Mariano Ubeda

Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.

CHOP (GADD153) is a mammalian nuclear protein that dimerizes with members of the C/EBP family of transcriptional factors. Absent under normal conditions, CHOP is induced by the stress encountered during nutrient deprivation, the acute-phase response, and treatment of cells with certain toxins. The basic region of CHOP deviates considerably in sequence from that of other C/EBP proteins, and CHOP-C/EBP heterodimers are incapable of binding to a common class of C/EBP sites. With respect to such sites, CHOP serves as an inhibitor of the activity of C/EBP proteins. However, recent studies indicate that certain functions of CHOP, such as the induction of growth arrest by overexpression of the wild-type protein and oncogenic transformation by the TLS-CHOP fusion protein, require an intact basic region, suggesting that DNA binding by CHOP may be implicated in these activities. In this study an in vitro PCR-based selection assay was used to identify sequences bound by CHOP-C/EBP dimers. These sequences were found to contain a unique core element PuPuPuTGCAAT(A/C)CCC. Competition in DNA-binding assays, DNase 1 footprint analysis, and methylation interference demonstrate that the binding is sequence specific. Deletions in the basic region of CHOP lead to a loss of DNA binding, suggesting that CHOP participates in this process. Stress induction in NIH 3T3 cells leads to the appearance of CHOP-containing DNA-binding activity. CHOP is found to contain a transcriptional activation domain which is inducible by cellular stress, lending further support to the notion that the protein can function as a positively acting transcription factor. We conclude that CHOP may serve a dual role both as an inhibitor of the ability of C/EBP proteins to activate some target genes and as a direct activator of others.

CHOP Enhancement of Gene Transcription by Interactions with Jun/Fos AP-1 Complex Proteins

ABSTRACT The transcription factor CHOP (C/EBP homologous protein 10) is a bZIP protein induced by a variety of stimuli that evoke cellular stress responses and has been shown to arrest cell growth and to promote programmed cell death. CHOP cannot form homodimers but forms stable heterodimers with the C/EBP family of activating transcription factors. Although initially characterized as a dominant negative inhibitor of C/EBPs in the activation of gene transcription, CHOP-C/EBP can activate certain target genes. Here we show that CHOP interacts with members of the immediate-early response, growth-promoting AP-1 transcription factor family, JunD, c-Jun, and c-Fos, to activate promoter elements in the somatostatin, JunD, and collagenase genes. The leucine zipper dimerization domain is required for interactions with AP-1 proteins and transactivation of transcription. Analyses by electrophoretic mobility shift assays and by an in vivo mammalian two-hybrid system for protein-protein interactions indicate that CHOP interacts with AP-1 proteins inside cells and suggest that it is recruited to the AP-1 complex by a tethering mechanism rather than by direct binding of DNA. Thus, CHOP not only is a negative or a positive regulator of C/EBP target genes but also, when tethered to AP-1 factors, can activate AP-1 target genes. These findings establish the existence of a new mechanism by which CHOP regulates gene expression when cells are exposed to cellular stress.

Inhibition of cyclin-dependent kinase 5 activity protects pancreatic beta cells from glucotoxicity.

Type 2 diabetes (T2D) and Alzheimer disease are degenerative diseases that may share common pathophysiologic mechanisms. Neuronal dysfunction in Alzheimer patients has been linked to overactivity of the cyclin-dependent kinase 5 (CDK5) and its activator p35. Both of these proteins are expressed in the insulin-producing beta cells of the pancreas. Further, glucose enhances p35 gene expression, promoting the formation of active p35/CDK5 complexes that regulate the expression of the insulin gene. In T2D, chronic elevations of glucose, glucotoxicity, impair beta cell function. We therefore postulated that CDK5 and p35 may be responsible for this beta cell impairment and that inhibition of CDK5 might have a beneficial effect. To test this hypothesis, the pancreatic cell line INS-1 was selected as a known in vitro model of glucotoxicity, and roscovitine (10 μm) was used as a CDK5 inhibitor. Chronic exposure of INS-1 cells to high glucose (20-30 mm) reduced both insulin mRNA levels and the activity of an insulin promoter reporter gene. Inhibition of CDK5 prevented this decrease of insulin gene expression. We used DNA binding (gel shift) assays and Western immunoblots to demonstrate that cellular levels of the transcription factor PDX-1, normally decreased by glucotoxicity, were preserved with CDK5 inhibition, as was the binding of PDX-1 to the insulin promoter. Analyses of nuclear and cytoplasmic PDX-1 protein levels revealed that CDK5 inhibition restores nuclear PDX-1, without affecting its cytoplasmic concentration, suggesting that CDK5 regulates the nuclear/cytoplasm partitioning of PDX-1. Using a Myc-tagged PDX-1 construct, we showed that the translocation of PDX-1 from the nucleus to the cytoplasm during glucotoxic conditions was prevented when CDK5 was inhibited. These studies indicate that CDK5 plays a role in the loss of beta cell function under glucotoxic conditions and that CDK5 inhibitors could have therapeutic value for T2D.

Identification and functional characterization of melatonin Mel 1a receptors in pancreatic β cells: potential role in incretin-mediated cell function by sensitization of cAMP signaling

Melatonin receptors are expressed within the pancreatic islets of Langerhans, and melatonin induces a direct effect on insulin secretion ex-vivo. Here, we report the endogenous expression of the melatonin Mel 1a receptor in the INS-1 pancreatic beta cell line. Pharmacological characterization of the receptor using a CRE-luciferase reporter gene demonstrated its functional activity in INS-1 cells, displaying the characteristic signaling properties of the G(i/o) coupled receptor. Acute melatonin treatment of INS-1 cells in the presence of either forskolin or the incretin hormone glucagon-like peptide 1 (GLP-1) caused an attenuation of the responses in insulin secretion, insulin promoter activity, and CRE mediated gene expression, consistent with its effects in inhibiting cAMP mediated signal transduction. However, prolonged exposure (12 h) of INS-1 cells to melatonin treatment resulted in a sensitization of cAMP mediated responses to forskolin and GLP-1. Insulin secretion, insulin promoter activity and CRE mediated gene expression levels were augmented compared with responses without melatonin pre-treatment in INS-1 cells. In isolated rat islets, insulin secretion was enhanced following melatonin pre-treatment both in the absence and presence of GLP-1 or forskolin. This phenomenon reflects observations reported in other cell types expressing the melatonin Mel 1a receptor, and may represent the first evidence of a specific physiological role for melatonin-induced sensitization.

Human Pancreatic Islet-Derived Progenitor Cell Engraftment in Immunocompetent Mice

The potential for the use of stem/progenitor cells for the restoration of injured or diseased tissues has garnered much interest recently, establishing a new field of research called regenerative medicine. Attention has been focused on embryonic stem cells derived from human fetal tissues. However, the use of human fetal tissue for research and transplantation is controversial. An alternative is the isolation and utilization of multipotent stem/progenitor cells derived from adult donor tissues. We have previously reported on the isolation, propagation, and partial characterization of a population of stem/progenitor cells isolated from the pancreatic islets of Langerhans of adult human donor pancreata. Here we show that these human adult tissue-derived cells, nestin-positive islet-derived stem/progenitor cells, prepared from human adult pancreata survive engraftment and produce tissue chimerism when transplanted into immunocompetent mice either under the kidney capsule or by systemic injection. These xenografts seem to induce immune tolerance by establishing a mixed chimerism in the mice. We propose that a population of stem/progenitor cells isolated from the islets of the pancreas can cross xenogeneic transplantation immune barriers, induce tissue tolerance, and grow.

CHOP transcription factor phosphorylation by casein kinase 2 inhibits transcriptional activation.

The CAAT/enhancer binding protein homologous transcription factor CHOP, also known as GADD153, is involved in DNA damage, growth arrest, and the induction of apoptosis after endoplasmic reticulum stress and nutrient deprivation. CHOP dimerizes with and inhibits the binding of C/EBP-related transcription factors to their consensus DNA target sequences and also forms novel complexes with other transcriptional proteins (e.g. c-Jun, c-Fos). The transcriptional activation of these complexes is modified by their phosphorylation. Phosphorylation of CHOP at serine 79 and serine 81 by p38-MAP kinase enhances its transcriptional activity. Here we show that an interactive association between CHOP and casein kinase II (CK2) results in the phosphorylation of its amino-terminal transactivation domain. Mapping of the functional domains of CHOP indicates that the region in CHOP required for association with CK2 differs from that required for its phosphorylation. Th binding of CK2 to CHOP requires only the carboxylterminal bZip domain of CHOP, whereas phosphorylation involves residues located in the amino-terminal domain. The presence of the bZip domain, however, facilitates the phosphorylation of CHOP. Analyses of the effect of point mutations of CHOP on its transcriptional activity and the effect of specific inhibitors of CK2 lead us to conclude that CK2-mediated phosphorylation of CHOP inhibits its transcriptional activity. Our findings suggest that inhibition of the proapoptotic functions of CHOP by CK2 may be a mechanism by which CK2 prevents apoptosis and promotes cellular proliferation.

The Large Subunit of the DNA Replication Complex C (DSEB/RF-C140) Cleaved and Inactivated by Caspase-3 (CPP32/YAMA) during Fas-induced Apoptosis

We report the identification of the large subunit of the DNA replication factor, DSEB/RF-C140, as a new substrate for caspase-3 (CPP32/YAMA), or a very closely related protease activated during Fas-induced apoptosis in Jurkat T cells. DSEB/RF-C140 is a multifunctional DNA-binding protein with sequence homology to poly(ADP-ribose) polymerase (PARP). This similarity includes a consensus DEVD/G cleavage site for caspase-3. Cleavage of DSEB/RF-C140 is predicted to occurs between Asp706 and Gly707, generating 87-kDa and 53-kDa fragments. An antiserum raised against the amino-terminal domain of DSEB/RF-C140 detects a new 87-kDa protein in Jurkat T cells in which apoptosis is activated by a monoclonal antibody to Fas. This cleavage occurs shortly after PARP cleavage. In vitro translated DSEB/RF-C140 is specifically cleaved into the predicted fragments when incubated with a cytoplasmic extract from Fas antibody-treated cells. Proteolytic cleavage was prevented by substituting Asp706 by an alanine in the DEVD706/G caspase-3 cleavage site. The cleavage of DSEB/RF-C140 is prevented by iodoacetamide and the specific caspase-3 inhibitor, tetrapeptide aldehyde Ac-DEVD-CHO, but not by the specific ICE (interleukin-1-converting enzyme) inhibitors: CrmA and Ac-YVAD-CHO, indicating that the protease responsible for the cleavage of DSEB/RF-C140 during Fas-induced apoptosis in Jurkat cells is caspase-3, or a closely related protease. This conclusion is reinforced by the fact that recombinant caspase-3 but not caspase-1 reproduced the “in vivo” cleavage. Inasmuch as the cleavage of DSEB/RF-C140 separates its DNA binding from its association domain, required for replication complex formation, we propose that such a cleavage will impair DNA replication. Recent in vitromutagenesis support this proposal (Uhlmann, F., Cai, J., Gibbs, E., O’Donnel, M., and Hurwitz, J. (1997) J. Biol. Chem. 272, 10058–10064).

NRSF/REST confers transcriptional repression of the GPR10 gene via a putative NRSE/RE-1 located in the 5' promoter region.

The G protein‐coupled receptor GPR10 is highly localized to areas of the brain. In an effort to reveal transcriptional determinants of this tissue specificity, we recognized a putative NRSE (neuron‐restrictive silencer element) located in the 5′ promoter region of the gene. The cognate NRSE binding protein NRSF (neuron‐restrictive silencer factor) restricts gene expression to mature neurons and endocrine cells by repressing their transcription in non‐neuronal/‐endocrine cells. In cell lines where NRSF‐mediated gene repression has been functionally established, the activity of the GPR10 promoter was repressed in a manner consistent with NRSE‐dependent regulation. A specific point mutation to confer non‐functionality of the NRSE revealed a 10‐fold de‐repression of reporter gene expression. In contrast, in the GPR10‐expressing cell line GH3, mRNA transcripts of NRSF were undetectable and suppression of promoter activity was not observed. However, transfection of a rat NRSF expression vector resulted in significant repression of transcription, which was reversed by mutation of the NRSE. In conclusion, we demonstrate that the GPR10 gene is specifically regulated by NRSF, and suggest this to be a contributory factor in the tissue‐specific distribution of GPR10 in vivo.