Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts - PubMed (original) (raw)

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

Robert F Spurney et al. J Clin Invest. 2002 May.

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.

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Figures

Figure 1

Expression of the GRK2-CT enhances PTH/PTHrP receptor responsiveness and inhibits agonist-induced phosphorylation of the PTH/PTHrP receptor. HEK293 cells were cotransfected with the rat PTH/PTHrP receptor cDNA and either the GRK2-CT or empty vector. Two days after transfection, expression of GRK2 and the GRK2-CT was assessed by immunoblotting as described in Methods using an Ab that recognizes the C terminus of both GRK2 and GRK3 (25). In parallel experiments, we investigated the effect of the GRK2-CT on PTH/PTHrP receptor responsiveness by measuring cAMP generation and agonist-induced phosphorylation of the PTH/PTHrP receptor by immunoprecipitation of 12CA5-tagged PTH/PTHrP receptors, as described in Methods. Apparent molecular mass is indicated in kilodaltons. (a) In lane 1, HEK293 cells express predominantly GRK2. A slightly smaller protein corresponding to the GRK3 was detected by immunoblotting only after prolonged exposure of the radiographic film. Lane 2 is a positive control prepared from HEK293 cells transfected with the GRK2 cDNA. (b) Cotransfection of the GRK2-CT significantly enhanced PTH/PTHrP receptor responsiveness compared with cells cotransfected with empty vector. The inset shows that the increase in PTH/PTHrP receptor responsiveness was associated with high levels of GRK2-CT expression in this model system. (c) Cotransfection of the GRK2-CT inhibited agonist-induced phosphorylation of the PTH/PTHrP receptor. (d) The amount of PTH/PTHrP receptor in the immunoprecipitates was assessed by immunoblotting using the 12CA5 Ab. Similar amounts of PTH/PTHrP receptor were immunoprecipitated from cells cotransfected with either the GRK2-CT or empty vector. *P < 0.05 vs. vector.

Figure 2

Expression of the GRK2-CT enhances PTH/PTHrP receptor responsiveness in cells that express an endogenous PTH/PTHrP receptor. The GRK2-CT was expressed in ROS 17/2.8 cells using a retroviral system as described in Methods. Two days after infection, expression of GRK2, GRK3, and the GRK2-CT was assessed by immunoblotting as described in Methods, using an Ab that recognizes the C terminus of both GRK2 and GRK3 (25). In parallel experiments, cAMP generation was measured to determine the effect of the GRK2-CT on PTH/PTHrP receptor responsiveness. (a) In lane 1, ROS 17/2.8 cells express GRK2 and lesser amounts of GRK3. Lane 2 and lane 3 are positive controls prepared from HEK293 cells transfected with either the GRK2 cDNA (lane 2) or the GRK3 cDNA (lane 3). (b) The GRK2-CT significantly enhanced PTH/PTHrP receptor responsiveness compared with cells infected with empty vector. (c) The increase in PTH/PTHrP receptor responsiveness was associated with high levels of GRK2-CT expression in this model system. *P < 0.05 vs. vector.

Figure 3

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.

Figure 4

Bone histomorphology in transgenic mice and in nontransgenic littermate controls. Mice were given an intraperitoneal injection of tetracycline HCl followed by an injection of calcein on days 3 and 8 prior to sacrifice, respectively. After harvesting, nondecalcified sections of tibias were prepared for histomorphologic analysis as described in Methods. (a and b) Representative 5-μm sections of trabecular bone visualized under transmitted light from nontransgenic controls and transgenic mice, respectively. (c and d) Representative 10-μm sections of trabecular bone visualized under fluorescent light from control animals and transgenic mice, respectively. Transgenic mice demonstrated an increase in trabecular thickness compared with nontransgenic littermate controls as indicated by the distance between the black arrowheads in a and b. Under fluorescent light, there was an increase in the distance between the fluorescent labels in transgenic mice compared with control animals as indicated by the distance between the white arrowheads in c and d.

Figure 5

Effect of the transgene on urinary excretion of DPD and expression OPG and OPGL mRNA in mouse calvaria. (a) Excretion of DPD in urine was measured as an index of osteoclast-mediated bone resorption in vivo (40). Excretion of urinary DPD was significantly enhanced in transgenic (TG) mice compared with nontransgenic (Non-TG) littermate controls. (b–d) Total cellular RNA was prepared from mouse calvaria prior to performing semiquantitative RT-PCR as described in Methods. Molecular size is indicated in base pairs. (b) The OPG PCR product was decreased in transgenic mice compared with nontransgenic littermate controls. (c) The OPGL PCR product was increased in transgenic animals compared with control animals. The GAPDH control PCR reaction shown in c confirmed that the RT reaction was successful in the animals studied. *P < 0.025 vs. nontransgenic littermate controls.

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Anabolic effects of a G protein-coupled receptor kinase inhibitor expressed in osteoblasts - PubMed (original) (raw)