Rho and Rho kinase are involved in parathyroid hormone-stimulated protein kinase C alpha translocation and IL-6 promoter activity in osteoblastic cells.
The role of small G-proteins in PTH-stimulated PKC translocation and IL-6 promoter expression in UMR-106 cells was determined. The effects of PTH(1-34) and PTH(3-34) in stimulating PKCalpha translocation and IL-6 were inhibited by agents that interfere with the activity of small G-proteins of the Rho family and with the downstream kinase Rho kinase.


BACKGROUND
Activation of protein kinase C (PKC) is a signaling mechanism by which parathyroid hormone (PTH) modulates interleukin-6 (IL-6) in osteoblasts, leading to osteoclastogenesis and bone resorption. PKCalpha and PKCbetaI are translocated after treatment with PTH in UMR-106 osteoblastic cells; however, the pathway leading to PKC isozyme translocation is not established. Diacylglycerol (DAG) generation from phospholipase D (PLD) is one pathway of PKC activation, and PTH-mediated PLD activity is dependent on small G-proteins of the Rho family. This study investigated whether Rho proteins modulate the PKCalpha translocation and IL-6 promoter activity stimulated by PTH in UMR-106 cells.


METHODS
UMR-106 cells were treated with PTH(1-34) or PTH(3-34). PKC translocation was determined by immunofluorescence, Rho A activation by Rhotekin assay and by translocation assessed by Western blotting in membrane and cytosol fractions, and IL-6 promoter expression by luciferase assay.


CONCLUSIONS
Inhibition of Rho proteins with Clostridium difficile toxin B or inhibition of Rho prenylation with GGTI attenuated PTH(1-34)- and PTH(3-34)-stimulated translocation of endogenous PKCalpha and IL-6 promoter activity. Expression of a constitutively active RhoA (RhoA63L) mimicked the effect of PTH(1-34) or PTH(3-34) to promote membrane localization of PKCalpha, whereas cells expressing a dominant negative RhoA (RhoA19N) did not respond to PTH(1-34) or PTH(3-34). The Rho kinase inhibitor Y27632 attenuated PTH(1-34)- and PTH(3-34)-stimulated PKCalpha translocation and IL-6 promoter activation. Rho seemed to be acting at a step before production of diacylglycerol (DAG), because the stimulation of PKCalpha translocation by the DAG mimetic phorbol 12,13 dibutyrate (PDBu) was unaffected by C. difficile toxin B or Y27632. These results indicate that Rho proteins are an important component of PTH signaling in osteoblastic cells and provide further demonstration of convergence between PKC and small G-protein signaling pathways.
Department of Molecular Pharmacology and Biological Chemistry, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
Julie M
JM
Radeff
Zsolt
Z
Nagy
Paula H
PH
Stern
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism; D010281:Parathyroid Hormone / Q000378:metabolism*; D010739:Phospholipase D / Q000737:chemistry; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction; D013347:Subcellular Fractions; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases
D001427:Bacterial Toxins; C488583:Clostridium difficile lethal toxin B; D004075:Diglycerides; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D010281:Parathyroid Hormone; D020033:Protein Isoforms; C498396:Rtkn protein, rat; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins