Poster abstracts

Poster number 63 submitted by Mamata Singh

Regulation of Anti-apoptotic mRNAs by HuR during Stress-induced Apoptosis

Mamata Singh (Physiology and cell biology), Beth S. Lee (Physiology and cell biology)

Abstract:
HuR is a ubiquitous RNA-binding protein that resides primarily in nuclei under normal conditions. Under stress, HuR translocates to the cytoplasm, where it binds and stabilizes a subset of mRNAs bearing adenine and uridine rich sequences in their 3’ untranslated regions. We previously showed that in ATP-depleted proximal tubule cells and rat proximal tubules subjected to ischemia-reperfusion injury, HuR not only translocated from nucleus to cytoplasm, but its protein levels increased. Also, we confirmed an anti-apoptotic role for HuR in cultured proximal tubule cells, suggestive of its protective function in native proximal tubules against ischemic stress. Here we used PCR array analysis to determine how suppression of HuR levels might affect expression of mRNAs within apoptotic pathways. siRNA- or control-treated proximal tubule cells were ATP-depleted for 6 hours and subjected to PCR array analysis. mRNAs with at least 2-fold increase or decrease in response to HuR knockdown were taken into consideration.
The mRNAs that were downregulated in siRNA-treated cells under ATP depletion conditions were transcripts corresponding primarily to anti-apoptotic proteins such as Akt, Bcl-2 family, Mcl-1, NAIP, NOD-1, and BFAR. The role of Akt in our model was confirmed by Western analysis in which we found its active phosphorylated state to be increased during ATP depletion. Further, inhibition of the PI3K/Akt pathway with PI3K inhibitor LY294002 increased apoptosis during this stress. Also, the down-regulation of most of anti-apoptotic protein members of PI3/Akt pathway during Apoptosis PCR Array was noticed. In the light of above results, PI3K/Akt pathway was studied using PCR array technique, where Grb 10, a member of Grb 7 family of adaptor proteins, found to be radically down-regulated in HuR knock down cells. Grb 10, downstream of PI3K/Akt, is implicated in anti-apoptotic signaling by its direct interaction with Raf-1 and MEK. HuR is known to bind and stabilize ARE containing mRNA’s and Grb 10 having ARE bearing mRNA, made it a putative candidate for HuR mediated mRNA stabilization. Hence, Grb 10 was found to be a novel binding target of HuR, which plays a key role in regulating IGFI-R and Akt pathway for cell survival.

Keywords: Grb 10,, HuR,, Apoptosis