Abstract
Apoptosis is an essential mechanism for the maintenance of somatic tissues, and when dysregulated can lead to numerous pathological conditions. G proteins regulate apoptosis in addition to other cellular functions, but the roles of specific G proteins in apoptosis signaling are not well characterized. Galpha12 stimulates protein phosphatase 2A (PP2A), a serine/threonine phosphatase that modulates essential signaling pathways, including apoptosis. Herein, we examined whether Galpha12 regulates apoptosis in epithelial cells. Inducible expression of Galpha12 or constitutively active (QL)alpha12 in Madin-Darby canine kidney cells led to increased apoptosis with expression of QLalpha12, but not Galpha12. Inducing QLalpha12 led to degradation of the anti-apoptotic protein Bcl-2 (via the proteasome pathway), increased JNK activity, and up-regulated IkappaBalpha protein levels, a potent stimulator of apoptosis. Furthermore, the QLalpha12-stimulated activation of JNK was blocked by inhibiting PP2A. To characterize endogenous Galpha12 signaling pathways, non-transfected MDCK-II and HEK293 cells were stimulated with thrombin. Thrombin activated endogenous Galpha12 (confirmed by GST-tetratricopeptide repeat (TPR) pull-downs) and stimulated apoptosis in both cell types. The mechanisms of thrombin-stimulated apoptosis through endogenous Galpha12 were nearly identical to the mechanisms identified in QLalpha12-MDCK cells and included loss of Bcl-2, JNK activation, and up-regulation of IkappaBalpha. Knockdown of the PP2A catalytic subunit in HEK293 cells inhibited thrombin-stimulated apoptosis, prevented JNK activation, and blocked Bcl-2 degradation. In summary, Galpha12 has a major role in regulating epithelial cell apoptosis through PP2A and JNK activation leading to loss of Bcl-2 protein expression. Targeting these pathways in vivo may lead to new therapeutic strategies for a variety of disease processes.
Highlights
Signaling through G proteins2 is an important mechanism regulating apoptosis, a highly conserved process of pro
Using a well characterized MDCK cell culture model of inducible expression of G␣12, we report that G␣12 is a potent activator of apoptosis in epithelial cells and identify critical regulation of Bcl-2 protein levels by G␣12, phosphatase 2A (PP2A), and JNK
We demonstrate for the first time that activation of endogenous G␣12-coupled signaling pathways stimulates apoptosis in epithelial cells and recapitulates the signaling pathways identified in the inducible G␣12-MDCK cell model
Summary
Reagents—Rabbit polyclonal anti-G␣12, anti-JNK1, antiERK, anti-NF-B, anti-IB␣, and goat anti-PP2A catalytic subunit were from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit anti-pThr183/pTyr185-JNK, anti-pThr202/pTyr204-ERK, and antiBcl-xL were from Cell Signaling Technology (Danvers, MA), mouse anti-Bcl-2 was from BD Biosciences (San Jose, CA), and rabbit anti-phospho-cJun was from Biovision (Mountain View, CA). DNA Fragmentation Assay—G␣12- or QL␣12-expressing MDCK cells were cultured Ϯ dox for 72 h. Adherent cells were collected by trypsinization and pooled with floating cells by low speed centrifugation, washed with PBS, and lysed with genomic DNA extraction buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K). Kinexus KinetworksTM Phospho-site Phosphorylation Screen— 1 ϫ 107 QL␣12-expressing MDCK cells were grown to confluence, cultured Ϯ dox for 72 h. Samples were eluted in Laemmli sample buffer and analyzed by SDS-PAGE and Western blot for phospho-cJun. RNA Isolation and Semiquantitiative RT-PCR Analysis—G␣12- or QL␣12-expressing MDCK cells were cultured Ϯ dox for 72 h and lysed with TRIzol (Invitrogen). Immunoblot Analysis—G␣12- or QL␣12-expressing MDCK cells were cultured Ϯ dox for 72 h
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call