The Forkhead Transcription Factor Foxi1 Is a Master Regulator of Vacuolar H+-ATPase Proton Pump Subunits in the Inner Ear, Kidney and Epididymis

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

Foxf2 and Foxc2, two transcription factors that regulate adipocyte metabolism

Type 2 diabetes is the most common metabolic disorder today and has reached epidemic proportions in many countries. Diet-induced insulin resistance plays a central role in the development of type 2 diabetes. Studies included in this thesis describe findings regarding two forkhead genes, Foxf2 and Foxc2, and their involvement in the development of insulin resistance. We produced a mouse in which the forkhead factor FOXF2 is overexpressed in an adipose tissue-restricted fashion, such mice display induced insulin secretion in response to an intravenous glucose load. In addition we could demonstrate that adipocytes from FOXF2 transgenic mice have an impaired insulin-mediated glucose uptake. We argue this to be, at least in part, due to lower expression of insulin receptor substrate 1. Mice overexpressing forkhead factor FOXC2 in adipose tissue have previously been shown to be protected from diet-induced obesity and glucose intolerance. In hyperinsulinemic-euglycemic clamp experiments, we demonstrated that FOXC2 transgenic mice are protected from diet-induced insulin resistance in liver and skeletal muscle. Furthermore, on high-fat diet, FOXC2 transgenic mice displayed decreased intramuscular levels of fatty acyl CoA compared with wild-type littermates. Expansion and regression of adipose tissue requires continuous remodelling of the vasculature in order to meet demands of metabolism. We have shown that the adipose tissue of FOXC2 transgenic mice exhibit a higher vascular density and altered pattering of vascular smooth muscle cells and pericytes. Furthermore, we could show this, at least in part, to be dependent on the role of FOXC2 as a direct regulator of Angiopoietin 2

Overexpression of Foxf2 in adipose tissue is associated with lower levels of IRS1 and decreased glucose uptake in vivo

Westergren R, Nilsson D, Heglind M, Arani Z, Grande M, Cederberg A, Ahren B, Enerback S. Overexpression of Foxf2 in adipose tissue is associated with lower levels of IRS1 and decreased glucose uptake in vivo. Am J Physiol Endocrinol Metab 298: E548-E554, 2010. First published December 15, 2009; doi:10.1152/ajpendo.00395.2009.-Many members of the forkhead genes family of transcription factors have been implicated as important regulators of metabolism, in particular, glucose homeostasis, e. g., Foxo1, Foxa3, and Foxc2. The purpose of this study was to exploit the possibility that yet unknown members of this gene family play a role in regulating glucose tolerance in adipocytes. We identified Foxf2 in a screen for adipose-expressed forkhead genes. In vivo overexpression of Foxf2 in an adipose tissue-restricted fashion demonstrated that such mice display a significantly induced insulin secretion in response to an intravenous glucose load compared with wild-type littermates. In response to increased Foxf2 expression, insulin receptor substrate 1 (IRS1) mRNA and protein levels are significantly downregulated in adipocytes; however, the ratio of serine vs. tyrosine phosphorylation of IRS1 seems to remain unaffected. Furthermore, adipocytes over-expressing Foxf2 have a significantly lower insulin-mediated glucose uptake compared with wild-type adipocytes. These findings argue that Foxf2 is a previously unrecognized regulator of cellular and systemic whole body glucose tolerance, at least in part, due to lower levels of IRS1. Foxf2 and its downstream target genes can provide new insights with regard to identification of novel therapeutic targets

mRNA in situ hybridization and immunohistochemistry on kidney sections from wt and Foxi1−/− mice.

<p>Combined <i>in situ</i> hybridization and immunofluorescence. DIG labeled cRNA probes for A1 and E2 were hybridized to wt and Foxi1−/− kidney sections and the same sections were then subjected to immunofluorescent staining with an anti-CAII specific antibody (green) and the nuclear marker Topro3 (red). CAII expressing cells in wt kidney displayed positive hybridization signal for both A1 and E2 probes while in Foxi1−/− kidney sections, no hybridization signal could be detected. White arrows point to the same CAII positive cells in images from in situ hybridization and immunofluorescence. Scale bars 10 µm.</p

Chromatin Immuoprecipitation (ChIP) analysis.

<p>Chromatin immunoprecipitation confirm presence of Foxi1- ATP6V0A4 promoter interaction at FK1-3. Transfected 3T3-L1 cells were fixed, and chromatin was prepared by sonication. After preclearance with Protein G Sepharose the chromatin was immunoprecipitated with or without an Anti-6X His tag antibody (Foxi1, No Ab). The purified precipitated DNA was used as template for PCR reactions with specific primers covering sequence with Foxi1 binding sites (Fk1-3) or unspecific primers (Random). ChIP analysis reveal a strong interaction of the ATP6V0A4 promoter with wt Foxi1 (Lane 1). The interaction is weakened using promoter with mutated FK1 (Lane2) and even more weakened with Fk2 and 3 (Lanes 3–4). Using the triple mutated promoter Fk1-3 (Lane 5) no interaction is detected.</p

In silico identification of forkhead sites in genes encoding subunits A1 and E2.

<p>Representation of potential forkhead binding sites in the upstream regions of genes encoding the A1 and E2 subunit. In the depicted 3 kb region 14 and 25 sites where identified for A1 and E2, respectively.</p

Confocal analysis of Foxi1 and H⁺-ATPase subunits A1, B1, E2 and a4 expression in wt and Foxi1−/− endolymphatic sac (ES) epithelium.

<p>Confocal images of inner ear sections from wt and Foxi1−/− mouse embryos (E16.5). Fluorescence images of wt ES tissue sections stained with a specific antibody against Foxi1 (green) and the H⁺-ATPase subunit A1 (red; A–B), subunit B1 (red; D–E), subunit E2 (red; G–H) and subunit a4 (red; J–K). The nuclei (blue) were visualized using To-Pro 3. Merged images reveal that cells with a strong staining of each of the H⁺-ATPase subunits also are positive for nuclear Foxi1 staining. In sections from Foxi1−/− ES no staining could be identified for A1 (C), B1 (F), E2 (I) or a4 (L). Scale bars 20 µm (A, D, G, J, C, F, I and L) and 10 µm (B, E, H and K) (L: lumen).</p

Foxi1 interact and activate <i>ATP6V0A4</i> promoter reporter construct at putative forkhead binding sites Fk1-3.

<p>(A) Core TTT-triplets of potential forkhead sites were mutated to GGG according to schematic picture. (B) Mutations reduce Foxi1-depedent reporter activation. While Fk3 mutations seem to abolish this activation the effects on Fk1-2 are intermediate and Fk4 seems to contribute very little to Foxi1-dependent reporter gene activation. (C) EMSA using <i>in vitro</i> transcribed/translated Foxi1 demonstrates interactions with [³²P] labeled oligonucleotides harboring the −561/−547 (Fk1-3) and −358/−352 (Fk4) regions. The comparatively weak interaction demonstrated for Fk4 correlates well with the modest effects on reporter gene activation seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004471#pone-0004471-g005" target="_blank">Fig. 5 B</a>. (D–F) EMSA showing the −561/−547 interaction is competed for with either wt or mutated oligonucleotides specific for the Fk1, 2 and 3 interactions. As can be deduced, there is a higher sensitivity in EMSA competition experiments using wt probes as compared with mutated probes – typical of a sequence specific significant interaction.</p