N‐cadherin Regulation of Bone Growth and Homeostasis Is Osteolineage Stage–Specific

N‐cadherin inhibits osteogenic cell differentiation and canonical Wnt/β‐catenin signaling in vitro. However, in vivo both conditional Cdh2 ablation and overexpression in osteoblasts lead to low bone mass. We tested the hypothesis that N‐cadherin has different effects on osteolineage cells depending upon their differentiation stage. Embryonic conditional osteolineage Cdh2 deletion in mice results in defective growth, low bone mass, and reduced osteoprogenitor number. These abnormalities are prevented by delaying Cdh2 ablation until 1 month of age, thus targeting only committed and mature osteoblasts, suggesting they are the consequence of N‐cadherin deficiency in osteoprogenitors. Indeed, diaphyseal trabecularization actually increases when Cdh2 is ablated postnatally. The sclerostin‐insensitive Lrp5^A214V mutant, associated with high bone mass, does not rescue the growth defect, but it overrides the low bone mass of embryonically Cdh2‐deleted mice, suggesting N‐cadherin interacts with Wnt signaling to control bone mass. Finally, bone accrual and β‐catenin accumulation after administration of an anti‐Dkk1 antibody are enhanced in N‐cadherin–deficient mice. Thus, although lack of N‐cadherin in embryonic and perinatal age is detrimental to bone growth and bone accrual, in adult mice loss of N‐cadherin in osteolineage cells favors bone formation. Hence, N‐cadherin inhibition may widen the therapeutic window of osteoanabolic agents. © 2017 American Society for Bone and Mineral Research.     

Prolonged Pharmacokinetic and Pharmacodynamic Actions of a Pegylated Parathyroid Hormone (1‐34) Peptide Fragment

Polyethylene glycol (PEG) addition can prolong the pharmacokinetic and pharmacodynamic actions of a bioactive peptide in vivo, in part by impeding rates of glomerular filtration. For parathyroid hormone (PTH) peptides, pegylation could help in exploring the actions of the hormone in the kidney; e.g., in dissecting the relative roles that filtered versus blood‐borne PTH play in regulating phosphate transport. It could also lead to potential alternate forms of treatment for hypoparathyroidism. We thus synthesized the fluorescent pegylated PTH derivative [Lys¹³(tetramethylrhodamine {TMR}), Cys³⁵(PEG‐20,000 Da)]PTH(1‐35) (PEG‐PTH^TMR) and its non‐pegylated counterpart [Lys¹³(TMR), Cys³⁵]PTH(1‐35) (PTH^TMR) and assessed their properties in cells and in mice. In PTHR1‐expressing HEK‐293 cells, PEG‐PTH^TMR and PTH^TMR exhibited similar potencies for inducing cAMP signaling, whereas when injected into mice, the pegylated analog persisted much longer in the circulation (>24 hours versus ～ 1 hour) and induced markedly more prolonged calcemic and phosphaturic responses than did the non‐pegylated control. Fluorescence microscopy analysis of kidney sections obtained from the injected mice revealed much less PEG‐PTH^TMR than PTH^TMR on the luminal brush‐border surfaces of renal proximal tubule cells (PTCs), on which PTH regulates phosphate transporter function, whereas immunostained phosphorylated PKA substrate, a marker of cAMP signaling, was increased to similar extents for the two ligands and for each, was localized to the basolateral portion of the PTCs. Pegylation of a bioactive PTH peptide thus led to prolonged pharmacokinetic/pharmacodynamic properties in vivo, as well as to new in vivo data that support a prominent role for PTH action at basolateral surfaces of renal proximal tubule cells. © 2016 American Society for Bone and Mineral Research.     

Thyroid hormone‐mediated growth and differentiation of growth plate chondrocytes involves IGF‐1 modulation of β‐catenin signaling

Thyroid hormone regulates terminal differentiation of growth plate chondrocytes in part through modulation of the Wnt/β‐catenin signaling pathway. Insulin‐like growth factor 1 (IGF‐1) has been described as a stabilizer of β‐catenin, and thyroid hormone is a known stimulator of IGF‐1 receptor expression. The purpose of this study was to test the hypothesis that IGF‐1 signaling is involved in the interaction between the thyroid hormone and the Wnt/β‐catenin signaling pathways in regulating growth plate chondrocyte proliferation and differentiation. The results show that IGF‐1 and the IGF‐ receptor (IGF1R) stimulate Wnt‐4 expression and β‐catenin activation in growth plate chondrocytes. The positive effects of IGF‐1/IGF1R on chondrocyte proliferation and terminal differentiation are partially inhibited by the Wnt antagonists sFRP3 and Dkk1. T₃ activates IGF‐1/IGF1R signaling and IGF‐1‐dependent PI3K/Akt/GSK‐3β signaling in growth plate chondrocytes undergoing proliferation and differentiation to prehypertrophy. T₃‐mediated Wnt‐4 expression, β‐catenin activation, cell proliferation, and terminal differentiation of growth plate chondrocytes are partially prevented by the IGF1R inhibitor picropodophyllin as well as by the PI3K/Akt signaling inhibitors LY294002 and Akti1/2. These data indicate that the interactions between thyroid hormone and β‐catenin signaling in regulating growth plate chondrocyte proliferation and terminal differentiation are modulated by IGF‐1/IGF1R signaling through both the Wnt and PI3K/Akt signaling pathways. While chondrocyte proliferation may be triggered by the IGF‐1/IGF1R‐mediated PI3K/Akt/GSK3β pathway, cell hypertrophy is likely due to activation of Wnt/β‐catenin signaling, which is at least in part initiated by IGF‐1 signaling or the IGF‐1‐activated PI3K/Akt signaling pathway. © 2010 American Society for Bone and Mineral Research     

Use of a Parathyroid Hormone Peptide (PTH1−34)‐enriched Fibrin Hydrogel for the Treatment of a Subchondral Cystic Lesion in the Proximal Interphalangeal Joint of a Warmblood Filly

To describe the treatment of a subchondral bone cyst in the proximal phalanx with parathyroid hormone peptide‐enriched fibrin hydrogel in a warmblood filly. The cyst was localized with computer‐assisted orthopaedic surgery, then curetted and finally filled with parathyroid hormone fragment peptide 1–34 (PTH₁₋₃₄) covalently attached to a fibrin hydrogel. The cyst healed quickly without any complications. This result supports the hypothesis that PTH₁₋₃₄ delivered locally in a fibrin hydrogel may improve the postoperative prognosis of surgical management of subchondral bone cysts in horses. Subchondral bone cysts are fairly common in horses. Especially in older horses, the prognosis is poor, even after surgical curettage. Therefore, different management protocols have been investigated in conjunction with surgical curettage to improve prognosis. Locally delivered PTH₁₋₃₄ seems to be a new method in the treatment of subchondral bone cysts.     

Parathyroid hormone signaling via Gαs is selectively inhibited by an NH2‐terminally truncated Gαs: Implications for pseudohypoparathyroidism

Pseudohypoparathyroid patients have resistance predominantly to parathyroid hormone (PTH), and here we have examined the ability of an alternative Gαs‐related protein to inhibit Gαs activity in a hormone‐selective manner. We tested whether the GNAS exon A/B‐derived NH₂‐terminally truncated (Tr) αs protein alters stimulation of adenylate cyclase by the PTH receptor (PTHR1), the thyroid‐stimulating hormone (TSH) receptor (TSHR), the β₂‐adrenergic receptor (β₂AR), or the AVP receptor (V2R). HEK293 cells cotransfected with receptor and full‐length (FL) Gαs ± Tr αs protein expression vectors were stimulated with agonists (PTH [10^?7 to 10^?9 M], TSH [1 to 100 mU], isoproterenol [10^?6 to 10^?8 M], or AVP [10^?6 to 10^?8 M]). Following PTH stimulation, HEK293 cells cotransfected with PTHR1 + FL Gαs + Tr αs had a significantly lower cAMP response than those transfected with only PTHR1 + FL Gαs. Tr αs also exerted an inhibitory effect on the cAMP levels stimulated by TSH via the TSHR but had little or no effect on isoproterenol or AVP acting via β₂AR or V2R, respectively. These differences mimic the spectrum of hormone resistance in pseudohypoparathyroidism type 1a (PHP‐1a) and type 1b (PHP‐1b) patients. In opossum kidney (OK) cells, endogenously expressing the PTHR1 and β₂AR, the exogenous expression of Tr αs at a level similar to endogenous FL Gαs resulted in blunting of the cAMP response to PTH, whereas that to isoproterenol was unaltered. A pseudopseudohypoparathyroid patient with Albright hereditary osteodystrophy harbored a de novo paternally inherited M1I Gαs mutation. Similar maternally inherited mutations at the initiation codon have been identified previously in PHP‐1a patients. The M1I αs mutant (lacking the first 59 amino acids of Gαs) blunted the increase in cAMP levels stimulated via the PTHR1 in both HEK293 and OK cells similar to the Tr αs protein. Thus NH₂‐terminally truncated forms of Gαs may contribute to the pathogenesis of pseudohypoparathyroidism by inhibiting the activity of Gαs itself in a GPCR selective manner. © 2011 American Society for Bone and Mineral Research     

p130Cas,E‐cadherin and β‐catenin in human transitional cell carcinoma of the bladder: Expression and clinicopathological significance

Objectives: To investigate the effects of junction protein, p130 Crk‐associated substance (p130Cas), and adhesion molecules, E‐cadherin and β‐catenin, on the biological behavior of transitional cell carcinoma of the bladder. Methods: In 72 paraffin embedded specimens of transitional cell carcinoma of the bladder and 20 normal controls, the expression of p130Cas, E‐cadherin and β‐catenin was examined by quantum dot‐based immunofluorescence histochemistry (QD‐IHC) and conventional immunohistochemistry (IHC). Results: QD‐IHC was consistent with IHC in detecting the expression of the three molecules (P > 0.05 for all comparisons). The positive expression rate of p130Cas in bladder cancer tissues increased more significantly than that in normal bladder tissues (P < 0.001). Similarly, the aberrant expression rates of E‐cadherin and β‐catenin in bladder cancer tissues were significantly higher than those in normal bladder tissues (P < 0.001 for both comparisons). The expression of each molecule was correlated with tumor pathological grade and clinical stage (P < 0.05 for all comparisons), but not with tumor number and size (P > 0.05 for all comparisons). Furthermore, negative correlations were found between the expression intensities of p130Cas and E‐cadherin or β‐catenin in transitional cell carcinoma of the bladder (P < 0.05 for both comparisons). Conclusions: p130Cas, E‐cadherin and β‐catenin might represent useful predictors of malignant degree of transitional cell carcinoma of the bladder.     

Activation of β‐Catenin Signaling in Articular Chondrocytes Leads to Osteoarthritis‐Like Phenotype in Adult β‐Catenin Conditional Activation Mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/β‐catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β‐catenin in this disease has not been reported. Because tissue‐specific activation of the β‐catenin gene (targeted by Col2a1‐Cre) is embryonic lethal, we specifically activated the β‐catenin gene in articular chondrocytes in adult mice by generating β‐catenin conditional activation (cAct) mice through breeding of β‐catenin^{fx(Ex3)/fx(Ex3)} mice with Col2a1‐CreER^T2 transgenic mice. Deletion of exon 3 of the β‐catenin gene results in the production of a stabilized fusion β‐catenin protein that is resistant to phosphorylation by GSK‐3β. In this study, tamoxifen was administered to the 3‐ and 6‐mo‐old Col2a1‐CreER^T2;β‐catenin^fx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β‐catenin protein was detected by immunostaining in articular cartilage tissues of β‐catenin cAct mice. In 5‐mo‐old β‐catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8‐mo‐old β‐catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in β‐catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp‐9, Mmp‐13, Alp, Oc, and colX, was significantly increased (3‐ to 6‐fold) in articular chondrocytes derived from β‐catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6‐fold increase) in these cells. In addition, we also observed overexpression of β‐catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β‐catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA‐like phenotype. This study provides direct and definitive evidence about the role of β‐catenin in the development of OA.     

Mechanical induction of PGE2 in osteocytes blocks glucocorticoid‐induced apoptosis through both the β‐catenin and PKA pathways

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid‐flow shear stress blocks dexamethasone‐induced apoptosis of osteocyte‐like cells (MLO‐Y4). Prostaglandin E₂ (PGE₂), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone‐induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3‐kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of β‐catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/β. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid‐induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase‐2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce β‐catenin protein expression. β‐catenin siRNA knockdown experiments abrogated the protective effects of PGE₂, confirming the central role of β‐catenin in mediating the protection against dexamethasone‐induced cell death. Our data support a central role for PGE₂ acting through the cAMP/PKA and β‐catenin signaling pathways in the protection of osteocyte apoptosis by fluid‐flow shear stress. © 2010 American Society for Bone and Mineral Research.     

Autocrine and Paracrine Regulation of the Murine Skeleton by Osteocyte‐Derived Parathyroid Hormone‐Related Protein

Parathyroid hormone–related protein (PTHrP) and parathyroid hormone (PTH) have N‐terminal domains that bind a common receptor, PTHR1. N‐terminal PTH (teriparatide) and now a modified N‐terminal PTHrP (abaloparatide) are US Food and Drug Administration (FDA)‐approved therapies for osteoporosis. In physiology, PTHrP does not normally circulate at significant levels, but acts locally, and osteocytes, cells residing within the bone matrix, express both PTHrP and the PTHR1. Because PTHR1 in osteocytes is required for normal bone resorption, we determined how osteocyte‐derived PTHrP influences the skeleton. We observed that adult mice with low PTHrP in osteocytes (targeted with the Dmp1(10kb)‐Cre) have low trabecular bone volume and osteoblast numbers, but osteoclast numbers were unaffected. In addition, bone size was normal, but cortical bone strength was impaired. Osteocyte‐derived PTHrP therefore stimulates bone formation and bone matrix strength, but is not required for normal osteoclastogenesis. PTHrP knockdown and overexpression studies in cultured osteocytes indicate that osteocyte‐secreted PTHrP regulates their expression of genes involved in matrix mineralization. We determined that osteocytes secrete full‐length PTHrP with no evidence for secretion of lower molecular weight forms containing the N‐terminus. We conclude that osteocyte‐derived full‐length PTHrP acts through both PTHR1 receptor‐mediated and receptor‐independent actions in a paracrine/autocrine manner to stimulate bone formation and to modify adult cortical bone strength. © 2017 American Society for Bone and Mineral Research.     

A Homozygous [Cys25]PTH(1‐84) Mutation That Impairs PTH/PTHrP Receptor Activation Defines a Novel Form of Hypoparathyroidism

Hypocalcemia and hyperphosphatemia are encountered in idiopathic hypoparathyroidism (IHP) and pseudohypoparathyroidism type Ib (PHP1B). In contrast to PHP1B, which is caused by resistance toward parathyroid hormone (PTH), the genetic defects leading to IHP impair production of this important regulator of mineral ion homeostasis. So far, only five PTH mutations were shown to cause IHP, each of which is located in the hormone's pre‐pro leader segment and thus impair hormone secretion. In three siblings affected by IHP, we now identified a homozygous arginine‐to‐cysteine mutation at position 25 (R25C) of the mature PTH(1‐84) polypeptide; heterozygous family members are healthy. Depending on the assay used for evaluating these patients, plasma PTH levels were either low or profoundly elevated, thus leading to ambiguities regarding the underlying diagnosis, namely IHP or PHP1B. Consistent with increased PTH levels, recombinant [Cys25]PTH(1‐84) and wild‐type PTH(1‐84) were secreted equally well by transfected COS‐7 cells. However, synthetic [Cys25]PTH(1‐34) was found to have a lower binding affinity for the PTH receptor type‐1 (PTH1R) than PTH(1‐34) and consequently a lower efficiency for stimulating cAMP formation in cells expressing this receptor. Consistent with these in vitro findings, long‐term infusion of [Cys25]PTH(1‐34) resulted only in minimal calcemic and phosphaturic responses, despite readily detectable levels of [Cys25]PTH(1‐34) in plasma. The mineral ion abnormalities observed in the three IHP patients are thus most likely caused by the inherited homozygous missense PTH mutation, which reduces bioactivity of the secreted hormone. Based on these findings, screening for PTH(1‐84) mutations should be considered when clinical and laboratory findings are consistent with PHP1B, but GNAS methylation changes have been excluded. Differentiating between IHP and PHP1B has considerable implications for genetic counseling, therapy, and long‐term outcome because treatment of IHP patients with inappropriately high doses of active vitamin D and calcium can contribute to development of nephrocalcinosis and chronic kidney disease. © 2015 American Society for Bone and Mineral Research.     

The survival effect of E‐cadherin and catenins in colorectal carcinomas

Aim The E‐cadherin/catenin complex plays an important role in epithelial tissue architecture. Decreased expression of cell adhesion molecules (E‐cadherin, α‐, β‐ and γ‐catenin) have been reported to correlate with invasive behaviour. The aim of this study was to investigate the relation between the expression of adhesion molecules and clinicopathological characteristics and survival in colorectal carcinoma. Method The expression of adhesion molecules were studied by immunohistochemistry in 138 colorectal carcinomas. Results The mean age of the patients was 65 years (range: 21–89 years). In primary carcinomas, a reduction in membranous expression of E‐cadherin, α‐catenin, β‐catenin, γ‐catenin was demonstrated (70%, 68%, 73%, 77%, respectively). Nuclear expression of β‐catenin was found in eight (5%) patients. Decreased membranous β‐ and γ‐catenin expression significantly correlated with tumour differentiation (P = 0.013, P = 0.03, respectively). There was a significant association between advanced stage of the tumour and decreased membranous α‐catenin expression (P = 0.012). Decreased E‐cadherin and β‐catenin membranous expression correlated with short survival following curative resection of the primary tumour (P = 0.04, P = 0.03, respectively). Conclusion The decreased membranous expression of E‐cadherin and β‐catenin and increased cytoplasmic expression of β‐catenin might be used as a prognostic marker to monitor patients with colorectal cancer.     

A comparison of parathyroid hormone‐related protein (1‐36) and parathyroid hormone (1‐34) on markers of bone turnover and bone density in postmenopausal women: The PrOP study

Parathyroid hormone‐related protein (PTHrP)(1‐36) increases lumbar spine (LS) bone mineral density (BMD), acting as an anabolic agent when injected intermittently, but it has not been directly compared with parathyroid hormone (PTH)(1‐34). We performed a 3‐month randomized, prospective study in 105 postmenopausal women with low bone density or osteoporosis, comparing daily subcutaneous injections of PTHrP(1‐36) to PTH(1‐34). Thirty‐five women were randomized to each of three groups: PTHrP(1‐36) 400 µg/day; PTHrP(1‐36) 600 µg/day; and PTH(1‐34) 20 µg/day. The primary outcome measures were changes in amino‐terminal telopeptides of procollagen 1 (PINP) and carboxy‐terminal telopeptides of collagen 1 (CTX). Secondary measures included safety parameters, 1,25(OH)₂ vitamin D, and BMD. The increase in bone resorption (CTX) by PTH(1‐34) (92%) (p < 0.005) was greater than for PTHrP(1‐36) (30%) (p < 0.05). PTH(1‐34) also increased bone formation (PINP) (171%) (p < 0.0005) more than either dose of PTHrP(1‐36) (46% and 87%). The increase in PINP was earlier (day 15) and greater than the increase in CTX for all three groups. LS BMD increased equivalently in each group (p < 0.05 for all). Total hip (TH) and femoral neck (FN) BMD increased equivalently in each group but were only significant for the two doses of PTHrP(1‐36) (p < 0.05) at the TH and for PTHrP(1‐36) 400 (p < 0.05) at the FN. PTHrP(1‐36) 400 induced mild, transient (day 15) hypercalcemia. PTHrP(1‐36) 600 required a dose reduction for hypercalcemia in three subjects. PTH(1‐34) was not associated with hypercalcemia. Each peptide induced a marked biphasic increase in 1,25(OH)₂D. Adverse events (AE) were similar among the three groups. This study demonstrates that PTHrP(1‐36) and PTH(1‐34) cause similar increases in LS BMD. PTHrP(1‐36) also increased hip BMD. PTH(1‐34) induced greater changes in bone turnover than PTHrP(1‐36). PTHrP(1‐36) was associated with mild transient hypercalcemia. Longer‐term studies using lower doses of PTHrP(1‐36) are needed to define both the optimal dose and full clinical benefits of PTHrP. © 2013 American Society for Bone and Mineral Research. © 2013 American Society for Bone and Mineral Research.     

Suppression of p38α MAPK Signaling in Osteoblast Lineage Cells Impairs Bone Anabolic Action of Parathyroid Hormone

Intermittent parathyroid hormone administration (iPTH) increases bone mass and strength by stimulating osteoblast number and activity. PTH exerts its anabolic effects through cAMP/protein kinase A (PKA) signaling pathway in mature osteoblasts and osteocytes. Here, we show that inactivation of the p38α MAPK‐encoding gene with the use of an osteocalcin‐cre transgene prevents iPTH bone anabolic action. Indeed, iPTH fails to increase insulin‐like growth factor 1 expression, osteoblast number and activity, and bone formation in mice lacking p38α in osteoblasts and osteocytes. Moreover, iPTH‐induced expression of receptor activator of NF‐κB ligand (RANKL) and subsequent increased bone resorption are suppressed in those mice. Finally, we found that PTH activates p38α MAPK downstream of cAMP/PKA signaling pathway in mature osteoblasts. Our findings identify p38α MAPK as a key component of PTH signaling in osteoblast lineage cells and highlight its requirement in iPTH osteoanabolic activity. © 2015 American Society for Bone and Mineral Research.     

Orphan Adhesion GPCR GPR64/ADGRG2 Is Overexpressed in Parathyroid Tumors and Attenuates Calcium‐Sensing Receptor‐Mediated Signaling

Abnormal feedback of serum calcium to parathyroid hormone (PTH) secretion is the hallmark of primary hyperparathyroidism (PHPT). Although the molecular pathogenesis of parathyroid neoplasia in PHPT has been linked to abnormal expression of genes involved in cell growth (e.g., cyclin D1, retinoblastoma, and β‐catenin), the molecular basis of abnormal calcium sensing by calcium‐sensing receptor (CaSR) and PTH hypersecretion in PHPT are incompletely understood. Through gene expression profiling, we discovered that an orphan adhesion G protein‐coupled receptor (GPCR), GPR64/ADGRG2, is expressed in human normal parathyroid glands and is overexpressed in parathyroid tumors from patients with PHPT. Using immunohistochemistry, Western blotting, and coimmunoprecipitation, we found that GPR64 is expressed on the cell surface of parathyroid cells, is overexpressed in parathyroid tumors, and physically interacts with the CaSR. By using reporter gene assay and GPCR second messenger readouts we identified Gαs, 3′,5′‐cyclic adenosine monophosphate (cAMP), protein kinase A, and cAMP response element binding protein (CREB) as the signaling cascade downstream of GPR64. Furthermore, we found that an N‐terminally truncated human GPR64 is constitutively active and a 15–amino acid–long peptide C‐terminal to the GPCR proteolysis site (GPS) of GPR64 activates this receptor. Functional characterization of GPR64 demonstrated its ability to increase PTH release from human parathyroid cells at a range of calcium concentrations. We discovered that the truncated constitutively active, but not the full‐length GPR64 physically interacts with CaSR and attenuates the CaSR‐mediated intracellular Ca²⁺ signaling and cAMP suppression in HEK293 cells. Our results indicate that GPR64 may be a physiologic regulator of PTH release that is dysregulated in parathyroid tumors, and suggest a role for GPR64 in pathologic calcium sensing in PHPT. © 2016 American Society for Bone and Mineral Research.     

The VDR, COL1A1, PTH, and PTHR1 gene polymorphisms are not associated with bone size and height in Chinese nuclear families

We tested the relationship of the ApaI, Eco31I, BstBI, and (AAAG)n polymorphisms in the vitamin D receptor (VDR), collagen type I α-1 (COL1A1), parathyroid hormone (PTH), and parathyroid hormone (PTH)/PTH-related peptide receptor (PTHR1) genes with variations in bone size (BS) and height. Population stratification, total-family association, and within-family association were used to test these relationships in 400 Chinese nuclear families with a total of 1256 individuals. The BS at hip and spine was measured using a Hologic QDR 2000 dual-energy X-ray absorptiometry (DXA) scanner. The minor allele frequencies were 29.2%, 36.0%, and 14.0% for the VDR-ApaI, COL1A1-Eco31I, and PTH-BstBI markers, respectively. (AAAG)₅ and (AAAG)₆ of the PTHR1 gene are two major alleles in the Chinese people. Significant population stratification was found between the spine BS and PTHR1-(AAAG)₅ (P = 0.048) and PTHR1-(AAAG)₆ (P = 0.023), as well as between PTHR1-(AAAG)₅ and height (P = 0.048), but we did not detect any significant within-family association or total-family association between the VDR, COL1A1, PTH, and PTHR1 gene polymorphisms and the variations in BS and height in our sample. Our results do not support that the VDR, COL1A1, PTH, and PTHR1 genes have an important influence on the variation in BS and height in our Chinese population.     

Role of TGF‐β in a Mouse Model of High Turnover Renal Osteodystrophy

Altered bone turnover is a key pathologic feature of chronic kidney disease‐mineral and bone disorder (CKD‐MBD). Expression of TGF‐β1, a known regulator of bone turnover, is increased in bone biopsies from individuals with CKD. Similarly, TGF‐β1 mRNA and downstream signaling is increased in bones from jck mice, a model of high‐turnover renal osteodystrophy. A neutralizing anti‐TGF‐β antibody (1D11) was used to explore TGF‐β's role in renal osteodystrophy. 1D11 administration to jck significantly attenuated elevated serum osteocalcin and type I collagen C‐telopeptides. Histomorphometric analysis indicated that 1D11 administration increased bone volume and suppressed the elevated bone turnover in a dose‐dependent manner. These effects were associated with reductions in osteoblast and osteoclast surface areas. Micro‐computed tomography (µCT) confirmed the observed increase in trabecular bone volume and demonstrated improvements in trabecular architecture and increased cortical thickness. 1D11 administration was associated with significant reductions in expression of osteoblast marker genes (Runx2, alkaline phosphatase, osteocalcin) and the osteoclast marker gene, Trap5. Importantly, in this model, 1D11 did not improve kidney function or reduce serum parathyroid hormone (PTH) levels, indicating that 1D11 effects on bone are independent of changes in renal or parathyroid function. 1D11 also significantly attenuated high‐turnover bone disease in the adenine‐induced uremic rat model. Antibody administration was associated with a reduction in pSMAD2/SMAD2 in bone but not bone marrow as assessed by quantitative immunoblot analysis. Immunostaining revealed pSMAD staining in osteoblasts and osteocytes but not osteoclasts, suggesting 1D11 effects on osteoclasts may be indirect. Immunoblot and whole genome mRNA expression analysis confirmed our previous observation that repression of Wnt/β‐catenin expression in bone is correlated with increased osteoclast activity in jck mice and bone biopsies from CKD patients. Furthermore, our data suggest that elevated TGF‐β may contribute to the pathogenesis of high‐turnover disease partially through inhibition of β‐catenin signaling. © 2014 American Society for Bone and Mineral Research.     

Ift88 regulates Hedgehog signaling,Sfrp5 expression,and β‐catenin activity in post‐natal growth plate

Primary cilia are present on most cell types including chondrocytes. Dysfunction of primary cilia results in pleiotropic symptoms including skeletal dysplasia. Previously, we showed that deletion of Ift88 and subsequent depletion of primary cilia from chondrocytes resulted in disorganized columnar structure and early loss of growth plate. To understand underlying mechanisms whereby Ift88 regulates growth plate function, we compared gene expression profiles in normal and Ift88 deleted growth plates. Pathway analysis indicated that Hedgehog (Hh) signaling was the most affected pathway in mutant growth plate. Expression of the Wnt antagonist, Sfrp5, was also down‐regulated. In addition, Sfrp5 was up‐regulated by Shh in rib chondrocytes and regulation of Sfrp5 by Shh was attenuated in mutant cells. This result suggests Sfrp5 is a downstream target of Hh and that Ift88 regulates its expression. Sfrp5 is an extracellular antagonist of Wnt signaling. We observed an increase in Wnt/β‐catenin signaling specifically in flat columnar cells of the growth plate in Ift88 mutant mice as measured by increased expression of Axin2 and Lef1 as well as increased nuclear localization of β‐catenin. We propose that Ift88 and primary cilia regulate expression of Sfrp5 and Wnt signaling pathways in growth plate via regulation of Ihh signaling. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 350–356, 2013     

PTH(1‐34) Treatment Increases Bisphosphonate Turnover in Fracture Repair in Rats

Bisphosphonates (BP) are antiresorptive drugs with a high affinity for bone. Despite the therapeutic success in treating osteoporosis and metabolic bone diseases, chronic BP usage has been associated with reduced repair of microdamage and atypical femoral fracture (AFF). The latter has a poor prognosis, and although anabolic interventions such as teriparatide (PTH_(1–34)) have been suggested as treatment options, there is a limited evidence base in support of their efficacy. Because PTH_(1–34) acts to increase bone turnover, we hypothesized that it may be able to increase BP in turnover in the skeleton, which, in turn, may improve bone healing. To test this, we employed a mixture of fluorescent Alexa647‐labelled pamidronate (Pam) and radiolabeled ¹⁴C‐ZA (zoledronic acid). These traceable BPs were dosed to Wistar rats in models of normal growth and closed fracture repair. Rats were cotreated with saline or 25 μg/kg/d PTH_(1–34), and the effects on BP liberation and bone healing were examined by X‐ray, micro‐CT, autoradiography, and fluorescent confocal microscopy. Consistent with increased BP remobilization with PTH_(1–34), there was a significant decrease in fluorescence in both the long bones and in the fracture callus in treated animals compared with controls. This was further confirmed by autoradiography for ¹⁴C‐ZA. In this model of acute BP treatment, callus bone volume (BV) was significantly increased in fractured limbs, and although we noted significant decreases in callus‐bound BP with PTH_(1–34), these were not sufficient to alter this BV. However, increased intracellular BP was noted in resorbing osteoclasts, confirming that, in principle, PTH_(1–34) increases bone turnover as well as BP turnover. © 2015 American Society for Bone and Mineral Research.