Pedro Esbrit

Changes in osteocalcin response to 1,25-dihydroxyvitamin D3 stimulation and basal vitamin D receptor expression in human osteoblastic cells according to donor age and skeletal origin

Age-related osteopenia is known to occur differently throughout the skeleton. In the present study, we examine the influence of donor age (<50 and >50 years), and bone structure (cortical vs. trabecular) on osteocalcin and vitamin D receptor (VDR) expression in primary cultures of human osteoblastic cells (hOB) cells. Cells were isolated from trabecular bone samples obtained from donors undergoing either knee (mainly trabecular) (n = 22; 4 <50 years, 18 >50 years) or hip (mainly cortical) (n = 16; 6 <50 years, 10 >50 years) arthroplasty. Pooling the results from knee and hip hOB cell cultures, we found that secreted osteocalcin was higher in hOB cells from the younger donors, compared with that in older donors, and peaked after stimulation with 10(-6)--10(-8) mol/L 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. In cells from the latter donors, this secretion was maximal after 10(-6) mol/L 1,25(OH)(2)D(3) treatment. On the other hand, using reverse transcription followed by polymerase chain reaction, baseline osteocalcin mRNA was found to be lower in hOB cells from the older donors than in those from younger donors. After treatment with 10(-6)--10(-8) mol/L 1,25(OH)(2)D(3), osteocalcin mRNA increased over baseline in all groups of hOB cells studied. In age-matched cultures, both basal and 10(-6)--10(-8) mol/L 1,25(OH)(2)D(3)-stimulated osteocalcin mRNA showed similar values in hOB cells from both skeletal sites in younger donors. However, in the older donors, baseline as well as 10(-8) mol/L 1,25(OH)(2)D(3)-stimulated osteocalcin mRNA were higher in knee hOB cells than in hip hOB cells. Furthermore, baseline VDR mRNA expression was also higher in the former cells than in the latter cells in the older group. Considering the influence of donor age at each skeletal site of origin, we found lower baseline osteocalcin and VDR mRNA levels in hip hOB cells in the older group than in the younger group. Our findings indicate that the response of osteocalcin secretion and its mRNA to physiological doses of 1,25(OH)(2)D(3) decreases with aging and is associated with decreased VDR mRNA expression in hOB cells from mainly cortical bone.

Immunohistochemical detection of parathyroid hormone-related protein in a rare variant of hepatic neoplasm (sclerosing hepatic carcinoma).

Sclerosing hepatic carcinoma represents an uncommon subtype of hepatic malignancy, frequently associated with hypercalcemia. We applied immunohistochemistry using the avidin-biotin-complex technique to examine the presence of parathyroid hormone-related protein (PTHrP) in formalin-fixed and paraffin-embedded sections of tissue from a case of sclerosing hepatic carcinoma obtained at autopsy. Two polyclonal antibodies against the regions 24 to 35 and 107 to 111 of human PTHrP, and a monoclonal antibody that recognizes the human sequence 38 to 64 of this protein, were used. Preabsortion tests using the corresponding synthetic peptide as antigen were done with these antibodies. The neoplastic tissue displayed cytoplasmic immunostaining, diffuse with the antibodies against the amino- or carboxy-terminal regions of PTHrP, and with a predominant peripheral pattern when using the antibody to the midregion of the molecule. Tumor cells positive for PTHrP were also positive for hepatocellular markers cytokeratins 10, 17, and 18, but negative for chromogranin A. Our findings provide the first evidence for PTHrP production in the sclerosing subtype of hepatic carcinoma.

Parathyroid hormone-related protein, parathyroid hormone, and vitamin D in hypercalcemia of malignancy.

The pathogenesis of cancer-associated hypercalcemia is not yet completely understood. In the majority of cancer patients, hypercalcemia appears to be a consequence of the tumor production of parathyroid hormone (PTH)-related protein (PTHrP). However, patients with humoral hypercalcemia of malignancy, in contrast to those with primary hyperparathyroidism, have an uncoupled bone turnover, and they usually have low circulating levels of 1.25(OH)2D3. We performed a case-control study to assess the relationship of plasma PTHrP, PTH and 1.25(OH)2D3 with hypercalcemia in cancer patients with a variety of tumors. Sixty of these patients had hypercalcemia, and 45 were normocalcemic. We measured PTHrP and PTH by immunoradiometric assay (Nichols), and 1.25(OH)2D3 by radioreceptor assay (Nichols), in plasma in both groups of cancer patients. Using a logistic regression analysis, we found that the higher PTHrP in plasma, the higher association with hypercalcemia occurred in these patients. In addition, the decreased plasma levels of PTH and 1.25(OH)2D3 in the majority of cancer patients were found to be significantly associated with hypercalcemia. Our results indicate that the combined determination of PTH, PTHrP and 1.25(OH)2D3 in plasma represents a more comprehensive approach to the investigation of hypercalcemia in cancer patients. Our data also support the role of PTHrP as a humoral factor responsible for hypercalcemia in these patients.

Co-purification of calcium transport-stimulating and DNA synthesis-stimulating agents with parathormone-like activity isolated from the hypercalcaemic strain of the Walker 256 tumour

One of the strains of the Walker 256 carcinosarcoma induces in the rat a humoral hypercalcaemia of malignancy (HHM) syndrome which is similar to that reported in human patients. We have isolated from this tumour a chromatographic fraction which displays an adenylate cyclase stimulating activity in dog kidney cortical membranes, similar to that of a parathormone (PTH) related protein isolated from various HHM related tumours. In addition, this fraction stimulated initial calcium (Ca) uptake in confluent Madin-Darby canine kidney (MDCK) cell monolayers in a dose-dependent manner. Maximal stimulation of Ca uptake was associated with an enhanced Ca efflux from MDCK cells preloaded with the cation, and with an increased DNA synthesis in these cells. These activities might be involved in development of increased tubular calcium reabsorption in Walker 256 tumour-bearing rats.

Relationship of plasma bone cytokines with hypercalcemia in cancer patients.

The pathogenesis of cancer-associated hypercalcemia is not yet completely understood. This syndrome appears to be a consequence of the tumor production of humoral factors, mainly parathyroid hormone related protein (PTHrP). However, patients with humoral hypercalcemia of malignancy have features suggesting that factors other than PTHrP might play a role in this syndrome. We performed a case-control study in cancer patients with and without hypercalcemia. A total of 105 patients with a variety of tumors, 60 of them with hypercalcemia (corrected serum calcium over 2.6 mmol/l), and 45 without hypercalcemia. In a previous study, we demonstrated that plasma PTHrP was highly associated with hypercalcemia in these patients. In the present study, we measured the plasma levels of various bone cytokines: interleukin-1beta (IL-1beta), interleukin-6 (IL-6), transforming growth factor (TGF) alpha, and tumor necrosis factor (TNF) alpha, in these cancer patients. We also determined C-terminal type I procollagen (PICP) and C-terminal telopeptide of type I collagen (ICTP), bone formation and bone resorption markers, respectively, in serum in these patients. We found that these osteolytic cytokines do not increase in plasma by the presence of hypercalcemia. In fact, using a logistic regression analysis, a significant (P<0.02) association was found between the low plasma levels of IL-1beta and TGFalpha and hypercalcemia, independent of plasma PTHrP and the presence of bone metastasis, in these patients. No significant association between the plasma levels of IL-6 or TNFalpha and hypercalcemia was found in these cancer patients. Serum ICTP correlated (r=0.35; P=0.008) with hypercalcemia in these patients, but none of the cytokines studied in plasma correlated with either ICTP or PICP in these hypercalcemic patients. Our data indicate that the circulating levels of several bone cytokines are not enhanced by PTHrP in hypercalcemic cancer patients. The mechanism responsible for the association between the low plasma levels of some of these cytokines and hypercalcemia in these patients remains obscure. However, this finding does not rule out the possible local bone effects of these cytokines, contributing to hypercalcemia in cancer patients.

Parathyroid Hormone-Related Protein (107-139) Decreases Alkaline Phosphatase in Osteoblastic Osteosarcoma Cells UMR 106 by a Protein Kinase C-Dependent Pathway

Abstract. The C-terminal (107-111) region of parathyroid hormone-related protein (PTHrP) appears to inhibit osteoclastic bone resorption, and to affect osteoblastic growth and differentiation. We tested the effect of human PTHrP (107-139) on alkaline phosphatase (ALP) activity in osteoblastic osteosarcoma UMR 106 cells. We found that this C-terminal PTHrP peptide, between 10 nM and 10 fM, inhibited ALP activity in these cells during the log phase of growth. Human PTHrP (1-34) amide and human PTHrP (1-141) were as potent as PTHrP (107-139) in growing UMR 106 cells. This inhibitory effect of 10 nM PTHrP (107-139) on ALP activity was also observed in serum-depleted cells, and in the presence of 10 nM dexamethasone, which increased ALP activity by 40% in these cells. In addition, this effect of PTHrP (107-139) was blunted by 25 nM bisindolylmaleimide I, a protein kinase C inhibitor. These results support a role for the C-terminal region of PTHrP as a modulator of bone formation.

Comparison of two immunoradiometric assays for parathyroid hormone-related protein in the evaluation of cancer patients with and without hypercalcemia.

Hypercalcemia is a common paraneoplastic syndrome due to the secretion by tumors of parathyroid hormone-related protein (PTHrP) and/or other osteolytic factors. In the present study, we have measured plasma PTHrP using two immunoradiometric assays for PTHrP, assay N (Nichols) and assay I (INCSTAR), recognizing the 1-86 domain of PTHrP, for the evaluation of malignancy-associated hypercalcemia. The study included 25 tumor patients with hypercalcemia (HCa) [corrected serum calcium (SCa) > or = 2.70 mmol/L], 20 normocalcemic patients with cancer (NCa), and ten healthy control subjects. Plasma PTHrP was either undetectable or within the respective normal range in the majority of NCa patients and in the control subjects, with both assays. Plasma PTHrP was increased in 13 and 15 of HCa cases with assay N and assay I, respectively. PTHrP was elevated in plasma in 5/6 (assay N) and 3/6 (assay I) HCa patients with squamous tumors. However, plasma PTHrP was high in only 2/9 (assay N) and 1/9 (assay I) HCa cases with hematological tumors. Less than 40% of HCa patients with bone metastases, and >75% of those without bone involvement, had elevated plasma PTHrP with both assays. Detectable plasma PTHrP and SCa were significantly correlated using assay N (p = 0.025) and assay I (p = 0.01), in the HCa group. A highly significant correlation (p <0.001) was found between detectable plasma PTHrP with both assays, and a high agreement between them based on simple kappa statistics (p < 0.001), in the latter group. Our results indicate that each assay may be similarly useful in detecting PTHrP hyperproduction in cancer patients.

Isolation of a 18 000-Da molecular weight form of parathyroid hormone-related protein from the rat walker carcinosarcoma 256

The hypercalcaemic Walker carcinosarcoma 256 is a rat model for humoral hypercalcaemia of malignancy (HHM). Tumour products such as parathyroid hormone-related proteins (PTHRP) and various growth factors appear to be responsible for this syndrome. Recently, PTHRP immunoreactivity has been detected in the conditioned medium of Walker tumour cells. The present report describes the isolation of a 18,000-Da molecular weight form of PTHRP from Walker tumour homogenates, by using a relatively simple immunoaffinity purification method. Our results suggest that this PTHRP form is similar to that purified from other HHM-related tumours.

Evidence of 1,25-dihydroxyvitamin D synthesis by the rat walker carcinosarcoma 256

We tested the existence of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in the Walker carcinosarcoma 256 implanted in rats. This tumour has been shown to induce hypercalcaemia in the host animal. We found this enzyme activity in tumour homogenates, which was in the same range as that in the kidney of tumour-bearing rats. Our results suggest that 1,25-dihydroxyvitamin D synthesized by the Walker tumour might be involved in the mechanism responsible for the hypercalcaemia in the host rat.

Role of Oxidative Stress in Bone Ageing

Oxidative stress (OS) plays a major role in ageing process. Aerobic metabolism involves the production of reactive oxygen species (ROS) whose accumulation produces irreversible cell damage. Nature has developed various antioxidant mechanisms as defence against oxidative stress, but their efficacy decreases with ageing, which leads to homeostasis alterations. There is evidence showing that OS plays a major role in the development of age-related osteopenia. In bone, loss of oestrogen production and chronic illnesses such as diabetes xadmellitus contribute to the increased ROS levels with age. However, typical xadantioxidants such as N-acetyl cysteine or catalase are not effective to prevent the deleterious effects of high OS in this tissue, probably due to their concomitant anti-remodelling action. Of interest, it has very recently been reported that parathyroid hormone, the only currently available anabolic agent for osteoporosis, exerts various osteogenic effects including anti-OS features. Unravelling the mechanisms underlying the increase of OS and its relation to bone loss with age would be important to design novel strategies to prevent the development of osteoporosis in ageing subjects.