Gα12/Gα13 Subunits of Heterotrimeric G Proteins Mediate Parathyroid Hormone Activation of Phospholipase D in UMR-106 Osteoblastic Cells

Abstract

PTH, a major regulator of bone remodeling and a therapeutically effective bone anabolic agent, stimulates several signaling pathways in osteoblastic cells. Our recent studies have revealed that PTH activates phospholipase D (PLD) -mediated phospholipid hydrolysis through a RhoA-dependent mechanism in osteoblastic cells, raising the question of the upstream link to the PTH receptor. In the current study, we investigated the role of heterotrimeric G proteins in mediating PTH-stimulated PLD activity in UMR-106 osteoblastic cells. Transfection with antagonist minigenes coding for small peptide antagonists to G alpha 12 and G alpha13 subunits of heterotrimeric G proteins prevented PTH-stimulated activation of PLD, whereas an antagonist minigene to G alphas failed to produce this effect. Effects of pharmacological inhibitors (protein kinase inhibitor, Clostridium botulinum exoenzyme C3) were consistent with a role of Rho small G proteins, but not of cAMP, in the effect of PTH on PLD. Expression of constitutively active G alpha12 and G alpha13 activated PLD, an effect that was inhibited by dominant-negative RhoA. The results identify G alpha12 and G alpha13 as upstream transducers of PTH effects on PLD, mediated through RhoA in osteoblastic cells.

Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

“…Because PTH treatment has a positive effect on osteoblast surface in G s ␣-deficient mice, it is likely that PTH also stimulates signaling pathways downstream of PTH1R independent of G s ␣. These may include pathways activated by other PTH1R-coupled G proteins including G q /G 11 and G 12 /G 13 (29,30). We found that although intermittent PTH in older mice did not significantly increase trabecular bone volume in long bones of either WT or D/D mice within the time frame of this experiment it did significantly increase almost all parameters of bone formation in both WT and D/D mice.…”

“…Furthermore, constitutive activation of G s -dependent signaling by an engineered G scoupled receptor also significantly increases trabecular bone mass (28). In addition to G s , PTH1R couples to several other G proteins, activating phospholipase C␤ via G q /G 11 to stimulate protein kinase C and G 12 /G 13 to stimulate phospholipase D (29,30). We have previously reported that phospholipase C (PLC) signaling via PTH1R is essential for stromal cell response to PTH in growing mice (31).…”

A-Raf and B-Raf Are Dispensable for Normal Endochondral Bone Development, and Parathyroid Hormone-Related Peptide Suppresses Extracellular Signal-Regulated Kinase Activation in Hypertrophic Chondrocytes

“…The PPR is a member of the B subfamily of G proteincoupled receptors that can activate multiple G proteins (38,44): Gs, which activates the adenylyl cyclase/protein kinase A (PKA) pathway; the Gq/G11 family, which activates the phos-pholipase C/protein kinase C pathway; Gi, which inhibits adenylyl cyclase and exerts other actions as well; and G12/G13 (41). Through the activation of these pathways, PPR generates multiple intracellular secondary messengers, including cyclic AMP (cAMP), inositol triphosphate, diacylglycerol, and cytosolic Ca 2ϩ , which then mediate various cellular responses.…”

PTH and PTHR1 in osteocytes. New insights into old partners

“…After ligand coupling, PTHR1 activates four different intracellular signalling cascades: a) GαS-adenylyl cyclise-cAMP-protein kinase A (PKA), b) Gαq-phospholipase C (PLC) β-inositol triphosphate-cytoplasmic Ca 2+ -protein kinase C, 18 c) Gα12/13-hospholipase D-transforming protein RhoA 19 and d) β-arrestin-xtracellular signal-regulated kinase 1/2 (ERK1/2) (Figure 1). 20,21 In the skeleton, PTHR1 is expressed on the surface of osteoblasts and osteocytes.…”