title: Galpha12 stimulates apoptosis in epithelial cells through JNK1-mediated Bcl-2 degradation and up-regulation of IkappaBalpha.
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.
authors.affiliation: Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Vijay
authors.initials: V
authors.lastname: Yanamadala
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Negoro
authors.forename: Lakshman
authors.initials: L
authors.lastname: Gunaratnam
authors.forename: Tianqing
authors.initials: T
authors.lastname: Kong
authors.forename: Bradley M
authors.initials: BM
authors.lastname: Denker
mesh_terms: D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000502:physiology*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics*; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000378:metabolism*
chemical_list: D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D000072000:NF-KappaB Inhibitor alpha; D048055:Mitogen-Activated Protein Kinase 8; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D044365:GTP-Binding Protein alpha Subunits, G12-G13