Protein-tyrosine phosphatase PTPepsilon C inhibits Jak-STAT signaling and differentiation induced by interleukin-6 and leukemia inhibitory factor in M1 leukemia cells

We engineered and expressed both a wild-type and mutant cytosolic isoform of PTPepsilon (PTPepsilonC) in murine M1 leukemic cells, which can be induced to growth arrest and monocytic differentiation by interleukin (IL)-6 and leukemia inhibitory factor (LIF). Forced expression of PTPepsilonC inhibited IL-6- and LIF-induced monocytic differentiation and apoptosis in M1 cells, whereas expression of PTPepsilonM, a transmembrane isoform of PTPepsilon, did not. PTPepsilonC expression resulted in lower levels of IL-6-induced tyrosine phosphorylation of Jak1, Tyk2, gp130, and Stat3 compared with parent cells. In M1 transfectants expressing an inactive mutant of PTPepsilonC, both tyrosine phosphorylation and apoptosis induced by IL-6 and LIF were potentiated rather than inhibited. These results suggest an important role for PTPepsilonC in negative regulation of IL-6- and LIF-induced Jak-STAT signaling.     

Production of interleukin 6 and its relation to the macrophage differentiation of mouse myeloid leukemia cells (M1) treated with differentiation-inducing factor and 1 alpha,25-dihydroxyvitamin D3

We have studied the production of interleukin 6 (IL-6) and its relation to the macrophage differentiation in murine myeloid leukemia cells (M1). As has been reported, differentiation-inducing factor (D-factor), 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3], and recombinant IL-6 similarly induced differentiation of M1 cells into macrophages. The three compounds also induced mRNA expression of IL-6 in M1 cells. M1 cells treated with D-factor or 1 alpha, 25(OH)2D3 produced biologically active IL-6, but the amounts of IL-6 secreted into culture media did not appear to be enough to induce differentiation of M1 cells. Furthermore, simultaneous addition of anti-IL-6 antibody did not suppress the differentiation of M1 cells induced by D-factor or 1 alpha, 25(OH)2D3. These results show that IL-6 production is an essential property associated with the macrophage differentiation of M1 cells, but it may not be responsible for the D-factor- and 1 alpha, 25(OH)2D3-induced differentiation.     

Biological activity of 24,24-difluoro-1 alpha, 25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone in inducing differentiation of human myeloid leukemia cells

Vitamin D compounds added to the culture medium induce differentiation of human myeloid leukemia cells (HL-60 cells) by binding to a specific cytosol receptor protein. This system provides a biologically relevant and technically simple assay to examine the relationship between molecular structure and biological activity of vitamin D compounds. Using this culture system, the biological activity of 24,24-F2-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3-26,23-lactone was assayed. 24,24-F2-1 alpha,25(OH)2D3 was four to seven times more potent than 1 alpha,25(OH)2D3 in inducing phagocytosis and C3 rosette formation of HL-60 cells, though both compounds bound equally well to the cytosol receptor, suggesting that the defuorination at the 24-carbon position may stimulate membrane permeability of the compound. 1 alpha,25(OH)2D3-26,23-lactone, on the other hand, was only 1/200th as active as 1 alpha,25(OH)2D3. The binding affinity of the lactone for the cytosol receptor was identical with that of 1 alpha (OH)D3, suggesting that the lactone formation between the 26 and 23 positions masks the function of the 25-hydroxyl group. The binding affinity of vitamin D3 derivatives to the specific cytosol receptor of HL-60 cells was well correlated with that of intestinal cytosol protein specifically bound to 1 alpha,25(OH)2D3.     

Fusion of mouse alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3 involves extracellular, but not intracellular, calcium

We have reported that the active form of vitamin D3, 1 alpha, 25-dihydroxy-vitamin D3 [1 alpha, 25(OH)2D3], directly induces the fusion of mouse alveolar macrophages (Abe et al: Proc. Natl. Acad. Sci. USA 80:5583-5587, 1983). The fusion process can be divided into two phases: the 1 alpha,25(OH)2D3-dependent priming phase (0-18 hr) and the calcium-dependent progression phase (18-72 hr) (Jin et al: J. Cell. Physiol. 137:110-116, 1988). In the present study, we examined the role of calcium in the progression phase of macrophage fusion induced by 1 alpha,25(OH)2D3. Macrophages pretreated with 1 alpha,25(OH)2D3 for 48 hr in a low-calcium (0.13 mM) medium began to fuse quickly 30 min after the culture medium was switched to a normal calcium (1.85 mM) medium. Of various cations tested, calcium was the most effective in inducing fusion, followed by strontium and manganese. Magnesium, potassium, and sodium had no effect. Calcium ionophores such as A23187 and ionomycin did not induce fusion in the low-calcium medium, nor did they potentiate fusion in the media containing higher concentrations of calcium. The intracellular concentration of free Ca2+, measured by a fluorescent method using fura-2 AM, was 116 +/- 1 nM in the macrophages pretreated with 1 alpha,25(OH)2D3 for 48 hr in the low-calcium medium. When calcium chloride was added to the assay system at a final concentration of 1.85 mM, the cytosolic free Ca2+ concentration did not increase appreciably (from 116 to 144 nM). But the macrophages began to fuse quickly when CaCl2 was added. In contrast, adding ionomycin increased cytosolic free Ca2+ from 116 to 440 nM, but no fusion occurred. These results clearly indicate that the extracellular, but not the intracellular, calcium is involved in the progression phase of the fusion of mouse alveolar macrophages primed by 1 alpha,25(OH)2D3.     

Antagonistic action of novel 1alpha,25-dihydroxyvitamin D3-26, 23-lactone analogs on differentiation of human leukemia cells (HL-60) induced by 1alpha,25-dihydroxyvitamin D3.

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D3-26,23-lactone (1alpha,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analogues. We found that the 1alpha,25-lactone analogues (23S)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S, 25R)-1alpha,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10(-6) M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10(-7) M) was found to be an effective antagonist of both 1alpha,25(OH)2D3 (10(-8) M) mediated induction of p21(WAF1, CIP1) in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha, 25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR.     

Basic fibroblast growth factor inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D(3) through suppressing the production of osteoclast differentiation factor.

Basic fibroblast growth factor (bFGF) inhibited osteoclast-like cell (OCL) formation in cocultures of mouse spleen cells with either osteoblasts or a stromal cell line, ST2, in the presence of 1alpha, 25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. bFGF directly acted on osteoblasts/stromal cells, but not osteoclast progenitors, to inhibit 1,25(OH)(2)D(3)-induced OCL formation. bFGF suppressed the mRNA expression of osteoclast differentiation factor (ODF) but did not affect that of osteoclastogenesis inhibitory factor (OCIF) in ST2 cells treated with 1,25(OH)(2)D(3) and dexamethasone. Enzyme-linked immunosorbent assay showed that bFGF hardly affected OCIF production in the treated ST2 cells. A genetically engineered soluble form of ODF, but not anti-OCIF neutralizing antibody, abolished bFGF-mediated inhibition of OCL formation. bFGF suppressed the binding of (125)I-labeled OCIF to both ST2 cells and osteoblasts treated with 1,25(OH)(2)D(3). These findings indicate that bFGF inhibits 1,25(OH)(2)D(3)-induced OCL formation via suppression of ODF production by osteoblasts/stromal cells.     

1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL-60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells).

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D(3)-26,23-lactone (1alpha,25-(OH)(2)D(3)-26,23-lactone) analogs on 1alpha,25(OH)(2)D(3)-induced differentiation of human leukemia HL-60 cells thought to be mediated by the genomic action of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non-genomic actions of 1alpha,25-(OH)(2)D(3). We found that the 1alpha,25-(OH)(2)D(3)-26,23-lactone analogs, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9648), inhibited differentiation of HL-60 cells induced by 1alpha,25-(OH)(2)D(3). However, 1beta-hydroxyl diastereomers of these analogs, i.e. (23S)-25-dehydro-1beta-hydroxyvitamin D(3)-26, 23-lactone (1beta-TEI-9647) and (23R)-25-dehydro-1beta-hydroxyvitamin D(3)-26,23-lactone (1beta-TEI-9648), did not inhibit differentiation of HL-60 cells caused by 1alpha,25-(OH)(2)D(3). A separate study showed that the nuclear vitamin D receptor (VDR) binding affinities of the 1-hydroxyl diastereomers were about 200 and 90 times weaker than that of 1alpha-hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In contrast, 1beta,25-dihydroxyvitamin D(3) (1beta,25-(OH)(2)D(3)) and 1beta,24R-dihydroxyvitamin D(3) (1beta,24R-(OH)(2)D(3)) inhibited NB4 cell differentiation but not HL-60 cell differentiation. Collectively, the results suggested that 1-hydroxyl lactone analogs, i.e. TEI-9647 and TEI-9648, are antagonists of 1alpha,25-(OH)(2)D(3), specifically for the nuclear VDR-mediated genomic actions, but not for non-genomic actions.     

Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 from pagetic bone marrow cells

(23S)-25-Dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) functions an antagonist of the 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells [J. Biol. Chem. 274 (1999) 16392]. We examined the effect of vitamin D antagonist, TEI-9647, on osteoclast formation induced by 1alpha,25-(OH)(2)D(3) from bone marrow cells of patients with Paget's disease. TEI-9647 itself never induced osteoclast formation even at 10(-6)M, but dose-dependently (10(-10) to 10(-6)M) inhibited osteoclast formation induced by physiologic concentrations of 1alpha,25-(OH)(2)D(3) (41 pg/ml, 10(-10)M) from bone marrow cells of patients with Paget's disease. At the same time, 10(-8)M of TEI-9647 alone did not cause 1alpha,25-(OH)(2)D(3) dependent gene expression, but almost completely suppressed TAF(II)-17, a potential coactivator of VDR and 25-hydroxyvitamin D(3)-24-hydroxylase (25-OH-D(3)-24-hydroxylase) gene expression induced by 10(-10)M 1alpha,25-(OH)(2)D(3) in bone marrow cells of patients with Paget's disease. Moreover, TEI-9647 dose-dependently inhibited bone resorption induced by 10(-9)M 1alpha,25-(OH)(2)D(3) by osteoclasts produced by RANKL and M-CSF treatment of measles virus nucleocapsid gene transduced bone marrow cells. These results suggest that TEI-9647 acts directly on osteoclast precursors and osteoclasts, and that TEI-9647 may be a novel agent to suppress the excessive bone resorption and osteoclast formation in patients with Paget's disease.     

Tumor necrosis factor as an interleukin 1-dependent differentiation inducing factor (D-factor) for mouse myeloid leukemic cells

Experiments were conducted to purify the differentiation-inducing factor (D-factor), which induces differentiation of mouse myeloid leukemic cell line, Ml, into macrophage-like cells, in a conditioned medium of guinea pig peritoneal macrophages stimulated with lipopolysaccharide. On gel filtration under high performance liquid column chromatography (HPLC), D-factor eluted at the position of 45-15 KD. By the subsequent separation on DEAE HPLC the D-factor activity disappeared. However, in the presence of recombinant human IL 1 alpha the D-factor activity appeared at a position where tumor necrosis factor (TNF) eluted. Even after fractionation on hydroxyapatite HPLC the IL 1-dependent D-factor was co-chromatographed with TNF. Recombinant human TNF as well as the partially purified guinea pig TNF induced differentiation of Ml cells in conjunction with either the partially purified guinea pig IL 1 or recombinant human IL 1 alpha, although these factors by themselves did not induce differentiation. These findings suggest that a part of D-factor activity in the conditioned medium resulted from the cooperative effects between TNF and IL 1.     

Inhibition by 1 alpha,25-dihydroxyvitamin D3 of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Regulation of erythroid differentiation by vitamin D3 derivatives was examined in Friend erythroleukemia cells. After Friend cells were cultured for 5 days with 1.5% dimethyl sulfoxide (DMSO), as much as 70% of the cells became benzidine-positive and the hemoglobin content increased in parallel with the increase of benzidine-positive cells. The DMSO-induced erythroid differentiation was markedly inhibited by concurrent addition of the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Of the vitamin D3 derivatives tested, 1 alpha,25(OH)2D3 was the most potent in inhibiting DMSO-induced erythroid differentiation. 1 alpha,25(OH)2D3 alone was totally ineffective in both cell growth and erythroid differentiation. These results together with our previous reports indicate that 1 alpha,25(OH)2D3 is somehow involved not only in myeloid differentiation, but also in erythroid differentiation.     

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.     

Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in rat kidney but not in intestine.

Using a cDNA probe for rat renal 24-hydroxylase, expression of its mRNA was compared in the rat kidney and intestine. Vitamin D-deficient rats received a single injection of 1 alpha,25-dihydroxyvitamin D3. Expression of 24-hydroxylase mRNA was first detected in the kidney at 3-h post-injection and increased thereafter. Similarly, 24-hydroxylase mRNA was expressed in the intestine after 1 alpha,25-dihydroxyvitamin D3 injection. However, the dose level of 1 alpha,25-dihydroxyvitamin D3 required to induce the intestinal 24-hydroxylase mRNA expression was only 1/100 the amount required to induce renal 24-hydroxylase mRNA. Induction of intestinal 24-hydroxylase mRNA expression by 1 alpha,25-dihydroxyvitamin D3 was far more rapid than that of renal 24-hydroxylase mRNA. Thyroparathyroidectomy shortened the time required to induce expression of renal, but not intestinal, 24-hydroxylase mRNA. Administration of either parathyroid hormone or cAMP to vitamin D-deficient rats greatly reduced the expression of 24-hydroxylase mRNA in the kidney but not in the intestine. When rats were fed a vitamin D-repleted diet containing 0.7% (adequate) or 0.03% (low) calcium for 2 weeks, intestinal expression of 24-hydroxylase mRNA could be induced only in the low calcium group. In contrast, renal mRNA expression was preferentially stimulated in the adequate calcium group. These results clearly demonstrate that the expression of 24-hydroxylase mRNA is down-regulated by parathyroid hormone in the kidney but not in the intestine.     

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.     

Calcium is essential in the fusion of mouse alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3

We have reported that the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], directly induces activation and fusion of mouse alveolar macrophages (Abe et al., 1983, 1984). The activated state appeared to be a prerequisite to the fusion of macrophages. Macrophages began to fuse 36 hr after adding 1 alpha,25(OH)2D3; the fusion rate attained a maximum of 70-80% at 72 hr. During the course of further investigating the mechanisms of fusion induced by the vitamin, we found that the calcium ion is closely involved in the fusion process of macrophages induced by 1 alpha,25(OH)2D3. When alveolar macrophages were cultured with 1 alpha,25(OH)2D3 in medium with graded concentrations (0.13-1.85 mM) of calcium, the fusion rate went down in parallel with the decrease of medium calcium. Neither calcium ionophore A23187 nor 12-O-tetradecanoylphorbol-13-acetate (TPA) induced fusion of freshly isolated macrophages, but the two compounds greatly promoted fusion of the macrophages pretreated for 18 hr with 1 alpha,25(OH)2D3. The vitamin effect for the first 18 hr was similar, irrespective of the medium calcium concentration. In contrast, millimolar amounts of calcium were essential in the subsequent period of incubation(18-72 hr) for inducing fusion. The activation of macrophages measured by the induction of cytotoxicity and the enhancement of glucose consumption by 1 alpha,25(OH)2D3 occurred similarly, irrespective of the medium calcium concentration. These results clearly indicate that the fusion process of alveolar macrophages induced by 1 alpha,25(OH)2D3 can be divided into two phases: 1) the calcium-independent priming phase (0-18 hr) and 2) the calcium-dependent progression phase (18-72 hr). 1 alpha,25(OH)2D3 is necessary only in the priming phase; A23187 and TPA can be substituted for 1 alpha,25(OH)2D3 in the progression phase.     

1 alpha,25-dihydroxyvitamin D3 inhibits productive infection of human monocytes by HIV-1.

Pretreatment of freshly isolated human peripheral blood monocytes with the steroid hormone, 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)D), markedly reduced (by 95%) productive infection of human monocytes by HIV-1. Equivalent concentrations (10nM) of 25-hydroxyvitamin D3 (25(OH)D), the biologic precursor of 1,25(OH)D, were ineffective at reducing either CD4 expression or HIV-1 production. Pretreatment was required for modulation of HIV-1 infection by 1,25(OH)D. Interestingly, 1,25(OH)D-mediated decreases in p24 antigen production were observed prior to any observed reduction in CD4 expression, suggesting that 1,25(OH)D treatment may modulate HIV-1 infection of monocytes through additional factors besides decreased HIV-1 binding. These data raise the possibility that 1,25(OH)D compounds may be important in host resistance to HIV-1.     

Study on the metabolites of 1alpha,25-dihydroxyvitamin D4

Three major metabolites of 1alpha,25-dihydroxyvitamin D(4) were isolated from the bile of rat and the structures were elucidated on the basis of spectral data and the periodate oxidative cleavage of the diol structures of the metabolites. One of the metabolites was the known calcitroic acid. Another two metabolites were isomers and identified as 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-26-oic acid and 9,10-secoergosta-5,7,10(19)-triene-1alpha,3beta,24,25-tetrahydroxy-28-oic acid. It was found that 1alpha,25-dihydroxyvitamin D(4) is metabolized in a similar manner in vivo to that of 1alpha,25-dihydroxyvitamin D(2) but differently from 1alpha,25-dihydroxyvitamin D(3).