Anabolic effects of a G protein–coupled receptor kinase inhibitor expressed in osteoblasts
Robert F. Spurney, … , Farshid Guilak, L. Darryl Quarles
Robert F. Spurney, … , Farshid Guilak, L. Darryl Quarles
Published May 15, 2002
Citation Information: J Clin Invest. 2002;109(10):1361-1371. https://doi.org/10.1172/JCI14663.
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Article Endocrinology

Anabolic effects of a G protein–coupled receptor kinase inhibitor expressed in osteoblasts

Abstract

G protein–coupled receptors (GPCRs) play a key role in regulating bone remodeling. Whether GPCRs exert anabolic or catabolic osseous effects may be determined by the rate of receptor desensitization in osteoblasts. Receptor desensitization is largely mediated by direct phosphorylation of GPCR proteins by a family of enzymes termed GPCR kinases (GRKs). We have selectively manipulated GRK activity in osteoblasts in vitro and in vivo by overexpressing a GRK inhibitor. We found that expression of a GRK inhibitor enhanced parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor-stimulated cAMP generation and inhibited agonist-induced phosphorylation of this receptor in cell culture systems, consistent with attenuation of receptor desensitization. To determine the effect of GRK inhibition on bone formation in vivo, we targeted the expression of a GRK inhibitor to mature osteoblasts using the mouse osteocalcin gene 2 (OG2) promoter. Transgenic mice demonstrated enhanced bone remodeling as well as enhanced urinary excretion of the osteoclastic activity marker dexoypyridinoline. Both osteoprotegrin and OPG ligand mRNA levels were altered in calvaria of transgenic mice in a pattern that would promote osteoclast activation. The predominant effect of the transgene, however, was anabolic, as evidenced by an increase in bone density and trabecular bone volume in the transgenic mice compared with nontransgenic littermate controls.

Authors

Robert F. Spurney, Patrick J. Flannery, Sanford C. Garner, Krairerk Athirakul, Shiguang Liu, Farshid Guilak, L. Darryl Quarles

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Figure 3

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Transgene construction and expression in ROS 17/2.8 cells as well as in ...
Transgene construction and expression in ROS 17/2.8 cells as well as in mouse tissues. (a) The GRK2-CT transgene containing the 1.3-kb mouse OG2 promoter, the GRK2-CT, and the human β-globulin polyadenylation signal. Also shown are the approximate locations of the PCR primers (primer 1 and primer 2). (b) ROS 17/2.8 cells were transfected with a mammalian expression vector containing our transgene and a neomycin-resistant cassette. The constructs was designed so that GRK2-CT expression was driven solely by the OG2 promoter (see Methods). Following G418 selection, GRK2-CT expression was investigated by immunoblotting. The GRK2-CT was expressed by ROS 17/2.8 cells transfected with the vector containing our transgene, but not in cells transfected with empty vector. Apparent molecular mass is indicated in kDa. (c and d) PCR or RT-PCR was performed using total cellular RNA prepared from the indicated mouse tissues as described in Methods. Molecular size is indicated in base pairs. (c) A PCR product of the appropriate size was detected in bone from transgenic mice when an RT reaction was performed prior to PCR. A small amount of GRK2-CT PCR product was also detected in the brain, as has been reported by other investigators (40). (d) No GRK2-CT PCR products were detected in tissues from nontransgenic littermate controls. Control PCR reactions revealed a PCR product of the appropriate size in all tissues using the GAPDH primers when an RT reaction was performed prior to PCR.