Author

Bhaskar Roy

Date of Award

January 2012

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Bryon D. Grove

Abstract

ABSTRACT

Gravin (AKAP12), a 300 kDa AKAP, is widely distributed in cells and binds to several signaling molecules including PKA-RII subunits, PKCα, PDE4, β2 adrenoreceptor and actin cytoskeleton. Initial characterization of gravin has revealed that this protein is undetectable in endothelia in vivo but expressed at the periphery of cultured endothelial cell (EC). Although the precise role of gravin in human ECs is not yet known, the cortical distribution of gravin in EC suggests that it may be involved in cAMP regulation by targeting PKA and PDE4 to plasma membrane. Several studies indicate that localization of PKA activity by A-kinase anchoring proteins (AKAPs) to the leading edge and other cytoskeletal regulators is an important factor in cell migration. SSeCKS, the rodent orthologue of gravin is reported to be involved in cell migration and regulation of actin cytoskeleton in cancer cell lines. Further, Western blot analysis, in the current study reveals that gravin upregulation in EC is associated with an active state, when cells are at a low cell density culture condition that promotes migration and proliferation, whereas downregulation occurs when cells became confluent and quiescent, indicating that gravin may have a functional role in ECs during their proliferative and migratory stage. Therefore, based on these findings, we hypothesized that gravin may play a role in EC migration.

To test this hypothesis, following experiments were conducted. First, the effect of gravin knockdown on EC migration was determined in a scratch wound and a 96-well based cell migration assay using antisense oligonucleotide and siRNA treatments. The

effect of antisense oligonucleotide and siRNA treatments on gravin expression was assessed by Western blotting and cell based ELISA. Finally, the reorganization of cortical actin band was quantitatively analyzed in antisense oligonucleotide treated cells to determine a role for gravin in actin cytoskeleton remodeling.

A significantly reduced distance, that wound edge moved after 18-20hr, was observed in both antisense oligonucleotide and siRNA treated cells in a scratch wound model. Consistent with this, the number of cells present in the cell free zone after 42-44hr was also reduced significantly in the cells treated with antisense oligonucleotide and siRNA treatments in a 96-well cell migration assay. Finally, antisense oligonucleotide treatment also reduced the length of cortical actin band normalized with the free edge (edge not touching adjacent cells) of cells at the wound edge, which further revealed that gravin was also involved in cortical actin remodeling in migrating EC.

This study proposes a possible mechanism for gravin mediated EC migration which involves the formation of a gravin-PKA-PDE4 functional complex, facilitating a compartmentalized regulation of cAMP/PKA dynamics that induces cortical actin remodeling and EC migration. In summary, the present study provides a new insight into the control of gravin expression in cultured EC and the role of gravin in EC migration and wound healing.

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