Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor

The scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1-35(tyr))PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10(-8) M) based on the N-terminal 1-34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3H]myo-inositol incorporation in sea bream scales. However, peptides (10(-8) M) with N-terminal deletions, such as (2-34), (3-34) and (7-34)PTHrP, were defective in stimulating cAMP production but stimulated [3H]myo-inositol incorporation. (1-34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7-34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1-34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1-34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.     

Periostin is a collagen associated bone matrix protein regulated by parathyroid hormone

Periostin is a 90 kDa secreted protein, originally identified in murine osteoblast-like cells, with a distribution restricted to collagen-rich tissues and certain tumors. In this paper, we first analyzed the expression of periostin mRNA and protein in human fetal osteoblasts (hFOB) and human osteosarcoma (hOS) cell lines by RT real-time PCR and Western blot, respectively. The hFOB 1.19 and three hOS (MHM, KPDXM and Eggen) showed highly variable periostin mRNA levels and protein. Second, we showed that the expression of periostin mRNA was inversely related to the cells' abilities to differentiate and mineralize. Then, we investigated the regulation of periostin mRNA in hFOB after siRNA treatment and in mouse primary osteoblasts (mOB) treated with PTH. Knock-down of periostin mRNA, down-regulated PTHrP, but did not affect the expression of other important markers of differentiation such as RUNX2. In addition, periostin mRNA was transiently up-regulated in osteoblasts by PTH. Finally, the localization of periostin and its partially co-localization with collagen 1a1 mRNA and protein was studied in mouse embryos and postnatal pups using in situ hybridization and immunohistochemistry, respectively. In conclusion, the present study provides novel observations related to the expression, distribution and regulation of periostin in bone cells and extracellular matrix.     

Differential regulation of osteoblast activity by Th cell subsets mediated by parathyroid hormone and IFN-gamma

Bone loss is a typical pathological feature of chronic inflammatory bone diseases including rheumatoid arthritis, in which CD4 effector T cells play critical roles. We found that activated mouse Th2 and not Th1 cells produced the parathyroid hormone (PTH). Unlike in the parathyroid cells, PTH expression in Th2 cells was not regulated by the fluctuation of calcium level, but rather it required the full activation of the T cells. Although PTH was expressed in immature Th2 cells, and its receptor was transiently expressed during Th1 and Th2 cell differentiation, PTH did not significantly affect the outcome of the differentiation. In primary osteoblasts cultured in Th2 cell condition medium, the alkaline phosphatase (ALP) activity was maintained at a basal level. However, antagonizing PTH in the condition medium resulted in a significant reduction of the ALP activity. These results demonstrated an important role of the Th2 cell-derived PTH in maintaining the bone-forming activity of the osteoblasts under inflammatory conditions. In osteoblasts cultured in the Th1 cell condition medium, the ALP activity was significantly suppressed. Neutralizing IFN-gamma alleviated the suppression. Conversely, treatment of osteoblasts with IFN-gamma suppressed the ALP activity. Unlike ALP, expression of the major bone matrix proteins by the osteoblasts was only minimally affected by either Th1 or Th2 cytokine environment. In addition, the Th2 cytokine environment also regulated to expression of receptor activator of NF-kappaB ligand and osteoprotegerin through both PTH-dependent and -independent mechanisms. Our study therefore identified new regulatory events in bone remodeling under inflammatory conditions.     

Amelioration of denervation-induced atrophy by clenbuterol is associated with increased PKC-alpha activity

Rat soleus muscle was denervated for 3 or 7 days, and total membrane protein kinase C (PKC) activity and translocation and immunocytochemical localization of PKC isoforms were examined. Dietary administration of clenbuterol concomitant with denervation ameliorated the atrophic response and was associated with increased membrane PKC activity at both 3 (140%) and 7 (190%) days. Of the five PKC isoforms (alpha, epsilon, theta, zeta, and mu) detected in soleus muscle by Western immunoblotting, clenbuterol treatment affected only the PKC-alpha and PKC-theta forms. PKC-alpha was translocated to the membrane fraction upon denervation, and the presence of clenbuterol increased membrane-bound PKC-alpha and active PKC-alpha as assayed by Ser(657) phosphorylation. PKC-theta protein was downregulated upon denervation, and treatment with clenbuterol further decreased both cytosolic and membrane levels. Immunolocalization of PKC-theta showed differences for regulatory and catalytic domains, with the latter showing fast-fiber type specificity. The results suggest potential roles of PKC-alpha and PKC-theta in the mechanism of action of clenbuterol in alleviating denervation-induced atrophy.     

Separation of inhibitory activity from biologically active parathyroid hormone in patients with pseudohypoparathyroidism type I

Patients with pseudohypoparathyroidism type I have the symptoms of hypoparathyroidism despite elevated levels of immunoreactive parathyroid hormone (PTH). However, the circulating levels of bioactive PTH, as measured in a cytochemical bioassay, are generally within the normal range suggesting that the high levels of immunoreactive PTH are either due to the presence of biologically inactive fragments of parathyroid hormone or to the presence of an 'inhibitor' of PTH bioactivity. Gel-permeation chromatography has been used to fractionate plasma from patients with pseudohypoparathyroidism type I and revealed the presence of high levels of bioactive PTH and of an 'inhibitor'. This inhibitory activity was absent or much lower in plasma from control subjects. These results indicate, therefore, that in pseudohypoparathyroidism type I the expression of the biological activity of PTH at the level of the kidney is affected by the presence of a circulating inhibitor which can be separated from intact PTH by gel-permeation chromatography.     

TH1 predominance is associated with improved survival in pediatric medulloblastoma patients

Medulloblastoma, a primitive neuro-ectodermal tumor that arises in the posterior fossa, is the most common malignant brain tumor occurring in childhood. Even though 60–70% of children with medulloblastoma will be cured with intensive multimodal therapy, including surgery, radiotherapy, and chemotherapy, a significant proportion of surviving patients may suffer from long-term treatment-related sequelae. Therapeutic success is limited especially in younger children by radiotherapy-induced neurocognitive longterm deficits. In order to avoid or delay craniospinal radiotherapy, high-dose chemotherapy followed by autologous stem cell transplantation (HSCT) has become an established treatment modality. Data on the host immunologic environment in medulloblastoma patients are rare, notably data on cytokine expression and immune reconstitution in patients with medulloblastoma undergoing HSCT are lacking. In this present study, we therefore decided to prospectively assess immune function following 24 consecutive autologous HSCT in 17 children with medulloblastoma treated according to the German-Austrian-Swiss HIT-2000-protocol. TH1 predominance was found to be the most important factor for probability of survival. Already before HSCT, survivors showed higher IFNγ levels in sera as well as higher numbers of IFNγ-positive T-cells. After transplantation, this effect was even more pronounced. Patients with higher numbers of IFNγ- and TNFα-positive T-cells had a more favorable outcome at all analyzed time points. In addition, patients in complete remission (CR) before transplantation, known to have a better prognosis a priori, showed higher expression of IFNγ in T-cells. Taken together, this is the first report to demonstrate that high expression of IFNγ and TNFα in T-cells of medulloblastoma patients in the early post-transplant period correlates with a better prognosis. Our data point toward a potentially important influence of TH1-cytokine expression before and after transplantation on the survival of pediatric medulloblastoma patients.     

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.     

Conformational changes in the parathyroid hormone receptor associated with activation by agonist

Binding of hormones to their cognate G protein-coupled receptors (GPCRs) induces conformational shifts within the receptor based on evidence from a few hormone-receptor systems. Employing an engineered disulfide bond formation strategy and guided by a previously established model of the PTH-PTH receptor (PTHR)1 bimolecular complex, we set out to document and characterize the nature of agonist-induced changes in this family B GPCR. A mutant PTHR1 was generated which incorporates a Factor Xa cleavage site in the third intracellular loop. Treatment with Factor Xa fragments the receptor. However, if a new disulfide bond was formed before exposure to the enzyme, the fragments remain held together. A set of double cysteine-containing mutants were designed to probe the internal relative movements of transmembrane (TM) helices 2 and TM7. PTH enhanced formation of disulfide bonds in the K240C/F447C and A242C/F447C mutants. For the F238C/F447C mutant, a disulfide bond is formed in the basal state, but is disrupted by interaction with PTH. For the D241C/F447C PTHR1 construct, no disulfide bond formation was observed in either the basal or hormone-bound state. These findings demonstrate that the conformation of PTHR1 is altered from the basal state when PTH is bound. Novel information regarding spatial proximities between TM2 and TM7 of PTHR1 and the nature of relative movements between the two transmembrane regions was revealed. The data confirm and extend the experimentally derived model of the PTH-PTHR1 complex and provide insights at a new level of detail into the early events in PTHR1 activation by PTH.     

The role of parathyroid hormone in the management of osteoporosis

It has been over 2 years since parathyroid hormone (PTH 1-34) was approved in the US and Europe for the treatment of osteoporosis in postmenopausal women and men. Clinical experience with this peptide has enhanced confidence in its use and its application in specific clinical scenarios. There is no doubt that PTH 1-34 is safe and effective in reducing spine and non-vertebral fractures in men and women. However, the lack of several randomized placebo-controlled trials and their relatively short duration raise several questions that still need to be answered. This paper reviews three major areas of uncertainty: (1) Is there significant heterogeneity in the bone density response of individuals to PTH? If so, what factors are important predictors? (2) What other regimens are available for PTH use? (3) What, if anything, should the clinician do after PTH is discontinued? Answers to these questions will undoubtedly lead to even greater utilization of this drug and some of its future derivatives.     

The activation of cAMP-dependent protein kinase is directly linked to the regulation of osteoblast proliferation (UMR-106) by parathyroid hormone.

In order to characterize the direct involvement of cAMP in the change of osteoblast proliferation by parathyroid hormone (PTH), we employed the diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, Sp-cAMPS and Rp-cAMPS, which have been recently shown to act directly as agonist and antagonist, respectively in the activation of cAMP-dependent protein kinase (PKA). Dibutyryl cAMP (dbcAMP) and cholera toxin as well as human(h) PTH-(1-34) significantly inhibited [3H]thymidine incorporation (TdR) in osteoblastic osteosarcoma cells, UMR-106. Sp-cAMPS (10(-6)-10(-4) M) inhibited TdR in a dose-dependent manner. Although Rp-cAMPS (10(-6)-10(-4) M) itself did not affect TdR, it significantly blocked dbcAMP-, cholera toxin- and Sp-cAMPS-induced suppression of TdR. Moreover, Rp-cAMPS (10(-6)-10(-4) M) dose-dependently antagonized hPTH-induced suppression of TdR. Present studies first indicated that the activation of PKA is directly linked to the change of osteoblast proliferation by PTH.     

Humoral hypercalcemia caused by a rat Leydig-cell tumor is associated with suppressed parathyroid hormone secretion and increased urinary cAMP excretion

We studied the effect of a transplantable Leydig-cell tumor (Rice H-500) on serum calcium, parathyroid hormone (PTH), and urinary cAMP in intact Fischer-344 rats. The tumor caused rapid and severe hypercalcemia (control = 10.5 +/- 0.1 mg/dl [mean +/- S.E.] vs. 14.6 +/- 0.9 at day 12 post tumor inoculation) without evidence of metastasis. Progressive renal impairment and death generally occurred within 15 days of tumor inoculation. Serum PTH declined from control values before hypercalcemia occurred and was significantly reduced in tumor-bearing hypercalcemic rats (mean = 60 +/- 8% of control values). Urinary cAMP excretion was increased in tumor-bearing rats (mean at day 12 post inoculation = 12.2 +/- 1.4 nmol/dl creatinine clearance vs. control = 6.2 +/- 0.2) and correlated positively with serum calcium. The Rice H-500 Leydig-cell tumor appears to secrete a humoral factor capable of causing hypercalcemia. This factor may also increase urinary cAMP excretion in a manner analogous to PTH, but it is not detected by PTH radioimmunoassay.     

The parathyroid hormone-related protein associated with malignancy is secreted by neuroendocrine tumors

We have demonstrated the production of the PTH-related protein (PTHrP) associated with hypercalcemia of malignancy by human neuroendocrine cell lines that also produce calcitonin gene products and chromogranin A. PTHrP was demonstrable in the cells by immunocytochemistry and immunoassay and Northern analysis of the cells revealed the presence of multiple mRNAs for PTHrP. The cell lines also secreted PTHrP in a regulated fashion, with the most potent secretagogue being phorbol. Thus, PTHrP is secreted by neuroendocrine cells and it may have neuroectodermal lineage. The coexpression of calcitonin gene products and chromogranin A, also neuroendocrine, with PTHRP may influence its secretion and ultimate biological effects in vivo.     

Nuclear vitamin D receptor expression is associated with improved survival in non-small cell lung cancer

Vitamin D has been shown to have anti-proliferative effects in a wide variety of cancers including lung cancer. The anticancer effects of vitamin D are mediated primarily by its active metabolite, 1,25-dihydroxyvitamin D (calcitriol), through vitamin D receptor (VDR) signaling. However, thus far there have been no studies evaluating the association between VDR expression and survival outcome in lung cancer. Using immunohistochemical analysis, we evaluated VDR expression, separately in the nucleus and cytoplasm, in lung cancer samples from 73 non-small cell lung carcinoma (NSCLC) patients with no prior therapy, and investigated the association between VDR expression and overall survival (OS). Cox proportional hazard models were used for our primary analyses. There were 44 deaths during a median follow-up of 51 months (range 13-93 months). High nuclear VDR expression was associated with improved OS after adjusting for age, gender, stage, smoking status, and histology (adjusted hazard ratio, 0.36; 95% confidence interval, 0.17-0.79). There was no association between cytoplasmic VDR expression and OS. Our results suggest that nuclear VDR status may be a prognostic marker in NSCLC. Future large studies to replicate our findings and to assess the impact of VDR gene polymorphisms on VDR expression are required as therapies targeting the vitamin D signaling pathway may be influenced by VDR status in the target lung cancer tissue.