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To investigate the effects of chitosan on the redifferentiation of dedifferentiated chondrocytes, we used chondrocytes obtained from a micromass culture system. Micromass cultures of chick wing bud mesenchymal cells yielded differentiated chondrocytes, but these dedifferentiated during serial monolayer subculture. When the dedifferentiated chondrocytes were cultured on chitosan membranes they regained the phenotype of differentiated chondrocytes. Expression of protein kinase Cα (PKCα) increased during chondrogenesis, decreased during dedifferentiation, and increased again during redifferentiation. Treatment of the cultures with phorbol 12- myristate 13-acetate (PMA) inhibited redifferentiation and down-regulated PKCα. In addition, the expression of p38 mitogen-activated protein (MAP) kinase increased during redifferentiation, and its inhibition suppressed redifferentiation. These findings establish a culture system for producing chondrocytes, point to a new role of chitosan in the redifferentiation of dedifferentiated chondrocytes, and show that PKCα and p38 MAP kinase activities are required for chondrocyte redifferentiation in this model system. [ABSTRACT FROM AUTHOR]

Human cord blood-derived mast cells (HCMC) grown in medium with serum and recombinant human stem cell factor (rhSCF) with or without interleukin (IL)-6 are less mature than human skin mast cells (HSMC). We found that e-kit-positive HCMC cultured for 8-10 weeks with rhSCF in serum-free medium became sensitive to basic secretatogues and expressed the serine protease, chymase, which is preferentially expressed in HSMC. The HCMC release β-hexosaminidase (β-HEX) within 1 min of stimulation with compound 48/80 or substance P, and release was suppressed by pertussis toxin. Approximately 34% of the HCMC in the serum-free culture stained positively with chymase antibody. Chymase and e-kit levels, and responsiveness to basic secretagogues, increased substantially after an additional 2 weeks in a serum-free environment with rhIL-6 and rhSCF. Moreover, FCεRI-dependent activation of the HCMC resulted in induction of cytokines and cyclooxygenase-2. These results show that HCMC can differentiate into a phenotype morphologically and functionally similar to HSMC if exposed to SCF in serum-free medium. [ABSTRACT FROM AUTHOR]

Secretory phospholipase A2 (sPLA2), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA2 increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA2 in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA2 decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1- MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA2-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H+-ATPase, sustained MMP-2 activation by sPLA2. The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA2. Expression of p85α and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA2-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA2 increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA2-promoted fibroblasts migration. [ABSTRACT FROM AUTHOR]

Macrophages are pivotal effector cells in the innate immune system. When microbial products bind to pathogen recognition receptors, macrophages are activated and release a broad array of mediators, such as cytokines, that orchestrate the inflammatory responses of the host. Phosphatidic acid (PA) has been implicated as an important metabolite of phospholipid biosynthesis and in membrane remodeling and has been further suggested to be a crucial second messenger in various cellular signaling events. Here we show that PA is an essential regulator of inflammatory response. Deleterious effects of PA are associated with the secretion of proinflammatory cytokines, such as tumor necrosis factor-α, interleukin-1β, interleukin-6, and the production of nitric oxide, prostaglandin E[sub 2], which are predominantly released by macrophage Raw264.7 cells. Furthermore, the administration of PA to mice increased the serum cytokine level. Moreover, direct or lipopolysaccharide-induced PA accumulation by macrophages led to the Akt-dependent activation of the mammalian target of rapamycin-p70 S6 kinase 1, a process required for the induction of inflammatory mediators. These findings demonstrate the importance of the role of PA in systemic inflammatory responses, and provide a potential usefulness as specific targets for the development of therapies. [ABSTRACT FROM AUTHOR]

The metabolism of arachidonic acid, in particular the generation of prostaglandins (PGs), has been proposed to play a key role in the regulation of labor. Moreover, several extracellular proteins have been reported to modulate PG synthesis in amnion cells. In this study, we found that lipid components dissolved in the amniotic fluid modulate PG synthesis in WISH human amnion cells and identified one of these components as a sphingosine 1-phosphate (S1P). WISH cells express several SIP receptors including S1P[sub 1], S1P[sub 2], and S1P[sub 3]. When WISH cells were stimulated with SIP, PGE[sub 2] synthesis increased in a concentration-dependent manner, showlng maximal activity at around 100 nM. S1P treatment also caused the up-regulation of cyclooxygenase-2 (COX-2) mRNA and protein, which was apparent within 3-12 h of stimulation. In terms of the intracellular signaling pathway of S1P-induced WISH cell activation, we found that SIP stimulated two kinds of MAPK, ERK, and p38 kinase. We examined the roles of these two MAPKs in S1P-induced COX-2 expression. S1P-induced COX-2 expression was blocked completely by PD-98059 but not by SB-203580, suggesting that ERK has a critical role in the process. Transfection of S1P[sub 1] or S1P[sub 3] but not of S1P[sub 2] antisense oligonucleotide inhibited S1P-induced COX-2 expression and PGE[sub 2] production in WISH cells, indicating the involvements of S1P[sub 1] and S1P[sub 3] in the processes. This study demonstrates the physiological role of S1P in amniotic fluid and its effect on the modulation of COX-2 expression and PGs synthesis in WISH cells. [ABSTRACT FROM AUTHOR]