Hung Ying Kao

Histone deacetylase 7 promotes PML sumoylation and is essential for PML nuclear body formation

ABSTRACT Promyelocytic leukemia protein (PML) sumoylation has been proposed to control the formation of PML nuclear bodies (NBs) and is crucial for PML-dependent cellular processes, including apoptosis and transcriptional regulation. However, the regulatory mechanisms of PML sumoylation and its specific roles in the formation of PML NBs remain largely unknown. Here, we show that histone deacetylase 7 (HDAC7) knockdown reduces the size and the number of the PML NBs in human umbilical vein endothelial cells (HUVECs). HDAC7 coexpression stimulates PML sumoylation independent of its HDAC activity. Furthermore, HDAC7 associates with the E2 SUMO ligase, Ubc9, and stimulates PML sumoylation in vitro, suggesting that it possesses a SUMO E3 ligase-like activity to promote PML sumoylation. Importantly, HDAC7 knockdown inhibits tumor necrosis factor alpha-induced PML sumoylation and the formation of PML NBs in HUVECs. These results demonstrate a novel function of HDAC7 and provide a regulatory mechanism of PML sumoylation.

Acetylation of retinal histones in diabetes increases inflammatory proteins: effects of minocycline and manipulation of histone acetyltransferase (HAT) and histone deacetylase (HDAC).

Background: Processes that regulate inflammatory changes that contribute to the development of diabetic retinopathy are not known. Results: Hyperglycemia increases histone acetylation, and HAT and HDAC inhibit this acetylation and induction of inflammatory proteins in retina. Conclusion: Protein acetylation regulates the hyperglycemia-induced up-regulation of proinflammatory proteins. Significance: Identification of acetylation as a regulator of proinflammatory processes in diabetes offers new therapeutic targets to inhibit the retinopathy. Histone acetylation was significantly increased in retinas from diabetic rats, and this acetylation was inhibited in diabetics treated with minocycline, a drug known to inhibit early diabetic retinopathy in animals. Histone acetylation and expression of inflammatory proteins that have been implicated in the pathogenesis of diabetic retinopathy were increased likewise in cultured retinal Müller glia grown in a diabetes-like concentration of glucose. Both the acetylation and induction of the inflammatory proteins in elevated glucose levels were significantly inhibited by inhibitors of histone acetyltransferase (garcinol and antisense against the histone acetylase, p300) or activators of histone deacetylase (theophylline and resveratrol) and were increased by the histone deacetylase inhibitor, suberolylanilide hydroxamic acid. We conclude that hyperglycemia causes acetylation of retinal histones (and probably other proteins) and that the acetylation contributes to the hyperglycemia-induced up-regulation of proinflammatory proteins and thereby to the development of diabetic retinopathy.

The actin-binding protein, actinin alpha 4 (ACTN4), is a nuclear receptor coactivator that promotes proliferation of MCF-7 breast cancer cells

Alpha actinins (ACTNs) are known for their ability to modulate cytoskeletal organization and cell motility by cross-linking actin filaments. We show here that ACTN4 harbors a functional LXXLL receptor interaction motif, interacts with nuclear receptors in vitro and in mammalian cells, and potently activates transcription mediated by nuclear receptors. Whereas overexpression of ACTN4 potentiates estrogen receptor α (ERα)-mediated transcription in transient transfection reporter assays, knockdown of ACTN4 decreases it. In contrast, histone deacetylase 7 (HDAC7) inhibits estrogen receptor α (ERα)-mediated transcription. Moreover, the ACTN4 mutant lacking the CaM (calmodulin)-like domain that is required for its interaction with HDAC7 fails to activate transcription by ERα. Chromatin immunoprecipitation (ChIP) assays demonstrate that maximal associations of ACTN4 and HDAC7 with the pS2 promoter are mutually exclusive. Knockdown of ACTN4 significantly decreases the expression of ERα target genes including pS2 and PR and also affects cell proliferation of MCF-7 breast cancer cells with or without hormone, whereas knockdown of HDAC7 exhibits opposite effects. Interestingly, overexpression of wild-type ACTN4, but not the mutants defective in interacting with ERα or HDAC7, results in an increase in pS2 and PR mRNA accumulation in a hormone-dependent manner. In summary, we have identified ACTN4 as a novel, atypical coactivator that regulates transcription networks to control cell growth.

Membrane permeable cyclic peptidyl inhibitors against human Peptidylprolyl Isomerase Pin1.

Peptidylprolyl isomerase Pin1 regulates the function and/or stability of phosphoproteins by altering the conformation of specific pSer/pThr-Pro peptide bonds. In this work, a cyclic peptide library was synthesized and screened against the catalytic domain of human Pin1. The selected inhibitors contained a consensus motif of D-pThr-Pip-Nal (where Pip is L-piperidine-2-carboxylic acid and Nal is L-2-naphthylalanine). Representative compounds were tested for binding to Pin1 by isothermal titration calorimetry and inhibition of Pin1 activity, and the most potent inhibitors had K(D) (and K(I)) values in the low nanomolar range. Treatment of breast cancer cells with the inhibitors, which were rendered membrane permeable by attachment of an octaarginine sequence, inhibited cell proliferation and increased the protein levels of two previously established Pin1 substrates, PML and SMRT. Finally, a second generation of cell permeable Pin1 inhibitors was designed by replacing the noncritical residues within the cyclic peptide ring with arginine residues and shown to have antiproliferative activity against the cancer cells.

The epigenetic regulator UHRF1 promotes ubiquitination-mediated degradation of the tumor-suppressor protein promyelocytic leukemia protein

The promyelocytic leukemia (PML) protein is a tumor suppressor originally identified in acute promyelocytic leukemia and implicated in tumorigenesis in multiple forms of cancer. Here, we demonstrate that the PML protein undergoes ubiquitination-mediated degradation facilitated by an E3 ligase UHRF1 (ubiquitin-like with PHD and RING finger domains 1), which is commonly upregulated in various human malignancies. Furthermore, UHRF1 negatively regulates PML protein accumulation in primary human umbilical vein endothelial cells (HUVECs), HEK 293 cells and cancer cells. Knockdown of UHRF1 upregulates whereas ectopic overexpression of UHRF1 downregulates protein abundance of endogenous or exogenous PML, doing so through its binding to the N-terminus of PML. Overexpression of wild-type UHRF1 shortens PML protein half-life and promotes PML polyubiquitination, whereas deletion of the RING domain or coexpression of the dominant-negative E2 ubiquitin-conjugating enzyme, E2D2, attenuates this modification to PML. Finally, knockdown of UHRF1 prolongs PML half-life and increases PML protein accumulation, yet inhibits cell migration and in vitro capillary tube formation, whereas co-knockdown of PML compromises this inhibitory effect. These findings suggest that UHRF1 promotes the turnover of PML protein, and thus targeting UHRF1 to restore PML-mediated tumor suppression represents a promising, novel, anticancer strategy.

Signal-dependent Regulation of Transcription by Histone Deacetylase 7 Involves Recruitment to Promyelocytic Leukemia Protein Nuclear Bodies

Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes, including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase (HDAC) 7 is a potent corepressor that inhibits transcription by myocyte enhancer factor 2 (MEF2) transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor (TNF)-alpha treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells. Consequently, TNF-alpha promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase (MMP)-10, leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, small interfering RNA knockdown of PML decreases basal and TNF-alpha-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression.

Promyelocytic Leukemia Protein Controls Cell Migration in Response to Hydrogen Peroxide and Insulin-like Growth Factor-1

Promyelocytic leukemia protein (PML) was originally identified as part of a chromosomal translocation that contributes to the development of acute promyelocytic leukemia (APL). Since its discovery, PML has been found to play diverse roles in different cellular processes. Notably, PML has anti-proliferative and pro-apoptotic activity that supports its role as a tumor suppressor. We have previously shown that the peptidyl-prolyl isomerase Pin1 is able to affect cell proliferation and hydrogen peroxide (H2O2)-mediated cell death through modulation of the steady-state levels of PML. We have extended these studies to show that the interaction between PML and Pin1 is targeted by multiple extracellular signals in the cell. We show that H2O2 up-regulates and IGF-1 down-regulates PML expression in a Pin1-dependent manner. Interestingly, we found that H2O2- and IGF-1-mediated alteration in PML accumulation regulate MDA-MB-231 cell migration. Furthermore, we show that the control of cell migration by PML, and thus H2O2 and IGF-1, results from PML-dependent decreased expression of integrin β1 (ITGB1). Knockdown of Pin1 leads to decreased cell migration, lower levels of ITGB1 expression and resistance to IGF-1- and H2O2-induced changes in cell migration and ITGB1 expression. Taken together, our work identifies PML as a common target for H2O2 and IGF-1 and supports a novel tumor suppressive role for PML in controlling cell migration through the expression of ITGB1.

Promyelocytic Leukemia Protein (PML) Regulates Endothelial Cell Network Formation and Migration in Response to Tumor Necrosis Factor α (TNFα) and Interferon α (IFNα)

Background: PML is highly expressed in endothelial cells (ECs), but its role in ECs remains largely unexplored. Results: PML is critical for TNFα- and IFNα-mediated inhibition of EC network formation and migration. Conclusion: PML is an angiogenesis inhibitor following inflammation-associated cytokine signaling. Significance: PML is a novel regulator of endothelial cell physiology. Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in vascular endothelium and inflamed tissues, yet its role in inflammation-associated cytokine-regulated angiogenesis and underlying mechanism remains largely unclear. We show that tumor necrosis factor α (TNFα) and interferon α (IFNα) stimulate PML expression while suppressing EC network formation and migration, two key events during angiogenesis. By a knockdown approach, we demonstrate that PML is indispensable for TNFα- and IFNα-mediated inhibition of EC network formation. We further demonstrate that signal transducer and activator of transcription 1 (STAT1) binds PML promoter and that is an important regulator of PML expression. Knockdown of STAT1 reduces endogenous PML and blocks TNFα- and IFNα-induced PML accumulation and relieves TNFα- and IFNα-mediated inhibition of EC network formation. Our data also indicate that PML regulates EC migration, in part, by modulating expression of downstream genes, such as negatively regulating integrin β1 (ITGB1). In addition, knockdown of STAT1 or PML alleviates TNFα- and IFNα-mediated inhibition of ITGB1 expression. Antibody blockade demonstrates that ITGB1 is functionally important for PML- and STAT1-regulated EC migration. Taken together, our data provide novel mechanistic insights that PML functions as a negative regulator in EC network formation and migration.

Familial Focal Segmental Glomerulosclerosis (FSGS)-linked α-Actinin 4 (ACTN4) Protein Mutants Lose Ability to Activate Transcription by Nuclear Hormone Receptors

Background: Mutations in α-ACTN4 are linked with familial FSGS. Results: FSGS linked α-ACTN4 mutants fail to activate transcription mediated by nuclear hormone receptors. Conclusion: The inability of FSGS-linked ACTN4 mutants to potentiate transcriptional activation might be because of their more cytoplasmic localization compared with wild type. Significance: Our results may have implications for understanding the role of α-ACTN4 in the pathophysiology of the kidney disease. Mutations in α-actinin 4 (ACTN4) are linked to familial forms of focal segmental glomerulosclerosis (FSGS), a kidney disease characterized by proteinuria due to podocyte injury. The mechanisms underlying ACTN4 mutant-associated FSGS are not completely understood. Although α-actinins are better known to cross-link actin filaments and modulate cytoskeletal organization, we have previously shown that ACTN4 interacts with transcription factors including estrogen receptor and MEF2s and potentiates their transcriptional activity. Nuclear receptors including retinoic acid receptor (RAR) have been proposed to play a protective role in podocytes. We show here that ACTN4 interacts with and enhances transcriptional activation by RARα. In addition, FSGS-linked ACTN4 mutants not only mislocalized to the cytoplasm, but also lost their ability to associate with nuclear receptors. Consequently, FSGS-linked ACTN4 mutants failed to potentiate transcriptional activation by nuclear hormone receptors in podocytes. In addition, overexpression of these mutants suppressed the transcriptional activity mediated by endogenous wild-type ACTN4 possibly by a cytoplasmic sequestration mechanism. Our data provide the first link between FSGS-linked ACTN4 mutants and transcriptional activation by nuclear receptor such as RARα and peroxisome proliferator-activated receptor γ.

Deacetylation of the tumor suppressor protein PML regulates hydrogen peroxide-induced cell death

The promyelocytic leukemia protein (PML) is a tumor suppressor that is expressed at a low level in various cancers. Although post-translational modifications including SUMOylation, phosphorylation, and ubiquitination have been found to regulate the stability or activity of PML, little is known about the role of its acetylation in the control of cell survival. Here we demonstrate that acetylation of lysine 487 (K487) and SUMO1 conjugation of K490 at PML protein are mutually exclusive. We found that hydrogen peroxide (H2O2) promotes PML deacetylation and identified SIRT1 and SIRT5 as PML deacetylases. Both SIRT1 and SIRT5 are required for H2O2-mediated deacetylation of PML and accumulation of nuclear PML protein in HeLa cells. Knockdown of SIRT1 reduces the number of H2O2-induced PML-nuclear bodies (NBs) and increases the survival of HeLa cells. Ectopic expression of wild-type PML but not the K487R mutant rescues H2O2-induced cell death in SIRT1 knockdown cells. Furthermore, ectopic expression of wild-type SIRT5 but not a catalytic defective mutant can also restore H2O2-induced cell death in SIRT1 knockdown cells. Taken together, our findings reveal a novel regulatory mechanism in which SIRT1/SIRT5-mediated PML deacetylation plays a role in the regulation of cancer cell survival.