Role of the LPA2 Receptor in Protecting Against Apoptosis

<p>Lysophosphatidic acid (LPA) is a naturally occurring lipid mediator. It exists abundantly in biological fluids such as serum, saliva, follicular fluid, seminal fluid and malignant effusions and induces a vast array of biological responses affecting cell growth, survival, differentiation, migration and morphology. We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. Based on computational modeling octadecenyl thiophosphate (OTP) was synthesized: a novel rationally designed, metabolically stabilized LPA mimic. OTP was more efficacious than LPA in reducing g-irradiation-, camptothecin-, or TNF-a/cycloheximide-induced apoptosis and caspase 3, 8 and 9 activity in the IEC-6 cell line. The OTP- and LPA-elicited antiapoptotic effects were completely blocked by the MEK inhibitor PD98059 and the PI3K inhibitor LY294002. Pertussis toxin partially abolished OTP-induced ERK1/2 and apoptotic protection. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA₂ but not LPA₁ and LPA₃ transfectants. In C57BL/6 and LPA₁ knockout mice exposed to 15 Gy g-irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase 3 positive cells but was ineffective in LPA₂ knockouts. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but had no effect in LPA₂knockouts. Intraperitonealy administered OTP reduced death caused by LD_100/30 radiation by 50%. Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA₂ receptor subtype. </p> <p>Unique sequence motifs in the LPA₂ carboxyl-terminal (CT) enable it to form macromolecular complexes with PDZ and LIM domain proteins, which link it to G protein-independent signaling networks. Using deletion and site-specific mutagenesis, we mapped out C311xxC314 motif of LPA₂-CT that is required for interaction with LIM domain proteins thyroid hormone receptor interactive protein 6 (TRIP6) and the proapoptotic molecule Siva-1 <em>in vitro</em> and <em>in vivo</em>. Palmitoylation that occurs on these cysteine residues, however, did not affect the association with TRIP6 or Siva-1. The L351A mutation in the PDZ motif weakened but did not abolish interaction with LIM proteins. Alanine mutation of the LIM binding motif or PDZ domain binding motif attenuated LPA-induced activation of the prosurvival ERK1/2 and Akt pathways in mouse embryonic fibroblasts (MEF) derived from LPA₁and LPA₂ double knockout mice and reconstituted with mutants of LPA₂. Neither of these mutations alone nor in combination had a detectable effect on G-protein-linked activation of Ca²⁺mobilization. Triple alanine mutations modifying residues Cys311, Cys314, and Leu351 abolished the antiapoptotic effect of LPA. Together, these findings suggest the macromolecular complex formed between LPA₂, TRIP6/Siva-1, and PDZ domain proteins plays an important role in mediating the anti-apoptotic effects of LPA₂.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Physiology

Year dc:date.available

2008

Author dc:creator

• E, Shuyu

Contributors dc:contributor

• Gabor Tigyi, M.D., Ph.D.

• LPA
• LPA2 receptor
• apoptosis
• irradiation
• protein-protein interaction
• LIM domain PDZ domain
• Medical Physiology
• Medical Sciences
• Medicine and Health Sciences

Repository record dc:identifier

https://dc.uthsc.edu/dissertations/336

OAI identifier oai:identifier

oai:dc.uthsc.edu:dissertations-1338