Rho GTPase signaling and PTH 3-34, but not PTH 1-34, maintain the actin cytoskeleton and antagonize bisphosphonate effects in mouse osteoblastic MC3T3-E1 cells.
Cytoskeletal elements are critical for cell morphology and signal transduction, and are involved in many cellular processes including motility, intracellular transport, and differentiation. Small GTP-binding proteins (G proteins) of the Ras family, such as RhoA, influence various elements of the cytoskeleton. RhoA stabilizes the actin cytoskeleton and promotes formation of focal adhesions. We found previously that RhoA is expressed in osteoblastic cells and is translocated to the plasma membrane and activated by PTH 1-34 as well as by Nleu(8,18) Tyr(34) PTH 3-34 amide, a PTH analog that does not increase cAMP. We therefore investigated effects of manipulating RhoA on the actin cytoskeleton of osteoblastic MC3T3-E1 cells. Three inhibitors were used: 1) GGTI-2166, a geranylgeranyl transferase I inhibitor that prevents the isoprenylation and membrane translocation of RhoA, 2) Y-27632, a Rho kinase inhibitor, and 3) alendronate, a nitrogen (N)-containing bisphosphonate that reduces intracellular geranylgeranylpyrophosphate through inhibiting farnesyl pyrophosphate synthase. To increase RhoA activity, we used the geranylgeranyl group donor geranylgeraniol (GGOH), and a constitutively active RhoA. The F-actin cytoskeleton and focal adhesions (FA) were visualized with rhodamine-phalloidin and fluorescent anti-vinculin antibodies, respectively. Cells were imaged with confocal microscopy. Actin stress fiber density, edge actin bundle density, focal adhesion density, cellular area and circularity (a morphological descriptor relating area and perimeter) were quantified by a program developed with Matlab software. GGTI-2166, Y-27632, and alendronate reduced actin stress fibers, FA density, and FA size, but had no effect on edge actin bundle density, cellular area, or circularity. GGOH completely antagonized the effects of alendronate, but did not significantly affect responses to GGTI-2166 or Y-27632. Constitutively active RhoA antagonized the effects of alendronate and GGTI-2166, but not those of Y-27632. The effects of alendronate were also antagonized by Nleu(8,18) Tyr(34) PTH 3-34 amide, but not by PTH 1-34. The results indicate that RhoA is involved in the maintenance of stress fibers and focal adhesions in osteoblastic cells, that PTH can affect this pathway independently of cAMP, and that a N-containing bisphosphonate can affect the actin cytoskeleton and focal adhesions through actions on geranylgeranyl groups and potentially through RhoA. In view of the importance of the actin cytoskeleton, the findings constitute evidence that N-containing bisphosphonates, when they attain certain concentrations, have effects on osteoblasts that could influence bone remodeling.
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
Nikolas H
NH
Kazmers
Sophia A
SA
Ma
Tomohiko
T
Yoshida
Paula H
PH
Stern
D000199:Actins / Q000378:metabolism*; D019386:Alendronate / Q000494:pharmacology; D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D004224:Diterpenes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D021621:Imaging, Three-Dimensional; D051379:Mice; D008954:Models, Biological; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
D000199:Actins; D000577:Amides; D004164:Diphosphonates; D004224:Diterpenes; D004791:Enzyme Inhibitors; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011725:Pyridines; C108830:Y 27632; C031617:parathyroid hormone (3-34); C017338:geranylgeraniol; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins; D019386:Alendronate