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Estrogen receptors (ERα and ERβ), the vitamin D receptor (VDR) and 25 hydroxyy vitamin D 1-α hydroxylase (1OHase) mRNA are expressed in vascular smooth muscle cells (VSMC). In these cells estrogenic hormones modulate cell proliferation as measured by DNA synthesis (DNA). In the present study we determined whether or not the calciotrophic hormones PTH 1-34 (PTH) and less- calcemic vitamin D analog QW as well as hyperglycemia can regulate DNA synthesis and CK. E2 had a bimodal effect on VSMC DNA synthesis, such that proliferation was inhibited at 30nM but stimulated at 0.3nM. PTH at 50nM increased, whereas QW at 10nM inhibited DNA synthesis. Hyperglycemia inhibited the effects on high E2, QW and PTH on DNA only. Both QW and PTH increased ERα mRNA expression, but only PTH increased ERβ expression. Likewise, both PTH and QW stimulated VDR and 1OHase expression and activity. ERβ, VDR and 1OHase expression and activity were inhibited by hyperglycemia, but ERα expression was unaffected by hyperglycemia. In conclusion, calcitrophic hormones modify VSMC growth and concomitantly affect ER expression in these cells as well as the endogenous VSMC vitamin D system elements, including VDR and 1OHase. Some of the later changes may likely participate in growth effects. Of importance in the observation is that several regulatory effects are deranged in the presence of hyperglycemia, particularly the PTH- and vitamin D-dependent up regulation of VDR and 1OHase in these cells. The implications of these effects require further studies. This article is part of a Special Issue entitled '17th Vitamin D Workshop'., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Vitamin D less-calcemic analog JKF 1624 F2-2 (JKF) and PTH 1-34 stimulate in human female cultured osteoblasts (Ob) DNA synthesis (DNA), creatine kinase specific activity (CK), 1α, 25 vitamin D hydroxylase mRNA (1OHase) expression and 1,25(OH)2D3 (1,25) production, estrogen receptors (ER) mRNA expression and intracellular and membranal estrogen binding. In the present study, cultured Ob from different ages were subjected to hormonal stimulations and analyzed for different parameters. We found: 1) ERα expression is higher and ERβ expression is lower in pre-meno - pausal Ob (prOb), with similar intracellular and membranal binding. 2) JKF and PTH up-regulated ERα and JKF downregulated ERβ in both Ob, while PTH stimulated it in post- (poOb) and inhibited it in prOb. 3) There is higher expression of 1OHase mRNA in prOb, but 1,25 production is similar. Both parameters were hormonally stimulated to higher extent in prOb. 4) Ob express 12 and 15 lipoxygenase (LO) mRNA and produce 12- and 15-hydroxyeicosatetraenoic acid (H). 12LO expression is higher and 15LO is lower in prOb, while 12H is higher in prOb and 15H is similar in both. JKF inhibited 12LO expression in prOb and stimulated in poOb, whereas PTH stimulated it to higher extent in prOb. JKF stimulated and PTH inhibited 15LO expression in both; 12 and 15H were stimulated by both hormones in both Ob. 5. PTH and JKF stimulated DNA and CK in both Ob. In conclusion Ob demonstrate some age-dependent response to calciotrophic hormones, but the mechanism and beneficial outcome for human is unclear.

PTH-induced osteoblast proliferation may contribute to its anabolic effects in bone. Since PTH-dependent osteoblast-like cell (Ob) growth is mediated via protein kinase C (PKC) and MAP kinase-kinase (MEK) and since lipoxygenase (LO) products activate PKC in a number of cell types, we assessed the expression of LO pathways in primary human cultured Ob. Ob from pre- or post-menopausal women were cultured and were treated with PTH and assayed for the expression of 12-LO and both type I and type II 15-LO mRNA and for the release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Cells were also treated with PTH for stimulation DNA synthesis. First, Ob express platelet type- 12-LO and both type I and type II 15-LO mRNA and release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Second, in female Ob, PTH induced a rapid increase in 12-HETE (50 fold increase) and 15-HETE (80 fold increase) and increased the expression of 12-LO mRNA but not of the two isoforms of 15-LO. PTH as well as 12 and 15-HETE stimulated DNA synthesis in Ob. The LO inhibitor baicalein inhibited PTH-stimulated DNA synthesis, which was reversed in the presence of either 12- or 15-HETE. A PKC inhibitor (bisindolylmaleimide I) as well as a MEK inhibitor (PD 98059) completely inhibited the stimulation of DNA synthesis by PTH, 12-HETE and the combination of PTH and 12-HETE. In contrast, 15-HETE-induced DNA synthesis was not abolished by these inhibitors. Further, 15-HETE partially restored the stimulatory effect of PTH on DNA synthesis in cells treated with PKC or MEK inhibitors. Finally, PTH- induced ERK1/2 phosphorylation, was blocked by a MEK inhibitor. These results demonstrate a novel mechanism of PTH-induced human bone cell proliferation operating through LO enzymes.

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.

Background: 1,25(OH)2 vitamin D3 exerts multiple effects in human vascular smooth muscle cells (VSMCs). We therefore tested the possibility that VSMCs possess an endogenous 25-hydroxyvitamin D3-1alpha-hydroxylase system, the final enzyme in the biosynthetic pathway of 1,25(OH)2D3., Methods and Results: We assessed the expression and activity of 25-hydroxyvitamin D3-1alpha-hydroxylase by real-time polymerase chain reaction and the conversion of 25(OH)D3 into 1,25(OH)2D3. First, 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA was identified in cultured VSMCs by real-time polymerase chain reaction. Second, in cells treated daily (3 days) with parathyroid hormone (66 nmol/L), estradiol-17beta (30 nmol/L), raloxifene (3 micromol/L), and the phytoestrogens genistein (3 micromol/L), biochainin A (3 micromol/L), and 6-carboxy biochainin A (30 nmol/L), 25-hydroxyvitamin D3-1alpha-hydroxylase mRNA increased by 43+/-13%, (P<0.05) 7+/-24% (P=NS), 176+/-28% (P<0.01), 65+/-11% (P<0.05), 152+/-24% (P<0.01), and 71+/-9% (P<0.05), respectively. Third, production of 1,25(OH)2D3 from 25(OH)D3 was seen with a Km of 25 ng/mL and increased dose dependently after treatment with parathyroid hormone, genistein, and the phytosetrogen derivative 6-carboxy biochainin A. Estradiol-17beta and biochainin A also increased the generation of 1,25(OH)2D3 by 40+/-23% (P<0.05) and 55+/-13% (P<0.05), respectively., Conclusions: We provide here the first evidence for the expression of an enzymatically active 25(OH)D3-1alpha-hydroxylase system in human VSMCs, which can be upregulated by parathyroid hormone and estrogenic compounds. Because exogenous vitamin D inhibits VSMC proliferation, the role of this system as an autocrine mechanism to curb changes in VSMC proliferation and phenotype is a subject for future investigation.

We have demonstrated the net anabolic potential of a mid-region fragment of human parathyroid hormone (hPTH), and a protease resistant mutein derived from it, to stimulate growth of skeletal-derived tissues. The fragment hPTH (28-48), lacking the N-terminal amino acids necessary for stimulation of adenylate cyclase, and therefore unable to stimulate bone resorption by osteoclasts, was compared with the protease-resistant double-mutein hPTH (28-48) F34M L37T, full-length hPTH (1-84), the protease resistant form hPTH (1-84) L37T, 17beta estradiol (E(2)), and the combination of mid-region fragments of PTH and E(2). The hormones, at concentrations spanning a 100-fold range, were given by 14 injections (6/week, excluding Saturday), to 17-day-old female Wistar-derived rats. At the low concentration of 200 ng/day of PTH (1-84), or the molar equivalent of the fragment, and 50 ng E(2), all the hormones increased significantly the specific activity of creatine kinase (CK; a marker of skeletal cell proliferation) in tibial diaphysis and epiphysis, the width of the cortical bone in the humeral diaphysis, and the number of cells in the proliferating zone of the humeral epiphyseal growth plate. At a 10-fold lower concentration of both PTH and E(2), CK specific activity was synergistically stimulated in both diaphyseal bone and epiphyseal cartilage. However, PTH mid-region fragments at a dose of 1 microg/day did not increase trabecular bone volume.

Collagenase digestion of young rat condyles released cells which were grown in culture during two weeks. Morphologically, two populations of cells were distinguished, one of which reacted to alkaline phosphatase and resembled chondroblast or osteoblast-like cells. Parathyroid hormone stimulated a 2-fold increase in cellular cyclic AMP, whereas calcitonin had no effect. Physical forces activated cellular cyclic AMP to a 2.5-fold of control levels and increased the incorporation of radioactive thymidine into DNA by 50 per cent. In contrast to cultured bone cells, the response to physical forces was not inhibited by indomethacin in cultured condyle cells. It seems, therefore, that condyle cells are specific in their response to bone-seeking hormones and physical forces.

Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJb medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E2 (PGE2) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE2 (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)2D3 increased significantly the [3H]thymidine incorporation into DNA, whereas 1,25(OH)2D3 effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.