Proliferative status regulates HDAC11 mRNA abundance in nontransformed fibroblasts

Abstract

Histone deacetylases (HDACs) are important determinants of gene transcription and other biological processes. HDAC11 is one of the least characterized HDACs and is the only member of the class IV HDAC family. Our studies examined the events that control the expression of the HDAC11 transcript. We show that platelet-derived growth factor (PDGF) rapidly reduces the abundance of HDAC11 mRNA when added to density-arrested Balb/c-3T3 cells, which are nontransformed fibroblasts. Reduction required mRNA and protein synthesis, but not AKT or ERK activity, and resulted from accelerated turnover of the HDAC11 transcript. Reduction was transient in cells receiving PDGF alone but sustained in cells receiving both PDGF and platelet-poor plasma, which together promote G₀/G₁ traverse and S phase entry. Plasma alone did not appreciably reduce HDAC11 mRNA abundance, nor did epidermal growth factor, insulin-like growth factor, or insulin. HDAC11 mRNA accumulated in Balb/c-3T3 cells exiting the cell cycle due to density-dependent growth inhibition or serum deprivation. Of note, HDAC11 mRNA did not accumulate in a spontaneously transformed Balb/c-3T3 clonal variant (clone 2) that does not density arrest. The HDAC11 promoter was active in Balb/c-3T3 but not clone 2 cells; inactivity in clone 2 cells did not result from methylation of CpG islands. Overexpression of HDAC11 inhibited the cell cycle progression of both transformed and nontransformed fibroblasts. Our studies identify the HDAC11 transcript as a PDGF target and show that HDAC11 mRNA abundance correlates inversely with proliferative status.

Keywords: cell cycle; clone 2; fibroblasts; histone deacetylase 11; platelet-derived growth factor.

Figure 1. PDGF reduces HDAC11 mRNA abundance in Balb/c-3T3 cells. (A) Density-arrested Balb/c-3T3 cells received 10 ng/ml PDGF and 10% PPP for 1, 3, or 7 h. A microarray analysis was performed as described in “Materials and Methods”. (B) Density-arrested Balb/c-3T3 cells were co-treated with 10 ng/ml PDGF and 10% PPP for the indicated times. Amounts of HDAC11 mRNA were determined by RT-qPCR. Data are expressed as percentage of control (no treatment, diagonal bar). Error bars show standard deviation. (C) Density-arrested Balb/c-3T3 cells received 10 ng/ml PDGF (left panel) or 10% PPP (right panel) for the indicated times. Amounts of HDAC11 mRNA were determined by RT-qPCR. Data are expressed as percentage of control (no treatment, diagonal bar). Error bars show standard deviation.

Figure 2. PDGF-mediated downregulation of HDAC11 mRNA requires protein synthesis. (A) Density-arrested Balb/c-3T3 cells received 10 ng/ml PDGF, 50 ng/ml EGF, 40 ng/ml IGF-1, or 5 μg/ml insulin for 1, 2, or 3 h. Amounts of HDAC11 mRNA were determined by RT-qPCR. Data are expressed as percentage of control (no treatment, diagonal bar). Error bars show standard deviation. (B) Density-arrested Balb/c-3T3 cells received 1 μM LY294002 or 10 μM U0126 for 25 min followed by 10 ng/ml PDGF for 2.5 h. Left panel: Amounts of HDAC11 mRNA were determined by RT-qPCR. Data are expressed as percentage of control (no treatment, diagonal bar). Error bars show standard deviation. Right panel: Amounts of phosphophorylated AKT (pAKT) and phosphorylated ERK (pERK) were determined by western blotting of cell extracts with phospho-specific antibodies. (C) Density-arrested Balb/c-3T3 cells received 10 ng/ml PDGF, 20 µg/ml cycloheximide (CHX), or both for 0.5, 1 or, 2 h. Amounts of HDAC11 mRNA were determined by RT-qPCR. Data are expressed as percentage of control (no treatment, diagonal bar).

Figure 3. PDGF destabilizes the HDAC11 transcript in a transcription-dependent manner. (A) Density-arrested Balb/c-3T3 cells received 10 ng/ml PDGF, 50 μM DRB or both for the indicated times. Amounts of HDAC11 mRNA were determined by RT-qPCR. Decay curves were generated by linear regression. (B) Growing Balb/c-3T3 cells (approximately 80% confluent) were co-transfected with pRL-CMV and either pGL3-Basic or pGL3-HDAC11. Cells received 10 ng/ml PDGF 38 h after transfection (0 h) and were harvested at 3, 6, or 9 h thereafter for determination of luciferase activity. Cells were quiescent and confluent at the time of PDGF addition. Firefly luciferase activity (pGL3) is normalized to Renilla luciferase activity (pRL-CMV) (RLU1/RLU2). Error bars show standard deviation.

Figure 4. Accumulation of HDAC11 mRNA in confluent Balb/c-3T3 but not clone 2 cells (A) Amounts of HDAC11 mRNA were determined by RT-qPCR on the days indicated (top panel). Percentages of cells in G₀/G₁, S, and G₂/M were determined by FACS analysis of propidium iodide-stained cells (bottom panel). Error bars show standard deviation. (B) Cultures were photographed on the days indicated. Magnification is 10×. (C) Sparse (day 2) and confluent (day 7) Balb/c-3T3 cells received 10 ng/ml PDGF for 3 h. Amounts of HDAC11 mRNA were determined. Error bars show standard deviation.

Figure 5. The HDAC11 promoter is active in Balb/c-3T3 but not clone 2 cells. (A) Proliferating Balb/c-3T3 and clone 2 cells (approximately 80% confluent) were transfected with pRL-CMV (for normalization) and either pGL3-Basic (–) or pGL3-HDAC11 (+). Cells were harvested 38 h later for determination of luciferase activity and were confluent at the time. Error bars show standard deviation. Data are expressed as fold increase in HDAC11 promoter activity. RLU1/RLU2 values for pGL3-Basic in Balb/c-3T3 and clone 2 cells were 0.011 and 0.035, respectively. (B) Genomic DNA was isolated from confluent Balb/c-3T3 and clone 2 cells, and CpG island methylation was determined as described in “Materials and Methods”.

Figure 6. Serum deprivation increases amounts of HDAC11 mRNA in Balb/c-3T3 cells. (A–C) Sparse, cycling cells in 10% serum were refed with medium containing 10% or 0.5% serum (day 0). (A) Cultures were photographed on the days indicated. Magnification is 10×. (B) Percentages of cells in G₀/G₁, S, and G₂/M were determined by FACS analysis of propidium iodide-stained cells. (C) Amounts of HDAC11 mRNA were determined by RT-qPCR on the days indicated. Error bars show standard deviation.

Figure 7. Overexpression of HDAC11 inhibits the cell cycle progression of nontransformed and transformed fibroblasts. (A) Sparse proliferating Balb/c-3T3, MEFs, and Clone-2 cells were transfected with plasmids encoding Flag-tagged HDAC11 and GFP. Thirty hours after transfection, cells were pulsed with 30 μM BrdU for 1 h and sorted for GFP expression. The percentage of BrdU-labeled cells was determined for cells expressing (+) or not expressing (–) ectopic proteins. (B) Balb/c-3T3 cells were mock transfected or transfected with a plasmid encoding HDAC11. Cell extracts were western blotted with a monoclonal antibody to HDAC11 prepared for us by Abpro Labs.