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Characterization and regulation of Wnt5a alternate promoters A and B

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Nicole Buna Joyner-Powell (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/
Advisor
Karen Katula

Abstract: WNT5A is an extracellular glycoprotein that activates several Wnt signaling pathways important in cancer. Significantly, Wnt5a expression is altered in numerous cancers. Little is known about Wnt5a gene regulation but current data indicate that misregulation of Wnt5a expression involves non genetic changes. The goal of this study was to characterize transcriptional regulation from two alternate Wnt5a promoters, A and B. To analyze the relative level of expression from the human Wnt5a alternative promoters, promoter-luciferase reporter constructs containing different amounts of upstream sequence from promoters A and B were transiently transfected into NIH3T3 and Caco-2 cell lines. The relative level of promoter activity was compared by measuring luciferase activity in the transfected cells. Both Wnt5a promoters A and B were functional but found to have differential expression patterns in NIH3T3 and Caco-2 cells, indicating both positive and negative regulatory sequences. The transcription factor NFkappaB and the MAPK signaling pathway were studied to determine if they influence the transcriptional activity of Wnt5a promoters A and B. Stable lines of NIH3T3 cells with Wnt5a promoters A and B were treated for 6 and 24 hours with TNFalpha, a known inducer of NFkappaB activity, and inhibitors of MAPK pathway kinases (MEK1/2 and ERK). The cells were collected and assayed for firefly luciferase activity (relative light units) and standardized to DNA content. TNFalpha slightly increased promoters A and B activity at 6 hrs. TNFalpha had little or no effect on promoter A at 24 hours, whereas promoter B activity increased between 1.28 and 2.84 fold. The NFkappaB inhibitor, JSH-23, confirmed that NFkappaB is involved in the response of promoter B but not promoter A to TNFalpha. MEK1/2 inhibitor had inconsistent effects on promoter A, whereas promoter B activity decreases at both time points. ERK inhibitor increased promoter A activity for both 6 and 24 hours, whereas activity only increased for promoter B at 24 hours. These findings were further examined by measuring the relative contribution of Wnt5a alternative promoter A and B endogenous transcripts in both NIH3T3 and GM03349. Custom primers that amplify and detect human and mouse Wnt5a promoters A and B specific transcripts were created to analyze the relative amount of each transcript by qRT-PCR. Results show that promoter A generates more transcripts than promoter B and that TNFalpha increases the activity of both promoters in mouse and human fibroblasts. Overall, these data indicate that Wnt5a promoters A and B are differentially regulated and that NFkappaB influences transcriptional activity of Wnt5a promoter B rather than promoter A.

Additional Information

Publication
Thesis
Language: English
Date: 2011
Keywords
Alternative Promoters, Cancer, Transcription Regulation, Wnt5a
Subjects
Wnt genes
Wnt proteins