Mammalian Sterile 20-like Kinase 1 (Mst1) Enhances the Stability of Forkhead Box P3 (Foxp3) and the Function of Regulatory T Cells by Modulating Foxp3 Acetylation.

Regulatory T cells (Tregs) play crucial roles in maintaining immune tolerance. The transcription factor Foxp3 is a critical regulator of Treg development and function, and its expression is regulated at both transcriptional and post-translational levels. Acetylation by lysine acetyl transferases/lysine deacetylases is one of the main post-translational modifications of Foxp3, which regulate Foxp3's stability and transcriptional activity. However, the mechanism(s) by which the activities of these lysine acetyl transferases/lysine deacetylases are regulated to preserve proper Foxp3 acetylation during Treg development and maintenance of Treg function remains to be determined. Here we report that Mst1 can enhance Foxp3 stability, its transcriptional activity, and Treg function by modulating the Foxp3 protein at the post-translational level. We discovered that Mst1 could increase the acetylation of Foxp3 by inhibiting Sirt1 activity, which requires the Mst1 kinase activity. We also found that Mst1 could attenuate Sirt1-mediated deacetylation of Foxp3 through directly interacting with Foxp3 to prevent or interfere the interaction between Sirt1 and Foxp3. Therefore, Mst1 can regulate Foxp3 stability in kinase-dependent and kinase-independent manners. Finally, we showed that treatment of Mst1(-/-) Tregs with Ex-527, a Sirt1-specific inhibitor, partially restored the suppressive function of Mst1(-/-) Tregs. Our studies reveal a novel mechanism by which Mst1 enhances Foxp3 expression and Treg function at the post-translational level.

PP6 controls T cell development and homeostasis by negatively regulating distal TCR signaling.

T cell development and homeostasis are both regulated by TCR signals. Protein phosphorylation and dephosphorylation, which are catalyzed by protein kinases and phosphatases, respectively, serve as important switches controlling multiple downstream pathways triggered by TCR recognition of Ags. It has been well documented that protein tyrosine phosphatases are involved in negative regulation of proximal TCR signaling. However, how TCR signals are terminated or attenuated in the distal TCR signaling pathways is largely unknown. We investigated the function of Ser/Thr protein phosphatase (PP) 6 in TCR signaling. T cell lineage-specific ablation of PP6 in mice resulted in enhanced thymic positive and negative selection, and preferential expansion of fetal-derived, IL-17-producing Vγ6Vδ1(+) T cells. Both PP6-deficient peripheral CD4(+) helper and CD8(+) cytolytic cells could not maintain a naive state and became fast-proliferating and short-lived effector cells. PP6 deficiency led to profound hyperactivation of multiple distal TCR signaling molecules, including MAPKs, AKT, and NF-κB. Our studies demonstrate that PP6 acts as a critical negative regulator, not only controlling both αß and γδ lineage development, but also maintaining naive T cell homeostasis by preventing their premature activation before Ag stimulation.

Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO.

Excessive activation of dendritic cells (DCs) leads to the development of autoimmune and inflammatory diseases, which has prompted a search for regulators of DC activation. Here we report that Rhbdd3, a member of the rhomboid family of proteases, suppressed the activation of DCs and production of interleukin 6 (IL-6) triggered by Toll-like receptors (TLRs). Rhbdd3-deficient mice spontaneously developed autoimmune diseases characterized by an increased abundance of the TH17 subset of helper T cells and decreased number of regulatory T cells due to the increase in IL-6 from DCs. Rhbdd3 directly bound to Lys27 (K27)-linked polyubiquitin chains on Lys302 of the modulator NEMO (IKKγ) via the ubiquitin-binding-association (UBA) domain in endosomes. Rhbdd3 further recruited the deubiquitinase A20 via K27-linked polyubiquitin chains on Lys268 to inhibit K63-linked polyubiquitination of NEMO and thus suppressed activation of the transcription factor NF-κB in DCs. Our data identify Rhbdd3 as a critical regulator of DC activation and indicate K27-linked polyubiquitination is a potent ubiquitin-linked pattern involved in the control of autoimmunity.

Mst1 and mst2 are essential regulators of trophoblast differentiation and placenta morphogenesis.

The placenta is essential for survival and growth of the fetus because it promotes the delivery of nutrients and oxygen from the maternal circulation as well as fetal waste disposal. Mst1 and Mst2 (Mst1/2), key components of the mammalian hpo/Mst signaling pathway, encode two highly conserved Ser/Thr kinases and play important roles in the prevention of tumorigenesis and autoimmunity, control of T cell development and trafficking, and embryonic development. However, their functions in placental development are not fully understood, and the underlying cellular and molecular mechanisms remain elusive. Here, we investigated the functions of Mst1/2 in mouse placental development using both conventional and conditional (endothelial) Mst1/2 double knockout mice. We found that the number of trophoblast giant cells dramatically increased while spongiotrophoblast cells almost completely disappeared in Mst1/2 deficient placentas. We showed that Mst1/2 deficiency down regulated the expression of Mash2, which is required for suppressing the differentiation of trophoblast giant cells. Furthermore, we demonstrated that endothelial-specific deletion of Mst1/2 led to impaired placental labyrinthine vasculature and embryonic lethality at E11.5, but neither affected vasculature in yolk sac and embryo proper nor endocardium development. Collectively, our findings suggest that Mst1/2 regulate placental development by control of trophoblast cell differentiation and labyrinthine vasculature at midgestation and Mst1/2 control labyrinth morphogenesis in trophoblast- and fetal endothelial-dependent manners. Thus, our studies have defined novel roles of Mst1/2 in mouse placental development.

Dlic1 deficiency impairs ciliogenesis of photoreceptors by destabilizing dynein.

Cytoplasmic dynein 1 is fundamentally important for transporting a variety of essential cargoes along microtubules within eukaryotic cells. However, in mammals, few mutants are available for studying the effects of defects in dynein-controlled processes in the context of the whole organism. Here, we deleted mouse Dlic1 gene encoding DLIC1, a subunit of the dynein complex. Dlic1(-/-) mice are viable, but display severe photoreceptor degeneration. Ablation of Dlic1 results in ectopic accumulation of outer segment (OS) proteins, and impairs OS growth and ciliogenesis of photoreceptors by interfering with Rab11-vesicle trafficking and blocking efficient OS protein transport from Golgi to the basal body. Our studies show that Dlic1 deficiency partially blocks vesicle export from endoplasmic reticulum (ER), but seems not to affect vesicle transport from the ER to Golgi. Further mechanistic study reveals that lack of Dlic1 destabilizes dynein subunits and alters the normal subcellular distribution of dynein in photoreceptors, probably due to the impaired transport function of dynein. Our results demonstrate that Dlic1 plays important roles in ciliogenesis and protein transport to the OS, and is required for photoreceptor development and survival. The Dlic1(-/-) mice also provide a new mouse model to study human retinal degeneration.

Ablation of vacuole protein sorting 18 (Vps18) gene leads to neurodegeneration and impaired neuronal migration by disrupting multiple vesicle transport pathways to lysosomes.

Intracellular vesicle transport pathways are critical for neuronal survival and central nervous system development. The Vps-C complex regulates multiple vesicle transport pathways to the lysosome in lower organisms. However, little is known regarding its physiological function in mammals. We deleted Vps18, a central member of Vps-C core complex, in neural cells by generating Vps18(F/F); Nestin-Cre mice (Vps18 conditional knock-out mice). These mice displayed severe neurodegeneration and neuronal migration defects. Mechanistic studies revealed that Vps18 deficiency caused neurodegeneration by blocking multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis, and biosynthetic pathways. Our study also showed that ablation of Vps18 resulted in up-regulation of ß1 integrin in mouse brain probably due to lysosome dysfunction but had no effects on the reelin pathway, expression of N-cadherin, or activation of JNK, which are implicated in the regulation of neuronal migration. Finally, we demonstrated that knocking down ß1 integrin partially rescued the migration defects, suggesting that Vps18 deficiency-mediated up-regulation of ß1 integrin may contribute to the defect of neuronal migration in the Vps18-deficient brain. Our results demonstrate important roles of Vps18 in neuron survival and migration, which are disrupted in multiple neural disorders.

Adam10 is essential for early embryonic cardiovascular development.

Notch pathway has been demonstrated to regulate cardiovascular development. One important step in Notch pathway is the cleavage of Notch receptor, during which an intracellular fragment of Notch protein is released to activate downstream genes. It is still uncertain whether Adam10, the mammalian homologue of Kuzbanian in Drosophila, is required to activate the Notch pathway during cardiovascular development. To further understand the physiological function of Adam10 in vascular and cardiac development, we generated mice lacking the Adam10 gene primarily in the endothelial compartment. We found that disruption of Adam10 in endothelial cells resulted in embryonic death after embryonic day 10.5 due to multiple cardiac and vascular defects similar to Notch1 mutants. We further showed that the expression of Notch target genes Snail and Bmp2 are impaired in Adam10-deficient cardiac tissues. Finally, we provide experimental evidence to support that Adam10 functions in a cell autonomous manner during mammalian cardiac development.

Ras promotes cell survival by antagonizing both JNK and Hid signals in the Drosophila eye.

BACKGROUND: Programmed cell death, or apoptosis, is a fundamental physiological process during normal development or in pathological conditions. The activation of apoptosis can be elicited by numerous signalling pathways. Ras is known to mediate anti-apoptotic signals by inhibiting Hid activity in the Drosophila eye. Here we report the isolation of a new loss-of-function ras allele, rasKP, which causes excessive apoptosis in the Drosophila eye. RESULTS: This new function is likely to be mediated through the JNK pathway since the inhibition of JNK signalling can significantly suppress rasKP-induced apoptosis, whereas the removal of hid only weakly suppresses the phenotype. Furthermore, the reduction of JNK signalling together with the expression of the baculovirus caspase inhibitor p35, which blocks Hid activity, strongly suppresses the rasKP cell death. In addition, we find a strong correlation between rasKP-induced apoptosis in the eye disc and the activation of JNK signalling. CONCLUSION: In the Drosophila eye, Ras may protect cells from apoptosis by inhibiting both JNK and Hid activities. Surprisingly, reducing Ras activity in the wing, however, does not cause apoptosis but rather affects cell and organ size. Thus, in addition to its requirement for cell viability, Ras appears to mediate different biological roles depending on the developmental context and on the level of its expression.

A large-scale functional approach to uncover human genes and pathways in Drosophila.

We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding to 236 human genes have been established. In all, 51 lines are capable of eliciting a phenotype suggesting that the human genes are functional. These heterologous genes are functionally relevant as we have found a similar mutant phenotype caused either by a dominant negative mutant form of the human ribosomal protein L8 gene or by RNAi downregulation of the Drosophila RPL8. Significantly, the Drosophila RPL8 mutant can be rescued by wild-type human RPL8. We also provide genetic evidence that Drosophila RPL8 is a new member of the insulin signaling pathway. In summary, the functions of many human genes appear to be highly conserved, and the ability to identify them in Drosophila represents a powerful genetic tool for large-scale analysis of human transcripts in vivo.

ADAM23 plays multiple roles in neuronal differentiation of P19 embryonal carcinoma cells.

ADAM23, belonging to ADAM (A Disintegrin And Metalloprotease) protein family, is mainly expressed in brain. P19 cells could differentiate into neuroectodermal cell lineage after cell aggregates have been induced by retinoic acid (RA). In this report, we show that the post-transcriptional and post-translational processes of ADAM23 are regulated during the differentiation of P19 cells. In P19-derived neurons, ADAM23 is polarized distributed in the proximal part. To explore the possible roles of ADAM23 during P19 cell neuronal differentiation, ADAM23-RNAi P19 cell lines were established. These transfected cells could differentiate into neurofilament-expression neurons in the absence of RA, whereas wild-type P19 cell can not. These results suggest ADAM23 may play roles in both early and later stage of neuronal differentiation.

Overexpression of human genes in Drosophila melanogaster by using GAL4 UAS system.

Many human genes determined by genomic sequencing have only few information about their functions. To fill this knowledge gap, the powerful Drosophila genetics was set as a model to elucidate human gene functions effectively. By using germline transformation together with GAL4-UAS system, we studied the possibility of expressing and functionally characterization of human genes in Drosophila. Fifty-four transgenic fly lines corresponding to 10 human genes have been established. When expressed individually by crossing to an array of 6 different GAL4 driver lines, one of these genes, the translation elongation factor 1 alpha 1 (EF1 alpha-1), resulted in abnormal notum and rough eye phenotypes. This study implies the feasibility of systematically screening human gene functions by overexpression in Drosophila.