Nitric oxide regulates cartilage destruction by causing dedifferentiation and apoptosis of chondrocytes. We investigated the role of the mitogen-activated protein kinase subtypes, extracellular signal-regulated protein kinase (ERK)-1/2, and p38 kinase in NO-induced apoptosis of rabbit articular chondrocytes and their involvement in dedifferentiation. Generation of NO with sodium nitroprusside (SNP) caused dedifferentiation, as indicated by the inhibition of type II collagen expression and proteoglycan synthesis. NO additionally caused apoptosis, accompanied by p53 accumulation and caspase-3 activation. SNP treatment stimulated activation of ERK-1/2 and p38 kinase. Inhibition of ERK-1/2 with PD98059 rescued SNP-induced dedifferentiation but enhanced apoptosis up to 2-fold, whereas inhibition of p38 kinase with SB203580 enhanced dedifferentiation, with significant blockage of apoptosis. The stimulation of apoptosis by ERK inhibition was accompanied by increased p53 accumulation and caspase-3 activity, whereas the inhibitory effect of p38 kinase blockade was associated with reduced p53 accumulation and caspase-3 activity. Our results indicate that NO-induced p38 kinase functions as an induction signal for apoptosis and in the maintenance of chondrocyte phenotype, whereas ERK activity causes dedifferentiation and operates as an anti-apoptotic signal. NO generation is less proapoptotic in chondrocytes that are dedifferentiated by serial monolayer culture or phorbol ester treatment. NO-induced p38 kinase activity is low in dedifferentiated cells compared with that in differentiated chondrocytes, with lower levels of p53 accumulation and caspase-3 activity. Our findings collectively suggest that ERK-1/2 and p38 kinase oppositely regulate NO-induced apoptosis of chondrocytes, in association with p53 accumulation, caspase-3 activation, and differentiation status.

A putative Akt kinase phosphorylation site (⁶⁴ydRIRplSYp⁷³) was found in Rac1/CDC42 and Rho family proteins (RhoA, RhoB, RhoC, and RhoG). Phosphorylation of Rac1 by Akt kinase was assayed with recombinant Rac1 protein and the fluorescein-labeled Rac1 peptide. It was shown that the Rac1 peptide and the recombinant protein were phosphorylated by the activated recombinant Akt kinase and the lysate of SK-MEL28 cells, a human melanoma cell line. The phosphorylation of Rac1 inhibited its GTP-binding activity without any significant change in GTPase activity. Both the GTP-binding and GTPase activities of Rac1 S71A protein (with the serine residue to be phosphorylated replaced with alanine) were abolished regardless of the treatment of Akt kinase. Akt kinase activity and Rac1 peptide phosphorylation were down-regulated by the treatment of SK-MEL28 cells with wortmannin or LY294002 (a phosphoinositide 3-kinase inhibitor), but JNK/SAPK kinase activity was up-regulated. Thus, the results suggest that Akt kinase of the phosphoinositide 3-kinase signal transduction pathway phosphorylates serine 71 of Rac1 as one of its authentic substrates and modulates the Rac1 signal transduction pathway through phosphorylation.

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, although the events through which TNF-alpha induces ROS generation are not yet well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of Rac in that process, stable expression of Rac(Asn-17), a dominant negative Rac1 mutant, completely blocked TNF-alpha-induced ROS generation. To understand better the mediators downstream of Rac, we investigated the involvement of cytosolic phospholipase A(2) (cPLA(2)) activation and metabolism of the resultant arachidonic acid (AA) by 5-lipoxygenase (5-LO). TNF-alpha-induced ROS generation was blocked by inhibition of cPLA(2) or 5-LO, but not cyclooxygenase, suggesting that TNF-alpha-induced ROS generation is dependent on synthesis of AA and its subsequent metabolism to leukotrienes. Consistent with that hypothesis, TNF-alpha Rac-dependently stimulated endogenous production of leukotriene B(4) (LTB(4)), while exogenous application of LTB(4) increased levels of ROS. In contrast, application of leukotrienes C(4), D(4), and E(4) or prostaglandin E(2) had little effect. Our findings suggest that LTB(4) production by 5-LO is situated downstream of the Rac-cPLA(2) cascade, and we conclude that Rac, cPLA(2), and LTB(4) play pivotal roles in the ROS-generating cascade triggered by TNF-alpha.

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A₂ (cPLA₂). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B₄ (LTB₄), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.

Serum response factor (SRF) gene expression in avian embryonic muscle lineages plays a central role in activating alpha-actin gene activity. In early stage HH 6 avian embryos, SRF mRNA expression showed strong localization to the neural groove, primitive streak, lateral plate mesoderm, and Hensen's node, while distinct SRF expression was seen later in the neural folds and the somites by HH stage 8. SRF transcripts appeared in the precardiac splanchnic mesoderm in stage HH 9 embryos and was detected at higher levels in the myocardium, somites, and lateral mesoderm of HH 11 embryos. SRF antibody staining demonstrated significant SRF protein accumulation in the myocardium of the developing heart and the myotomal portion of somites. During primary myogenesis in culture, SRF transcripts and nuclear SRF protein content increased about 40-fold, as primary myoblasts withdrew from the cell cycle, reaching their highest levels prior to the upregulation of the skeletal alpha-actin gene. A dominant-negative SRF mutant, SRFpm1, which inhibited DNA binding, but not dimerization of monomeric SRF subunits, blocked transcriptional activation of a skeletal alpha-actin promoter-luciferase reporter gene during myogenesis. Transcriptional blockade was reversed by co-transfections of a wild-type SRF expression vector, but was not rescued by the expression of other myogenic factors, such as MyoD and Mef-2C. Thus, SRF displayed an embryonic expression pattern restricted primarily to striated muscle cell lineages, in which increased mass of nuclear SRF was obligatory for alpha-actin gene transcription.