Sirtuins are homologues of the yeast transcriptional repressor Sir2p and are conserved from bacteria to humans. We report that human SIRT4 is localized to the mitochondria. SIRT4 is a matrix protein and becomes cleaved at amino acid 28 after import into mitochondria. Mass spectrometry analysis of proteins that coimmunoprecipitate with SIRT4 identified insulindegrading enzyme and the ADP/ATP carrier proteins, ANT2 and ANT3. SIRT4 exhibits no histone deacetylase activity but functions as an efficient ADP-ribosyltransferase on histones and bovine serum albumin. SIRT4 is expressed in islets of Langerhans and colocalizes with insulin-expressing ␤ cells. Depletion of SIRT4 from insulin-producing INS-1E cells results in increased insulin secretion in response to glucose. These observations define a new role for mitochondrial SIRT4 in the regulation of insulin secretion.Histone deacetylases are enzymes that catalyze the removal of acetyl groups from the ⑀-amino group of lysine residues and are separated into three classes. Sirtuins, the class III histone deacetylases, are homologous to the yeast transcriptional repressor, Sir2p, and are NAD ϩ -dependent enzymes (1-3). Seven sirtuins have been identified in the human genome (4, 5). They share a conserved Sir2 catalytic core domain and exhibit variable amino-and carboxyl-terminal extensions that contribute to their unique subcellular localization and may also regulate their catalytic activity.The subcellular distribution, substrate specificity, and cellular functions of sirtuins are quite diverse (reviewed in Refs. 1-3). SIRT1 is found in the nucleus, where it functions as a transcriptional repressor via histone deacetylation. SIRT1 can also regulate transcription by modifying the acetylation levels of transcription factors, such as MyoD, FOXO, p53, and NF-B (6 -12). The SIRT2 protein is found in the cytoplasm, where it associates with microtubules and deacetylates lysine 40 of ␣-tubulin (13). The SIRT3 protein is localized in the mitochondrial matrix (14, 15), where it is proteolytically processed at its NH 2 terminus, yielding a mature protein that has protein deacetylase activity (14). These observations indicate that the targets of sirtuins are not restricted to histone proteins but extend to acetylated proteins in other subcellular compartments.Sirtuins also differ in their substrate specificities. For instance, SIRT1, -2, and -3 have robust activity on chemically acetylated histone H4 peptides, whereas SIRT5 has weak but detectable activity, and SIRT4, -6, and -7 have no detectable activity on the same substrate (13). Interestingly, a sirtuin from Archaeoglobus fulgidus, Sir2-Af1, which has close homology with SIRT5, also has weak activity on a histone peptide but significantly stronger activity on an acetylated bovine serum albumin substrate (16,17). Similarly, both SIRT1 and SIRT2 can deacetylate p53; however, only SIRT2 deacetylates lysine 40 of ␣-tubulin (13, 17).Recently, SIRT6 was demonstrated to be a nuclear ADP-ribosyltransferase (18), whereas a T. brucei SI...

The high-affinity cellular receptor for the basement membrane component laminin is differentially expressed during tumor invasion and metastasis. A cDNA clone encoding the murine laminin receptor was isolated and identified on the basis of sequence homology to the human laminin receptor [Wewer et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7137-7141]. Primer extension experiments demonstrated that the clone contained the complete 5' sequence of the murine laminin receptor mRNA. RNA blot data demonstrated a single-sized laminin receptor mRNA, approximately 1400 bases long, in human, mouse, and rat. The nascent laminin receptor predicted from the cDNA sequence is 295 amino acids long, with a molecular weight of 33,000, and contains one intradisulfide bridge, a short putative transmembrane domain, and an extracellular carboxy-terminal region which has abundant glutamic acid residues and multiple repeat sequences. The precursor of the laminin receptor is apparently smaller than the 67-kilodalton protein isolated from tissue. The apparent molecular weight on SDS-polyacrylamide gels of the rabbit reticulocyte cell-free translation product of selectively hybridized laminin receptor mRNA is 37,000. Antisera to three different domains of the cDNA-predicted receptor were used to study the relationship between the 37- and 67-kilodalton polypeptides. Antisera to cDNA-deduced synthetic peptides of the receptor immunoprecipitated a 37-kilodalton band both from cell-free translation products and from pulse-labeled cell extracts. On immunoblots of cell extracts, one antisynthetic peptide antiserum recognized only the 67-kilodalton receptor, while another antiserum identified both 37- and 67-kilodalton polypeptides, suggesting a precursor-product relationship between the two polypeptides.

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Abstract-Global inhibition of class I and II histone deacetylases (HDACs) impairs angiogenesis. Herein, we have undertaken the identification of the specific HDAC(s) with activity that is necessary for the development of blood vessels. Using small interfering RNAs, we observed that HDAC7 silencing in endothelial cells altered their morphology, their migration, and their capacity to form capillary tube-like structures in vitro but did not affect cell adhesion, proliferation, or apoptosis. Among several factors known to be involved in angiogenesis, platelet-derived growth factor-B (PDGF-B) and its receptor (PDGFR-␤) were the most upregulated genes following HDAC7 silencing. We demonstrated that their increased expression induced by HDAC7 silencing was partially responsible for the inhibition of endothelial cell migration. In addition, we have also shown that treatment of endothelial cells with phorbol 12-myristate 13-acetate resulted in the exportation of HDAC7 out of the nucleus through a protein kinase C/protein kinase D activation pathway and induced, similarly to HDAC7 silencing, an increase in PDGF-B expression, as well as a partial inhibition of endothelial cell migration. Collectively, these data identified HDAC7 as a key modulator of endothelial cell migration and hence angiogenesis, at least in part, by regulating PDGF-B/PDGFR-␤ gene expression. Because angiogenesis is required for tumor progression, HDAC7 may represent a rational target for therapeutic intervention against cancer. A ngiogenesis is a crucial biologic event in physiologic conditions but also in many pathologic situations such as inflammation, tumor growth, and metastasis. This process depends on the activation/inactivation of genes associated with proliferation, adhesion, migration, and invasion of endothelial cells. 1,2 Histone deacetylases (HDACs) constitute a family of enzymes that regulate gene transcription by modifying the acetylation level of histones and nonhistone proteins. 3,4 Global inhibition of HDAC activity inhibits angiogenesis and tumor growth both in vitro and in vivo both by reducing expression of proangiogenic factors and enhancing expression of angiogenic inhibitors. 5-10 At the time our study was initiated, no information was available regarding the identity of the HDAC(s) that could intervene in the control of angiogenesis. However, while we were preparing this report, a study was published that indicates a role for HDAC7 in the maintenance of vascular integrity. 11 Here, we have applied a gene-silencing strategy using small interfering (si)RNAs efficiently targeting HDAC1 through HDAC7 in endothelial cells stimulated to form capillary-like structures. Our results indicate that HDAC7 is necessary for the assembly of endothelial cell in tube-like structures in vitro. We further show HDAC7 silencing causes cell shape changes and decreases endothelial cell migration, at least in part, through an upregulation of platelet-derived growth factor (PDGF)-B and its ␤ receptor (PDGFR-␤). Moreover, phorbol 12-myristate 13-acetate...

Transforming Growth Factor beta1 (TGFbeta1) is a crucial cytokine triggering myofibroblastic (MF) differentiation, a process involved in tissue healing as well as in pathologic conditions such as fibrosis and cancer. Together with cell shape modifications, TGFbeta1-mediated differentiation of fibroblasts into myofibroblasts is characteristically associated with the neo-expression of smooth muscle alpha-actin (alpha-SMA), a cytoskeletal protein that enhances their contractile activity. Several cellular differentiation programs have been linked to epigenetic regulation of gene expression, including gene methylation and histone acetylation. Herein, we sought to investigate the role of histone deacetylases (HDAC) in TGFbeta1-induced MF differentiation. We found that TSA, a global inhibitor of class I and class II HDACs, prevented alpha-SMA transcript and protein expression and morphological changes mediated by TGFbeta1 in cultured human skin fibroblasts. In order to identify the HDAC(s) participating in MF differentiation, the impact of specific HDAC silencing (HDAC1 through HDAC8) using RNA interference was investigated in fibroblasts exposed to TGFbeta1. Among the eight HDACs tested, silencing of HDAC4, HDAC6, and HDAC8 expression impaired TGFbeta1-induced alpha-SMA expression. HDAC4 silencing most efficiently abrogated alpha-SMA expression and also prevented TGFss1-mediated morphological changes. Forced down-regulation of HDAC4 stimulated the expression of 5'-TG-3'-Interacting Factor (TGIF) and TGIF2 homeoproteins, two known endogenous repressors of the TGFbeta signaling pathway, but not of the inhibitory Smad7. Collectively, these data suggest that HDAC4 is an essential epigenetic regulator of MF differentiation and unveil HDAC4 as a potential target for treating MF-related disorders.