Defense Date

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biochemistry

First Advisor

XIANJUN FANG

Abstract

Lysophosphatidic acid (LPA), a naturally occurring phospholipid present in serum and malignant effusions, elicits diverse biological functions through binding and activating specific cell surface G-protein coupled receptors. In addition to the conventional LPA1/Edg2, LPA2/Edg4 and LPA3/Edg7 receptors of the endothelial differentiation gene (Edg) family, LPA4/p2y9/GPR23 of the purinergic receptor family and the related LPA5/GPR92 and LPA6/p2y5 have been identified as novel LPA receptors. These newly identified LPA receptors are structurally distant from the Edg LPA1-3 receptors and couple to Gq, G12/13 and probably Gs subunits. However, the roles of the LPA4-6 receptors in LPA signal transduction and physiology are poorly understood. This project has used biochemical and genetic approaches to study biological functions of LPA4. In the first part of the study, we confirmed that LPA4 is indeed a functional LPA receptor mediating some cellular and biochemical responses to LPA including stimulation of neurite retraction, protein tyrosine phosphorylation. LPA4 also physically binds to LPA when ectopically expressed in cell lines. Mammalian cells usually express multiple LPA receptor subtypes and respond to LPA, making it difficult to link LPA receptors to specific responses. Targeted deletion has become a necessary approach to probe functions of individual LPA receptors. We therefore disrupted LPA4-encoding gene (lpa4/p2y9/gpr23) in mice. LPA4-deficient mice were born at the expected frequency and displayed no apparent abnormalities at least at early ages, indicating that LPA4 is not required for fertility, embryonic development or normal physiology. This is similar to knockouts of other LPA receptors. The backup and/or redundant receptor subtypes of LPA may suffice to compensate for the loss of individual LPA receptors in vivo. Alternatively, LPA may not be the only or rate-limiting mediator physiologically required in vivo. LPA signaling may be more critical in pathophysiological conditions when levels of the lipid mediator are locally and temporally altered. The availability of LPA4-null mice provides a valued model to analyze the roles of LPA4 in pathophysiological processes. Despite the lack of apparent phenotypes in mice, we took advantage of the LPA4- negative mouse embryonic fibroblasts (MEFs) to evaluate the effects of lpa4 deletion on cellular responses to LPA. Strikingly, LPA4-deficient MEFs were hypersensitive to LPA induced migration. Consistent with negative modulation of the phosphatidylinositol 3 kinase (PI3K) pathway by LPA4, LPA4 deficiency potentiated AKT and Rac but decreased Rho activation induced by LPA. Reconstitution of LPA4 converted LPA4-negative cells into a less motile phenotype. In support of the biological relevance of these observations, ectopic expression of LPA4 strongly inhibited migration and invasion of human cancer cells. When coexpressed with LPA1 in B103 neuroblastoma cells devoid of endogenous LPA receptors, LPA4 attenuated LPA1-driven migration and invasion, indicating functional antagonism between the two subtypes of LPA receptors. These results provide genetic and biochemical evidence that LPA4 is a suppressor of LPA-dependent cell migration and invasion. LPA4 may thus play a role in negative regulation of LPA signal transduction and specific cellular responses.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

April 2009

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