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Engineering a heterologous transforming growth factor-alpha promoter that is glucose-responsive in 293T cells / Virginia Danielle Hikel Reale

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Identifier	Thesis 1655
Author	Reale, Virginia Danielle Hikel
Title	Engineering a heterologous transforming growth factor-alpha promoter that is glucose-responsive in 293T cells / Virginia Danielle Hikel Reale
Publisher	Central Connecticut State University
Date	2002
Resource Type	Master's Thesis
Notes	Transforming growth factor-alpha (TGFa), a member of the endothelial growth factor (EGF) family of mitogens, was previously thought to exist solely in neoplastic cells and the quickly dividing cells of a developing fetus. Since then, TGFa expression has been confirmed in several normal adult cell types such as brain, skin and macrophages. High levels of TGFa expression have been reported in several disease states, including virus-infected cells and the vascular smooth muscle cells (VSMCs) of hyperglycemic individuals. Previous studies have provided evidence for regulation of TGFa expression at the transcriptional and post-translational levels. Transcriptional level regulation by glucose, as well as by other endogenous and exogenous factors such as hormones and phorbol esters, has been reported. Earlier studies identified a putative glucose response element in the TGFa promoter, localizing it to a 3 15 basepair (bp) element upstream of the transcription start site. The purpose of the present study was to confirm a minimal TGFa promoter that is necessary and sufficient for glucose-responsiveness, and to then investigate if the glucose-responsiveness of a minimal promoter could be enhanced through use of a heterologous promoter by inserting putative enhancer elements from other glucoseresponsive promoters into the minimal promoter-reporter construct. This minimal promoter was identified using deletion constructs of the TGFa promoter. The kilobase (kb)proximal elements, comprising the 59th bp through the 1,139'" bp 5' of the TGFa transcription start site, was designated as the reference sequence and served as the full length promoter for this study. The 5' deletion constructs made from the 1.1 kb reference sequence were 3 15 bp, 347 bp and 442 bp. The deletion constructs fused to a luciferase reporter gene were transiently expressed in 293T cells. The expression of the luciferase reporter driven by the deletion constructs was assessed in 293T cells in low (1.0 mM) and high (16.7 mM) glucose conditions. Of the three TGFa promoter deletion constructs, only the 3 15 bp elements drove glucose-responsive luciferase expression, resulting in a 1 -3-fold increase in luciferase activity when the element was expressed in 293T cells in 16.7 mM glucose media, This increase was comparable to that induced by the full-length construct, confirming the previously reported results. Expressing the 347 bp and 442 bp 5' deletion constructs in high glucose conditions resulted in no appreciable glucose-responsive luciferase expression. The 3 15 bp elements were selected as a minimal glucose-responsive TGFa promoter. Having confirmed the minimal glucose-responsive TGFa promoter, the heterologous promoters were constructed by inserting glucose-responsive enhancer elements upstream of the minimal TGFa promoter-luciferase reporter construct. The following nine enhancer elements, reported in the literature as having glucose-sensitivity, were used for this study: the S 14 gene glucose sensitive element (GSE), the fatty acid synthase 2 (FAS2) gene GSE, the adenoviral major late transcription factor (Ad-MLTF) from the major late adenovirus promoter, the L4 element from the L-pyruvate kinase (L-PK) gene, the A2 and E 1 elements (HA2E 1) from the human insulin promoter, the E 1 element (HE1) from the human insulin promoter, the A2 element (HA2) from the human insulin promoter, the A2 element (RA2) from the rat insulin I gene and the R2 element from the rat insulin I gene. The ability of the heterologous promoters to increase glucoseresponsive luciferase expression was assessed in the same manner as the deletion constructs were tested. Of the nine heterologous promoters, the one with the HA2E1 elements showed an appreciable increase in luciferase expression, resulting in a 3.7-fold increase in luciferase expression when in 16.7 mM glucose media. This study confirmed the 3 15 bp 5' deletion construct as a minimal glucoseresponsive TGFa promoter. Further, this study confirmed that the glucoseresponsiveness of a minimal promoter can be enhanced through the use of a heterologous promoter, specifically, the HA2E1-315 bp TGFa promoter construct.
Subject	Cytology
Department	Department of Biological Sciences
Advisor	King, Thomas R.
Type	Text
Digital Format	application/pdf
Language	eng
OCLC number	50678896
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Engineering a heterologous transforming growth factor-alpha promoter that is glucose-responsive in 293T cells / Virginia Danielle Hikel Reale

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