The authors have no conflict of interest.
β-Arrestin2 Regulates the Differential Response of Cortical and Trabecular Bone to Intermittent PTH in Female Mice†
Article first published online: 6 DEC 2004
Copyright © 2005 ASBMR
Journal of Bone and Mineral Research
Volume 20, Issue 4, pages 635–643, April 2005
How to Cite
Bouxsein, M. L., Pierroz, D. D., Glatt, V., Goddard, D. S., Cavat, F., Rizzoli, R. and Ferrari, S. L. (2005), β-Arrestin2 Regulates the Differential Response of Cortical and Trabecular Bone to Intermittent PTH in Female Mice. J Bone Miner Res, 20: 635–643. doi: 10.1359/JBMR.041204
- Issue published online: 4 DEC 2009
- Article first published online: 6 DEC 2004
- Manuscript Accepted: 4 NOV 2004
- Manuscript Revised: 29 SEP 2004
- Manuscript Received: 12 MAY 2004
- bone architecture;
- bone remodeling
Cytoplasmic arrestins regulate PTH signaling in vitro. We show that female β-arrestin2−/− mice have decreased bone mass and altered bone architecture. The effects of intermittent PTH administration on bone microarchitecture differed in β-arrestin2−/− and wildtype mice. These data indicate that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH at endosteal and periosteal bone surfaces.
Introduction: The effects of PTH differ at endosteal and periosteal surfaces, suggesting that PTH activity 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 molecule β-arrestin2 plays a central role in this signaling regulation. Thus, we hypothesized that arrestins would modulate the effects of PTH on bone in vivo.
Materials and Methods: We used pDXA, μCT, histomorphometry, and serum markers of bone turnover to assess the skeletal response to intermittent PTH (0, 20, 40, or 80 μg/kg/day) in adult female 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 bone volume fraction (BV/TV), and femoral cross-sectional area compared with WT. In WT females, PTH increased total body BMD, trabecular bone parameters, and cortical thickness, with a trend toward decreased 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 femoral cross-sectional area and medullary area. Histomorphometry showed that PTH-stimulated periosteal bone formation was 2-fold higher in β-arr2−/− compared with WT. Osteocalcin levels were significantly lower in β-arr2−/− mice, but increased dose-dependently with PTH in both β-arr2−/− and WT. In contrast, whereas the resorption marker TRACP5B increased dose-dependently in WT, 20-80 μg/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-replete female mice, the ability of intermittent PTH to stimulate periosteal bone apposition and endosteal resorption is inhibited by arrestins. We therefore infer that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH on cancellous and cortical bone.
INTERMITTENT ADMINISTRATION OF an amino-terminal fragment of PTH(1-34) increases hip, spine, and total body BMD, improves iliac trabecular bone microarchitecture, and reduces vertebral and nonvertebral fracture risk in postmenopausal osteoporotic women.(1–3) However, the effects of PTH on cortical bone remain controversial.(1,4–7) Notably, the antifracture efficacy of PTH analogs at skeletal sites comprised predominantly of cortical bone, such as the hip and distal radius, is not known. Indeed, BMD of the radial shaft declined after a median of 21 months of treatment with intermittent PTH(1-34).(1,8) Similarly, volumetric BMD of the femoral neck cortex 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 periosteal surface(8–11); however, the ability of intermittent PTH to induce periosteal bone apposition at clinically relevant doses in humans or monkeys remains controversial. Longitudinal QCT measurements revealed no increase in femoral neck or vertebral cross-sectional area after administration 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 women treated concurrently with estrogen showed increased cortical thickness, which was attributed to endocortical bone apposition, but there was no change in bone formation rate on the subperiosteal surface.(2) The cortical bone response to intermittent PTH in nonhuman primates also revealed marked endosteal bone apposition accompanied by increased intracortical porosity, principally near the endocortical surface.(5,12) In these studies, where PTH was administered at doses equal to and five times greater than that used in human clinical trials, no significant increase in total cross-sectional area of the proximal humerus or femoral neck was reported.
In contrast, other studies have reported that intermittent PTH administration induces periosteal bone formation. For example, vertebral cross-sectional area, assessed by QCT, increased after intermittent PTH administration in individuals with steroid-induced osteoporosis.(13) In addition, a cross-sectional study reported a greater periosteal circumference at the distal radius in postmenopausal women treated with PTH(1-34) compared with those who received placebo.(8)
Rabbits, rats, and mice also predominantly exhibit endosteal bone apposition in response to intermittent PTH. However, in contrast to humans and primates, they frequently exhibit increased periosteal bone apposition as well.(9,10,14) Altogether, the factors that govern periosteal bone formation in response to intermittent PTH are poorly understood, although clinical observations and animals studies suggest it may depend on the dose and duration of PTH exposure.(14–17) Improved knowledge regarding the mechanisms that determine the ability of intermittent PTH to induce bone formation on periosteal surfaces is important because periosteal bone apposition is theoretically an important mechanism to improve whole bone strength.(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 cAMP and IP3/iCa pathways. In turn, stimulation of cAMP signaling seems to play a fundamental role in mediating the biologic activity of PTH in bone.(19–21) Activity of PTH1R and its agonists in 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 complexes and inhibit cAMP signaling in response to agonists.(22) These processes allow for rapid desensitization and later resensitization to PTH stimulation in vitro.(23,24) Additional mechanisms whereby arrestins may influence GPCR signaling include their ability to activate cAMP phosphodiesterase (PDE), thereby promoting degradation of intracellular cAMP,(25) and to serve as scaffolds linking GPCRs to other signaling proteins, including the scr-family kinases and members of the mitogen-activated protein kinase (MAPK) family.(22) Because receptor desensitization and resensitization modulate the cellular responses to both acute and chronic stimulation, an important implication is that not only signal transduction per se, but also the mechanisms regulating signal transduction may influence the physiological processes mediated by GPCRs, including PTH1R. In support of this view, mice null for β-arr2, which appear normal and are viable and fertile, exhibit a sustained response to opiate receptor agonists.(26,27)
Arrestins are expressed in osteoblasts.(28,29) Hence, we hypothesized that arrestins may play a role in regulating the anabolic effects of PTH on the skeleton. More specifically, we hypothesized that, in the absence of β-arr2, remodeling of endosteal and periosteal bone surfaces in response to PTH would be increased. To test this hypothesis, we evaluated bone mass and architecture of adult β-arr2 null and wildtype female mice in response to increasing doses of intermittent hPTH(1-34).
MATERIALS AND METHODS
β-arr2 null mice (β-arr2−/−) were initially generated by Bohn et al.(26) and subsequently backcrossed six generations onto a C57BL/6J background, resulting in mice whose genetic composition was >98% C57BL/6J. At the N6 generation, mice were genotyped using PCR analysis of tail DNA, and separately bred as homozygous wildtype (WT) or β-arr2−/− for the described experiments. Mice were maintained under standard nonbarrier conditions and had access 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 were approved by the ethics committees on animal care and use at Beth Israel Deaconess Medical Center, Boston, MA.
We tested the response to intermittent PTH administration in β-arr2−/− and WT mice by assigning 13-week old, intact female β-arr2−/− and WT mice to receive either vehicle or one of three 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) and 2% heat inactivated mouse serum. Mice received subcutaneous injections of vehicle or PTH 5 days/week for 4 weeks. Body weight was measured weekly, and the dose was adjusted accordingly. After 4 weeks, blood was collected retro-orbitally, mice were killed by CO2 inhalation, and the fifth lumbar vertebrae and right femurs were collected for μCT and histomorphometric evaluation.
Measurement of BMD, morphology, and microarchitecture
Total body BMD (TBBMD, g/cm2) was measured in vivo at baseline and at 4 weeks by peripheral DXA (PIXImus; GE Lunar, Madison, WI, USA).(30–32) μCT (UCT40; Scanco Medical AG, Basserdorf, Switzerland) was used to assess trabecular bone volume fraction and microarchitecture in the excised fifth lumber vertebral body and distal femur (12-μm isotropic voxels), and cortical bone geometry at a 1-mm thick section of the midfemoral diaphysis (34 μm isotropic voxels). For the vertebral trabecular region, we evaluated ∼300 transverse CT slices between the cranial and caudal end plates, excluding 100 μm near each endplate. Femoral cortical geometry was assessed in a 1-mm-long region centered at the femoral midshaft. CT images were reconstructed in 1024 × 1024 pixel matrices using a standard convolution-backprojection procedure, and the resulting gray-scale images were segmented using a constrained 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 computed from the binarized images using direct, 3D techniques that do not rely on any prior assumptions about the underlying structure.(33–35) For trabecular bone regions, we assessed the bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, mm−1), and trabecular separation (Tb.Sp, μm). For cortical bone at the femoral midshaft, we measured the average total cross-sectional area inside the periosteal envelope (CSA, mm2), the cortical bone area 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).
Femurs were dehydrated in graded ethanol and embedded in methylmethacrylate. Five- and 8-μm-thick sagittal sections were cut with a Polycut E microtome (Leica Corp. Microsystems AG, Glattbrugg, Switzerland) and stained with modified Goldner's trichrome (5-μm sections) for assessment of static histomorphometric variables or left unstained (8-μm sections) for assessment of calcein fluorescence and dynamic indices of bone formation. Histomorphometric measurements were performed on the secondary spongiosa of the distal femoral metaphysis (beginning 690 μm proximal to the growth plate and extending 344 μm proximally) and on the endocortical and cortical bone surfaces (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 and expressed according to standard formulas and nomenclatures.(36)
Serum biochemistry and bone turnover markers:
Serum concentration of calcium (Ca) and inorganic phosphate (Pi) was measured using atomic spectrometry and colorimetric methods, respectively, as previously described.(37) Serum osteocalcin (OC) was measured by RIA with a goat anti-mouse osteocalcin antibody and donkey anti-goat secondary antibody (Biomedical Technologies, Stoughton, MA, USA). Serum TRACP-5b was measured according to manufacturer's instructions (SBA Sciences, Turku, Finland).
Standard 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 ANOVA was used to assess the effect of PTH dose and β-arr deficiency on skeletal morphology. As appropriate, 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 considered significant at p < 0.05. Data are presented as mean ± SE, unless otherwise noted.
Total body BMD in vivo
At baseline (i.e., 13 weeks of age), female β-arr2−/− mice had modestly but significantly lower total body BMD compared with WT (47.9 ± 0.5 versus 50.0 ± 0.3 mg/cm2, p = 0.0008). Total body BMD increased markedly in response to PTH in both WT and β-arr2−/− mice (+7.3% to 10.1% 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).
Trabecular bone volume fraction and microarchitecture
In both the lumbar vertebral body and distal femoral metaphysis, VEH-treated β-arr2−/− had lower 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 thickness was 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 WT mice (Figs. 2A-2C). At the distal femur, PTH80 maximally increased trabecular BV/TV and thickness in both genotypes, whereas the lower PTH doses induced significant gains in β-arr2−/− mice only (Figs. 2D-2F).
Femoral cortical bone
Midfemoral 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 lower in VEH-treated β-arr2−/− compared with WT mice (Figs. 3A-3D). Accordingly, the area moment of inertia about the medio-lateral and antero-posterior axes were also lower in β-arr2−/− than WT mice (−26.8% and −18.5%, respectively, p < 0.0005 for both).
The cortical bone response to intermittent PTH at the femoral midshaft differed dramatically between 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 corresponding changes were smaller or absent in WT (Fig. 3). The most prominent differences were seen in cross-sectional area and medullary area, which both increased dose-dependently with PTH in β-arr2−/− mice. In contrast, cross-sectional area was unchanged and medullary area decreased in 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).
Dynamic histomorphometry data were consistent with the μCT observations of trabecular and cortical bone morphology. In cancellous bone, there was a trend for lower mineralizing surface and 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 bone formation, as well as osteoblast and osteoclast surfaces, increased with PTH treatment in both β-arr2−/− and WT (Table 1), consistent with the observed response in BV/TV (Fig. 2). At the endocortical surface, PTH increased osteoclastic number and surface increased significantly in β-arr2−/−, with a nonsignificant trend to increase in WT. In comparison, endocortical osteoblast 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 in WT, but increased in β-arr2−/− (Fig. 3). On the periosteal surface, no calcein double-labeled surfaces were detected in VEH-treated mice of either genotype. PTH treatment led to measurable double-labeled periosteal surfaces, with mineral apposition and bone formation rates that were twice as high in β-arr2−/− than WT mice (Table 1). This overall pattern of cortical bone response to PTH is exemplified in Fig. 4, which shows extensive PTH-stimulated calcein double-labeling on the endocortical surface in WT, with little evidence of bone formation on the periosteal surface. In stark contrast, PTH-stimulated calcein double-labeling is seen predominantly on the periosteal surface in β-arr2−/−, with limited evidence of endocortical bone formation, but rather a region of bone resorption. Altogether these histomorphometric data are consistent with the observed increase in cross-sectional area and medullary expansion in PTH-treated β-arr2−/− mice.
Serum biochemistry and markers of bone turnover
At 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, baseline OC values were slightly but significantly lower in β-arr2−/− mice (62.9 ± 2.6 versus 72.8 ± 3.3 ng/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 this decline, 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 bone resorption, did not differ between β-arr2−/− and WT. PTH led to a dose-dependent increase in TRACP-5b in WT, but all PTH doses were equipotent with regard to increasing TRACP-5b in β-arr2−/− mice (Table 2).
In this study, we tested the hypothesis that mice null for β-arr2 would have altered bone modeling/remodeling and a different response to increasing doses of intermittent PTH compared with wildtype mice. We found that adult female β-arr2−/− mice have a lower total body 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 the anabolic effects of PTH on cortical bone. Specifically, μCT measurements showed that, in the absence of β-arr2, intermittent PTH led to an increase in midfemoral cross-sectional area and medullary area. These findings were supported further by histomorphometric evidence of periosteal bone apposition and endocortical resorption in β-arr2−/− mice. Moreover, serum bone resorption markers suggested that β-arr2−/− mice were more responsive than WT to lower doses of PTH. Altogether, these observations indicate that PTH-stimulated bone remodeling (and perhaps “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 is useful to examine the endocortical and periosteal response to increasing doses of PTH. The cortical bone response to PTH was weak in WT mice, with marginally increased cortical thickness 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 trabecular bone seen at the same PTH doses, these observations suggest that the anabolic effects of PTH on various bone compartments occur at different dose and/or activity “thresholds.” Thus, it seems that a greater PTH dose and/or activity is required to induce bone formation at periosteal surfaces 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 direct opposition to the trend for a PTH-induced decline in medullary area in WT mice and was consistent with the lower endocortical bone formation rate and higher osteoclast surface in PTH-treated β-arr2−/− compared with WT. These data suggest that by inhibiting cAMP signaling, 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 the established inhibitory effects of estrogen on bone remodeling at both endosteal and periosteal surfaces,(38) studies in male mice or ovariectomized females may show different responses to PTH 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 bone gains, but reduced trabecular bone gains compared with male WT.(39) Taken together, these findings provide evidence that β-arrestin influences bone remodeling, yet they suggest that gonadal 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 mice had significantly lower total body bone mass, trabecular bone parameters, and midfemoral cross-sectional area compared with WT mice, also merits attention. In comparison, transgenic mice expressing a constitutively active PTH1R in osteoblasts also exhibit decreased periosteal bone formation, but increased bone formation in the trabecular compartment.(17) The differential effects of sustained cAMP signaling on periosteal and endosteal osteoblasts highlight the complex nature of compartment-specific responses to PTH activity and support the notion that factors present in the local bone microenvironment and/or regulated locally impact bone cell function. Furthermore, it must be acknowledged that absence of βarr2 may influence many GPCRs and signaling pathways that contribute to skeletal growth and homeostasis, and thus it is unlikely that increased PTH-stimulated cAMP signaling by itself is sufficient to explain the smaller femoral cross-sectional area and decreased trabecular bone volume in adult β-arr2−/− mice. For example, lower trabecular bone mass (particularly trabecular number) and OC levels would be compatible with increased β-adrenergic activity in osteoblasts in the absence of β-arrestins.(40) Moreover, in addition to their known effects on regulating GPCR signaling, arrestins have also been implicated in regulation of TGF-β and insulin-like growth factor (IGF)-I signaling in vitro.(41,42) Coupled with the known effects of TGF-β on skeletal development and the influence of IGF-1 on bone,(43–47) this prompts consideration that multiple factors may contribute to the observed alterations of bone modeling/remodeling in β-arr2 null mice.
Although the interpretation of different studies in mice is confounded by the fact that the response to PTH may vary with the genetic background, age, sex, and hormonal status, our observations generally confirm previous reports that intermittent PTH consistently induces increased bone mass in the trabecular compartment, whereas its effects on cortical bone are more variable.(48–52) Few studies have examined skeletal responses to various doses of intermittent PTH in mice. However, our results are consistent with those reported previously in 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 cancellous bone area fraction at all doses, whereas trabecular thickness increased and trabecular number tended to decreased with increasing PTH dose.(14) Moreover, these authors, as well as several others, reported that increasing PTH doses and/or activity may be necessary to induce periosteal bone apposition.(5,9,10,12,14)
It is tempting to consider the potential implications of our results with regard to development of PTH analogs for treatment of osteoporosis and other bone disorders. Normally, binding of PTH to the PTH1R promotes translocation of β-arrestins from the cytoplasm to the cell membrane, where they promote rapid endocytosis of ligand-receptor complexes and inhibit cAMP signaling.(23,53) Although speculative, our findings imply that PTH analogs that do not recruit β-arrestins(54) may have marked effects on bone strength by inducing both trabecular bone 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 currently used analogs, although this hypothesis would need further testing, particularly in models of estrogen deficiency.
In conclusion, our results show that arrestins influence not only normal bone modeling and/or remodeling, but also the skeletal response to intermittent PTH. In particular, these findings indicate that the differential effects of intermittent PTH on trabecular and cortical bone surfaces are attributable in part to the regulation of PTH activity by arrestins.
The authors thank Fanny Lin and Robert Lefkowitz for providing breeding pairs of β-arrestin2-deficient mice. Funding for this project was provided by a Mazess Fellowship from the National Osteoporosis Foundation (DSG), the American Federation for Aging Research (MLB), the NIH Office for Research on Women's Health (NIAMS AR049265), Swiss National Science Foundation Grant 631-62937 (SLF), and the Roche Research Foundation (DDP). We also acknowledge support of the Metabolic Physiology Core of the Diabetes Endocrinology Research Center at Beth Israel Deaconess Medical Center (NIDDK P30 DK57521).
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