Tumor necrosis factor-alpha augments matrix metalloproteinase-9 production in skeletal muscle cells through the activation of transforming growth factor-beta-activated kinase 1 (TAK1)-dependent signaling pathway.

We have investigated the effect of tumor necrosis factor-alpha (TNF-alpha) on the production of extracellular matrix-degrading proteases in skeletal muscles. Using microarray, quantitative PCR, Western blotting, and zymography, we found that TNF-alpha drastically increases the production of matrix metalloproteinase (MMP)-9 from C2C12 myotubes. In vivo administration of TNF-alpha in mice increased the transcript level of MMP-9 in skeletal muscle tissues. Although TNF-alpha activated all the three MAPKs (i.e. ERK1/2, JNK, and p38), inhibition of ERK1/2 or p38 but not JNK blunted the TNF-alpha-induced production of MMP-9 from myotubes. Inhibition of Akt also inhibited the TNF-alpha-induced production of MMP-9. TNF-alpha increased the activation of transcription factors NF-kappaB and AP-1 but not SP-1 in myotubes. Overexpression of a dominant negative inhibitor of NF-kappaB or AP-1 blocked the TNF-alpha-induced expression of MMP-9 in myotubes. Similarly, point mutations in AP-1- or NF-kappaB-binding sites in MMP-9 promoter inhibited the TNF-alpha-induced expression of a reporter gene. TNF-alpha increased the activity of transforming growth factor-beta-activating kinase-1 (TAK1). Furthermore, overexpression of a dominant negative mutant of TAK1 blocked the TNF-alpha-induced expression of MMP-9 and activation of NF-kappaB and AP-1. Our results also suggest that TNF-alpha induces MMP-9 expression in muscle cells through the recruitment of TRAF-2, Fas-associated protein with death domain, and TNF receptor-associated protein with death domain but not NIK or TRAF-6 proteins. We conclude that TAK1-mediated pathways are involved in TNF-alpha-induced MMP-9 production in skeletal muscle cells.

Costimulation requirements of induced murine systemic autoimmune disease.

Costimulation between T cells and APC is required for productive immune responses. A number of receptor/ligand pairs have been shown to mediate costimulation, including CD28/B7 molecules (CD80 and CD86), CD40/CD40 ligand (CD40L, CD154), and LFA-1 (CD18)/ICAM-1 (CD54). T-B cell costimulation also plays a significant role in autoimmune diseases such as systemic lupus erythematosus. Murine HgCl2-induced autoimmunity (mHgIA) is a T cell-dependent systemic autoimmune disease that shares a number of common pathogenic mechanisms with idiopathic lupus. In this report, the significance of costimulation in mHgIA is examined by attempting to induce disease in mice deficient in either CD40L, CD28, or ICAM-1. Unlike absence of ICAM-1, homozygous deficiencies in either CD40L or CD28 significantly reduced the development of mHgIA. CD40L displayed a gene dosage effect as heterozygous mice also showed reduction of autoantibody responses and immunopathology. Markers of T cell activation such as CD44 and CTLA-4 were associated with disease expression in wild-type and ICAM-1-deficient mice but not in CD40L- or CD28-deficient mice. Absence of CTLA-4 expression in CD40L-/- mice suggests that signaling via both CD28 and CD40L is important for T cell activation and subsequent autoimmunity in mHgIA. Attempts to circumvent the absence of CD40L by increasing CD28 signaling via agonistic Ab failed to elicit CTLA-4 expression. These findings indicate that breaking of self-tolerance in mHgIA requires signaling via both the CD28/B7 and CD40/CD40L pathways.

Cadmium-induced damage to primary cultures of rat Leydig cells.

The mechanism of testicular toxicity of cadmium is poorly understood. Previous studies focusing on cadmium-related changes in testicular histopathology have implicated testicular blood vessel damage as the main cause of cadmium toxicity. To further explore the toxic effects of cadmium on testis, we isolated and cultured rat Leydig cells, exposed to 10, 20, and 40 microM of cadmium chloride (base doses). After 24 h of exposure, cells and supernatants were harvested to examine cytotoxicity and genotoxicity of cadmium. The results show that both cell viability and concentration of testosterone excretion in primary Leydig cells are significantly lower in cadmium-exposed groups compared to the controls. Changes in testosterone excretion with human chorionic gonadotropin (hCG) stimulation is especially profound. The contents of malondialdehyde (MDA) and the activity of glutathione peroxidase (GSH-Px) in exposed groups are significantly higher than those in the control group, but the activity of superoxide dismutase (SOD) is lower. The number of cells with DNA single strand breaks and the levels of cellular DNA damage in all three exposure groups are significantly higher than in controls. These results indicate that cadmium is directly toxic to primary Leydig cells, and that the decreased percentage of normal cells and the increased level of DNA damage in cadmium-exposed Leydig cells may be responsible for decreased testosterone secretion.