Role of EVC2 in hedgehog signaling and chondrocyte proliferation
Date
2016
DOI
Authors
Venkitapathi, Sundharamani
Version
OA Version
Citation
Abstract
Ellis-van Creveld syndrome (EvC) is an autosomal recessive chondrodysplasia with dwarfism, characterized by short ribs/limbs, postaxial polydactyly, dysplastic nails, dysplastic teeth, delayed eruption of teeth and hyperplastic frena. Weyer's Acrofacial Dysostosis (WAD) is an autosomal dominant form of EvC with a milder phenotype caused due to mutations in EVC2. Mutations in either of the genes EVC or EVC2 lead to the manifestation of the same clinical phenotype identified as EvC. Evc2 knockout (KO) mice model characterized in our lab also demonstrated phenotypes similar to EvC. Reports suggest that EVC2 plays a key role in the processing of GLl3 to activator and repressor forms thereby modulating hedgehog signaling. Previous reports also show a decrease in BrdU labelled chondrocytes in the growth plates of Evc2 KO mice suggestive of decreased chondrocyte proliferation. The objective of the study is to investigate if EVC2 is required for GLl3 expression and analyze proliferation of Evc2 knockdown (KO) chondrogenic ATDC5 cells EVC and EVC2 proteins co-localized with the centrosomal marker pericentrin and localized to the base of cilia.
Our results show that EVC interacts with EVC2 wild type (WT) and mutant proteins. EVC2's interaction with SMO to form a ciliary complex in the presence of WT and mutant EVC proteins was investigated by overexpression of plasmids in HEK 293 cells. Immunoblotting results from our study showed that EVC2 and EVC form a ciliary complex with SMO. To investigate the role of EVC2 in determining GLl3 levels, the expression of full length and truncated GLl3 proteins was analyzed in WT and Evc2 KO mouse embryonic fibroblasts (MEF) and chondrocytes by immunoblotting. Our data showed a decrease in the cytosolic expression of full length form of GLl3 in Evc2 KO MEF and chondrocytes demonstrating the importance of EVC2 in determining GLl3 levels. Our results show that stable knock down of Evc2 in ATDC5 cells leads to a decrease in cell proliferation in serum starvation/stimulation condition in comparison to control clones and parental ATDC5 cells. Our study shows no significant difference in cell proliferation between control and Evc2 knock down clones in the presence of serum. Also analysis of cell cycle progression by propidium iodide staining after serum starvation/stimulation revealed an absence of a distinct peak in "S" phase in Evc2 knock down clones in comparison to the control suggestive of a defect in the ability of the Evc2 knock down cells to progress through the G1-S phase of the cell cycle. Our data shows that EVC2 is required for expression of GLl3 protein and chondrocyte proliferation.
Description
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Thesis (MSD)--Boston University, Henry M. Goldman School of Dental Medicine, 2016 (Department of Oral Biology)
Presented in two parts.
Includes bibliographic references: leaves 78-82 (part 1), 128-130 (part 2).
Thesis (MSD)--Boston University, Henry M. Goldman School of Dental Medicine, 2016 (Department of Oral Biology)
Presented in two parts.
Includes bibliographic references: leaves 78-82 (part 1), 128-130 (part 2).
License
This work is protected by copyright. Downloading is restricted to the BU community. If you are the author of this work and would like to make it publicly available, please contact open-help@bu.edu.