Presence Of Parathyroid Hormone Research Articles

Endogenous ligand recognition and structural transition of a human PTH receptor

Endogenous parathyroid hormone (PTH) and PTH-related peptide (PTHrP) bind to the parathyroid hormone receptor 1 (PTH1R) and activate the stimulatory G-protein (Gs) signaling pathway. Intriguingly, the two ligands have distinct signaling and physiological properties: PTH evokes prolonged Gs activation, whereas PTHrP evokes transient Gs activation with reduced bone-resorption effects. The distinct molecular actions are ascribed to the differences in ligand recognition and dissociation kinetics. Here, we report cryoelectron microscopic structures of six forms of the human PTH1R-Gs complex in the presence of PTH or PTHrP at resolutions of 2.8 -4.1Å. A comparison of the PTH-bound and PTHrP-bound structures reveals distinct ligand-receptor interactions underlying the ligand affinity and selectivity. Furthermore, five distinct PTH-bound structures, combined with computational analyses, provide insights into the unique and complex process of ligand dissociation from the receptor and shed light on the distinct durations of signaling induced by PTH and PTHrP.

Purinergic Signaling Mediates PTH and Fluid Flow-Induced Osteoblast Proliferation.

Both parathyroid hormone (PTH) and mechanical signals are able to regulate bone growth and regeneration. They also can work synergistically to regulate osteoblast proliferation, but little is known about the mechanisms how PTH and mechanical signals interact with each other during this process. In this study, we investigated responses of MC3T3-E1 osteoblasts to PTH and oscillatory fluid flow. We found that osteoblasts are more sensitive to mechanical signals in the presence of PTH according to ERK1/2 phosphorylation, ATP release, CREB phosphorylation, and cell proliferation. PTH may also reduce the osteoblast refractory period after desensitization due to mechanical signals. We further found that the synergistic responses of osteoblasts to fluid flow or ATP with PTH had similar patterns, suggesting that synergy between fluid flow and PTH may be through the ATP pathway. After we inhibited ATP effects using apyrase in osteoblasts, their synergistic responses to mechanical stimulation and PTH were also inhibited. Additionally, knocking down P2Y2 purinergic receptors can significantly attenuate osteoblast synergistic responses to mechanical stimulation and PTH in terms of ERK1/2 phosphorylation, CREB phosphorylation, and cell proliferation. Thus, our results suggest that PTH enhances mechanosensitivity of osteoblasts via a mechanism involving ATP and P2Y2 purinergic receptors.

Surgery for Primary Hyperparathyroidism with Normal Non-suppressed Parathyroid Hormone can be Both Challenging and Successful.

Criteria for diagnosing primary hyperparathyroidism (PHPT) include hypercalcemia in the presence of parathyroid hormone (PTH) levels that are either elevated (classic PHPT) or normal but non-suppressed. However, there is no standard definition of what constitutes normal non-suppressed levels, and data are lacking regarding the potential for surgical cure in these patients. A retrospective review of patients undergoing parathyroidectomy for sporadic PHPT between 2012 and 2014 was performed. Patients with normal PTH were compared to classic PHPT patients to assess demographics, imaging, operative findings, and outcomes. In total, 332 patients met study criteria, and 60 (18%) had normal PTH levels. Negative sestamibi scans were seen more often with normal PTH levels (18.3 vs. 4.8%, p<0.001). Patients with normal PTH were more likely to have ≥2 glands removed (26.7 vs. 14.3%, p=0.02), and the specimens were more likely to be classified as only mildly hypercellular or normocellular (20 vs. 2.9%, p<0.001). Average follow-up was 24months (range 6-55). Cure rate was 88% in the normal PTH group, compared to 96% in classic PHPT (p=0.02). Among patients with normal PTH, those with PTH≤55pg/mL had an 83% cure rate, whereas those with PTH 56-65 had a 96% cure rate (p=0.12). Parathyroidectomy can have a high cure rate in the context of normal PTH levels despite an increased likelihood of negative imaging and multigland resection. Operative success is equivalent to classic PHPT when PTH levels are >55pg/mL.

Electrochemistry Based Platform for the Highly Sensitive Detection of Human Parathyroid Hormone Using Au Electrode with MoS2/Graphene Matrix

A self-assembled novel nanocomposite composed of MoS2-Graphene(MoS2-Gr) composites were electrostatically formed on top of gold (Au) electrode and their biochemical amplification responses were reported. The properties of the MoS2-Gr were characterized by scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), and Fourier transform infrared spectrometer (FT-IR). Cyclic voltammetry was performed in the presence of Parathyroid Hormone (PTH) and its linearity was obtained by measuring the anodic current signals coming from interactions between MoS2-Gr-Ab and different concentrations of PTH with the aid of substrate and enzyme conjugated secondary antibodies. With this complex and Au as working electrode, we have developed a portable biosensing platform which shows good sensitivity in detecting PTH as low as 10 pg/mL and high reproducibility

Parathyroid hormone regulation of hypoxia-inducible factor signaling in osteoblastic cells

Osteoblasts perceive and respond to changes in their pericellular environment, including biophysical signals and oxygen availability, to elicit an anabolic or catabolic response. Parathyroid hormone (PTH) affects each arm of skeletal remodeling, with net anabolic or catabolic effects dependent upon duration of exposure. Similarly, the capacity of osteoblastic cells to perceive pericellular oxygen has a profound effect on skeletal mass and architecture, as mice expressing stable hypoxia-inducible factor (HIF)-1α and -2α demonstrate age-dependent increases in bone volume per tissue volume and osteoblast number. Further, HIF levels and signaling can be influenced in an oxygen-independent manner. Because the cellular mechanisms involved in PTH regulation of the skeleton remain vague, we sought whether PTH could influence HIF-1α expression and HIF-α-driven luciferase activity independently of altered oxygen availability. Using UMR106.01 mature osteoblasts, we observed that 100nM hPTH(1–34) decreased HIF-1α and HIF-responsive luciferase activity in a process involving heat shock protein 90 (Hsp90) and cyclic AMP but not intracellular calcium. Altering activity of the small GTPase RhoA and its effector kinase ROCK altered HIF-α-driven luciferase activity in the absence and presence of PTH. Taken together, these data introduce PTH as a regulator of oxygen-independent HIF-1α levels through a mechanism involving cyclic AMP, Hsp90, and the cytoskeleton.

EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone

Previous reports indicate that ephrinB2 expression by osteoblasts is stimulated by parathyroid hormone (PTH) and its related protein (PTHrP) and that ephrinB2/EphB4 signaling between osteoblasts and osteoclasts stimulates osteoblast differentiation while inhibiting osteoclast differentiation. To determine the role of the ephrinB2/EphB4 interaction in the skeleton, we used a specific inhibitor, soluble EphB4 (sEphB4), in vitro and in vivo. sEphB4 treatment of cultured osteoblasts specifically inhibited EphB4 and ephrinB2 phosphorylation and reduced mRNA levels of late markers of osteoblast/osteocyte differentiation (osteocalcin, dentin matrix protein-1 [DMP-1], sclerostin, matrix-extracellular phosphoglycoprotein [MEPE]), while substantially increasing RANKL. sEphB4 treatment in vivo in the presence and absence of PTH increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1α1, and PTH receptor [PTHR1]), but despite a substantial increase in osteoblast numbers, there was no significant change in bone formation rate or in late markers of osteoblast/osteocyte differentiation. Rather, in the presence of PTH, sEphB4 treatment significantly increased osteoclast formation, an effect that prevented the anabolic effect of PTH, causing instead a decrease in trabecular number. This enhancement of osteoclastogenesis by sEphB4 was reproduced in vitro but only in the presence of osteoblasts. These data indicate that ephrinB2/EphB4 signaling within the osteoblast lineage is required for late stages of osteoblast differentiation and, further, restricts the ability of osteoblasts to support osteoclast formation, at least in part by limiting RANKL production. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis.

The electrophysiological study of changes in the activities of locus coeruleus neurons under conditions of parathyroidectomy and parathyroid hormone administration

The evoked activity of single neurons in the locus coeruleus (LC) in rats was studied in the norm, after parathyroidectomy (PTX), and after the administration of parathyroid hormone (PTH) during high frequency-stimulation of the supraoptic nucleus of the hypothalamus (SON). Spike activity was analyzed using mathematical software. The excitatory poststimulus neuronal activity in the LC with PTH decreases the average frequency of activity compared to the norm, but it approaches the norm after the introduction of PTH. The poststimulus inhibitory responses of neurons in the LC with PTH were lowered, as compared to the norm; they recovered to the normal level after the introduction of PTH. Calculation of the ratio between the sum of spikes in excitatory poststimulus responses with PTX and the sum in the norm showed a twofold decrease in the responses; after administration of PTH, this sum returned back to normal. However, after PTX we observed an extreme reduction of neurons that responded by excitation as compared to the norm; after PTH administration we found a twofold increase in the number of these neurons as compared to the norm. For the sum of spikes in depression tetanic responses, the introduction of PTH was accompanied by their identical decrease compared to the norm and by an increase in the number of responding neurons after treatment with PTH as compared to both the norm and PTX. Our results led us to conclude that the greater intensity of tetanic responses of poststimulus inhibitory activity apparently results from an activation of the protective inhibitory GABAergic mechanism in intact animals, a sharp deficit of tetanic excitatory effects under conditions of PTX, and the return of these effects to the norm in the presence of PTH.

Parathyroid hormone and the regulation of cell cycle in colon adenocarcinoma cells

Parathyroid hormone (PTH) functions as a major mediator of bone remodeling and as an essential regulator of calcium homeostasis. In this study, we investigated the role of PTH in the regulation of the cell cycle in human colon adenocarcinoma Caco-2 cells. Flow cytometry analysis revealed that PTH (10−8M, 12–24h) treatment increases the number of cells in the G0/G1 phase and diminishes the number in both phases S and G2/M. In addition, analysis by Western blot showed that the hormone increases the expression of the inhibitory protein p27Kip1 and diminishes the expression of cyclin D1, cyclin D3 and CDK6. However, the amounts of CDK4, p21Cip1, p15INK4B and p16INK4A were not different in the absence or presence of PTH. Inhibitors of PKC (Ro-318220, bisindolylmaleimide and chelerythine), but not JNK (SP600125) and PP2A (okadaic acid and calyculin A), reversed PTH response in Caco-2 cells. Taken together, our results suggest that PTH induces G0/G1 phase arrest of Caco-2 intestinal cells and changes the expression of proteins involved in cell cycle regulation via the PKC signaling pathway.

Analysis of dynamics of degenerative and regenerative processes in the flexor and extensor collaterals of crushed sciatic nerve: Effects of parathyroid hormone

We performed morphological, histochemical, and electrophysiological analysis of changes in the state of the rat sciatic nerve after nerve crushing. The activity of Ca2+-dependent acidic phosphatase (AP) was measured in sections of the nerve fibers to trace the recovery of the nerve after its injury. The systemic application of parathyroid hormone (PTH) resulted in the acceleration of injured nerve recovery. Proliferation of both endoneurium and Schwann cells was observed on the 3rd day after the crushing of the ipsilateral extensor (N. peroneus communis) and flexor (N. gastrocnemius) nerve bundles. A complete recovery of the AP enzymatic activity was found on the 7–9th days after the crushing. From the 5th to 35th days after the crushing, we performed online registration and mathematical analysis of pulse activity. In the control experiments, from the 35th to 70th days, high-frequency stimulation of the nerves showed the regeneration of the extensor nerve only accompanied by alterations in fiber direction. In the presence of PTH, the regeneration was significantly accelerated and it was associated with early regeneration of the flexor nerve and more structured fiber morphology. We found a correlation between the electrophysiological and morphological data.

Effect of Parathyroid Hormone on Type X and Type II Collagen Expression in Mesenchymal Stem Cells from Osteoarthritic Patients

A major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express type X collagen (COL10), a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). Parathyroid hormone (PTH) regulates endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. In this study, we investigated the effect of PTH on expression of COL10 in MSCs from OA patients and analyzed the potential mechanisms related to its effect. MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Expanded cells were then incubated for 0-48 h without (control) or with 100 nM PTH (1-34). Protein expression and phosphorylation were measured by Western blot. Results showed that PTH (1-34) inhibited expression of COL10 in MSCs from OA patients in a time-dependent manner. In parallel, PTH (1-34) stimulated expression of COL2, a marker of chondrogenic differentiation. Results also showed that PTH (1-34) inhibited in a sustained manner the phosphorylation of p38 and AKT protein kinase signaling pathways. Interestingly, the modulation of COL2 and COL10 gene expression was significant as rapidly as after 1 h in the presence of PTH (1-34); changes in the phosphorylation of p38 and AKT were significant only after 6 h. This suggests that while p38 and AKT protein kinase signaling pathways may not be required to initiate the regulation of expression of COL2 and COL10 by PTH (1-34), these pathways may modulate later events necessary for preventing precocious MSC hypertrophy.

PTH(1-34)-induced changes in RANKL and OPG expression by human PDL cells modify osteoclast biology in a co-culture model with RAW 264.7 cells

Parathyroid hormone (PTH) is widely accepted as an anabolic agent when administered intermittently. Here, we explored the influence of intermittent PTH(1-34) on the expression of local factors by human periodontal ligament (PDL) cells that modify osteoclast biology. This approach aimed at a further elucidation of the role of the hormone and of PDL cells in the regulation of periodontal tissue homeostasis and of repair processes. In a co-culture model of mature PDL cells and RAW 264.7 cells, intermittent PTH(1-34) induced an increased gene expression for tartrate-resistant acid phosphatase (+84%), cathepsin K (+56%), and vitronectin-receptor (+56%); and an enhanced resorptive activity of differentiated osteoclasts (+154%). These findings were correlated with a reduction of the osteoprotegerin (OPG)/receptor activator of nuclear factor kappaB ligand (RANKL) ratio in the presence of PTH(1-34; -44%). Similar results were obtained when RAW cells were cultured with the conditioned medium of PTH(1-34)-stimulated PDL cells. In contrast, when less mature PDL cells were co-cultured with RAW cells, PTH(1-34) induced an inhibition of osteoclastic differentiation (TRAP, -35%; cathepsin K, -28%; vitronectin-receptor, -35%), a reduction of the resorbed substrate area (-77%) and an increase of the OPG/RANKL ratio (+11%). The conditioned medium of PTH(1-34)-pretreated less mature PDL cells led to a down-regulation of the number and activity of multinucleated cells. These data indicate that intermittent PTH(1-34) modifies the expression of membrane-bound and secreted factors by PDL cells which then in turn alter osteoclast biology. The PDL cell response to PTH(1-34) is specific in terms of cell maturation and the mechanism involved.

Effect of parathyroid hormone and teriparatide on immune function of human adherent and non-adherent leukocytes

Clinical data indicate that patients with hyperparathyroidism due to chronic kidney disease have abnormal immune function. We evaluated the direct effect of parathyroid hormone (PTH) and two 1-34 peptide fragments of PTH on immune function in activated leukocytes from healthy donors. IL-6 and IL-8 were measured from supernatants from phytohemagglutinin-activated non-adherent and lipopolysaccharide-activated adherent leukocytes in the presence of PTH and the two peptides using ELISA. Data showed no significant change in IL-6 and IL-8 production using PTH (0.1 - 0.8 microM) or two peptides (0.2 - 1.6 microM). Lymphocyte proliferation using 3H-thymidine was not inhibited with 0.1 - 0.4 microM of PTH. However, lymphocyte proliferation was significantly inhibited at the highest dose (0.8 microM) of PTH. There was no effect of two peptides on lymphocyte proliferation. Except for inhibition of lymphocyte proliferation at the highest dose of PTH tested, our data demonstrate no direct effect of PTH and two peptide fragment on immune function.

The gingipains from Porphyromonas gingivalis do not directly induce osteoclast differentiation in primary mouse bone marrow cultures

Porphyromonas gingivalis is a major aetiological agent in the development of periodontitis, the major clinical hallmark of which is bone resorption. The cysteine proteases (gingipains) produced by P. gingivalis have a critical role in the pathogenesis of the disease, and previous studies on whole bacteria have implicated these enzymes in osteoclastogenesis, a process which serves to upregulate bone resorption. The effects of the gingipains from P. gingivalis on osteoclast differentiation were investigated here to determine whether the enzymes directly contribute to osteoclastogenesis and thus to bone resorption. The effects of the gingipains on osteoclast differentiation were investigated in primary mouse bone marrow cultures. The cultures harvested from C57BL6/J mice were incubated in the presence of parathyroid hormone, a known osteoclastogenic factor, or active/inactivated forms of three gingipains. Osteoclast differentiation was quantified by counting the number of multinucleated cells positive for tartrate-resistant acid phosphatase, an enzyme marker for these cells. After 10 days of culture, the gingipains, either active or inactive, failed to stimulate osteoclast differentiation in comparison to the parathyroid hormone. The data presented here demonstrate that the gingipains do not induce osteoclast differentiation in this system, indicating that the bacterium uses other mechanisms to induce bone loss.

The regulation of osteoclastic development and function.

Cells of the osteoblastic lineage exert a dominant influence on osteoclastic bone resorption. They form a communicating network of osteocytes, surface osteocytes and osteoblasts that seems well placed to monitor the structure and performance of bone and to judge where bone formation or resorption is appropriate. Osteoblasts produce prostaglandins (PGs) which strongly inhibit osteoclastic resorption. None of the agents that stimulate resorption in intact bone, such as parathyroid hormone (PTH), interleukin 1 (IL-1), 1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) or tumour necrosis factors, affects isolated osteoclasts, but all induce osteoblastic cells to produce osteoclastic resorption stimulatory activity (ORSA) that acts directly on osteoclasts. Osteoblasts seem to initiate resorption as well as stimulating or inhibiting it. Contact with bone mineral appears to be necessary: osteoclasts resorb mineralized but not unmineralized bone. All bone surfaces are lined by unmineralized organic material. Osteoblastic cells secrete neutral proteases, including collagenase, in response to hormonal stimulators of bone resorption. Incubation of osteoblasts, in the presence of PTH, on such surfaces or preincubation of the bone with collagenase predisposes bone to osteoclastic resorption. Agents that stimulate resorption in organ cultures seem to share these osteoblast-mediated mechanisms for induction and stimulation of resorption but 1,25-(OH)2D3 stimulates it through an additional mechanism. We have found that osteoclasts can be induced from haemopoietic tissue (including haemopoietic spleen cells) in the presence of 1,25-(OH)2D3--PTH and IL-1 have no effect in this system. Because osteoclasts lack receptors for 1,25-(OH)2D3 these results suggest either that osteoclast precursors lose 1,25-(OH)2D3 receptors during differentiation, or that a 1,25-(OH)2D3-responsive accessory cell in bone marrow induces osteoclastic differentiation in the presence of 1,25-(OH)2D3.

Differential effects of selective oestrogen receptor modulators (SERMs) tamoxifen, ospemifene and raloxifene on human osteoclasts in vitro

Several selective oestrogen receptor modulators (SERMs) with oestrogen agonist effects in bone cells and without increased risk of breast and endometrial cancer have been developed. Here, we have investigated the effects of different types of SERMs on osteoclast differentiation, bone resorption and apoptosis in vitro. Human peripheral blood-derived CD14+ monocytes were cultured on bovine bone slices in the presence of RANKL, M-CSF, TNF-alpha and dexamethasone for seven days. Also, CD14+ monocytes were co-cultured either with human SaOS-2 or MG-63 osteosarcoma cells, in the presence of parathyroid hormone. Osteoclast cultures were treated with different SERMs. TRACP+ multinucleated cells and C-terminal telopeptide of type I collagen were used as markers for osteoclast formation and bone resorption, respectively. In CD14+ monocyte cultures, tamoxifen directly inhibited human osteoclast formation and bone resorption, while raloxifene and ospemifene had no inhibitory effect. In the co-cultures either with SaOS-2 or MG-63 cells, ospemifene and raloxifene as well as tamoxifen inhibited osteoclast formation in a concentration-dependent manner. The inhibitory effect was associated with an increased production of osteoprotegerin. The anti-oestrogen ICI 182 780 completely reversed the effects of these SERMs. Tamoxifen had an oestrogen receptor dependent, direct, inhibitory effect on human osteoclast differentiation and bone resorption, whereas ospemifene and raloxifene required osteoblastic cells to achieve a similar inhibition. The effects of ospemifene and raloxifene were mediated by oestrogen receptors by a mechanism involving paracrine induction of osteoprotegerin in cultures with osteoblast derived osteosarcoma cells.

Effects of flavonoid on calcium content in femoral tissue culture and parathyroid hormone-stimulated osteoclastogenesis in bone marrow culture in vitro

The effect of various flavonoids, which are present in food and plants, on bone calcium content and osteoclastogenesis were investigated to compare action of flavonoid on bone formation and bone resorption in vitro. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose) supplemented with antibiotics and bovine serum albumin. Amoung quercetin, myricetin, kaempferol, isorhamnetin, curcumin, hesperidin, or astaxanthin in the range of 10(-7)-10(-5)M, culture with quercetin (10(-6) or 10(-5)M) caused a significant increase in diaphyseal calcium content. Such an effect was not seen in other compounds. Mouse bone marrow cells were cultured for 7 days in the presence of parathyroid hormone (PTH; 10(-7)M), a bone-resorbing factor, in vitro. Culture with PTH caused a significant increase in osteoclast-like cell formation. This increase was significantly inhibited in the presence of quercetin, myricetin, kaempferol, isorhamnetin, or curcumin in the range of 10(-8)-10(-6)M. Such an effect was not seen in the case of hesperidin or astaxanthin. In addition, culture with PTH (10(-7)M) caused a significant decrease in diaphyseal calcium content. This decrease was completely prevented in the presence of quercetin, myricetin, kaempferal, or isorhamnetin of 10(-6)M. This study demonstrates that various flavonoids have a potent inhibitory effect on osteoclastogenesis and bone resorption rather than bone formation in vitro. Among various flavonoids, quercetin had a stimulatory effect on bone formation and an inhibitory effect on bone resorption in vitro.

Phytocomponent p-hydroxycinnamic acid stimulates bone formation and inhibits bone resorption in rat femoral tissues in vitro

The effect of cinnamic acid or its related compounds, which is present in many plants, on bone metabolism has not been clarified yet. The effect of cinnamic acid, p-hydroxycinnamic acid (HCA), ferulic acid, caffeic acid, or 3,4-dimethoxycinnamic acid (DCA) on bone calcium content in vitro was investigated. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48,h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of HCA (10(-5) or 10(-4),M) caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. Such an effect was not observed in the presence of cinnamic acid or other compounds at the concentration of 10(-5) or 10(-4),M. Alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in the presence of HCA (10(-5) or 10(-4),M). The effect of HCA (10(-4),M) in increasing calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal or metaphyseal tissues was completely prevented in the presence of cycloheximide (10(-6),M), an inhibitor of protein synthesis. Thus HCA had anabolic effects on bone components. The presence of parathyroid hormone (PTH; 10(-7),M), a bone-resorbing factor, caused a significant decrease in calcium content and a corresponding elevation in medium glucose consumption, lactic acid production or tartrate-resistant acid phosphatase (TRACP) activity in the diaphyseal or metaphyseal tissues. These alterations were completely prevented in the presence of HCA (10(-5) or 10(-4),M). This study demonstrates that p-hydroxycinnamic acid (HCA) has stimulatory effects on bone formation and inhibitory effects on bone resorption in tissue culture in vitro.

Overexpression of regucalcin suppresses Ca2+‐sensing receptor and L‐type Ca2+ channel mRNA expression in cloned rat kidney proximal tubular NRK52E cells

The effect of regucalcin, a regulatory protein in intracellular signaling pathway [Yamaguchi M, Int J Mol Med 15:372–389, 2005 in Review], on various gene expressions was investigated using the cloned normal rat kidney proximal tubular epithelial NRK52E cells overexpressing regucalcin. NRK52E cells (wild type) and stable regucalcin /pCXN2 transfectants were cultured for 72 hours in medium containing 5% bovine serum to obtain subconfluent monolayers. After culture, cells were further cultured for 24–72 hours in serum free medium. Regucalcin was localized in the nucleus of wild type cells. This localization was markedly increased in transfectants. Overexpression of regucalcin caused a significant increase in rat outer medullary K+ channel mRNA expression, while it did not cause epithelial sodium channel, Na+-K+-ATPase, type II Na-Pi cotransporter, angiotensinogen and Na+/Ca+ exchanger mRNA expressions in transfectants. The expression of Ca2+-sensing receptor and L-type Ca2+ channel mRNA was markedly suppressed in transfectants. Thses decreases were changed in the presence of parathyroid hormone that can suppress the gene expression. This study demonstrates that regucalcin has a role in the regulation of gene expression for Ca2+-sensing receptor and L-type Ca2+ channel that is involved in intracellular Ca2+ transport in kidney cells.

Inhibition of Hormone and Cytokine-stimulated Osteoclastogenesis and Bone Resorption by Interleukin-4 and Interleukin-13 Is Associated with Increased Osteoprotegerin and Decreased RANKL and RANK in a STAT6-dependent Pathway

Interleukin (IL)-4 and IL-13 are cytokines that inhibit bone resorption. Data showing an inhibitory effect of IL-4 and IL-13 on RANK mRNA in mouse calvariae were first reported at the 22nd American Society for Bone and Mineral Research Meeting (Lerner, U.H., and Conaway, H. H. 2000) J. Bone Min. Res. 15, Suppl. 1, Abstr. SU 230). In the present study, release of 45Ca from cultured mouse calvarial bones stimulated by different cytokines, peptides, and steroid hormones was inhibited by IL-4 and IL-13. IL-4 and IL-13 decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and RANK mRNA and increased osteoprotegerin (OPG) mRNA in calvariae. Additionally, the cytokines decreased RANKL protein and increased OPG protein in calvarial bones. In osteoblasts isolated from calvariae, both an increase in RANKL mRNA and a decrease in OPG mRNA and protein elicited by vitamin D3 were reversed by IL-4 and IL-13. IL-4 and IL-13 decreased the number of tartrate-resistant acid phosphatase positive multinucleated cells and the mRNA expression of calcitonin receptor, tartrate-resistant acid phosphatase, and cathepsin K in mouse spleen cells and bone marrow macrophages (BMM) treated with macrophage colony-stimulating factor and RANKL. Inhibition of mRNA for RANK and the transcription factor NFAT2 was also noted in spleen cell and BMM cultures treated with IL-4 and IL-13. In addition, RANK mRNA and RANK protein were decreased by IL-4 and IL-13 in RAW 264.7 cells. Osteoblasts, spleen cells, and BMM expressed mRNA for the four proteins making up the IL-4 and IL-13 receptors. No effects by IL-4 on bone resorption and osteoclast formation or on RANKL and RANK mRNA expression were seen in Stat6-/- mice. The data indicate that IL-4 and IL-13, via a STAT6-dependent pathway, inhibit osteoclast differentiation and bone resorption by activating receptors on osteoblasts and osteoclasts that affect the RANKL/RANK/OPG system.

Release of Intact and Fragmented Osteocalcin Molecules from Bone Matrix during Bone Resorption in Vitro

Osteocalcin detected from serum samples is considered a specific marker of osteoblast activity and bone formation rate. However, osteocalcin embedded in bone matrix must also be released during bone resorption. To understand the contribution of each type of bone cell in circulating osteocalcin levels, we used immunoassays detecting different molecular forms of osteocalcin to monitor bone resorption in vitro. Osteoclasts were obtained from rat long bones and cultured on bovine bone slices using osteocalcin-depleted fetal bovine serum. In addition, human osteoclasts differentiated from peripheral blood mononuclear cells were used. Both rat and human osteoclasts released osteocalcin from bovine bone into medium. The amount of osteocalcin increased in the presence of parathyroid hormone, a stimulator of resorption, and decreased in the presence of bafilomycin A1, an inhibitor of resorption. The amount of osteocalcin in the medium correlated with a well characterized marker of bone resorption, the C-terminal telopeptide of type I collagen (r > 0.9, p < 0.0001). The heterogeneity of released osteocalcin was determined using reverse phase high performance liquid chromatography, and several molecular forms of osteocalcin, including intact molecule, were identified in the culture medium. In conclusion, osteocalcin is released from the bone matrix during bone resorption as intact molecules and fragments. In addition to the conventional use as a marker of bone formation, osteocalcin can be used as a marker of bone resorption in vitro. Furthermore, bone matrix-derived osteocalcin may contribute to circulating osteocalcin levels, suggesting that serum osteocalcin should be considered as a marker of bone turnover rather than bone formation.