Jorge L. Sepulveda

GATA-4 and Nkx-2.5 Coactivate Nkx-2 DNA Binding Targets: Role for Regulating Early Cardiac Gene Expression

ABSTRACT The cardiogenic homeodomain factor Nkx-2.5 and serum response factor (SRF) provide strong transcriptional coactivation of the cardiac α-actin (αCA) promoter in fibroblasts (C. Y. Chen and R. J. Schwartz, Mol. Cell. Biol. 16:6372–6384, 1996). We demonstrate here that Nkx-2.5 also cooperates with GATA-4, a dual C-4 zinc finger transcription factor expressed in early cardiac progenitor cells, to activate the αCA promoter and a minimal promoter, containing only multimerized Nkx-2.5 DNA binding sites (NKEs), in heterologous CV-1 fibroblasts. Transcriptional activity requires the N-terminal activation domain of Nkx-2.5 and Nkx-2.5 binding activity through its homeodomain but does not require GATA-4’s activation domain. The minimal interactive regions were mapped to the homeodomain of Nkx-2.5 and the second zinc finger of GATA-4. Removal of Nkx-2.5’s C-terminal inhibitory domain stimulated robust transcriptional activity, comparable to the effects of GATA-4 on wild-type Nkx-2.5, which in part facilitated Nkx-2.5 DNA binding activity. We postulate the following simple model: GATA-4 induces a conformational change in Nkx-2.5 that displaces the C-terminal inhibitory domain, thus eliciting transcriptional activation of promoters containing Nkx-2.5 DNA binding targets. Therefore, αCa promoter activity appears to be regulated through the combinatorial interactions of at least three cardiac tissue-enriched transcription factors, Nkx-2.5, GATA-4, and SRF.

Cardiac Tissue Enriched Factors Serum Response Factor and GATA-4 Are Mutual Coregulators

ABSTRACT Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal α-actin promoter. GATA-4s C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

Products of the Colonic Microbiota Mediate the Effects of Diet on Colon Cancer Risk

It is estimated that most colon cancers can be attributed to dietary causes. We have hypothesized that diet influences the health of the colonic mucosa through interaction with the microbiota and that it is the milieu interior that regulates mucosal proliferation and therefore cancer risk. To validate this further, we compared colonic contents from healthy 50- to 65-y-old people from populations with high and low risk, specifically low risk Native Africans (cancer incidence <1:100,000; n = 17), high risk African Americans (risk 65:100,000; n = 17), and Caucasian Americans (risk 50:100,000; n = 18). Americans typically consume a high-animal protein and -fat diet, whereas Africans consume a staple diet of maize meal, rich in resistant starch and low in animal products. Following overnight fasting, rapid colonic evacuation was performed with 2 L polyethylene glycol. Total colonic evacuants were analyzed for SCFA, vitamins, nitrogen, and minerals. Total SCFA and butyrate were significantly higher in Native Africans than in both American groups. Colonic folate and biotin content, measured by Lactobacillus rhamnoses and Lactobacillus plantarum ATCC 8014 bioassay, respectively, exceeded normal daily dietary intakes. Compared with Africans, calcium and iron contents were significantly higher in Caucasian Americans and zinc content was significantly higher in African Americans, but nitrogen content did not differ among the 3 groups. In conclusion, the results support our hypothesis that the microbiota mediates the effect diet has on colon cancer risk by their generation of butyrate, folate, and biotin, molecules known to play a key role in the regulation of epithelial proliferation.

Identification of a Second MutL DNA Mismatch Repair Complex (hPMS1 and hMLH1) in Human Epithelial Cells

Deficiencies of MutL DNA mismatch repair-complex proteins (hMLH1, hPMS2, and hPMS1) typically result in microsatellite instability in human cancers. We examined the association patterns of MutL proteins in human epithelial cancer cell lines with (HCT-116, N87, SNU-1, and SNU-638) and without microsatellite instability (HeLa, AGS, KATO-III, and SNU-16). The analysis of hMLH1, hPMS2, and hPMS1 was performed using Northern blot, Western blot, and co-immunoprecipitation studies. Our data provide evidence that MutL proteins form two different complexes, MutL-α (hPMS2 and hMLH1) and MutL-β (hPMS1 and hMLH1). Gastric and colorectal cancer cells lines with microsatellite instability lacked detectable hMLH1. Decreased levels of hMLH1 protein were associated with markedly reduced levels of hPMS2 and hPMS1 proteins, but the RNA levels of hPMS1 and hPMS2 were normal. In this study, we describe the association of hPMS1 with hMLH1 as a heterodimer, in human cells. Furthermore, normal levels of hMLH1 protein appear to be important in maintaining normal levels of hPMS1 and hPMS2 proteins.

Role of the integrin-linked kinase/PINCH1/alpha-parvin complex in cardiac myocyte hypertrophy

Outside-in signaling from fibronectin (FN) through integrin receptors has been shown to play an important role in promoting cardiac myocyte hypertrophy and synergizes with other hypertrophic stimuli such as the alpha-adrenergic agonist phenylephrine (PE) and mechanical strain. The integrin-linked kinase (ILK) is a critical molecule involved in cell adhesion, motility and survival in nonmyocytes such as fibroblasts and epithelial cells. Its role in cardiac myocytes is unclear. In this study, we demonstrate that (1) ILK forms a complex with PINCH1 and alpha-parvin proteins (IPAP1 complex) in neonatal rat ventricular myocytes; (2) localization of IPAP1 complex proteins to costameres in cardiac myocytes is stimulated by FN, PE and synergistically by the combination of FN and PE in an integrin β1-dependent manner; (3) a dominant-negative mutant lacking the PINCH-binding N-terminus of ILK (ILK-C) prevents costamere association of ILK and alpha-parvin, but not PINCH1; (4) FN- and PE-induced hypertrophy, measured by increased protein/DNA ratio, beating frequency and atrial natriuretic peptide expression, is stimulated by low levels of ILK-C but repressed by high ILK-C expression; and (5) overexpression of ILK-C, as well as deletion of the ILK gene in mouse neonatal ventricular myocytes, induces marked apoptosis of cardiac myocytes. These results suggest that the IPAP1 complex plays an important role in mediating integrin-signaling pathways that regulate cardiac myocyte hypertrophy and resistance to apoptosis.

Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells.

Background:  Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability‐type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability‐positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection.

Functional identification of LZTS1 as a candidate prostate tumor suppressor gene on human chromosome 8p22

Deletions in the 8p21-22 region of the human genome are among the most common genetic alterations in prostate carcinomas. Several studies in different tumor tissues, including prostate, indicate that there are probably multiple tumor suppressor genes (TSGs) present in this region. To identify candidate TSGs on 8p22 a YAC contig spanning this region was assembled and YAC clones retrofitted with a selectable marker (neo) were transferred into rat prostate AT6.2 cells. Two overlapping YAC clones showed greatly reduced colony-forming efficiency, indicating they may carry a TSG. Two BAC clones encompassing the overlapping region also appeared to exert suppressive effects on the growth of AT6.2 cells. Database searches for genes mapped to the critical region identified a gene known as FEZ1 (LZTS1) as a potential candidate suppressor gene. Subsequent experiments showed that over-expression of LZTS1 cDNA inhibited stable colony-forming efficiencies of AT6.2, HEK-293 and LNCaP cells. In contrast, LZTS1-transfected Rat-1 and RM1 cells were growth-stimulated. Database searches also identified additional isoforms of the LZTS1 mRNA, as well as LZTS1 protein domains reminiscent of those found in transcription factors. Together these data suggest that the LZTS1 gene is involved in the regulation of cell growth and its loss of function may contribute to the development of prostatic carcinomas, as well as other cancers.

Assembly and signaling of adhesion complexes.

Cell-extracellular matrix (ECM) adhesion is crucial for control of cell behavior. It connects the ECM to the intracellular cytoskeleton and transduces bidirectional signals between the extracellular and intracellular compartments. The subcellular machinery that mediates cell-ECM adhesion and signaling is complex. It consists of transmembrane proteins (e.g., integrins) and at least several dozens of membrane-proximal proteins that assemble into a network through multiple protein interactions. Furthermore, despite sharing certain common components, cell-ECM adhesions exhibit considerable heterogeneity in different types of cells (e.g., the cell-ECM adhesions in cardiac myocytes are considerably different from those in fibroblasts). Here, we will first briefly describe the general properties of the integrin-mediated cell-ECM adhesion and signal transduction. Next, we will focus on one of the recently discovered cell-ECM adhesion protein complexes consisting of PINCH, integrin-linked kinase (ILK), and Parvin and use it as an example to illustrate the molecular basis underlying the assembly and functions of cell-ECM adhesions. Finally, we will discuss in detail the structure and regulation of cell-ECM adhesion complexes in cardiac myocytes, which illustrate the importance and complexity of the cell-ECM adhesion structures in organogenesis and diseases.

LAPSER1: a novel candidate tumor suppressor gene from 10q24.3.

Numerous LOH and mutation analysis studies in different tumor tissues, including prostate, indicate that there are multiple tumor suppressor genes (TSGs) present within the human chromosome 8p21–22 and 10q23–24 regions. Recently, we showed that LZTS1 (or FEZ1), a putative TSG located on 8p22, has the potential to function as a cell growth modulator. We report here the cloning, gene organization, cDNA sequence characterization and expression analysis of LAPSER1, an LZTS1-related gene. This gene maps within a subregion of human chromosome 10q24.3 that has been reported to be deleted in various cancers, including prostate tumors, as frequently as the neighboring PTEN locus. The complete LAPSER1 cDNA sequence encodes a predicted protein containing various domains resembling those typically found in transcription factors (P-Box, Q-rich and multiple leucine zippers). LAPSER1 is expressed at the highest levels in normal prostate and testis, where multiple isoforms are seen, some of which are either undetectable or differentially expressed in some prostate tumor tissues and cell lines. Over-expression of LAPSER1 cDNA strongly inhibited cell growth and colony-forming efficiencies of most cancer cells assessed. Together these data suggest that LAPSER1 is another gene involved in the regulation of cell growth whose loss of function may contribute to the development of cancer.

The Bladder Tumor Suppressor Protein TERE1 (UBIAD1)Modulates Cell Cholesterol: Implications for Tumor Progression

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyders corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression.