Growth inhibition and apoptosis induction by tumor necrosis factor-α in human urethral rhabdosphincter satellite cells

Abstract

Purpose: A decrease in the human urethral rhabdosphincter is reported with aging due to apoptosis, which may be a cause of urinary incontinence in the elderly population. To explore this mechanism we investigated the effects of tumor necrosis factor-alpha (Upstate, Temecula, California) on human urethral rhabdosphincter satellite cells.

Materials and methods: Human urethral rhabdosphincter satellite cells were cultured and selected by magnetic affinity cell sorting, extended their life span. Apoptosis induction was examined by flow cytometry and immunocytochemistry. Caspase cascade activation was determined by Western blot analysis. After tumor necrosis factor receptor expression was confirmed we determined the tumor necrosis factor signaling pathway.

Results: Tumor necrosis factor-alpha inhibited human urethral rhabdosphincter satellite cell proliferation. It caused some cells to stain positive for annexin V-fluorescein isothiocyanate but not for propidium iodide, suggesting the induction of early phase apoptosis. Flow cytometry revealed an increased sub-G1 fraction. Western blot analysis showed activation of caspase-8 and 3, and cleavage of poly (adenosine diphosphate-ribose) polymerase. Tumor necrosis factor receptor expression at the mRNA and protein levels was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. IkappaBalpha phosphorylation was noted within 2 to 5 minutes after tumor necrosis factor-alpha treatment. The tumor necrosis factor-alpha antagonist etanercept (Wyeth, Collegeville, Pennsylvania) inhibited IkappaBalpha activation and reversed tumor necrosis factor-alpha effects on human urethral rhabdosphincter satellite cells.

Conclusions: Since tumor necrosis factor-alpha induces growth inhibition and apoptosis of human urethral rhabdosphincter satellite cells via tumor necrosis factor receptor activation, it may be involved in age related decreases in the number of human urethral rhabdosphincter cells and be a causative factor for urinary incontinence in the elderly population.