Erin L. Reineke

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.

TNF receptor 1 signaling is critically involved in mediating angiotensin-II-induced cardiac fibrosis

Angiotensin-II (Ang-II) is associated with many conditions involving heart failure and pathologic hypertrophy. Ang-II induces the synthesis of monocyte chemoattractant protein-1 that mediates the uptake of CD34(+)CD45(+) monocytic cells into the heart. These precursor cells differentiate into collagen-producing fibroblasts and are responsible for the Ang-II-induced development of non-adaptive cardiac fibrosis. In this study, we demonstrate that in vitro, using a human monocyte-to-fibroblast differentiation model, Ang-II required the presence of tumor necrosis factor-alpha (TNF) to induce fibroblast maturation from monocytes. In vivo, mice deficient in both TNF receptors did not develop cardiac fibrosis in response to 1week Ang-II infusion. We then subjected mice deficient in either TNF receptor 1 (TNFR1-KO) or TNF receptor 2 (TNFR2-KO) to continuous Ang-II infusion. Compared to wild-type, in TNFR1-KO, but not in TNFR2-KO hearts, collagen deposition was greatly attenuated, and markedly fewer CD34(+)CD45(+) cells were present. Quantitative RT-PCR demonstrated a striking reduction of key fibrosis-related, as well as inflammation-related mRNA expression in Ang-II-treated TNFR1-KO hearts. TNFR1-KO animals also developed less cardiac remodeling, cardiac hypertrophy, and hypertension compared to wild-type and TNFR2-KO in response to Ang-II. Our data suggest that TNF induced Ang-II-dependent cardiac fibrosis by signaling through TNFR1, which enhances the generation of monocytic fibroblast precursors in the heart.

α-Actinin 4 Potentiates Myocyte Enhancer Factor-2 Transcription Activity by Antagonizing Histone Deacetylase 7

Histone deacetylase 7 (HDAC7) is a member of class IIa HDACs that regulate myocyte enhancer factor-2 (MEF2)-mediated transcription and participate in multiple cellular processes such as T cell apoptosis. We have identified α-actinin 1 and 4 as class IIa HDAC-interacting proteins. The interaction domains are mapped to C terminus of α-actinin 4 and amino acids 72-172 of HDAC7. A point mutation in HDAC7 that disrupts its association with MEF2A also disrupts its association with α-actinin 4, indicating that MEF2A and α-actinin 4 binding sites largely overlap. We have also isolated a novel splice variant of α-actinin 4 that is predominantly localized in the nucleus, a pattern distinct from the full-length α-actinin 4, which is primarily distributed in the cytoplasm and plasma membrane. Using small interfering RNA, chromatin immunoprecipitation, and transient transfection assays, we show that α-actinin 4 potentiates expression of TAF55, a putative MEF2 target gene. Loss of MEF2A interaction correlates with loss of the ability of α-actinin 4 to potentiate TAF55 promoter activity. Ectopic expression of α-actinin 4, but not the mutant defective in MEF2A association, leads to disruption of HDAC7·MEF2A association and enhancement of MEF2-mediated transcription. Taken together, we have identified a novel mechanism by which HDAC7 activity is negatively regulated and uncovered a previously unknown function of α-actinin 4.

A Sleeping Beauty mutagenesis screen reveals a tumor suppressor role for Ncoa2/Src-2 in liver cancer

The Sleeping Beauty (SB) transposon mutagenesis system is a powerful tool that facilitates the discovery of mutations that accelerate tumorigenesis. In this study, we sought to identify mutations that cooperate with MYC, one of the most commonly dysregulated genes in human malignancy. We performed a forward genetic screen with a mouse model of MYC-induced liver cancer using SB-mediated mutagenesis. We sequenced insertions in 63 liver tumor nodules and identified at least 16 genes/loci that contribute to accelerated tumor development. RNAi-mediated knockdown in a liver progenitor cell line further validate three of these genes, Ncoa2/Src-2, Zfx, and Dtnb, as tumor suppressors in liver cancer. Moreover, deletion of Ncoa2/Src-2 in mice predisposes to diethylnitrosamine-induced liver tumorigenesis. These findings reveal genes and pathways that functionally restrain MYC-mediated liver tumorigenesis and therefore may provide targets for cancer therapy.

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.

Ablation of Steroid Receptor Coactivator-3 Resembles the Human CACT Metabolic Myopathy

Oxidation of lipid substrates is essential for survival in fasting and other catabolic conditions, sparing glucose for the brain and other glucose-dependent tissues. Here we show Steroid Receptor Coactivator-3 (SRC-3) plays a central role in long chain fatty acid metabolism by directly regulating carnitine/acyl-carnitine translocase (CACT) gene expression. Genetic deficiency of CACT in humans is accompanied by a constellation of metabolic and toxicity phenotypes including hypoketonemia, hypoglycemia, hyperammonemia, and impaired neurologic, cardiac and skeletal muscle performance, each of which is apparent in mice lacking SRC-3 expression. Consistent with human cases of CACT deficiency, dietary rescue with short chain fatty acids drastically attenuates the clinical hallmarks of the disease in mice devoid of SRC-3. Collectively, our results position SRC-3 as a key regulator of β-oxidation. Moreover, these findings allow us to consider platform coactivators such as the SRCs as potential contributors to syndromes such as CACT deficiency, previously considered as monogenic.

Mitogen-activated Protein Kinase Extracellular Signal-regulated Kinase 2 Phosphorylates and Promotes Pin1 Protein-dependent Promyelocytic Leukemia Protein Turnover

Background: The degradation of promyelocytic leukemia (PML) protein is regulated by phosphorylation-dependent manner. Results: The inhibition or knockdown of ERK2 increase PML expression level and decrease the interaction between PML and Pin1. Conclusion: The activation of ERK2 promotes Pin1-mediated degradation of PML in response to EGF. Significance: Understanding the mechanism by which mitogens promote PML protein turnover will have implication in developing therapeutic agents in treating cancer. The promyelocytic leukemia (PML) protein is a tumor suppressor that has an important role in several cellular processes, including apoptosis, viral infection, DNA damage repair, cell cycle regulation, and senescence. PML is an essential component of sub-nuclear structures called PML nuclear bodies (NBs). Our laboratory has previously demonstrated that the peptidyl-prolyl cis-trans isomerase, Pin1, binds and targets PML for degradation in a phosphorylation-dependent manner. To further elucidate the mechanisms underlying Pin1-mediated PML degradation, we aimed to identify one or more factors that promote PML phosphorylation. Here we show that treatment with U0126, an inhibitor of the ERK2 upstream kinases MEK1/2, leads to an increase in PML protein accumulation and an inhibition of the interaction between Pin1 and PML in MDA-MB-231 breast cancer cells. Consistent with this observation, phosphorylated ERK2 partially co-localized with PML NBs. Although U0126 up-regulated exogenous wild-type PML levels, it did not have an effect on the steady-state level of a mutant form of PML that is defective in binding Pin1. In addition, exogenous wild-type, but not Pin1 binding-defective PML protein expression levels were decreased by overexpression of ERK2. In contrast, knockdown of ERK2 by siRNA resulted in an increase in PML protein levels and an increase in the formation of PML NBs. Using phospho-specific antibodies, we identified Ser-403 and Ser-505 as the ERK2 targets that promote Pin1-mediated PML degradation. Finally, we demonstrated that EGF induced activation of ERK and interaction between PML and phosphorylated ERK resulting in a decrease in PML protein levels. Taken together, our results support a model in which Pin1 promotes PML degradation in an ERK2-dependent manner.

Research Resource: Tissue- and Pathway-Specific Metabolomic Profiles of the Steroid Receptor Coactivator (SRC) Family

The rapidly growing family of transcriptional coregulators includes coactivators that promote transcription and corepressors that harbor the opposing function. In recent years, coregulators have emerged as important regulators of metabolic homeostasis, including the p160 steroid receptor coactivator (SRC) family. Members of the SRC family have been ascribed important roles in control of gluconeogenesis, fat absorption and storage in the liver, and fatty acid oxidation in skeletal muscle. To provide a deeper and more granular understanding of the metabolic impact of the SRC family members, we performed targeted metabolomic analyses of key metabolic byproducts of glucose, fatty acid, and amino acid metabolism in mice with global knockouts (KOs) of SRC-1, SRC-2, or SRC-3. We measured amino acids, acyl carnitines, and organic acids in five tissues with key metabolic functions (liver, heart, skeletal muscle, brain, plasma) isolated from SRC-1, -2, or -3 KO mice and their wild-type littermates under fed and fasted conditions, thereby unveiling unique metabolic functions of each SRC. Specifically, SRC-1 ablation revealed the most significant impact on hepatic metabolism, whereas SRC-2 appeared to impact cardiac metabolism. Conversely, ablation of SRC-3 primarily affected brain and skeletal muscle metabolism. Surprisingly, we identified very few metabolites that changed universally across the three SRC KO models. The findings of this Research Resource demonstrate that coactivator function has very limited metabolic redundancy even within the homologous SRC family. Furthermore, this work also demonstrates the use of metabolomics as a means for identifying novel metabolic regulatory functions of transcriptional coregulators.

SRC-2 Coactivator Deficiency Decreases Functional Reserve in Response to Pressure Overload of Mouse Heart

A major component of the cardiac stress response is the simultaneous activation of several gene regulatory networks. Interestingly, the transcriptional regulator steroid receptor coactivator-2, SRC-2 is often decreased during cardiac failure in humans. We postulated that SRC-2 suppression plays a mechanistic role in the stress response and that SRC-2 activity is an important regulator of the adult heart gene expression profile. Genome-wide microarray analysis, confirmed with targeted gene expression analyses revealed that genetic ablation of SRC-2 activates the “fetal gene program” in adult mice as manifested by shifts in expression of a) metabolic and b) sarcomeric genes, as well as associated modulating transcription factors. While these gene expression changes were not accompanied by changes in left ventricular weight or cardiac function, imposition of transverse aortic constriction (TAC) predisposed SRC-2 knockout (KO) mice to stress-induced cardiac dysfunction. In addition, SRC-2 KO mice lacked the normal ventricular hypertrophic response as indicated through heart weight, left ventricular wall thickness, and blunted molecular signaling known to activate hypertrophy. Our results indicate that SRC-2 is involved in maintenance of the steady-state adult heart transcriptional profile, with its ablation inducing transcriptional changes that mimic a stressed heart. These results further suggest that SRC-2 deletion interferes with the timing and integration needed to respond efficiently to stress through disruption of metabolic and sarcomeric gene expression and hypertrophic signaling, the three key stress responsive pathways.

Steroid Receptor Coactivator-2 Is a Dual Regulator of Cardiac Transcription Factor Function

Background: An efficient cardiac stress response requires a series of coordinated molecular changes. Results: Steroid receptor coactivator-2 (SRC-2) controls the expression and activity of key cardiac transcription factors. Conclusion: Dual regulation of cardiac transcription poises SRC-2 as a novel coordinator of the stress-responsive molecular network. Significance: Identification of factors responsible for coordination of the cardiac stress response is critical for improved treatment. We have previously demonstrated the potential role of steroid receptor coactivator-2 (SRC-2) as a co-regulator in the transcription of critical molecules modulating cardiac function and metabolism in normal and stressed hearts. The present study seeks to extend the previous information by demonstrating SRC-2 fulfills this role by serving as a critical coactivator for the transcription and activity of critical transcription factors known to control cardiac growth and metabolism as well as in their downstream signaling. This knowledge broadens our understanding of the mechanism by which SRC-2 acts in normal and stressed hearts and allows further investigation of the transcriptional modifications mediating different types and degrees of cardiac stress. Moreover, the genetic manipulation of SRC-2 in this study is specific for the heart and thereby eliminating potential indirect effects of SRC-2 deletion in other organs. We have shown that SRC-2 is critical to transcriptional control modulated by MEF2, GATA-4, and Tbx5, thereby enhancing gene expression associated with cardiac growth. Additionally, we describe SRC-2 as a novel regulator of PPARα expression, thus controlling critical steps in metabolic gene expression. We conclude that through regulation of cardiac transcription factor expression and activity, SRC-2 is a critical transcriptional regulator of genes important for cardiac growth, structure, and metabolism, three of the main pathways altered during the cardiac stress response.