A recurrent missense variant in EYA3 gene is associated with oculo-auriculo-vertebral spectrum.

Goldenhar syndrome or oculo-auriculo-vertebral spectrum (OAVS) is a complex developmental disorder characterized by asymmetric ear anomalies, hemifacial microsomia, ocular and vertebral defects. We aimed at identifying and characterizing a new gene associated with OAVS. Two affected brothers with OAVS were analyzed by exome sequencing that revealed a missense variant (p.(Asn358Ser)) in the EYA3 gene. EYA3 screening was then performed in 122 OAVS patients that identified the same variant in one individual from an unrelated family. Segregation assessment in both families showed incomplete penetrance and variable expressivity. We investigated this variant in cellular models to determine its pathogenicity and demonstrated an increased half-life of the mutated protein without impact on its ability to dephosphorylate H2AFX following DNA repair pathway induction. Proteomics performed on this cellular model revealed four significantly predicted upstream regulators which are PPARGC1B, YAP1, NFE2L2 and MYC. Moreover, eya3 knocked-down zebrafish embryos developed specific craniofacial abnormalities corroborating previous animal models and supporting its involvement in the OAVS. Additionally, EYA3 gene expression was deregulated in vitro by retinoic acid exposure. EYA3 is the second recurrent gene identified to be associated with OAVS. Moreover, based on protein interactions and related diseases, we suggest the DNA repair as a key molecular pathway involved in craniofacial development.

Biallelic loss-of-function HACD1 variants are a bona fide cause of congenital myopathy.

Congenital myopathies include a wide range of genetically determined disorders characterized by muscle weakness that usually manifest shortly after birth. To date, two different homozygous loss-of-function variants in the HACD1 gene have been reported to cause congenital myopathy. We identified three patients manifesting with neonatal-onset generalized muscle weakness and motor delay that carried three novel homozygous likely pathogenic HACD1 variants. The two of these changes (c.373_375+2delGAGGT and c.785-1G>T) were predicted to introduce splice site alterations, while one is a nonsense change (c.458G>A). The clinical presentation of our and the previously reported patients was comparable, including the temporally progressive improvement that seems to be characteristic of HACD1-related myopathy. Our findings conclusively confirm the implication of HACD1 in the pathogenesis of congenital myopathies, corroborate the main phenotypic features, and further define the genotypic spectrum of this genetic form of myopathy. Importantly, the genetic diagnosis of HACD1-related myopathy bears impactful prognostic value.

Phagocytes from Mice Lacking the Sts Phosphatases Have an Enhanced Antifungal Response to Candida albicans.

Mice lacking expression of the homologous phosphatases Sts-1 and Sts-2 (Sts-/- mice) are resistant to disseminated candidiasis caused by the fungal pathogen Candida albicans To better understand the immunological mechanisms underlying the enhanced resistance of Sts-/- mice, we examined the kinetics of fungal clearance at early time points. In contrast to the rapid C. albicans growth seen in normal kidneys during the first 24 h postinfection, we observed a reduction in kidney fungal CFU within Sts-/- mice beginning at 12 to 18 h postinfection. This corresponds to the time period when large numbers of innate leukocytes enter the renal environment to counter the infection. Because phagocytes of the innate immune system are important for host protection against pathogenic fungi, we evaluated responses of bone marrow leukocytes. Relative to wild-type cells, Sts-/- marrow monocytes and bone marrow-derived dendritic cells (BMDCs) displayed a heightened ability to inhibit C. albicans growth ex vivo This correlated with significantly enhanced production of reactive oxygen species (ROS) by Sts-/- BMDCs downstream of Dectin-1, a C-type lectin receptor that plays a critical role in stimulating host responses to fungi. We observed no visible differences in the responses of other antifungal effector pathways, including cytokine production and inflammasome activation, despite enhanced activation of the Syk tyrosine kinase downstream of Dectin-1 in Sts-/- cells. Our results highlight a novel mechanism regulating the immune response to fungal infections. Further understanding of this regulatory pathway could aid the development of therapeutic approaches to enhance protection against invasive candidiasis.IMPORTANCE Systemic candidiasis caused by fungal Candida species is becoming an increasingly serious medical problem for which current treatment is inadequate. Recently, the Sts phosphatases were established as key regulators of the host antifungal immune response. In particular, genetic inactivation of Sts significantly enhanced survival of mice infected intravenously with Candida albicans The Sts-/-in vivo resistance phenotype is associated with reduced fungal burden and an absence of inflammatory lesions. To understand the underlying mechanisms, we studied phagocyte responses. Here, we demonstrate that Sts-/- phagocytes have heightened responsiveness to C. albicans challenge relative to wild-type cells. Our data indicate the Sts proteins negatively regulate phagocyte activation via regulating selective elements of the Dectin-1-Syk tyrosine kinase signaling axis. These results suggest that phagocytes lacking Sts respond to fungal challenge more effectively and that this enhanced responsiveness partially underlies the profound resistance of Sts-/- mice to systemic fungal challenge.

Knockdown of LMW-PTP enhances stress resistance in Caenorhabditis elegans.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is highly conserved across almost all living organisms and is involved in the modulation of a number of cellular proteins related to important signaling pathways. In this study, we isolated lmwptp (Y94H6A.7) of Caenorhabditis elegans, the homolog of human ACP1, and set up an effective feeding-based RNA interference (RNAi) knockdown against this gene. We found that knockdown of lmwptp decreased damage associated with heat shock, oxidative stress and UV irradiation in wild-type worms, however, its deficiency didn't further reduce the stress resistance of daf-16 or hsf-1 mutants and didn't further increase the stress sensitivity associated with age-1, akt-1 or akt-2 mutants, but it enhanced the stress resistance of daf-2 mutants. Further studies demonstrated that this stress tolerance could be attributed to increased daf-16 nuclear accumulation and enhanced expression of both superoxide dismutase-3 protein (SOD-3) and heat shock protein-16.2 (HSP-16.2) in response to stress. Additionally, quantitative real-time PCR results showed that the expression of hsf-1 and its target genes were up-regulated in lmwptp-knockdown worms under stress conditions. Together these results indicated that lmwptp is related to stress resistance of worms, and it is likely associated with the insulin/IGF-1-like signaling (IIS) pathway.

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis.

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4-/- mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-ß with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

Galectin-1-Driven Tolerogenic Programs Aggravate Yersinia enterocolitica Infection by Repressing Antibacterial Immunity.

Yersinia enterocolitica is an enteropathogenic bacterium that causes gastrointestinal disorders, as well as extraintestinal manifestations. To subvert the host's immune response, Y. enterocolitica uses a type III secretion system consisting of an injectisome and effector proteins, called Yersinia outer proteins (Yops), that modulate activation, signaling, and survival of immune cells. In this article, we show that galectin-1 (Gal-1), an immunoregulatory lectin widely expressed in mucosal tissues, contributes to Y. enterocolitica pathogenicity by undermining protective antibacterial responses. We found higher expression of Gal-1 in the spleen and Peyer's patches of mice infected orogastrically with Y. enterocolitica serotype O:8 compared with noninfected hosts. This effect was prevented when mice were infected with Y. enterocolitica lacking YopP or YopH, two critical effectors involved in bacterial immune evasion. Consistent with a regulatory role for this lectin during Y. enterocolitica pathogenesis, mice lacking Gal-1 showed increased weight and survival, lower bacterial load, and attenuated intestinal pathology compared with wild-type mice. These protective effects involved modulation of NF-κB activation, TNF production, and NO synthesis in mucosal tissue and macrophages, as well as systemic dysregulation of IL-17 and IFN-γ responses. In vivo neutralization of these proinflammatory cytokines impaired bacterial clearance and eliminated host protection conferred by Gal-1 deficiency. Finally, supplementation of recombinant Gal-1 in mice lacking Gal-1 or treatment of wild-type mice with a neutralizing anti-Gal-1 mAb confirmed the immune inhibitory role of this endogenous lectin during Y. enterocolitica infection. Thus, targeting Gal-1-glycan interactions may contribute to reinforce antibacterial responses by reprogramming innate and adaptive immune mechanisms.

Increased Resistance to Intradermal Francisella tularensis LVS Infection by Inactivation of the Sts Phosphatases.

The Suppressor of TCR signaling proteins (Sts-1 and Sts-2) are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic lineages, including T lymphocytes. Mice lacking Sts expression are characterized by enhanced T cell responses. Additionally, a recent study demonstrated that Sts-/- mice are profoundly resistant to systemic infection by Candida albicans, with resistance characterized by enhanced survival, more rapid fungal clearance in key peripheral organs, and an altered inflammatory response. To investigate the role of Sts in the primary host response to infection by a bacterial pathogen, we evaluated the response of Sts-/- mice to infection by a Gram-negative bacterial pathogen. Francisella tularensis is a facultative bacterial pathogen that replicates intracellularly within a variety of cell types and is the causative agent of tularemia. Francisella infections are characterized by a delayed immune response, followed by an intense inflammatory reaction that causes widespread tissue damage and septic shock. Herein, we demonstrate that mice lacking Sts expression are significantly resistant to infection by the live vaccine strain (LVS) of F. tularensis Resistance is characterized by reduced lethality following high-dose intradermal infection, an altered cytokine response in the spleen, and enhanced bacterial clearance in multiple peripheral organs. Sts-/- bone marrow-derived monocytes and neutrophils, infected with F. tularensis LVS ex vivo, display enhanced restriction of intracellular bacteria. These observations suggest the Sts proteins play an important regulatory role in the host response to bacterial infection, and they underscore a role for Sts in regulating functionally relevant immune response pathways.

MAP kinase phosphatase 2 deficient mice develop attenuated experimental autoimmune encephalomyelitis through regulating dendritic cells and T cells.

Mitogen-activated protein kinase phosphatases (MKPs) play key roles in inflammation and immune mediated diseases. Here we investigated the mechanisms by which MKP-2 modulates central nervous system (CNS) inflammation in experimental autoimmune encephalomyelitis (EAE). Our results show that MKP-2 mRNA levels in the spinal cord and lymphoid organs of EAE mice were increased compared with naive controls, indicating an important role for MKP-2 in EAE development. Indeed, MKP-2-/- mice developed reduced EAE severity, associated with diminished CNS immune cell infiltration, decreased proinflammatory cytokine production and reduced frequency of CD4+ and CD8+ T cells in spleens and lymph nodes. In addition, MKP-2-/- CD11c+ dendritic cells (DCs) had reduced expression of MHC-II and CD40 compared with MKP-2+/+ mice. Subsequent experiments revealed that CD4+ T cells from naïve MKP-2-/- mice had decreased cell proliferation and IL-2 and IL-17 production relative to wild type controls. Furthermore, co-culture experiments showed that bone marrow derived DCs of MKP-2-/- mice had impaired capability in antigen presentation and T cell activation. While MKP-2 also modulates macrophage activation, our study suggests that MKP-2 is essential to the pathogenic response of EAE, and it acts mainly via regulating the important antigen presenting DC function and T cell activation.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

OBJECTIVE: The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). APPROACH AND RESULTS: HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2-/- mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2-/- murine platelets. Compared with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. CONCLUSIONS: Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling.

Dual-Specificity Phosphatase 4 Regulates STAT5 Protein Stability and Helper T Cell Polarization.

Immune responses are critically regulated by the functions of CD4 helper T cells. Based on their secreted cytokines, helper T cells are further categorized into different subsets like Treg or Th17 cells, which suppress or promote inflammatory responses, respectively. Signals from IL-2 activate the transcription factor STAT5 to promote Treg but suppress Th17 cell differentiation. Our previous results found that the deficiency of a dual-specificity phosphatase, DUSP4, induced STAT5 hyper-activation, enhanced IL-2 signaling, and increased T cell proliferation. In this report, we examined the effects of DUSP4 deficiency on helper T cell differentiation and STAT5 regulation. Our in vivo data showed that DUSP4 mice were more resistant to the induction of autoimmune encephalitis, while in vitro differentiations revealed enhanced iTreg and reduced Th17 polarization in DUSP4-deficient T cells. To study the cause of this altered helper T cell polarization, we performed luciferase reporter assays and confirmed that, as predicted by our previous report, DUSP4 over-expression suppressed the transcription factor activity of STAT5. Surprisingly, we also found that DUSP4-deficient T but not B cells exhibited elevated STAT5 protein levels, and over-expressed DUSP4 destabilized STAT5 in vitro; moreover, this destabilization required the phosphatase activity of DUSP4, and was insensitive to MG132 treatment. Finally, domain-mapping results showed that both the substrate-interacting and the phosphatase domains of DUSP4 were required for its optimal interaction with STAT5, while the coiled-coil domain of STAT5 appeared to hinder this interaction. Our data thus provide the first genetic evidence that DUSP4 is important for helper T cell development. In addition, they also help uncover the novel, DUSP4-mediated regulation of STAT5 protein stability.

Protein tyrosine phosphatase deficiency protects against the induction of the early apoptosis by paracetamol in hepatocytes

Acetaminophen (paracetamol, APAP) is a widely used analgesic and antipyretic drug safe at therapeutic doses but its overdose causes liver injury. Our goal was to explore whether protein tyrosine phosphatase 1B (PTP1B), a negative modulator of survival signaling pathways, plays a role in APAP-induced cell death in hepatocytes. Hepatotoxicity was evaluated in immortalized hepatocytes generated from wild-type (PTP1B+/+) and PTP1B-deficient (PTP1B-/-) mice. Apoptosis occurred as an early event only in APAP-treated PTP1B+/+ hepatocytes. PTP1B deficiency conferred protection against cell cycle arrest and loss of cellular viability. These data suggest that PTP1B as a target against APAP-induced liver failure

El paracetamol es un analgésico/antipirético hepatotóxico a dosis altas. Investigamos el papel de la proteína tirosina fosfatasa 1B (PTP1B), un modulador negativo de señalización de supervivencia celular, en la muerte celular temprana (apoptosis) inducida por paracetamol en hepatocitos. En hepatocitos controles se inducía apoptosis en respuesta al paracetamol. Este efecto se encontraba disminuido en hepatocitos deficientes en PTP1B. La falta de PTP1B protegía a los hepatocitos de la parada del ciclo celular y la pérdida de la viabilidad celular tras el tratamiento con paracetamol. Proponemos a la PTP1B como diana terapéutica frente al fallo hepático inducido por sobredosis de paracetamol

Integrin-mediated first signal for inflammasome activation in intestinal epithelial cells.

How intestinal epithelial cells (IECs) recognize pathogens and activate inflammasomes at intestinal surfaces is poorly understood. We hypothesized that IECs use integrin receptors to recognize pathogens and initiate inflammation within the intestinal tract. We find that IECs infected with Yersinia enterocolitica, an enteric pathogen, use ß1 integrins as pathogen recognition receptors detecting the bacterial adhesin invasin (Inv). The Inv-integrin interaction provides the first signal for NLRP3 inflammasome activation with the type three secretion system translocon providing the second signal for inflammasome activation, resulting in release of IL-18. During infection, Yersinia employs two virulence factors, YopE and YopH, to counteract Inv-mediated integrin-dependent inflammasome activation. Furthermore, NLRP3 inflammasome activation in epithelial cells requires components of the focal adhesion complex signaling pathway, focal adhesion kinase, and rac1. The binding of Inv to ß1 integrins rapidly induces IL-18 mRNA expression, suggesting integrins provide a first signal for NLRP3 inflammasome activation. These data suggest integrins function as pathogen recognition receptors on IECs to rapidly induce inflammasome-derived IL-18-mediated responses.

Mice deficient in transmembrane prostatic acid phosphatase display increased GABAergic transmission and neurological alterations.

Prostatic acid phosphatase (PAP), the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG), but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP) in the brain by utilizing mice deficient in TMPAP (PAP-/- mice). Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.

Transmembrane prostatic acid phosphatase (TMPAP) interacts with snapin and deficient mice develop prostate adenocarcinoma.

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/-)) with C57BL/6J background. The PAP(-/-) mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

Abrogation of Eya1/Six1 disrupts the saccular phase of lung morphogenesis and causes remodeling.

The Eya1 gene encodes a transcriptional co-activator that acts with Six1 to control the development of different organs. However, Six1-Eya1 interactions and functional roles in mesenchymal cell proliferation and differentiation as well as alveolarization during the saccular stage of lung development are still unknown. Herein, we provide the first evidence that Six1 and Eya1 act together to regulate mesenchymal development as well as alveolarization during the saccular phase of lung morphogenesis. Deletion of either or both Six1 and Eya1 genes results in a severe saccular phenotype, including defects of mesenchymal cell development and remodeling of the distal lung septae and arteries. Mutant lung histology at the saccular phase shows mesenchymal and saccular wall thickening, and abnormal proliferation of α-smooth muscle actin-positive cells, as well as increased mesenchymal/fibroblast cell differentiation, which become more sever when deleting both genes. Our study indicates that SHH but not TGF-ß signaling pathway is a central mediator for the histologic alterations described in the saccular phenotype of Eya1(-/-) or Six1(-/-) lungs. Indeed, genetic reduction of SHH activity in vivo or inhibition of its activity in vitro substantially rescues lung mesenchymal and alveolar phenotype of mutant mice at the saccular phase. These findings uncover novel functions for Six1-Eya1-SHH pathway during the saccular phase of lung morphogenesis, providing a conceptual framework for future mechanistic and translational studies in this area.

Eyes absent tyrosine phosphatase activity is not required for Drosophila development or survival.

Eyes absent (Eya) is an evolutionarily conserved transcriptional coactivator and protein phosphatase that regulates multiple developmental processes throughout the metazoans. Drosophila eya is necessary for survival as well as for the formation of the adult eye. Eya contains a tyrosine phosphatase domain, and mutations altering presumptive active-site residues lead to strongly reduced activities in ectopic eye induction, in vivo genetic rescue using the Gal4-UAS system, and in vitro phosphatase assays. However, these mutations have not been analyzed during normal development with the correct levels, timing, and patterns of endogenous eya expression. To investigate whether the tyrosine phosphatase activity of Eya plays a role in Drosophila survival or normal eye formation, we generated three eya genomic rescue (eyaGR) constructs that alter key active-site residues and tested them in vivo. In striking contrast to previous studies, all eyaGR constructs fully restore eye formation as well as viability in an eya null mutant background. We conclude that the tyrosine phosphatase activity of Eya is not required for normal eye development or survival in Drosophila. Our study suggests the need for a re-evaluation of the mechanism of Eya action and underscores the importance of studying genes in their native context.

T cell hypo-responsiveness against Leishmania major in MAP kinase phosphatase (MKP) 2 deficient C57BL/6 mice does not alter the healer disease phenotype.

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished T(H)1 response. By contrast, in the present study footpad infection of MKP-2(-/-) mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2(+/+) mice. Analysis of immune responses following infection demonstrated a reduced T(H)1 response in MKP-2(-/-) mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4(+) and CD8(+) T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2(-/-) mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2(+/+) mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall T(H)1/T(H)2 balance was unaltered in MKP-2(-/-) compared with wild-type mice. Although non-stimulated MKP-2(-/-) macrophages were more permissive to L. major growth than macrophages from MKP-2(+/+) mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2(-/-) and MKP-2(+/+) macrophages. Consequently, in the absence of any switch in the T(H)1/T(H)2 balance in MKP-2(-/-) mice, no significant change in disease phenotype was observed.

Eya1 protein phosphatase regulates tight junction formation in lung distal epithelium.

Little is known about the regulatory mechanisms underlying lung epithelial tight junction (TJ) assembly, which is inextricably linked to the preservation of epithelial polarity, and is highly coordinated by proteins that regulate epithelial cell polarity, such as aPKCζ. We recently reported that Eya1 phosphatase functions through aPKCζ-Notch1 signaling to control cell polarity in the lung epithelium. Here, we have extended these observations to TJ formation to demonstrate that Eya1 is crucial for the maintenance of TJ protein assembly in the lung epithelium, probably by controlling aPKCζ phosphorylation levels, aPKCζ-mediated TJ protein phosphorylation and Notch1-Cdc42 activity. Thus, TJs are disassembled after interfering with Eya1 function in vivo or during calcium-induced TJ assembly in vitro. These effects are reversed by reintroduction of wild-type Eya1 or partially inhibiting aPKCζ in Eya1siRNA cells. Moreover, genetic activation of Notch1 rescues Eya1(-/-) lung epithelial TJ defects. These findings uncover novel functions for the Eya1-aPKCζ-Notch1-Cdc42 pathway as a crucial regulatory mechanism of TJ assembly and polarity of the lung epithelium, providing a conceptual framework for future mechanistic and translational studies in this area.