Expression of antioxidant molecules after peripheral nerve injury and regeneration.

Oxidative stress is considered to be one of the main causes of neural damage after injury. However, little is known about the changes in mRNA expression of antioxidant molecules that occur after injury and regeneration of the peripheral nerve. In the present study, the rat median nerve was transected, and transcriptional changes were studied at day 6 and day 12 after injury in both the proximal and the distal stumps, in the absence or presence of microsurgical repair. The expression profiles of the following genes were investigated: three metallothionein isoforms (MT-1, MT-2, and MT-3), the main antioxidant enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), and the marker of cellular damage poly(ADP-ribose) polymerase-1 (PARP-1). The results showed that, in the proximal nerve stump, MT-3 mRNA expression was significantly and markedly up-regulated in the absence of surgical repair, whereas MT-1 and MT-2 showed significant down-regulation. In the distal nerve portion, mRNA expression of all MT isoforms decreased significantly in the absence of microsurgical reconstruction, whereas, after repair, MT-3 mRNA expression alone was up-regulated. Expression of all the antioxidant enzymes decreased significantly after repair in the proximal nerve portion, but a significant general increase in their mRNA expression was revealed in the distal nerve stump. PARP-1 expression was significantly up-regulated in the proximal nerve portion without repair but dramatically reduced after reconstruction. In contrast, PARP-1 expression increased markedly in the distal stump after surgical repair. Taken together, these findings indicate that antioxidant molecules are differentially modulated and might, therefore, play an important role in peripheral nerve injury and regeneration.

Expression of antioxidant defense and poly(ADP-ribose) polymerase-1 in rat developing Sertoli cells.

Sertoli cells play an essential role in the development of a functional testis. ROS (reactive oxygen species) are normally produced by the developing testicular cells and may be dangerous to spermatogenesis. The aim of this study was to investigate the developmental expression of genes involved in antioxidant defense as well as in the DNA damage response in rat Sertoli cells. As revealed by quantitative RT-PCR analysis, the expression pattern of the antioxidant enzymes GST (glutathione-S-transferase), CAT (catalase) and SOD (superoxide dismutase) showed a progressive decrease from birth to puberty. The expression level of the oncosuppressor p53 revealed a net reduction as well. We next focused on PARP-1 [poly(ADP-ribose) polymerase-1], a 'guardian of the genome' that combats stress conditions. At both the mRNA and protein level, PARP-1 expression was low at the early stage of development and increased later on. Maximal PARP-1 expression was preceded by a rise in the transcript level for MTs (metallothioneins), which provide zinc to zinc-dependent enzymes and proteins, including PARP-1. Our results showed an increased expression of PARP-1 during Sertoli cell development, together with a decrease in the expression of antioxidant enzymes. In conclusion, a role of PARP-1 in protecting the testicular differentiation is suggested.

Neuroprotective mesenchymal stem cells are endowed with a potent antioxidant effect in vivo.

Experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis, is characterized by demyelination, inflammation and neurodegeneration of CNS in which free radicals play a role. Recently, the efficacy of murine mesenchimal stem cells (MSCs) as treatment of EAE induced in mice by the encephalitogenic peptide MOG(35-55) was demonstrated. The present study analyzed some markers of oxidative stress, inflammation/degeneration and apoptosis such as metallothioneins (MTs), antioxidant enzymes (superoxide dismutase, catalase and glutathione-S-transferase), poly(ADP-ribose) polymerase-1 and p53 during EAE progression and following MSC treatment. Expression of the three brain MT isoforms increased significantly in EAE mice compared with healthy controls, but while expression of MT-1 and MT-3 increased along EAE course, MT-2 was up-regulated at the onset, but returned to levels similar to those of controls in chronic phase. The changes in the transcription and activity of the antioxidant enzymes and in expression of poly(ADP-ribose) polymerase-1 and p53 showed the same kinetics observed for MT-1 and MT-3 during EAE. Interestingly, i.v. administration of MSCs reduced the EAE-induced increases in levels/activities of all these proteins. These results support an antioxidant and neuroprotective activity for MSCs that was also confirmed in vitro on neuroblastoma cells exposed to an oxidative insult.

Heavy metal and growth hormone pathways in metallothionein regulation in fish RTH-149 cell line.

Interference between heavy metals and growth hormone (GH) on cell signaling has been previously demonstrated in fish cells. This study was aimed at assessing their effects on expression of the metallothionein isoforms MT-A and MT-B. The results indicate that all heavy metals induce MT-A more markedly than MT-B, but differences appeared when metals were combined with GH. For MT-B induction, a positive interference between metals and GH was observed for Zn(2+)/GH and Cd(2+)/GH, a negative interference for Hg(2+)/GH. With regards to MT-A, no interference was observed for Zn(2+)/GH and Hg(2+)/GH, while a negative interference occurred with Cu(2+)/GH and a positive interference with Cd(2+)/GH. The possible mechanisms underlying the differential regulation of metallothioneins include different signaling pathways. The results show that STAT5 and ERKs responded differently to different combinations, and Zn(2+)/GH and Cd(2+)/GH exerted a slight positive interference on ERK activation. On the other hand, a synergic rise in [Ca(2+)](i) occurred for all combinations except for Cu(2+)/GH. Our data suggest that the cross-talk between heavy metals and GH resulting in MT transcription modulation does not strictly depend on Ca(2+) signalling; (ii)ERK activation may represent the point of cross-talk between Zn(2+) or Cd(2+) and GH, converging on MT-B transcription, probably through a differential recruitment of transcription factors.

Efects of growth hormone and cadmium on the transcription regulation of two metallothionein isoforms.

The effect of growth hormone (GH) and cadmium (Cd) on metallothionein (MT) expression was investigated in hepatoma cells. In fish the constitutive isoform MT-B and the metal-responsive MT-A are expressed. Real-time RT-PCR revealed that: Cd up-regulates mostly MT-A, GH slightly induces MT-B and the GH/Cd combination induces synergistically both MTs. Perturbations in Ca2+ levels suppressed or reduced the Cd-induction of MTs and abolished the GH/Cd synergy. Similar results were obtained by inhibition of tyrosine kinases. Also the signaling molecules recruited by the GH receptor responded differently to GH and Cd, with ERKs showing a synergistic activation upon GH/Cd. The following conclusions can be drawn: (1) cytosolic Ca2+ is mainly involved in MT-A regulation; (2) both Ca2+ and tyrosine phosphorylation are essential for Cd-induction and GH/Cd synergy on MTs. The synergy could depend on interactions in different signaling pathways, leading to a differential recruitment of MTF-1 and AP-1 transcription factors.