1,25-dihydroxyvitamin D(3)-induced retardation of the G(2)/M traverse is associated with decreased levels of p34(cdc2) in HL60 cells.

Cellular differentiation of neoplastic cells after exposure to 1, 25-dihydroxyvitamin D(3) (1,25 D(3)) is accompanied by altered cell cycle regulation. In previous studies, blocks in both G(1)/S and G(2)/M checkpoints have been observed in 1,25D(3)-treated HL60 cells, but the mechanism of the 1,25D(3)-induced G(2)/M block has not been previously reported. In this study, we show by cell cycle analysis, using bromodeoxyuridine pulse-chase labeling, that the G(2)/M block in 1,25D(3)-treated HL60 cells is incomplete. We also demonstrate that although the 1,25D(3)-treated cells exhibit elevated levels of cyclin B1, Cdc25C, and Cdk7, which are positive regulators of the G(2)/M traverse, these cells have decreased protein levels of p34(cdc2) and decreased p34(cdc2) kinase activity. This provides potential mechanisms for the observed accumulation of cells in the G(2) cell cycle compartment and occasional polyploidization following treatment of HL60 cells with 1,25D(3). The data also suggest that the ability of some cells to traverse this block may be the result of cellular compensatory mechanisms responding to decreased p34(cdc2) activity by increasing the levels of other regulators of the G(2) traverse, such as cyclin B1, Cdc25C, and Cdk7.     

Insulin-like growth factor binding protein-5 interacts with the vitamin D receptor and modulates the vitamin D response in osteoblasts

The 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]-induced differentiation of osteoblasts comprises the sequential induction of cell cycle arrest at G0/G1 and the expression of bone matrix proteins. Reports differ on the effects of IGF binding protein (IGFBP)-5 on bone cell growth and osteoblastic function. IGFBP-5 can be growth stimulatory or inhibitory and can enhance or impair osteoblast function. In previous studies, we have shown that IGFBP-5 localizes to the nucleus and interacts with the retinoid receptors. We now show that IGFBP-5 interacts with nuclear vitamin D receptor (VDR) and blocks retinoid X receptor (RXR):VDR heterodimerization. VDR and IGFBP-5 were shown to colocalize to the nuclei of MG-63 and U2-OS cells and coimmunoprecipitate in nuclear extracts from these cells. Induction of osteocalcin promoter activity and alkaline phosphatase activity by 1,25(OH)2D3 were significantly enhanced when IGFBP-5 was down-regulated in U2-OS cells. Moreover, we found IGFBP-5 increased basal alkaline phosphatase activity and collagen alpha1 type 1 expression, and that 1,25(OH)2D3 was unable to further induce the expression of these bone differentiation markers in MG-63 cells. Expression of IGFBP-5 inhibited MG-63 cell growth and caused cell cycle arrest at G0/G1 and G2/M. Furthermore, IGFBP-5 reduced the effects of 1,25(OH)2D3 in blocking cell cycle progression at G0/G1 and decreased the expression of cyclin D1. These results demonstrate that IGFBP-5 can interact with VDR to prevent RXR:VDR heterodimerization and suggest that IGFBP-5 may attenuate the 1,25(OH)2D3-induced expression of bone differentiation markers while having a modest effect on the 1,25(OH)2D3-mediated inhibition of cell cycle progression in bone cells.     

Activation of extracellular signal-regulated kinases (ERKs) defines the first phase of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells

Activation of ERK1 and ERK2 protein kinases has been implicated in diverse cellular processes, including the control of cell proliferation and cell differentiation (Marshall [1995] Cell 80:179). In human myeloblastoid leukemia HL60 cells rapid (ca. 15 min) but transient activation of ERK1/2 has been reported following induction of macrophage/monocyte differentiation by phorbol esters, or by very high (10(-6) M) concentrations of 1,25-dihydroxyvitamin D(3) (1,25D3), while retinoic acid-induced granulocytic differentiation was accompanied by sustained activation of ERK1/2. We report here that monocytic differentiation of HL60 cells induced by moderate (10(-9) to 10(-7) M) concentrations of 1,25D3 could be divided into at least two stages. In the first phase, which lasts 24-48 h, the cells continued in the normal cell cycle while expressing markers of monocytic phenotype, such as CD14. In the next phase the onset of G1 cell cycle block became apparent and expression of CD11b was prominent, indicating a more mature myeloid phenotype. The first phase was characterized by high levels of ERKs activated by phosphorylation, and these decreased as the cells entered the second phase, while the levels of p27/Kip1 increased at that time. Serum-starved or PD98059-treated HL60 cells had reduced growth rate and slower differentiation, but the G1 block also coincided with decreased levels of activated ERK1/2. The data suggest that the MEK/ERK pathway maintains cell proliferation during 1,25D3-induced monocytic differentiation of HL60 cells, but that ERK1/2 activity becomes suppressed during the later stages of differentiation, and the consequent G1 block leads to "terminal" differentiation.     

Inhibition of p38MAP kinase potentiates the JNK/SAPK pathway and AP-1 activity in monocytic but not in macrophage or granulocytic differentiation of HL60 cells

Monocytic differentiation of HL60 cells induced by 1,25-dihydroxyvitamin D(3) (1,25 D(3)) has been recently shown (Exp Cell Res 258, 425, 2000) to be enhanced by an exposure to SB203580 or to SB202190, specific inhibitors of p38MAP kinase, with concomitant up-regulation of the c-jun N terminal kinase (JNK) pathway. In the present study we inquired if this enhancement and the JNK up-regulation are limited to 1,25 D(3)-induced differentiation, or if they occur more generally in HL60 cell differentiation. We found that dimethylsulfoxide (DMSO)-induced differentiation, and to a lesser extent tetradecanoylphorbol acetate (TPA)-induced macrophage differentiation were also potentiated by the p38MAPK inhibitors, but that granulocytic differentiation in response to all-trans retinoic acid (RA) was not. The enhancement of differentiation by p38MAPK inhibitors was accompanied by an activation of the JNK MAPK pathway, as shown by the phosphorylation levels of these kinases and by AP-1 binding, but only in 1,25 D(3)-treated cells. This shows that an up-regulation of the JNK stress pathway during 1,25 D(3)-induced monocytic differentiation occurs selectively in this lineage of differentiation and is not necessary for the expression of the differentiated phenotype.     

1alpha,25-dihydroxyvitamin D3 stimulates phosphorylation of IkappaBalpha and synergizes with TPA to induce nuclear translocation of NFkappaB during monocytic differentiation of NB4 leukemia cells.

Treatment of NB4 acute promyelocytic leukemia cells with 1,25-dihydroxyvitamin D3 (1,25D3) or analogs 20-epi-22-oxa-24a,26a,27a-trihomo-1alpha,25-dihydroxyvitamin D3, 1,24-dihydroxy-22-ene-24-cyclopropylvitamin D3, 1alpha,25-dihydroxylumisterol3, or 1alpha,25(OH)2-d5-previtamin D3 in combination with TPA induces monocytic differentiation. The role of 1,25D3 in the induction of maturation has been shown to be a priming effect. Differentiation in response to these agents requires VDR-independent signaling of 1,25D3, PKC signaling, intracellular calcium, and calpain activity. In this study we identify the NFkappaB/IkappaB signaling pathway as a target of 1,25D3 and TPA action. One of the priming effects of 1,25D3 appears to be the rapid phosphorylation of serine residues on IkappaBalpha. On their own, 1,25D3, its analogs, and TPA do not alter IkappaBalpha expression; however, combinations of analogs with TPA result in a synergistic decrease in IkappaBalpha expression. Decreased expression of IkappaBalpha likely results from enhanced degradation, which allows the observed subsequent nuclear translocation of NFkappaB subunit p65. Since nuclear-localized NFkappaB was observed only in combination-treated cells, it is proposed that nuclear targets of NFkappaB are required for monocytic differentiation. Intracellular calcium and proteolytic activity are both necessary for the induction of IkappaB regulation and translocation of NFkappaB and are critical components of the nongenomic signaling cascades of the 1,25D3-induced differentiation pathway.     

Induction of differentiation of human leukemia cells by combinations of COX inhibitors and 1, 25-dihydroxyvitamin D3 involves Raf1 but not Erk 1/2 signaling

Differentiation therapy of cancer is being explored as a potential modality for treatment of myeloid leukemia, and derivatives of vitamin D are gaining prominence as agents for this form of therapy. Cyclooxygenase (COX) inhibitors have been reported to enhance 1,25-dihydroxyvitamin D3 (1,25D)-induced monocytic differentiation of promyeloblastic HL60 cells, but the mechanisms of this effect are not fully elucidated, and whether this potentiation can occur in other types of myeloid leukemia is not known. We found that combination treatment with 1,25D and non-specific COX inhibitors acetyl salicylic acid (ASA) or indomethacin can robustly potentiate differentiation of other types of human leukemia cells, i.e. U937, THP-1, and that ASA +/- 1,25D is effective in primary AML cultures. Increased cell differentiation is paralleled by arrest of the cells in the G1 phase of the cell cycle, and by increased phosphorylation of Raf1 and p90RSK1 proteins. However, there is no evidence that this increase in phosphorylation of Raf1 is transmitted through the ERK module of the MAPK signaling cascade. Transfection of small interfering (si) RNA to Raf1 decreased differentiation of U937 cells induced by a combination of ASA or indomethacin with 1,25D. However, phosphorylation levels of ERK1/2, though not of p90RSK, were increased when P-Raf1 levels were decreased by the siRNA, suggesting that in this system the ERK module does not function in the conventional manner. Identification of the strong antiproliferative activity of ASA/1,25D combinations associated with monocytic differentiation has implications for cancer chemoprevention in individuals who have a predisposition to myeloid leukemia.     

Vitamin D inhibits G1 to S progression in LNCaP prostate cancer cells through p27Kip1 stabilization and Cdk2 mislocalization to the cytoplasm

1,25-(OH)2 vitamin D3 (1,25-(OH)2D3) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LN-CaP, 1,25-(OH)2D3 mediates an increase in cyclin-dependent kinase inhibitor p27Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G1. In this study, we investigated the mechanism whereby 1,25-(OH)2D3 increases p27 levels. 1,25-(OH)2D3 had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)2D3 as measured by luciferase activity driven by the 5'- and 3'-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of 35S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)2D3 treatment. Because Cdk2-mediated phosphorylation of p27 at Thr187 targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)2D3-treated cells. 1,25-(OH)2D3 decreased levels of Thr187 phosphorylated p27, consistent with inhibition of Thr187 phosphorylation-dependent p27 degradation. In addition, 1,25-(OH)2D3 reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr160 phosphorylation and by cdc25A phosphatase via Thr14 and Tyr15 dephosphorylation. Interestingly, 1,25-(OH)2D3 decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH)2D3 may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)2D3.     

MicroRNAs181 regulate the expression of p27Kip1 in human myeloid leukemia cells induced to differentiate by 1,25-dihydroxyvitamin D3

Human myeloid leukemia cells exposed to 1,25-dihydroxyvitamin D3 (1,25D), a major cancer chemopreventive agent, acquire features of normal monocytes and arrest in the G1 phase of the cell cycle, due to the upregulation of p27Kip1 and p21Cip1, but the mechanism is not clear. Here evidence is provided that an exposure of HL60 and U937 cells to low (1-10 nM) concentrations of 1,25D decreases the expression of miR181a and miR181b in a concentration and time-dependent manner. Since the predicted miR181 targets include the 3’-UTR of p27Kip1, we expressed pre-miR181a in these cells, and found that the elevation of cellular miR181a levels abrogates the 1,25D-induced increase in p27Kip1 at both mRNA and protein levels. In contrast, transfection of pre-miR181a resulted in a slight elevation of p21Cip1 expression. Importantly, transfection of pre-miR181a blunted the effect of 1,25D on the expression of monocytic differentiation markers, and reduced the G1 block in 1,25D-treated cells, while transfection of anti-miR181a increased 1,25D-induced differentiation. Together, these data show that miR181a participates in 1,25D-induced differentiation of HL60 and U937 cells, and suggest that a high constitutive expression of members of miR181 family may contribute to the malignant phenotype in the myeloid lineage.     

Kinase suppressor of RAS (KSR) amplifies the differentiation signal provided by low concentrations 1,25-dihydroxyvitamin D3

The activity of kinase suppressor of ras (KSR), a kinase or a molecular scaffold upstream from Raf-1, is involved in the MEK/ERK MAP kinase cascade which can signal cell growth, survival, or differentiation, depending on the cellular context. We provide evidence here that KSR is upregulated in HL60 cells undergoing differentiation induced by low (0.3-3 nM) concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)), and an antisense oligo (AS), but not a sense oligo, to KSR inhibits this differentiation. The inhibition of differentiation by AS-KSR oligo was less apparent when the concentration of 1,25D(3) was increased, suggesting that at the higher concentrations of 1,25D(3) KSR is not essential for the signaling of the differentiated phenotype. The reduced differentiation of HL60 cells exposed to AS-KSR was paralleled by reduced phosphorylation of Raf-1 Ser 259, and of p90RSK, used here as read-out for MAPK cascade activity. Conversely, ectopic expression of Flag-tagged wild type KSR potentiated the differentiation-inducing effects of low concentrations of 1,25D(3). Additional data suggest that the kinase activity of KSR is required for these effects, as transfection of a kinase inactive KSR construct did not significantly increase the 1,25D(3)-induced differentiation. Enzyme assays performed with KSR immunoprecipitated from 1,25D(3)-treated cells showed kinase activity when recombinant Raf-1 was used as the substrate, but not when the 1,25D(3)-treated cells were pretreated with AS-KSR oligos. Taken together, these data suggest that KSR participates in signaling of monocytic differentiation by augmenting the strength of the signal transmitted through Raf-1 to downstream targets.     

Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.     

Monocytically differentiating HL60 cells proliferate rapidly before they mature

1alpha,25-Dihydroxyvitamin D(3) (D(3)) provokes growth arrest and monocytic differentiation in myeloid cells. Although it is usually assumed that the cellular events leading to growth arrest start within one cell cycle of D(3) addition, there is also evidence that D(3) provokes the expression of proliferation-related genes and accelerates cell division. Herein we clarify the relationship between proliferation and maturation in differentiating HL60 cells. Cells were cultured singly, D(3) was added at various stages of the cell cycle, the progeny were counted, and the proportions of mature monocytes were determined. Initially, the D(3)-treated cells proliferated at an accelerated rate, and they matured only later. If cells encountered D(3) early in G1 they divided two to four times before maturing, and if they encountered D(3) later in the cell cycle they underwent an extra division. Indomethacin slows HL60 cell multiplication by prolonging G1, and when these slower-growing cells were exposed to D(3), they matured after the usual period but underwent one division less than indomethacin-free cells. Contrary to common assumptions, we conclude that promyeloid cells do not initiate growth arrest or monocytic maturation immediately after exposure to D(3). Instead, an encounter with D(3) early in G1 sets in train a complex differentiation program. This consists of 2-3 days of rapid proliferation-probably employing cell cycles with a shortened G1 phase-that is followed by growth arrest and maturation. As a result, a single D(3)-treated promyeloid cell gives rise to 10 or more mature monocytes. These observations not only explain why "differentiating" cells express proliferation-related characteristics soon after D(3) addition, but they also show that the process of D(3)-induced monocytic differentiation is much more complex than has previously been realized.     

The expression and activity of D-type cyclins in F9 embryonal carcinoma cells: modulation of growth by RXR-selective retinoids

The growth rate of malignant F9 embryonal carcinoma cells slows considerably following all-trans-retinoic acid-induced differentiation into benign parietal endoderm. To determine the mechanism of this process, we examined the expression of cyclins D1, D2, and D3 and the activity of their associated kinases. Cyclin D1 and D3 mRNA levels decreased during complete differentiation induced by all-trans-retinoic acid and dibutyryl cAMP, while the levels of cyclin D2 and the cyclin-dependent kinase (Cdk) inhibitor p27 mRNAs increased. Ultimately, terminally differentiated cells possessed 50% of the Cdk4-associated kinase activity observed in undifferentiated cells. Since numerous genes are differentially regulated during parietal endoderm differentiation, it is difficult to determine whether retinoic acid affects cell cycle gene expression directly or if these changes are caused by differentiation. We found that the retinoid X receptor (RXR)-selective agonists LG100153 and LG100268 significantly inhibited F9 cell growth without causing overt terminal differentiation as assessed by anchorage-independent growth and differentiation-associated gene expression. As seen in cells induced to differentiate by the RAR agonist all-trans-retinoic acid, RXR activation led to an increase in the number of cells in G1 phase. RXR agonists also sharply induced the levels of the Cdk regulatory subunits, cyclin D2 and D3. However, Cdk4-dependent kinase activity was reduced by RXR-selective retinoid treatment. These observations suggest that some retinoids can directly inhibit proliferation and regulate Cdk4-dependent kinase activity without inducing terminal differentiation.     

Interferon-gamma-induced IA expression in WEHI-3 cells is enhanced by the presence of 1,25-dihydroxyvitamin D3

It has been suggested recently that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is involved in the regulation of the immune functions of lymphocytes and in the differentiation of monocytic cells. This report examined the possibility that 1,25(OH)2D3 influences immune functions mediated by monocytic cells by studying its effect on the murine myelomonocytic line WEHI-3. We found that WEHI-3 cells possess 3.3S receptor proteins with high affinity (Kd = 3.3 X 10(-10) M) for 1,25(OH)2D3 that are capable of binding to DNA. Also we found that 1,25(OH)2D3 enhances the interferon-gamma (IFN-gamma)-induced expression of the class II major histocompatibility complex antigens (Ia molecules), and such enhancement leads to increased capacity of the WEHI-3 cells to stimulate antigen-specific Ia-restricted T cell activation. Finally, 1,25(OH)2D3 inhibits the proliferation of WEHI-3 cells, and this inhibition is enhanced in the presence of IFN-gamma. The 1,25(OH)2D3 modulation of IFN-gamma induction of Ia antigens suggests that the hormone might promote monocytes to function more efficiently as antigen-presenting cells.