Niacin decreases removal of high-density lipoprotein apolipoprotein A-I but not cholesterol ester by Hep G2 cells. Implication for reverse cholesterol transport

Increasing the expression of c-FMS (colony-stimulating factor 1 receptor) by introduction of a transgene reduced the concentration of retinoic acid or 1,25-dihydroxy vitamin D3 needed to cause myeloid or monocytic cell differentiation and hypophosphorylation of the retinoblastoma tumor suppressor protein (RB) typically associated with cell cycle G0 arrest and differentiation of HL-60 human myelo-monoblastic precursor cells. The data are consistent with a model in which signals originating with retinoic acid and c-FMS integrate to cause differentiation, RB hypophosphorylation, and G0 arrest. Furthermore, these two signals can compensate for each other. Three HL-60 sublines described previously (A. Yen et al., Exp. Cell Res., 229: 111-125, 1996) expressing low (wild-type HL-60), intermediate, and high cell surface c-FMS were treated with various concentrations of retinoic acid. The lowest concentration tested, 10(-8) M, induced significant differentiation of only the high c-FMS-expressing cells, with no accompanying hypophosphorylated RB or G0 arrest. The low and intermediate c-FMS expressing cells showed no induced differentiation, hypophosphorylation of RB, or G0 arrest. A 10-fold higher retinoic acid concentration, 10(-1) M, induced significant differentiation of both intermediate and high c-FMS-expressing cells. It induced RB hypophosphorylation only in high c-FMS-expressing cells but with no accompanying G0 arrest in any of the cells. The highest retinoic acid concentration, 10(-6) M, elicited differentiation, hypophosphorylation of RB, and G0 arrest in low, intermediate, and high c-FMS-expressing cells. As the concentration of retinoic acid increased, cell differentiation, hypophosphorylation of RB, and G0 arrest were progressively elicited within this ensemble of cells with different c-FMS expression levels. Thus, for example, at the lowest concentration of retinoic acid, expression of high enough c-FMS still allowed differentiation. At higher concentrations, progressively less c-FMS was needed for differentiation. The apparent threshold for the sum of the retinoic acid plus c-FMS originated signals to elicit differentiation, hypophosphorylation of RB, and G0 arrest increased, in that order. Thus retinoic acid-induced cell differentiation, RB hypophosphorylation, and G0 arrest have different signal threshold requirements. 1,25-Dihydroxy vitamin D3, also a ligand for a member of the steroid thyroid hormone receptor superfamily, caused monocytic differentiation with a similar c-FMS dependency, indicating that these effects characterize both myeloid and monocytic differentiation.     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.     

Protein kinase C is not necessary for transforming growth factor beta-induced growth-arrest in leukemia cell lines

Growth and differentiation of blood cell precursors are regulated by cytokines and hormones by mechanisms which are incompletely understood. Protein kinase C (PKC) isozymes are widely regarded as being important in signal transduction pathways. We have shown that one isozyme, PKC beta, is uniquely important in mediating phorbol ester-induced growth-arrest in the HL-60 myeloid cell line. 1,25-dihydroxyvitamin D3 induces differentiation and growth-arrest in many cells. It upregulates the expression of PKC beta, potentiating the action of phorbol ester. We tested the hypotheses that cytokines, which arrest the growth of hematopoietic cells, do so by activating PKC beta, and that differentiation and growth-arrest induced by 1,25-dihydroxyvitamin D3 is caused by upregulation of PKC beta isozyme gene expression. The influence on growth of combinations of five cytokines (TNF alpha, TGF beta 1, gamma-IFN, IL-1, and G-CSF) and 1,25-dihydroxyvitamin D3 on ten human leukemia cell lines (THP-1, HL-60 S, HL-60 PET, U937, K562, Jurkat, MOLT-4, RPM1 8402, KG-1, and KG-1a) was determined. Four cell lines (THP-1, HL-60 S and PET, and U937) exhibited total growth-arrest when incubated with 1,25-dihydroxyvitamin D3 followed by TGF beta 1. The expression by each cell line of mRNA encoding PKC alpha, beta, and delta, both before and after 24 or 48 h of incubation with 1,25-dihydroxyvitamin D3, was determined. Cell lines sensitive to TGF beta 1 each expressed PKC delta endogenously, or expression was up-regulated with 1,25-dihydroxyvitamin D3. U937 cells underexpressed PKC alpha, and HL-60 PET cells underexpressed PKC beta. These data suggested that PKC delta could be responsible for mediating growth-arrest by TGF beta 1. To test this hypothesis directly, we incubated the cells with two bisindolylmaleimide PKC inhibitors during the addition of 1,25-dihydroxyvitamin D3 and TGF beta 1. Surprisingly, the PKC inhibitors did not block the growth-arrest induced by 1,25-dihydroxyvitamin D3 and TGF beta 1. This experiment strongly suggests that neither growth-arrest induced by TGF beta 1 nor the potentiation of this growth-arrest by 1,25-dihydroxyvitamin D3 is mediated by a PKC isozyme which is inhibitable by the bisindolymaleimides.     

Vitamin D metabolism: update for the clinician

Vitamin D3 must undergo two hydroxylation steps before it becomes fully active: 25-hydroxylation in the liver and 1- or 24-hydroxylation in the kidney. Parathyroid hormone, serum phosphate, and serum calcium are important in regulation of renal production of 1,25-dihydroxy vitamin D3 (1,25-[OH]2D3) and 24,25-dihydroxy vitamin D3. An enzyme involved in renal hydroxylation is deficient or defective in patients with chronic renal failure, the Fanconi syndrome, vitamin D-dependent rickets, hypoparathyroidism, and pseudohypoparathyroidism. Altered vitamin D metabolism also occurs in various hepatic diseases, postmenopausal osteoporosis, and anticonvulsant osteomalacia. Recently, 1,25-(OH)2D3 was approved for treatment of renal osteodystrophy. In physiologic doses, it predictably corrects many of the clinical and biochemical abnormalities associated with this disorder.     

1,25-Dihydroxyvitamin D induces lipoprotein lipase expression in 3T3-L1 cells in association with adipocyte differentiation

1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is known to modulate the development of bone and other mesenchymal cell types. Since osteoblasts and adipocytes are thought to arise in bone marrow from a common progenitor, this work examined the effects of 1,25-(OH)2D3 on adipocyte development, and in particular on the expression of lipoprotein lipase (LPL), which is an early marker for the differentiated adipocyte. 3T3-L1 preadipocytes were cultured in the presence of 1,25-(OH)2D3 (10(-9) to 10(-7) M) for up to 7 days. LPL activity was measured in the medium and cell extracts, and LPL messenger RNA levels were measured by Northern blotting. When compared to control cells, 10(-7) M 1,25-(OH)2D3 increased medium LPL activity by 2- to 3-fold and cellular LPL by 1.5-fold. Significant increases in medium and cellular LPL were observed at 10(-9) M and were maximal at 10(-7) M. Along with the increase in LPL activity, there was an increase in LPL messenger RNA by 2-fold at 5 days, and by 5-fold at 7 days. In addition to an increase in LPL, 1,25-(OH)2D3 increased expression of aP2, an adipocyte-specific marker associated with differentiation. After the addition of 1,25-(OH)2D3, there was a decrease in 3T3-L1 cell number, which is consistent with differentiation, and a decrease in vitamin D receptors. Finally, these cells developed a different morphology. 1,25-(OH)2D3-treated cells assumed a rounded appearance, although without detachment from the dish and without the degree of lipid accumulation usually associated with the addition of insulin, isbutylmethylxanthine, and dexamethasone. It is concluded that 1,25-(OH)2D3 induced LPL expression in 3T3-L1 cells through an induction of differentiation-dependent mechanism(s). These findings suggest an important role for 1,25-(OH)2D3 in normal adipocyte differentiation.     

The E1A oncogene induces resistance to the effects of 1,25-dihydroxyvitamin D3 on inhibition of growth of mouse keratinocytes

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited DNA synthesis in transformed mouse keratinocytes (Pam212) in a time- and dose-dependent manner as measured by [3H]thymidine incorporation. To investigate the mechanism through which 1,25-(OH)2D3 acts, we examined its effects on Pam212 cells further transformed with the E1A oncogene. Here, we show that transformation of the cells with the E1A oncogene induced resistance to the effects of 1,25-(OH)2D3 on inhibition of growth of Pam212 cells. While 1,25-(OH)2D3 treatment increased the level of expression of vitamin D receptor mRNA 20-fold in parental cells, the E1A-transformed cells failed to express vitamin D receptor mRNA even after treatment with 1,25-(OH)2D3. Transfection of the E1A-transformed cell line with an expression construct encoding the vitamin D receptor restored receptor expression as well as the inhibition of growth by 1,25-(OH)2D3. These results suggest that one of the mechanisms for acquisition of 1,25-(OH)2D3 resistance induced by E1A may involve loss of vitamin D receptor inducibility by 1,25-(OH)2D3.     

1,25-dihydroxyvitamin D3 and 9-cis-retinoic acid act synergistically to inhibit the growth of LNCaP prostate cells and cause accumulation of cells in G1

Recent studies have suggested that the active metabolite of vitamin D3, 1,25-dihydroxyvitamin D3, can inhibit the growth and/or induce the differentiation of a variety of cell types and that these characteristics might be useful in the treatment of some cancers. Retinoids also promote the differentiation and inhibit the growth of some cells. That the vitamin D receptor acts as a heterodimer with the retinoid X receptor (RXR) suggests that there may be functional interactions between 1,25-dihydroxyvitamin D3 and retinoids. In this study, we show that the combination of 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid synergistically inhibits the growth of LNCaP prostate cancer cells. That this effect is mediated by RXR rather than retinoic acid receptors was shown using RXR- and retinoic acid receptor-specific ligands. The vitamin D3 analog, EB1089, inhibited growth more effectively than 1,25-dihydroxyvitamin D3 and also acted synergistically with 9-cis-retinoic acid. These treatments caused cells to accumulate in the G1 phase of the cell cycle, suggesting that 1,25-dihydroxyvitamin D3 can regulate one or more factors critical for the G1/S transition.     

Effect of growth hormone on urine calcium and serum vitamin D metabolites in renal failure

Growth hormone (GH) causes a modest increase in urine calcium excretion in normal adults, but uremic rats given both GH and calcitriol developed hypercalciuria. Ten short prepubertal children with renal insufficiency treated with recombinant human GH (rhGH) had urine calcium to creatinine (Ca/Cr) ratios and serum vitamin D metabolite concentrations monitored prospectively for up to 24 months. Six were also treated with calcitriol and two with other vitamin D preparations. Mean urine Ca/Cr ratios or mean serum concentrations of 1,25-dihydroxy vitamin D, 24,25-dihydroxy vitamin D, and 25-hydroxy vitamin D did not change significantly during treatment with rhGH. The risk for rhGH-induced hypercalciuria is small in children with renal insufficiency, even when treated concomitantly with a vitamin D preparation.     

Detection of a human chromosomal translocation t(8;9) in a baby with multiple malformations using two-color fluorescence in situ hybridization

In human fibroblast cultures TPA increased IL-6 and IL-8 production. This was reduced by vitamin D3 metabolites and analogs. The two analogs employed: 1,25 (OH)2-22 (E)-dehydro-24-monohomo vitamin D3 (Compound A) and 1,25 (OH)2 -22 (E)-dehydro-24 dihomo-vitamin D3 (Compound B) may be useful in the therapy of pathologic proliferative disorders including psoriasis, particularly since they are less toxic and have less effect on calcium metabolism than vitamin D3.     

Antisense oligonucleotides targeted against protein kinase Cbeta and CbetaII block 1,25-(OH)2D3-induced differentiation

It is now recognized that protein kinase C (PKC) plays a critical role in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) promotion of HL-60 cell differentiation. In this study, the effects of phosphorothioate antisense oligonucleotides directed against PKCalpha, PKCbeta, PKCbetaI, and PKCbetaII on HL-60 promyelocyte cell differentiation and proliferation were examined. Cellular differentiation was determined by nonspecific esterase activity, nitro blue tetrazolium reduction, and CD14 surface antigen expression. Differentiation promoted by 1,25-(OH)2D3 (20 nM for 48 h) was inhibited similarly in cells treated with PKCbeta antisense (30 microM) 24 h prior to or at the same time as hormone treatment (86 +/- 9% inhibition; n = 4 versus 82 +/- 8% inhibition; n = 4 (mean +/- S.E.), respectively). In contrast, cells treated with PKCbeta antisense 24 h after 1, 25-(OH)2D3 were unaffected and fully differentiated. PKCalpha antisense did not block 1,25-(OH)2D3 promotion of HL-60 cell differentiation. Next, the ability of PKCbetaI- and PKCbetaII-specific antisense oligonucleotides to block 1,25-(OH)2D3 promotion of cell differentiation was examined. PKCbetaII antisense (30 microM) completely blocked CD14 expression induced by 1, 25-(OH)2D3, whereas PKCbetaI antisense had little effect. Interestingly, PKCbetaII antisense blocked differentiation by 87 +/- 7% (n = 2, mean +/- S.D.) but had no effect on 1,25-(OH)2D3 inhibition of cellular proliferation. These results indicate that the effects of 1,25-(OH)2D3 on HL-60 cell differentiation and proliferation can be dissociated by blocking PKCbetaII expression.     

EB 1089, a novel vitamin D analogue, has strong antiproliferative and differentiation inducing effects on cancer cells

EB 1089 is a novel vitamin D analogue which in vitro strongly inhibits the proliferation of U937 histiocytic lymphoma cells and MCF-7 breast cancer cells, with a potency of 50 to 100 times that of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Studies of c-myc and c-fos expression in MCF-7 cells and of differentiation markers in U937 cells show that growth inhibition by EB 1089 is accompanied by induction of differentiation. The ability of EB 1089 to affect calcium metabolism in vivo in rats is decreased, compared to 1,25(OH)2D3. This low calcemic effect combined with the strong biological effect on cancer cells in vitro, makes EB 1089 an interesting candidate for treatment of cancer.     

The effect of 1,25-vitamin D3 on maturation of monocytes from HIV-infected patients varies with degree of immunodeficiency

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has been shown to induce monocyte-to-macrophage maturation in vitro as well as monocytic differentiation of bone marrow precursors and monocytic leukaemic cell lines. In this study we assessed whether 1,25D could improve the maturation defect we have previously demonstrated in monocytes from AIDS patients. In vitro growth and maturation of monocytes from 10 controls, 15 asymptomatic HIV positives (CDC group II or III) and 13 symptomatic HIV positives (CDC group IV) was examined by assessing cellular morphology, differentiation, adherence and protein content. Cells were cultured for 10 days with or without addition of 1,25D at a concentration of 100 pg/ml. In addition, patients were monitored clinically and by immunological parameters and HIV p24 antigen in serum. The present study showed that addition of 1,25D significantly improved the growth and maturation in both patient and control groups. There was a significant negative correlation between response to 1,25D and CD4+ lymphocyte count in blood in HIV-infected patients. A greater response to 1,25D was seen in monocytes from patients with advanced immunodeficiency and symptomatic disease than in monocytes from asymptomatic patients. However, in the most advanced cases of HIV infection with serious ongoing opportunistic infections the response to 1,25D was very poor, possibly reflecting profound and incorrigible dysfunction of monocytes.     

Lowering of p27Kip1 levels by its antisense or by development of resistance to 1,25-dihydroxyvitamin D3 reverses the G1 block but not differentiation of HL60 cells

Cyclin-dependent kinase inhibitors are proteins with functions which appear to involve regulation of cell cycle traverse, and have been suggested to have a role in cell differentiation. However, there is as yet no rigorous proof that this is the case. We have addressed the participation of one of these inhibitors, p27Kip1, in the induction of differentiation and the subsequent G1 block induced in HL60 cells by 1,25-dihydroxyvitamin D3 (1,25D3). First, it was noted that sublines of HL60 cells able to grow rapidly in the presence of 1,25D3 have protein levels of p27Kip1 lower than the levels in cells subjected to 1,25D3-induced growth inhibition, but higher than in untreated parental cells. In contrast, there was no discernible relationship between the levels of p27Kip1 and the expression of differentiation markers. Further, HL60 cells treated with 1,25D3 and an oligonucleotide antisense, but not mismatched, to p27Kip1 showed an almost complete elimination of the 1,25D3-induced G1 block, but no decrease in the expression of differentiation markers. Similar results were obtained following transient transfection with an expression vector bearing the entire p27Kip1 coding sequence in the anti-sense orientation. This is the first direct demonstration that p27Kip1 plays a role in the 1,25D3-induced G1 arrest, and that partial reduction in its levels has no effect on the induction of differentiation in HL60 cells.