β-Arrestin2 Regulates the Differential Response of Cortical and Trabecular Bone to Intermittent PTH...

β-Arrestin2 Regulates the Differential Response of Cortical andTrabecular Bone to Intermittent PTH in Female Mice

Mary L Bouxsein1, Dominique D Pierroz2, Vaida Glatt1, Deborah S Goddard1, FannyCavat2, René Rizzoli2, and Serge L Ferrari21Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center and Harvard MedicalSchool, Boston, Massachusetts, USA

2Service of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva UniversityHospital, Geneva, Switzerland

AbstractCytoplasmic arrestins regulate PTH signaling in vitro. We show that female β-arrestin2-/- mice havedecreased bone mass and altered bone architecture. The effects of intermittent PTH administrationon bone microarchitecture differed in β-arrestin2-/- and wildtype mice. These data indicate thatarrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTHat endosteal and periosteal bone surfaces.

Introduction: The effects of PTH differ at endosteal and periosteal surfaces, suggesting that PTHactivity in these compartments may depend on some yet unidentified mechanism(s) of regulation.The action of PTH in bone is mediated primarily by intracellular cAMP, and the cytoplasmic moleculeβ-arrestin2 plays a central role in this signaling regulation. Thus, we hypothesized that arrestins wouldmodulate the effects of PTH on bone in vivo.

Materials and Methods: We used pDXA, μCT, histomorphometry, and serum markers of boneturnover to assess the skeletal response to intermittent PTH (0, 20, 40, or 80 μg/kg/day) in adultfemale mice null for β-arrestin2 (β-arr2-/-) and wildtype (WT) littermates (7-11/group).

Results and Conclusions: β-arr2-/- mice had significantly lower total body BMD, trabecular bonevolume fraction (BV/TV), and femoral cross-sectional area compared with WT. In WT females, PTHincreased total body BMD, trabecular bone parameters, and cortical thickness, with a trend towarddecreased midfemoral medullary area. In β-arr2-/- mice, PTH not only improved total body BMD,trabecular bone architecture, and cortical thickness, but also dose-dependently increased femoralcross-sectional area and medullary area. Histomorphometry showed that PTH-stimulated periostealbone formation was 2-fold higher in β-arr2-/- compared with WT. Osteocalcin levels weresignificantly lower in β-arr2-/- mice, but increased dose-dependently with PTH in both β-arr2-/- andWT. In contrast, whereas the resorption marker TRACP5B increased dose-dependently in WT, 20-80μg/kg/day of PTH was equipotent with regard to stimulation of TRACP5B in β-arr2-/-. In summary,β-arrestin2 plays an important role in bone mass acquisition and remodeling. In estrogen-repletefemale mice, the ability of intermittent PTH to stimulate periosteal bone apposition and endostealresorption is inhibited by arrestins. We therefore infer that arrestin-mediated regulation ofintracellular signaling contributes to the differential effects of PTH on cancellous and cortical bone.

Address reprint requests to: Mary L Bouxsein, PhD, Orthopedic Biomechanics Laboratory, RN115 Beth Israel Deaconess Medical Center,330 Brookline Avenue, Boston, MA 02215, USA, E-mail: [email protected] authors have no conflict of interest.

NIH Public AccessAuthor ManuscriptJ Bone Miner Res. Author manuscript; available in PMC 2006 October 2.

Published in final edited form as:J Bone Miner Res. 2005 April ; 20(4): 635–643.

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Keywordsβ-arrestin; PTH; knockout; bone architecture; bone remodeling

INTRODUCTIONINTERMITTENT ADMINISTRATION OF an amino-terminal fragment of PTH(1-34) increases hip, spine, andtotal body BMD, improves iliac trabecular bone microarchitecture, and reduces vertebral andnonvertebral fracture risk in postmenopausal osteoporotic women.(1-3) However, the effectsof PTH on cortical bone remain controversial.(1,4-7) Notably, the antifracture efficacy of PTHanalogs at skeletal sites comprised predominantly of cortical bone, such as the hip and distalradius, is not known. Indeed, BMD of the radial shaft declined after a median of 21 months oftreatment with intermittent PTH(1-34).(1,8) Similarly, volumetric BMD of the femoral neckcortex declined after 1 year of intermittent PTH(1-84) administration.(6)

These declines in cortical BMD may be offset by apposition of new bone on the periostealsurface(8-11); however, the ability of intermittent PTH to induce periosteal bone apposition atclinically relevant doses in humans or monkeys remains controversial. Longitudinal QCTmeasurements revealed no increase in femoral neck or vertebral cross-sectional area afteradministration of PTH(1-84) for 1 year (D Black, personal communication, 2004). Moreover,iliac crest biopsies taken before and after intermittent administration of hPTH(1-34) in womentreated concurrently with estrogen showed increased cortical thickness, which was attributedto endocortical bone apposition, but there was no change in bone formation rate on thesubperiosteal surface.(2) The cortical bone response to intermittent PTH in nonhuman primatesalso revealed marked endosteal bone apposition accompanied by increased intracorticalporosity, principally near the endocortical surface.(5,12) In these studies, where PTH wasadministered at doses equal to and five times greater than that used in human clinical trials, nosignificant increase in total cross-sectional area of the proximal humerus or femoral neck wasreported.

In contrast, other studies have reported that intermittent PTH administration induces periostealbone formation. For example, vertebral cross-sectional area, assessed by QCT, increased afterintermittent PTH administration in individuals with steroid-induced osteoporosis.(13) Inaddition, a cross-sectional study reported a greater periosteal circumference at the distal radiusin postmenopausal women treated with PTH(1-34) compared with those who receivedplacebo.(8)

Rabbits, rats, and mice also predominantly exhibit endosteal bone apposition in response tointermittent PTH. However, in contrast to humans and primates, they frequently exhibitincreased periosteal bone apposition as well.(9,10,14) Altogether, the factors that governperiosteal bone formation in response to intermittent PTH are poorly understood, althoughclinical observations and animals studies suggest it may depend on the dose and duration ofPTH exposure.(14-17) Improved knowledge regarding the mechanisms that determine the abilityof intermittent PTH to induce bone formation on periosteal surfaces is important becauseperiosteal bone apposition is theoretically an important mechanism to improve whole bonestrength.(18)

The biological actions of PTH in bone are mediated by the PTH/PTH-related peptide (PTHrP)receptor (PTH1R), a G protein-coupled receptor (GPCR) that signals through both the cAMPand IP3/iCa pathways. In turn, stimulation of cAMP signaling seems to play a fundamentalrole in mediating the biologic activity of PTH in bone.(19-21) Activity of PTH1R and its agonistsin vitro is in part regulated by cytoplasmic arrestins, namely β-arrestin1 (β-arr1) and β-arrestin2(β-arr2), through several mechanisms. Arrestins promote rapid endocytosis of ligand-receptor

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complexes and inhibit cAMP signaling in response to agonists.(22) These processes allow forrapid desensitization and later resensitization to PTH stimulation in vitro.(23,24) Additionalmechanisms whereby arrestins may influence GPCR signaling include their ability to activatecAMP phosphodiesterase (PDE), thereby promoting degradation of intracellular cAMP,(25)

and to serve as scaffolds linking GPCRs to other signaling proteins, including the scr-familykinases and members of the mitogen-activated protein kinase (MAPK) family.(22) Becausereceptor desensitization and resensitization modulate the cellular responses to both acute andchronic stimulation, an important implication is that not only signal transduction per se, butalso the mechanisms regulating signal transduction may influence the physiological processesmediated by GPCRs, including PTH1R. In support of this view, mice null for β-arr2, whichappear normal and are viable and fertile, exhibit a sustained response to opiate receptoragonists.(26,27)

Arrestins are expressed in osteoblasts.(28,29) Hence, we hypothesized that arrestins may playa role in regulating the anabolic effects of PTH on the skeleton. More specifically, wehypothesized that, in the absence of β-arr2, remodeling of endosteal and periosteal bonesurfaces in response to PTH would be increased. To test this hypothesis, we evaluated bonemass and architecture of adult β-arr2 null and wildtype female mice in response to increasingdoses of intermittent hPTH(1-34).

MATERIALS AND METHODSAnimals

β-arr2 null mice (β-arr2-/-) were initially generated by Bohn et al.(26) and subsequentlybackcrossed six generations onto a C57BL/6J background, resulting in mice whose geneticcomposition was >98% C57BL/6J. At the N6 generation, mice were genotyped using PCRanalysis of tail DNA, and separately bred as homozygous wildtype (WT) or β-arr2-/- for thedescribed experiments. Mice were maintained under standard nonbarrier conditions and hadaccess to mouse chow (Harlan Teklad 5542, 2.5% Ca, 1.2% Pi) and water ad libitum. Calcein(15 mg/kg) was injected subcutaneously 9 and 2 days before death. All animal procedures wereapproved by the ethics committees on animal care and use at Beth Israel Deaconess MedicalCenter, Boston, MA.

Intermittent PTHWe tested the response to intermittent PTH administration in β-arr2-/- and WT mice byassigning 13-week old, intact female β-arr2-/- and WT mice to receive either vehicle or one ofthree PTH doses (20, 40, or 80 μg/kg/day, n = 7-11/group per genotype). Synthetic human PTH(1-34) (Bachem, Torrance, CA, USA) was dissolved in a vehicle of acidified saline (0.1N) and2% heat inactivated mouse serum. Mice received subcutaneous injections of vehicle or PTH5 days/week for 4 weeks. Body weight was measured weekly, and the dose was adjustedaccordingly. After 4 weeks, blood was collected retro-orbitally, mice were killed by CO2inhalation, and the fifth lumbar vertebrae and right femurs were collected for μCT andhistomorphometric evaluation.

Measurement of BMD, morphology, and microarchitectureTotal body BMD (TBBMD, g/cm2) was measured in vivo at baseline and at 4 weeks byperipheral DXA (PIXImus; GE Lunar, Madison, WI, USA).(30-32) μCT (UCT40; ScancoMedical AG, Basserdorf, Switzerland) was used to assess trabecular bone volume fraction andmicroarchitecture in the excised fifth lumber vertebral body and distal femur (12-μm isotropicvoxels), and cortical bone geometry at a 1-mm thick section of the midfemoral diaphysis (34μm isotropic voxels). For the vertebral trabecular region, we evaluated ∼300 transverse CTslices between the cranial and caudal end plates, excluding 100 μm near each endplate. Femoral



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cortical geometry was assessed in a 1-mm-long region centered at the femoral midshaft. CTimages were reconstructed in 1024 × 1024 pixel matrices using a standard convolution-backprojection procedure, and the resulting gray-scale images were segmented using aconstrained 3D Gaussian filter (σ = 0.8, support = 1.0) to remove noise, and a fixed threshold(22% and 30% of maximal gray scale value for trabecular bone and cortical bone, respectively)was used to extract the structure of mineralized tissue. Morphometric variables were computedfrom the binarized images using direct, 3D techniques that do not rely on any prior assumptionsabout the underlying structure.(33-35) For trabecular bone regions, we assessed the bone volumefraction (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, mm-1), andtrabecular separation (Tb.Sp, μm). For cortical bone at the femoral midshaft, we measured theaverage total cross-sectional area inside the periosteal envelope (CSA, mm2), the cortical bonearea and medullary area within this same envelope (BA, mm2 and MA, mm2, respectively),the average cortical thickness (CortTh, μm), and the area moment of inertia about the medio-lateral (Iml, mm4) and antero-posterior axes (Iap, mm4).

Bone histomorphometry: Femurs were dehydrated in graded ethanol and embedded inmethylmethacrylate. Five- and 8-μm-thick sagittal sections were cut with a Polycut Emicrotome (Leica Corp. Microsystems AG, Glattbrugg, Switzerland) and stained withmodified Goldner’s trichrome (5-μm sections) for assessment of static histomorphometricvariables or left unstained (8-μm sections) for assessment of calcein fluorescence and dynamicindices of bone formation. Histomorphometric measurements were performed on thesecondary spongiosa of the distal femoral metaphysis (beginning 690 μm proximal to thegrowth plate and extending 344 μm proximally) and on the endocortical and cortical bonesurfaces (beginning 2.8 mm proximal to the growth plate and extending for 1.1 mm proximally)using a Leica Q image analyzer at 40× magnification. All parameters were calculated andexpressed according to standard formulas and nomenclatures.(36)

Serum biochemistry and bone turnover markers: Serum concentration of calcium (Ca) andinorganic phosphate (Pi) was measured using atomic spectrometry and colorimetric methods,respectively, as previously described.(37) Serum osteocalcin (OC) was measured by RIA witha goat anti-mouse osteocalcin antibody and donkey anti-goat secondary antibody (BiomedicalTechnologies, Stoughton, MA, USA). Serum TRACP-5b was measured according tomanufacturer’s instructions (SBA Sciences, Turku, Finland).

Data analysisStandard descriptive statistics were computed, and data were checked for normality. Repeated-measures ANOVA was used to assess the longitudinal data from pDXA. A two-factor ANOVAwas used to assess the effect of PTH dose and β-arr deficiency on skeletal morphology. Asappropriate, posthoc testing was performed using Fisher’s protected least squares difference(PLSD) or unpaired Student’s t-tests. All tests were two-tailed, with differences consideredsignificant at p < 0.05. Data are presented as mean ± SE, unless otherwise noted.

RESULTSTotal body BMD in vivo

At baseline (i.e., 13 weeks of age), female β-arr2-/- mice had modestly but significantly lowertotal body BMD compared with WT (47.9 ± 0.5 versus 50.0 ± 0.3 mg/cm2, p = 0.0008). Totalbody BMD increased markedly in response to PTH in both WT and β-arr2-/- mice (+7.3% to10.1% versus baseline, p < 0.005 for all, Fig. 1). Total body BMD tended to increase dose-dependently in WT (p = 0.07 for PTH40 versus PTH20 and p = 0.10 for PTH80 versus PTH20).In contrast, PTH was equipotent with regard to increasing total body BMD in β-arr2-/- (Fig.1).



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Trabecular bone volume fraction and microarchitectureIn both the lumbar vertebral body and distal femoral metaphysis, VEH-treated β-arr2-/- hadlower trabecular BV/TV (-12.7%, p = 0.03 and -26.7%, p = 0.02, respectively), and number(-7.9%, p = 0.02 and -11.4%, p = 0.02, respectively) than WT, although trabecular thicknesswas similar (Fig. 2). Intermittent PTH significantly increased vertebral trabecular BV/TV(+17% to 38%), thickness (9.4% to 14.1%), and number above VEH in both β-arr2-/- and WTmice (Figs. 2A-2C). At the distal femur, PTH80 maximally increased trabecular BV/TV andthickness in both genotypes, whereas the lower PTH doses induced significant gains in β-arr2-/- mice only (Figs. 2D-2F).

Femoral cortical boneMidfemoral cross-sectional area (-13.7%, p < 0.0001), cortical bone area (-14.1%, p < 0.0001),medullary area(-13.5%, p = 0.0002), and cortical thickness (-6.5%, p = 0.0043) were all lowerin VEH-treated β-arr2-/- compared with WT mice (Figs. 3A-3D). Accordingly, the area momentof inertia about the medio-lateral and antero-posterior axes were also lower in β-arr2-/- thanWT mice (-26.8% and -18.5%, respectively, p < 0.0005 for both).

The cortical bone response to intermittent PTH at the femoral midshaft differed dramaticallybetween β-arr2-/- and WT mice (pinteraction for genotype × treatment = 0.02-0.005, Fig. 3). β-arr2-/- exhibited a consistent dose-dependent anabolic response to PTH, whereas correspondingchanges were smaller or absent in WT (Fig. 3). The most prominent differences were seen incross-sectional area and medullary area, which both increased dose-dependently with PTH inβ-arr2-/- mice. In contrast, cross-sectional area was unchanged and medullary area decreasedin WT mice. Accordingly, the area moments of inertia about the medio-lateral and antero-posterior axes increased significantly in PTH-treated β-arr2-/- mice, but not WT (+22.4%, p =0.0034 and +34%, p < 0.0001, respectively, all doses combined).

HistomorphometryDynamic histomorphometry data were consistent with the μCT observations of trabecular andcortical bone morphology. In cancellous bone, there was a trend for lower mineralizing surfaceand bone formation rate in VEH-treated β-arr2-/- than WT mice (p = 0.06 and 0.07,respectively), potentially explaining their significantly lower BV/TV. Indices of boneformation, as well as osteoblast and osteoclast surfaces, increased with PTH treatment in bothβ-arr2-/- and WT (Table 1), consistent with the observed response in BV/TV (Fig. 2). At theendocortical surface, PTH increased osteoclastic number and surface increased significantlyin β-arr2-/-, with a nonsignificant trend to increase in WT. In comparison, endocorticalosteoblast indices and bone formation rate increased more markedly, although not significantly,in WT compared with β-arr2-/-. This may potentially explain why medullary area decreased inWT, but increased in β-arr2-/- (Fig. 3). On the periosteal surface, no calcein double-labeledsurfaces were detected in VEH-treated mice of either genotype. PTH treatment led tomeasurable double-labeled periosteal surfaces, with mineral apposition and bone formationrates that were twice as high in β-arr2-/- than WT mice (Table 1). This overall pattern of corticalbone response to PTH is exemplified in Fig. 4, which shows extensive PTH-stimulated calceindouble-labeling on the endocortical surface in WT, with little evidence of bone formation onthe periosteal surface. In stark contrast, PTH-stimulated calcein double-labeling is seenpredominantly on the periosteal surface in β-arr2-/-, with limited evidence of endocortical boneformation, but rather a region of bone resorption. Altogether these histomorphometric data areconsistent with the observed increase in cross-sectional area and medullary expansion in PTH-treated β-arr2-/- mice.



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Serum biochemistry and markers of bone turnoverAt baseline, there was no difference in serum Ca or Pi levels between β-arr2-/- and WT mice,and these levels remained stable after PTH treatment (data not shown). In contrast, baselineOC values were slightly but significantly lower in β-arr2-/- mice (62.9 ± 2.6 versus 72.8 ± 3.3ng/ml, p = 0.019), consistent with their lower histomorphometrical indices of bone formation.OC levels declined in the VEH-treated groups, whereas PTH20 and PTH40 inhibited thisdecline, and PTH80 significantly increased osteocalcin compared with baseline in both β-arr2-/- and WT mice (Table 2). Baseline values of serum TRACP-5b, a marker of boneresorption, did not differ between β-arr2-/- and WT. PTH led to a dose-dependent increase inTRACP-5b in WT, but all PTH doses were equipotent with regard to increasing TRACP-5bin β-arr2-/- mice (Table 2).

DISCUSSIONIn this study, we tested the hypothesis that mice null for β-arr2 would have altered bonemodeling/remodeling and a different response to increasing doses of intermittent PTHcompared with wildtype mice. We found that adult female β-arr2-/- mice have a lower totalbody BMD, trabecular bone volume, and femoral cross-sectional area compared with WT.These observations indicate that arrestins influence normal bone mass acquisition (modeling)and/or maintenance (remodeling). Importantly, we also found that arrestins restrain theanabolic effects of PTH on cortical bone. Specifically, μCT measurements showed that, in theabsence of β-arr2, intermittent PTH led to an increase in midfemoral cross-sectional area andmedullary area. These findings were supported further by histomorphometric evidence ofperiosteal bone apposition and endocortical resorption in β-arr2-/- mice. Moreover, serum boneresorption markers suggested that β-arr2-/- mice were more responsive than WT to lower dosesof PTH. Altogether, these observations indicate that PTH-stimulated bone remodeling (andperhaps “renewed modeling”(15)) is increased in absence of β-arr2.

To further explore the relationship between PTH activity and its anabolic effects on bone, it isuseful to examine the endocortical and periosteal response to increasing doses of PTH. Thecortical bone response to PTH was weak in WT mice, with marginally increased corticalthickness at the higher PTH dose because of a trend for endosteal apposition only (i.e.,decreased medullary area, Fig. 3). Considered together with the marked gains in trabecularbone seen at the same PTH doses, these observations suggest that the anabolic effects of PTHon various bone compartments occur at different dose and/or activity “thresholds.” Thus, itseems that a greater PTH dose and/or activity is required to induce bone formation at periostealsurfaces compared with trabecular and endocortical surfaces.(14)

This suggestion is further supported by our observations that intermittent PTH induced dose-dependent increases in midfemoral cross-sectional area, bone area, and medullary area in β-arr2-/- (Fig. 3). Notably, the PTH-induced increase in medullary area in β-arr2-/- was in directopposition to the trend for a PTH-induced decline in medullary area in WT mice and wasconsistent with the lower endocortical bone formation rate and higher osteoclast surface inPTH-treated β-arr2-/- compared with WT. These data suggest that by inhibiting cAMPsignaling, one consequence of the expression of β-arr2 in osteoblasts is the restraint of PTH-induced periosteal bone formation and endocortical bone resorption. It is important to note,however, that our experiment was conducted in estrogen-replete female mice. In view of theestablished inhibitory effects of estrogen on bone remodeling at both endosteal and periostealsurfaces,(38) studies in male mice or ovariectomized females may show different responses toPTH in absence of arrestins. In this regard, despite a similar skeletal phenotype as female β-arr2-/- mice at baseline, in male β-arr2-/- mice, intermittent PTH led to similar cortical bonegains, but reduced trabecular bone gains compared with male WT.(39) Taken together, these



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findings provide evidence that β-arrestin influences bone remodeling, yet they suggest thatgonadal steroids may further modulate the response to PTH in different skeletal compartments.

The finding that, in the absence of intermittent PTH administration, adult β-arr2 knockout micehad significantly lower total body bone mass, trabecular bone parameters, and midfemoralcross-sectional area compared with WT mice, also merits attention. In comparison, transgenicmice expressing a constitutively active PTH1R in osteoblasts also exhibit decreased periostealbone formation, but increased bone formation in the trabecular compartment.(17) Thedifferential effects of sustained cAMP signaling on periosteal and endosteal osteoblastshighlight the complex nature of compartment-specific responses to PTH activity and supportthe notion that factors present in the local bone microenvironment and/or regulated locallyimpact bone cell function. Furthermore, it must be acknowledged that absence of βarr2 mayinfluence many GPCRs and signaling pathways that contribute to skeletal growth andhomeostasis, and thus it is unlikely that increased PTH-stimulated cAMP signaling by itself issufficient to explain the smaller femoral cross-sectional area and decreased trabecular bonevolume in adult β-arr2-/- mice. For example, lower trabecular bone mass (particularly trabecularnumber) and OC levels would be compatible with increased β-adrenergic activity in osteoblastsin the absence of β-arrestins.(40) Moreover, in addition to their known effects on regulatingGPCR signaling, arrestins have also been implicated inregulation of TGF-β and insulin-likegrowth factor (IGF)-I signaling in vitro.(41,42) Coupled with the known effects of TGF-β onskeletal development and the influence of IGF-1 on bone,(43-47) this prompts considerationthat multiple factors may contribute to the observed alterations of bone modeling/remodelingin β-arr2 null mice.

Although the interpretation of different studies in mice is confounded by the fact that theresponse to PTH may vary with the genetic background, age, sex, and hormonal status, ourobservations generally confirm previous reports that intermittent PTH consistently inducesincreased bone mass in the trabecular compartment, whereas its effects on cortical bone aremore variable.(48-52) Few studies have examined skeletal responses to various doses ofintermittent PTH in mice. However, our results are consistent with those reported previouslyin rats. In estrogen-deficient rats treated with 25, 50, or 100 μg/kg hPTH(1-34) or with 12.5,25, 50, or 100 μg/kg of the PTH analog SDZ PTH398, PTH induced a similar rise in cancellousbone area fraction at all doses, whereas trabecular thickness increased and trabecular numbertended to decreased with increasing PTH dose.(14) Moreover, these authors, as well as severalothers, reported that increasing PTH doses and/or activity may be necessary to induce periostealbone apposition.(5,9,10,12,14)

It is tempting to consider the potential implications of our results with regard to developmentof PTH analogs for treatment of osteoporosis and other bone disorders. Normally, binding ofPTH to the PTH1R promotes translocation of β-arrestins from the cytoplasm to the cellmembrane, where they promote rapid endocytosis of ligand-receptor complexes and inhibitcAMP signaling.(23,53) Although speculative, our findings imply that PTH analogs that do notrecruit β-arrestins(54) may have marked effects on bone strength by inducing both trabecularbone gains and periosteal bone apposition at lower doses than would be normally required.Theoretically this could improve the safety profile and risk-to-benefit ratio relative to currentlyused analogs, although this hypothesis would need further testing, particularly in models ofestrogen deficiency.

In conclusion, our results show that arrestins influence not only normal bone modeling and/orremodeling, but also the skeletal response to intermittent PTH. In particular, these findingsindicate that the differential effects of intermittent PTH on trabecular and cortical bone surfacesare attributable in part to the regulation of PTH activity by arrestins.



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ACKNOWLEDGMENTS

The authors thank Fanny Lin and Robert Lefkowitz for providing breeding pairs of β-arrestin2-deficient mice. Fundingfor this project was provided by a Mazess Fellowship from the National Osteoporosis Foundation (DSG), the AmericanFederation for Aging Research (MLB), the NIH Office for Research on Women’s Health (NIAMS AR049265), SwissNational Science Foundation Grant 631-62937 (SLF), and the Roche Research Foundation (DDP). We alsoacknowledge support of the Metabolic Physiology Core of the Diabetes Endocrinology Research Center at Beth IsraelDeaconess Medical Center (NIDDK P30 DK57521).

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FIG. 1.Mean percent change (vs. baseline) in total body BMD in VEH- and PTH-treated β-arr2-/-andWT mice, assessed by peripheral DXA. Error bars represent SE. All increases were significantcompared with baseline values (p < 0.01). *p < 0.05, **p < 0.001 vs. VEH within each genotype.



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FIG. 2.Trabecular bone parameters after intermittent PTH or VEH administration in β-arr2-/- and WTmice, including trabecular bone volume fraction, thickness, and number in the fifth lumbarvertebral body (A, B, C) and distal femoral metaphysis (D, E, F), assessed by μCT. Error barsrepresent SE. *p < 0.05, **p < 0.005 vs. vehicle within each genotype; #p < 0.05 β-arr2-/- vs.WT within VEH-treated group.



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FIG. 3.Mean values for midfemoral (A) total cross-sectional area (mm2), (B) cortical bone area(mm2), (C) medullary area (mm2), and (D) cortical thickness (μm) in VEH- and PTH-treatedWT and β-arr2-/- mice, assessed by μCT. Error bars represent SE. *p < 0.05, **p < 0.005 vs.vehicle within each genotype; #p < 0.05 β-arr2-/- vs. WT within the VEH-treated group.



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FIG. 4.Representative histologic image (sagittal section) of femoral cortical bone in WT and β-arr2-/- after administration of intermittent PTH (80 μg/kg/day) or VEH. Note the calceindouble-labeled endocortical surface (ES) in PTH-treated WT and calcein double-labeledperiosteal surface (PS) in β-arr2-/-. Also note the region of bone resorption on the endocorticalsurface in PTH-treated β-arr2-/- (shown by arrows). Magnification, ×200.



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Table 1.Quantitative Histomorphometric Indices at Trabecular, Endocortical, and Periosteal Bone Surfaces in the DistalFemoral Metaphysis of β-arrestin2−/− and WT mice Treated With VEH or PTH (80 mg/kg/day) for 4 Weeks(Mean ± SE)

β-arrestin2−/− WT

VEH PTH80 VEH PTH80

Trabecular OcS/BS (%) 6.00 ± 1.27 10.25 ± 0.81

* 6.82 ± 2.58 9.84 ± 0.93 NOc/B.Pm (mm−1) 2.7 ± 0.6 4.3 ± 0.4 2.8 ± 1.1 4.1 ± 0.3 ObS/BS (%) 9.36 ± 1.51 14.15 ± 1.72

* 6.82 ± 2.18 11.49 ± 0.62*

NOb/B.Pm (mm−1) 8.0 ± 1.3 12.2 ± 1.4* 5.9 ± 1.6 9.9 ± 0.8

*

MS/BS (%) 26.9 ± 2.9 35.5 ± 3.0 38.6 ± 4.2 44.8 ± 1.4 MAR (μm/day) 1.56 ± 0.09 2.03 ± 0.19 1.54 ± 0.12 1.99 ± 0.06

*

BFR/BS (μm2/μm3/day) 0.42 ± 0.05 0.71 ± 0.08* 0.59 ± 0.06 0.89 ± 0.05

*

Endocortical ObS/BS (%) 2.45 ± 1.09 6.11 ± 1.59

* 1.80 ± 0.70 4.78 ± 1.11 NOc/B.Pm (mm−1) 0.98 ± 0.41 2.41 ± 0.59

* 0.74 ± 0.28 1.96 ± 0.46 ObS/BS (%) 5.9 ± 4.2 6.7 ± 1.7 6.2 ± 4.0 11.1 ± 2.1 Nob/B.Pm (mm−1) 4.9 ± 3.3 5.5 ± 1.4 5.2 ± 3.5 9.3 ± 1.8 MAR (μm/day) 0.55 ± 0.34 0.83 ± 0.06 0.87 ± 0.44 1.11 ± 0.19 BFR/BS (μm2/μm3/day) 0.41 ± 0.34 0.31 ± 0.10 0.45 ± 0.31 0.85 ± 0.31Periosteal MAR (μm/day) 0 0.80 ± 0.14

† 0 0.48 ± 0.23 BFR/BS (μm2/μm3/day) 0 0.93 ± 0.16

† 0 0.39 ± 0.20

Histomorphometric indices were measured using sagittal sections of the distal femoral metaphysis: 0 = unmeasurable; mineral apposition rate (MAR);bone formation rate, surface referent (BFR/BS); osteoclast surface (OcS/BS); osteoclast number (OcN/B.Pm); osteoblast surface (ObS/BS); osteoblastnumber (ObN/B.Pm); and mineralizing surface (MS/BS).

*p < 0.05 and

†p < 0.01 for PTH vs. VEH within a genotype by unpaired t-test or Mann-Whitney U test.


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Table 2.Effect of Intermittent PTH or VEH on OC (ng/ml) and TRACP-5b (U/liter) in β-arrestin2−/− and WT mice (n =5-11/group, mean ± SE)

β-Arrestin2−/− WT

Baseline 4 weeks Baseline 4 weeks

OC VEH 71.3 ± 3.3 49.1 ± 3.6

* 75.3 ± 3.3 55.1 ± 5.0*

20 μg/kg/day 64.3 ± 4.3 63.8 ± 3.9† 73.6 ± 4.4 73.7 ± 3.3

†

40 μg/kg/day 64.4 ± 4.9 74.1 ± 6.1‡ 80.2 ± 5.9 68.9 ± 4.4

80 μg/kg/day 49.1 ± 4.8 87.3 ± 5.3*‡ 57.4 ± 12.5 108.7 ± 13.3

*‡

TRACP-5b VEH 2.3 ± 0.2 6.1 ± 0.4

* 2.8 ± 0.3 7.5 ± 0.7*

20 μg/kg/day 2.1 ± 0.5 18.0 ± 3.9*‡ 2.1 ± 0.2 13.2 ± 1.0

*

40 μg/kg/day 1.7 ± 0.1 17.5 ± 1.3*‡ 1.6 ± 0.1 16.4 ± 3.0

*‡

80 μg/kg/day 2.5 ± 0.2 14.2 ± 1.5*‡ 2.6 ± 0.2 17.0 ± 3.3

*‡

*p < 0.005 vs. baseline by paired t-test.

†p < 0.01 and

‡p < 0.001 vs. vehicle after 4-week treatment with PTH, by ANOVA.


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β-Arrestin2 Regulates the Differential Response of Cortical and Trabecular Bone to Intermittent PTH...

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