Lysophosphatidic Acid Induced Apoptosis, DNA Damage, and Oxidative Stress in Spinal Cord Neurons by Upregulating LPA4/LPA6 Receptors.

Lysophosphatidic acid (LPA) has disruptive effects on lumbar spinal stenosis (LSS). Recently, LPA has been reported to be involved in spinal cord neuronal injury and toxicity, promoting the pathogenesis of LSS. However, the exact effects of LPA on spinal cord neurons remain unknown. The purpose of this study is to investigate the effects of LPA (18 : 1) on spinal cord neuronal cytotoxicity, apoptosis, DNA damage, and oxidative stress. After clinical detection of LPA secretion, spinal cord neurons were treated with LPA (18 : 1); cell viability was analyzed by MTT assay, and LDH leakage was detected by LDH kit; cell apoptosis was detected by flow cytometry; ROS production was measured by DCFDA staining and MitoSOX Red Staining; the activation of the Gα12/Gα13 signaling pathway was detected by serum response factor response element (SRF-RE) luciferase reporter gene; the relationship among LPA, LPA4/6, and ROCK was examined by western blotting. In spinal cord neurons treated with LPA (18 : 1), cellular activity decreased and LDH release increased. The Rho kinase inhibitor (Y-27632) can attenuate LPA-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons. Moreover mechanistic investigation indicated that LPA (18 : 1) activates Gα12/13-Rho-ROCK2-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons by upregulating LPA4/LPA6 receptors. Further, the Rho kinase inhibitor Y-27632 attenuates the effects of LPA by downregulating LPA4/LPA6 receptors. Taken together, the possible mechanism by which LPA secretion in LSS patients aggravates patient injury was further elucidated using an LPA-induced spinal cord neuronal injury cell model in vitro.

Signaling through G(alpha)13 switch region I is essential for protease-activated receptor 1-mediated human platelet shape change, aggregation, and secretion.

This study investigated the involvement of Galpha(13) switch region I (SRI) in protease-activated receptor 1 (PAR1)-mediated platelet function and signaling. To this end, myristoylated peptides representing the Galpha(13) SRI (Myr-G(13)SRI(pep)) and its random counterpart were evaluated for their effects on PAR1 activation. Initial studies demonstrated that Myr-G(13)SRI(pep) and Myr-G(13)SRI(Random-pep) were equally taken up by human platelets and did not interfere with PAR1-ligand interaction. Subsequent experiments revealed that Myr-G(13)SRI(pep) specifically bound to platelet RhoA guanine nucleotide exchange factor (p115RhoGEF) and blocked PAR1-mediated RhoA activation in platelets and human embryonic kidney cells. These results suggest a direct interaction of Galpha(13) SRI with p115RhoGEF and a mechanism for Myr-G(13)SRI(pep) inhibition of RhoA activation. Platelet function studies demonstrated that Myr-G(13)SRI(pep) specifically inhibited PAR1-stimulated shape change, aggregation, and secretion in a dose-dependent manner but did not inhibit platelet activation induced by either ADP or A23187. It was also found that Myr-G(13)SRI(pep) inhibited low dose, but not high dose, thrombin-induced aggregation. Additional experiments showed that PAR1-mediated calcium mobilization was partially blocked by Myr-G(13)SRI(pep) but not by the Rho kinase inhibitor Y-27632. Finally, Myr-G(13)SRI(pep) effectively inhibited PAR1-induced stress fiber formation and cell contraction in endothelial cells. Collectively, these results suggest the following: 1) interaction of Galpha(13) SRI with p115RhoGEF is required for G(13)-mediated RhoA activation in platelets; 2) signaling through the G(13) pathway is critical for PAR1-mediated human platelet functional changes and low dose thrombin-induced aggregation; and 3) G(13) signaling elicits calcium mobilization in human platelets through a Rho kinase-independent mechanism.

Role of Lbc RhoGEF in Galpha12/13-induced signals to Rho GTPase.

Heterotrimeric Galpha12/13 signals induce cellular responses such as serum response element (SRE)-mediated gene transcription via Rho GTPase. Guanine nucleotide exchange factors (GEFs) are strong candidates for linking Galpha signals to Rho. For example, p115 RhoGEF transduces Galpha13 signals to Rho and inhibits Galpha12/13 signals via the RhoGEF LH domain which links to Galpha subunits. Here, we have evaluated the signaling capacity of Lbc RhoGEF in the context of Galpha12/13 signals. In vitro GEF assays indicate that baculoviral-expressed proto-Lbc has minimal exchange activity, implying that a stimulus is required for Lbc activity in vivo. Expression of a catalytically inactive proto-Lbc mutant in HEK293T cells attenuates Galpha12- and thrombin-induced activation of an SRE transcriptional reporter, and the levels of inhibition observed is similar to that obtained with an analogous p115 RhoGEF mutant. proto-Lbc mutant expression also led to decreased levels of Galpha12-induced RhoA activation in vivo. Complex formation between Galpha12 and Lbc forms was detected. Analysis of the Lbc peptide sequence reveals a previously undetected region which may link to Galpha subunit signals. These findings support a role for Lbc in Galpha12-induced signaling pathways to Rho.