title: GATA4 Regulates Blood-Testis Barrier Function and Lactate Metabolism in Mouse Sertoli Cells.
abstract: Conditional deletion of Gata4 in Sertoli cells (SCs) of adult mice has been shown to increase permeability of the blood-testis barrier (BTB) and disrupt spermatogenesis. To gain insight into the molecular underpinnings of these phenotypic abnormalities, we assessed the impact of Gata4 gene silencing in cell culture models. Microarray hybridization identified genes dysregulated by siRNA-mediated inhibition of Gata4 in TM4 cells, an immortalized mouse SC line. Differentially expressed genes were validated by quantitative RT-PCR analysis of primary cultures of Gata4(flox/flox) mouse SCs that had been subjected to cre-mediated recombination in vitro. Depletion of GATA4 in TM4 cells and primary SCs was associated with altered expression of genes involved in key facets of BTB maintenance, including tight/adherens junction formation (Tjp1, Cldn12, Vcl, Tnc, Csk) and extracellular matrix reorganization (Lamc1, Col4a1, Col4a5, Mmp10, Mmp23, Timp2). Western blotting and immunocytochemistry demonstrated reduced levels of tight junction protein-1, a prototypical tight junction protein, in GATA4-depleted cells. These changes were accompanied by a loss of morphologically recognizable junctional complexes and a decline in epithelial membrane resistance. Furthermore, Gata4 gene silencing was associated with altered expression of Hk1, Gpi1, Pfkp, Pgam1, Gls2, Pdk3, Pkd4, and Ldhb, genes regulating the production of lactate, a key nutrient that SCs provide to developing germ cells. Comprehensive metabolomic profiling demonstrated impaired lactate production in GATA4-deficient SCs. We conclude that GATA4 plays a pivotal role in the regulation of BTB function and lactate metabolism in mouse SCs.
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Anja
authors.initials: A
authors.lastname: Schrade
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Antti
authors.initials: A
authors.lastname: Kyrönlahti
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Oyediran
authors.initials: O
authors.lastname: Akinrinade
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Marjut
authors.initials: M
authors.lastname: Pihlajoki
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Simon
authors.initials: S
authors.lastname: Fischer
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Verena Martinez
authors.initials: VM
authors.lastname: Rodriguez
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Otte
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Vidya
authors.initials: V
authors.lastname: Velagapudi
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Jorma
authors.initials: J
authors.lastname: Toppari
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: David B
authors.initials: DB
authors.lastname: Wilson
authors.affiliation: Children's Hospital (A.S., A.K., O.A., M.P., M.H.), University of Helsinki and Helsinki University Central Hospital, Helsinki 00014, Finland; Institute of Applied Biotechnology (S.F., K.O.), University of Applied Sciences Biberach, Biberach 88400, Germany; Metabolomics Unit (V.V.), Institute for Molecular Medicine Finland, University of Helsinki 00014, Helsinki, Finland; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, Turku 20520, Finland; and Departments of Pediatrics (A.S., V.M.R., D.B.W., M.H.) and Developmental Biology (D.B.W.), Washington University, St Louis, Missouri 63110.
authors.forename: Markku
authors.initials: M
authors.lastname: Heikinheimo
mesh_terms: D000818:Animals; D001814:Blood-Testis Barrier / Q000378:metabolism; D015153:Blotting, Western; D024141:Collagen Type IV / Q000378:metabolism; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D007773:Lactates / Q000378:metabolism*; D007797:Laminin / Q000378:metabolism; D008297:Male; D053506:Matrix Metalloproteinase 10 / Q000378:metabolism; D053504:Matrix Metalloproteinases, Membrane-Associated / Q000378:metabolism; D051379:Mice; D034741:RNA, Small Interfering; D012708:Sertoli Cells / Q000378:metabolism*; D013091:Spermatogenesis / Q000502:physiology; D013737:Testis / Q000378:metabolism*; D019108:Tight Junctions / Q000378:metabolism; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: C000619517:Col4a1 protein, mouse; C549554:Col4a5 protein, mouse; D024141:Collagen Type IV; D050991:GATA4 Transcription Factor; C484171:Gata4 protein, mouse; D007773:Lactates; D007797:Laminin; D034741:RNA, Small Interfering; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; C089141:laminin gamma 1; D053504:Matrix Metalloproteinases, Membrane-Associated; C118502:Mmp24 protein, mouse; D053506:Matrix Metalloproteinase 10