A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation - PubMed (original) (raw)

A beta-arrestin-biased agonist of the parathyroid hormone receptor (PTH1R) promotes bone formation independent of G protein activation

Diane Gesty-Palmer et al. Sci Transl Med. 2009.

Erratum in

Abstract

About 40% of the therapeutic agents in use today exert their effects through seven-transmembrane receptors (7TMRs). When activated by ligands, these receptors trigger two pathways that independently transduce signals to the cell: one through heterotrimeric GTP-binding proteins (G proteins) and one through beta-arrestins; so-called biased agonists can selectively activate these distinct pathways. Here, we investigate selective activation of these pathways through the use of a biased agonist for the type 1 parathyroid hormone (PTH)-PTH-related protein receptor (PTH1R), (D-Trp(12),Tyr(34))-PTH(7-34) (PTH-betaarr), which activates beta-arrestin but not classic G protein signaling. In mice, PTH-betaarr induces anabolic bone formation, as does the nonselective agonist PTH(1-34), which activates both mechanisms. In beta-arrestin2-null mice, the increase in bone mineral density evoked by PTH(1-34) is attenuated and that stimulated by PTH-betaarr is ablated. The beta-arrestin2-dependent pathway contributes primarily to trabecular bone formation and does not stimulate bone resorption. These results show that a biased agonist selective for the beta-arrestin pathway can elicit a response in vivo distinct from that elicited by nonselective agonists. Ligands with these properties may form the basis for improved 7TMR-directed pharmacologic agents with enhanced therapeutic specificity.

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Conflict of interest statement

Competing interests: R.J.L. is a founder and member of the Scientific Advisory Board for Trevena, Inc., a company that discovers and develops novel G protein–coupled receptor–targeted medicines. R.J.L., D.G.-P., and L.M.L. have filed a patent related to the results reported in this paper.

Figures

Fig. 1

Fig. 1

PTH-βarr stimulates β-arrestin–mediated ERK1/2 activation, independent of G protein signaling, in osteoblasts. (A) cAMP activation in response to PTH(1–34) and PTH-βarr stimulation of endogenously expressed PTH1R in POBs isolated from β-arrestin2−/− and WT C57BL/6 mice. cAMP values were normalized to 10 μM forskolin–induced concentrations (2.24 ± 0.2 μM). Data correspond to the mean ± SEM from four independent experiments. ***P < 0.001 compared with the vehicle-stimulated WT POBs. †††P < 0.001; ††P < 0.01 compared with the vehicle-stimulated β-arrestin2−/− POBs; direct comparisons were made with two-tailed unpaired t test. Veh, vehicle. (B) PTH(1–34) and PTH-βarr stimulated ERK1/2 activation in POBs isolated from β-arrestin2−/− and WT C57BL/6 mice. Values presented are the fold ERK1/2 phosphorylation over vehicle-stimulated controls. Data represent the mean ± SEM from four independent experiments. **P < 0.01 compared with the vehicle-stimulated WT POBs. ††P < 0.01 compared with the vehicle-stimulated β-arrestin2−/− POBs; direct comparisons were made with two-tailed unpaired t test.

Fig. 2

Fig. 2

PTH-βarr increases lumbar spine BMD. (A and B) Lumbar spine and (C and D) femoral BMD of male WT and β-arrestin2−/− mice treated with vehicle (Veh), 1-34), or PTH-βarr was determined by dual-energy x-ray absorption. Mice were treated starting at 9 weeks of age. Data represent the mean percent change from baseline BMD ± SEM of measurements taken from at least seven male mice. *P < 0.05; **P < 0.01 compared with vehicle-treated WT mice. †P < 0.05, ††P < 0.01, †††P < 0.001 compared with vehicle-treated β-arrestin2−/− mice; significance was determined with one-way ANOVA with Bonferroni multiple comparisons post test.

Fig. 3

Fig. 3

β-Arrestin2–dependent signaling contributes to increases in trabecular but not cortical bone. (A) Representative qCT of lumbar vertebrae isolated from male WT and β-arrestin2−/− mice treated for 8 weeks with vehicle, PTH(1–34), or PTH-βarr. Scale bar, 1.0 mm. Mice were treated starting at 9 weeks of age. qCT of the lumbar spine was used to determine the effects on (B) trabecular bone (Tb) volume fraction (BV/TV), (C) Tb thickness, and (D) Tb number. (E) Representative qCT of proximal tibia from male WT and β-arrestin2−/− mice treated for 8 weeks with vehicle, PTH(1–34), or PTH-βarr. Scale bar, 1.0 mm. qCT of proximal tibia was used to determine the effects on (F) Tb volume fraction (BV/TV), (G) Tb thickness, and (H) Tb number. qCT of the mid-femoral shaft was used to determine (I) periosteal circumference and (J) cortical thickness. Data represent the mean ± SEM of measurements taken from at least seven male mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle-treated WT mice. †P < 0.05; ††P < 0.01; †††P < 0.001 compared with vehicle-treated β-arrestin2−/− mice; significance was determined with one-way ANOVA with Bonferroni multiple comparisons post test.

Fig. 4

Fig. 4

Bone histomorphometry and dynamic indices of bone formation in WT and β-arrestin2−/− mice. (A) Representative nondecalcified, 5-μm sections of lumbar vertebrae isolated from male WT and β-arrestin2−/− mice treated at 9 weeks of age for 8 weeks with vehicle, PTH(1–34), or PTH-βarr. Scale bar, 100 μm. ob, osteoblasts; oc, osteoclasts; os, osteoid. Quantitated histomorphometric analysis of (B) OBS, (C) OS, and (D) OCS after treatment with either vehicle, PTH(1–34), or PTH-βarr. Data represent the mean ± SEM of measurements from four mice. (E) Representative calcein double-labeled, nondecalcified, 10-μm sections of lumbar vertebrae isolated from male WT and β-arrestin2−/− mice treated for 8 weeks with either vehicle, PTH, or PTH-βarr. Scale bar, 100 μm. Bone growth is determined by measuring the distance between calcein-labeled layers (arrows). Quantitation of the (F) mineral apposition rate and (G) bone formation rates from calcein-labeled trabecular bone. Data represent the mean ± SEM of measurements from four mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle-treated mice. †P < 0.05; ††P < 0.01; †††P < 0.001 compared with vehicle-treated β-arrestin2−/− mice; significance determined with one-way ANOVA with Bonferroni correction.

Fig. 5

Fig. 5

PTH-βarr increases serum osteocalcin but has no effect on urine DPD excretion. Osteocalcin was measured in serum obtained from male (A) WT and (B) β-arrestin2−/− mice after 4 and 8 weeks of treatment with vehicle, PTH(1–34), or PTH-βarr. Twenty-four-hour urine DPD was measured in male (C) WT and (D) β-arrestin2−/− mice after 4 and 8 weeks of treatment with vehicle, PTH(1–34), or PTH-βarr. Mice were treated starting at 9 weeks of age. Data represent the mean ± SEM of measurements taken from at least seven male mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle-treated WT mice. †P < 0.05; ††P < 0.01; †††P < 0.001 compared with vehicle-treated β-arrestin2−/− mice; significance was determined with one-way ANOVA with Bonferroni multiple comparisons post test.

Fig. 6

Fig. 6

PTH-βarr induces β-arrestin–dependent expression of osteocalcin but not RANKL or OPG. mRNA was isolated from the calvaria of male WT and β-arrestin2−/− mice treated with vehicle, PTH(1–34), or PTH-βarr, and quantitative RT-PCR was used to determine relative gene expression of protein modulators of bone metabolism: (A) osteocalcin, (B) RANKL, and (C) OPG. Data represent the mean ± SEM from at least six mice. *P < 0.05; **P < 0.01; ***P < 0.001 compared with vehicle-treated WT mice. †P < 0.05; †††P < 0.001 compared with vehicle-treated β-arrestin2−/− mice; significance was determined with one-way ANOVA with Bonferroni multiple comparisons post test.

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