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

Design and synthesis of soluble and cell-permeable PI3Kδ inhibitors for long-acting inhaled administration

PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesised to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimisation for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC50 >9) and by careful manipulation of the pKa and lipophilicity we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in-vivo studies where significant target engagement could be demonstrated

The importance of Foxi1 expression in kidney and inner ear

The forkhead (FOX) genes comprise a family of transcription factors involved in generegulation in both embryonic and adult tissue. Foxi1 is expressed in the kidney andthe developing inner ear. We examined the significance of Foxi1 in these two organsand explain the molecular mechanisms behind the phenotype seen in Foxi1 deficientmice.The kidney collecting duct epithelium is heterogeneous, composed of intercalatedand principal cells. By dual labelling of kidney sections with Foxi1 cRNA in situhybridisation and immunostaining with various kidney collecting duct cell markers,we localised Foxi1 expression exclusively to intercalated cells. Foxi1 deficientcollecting ducts have lost the expression of several proteins involved in bicarbonateor proton transport. Transmission electron microscopy of collecting ducts revealedthat the epithelium from Foxi1 null tubules was homogeneous and completely lackeda protruding, microvilli-rich cell type. Instead, this epithelium was found to becomposed of a single cell type that expressed both principal (aquaporin 2) andintercalated (carbonic anhydrase II) cell markers. This hybrid cell is unable tomaintain systemic acid/base homeostasis and Foxi1 deficiency consequently resultsin distal renal tubular acidosis.Foxi1 and the co-activator Eya1 are expressed in the developing inner ear. The earlymorphogenesis and patterning of the otic vesicle (OV) in Foxi1 deficient mice appearto proceed normally, while Eya1 deficiency leads to an arrest in development at theOV stage. Our results indicate that Eya1 is required for the establishment of regionalspecification of the OV. Eya1 also regulates Bmp- and Fgf-signalling pathways duringearly inner ear development. At E16.5, Foxi1 is exclusively expressed in theendolymphatic duct and sac. Without Foxi1, the inner ear develops an endolymphaticepithelium with no FORE cells. The loss of this cell type and consequently the genesnormally expressed here, leads to defective endolymph fluid handling and inner earexpansion. This causes rupture of inner ear membranes that makes it impossible topreserve an endocochlear potential and the Foxi1 deficient mice therefore becomedeaf.In summary, Foxi1 is significant for complete maturation of the collecting duct andendolymphatic duct and sac epithelia, including the formation of the intercalated andFORE cells, which are responsible for acid/base regulation. Our data suggest thatmutations in human FOXI1 might cause a sensorineural deafness syndrome withdistal renal tubular acidosis

Transcriptional Control of SLC26A4 Is Involved in Pendred Syndrome and Nonsyndromic Enlargement of Vestibular Aqueduct (DFNB4)

Although recessive mutations in the anion transporter gene SLC26A4 are known to be responsible for Pendred syndrome (PS) and nonsyndromic hearing loss associated with enlarged vestibular aqueduct (EVA), also known as “DFNB4,” a large percentage of patients with this phenotype lack mutations in the SLC26A4 coding region in one or both alleles. We have identified and characterized a key transcriptional regulatory element in the SLC26A4 promoter that binds FOXI1, a transcriptional activator of SLC26A4. In nine patients with PS or nonsyndromic EVA, a novel c.−103T→C mutation in this regulatory element interferes with FOXI1 binding and completely abolishes FOXI1-mediated transcriptional activation. We have also identified six patients with mutations in FOXI1 that compromise its ability to activate SLC26A4 transcription. In one family, the EVA phenotype segregates in a double-heterozygous mode in the affected individual who carries single mutations in both SLC26A4 and FOXI1. This finding is consistent with our observation that EVA occurs in the Slc26a4(+/−); Foxi1(+/−) double-heterozygous mouse mutant. These results support a novel dosage-dependent model for the molecular pathogenesis of PS and nonsyndromic EVA that involves SLC26A4 and its transcriptional regulatory machinery

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

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

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

Confocal analysis of Foxi1 and H⁺-ATPase subunits A1, E2 and a4 expression in wt and Foxi1−/− epididymides.

<p>Confocal images of wt and Foxi1 −/− epididymal sections. In confocal images of wt epididymal sections, all three ATPase subunits (red) were exclusively found in Foxi1 (green) immunoreactive epididymal cells of caput (A, G, M), corpus (B, H, N) and cauda (C, I, O). Foxi1 (green) is localized to the nuclei (blue, TOPRO3), while the ATPase subunits are found on the apical/luminal (L) side of the cells. Staining of Foxi1−/− tissue sections showed no detectable A1 (D, E, F), E2 (J, K, L) nor a4 (P, Q, R) subunit expression in Foxi1 deficient epididymal tissue sections. Scale bars 20 µm (L: lumen).</p