C/EBPbeta is known to be important for monocytic differentiation and macrophage function. Here, we found that expression of all three C/EBPbeta isoforms induced in HL60 cells by 1,25-dihydroxyvitamin D3 (1,25D) was upregulated in a sustained manner that correlates with the appearance of monocytic phenotype and with the G1 phase cell cycle arrest. In 1,25D-resistant HL60-40AF cells, isoforms beta-1 and beta-3 were expressed at levels comparable to 1,25D-sensitive HL60-G cells, but isoform beta-2 was difficult to detect. Treatment of sensitive HL60 cells with 1,25D resulted in predominantly nuclear localization of C/EBP isoforms beta-2 and beta-3, while a large proportion of C/EBPbeta-1 remained in the cytoplasm. Attenuation of the MEK-ERK MAPK pathway by the inhibitor PD98059 markedly reduced the expression, 1,25D-induced phosphorylation and nuclear localization of C/EBPbeta-2 and C/EBPbeta-3. Interestingly, only the lower molecular mass isoforms of C/EBPbeta phosphorylated on Thr235 were found in the nuclei, while C/EBPbeta-1 was constitutively phosphorylated and was detected principally in the cytoplasmic fraction. Although the role of C/EBPbeta isoforms in 1,25D-induced differentiation is complex, our results taken together strongly suggest that the phosphorylation of C/EBPbeta isoforms on Thr235 takes place mainly via the MEK-ERK pathway and that C/EBPbeta-2 is the principal transcription factor in this cell system.

1,25-dihydroxyvitamin D 3 (1,25D) induces differentiation of myeloid leukemia cells, but resistant cells are also encountered. We studied the mechanistic basis for the resistance in a model system using enhancers of 1,25D, the antioxidant carnosic acid and a kinase inhibitor SB202190. Knockdown (KD) of JNK2p54 unexpectedly increased the intensity of differentiation induced by the 1,25D, carnosic acid and SB202190 (DCS) combination. This was associated with upregulation of activated JNK1p46, and the transcription factors regulated by the JNK pathway, c-Jun, ATF2 and JunB, as well as C/EBP β. In contrast, KD of JNK1p46 reduced the intensity of DCS-induced differentiation, and partially abrogated activation of c-Jun/AP-1 transcription factors.

Among the many important physiological functions of the activated vitamin D receptor (VDR) is the signaling of monocytic differentiation, first demonstrated by conversion of malignant myeloid leukemia cells to nonproliferating cells with mature monocyte/macrophage appearance. However, the understanding of how 1, 25-dihydroxyvitamin D3 (1,25D) signals monocytic differentiation is still developing. Recent advances summarized here include the role of the principal "mitogen-activated protein kinase" (MAPK) pathways, their potential downstream target the CCAAT/enhancer binding protein beta (C/EBP beta), cell cycle related proteins, and cyclin-dependent kinase 5 (Cdk5) in 1,25D-induced differentiation. The precise steps by which activated VDR signals differentiation are incompletely understood in any of the cell types known to respond to 1,25D. We have focused our studies on HL60 cells, a widely available cell line derived from a patient with promyeloblastic leukemia, with the goal of achieving as clear a picture as possible with the currently available tools. In this model, outlined in Fig. 1, a plausible sequence of events is presented, with the caveats that these are not the only pathways activated by liganded VDR, and that several other pathways, also operative, remain to be convincingly demonstrated. The details of the scheme will be discussed in the sections below.

The anti-neoplastic effects of 1,25-dihydroxyvitamin D 3 (1,25D) are well documented in numerous tumor cell systems and animal models of cancer. However, despite this pre-clinical success, the clinical use of 1,25D is currently impeded by the dose-limiting hypercalcemia, and the risk of development of resistance to 1,25D. In this study, we investigated the mechanism of resistance to 1,25D of HL60-40AF cells, a model of drug-resistant acute myeloid leukemia, derived from HL60 cells by cultivation in the presence of 1,25D. The data indicate that transcriptional activity of vitamin D receptor (VDR) in 40AF cells increases only briefly when the cells are treated with 1,25D, despite greater basal cellular levels of VDR protein in the resistant than in the 1,25D-sensitive cells. Analysis of the 40AF VDR mRNA sequence indicated alterations in the 5 0 untranslated region (UTR), but coding domain variations were not observed. When resistance to 1,25D-induced differentiation of 40AF cells was reversed by a combination of 1,25D with potentiators of differentiation (plant derived antioxidants and a p38MAPK inhibitor), an increase in the level of nuclear VDR, as well as an increase in CYP24 mRNA expression was observed. These data suggest that decreased ability of 1,25D to induce VDR nuclear localization and the consequent VDR target gene transcription may be an important reason for the resistance of 40AF cells to 1,25D. Further, our data show that VDR localization and phosphorylation can be increased by combining 1,25D with potentiators of differentiation. Analysis of the mechanisms that underlie the reduction and potentiation of 1,25D-mediated changes in VDR activity may lead to the identification of new cellular targets that enhance 1,25D-induced monocytic differentiation.

Signaling of cell differentiation is one of the important physiological functions of the activated vitamin D receptor (VDR). Activation of the VDR can be achieved not only by 1α,25-dihydroxyvitamin D 3 (1,25D), the natural ligand, but also by a large number of its analogs. These include a category containing two side chains emanating at C-20, generally referred to as Gemini. The introduction of a cyclopropyl moiety as part of the pro-R side chain provides modified Gemini compounds with increased steric requirement and decreased chain flexibility; the biological consequences of this novel structural variant are subject of this investigation. In general, the resulting 1α,25-dihydroxy-(4-hydroxy-4-methyl-pentyl)-21,22-cis-cyclo-cholecalciferols reduced had differentiation and transcriptional potency and induced cell cycle arrest less effciently, as shown by a decrease in G1/S ratio, when compared to 1,25D. Modifying their calcitriol side chain in the form of a 4-hydroxy-4-trifluoromethyl-5,5,5-trifluoropent-2-ynyl moiety, however, resulted in pronounced induction of differentiation in 1,25D-sensitive and moderate level of differentiation in 1,25D-resistant leukemia cells.