Gipsy Majumdar

Insulin dynamically regulates calmodulin gene expression by sequential o-glycosylation and phosphorylation of sp1 and its subcellular compartmentalization in liver cells.

O-Glycosylation and phosphorylation of Sp1 are thought to modulate the expression of a number of genes in normal and diabetic state. Sp1 is an obligatory transcription factor for constitutive and insulin-responsive expression of the calmodulin gene (Majumdar, G., Harmon, A., Candelaria, R., Martinez-Hernandez, A., Raghow, R., and Solomon, S. S. (2003) Am. J. Physiol. 285, E584-E591). Here we report the temporal dynamics of accumulation of total, O-GlcNAc-modified, and phosphorylated Sp1 in H-411E hepatoma cells by immunohistochemistry with monospecific antibodies, confocal microscopy, and matrix-assisted laser desorption and ionization-time of flight mass spectrometry. Insulin elicited sequential and reciprocal post-translational modifications of Sp1. The O-glycosylation of Sp1 and its nuclear accumulation induced by insulin peaked early (∼30 min), followed by a steady decline of O-GlcNAc-modified Sp1 to negligible levels by 240 min. The accumulation of phosphorylated Sp1 in the nuclei of insulin-treated cells showed an opposite pattern, increasing steadily until reaching a maximum around 240 min after treatment. Analyses of the total, O-GlcNAc-modified, or phosphorylated Sp1 by Western blot and mass spectrometry corroborated the sequential and reciprocal control of post-translational modifications of Sp1 in response to insulin. Treatment of cells with streptozotocin (a potent inhibitor of O-GlcNAcase) led to hyperglycosylation of Sp1 that failed to be significantly phosphorylated. The mass spectrometry data indicated that a number of common serine residues of Sp1 undergo time-dependent, reciprocal O-glycosylation and phosphorylation, paralleling its rapid translocation from cytoplasm to the nucleus. Later, changes in the steady state levels of phosphorylated Sp1 mimicked the enhanced steady state levels of calmodulin mRNA seen after insulin treatment. Thus, O-glycosylation of Sp1 appears to be critical for its localization into the nucleus, where it undergoes obligatory phosphorylation that is needed for Sp1 to activate calmodulin gene expression.

Pan-histone deacetylase inhibitors regulate signaling pathways involved in proliferative and pro-inflammatory mechanisms in H9c2 cells

BackgroundWe have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA.ResultsWe incubated H9c2 cardiac myocytes in growth medium containing either of the two HDACIs for 6h and 24h and analyzed changes in gene expression using Illumina microarrays. H9c2 cells exposed to TSA for 6h and 24h led to differential expression of 468 and 231 genes, respectively. In contrast, cardiac myocytes incubated with CBHA for 6h and 24h elicited differential expression of 768 and 999 genes, respectively. We analyzed CBHA- and TSA-induced differentially expressed genes by Ingenuity Pathway (IPA), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Core_TF programs and discovered that CBHA and TSA impinged on several common gene networks. Thus, both HDACIs induced a repertoire of signaling kinases (PTEN-PI3K-AKT and MAPK) and transcription factors (Myc, p53, NFkB and HNF4A) representing canonical TGFβ, TNF-α, IFNγ and IL-6 specific networks. An overrepresentation of E2F, AP2, EGR1 and SP1 specific motifs was also found in the promoters of the differentially expressed genes. Apparently, TSA elicited predominantly TGFβ- and TNF-α-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-α and IFNγ specific networks at 6 h, followed by elicitation of IL-6 and IFNγ-centered gene networks at 24h.ConclusionsOur data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene networks in H9c2 cardiac myocytes. Initially, both HDACIs impinged on numerous genes associated with adipokine signaling, intracellular metabolism and energetics, and cell cycle. A continued exposure to either CBHA or TSA led to the emergence of a number of apoptosis- and inflammation-specific gene networks that were apparently suppressed by both HDACIs. Based on these data we posit that the anti-inflammatory and anti-proliferative actions of HDACIs are myocyte-intrinsic. These findings advance our understanding of the mechanisms of actions of HDACIs on cardiac myocytes and reveal potential signaling pathways that may be targeted therapeutically.

Panhistone deacetylase inhibitors inhibit proinflammatory signaling pathways to ameliorate interleukin-18-induced cardiac hypertrophy

We investigated the genome-wide consequences of pan-histone deacetylase inhibitors (HDACIs) trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA) in the hearts of BALB/c mice eliciting hypertrophy in response to interleukin-18 (IL-18). Both TSA and CBHA profoundly altered cardiac chromatin structure that occurred concomitantly with normalization of IL-18-induced gene expression and amelioration of cardiac hypertrophy. The hearts of mice exposed to IL-18+/-TSA or CBHA elicited distinct gene expression profiles. Of 184 genes that were differentially regulated by IL-18 and TSA, 33 were regulated in an opposite manner. The hearts of mice treated with IL-18 and/or CBHA elicited 147 differentially expressed genes (DEGs), a third of which were oppositely regulated by IL-18 and CBHA. Ingenuity Pathways and Kyoto Encyclopedia of Genes and Genomes analyses of DEGs showed that IL-18 impinged on TNF-α- and IFNγ-specific gene networks relegated to controlling immunity and inflammation, cardiac metabolism and energetics, and cell proliferation and apoptosis. These TNF-α- and IFNγ-specific gene networks, extensively connected with PI3K, MAPK, and NF-κB signaling pathways, were oppositely regulated by IL-18 and pan-HDACIs. Evidently, both TSA and CBHA caused a two- to fourfold induction of phosphatase and tensin homolog expression to counteract IL-18-induced proinflammatory signaling and cardiac hypertrophy.

A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle

We combined the TRIzol method of nucleic acid extraction with QIAamp columns to achieve coextraction of RNA and genomic DNA from peripheral blood mononuclear cells (PBMCs) and biopsied skeletal muscle, both stored at -80 °C for many months. Total RNA was recovered from the upper aqueous phase of TRIzol. The interphase and organic phases were precipitated with ethanol, digested with proteinase K, and filtered through QIAamp MinElute columns to recover DNA. The combined protocol yielded excellent quality and quantity of nucleic acids from archived human PBMCs and muscle and may be easily adapted for other tissues.

A systems biology approach to elucidating epigenetic regulation of cardiac hypertrophy

Background Reversible acetylation of histone and non-histone proteins is involved in cardiac hypertrophy that may be attenuated by histone deacetylase inhibitors (HDACIs). Since the mechanistic underpinnings of how pan-HDACIs ameliorate pathological cardiac hypertrophy remain elusive, we investigated genome-wide consequences of HDACIs, trichostatin A (TSA) and m-carboxycinnamic acid bis-hydroxamide (CBHA), in Balb/c mice eliciting cardiac hypertrophy in response to interleukin-18 (IL-18).

Epigenetic control of signaling networks involved in interleukin-18-induced cardiac hypertrophy and its attenuation by pan-histone deacetylase inhibitors

Background Daily intra-peritoneal injection of IL-18 for one week in Balb/c mice led to the development of severe cardiac hypertrophy that was accompanied by induction of fetal gene expression. Both anatomical and molecular features of cardiac hypertrophy were ameliorated by pan-histone deacetylase inhibitors (HDACIs), m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA). Materials and methods Treatment of mice with IL-18, with or without CBHA or TSA, caused dynamic remodeling of cardiac chromatin as judged by altered posttranslational modifications of the constituent histones. Administration of either TSA or CBHA in mice, even in the presence of IL-18, induced hyper-acetylation of histone H3 at lysine 9 demonstrating the unique action of HDAC inhibitors. Epigenetic changes in chromatin architecture were accompanied by reprogramming of cardiac hypertrophy-specific gene expression. Comparative profiles of cardiac gene expression of vehicle-treated mice with animals that received IL-18±CBHA or TSA revealed that 184 genes were differentially expressed genes (DEGs) and a subset of 38 genes was oppositely regulated by IL-18 and TSA. In contrast, the hearts of mice treated with IL-18 and/or CBHA elicited 147 DEGs; IL-18 robustly up-regulated 58 genes that were blocked by CBHA or IL-18+CBHA. Results and conclusion Ingenuity Pathway Analysis of DEGs revealed that both HDACIs profoundly impacted a number of intracellular signaling pathways. Global gene expression analyses of H9c2 cardiac myocytes incubated with CBHA or TSA for 6 and 24 hr were also carried out. We observed that the signatures of DEGs evoked by TSA and CBHA after 6 and at 24h treatment were unique. Nevertheless, it appeared that both HDACIs altered the course of PI3KPTEN-Akt/PKB, Ca ++ /calmodulin and p38-ERK-MAPKJNK signaling pathways to regulate gene expression via activation of Ap1, HNF4A, Myc and NFkB. Informed by the putative signaling networks deduced from the DEGs seen in the heart, we investigated the expression of phosphatase and tensin homolog (PTEN), a key regulator of the PI3K-AKT/PKB-NFkB pathway. Both TSA and CBHA induced robust expression of PTEN mRNA and protein in the heart. Based on these data we conclude that both TSA and CBHA attenuated anatomical and functional manifestations of IL-18-induced cardiac hypertrophy by epigenetic induction of PTEN to selectively block the PI3K-AKT/PKB-NFkB signaling.

Molecular dissection of cardiac hypertrophy induced by interleukin-18

Background Cardiac hypertrophy represents a primary mechanism by which heart is remodeled in response to a variety of intrinsic and extrinsic stimuli. The underlying role of inflammation in the molecular mechanisms of cardiac hypertrophy remains incompletely elucidated. We are investigating the role of epigenetic mechanisms in the regulation of cardiac hypertrophy induced by interleukin 18 (IL-18) using in vivo and in vitro models. Daily intra-peritoneal administration of interleukin 18 for one week in Balb/c mice induced cardiac hypertrophy as judged by increased ratios of their heart and lung weights to total body weights, and enhanced thickness of their ventricular walls. The hearts of IL-18-treated mice also elicited an increased expression of atrial naturetic factor, desmin and skeletal α-actin genes and a concomitant switch in the rate of expression of αand β-myosin heavy chain genes. Both the gross histological manifestations of cardiac hypertrophy and altered gene expression were greatly normalized by co-administration of histone deacetylase (HDAC) inhibitors, m-carboxycinnamic acid bis-hydroxamide (CBHA) or trichostatin A (TSA). Evidently, IL-18 and HDAC inhibitors modulated the signal transduction pathways that elicit the program of hypertrophy-specific gene expression. Our data revealed that chromatin remodeling and expression of PTEN (phosphatase and tensin homolog) by CBHA and TSA was mechanistically related to amelioration of cardiac hypertrophy. A brisk induction of PTEN by inhibitors of HDACs led to attenuation of IL-18induced PI3K-Akt/protein kinase B signal transduction pathway.

175 INSULIN STIMULATES GENE TRANSCRIPTION BY SEQUENTIAL MODIFICATIONS OF O-GLYCOSYLATION AND PHOSPHORYLATION OF SP1.

Insulin activates calmodulin (CaM) gene transcription, which leads to activation of low Km cAMP phosphodiesterase. This results in reversal of diabetic ketoacidosis. We have previously shown by 32 P labeling experiments and Western blots with antibodies highly specific for Sp1, O-GlcNAc, and phosphoserine that insulin first stimulates synthesis of Sp1 and then O-glycosylates it (early), followed by phosphorylation (later). Transcription of the CaM gene then occurs. H-411E liver cells in tissue culture were incubated with insulin (10,000 μU/mL) and processed at 0, 30, and 240 min. After incubation, cell extracts were prepared and run on 7.5% SDS polyacrylamide gel. The Sp1 band, localized by SYPRO stain and Western blot using specific anti-Sp1 antibody, was trypsin digested and then analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS). The data revealed that at least 3 peptide fragments containing serine/threonine sites are modified either by O-glycosylation or phosphorylation over the period of 4 hr of insulin exposure. The peptides are (1) 612-616; (2) 641-645; and (3) 699-704. The serine sites of these peptides were unmodified at 0 min and O-glycosylated at 30 min and later that same site was converted to a phosphate, eg, one of the three peptide fragments with mass 563.24 kDa (serine 613) is glycosylated at 30 min with a peptide mass of 766.31 kDa (563.24 + 203.19) and then at 240 min deglycosylated and phosphorylated with a peptide of mass of 644.26 (563.24 + 80.9) appearing. Insulin treatment at 0 min shows that the 4 serine sites in these 3 peptides initially are unmodified, but after 30 min all of these serine sites (100%) are O-GlcNAced. Following 4 hr insulin treatment, 3 out of 4 (75%) of the serine sites are phosphorylated. Conclusions MALDI TOF MS experiments support a yin-yang hypothesis, ie, the existence of a reciprocal relationship between O-glycosylation and phosphorylation of Sp1 and its role in translating insulins effect on CaM gene transcription.

100 INSULIN REGULATES GENE TRANSCRIPTION BY O-GLYCOSYLATION AND PHOSPHORYLATION OF Sp1

Insulin (INS) stimulates steady state levels of Sp1 transcription factor, which leads to increased calmodulin (CaM) gene expression. We have investigated the mechanism(s) involved and find that INS stimulates O-glycosylation (O-GlcNAc) of Sp1 as an obligatory precursor event. O-GlcNAcation-, followed by phosphorylation (PO4) of Sp1, regulates both its intracellular mobility and activity. To elucidate the mechanistic basis of these post-translational changes of Sp1 in response to insulin, we assessed temporal dynamics of accumulation of total, O-GlcNAc-modified and phosphorylated-Sp1 in H-411E liver cells exposed to 10,000 μU/mL of insulin. Extracts from untreated and timed insulin treated cells were analyzed by Western blotting (Wb) using specific antibodies. The steady state levels of total and modified Sp1 were also investigated by confocal microscopy of H411E cells probed with Sp1-, O-GlcNAc-, and phosphoserine-specific antibodies that were detected with secondary antibodies labeled with various fluorochromes. The results from both Wb and confocal microscopy demonstrate that: INS stimulates (1) Sp1; (2) O-glycosylation of Sp1 early (30 min), which declines by 4 hours; and (3) phosphorylation of Sp1 in a steady increase through 4 hours. STZ, which inhibits O-GlcNAcase, and leaves Sp1 O-glycosylated, not only lead to intensified nuclear staining for O-GlcNAc-Sp1 but inhibited the insulin-driven expected nuclear staining for phosphorylated Sp1. Thus, reciprocal changes in O-GlcNAcation and phosphorylation of Sp1 in response to insulin appear to fine-tune calmodulin gene expression.