Robert W. Lim

Differential regulation of primary response gene expression in skeletal muscle cells through multiple signal transduction pathways

One of the earliest cellular responses to growth factors is the rapid induction of primary response genes. One group of such genes was originally isolated as tetradecanoyl phorbol acetate (TPA) inducible sequences (TIS genes) from mouse 3T3 cells. Proteins encoded by the TIS genes include two transcription factors: TIS8 (also known as egr1/NGFIA/zif268) and TIS1 (also known as NGFIB/nur77/N10). We have examined the inducibility of these two genes in a skeletal muscle cell line in response to agents that have been reported to block muscle differentiation. We report here that basic fibroblast growth factor (bFGF) induced the expression of both TIS1 and TIS8 in mouse C2C12cells. Both genes were also inducible by TPA while forskolin which activates the cAMP-dependent pathway induced TIS1 but not TIS8. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment repressed the bFGF induction of TIS1 but had little effect on the bFGF-stimulated expression of TIS8. Moreover, while both TPA and bFGF stimulated the hyperphosphorylation of c-RAF and the activity of MAP kinase, TPA pretreatment failed to block RAF phosphorylation or the stimulation of MAP kinase activity by bFGF. Induction of the two TIS genes in skeletal myoblasts therefore appeared to be dependent to different extents on the activation of protein kinase A (PKA), PKC and MAP kinase.

CLONING, EXPRESSION, AND CHARACTERIZATION OF A NOVEL GUANYLATE-BINDING PROTEIN, GBP3 IN MURINE ERYTHROID PROGENITOR CELLS

We report the molecular cloning of a novel guanylate-binding protein (GBP), termed mouse GBP3 (mGBP3) in Friend virus-induced mouse erythroid progenitor (FVA) cells. The 71-kDa mGBP3 belongs to a family of known GBPs that contain the first two consensus motifs, GXXXXGK(S/T) and DXXG, but lack the third element, (N/T)KXD, found in typical GTP-binding proteins. Recombinant mGBP3 protein, expressed using a baculovirus expression system, binds to agarose-immobilized guanine nucleotides (GTP, GDP and GMP). Moreover, mGBP3 has been found to have an intrinsic GTPase activity with K(m) and Vmax values of 77 +/- 4 microM and 21 +/- 0.5 pmol min-1 microgram-1 of protein, respectively. The mGBP3 is distinct from the other GBPs, in that it does not have an isoprenylation/methylation motif CAAX at the carboxyl terminus. The mGBP3 appears to be localized in the cytosol based on immunofluorescence staining. Although the mGBP3 transcript is expressed to a varying degree in numerous mouse tissues, the message is most abundant in FVA cells. The mGBP3 transcript increases in FVA cells undergoing differentiation to a maximum within a few hours and then decreases to an undetectable level by 24 h. These results, taken together, suggest that mGBP3 is a novel member of a family of guanylate-binding proteins, which plays a role in the erythroid differentiation. The nucleotide sequence reported in this paper has been submitted to the GenBank with accession number U44731.

Platelet activating factor induces expression of early response genes c-fos and TIS-1 in human epidermoid carcinoma A-431 cells

The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. It is concluded that PAF causes induction of the early response genes c-fos and TIS-1 in a structurally specific and receptor dependent manner. This finding offers a new role for PAF at the nuclear level and may have important implications in the long term effects of PAF in pathophysiological conditions.

Physical and Functional Interactions between the Transcriptional Inhibitors Id3 and ITF-2b EVIDENCE TOWARD A NOVEL MECHANISM REGULATING MUSCLE-SPECIFIC GENE EXPRESSION

We have used an interaction cloning strategy to identify an inhibitory isoform of the ITF-2 transcription factor, ITF-2b, that interacts with the transcriptional inhibitor Id3/HLH462. The interaction was confirmed in vitro, and inside intact myogenic C2C12 cells. As expected, overexpression of either Id3/HLH462 or ITF-2b effectively inhibited the activation of the muscle-specific creatine kinase promoter by the myogenic transcription factor MyoD. However, when overexpressed simultaneously, ITF-2b and Id3/HLH462 counteracted each others inhibitory effect to produce a reduced overall inhibition. Moreover, while ITF-2b inhibited the creatine kinase promoter, it acted as a weak transactivator on an artificial promoter consisting of three tandem copies of the consensus myogenic factor DNA binding site. Further investigation indicated that the ITF-2b/MyoD heterodimer bound to its specific DNA binding site in vitro, and the DNA binding was effectively blocked by Id3/HLH462. Additional analysis revealed the presence of transcripts for both the activating (ITF-2a) and inhibitory (ITF-2b) isoforms in differentiating C2C12 cultures, suggesting that both isoforms might participate in regulating the differentiation process. Taken together, this study reveals a more complex pattern of regulatory interactions involving the helix-loop-helix proteins than was previously anticipated.

Histone H3 Phosphorylation (Ser10, Ser28) and Phosphoacetylation (K9S10) Are Differentially Associated with Gene Expression in Liver of Rats Treated In Vivo with Acute Ethanol

The epigenetic histone modification by ethanol is emerging as one of the mechanisms for its deleterious effects in the liver. In this context, we have investigated the role of histone H3 phosphorylation at Ser10 (P-H3-Ser10), and Ser28 (P-H3-Ser28) in liver after acute ethanol treatment in vivo. Ethanol was administered intraperitoneally in male Sprague-Dawley rats. Ethanol dose-response (1–5 g/kg body weight) and time-course (1–4 h) experiments were conducted, and various parameters were monitored. Steatosis and necrosis (serum alanine aminotransferase) of the liver increased in 4 h, suggesting liver injury. There were differences between P-H3-Ser10 and P-H3-Ser28 at 1 h, with the latter being more sensitive to lower ethanol doses. It was noteworthy that phosphorylation of both serines disappeared at the highest dose used (5 g/kg). We also examined phosphoacetylation of histone H3 at K9S10 and observed a dramatic increase. The changes in histone H3 phosphorylation and phosphoacetylation were also accompanied with expression of early response genes (c-fos, c-jun, mitogen-activated protein kinase phosphatase-1). Chromatin immunoprecipitation assays in samples from 1.5 and 4 h of ethanol administration indicated that increased histone H3 phosphorylation at Ser28 was associated with the promoters of c-jun and plasminogen activator inhibitor-1. In conclusion, this study demonstrates for the first time that in vivo exposure of liver to acute ethanol induced phosphorylation and phosphoacetylation of histone H3, and these modifications are differentially involved in the mRNA expression of genes.

Gene-Selective Histone H3 Acetylation in the Absence of Increase in Global Histone Acetylation in Liver of Rats Chronically Fed Alcohol

AIMS The aim of this study was to determine the effect of chronic ethanol feeding on acetylation of histone H3 at lysine 9 (H3-Lys9) at promoter and coding regions of genes for class I alcohol dehydrogenase (ADH I), inducible nitric oxide synthase (iNOS), Bax, p21, c-met and hepatocyte growth factor in the rat liver. METHODS Rats were fed ethanol-containing liquid diet (5%, w/v) for 1-4 weeks. The global level of acetylation of H3-Lys9 in the liver was examined by western blot analysis. The levels of mRNA for various genes were measured by real-time reverse transcriptase-polymerase chain reaction. The association of acetylated histone H3-Lys9 with the different regions of genes was monitored by chromatin immunoprecipitation assay. RESULTS Chronic ethanol treatment increased mRNA expression of genes for iNOS, c-jun and ADH 1. Chronic ethanol treatment did not cause increase in global acetylation of H3-Lys9, but significantly increased the association of acetylated histone H3-Lys9 in the ADH I gene, both in promoter and in coding regions. In contrast, chronic ethanol treatment did not significantly increase the association of acetylated histone H3-Lys9 with iNOS and c-jun genes. CONCLUSION Chronic ethanol exposure increased the gene-selective association of acetylated H3-Lys9 in the absence of global histone acetylation. Thus, not all genes expressed by ethanol are linked to transcription via histone H3 acetylation at Lys9.

Knock down of GCN5 histone acetyltransferase by siRNA decreases ethanol-induced histone acetylation and affects differential expression of genes in human hepatoma cells

We have investigated whether Gcn5, a histone acetyltransferase (HAT), is involved in ethanol-induced acetylation of histone H3 at lysine 9 (H3AcK9) and has any effect on the gene expression. Human hepatoma HepG2 cells transfected with ethanol-metabolizing enzyme alcohol dehydrogenase 1 (VA 13 cells) were used. Knock down of Gcn5 by siRNA silencing decreased mRNA and protein levels of general control nondepressible 5 (GCN5), HAT activity, and also attenuated ethanol-induced H3AcK9 in VA13 cells. Illumina gene microarray analysis using total RNA showed 940 transcripts affected by GCN5 silencing or ethanol. Silencing caused differential expression of 891 transcripts (≥1.5-fold upregulated or downregulated). Among these, 492 transcripts were upregulated and 399 were downregulated compared with their respective controls. Using a more stringent threshold (≥2.5-fold), the array data from GCN5-silenced samples showed 57 genes differentially expressed (39 upregulated and 18 downregulated). Likewise, ethanol caused differential regulation of 57 transcripts with ≥1.5-fold change (35 gene upregulated and 22 downregulated). Further analysis showed that eight genes were differentially regulated that were common for both ethanol treatment and GCN5 silencing. Among these, SLC44A2 (a putative choline transporter) was strikingly upregulated by ethanol (three fold), and GCN5 silencing downregulated it (1.5-fold). The quantitative real-time polymerase chain reaction profile corroborated the array findings. This report demonstrates for the first time that (1) GCN5 differentially affects expression of multiple genes, (2) ethanol-induced histone H3-lysine 9 acetylation is mediated via GCN5, and (3) GCN5 is involved in ethanol-induced expression of the putative choline transporter SLC44A2.

Molecular mechanisms regulating expression and function of transcription regulator “inhibitor of differentiation 3”

AbstractThe transcription factor antagonist inhibitor of differentiation 3 (Id3) has been implicated in many diverse developmental, physiological and pathophysiological processes. Its expression and function is subjected to many levels of complex regulation. This review summarizes the current understanding of these mechanisms and describes how they might be related to the diverse functions that have been attributed to the Id3 protein. Detailed understanding of these mechanisms should provide insights towards the development of therapeutic approaches to various diseases, including cancer and atherogenesis.

Involvement of p27kip1 and cyclin D3 in the regulation of cdk2 activity during skeletal muscle differentiation

Abstract Terminal myogenic differentiation involves an irreversible transition from a proliferative state to a post-mitotic quiescent state. We showed here that in addition to the previously reported down regulation of G 1 -related cyclin-associated kinase activities, this transition was also accompanied by an extensive reorganization of the cyclin–cdk complexes, including a dramatic shift of cdk2 from cyclin A to cyclin D3. Moreover, the inhibition of cdk activity also correlated with an increase in the expression of the p27 kip1 cdk inhibitor and in its association with the cyclin–cdk2 complexes. Since depletion of p27 substantially reduced the cdk inhibitor activity present in differentiated muscle cells, we believe that the increase in p27 expression along with the reorganization of the cyclin–cdk2 complexes may play an important role in the inhibition of cdk2 activity during the differentiation process.

Different Mechanisms for Histone Acetylation by Ethanol and Its Metabolite Acetate in Rat Primary Hepatocytes

Ethanol and its major metabolite acetate both induced histone H3 acetylation in primary culture of rat hepatocytes. The acetylation by ethanol was dependent on the reactive oxygen species and mitogen-activated protein kinase pathway, whereas that by acetate was independent of both pathways. Ethanol increased CYP2E1 protein expression but acetate had negligible effect. The level of phospho-H2AX, an indicator of DNA breaks, was elevated by ethanol but not by acetate. Ethanol and acetate differentially activated mRNA expression for different genes, e.g., IL-6, PPARγ, c-Fos, Egr-1, and PNPLA3 in hepatocytes. The most striking increase (3-fold) was in PNPLA3 mRNA by ethanol with little change by acetate. It was further shown that acetate inhibited histone deacetylase activity. Taken together, these data establish for the first time that ethanol and acetate exhibit differences in their effects on hepatocytes in gene expression, P-H2AX levels, and the mechanism of histone H3 acetylation. The implications of these differences in the actions of ethanol in liver are discussed.