Tony F. Cruz

The cytokine tumor necrosis factor alpha (TNF alpha) and the growth factor basic fibroblast growth factor (bFGF) are known to induce early response genes such as c-fos and c-jun in various cell types. Activation of AP-1, a heterodimeric complex of Fos and Jun proteins, is required for matrix metalloproteinase production and cell proliferation. However, the signaling pathways by which these two factors influence the expression and activities of AP-1 remain currently poorly characterized. Several studies have shown that cytokines induce reactive oxygen species (ROS) production, but growth factor induction of ROS has not been reported. In the present study we demonstrate that both TNF alpha and bFGF induce ROS production, and that this is a common signaling event involved in the stimulation of c-fos gene expression in chondrocytes. To our knowledge, this is the first report directly demonstrating ROS production upon stimulation with a growth factor. TNF alpha and bFGF induction of ROS production is mediated through flavonoid-containing enzymes such as NADPH oxidase. Moreover, the ROS nitric oxide is not responsible for the induction of c-fos expression by TNF alpha and bFGF. In addition, the inhibitory effects of antioxidants on c-fos expression may account for their protective roles against proliferative and inflammatory diseases such as cancer, cardiovascular diseases, and arthritis.

Interleukin 1 (IL-1) and tumor necrosis factor α (TNFα) are known to induce production of reactive oxygen species (ROS), which have been suggested to act as second messengers. Here we demonstrate that ROS production by bovine chondrocytes upon cytokine stimulation induces c-jun expression. Since c-jun expression is regulated by its own gene product via phosphorylation by c-Jun NH2-terminal kinases (JNKs), we investigated if cytokines and ROS could modulate JNK activity in chondrocyte monolayer cultures. Treatment of bovine chondrocytes with both IL-1 and TNFα leads to rapid induction of JNK activity, stimulating JNK activity 7- and 20-fold, respectively. Importantly, the observation that antioxidant treatment antagonizes IL-1 and TNFα activation of JNK provides strong evidence that ROS can act as mediators of JNK activity. Moreover, potent activation of JNK is also observed by direct addition of the ROS hydrogen peroxide (H2O2) to the chondrocyte cultures. Nitric oxide (NO), a multifunctional ROS, also appears to simulate JNK, albeit to a lesser extent. These findings identify JNK as another molecular target for the actions of NO and H2O2. In addition, the inhibitory effect of diphenyleneiodonium on JNK activation implicates the involvement of flavonoid-containing enzymes in the ROS-mediated signaling process. Overstimulation of JNK activity by excessive production of ROS may, therefore, underlie pathological conditions such as arthritis and cancer. Interleukin 1 (IL-1) and tumor necrosis factor α (TNFα) are known to induce production of reactive oxygen species (ROS), which have been suggested to act as second messengers. Here we demonstrate that ROS production by bovine chondrocytes upon cytokine stimulation induces c-jun expression. Since c-jun expression is regulated by its own gene product via phosphorylation by c-Jun NH2-terminal kinases (JNKs), we investigated if cytokines and ROS could modulate JNK activity in chondrocyte monolayer cultures. Treatment of bovine chondrocytes with both IL-1 and TNFα leads to rapid induction of JNK activity, stimulating JNK activity 7- and 20-fold, respectively. Importantly, the observation that antioxidant treatment antagonizes IL-1 and TNFα activation of JNK provides strong evidence that ROS can act as mediators of JNK activity. Moreover, potent activation of JNK is also observed by direct addition of the ROS hydrogen peroxide (H2O2) to the chondrocyte cultures. Nitric oxide (NO), a multifunctional ROS, also appears to simulate JNK, albeit to a lesser extent. These findings identify JNK as another molecular target for the actions of NO and H2O2. In addition, the inhibitory effect of diphenyleneiodonium on JNK activation implicates the involvement of flavonoid-containing enzymes in the ROS-mediated signaling process. Overstimulation of JNK activity by excessive production of ROS may, therefore, underlie pathological conditions such as arthritis and cancer.

Myocyte enhancer factor 2 (MEF2) has been implicated in the complex hierarchical regulation of muscle-specific gene expression and differentiation. While the MyoD family members are able to initiate the skeletal muscle differentiation program, whether MEF2 is sufficient in directing skeletal muscle differentiation is still controversial. Furthermore, how MEF2 transactivates its target genes is not fully understood. It has been suggested that the interactions of MEF2 with other factors modify its transcriptional activity. Therefore, the identification of MEF2-interacting factors may be important in understanding the mechanism by which MEF2 activates its target genes. In this study, a mitogen-activated protein kinase (MAP kinase), ERK5/BMK1 was found to interact with MEF2 in a yeast two hybrid screen. The interaction was confirmed by a glutathione S -transferase-pull down assay and a co-immunoprecipitation study indicating that endogenous ERK5 and MEF2 interact with each other in vivo . The interacting domain of MEF2 was mapped to the N-terminus which contains the highly conserved MADS and MEF2 domains. Functionally, ERK5/BMK1 was able to phosphorylate MEF2 in vitro . Furthermore, when cotransfected with ERK5/BMK1, the transactivation capacity of MEF2 was enhanced. These results suggest that the functions of MEF2 could be regulated through ERK5/BMK1.

Interleukin-1 beta (IL-1) is implicated in cartilage destruction in arthritis through promotion of matrix metalloproteinase production. Upregulation of collagenase gene expression by IL-1 is known to require the transactivators Fos and Jun. Recently, reactive oxygen species (ROS) have been suggested to act as intracellular signaling molecules mediating the biological effects of cytokines. Here, we demonstrated ROS production by IL-1-stimulated bovine chondrocytes and that neutralizing ROS activity by the potent antioxidant, N-acetylcysteine, or inhibiting endogenous ROS production by diphenyleneiodonium (DPI), significantly attenuated IL-1-induced c-fos and collagenase gene expression. The inhibitory effect of DPI implicates enzymes such as NADPH oxidase in the endogenous production of ROS. Chondrocytes were also found to produce nitric oxide (NO) upon IL-1 stimulation. That NO may mediate part of the inducing effects of IL-1 was supported by the observation that L-NG-monomethylarginine, a NO synthase inhibitor, partially inhibited IL-1-regulated collagenase expression. Moreover, treatment of chondrocytes with the NO-producing agent, S-nitroso-N-acetylpenicillamine, was sufficient to induce collagenase mRNA levels. In summary, our results suggest that ROS released in response to IL-1 may function as second messengers transducing extracellular stimuli to their targets in the nucleus, leading to augmentation of gene expression.

The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that of lymphocytes to endothelium. The CD44 antigen, reactive with monoclonal antibody (MAb) 44D10, has been shown previously to be expressed in normal human white matter homogenates and to be found at higher concentrations in brain homogenates of victims of multiple sclerosis (MS). The cellular localization of CD44 in human brain of normal individuals and in those afflicted with MS has now been determined. Monoclonal antibody 44D10 reacted with astrocyte-like cells in 40 microns thick paraformaldehyde-fixed sections but not in thin (6 microns) fixed sections. A double labeling experiment performed on a frozen brain section with MAb 44D10 and rabbit anti-glial fibrillary acidic protein (GFAP), a cytoplasmic marker of astrocytes, confirmed the co-localization of these two antigens. The reactivity with brain tissue sections of a rabbit antiserum produced against lymphocyte-CD44 could be absorbed by a preparation of the CD44 glycoprotein, purified 2,100-fold from a white matter homogenate. The antiserum was shown by Western blot analysis to be specific for p80 glycoprotein in brain extracts derived from a normal and MS patients. This antibody reacted with fibrous astrocytes predominantly in white matter; staining was also noted in subependymal and subpial regions. Inhibition studies using a cellular radioimmunoassay indicated that the highest concentrations of CD44 in three MS victims were found in plaques, followed by periplaques and non-involved areas of white matter which were higher than normal white matter. Reactive astrocytes, identified in active lesions, expressed high levels of CD44 on their surfaces. Thus, CD44 is associated with astrocytes in human brain and the increased expression observed in MS brain may reflect activation and/or proliferation of astrocytes implicated in the pathogenesis of this disease.