ca2+ channel. We propose that ATP, co‐released with neuropeptides, could act as a paracrine‐autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.

Objective. Microsomal prostaglandin E synthase 1 (mPGES-1) is the final enzyme of the cascade that produces prostaglandin E 2 (PGE 2 ), a key actor in arthritis. To study mPGES-1 synthesis in human cartilage and its regulation by interleukin-1␤ (IL-1␤), we used human cartilage and an immortalized human chondrocyte cell line. Furthermore, we investigated the signaling pathways involved in mPGES-1 expression.Methods. We used real-time quantitative reverse transcription-polymerase chain reaction, Northern blotting, and Western blotting to measure mPGES-1 messenger RNA (mRNA) and protein expression in human chondrocytes. PGE 2 production was measured by enzyme-linked immunosorbent assay.Results. Cartilage specimens from osteoarthritis (OA) patients contained far greater amounts of mPGES-1 and cyclooxygenase 2 (COX-2) mRNA than did normal cartilage. Incubation with IL-1␤ markedly increased mPGES-1 mRNA and protein in a dosedependent and time-dependent manner, in parallel with an increase in PGE 2 levels. Both PD98059, an ERK pathway inhibitor, and SB203580, a p38␣/␤ MAPK inhibitor, abolished the increases in mPGES-1 mRNA and protein in response to IL-1␤. The specific p38␣ MAPK inhibitor SC906 suppressed IL-1␤-induced COX-2 expression but not IL-1␤-induced mPGES-1 expression, suggesting preferential involvement of p38␤ MAPK in IL-1␤-induced mPGES-1 expression.Conclusion. This study is the first to show that mPGES-1 is stimulated in human chondrocytes by the proinflammatory cytokine IL-1␤ via activation of both ERK-1/2 and p38 MAPK in an isoform-specific manner. We postulate that mPGES-1 may be a novel target for OA therapy.Prostaglandin E 2 (PGE 2 ) plays an important role in cartilage metabolism. Its many effects on chondrocytes (for review, see ref. 1) include enhanced production of matrix metalloproteinase 3 (2), modulation of proteoglycan and collagen synthesis (2,3), and stimulation of chondrocyte apoptosis (4,5). The synovial fluid of patients with osteoarthritis (OA) and rheumatoid arthritis contains high concentrations of PGE 2 (4), and cartilage and chondrocytes from OA patients spontaneously release more PGE 2 than does normal cartilage (2,6,7). These results suggest that PGE 2 may be actively involved in cartilage breakdown in OA patients. PGE 2 is synthesized by the isoenzymes cyclooxygenase 1 (COX-1) and COX-2, both of which act on arachidonic acid to form the prostaglandin endoperoxide H 2 (PGH 2 ). The prostanoid synthase prostaglandin E synthase (PGES) produces PGE 2 from PGH 2 . Three

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Physiological regulations of energy balance and body weight imply highly adaptive mechanisms which match caloric intake to caloric expenditure. In the central nervous system, the regulation of appetite relies on complex neurocircuitry which disturbance may alter energy balance and result in anorexia or obesity. Deoxynivalenol (DON), a trichothecene, is one of the most abundant mycotoxins found on contaminated cereals and its stability during processing and cooking explains its widespread presence in human food. DON has been implicated in acute and chronic illnesses in both humans and farm animals including weight loss. Here, we provide the first demonstration that DON reduced feeding behavior and modified satiation and satiety by interfering with central neuronal networks dedicated to food intake regulation. Moreover, our results strongly suggest that during intoxication, DON reaches the brain where it modifies anorexigenic balance. In view of the widespread human exposure to DON, the present results may lead to reconsider the potential consequences of chronic DON consumption on human eating disorders.

The culture of chondrocytes is one of the most powerful tool for exploring the intracellular and molecular features of chondrocyte differentiation and activation. However, chondrocytes tend to dedifferentiate to fibroblasts when they are subcultured, which is a major problem. This chapter describes several protocols for culturing chondrocytes of different anatomical origins (articular and costal chondrocytes) from various species (humans, mice, rabbits, and cattle). All these protocols involve primary cultures in order to limit dedifferentiation. This chapter also describes a new protocol for culturing mouse articular chondrocytes.

Extracellular ATP is a pro-inflammatory mediator involved in the release of prostaglandin from articular chondrocytes, but little is known about its effects on intracellular signaling. ATP triggered the rapid release of prostaglandin E 2 (PGE 2 ) by acting on P2Y 2 receptors in rabbit articular chondrocytes. We have explored the signaling events involved in this synthesis. ATP significantly increased arachidonic acid production, which involved the activation of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) but not a secreted form of PLA 2 , as demonstrated by various PLA 2 inhibitors and translocation experiments. We also showed that ATP induced the phosphorylation of p38 and ERK1/2 mitogen-activatedprotein kinases (MAPKs). Both PD98059, an inhibitor of the ERK pathway, and SB203580, an inhibitor of p38 MAPK, completely inhibited the ATP-induced release of PGE 2 . Finally, dominant-negative plasmids encoding p38 and ERK transfected alone into the cells impaired the ATP-induced release of PGE 2 to about the same extent as both plasmids transfected together. These results suggest that PGE 2 production induced by ATP requires the activation of both ERK1/2 and p38 MAPKs. Thus, ATP acts via P2Y 2 -purine receptors to recruit cPLA 2 by activating both ERK1/2 and p38 MAPKs and stimulates the release of PGE 2 from articular chondrocytes.