Insertion of an Alu sequence in the Ca(2+)-sensing receptor gene in familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism.

Missense mutations in the calcium-sensing receptor (CaR) gene have previously been identified in patients with familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT). We studied family members of a Nova Scotian deme expressing both FHH and NSHPT and found, by PCR amplification of CaR gene exons, that FHH individuals were heterozygous and NSHPT individuals were homozygous for an abnormally large exon 7. This is due to an insertion at codon 877 of an Alu-repetitive element of the predicted-variant/human-specific-1 subfamily. It is in the opposite orientation to the CaR gene and contains an exceptionally long poly(A) tract. Stop signals are introduced in all reading frames within the Alu sequence, leading to a predicted shortened mutant CaR protein. The loss of the majority of the CaR carboxyl-terminal intracellular domain would dramatically impair its signal transduction capability. Identification of the specific mutation responsible for the FHH/NSHPT phenotype in this community will allow rapid testing of at-risk individuals.     

Distinct roles for mitogen-activated protein kinase phosphatase-1 (MKP-1) and ERK-MAPK in PTH1R signaling during osteoblast proliferation and differentiation

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) activate one single receptor (PTH1R) which mediates catabolic and anabolic actions in the bone. Activation of PTH1R modulates multiple intracellular signaling responses. We previously reported that PTH and PTHrP down-regulate pERK1/2 and cyclin D1 in differentiated osteoblasts. In this study we investigate the role of MAPK phosphatase-1 (MKP-1) in PTHrP regulation of ERK1/2 activity in relation to osteoblast proliferation, differentiation and bone formation. Here we show that PTHrP increases MKP-1 expression in differentiated osteoblastic MC3T3-E1 cells, primary cultures of differentiated bone marrow stromal cells (BMSCs) and calvarial osteoblasts. PTHrP had no effect on MKP-1 expression in proliferating osteoblastic cells. Overexpression of MKP-1 in MC-4 cells inhibited osteoblastic cell proliferation. Cell extracts from differentiated MC-4 cells treated with PTHrP inactivate/dephosphorylate pERK1/2 in vitro; immunodepletion of MKP-1 blocked the ability of the extract to dephosphorylate pERK1/2; these data indicate that MKP-1 is involved in PTHrP-induced pERK1/2 dephosphorylation in the differentiated osteoblastic cells. PTHrP regulation of MKP-1 expression is partially dependent on PKA and PKC pathways. Treatment of nude mice, bearing ectopic ossicles, with intermittent PTH for 3 weeks, up-regulated MKP-1 and osteocalcin, a bone formation marker, with an increase in bone formation. These data indicate that PTH and PTHrP increase MKP-1 expression in differentiated osteoblasts; and that MKP-1 induces growth arrest of osteoblasts, via inactivating pERK1/2 and down-regulating cyclin D1; and identify MKP-1 as a possible mediator of the anabolic actions of PTH1R in mature osteoblasts.     

Use of genetically modified mice to examine the skeletal anabolic activity of vitamin D

We employed genetically modified mice to examine the role of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on skeletal and calcium homeostasis. In mice expressing the null mutation for 25-hydroxyvitamin D 1 hydroxylase (1OHase^−/−), or the vitamin D receptor (VDR^−/−), 1,25(OH)₂D₃ and calcium were both required for optimal epiphyseal growth plate development, serum calcium and phosphorus alone were sufficient to mineralize skeletal tissue independent of 1,25(OH)₂D₃ and the VDR, and endogenous 1,25(OH)₂D₃ and the VDR were essential for baseline bone formation. In 2-week-old 1OHase^−/− mice and in 2-week-old mice homozygous for the PTH null mutation(PTH^−/−), PTH and 1,25(OH)₂D₃ were each found to exert independent and complementary effects on skeletal anabolism, with PTH predominantly affecting appositional trabecular bone growth and 1,25(OH)₂D₃ influencing both endochondral bone formation and appositional bone growth. Endogenous 1,25(OH)₂D₃ maintained serum calcium homeostasis predominantly by modifying intestinal and renal calcium transporters but not by producing net bone resorption. Administration of exogenous 1,25(OH)₂D₃ to double mutant PTH^−/−1OHase^−/− mice produced skeletal effects consistent with the actions of endogenous 1,25(OH)₂D₃. These studies reveal an important skeletal anabolic role for both endogenous and exogenous 1,25(OH)₂D₃ and point to a potential role for 1,25(OH)₂D₃ analogs in the treatment of disorders of bone loss.     

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.     

Recent studies on the biological action of parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor in cartilage and bone

Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and premature maturation of chondrocytes. Abnormal, heterogeneous populations of chondrocytes at different stages of differentiation were seen in the hypertrophic zone of the mutant growth plate. Although the homozygous null animals die within several hours of birth, mice heterozygous for PTHrP gene deletion reach adulthood, at which time they show evidence of osteopenia. Therefore, PTHrP appears to modulate cell proliferation and differentiation in both the pre and post natal period. PTH/PTHrP receptor expression in the mouse is controlled by two promoters. We recently found that, while the downstream promoter controls PTH/PTHrP receptor gene expression in bone and cartilage, it is differentially regulated in the two tissues. 1alpha,25-dihydroxyvitamin D3 downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes, both in vivo and in vitro. Most of the biological activity of PTHrP is thought to be mediated by binding of its amino terminus to the PTH/PTHrP receptor. However, recent evidence suggests that amino acids 87-107, outside of the amino terminal binding domain, act as a nucleolar targeting signal. Chondrocytic cell line, CFK2, transfected with wild-type PTHrP cDNA showed PTHrP in the nucleoli as well as in the secretory pathway. Therefore, PTHrP appears to act as a bifunctional modulator of both chondrocyte proliferation and differentiation, through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus.     

The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.     

Hypercalcemia produced by parathyroid hormone suppresses experimental autoimmune encephalomyelitis in female but not male mice

Besides its role in regulating serum levels of calcium and phosphorus, 1alpha, 25-dihydroxyvitamin D3 (1,25-(OH)2D3) has potent effects on the immune system and suppresses disease in several animal models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. While the amount of 1,25-(OH)2D3 needed to prevent EAE is dependent on the gender of the mouse and amount of calcium available in the diet, the minimum levels of 1,25-(OH)2D3 sufficient to prevent disease cause hypercalcemia. To test if hypercalcemia independent of high levels of 1,25-(OH)2D3 can suppress EAE, we used a 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-hydroxylase) knockout mouse strain. Because these 1alpha-hydroxylase knockout mice lack the parathyroid hormone (PTH)-regulated enzyme that synthesizes 1,25-(OH)2D3, hypercalcemia from increased bone turnover was created by continuous administration of PTH without changing the circulating levels of 1,25-(OH)2D3. This PTH-mediated hypercalcemia generated after EAE induction prevented disease in female mice but not male mice. When hypercalcemia was prevented by diet manipulation, PTH administration no longer prevented EAE. We conclude that hypercalcemia is able to prevent EAE after disease induction in female mice.     

The PTH/PTHrP receptor can delay chondrocyte hypertrophy in vivo without activating phospholipase C

One G protein-coupled receptor (GPCR) can activate more than one G protein, but the physiologic importance of such activation has not been demonstrated in vivo. We have generated mice expressing exclusively a mutant form of the PTH/PTHrP receptor (DSEL) that activates adenylyl cyclase normally but not phospholipase C (PLC). DSEL mutant mice exhibit abnormalities in embryonic endochondral bone development, including delayed ossification and increased chondrocyte proliferation. Analysis of the differentiation of embryonic metatarsals in vitro shows that PTH(1-34) and forskolin inhibit, whereas active phorbol ester stimulates, hypertrophic differentiation. Thus, PLC signaling via the PTH/PTHrP receptor normally slows the proliferation and hastens the differentiation of chondrocytes, actions that oppose the dominant effects of PTH/PTHrP receptors and that involve cAMP-dependent signaling pathways.     

The Paracrine Feedback Loop Between Vitamin D3 (1,25(OH)2D3) and PTHrP in Prehypertrophic Chondrocytes

The endocrine feedback loop between vitamin D₃ (1,25(OH)₂D₃) and parathyroid hormone (PTH) plays a central role in skeletal development. PTH‐related protein (PTHrP) shares homology and its receptor (PTHR1) with PTH. The aim of this study was to investigate whether there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate, in parallel with the endocrine feedback loop between 1,25(OH)₂D₃ and PTH. This was investigated in ATDC5 cells treated with 10^?8 M 1,25(OH)₂D₃ or PTHrP, Col2‐pd2EGFP transgenic mice, and primary Col2‐pd2EGFP growth plate chondrocytes isolated by FACS, using RT‐qPCR, Western blot, PTHrP ELISA, chromatin immunoprecipitation (ChIP) assay, silencing of the 1,25(OH)₂D₃ receptor (VDR), immunofluorescent staining, immunohistochemistry, and histomorphometric analysis of the growth plate. The ChIP assay confirmed functional binding of the VDR to the PTHrP promoter, but not to the PTHR1 promoter. Treatment with 1,25(OH)₂D₃ decreased PTHrP protein production, an effect which was prevented by silencing of the VDR. Treatment with PTHrP significantly induced VDR production, but did not affect 1α‐ and 24‐hydroxylase expression. Hypertrophic differentiation was inhibited by PTHrP and 1,25(OH)₂D₃ treatment. Taken together, these findings indicate that there is a functional paracrine feedback loop between 1,25(OH)₂D₃ and PTHrP in the growth plate. 1,25(OH)₂D₃ decreases PTHrP production, while PTHrP increases chondrocyte sensitivity to 1,25(OH)₂D₃ by increasing VDR production. In light of the role of 1,25(OH)₂D₃ and PTHrP in modulating chondrocyte differentiation, 1,25(OH)₂D₃ in addition to PTHrP could potentially be used to prevent undesirable hypertrophic chondrocyte differentiation during cartilage repair or regeneration. J. Cell. Physiol. 229: 1999–2014, 2014. © 2014 Wiley Periodicals, Inc.

     

Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.     

Deletion of PTH rescues skeletal abnormalities and high osteopontin levels in Klotho-/- mice

Maintenance of normal mineral ion homeostasis is crucial for many biological activities, including proper mineralization of the skeleton. Parathyroid hormone (PTH), Klotho, and FGF23 have been shown to act as key regulators of serum calcium and phosphate homeostasis through a complex feedback mechanism. The phenotypes of Fgf23(-/-) and Klotho(-/-) (Kl(-/-)) mice are very similar and include hypercalcemia, hyperphosphatemia, hypervitaminosis D, suppressed PTH levels, and severe osteomalacia/osteoidosis. We recently reported that complete ablation of PTH from Fgf23(-/-) mice ameliorated the phenotype in Fgf23(-/-)/PTH(-/-) mice by suppressing serum vitamin D and calcium levels. The severe osteomalacia in Fgf23(-/-) mice, however, persisted, suggesting that a different mechanism is responsible for this mineralization defect. In the current study, we demonstrate that deletion of PTH from Kl(-/-) (Kl(-/-)/PTH(-/-) or DKO) mice corrects the abnormal skeletal phenotype. Bone turnover markers are restored to wild-type levels; and, more importantly, the skeletal mineralization defect is completely rescued in Kl(-/-)/PTH(-/-) mice. Interestingly, the correction of the osteomalacia is accompanied by a reduction in the high levels of osteopontin (Opn) in bone and serum. Such a reduction in Opn levels could not be observed in Fgf23(-/-)/PTH(-/-) mice, and these mice showed sustained osteomalacia. This significant in vivo finding is corroborated by in vitro studies using calvarial osteoblast cultures that show normalized Opn expression and rescued mineralization in Kl(-/-)/PTH(-/-) mice. Moreover, continuous PTH infusion of Kl(-/-) mice significantly increased Opn levels and osteoid volume, and decreased trabecular bone volume. In summary, our results demonstrate for the first time that PTH directly impacts the mineralization disorders and skeletal deformities of Kl(-/-), but not of Fgf23(-/-) mice, possibly by regulating Opn expression. These are significant new perceptions into the role of PTH in skeletal and disease processes and suggest FGF23-independent interactions of PTH with Klotho.     

Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed that the CaR inhibits PTHrP production by MMECs but stimulates PTHrP production by Comma-D cells (immortalized murine mammary cells) and MCF-7 human breast cancer cells. We found that changes in intracellular cAMP, but not phospholipase C or MAPK signaling, correlated with the opposing effects of the CaR on PTHrP production. Pharmacologic stimulation of cAMP accumulation increased PTHrP production by normal and transformed breast cells. Inhibition of protein kinase A activity mimicked the effects of CaR activation on inhibiting PTHrP secretion by MMECs and blocked the effects of the CaR on stimulating PTHrP production in Comma-D and MCF-7 cells. We found that the CaR coupled to Galpha(i) in MMECs but coupled to Galpha(s) in Comma-D and MCF-7 cells. Thus, the opposing effects of the CaR on PTHrP production are because of alternate G-protein coupling of the receptor in normal versus transformed breast cells. Because PTHrP contributes to hypercalcemia and bone metastases, switching of G-protein usage by the CaR may contribute to the pathogenesis of breast cancer.     

Role of protein kinase A, phospholipase C and phospholipase D in parathyroid hormone receptor regulation of protein kinase Calpha and interleukin-6 in UMR-106 osteoblastic cells

Parathyroid hormone (PTH) stimulates both bone formation and resorption by activating diverse osteoblast signalling pathways. Upstream signalling for PTH stimulation of protein kinase C-alpha (PKCalpha) membrane translocation and subsequent expression of the pro-resorptive cytokine interleukin-6 (IL-6) was investigated in UMR-106 osteoblastic cells. PTH 1-34, PTH 3-34, PTHrP and PTH 1-31 stimulated PKCalpha translocation and IL-6 promoter activity. Pharmacologic intervention at the adenylyl cyclase (AC) pathway (forskolin, IBMX, PKI) failed to alter PTH 1-34- or PTH 3-34-stimulated PKCalpha translocation. The phosphoinositol-phospholipase C (PI-PLC) antagonist U73122 slightly decreased PTH 1-34-stimulated PKCalpha translocation; however, the control analogue U73343 acted similarly. Propranolol, an inhibitor of phosphatidic acid (PA) phosphohydrolase, decreased diacylglycerol (DAG) formation and attenuated PTH 1-34- and PTH 3-34-stimulated PKCalpha translocation and IL-6 promoter activity, suggesting a phospholipase D (PLD)-dependent mechanism. This is the first demonstration that PLD-mediated signalling leads to both PKC-alpha translocation and IL-6 promoter activation in osteoblastic cells.     

Effects of calcium-sensing receptor on the secretion of parathyroid hormone-related peptide and its impact on humoral hypercalcemia of malignancy

The extracellular calcium-sensing receptor (CaR) plays a key role in the defense against hypercalcemia by "sensing" extracellular calcium (Ca2+(o)) levels in the parathyroid and kidney, the key organs maintaining systemic calcium homeostasis. However, CaR function can be aberrant in certain pathophysiological states, e.g., in some types of cancers known to produce humoral hypercalcemia of malignancy (HHM) in humans and animal models in which high Ca2+(o), via the CaR, produces a homeostatically inappropriate stimulation of parathyroid hormone-related peptide (PTHrP) secretion from these tumors. Increased levels of PTHrP set a cycle in motion whereby elevated systemic levels of Ca2+(o) resulting from its increased bone-resorptive and positive renal calcium-reabsorbing effects give rise to hypercalcemia, which in turn begets worsening hypercalcemia by stimulating further release of PTHrP by the cancer cells. I review the relationship between CaR activation and PTHrP release in normal and tumor cells giving rise to HHM and/or malignant osteolysis and the actions of the receptor on key cellular events such as proliferation, angiogenesis, and apoptosis of cancer cells that will favor tumor growth and osseous metastasis. I also illustrate diverse signaling mechanisms underlying CaR-stimulated PTHrP secretion and other cellular events in tumor cells. Finally, I raise several necessary questions to demonstrate the roles of the receptor in promoting tumors and metastases that will enable consideration of the CaR as a potential antagonizing/neutralizing target for the treatment of HHM.     

Clinical use of calcimimetics in the treatment of hyperparathyroidisms

Recognition of the role of the extracellular calcium sensing receptor (CaR) in mineral metabolism has greatly improved our understanding of calcium homeostasis. The activation of this receptor by small changes in the extracellular ionized calcium concentration (Ca(2+)ec) regulates parathormone (PTH) and calcitonin secretion, urinary calcium excretion and ultimately bone turnover. Cloning of CaR and discovery of mutations making the receptor less or more sensitive to calcium allowed a better understanding of several hereditary disorders characterized either by hyperparathyroidism or hypoparathyroidism. CaR became an ideal target for the development of compounds able to modulate the activity of CaR, activators (calcimimetics) as well as inhibitors (calcilytics). The calcimimetics are able to amplify the sensitivity of the CaR to Ca(2+)ec, suppressing PTH levels with a resultant fall in blood Ca2+. They dose-dependently reduce the secretion of PTH in vitro in cultured parathyroid cells, in animal models and in humans. In uremic animals, these compounds prevent parathyroid cell hyperplasia, normalize plasma PTH levels and bone remodelling. In uremic patients undergoing hemodialysis, the calcimimetics reduce plasma PTH concentration at short-term (12 weeks) as well as at long-term (2 years), serum calcium-phosphorus product and bone remodelling. After one year of treatment, these patients show a gain of bone mass of 2-3% at the femoral neck and at the total body. Contrarily, the calcilytics, by inhibiting CaR, can intermittently stimulate the secretion and the serum concentration of PTH. This results in an skeletal anabolic effect with a substantial increase in bone mineral density. They are potentially very interesting for the treatment of post-menopausal osteoporosis.