Different signal-regulated serine/threonine kinases phosphorylate class II histone deacetylases (HDACs) to promote nuclear export, cytosolic accumulation, and activation of gene transcription. However, little is known about mechanisms operating in the opposite direction, which, possibly through phosphatases, should promote class II HDACs nuclear entry and subsequent gene repression. Here we show that HDAC4 forms a complex with the PP2A holoenzyme C alpha, A alpha, B/PR55 alpha. In vitro and in vivo binding studies demonstrate that the N-terminus of HDAC4 interacts with the catalytic subunit of PP2A. HDAC4 is dephosphorylated by PP2A and experiments using okadaic acid or RNA interference have revealed that PP2A controls HDAC4 nuclear import. Moreover, we identified serine 298 as a putative phosphorylation site important for HDAC4 nuclear import. The HDAC4 mutant mimicking phosphorylation of serine 298 is defective in nuclear import. Mutation of serine 298 to alanine partially rescues the defect in HDAC4 nuclear import observed in cells with down-regulated PP2A. These observations suggest that PP2A, via the dephosphorylation of multiple serines including the 14-3-3 binding sites and serine 298, controls HDAC4 nuclear import.

Inhibitors of the ubiquitin-proteasome system (UPSIs) promote apoptosis of cancer cells and show encouraging anti-tumor activities in vivo. In this study, we evaluated the death activities of two different UPSIs: bortezomib and the isopeptidase inhibitor G5. To unveil whether these compounds elicit different types of death, we compared their effect both on apoptosis-proficient wild type mouse embryo fibroblasts and on cells defective for apoptosis (double-deficient Bax/Bak mouse embryo fibroblasts) (double knockout; DKO). We have discovered that (i) both inhibitors induce apoptosis in a Bax and Bak-dependent manner, (ii) both inhibitors elicit autophagy in WT and DKO cells, and (iii) only G5 can kill apoptosis-resistant DKO cells by activating a necrotic response. The induction of necrosis was confirmed by different experimental approaches, including time lapse analysis, HMGB1 release, and electron microscopy studies. Neither treatment with antinecrotic agents, such as antioxidants, poly(ADP-ribose) polymerase and JNK inhibitors, necrostatin, and the intracellular Ca 2؉ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester, nor overexpression of Bcl-2 and Bcl-xL prevented necrosis induced by G5. This necrotic death is characterized by the absence of protein oxidation and by the rapid cyclosporin A-independent dissipation of the mitochondrial membrane potential. Notably, a peculiar feature of the G5-induced necrosis is an early and dramatic reorganization of the actin cytoskeleton, coupled to an alteration of cell adhesion. The importance of cell adhesion impairment in the G5-induced necrotic death of DKO cells was confirmed by the antagonist effect of the extracellular matrixadhesive components, collagen and fibronectin.

The regulation of the necrotic death and its relevance in anticancer therapy are largely unknown. Here, we have investigated the proapoptotic and pronecrotic activities of two ubiquitin-proteasome system inhibitors: bortezomib and G5. The present study points out that the glioblastoma cell lines U87MG and T98G are useful models to study the susceptibility to apoptosis and necrosis in response to ubiquitin-proteasome system inhibitors. U87MG cells show resistance to apoptosis induced by bortezomib and G5, but they are more susceptible to necrosis induced by G5. Conversely, T98G cells are more susceptible to apoptosis induced by both inhibitors but show some resistance to G5-induced necrosis. No overt differences in the induction of Noxa and Mcl-1 or in the expression levels of other components of the apoptotic machinery were observed between U87MG and T98G cells. Instead, by comparing the transcriptional profiles of the two cell lines, we have found that the resistance to G5-induced necrosis could arise from differences in glutathione synthesis/utilization and in the microenvironment. In particular, collagen IV, which is highly expressed in T98G cells, and fibronectin, whose adhesive function is counteracted by tenascin-C in U87MG cells, can restrain the necrotic response to G5. Collectively, our results provide an initial characterization of the molecular signals governing cell death by necrosis in glioblastoma cell lines.

From the nucleus, histone deacetylase 4 (HDAC4) regulates a variety of cellular processes, including growth, differentiation, and survival, by orchestrating transcriptional changes. Extracellular signals control its repressive influence mostly through regulating its nuclear-cytoplasmic shuttling. In particular, specific posttranslational modifications such as phosphorylation and caspase-mediated proteolytic processing operate on HDAC4 to promote its nuclear accumulation or export. To understand the signaling properties of this deacetylase, we investigated its cell death-promoting activity and the transcriptional repression potential of different mutants that accumulate in the nucleus. Here we show that, compared to that of other nuclear forms of HDAC4, a caspase-generated nuclear fragment exhibits a stronger cell death-promoting activity coupled with increased repressive effect on Runx2-or SRF-dependent transcription. However, this mutant displays reduced repressive action on MEF2C-driven transcription. Photobleaching experiments and quantitative analysis of the raw data, based on a two-binding-state compartmental model, demonstrate the existence of two nuclear pools of HDAC4 with different chromatin-binding properties. The caspase-generated fragment is weakly bound to chromatin, whereas an HDAC4 mutant defective in 14-3-3 binding or the wild-type HDAC5 protein forms a more stable complex. The tightly bound species show an impaired ability to induce cell death and repress Runx2-or SRF-dependent transcription less efficiently. We propose that, through specific posttranslation modifications, extracellular signals control two distinct nuclear pools of HDAC4 to differentially dictate cell death and differentiation. These two nuclear pools of HDAC4 are characterized by different repression potentials and divergent dynamics of chromatin interaction.