Influence of aluminum on the regulation of PTH- and 1,25(OH)2D3-dependent pathways in the rat osteosarcoma cell line ROS 17/2.8

The role of hormonal status in the development of aluminum (Al)-dependent renal osteodystrophy, which is characterized by reduced bone matrix deposition, still remains largely unknown. To address this question, we used the osteoblast-like osteosarcoma cell line ROS 17/2.8 to evaluate the role of Al on parathyroid hormone (PTH)- and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-dependent activities in these cells. Al (1 microM) caused an inhibition of basal and 1,25(OH)2D3-induced alkaline phosphatase, but only at low doses (< 1 nM) of the steroid. Al partly inhibited basal osteocalcin (OC) secretion in ROS cells (p < 0.001), and the dose-dependent increase in 1,25(OH)2D3-induced OC release by these cells was also reduced by 1 microM Al at low concentrations of the steroid (< or = 1 nM), whereas high doses of 1,25(OH)2D3 (> or = 5 nM) totally prevented the inhibiting effects of Al. Al also had strong inhibitory actions on PTH-dependent cAMP production by ROS cells over the concentration range tested (0.5-50 nM). This inhibitory action of Al was also observed for PTH-related peptide- (PTHrp, 50 nM) but not for Isoproterenol-dependent (100 nM) cAMP formation. To evaluate more fully the mechanism of this inhibition of cAMP formation, we investigated the effect of Al on toxin-modulated, G protein-dependent regulation of cAMP formation and on the activation of adenylate cyclase by Forskolin. Cholera toxin (CT, 10 micrograms/ml), applied to cells for 4 h prior to PTH challenge, enhanced cAMP production about 2-fold above PTH alone (p < 0.001), a process that was further stimulated by Al. Pertussis toxin (PT, 1 microgram/ml, 4 h) did not modify basal PTH-dependent cAMP formation by ROS cells. However, PT treatment prevented the inhibitory effect of Al on cAMP formation by these cells (p < 0.025). The stimulation of adenylate cyclase by Forskolin (0.1 and 1 microM), which bypasses G protein regulation, was not modified by Al, indicating that Al does not affect adenylate cyclase directly. Northern blot analysis of PTH receptor mRNA levels showed that Al did not modify PTH receptor message in ROS cells. Likewise, Western blot analyses of G protein subunits showed that Al did not significantly alter Gs alpha subunit levels, in accordance with the results obtained for cAMP-dependent formation in response to CT. In contrast, Gi alpha-1 and Gi alpha-2 subunits were decreased by Al treatment, consistent with PT-restricted increases in cAMP formation in Al-treated ROS cells. Taken together, these results suggest that Al has multiple actions in osteoblast-like ROS cells. The effects of Al are modulated by hormonal control of the pathways investigated. Al affects 1,25(OH)2D3-regulated functions only when this steroid is low. Al has large inhibitory effects on PTH- and PTHrp-dependent cAMP formation. This last feature is related to the ability of Al to alter the G protein transducing pathway for PTH/PTHrp-dependent formation of cAMP since it does not affect adenylate cyclase activity directly and does not affect the PTH receptor message level. Thus, Al has stronger deleterious effects in osteoblast-like cells with an already compromised 1,25(OH)2D3 status and can modulate specifically PTH/PTHrp-mediated cAMP formation at the postreceptor level.     

In vivo regulation of chromogranin A messenger ribonucleic acid in the parathyroid by 1,25-dihydroxyvitamin D: studies in normal rats and in chronic renal insufficiency

Chromogranin-A (CgA) and PTH are the two major secretory products of the parathyroid gland. In vitro, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] increases CgA, but decreases PTH messenger RNA (mRNA) levels. We investigated the physiological significance of the induced changes in CgA expression by examining the effects of 1,25-(OH)2D3 on parathyroid CgA mRNA levels in vivo. Normal rats were injected with 1,25-(OH)2D3 at 48 and 24 h before blood sampling and isolation of both parathyroid glands. Parathyroid total RNA was extracted and CgA and PTH mRNA quantified by Northern blot analysis. CgA mRNA levels increased 1.6-, 3.2- and 5.6-fold, whereas PTH mRNA levels decreased by 37, 63 and 97%, respectively, with 1,25-(OH)2D3 doses of 10, 50, and 250 pmol/100 g BW. Parathyroid gland CgA expression also was examined in rats with mild chronic renal insufficiency, induced by a 5/6 nephrectomy 5 weeks earlier. Chronic renal insufficiency rats, fed normal chow, had elevated serum urea, creatinine, and PTH levels and reduced 1,25-(OH)2D3 but normal serum levels of calcium and phosphate. PTH mRNA levels were elevated 4-fold and CgA mRNA levels were 50% lower in the uremic animals. This indicates that the regulation of CgA expression in normocalcemic rats occurs at physiological 1,25-(OH)2D3 concentrations. In summary, increases and decreases in serum 1,25-(OH)2D3 levels are associated with corresponding increases and decreases in CgA mRNA levels in the parathyroid glands of rats. Therefore, this study is the first to demonstrate the physiological relevance of the earlier in vitro observations.     

Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.     

Human osteoclast-like cells are formed from peripheral blood mononuclear cells in a coculture with SaOS-2 cells transfected with the parathyroid hormone (PTH)/PTH-related protein receptor gene

Subclones of the human osteosarcoma cell line SaOS-2 were established by transfecting with an expression vector containing the human PTH/PTH-related protein (PTHrP) receptor, and their abilities to support osteoclast-like multinucleated cell (OCL) formation were examined in coculture with mouse or human hemopoietic cells. Of four subclones examined, SaOS-2/4 and SaOS-4/3 bound high levels of [125I]-PTH and produced a significant amount of cAMP in response to PTH. OCLs were formed in response to PTH in the cocultures of mouse bone marrow cells with either SaOS-2/4 cells or SaOS-4/3 cells. Human OCLs were also formed in response to PTH in the coculture of SaOS-4/3 cells and human peripheral blood mononuclear cells. Adding dexamethasone together with PTH greatly enhanced PTH-induced human OCL formation. Like mouse OCLs, human OCLs formed in response to PTH were tartrate-resistant acid phosphatase positive, expressed abundant calcitonin receptors and vitronectin receptors, and formed resorption pits on dentine slices. Other osteotropic factors such as 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, and interleukin 6 plus soluble interleukin 6 receptors failed to induce mouse and human OCLs in cocultures with SaOS-4/3 cells. Both mouse and human OCL formation supported by SaOS-4/3 cells were inhibited by either adding an antibody against macrophage-colony stimulating factor or adding granulocyte/macrophage-colony stimulating factor. Thus, it is likely that human and mouse OCL formation supported by SaOS-4/3 cells are similarly regulated. These results indicate that the target cells of PTH for inducing osteoclast formation are osteoblast/stromal cells but not osteoclast progenitor cells in the coculture. This coculture model will be useful for investigating the abnormalities ofosteoclast differentiation and function in human metabolic bone diseases.     

Recombinant human bone morphogenetic protein-2 stimulates osteoblast differentiation and suppresses matrix metalloproteinase-1 production in human bone cells isolated from mandibulae

Bone morphogenetic protein (BMP), a member of the transforming growth factor superfamily, is one of the most potent growth factors that stimulate osteoblast differentiation and bone formation. We investigated the effects of recombinant human BMP-2 (rhBMP-2) on osteoblast differentiation and matrix metalloproteinase-1 (MMP-1) production in human bone cells (HBC) isolated from mandibulae of 3 adult patients. rhBMP-2 at concentrations over 50 ng/ml significantly stimulated alkaline phosphatase activity and parathyroid hormone (PTH)-dependent 3', 5'-cyclic adenosine monophosphate accumulation, which are early markers of osteoblast differentiation, in HBCs. rhBMP-2 (500 ng/ml) also enhanced the level of PTH/PTH related-peptide receptor mRNA expression in HBCs. Although neither HBCs untreated nor treated with rhBMP-2 produced measurable amounts of osteocalcin, which is a marker of more mature osteoblasts, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced ostocalcin mRNA expression and its protein synthesis in these cells. rhBMP-2 inhibited 1,25(OH)2D3-induced osteocalcin synthesis in HBCs at both the mRNA and protein level. rhBMP-2 also significantly suppressed MMP-1 production and MMP-1 mRNA expression at concentrations over 500 ng/ml. These results suggest that rhBMP-2 exerts anabolic effects on human osteoblastic cells derived from mandibulae by stimulation of osteoblast differentiation and down-regulation of MMP-1 synthesis.     

The E1A oncogene induces resistance to the effects of 1,25-dihydroxyvitamin D3 on inhibition of growth of mouse keratinocytes

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited DNA synthesis in transformed mouse keratinocytes (Pam212) in a time- and dose-dependent manner as measured by [3H]thymidine incorporation. To investigate the mechanism through which 1,25-(OH)2D3 acts, we examined its effects on Pam212 cells further transformed with the E1A oncogene. Here, we show that transformation of the cells with the E1A oncogene induced resistance to the effects of 1,25-(OH)2D3 on inhibition of growth of Pam212 cells. While 1,25-(OH)2D3 treatment increased the level of expression of vitamin D receptor mRNA 20-fold in parental cells, the E1A-transformed cells failed to express vitamin D receptor mRNA even after treatment with 1,25-(OH)2D3. Transfection of the E1A-transformed cell line with an expression construct encoding the vitamin D receptor restored receptor expression as well as the inhibition of growth by 1,25-(OH)2D3. These results suggest that one of the mechanisms for acquisition of 1,25-(OH)2D3 resistance induced by E1A may involve loss of vitamin D receptor inducibility by 1,25-(OH)2D3.     

Vitamin D derivatives inhibit the mitogenic effects of IGF-I on MCF-7 human breast cancer cells

The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and four novel synthetic analogues (EB1089, KH1060, KH1230 and CB1093) on IGF-I-stimulated growth of MCF-7 human breast cancer cells have been determined. A significant time- and dose-dependent inhibition of IGF-I-stimulated cell growth was seen with EB1089, such that after 7 days of treatment with 10(-8) M EB1089, the mitogenic effect of IGF-I (30 ng/ml) was negated. Comparison with 1,25(OH)2D3 showed the synthetic analogues to be more potent. The anti-oestrogen ICI 182,780 similarly inhibited IGF-I-stimulated growth of these cells and in combination with EB1089 exerted additional inhibitory effects. Retinoids (all-trans-retinoic acid or the isomer 9-cis-retinoic acid) were less effective in limiting MCF-7 cell responsiveness to IGF-I but, in combination with EB1089, a co-operative effect was achieved. Using radioligand-binding techniques, we observed that 1,25(OH)2D3 and EB1089 down-regulated the levels of 125I-IGF-I binding to MCF-7 cell membranes. Scatchard analysis showed that EB1089 decreased maximal binding approximately 2-fold. Vitamin D derivatives were also demonstrated to reduce IGF-I receptor expression in MCF-7 cells by Western analysis. Our findings demonstrate that vitamin D derivatives limit responsiveness of MCF-7 cells to the mitogenic effects of IGF-I, which may be mediated by reduction of IGF-I receptor expression.     

Effects of triiodothyronine on DNA synthesis and differentiation markers of normal human osteoblast-like cells in vitro

To study the direct effects of thyroid hormones on human osteoblasts we examined the effects of triiodothyronine (T3) on proliferation and differentiation of human osteoblast-like (hOB) cells in vitro. T3 increased 3H-thymidine incorporation in DNA of hOB cells (p < 0.05, n = 10). Half maximal effects obtained at a T3 concentration of 1-10 nM which lies within the physiological concentration of the hormone. In addition, T3 increased alkaline phosphatase production (p < 0.05, n = 13) and inhibited procollagen type I carboxyterminal propeptide (PICP) production (p < 0.05, n = 13). T3 interaction with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was also studied. 1,25-(OH)2D3 (10(-9)M) alone doubled AP production and induced osteocalcin expression by hOB cells. Concurrent addition of T3 and 1,25-(OH)2D3 did not further increase production of AP, PICP or osteocalcin by hOB cells. In conclusion, T3 exerts significant effects on osteoblast proliferation and differentiation, suggesting that human osteoblasts are targets for thyroid hormones.     

Cyclosporin A in resistant chronic inflammatory demyelinating polyradiculoneuropathy

Previous in vivo studies have shown that growth hormone (GH) affects vitamin D and mineral metabolism. Insulin-like growth factor-I (IGF-I) was recently reported to be a regulator of renal 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production, suggesting that it mediates the effects of GH on vitamin D metabolism. However, there is no direct evidence to support this. The present study was designed to investigate the in vitro effects of GH and IGF-I on the renal production of 1,25-(OH)2D3 and 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) in a pig kidney cell line, LLC-PK1. Confluent cells were preincubated in serum-free medium with hormone (GH or IGF-I) or vehicle, and then incubated with 25-[3H]OHD3. The levels of 1,25-[3H](OH)2D3 and 24,25-[3H](OH)2D3 produced were determined after lipid extraction and HPLC purification. Production of 1,25-(OH)2D3 and 24,25-(OH)2D3 was increased after both IGF-I and GH preincubation in a dose-dependent manner. Significant increases were found after preincubation with 13 nmol/l IGF-I (1,25-(OH)2D3, 1.8-fold: 24,25-(OH)2D3, 1.5-fold)or 0.9 or 9 nmol/lGH (1,25-(OH)2D3, 1.3-fold and 1.5-fold; 24.25-(OH)2D3, 1.4-fold and 1.5-fold respectively). Furthermore, the effect of 9 nmol/l GH on 1.25-(OH)2D3 and 24,25-(OH)2D3 production was blocked in the presence of IGF-I receptor monoclonal antibody. These results confirm that IGF-I acts on renal tubules, resulting in induction of 1,25-(OH)2D3 and 24,25-(OH)2D3 production, and the findings suggest that GH stimulates 1.25-(OH)2D3 and 24,2 5-(OH)2D3 production by increasing local IGF-I production in the kidney.