The present studies were performed to determine subtype-specific roles of mitogen-activated protein kinase in chondrogenesis. Erk-1/2 activities, downstream of protein kinase C, decreased as chondrogenesis proceeded, whereas p38 activities, independent of protein kinase C, continuously increased during chondrogenesis. Inhibition of Erk-1/2 with PD98059 enhanced chondrogenesis up to 1.7-fold, whereas inhibition of p38 with SB203580 reduced it to about 30% of the control level. Inhibition of Erk-1/2 or p38 did not affect precartilage condensation. However, cartilage nodule formation was significantly blocked by the inhibition of p38, whereas Erk-1/2 inhibition did not affect it. Modulation of chondrogenesis by the inhibition of Erk-1/2 and p38 was accompanied by altered expression of adhesion molecules in an opposite way. Expression of N-cadherin was reduced as chondrogenesis proceeded. Inhibition of p38 caused sustained expression of N-cadherin, whereas Erk-1/2 inhibition accelerated the reduction of N-cadherin expression. Expression of integrin ␣5␤1 and fibronectin were found to transiently increase during chondrogenesis. Inhibition of p38 caused continuous increase of expression of these molecules, whereas Erk-1/2 inhibition accelerated the decrease of expression of these molecules at a later period of chondrogenesis. Because temporal expression of these adhesion molecules regulates chondrogenesis, the above results indicate that Erk-1/2 and p38 conversely regulate chondrogenesis at post-precartilage condensation stages by modulating expression of adhesion molecules.Formation of cartilage is initiated by differentiation of mesenchymes into chondrocytes. The differentiation process requires proliferation of chondrogenic competent cells that subsequently undergo precartilage condensation (1-3). Precartilage condensation is characterized by cells that are more densely packed in specific regions. This precartilage condensation is an important prerequisite for the initiation of chondrogenesis in mesenchymes in vivo. It becomes evident that several adhesion molecules including cadherins (4-7), integrins (8, 9), and extracellular matrix (ECM) 1 components (9-12) regulate precartilage condensation. Limb mesenchymes undergo spontaneous differentiation to chondrocytes in vitro when cultured at a high seeding density such as micromass culture in the presence of serum. Precartilage condensation similar to that formed in vivo during chondrogenesis occurs in micromass cultures of limb mesenchymes (13). The cellular aggregates that form in early micromass cultures subsequently differentiate into cartilage nodules, which express cartilage-specific molecules such as type II collagen (6, 14-16). However, the signal transduction pathways involved in precartilage condensation and initiated by condensation are not well characterized.We have previously shown that protein kinase C (PKC) positively regulates chondrogenesis of mesenchymes (17,18). PKC is a multigene family composed of 11 known isoforms (19,20). Multiple PKC isoforms su...

Bacteroides fragilis produces an approximately 20-kDa heat-labile toxin (B. fragilis enterotoxin, BFT) which is known to be associated with diarrhea. To determine whether cyclooxygenase (COX)-2, via NF-jB activation, can contribute to BFT-induced diarrhea, the relationship between COX-2 expression and fluid secretion in BFT-stimulated human intestinal epithelial cells was examined. BFT stimulation increased the expression of COX-2, but not COX-1, in human intestinal epithelial cells. Suppression of the NF-jB signal significantly decreased COX-2 expression in response to BFT stimulation. Prostaglandin E 2 (PGE 2 ) levels were increased in parallel with COX-2 expression, and, conversely, PGE 2 production was significantly inhibited when COX-2 or NF-jB activities were suppressed using COX-2 small interfering RNA (siRNA), p65 NF-jB subunit siRNA, or a retrovirus encoding the IjBa superrepressor. In addition, a selective COX-2 inhibitor, NS-398, significantly inhibited the increased cAMP level induced by BFT stimulation. Furthermore, a selective COX-2 inhibitor prevented BFTinduced PGE 2 production and ileal fluid secretion in a mouse ileal loop model. These results suggest that the secretory response to BFT stimulation may be mediated by the production of PGE 2 , through NF-jB activation and the up-regulation of COX-2 in intestinal epithelial cells. IntroductionEnterotoxigenic Bacteroides fragilis (ETBF) has been shown to be associated with non-invasive diarrheal diseases in animals and young children [1][2][3][4][5], and B. fragilis enterotoxin (BFT), an approximately 20-kDa heat-labile metalloprotease, is regarded as a virulence factor for this diarrheal disease. BFT stimulates NF-jB activation and IL-8 secretion [6][7][8][9], which is predicted to lead to mucosal transmigration of neutrophils with release of prostaglandins (PG). In addition, animal loop experiments and gnotobiotic piglet infection indicate that BFT is proinflammatory and BFT stimulates a brief chloride current and a loss of barrier function in vitro [3,10], suggesting that these findings are potential mechanisms accounting for ETBF-induced diarrhea. Several studies have shown that secretion of PG from intestinal mucosal epithelial cells is closely associated with diarrheal diseases [11,12], and administration of PG analogues, such as PGE 2 , causes increased fluid secretion in human subjects [13,14]. In addition, the roles of PG in the secretory response to cholera toxin and enterotoxigenic Escherichia coli have been demonstrated [15][16][17]. These results suggest the hypothesis that the diarrhea associated with ETBF may be PG-derived. PGE 2 is a metabolite of arachidonic acid and is synthesized by cyclooxygenase (COX). There are two isoforms of this enzyme [18]: COX-1, which is constitutively expressed in crypt epithelial cells, and COX-2, which can be induced in a variety of cell types, including epithelial cells, macrophages and fibroblasts. COX-2 is induced by proinflammatory cytokines, lipopolysaccharide and infectious agents [19]. The expr...

Intestinal epithelial cells are known to upregulate the expression of several chemokines in response to stimulation with bacterial toxin. However, the cellular mechanisms of Clostridium difficile toxin A-induced mucosal inflammation have not yet been fully elucidated. In this study, we investigated whether nuclear factor-kappa B (NF-kB) could regulate chemokine expression in intestinal epithelial cells. Toxin A increased the levels of NF-kB complexes containing p65/p50 heterodimers and p65/p65 homodimers. Concurrently, toxin A decreased the levels of IkBa. Toxin A stimulation also increased the signals of phosphorylated IkB kinase (IKK)a/b and NF-kB-inducing kinase (NIK). In the toxin A-stimulated HT-29 cells, the suppression of IKK or NIK inhibited the upregulation of downstream target genes of NF-kB such as IL-8 and monocytechemotactic protein (MCP)-1 and similarly, inhibition of NF-kB also downregulated the expression of IL-8, growth-related oncogene-a, and MCP-1. These results suggest that NF-kB signalling events may be involved in the inflammatory responses to toxin A produced by toxigenic C. difficile.

Enterotoxigenic Bacteroides fragilis (ETBF) produces an approximately 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in mucosal inflammation. Although spontaneous disappearance of ETBF infection is common, little information is available on regulated expression of antibacterial factors in response to BFT stimulation. This study investigates the role of BFT in human β-defensin 2 (hBD-2) induction from intestinal epithelial cells. Stimulation of HT-29 and Caco-2 intestinal epithelial cell lines with BFT resulted in the induction of hBD-2. Activation of a reporter gene for hBD-2 was dependent on the presence of NF-κB binding sites. In contrast, suppression of AP-1 did not affect hBD-2 expression in BFT-stimulated cells. Inhibition of p38 mitogen-activated protein kinase (MAPK) using SB203580 and small interfering RNA (siRNA) transfection resulted in a significant reduction in BFT-induced IκB kinase (IKK)/NF-κB activation and hBD-2 expression. Our results suggest that a pathway including p38 MAPK, IKK, and NF-κB activation is required for hBD-2 induction in intestinal epithelial cells exposed to BFT, and may be involved in the host defense following infection with ETBF.

Targeted delivery of self-antigens to the immune system in a mode that stimulates a tolerance-inducing pathway has proven difficult. To address this hurdle, we developed a vaccine based-approach comprised of two synthetic controlled-release biomaterials, poly(lactide-co-glycolide; PLGA) microparticles (MPs) encapsulating denatured insulin (key self-antigen in type 1 diabetes; T1D), and PuraMatrixTM peptide hydrogel containing granulocyte macrophage colony-stimulating factor (GM-CSF) and CpG ODN1826 (CpG), which were included as vaccine adjuvants to recruit and activate immune cells. Although CpG is normally considered pro-inflammatory, it also has anti-inflammatory effects, including enhancing IL-10 production. Three subcutaneous administrations of this hydrogel (GM-CSF/CpG)/insulin-MP vaccine protected 40% of NOD mice from T1D. In contrast, all control mice became diabetic. In vitro studies indicate CpG stimulation increased IL-10 production, as a potential mechanism. Multiple subcutaneous injections of the insulin containing formulation resulted in formation of granulomas, which resolved by 28 weeks. Histological analysis of these granulomas indicated infiltration of a diverse cadre of immune cells, with characteristics reminiscent of a tertiary lymphoid organ, suggesting the creation of a microenvironment to recruit and educate immune cells. These results demonstrate the feasibility of this injectable hydrogel/MP based vaccine system to prevent T1D.