PTH receptors and apoptosis in osteocytes

Osteocytes comprise a heterogenous population of terminally differentiated osteoblasts that direct bone remodeling in response to applied mechanical loading of bone. Increased osteocyte density accompanies the anabolic effect of PTH in vivo, whereas accelerated osteocyte death may be precipitated by estrogen deficiency or excess glucocorticoid exposure (conditions benefitted by intermittent PTH therapy) and by renal failure (where circulating intact PTH and, especially, PTH carboxylfragments are elevated). Osteocytes express type-1 PTH/ PTHrP receptors (PTH1Rs), which are fully activated by aminoterminal PTH fragments and couple to multiple signal transducers, including adenylyl cyclase and phospholipase C. Activation of PTH1Rs in osteocytes promotes gap junction-mediated intercellular coupling, increases expression of MMP-9, potentiates calcium influx via stretch-activated cation channels, amplifies the osteogenic response to mechanical loading in vivo, and regulates apoptosis. Control of osteocyte apoptosis by PTH1Rs is complex, in that intermittent PTH(1-34) administration reduces the fraction of vertebral apoptotic osteocytes at 1 month in adult mice but increases femoral metaphyseal osteocyte apoptosis at 1-2 weeks in young rats. In MLO-Y4 cells, PTH(1-34) prevents apoptosis otherwise induced within 6 hr by dexamethasone. In older studies, large doses of intact PTH(1-84) caused rapid "degenerative" morphologic changes in osteocytes, similar to those described in renal osteodystrophy. We isolated clonal conditionally immortalized osteocytic (OC) cell lines from mice homozygous for targeted ablation of the PTH1R gene. OC cells express abundant (2-3 x 10(6) per cell) receptors specific for the carboxyl(C)-terminus of intact PTH(1-84) ("CPTHRs") but, as expected, do not express PTH1Rs or respond to PTH(1-34). CPTHRs are expressed at much lower levels by other skeletally-derived cell lines. Several highly conserved ligand determinants of CPTHR binding have been identified, including PTH(24-27), PTH(53-54) and the sequence PTH(55-84), loss of which reduces binding affinity by over 100-fold. Human PTH(53-84), like PTH(1-84), PTH(24-84), and PTH(39-84), increases OC cell apoptosis. Ala-scanning mutagenesis to define sequences within PTH(55-84) important for binding and bioactivity is underway. We conclude that osteocytes may be important targets for CPTH fragments that are secreted by the parathyroid glands or generated by peripheral metabolism of intact PTH and that accumulate in blood, especially in renal failure. Studies of functional interplay between responses to CPTHRs and (transfected) PTH1Rs, using receptor-specific ligands in OC cells, should provide new insight into PTH regulation of osteocyte function and survival.     

Role of calcium channels in carboxyl-terminal parathyroid hormone receptor signaling

Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(1–84) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([Ca²⁺]_i) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca²⁺]_i was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca²⁺]_i was observed in OC59 cells in response to the CPTH fragment hPTH-(53–84) (250 nM). No [Ca²⁺]_i signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(1–34) nor a mutant CPTH analog, [Ala^55–57]hPTH-(53–84), that does not to bind to CPTHRs, increased [Ca²⁺]_i in OC59 cells. The [Ca²⁺]_i response to hPTH-(53–84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), -agatoxin IVA (10 nM), and -conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs. calcium influx; osteocytes     

Parathyroid hormone binding sites in the brain

Parathyroid hormone (PTH) has been shown to have actions within the brain, suggesting the presence of central PTH receptors. This possibility was examined by determining the binding of ¹²⁵I-labeled [Nle^8,18,Tyr³⁴]bovine PTH to the plasma membranes of rat and rabbit brains. Specific binding of the tracer to membranes of the whole brain was time and tissue dependent, and was greater with membranes from the hypothalamus than with membranes from the cerebellum, cerebrum, or brain stem. The binding of the tracer to rat hypothalamic membranes was saturable and competitively displaced by unlabeled PTH(1–34), PTH(3–34), [Nle^8,18,Tyr³⁴]PTH(1–34), and by PTH-related protein, indicating the presence of a single class of high-affinity (dissociation constant = 2–5 nM), low-capacity (maximum binding capacity, B_max = 110–250 fmol/mg protein) binding site. The binding of radiolabeled PTH to these sites was not displaced by unrelated peptides of comparable molecular size (calcitonin, calcitonin-gene related peptide, adrenocorticotropin). The binding of PTH to these sites did not, however, appear to stimulate adenylate cyclase activity, as in peripheral PTH target sites. Thus, although these results indicate the presence of PTH receptors in the brain, these binding sites have a lower affinity than those in peripheral tissues and may utilize a different signal transduction system.     

Extracellular calcium is a direct effecter of VDR levels in proximal tubule epithelial cells that counter-balances effects of PTH on renal Vitamin D metabolism

In renal proximal tubules, VDR is transiently decreased by parathyroid hormone (PTH) during times of hypocalcemia and returns to normal levels with the rise in serum calcium (Ca). In this study we tested the hypothesis that elevated extracellular Ca induces VDR in a human renal proximal cell line (HK-2G) stably expressing PTH receptor type I. Exposure of HK-2G cells to increasing Ca concentration, up to 3 mM, induced the expression of VDR. The increase in VDR occurred within 1 h and was sustained over 24 h. The increase in VDR was also dose-dependently increased using 20–100 nM gadolinium, suggesting the induction of VDR is regulated via the extracellular Ca sensing receptor (CaSR) with is naturally expressed in HK-2G cells. In conclusion, an extracellular Ca concentration in the physiological range is capable of direct increase of renal proximal VDR expression, and the induction mechanism represents a strategy the body may use to counterbalance effects of PTH on renal Vitamin D metabolism.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone

Summary Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm² show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.     

Parathyroid Hormone (PTH)/PTH-related Peptide Type 1 Receptor (PPR) Signaling in Osteocytes Regulates Anabolic and Catabolic Skeletal Responses to PTH

Parathyroid hormone (PTH) is the only Food and Drug Administration-approved anabolic agent to treat osteoporosis; however, the cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH-related peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, the physiological relevance of PPR signaling in osteocytes remains to be elucidated. Toward this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decreased collagen type I α1 mRNA and receptor activator of NF-κB ligand (RANKL) expression. Importantly, when subjected to an anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared with controls. In vitro, osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling, and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.     

Isolation and characterization of an estrogen binding protein which may integrate the plethora of estrogenic actions in non-reproductive organs

A putative estrogen receptor (pER) from mouse liver has been characterized. The heterodimer protein (81–84 kDa) consists of two covalently bound subunits (61–67 and 17–27 kDa) with following characteristics: sedimentation constant — 4.9 S; IP — 4.8; dissociation constant (K_d) for estradiol-17β binding — 0.7 nmol; binding sites — 0.746 pmol/mg protein; relative binding affinity — estradiol-17β — 100, estrone — 80 and estriol — 30; specificity — does not bind, other natural steroids, synthetic estrogens, antiestrogens and bioflavonoids. Importantly, immunosuppressants, neuroleptic and carcinogens influence ³H-estradiol-17β binding to pER. Interestingly, pER is a serine phosphatase and this may have relevancy to estrogen action in Alzheimer's disease. The polyclonal anti-pER antibody does not react with estrogen receptors (ER). ER antibody does not react with pER. Remarkably, anti-pER antibody reacts with calcineurin, a brain phosphatase and anti-calcineurin antibody reacts with pER. Immunohistochemical analyses showed that pER is undetectable in reproductive organs (except ovary). It is localized on the plasma or the nuclear membranes in some, in cytoplasm and/or nucleus in other cells of non-reproductive organs (skeletal, neural, vascular, hair and retina), and in tumors (mammary, endometrial and prostate cancers, and prostatic hyperplasia). The information presented justifies the proposition that pER may mediate the estrogenic actions in non-reproductive organs.     

Recombinant production of TEV cleaved human parathyroid hormone

The parathyroid hormone, PTH, is responsible for calcium and phosphate ion homeostasis in the body. The first 34 amino acids of the peptide maintain the biological activity of the hormone and is currently marketed for calcium imbalance disorders. Although several methods for the production of recombinant PTH(1‐34) have been reported, most involve the use of cleavage conditions that result in a modified peptide or unfavorable side products. Herein, we detail the recombinant production of ¹⁵N‐enriched human parathyroid hormone, ¹⁵N PTH(1‐34), generated via a plasmid vector that gives reasonable yield, low‐cost protease cleavage (leaving the native N‐terminal serine in its amino form), and purification by affinity and size exclusion chromatography. We characterize the product by multidimensional, heteronuclear NMR, circular dichroism, and LC/MS. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Apical and basolateral effects of PTH in OK cells: Transport inhibition,messenger production,effects of pertussis toxin,and interaction with a PTH analog

Summary The cellular distribution (apicalvs. basolateral) of parathyroid hormone (PTH) signal transduction systems in opossum kidney (OK) cells was evaluated by measuring the action of PTH on apically located transport processes (Na/P_i cotransport and Na/H exchange) and on the generation of intracellular messengers (cAMP and IP₃).PTH application led to immediate inhibition of Na/H-exchange without a difference in dose/response relationships between apical and basolateral cell-surface hormone addition (halfmaximal inhibition at 5×10^–10 m). PTH required 2–3 hr for maximal inhibition of Na/P_i cotransport with a half-maximal inhibition occurring at ×10^–12 m for apical application. PTH addition to either side of the monolayer produced a dose-dependent production of both cAMP and IP₃. Half-maximal activation of IP₃ was at about 7×10^–12 m PTH and displayed no differences between apical and basolateral hormone addition, while cAMP was produced with a half maximal concentration of 7×10^–9 m for apical PTH application and 10^–9 m for basolateral administration.The PTH analog [nle^8.18, tyr³⁴]PTH(3-34), (nlePTH), produced partial inhibition of Na/P_i cotransport (agonism) with no difference between apical and basolateral application. When applied as a PTH antagonist, nlePTH displayed dose-dependent antagonism of PTH inhibition of Na/P_i cotransport on the apical surface, failing to have an effect on the basolateral surface. Independent of addition to the apical or basolateral cell surface, nlePTH had only weak stimulatory effect on production of cAMP, whereas high levels of IP₃ could be measured after addition of this PTH analog to either cell surface. Also an antagonistic action of nlePTH on PTH-dependent generation of the internal messengers, cAMP and IP₃, was observed; at the apical and basolateral cell surface nlePTH reduced PTH-dependent generation of cAMP, while PTH-dependent generation of IP₃ was only reduced by nlePTH at the apical surface.Pertussis toxin (PT) preincubation produced an attenuation of both PTH-dependent inhibition of Na/P_i cotransport and IP₃ generation while producing an enhancement of PTH-dependent cAMP generation; these effects displayed no cell surface polarity, suggesting that PTH action through either adenylate cyclase or phospholipase C was transduced through similar sets of G-proteins at each cell surface.It is concluded that apparent receptor activities with high and low affinity for PTH exist on both cell surfaces; those with apparent high affinity seem to be coupled preferentially to phospholipase C and those with apparent low affinity to adenylate cyclase. The differences in apparent affinity of receptor events coupled to adenylate cyclase and the differences in PTH/nlePTH interaction on the two cell surfaces are suggestive of the existence of differences in apparent PTH-receptor activities on the two cell surfaces.     

Characterization of the interaction of parathyroid hormone with the mitochondrial ATPase

Parathyroid hormone (PTH) has been shown to bind specifically to the beta subunit of the mitochondrial ATPase on nitrocellulose blots. We have now examined this interaction further, using intact mitochondria, submitochondrial particles, and the purified F1 ATPase. With intact mitochondria, 1 microM concentrations of PTH and its biologically active 1-34 fragment activate the ATPase about 3-fold. This effect was reduced to a 1.4-fold activation with 3-34 and 7-34 fragments of the hormone, and oxidized PTH gave no detectable activity. Activation could only be observed below pH 7. PTH had no significant effect on the activity of the purified enzyme or on submitochondrial particles. However, specific binding of an iodinated PTH analog, [Nle 8,18-Tyr 34] bPTH (1-34) amide, was found with submitochondrial particles and the purified ATPase. Binding affinity with the purified enzyme was about 10(-3) that of the plasma membrane receptor, and the molar stoichiometry was close to 1:1 (PTH:intact enzyme). With submitochondrial particles the affinity was about 10-fold higher than with the purified enzyme. This binding was further examined with PTH derivatives and fragments, and compared to that seen in the plasma membrane receptor. Oxidation of methionine 18 in PTH reduced the affinity about 50%, oxidation of methionine 8 reduced the affinity 95%, and oxidation of both methionines further decreased affinity in both membranes and submitochondrial particles. However, when compared to the native hormone, the 3-34 and 7-34 PTH fragments had much higher affinity for the submitochondrial particles than for the plasma membranes. PTH also reduced chemical crosslinking of the ATP analog, p-fluorosulfonyl benzoyl 5'-adenosine, to the alpha subunit of this enzyme, but did not alter labeling of the enzyme with 3'-O-(4'-benzoyl) benzoyl ATP, suggesting that the hormone binds near a regulatory nucleotide binding site. Direct chemical crosslinking of PTH to the beta-subunit of the enzyme was attained with a cleavable, photoactivate crosslinker, sulfosuccinimidyl 2-(p-azidosalicylamido) ethyl-1,3-dithiopropionate. The crosslinked protein was cleaved with cyanogen bromide and the labeled fragments were sequenced. The labeled fragments were found to be segments of the protein which have previously been implicated as being close to the noncatalytic ATP binding sites.     

Serum proteome profiles identifies parathyroid hormone physiologic response

Parathyroid hormone (amino acids 1-34) (PTH) regulates bone and calcium homeostasis. The magnitude of the effects of PTH on bone varies in osteoporosis patients. We employed ProteinChip technology to generate protein profiles from sera of mice treated once daily with PTH or vehicle for 3 or 11 days. Data analyses on selected arrays indicated significant increases in serum proteins or peptides in PTH-treated groups, compared to vehicle-controls. The magnitude of change increased with duration of treatment. Anion-exchange fractionation of sera prior to profiling on array surfaces increased the number of proteins detected that were regulated by PTH. The optimized purification conditions developed "on-chip" for subsets of proteins, reflected corresponding behavior with process-compatible chromatographic resins under elution chromatography. We have identified and evaluated subsets of serum proteins regulated by PTH treatment, using a combination of ProteinChip technology, column chromatography, PAGE and LC-MS/MS. Our data demonstrate the feasibility of using a panel of serum proteins to detect PTH responsiveness in humans.     

Effect of methionine oxidation and deletion of amino-terminal residues on the conformation of parathyroid hormone. Circular dichroism studies

Circular dichroism (CD) studies of parathyroid hormone (PTH), its oxidized forms, and some fragments of the hormone are described. The CD spectrum of native PTH (84 amino acids) and the active fragment, 1-34 PTH, suggests that most of the secondary structure resides in the amino-terminal segment of this hormone. Oxidation of the methionine residue at position 18 has a small impact on secondary structure, whereas oxidation of the methionine at position 8 produces substantial changes. Oxidation of both methionines produces secondary structure changes that are greater than the sum of those seen upon oxidation of the individual methionines. The CD spectrum for the 3-34 fragment of PTH is identical to that of the 1-34 fragment, and that of the 7-34 fragment is only slightly different. The spectra of the 13-34 and 19-34 fragments are markedly altered from that of the 1-34 peptide, and those of the 9-84 and 19-84 fragments of native PTH are significantly different from the intact hormone. Computer-assisted estimates of secondary structure content, and difference spectra, were utilized to evaluate the secondary structure content of the peptides. These results suggest that residues 6-12 are important in formation of helical secondary structure and that a reverse turn may be important for the folding of PTH into a conformation with high affinity for receptors. Residues 1 and 2 appear to make no contribution to the secondary structure and may be directly involved in activation of receptors.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

Evaluation of a novel bifunctional xylanase–cellulase constructed by gene fusion

An artificial bifunctional enzyme, xylanase–cellulase, has been prepared by gene fusion. Three chimeric genes were constructed that encoded fusion proteins of different lengths. The fusion proteins exhibited both xylanase (XynX) and cellulase (Cel5Z::Ω) activity when cel5Z::Ω was fused downstream of xynX, but not when xynX was fused downstream of cel5Z::Ω. Activities of bifunctional enzymes decreased when a shorter xylanase peptide was fused. Three fusion enzymes were purified, and the molecular weights of the enzymes were estimated by CMC-SDS-PAGE and XYN-SDS-PAGE to be 149, 129, and 87 kDa, respectively. The fusion enzymes displayed optimum cellulase activity at pH 8.0 and 50 °C and optimum xylanase activity at pH 8.0 and 70 °C.     

Parathyroid hormone(1–34) mediates proliferative and apoptotic signaling in human periodontal ligament cells in vitro via protein kinase C-dependent and protein kinase A-dependent pathways

Periodontal ligament (PDL) cells exhibit several osteoblastic traits and are parathyroid hormone (PTH)-responsive providing evidence for a role of these cells in dental hard-tissue repair. To examine the hypothesis that PDL cells respond to PTH stimulation with changes in proliferation and apoptotic signaling through independent but convergent signaling pathways, PDL cells were cultured from human bicuspids obtained from six patients. PDL cells at different states of maturation were challenged with PTH(1–34) intermittently for 0, 1, or 24 h/cycle or exposed continuously. Specific inhibitors to protein kinases A and C (PKA, PKC) and the mitogen-activated protein kinase cascade (MAPK) were employed. At harvest, the cell number, BrdU incorporation, and DNA fragmentation were determined by means of cell counting and immunoassays. Intermittent PTH(1–34) caused a significant increase in cell number in confluent cells as opposed to a reduction in pre-confluent cells. In confluent cells, the effect resulted from a significant increase in proliferation, whereas DNA fragmentation was reduced when PTH(1–34) was administered for 1 h/cycle but increased after PTH(1–34) for 24 h/cycle. Inhibition of PKC inhibited PTH(1–34)-induced proliferation but enhanced apoptosis. Inhibition of PKA enhanced proliferation and DNA fragmentation. Similar results were obtained in less mature cells, although, in the presence of the PKA inhibitor, the PTH(1–34)-induced changes were more pronounced than in confluent cells. In the presence of the MAPK inhibitor, all of the parameters examined were reduced significantly in both maturation states. Thus, PTH(1–34) mediates proliferative and apoptotic signaling in human PDL cells in a maturation-state-dependent manner via PKC-dependent and PKA-dependent pathways.This research was supported by research grants from the BONFOR program (O-135.0006) of the University of Bonn, Bonn, Germany and the Deutsche Forschungsgemeinschaft (DFG; LO-1181/1-1).     

Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells

The specific localization and the characterization of the parathyroid hormone (PTH) receptor in bone have been studied using 18-d embryonic chick calvariae and biologically active, electrolytically labeled [125I] bovine PTH(1-34). Binding was initiated by adding [125I]-bPTH(1-34) to bisected calvariae at 30 degrees C. Steady state binding was achieved at 90 min at which time 10 mg drg wt of calvaria specifically bound 17% of the added [125I]bPTH(1-34). Nonspecific binding in the presence of 244 nM unlabeled bPTH(1-34) was less than 2%. Insulin, glucagon, and calcitonin (1 microgram/ml) did not compete for PTH binding sites. Half-maximal inhibition of binding was achieved at concentrations of unlabeled bPTH(1-34) or bPTH(1-84) of about 10 nM. The range of concentration (2-100 nM) over which bPTH(1-34) and bPTH(1-84) stimulated cyclic 3'5'adenosine monophosphate (cAMP) production was similar to that which inhibited the binding of [125I]bPTH(1-34). Light microscope autoradiograms showed that grains were concentrated over cells (osteoblasts and progenitor cells) at the external surface of the calvariae and in trabeculae. In the presence of excess unlabeled PTH, labeling of control autoradiograms was reduced to near background levels. No labeling of osteocytes or osteoclasts was observed. At the electron microscopic level, grains were localized primarily over cell membranes. A quantitative analysis of grain distribution suggested that cellular internalization of PTH occurred.     

肿瘤坏死因子α对ROS17/2.8细胞甲状旁腺素受体的下行调节

肿瘤坏死因子α(TNFα)是激活的单核巨噬细胞分泌的蛋白质,分子量17kD。其多功能性和选择性抑制肿瘤细胞生长的作用受到高度重视。我们的实验表明:TNFα(3×10~(-10)-1×10~(-7)mol/L)能显著降低大鼠成骨肉瘤细胞株ROS17/2.8的甲状旁腺素(PTH)受体总结合率,比对照降低7.47-37.45％,且与TNFα的浓度呈正相关。时间曲线显示,TNFα作用时间越长,受体总结合率降低越明显。Scatchard作图表明PTH受体数目降低而其亲和力无显著变化。细胞周期分析显示,TNFα(3.83×10~(-10) mol/L作用3天)能抑制S期DNA合成。可见TNFα通过减少PTH受体数目以调节骨代谢。同时通过抑制DNA的合成以调节骨细胞的增殖。