Up-Regulation of Inducible Nitric Oxide (NO) Synthase and NO Production in HL-60 Cells Stimulated to Differentiate by Phorbol 12-Myristate 13-Acetate Plus 1,25-Dihydroxyvitamin D3 Is Not Obtained With Dimethylsulfoxide Plus 1,25-Dihydroxyvitamin D3

In previous studies we found that the calciotropic hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] augments the action of either prostaglandin E₁ (PGE₁) or NaF to induce differentiation of human promyelocytic HL-60 cells, a process that features increased generation of nitric oxide (NO) via up-regulation of inducible nitric oxide synthase (iNOS). We have now examined the short-term interaction of 1,25(OH)₂D₃ with phorbol 12-myristate 13-acetate (PMA) and dimethylsulfoxide (DMSO) in these cells. PMA (100 nM) alone generally up-regulated several classical indices of macrophagic differentiation and stimulated cellular production of interleukin (IL)-1α, IL-6, tumor-necrosis factor (TNF)-α, PGE₂, and NO. Increased generation of NO primarily resulted from increased expression of cellular iNOS. When 1,25(OH)₂D₃ (10 nM) was added to PMA treatments, most PMA-induced changes, particularly its effects to up-regulate iNOS-dependent NO production and change cell morphology, were multiplicatively augmented. In contrast, DMSO (1.3%) alone, an inducer of granulocytic differentiation, increased cytokine production, but failed to stimulate NO production or induce iNOS. In contrast to its striking interaction with PMA, 1,25(OH)₂D₃ could not augment DMSO's differentiative effects. Changes in cellular cytokine production were eliminated as the driving force in HL-60 differentiation when specific neutralizing antibodies failed to produce any attenuation of iNOS up-regulation or of the shifts in cell morphology. However, indomethacin (30 μM) blocked the synergistic interaction between 1,25(OH)₂D₃+ PMA to shift cell morphology and stimulate NO production. Subsequently adding PGE₂ (1 ng/ml) to indomethacin-treated cells restored the ability of 1,25(OH)₂D₃+ PMA to interactively increase cellular NO production, but failed to fully replicate the strong shift in cell morphology typical of PMA + 1,25(OH)₂D₃ treatments. Our findings suggest that interaction between 1,25(OH)₂D₃ and PMA to induce macrophagic differentiation increases iNOS-dependent NO production by a mechanism involving a cyclooxygenase product(s), possibly PGE₂. Received: 13 March 1997 / Accepted: 14 November 1997     

Effect of Vitamin D Receptor Genotypes on Calcium Absorption,Duodenal Vitamin D Receptor Concentration,and Serum 1,25 Dihydroxyvitamin D Levels in Normal Women

It is well established that bone mineral density is under strong genetic control. Recently it was reported that the Bsm I restriction fragment length polymorphism of the vitamin D receptor (VDR) gene could account for up to 75% of the genetic variance in bone mineral density. However, the physiological basis for such an effect has not been established. The VDR gene codes for the vitamin D receptor protein which regulates intestinal calcium absorption. In order to assess the biochemical basis we studied the effect of common allelic variation of the VDR gene on intestinal VDR protein concentration, calcium absorption, and serum 1,25 dihydroxyvitamin D (1,25(OH)₂D). Ninety-two Caucasian women were genotyped for Bsm I and Taq I polymorphism at the VDR gene locus. From these we compared 49 young women aged 25–35 years and 43 elderly women aged 65–83 years, who had all three measurements performed. There were no significant differences in intestinal VDR protein concentration, serum 1,25(OH)₂D, or radioactive calcium absorption among VDR genotype groups. Therefore, the small intestine does not seem to be a target for VDR gene polymorphism. Received: 12 August 1996 / Accepted: 3 January 1997     

Effects of Postmenopausal Hormone Replacement Therapy With and Without Vitamin D3 on Circulating Levels of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D

The effects of postmenopausal hormone replacement therapy (HRT) and vitamin D₃ on vitamin D metabolites (25OHD and 1,25(OH)₂D) were studied in a population-based prospective 1-year study. The serum concentrations of intact parathyroid hormone (PTH), calcium, and phosphate were also studied. A total of 72 women were randomized into four treatment groups: HRT group (sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate), Vit D₃ group (vitamin D₃ 300 IU/day + calcium lactate 500 mg/day), HRT + Vit D₃ group (both above) and placebo group (calcium lactate 500 mg/day). Serum samples were taken in March–April, when vitamin D formation from sunlight in Finland is minimal after the dark winter. Serum concentrations of 25OHD increased in the Vit D₃ group (33.5%, P < 0.001) and in the HRT + Vit D₃ group (38.2%, P < 0.001) but had not changed significantly in the HRT and placebo groups at the 1-year follow-up examination. Serum concentrations of calcitriol (1,25(OH)₂D) increased, however, only in the HRT group (23.7%, P < 0.05), and remained unchanged in other groups. Serum concentrations of PTH decreased by 23.2% (P < 0.05) in the placebo group, but did not change significantly in the other three groups. The concentrations of serum calcium increased in the nonhormone groups (P < 0.001), whereas serum phosphate concentrations decreased in the hormone groups (P < 0.05 and 0.001). Our results confirm the positive effect of 1 year of HRT on serum calcitriol. Vitamin D₃ supplementation increased 25OHD concentrations, but did not affect calcitriol concentrations even though the initial levels were low. Interestingly, the combination of HRT and vitamin D₃ did not increase serum calcitriol concentrations as much as HRT alone. Received: 14 June 1996 / Accepted: 17 June 1997     

Bone Mass and Markers of Bone and Calcium Metabolism in Postmenopausal Women Treated with 1,25-Dihydroxyvitamin D (Calcitriol) for Four Years

To evaluate the long-term effect of calcitriol treatment on bone mineral density (BMD) of the femoral neck and lumbar spine and the parameters of calcium and bone metabolism in elderly women, 55 healthy, postmenopausal women, all aged 66 years, were enrolled in the study. Eighteen started a 4-year supplementation with 0.5 μg of calcitriol daily and 37 served as controls. Calcium intake of all the subjects was adjusted to 800 mg daily. In 4 years femoral neck BMD increased by 3.0% in the calcitriol group, but decreased by 1.6% in the control group (P= 0.009). The respective changes in lumbar spine BMD were +2.3% and +0.9% (P= 0.067). Two years' treatment with calcitriol increased the intestinal absorption of strontium by 57% (P < 0.001), doubled the urinary excretion of calcium (P < 0.001), and decreased the mean parathyroid hormone (PTH) level by 32% (P < 0.01). In the calcitriol group the marker of bone formation, serum osteocalcin, decreased by 27% (P < 0.01), and the marker of bone resorption, serum C-telopeptide of type I collagen (CTx), by 33% (P= 0.05) after 2 years. In two subjects the calcitriol dose had to be reduced because of hypercalciuria. We conclude that calcitriol treatment increases bone mass at the femoral neck and lumbar spine, the increases being maintained for up to 4 years. The gain in bone mass results from reduced bone turnover which is partly a consequence of the enhanced intestinal absorption of calcium and suppressed serum PTH levels. Received: 8 January 1999 / Accepted: 29 February 2000     

Inhibitory Effects of 1,25(OH)2D3 on Membrane-Associated and Cytosolic Casein Kinase Activity in MC3T3-E1 Osteoblast-like Cells

The secretion of phosphorylated matrix proteins is high in osteoblasts. Phosphorylation of these proteins may be catalyzed by casein kinases (CK), and CK may play an important role in the site of bone mineralization. In this study, we examined the effects of 1,25(OH)₂D₃ on CK activities in MC3T3-E1 osteoblast-like cells. Different concentrations (ranging from 10⁻⁷ to 10⁻¹¹M) of 1,25(OH)₂D₃ were included in a culture medium. After incubation for various lengths of time, MC3T3-E1 cells were homogenized and segregated into cytosolic (c) and microsomal (m) fractions. To measure CK activity, each fraction was used as an enzyme source to phosphorylate casein. MC3T3-E1 cells showed the highest cCK activity after incubation for 21 days, and showed the highest mCK activity after incubation for 14 days. 1,25(OH)₂D₃ inhibited mCK activity at the early stage of culture, but inhibited cCK activity at the late stage of culture. In contrast, 1,25(OH)₂D₃ had a slight stimulatory effect on CK activity in the culture medium of MC3T3-E1 cells. Our data suggest that cCK and mCK may play different roles in the function of osteoblasts, and 1,25(OH)₂D₃ regulates intracellular and extracellular casein kinase activities related to the function of osteoblasts. Received: 26 June 1997 / Accepted: 23 March 1998     

Parathyroid Hormone,Prostaglandin E2, and 1,25-Dihydroxyvitamin D3 Decrease the Level of Na+-Ca2+ Exchange Protein in Osteoblastic Cells

We previously described Na⁺-Ca²⁺ exchange in osteoblastic rat osteosarcoma cells (UMR-106) and demonstrated that Na⁺-dependent Ca²⁺ transport was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E₂ (PGE₂), or 1,25(OH)₂D₃. To determine whether this inhibition of Na⁺-Ca²⁺ exchange is at the level of exchanger protein synthesis we have examined exchanger protein levels using immunoblot analysis. UMR-106 cells were treated for 24 hours with or without PTH, PGE₂, or 1,25(OH)₂D₃. Plasma membrane fractions (7500 g) were obtained and proteins were separated by SDS-PAGE, transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na⁺-Ca²⁺ exchanger. In rat cardiac membranes, we detected 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected at 90 kD that decreased 65% after treatment of cells with PTH. Inhibition by PTH was dose dependent, was maximal with 10⁻⁷ M PTH, and required 16–24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10⁻⁶ M PGE₂ or 10⁻⁸ M 1,25(OH)₂D₃. These results demonstrate the presence of a specific protein in UMR cells that cross-reacts with antibody directed against the cardiac Na⁺-Ca²⁺ exchanger. Thus, the previously reported inhibition of Na⁺-Ca²⁺ exchange activity by calcemic agents in osteoblasts appears to be due to regulation of exchanger protein levels in these osteoblastic cells. Received: 5 February 1996 / Accepted: 18 October 1996     

1,25 Dihydroxyvitamin-D3 Attenuates the Confluence-Dependent Differences in the Osteoblast Characteristic Proteins Alkaline Phosphatase, Procollagen I Peptide, and Osteocalcin

In the present study a cell culture model of primary human osteoblasts based on degrees of confluence was investigated by measuring basal and 1,25(OH)₂D₃stimulated levels of the osteoblast characteristic proteins alkaline phosphatase (AP), procollagen I-peptide (PICP), and osteocalcin (OC), as well as the corresponding gene expression. Primary osteoblast-like cell cultures from seven donors were treated in the second passage with 1,25(OH)₂D₃ (5 × 10⁻⁸ M for 48 hours) and investigated at four stages of confluence (stage I 50%, stage II 75%, stage III 100%, and stage IV 7 days postconfluence). In untreated cultures passing through the different stages of confluence, we saw a 1.8-fold increase of AP activity, a 2.3-fold increase of OC secretion, but a decrease of PICP levels to 0.36-fold. Gene expression showed only minor variation between the different confluence stages. 1,25(OH)₂D₃ did not significantly affect PICP production. Alkaline phosphatase protein was stimulated during proliferation until confluence, with no effect thereafter. Surprisingly, OC secretion and mRNA expression were stimulated in all four stages to the same absolute level independent of basal values. We conclude that our results correspond to other studies showing differentiation-stage dependent changes of basal levels of osteoblast-specific proteins. However, 1,25(OH)₂D₃ stimulation decreased the confluence-dependent difference for AP and abolished it for osteocalcin, thus leading to a more differentiated phenotype of the osteoblast. Therefore, 1,25(OH)₂D₃ stimulation might improve the reproducibility of results obtained at different confluence stages from cultures of clinical samples. Received: 25 November 1997 / Accepted: 2 September 1998     

1,25-Dihydroxyvitamin D3 promotes prostaglandin E1-induced differentiation of HL-60 cells

Human promyelocytic HL-60 cells can be induced by biochemical agents to differentiate in vitro towards divergent types of myelomonocytic cells. It has been reported that prostaglandin E₁ (PGE₁) can induce granulocytic differentiation and that 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) can induce monocytic differentiation. We have now examined the effects of these compounds, both alone and in combination, on HL-60 cell differentiation. PGE₁ (1 g/ml) or 1,25(OH)₂D₃ (10 nM) each inhibited cell proliferation over 48–96 hours of treatment, but combined treatment with both agents was necessary to produce a strong inhibition. The percentage of HL-60 cells that can reduce nitroblue tetrazolium (NBT) (a characteristic index of early monocytic or granulocytic differentiation) increased 13-fold within 72 hours of PGE₁ treatment, and 1,25(OH)₂D₃ produced a fivefold stimulation. However, combined treatment (PGE₁ plus 1,25(OH)₂D₃) produced a dramatic 35-fold increase. HL-60 cells did not produce significant levels of nitric oxide (NO) before 48 hours in culture, and treatment with PGE₁ or 1,25(OH)₂D₃ did not significantly increase cellular NO elaboration over control levels. However, combined treatment produced a striking 12-fold increase over control levels. Similarly, combined treatment was necessary to obtain the maximal time-dependent stimulation of cellular lactate dehydrogenase (LDH) activity (a marker of granulocytic differentiation) as well as acid phosphatase (ACP) activity. During this same period of time, PGE₁, but not 1,25(OH)₂D₃, markedly stimulated cellular claboration of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-, and 1,25(OH)₂D₃ cotreatment strongly augmented these effects. Thus, combined treatment with 1,25(OH)₂D₃ plus PGE₁ generally augmented the apparent conversion of these cells, producing synergistic (multiplicative) or additive effects. Furthermore, PGE₁ induced within 48 hours the more general phenotypic changes classically associated with the differentiation of these cells: increased expression of chloroacetate esterase (ChAE) (a granulocytic marker), decreases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in morphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)₂D₃ had little effect itself on most of these parameters, but augmented the morphological changes induced by PGE₁ treatment. Within 48 hours, the ability of these cells to reduce the tetrazolium salt WST-1, a general measure of cellular metabolic activity, was increased by PGE₁, but not by 1,25(OH)₂D₃; however, the combination of 1,25(OH)₂D₃ and PGE₁ again produced the strongest stimulation. Similarly, only PGE₁ significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. In summary, our findings suggest that PGE₁, not 1,25(OH)₂D₃, is sufficient to promote rapid in vitro differentiation of HL-60 cells along the granulocytic pathway; however, the PGE₁-induced conversion of these cells is markedly augmented by cotreatment with 1,25(OH)₂D₃. In addition, these converted HL-60 cells preferentially utilize the glycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mature granulocytes.     

In Vivo Treatment with Calcitriol (1,25(OH)2D3) Reverses Age-Dependent Alterations of Intestinal Calcium Uptake in Rat Enterocytes

The vitamin D endocrine system has been involved in the impairment of intestinal calcium absorption during aging. Alterations in the nongenomic mechanism of calcitriol (1,25-dihydroxy-vitamin D₃; [1,25(OH)₂D₃] have been recently evidenced. In enterocytes isolated from aged rats, 1,25(OH)₂D₃ stimulation of Ca²⁺ channels through the cAMP/PKA pathway is blunted. We have now investigated whether in vivo administration of calcitriol to senescent rats reverses the absence of hormonal effects in isolated intestinal cells. In enterocytes from 20–24-month-old rats given 1,25(OH)₂D₃ for 3 days (30 ng/100 g bw/day), calcitriol (10⁻¹⁰ M, 3–5 minutes) stimulated Ca²⁺ uptake and intracellular cAMP to the same degree and protein quinase A (PKA) activity to a lesser degree than in enterocytes from young animals. Significantly higher basal levels of cAMP and PKA detected in enterocytes from old rats were not affected by prior injection of animals with 1,25(OH)₂D₃. When the aged rats were injected with 25(OH)D₃, similar Ca²⁺ influx, cAMP, and PKA responses to in vitro stimulation with calcitriol were obtained. 1,25(OH)₂D₃-dependent changes in Ca²⁺ uptake by enterocytes from both young and old rats treated with calcitriol were totally suppressed by the cAMP antagonist Rp-cAMPS, whereas the response to the agonist Sp-cAMPS was markedly depressed in aged animals. These results suggest that intestinal resistance to nongenomic 1,25(OH)₂D₃ stimulation of duodenal cell Ca²⁺ uptake develops in rats upon aging and show that in vivo administration of 1,25(OH)₂D₃ or its precursor to senescent rats restores the ability of the hormone to stimulate duodenal cell calcium influx through the cAMP messenger system. Received: 26 December 1997 / Accepted: 12 May 1998     

Increased Serum 1,25-Dihydroxyvitamin D after Growth Hormone Administration is not Parathyroid Hormone-Mediated

To assess the effects of growth hormone (GH) on serum 1,25-dihydroxyvitamin D [1,25(OH)₂D], we performed the following prospective crossover study in six healthy, young, adult, white men. During each of two admissions for 2? days to a general clinical research center, subjects were placed on a daily dietary calcium intake of 400 mg. Serum calcium, phosphorus, 1,25(OH)₂D, immunoreactive intact parathyroid hormone (PTH), insulin-like growth factor I (IGF-I), IGF binding protein 3 (IGFBP3), tubular reabsorption of phosphate (TRP), and maximum tubular reabsorption of phosphate (TMP/GFR) were measured. Recombinant human GH (rhGH, Humatrope) (25 μg/kg/day subcutaneously for 1 week) was administered prior to and during one of the admissions. Results are expressed as mean ± SEM. Whereas serum 1,25(OH)₂D (58.9 ± 7.7 versus 51.6 ± 7.4 pg/ml, P < 0.01), serum phosphorus (4.5 ± 0.1 versus 3.7 ± 0.1 mg/dl, P < 0.01), TRP (92.0 ± 0.5 versus 87.8 ± 0.7 mg/dl, P < 0.005), TMP/GFR (4.6 ± 0.1 versus 3.5 ± 0.2, P < 0.005), and urinary calcium (602 ± 49 versus 346 ± 25 mg/day, P < 0.001) increased significantly, serum PTH decreased significantly (19.9 ± 1.9 versus 26.8 ± 4.0 pg/ml, P < 0.05) and serum calcium did not change when subjects received rhGH. These findings indicate that in humans, GH affects serum 1,25(OH)₂D independently of circulating PTH and that this effect is mediated by IGF-I. We propose, therefore, that one potential mechanism by which GH stimulates increases in bone mass is via modest increases in serum 1,25(OH)₂D. Received: 2 May 1996 / Accepted: 18 October 1996     

Hormonal Regulation of the Osteoblastic Phenotype Expression in Neonatal Murine Calvarial Cells

Osteoblastic cell cultures from fetal rat calvariae have provided a popular model for studying the effects of dexamethasone (DEX) and 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] on gene expression but data from murine calvarial cells are scarce. Species-specific responses of rat and mouse osteoblastic cells to these hormones have been reported previously. In the present study, we investigated the effects of DEX and 1,25(OH)₂D₃ on expression of the osteoblastic phenotype by mouse calvarial cells. These murine osteoblast-like (MOB) cells expressed alkaline phosphatase (ALP) activity and osteocalcin and formed calcified nodules. Unlike the rat calvarial cells, ALP activities and nodule formation in MOB were inhibited by DEX. 1,25(OH)₂D₃ enhanced and DEX lowered the amount of osteocalcin synthesized by MOB. 1,25(OH)₂D₃ did not affect the number of nodules, but increased their sizes. Treating the cells for 2 days with only DEX at the beginning of the culture enhanced the effect of 1,25(OH)₂D₃ on ALP. We found that in murine calvarial cells, DEX inhibits and 1,25(OH)₂D₃ enhances ALP activity, osteocalcin synthesis, and calcified nodule formation. This is in contrast to previous reports of rat calvarial cells where DEX is a positive and 1,25(OH)₂D₃ can be a negative regulator of the osteoblastic phenotype. These results suggest that profound species-specific differences exist between mice and rats in the regulation of the osteoblastic phenotype. Received: 15 October 1997 / Accepted: 16 June 1998     

Avian Osteoclast Cells are Stimulated to Resorb Calcified Matrices by and Possess Receptors for Leukotriene B4

Leukotriene B₄ (LTB₄) is elevated in inflammatory conditions and appears to be a potential mediator of inflammation. We have recently shown that this 5-lipoxygenase metabolite of arachidonic acid stimulates bone resorption in vitro and in vivo. In order to determine the mechanism whereby LTB₄ causes bone resorption, avian osteoclasts were examined for the effects of LTB₄ and for the presence of LTB₄ receptors. Isolated avian osteoclast mononuclear precursor cells, which fuse in culture to form multinucleated cells, were chosen for receptor binding studies because this population is a morphologically similar source of osteoclasts, and large numbers of these cells can be obtained from egg-laying hens. Binding of LTB₄ and activation would support the hypothesis of a direct effect of this compound on osteoclasts. LTB₄ stimulated isolated avian osteoclasts to form resorption lacunae on calcified matrices and to increase their content of tartrate-resistant acid phosphatase (TRAP), a marker of activated osteoclasts. Receptor binding studies were performed at day 1, when the cells were mononuclear, at day 4, when mononuclear precursors were actively fusing, and at day 7, when fusion has slowed. Scatchard analysis of saturation binding data showed two classes of binding sites, a high- and low-affinity binding site with dissociation constants (K_D) of 0.2–0.4 nM and 5.6–24 nM. Association studies showed rapid binding of LTB₄ to the cells within 10 minutes. These data show that LTB₄ accelerates fusion and activates highly enriched populations of avian osteoclasts and that LTB₄ receptors are present in this cell population. Received: 30 November 1997 / Accepted: 12 May 1998     

1,25-Dihydroxyvitamin D3 Promotes Vitamin K2 Metabolism in Human Osteoblasts

It has been reported that vitamin K₂ (menaquinone-4) promoted 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)-induced mineralization and enhanced γ-carboxyglutamic acid (Gla)-containing osteocalcin accumulation in cultured human osteoblasts. In the present study, we investigated whether menaquinone-4 (MK-4) was metabolized in human osteoblasts to act as a cofactor of γ-glutamyl carboxylase. Both conversions of MK-4 to MK-4 2,3-epoxide (epoxide) and epoxide to MK-4 were observed in cell extracts of cultured human osteoblasts. The effect of 1,25(OH)₂D₃ and warfarin on the vitamin K cycle to cultured osteoblasts were examined. With the addition of 1 nM 1,25(OH)₂D₃ or 25 μM warfarin in cultured osteoblasts, the yield of epoxide from MK-4 increased. However, the conversion of epoxide to MK-4 was strongly inhibited by the addition of warfarin (2.5–25 μM), whereas it was almost not inhibited by 1,25(OH)₂D₃ (0.1–10 nM). To clarify the mechanism for this phenomenon, a cell-free assay system was studied. Osteoblast microsomes were incubated with 10 μM epoxide in the presence or absence of warfarin and 1,25(OH)₂D₃. Epoxide reductase, one of the enzymes in the vitamin K cycle was strongly inhibited by warfarin (2.5–25 μM), whereas it was not affected by 1,25(OH)₂D₃ (0.1–1 nM). Moreover, there was no effect of pretreatment of osteoblasts with 1 nM 1,25(OH)₂D₃ on the activity of epoxide reductase. However, the activity of epoxidase, that is the γ-glutamyl carboxylase was induced by the pretreatment of osteoblasts with 1 nM 1,25(OH)₂D₃. In the present study, it was demonstrated that the vitamin K metabolic cycle functions in human osteoblasts as well as in the liver, the post-translational mechanism, by which 1,25(OH)₂D₃ caused mineralization in cooperation with vitamin K₂ was clarified. Received: 20 September 2000 / Accepted: 19 February 2001     

Odontoblast Differentiation of Human Dental Pulp Cells in Explant Cultures

In order to elucidate the mechanisms involved in human dentin formation, we developed a cell culture system to promote differentiation of dental pulp cells into odontoblasts. Explants from human teeth were cultured in Eagle's basal medium supplemented with 10% or 15% fetal calf serum, with or without β-glycerophosphate (βGP). Addition of βGP to the culture medium induced odontoblast features in the cultured pulp cells. Cells polarized and some of them exhibited a typical cellular extension. In some cases, cells aligned with their processes oriented in the same direction and developed junctional complexes similar to the terminal web linking odontoblasts in vivo. Fine structural analyses showed the presence of typical intracellular organelles of the odontoblast body, whereas the process contained only cytoskeleton elements and secretory vesicles. Polarized cells deposited onto the plastic dishes an abundant and organized type I collagen-rich matrix with areas of mineralization appearing thereafter. X-ray microanalysis showed the presence of calcium and phosphorus and the electron diffraction pattern confirmed the apatitic crystal structure of the mineral. High expression of α1(l) collagen mRNAs was detected in all polarized cells whereas dentin sialoprotein gene was mainly expressed in mineralizing areas. This cell culture system allowed for the differentiation of pulp cells into odontoblasts, at both the morphological and functional level. Moreover, these cells presented a spatial organization similar to the odontoblastic layer. Received: 8 January 1999 / Accepted: 13 August 1999     

Acute Responses of Parathyroid Hormone and 1,25 Dihydroxyvitamin D3 to Unilateral Nephrectomy in Healthy Donors

Plasma immunoreactive parathyroid hormone (iPTH), 1,25(OH)₂D3calcium and phosphate and urinary creatinine, calcium and phosphatewere measured before and following unilateral nephrectomy insix kidney donors. Unexpectedly, plasma calcium rose, from 2.27±0.02mmol/l (mean±SEM) to 2.41±0.03 mmol/l on day 7and to 2.37±0.02 mmol/l on day 30 (P<0.02). A parallelrise in iPTH occurred, from 0.61±0.16 ng/ml initially,to 1.83±0.54 ng/ml on day 7 (P<0.05) and to 1.18±0.18on day 30 (P<0.01). The ratio of maximal tubular reabsorptionof phosphate to GFR (TmP/GFR) fell by day 2 (P<0.001), remainingreduced on day 30 (P<0.05). The significance of elevated iPTH in renal insufficiency wasfurther assessed by determining the time course of the disappearanceof iPTH after parathyroidectomy in three haemodialysis subjects.Fifty per cent baseline iPTH level occurred after an averageof 104.7 min, suggesting that the assay did not predominantlyrecognize C-terminal PTH fragments. By day 2, plasma 1,25(OH)₂D3had fallen from 34.3±4.5 pg/ml to 22.8±3.8 pg/ml(P<0.001), but by day 4 had regained its pre-nephrectomyvalue. Our results suggest that hypocalcaemia may not be thesole stimulus to parathyroid hormone secretion. It is speculatedthat reduction in circulating 1,25(OH)₂D3 may be involved.     

Loss of the Differentiated Phenotype Precedes Apoptosis of ROS 17/2.8 Osteoblast-Like Cells

Osteoblast cells, recruited from mesenchymal precursors, initiate the final phase of bone remodeling by secreting the protein components of the bone matrix. Upon completion of remodeling, some of these osteoblasts may further differentiate, giving rise to matrix-embedded osteocytes and bone lining cells. The fate of the remaining osteoblasts is unknown, although by analogy with other cell systems, apoptotic cell death may be involved. We induced and characterized the apoptotic process in ROS 17/2.8 osteosarcoma cells by growing and maintaining confluent cultures in low serum medium. At confluence, but prior to apoptosis, the levels of collagen type I, alkaline phosphatase, and osteocalcin mRNAs declined abruptly. Expression of two housekeeping genes (ribosomal protein RPS6 and GAPDH) remained unchanged. Some 72 hours later cells began to show morphological and biochemical features of apoptosis, namely, chromatin condensation, membrane budding, and internucleosomal degradation of genomic DNA. We conclude that serum starvation-induced apoptosis of ROS 17/2.8 cells can serve as a model for investigating the mechanisms of osteoblastic apoptosis. Received: 20 November 1996 / Accepted: 8 January 1998     

Vitamin D Therapy of Osteoporosis: Plain Vitamin D Therapy Versus Active Vitamin D Analog (D-Hormone) Therapy

Normal intestinal calcium (Ca) absorption is an essential feature of bone homeostasis. As with many other organ systems, intestinal Ca absorption declines with aging, and this is one pathological factor that has been identified as a cause of senile osteoporosis in the elderly. This abnormality leads to secondary hyperparathyroidism, which is characterized by high serum parathyroid hormone (PTH) and an increase in bone resorption. Secondary hyperparathyroidism due to poor intestinal Ca absorption has been implicated not only in senile osteoporosis but also in age-related bone loss. Accordingly, in population-based studies, there is a gradual increase in serum PTH from about 20 years of age onward, which constitutes a maximum increase at 80 years of age of 50% of the basal value seen at 30 years of age. The cause of the increase in PTH is thought to be partly due to impaired intestinal Ca absorption that is associated with aging, a cause that is not entirely clear but at least in some instances is related to some form of vitamin D deficiency. There are three types of vitamin D deficiency: (1) primary vitamin D deficiency, which is due to a deficiency of vitamin D, the parent compound; (2) a deficiency of 1,25(OH)₂D₃ resulting from decreased renal production of 1,25(OH)₂D₃; and (3) resistance to 1,25(OH)₂D₃ action owing to decreased responsiveness to 1,25(OH)₂D₃ of target tissues. The cause for the resistance to 1,25(OH)₂D₃ could be related to the finding that the vitamin D receptor level in the intestine tends to decrease with age. All three types of deficiencies can occur with aging, and each has been implicated as a potential cause of intestinal Ca malabsorption, secondary hyperparathyroidism, and senile osteoporosis. There are two forms of vitamin D replacement therapies: plain vitamin D therapy and active vitamin D analog (or D-hormone) therapy. Primary vitamin D deficiency can be corrected by vitamin supplements of 1000 U a day of plain vitamin D whereas 1,25(OH)₂D₃ deficiency/resistance requires active vitamin D analog therapy [1,25(OH)₂D₃ or 1α(OH)D₃] to correct the high serum PTH and the Ca malabsorption. In addition, in the elderly, there are patients with decreased intestinal Ca absorption but with apparently normal vitamin D metabolism. Although the cause of poor intestinal Ca absorption in these patients is unclear, these patients, as well as all other patients with secondary hyperparathyroidism (not due to decreased renal function), show a decrease in serum PTH and an increase in Ca absorption in response to therapy with 1,25(OH)₂D₃ or 1α(OH)D₃. In short, it is clear that some form of vitamin D therapy, either plain vitamin D or 1,25(OH)₂D₃ or 1α(OH)D₃, can be used to correct all types of age-dependent impairments in intestinal Ca absorption and secondary hyperparathyroidism during aging. However, from a clinical standpoint, it is important to recognize the type of vitamin D deficiency in patients with senile osteoporosis so that primary vitamin D deficiency can be appropriately treated with plain vitamin D therapy, whereas 1,25(OH)₂D₃ deficiency/resistance will be properly treated with 1,25(OH)₂D₃ or 1α(OH)D₃ therapy. With respect to postmenopausal osteoporosis, there is strong evidence that active vitamin D analogs (but not plain vitamin D) may have bone-sparing actions. However, these effects appear to be results of their pharmacologic actions on bone formation and resorption rather than through replenishing a deficiency.