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The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis.

Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, Enerbäck S - PLoS ONE (2009)

Bottom Line: Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner.Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter.Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

View Article: PubMed Central - PubMed

Affiliation: Center of Medical Genetics, Institute of Biomedicine, The Sahlgrenska Academy, Göteborg University, Göteborg, Sweden.

ABSTRACT
The vacuolar H(+)-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

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Confocal analysis of Foxi1 and the H+-ATPase subunits A1, E2 and a4 expression in wt and Foxi1−/− distal nephrons.Confocal images of wt and Foxi1−/− kidney sections. Double staining with Foxi1 and anti H+-ATPase antibodies showed exclusive expression of Foxi1 protein (green) in cells of distal nephron cells co-expressing subunits A1 (red; A–D), E2 (red; G–J) and a4 (red; M–P), in both cortex as well as medulla. Foxi1 is localized to the nuclei, which are stained blue (TOPRO 3), while the ATPase subunits are found apically, a4 (O–P) or both apically and in the cytosol, A1 (C–D) and E2 (I–J). No expression of ATPase subunits were detected in epithelial cells from Foxi1−/− kidneys, neither in medulla (E, K, Q) nor cortex (F, L, R). Scale bars 20 µm (L: lumen).
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pone-0004471-g002: Confocal analysis of Foxi1 and the H+-ATPase subunits A1, E2 and a4 expression in wt and Foxi1−/− distal nephrons.Confocal images of wt and Foxi1−/− kidney sections. Double staining with Foxi1 and anti H+-ATPase antibodies showed exclusive expression of Foxi1 protein (green) in cells of distal nephron cells co-expressing subunits A1 (red; A–D), E2 (red; G–J) and a4 (red; M–P), in both cortex as well as medulla. Foxi1 is localized to the nuclei, which are stained blue (TOPRO 3), while the ATPase subunits are found apically, a4 (O–P) or both apically and in the cytosol, A1 (C–D) and E2 (I–J). No expression of ATPase subunits were detected in epithelial cells from Foxi1−/− kidneys, neither in medulla (E, K, Q) nor cortex (F, L, R). Scale bars 20 µm (L: lumen).

Mentions: The subunits A1, E2 and a4 are expressed in a subset of epithelial kidney cells (Fig. 2). Based on previous findings in which Foxi1 has been established as a marker for intercalated cells [12] we can based on co-localization with Foxi1 determine that A1, E2 and a4 all are expressed in intercalated cells. In sections from both medulla and cortex we demonstrate a complete co-localization between Foxi1 and the subunits A1, E2 and a4. These subunits display a luminal staining pattern while the Foxi1 signals reside in the nuclei (Fig. 2 A–D, G–J, M–P). Kidneys from mice lacking Foxi1 have no detectable A1, E2 or a4 (Fig. 2 E, F, K, L, Q, R). The overlap in expression between these subunits and Foxi1 appears to be complete i.e. all Foxi1 cells studied are positive for A1, E2 and a4. It is known from previous work that the B1 subunit is expressed in intercalated cells and that its expression requires Foxi1 [12].


The forkhead transcription factor Foxi1 is a master regulator of vacuolar H-ATPase proton pump subunits in the inner ear, kidney and epididymis.

Vidarsson H, Westergren R, Heglind M, Blomqvist SR, Breton S, Enerbäck S - PLoS ONE (2009)

Confocal analysis of Foxi1 and the H+-ATPase subunits A1, E2 and a4 expression in wt and Foxi1−/− distal nephrons.Confocal images of wt and Foxi1−/− kidney sections. Double staining with Foxi1 and anti H+-ATPase antibodies showed exclusive expression of Foxi1 protein (green) in cells of distal nephron cells co-expressing subunits A1 (red; A–D), E2 (red; G–J) and a4 (red; M–P), in both cortex as well as medulla. Foxi1 is localized to the nuclei, which are stained blue (TOPRO 3), while the ATPase subunits are found apically, a4 (O–P) or both apically and in the cytosol, A1 (C–D) and E2 (I–J). No expression of ATPase subunits were detected in epithelial cells from Foxi1−/− kidneys, neither in medulla (E, K, Q) nor cortex (F, L, R). Scale bars 20 µm (L: lumen).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637605&req=5

pone-0004471-g002: Confocal analysis of Foxi1 and the H+-ATPase subunits A1, E2 and a4 expression in wt and Foxi1−/− distal nephrons.Confocal images of wt and Foxi1−/− kidney sections. Double staining with Foxi1 and anti H+-ATPase antibodies showed exclusive expression of Foxi1 protein (green) in cells of distal nephron cells co-expressing subunits A1 (red; A–D), E2 (red; G–J) and a4 (red; M–P), in both cortex as well as medulla. Foxi1 is localized to the nuclei, which are stained blue (TOPRO 3), while the ATPase subunits are found apically, a4 (O–P) or both apically and in the cytosol, A1 (C–D) and E2 (I–J). No expression of ATPase subunits were detected in epithelial cells from Foxi1−/− kidneys, neither in medulla (E, K, Q) nor cortex (F, L, R). Scale bars 20 µm (L: lumen).
Mentions: The subunits A1, E2 and a4 are expressed in a subset of epithelial kidney cells (Fig. 2). Based on previous findings in which Foxi1 has been established as a marker for intercalated cells [12] we can based on co-localization with Foxi1 determine that A1, E2 and a4 all are expressed in intercalated cells. In sections from both medulla and cortex we demonstrate a complete co-localization between Foxi1 and the subunits A1, E2 and a4. These subunits display a luminal staining pattern while the Foxi1 signals reside in the nuclei (Fig. 2 A–D, G–J, M–P). Kidneys from mice lacking Foxi1 have no detectable A1, E2 or a4 (Fig. 2 E, F, K, L, Q, R). The overlap in expression between these subunits and Foxi1 appears to be complete i.e. all Foxi1 cells studied are positive for A1, E2 and a4. It is known from previous work that the B1 subunit is expressed in intercalated cells and that its expression requires Foxi1 [12].

Bottom Line: Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner.Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter.Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

View Article: PubMed Central - PubMed

Affiliation: Center of Medical Genetics, Institute of Biomedicine, The Sahlgrenska Academy, Göteborg University, Göteborg, Sweden.

ABSTRACT
The vacuolar H(+)-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position -561/-547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H(+)-ATPase complex at these locations.

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