Role of vitamin D and calcium signaling in epidermal wound healing.

PURPOSE: This review will discuss the role of vitamin D and calcium signaling in the epidermal wound response with particular focus on the stem cells of the epidermis and hair follicle that contribute to the wounding response. METHODS: Selected publications relevant to the mechanisms of wound healing in general and the roles of calcium and vitamin D in wound healing in particular were reviewed. RESULTS: Following wounding the stem cells of the hair follicle and interfollicular epidermis are activated to proliferate and migrate to the wound where they take on an epidermal fate to re-epithelialize the wound and regenerate the epidermis. The vitamin D and calcium sensing receptors (VDR and CaSR, respectively) are expressed in the stem cells of the hair follicle and epidermis where they play a critical role in enabling the stem cells to respond to wounding. Deletion of Vdr and/or Casr from these cells delays wound healing. The VDR is regulated by co-regulators such as the Med 1 complex and other transcription factors such as Ctnnb (beta-catenin) and p63. The formation of the Cdh1/Ctnn (E-cadherin/catenin) complex jointly stimulated by vitamin D and calcium plays a critical role in the activation, migration, and re-epithelialization processes. CONCLUSION: Vitamin D and calcium signaling are critical for the ability of epidermal and hair follicle stem cells to respond to wounding. Vitamin D deficiency with the accompanying decrease in calcium signaling can result in delayed and/or chronic wounds, a major cause of morbidity, loss of productivity, and medical expense.

Hypercalcemia in non-Hodgkin's lymphoma due to cosecretion of PTHrP and 1,25-dihydroxyvitamin D.

Hypercalcemia occurs in up to 30% of patients with malignancies and can be due to osteolysis by metastases, parathyroid hormone-related protein (PTHrP), excess 1,25-dihydroxyvitamin D (1,25(OH)2D) production or, rarely, ectopic parathyroid hormone (PTH) secretion. Hypercalcemia in non-Hodgkin's lymphoma has been described with elevations in PTHrP or, more commonly, excess 1,25(OH)2D production. We present the first case of a patient with new diagnosis of non-Hodgkin's lymphoma and severe hypercalcemia who was found to have concurrently elevated PTHrP and 1,25(OH)2D. In human studies, PTHrP has shown limited ability to stimulate 1,25(OH)2D production. To demonstrate that both PTHrP and 1,25(OH)2D were of tumor origin in our patient, tissue from her tumor underwent histochemical staining, demonstrating expression of both PTHrP and CYP27B1, indicating the presence of 1,25(OH)2D production in the tumor tissue. Our case illustrates the complexity of hypercalcemia in patients with underlying malignancy and highlights the importance of a thorough diagnostic workup for achievement of a successful therapeutic approach. In our patient, definitive chemotherapeutic treatment resulted in achievement and maintenance of normal calcium, PTHrP and 1,25(OH)2D levels 18 months after initial diagnosis. Hypercalcemia occurs in up to 30% of malignancies and can be due to several mechanisms. We present the first case of cosecretion of parathyroid hormone related peptide (PTHrP) and 1,25-dihydroxyvitamin D (1,25(OH)2D) in a patient with non-Hodgkin's lymphoma and demonstrate that both PTHrP and 1,25(OH)2D were of tumor origin by immunohistochemical staining.

Endoplasmic reticulum Ca2+ depletion activates XBP1 and controls terminal differentiation in keratinocytes and epidermis.

BACKGROUND: Endoplasmic reticulum (ER) Ca(2+) depletion, previously shown to signal pathological stress responses, has more recently been found also to trigger homeostatic physiological processes such as differentiation. In keratinocytes and epidermis, terminal differentiation and barrier repair require physiological apoptosis and differentiation, as evidenced by protein synthesis, caspase 14 expression, lipid secretion and stratum corneum (SC) formation. OBJECTIVES: To investigate the role of Ca(2+) depletion-induced ER stress in keratinocyte differentiation and barrier repair in vivo and in cell culture. METHODS: The SERCA2 Ca(2+) pump inhibitor thapsigargin (TG) was used to deplete ER calcium both in cultured keratinocytes and in mice. Levels of the ER stress factor XBP1, loricrin, caspase 14, lipid synthesis and intracellular Ca(2+) were compared after both TG treatment and barrier abrogation. RESULTS: We showed that these components of terminal differentiation and barrier repair were signalled by physiological ER stress, via release of stratum granulosum (SG) ER Ca(2+) stores. We first found that keratinocyte and epidermal ER Ca(2+) depletion activated the ER-stress-induced transcription factor XBP1. Next, we demonstrated that external barrier perturbation resulted in both intracellular Ca(2+) emptying and XBP1 activation. Finally, we showed that TG treatment of intact skin did not perturb the permeability barrier, yet stimulated and mimicked the physiological processes of barrier recovery. CONCLUSIONS: This report is the first to quantify and localize ER Ca(2+) loss after barrier perturbation and show that homeostatic processes that restore barrier function in vivo can be reproduced solely by releasing ER Ca(2+), via induction of physiological ER stress.

Integrins, insulin like growth factors, and the skeletal response to load.

Bone loss during skeletal unloading, whether due to neurotrauma resulting in paralysis or prolonged immobilization due to a variety of medical illnesses, accelerates bone loss. In this review the evidence that skeletal unloading leads to bone loss, at least in part, due to disrupted insulin like growth factor (IGF) signaling, resulting in reduced osteoblast proliferation and differentiation, will be examined. The mechanism underlying this disruption in IGF signaling appears to involve integrins, the expression of which is reduced during skeletal unloading. Integrins play an important, albeit not well defined, role in facilitating signaling not only by IGF but also by other growth factors. However, the interaction between selected integrins such as alphaupsilonbeta3 and beta1 integrins and the IGF receptor are of especial importance with respect to the ability of bone to respond to mechanical load. Disruption of this interaction blocks IGF signaling and results in bone loss.

1,25-Dihydroxyvitamin D increases calmodulin binding to specific proteins in the chick duodenal brush border membrane.

In previous studies we demonstrated that the biologically active vitamin D metabolite 1,25-dihydroxyvitamin D [1,25(OH)2D] increased the calmodulin (CaM) content of chick duodenal brush border membranes (BBM) without increasing the total cellular CaM content. Therefore, we evaluated the binding of CaM to discrete proteins in the BBM and determined whether 1,25(OH)2D could influence such binding. We observed one major and several minor CaM-binding bands on autoradiograms of sodium dodecyl sulfate polyacrylamide gels incubated with [125I]CaM. The major band had a molecular weight of 102,000-105,000. It bound CaM even in the presence of EGTA, but not in the presence of trifluoperazine or excess nonradioactive CaM. The administration of 1,25(OH)2D increased the apparent binding of CaM to this protein as assessed by densitometry of the autoradiogram. This increase in CaM binding coincided with the increased ability of the same BBM vesicles to accumulate calcium. Cycloheximide in doses that markedly reduced the incorporation of [35S]methionine into BBM proteins did not reduce the ability of 1,25-dihydroxyvitamin D3 to stimulate either calcium uptake by the BBM vesicles or CaM binding to the 102,000-105,000-mol-wt protein. These results suggest that 1,25(OH)2D administration increases the CaM content of duodenal BBM by increasing the ability of a 102,000-105,000-mol-wt protein to bind CaM. This mechanism may underlie the ability of 1,25(OH)2D to stimulate calcium movement across the intestinal BBM.

Adenosine triphosphate stimulates phosphoinositide metabolism, mobilizes intracellular calcium, and inhibits terminal differentiation of human epidermal keratinocytes.

During wound healing, release of ATP from platelets potentially exposes the epidermis to concentrations of ATP known to alter cellular functions mediated via changes in inositol trisphosphate (IP3) and intracellular calcium (Cai) levels. Therefore, we determined whether keratinocytes respond to ATP with a rise in Cai and IP3 and whether such increases are accompanied by a change in their proliferation and differentiation. Changes in Cai were measured in Indo-1-loaded neonatal human foreskin keratinocytes after stimulation with extracellular ATP. Extracellular ATP evoked a transient and acute increase in Cai of keratinocytes both in the presence and in the absence of extracellular calcium. ATP also induced the phosphoinositide turnover of keratinocytes, consistent with its effect in releasing calcium from intracellular sources. ATP did not permeabilize keratinocytes, nor did it promote Ca influx into the cells. The half-maximal effect of ATP was at 10 microM, and saturation was observed at 30-100 microM. UTP, ITP, and ATP gamma S were as effective as ATP in releasing Cai from intracellular stores and competed with ATP for their response, whereas AMP and adenosine were ineffective, suggesting the specificity of P2 purinergic receptors in mediating the ATP response in keratinocytes. Single cell measurements revealed heterogeneity in the calcium response to ATP. This heterogeneity did not appear to be due to differences in the initial Cai response but to subsequent removal of increased Cai by these cells. ATP inhibited terminal differentiation of keratinocytes as measured by [35S]methionine incorporation into cornified envelopes and modestly stimulated incorporation of [3H]thymidine into DNA. Chelation of Cai by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid reduced basal Cai, blocked the Cai response to ATP, inhibited the basal rate of DNA synthesis, and blocked the ATP-induced increase in DNA synthesis. We conclude that extracellular ATP may be an important physiological regulator of epidermal growth and differentiation acting via IP3 and Cai.

The ionic control of 1,25-dihydroxyvitamin D3 production in isolated chick renal tubules.

Isolated renal tubules prepared from vitamin D-deficient chicks catalyze the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 (250HD3) in vitro. The effect of calcium and phosphate on the rate of synthesis of the product, 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3), was studied at two levels: the long-term effects of various dietary calcium and phosphate contents on the ability of the tubules to produce 1, 25 (OH)2D3, and the acute effects of different calcium and phosphate concentrations in the incubation medium on the rate of synthesis of 1,25(OH)2D3 by the tubules. Manipulation of dietary calcium and phosphate sufficient to produce marked changes in the concentration of calcium and phosphate in the serum led to altered rates of 1,25(OH)2D3 synthesis by the isolated renal tubules. The renal tubules isolated from chicks raised on a vitamin D-deficient diet containing 0.43% calcium and 0.3% P as inorganic phosphate showed the highest rate of synthesis of 1,25(OH)2D3. Diets containing more or less of either calcium or phosphate produced chicks whose renal tubules had a slower rate of 1,25(OH)2D3 production. The calcium, phosphate, and hydrogen ion content of the incubation medium were manipulated to determine the possible factors concerned with the immediate regulation of 1,25(OH)2D3 production. A calcium concentration of 0.5-1.0 mM was necessary for optimal enzymatic activity. Concentrations of calcium greater than this optimal concentration inhibited 1,25(OH)2D3 production if phosphate was also present, and this inhibition was more pronounced as the phosphate concentration was increased. The stimulation of 1,25(OH)2D3 production by calcium was less at pH 6.7 than at 7.4. Raising the phosphate concentration from 0 to 6 mM in the absence of calcium also stimulated the rate of synthesis of 1,25(OH)2D3. This stimulatory effect was blocked by 4 mM calcium. However, at 1-2 mM calciu, phosphate had a biphasic influence on 1,25(OH)2D3 production; extracellular concentrations of phosphate from 0.6 to 1.2 mM resulted in less 1,25(OH)2D3 production than higher or lower phosphate concentrations. This biphasic effect was seen both at pH 7.4 and 6.8.

The ionic control of 1,25-dihydroxyvitamin D3 synthesis in isolated chick renal mitochondria. The role of calcium as influenced by inorganic phosphate and hydrogen-ion.

Isolated kidney mitochondria prepared from Vitamin D-deficient chicks catalyze the conversion of 25-hydroxyvitamin D3 to 1,25 dihydroxyvitamin D3. It wasfound that changes in the concentrations of Ca-2plus, HPO4-2minus, and Hplus altered synthesis in an interrelated fashion. Increasing the Ca-2plus concentration from 10-6 to 10-5 M caused a four- to fivefold increase in 1 alpha-hydroxylase activity when the medium pH was between 6.5 and 7.0. increasing the [Ca2+] to 10-4 M caused to furhter stimulation. At higher pH values, Ca-2plus had little effect upon 1 alpha-hydroxylase activity. In the absence of calcium [Ca2+] less than or equal to 10-7 M), a change in pH from 6.5 to 7.1 had no effect upon 1 alpha-hydroxylase activity in the presence of 10-5 M calcium, increasing the medium pH had a biphasic effect. An increase in pH from 6.5 to 6.9 caused a 1.5-fold increase in 1 alpha-hydroxylase activity, but a further increase of the pH to 7.1 caused a profound decrease in rate of hydroxylation to approximately 20% of the peak value. Neither 10-5 M LaC13 nor 10 mug/ml of oligomycin altered the effects of Ca2+ upon hydroxylate activity. However, the effect of calcium was blocked by 2.5 times 10-5 M ruthenium red, 0.83 mug/ml of antimycin A, and 500 muM dinitrophenol. The clcium ionophore, A23187, decreased but did not prevent the stimulatory effect of calcium. These data are consistent with the concept that the [Ca2+ in the mitochondrial matrix space is of importance in regulating the 1 alpha-hydroxylase. Phosphate exerted a biphasic effect on 1,25(OH)2D3 production with maximal stimulation (approximately twofold) at 1-3 mM. Calcium enhanced the stimulation by phosphate at all concentrations studied. The presence of potassium modified the interrelated effects of calcium and phosphate in two ways: 10-3 M calcium blocked the stimulation by phosphate; and in the presence of phosphate, 10-3 M calcium resulted in less 1,25(OH)2D3 production by production by isolated mitochondria are qualitatively similar to the effects of these ions on 1,25(OH)2D3 production yb isolated renal tubules.

1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation.

Human foreskin keratinocytes in vitro metabolize 25-hydroxyvitamin D3 to a number of metabolites, including 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This metabolite remains mostly within the cell and does not accumulate in the medium under the conditions of these experiments. With time, 1,25(OH)2D3 is catabolized, and more polar metabolites appear in both the cells and the medium. The production of 1,25(OH)2D3 has an apparent Michaelis constant (Km) for 25-hydroxyvitamin D3 of 5.4 X 10(-8) M. The levels of 1,25(OH)2D3 within the cell are increased both by increased production and decreased catabolism when parathyroid hormone(1-34) and isobutylmethylxanthine are added. Exogenously added 1,25(OH)2D3 at concentrations as low as 10(-12) M reduces endogenous 1,25(OH)2D3 production, increases 1,25(OH)2D3 catabolism, and increases 24,25-dihydroxyvitamin D3 production by an actinomycin D-sensitive process. These data indicate that the regulation of 1,25(OH)2D3 production by keratinocytes is similar to, but not identical to the regulation of 1,25(OH)2D3 by the kidney.

Free 25-hydroxyvitamin D levels are normal in subjects with liver disease and reduced total 25-hydroxyvitamin D levels.

We determined the free fraction of 25-dihydroxyvitamin D (25OHD) in the serum of subjects with clinical evidence of liver disease and correlated these measurements to the levels of vitamin D binding protein and albumin. These subjects when compared to normal individuals had lower total 25OHD levels, higher percent free 25OHD levels, but equivalent free 25OHD levels. These subjects also had reduced vitamin D binding protein and albumin concentrations. The total concentration of 25OHD correlated positively with both vitamin D binding protein and albumin, whereas the percent free 25OHD correlated negatively with vitamin D binding protein and albumin. The free 25OHD levels did not correlate with either vitamin D binding protein or albumin. We conclude that total vitamin D metabolite measurements may be misleading in the evaluation of the vitamin D status of patients with liver disease, and recommend that free 25OHD levels also be determined before making a diagnosis of vitamin D deficiency.

Transpleural gradient of 1,25-dihydroxyvitamin D in tuberculous pleuritis.

We used tuberculous pleuritis as a model to study the compartmentalization and potential immunoregulatory role of 1,25-dihydroxyvitamin D [1,25-(OH)2-D] in human granulomatous disease. In tuberculous pleuritis, mean concentrations of total 1,25-(OH)2-D were elevated in pleural fluid, compared to blood (67 pg/ml vs. 35 pg/ml). Concentrations of albumin, protein and 25-hydroxyvitamin D (25-OH-D) were lower in pleural fluid than blood, suggesting that accumulation of binding proteins does not explain the transpleural gradient of 1,25-(OH)2-D. The mean free 1,25-(OH)2-D concentration in pleural fluid was increased 5.3-fold over that in serum. 1,25-(OH)2-D3 inhibited PPD-induced proliferation of pleural fluid mononuclear cells, antigen-reactive lines and T lymphocyte clones derived from a single cell. Patient-derived PPD-reactive lines expressed a high-affinity intracellular binding moiety for 1,25-(OH)2-D3. Pleural fluid mononuclear cells and PPD-reactive lines did not metabolize 25-OH-D3 to 1,25-(OH)2-D3. The sum of these data suggests that concentration of 1,25-(OH)2-D in pleural fluid of tuberculosis patients is probably due to local hormone production by pleural tissue-based inflammatory cells that are not present in significant numbers in pleural fluid. Elevated concentrations of 1,25-(OH)2-D in pleural fluid may exert receptor-mediated inhibition of antigen-induced proliferation by pleural fluid lymphocytes. Inhibition of lymphocyte proliferation and lymphokine production may prevent tissue destruction from an uncontrolled inflammatory response.

1,25-Dihydroxyvitamin D3 upregulates the phosphatidylinositol signaling pathway in human keratinocytes by increasing phospholipase C levels.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) induces the differentiation of normal human keratinocytes, in part by increasing their basal intracellular calcium levels (Cai) over a period of hours. Agonists such as ATP acting through membrane receptors cause an immediate but transient increase in Cai accompanied by an increase in inositol trisphosphate (IP3). Treatment of keratinocytes for 24 h with 1 nM 1,25(OH)2D3 resulted in a two- to four-fold potentiation of the Cai response of these cells to ATP. This potentiation was inhibitable with cycloheximide, unaccompanied by a change in total intracellular calcium pools, but associated with an increase in basal IP3 levels and ATP-stimulated IP3 production. Treatment with 1,25(OH)2D3 raised the protein and mRNA levels of phospholipase C isoenzymes, particularly phospholipase C-beta 1 in a dose-dependent manner. These studies indicate that 1,25(OH)2D3 modulates the keratinocyte signal transduction pathway by induction of phospholipase isoenzymes, a previously undescribed action for this hormone.

Changes in calcium responsiveness and handling during keratinocyte differentiation. Potential role of the calcium receptor.

Extracellular calcium concentrations (Cao) > 0.1 mM are required for the differentiation of normal human keratinocytes in culture. Increments in Cao result in acute and sustained increases in the intracellular calcium level (Cai), postulated to involve both a release of calcium from intracellular stores and a subsequent increase in calcium influx through nonspecific cation channels. The sustained rise in Cai appears to be necessary for keratinocyte differentiation. To understand the mechanism by which keratinocytes respond to Cao, we measured the acute effects of Cao on Cai and calcium influx in keratinocytes at various stages of differentiation. We then demonstrated the existence of the calcium receptor (CaR) in keratinocytes and determined the effect of calcium-induced differentiation on its mRNA levels. Finally, we examined the role of Cai in regulating both the initial rise in Cai after the switch to higher Cao and the activity of the nonspecific cation channel through which calcium influx occurs. Our data indicate that the acute Cai response to Cao is lost as the cells differentiate and increase their basal Cai. These data correlated with the decrease in CaR mRNA levels in cells grown in low calcium. However, calcium influx as measured by 45Ca uptake increased with differentiation in 1.2mM calcium, consistent with the increase in CaR mRNA in these cells as well as the calcium-induced opening of the nonspecific cation channels. We conclude that the keratinocyte contains a CaR that regulates both the initial release of Cai from intracellular stores and the subsequent increase in calcium flux through nonspecific calcium channels. A rising level of Cai may turn off the release of calcium from intracellular stores while potentiating the influx through the nonspecific cation channels. Differentiation of keratinocytes appears to increase the CaR, which may facilitate the maintenance of the high Cai required for differentiation.

Free 1,25-dihydroxyvitamin D levels in serum from normal subjects, pregnant subjects, and subjects with liver disease.

We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration, and the level of vitamin D-binding protein (DBP) in 24 normal subjects, 17 pregnant subjects, and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus, the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl, respectively, P less than 0.001), whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml, respectively). In contrast, in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%, P less than 0.001), the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml, P less than 0.001). When data from all three groups were combined, there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels, (b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels, and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions.

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes.

Tumor necrosis factor alpha (TNF alpha) localizes to the epidermis when injected in vivo, but its role in the skin has heretofore not been evaluated. As a first approach to assessing the role of TNF alpha in the skin, we evaluated the binding and biological effects of TNF alpha on human neonatal foreskin keratinocytes maintained in culture. We found that TNF alpha at 0.3-1.0 nM inhibited proliferation of keratinocytes in a reversible fashion as demonstrated by a reduction in total DNA content and clonal growth. The antiproliferative effects were most marked when TNF alpha was added in the preconfluent stages of cell growth. Accompanying this antiproliferative effect was a stimulation by TNF alpha of differentiation of keratinocytes as indicated by the stimulation of cornified envelope formation. Keratinocytes specifically bound TNF alpha, reaching maximal binding in 2 h at 34 degrees C or 8 h at 4 degrees C. Much of the apparent binding at 34 degrees C was due to internalization of the TNF alpha. At 4 degrees C the rate of internalization was much less. Confluent keratinocytes showed a single class of high-affinity receptors with 1,250 receptors/cell and a Kd of 0.28 nM. These data suggest a role for TNF alpha in the growth and differentiation of the epidermis.

Chronic 1,25-dihydroxyvitamin D3 administration in the rat reduces the serum concentration of 25-hydroxyvitamin D by increasing metabolic clearance rate.

Administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can lower the serum concentration of 25-hydroxyvitamin (25-OH-D). To determine if 1,25(OH)2D3 lowers serum 25-OH-D by increasing clearance or reducing production, we directly measured the metabolic clearance rate (MCR) of 25-OH-D in rats chronically infused with 1,25(OH)2D3. Chronic 1,25(OH)2D3 administration (0 to 75 pmol/d) reduced, in a time- and dose-dependent fashion, the serum concentrations of 25-OH-D3 and 24,25(OH)2D3 from 18 +/- 2 to 9 +/- 1 ng/ml and from 4.8 +/- 0.7 to 1.3 +/- 0.3 ng/ml, respectively, and increased sevenfold the in vitro conversion of 25-OH-D to 24,25(OH)2D3 by kidney homogenates. The reduction in serum 25-OH-D3 was completely accounted for by an increase in MCR. No change in production occurred. The influence of 1,25(OH)2D3 on serum 25-OH-D3 and 24,25(OH)2D3 was shown not to be dependent on induction of hypercalcemia. These data suggest that chronic 1,25(OH)2D3 administration lowers serum 25-OH-D by increasing the metabolic clearance of 25-OH-D3 and not by decreasing its production.

Antiestrogen potentiation of antiproliferative effects of vitamin D3 analogues in breast cancer cells.

[3H]thymidine incorporation and DNA content were used to investigate the antiproliferative effects of 1,25(OH)2D3 and four analogues [16-ene-1,25(OH)2D3 (16-ene)]; 16-ene,23-yne-1,25(0H)2,D3; EB1089; and 22 oxa-1,25(OH)2D3] on MCF-7, BT-474, and MDA-MB-453 breast cancer cell lines. 1,25(OH)2D3 and the analogues elicited a biphasic response from MCF-7 and BT-474 estrogen receptor (ER)-positive cells, in the presence of estradiol (E2), with lower doses (between 10(-12) and 10(-10) M) tending to stimulate proliferation and higher doses (between 10(-9) and 10(-6) M) inhibiting proliferation by as much as 65%. In the absence of E2, the stimulatory effect was abrogated. Proliferation of MDA-MB-453, estrogen receptor-negative (ER-) cells, was stimulated by these compounds only at 10(-12) M, and inhibited by all higher doses, by as much as 83%. All three cell lines were shown to be vitamin D receptor (VDR) positive, and 1,25(OH)2D3 and all four analogues bound to the VDR with high affinities in each cell line. The antiestrogen ICI 164,384 inhibited the proliferation of all three cell lines. ICI 164,384 at 10(-8) M in combination with 1,25(OH)2D, or EB1089 converted biphasic response of the ER+ cells to one resembling the response of the ER- cells, by eliminating the stimulatory response elicited by 1,25(OH)2D3 at low doses and enhancing the antiproliferative effects of higher doses by as much as 1000-fold. These data are consistent with the hypothesis that E2 in the ER+ cells blocks the antiproliferative effects of the analogues and suggest the potential usefulness of combined antiestrogen and 1,25(OH)2D3 analogues in ER+ breast tumors, whereas 1,25(OH)2D3 analogues alone might suffice in ER- breast tumors.

Studies of the chick renal mitochondrial 25-hydroxyvitamin D-3 24-hydroxylase.

The vitamin D metabolite, 24,25-dihydroxyvitamin D-3, is made from 25-hydroxyvitamin D-3 by an enzyme (25-hydroxyvitamin D-3 24-hydroxylase) located in the renal mitochondria of vitamin D and calcium-replete chicks. The apparent Km for 25-hydroxyvitamin D-3 for this reaction was found to be 3 x 10(-7) M, although simple Michaelis-Menten kinetics were not observed at the higher substrate concentrations. Enzymatic activity was reduced by the mitochondrial metabolic inhibitors, antimycin A and dinitrophenol, as well as the inhibitors of mixed function oxidases, 2,4-dichloro-6-phenylphenoxyethylamine, 2-diethylaminoethyl-2,2-diphenylvalerate and metyrapone. Increasing the calcium concentration from 0 to 1 x 10(-5) M or the potassium concentration from 0 to 50 mM stimulated enzymatic activity 4- to 8-fold. Increasing the phosphate or acetate concentration from 0 to 50 mM stimulated enzymatic activity 6-fold. Raising the CO2/HCO3(-) content from 0/0 to 10%/10 mM stimulated enzymatic activity 5-fold. The effects of the cations were additive to those of the anions except in the presence of 50 mM phosphate or acetate. The effect of ions on 24,25-hydroxyvitamin D-3 production by renal mitochondria from vitamin D-replete chicks resembles the effect of these ions on production of 1,25-dihydroxyvitamin D-3 by renal mitochondria from vitamin D-deficient chicks, suggesting that the same mechanisms may be utilized.

Regulation of calmodulin binding to the ATP extractable 110 kDa protein (myosin I) from chicken duodenal brush border by 1,25-(OH)2D3.

In earlier studies we observed that the active vitamin D metabolite 1,25-(OH)2D3 increased the calmodulin content of purified duodenal brush-border membrane vesicles where it bound principally to the 110 kDa protein myosin I. In this study we further evaluated the regulation of calmodulin binding to ATP releasable myosin I. Whole brush borders (BB) or purified brush-border membrane vesicles (BBMV) were prepared from duodena of vitamin D-deficient rachitic chicks treated 12-18 h before killing with either 625 pmol 1,25-(OH)2D3 or vehicle. The ATP extractable myosin I from BB resulted in an 1.6-fold increase of calmodulin binding to the 110 kDa band after treatment with 1,25-(OH)2D3. In contrast to BB, ATP extraction of myosin I from purified BBMV required alamethicin for ATP entry. As for BB extracts, calmodulin binding to the 110 kDa band in BBMV extracts was also increased about 2.4-fold by 1,25-(OH)2D3. It was concluded that both intact BB and purified BBMV showed the same type of increase in calmodulin binding to ATP releasable myosin I by 1,25-(OH)2D3. To see whether 1,25-(OH)2D3 increased the intrinsic affinity of calmodulin binding to myosin I, the ATP extractable myosin I from BB was purified from rachitic chicks treated with 1,25-(OH)2D3 or vehicle. In contrast to ATP extracts of BB or BBMV, calmodulin binding to the purified myosin I was not different between preparations from 1,25-(OH)2D3- or vehicle-treated chicks. We conclude that 1,25-(OH)2D3 does not change the affinity of calmodulin binding to myosin I but increases the amount of myosin I in the membrane or alters its ATP releasability. It was further investigated whether phosphorylation is involved in these 1,25-(OH)2D3 dependent posttranslational changes of myosin I. Phosphorylation of brush-border membrane proteins in vivo was performed by incubation of [32P]P(i) in the lumen of a ligated duodenal loop in situ for 15 min. Brush-border membrane proteins were phosphorylated in vitro by incubating BB or BBMV with [gamma-32P]ATP for 1 min. Incubation experiments in vivo and in vitro in fact resulted in phosphorylation of several proteins including 110 kDa proteins. However, there was no specific effect of 1,25-(OH)2D3 on phosphorylation of 110 kDa proteins. We conclude that the effects of 1,25-(OH)2D3 on protein phosphorylation are minimal and not likely to explain 1,25-(OH)2D3 stimulated calmodulin binding to ATP extractable brush-border membrane myosin I and 1,25-(OH)2D3 stimulated changes of calcium uptake across the brush-border membrane.