Short-chain fatty acid derivatives stimulate cell proliferation and induce STAT-5 activation

Abstract

Current chemotherapeutic and butyrate therapeutics that induce fetal hemoglobin expression generally also suppress erythropoiesis, limiting the production of cells containing fetal hemoglobin (F cells). Recently, selected short-chain fatty acid derivatives (SCFADs) were identified that induce endogenous gamma-globin expression in K562 cells and human burst-forming units-erythroid and that increase proliferation of human erythroid progenitors and a multilineage interleukin-3-dependent hematopoietic cell line. In this report, gamma-globin inducibility by these SCFADs was further demonstrated in mice transgenic for the locus control region and the entire beta-globin gene locus in a yeast artificial chromosome and in 2 globin promoter-reporter assays. Conditioned media experiments strongly suggest that their proliferative activity is a direct effect of the test compounds. Investigation of potential mechanisms of action of these SCFADs demonstrates that these compounds induce prolonged expression of the growth-promoting genes c-myb and c-myc. Both butyrate and specific growth-stimulatory SCFADs induced prolonged signal transducer and activator of transcription (STAT)-5 phosphorylation and activation, and c-cis expression, persisting for more than 120 minutes, whereas with IL-3 alone phosphorylation disappeared within minutes. In contrast to butyrate treatment, the growth-stimulating SCFADs did not result in bulk histone H4 hyperacetylation or induction of p21(Waf/Cip), which mediates the suppression of cellular growth by butyrate. These findings suggest that the absence of bulk histone hyperacetylation and p21 induction, but prolonged induction of cis, myb, myc, and STAT-5 activation, contribute to the cellular proliferation induced by selected SCFADs.

Figure 1. Induction of γ-globin gene transcription by the SCFADs

K562 cells stably transtected with a construct containing HS2 linked to the γ-globin gene promoter and the reporter gene EGFP were cultured with certain SCFADs for 24 hours. The total fluorescence of 2.5 × 10⁵ cells per well was measured in a cytofluorometric plate reader and is expressed in arbitrary units including the standard deviation of each condition performed in quadruplicates. Test compounds were added at 1 mM. C, untreated control cells; AB, arginine butyrate; 1, α-methylhy-drocinnamic acid; 2, 2,2 dimethyl butyric acid; 3, 3-(3,4-dimethoxyphenyl) propionic acid; 4. DLα–amino-n-butyric acid.

Figure 2. Relative steady-state levels of c-myb, c-myc, and β-actin transcripts in 32D cells with hematopoietic growth factors or SCFADs

(A) Cells were cultured for 11 days in high IL-3 (25 U/mL, proliferating conditions), low IL-3 (0.5 U/mL, growth-arresting conditions) or no IL-3 (—), death-inducing conditions. SCFADs were tested at 1mM concentrations with 0.5 U/mL IL-3, growth-arresting conditions. Cells were harvested for RN A analysis at days 1 and 11. The first lane of each pair represents RNA from day 1 cultures, the second lane, RNA derived at day 11 (except with arginine butyrate and no IL-3, where RNA was available only at day 1, with no cells surviving to day 11). Total cellular RNA was separated by agarose gel electrophoresis, transferred to nitrocellulose and hybridized with [³²P]-labeled probes specific for c-myc, c-myb, or β-actin, as a control for loading. The autoradiograms are shown here. Experimental conditions: 25U IL-3, cells cultured in 25 U/mL IL-3; —, cultured without any IL-3; in all the other conditions, cells were cultured in 0.5 U/mL IL-3, which is sufficient for survival, but not proliferation. The control was 0.5 U IL-3, with 0.5 U/mL IL-3; B, 0.5 U/mL IL-3 plus 1mM arginine butyrate; G-CSF, 0.5 U/mL IL-3 plus 100 U/mL G-CSF; 1, 0.5 U/mL IL-3 plus 1 mM α melhyl hydrocinnamic acid; 2, 0.5 U/mL IL-3 plus 1mM 2,2 dimethyl butyric acid; 3, 0.5 U/mL IL-3 plus 1mM 2–2 dimethylmethoxyacelic acid; 4, 0.5 U/mL IL-3 plus 1 mM phenoxyacetic acid; 5, 0.5 U/mL IL-3 plus 1mM DL-α-amino-n-butyric acid; EPO, 0.5 U/mL IL-3 plus 3 U/mL erythropoietin. (B) Quantitation of c-myc, c-myb, and β-actin transcript expression levels by densitometric analysis. The treatment conditions identified by number correspond to those described in panel A, and a represents mRNA levels from day 1 cultures; b, day 11 cultures; *, no sample available due lo death of the cells by day 11. The dala for each transcript are expressed as relative to the levels of that specific transcript found in RNA from day 11 cultures under control conditions (low IL-3, 0.5 U/mL), which were arbitrarily given a value of one. The dashed line in each graph represents the levels of c-myc, c-myb, or β-actin transcripts in the cells at day 11 under control conditions. The conditions were the same as in part A, and conditions 3–10 had 0.5U/mL IL-3 in addition lo 1mM of the SCFA derivatives or cytokines added. 1, 25 U/mL IL-3; 2, no IL-3; 3, arginine butyrate; C, control had 0.5 U/mL IL-3 alone; 4, 100U/mL G-CSF; 5, α methylhydrocinnamic acid; 6, 2,2 dimethylbutyric acid; 7, 2,2 dimethylmethoxyacelic acid; 8, phenoxyacetic acid; 9, α-amino-n-butyric acid; 10, 3U/mL erythropoietin.

Figure 3. Activation of STAT-5 by treatment with SCFADs

(A) Immunoblot analysis of total STAT-5 protein and phosphorylated STAT-5 protein in 32D cells. 32D cells were deprived of IL-3 for 18 hours, in the presence of various SCFADs, and then stimulated with 25 U/mL IL-3. Total cellular protein extracts harvested at 0, 5, 30, 60, and 120 minutes after IL-3 stimulation were treated with anti-Stat5 antibody, and the immunoprecipitated complexes were separated by SDS-PAGE and transferred to nitrocellulose. Filters were probed first with an antiphospholyrosine, developed, stripped, and subsequently probed with an anti–STAT-5 antibody and developed. The figure is an autoradiogram of the ECL exposures. Treatments included Control (C); arginine butyrale at 1mM (AB); phenoxyacetic acid at 1mM (1); α methylhydrocinnamic acid at 1mM (2); 2,2 dimethyl butyric acid at 1mM (3); and 3-(3,4-dimethoxyphenyl) propionic acid at 1mM (4). (B) Densitometric quantitation of the films of the ECL immunoblots, expressed as arbitrary units, after subtraction of background and normalization for total Slat5 protein present in the sample. The dashed lines in all bar graphs indicate the level of phosphorylated STAT-5 protein remaining in the IL-3–only control at each specific time point. C, control; AB, arginine butyrale at 1mM; 1, phenoxyacetic acid at 1mM; 2, α-methyl hydrocinnamic acid at 1 mM; 3, 2,2 dimethyl butyric acid at 1 mM; and 4, 3-(3,4-dimethoxyphenyl) propionic acid at 1mM.

Figure 4. Northern blot analysis of c-cis expression in 32D cells with and without treatment with the SCFA derivatives

After the addition of 0.5 U/mL IL-3 at time zero, mRNA was prepared from the 32D cells at 0, 1, and 3 hours, as labeled above each lane. In the control lane (IL-3) 0.5 U/mL IL-3 was added at time 0, and in the EPO lane 3 U/mL was added at time 0 after starvation overnight. AB lanes had a concentration of 1 mM arginine butyrate; 1, 1 mM α methylhydrocinnamic acid; 2, 1 mM 2,2 dimethylbutyric acid; the cells were starved overnight in these concentrations and at time zero 0.5 U/mL IL-3 was added.

Figure 5. Histone deacetylase inhibitory activity of butyrate compared to SCFA derivatives

Immunoblot analysis of total nuclear proteins from K562 cells separated on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antibody to acetylated histone H4. Cells were left untreated (C) or treated for 18 hours with the following test compounds at 1 mM: arginine butyrate (B); α methyl hydrocinnamic acid (1); 2,2 dimethylbutyric acid (2); phenoxyacetic acid (3); or 3-(3,4-dimethoxyphe-nyl) propionic acid (4).

Figure 6. Northern blot analysis of p21 expression in 32D cells with and without treatment with SCFADs

The mRNA was prepared from cells cultured for 1 day. p21 expression was induced 3-fold with arginine butyrate (AB) compared to control (C) 0.5 U/mL IL-3 alone. In contrast, the short-chain fatty acid derivatives, 2,2 dimethylbutyric acid (1); α-methylhydrocinnamic acid (2); DL-α amino-n-butyric acid (3); phenoxyacetic acid (4); and 2,2 dimethylmethoxyacetic acid (5) were not significantly different from the control cells treated with 0.5 U/mL IL-3 alone.

Figure 7. Schema of signaling pathways

Butyrate induces components of both signaling pathways, growth inhibitory and growth stimulatory. Treatment of 32D cells with the selected short-chain fatty acid derivatives results in activation of the growth-stimulatory signaling events shown.