Are the WNT5A isoforms functionally distinct? - promoter and signaling pathway analyses

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Ahmed G. Elshaarrawi (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Karen Katula

Abstract: WNT5A is a secreted glycoprotein that binds to both canonical and non-canonical Wnt receptors and has important roles in morphogenesis (e.g., anterior-posterior axis elongation and limb formation) and differentiation (e.g., bone and cartilage). On a cellular level WNT5A functions in proliferation, adhesion, migration, and cell polarity. Altered WNT5A expression is associated with various human diseases, particularly cancer, but has also been linked with the inflammatory response. WNT5A has two isoforms that are derived from distinct promoters; the proteins isoforms referred to as L(A) and S(B) differ by 18 amino acids. In this project I examined the functional differences between the proteins isoforms L(A) and S(B) and the regulation of their promoters. The isoforms may have differential affinity for non-canonical receptors, selectively activating particular signaling pathways. They may also display distinct patterns of expression in particular cells, during differentiation and in development, as a consequence of their unique promoters. Conditioned medium (CM) was prepared from CHO cells expressing either isoform L(A) or S(B). Using a TOPFlash system the CML(A) and CM-S(B) were shown to be active. The CM was used to analyze the effects of the isoforms on the non-canonical Wnt signaling pathways Ca2+ and PCP/CE in HCT 116 (colon cancer) and hFOB1.19 (normal human osteoblast) cell lines by measuring levels of phospho (p)PKC and phospho (p)JNK, downstream targets of each pathway. Results showed that the CM-S(B) activated both pPKC and pJNK in HCT 116 cells whereas CML(A) had less of an effect. There was little or no effect of both CM’s in hFOB1.19 cells. AP-1 and NFAT luciferase reporter assays in HCT 116 cells confirmed the effect of CMS(B) on pJNK. Next the effect of the CM’s on apoptosis, proliferation, and migration were analyzed. Neither CMs affected the level of apoptosis in HCT 116 cells. Both isoforms were found to decrease proliferation in HCT 116 cells but CM-S(B) had a more consistent effect. CM-S(B) was found to increase migration and CM-L(A) decrease migration in HCT 116 cells. In a mouse embryonic fibroblast (MEF)-with a PORCN gene knock-out, both CMs caused a decrease in migration. Next, isoform L(A) and S(B) luciferase promoter constructs were transfected into HCT 116 and hFOB1.19 cells. The pattern of expression in the two cell types was similar for promoter L(A) and promoter S(B) but promoter S(B) showed a higher level of expression. 1707bp of upstream sequence showed a maximal expression for promoter L(A) and 1257 bp showed a maximal expression for promoter S(B) in both proliferating cell types. Analysis of the promoter L(A) and promoter S(B) reporter constructs during hFOB1.19 differentiation indicated that promoter L(A) is more highly activated than promoter S(B). Sequences within 420bp and 187bp for promoter L(A) and promoter S(B), respectively, were sufficient for activation. Putative transcription factor binding sites were identified in promoter L(A) and promoter S(B) upstream sequences. Some of these factors are known to be involved in osteogenesis. In summary, my results suggest that the WNT5A isoform proteins have distinct and cell type dependent. The isoform promoters have distinct sequences and include similar and unique putative transcription factor binding sites but both promoters are activated during osteogenesis.

Additional Information

Language: English
Date: 2018
HCT-116, hFOB1.19, Isoforms, Non-canonical, Osteogenesis, WNT5A
Wnt proteins
Promoters (Genetics)
Genetic transcription
Genetic regulation
Bones $x Growth

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