Inhibitory effects of 1,25(OH)2 vitamin D3 on collagen type I,osteopontin, and osteocalcin gene expression in chicken osteoblasts

Seventeen day chicken embryonic osteoblasts treated over a 30-day period with 1,25(OH)₂ D₃ showed a 2–10-fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for α₁(I) and α₂(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)₂ D₃ at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5-day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)₂ D₃ treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)₂ D₃ in primary avian osteoblast cultures derived from 17-day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.     

24,25(OH)2D3 enhances the calcemic effect of 1,25(OH)2D3

The effect of 24,25(OH)2D3 on 1,25(OH)2D3-induced hypercalcemia was studied in normal rats. Serum (S) levels and urinary excretion of Ca2+ (UCaV) were measured in (a) control rats, (b) rats receiving a daily sc injection of 54 ng 1,25(OH)2D3, (c) rats receiving 24,25(OH)2D3 in the same dose and same manner, and (d) rats receiving 1,25(OH)2D3 + 24,25(OH)2D3. The animals were housed in metabolic cages and 24-hr urine specimens were collected. After 24 hr SCa2+ increased similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3, while 24,25(OH)2D3 alone did not change SCa2+. UCaV after 24 hr increased significantly less (P less than 0.025) with 1,25(OH)2D3 + 24,25(OH)2D3 than with 1,25(OH)2D3 alone. After 5 days of 1,25(OH)2D3, SCa2+ rose from 5.1 +/- 0.15 to 6.29 +/- 0.08 whereas 1,25(OH)2D3 + 24,25(OH)2D3 effected a greater increase in SCa2+ up to 6.63 +/- 0.09 (P less than 0.01). 24,25(OH)2D3 alone did not change SCa2+. UCaV after 5 days of treatment rose similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3. After 10 days of 1,25(OH)2D3 SCa2+ was 6.17 +/- 0.15 meq/liter while with the combination SCa2+ rose to 6.74 +/- 0.2 (P less than 0.025). 24,25(OH)2D3 alone did not change SCa2+. These results show that (a) 24,25(OH)2D3 alone does not alter SCa2+ in normal rats, (b) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 enhances the hypercalcemic response to 1,25(OH)2D3 without a parallel increase in UCaV, and (c) it is suggested that the effect of 24,25(OH)2D3 on serum Ca2+ level, at least partly, may result from its hypocalciuric effect.     

Transforming growth factor-beta 1 signaling contributes to Caco-2 cell growth inhibition induced by 1,25(OH)(2)D(3)

Growth of Caco-2 and many cancer cells is inhibited by 1,25(OH)(2)D(3). Whereas TGF-beta 1 inhibits normal colonic epithelial cell growth, most human colon cancer-derived cells, including Caco-2 and SW480 cells, are resistant to it. The mechanisms underlying these antiproliferative actions and resistance to TGF-beta growth inhibition are largely unknown. We observed that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] sensitized Caco-2 and SW480 cells to TGF-beta 1 growth inhibitory effects. Versus 1,25(OH)(2)D(3) alone, the combination of 1,25(OH)(2)D(3) and TGF-beta 1 significantly reduced cell numbers. Also, the amount of active TGF-beta 1 was increased (~4-fold) by this secosteroid in conditioned media from Caco-2 cells. The 1,25(OH)(2)D(3) increased the expression of IGF-II receptors (IGF-IIR), which facilitated activation of latent TGF-beta 1, and was found to activate TGF-beta signaling in Caco-2 cells. By using neutralizing antibodies to human TGF-beta 1, we showed that this cytokine contributes to secosteroid-induced inhibition of Caco-2 cell growth. Also, 1,25(OH)(2)D(3) was found to enhance the type I TGF-beta receptor mRNA and protein abundance in Caco-2 cells. Whereas the 1,25(OH)(2)D(3)-induced sensitization of Caco-2 cells to TGF-beta 1 was IGF-IIR independent, the type I TGF-beta 1 receptor was required for this sensitization. Thus 1,25(OH)(2)D(3) treatment of Caco-2 cells results in activation of latent TGF-beta 1, facilitated by the enhanced expression of IGF-IIR by this secosteroid. Also, 1,25(OH)(2)D(3) sensitized Caco-2 cells to growth inhibitory effects of TGF-beta 1, contributing to the inhibition of Caco-2 cell growth by this secosteroid.     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

1,25-二羟维生素D3的生物学效应

1,25-二羟维生素D3是维生素D3的活性形式,其生物学效应是由基因组与非基因组两种机制介导的。维生素D3除了具有钙磷代谢调节作用外,还具有其他更为广泛的生物学效应。1,25-二羟维生素D3能够抑制多种类型细胞的增殖,诱导细胞的凋亡和分化,调节机体免疫系统功能,保护中枢神经系统,以及保护基因等。     

Effect of 1,25(OH)(2)D(3) on rat peritoneal mesothelial cells treated with high glucose plus lipopolysaccharide

1,25(OH)₂D₃, the active metabolite of vitamin D₃, its activity is not limited to mineral and skeletal homeostasis. In recent years, there has been increasing evidence pointing to the role of its activity in the regulation of cell proliferation, cell differentiation and immunomodulation. Here we report lipopolysaccharide (LPS), a glycolipid that is produced and secreted by gram-negative bacteria during peritonitis, plus high glucose (HG) can significantly inhibit mesothelial cell viability while induce more apoptosis in rat peritoneal mesothelial cells (RPMC). Pretreatment with 1,25(OH)₂D₃ can reverse the above effect in a concentration dependent manner. HG plus LPS can down-regulate the levels of both mRNA and protein of VDR, and up-regulate the expression of TGF-β1 and TNF-α in RPMC, which can also be effectively reversed by pretreatment with 1,25(OH)₂D₃. The above results suggest that HG plus LPS may induce changes in RPMC’s viability and apoptosis, leading to peritoneal injury. 1,25(OH)₂D₃ can reverse the inhibition of cell viability, the increase of apoptotic rate and induction of fibrosis related cytokine TGF-β1 and TNF-α by HG plus LPS in RPMC, thus protect peritoneal membrane.     

1,25(OH)2 vitamin D3 inhibits the CD23 gene expression by human peripheral blood monocytes

A biologically active form of vitamin D, 1,25(OH)2D3, and a leukocyte surface molecule, CD23, play important roles in immune regulation. The effect of 1,25(OH)2D3 on the CD23 gene expression was examined in this study. The results show that 1,25(OH)2D3 suppresses spontaneous and IL-4-stimulated CD23 synthesis by peripheral blood monocytes. The inhibition occurs at both the protein and mRNA levels. However, 1,25(OH)2D3 has no inhibitory effect on CD23 production by either resting or activated tonsillar B cells. Our results suggest a possible role of 1,25(OH)2D3 in the regulation of certain immune and inflammatory responses via its effect on CD23 production.     

24,25(OH)2D3 enhances the calcemic effect of 1,25(OH)2D3 in PTX rats

The effect of 24,25(OH)2D3 on 1,25(OH)2D3-induced hypercalcemia was studied in parathyroidectomized (PTX) rats for 10 days. Serum (S) and urinary Ca excretion (UCaV) were measured in (a) control rats, (b) rats receiving a daily sc injection of 54 ng 1,25(OH)2D3, (c) rats receiving 24,25(OH)2D3 in the same dose and same manner, and (d) rats receiving 1,25(OH)2D3 + 24,25(OH)2D3. Our results show that (i) 24,25(OH)2D3 alone does not increase SCa2+ in PTX rats, (ii) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 enhances the hypercalcemic response to 1,25(OH)2D3 without a parallel increase in UCaV, (iii) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 reduces the rise in urinary excretion of Ca2+ compared with that of rats receiving 1,25(OH)2D3 alone for 10 days, and (iv) these alterations are independent of parathyroid hormone.     

Synergistic effects of 1,25(OH)2D3 and 24,25(OH)2D3 on duodenal CaBP in rachitic chicks and on eggshell weight in Japanese quails

The role of 24,25(OH)2D3 in calcium homeostasis is still controversial. In the present study the administration of low doses of 1,25(OH)2D3 and of higher doses of 24,25(OH)2D3 either alone or in conjunction with each other, were studied in rachitic chicks and in Japanese quails. Whereas 24,25(OH)2D3 alone had no significant effect on duodenal CaBP and on alkaline phosphatase in chick serum, it increased the influence of 1,25(OH)2D3 on these two parameters strongly. Also, when 1,25(OH)2D3 and 24,25(OH)2D3 were given simultaneously to Japanese quails, calcium excretion via the egg shell was clearly higher than when either metabolite had been administered alone. These results indicate that 1,25(OH)2D3 and 24,25(OH)2D3 exert a strong synergistic effect in rachitic animals.     

Enhanced effects of guggulsterone plus 1,25(OH)2D3 on 3T3-L1 adipocytes

Guggulsterone (GS) and 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] have been shown to influence adipogenesis in 3T3-L1 cells. We investigated the ability of GS and 1,25(OH)₂D₃, alone and in combination to inhibit adipogenesis and induce apoptosis in 3T3-L1 adipocytes. Maturing preadipocytes were treated with 1,25(OH)₂D₃ in combination with GS for 6 days during differentiation. GS and 1,25(OH)₂D₃ each inhibited lipid accumulation, but the combination potentiated the inhibition of lipid accumulation. Apoptosis was increased by 1,25(OH)₂D₃ while GS had no effect, but GS + 1,25(OH)₂D₃ increased apoptosis more than either compound alone. Furthermore, GS + 1,25(OH)₂D₃ caused a potentiated decrease in the expression of aP2 and farnesoid X receptor expression more than either compound alone. In addition, 1,25(OH)₂D₃ increased vitamin D receptor expression after 6 days, while GS had no effect. GS + 1,25(OH)₂D₃, however, caused a potentiated increase in the expression of VDR. These findings show that GS potentiates 1,25(OH)₂D₃’s anti-adipogenic and pro-apoptotic effects in maturing 3T3-L1 preadipocytes.     

The effect of donor age on the sensitivity of osteoblasts to the proliferative effects of TGF(beta) and 1,25(OH(2)) vitamin D(3)

The loss of osteoblast function in aging bone is one of the major causes of osteopenia, or loss of bone mass. In this study, this loss of function was investigated by examining the proliferative response of rat long bone periosteal osteoblasts to TGF(beta1) and 1,25-dihydroxy vitamin D(3) (1,25-D(3)) as a function of donor age. Using a DNA binding fluorescent dye, DNA levels were measured in osteoblast cultures derived from either young adult (3-4 months) or old (14-15 months) rats following treatment with two concentrations (10(-9) M or 10(-12) M) of either 1,25-D(3) or TGF(beta1) or with vehicle. Cells from young rat bone, when treated with 1, 25-D(3), showed a dose-dependent increase in proliferation when treated with the higher dose and a decrease in proliferation when treated with the lower dose. Osteoblasts isolated from old rats did not respond to 1, 25-D(3) treatment. A similar pattern of response to TGF(beta1) was found. When treated with 10(-9) M TGF(beta1), the rate of proliferation increased for young rat osteoblasts, but the old rat derived cells were unresponsive. The 10(-12) M dose of TGF(beta1) was ineffective for both young and old cells. This study has shown that osteoblasts derived from old donors are impaired in their ability to respond to vitamin D and TGF(beta), two of the major controlling factors of skeletal development and maintenance.     

Identification of novel mediators of Vitamin D signaling and 1,25(OH)2D3 resistance in mammary cells

Since the discovery of the Vitamin D receptor (VDR) in mammary cells, the role of the Vitamin D signaling pathway in normal glandular function and in breast cancer has been extensively explored. In vitro studies have demonstrated that the VDR ligand, 1,25(OH)₂D₃, modulates key proteins involved in signaling proliferation, differentiation and survival of normal mammary epithelial cells. Anti-proliferative and pro-differentiating effects of 1,25(OH)₂D₃ have also been observed in VDR positive breast cancer cells, indicating that transformation per se does not abolish Vitamin D signaling. However, many breast cancer cell lines are less sensitive to 1,25(OH)₂D₃ than normal mammary epithelial cells. Reduced sensitivity to 1,25(OH)₂D₃ has been linked to alterations in Vitamin D metabolizing enzymes as well as down regulation of VDR expression or function. In this report, we describe results from a proteomics screening approach used to search for proteins involved in dictating sensitivity or resistance to Vitamin D mediated apoptosis in breast cancer cells. Several proteins not previously linked to 1,25(OH)₂D₃ signaling were identified with this approach, and a distinct subset of proteins was linked to 1,25(OH)₂D₃ resistance. Follow-up studies to determine the relevance of these proteins to Vitamin D signaling in general are in progress.     

Sodium butyrate (SB) augments the effects of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) on neoplastic and osteoblastic phenotype in clonal rat osteosarcoma cells

1,25 Dihydroxyvitamin D3 suppressed colony formation in soft agar and increased alkaline phosphatase activity in clonal rat osteosarcoma cells. Sodium butyrate enhanced these effects of the hormone partly through a mechanism involving an alteration of nuclear binding of the hormone. It is suggested that 1,25 dihydroxyvitamin D3 in conjunction with sodium butyrate might be able to regulate differentiation and proliferation of neoplastic cells.     

Effects of 24,25(OH)2D3, 1,25(OH)2D3 and 25(OH)D3 on alkaline and tartrate-resistant acid phosphatase activities in fetal rat calvaria

The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).     

24,25(OH)2D3 attenuates the calcemic effect of 1,25(OH)2D3 in rats with reduced renal mass

The present study was undertaken to evaluate the effect of 24,25(OH)2D3 on serum calcium concentration in rats with reduced renal mass. Adult 5/6 nephrectomized male rats were divided into four groups: (i) control rats, (ii) rats treated with 1,25(OH)2D3, (iii) rats treated with 24,25(OH)2D3, and (iv) rats treated with 1,25(OH)2D3 and 24,25(OH)2D3. After 4 days, serum calcium in the 1,25(OH)2D3-treated group was 7.13 +/- 0.32 meq/liter (P less than 0.001 vs control). With the combination of 1,25(OH)2D3 and 24,25(OH)2D3 serum calcium was higher than that in control, 6.25 +/- 0.5 meq/liter (P less than 0.001 vs control), but lower than that in rats receiving 1,25(OH)2D3 alone (P less than 0.05). No change in serum calcium was seen in animals treated with 24,25(OH)2D3 alone. On the eighth day serum calcium in the 1,25(OH)2D3-treated group, 6.52 +/- 0.25, was higher than in the 1,25(OH)2D3 + 24,25(OH)2D3 group, 5.87 +/- 0.17 meq/liter, P less than 0.05, P less than 0.001 vs control. In both 1,25(OH)2D3- and 1,25(OH)2D3 + 24,25(OH)2D3-treated rats, hypercalciuria of similar magnitude occurred on the fourth and eighth day of treatment. No change in urinary calcium was seen in the control and 24,25(OH)2D3-treated rats. Thus, in 5/6 nephrectomized rats combined administration of 1,25(OH)2D3 and 24,25(OH)2D3 attenuates the calcemic response to 1,25(OH)2D3 without changes in urinary calcium excretion. These observations suggest that the effect of 24,25(OH)2D3 on serum calcium is different in 5/6 nephrectomized rats as compared to normal rats, in which an augmentation of serum calcium was observed following administration of both vitamin D metabolites. The effect of 24,25(OH)2D3 on serum calcium in rats with reduced renal mass may result from a direct effect of 24,25(OH)2D3 on the bone.     

1,25(OH)2 vitamin D3 signalling on immature rat Sertoli cells: gamma-glutamyl transpeptidase and glucose metabolism

1α,25-Dihydroxyvitamin D₃ (1,25-D₃) is critical for the maintenance of normal male reproduction since reduced fertility is observed in vitamin D-deficient rats. Gamma-glutamyl transpeptidase (GGT) is a membrane-bound enzyme that is localized on Sertoli cells and catalyses the transfer of the gamma-glutamyl residues to an amino acid or peptide acceptor. Sertoli cells are also responsible for providing nutrients, as lactate, to the development of germ cells. The aim of this study was to investigate the effect and the mechanism of action of 1,25-D₃ on GGT on Sertoli cell functions from 30-day-old immature rat testis. Results demonstrated that 1,25-D₃ stimulates GGT activity at Sertoli cells plasma membrane through a PKA-dependent mechanism of action, which was not dependent of active de novo protein synthesis. The hormone increases glucose uptake, as well as lactate production and release by Sertoli cells without altering the reactive oxygen species (ROS) generation. In addition, 1,25-D₃ did not change reduced glutathione (GSH) amount or oxygen consumption, and diminished Sertoli cell death. These findings demonstrate that 1,25-D₃ stimulatory effect on GGT activity, glucose uptake, LDH activity and lactate production seem to be an important contribution of Sertoli cells for germ cells nutrition and for a full and active ongoing spermatogenesis.     

Regulation of 1,25(OH)2D3 (calcitriol) receptor by vitamin B6

In vitamin B6-deficient rats the concentration of in vivo occupied nuclear and total cellular receptors of 1.25(OH)2D3 increases 1.3-1.7 times, whereas the binding of in vitro occupied receptors to DNA-cellulose increases by 40%. Pyridoxal-5'-phosphate (PLP) added in vitro to solubilized receptors of 1.25(OH)2D3 lowers the ligand binding by 15-25% but causes no dissociation of hormone-receptor complexes formed in vivo. The association of in vitro occupied receptors of 1.25(OH)2D3 with DNA-cellulose is suppressed by PLP (3.5-4.5-fold). It has been shown for the first time that vitamin B6 is a physiological regulator of 1.25(OH)2D3 receptor binding by chromatin and DNA which diminish the concentration of occupied receptors and thus suppress the hormonal response.     

Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.     

Regulation of the CAMP gene by 1,25(OH)2D3 in various tissues

The induction of antimicrobial peptides such as the human cathelicidin, CAMP/hCAP18, by 1,25(OH)₂D₃ provides a very exciting therapeutic approach in boosting immunity against infectious diseases. To explore the range of cell types and expand the number of cell models for studying the regulation of CAMP gene expression by 1,25(OH)₂D₃, we treated cell lines from various tissue types and determined CAMP gene expression. Also, we tested additional compounds together with 1,25(OH)₂D₃ to look for possible cooperative activation of the gene. We identified 1,25(OH)₂D₃-mediated induction of the CAMP gene in B-cell lymphomas, prostate and endometrial cancer lines and found cooperative activation with the histone deacetylase inhibitor sodium butyrate. The data suggest that regulation of CAMP by 1,25(OH)₂D₃ is potentially important in a wide range of tissues.