Parathyroid hormone: anabolic and catabolic actions on the skeleton

The skeleton is a highly dynamic tissue whose structure relies on the balance between bone deposition and resorption. This equilibrium, which depends on osteoblast and osteoclast functions, is controlled by multiple factors that can be modulated post-translationally. Some of the modulators are Mitogen-activated kinases (MAPKs), whose role has been studied in vivo and in vitro. p38-MAPK modifies the transactivation ability of some key transcription factors in chondrocytes, osteoblasts and osteoclasts, which affects their differentiation and function. Several commercially available inhibitors have helped to determine p38 action on these processes. Although it is frequently mentioned in the literature, this chemical approach is not always as accurate as it should be. Conditional knockouts are a useful genetic tool that could unravel the role of p38 in shaping the skeleton. In this review, we will summarize the state of the art on p38 activity during osteoblast differentiation and function, and emphasize the triggers of this MAPK.

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p38 MAPK Signaling in Osteoblast Differentiation

PTH, an 84-amino acid peptide hormone synthesized by the parathyroid glands, is essential for the maintenance of calcium homeostasis. While in its traditional metabolic role, PTH helps to maintain the serum calcium concentration within narrow, normal limits and participates as a determinant of bone remodeling, more specific actions, described as catabolic and anabolic are also well known. Clinically, the catabolic effect of PTH is best represented by primary hyperparathyroidism (PHPT), while the osteoanabolic effect of PTH is best seen when PTH or its biological amino-terminal fragment [PTH(1–34)] is used as a therapy for osteoporosis. These dual functions of PTH are unmasked under very specific pathological (PHPT) or therapeutic conditions. At the cellular level. PTH favors bone resorption, mostly by affecting the receptor activator of nuclear factor κ-B (RANK) ligand (RANKL)-osteoprotegerin-RANK system, leading to an increase in osteoclast formation and activity. Increased bone formation due to PTH therapy is explained best by its ability to enhance osteoblastogenesis and/or osteoblast survival. The PTH-induced bone formation is mediated, in part, by a decrease in SOST/sclerostin expression in osteocytes. This review focuses on the dual anabolic and catabolic actions of PTH on bone, situations where one is enhanced over the other, and the cellular and molecular mechanisms by which these actions are mediated.

Catabolic and anabolic actions of parathyroid hormone on the skeleton.

The balance of protein phosphorylation is achieved through the actions of a family of protein serine/threonine kinases called the mitogen-activated protein kinases (MAPKs). The propagation of MAPK signals is attenuated through the actions of the MAPK phosphatases (MKPs). The MKPs specifically inactivate the MAPKs by direct dephosphorylation. The archetypal MKP family member, MKP-1 has garnered much of the attention amongst its ten other MKP family members. Initially viewed to play a redundant role in the control of MAPK signaling, it is now clear that MKP-1 exerts profound regulatory functions on the immune, metabolic, musculoskeletal and nervous systems. This review focuses on the physiological functions of MKP-1 that have been revealed using mouse genetic approaches. The implications from studies using MKP-1-deficient mice to uncover the role of MKP-1 in disease will be discussed.

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Diversity and specificity of the mitogen-activated protein kinase phosphatase-1 functions

Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting “knock-in” mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1–34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.

https://www.pnas.org/content/pnas/110/15/5864.full.pdf

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH

Parathyroid hormone (PTH) signaling via PTH 1 receptor (PTH1R) involves mitogen-activated protein kinase (MAPK) pathways. MAPK phosphatase 1 (MKP1) dephosphorylates and inactivates MAPKs in osteoblasts, the bone-forming cells. We previously showed that PTH1R activation in differentiated osteoblasts upregulates MKP1 and downregulates pERK1/2–MAPK and cyclin D1. In this study, we evaluated the skeletal phenotype of Mkp1 knockout (KO) mice and the effects of PTH in vivo and in vitro. Microcomputed tomography analysis of proximal tibiae and distal femora from 12-week-old Mkp1 KO female mice revealed osteopenic phenotype with significant reduction (8–46%) in bone parameters compared with wild-type (WT) controls. Histomorphometric analysis showed decreased trabecular bone area in KO females. Levels of serum osteocalcin (OCN) were lower and serum tartrate-resistant acid phosphatase 5b (TRAP5b) was higher in KO animals. Treatment of neonatal mice with hPTH (1–34) for 3 weeks showed attenuated anabolic responses in the distal femora of KO mice compared with WT mice. Primary osteoblasts derived from KO mice displayed delayed differentiation determined by alkaline phosphatase activity, and reduced expressions of Ocn and Runx2 genes associated with osteoblast maturation and function. Cells from KO females exhibited attenuated PTH response in mineralized nodule formation in vitro. Remarkably, this observation was correlated with decreased PTH response of matrix Gla protein expression. Expressions of pERK1/2 and cyclin D1 were inhibited dramatically by PTH in differentiated osteoblasts from WT mice but much less in osteoblasts from Mkp1 KO mice. In conclusion, MKP1 is important for bone homeostasis, osteoblast differentiation and skeletal responsiveness to PTH.

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Mitogen activated protein kinase phosphatase-1 regulates bone mass, osteoblast gene expression, and responsiveness to parathyroid hormone

Phosphorylation, internalization, and desensitization of G protein-coupled receptors, such as the parathyroid hormone (PTH) and PTH-related peptide (PTHrP) receptor (PTH1R), are well characterized and known to regulate the cellular responsiveness in vitro. However, the role of PTH1R receptor phosphorylation in bone formation and osteoblast functions has not yet been elucidated. In previous studies, we demonstrated impaired internalization and sustained cAMP stimulation of a phosphorylation-deficient (pd) PTH1R in vitro, and exaggerated cAMP and calcemic responses to s.c. PTH infusion in pdPTH1R knock-in mouse model. In this study, we examined the impact of impaired PTH1R phosphorylation on the skeletal phenotype of mice maintained on normal, low, and high calcium diets. The low calcium diet moderately reduced (P<0.05) bone volume and trabecular number, and increased trabecular spacing in both wild-type (WT) and pd mice. The effects, however, seem to be less pronounced in the female pd compared to WT mice. In primary calvarial osteoblasts isolated from 2-week-old pd or WT mice, PTH and PTHrP decreased phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2), a member of mitogen-activated protein kinase, and cyclin D1, a G1/S phase cyclin, in vitro. In contrast to WT osteoblasts, down-regulation of cyclin D1 was sustained for longer periods of time in osteoblasts isolated from the pd mice. Our results suggest that adaptive responses of intracellular signaling pathways in the pd mice may be important for maintaining bone homeostasis.

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Role of PTH1R internalization in osteoblasts and bone mass using a phosphorylation-deficient knock-in mouse model.

Limited information is available on the role of MAPK phosphatase 1 (MKP1) signaling in osteoblasts. We have recently reported distinct roles for MKP1 during osteoblast proliferation, differentiation, and skeletal responsiveness to parathyroid hormone (PTH). As MKP1 regulates the phosphorylation status of MAPKs, we investigated the involvement of P-ERK and P-p38 MAPKs in MKP1 knockout (KO) early and mature osteoblasts with respect to mineralization and PTH response. Calvarial osteoblasts from 9-14-week-old WT and MKP1 KO male and female mice were examined. Western blot analysis revealed downregulation and sustained expressions of P-ERK and P-p38 with PTH treatment in differentiated osteoblasts derived from KO males and females respectively. Exposure of early osteoblasts to p38 inhibitor, SB203580 (S), markedly inhibited mineralization in WT and KO osteoblasts from both genders as determined by von Kossa assay. In osteoblasts from males, ERK inhibitor U0126 (U), not p38 inhibitor (S), prevented the inhibitory effects of PTH on mineralization in early or mature osteoblasts. In osteoblasts from KO females, PTH sustained mineralization in early osteoblasts and decreased mineralization in mature cells. This effect of PTH was attenuated by S in early osteoblasts and by U in mature KO cells. Changes in matrix Gla protein expression with PTH in KO osteoblasts did not correlate with mineralization, indicative of MKP1-dependent additional mechanisms essential for PTH action on osteoblast mineralization. We conclude that PTH regulation of osteoblast mineralization in female mice is maturation stage specific and involves MKP1 modulation of P-ERK and P-p38 MAPKs.

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Chandrika D Mahalingam

Papers

Mitogen activated protein kinase phosphatase-1 regulates bone mass, osteoblast gene expression, and responsiveness to parathyroid hormone

Role of PTH1R internalization in osteoblasts and bone mass using a phosphorylation-deficient knock-in mouse model.

MKP1-dependent PTH modulation of bone matrix mineralization in female mice is osteoblast maturation stage specific and involves P-ERK and P-p38 MAPKs

Cell cycle and apoptosis regulatory protein (CARP)-1 is expressed in osteoblasts and regulated by PTH.