ABSTRACT
Ets1 is a member of the Ets transcription factor family. Alternative splicing of exon VII results in two naturally occurring protein isoforms: full-length Ets1 (p51-Ets1) and Ets1(DeltaVII) (p42-Ets1). These isoforms bear key distinctions regarding protein-protein interactions, DNA binding kinetics, and transcriptional target specificity. Disruption of both Ets1 isoforms in mice results in the loss of detectable NK and NKT cell activity and defects in B and T lymphocytes. We generated mice that express only the Ets1(DeltaVII) isoform. Ets1(DeltaVII) homozygous mice express no p51-Ets1 and elevated levels of the p42-Ets1 protein relative to the wild type and display increased perinatal lethality, thymomegaly, and peripheral lymphopenia. Proliferation was increased in both the thymus and the spleen, while apoptosis was decreased in the thymus and increased in the spleen of homozygotes. Significant elevations of CD8(+) and CD8(+)CD4(+) thymocytes were observed. Lymphoid cell (CD19(+), CD4(+), and CD8(+)) reductions were predominantly responsible for diminished spleen cellularity, with fewer memory cells and a failure of homeostatic proliferation to maintain peripheral lymphocytes. Collectively, the Ets1(DeltaVII) mutants demonstrate lymphocyte maturation defects associated with misregulation of p16(Ink4a), p27(Kip1), and CD44. Thus, a balance in the differential regulation of Ets1 isoforms represents a potential mechanism in the control of lymphoid maturation and homeostasis.
Subject(s)
Homeostasis , Lymphocytes/cytology , Lymphocytes/metabolism , Proto-Oncogene Protein c-ets-1/deficiency , Proto-Oncogene Protein c-ets-1/metabolism , Spleen/metabolism , Thymus Gland/metabolism , Animals , Base Sequence , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Gene Expression Regulation , Heterozygote , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Mice , Molecular Sequence Data , Phenotype , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proto-Oncogene Protein c-ets-1/genetics , Spleen/cytology , Thymus Gland/cytology , Transcription, Genetic/geneticsABSTRACT
Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters.
Subject(s)
Antiporters/deficiency , Chlorides/metabolism , Colon/metabolism , Colon/pathology , Animals , Antiporters/genetics , Antiporters/physiology , Base Sequence , Cell Proliferation , DNA Primers/genetics , Female , H(+)-K(+)-Exchanging ATPase/metabolism , Ion Transport , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Sodium-Hydrogen Exchanger 3 , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Sulfate Transporters , Up-RegulationABSTRACT
The down-regulated in adenoma (DRA) gene is significantly down-regulated in adenomas and adenocarcinomas of the colon as well as in colon cancer cell lines. It is also mutated in the disease congenital chloride diarrhea, which is characterized by loss of chloride transport and diarrhea. We now show a second function for DRA relevant to colon tumorigenesis, i.e., growth suppression. Transfection of full-length DRA into various cell lines (DLD-1, HT-29, HCT-15, SW837, SW480, MCF-7, NIH3T3, CaSki, and HeLa) that lack endogenous DRA expression results in a reduced number of drug-resistant colonies compared with vector control, suggesting growth suppression by DRA. In addition, expression of DRA under the control of an inducible promoter reduced the growth rate of DLD-1 cells compared with cells not expressing DRA. The COOH-terminal cytoplasmic domain of DRA is required for growth suppression, but an in-frame deletion (DeltaVal317) that causes congenital chloride diarrhea and results in a loss of anion transport had no effect on growth suppression, indicating that anion transport and growth suppression are independent functions of DRA. One cell line, adenovirus-transformed HEK293, exhibited significant resistance to DRA-induced growth suppression, whereas the human papillomavirus-transformed cell lines, CaSki and HeLa, did not. E1A is an adenoviral protein required to transform HEK293 cells. DLD-1 cells that stably express 12S E1A are resistant to growth suppression by DRA, similar to HEK293 cells.
