Evaluación de las asignaturas virtuales para la construcción de indicadores de

Histone deacetylase 4 (HDAC4) is a class IIa HDACs encoded by a gene located on 2q37.2. The amplification of this segment has been associated with oral squamous carcinomas (Wolff et al.1998). Abnormalities in the HDAC4 genomic region have been implicated in congenital malformation syndrome in a 12-year- old girl born with a de novo unbalanced t (2; 22) (q37; q11.2) and later displayed signs of neurologic impairment. HDAC4 is expressed in a tissue-specific manner mainly in skeletal muscle, brain, leukocyte, colon, small intestine, and ovary but not in liver, lung, and placenta. The predicated molecular weight for HDAC4 is 119-kD protein although it has been shown that the molecular weight of nuclear HDAC4 was higher slightly than cytosolic HDAC4 ( Wilson et al. 2008). HDAC4 contains 1084 amino acid residues, with a highly homologous conserved catalytic domain (HDAC domain) at amino acid 802 in the carboxy terminal region (C- terminal) which is believed to be responsible for the deacetylate activity and similar to the deacetylase domain of the yeast Hda1 (Wang et al.1999). The functional analysis of HDAC4 by Grozinger et al.1999 confirmed that HDAC4 has deacetylation activity against all 4 core histones in vitro. Bottomley MJ et al. 2008 demonstrated that the deacetylase domain of HDAC4 possesses intrinsically enzymatic activity toward acetylated lysines in vitro but activity lower than class I HDACs. The studies used trifluoroacetamide substrate for HDAC activity assay since no biological substrates yet have been identified for class IIa HDACs (Bertrand, 2010). In the end of the C-terminal there is nuclear export signal (NES) and nuclear localization signal sequences (NLS) is located in the N- terminal (Figure 13). These signal responsive elements in HDAC4 enable its shuttling between the nucleus and cytoplasm. The amino acid terminal (N- terminal) in HDAC4 has been reported to be involved in transcription repression, phosphorylation and nucleocytoplasmic shuttling (Bottomley et al. 2008; Lahm et

al. 2007).HDAC4 repression activity is regulated by its sub-cellular localization.

Phosphorylated HDAC4 binds to 14-3-3 proteins prevent HDAC4 nuclear localization. Thus, 14-3-3 proteins negatively regulate HDAC4 repression

function. 14-3-3 proteins are conserved with seven isoforms that present ubiquitously in approximately all the tissues in mammals. These proteins are known as cytoplasmic anchors for their binding partners (Sun et al. 2009). 14-3-3 proteins have the ability to recognise phosphorylated proteins and retain their partner protein in the cytoplasm (Muslin and Xing, 2000). It has been reported that 14-3-3 proteins have more than 200 binding partners and are involved in variety of cellular activity such as protein synthesis, transcription repression, cell cycle and metabolic pathway (Nishino et al. 2008). HDAC4 function as a repressor when inside the nucleus as it binds and represses the transcriptional activity of myocyte enhancer factor 2 (MEF2). Furthermore, it has been reported that HDAC4 is enzymatically inactive and the deacetylase activity arises from the presence of HDAC3 in the transcriptional corepressor SMRT (silencing mediator for retinoid and thyroid receptor) and NCoR (nuclear receptor corepressor) complex. The interaction between HDAC4 and the complex HDAC3 and SMRT/N-CoR happens inside the nucleus (Emiliani et al.1998). HDAC4 has been shown to exhibit functional activity on the NCoR/SMRT complex that did not contain HDAC3 using recombinant proteins (Huang et al. 2000). Although, the enzymatic activity of class II HDACs is associated with multi-proteins complex contains HDAC3 and SMRT/N-CoR. Wang et al. 1999 has reported that both the deacetylase domain and the amino acid-terminal domain (208 residues) have the potential to repress transcription. HDAC4 has a role in muscle differentiation. It interacts specifically with the myogenic MEF2 transcription factor and represses its activity. In myoblasts cells, phosphorylated HDAC4 binds to 14-3-3 proteins and held in the cytoplasm during myoblast differentiation, however it translocates to the nucleus once fusion has occurred. Nuclear HDAC4 suppresses the myogenic programme and MEF2-dependent transcription. Activation of the Ca2+/calmodulin signalling pathway by active CaMKIV prevents nuclear entry of HDAC4 and HDAC4-mediated inhibition of differentiation (Miska et al. 2001). HDAC4 also regulates chondrocyte hypertrophy and skeletogenesis through interaction with the chondrocyte hypertrophy transcription factor runt-related

osteoblast-specific genes. HDAC4-null mice display premature ossification of developing bones due to ectopic and early onset chondrocyte hypertrophy and over-expressed HDAC4 resulted in inhibition of chondrocyte hypertrophy with delayed ossification (Vega et al. 2004). Moreover, HDAC4 has been revealed to have a role in the DNA damage response. Kao GD et al. 2003 showed that endogenous HDAC4 is recruited to nuclear foci with kinetics similar to 53BP1, a p53 binding protein, following DNA damage meditated by -irradiation in human HeLa cells. 53BP1 is phosphorylated in response to DNA damage and then translocates to nucleus to form nuclear foci that co-localise with those formed by phosphorylated histone H2AX, a double strand break marker (Kao et al. 2003). 53BP1 also participates in the phosphorylation of p53 in the maintenance of S and G2 cell cycle checkpoints after DNA damage (Motoyama N, Naka K 2004). HDAC4 has been demonstrated to translate to the nucleus upon DNA damage caused by adriamycin treatment in NIH3T3 mouse fibroblasts. HDAC4 nuclear recruitment after DNA damage has been reported but was not associated with apoptosis (Basile et al. 2006). This could indicate a role of HDAC4 in DNA damage response. The role of HDAC4 in the development of cancer also has been reported. Immunohistochemical staining of prostate sections of benign tissue and primary and hormone refractory (HR) prostate cancer, as well as of the CWR22 mouse xenograft model showed that HDAC4 is recruited to the nuclei of HR cancer cells, where it might exhibit inhibitory effect on differentiation and contribute to the development of the aggressive phenotype of late stage prostate cancer (Halkidou et al. 2004). Thus, HDAC4 may participate in the development of HR prostate cancer and can be a potential therapeutic target in this disease.

Figure 13: HDAC4 contains amino acid terminal where nuclear localization signal sequences (NLS). This amino terminal region interacts with specific transcription factors such as MEF2, RUNX1 and 14-3-3 and possesses transcriptional suppressive activity. Carboxy terminal deacetylase region of HDAC4 contains a highly homologous conserved catalytic domain (HDAC domain) and nuclear export signal (NES) at the end. The signal responsive elements in HDAC4 affect its nucleocytoplamic shuttling. It is believed that both the deacetylase domain and the amino acid-terminal domain have the potential to repress transcription. Adapted from (Chen et al. 2009)

Likewise, in HeLa cells, HDAC4 knockdown resulted in cell cycle arrest, chromatid segregation defects, and caspase-dependent apoptosis. However, HDAC4 knockdown seems to be well tolerated in normal human dermal fibroblasts and murine embryonic fibroblasts (MEF) cells (Cadot et al. 2009). This might imply that the biological function of HDAC4 is different between normal and cancer cells. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK) pathway is an important signalling system that mediates ligand-stimulated signals for the initiation of cell proliferation, differentiation, and cell survival. The ERK1/2 pathway has shown to be active in ovarian cancer and might be involved in the pathogenesis of these tumours (Steinmetz et al. 2004). It has been reported that ERK1/2 interacts with HDAC4 by immunoprecipitation analysis and the activation of ERK1/2 by Ras- MAPK pathway via expression of oncogenic Ras or active MAPK/ERK kinase 1 increase the percentage of cells expressing HDAC4 in the nucleus in C2C12 myoblast cells (Zhou et al. 2000). The role of the interaction between HDAC4 and ERK1/2 in the regulation of HDAC4 localization is unclear however; the activated Ras seems to act on HDAC4 indirectly by enhancing its nuclear localization. Moreover, HDAC4 knockdown increased p21 expression in human cancer cell lines; ovarian carcinoma (IGROV-1), glioblastoma (U87-MG), cervical cancer cell lines (HeLa), colon cancer (Wilson et al. 2008) and Osteosarcoma cells (Saos-2) (Mottet D, et al. 2009).

Thus, HDAC4 is a key transcriptional regulator of p21 expression and a potential valuable target for anticancer therapies. Together the data show that HDAC4 not only plays a role in transcription repression via interaction with MEF2 and RUNX1 but also plays role in DNA damage response, cell cycle arrest, chromatid segregation, and apoptosis in human cancer cell lines. However, the biological activity of HDAC4 seems different between human cancer cells and normal cells. These specify the important role of HDAC4 in cancer cells.