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     

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     

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.     

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     

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     

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     

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     

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     

Age-Related Decline of Bone Mass and Intestinal Calcium Absorption in Normal Males

Although about 25% of all hip fractures occur in men, little is known about the pattern of their age-related bone loss and its main determinants. The aim of this cross-sectional study was to evaluate the age-related changes of intestinal calcium absorption, bone mass, and bone turnover in normal men. In 70 normal males (age 17–91 years), we measured spinal and forearm bone density (FBD) (by DXA), fractional intestinal calcium absorption (by oral test), serum immunoreactive parathyroid hormone (PTH), dietary calcium intake (diet records), biochemical markers of bone turnover (serum alkaline phosphatase (ALP), osteocalcin, urine calcium, creatinine, and hydroxyproline), and 1,25(OH)₂D₃ serum levels. Vertebral bone density (VBD) showed a modest decline before age 50 and a greater decline after age 50, whereas FBD presented a significant decrease with advancing age starting at age 40, suggesting a predominant age-related cortical bone loss. Intestinal calcium absorption (⁴⁷CaFA) and serum 1,25(OH)₂D₃ also presented an age-related decline similar to FBD. Simple correlation analysis revealed that age was significantly related to ⁴⁷CaFA (r = 0.60), calcium intake (r = 0.32), VBD and FBD (r = 0.79 and 0.63, respectively), serum 1,25(OH)₂D₃ (r = 0.69), and serum iPTH (r = 0.72). No significant correlation was found between age and biochemical markers of bone remodeling. Partial correlation and stepwise variable selection analyses, using ⁴⁷CaFA and bone mass as dependent variables, showed that in normal males, serum 1,25(OH)₂D₃ and dietary calcium intake were the main contributors (64%) to ⁴⁷CaFA variability, whereas only age accounted for 63% of VBD and age and dietary calcium accounted for 45% of FBD variability. These results indicate that bone loss in men accelerates after age 50 years and that among other factors, intestinal calcium malabsorption and 1,25(OH)₂D₃ serum levels play a role. Received: 19 November 1996 / Accepted: 26 January 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     

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     

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     

Factors Associated with Cortical and Trabecular Bone Loss as Quantified by Peripheral Computed Tomography (pQCT) at the Ultradistal Radius in Aging Women

Peripheral quantitative computed tomography (pQCT) allows the separate determination of cortical and trabecular bone mineral density in the peripheral skeleton. This cross-sectional study was designed to examine the effects of healthy aging on pQCT measurements at the ultradistal radius. In a well-defined sample of 129 community-based women, aged 70–87 years, the differences in cortical and trabecular density over the age range were equivalent to losses of −0.41% and −0.65% per year, respectively. To investigate the mechanism of this age-related decline, we assessed relationships between both parameters and height, weight, body mass index, dietary calcium intake, grip strength, and serum concentrations of insulin-like growth factor-I (IGF-I), calcidiol (25(OH)D₃), calcitriol (1,25(OH)₂D₃), parathyroid hormone (PTH), and sex hormone binding globulin (SHBG). Multiple regression was used to adjust for potential confounders. Age was not significant after controlling for other covariables. Body mass index, grip strength, serum IGF-I, 25(OH)D₃, and PTH (1–84) were found to be independent predictors of total bone density. Including (total or free) 1,25(OH)₂D₃ did not improve the model precision. These findings provide evidence that, among other factors, the activity of the growth hormone-IGF-I-axis is of importance for skeletal integrity. Grip strength, serum IGF-I, and PTH (1–84) were discovered to be significantly related to cortical but not to trabecular density, suggesting that different mechanisms may be involved in compact and cancellous bone loss. Received: 2 May 1996 / Accepted: 18 June 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     

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     

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     

Prophylactic Effects of 1,24,25-Trihydroxyvitamin D3 on Ovariectomy-Induced Cancellous Bone Loss in the Rat

Vitamin D metabolites can prevent estrogen depletion-induced bone loss in ovariectomized (OVX) rats. In this study, we investigated the bone-sparing effects of oral 1α,24R,25-trihydroxyvitamin D₃ (1,24,25(OH)₃D₃) in a wide dose range in aged OVX rats. Fifty-three female Fischer-344 rats (6 months old, 170 g BW) were either ovariectomized or sham-operated (SHAM). Eight rats served as baseline controls. Groups of OVX rats (n= 7–8 each) received vehicle alone or graded oral doses of 1,24,25(OH)₃D₃ (0.05, 0.1, 0.2, and 0.3 μg/kg BW/day), starting five days after surgery. Urine and blood samples were collected one, two, three, and four months after surgery. Serum samples were analyzed for total calcium and alkaline phosphatase. Calcium, hydroxyproline, and collagen crosslinks (HPLC) were determined in urine. After fluorochrome double labeling, the rats were sacrificed four months postsurgery and the first lumbar vertebrae and the proximal tibiae were processed undecalcified for bone histomorphometry. Ovariectomy induced a 28% and a 69% reduction in vertebral and tibial cancellous bone area, respectively. Osteopenia in OVX rats was associated with increased histomorphometric and biochemical indices of bone turnover. The administration of 1,24,25(OH)₃D₃ to OVX rats dose-dependently increased vertebral and tibial cancellous bone mass, serum calcium, and urinary calcium excretion, and reduced histomorphometric and biochemical indices of bone resorption. 1,24,25(OH)₃D₃ at doses of 0.2 and 0.3 μg/kg/day produced strong anabolic effects, especially on vertebral cancellous bone in OVX rats, and increased mineral apposition rate and wall width of completed remodeling units relative to vehicle-treated OVX rats. Even at high doses, 1,24,25(OH)₃D₃ did not impair bone mineralization. We conclude that oral administration of 1,24,25(OH)₃D₃ can effectively prevent estrogen depletion-induced cancellous bone osteopenia in the aged OVX rat model. The therapeutic window for 1,24,25(OH)₃D₃ in OVX rats, however, is also narrow, comparable to that for calcitriol. Received: 29 March 1996 / Accepted: 23 September 1996     

1,25(OH)2D3 inhibits hormone secretion and proliferation but not functional dedifferentiation of cultured bovine parathyroid cells

Summary Increasing the extracellular Ca²⁺ concentration from 0.5 to 3.0 mM induced marked increments in cytoplasmic Ca²⁺ concentration (Ca²⁺ _i) and inhibition of parathyroid hormone (PTH) release of freshly isolated bovine parathyroid cells. 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃; 0.1–100 ng/ml) did not affect (Ca²⁺ _i) and was also without acute effect on the secretion. During 4 days of monolayer culture, the parathyroid cells underwent significant increases in both number and size, and presence of 10–100 ng/ml 1,25(OH)₂D₃ almost completely inhibited the cell proliferation, whereas the hypertrophy was unaffected. One day of culture with 0.1–100 ng/ml 1,25(OH)₂D₃ was without effect on PTH release but after 4 days there was a dose-related reduction of recretion. At this time point and irrespective of the culture condition, PTH release was no longer suppressed by high extracellular Ca²⁺. Furthermore, Ca²⁺ _i increased little upon increments in the extracellular Ca²⁺ concentration as compared with freshly isolated cells. It is concluded that after prolonged exposure to 1,25(OH)₂D₃, PTH release is inhibited and, at high concentrations, the parathyroid cells cease to proliferate. However, 1,25(OH)₂D₃ does not affect the development of functional dedifferentiation of parathyroid cells during monolayer culture.