Mechanism of action of 1,25-dihydroxyvitamin D3-induced stimulation of alkaline phosphatase in cultured osteoblast-like cells

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates the alkaline phosphatase of rat and human osteoblast-like cells in culture. Here the mechanism of this effect was investigated using the rat osteogenic sarcoma cell line ROS 17/2-8. We found that 50% maximum alkaline phosphatase stimulation is elicited by 1,25(OH)2D3 at 7 X 10(-10) M. The concentration of serum in the culture medium influences inversely the effective 1,25(OH)2D3 concentration. Increased alkaline phosphatase appears after a lag period of cell exposure to 1,25(OH)2D3 which is between 8 and 24 h; during 96 h culture in the presence of 1,25(OH)2D3 the enzyme activity continues to rise. Cycloheximide (0.1-1 micrograms/ml) added in the cultures for 3 days or actinomycin-D (1-30 ng/ml) added for 24 h inhibit the 1,25(OH)2D3 effect on alkaline phosphatase in a dose-dependent fashion; withdrawal of cycloheximide restores the responsiveness of cells to 1,25(OH)2D3 completely, but withdrawal of actinomycin-D restores cell responsiveness only partially. These findings suggest that 1,25(OH)2D3-induced stimulation of alkaline phosphatase in the osteoblast-like cells involves genome activation and de novo protein synthesis.     

1alpha,25-dihydroxyvitamin D3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms

We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.     

Differential effects of calcium-regulating hormones on bone metabolism in weanling rats orally administered zinc sulfate

The effect of calcium-regulating hormones on bone metabolism was investigated in weanling rats orally administered zinc sulfate. Administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 (150 ng/100 g BW) or parathyroid hormone (1-34) (PTH) (10 U/100 g) produced significant increases in alkaline phosphatase activity and DNA content in the femoral diaphysis, while calcitonin (CT) (1.0 U/100 g) did not have a significant effect. Administration of zinc (1.0 mg/100 g) caused a significant elevation of alkaline phosphatase activity and DNA content in the femoral diaphysis. In combination with these hormones and zinc, both 1,25(OH)2D3 and zinc caused a synergistic increase in diaphyseal alkaline phosphatase activity and DNA content, while the combination with zinc and PTH, or zinc and CT did not cause a synergistic increase. The synergistic effects in combination with 1,25(OH)2D3 and zinc were completely inhibited by treatment of mitomycin C (10 micrograms/100 g). Meanwhile, the increase in diaphyseal zinc content following zinc administration was not altered by treatment with 1,25(OH)2D3. These results indicate that zinc synergistically enhances 1,25(OH)2D3-stimulated bone metabolism. This suggests a physiologic significance of zinc in the regulation of bone metabolism.     

The effects of vitamin D metabolites on the plasma and matrix vesicle membranes of growth and resting cartilage cells in vitro

This study compares the effects of vitamins 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 on populations of chondrocytes at different developmental stages. Confluent third passage chondrocytes derived from the resting zone and adjacent growth region of rat costochondral cartilage were cultured in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum and increasing concentrations of hormone. After determination of cell number, matrix vesicles and plasma membranes were isolated by differential centrifugation. The effects of hormone on alkaline phosphatase, 5'-nucleotidase, ouabain-sensitive Na+/K+-ATPase, and phospholipid composition were dependent on vitamin D metabolite and were cell specific. Growth cartilage chondrocytes responded primarily to 1,25-(OH)2D3, whereas resting zone cells responded primarily to 24,25-(OH)2D3. 1,25-(OH)2D3 inhibited growth cartilage cell number at pharmacological concentrations and had no effect on resting cartilage cell number. In contrast, 24,25-(OH)2D3 appeared to stimulate resting cartilage cell number at physiological concentrations and inhibit these cells at pharmacological doses, but had no effect on growth cartilage chondrocytes. These data were supported by [3H]thymidine incorporation studies. 1,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase activity, and Na+/K+-ATPase activity in the matrix vesicles of growth cartilage cells. 1,25-(OH)2D3 also stimulated Na+/K+-ATPase activity in the matrix vesicles and plasma membranes of resting zone cells. Incubation with 24,25-(OH)2D3 stimulated alkaline phosphatase, 5'-nucleotidase, and Na+/K+-ATPase in the matrix vesicles produced by resting zone cells. In addition, 24,25-(OH)2D3 stimulated Na+/K+-ATPase activity in the plasma membranes of resting zone cells as well as in both matrix vesicles and plasma membranes of growth cartilage cells.     

Differential response of duodenal epithelial cells to 1,25-dihydroxyvitamin D3 according to position on the villus: a comparison of calcium uptake, calcium-binding protein, and alkaline phosphatase activity

To determine which region of the intestinal villus was primarily responsible for calcium uptake and whether cells from the different regions of the villus differed in their response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], we studied cells eluted from the duodenal villus in a sequential fashion at various times after vitamin D-deficient chicks had received 1,25-(OH)2D3. The elution scheme employed removes cells from the villus tip first and cells from the villus base last, as was documented by the distribution of alkaline phosphatase activity, sucrase activity, and cytosolic calcium-binding protein (CaBP) in the eluted fractions. Brush border membrane vesicles (BBMV) were prepared from different fractions of the villus. Calcium uptake was greatest in BBMV from cells eluted from the villus tip and least in those from the villus base. The distribution of calcium uptake and alkaline phosphatase activity in the same BBMV were parallel. After 1,25-(OH)2D3 treatment, cytosolic CaBP was observed in the cells from the villus base by 4 h and in all fractions by 8 h; at all times (from 4-24 h), cells from the villus base contained more cytosolic CaBP than did cells from the villus tip. Alkaline phosphatase activity in BBMV was stimulated in all fractions by 4 h; at all times, alkaline phosphatase activity was greatest in BBMV from cells of the villus tip. In contrast, calcium uptake by BBMV was stimulated 2 h after 1,25-(OH)2D3 administration only in cells from the villus tip and was not stimulated even by 24 h in cells from the villus base. These results indicate that the cellular response to 1,25-(OH)2D3 depends on the location of the cell on the villus and that 1,25-(OH)2D3-stimulated calcium flux across the brush border can be dissociated from 1,25-(OH)2D3-stimulated alkaline phosphatase activity and CaBP production.     

Modulation by retinoic acid of 1,25-dihydroxyvitamin D3 effects on alkaline phosphatase activity and parathyroid hormone responsiveness in an osteoblast-like osteosarcoma cell line

Based on the finding that retinoic acid (RA) increases 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor number in ROS 17/2 cells, we investigated the effects of RA on the ability of 1,25-(OH)2D3 to regulate alkaline phosphatase activity and PTH-responsive adenylate cyclase in these cells. A maximally effective dose of 1,25-(OH)2D3 (10(-8) M) caused a 75-80% increase in alkaline phosphatase activity and an approximately 70-75% attenuation of the cAMP response to PTH, while RA (10(-6) M) decreased alkaline phosphatase activity by 30-45% and decreased PTH-stimulated cAMP levels by approximately 20%. Preincubation with RA did not enhance the 1,25-(OH)2D3-induced increases in alkaline phosphatase activity. The ED50 values for control and RA-treated cultures were approximately 8 X 10(-10) M and 6 X 10(-10) M, respectively. With regard to PTH responsiveness, the effects of RA preincubation on the 1,25-(OH)2D3 attenuation of cAMP response varied with the concentration of 1,25-(OH)2D3. At low doses (less than 10(-9) M), the effects of 1,25-(OH)2D3 and RA were additive. At higher doses of 1,25-(OH)2D3, the effects of RA and 1,25-(OH)2D3 were not additive, and there were no differences between control- and RA-treated cultures. The ED50 values for control- and RA-treated cultures were 10(-10) M and 3 X 10(-11) M, respectively. None of the above effects were observed using equimolar doses of the vitamin D3 metabolites 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The data show that pretreating ROS 17/2A cells with RA to increase 1,25-(OH)2D3 receptors does not correspond with a concomitant increase in the cellular responsiveness to 1,25-(OH)2D3, as measured by increases in alkaline phosphatase activity and decreases in PTH-responsive adenylate cyclase.     

Distinct target cells and effects of 1 alpha,25-dihydroxyvitamin D3 and glucocorticoids in the rat thymus gland

Thymocytes are known to possess receptors for glucocorticoids (GC) as well as for alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We have now investigated the distribution of the receptors for GC and 1,25-(OH)2D3 in rat thymocytes and compared the effects of the two steroid hormones on short term primary cultures of these cells. We report that in thymic cells, as in other tissues, 1,25-(OH)2D3 and GC bind specifically to distinct receptor molecules which exhibit sedimentation coefficients of 3.3S and 3.7S, respectively. Furthermore, the thymocytes that express the 1,25-(OH)2D3 receptor belong to a different and distinct subpopulation than the cells that express the glucocorticoid receptor. Specifically, by separating the thymocytes into two subsets by means of agglutination with the lectin peanut agglutinin (PNA), we have determined that the 1,25-(OH)2D3 receptor-positive cells belong to the PNA-negative medullary mature subset, whereas the GC receptor-positive cells belong to the PNA-positive cortical immature subset of thymocytes. Finally, we have compared the effects of the two steroid hormones on primary cultures of each of the two subsets as well as on unseparated thymocytes and found that GC act on PNA-positive cells to induce cell lysis; this leads to an enrichment in 1,25-(OH)2D3 receptor-positive thymocytes, as indicated by an apparent increase (6-fold) in the 1,25-(OH)2D3 binding in the cells surviving at the end of the culture. In contrast, we found that 1,25-(OH)2D3 acts on the PNA-negative cells to decrease the rate of cell lysis. These data indicates that the target cells for GC and 1,25-(OH)2D3 in the thymus are distinct and that these two hormones exert a different regulatory influence on the gland.     

Glucocorticoid and 1,25-dihydroxyvitamin D modulate the degree of adenosine 3',5'-monophosphate-dependent protein kinase isoenzyme I and II activation by parathyroid hormone in rat osteosarcoma cells

Glucocorticoid increases and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] decreases PTH activation of adenylate cyclase and cAMP-dependent protein kinase in rat osteosarcoma cells (ROS 17/2.8). Since selective cAMP-dependent protein kinase isoenzyme activation may account for specific physiological hormonal responses, we investigated steroid effects on activation of isoenzymes I and II in response to PTH using a new ion exchange separation procedure. Pretreatment of cells for 2 days with the glucocorticoid triamcinolone acetonide (TRM) or 1,25-(OH)2D3 altered the degree of cAMP-dependent protein kinase isoenzyme activation by PTH in accordance with their modulation of intracellular cAMP accumulation, but did not alter the amount of each isoenzyme present or the order in which isoenzymes I and II were activated. In all treatment groups isoenzyme I was preferentially activated by low doses of PTH, while high concentrations activated both isoenzymes, as predicted by the relative affinities of each isoenzyme for cAMP. Glucocorticoid reduced the concentration of bovine PTH-(1-34) required for maximal activation of isoenzyme I from 1 to 0.05 ng/ml and that required for activation of isoenzyme II from 10 to 1 ng/ml. This effect was abolished by simultaneous treatment of cells with 1,25-(OH)2D3. At doses of PTH that caused partial activation (0.05-0.1 ng/ml for isoenzyme I; 1 ng/ml for isoenzyme II), 1,25-(OH)2D3 treatment attenuated this activation. In all groups both isoenzymes were fully activated by 100 ng/ml PTH. Control experiments demonstrated that isoenzyme activation is not a result of cell disruption over the range of PTH doses that regulation by steroid hormone was observed. These results extend our studies on modulation of the cAMP pathway by steroid hormones and make it feasible to correlate selective isoenzyme activation with specific responses to PTH.     

1,25-Dihydroxyvitamin D3 and human bone-derived cells in vitro: effects on alkaline phosphatase, type I collagen and proliferation

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], but not 24,25-(OH)2D3 stimulates the alkaline phosphatase activity of cultured human bone cell populations. The stimulatory effect of the sterol was dose dependent (10(-10)-10(-7) M), evident by 24 h, and observed over a range of cell densities. Analysis of the radiolabeled collagens synthesised by human bone cell cultures indicated the synthesis of predominantly type I collagen. In the presence of 1,25-(OH)2D3, but not 24,25-(OH)2D3, there was a dose-dependent (10(-11)-10(-9) M) increase in radiolabeled proline incorporation into collagenase-digestible protein and in the amount of collagen synthesized, expressed as a percentage of the total protein synthesis. The effect of 1,25-(OH)2D3 was observed over a range of cell densities and appeared to be specific for the synthesis of type I collagen. The stimulatory effect of 1,25-(OH)2D3 on alkaline phosphatase activity and the increase in proline incorporation into collagenase-digestible protein were accompanied by a dose-dependent (5 X 10(-11) to 5 X 10(-8) M) inhibition of bone cell proliferation. These findings suggest that 1,25-(OH)2D3 is an important modulator of the growth and differentiation of human bone cells in vitro. They are also consistent with the possibility that 1,25-(OH)2D3 has direct effects on bone formation in vivo.     

Phenotype-associated changes in the effects of 1,25-dihydroxyvitamin D3 on alkaline phosphatase and bone GLA-protein of rat osteoblastic cells

The effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on alkaline phosphatase (AP) activity and the synthesis of gamma-carboxy-glutamic acid containing protein (BGP) were compared in phenotypically distinct cloned cell lines derived from the osteoblast-like rat osteogenic sarcoma line ROS 17/2-8. 1,25(OH)2D3 stimulated AP activity and BGP synthesis in phenotypes which exhibited relatively low basal AP activity and high basal BGP levels. In contrast 1,25(OH)2D3 inhibited AP activity in phenotypes that exhibited high basal AP activity. The latter cells had undetectable BGP levels and the synthesis of this protein failed to respond to the 1,25(OH)2D3 stimulus. In the cells that responded to 1,25(OH)2D3 with an increase in AP activity the effect of the hormone on AP could be blocked by actinomycin-D. However in the cells that responded to 1,25(OH)2D3 with inhibition of AP the effect of the hormone on AP was not influenced by actinomycin-D. The directly opposite effects of 1,25(OH)2D3 on the AP activity of the respective clones did not change qualitatively at different stages of culture and could not be accounted by differences in the 1,25(OH)2D3 receptor status nor by different effects of the hormone on cell proliferation. These data raise the possibility that the response of AP to 1,25(OH)2D3 in osteoblastic cells depends on their state of phenotypic differentiation. The stimulatory effect of the hormone in low AP-producing cells might be related to differentiation promoting properties of 1,25(OH)2D3. The inhibitory effect of 1,25(OH)2D3 on AP, unlike the stimulatory effect of the hormone does not appear to be mediated by the classical mechanism of 1,25(OH)2D3 action on the genome and might be associated with dedifferentiated osteoblastic cells.     

Effect of hormones and growth factors on alkaline phosphatase activity and collagen synthesis in cultured rat calvariae

Studies on the direct effects of hormones and growth factors on bone alkaline phosphatase have been limited to parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and have not been compared to other parameters of bone formation. Insulin, PTH, 1,25(OH)2D3, epidermal and fibroblast growth factors (EGF, FGF) were examined for their effects on alkaline phosphatase activity and type I, [alpha 1 (I)]2 alpha 2, collagen synthesis in cultures of 21-day fetal rat calvariae. After 24 hr and 96 hr of treatment, insulin increased whereas PTH, 1,25(OH)2D3, EGF and FGF inhibited calvarial alkaline phosphatase activity and the incorporation of 3H-proline into collagenase-digestible protein and type I collagen. The agents tested did not affect the release of alkaline phosphatase into the culture medium. Although type I collagen was the only collagen detected, a small amount of another collagen might have been also synthesized. The hormonal effects on alkaline phosphatase activity and type I collagen synthesis were of greater magnitude after 96 hr than after 24 hr of continuous exposure to the agents tested and the two parameters correlated well (r = 0.88 after 96 hr and r = 0.97 after 24 hr of treatment. These studies indicate that insulin increases bone alkaline phosphatase activity and type I collagen synthesis in calvariae whereas PTH, 1,25(OH)2D3, EGF and FGF have an inhibitory effect. The results suggest that these agents affect osteoblastic function.     

Differentiation of normal human bone cells by transforming growth factor-beta and 1,25(OH)2 vitamin D3.

To investigate the role of transforming growth factor-beta 1 (TGF beta) in bone metabolism, the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity, osteocalcin production, and mRNA levels for alkaline phosphatase, type I alpha 2-procollagen, and osteocalcin. The effect of TGF beta on alkaline phosphatase was not constant, but varied with the incubation conditions. At high cell density and in the presence of serum, TGF beta decreased alkaline phosphatase activity. However, at low cell density and under serum-free conditions, TGF beta stimulated alkaline phosphatase activity. The addition of 1,25(OH)2 vitamin D3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%, while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation, mRNA levels were measured at 24 hours. Individually, TGF beta and 1,25(OH)2D3 increased message levels for alkaline phosphatase and type I procollagen, but the greatest effect was produced by the combination of the two factors. 1,25(OH)2D3 increased osteocalcin mRNA levels, but TGF beta markedly inhibited this stimulation. TGF beta also inhibited production of osteocalcin by the human bone cells. TGF beta appears to modulate differentiation of human bone cells in combination with 1,25(OH)2D3 and other factors.     

Stimulation of creatine kinase activity by calcium-regulating hormones in explants of human amnion, decidua, and placenta

We have used stimulation of the activity of the brain type creatine kinase (CK) isoenzyme as a response marker to examine the effects of vitamin D metabolites, PTH, and calcitonin in cultured explants of placenta, decidua, and amnion from normal human deliveries. We found a biological response to PTH in placenta and amnion and to vitamin D metabolites in all three tissues. In the amnion, CK activity increased 2.3-fold after 24 h of incubation in 2.5 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 3.8-fold when incubated with 12.5 nM 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] and 2.7-fold when incubated with 10 U/ml bovine PTH. In the decidua, 24,25-(OH)2D3, but not 1,25-(OH)2D3 or bPTH caused a 1.7-fold increase in CK activity. In contrast, the placenta responded to 1,25-(OH)2D3 with a 1.6-fold increase in CK activity and to bPTH, with a 1.7-fold increase but did not respond to 24,25-(OH)2D3. Bovine calcitonin (100 ng/ml) had no effect on CK activity in any of the three tissues. Nearly all CK in both the unstimulated and stimulated explants was the brain type isoenzyme. CK activity increased significantly between 1 and 4 h after hormonal treatment in all experiments. The enzyme activity rose steeply with dose and reached a significant increase, and usually a plateau, at hormone concentrations considered to be physiological in vivo. [3H]Thymidine incorporation into DNA increased in parallel to stimulation of CK activity in all experiments, except that PTH did not increase DNA synthesis in the placenta. PTH did cause an increase in cAMP production in explants of amnion (1.5-fold) and placenta (2.6-fold).     

Regulation of cell growth, c-myc mRNA, and 1,25-(OH)2 vitamin D3 receptor in C3H/10T1/2 mouse embryo fibroblasts by calcipotriol and 1,25-(OH)2 vitamin D3.

Calcipotriol is a synthetic 1,25-(OH)2D3 analogue with high affinity for the 1,25-(OH)2D3 receptor, but with a lower affinity than 1,25-(OH)2D3 for vitamin D binding protein in serum. The inhibitory action of calcipotriol and 1,25-(OH)2D3 on proliferation of C3H/10T1/2 mouse embryo fibroblasts was examined in the non-transformed cell line Cl 8 and in the two transformed, tumorigenic cell lines Cl 16 and TPA 482. Upon exposure to 10 nmol/l calcipotriol or 1,25-(OH)2D3, the proliferation of Cl 8 cell line was almost completely suppressed, whereas both hormones had no effect on the cell lines Cl 16 and TPA 482. Calcipotriol was at least as effective as 1,25-(OH)2D3 in inducing up-regulation of the 1,25-(OH)2D3 receptor. Displacement studies showed no difference between calcipotriol and 1,25-(OH)2D3 in the affinity for the receptor present in Cl 8 or Cl 16 cell extracts. Furthermore, the inhibition of cell growth in Cl 8 cells by calcipotriol was not accompanied by any consistent change in the steady-state expression of c-myc mRNA. In conclusion, calcipotriol had potent growth inhibitory effect on the non-transformed cell line similar to 1,25-(OH)2D3. In the transformed cell lines, calcipotriol did not inhibit proliferation despite potent up-regulation of the 1,25-(OH)2D3 receptor.     

The effects of vitamin D metabolites on phospholipase A2 activity of growth zone and resting zone cartilage cells in vitro

Third passage confluent cultures of cartilage cells, initially derived from the growth zone (GC) and resting zone (RC) of rat costochondral cartilage, were incubated with either 10(-11)-10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or 10(-9)-10(-6) M 24,25-(OH)2D3. Plasma membranes and extracellular matrix vesicles were isolated, and specific activities of phospholipase A2 and alkaline phosphatase were determined. The results demonstrate that the response to hormone is both cell and membrane specific. 1,25-(OH)2D3 produces an increase in GC matrix vesicle alkaline phosphatase and phospholipase A2 specific activities at 10(-9) and 10(-8) M, but has no effect on these enzyme activities in RC membranes. RC cultured in 24,25-(OH)2D3 exhibit increased matrix vesicle alkaline phosphatase but decreased phospholipase A2 activities at 10(-7) and 10(-6) M hormone. No effect on the RC plasma membrane enzymes or on GC plasma membrane or matrix vesicle enzymes was observed. The data suggest that changes in membrane fluidity due to phospholipase A2 activity may play a role in regulating alkaline phosphatase activity in response to vitamin D metabolites and that this regulation in GC and RC may proceed by different mechanisms.     

Actions of recombinant human gamma-interferon and tumor necrosis factor alpha on the proliferation and osteoblastic characteristics of human trabecular bone cells in vitro

Using cultured human osteoblast-like cells, we studied the effects of tumor necrosis factor (TNF) and recombinant human gamma-interferon (gamma-IFN) on osteoblast growth and function, and demonstrated that TNF stimulated bone cell proliferation and prostaglandin production while inhibiting 1,25-(OH)2D3-stimulated alkaline phosphatase activity and osteocalcin release. In contrast, gamma-IFN inhibited proliferation and stimulated alkaline phosphatase activity of the cells, while inhibiting 1,25-(OH)2D3-stimulated osteocalcin production and having variable effects on the release of prostaglandins, depending on the presence of other factors. Our results suggest that TNF and gamma-IFN can act directly on bone-forming cells to affect both their proliferation and their differentiated function, and that changes in the ability of cells to produce these factors in disease states may contribute to alterations in the integrity of connective tissue matrices.     

Two distinct cell lines derived from a human osteosarcoma

Summary Two cell lines were established from a human osteosarcoma transplanted into athymic nude mice after the second (O9N2) and fifth passages (HuO9). Both cell lines expressed 1,25(OH)₂D₃-responsive alkaline phosphatase activity and produced tumors in the dorsum of nude mice that were histologically similar to the original tumor. However, the morphological and growth characteristics of the two cell lines differed. O9N2 cells were large and polygonal, whereas HuO9 cells showed spindle shapes. HuO9 cells had a higher growth rate and saturation density than O9N2 cells. The c-myc oncogene was amplified 4-to 8-fold in HuO9 cells but not in O9N2 cells. Both cell lines had a homozygous internal deletion, lacking the 7.4-kb HindIII fragment in the Rb gene. The results suggest the importance of the c-myc oncogene in the growth and morphological control of human osteosarcoma cells and of the Rb gene in the pathogenesis of the tumor.Abbreviations used ALP alkaline phosphatase - PBS phosphatebuffered saline - 1,25(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - Rb gene retinoblastoma gene     

Effects of diastereoisomers of 1,25-dihydroxyvitamin D3-26,23-lactone on alkaline phosphatase and collagen synthesis in osteoblastic cells

The effects of the four diastereoisomers of 1,25-dihydroxyvitamin D3-26,23-lactone (1,25-(OH)2D3-26,23-lactone) on alkaline phosphatase (AP) activity and collagen and noncollagen protein synthesis were examined in cultures of the osteoblastic clone MC3T3-E1 cell line. The four lactone diastereoisomers had little effect on the protein and DNA content of the cells. The 23(S),25(S)- and 23(R),25(R)-1,25-(OH)2D3-26,23-lactones increased AP activity in a linear dose-dependent fashion. Maximal effects were observed at 100 and 1000 pg/ml, respectively. In contrast, the naturally occurring 23(S),25(R)-, 1,25-(OH)2D3-26,23-lactone and the 23(R),25(S)-1,25-(OH)2D3-26,23-lactone showed biphasic stimulatory effects on AP activity. At both 80 and 10,000 pg/ml, they stimulated maximum increases in alkaline phosphatase activity. At 80 pg/ml the 23(S),25(R)- and 23(R),25(S)-isomers stimulated an increase in collagen synthesis, while at 10,000 pg/ml these isomers and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) did not. Moreover, these two isomers (at 10,000 pg/ml) plus insulin or dexamethasone had an additive effect on AP activity, but not at 80 pg/ml. At 80 pg/ml but not at 10,000 pg/ml, the 23(S),25(R)-isomer had an additive effect on AP activity with the simultaneous addition of 25-hydroxyvitamin D3. Relative to 1,25-(OH)2D3, the binding affinities of 23(S),25(S)-, 23(R),25(R)-, 23(S),25(R)- and 23(R),25(S)-1,25-(OH)2D3-26,23-lactones were calculated to be 1/13.0, 1/131.8, 1/805.2, and 1/1083.3, respectively. No metabolites could be detected in the medium when [1-3H]23(S),25(R)-1,25-(OH)2D3-26,23-lactone (the naturally occurring diastereoisomer) was added to the cultures. However, the stimulative effects of 1,25-(OH)2D3 and the 23(S),25(R)-isomer at both concentrations were completely abolished by L-1-tosyl-amido-2-phenylethyl chloromethyl ketone. These results indicate that 1,25-(OH)2D3-lactone has a stimulative effect on osteoblastic cell functions in vitro. The naturally occurring 23(S),25(R)-1,25-(OH)2D3-lactone acts biphasically and may act on bone metabolism in vivo, possibly through a 1,25-(OH)2D3-receptor-mediated pathway.     

Effects of 1,25-dihydroxyvitamin D3 on osteoblastic MC3T3-E1 cells

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] was examined for a possible stimulative effect on osteoblastic MC3T3-E1 cells. During the early period of culture, 1,25-(OH)2D3 had a stimulative effect. During the growth phase, however, the steroid had little effect on either the protein or DNA content of the cultures. 1,25-(OH)2D3 increased bone-liver-kidney-type alkaline phosphatase activity in a dose-related manner up to a concentration of 5 pg/ml; the increase was 2.2-fold over the control value. Studies on the effect of actinomycin D or cycloheximide treatment indicated that the vitamin may enhance de novo synthesis of ALP. The steroid also stimulated type I collagen production dose dependently via an increase in collagen synthesis rather than by inhibition of collagen degradation. MC3T3-E1 cells have a specific receptor for 1,25-(OH)2D3 which has a dissociation constant of 4.17 X 10(-11) M and a sedimentation coefficient of 3.67S. The receptor concentration varied with the period of culture, being higher during the growth phase and lower at confluence, but its affinity did not change. The results indicate that 1,25-(OH)2D3 has a direct specific anabolic effect on osteoblastic cells in vitro during the growth phase and that this effect is related to receptor concentration.     

Effect of a highly potent fluoro analog of 1,25-dihydroxyvitamin D3 on human bone-derived cells.

The fluorine introduced analog of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 [26,27-F6-1,25-(OH)2D3] is 5-10 times more potent than 1,25-(OH)2D3 in vitamin D-deficient rats and chicks. In this study we established cultures of human bone cells in order to elucidate the mechanisms responsible for the higher activity of this compound. The effects of 26,27-F6-1,25-(OH)2D3 and 26,26,26,27,27,27-hexafluoro-1,23(S),25-trihydroxyvitamin D3[26,27-F6-1,23(S),25-(OH)3D3], the postulated main metabolite of 26,27-F6-1,25-(OH)2D3, were assessed by the response of alkaline phosphatase (ALP) activity. 26,27-F6-1,25-(OH)2D3 increased ALP activity in a dose-related fashion, from a concentration of 10(-11) M and caused a 3-fold elevation at a concentration of 10(-9) M. To achieve the same stimulating effect on ALP activity, the required dose of 26,27-F6-1,25-(OH)2D3 was 100 times less than that of 1,25-(OH)2D3. Analysis of the receptors of these cells revealed that they have specific receptors for 1,25-(OH)2D3, which have a dissociation constant of 0.9 x 10(-10) M. The competitive binding assays of 26,27-F6-1,25-(OH)2D3 on these receptors showed that binding ability of 26,27-F6-1,25-(OH)2D3 is almost the same as that of 1,25-(OH)2D3. Therefore, receptor binding affinity does not account for the higher potency of 26,27-F6-1,25-(OH)2D3. The trihydroxylated compound, 26,27-F6-1,23(S),25-(OH)3D3 revealed almost the same stimulatory activity on ALP activity in these cells. The most likely explanation for the higher activity of 26,27-F6-1,25-(OH)2D3 than 1,25-(OH)2D3 is that 26,27-F6-1,25-(OH)2D3 is metabolized to 26,27-F6-1,23(S),25-(OH)3D3, which has almost the same activity as 26,27-F6-1,25-(OH)2D3 in target tissues, whereas 1,25-(OH)2D3 is degraded to less active metabolites such as 1,24,25-(OH)3D3.