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Insights into the Renal Protective Mechanisms of mRNA Binding Protein HuR

Singh, Mamata

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2011, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.

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. To clarify HuR’s role in renal stress, we knocked down or overexpressed it in proximal tubule cell lines that were subjected to ATP depletion, an in vitro procedure that mimics effects of ischemic injury to native kidneys. HuR was found to be protective against apoptosis that occurs when proximal tubule cells are ATP depleted. These results suggest that HuR’s function in native proximal tubules may be protective against ischemic stress. To understand what other cell survival signaling pathways might be induced by HuR, PCR array analysis was used to compare gene expression levels in ATP-depleted cells with normal or suppressed levels of HuR. Here, we show that in renal proximal tubule cells, HuR performs a central role in cell survival by amplifying Akt signaling in a positive feedback loop. Key to this feedback loop is HuR-mediated stabilization of mRNA encoding Grb10, an adaptor protein that enhances Akt activity by aiding in its transport to sites of activation. Under normal growth conditions, stimulation of Akt by HuR-Grb10 interactions then activates NFkappaB, which further enhances HuR expression at a transcriptional level. This study demonstrates a central role for HuR in cell survival mechanisms and reveals how only modest changes in HuR levels below or above normal can be amplified, resulting in cell death, or alternately, cellular transformation.

The data presented here show that the expression of HuR during ATP depletion and recovery is upregulated by both transcriptional and translational mechanisms. It was previously demonstrated that HuR mRNA is expressed as two isoforms with vastly different 5’ untranslated regions and different translatabilities. Here we have demonstrated that one of these mRNAs is responsive to the BMP-7/Smad 1/5/8 signaling pathway, is more highly expressed during recovery from ATP depletion, and is the only form capable of being translated during ATP depletion. We also demonstrated that translation of the alternate form occurs during normal growth and recovery, and occurs through regulation by a post-transcriptional control element (PCE) present in its 5’ untranslated region. We further hypothesize that the differential translation of the HuR isoforms may be due to their alternate targeting to polyribosomes or stress granules.

Stress granules are sites of aborted translation formed during various types of cell stress and recovery, including heat shock, arsenite poisoning, and UV-irradiation. We demonstrated that ATP-depleted epithelial cells form stress granules and activated their upstream kinases presumably for the purpose of regulating mRNA stability and translatability. During ATP depletion, some HuR mRNA is stored temporarily in stress granules. We hypothesize that this may be to avoid aberrant translation of HuR, which can lead to tumorigenicity. The presence of a PCE imposes another level of regulation on the translation of HuR and thus the translation of HuR is controlled during normal, stressed, and recovered conditions.

Beth S. Lee, PhD (Advisor)
Richard W. Burry, PhD (Committee Member)
Arthur R. Strauch, PhD (Committee Member)
Jiyan Ma, PhD (Committee Member)
127 p.

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Citations

  • Singh, M. (2011). Insights into the Renal Protective Mechanisms of mRNA Binding Protein HuR [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1300995188

    APA Style (7th edition)

  • Singh, Mamata. Insights into the Renal Protective Mechanisms of mRNA Binding Protein HuR. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1300995188.

    MLA Style (8th edition)

  • Singh, Mamata. "Insights into the Renal Protective Mechanisms of mRNA Binding Protein HuR." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1300995188

    Chicago Manual of Style (17th edition)