Antonio Martinez-Hernandez

A critical role of Sp1 transcription factor in regulating gene expression in response to insulin and other hormones

Specificity protein 1 (Sp1) belongs to a family of ubiquitously expressed, C(2)H(2)-type zinc finger-containing DNA binding proteins that activate or repress transcription of many genes in response to physiological and pathological stimuli. There is emerging evidence to indicate that in addition to functioning as housekeeping transcription factors, members of Sp family may be key mediators of gene expression induced by insulin and other hormones. The founding member of the family, Sp1, by virtue of its multi-domain organization, potential for posttranslational modifications and interactions with numerous transcription factors, represents an ideal mediator of nuclear signaling in response to hormones. Insulin regulates the sub-cellular localization, stability and trans-activation potential of Sp1 by dynamically modulating its post-translational modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) or phosphate residues. We briefly review the recent literature demonstrating that an involvement of Sp-family of transcription factors in the regulation of differential gene expression in response to hormones is more common than previously appreciated and may represent a key regulatory mechanism.

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.

CpG DNA-mediated Induction of Acute Liver Injury in d-Galactosamine-sensitized Mice THE MITOCHONDRIAL APOPTOTIC PATHWAY-DEPENDENT DEATH OF HEPATOCYTES

Unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce innate inflammatory responses, including rapid induction of proinflammatory cytokines. Although innate inflammatory responses induced by CpG DNA and other pathogen-associated molecular patterns are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is detrimental to the host. In this study, we demonstrate that CpG DNA, but not control non-CpG DNA, induces a fulminant liver failure with subsequent shock-mediated death by promoting massive apoptotic death of hepatocytes in d-galactosamine (d-GalN)-sensitized mice. Inhibition of mitochondrial membrane permeability transition pore opening or caspase 9 activity in vivo protects d-GalN-sensitized mice from the CpG DNA-mediated liver injury and death. CpG DNA enhanced production of proinflammatory cytokines in d-GalN-sensitized mice via a TLR9/MyD88-dependent pathway. In addition, CpG DNA failed to induce massive hepatocyte apoptosis and subsequent fulminant liver failure and death in d-GalN-sensitized mice that lack TLR9, MyD88, tumor necrosis factor (TNF)-α, or TNF receptor I but not interleukin-6 or -12p40. Taken together, our results provide direct evidence that CpG DNA induces a severe acute liver injury and shock-mediated death through the mitochondrial apoptotic pathway-dependent death of hepatocytes caused by an enhanced production of TNF-α through a TLR9/MyD88 signaling pathway in d-GalN-sensitized mice.

CpG DNA Prevents Liver Injury and Shock-mediated Death by Modulating Expression of Interleukin-1 Receptor-associated Kinases

Tumor necrosis factor-α (TNF-α) produced by macrophages in response to CpG DNA induces severe liver injury and subsequent death of d-galactosamine (d-GalN)-sensitized mice. In the present study we demonstrate that mice pre-exposed to CpG DNA are resistant to liver injury and death induced by CpG DNA/d-GalN. CpG DNA/d-GalN failed to induce TNF-α production and hepatocyte apoptosis in the mice pre-exposed to CpG DNA. In addition, macrophages isolated from the CpG DNA-pretreated mice showed suppressed activation of MAPKs and NF-κB and production of TNF-α in response to CpG DNA, indicating that the CpG DNA-mediated protection of CpG DNA/d-GalN-challenged mice is due to the hyporesponsiveness of macrophages to CpG DNA. CpG DNA pretreatment in vivo inhibited expression of interleukin-1 receptor-associated kinase (IRAK)-1 while inducing IRAK-M expression in macrophages. Suppressed expression of IRAK-1 was responsible for the macrophage hyporesponsiveness to CpG DNA. However, increased expression of IRAK-M was not sufficient to render macrophages hyporesponsive to CpG DNA but was required for induction of the optimal level of macrophage hyporesponsiveness. Taken together, reduced expression of IRAK-1 and increased expression of IRAK-M after CpG DNA pretreatment resulted in the hyporesponsiveness of macrophages that leads to the protection of mice from hepatic injury and death caused by CpG DNA/d-GalN.

Transplantation of a liver with the C282Y mutation into a recipient heterozygous for H63D results in iron overload.

Hemochromatosis is a common hereditary disease associated with progressive iron overload eventually leading to parenchymal damage of the liver, heart, pancreas, and other organs. Liver transplantation has been the single most important therapy to extend long-term survival in patients with a variety of acute and chronic liver diseases. We report a case of inadvertent transplantation of a hemochromatotic liver into a nonhemochromatotic recipient, resulting in rapid iron overload. Neither the recipient nor the donor had iron overload at the time of transplantation, but the donor liver was subsequently found to be homozygous for C282Y mutation. The report includes 8 years follow-up, serial biopsies, and molecular studies. Iron overload in our patient transplanted with a C282Y homozygous liver provides an in vivo model for the pathophysiology of hemochromatosis and further supports liver playing a primary role in the maintenance of iron hemostasis rather intestine being the sole regulatory site.

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.