Brain ventriculomegaly in Down syndrome mice is caused by Pcp4 dose-dependent cilia dysfunction.

Down syndrome is a leading cause of congenital intellectual disability caused by an additional copy of the chromosome 21. Patients display physiological and morphological changes affecting the brain and its function. Previously we showed that Ts1Cje and Ts2Cje, Down syndrome mouse models carrying overlapping trisomic segments of different length, show similar ventriculomegaly and neurogenesis dysfunction leading to the hypothesis of a cause-consequence relationship between these phenotypes. However, we here discovered that Ts1Rhr Down syndrome model, carrying an even shorter trisomic segment, was sufficient to trigger ventricular enlargement and ependymal cilia beating deficiency without affecting neurogenesis. We further found that Pcp4 gene on the Ts1Rhr trisomic segment is expressed in ependymal cells, and its resumption to two copies rescued both ventricular enlargement and cilia dysfunction in Ts1Rhr mice. This work underlines a Pcp4-dependent ciliopathy in Down syndrome brain affecting cerebrospinal fluid flow.

Oxidative stress-induced ubiquitination of RCAN1 mediated by SCFbeta-TrCP ubiquitin ligase.

A change in the protein level of RCAN1 (DSCR1/MCIP/Adapt78/CSP1) has been implicated in oxidative stress-induced cell death in neurons and in the pathogenesis of Alzheimer's disease. The pathogenic processes in neurodegenerative diseases are closely related to oxidative stress and the ubiquitin proteasome system (UPS). Therefore, we investigated whether oxidative stress induces a change in the protein level of RCAN1 through the UPS. H2O2 induced ubiquitination of RCAN1 at the same concentrations as those causing a decrease in RCAN1 in HEK293T cells. beta-TrCP, the F-box protein component of SCF ubiquitin ligase, interacted with RCAN1 in response to H2O2 stimulation. Although FBW4, another F-box protein, interacted with RCAN1, its interaction was independent of H2O2 stimulation. In vitro ubiquitination assay showed that SCFbeta-TrCP but not SCFFBW4 increased ubiquitination of RCAN1, dependent on H2O2 stimulation. In addition, knockdown of beta-TrCP by siRNA abolished the H2O2-induced decrease in RCAN1 in HEK293T cells. We further examined whether RCAN1 undergoes ubiquitination by H2O2 in primary neurons, similarly to that in HEK293T cells. An H2O2-induced decrease in RCAN1 was exhibited also in hippocampal and cortical neurons. Ubiquitination of RCAN1 was induced by 500 muM H2O2, the concentration at which H2O2 induced a decrease in RCAN1 in primary neurons. These results suggest that H2O2 induces SCF beta-TrCP-mediated ubiquitination of RCAN1, leading to a decrease in the protein level of RCAN1.

Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1.

The members of the transcription factor Foxo family regulate the expression of genes concerned with the stress response, cell cycle and gluconeogenesis. Foxo1 (FKHR) contains 15 consensus phosphorylation sites for the mitogen-activated protein kinase (MAPK) family. Therefore, we hypothesized that MAPKs could directly regulate the transcriptional activity of Foxo1 via phosphorylation. In vitro kinase assay showed that Foxo1 was phosphorylated by extracellular signal-regulated kinase (Erk) and p38 MAPK (p38) but not by c-jun NH2-terminal kinase (JNK). In NIH3T3 cells, epidermal growth factor or anisomycin increased phosphorylation of exogenous Foxo1, which was significantly inhibited by pretreatment with an MEK 1 inhibitor, PD98059, or a p38 inhibitor, SB203580. Two-dimensional phosphopeptide mapping using mutation of phosphorylation sites for MAPK revealed that the nine serine residues in Foxo1 are specifically phosphorylated by Erk and that five of the nine residues are phosphorylated by p38 in vivo. Moreover, we also found that Foxo1 interacts with Ets-1 and functions as a coactivator for Ets-1 on the fetal liver kinase (Flk)-1 promoter in bovine carotid artery endothelial cells. Mutation of the nine phosphorylation sites for Erk in Foxo1 was shown to lead to less binding and synergistic activity for Ets-1 on the Flk-1 promoter when compared with wild-type Foxo1. These results suggest that Foxo1 is specifically phosphorylated by Erk and p38, and that this phosphorylation regulates the function of Foxo1 as a coactivator for Ets-1.

G protein-coupled APJ receptor signaling induces focal adhesion formation and cell motility.

APJ, a G protein-coupled receptor, has an endogenous ligand called apelin. APJ and apelain are highly expressed in the cardiovascular system from embryo to adulthood. It has been shown that apelin elicited the migration of APJ-expressing cells, but details of the receptor signaling have not been identified. To address the signal transduction molecular mechanisms of the apelin/APJ-induced cell motility, we established human embryonic kidney 293T cells stably expressing the mouse APJ (APJ/293T). APJ/293T cells exhibited a specific [(Glp65, Nle75, Tyr77) [125I]]-Apelin13 binding activity (Kd = 4.45 nM). Apelin induced Akt/PKB phosphorylation in APJ/293T cells, but not in the intact 293T cells (-/293T cells). This APJ-dependent activation of Akt/PKB was significantly inhibited by the pretreatment of pertussis toxin (PTx) and a PI3K inhibitor, LY29004. In addition, apelin enhanced focal adhesion kinase (FAK) phosphorylation and increased focal adhesion formation with staining for F-actin in APJ/293T cells. PTx and LY29004 significantly suppressed these responses to apelin. Moreover, we examined the migration activity by using a scratch-test. Apelin strongly accelerated the cell motility in APJ/293T cells, and this activity was abolished by PTx and LY29004. These results indicated that the apelin/APJ signaling coupled with the PTx-sensitive G-protein activates Akt/PKB and FAK proteins through PI3K.

Cytodifferentiation enhances Erk activation induced by endothelin-1 in primary cultured astrocytes.

We have previously demonstrated that endothelin-1 (ET-1)-induced extracellular signal-regulated kinase (Erk) activity via the ETB receptor (EDNRB) is mediated through two independent pathways, a protein kinase C-dependent pathway and a pertussis toxin (PTX)-sensitive pathway, in astrocytes. In this study, we showed that the molar potency of ET-1 to induce Erk activation was two orders of magnitude higher in dibutyryl cAMP (DBcAMP)-treated astrocytes than in quiescent astrocytes. This DBcAMP-enhanced molar potency of ET-1 in Erk activation was selectively inhibited by pretreatment of astrocytes with PTX. The expression level of EDNRB in astrocytes was markedly upregulated by DBcAMP-induced cytodifferentiation. However, this up-regulation was simply attributed to the high expression of low-affinity sites. The molar potency of ET-1 to induce both stimulation of inositol trisphosphate production and activation of protein kinase C in DBcAMP-treated astrocytes was similar to that in quiescent astrocytes. On the contrary, the molar potency of ET-1 to induce accumulation of Ras-GTP was two orders of magnitude higher in DBcAMP-treated astrocytes than in quiescent astrocytes, which was consistent with the case of ET-1-induced Erk activation. Moreover, the ET-1-induced Ras activation was PTX sensitive. These results suggest that cytodifferentiation selectively enhances the PTXsensitive Ras/Erk pathway induced by ET-1 in astrocytes, and that cytodifferentiation-induced EDNRB up-regulation might not contribute to this selective potentiation of ET-1 signaling.

Hepatocyte nuclear factor-4 is a novel downstream target of insulin via FKHR as a signal-regulated transcriptional inhibitor.

Previous studies have shown that FKHR, a member of the forkhead family of transcription factors, acts as a DNA binding-independent cofactor of nuclear receptors, including estrogen, retinoid, and thyroid hormone receptors, in addition to the original function as a DNA binding transcription factor that redistributes from the nucleus to the cytoplasm by insulin-induced phosphorylation. Here, we demonstrated the physical interaction of FKHR with hepatocyte nuclear factor (HNF)-4, a member of steroid/thyroid nuclear receptor superfamily, and the repression of HNF-4 transactivation by FKHR. FKHR interacted with the DNA binding domain of HNF-4 and inhibited HNF-4 binding to the cognate DNA. Furthermore, the binding affinity of HNF-4 with phosphorylated FKHR significantly decreased in comparison to that with unphosphorylated FKHR. Therefore, a phosphorylation of FKHR by insulin followed by its dissociation from HNF-4 and the redistribution of FKHR from the nucleus to the cytoplasm would expect to induce the transcriptional activation of HNF-4 by facilitating to the access of HNF-4 to its DNA element. Indeed, most intriguingly, insulin stimulation reversed the repression of HNF-4 transcriptional activity by phosphorylation-sensitive (wild-type) FKHR, but not by phosphorylation-deficient FKHR. These results suggest that insulin regulates the transcriptional activity of HNF-4 via FKHR as a signal-regulated transcriptional inhibitor.

Essential role of p38 mitogen-activated protein kinase in contact hypersensitivity.

The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in the pathogenesis of inflammation, using a mouse contact hypersensitivity (CHS) model induced by 2,4-dinitro-1-fluorobenzene (DNFB). Ear swelling was induced by challenge with DNFB, accompanied by infiltration of mononuclear cells, neutrophils, and eosinophils and a marked increase in mRNA levels of cytokines such as interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-1beta, IL-18, and tumor necrosis factor-alpha in the challenged ear skin. Both ear swelling and the number of infiltrated cells in DNFB-challenged ear skin were significantly inhibited by treatment with SB202190, a p38 inhibitor. Furthermore, the DNFB-induced expression of all cytokines except IL-4 was significantly inhibited by treatment with SB202190. Ribonuclease protection assay revealed that the mRNA levels of chemokines such as IP-10 and MCP-1 in ear skin were markedly increased at 24 h after challenge with DNFB. The induction of these chemokines was significantly inhibited by treatment with SB202190. In p38alpha +/- mice, both ear swelling and infiltration of cells induced by DNFB were reduced compared with those in wild-type mice. However, induction of cytokines by DNFB was also observed in p38alpha +/- mice, although the induction of IFN-gamma, IL-5, and IL-18 was typically reduced compared with that in wild-type mice. Challenge with DNFB slightly induced IP-10 and MCP-1 mRNA in p38alpha +/- mice, with weaker signals than those in SB202190-treated wild-type mice. These results suggest that p38 plays a key role in CHS and is an important target for the treatment of CHS.