Class I histone deacetylases localize to the endoplasmic reticulum and modulate the unfolded protein response - PubMed (original) (raw)

Class I histone deacetylases localize to the endoplasmic reticulum and modulate the unfolded protein response

Soumen Kahali et al. FASEB J. 2012 Jun.

Abstract

Post-translational modification through protein acetylation is emerging as an important mode of cellular regulation. We have previously demonstrated the role that glucose-regulated protein 78 kDa (GRP78) acetylation and subsequent activation of the unfolded protein response (UPR) play in the antitumor activity of class I histone deacetylase (HDAC) inhibitors, which primarily target class I HDACs. In this study, we explored the contributory role these class I HDACs may play in UPR regulation. Binding studies were performed using immunoprecipitation/immunoblotting following dual-transfection with HA-tagged GRP78 and FLAG-tagged HDACs. Subcellular localization was performed using Western blot of fractionated cell lysates and confocal microscopy. Individual HDACs were inhibited using RNA interference. We identified the potential of HDACs 1, 2, and 3 to bind to GRP78. These HDACs colocalized with GRP78 in the endoplasmic reticulum (ER). Inhibition of individual HDACs resulted in GRP78 acetylation and selective activation of the UPR. Although traditionally viewed as nuclear enzymes, we demonstrate that Class I HDACs localize to the ER, bind to GRP78, and selectively activate the UPR, representing a novel mode of UPR regulation and mechanism of action of HDAC inhibitors.

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Figures

Figure 1.

Class 1 HDACs interact with GRP78. A) U251 cells were transiently transfected with an expression plasmid of HA-tagged GRP78 alone (HA-GRP78) or in combination with FLAG-tagged HDAC1, HDAC2, HDAC3, or HDAC8 (F-H1, H2, H3, H8) expression plasmids. Untransfected cells served as controls (B). Cells were lysed 48 h post-transfection in CelLytic M cell lysis reagent for immunoprecipitation with HA-tagged agarose beads followed by immunoblotting with anti-FLAG or with anti-HA antibody. The precision plus dual-color protein standard was used as protein molecular size marker (in kilodaltons). B) GRP78 binding study using HA-GRP78 stable cell lines for class 1 HDACs. U251 (B) and HA-GRP78 stable U251 cell extracts were prepared in CelLytic M cell lysis reagent. The lysates were immunoprecipitated with HA-tagged agarose beads and then immunoblotted and probed for anti-HDAC1, anti-HDAC2, or anti-HDAC3 and anti-HA.

Figure 2.

Subcellular fractionation of class I HDACs. Cell pellets were fractionated using sequential subcellular extraction steps, yielding protein fractions from the nucleus and cytoskeleton (fraction A) and cytosol and organelles (fraction B). Western blot analysis was performed using indicated antibodies. The typical nuclear marker lamin A/C, cytoskeletal marker vimentin, ER marker calreticulin, and mitochondrial marker Cox IV were used to verify the purity of each fraction.

Figure 3.

Confocal microscopy demonstrating colocalization of HDAC1, HDAC2, and HDAC3 with GRP78. U251 cells were fixed in paraformaldehyde and immunostained for GRP78 (green) and HDAC1, HDAC2, and HDAC3 (red). Cell nuclei were counterstained and mounted with antifade containing DAPI (blue). Colocalization of HDAC1, HDAC2, HDAC3, and GRP78 were visualized by confocal microscopy (×63 view). Merged images show the colocalization of HDAC1, HDAC2, or HDAC3 with GRP78 (yellow).

Figure 4.

Knockdown of HDACs using RNA interference leads to GRP78 acetylation. A) U251 cells were knocked down for HDAC1 (siH1), HDAC2 (siH2), HDAC3 (siH3), HDAC6 (siH6), or scrambled nontargeting siRNA (sc) for 48 h. Immunoblotting against HDAC1 (H1), HDAC2 (H2), HDAC3 (H3), and HDAC6 (H6) antibodies validated significant knockdown of the respective HDACs. Actin served as loading control. B) These lysates were then immunoprecipitated with anti-GRP78 and immunoblotted with either anti-acetyl lysine or anti-GRP78. Images were quantified using densitometric scanning, normalizing samples to GRP78 expression. C) Similar studies were performed on cells exposed to the HDAC inhibitor vorinostat at described concentrations for 24 h.

Figure 5.

Knockdown of HDACs using RNA interference activates the UPR. U251 cells were knocked down for HDAC1 (siH1), HDAC2 (siH2), HDAC3 (siH3), HDAC6 (siH6) or scrambled nontargeting siRNA (sc) for 48 h. Immunoblotting was then performed on cell lysates using antibodies specific for phospho-PERK (pPERK) and PERK (A), cleaved ATF6 and actin (B), and XBP-1 mRNA splicing and GAPDH was determined by RT-PCR (C). Cells treated with thapsigargin (1 μM) served as a positive control (+).

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Class I histone deacetylases localize to the endoplasmic reticulum and modulate the unfolded protein response - PubMed (original) (raw)