Deepak Bhatia

Date of Graduation

2007

Document Type

Dissertation/Thesis

Abstract

Previously, publications from our lab had demonstrated that arsenic can regulate Gadd45α through p53-dependent and -independent manner where we explored the regulation of Gadd45α by various transcription factors, including Foxo3a, NF-κB and through stress signaling pathways such as JNK. Here we analyzed whether arsenic has any impact on the biosynthesis of Egr-1, since Egr-1 has also been reported to regulate Gadd45α in keratinocytes in response to UVB. Our results show arsenic can induce Egr-1 synthesis in a dose- and time-dependent manner. To determine the regulatory sequence responsible for Egr-1 induction, we created deletion constructs in the Egr-1 promoter and found Elk-1 binding site was important in arsenic mediated regulation of Egr-1. Rendering BEAS2B cells deficient in Elk-1 expression abrogated the Egr-1 biosynthesis. Furthermore, Mitogen-activated protein kinase inhibitor U0126 completely blocked the activation of Egr-1. Since Elk-1 is known to be regulated through ERK, our data suggested a signaling event that follows the activation of ERK, Elk-1, Egr-1 and finally to Gadd45α in response to arsenic. Further studies suggested arsenic can also regulate Gadd45α at the translational level by favoring Gadd45α translation through Internal Ribosome Entry Site. In growth arrested cells, arsenic led to the accumulation of Gadd45α protein, activation of which did not change in the presence of cap-dependent inhibitor; rapamycin. Further analysis of Gadd45α 5'-UTR sequence revealed an IRES element which was confirmed by dicistronic reporter gene assay. Immunoprecipitation and proteomic studies suggested arsenic impairs assembly of cap-dependent initiation complex for general protein translation but increases association of eEF2 and hnRNP with this complex. Thus our result suggests arsenic can modulate the expression of Gadd45α through post-transcriptional regulation.

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