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Acetyl-CoA carboxylase (ACC) exists as two major isoforms originated from separate genes: ACC␣ (or ACC1) and ACC␤ (or ACC2). Previous data revealed that ACC␤ has two forms of mRNA with different 5-untranslated regions derived by different usage of promoters, I and II, in human. In this study, we revealed that ACC␤ expression in liver is markedly stimulated by food intake at the transcriptional level. In the process of this induction in rat liver, promoter II plays the major role in regulating the expression of ACC␤ gene. The transient transfection with promoter II-luciferase reporters elucidated that the region from ؊93 to ؊38 nucleotides is important for the responsiveness to sterol regulatory element-binding protein-1 (SREBP-1), which is known to be the principle mediator for the stimulation of gene transcriptions by insulin and diet. The Sp1-binding site (؊71 to ؊66) and neighboring two conserved SREs (؊62 to ؊44) play a critical role in the stimulation of ACC␤ gene expression by SREBP-1. In vivo chromatin immunoprecipitation assay revealed that SREBP-1 directly bound to ACC␤ promoter II in liver, and its binding was regulated by the diet. This study provides evidence that ACC␤ expression in liver is regulated at the transcriptional level by the direct interaction of SREBP-1 with promoter II. Acetyl-CoA carboxylase (ACC)1 catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which is served not only as the substrate for fatty acid biosynthesis but also as a signal molecule for metabolic control of fatty acid ␤-oxidation in skeletal muscle and insulin secretion in pancreatic ␤ cells (1). Two isoforms of ACC have been identified (ACC␣ and ACC␤). These two isoforms are encoded by the separate genes and display distinct tissue distribution. The ␣ isoform of ACC (also called ACC1, 265 kDa) is mainly distributed in liver and adipose tissue, where lipogenesis is active. In contrast, the ␤ isoform of ACC (also called as ACC2, 275 kDa) is the predominant carboxylase in skeletal muscle and heart, where fatty acid ␤-oxidation serves as the main energy source (2). ACC␤ shows considerable homology to ACC␣ except the additional NH 2 -terminal portion, comprised of about 200 amino acids, which is known to direct ACC␤ to the outer membrane of mitochondria (3, 4). The level of malonyl-CoA generated by ACC␤ around mitochondria functions as the important factor in regulating mitochondrial fatty acid ␤-oxidation through inhibition of carnitine palmitoyl-CoA transferase I. The activities of ACC␤ in skeletal muscle are mainly regulated by phosphorylation and dephosphorylation but not by changes of enzyme contents (5-8). For example, sympathetic nerve stimulation or exercise increases the phosphorylation of ACC␤, resulting in the inhibition of ACC␤ activities and the increase of fatty acid oxidation (7)(8)(9)(10)(11).ACC␤ is also expressed in liver and HepG2 cells (4, 12). Oxidation of fatty acid also occurs actively in liver, but its regulation is different from that in skeletal muscle. In liver, fatty acid oxidation is increa...

1 Zinc is an important trace element in the body and is involved in both the proliferation and growth arrest of many kinds of cells including colorectal epithelial cells. The aim of this study was to identify the molecular mechanism of the growth regulation of colorectal cancer cells by extracellular zinc. 2 Zinc-stimulated activation of the mitogen-activated protein kinase (MAPK) cascade was measured by immunoblotting and Elk-1 dependent trans-reporter gene expression, and zincstimulated p21 Cip/WAF1 activation by immunoblotting, Northern blot analysis and immunochemistry. Cell proliferation was measured by thymidine and bromodeoxyuridine (BrdU) incorporation. 3 By treating colorectal cancer cells with 100 mM ZnCl 2 , MAPKs were activated in two di erent phases, the initial weak activation occurred within 5 min and this was followed by a stronger and more prolonged activation. 4 Zinc concomitantly activated Raf-1-MEK-MAPK kinases, and induced Elk-1 dependent transreporter gene expression. 5 Prolonged activation of MAPKs by 100 mM of ZnCl 2 resulted in the induction and nuclear localization of p21 Cip/WAF1 and was related to the inhibition of both thymidine and BrdU incorporations. 6 These results not only suggest the presence of a mechanism for p21 Cip/WAF1 dependent negative regulation of colorectal cancer cell growth by zinc but also suggest potential usage of zinc to control the growth of colorectal cancer cells.

A partial C-terminal cDNA sequence of a novel Drosophila mitogen-activated protein kinase phosphatase (MKP), designated DMKP-3, was identified from an epitope expressed sequence tag database, and the missing N-terminal cDNA fragment was cloned from a Drosophila cDNA library. DMKP-3 is a protein of 411 amino acids, with a calculated molecular mass of 45.8kDa; the deduced amino acid sequence is most similar to that of mammalian MKP-3. Recombinant DMKP-3 produced in Escherichia coli retained intrinsic tyrosine phosphatase activity. In addition, DMKP-3 specifically inhibited extracellular-signal-regulated kinase (ERK) activity, but was without a significant affect on c-Jun N-terminal kinase (JNK) and p38 activities, when it was overexpressed in Schneider cells. DMKP-3 interacted specifically with Drosophila ERK (DERK) via its N-terminal domain. In addition, DMKP-3 specifically inhibited Elk-1-dependent trans-reporter gene expression in mammalian CV1 cells, and dephosphorylated activated mammalian ERK in vitro. DMKP-3 is uniquely localized in the cytoplasm within Schneider cells, and gene expression is tightly regulated during development. Thus DMKP-3 is a Drosophila homologue of mammalian MKP-3, and may play important roles in the regulation of various developmental processes.

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The liver-specific expression of the GLUT2 glucose transporter gene is suppressed in cultured hepatoma cell lines as well as in hepatocytes in primary culture. To understand the underlying mechanism involved in this process, we analysed the rat GLUT2 promoter region. A DNase I footprinting assay with rat liver nuclear extract revealed eight protected regions within a -500 bp region of the GLUT2 promoter (sites A to H). Three of these sites (B, F and H) were occupied by transcription factors that are considerably enriched in liver cells compared with spleen or kidney. The proteins binding to these sites were investigated by a combination of DNase I footprinting assay and electrophoretic mobility-shift assay with the addition of specific oligonucleotide competitors and specific antibody against known transcription factors. As a result it was revealed that hepatocyte nuclear factor 3 binds to site B (-120 to -70), and CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta bind to site F (-375 to -356) and site H (-500 to -471). The binding of C/EBP to sites F and H was markedly decreased within 4 h when liver cells were subjected to primary culture, suggesting that C/EBP might be responsible for the decreased expression of GLUT2 in this process. In contrast, Western blot analysis revealed that C/EBPalpha began to decrease after 1 h of hepatocyte culture, and C/EBPbeta was not changed significantly throughout the culture period, suggesting that C/EBP could be regulated at the transcriptional level as well as the post-translational level when hepatocytes were put in culture. To confirm the role of C/EBP in the regulation of GLUT2 promoter activity, sites F and H were ligated to a chloramphenicol acetyltransferase (CAT) reporter gene and co-transfected with a C/EBP expression vector into HepG2 cells. The co-expression of C/EBPalpha and C/EBPbeta resulted in 9.1-fold and 3. 8-fold increases of CAT activities in the site F-CAT and site H-CAT constructs respectively. These results indicate that C/EBPalpha and C/EBPbeta regulate the promoter activity of the GLUT2 gene and might be responsible for the down-regulation of the GLUT2 gene when hepatocytes are subjected to primary culture.

The incidence of prostate cancer (PC) is growing rapidly throughout the world, in probable association with the adoption of western style diets. Thus, understanding the molecular pathways triggering the development of PC is crucial for both its prevention and treatment. Here, we investigated the role of the metabolism-associated protein, CREB3L4, in the proliferation of PC cells. CREB3L4 was upregulated by the synthetic androgen, R1881, in LNCaP PC cells (an androgen-dependent cell line). Knockdown of CREB3L4 resulted in decreased androgen-dependent PC cell growth. LNCaP cells transfected with siCREB3L4 underwent G2/M arrest, with upregulation of the proteins cyclin B1, phospho-CDK1, p21Waf1/Cip1, and INCA1, and downregulation of cyclin D1. Moreover, depletion of CREB3L4 resulted in significantly decreased expression of a subset of androgen-receptor (AR) target genes, including PSA, FKBP5, HPGD, KLK2, and KLK4. We also demonstrated that CREB3L4 directly interacts with the AR, and increases the binding of AR to androgen response elements (AREs). We also identified a role for the unfolded protein response (and its surrogate, IRE1α), in activating CREB3L4. Cumulatively, we postulate that CREB3L4 expression is mediated by an AR-IRE1α axis, but is also directly regulated by AR-to-ARE binding. Thus, our study demonstrates that CREB3L4 plays a key role in PC cell proliferation, which is promoted by both AR and IRE1α.