Ascorbate Increases the 1,25 Dihydroxyvitamin D3-Induced Monocytic Differentiation of HL-60 Cells

1,25 Dihydroxyvitamin D₃ (calcitriol) induces differentiation of HL-60 leukemia cells. We studied the in vitro effect of a physiological concentration of ascorbate as potentiator of 1,25 dihydroxyvitamin D₃ [(OH)₂D₃] activity by determining different markers of differentiation: nitroblue tetrazolium reduction, nonspecific esterase activity, and the expression of CD11b and CD14 surface antigens. Nitroblue tetrazolium reduction and nonspecific esterase activity increased up to 50% in the presence of both 1,25 (OH)₂D₃ plus 0.2 mM ascorbate (ASC), compared with (OH)₂D₃ as a unique agent. ASC also increased the expression of specific surface antigens (CD11b and CD14) during differentiation induced by 1,25 (OH)₂D₃, the effect being more pronounced after 48 hours of treatment with 10⁻⁸ M 1,25 (OH)₂D₃. Furthermore, 1,25 (OH)₂D₃ alone increased intracellular cAMP level during differentiation, and the addition of ASC increased its concentration from 60 to 100% above the level reached with 1,25 (OH)₂D₃ as unique agent. ASC did not enhance the antiproliferative effect of calcitriol, suggesting that it only affects the ability of 1,25 (OH)₂D₃ to promote differentiation of HL-60 cells. Received: 9 June 1995 / Accepted: 19 February 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     

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     

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     

Parathyroid Hormone Modulates the Response of Osteoblast-Like Cells to Mechanical Stimulation

Mechanical loading stimulates many responses in bone and osteoblasts associated with osteogenesis. Since loading and parathyroid hormone (PTH) activate similar signaling pathways in osteoblasts, we postulate that PTH can potentiate the effects of mechanical stimulation. Using an in vitro four-point bending device, we found that expression of COX-2, the inducible isoform of cyclooxygenase, was dependent on fluid forces generated across the culture plate, but not physiologic levels of strain in MC3T3-E1 osteoblast-like cells. Addition of 50 nM PTH during loading increased COX-2 expression at both subthreshold and threshold levels of fluid forces compared with either stimuli alone. We also demonstrated that application of fluid shear to MC3T3-E1 cells induced a rapid increase in [Ca²⁺]_i. Although PTH did not significantly change [Ca²⁺]_i levels, flow and PTH did produce a significantly greater [Ca²⁺]_i response and increased the number of responding cells than is found in fluid shear alone. The [Ca²⁺]_i response to these stimuli was significantly decreased when the mechanosensitive channel inhibitor, gadolinium, was present. These studies indicate that PTH increases the cellular responses of osteoblasts to mechanical loading. Furthermore, this response may be mediated by alterations in [Ca²⁺]_i by modulating the mechanosensitive channel. Received: 5 October 1999 / Accepted: 15 February 2000     

Regulation of Osteogenic Differentiation of Human Bone Marrow Stromal Cells: Interaction Between Transforming Growth Factor-β and 1,25(OH)2 Vitamin D3 In Vitro

Bone marrow stromal cells are believed to play a major role in bone formation as a major source of osteoprogenitor cells, however, very little is known about how the osteogenic differentiation of these cells is regulated by systemic hormones and local growth factors. We examined the effects of TGF-β and its interaction with 1,25(OH)₂ Vitamin D₃ [1,25(OH)₂D₃] on the differentiation and proliferation of human bone marrow stromal cells (hBMSC) in secondary cultures. Alkaline phosphatase (ALP) activity was inhibited by TGF-β (0.1–10 ng/ml) and increased by 1,25(OH)₂D₃ (50 nM), however, co-treatment of TGF-β and 1,25(OH)₂D₃ synergistically enhanced ALP activity with maximal stimulation occurring at about 8 days after treatment. This synergistic effect was independent of proliferation because, in contrast to TGF-β alone, combined treatment with TGF-β and 1,25(OH)₂D₃ had no effect on hBMSC proliferation. As no synergistic effect was seen with combinations of 1,25(OH)₂D₃ and other osteotrophic growth factors, including BMP-2, IGF-I, and basic fibroblast growth factor (bFGF), it would seem likely that the synergistic interaction is specific for TGF-β. The increased ALP activity was due to an enhancement of 1,25(OH)₂D₃-induced ALP activity by TGF-β, rather than vice versa. In contrast, TGF-β inhibited 1,25(OH)₂D₃-induced osteocalcin production. Taken together, these results indicate that TGF-β and 1,25(OH)₂D₃ act synergistically to stimulate the recruitment of BMSC to the osteoblast lineage. This interaction may play an important role in bone remodeling. Received: 24 March 1998 / Accepted: 1 February 1999     

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     

Prostaglandin E2 (PGE2) Autoamplifies its Production Through EP1 Subtype of PGE Receptor in Mouse Osteoblastic MC3T3-E1 Cells

Prostaglandin E₂ (PGE₂) is known to autoamplify its production in the osteoblasts through the induction of prostaglandin G/H synthase-2 (PGHS-2), which is the inducible form of the rate-limiting enzyme in PG synthesis, PGHS. To elucidate the cellular mechanism mediating this process, we have employed the PGE₂ analogs, which are specific agonists for four subtypes of PGE receptor, and studied the potency of these analogs to induce PGHS-2 mRNA in mouse osteoblastic MC3T3-E1 cells. The induction was mainly observed by 17-phenyl-ω-trinor PGE₂ (EP₁ agonist) and sulprostone (EP₃/EP₁ agonist), but not by butaprost (EP₂ agonist) or 11-deoxy PGE₁ (EP₄/EP₂ agonist). Since EP₃ subtype was undetectable in MC3T3-E1 cells, these data indicate that PGHS-2 mRNA induction is mediated through EP₁ subtype of PGE receptor in MC3T3-E1 cells. PGE₂ production determined by radioimmunoassay was also increased by 17-phenyl-ω-trinor PGE₂ and sulprostone. The autoamplification of PGE₂ production is considered to be important in elongating the otherwise short-lived PGE₂ action in certain physiological conditions such as mechanical stress and fracture healing, as well as the pathological inflammatory bone loss. The observations in the present study provide us with the better understanding of these processes. Received: 29 April 1997 / Accepted: 22 August 1997     

Sustained Osteoblast Nuclear Receptor Binding of Converted 1α,25-Dihydroxyvitamin D3 after Administration of 3H-1α-Hydroxyvitamin D3: A Combined Receptor Autoradiography and Radioassay Time Course Study with Comparison to 3H-1α,25-Dihydroxyvitamin D3

The present study was undertaken to clarify the receptor distribution and the pharmacokinetics of ³H-1α(OH)D₃, and ³H-1α,25(OH)₂D₃ for comparison. Receptor autoradiography was used after intravenous injection to 3-day-old neonatal rats and radioassay-HPLC after oral application to young adult rats. Corresponding results were obtained from both receptor autoradiography and radioassay. After ³H-1α(OH)D₃ administration, uptake was delayed but sustained over a long period of time and the concentration of silver grains (autoradiography) or recovered ³H-1α,25(OH)₂D₃ (radioassay) peaked at a lower level. After ³H-1α,25(OH)₂D₃ administration, osteoblast nuclear, whole bone uptake and retention of radiolabeled compound were relatively rapid and short in duration. Nuclear uptake in osteoblasts after administration of ³H-1α(OH)D₃ was abolished in competition studies with 10-fold unlabeled 1α,25(OH)₂D₃. These results indicate that 1α(OH)D₃ continuously supplies osteoblasts with converted 1α,25(OH)₂D₃ and would not spread to the cells because of the low binding affinity of the receptor. Accordingly, 1α(OH)D₃ appears to have some therapeutic properties different from 1α,25(OH)₂D₃ because of a relatively slow and sustained accumulation of the receptor and less C_max (pharmacokinetics) compared with 1α,25(OH)₂D₃. Received: 26 August 1997 / Accepted: 20 February 1998     

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     

1α-Hydroxyvitamin D2 Partially Dissociates Between Preservation of Cancellous Bone Mass and Effects on Calcium Homeostasis in Ovariectomized Rats

Vitamin D metabolites can prevent estrogen depletion-induced bone loss in ovariectomized (OVX) rats. Our aim was to compare the bone-protective effects of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 1α,25-dihydroxyvitamin D₂ (1,25(OH)₂D₂), 1α-hydroxyvitamin D₃ (1α(OH)D₃), and 1α-hydroxyvitamin D₂ (1α(OH)D₂) in OVX rats. 1α(OH)D₃ and 1α(OH)D₂ are thought to be activated in the liver to form 1,25(OH)₂D₃ and 1,25(OH)₂D₂, respectively. Forty-four 12-week-old female Fischer-344 rats were either OVX or sham-operated (SHAM). Groups of OVX rats (n = 7 each) received vehicle alone, 1,25(OH)₂D₃, 1,25(OH)₂D₂, 1α(OH)D₃, or 1α(OH)D₂, starting 2 weeks after surgery. All vitamin D metabolites were administered orally at a dose of 15 ng/day/rat. Urine and blood samples were collected 6, 9, 12, and 16 weeks after surgery. Serum samples were analyzed for total calcium and phosphate. Calcium, phosphate, creatinine, and free collagen cross-links (ELISA) were determined in urine. After tetracycline double labeling, the rats were sacrificed 16 weeks postsurgery, and the proximal tibiae and the first lumbar vertebrae were processed undecalcified for static and dynamic bone histomorphometry. 1,25(OH)₂D₃ and, to a slightly lesser extent, 1,25(OH)₂D₂ elevated vertebral cancellous bone mass in OVX rats to a level beyond that observed in SHAM animals, and both compounds increased serum calcium and urinary calcium excretion to similar extents. 1α(OH)D₃ and 1α(OH)D₂ resulted in a 64% and 84%, respectively, inhibition of ovariectomy-induced vertebral cancellous bone loss. In the proximal tibial metaphysis, all vitamin D metabolites tested could only partially prevent post-OVX trabecular bone loss, with a tendency for 1α(OH)D₃ to be the least active compound. The effects of 1α(OH)D₃ and 1α(OH)D₂ on calcium homeostasis differed markedly, however. The mean increase in urinary calcium excretion over the whole experiment was fivefold for 1α(OH)D₃, whereas the corresponding increase for 1α(OH)D₂ was only twofold. We conclude that, compared with 1α(OH)D₃, 1α(OH)D₂ combined at least equal or higher bone-protective activity in OVX rats with distinctly less pronounced effects on calcium homeostasis. This effect was not due to a differential action of the corresponding main activation products, 1,25(OH)₂D₃ and 1,25(OH)₂D₂. Received: 2 May 1996 / Accepted: 18 October 1996     

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     

Oral calcium transiently increases calbindin9k gene expression in adult rat duodena

In rat intestine, the 9 kilodalton calbindin (CaBP_9K) is significantly increased in vivo by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) through a vitamin D (D) response element located in the 5′-flanking region of the gene. However, in vitro calcium has also been reported to increase CaBP_9K gene expression in fetal duodenal culture preparations. The aim of the studies was to investigate whether calcium feeding alone can influence CaBP_9K gene expression in vivo in adult rat duodena by evaluating the pattern of expression of its mRNA following short- or long-term exposure to oral calcium, comparing the data to exposure to the known inducer of the gene, 1,25(OH)₂D₃. Hypocalcemic D-depleted rats were acutely or chronically supplemented with calcium per os, or with 1,25(OH)₂D₃ in the presence or absence of oral calcium. Short-term calcium feeding was shown to significantly increase the expression of the CaBP_9K gene to a level similar to that observed in 1,25(OH)₂D₃-treated rats but no additive effect between oral calcium and 1,25(OH)₂D₃ on the level of its mRNA was observed. Moreover, the calcium effect on CaBP_9K gene expression was shown to be independent of the circulating ionized calcium concentration and, contrary to the effect of 1,25(OH)₂D₃, not sustained following long-term exposure. Our data clearly indicate that oral calcium alone has a significant but only transient effect of the expression of the adult rat intestinal CaBP_9K gene in vivo and that maintenance of its expression requires normalization of the D endocrine system. Received: 30 September 1995 / Accepted: 3 March 1996     

Poor Glycemic Control Impairs the Response of Biochemical Parameters of Bone Formation and Resorption to Exogenous 1,25-Dihydroxyvitamin D3 in Patients with Type 2 Diabetes

Osteoblast deficit plays a principal role in the development of diabetic osteopenia. We have previously reported that high glucose conditions impair the function of osteoblast-like MG-63 cells. This study was performed to assess the sensitivity of osteoblasts to 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in patients with type 2 diabetes without insulin deficiency or overt diabetic complications. During stimulation with 1,25(OH)₂D₃ at 2.0 mg/day for 6 consecutive days in 9 type 2 diabetic patients, serum levels of bone alkaline phosphatase (BALP), osteocalcin (OC) and the carboxyterminal propeptide of type 1 procollagen, and the urinary excretion of pyridinoline and deoxypyridinoline (DPYR), were monitored. As parameters of glycemic control, the mean level of fasting plasma glucose (mFPG) throughout the 1,25(OH)₂D₃ stimulation test and the level of HbA_1C were used. 1,25(OH)₂D₃ increased serum 1,25(OH)₂D significantly by day 2, which was followed by a significant reduction in the serum level of intact parathyroid hormone. The maximal increment of serum OC adjusted for that of 1,25(OH)₂D was negatively correlated with both mFPG and HbA_1C levels (p50.05). Furthermore, the magnitude of 1,25(OH)₂D₃-induced bone resorption, as reflected by the maximal increase in urinary DPYR excretion, was negatively correlated with the mFPG level (p50.05). Basal BALP tended to be negatively correlated with HbA_1C, although not to a significant extent. In conclusion, our findings would indicate that poor glycemic control impairs the responses of osteoblasts and osteoclasts to 1,25(OH)₂D₃ in normo-insulinemic type 2 diabetic patients. Received: 9 February 1998 / Accepted: 10 November 1998     

Effect of Vitamin D Metabolites and Analogs on Renal and Intestinal Calbindin-D in the Rat

The effects on renal and intestinal calbindin-D of vitamin D₃ metabolites and synthetic 20-epi-vitamin D₃ analogs with different calcemic actions were examined in Wistar rats. The compounds were administered intraperitoneally once daily for 5 days. The dosages of the metabolites were 1,25-(OH)₂D₃ 0.01, 0.05, 0.1, and 0.4 μg/kg × d, 24,25-(OH)₂D₃ 0.1, 1 and 10 μg/kg × d, and 25-(OH)D₃ 10 and 400 μg/kg × d. The dosage of the synthetic analogs were MC903 0.1, 10, and 100 μg/kg × d, EB1213 0.1 and 10 μg/kg × d, KH1060 0.1 and 0.4 μg/kg × d, and GS1725 0.01 and 0.1 μg/kg × d. Two control groups had either vehicle alone or no treatment. N= 8 in each group. 1,25-(OH)₂D₃ increased renal and intestinal calbindin-D levels, induced hypercalcemia, and suppressed plasma PTH and magnesium concentrations. 24,25-(OH)₂D₃ increased intestinal calbindin-D9k and plasma calcium, but had no effect on renal calbindin-D28k, plasma PTH, and magnesium. The dosage of 24,25-(OH)₂D₃ that was required to increase plasma calcium was larger than the dosage required to increase intestinal calbindin-D9k. 25-(OH)D₃ did not change the calcium metabolic parameters. MC903, a low calcemic analog with a relative high affinity for the vitamin D receptor and a short half-life, increased renal calbindin-D28k without increasing ionized calcium or intestinal calbindin-D9k. EB1213, an analog with a reduced calcemic action and short half-life, increased renal calbindin-D28k and ionized calcium without increasing intestinal calbindin-D9k. The effect of the high calcemic vitamin D analogs KH1060 and GS1725 on calbindin-D was directly related to their calcemic activity. In conclusion, these results demonstrate that 24,25-(OH)₂D₃ increases intestinal calbindin-D9k, but has no effect on renal calbindin-D28k, that low calcemic analogs may increase renal calbindin-D28k without increasing intestinal calbindin-D9k, and that the effect of high calcemic analogs on calbindin-D is directly related to their calcemic activity. Received: 26 May 1995 / Accepted: 29 February 1996     

Relationship Between Disease Activity and Serum Levels of Vitamin D Metabolites and PTH in Rheumatoid Arthritis

In several studies on patients with rheumatoid arthritis, an association of bone loss with a persistently high disease activity has been found. The aim of our study was to investigate the relation between disease activity and serum levels of vitamin D metabolites, parathyroid hormone (PTH), and parameters of bone turnover in patients with rheumatoid arthritis. A total of 96 patients (83 women and 13 men) were divided into three groups according to disease activity measured by serum levels of C-reactive protein (CRP). In the whole group, serum levels of 1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) (P < 0.001) and PTH (P < 0.05) were negatively correlated to disease activity. The urinary excretion of collagen crosslinks—pyridinoline (Pyd) (P < 0.001) and deoxypyridinoline (Dpd) (P < 0.05)—showed a positive correlation with disease activity. The inverse correlation between serum 1,25(OH)₂D₃ and disease activity was separately evident in patients with (P < 0.001) and without (P < 0.01) glucocorticoid treatment, in pre- (P < 0.01) and postmenopausal (P < 0.001) women, and in men (P < 0.01). 1,25(OH)₂D₃ and PTH serum levels were positively correlated to serum bone alkaline phosphatase (ALP) (P < 0.01). The results indicate that high disease activity in patients with rheumatoid arthritis is associated with an alteration in vitamin D metabolism and increased bone resorption. The decrease of 1,25(OH)₂D₃ levels in these patients may contribute to a negative calcium balance and inhibition of bone formation. Furthermore, low levels of 1,25(OH)₂D₃ as an endogenous immunomodulator suppressing activated T cells and the proliferation of cells may accelerate the arthritic process in rheumatoid arthritis. Received: 3 February 1997 / Accepted: 26 June 1997     

24,25-(OH)2D3 Regulation of Matrix Vesicle Protein Kinase C Occurs Both During Biosynthesis and in the Extracellular Matrix

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCα) and PKCζ, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)₂D₃ [14]. In the present study, we examined whether the effect of 24,25-(OH)₂D₃ on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10⁻⁹–10⁻⁷ M 24,25-(OH)₂D₃. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)₂D₃-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24,25-(OH)₂D₃-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)₂D₃-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)₂D₃ on PKC involved phospholipase A₂ (PA₂). Quinacrine (a PA₂ inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)₂D₃, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA₂ plays a role in the nongenomic regulation of PKC by 24,25-(OH)₂D₃. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)₂D₃ modulated the distribution of PKCα, β, and ζ between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24,25-(OH)₂D₃ regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation. Received: 11 October 1996 / Accepted: 25 April 1997