Mice with Disrupted Type I Protein Kinase A Anchoring in T Cells Resist Retrovirus-Induced Immunodeficiency

Type I protein kinase A (PKA) is targeted to the TCR-proximal signaling machinery by the A-kinase anchoring protein ezrin and negatively regulates T cell immune function through activation of the C-terminal Src kinase. RI anchoring disruptor (RIAD) is a high-affinity competitor peptide that specifically displaces type I PKA from A-kinase anchoring proteins. In this study, we disrupted type I PKA anchoring in peripheral T cells by expressing a soluble ezrin fragment with RIAD inserted in place of the endogenous A-kinase binding domain under the lck distal promoter in mice. Peripheral T cells from mice expressing the RIAD fusion protein (RIAD-transgenic mice) displayed augmented basal and TCR-activated signaling, enhanced T cell responsiveness assessed as IL-2 secretion, and reduced sensitivity to PGE2- and cAMP-mediated inhibition of T cell function. Hyperactivation of the cAMP–type I PKA pathway is involved in the T cell dysfunction of HIV infection, as well as murine AIDS, a disease model induced by infection of C57BL/6 mice with LP-BM5, a mixture of attenuated murine leukemia viruses. LP-BM5–infected RIADtransgenic mice resist progression of murine AIDS and have improved viral control. This underscores the cAMP–type I PKA pathway in T cells as a putative target for therapeutic intervention in immunodeficiency diseases.Peer reviewe

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

Epitaxial growth of perovskite oxide films facilitated by oxygen vacancies

The authors would like to thank P. Yudin for valuable discussions, N. Nepomniashchaia for VASE studies, and S. Cichon for XPS analysis. The authors acknowledge support from the Czech Science Foundation (Grant No. 19-09671S), the European Structural and Investment Funds and the Ministry of Education, Youth and Sports of the Czech Republic through Programme ‘‘Research, Development and Education’’ (Project No. SOLID21 CZ.02.1.01/0.0/0.0/16-019/0000760), and ERA NET project Sun2Chem (E. K. and L. R.). Calculations have been done on the LASC Cluster in the ISSP UL.Single-crystal epitaxial films of technologically important and scientifically intriguing multifunctional ABO3 perovskite-type metal oxides are essential for advanced applications and understanding of these materials. In such films, a film-substrate misfit strain enables unprecedented crystal phases and unique properties that are not available in their bulk counterparts. However, the prerequisite growth of strained epitaxial films is fundamentally restricted by misfit relaxation. Here we demonstrate that introduction of a small oxygen deficiency concurrently stabilizes epitaxy and increases lattice strain in thin films of archetypal perovskite oxide SrTiO3. By combining experimental and theoretical methods, we found that lattice distortions around oxygen vacancies lead to anisotropic local stresses, which interact with the misfit strain in epitaxial films. Consequently, specific crystallographic alignments of the stresses are energetically favorable and can facilitate epitaxial growth of strained films. Because anisotropic oxygen-vacancy stresses are inherent to perovskite-type and many other oxides, we anticipate that the disclosed phenomenon of epitaxial stabilization by oxygen vacancies is relevant for a very broad range of functional oxides.This work is licensed under CC BY, CC BY-NC licenses.Czech Science Foundation (Grant No. 19-09671S); European Structural and Investment Funds and the Ministry of Education, Youth and Sports of the Czech Republic through Programme ‘‘Research, Development and Education’’ (Project No. SOLID21 CZ.02.1.01/0.0/0.0/16-019/0000760), and ERA NET project Sun2Chem; Institute of Solid State Physics, University of Latvia as the Center of Excellence has received funding from the European Union’s Horizon 2020 Framework Programme H2020-WIDESPREAD-01-2016-2017-TeamingPhase2 under grant agreement No. 739508, project CAMART²

Lack of the Central Nervous System- and Neural Crest-Expressed Forkhead Gene Foxs1 Affects Motor Function and Body Weight

To gain insight into the expression pattern and functional importance of the forkhead transcription factor Foxs1, we constructed a Foxs1-β-galactosidase reporter gene “knock-in” (Foxs1(β-gal/β-gal)) mouse, in which the wild-type (wt) Foxs1 allele has been inactivated and replaced by a β-galactosidase reporter gene. Staining for β-galactosidase activity reveals an expression pattern encompassing neural crest-derived cells, e.g., cranial and dorsal root ganglia as well as several other cell populations in the central nervous system (CNS), most prominently the internal granule layer of cerebellum. Other sites of expression include the lachrymal gland, outer nuclear layer of retina, enteric ganglion neurons, and a subset of thalamic and hypothalamic nuclei. In the CNS, blood vessel-associated smooth muscle cells and pericytes stain positive for Foxs1. Foxs1(β-gal/β-gal) mice perform significantly better (P < 0.01) on a rotating rod than do wt littermates. We have also noted a lower body weight gain (P < 0.05) in Foxs1(β-gal/lβ-gal) males on a high-fat diet, and we speculate that dorsomedial hypothalamic neurons, expressing Foxs1, could play a role in regulating body weight via regulation of sympathetic outflow. In support of this, we observed increased levels of uncoupling protein 1 mRNA in Foxs1(β-gal/β-gal) mice. This points toward a role for Foxs1 in the integration and processing of neuronal signals of importance for energy turnover and motor function

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

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