Modulation of actomyosin contractility by myosin light chain phosphorylation/dephosphorylation through Rho GTPases signaling specifies axon formation in neurons

Actin depolymerization through Rho GTPases or exogenous mechanical tension has been suggested as a key determinant for the first step of neuronal polarization, the axonogenesis, in which one of the neurites starts to grow becoming the axon. The underlying mechanism and the relationship between two forces in the cells, however, are mostly unknown. Here, we report that the myosin-dependent contractility is a common effector between two forces and a critical determinant in axonogenesis and neuronal polarization. We have found that inhibition of myosin ATPase activity and modulation of myosin light chain phosphorylation/dephosphorylation through Rho GTPases signaling induced multiple axons. Moreover, overexpression of wild-type myosin light chain kinase dramatically increased filopodial structures and produced multi-axonal structures. Our results suggest that MLC phosphorylation/dephosphorylation through Rho GTPases signaling modulates the actomyosin contractility, and then in turn provides a physiological tension in neurons to induce axon.     

Impaired TGF-beta responses in peripheral T cells of G alpha i2-/- mice

Null mutation of heterotrimeric G protein alpha2 inhibitory subunit (Galphai2) induces Th1-skewed hyperimmune responses in the colon, leading to chronic colitis and the development of colonic adenocarcinoma. However, the underlying molecular mechanisms and cellular basis, in particular, for the role of Galphai2 in regulating immune responses, are poorly understood. We show here that peripheral T cells from Galphai2-deficient mice do not respond normally to the inhibitory effects of TGF-beta on proliferation and cytokine production, revealing a previously unappreciated cross-talk between these two signaling pathways. Lack of Galphai2 resulted in decreased phosphorylation of Smad2 and Smad3 in T cells at the basal levels as well as at the late but not early phase of TGF-beta stimulation, which appears to be ascribed to differential expression of neither cell surface TGF-beta receptors nor Smad7. The altered phosphorylation of Smad proteins involves phospholipase C-mediated signaling, a downstream signaling molecule of Galphai2, because phospholipase C inhibitors could restore Smad2 and Smad3 phosphorylation in Galphai2(-/-) T cells at levels comparable to that in wild-type T cells. Moreover, adoptive transfer of Galphai2-deficient T cells into immunocompromised mice rendered an otherwise resistant mouse strain susceptible to trinitrobenzesulfonic acid-induced colitis, suggesting that an impaired response of Galphai2-deficient T cells to TGF-beta may be one of the primary defects accounting for the observed colonic Th1-skewed hyperimmune responses. These findings shed new lights on the molecular and cellular basis of how Galphai2 down-regulates immune responses, contributing to the maintenance of mucosal tolerance.     

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.     

Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.     

The SAM Domains of Anks Family Proteins Are Critically Involved in Modulating the Degradation of EphA Receptors

We recently reported that the phosphotyrosine-binding (PTB) domain of Anks family proteins binds to EphA8, thereby positively regulating EphA8-mediated signaling pathways. In the current study, we identified a potential role for the SAM domains of Anks family proteins in EphA signaling. We found that SAM domains of Anks family proteins directly bind to ubiquitin, suggesting that Anks proteins regulate the degradation of ubiquitinated EphA receptors. Consistent with the role of Cbl ubiquitin ligases in the degradation of Eph receptors, our results revealed that the ubiquitin ligase c-Cbl induced the ubiquitination and degradation of EphA8 upon ligand binding. Ubiquitinated EphA8 also bound to the SAM domains of Odin, a member of the Anks family proteins. More importantly, the overexpression of wild-type Odin protected EphA8 and EphA2 from undergoing degradation following ligand stimulation and promoted EphA-mediated inhibition of cell migration. In contrast, a SAM domain deletion mutant of Odin strongly impaired the function of endogenous Odin, suggesting that the mutant functions in a dominant-negative manner. An analysis of Odin-deficient primary embryonic fibroblasts indicated that Odin levels play a critical role in regulating the stability of EphA2 in response to ligand stimulation. Taken together, our studies suggest that the SAM domains of Anks family proteins play a pivotal role in enhancing the stability of EphA receptors by modulating the ubiquitination process.Activation of Eph receptor tyrosine kinases (RTKs) by ephrin ligands stimulates intracellular signaling pathways that regulate diverse cell behaviors such as axon guidance, cell adhesion, and cell migration (1). Activated Eph receptors also initiate negative signaling events that counteract or alter positive signals, thereby modulating biological outcomes. Negative signaling events associated with Eph RTKs include metalloprotease-mediated cleavage of ephrins and trans endocytosis of Eph-ephrin complexes (9, 15, 24). These negative regulatory mechanisms may be important in the repulsive mechanism responsible for retraction of cellular processes. Some studies suggest that c-Cbl, a RING finger E3 ligase, participates in activated Eph receptor signal termination. Ligand stimulation induces the tyrosine phosphorylation of c-Cbl and facilitates the degradation of Eph receptors (19, 23). More recent studies have shown that the E3 ligase activity of c-Cbl is activated through tyrosine phosphorylation by Src family kinases and that c-Cbl is recruited to activated Eph receptors and induces the ubiquitination and degradation of the receptors (6, 14). These studies point to an important role for Cbl family ubiquitin (Ub) ligases in mediating the ubiquitination of activated Eph RTKs and in fine-tuning Eph receptor signaling pathways.Emerging evidence points to a critical role for Eph receptors in human diseases such as diabetes and cancer (2, 13, 17). For example, EphA2 overexpression has been found in many types of malignant tumors. Overexpression of EphA2 in nontransformed epithelial cells enhances tumorigenic and metastatic potential, whereas downregulation of EphA2 expression suppresses tumor growth and metastasis (4). In addition, either soluble ephrin-A ligand or a monoclonal antibody that activates and degrades EphA2 has been shown to inhibit the growth of human tumor xenografts in nude mice (5, 12). More recent evidence reveals that EphA2 cooperates with Erb2 (also known as Neu) to promote tumor progression in mice (3). These findings strongly suggest that EphA2 and possibly other Eph receptors function in tumor progression in the context of either specific oncogenes or tumor suppressors. In this respect, understanding the negative regulation of Eph receptors, such as their degradation, may have important implications in the design of effective antitumor therapeutics.Recently, we showed that Anks family proteins act as key scaffolding molecules in EphA8-mediated signaling pathways (20). Anks family proteins contain six ankyrin repeats at their N terminus, two SAM domains, and a phosphotyrosine-binding (PTB) domain at their C terminus (22). Odin and AβPP intracellular domain-associated protein 1b (AIDA-1b) belong to this protein family. Several isoforms of AIDA-1b have been described, and the regions encoding the PTB domain and the two SAM domains are very well conserved among all isoforms (7). Interestingly, AIDA-1 has been implicated in reducing AβPP processing through the inhibition of γ-secretase activity (7) and in increasing the global protein biosynthetic capacity in response to long-term neuronal stimulation through the regulation of nucleolar assembly (10). Functions attributed to Odin have been limited to its negative role in platelet-derived growth factor (PDGF)-mediated cell proliferation (16). In contrast to AIDA-1 proteins, Odin appears to be abundantly and ubiquitously expressed in many different mammalian cell lines, and its expression is restricted to the mouse embryonic brain rather than the adult brain (20). We recently reported that the PTB domains of Anks family proteins are crucial for the association of these proteins with the juxtamembrane (JM) domain of EphA8; however, an as-yet-unidentified motif in Anks family proteins also contributes to stable complex formation between these two proteins (20).While the SAM domains of Anks family proteins are highly conserved among all isoforms, the function of this domain is not well understood. In the current study, we identified a potential role for SAM domains in EphA signaling. We showed that while the ubiquitin ligase c-Cbl mediates the ubiquitination and degradation of EphA8 upon ligand binding, the SAM domains of Anks family proteins associate with ubiquitinated EphA8 receptor and are critically involved in inhibiting the degradation of EphA2 and EphA8 receptors. These results suggest that the fine-tuning of EphA RTK signaling is regulated by a delicate balance between the activity of c-Cbl E3 ligase and Anks family proteins.     

Reversible inactivation of bovine plasma amine oxidase by cysteamine and related analogs

Cysteamine (1) was reported many years ago to reversibly inhibit lentil seedling amine oxidase, through the formation of a complex with thioacetaldehyde, the turnover product of 1. Herein, cysteamine (1) and its analogs 2-(methylamino)ethanethiol (3) and 3-aminopropanethiol (6) were found to be reversible inhibitors of bovine plasma amine oxidase (BPAO), but 2-(methylthio)ethylamine (7) was determined to be a weak irreversible inhibitor of BPAO. Based on our results, indicating the necessity of a sulfhydryl-amine for reversible inactivation of BPAO, the failure of inhibited BPAO to recover activity after gel filtration, the first-order kinetics of activity recovery upon dialysis, and 2,4,6-trihydroxyphenylalanine quinine (TPQ) cofactor transformation which indicated from the results of phenylhydrazine titration and substrate protection, we propose a mechanism for the reversible inactivation of BPAO by 1 involving the formation of a cofactor adduct, thiazolidine, between BPAO and 1.     

Physiological and subjective responses to low relative humidity in young and elderly men

In order to compare the physiological and the subjective responses to low relative humidity of elderly and young men, we measured saccharin clearance time (SCT), frequency of blinking, hydration state of the skin, transepidermal water loss (TEWL), sebum level recovery and skin temperatures as physiological responses. We asked subjects to evaluate thermal, dryness and comfort sensations as subjective responses using a rating scale. Eight non-smoking healthy male students (21.7+/-0.8 yr) and eight non-smoking healthy elderly men (71.1+/-4.1 yr) were selected. The pre-room conditions were maintained at an air temperature (Ta) of 25 degrees C and a relative humidity (RH) of 50%. The test-room conditions were adjusted to provide 25 degrees C Ta and RH levels of 10%, 30% and 50%. RH had no effect on the activity of the sebaceous gland or change of mean skin temperature. SCT of the elderly group under 10% RH was significantly longer than that of the young group. In particular, considering the SCT change, the nasal mucous membrane seems to be affected more in the elderly than in the young in low RH. Under 30% RH, the eyes and skin become dry, and under 10% RH the nasal mucous membrane becomes dry as well as the eyes and skin. These findings suggested that to avoid dryness of the eyes and skin, it is necessary to maintain greater than 30% RH, and to avoid dryness of the nasal mucous membrane, it is necessary to maintain greater than 10% RH. On the thermal sensation of the legs, at the lower humidity level, the elderly group felt cooler than the young group. On the dry sensation of the eyes and throat, the young group felt drier than the elderly group at the lower humidity levels. From the above results, the elderly group had difficulty in feeling dryness in the nasal mucous membrane despite being easily affected by low humidity. On the other hand, the young group felt the change of humidity sensitively despite not being severely affected by low humidity. Ocular mucosa and physiology of skin by dryness showed no difference by age. In the effect of longer exposure (180 min.) to low RH, only TEWL showed a slight decrease after 120 minutes in 30% RH, and all the measured results showed no noticeable differences compared with the result at 120 minutes.