Aristolochic Acid I Induced Autophagy Extenuates Cell Apoptosis via ERK 1/2 Pathway in Renal Tubular Epithelial Cells

Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. However, limited information is available about autophagy in aristolochic acid (AA) nephropathy. In this study, we investigated the role of autophagy and related signaling pathway during progression of AAI-induced injury to renal tubular epithelial cells (NRK52E cells). The results showed that autophagy in NRK52E cells was detected as early as 3–6 hrs after low dose of AAI (10 µM) exposure as indicated by an up-regulated expression of LC3-II and Beclin 1 proteins. The appearance of AAI-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in NRK52E cells provided further evidence for autophagy. However, cell apoptosis was not detected until 12 hrs after AAI treatment. Blockade of autophagy with Wortmannin or 3-Methyladenine (two inhibitors of phosphoinositede 3-kinases) or small-interfering RNA knockdown of Beclin 1 or Atg7 sensitized the tubular cells to apoptosis. Treatment of NRK52E cells with AAI caused a time-dependent increase in extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity, but not c-Jun N-terminal kinase (JNK) and p38. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased AAI-induced autophagy that was accompanied by an increased apoptosis. Taken together, our study demonstrated for the first time that autophagy occurred earlier than apoptosis during AAI-induced tubular epithelial cell injury. Autophagy induced by AAI via ERK1/2 pathway might attenuate apoptosis, which may provide a protective mechanism for cell survival under AAI-induced pathological condition

The role of RelA (p65) threonine 505 phosphorylation in the regulation of cell growth, survival, and migration

The NF-κB family of transcription factors is a well-established regulator of the immune and inflammatory responses and also plays a key role in other cellular processes, including cell death, proliferation, and migration. Conserved residues in the trans-activation domain of RelA, which can be posttranslationally modified, regulate divergent NF-κB functions in response to different cellular stimuli. Using rela(−/−) mouse embryonic fibroblasts reconstituted with RelA, we find that mutation of the threonine 505 (T505) phospho site to alanine has wide-ranging effects on NF-κB function. These include previously described effects on chemotherapeutic drug-induced apoptosis, as well as new roles for this modification in autophagy, cell proliferation, and migration. This last effect was associated with alterations in the actin cytoskeleton and expression of cellular migration–associated genes such as WAVE3 and α-actinin 4. We also define a new component of cisplatin-induced, RelA T505–dependent apoptosis, involving induction of NOXA gene expression, an effect explained at least in part through induction of the p53 homologue, p73. Therefore, in contrast to other RelA phosphorylation events, which positively regulate NF-κB function, we identified RelA T505 phosphorylation as a negative regulator of its ability to induce diverse cellular processes such as apoptosis, autophagy, proliferation, and migration