Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-beta1-mediated gene activation - PubMed (original) (raw)

Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-beta1-mediated gene activation

Yan Zhuang et al. Biochem Biophys Res Commun. 2010.

Abstract

Histone deacetylase 6 (HDAC6) belongs to the family of class IIb HDACs and predominantly deacetylates non-histone proteins in the cytoplasm via the C-terminal deacetylase domain of its two tandem deacetylase domains. HDAC6 modulates fundamental cellular processes via deacetylation of alpha-tubulin, cortactin, molecular chaperones, and other peptides. Our previous study indicates that HDAC6 mediates TGF-beta1-induced epithelial-mesenchymal transition (EMT) in A549 cells. In the current study, we identify a novel splicing variant of human HDAC6, hHDAC6p114. The hHDAC6p114 mRNA arises from incomplete splicing and encodes a truncated isoform of the hHDAC6p114 protein of 114kDa when compared to the major isoform hHDAC6p131. The hHDAC6p114 protein lacks the first 152 amino acids from N-terminus in the hHDAC6p131 protein, which harbors a nuclear export signal peptide and 76 amino acids of the N-terminal deacetylase domain. hHDAC6p114 is intact in its deacetylase activity against alpha-tubulin. The expression hHDAC6p114 is elevated in a MCF-7 derivative that exhibits an EMT-like phenotype. Moreover, hHDAC6p114 is required for TGF-beta1-activated gene expression associated with EMT in A549 cells. Taken together, our results implicate that expression and function of hHDAC6p114 is differentially regulated when compared to hHDAC6p131.

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Figures

Figure 1

Manifestation of the p114 mRNA. A) The schemes to amplify p131 and p114. The shaded boxes indicate exons and the connecting lines indicate introns. B) Total cell RNA was extracted from A549 (A) and MCF-7 (M) cells. The p131 and p114 mRNAs were detected by RT-PCR using a pair of non-selective primers. C) Similar to B except that cDNA was amplified using a pair of the p114 mRNA-specific primers. D) The mRNA levels of p114 (p114), and p114 and p131 combined (p114+p131) were determined using quantitative RT-PCR. Ratios of p114/(p114+p131) were obtained after normalization to the Ct values of the housekeeping gene 36B4. Mean and standard deviations were obtained from 3 independent experiments. *, P value < 0.05.

Figure 2

Manifestation of the p114 protein. A) Comparison of the structure of the conceptual proteins of p131 and p114. B) Expression of potential isoforms of the hHDAC6 proteins and acetylated α-tubulin was examined in A549 cells stably expressing either scrambled (sc) or hHDAC6-targeting siRNA (HD6) using immunoblots. C) A549 and MCF-7 cells were transfected with either scrambled (−) or p114-targeting siRNA (+). The protein levels of p131, p114, and acetylated α-tubulin were examined using immunoblots. D) A549 cells were transfected with either the backbone or p114/p131 expression vectors. The protein levels of p131, p114, and acetylated α-tubulin were evaluated using immunoblots. The results are representative of at least 2 independent experiments.

Figure 3

Impact of p114-targeting RNAi on TGF-β1-induced deacetylation of α-tubulin. A) Total cell RNA was isolated from A549 cells transfected with either the scrambled (scramble) or p114-targeting (p114si) siRNA followed by exposure to TGF-β1 (5 ng/ml) for 48 hrs. The mRNA levels of p114 (p114) and the combined p114 and p131 were determined using quantitative RT-PCR. Ratios of the genes of interest over the housekeeping gene 36B4 were compared across the groups. B) The culture conditions were similar to part A except that the protein levels of p131, p114, and acetylated α-tubulin were determined using immunoblots. Mean and standard deviations were obtained from 4 independent transfections.

Figure 4

Requirement of p114 for TGF-β1-activated gene expression. A-D) Total cell RNA was isolated from A549 cells transfected with either the scrambled (scramble) or hHDAC6p114-targeting (p114si) siRNA followed by exposure to TGF-β1 (5 ng/ml) for 48 hrs. The mRNA levels of PAI-1 (A), PDGF-B (B), Collagen I (C), and E-cadherin (D) were determined using quantitative RT-PCR. Ratios of the genes of interest over the housekeeping gene 36B4 were compared across the groups. E) A549 cells were co-transfected with luciferase reporter constructs and siRNAs followed by exposure to TGF-β1 for 24 hrs. The ratios of TGF-β1 responsive 3TP-LUX activity over the co-transfected RL-TK activity were compared across the groups. Mean and standard deviations were obtained from 4 independent transfections. *, P value < 0.05.

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Requirement of a novel splicing variant of human histone deacetylase 6 for TGF-beta1-mediated gene activation - PubMed (original) (raw)