Interferon regulatory factor-1 binds c-Cbl, enhances mitogen activated protein kinase signaling and promotes retinoic acid-induced differentiation of HL-60 human myelo-monoblastic leukemia cells

Abstract

All-trans retinoic acid (RA) and interferons (IFNs) have efficacy in treating certain leukemias and lymphomas, respectively, motivating interest in their mechanism of action to improve therapy. Both RA and IFNs induce interferon regulatory factor-1 (IRF-1). We find that in HL-60 myeloblastic leukemia cells which undergo mitogen activated protien kinase (MAPK)-dependent myeloid differentiation in response to RA, IRF-1 propels differentiation. RA induces MAPK-dependent expression of IRF-1. IRF-1 binds c-Cbl, a MAPK related adaptor. Ectopic IRF-1 expression causes CD38 expression and activation of the Raf/MEK/ERK axis, and enhances RA-induced differentiation by augmenting CD38, CD11b, respiratory burst and G0 arrest. Ectopic IRF-1 expression also decreases the activity of aldehyde dehydrogenase 1, a stem cell marker, and enhances RA-induced ALDH1 down-regulation. Interestingly, expression of aryl hydrocarbon receptor (AhR), which is RA-induced and known to down-regulate Oct4 and drive RA-induced differentiation, also enhances IRF-1 expression. The data are consistent with a model whereby IRF-1 acts downstream of RA and AhR to enhance Raf/MEK/ERK activation and propel differentiation.

Figure 1

Interferon regulatory factor-1 (IRF-1) expression in wild-type HL-60 and IRF-1 stable transfectant cell lines. Western blot of IRF-1 expression in wild-type and IRF-1 stably transfected (IRF +) HL-60 cells, 48 h. IRF-1 protein expression level was higher in IRF-1 stable transfectants than in parental wild-type cells. RA induced IRF-1 expression in wild-type and even more in IRF-1 overexpressors. RA, C treated or untreated control; RA, 1 μM retinoic acid, 48 h.

Figure 2

IRF-1 transfectants underwent enhanced cell differentiation. (A) In untreated cells, the expression level of CD38 was significantly higher in IRF-1 stable transfectants (IRF+) than in HL-60 control (WT). In RA-treated cells, IRF-1 overexpressors propelled CD38 expression levels (flow cytometric assay of live cells was carried out setting the logical gate to exclude 95% of untreated cells using APC conjugated anti-CD38 antibody). (B) In RA-treated cells, the expression level of CD11b was higher in IRF-1 stable transfectants than in HL-60 control. (C) In D3-treated cells, the CD11b level was lower in IRF-1 overexpressors compared to wild-type cells (flow cytometric assay of live cells was carried out setting the logical gate to exclude 95% of untreated cells with APC conjugated anti-CD11b antibody). (D) IRF-1 enhanced RA-induced respiratory burst. The percentage of cells capable of PMA inducible oxidative metabolism was analyzed by flow cytometry. The threshold to determine percentage positive cells was set to exclude 95% of control cells. PMA stimulated inducible oxidative metabolism. DMSO, carrier control blank. (E) IRF-1 accelerated G0 arrest induced by RA treatment. Nuclei stained with hypotonic PI staining solution were analyzed by flow cytometry. (F) Growth curves during duration of experiment were constructed by counting cells. Different letters represent significantly different values (p ≤ 0.05). Results are mean ± SEM for at least three repeats.

Figure 3

Aldehyde dehydrogenase 1 activity was significantly lower (p = 0.01) in cells treated with RA than in untreated cells. IRF-1 overexpression enhanced this effect. Results of flow cytometric analysis of live cells are expressed as difference in mode phycoerythrin (PE) fluorescence of test sample and control sample (diethylaminobenzaldehyde [DEAB] inhibited). a and b, statistically significant difference between them and compared with rest of samples (p < 0.005 for IFR + RA vs. IRF + C or WT RA and p < 0.001 for WT C vs. WT RA).

Figure 4

IRF-1 caused enhanced Raf/MEK/ERK activation. Western blots of phospho-Raf S259 and -Raf S621, phospho-MEK and phospho-ERK1/2 in WT and IRF+, untreated vs. RA-treated cells for 48 h. GAPDH was used to check protein loading. C, untreated cells; RA, 1 μM RA treated cells for 48 h. Twenty-five micrograms of protein were loaded per well.

Figure 5

RA caused, in a MEK-dependent manner, enhanced IRF-1 and c-Cbl. IRF-1 and c-Cbl associated Western blots (48 h, first three lanes, and 72 h, last three lanes) of IRF, c-Cbl, c-Cbl immunoprecipitated with IRF-1, actin (lane loading control) and histone 3. Twenty-five micrograms of protein were loaded per well. For IP, 300 μg of protein were used. For treatment, RA final concentration was 1 μM and VPA was 1 mM. C, RA and PD/RA are control, RA-treated and RA plus PD 98059-treated. PD 98059 (1 μM from a 1 mM DMSO stock) treatment was done 4 h prior to RA treatment.

Figure 6

AhR expression induced IRF-1. AhR overexpressors and VPA-treated samples expressed an augmented amount of IRF-1 protein, as determined by Western blotting (shown at 48 h).