Poster abstracts

Poster number 30 submitted by Mamata Singh

HuR inhibits apoptosis in ATP-depleted proximal tubule cells

Mamata Singh (Physiology and cell biology), Dina Ayupova (Physiology and cell biology), Beth lee (Physiology and cell biology)

Abstract:
HuR inhibits apoptosis in ATP-depleted proximal tubule cells
Mamata Singh, Dina Ayupova, and Beth S. Lee
HuR is a ubiquitously expressed protein that resides primarily in nuclei under normal growth conditions but translocates to the cytosol during stress, where it binds and stabilizes a select subset of mRNAs bearing adenine and uridine-rich sequences in their 3’ untranslated regions. In non-renal cells, these mRNAs have been shown to include those encoding a variety of proteins involved in preventing apoptosis and promoting cell survival. We previously showed that HuR stabilizes target mRNAs in LLC-PK1 cells during ATP depletion, and that its expression during ATP depletion and recovery is upregulated by both transcriptional and translational mechanisms. Although HuR is generally regarded as an anti-apoptotic protein, recent studies have indicated that caspase cleavage of HuR may contribute to an amplified apoptotic response. To clarify HuR’s role in renal stress, we ATP-depleted LLC-PK1 cells in which HuR was stably overexpressed or knocked down via RNA interference. Knockdown of HuR (75%) caused a 50-100% increase in apoptosis as assayed by caspase 3/7 activation or nuclear condensation. Conversely, 2-3 fold over expression of HuR suppressed apoptosis by 50%, as measured by the same assays. The role of caspase activation in this process was confirmed with the use of caspase inhibitors. Additionally, siRNA-mediated knockdown of HuR resulted in decreased expression of the anti-apoptotic proteins like Bcl-2 and HSP70, which contains HuR binding sequences. To rule out a pro-apoptotic effect for HuR in ATP-depleted LLC-PK1 cells, Western analysis was performed to detect potential caspase-mediated HuR cleavage products. These products were not detectable in our model system. These results strongly indicate an anti-apoptotic role for HuR in cultured proximal tubules cells, and suggest that its function in native proximal tubules may be protective against ischemic stress. A similar anti-apoptotic role of HuR was noted in HK-2 cell lines (Human cortical kidney cell line). Our PCR-Array analysis also confirmed the anti-apoptotic nature of HuR, involved in up regulating anti-apoptotic genes like Bcl-2 family and down regulating pro-apoptotic genes like the caspase and TNF families.

Keywords: HuR, ATP depletion, Apoptosis