Galactomutarotase and other galactose-related genes are rapidly induced by retinoic acid in human myeloid cells

Abstract

Aldose-1-epimerase (mutarotase) catalyzes the interconversion of alpha and beta hexoses, which is essential for normal carbohydrate metabolism and the production of complex oligosaccharides. Galactose mutarotase (GALM) has been well characterized at the protein level, but information is lacking on the regulation of GALM gene expression. We report herein that all-trans-retinoic acid (RA), an active metabolite of vitamin A that is known to induce myeloid lineage cell differentiation into macrophage-like cells, induces a rapid and robust regulation of GALM mRNA expression in human myeloid cells. all-trans-RA at a physiological concentration (20 nM), or Am580, a ligand selective for the nuclear retinoid receptor RARalpha, increased GALM mRNA in THP-1 cells, with significantly increased expression in 2 h, increasing further to an approximately 8-fold elevation after 6-40 h (P < 0.005). In contrast, tumor necrosis factor-alpha did not increase GALM mRNA expression, although it is capable of inducing cell differentiation. RA also increased GALM mRNA in U937 and HL-60 cells. The increase in GALM mRNA by RA was blocked by pretreating THP-1 cells with actinomycin D but not by cycloheximide. GALM protein and mutarotase activity were also increased time dependently in RA-treated THP-1 cells. In addition to GALM, several other genes in the biosynthetic pathway of galactosyl-containing complex oligosaccharides were more highly expressed in RA-treated THP-1 cells, including B4GALT5, ST3GAL3, ST6GALNAC5, and GALNAC4S-6ST. Thus, the results of this study identify RA as a significant regulator of GALM and other galactose-related genes in myeloid-monocytic cells, which could affect energy utilization and synthesis of cell-surface glycoproteins or glycolipids involved in cell motility, adhesion, and/or functional properties.

Figure 1

GALM mRNA expression is increased by RA, but not by TNFα, in THP-1 monocytic cells. A, THP-1 cells were treated with control medium (n=6), retinoic acid (20 nM at time 0 and again after overnight incubation (24 h, n=5), TNFα (5 ng/ml, for 24 h, n=5), or RA overnight, followed by RA and TNFα for the final 24 h (n=6). The level of GALM mRNA was detected by microarray analysis of total RNA prepared from each well of cells. B, Time course of GALM mRNA expression in THP-1 cells. RA (20 nM) was applied at time 0 (n=6), and cells were analyzed after 2 h (n=2), 6 h (n=2), 16 h (n=3); after overnight incubation at 37°C, RA was added again (indicated by black triangles), up to 40 h (n=2). The mean ± SEM of the log(2) values of the array signal intensities are shown. Bars or points that do not share a superscript letter differed significantly (P values as shown).

Figure 2

Retinoic acid and Am580 increase GALM mRNA in myeloid cell lines, but not in human hepatoma HepG2 cells. A, GALM mRNA expression (relative to GAPDH) determined by real-time PCR in THP1 cells that were treated either with <0.01% ethanol (control), RA, or Am580 (20 or 100 nM as shown) for 6 h. HepG2 cells (not shown) that were treated similarly did not show significant regulation. Inset shows agarose gel electrophoresis of PCR products after the end of 40 cycles, indicating the appropriate 395 bp size of the amplified product. B, GALM mRNA relative to GAPDH (left axis) and relative to control cells (right axis) in U937 cells and, C, HL-60 cells that were treated RA (20 nM) for 24 h. Data are means ± SEM, n≥3/treatment; * are significantly different from control within each panel, P<0.05.

Figure 3

GALM mRNA is induced by RA, and completely inhibited in cells pretreated with actinomycin D, but not with cycloheximide. A, THP-1 cells were treated with either RA (20 nM), actinomycin D (20 nM), RA + actinomycin D, or vehicle (control) for 24 h. B. THP-1 cells were treated with either RA (20 nM), cycloheximide (20 nM), RA+ cycloheximide, or vehicle (control) for 24 h. After incubation, cells were harvested and GALM and GAPDH mRNAwas measured by real-time RT-PCR as described in Materials and Methods. Data are means (with control set to 1) ± SEM, n=3/treatment; * denotes different from control, P<0.05.

Figure 4

Regulation of GALM protein and mutorotase enzyme activity by RA. A, GALM protein levels were detected by immunoblot analysis in lysates of THP-1 cells that had been treated for 24 h with vehicle (control, C), RA (20 nM), TNFα (5ng/ml), or both RA and TNFα. B. GALM protein in THP-1 cells treated with vehicle or RA (20 nM) for 24 and 48 h, showing persistent increase in RA-treated cells. The uniform non-specific band above GALM (38 kDa) demonstrates equal protein loading in all lanes. C. Mutorotase assay in THP-1 cells and HepG2 cells (1.4 × 10⁶ cells/well) treated without (control, C) or with RA (100 nM) for 6 h, after which cells were harvested and homogenized. Bars show mean ± SEM, n=4/condition. The * denotes a significant difference between control and RA-treated cells (P < 0.002). D. THP-1 cells were treated with either RA or Am580 (20 nM) and incubated for 6 h. Results for each of n=3 independent experiments totaling n=12 for control cells, n=12 for RA-treated cells, and n=8 for Am580-treated cells were pooled and compared by setting the mean value for the control group to 1.0. Values shown are the mean ± SEM; * denotes differences from control, P < 0.001.

Figure 5

Time dependent up-regulation of galactose-related genes in THP-1 cells treated with RA. THP-1 cells were treated with RA (20 nM) at t=0 and again after overnight incubation (shown by black triangles); microarray analysis was conducted (as described in Fig. 1B and Materials and Methods). Median signal intensity for the arrays is 0 on a log(2) scale and the Y-axis displays the mean ± SEM of the log(2) values of the array signal intensities at each time. Bars or points that do not share a superscript letter differed significantly (P values as shown). Genes illustrated are A, B4GALT5 (Entrez Gene ID 9334; Feature IDs 221484_at, and 221485_at); B, ST3GAL3 (Entrez Gene ID 6487; Feature ID 1555702_a_at); C, ST6GALNAC5 (Entrez Gene ID 81849; Feature ID 220979_s_at); and D, GALNAC4S-6ST (Entrez Gene ID 51363; Feature ID 203066_at).

Figure 6