Activation of the nuclear factor kappa B pathway following ischemia-reperfusion of the murine testis.
Ischemia-reperfusion (IR) of the testis results in testicular oxidative stress and germ cell-specific apoptosis. Nuclear factor kappa B (NF-kappaB) is a nuclear transcription factor involved in the control of a number of cellular processes, and its activation is part of the cellular stress response to a variety of factors including cytokine stimulation, irradiation, and IR. The present study investigates NF-kappaB activation after IR of the murine testis and potential downstream target genes of that activation. Mice were subjected to a period of testicular ischemia followed by 0-4 hours of reperfusion. Activation of NF-kappaB was assessed by 1) Western blot analysis of the NF-kappaB inhibitory protein, IkappaBalpha; 2) immunohistochemistry for IkappaBalpha; and 3) TranSignal NF-kappaB target gene array (107 genes) analysis. Results demonstrate that IkappaBalpha is phosphorylated on serine 32 reaching a peak by 2 hours after IR of the testis. A decrease in total IkappaBalpha was also noted at 2 hours after IR, consistent with the rapid degradation of the phosphorylated protein. Phosphorylation and degradation of IkappaBalpha is indicative of NF-kappaB activation. Immunolocalization revealed IkappaBalpha specifically in Sertoli cells of the murine testis. Results of the TranSignal target gene array revealed that the expression of 9 genes was consistently changed 2 hours after IR of the testis, 3 of which increased in expression and 6 of which were down-regulated. Most notably, high-mobility group nucleosomal binding domain 1 increased in expression while platelet-derived growth factor B and Wilms tumor homolog decreased. These results suggest that testicular IR releases the suppression of NF-kappaB by IkappaBalpha in Sertoli cells. Activation of the NF-kappaB pathway in the testis resulted in an alteration of expression of potential NF-kappaB target genes, some increased while others decreased. The specific roles of these genes in the testicular response to IR remains to be determined.
Lysiak
Jeffrey J
JJ
Department of Urology Box 800422, University of Virginia Health System, Charlottesville, VA 22908, USA. jl6n@virginia.edu
Bang
Hyun J
HJ
Nguyen
Quoc An T
QA
Turner
Terry T
TT
D000818:Animals; D015153:Blotting, Western; D015870:Gene Expression; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation; D015427:Reperfusion Injury / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013737:Testis / Q000473:pathology / Q000502:physiology*
D016328:NF-kappa B