HDAC4… not only a histone deacetylase

HIF1α is a transcriptional regulator which plays an essential role in the cellular response to hypoxia. As discussed in last week’s blog, prolyl hydroxylases (PHDs) mark HIFα subunits for degradation, but HIF1α can also be regulated by reversible acetylation. Earlier work in a VHL-null human RCC cell line noted that histone deacetylases (HDACs), such as HDAC4 and HDAC6, are associated with this process (Qian et al., 2006). However, the mechanism by which HDAC4 regulates HIF1α was not completely clear. Now, a related study by Geng et al. (2011) suggests that HDAC4 inhibition increases HIF1α acetylation, which decreases HIF1α stability, and reduces HIF1-mediated gene expression in a prostate (C42B) and liver (Hep3Bc1) cancer cell line.

In this study, hypoxic Hep3Bc1 cells had significantly less HIF1α protein present after shRNA-mediated knockdown of HDAC4, with a similar result being obtained using CoCl2 as a hypoxic mimic in C42B cells. The proteasome inhibitor MG132 was able to rescue this effect and maintain levels of HIF1α in CoCl2-treated Hep3Bc1 cells after HDAC4 shRNA knockdown, and this HIF1α was also more acetylated. Using the protein synthesis inhibitor cyclohexamide, HIF1α also degraded faster in CoCl2-treated Hep3Bc1 cells after HDAC4 shRNA knockdown, when compared to shRNA controls. In contrast, shRNA knockdown of HDAC1 and HDAC3 did not decrease HIF1α protein levels in hypoxic Hek293 cells. Co-immunoprecipitation experiments in Hek293T cells also showed that HIF1α interacts with HDAC4, and that HDAC4 (but not HDAC1) overexpression reduced the levels of acetylated HIF1α observed by western blot. Together, these experiments suggest that HDAC4 specifically influences both HIF1α acetylation and stability.

Subsequent mass spectrometric analysis indicated that the first 30 amino acids of HIF1α may be acetylated, and site-directed mutagenesis of all 5 lysines in this region produced a HIF1α mutant that had significantly lower levels of acetylation than wild-type HIF1α. Overexpression of HDAC4 in Hek293T cells had no effect on the acetylation and protein levels of the HIF1α mutant, and HDAC4 shRNA knockdown in CoCl2-treated Hek293 cells also had no effect on this mutant’s protein levels. The HIF1α mutant was also resistant to HDAC inhibitor (SAHA)-mediated degradation in Hek293T cells, further emphasising the importance of these residues in the regulation of HIF1α.

Using qRT-PCR, it could be seen that the hypoxic upregulation of the HIF1α-target genes VEGFA, LDHA and GLUT1 was inhibited by shRNA knockdown of HDAC4 when compared to shRNA controls in both cancer cell lines. Genes such as LDHA and GLUT1 are involved in glycolysis, and accordingly, shRNA knockdown of HDAC4 in Hep3Bc1 and C42B cells significantly reduced hypoxia-induced lactate production when compared to controls. Additionally, after HDAC4 shRNA knockdown, Hep3Bc1 cells grew slower under long-term hypoxia, and hypoxic C42B cells were also more sensitive to the cytotoxic agent docetaxel. This latter result could have therapeutic implications, as HIF signalling has been shown to play a role in tumourigenesis in a number of disorders, including BHD syndrome (Preston et al., 2010).

Additionally, although the functional implications of HIF1α lysine acetylation are yet to be fully elucidated, it is useful to remember that HDACs and their inhibitors do not only augment transcription through chromatin regulation (as observed by Cash et al., 2011), but also through the direct modulation of transcription factors themselves.

 

  • Cash TP, Gruber JJ, Hartman TR, Henske EP, & Simon MC (2011). Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene, 30 (22), 2534-46. PMID: 21258407
  • Geng H, Harvey CT, Pittsenbarger J, Liu Q, Beer TM, Xue C, Qian DZ (2011). HDAC4 regulates HIF1{alpha} lysine acetylation and cancer cell response to hypoxia. J Biol Chem. Sep 14. [Epub ahead of print]. PMID: 21917920
  • Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, Harper KT, Brinkhuizen T, Menko FH, Davies DM, Land SC, Pause A, Baar K, van Steensel MA, & Tee AR (2011). Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene, 30 (10), 1159-73. PMID: 21057536
  • Qian DZ, Kachhap SK, Collis SJ, Verheul HM, Carducci MA, Atadja P, Pili R (2006). Class II histone deacetylases are associated with VHL-independent regulation of hypoxia-inducible factor 1 alpha. Cancer Res. Sep 1; 66(17): 8814-21. PMID: 16951198
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.
 
 

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