1,25-dihydroxyvitamin D-3 and 24,25-dihydroxyvitamin D-3 affect parathormone (PTH) -sensitive adenylate cyclase activity and alkaline phosphatase secretion of osteoblastic cells through different mechanisms of action

In UMR 106 rat osteosarcoma cells, parathormone (1-34hPTH) and calcitonin (sCT) stimulated adenylate cyclase (AC) activity 5.5-and 2.8-fold, respectively. AC in osteoblasts (OB) from collagenase-treated calvaria of 3-day-old rats responded similarly to 1-34hPTH. In contrast, fibroblasts (mouse fibroblastomas) displayed a marginal 1-34hPTH sensitive AC. Osteoclasts (OC) of collagenase-treated rat calvariae, rat monocytes and mouse macrophages did not demonstrate 1-34hPTH inducable AC activity. Physiological concentrations of 24,25-dihydroxyvitamin D-3 attenuated PTH-sensitive AC in OB and UMR 106 cells within 20 min, while 1,25-dihydroxyvitamin D-3 showed no such immediate effect. In contrast, the AC response to Gpp(NH)p was unaffected by 24,25-(OH)2D3, indicating that 24,25-(OH)2D3 interrupts the coupling of the PTH receptor to the GTP binding protein Gs. OB and UMR 106 cells were also subjected to long-term (48 h) incubation with vitamin D-3 metabolites, 1-34hPTH or 20% serum from patients with secondary hyperparathyroidism (sHBT-serum), respectively. PTH-sensitive AC was markedly attenuated by pre-exposure to both 1-34hPTH and 1,25-(OH)2D3, while minimally affected by corresponding 24,25-(OH)2D3 and 20% sHPT-serum treatment. The secretion of alkaline phosphatase (Alphos) from the two cell types was strongly increased by 1-34hPTH, the effect being abolished by the presence of 24,25-(OH)2D3. Iliac crest biopsies of normal individuals exhibited a clear negative correlation between PTH-sensitive AC and corresponding serum 24,25-(OH)2D3 levels. Basal AC activity was, however, negatively correlated to serum 1,25-(OH)2D3 concentrations. In summary, the results show that 24,25-(OH)2D3 reduces PTH-stimulated AC activity in and Alphos secretion from osteoblastic bone cells by rapidly and directly interfering with the plasma membrane. These data reinforce the probable in vivo significance of 24,25-(OH)2D3. Moreover, the negative correlation between basal AC activity and serum 1,25-(OH)2D3 levels indicates a possible role for 1,25-(OH)2D3 in regulating bone cell synthesis of AC components in vivo.     

Age, strain and species differences in circulating parathyroid hormone

A new, sensitive parathyroid hormone (PTH) radioimmunoassay that appears specific for the intact hormone, and its validation for measuring rat PTH are described. The assay is based on antibody C2-7 from chicken immunized with bovine PTH; it has a detection limit of 6 pg of bPTH per assay tube and measures basal PTH in most rats; it is responsive to provoked changes in endogenous PTH concentration, and the intra-assay and inter-assay coefficients of variation are 6.0% and 7.2%, respectively. Multiple dilutions of rat serum and parathyroid gland extract, result in competitive inhibition curves that are parallel to that of highly purified bPTH. Under our assay conditions the C2-7 antibody cross-reacts well with intact PTH but synthetic fragments of the hormone (1-34bPTH, 1-34hPTH, 28-48hPTH, 44-68hPTH, 53-84hPTH) do not depress tracer (125l-bPTH) binding to the antibody. Studies designed to validate the assay gave predictable results such as enhanced secretion of the hormone in response to EDTA infusion, and failure to detect the hormone in serum following thyroparathyroidectomy. In addition, we made the novel observation that in F344 rats circulating immunoreactive PTH increases progressively with aging.     

Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the O-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for O-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.     

人甲状旁腺激素的研究

人甲状旁腺激素(hPTH)是甲状旁腺分泌的多肽激素。它能与骨基质和肾细胞膜上专一性的受体相结合,将调节细胞中钙磷浓度的信号传导到膜内。hPTH活性片段在N端,其N端氨基酸序列与牛、猪PTH高度同源。hPTH及其活性片段在治疗骨及肌肉疾病方面有重要作用,重组hPTH已获成功。     

Existence of parathyroid hormone binding sites on murine hemopoietic blast cells

We demonstrated that 125I-labeled human parathyroid hormone (1-34;8,18-Nle,34-Tyr)[[125I]hPTH(1-34)] bound specifically to hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Half-maximal inhibition of binding was achieved at concentrations of unlabeled hPTH(1-34) of about 5 x 10(-9)M. Insulin and hPTH(39-68) did not compete for PTH binding sites. Specific binding of hPTH(1-34) was detected in neither macrophages nor multinucleated cells (MNC's). Furthermore, treatment of hemopoietic blast cells with hPTH(1-34) stimulated MNC formation, and the range of concentrations (10(-10)-10(-8)M) over which hPTH(1-34) caused these effects was similar to that which inhibited the binding of [125I]hPTH(1-34). These findings suggest the presence of a PTH receptor on osteoclast precursors and the direct effect of PTH on them, resulting in osteoclast-mediated bone resorption.     

Prevention of osteoporosis in ovariectomized mice by electroporational gene transfer of parathyroid hormone

Non-viral vector, pCMV-Pth, with full-length human parathyroid hormone (prepro-hPTH) cDNA was constructed and delivered into the ovariectomized mouse quadriceps to explore the effects of electroporational gene transfer of PTH on the bone. The expression of hPTH in the transfected mice was detected by RT-PCR and radioimmunoassay. Mechanical testing and the analysis of bone mineral content demonstrated the improvement of bone properties. These results suggest that the electroporational gene taransfer of parathyroid hormone might be a promising method to prevent the bone loss in the postmenopausal women.     

Effects of<Emphasis Type="Italic">Nigella sativa</Emphasis> and human parathyroid hormone on bone mass and strength in diabetic rats

Osteoporosis is a major complication in patients with diabetes mellitus (DM), particularly in those with insulin dependency. Recently, many therapeutic effects ofNigella sativa L. (NS) extracts have been exhibited such as anti-inflammatory, antitumor, and antidiabetic with clinical and experimental studies. Mechanical strength in the femur and vertebrae increases with human parathyroid hormone (hPTH) treatment. The aim of the present study was to test the hypothesis that combined treatment with NS and hPTH is more effective than treatment with NS or hPTH alone in improving bone mass, connectivity, and biomechanical behavior using the finite element method (FEM) in insulin-dependent diabetic rats. In the mechanical analysis, five rat bones (control, diabetic diabetic NS treated, diabetic hPTH treated, and diabetic NS + hPTH treated) have been studied for bending analysis using the finite element analysis program ANSYS. Combined treatment of NS and hPTH was more effective on bone histomorphometry and mechanical strength than treatment with NS or hPTH alone for streptozotocin-induced diabetic osteopenia, which notably decreased bone volume.     

Calcium rather than cyclic AMP is an intracellular messenger of parathyroid hormone action on glycogen metabolism in isolated rat hepatocytes.

The synthetic 1-34 fragment of human parathyroid hormone (1-34hPTH) stimulated glucose production in isolated rat hepatocytes. The effect of 1-34hPTH was dose-dependent and 10(10) M-1-34 hPTH elicited the maximum glucose output, which was approx. 80% of that by glucagon. Although 1-34hPTH induced a small increase in cyclic AMP production at concentrations higher than 10(-9) M, 10(-10) M-1-34hPTH induced the maximum glucose output without significant elevation of cyclic AMP. This is in contrast to the action of forskolin, which increased glucose output to the same extent as 10(-10) M-1-34hPTH by causing a 2-fold elevation of cyclic AMP. In addition to increasing cyclic AMP, 1-34hPTH caused an increase in cytoplasmic free calcium concentration ([Ca2+]c). When the effect of 1-34hPTH on [Ca2+]c was studied in aequorin-loaded cells, low concentrations of 1-34hPTH increased [Ca2+]c: the 1-34hPTH effect on [Ca2+]c was detected at as low as 10(-12) M and increased in a dose-dependent manner. 1-34hPTH increased [Ca2+]c even in the presence of 1 microM extracellular calcium, suggesting that PTH mobilizes calcium from an intracellular pool. In line with these observations, 1-34hPTH increased the production of inositol trisphosphate. These results suggest that: (1) PTH activates both cyclic AMP and calcium messenger systems and (2) PTH stimulates glycogenolysis mainly via the calcium messenger system.     

Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae

A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.     

Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae

A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (>/=0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases.     

NMR structure of a minimum activity domain of human parathyroid peptide hormone: structural origin of receptor activation.

Parathyroid hormone (PTH) which increases osteoblast numbers and bone formation by activating bone-lining cells to osteoblasts plays an important role in calcium and phosphate homeostasis and bone remodeling by activating PTH receptors. To determine the structural origin of a minimum activity domain of hPTH, we initiated a detailed structural determination of the hPTH(H14) in aqueous solution using NMR spectroscopy. Circular dichroism and NMR data demonstrated that hPTH(H14) maintains a typical helical conformation in both membrane-mimicking environments and 30% TFE solution. The solution structure clearly showed that the residues from Ser(3) to Leu(11) of hPTH(H14) formed a stable helical structure, especially having charged side-chains oriented in opposite directions relative to one another for optimum interaction with the receptor molecule.     

Efficient production of intact human parathyroid hormone in a Saccharomyces cerevisiae mutant deficient in yeast aspartic protease 3 (YAP3)

When human parathyroid hormone (hPTH) is expressed as a secretory product in yeast, the main problem is the aberrant proteolytic cleavage that reduces the yield of intact protein. To overcome this problem, we developed an hPTH expression system using a host strain in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 48 h of culture, most of the hPTH secreted by the yap3 disruptant remained intact, whereas more than 90% of the hPTH secreted by the wild-type strain was cleaved. When the authentic hPTH was incubated in each of the culture supernatants of untransformed yap3 disruptant and wild-type strain, the proteolysis proceeded much more slowly in the culture supernatant of yap3 disruptant than in that of the wild type. The extent of hPTH proteolysis was also significantly reduced by the addition of pepstatin A, a specific aspartic protease inhibitor. The results suggest that YAP3 is involved in the internal cleavage of hPTH expressed in yeast. The correct processing of the intact hPTH secreted in the yap3 disruptant demonstrates that the yeast mutant lacking the YAP3 activity is a suitable host for the high-level expression of intact hPTH. Received: 8 December 1997 / Received last revision: 3 March 1998 / Accepted: 19 April 1998     

Human parathyroid hormone as a secretory peptide in milk of transgenic mice

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t-antigen intron and polyadenylation site in the 3′ end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS-PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH. © 1995 Wiley-Liss, Inc.     

Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone

Human parathyroid hormone (hPTH) is involved in calcium metabolism, and the unique ability of this hormone to stimulate bone growth makes it a promising agent in the treatment of osteoporosis. We have engineered the methylotrophic yeast Pichia pastoris for the production of over 300 mg intact hPTH per liter growth medium. The presence of 10 mM EDTA in the culture medium was essential to obtain this high hormone yield, indicating that metallopeptidases are mainly responsible for the otherwise instability of hPTH. Furthermore, the secretion process of hPTH was considerably improved by coexpression of Saccharomyces cerevisiae protein disulphide isomerase (ScPDI). Since hPTH does not contain any cystein residues, this effect may be indirect or ascribed to the chaperone activity of PDI. Contrary to the situation in S. cerevisiae, use of a protease-deficient host strain provided no additional advantage. The hormone secreted by P. pastoris was not subjected to proteolytic processing by Kex2p in the two internal tribasic sites, nor were any C-terminal truncated hPTH forms detected. However, the P. pastoris hPTH producing transformants cosecreted ubiquitin to the culture medium, possibly as a result of a stress-related response.     

Anabolic effects of recombinant human parathyroid hormone (1 - 84) and synthetic human parathyroid hormone (1 - 34) on the mandibles of osteopenic ovariectomized rats with maxillary molar extraction.

In rodent osteoporosis models such as ovariectomized (OVX) rats, intermittently administered human parathyroid hormone (hPTH) has an anabolic effect in vertebrae and long bones. In the present experiments, subcutaneously injected hPTH(1 - 34) or hPTH(1 - 84) dose- and time-dependently increased bone mineral density (BMD) as measured by dual energy X-ray absorptiometry in mandibles, L2 to L4 vertebrae and femurs of such rats. The highest dose (15.9 nmol/kg, s. c.) of either peptide given four times weekly for 10 weeks completely reversed the effects of overiectomy on BMD. Significant elevation in lumbar BMD after 10 weeks was observed with hPTH(1 - 34) or hPTH(1 - 84) at 1.1 nmol/kg, whereas hPTH(1 - 34) at 1.1 and 4.2 nmol/kg significantly increased BMD of the whole bone and the metaphysis of the femur and the diaphysis of the bone, respectively. In contrast, significant effects of hPTH(1 - 84) administration on BMD increase in the femur were observed at 4.2 and 15.9 nmol/kg in the whole bone and the metaphysis, and in the diaphysis, respectively. Maxillary molar extraction left mandibular BMD in rats with intact ovaries unchanged, but significantly decreased mandibular BMD in OVX rats. Administration of hPTH(1 - 84) for 10 weeks in OVX rats without or with extraction significantly increased BMD in the mandibular molar region at doses of 15.9 and 4.2 nmol/kg, respectively, indicating that efficacy was increased by extraction. A significant BMD increase in the molar region in OVX rats with extraction occurred at only 1.1 nmol/kg of hPTH(1 - 34) and 4.2 nmol/kg of hPTH(1 - 84). Also, BMD of the ramus region was increased by administration of both peptides to a lesser extent than that of the molar region in these rats. Thus, intermittent administration of hPTH, especially hPTH(1 - 34), has an anabolic effect on bone, particularly alveolar bone. Such treatment may increase alveolar bone mass in postmenopausal women with osteoporosis.     

Human parathyroid hormone (1-34) transiently increases the excretion of lysosomal enzymes into urine and the size of renal lysosomes.

It has been reported that the urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases in human after treatment with human parathyroid hormone (hPTH)(1-34). We report here that hPTH(1-34) caused transient changes in the size and density of rat renal lysosomes following urinary excretion of NAG and other lysosomal enzymes tested. Percoll density gradient centrifugation revealed that hPTH(1-34) slightly but significantly increased the fraction of high density lysosomes (around 1.12 g/ml) 5-10 min after the treatment with hPTH(1-34), with a concomitant decrease in the fraction of intermediate density lysosomes (1.07-1.08 g/ml). On electron micrographs, some lysosomes in proximal tubules but not in distal tubules showed a change in morphology from circular to oval, and became enlarged and electron-dense 5-10 min after the treatment with hPTH(1-34). These responses to hPTH(1-34) were also reversible and transient. NAG excreted in urine after treatment with hPTH(1-34) had the molecular mass of a mature form in lysosomes and/or endosomes and was not a prepro-and/or pro-form of the enzyme. Thus, the changes in the density and size of renal lysosomes appear to be associated with the exocytosis of lysosomal enzymes by hPTH(1-34).