mTORC1 accelerates retinal development via the immunoproteasome.

The numbers and types of cells constituting vertebrate neural tissues are determined by cellular mechanisms that couple neurogenesis to the proliferation of neural progenitor cells. Here we identified a role of mammalian target of rapamycin complex 1 (mTORC1) in the development of neural tissue, showing that it accelerates progenitor cell cycle progression and neurogenesis in mTORC1-hyperactive tuberous sclerosis complex 1 (Tsc1)-deficient mouse retina. We also show that concomitant loss of immunoproteasome subunit Psmb9, which is induced by Stat1 (signal transducer and activator of transcription factor 1), decelerates cell cycle progression of Tsc1-deficient mouse retinal progenitor cells and normalizes retinal developmental schedule. Collectively, our results establish a developmental role for mTORC1, showing that it promotes neural development through activation of protein turnover via a mechanism involving the immunoproteasome.

Amino acid-dependent NPRL2 interaction with Raptor determines mTOR Complex 1 activation.

We assign a new function to a tumor suppressor NPRL2 that activates the mTOR complex 1 (mTORC1) activity. The positive regulation of mTORC1 activity by NPRL2 is mediated through NPRL2 interaction with Raptor. While NPRL2 interacts with Rag GTPases, RagD in particular, to interfere with mTORC1 activity in amino acid scarcity, NPRL2 interacts with Raptor in amino acid sufficiency to activate mTORC1. A reciprocal relationship exists between NPRL2 binding to Rag GTPases and Raptor. NPRL2 majorly locates in the lysosomal membranes and has a higher binding affinity to the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) that inactivates mTORC1. However, the binding affinity of NPRL2 with Raptor is much less pronounced in cells expressing the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) than in cells expressing the dominant positive mutant heterodimer, RagA(GTP)/RagD(GDP). The positive effect of NPRL2 on TORC1 pathway was also evidenced in Drosophila animal model. Here, we propose a 'seesaw' model in which the interactive behavior of NPRL2 with Raptor determines mTORC1 activation by amino acid signaling in animal cells.

Small interfering RNA nunchucks with a hydrophobic linker for efficient intracellular delivery.

The high stiffness and low spatial charge density of siRNA limit the effectiveness of the electrostatic condensation of siRNA with cationic polyelectrolytes. Here, a facile method to stabilize nanoscale siRNA/polyelectrolyte complexes by introducing a reductively cleavable alkyl chain to siRNA as a hybrophobic linker of dimeric siRNA conjugates is reported. The increased length of the hydrophobic linker increases the intracellular translocation and gene silencing activity of the dimeric siRNA conjugates when they are complexed with linear polyethylenimine (LPEI). The results suggest that the introduction of a hydrophobic linker in the dimeric siRNA conjugates can facilitate the intracellular delivery of siRNA through effective condensation with cationic polyelectrolytes.

Amelioration of behavioral abnormalities in BH(4)-deficient mice by dietary supplementation of tyrosine.

This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4)-deficient Spr (-/-) mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr (-/-) mice. We found that Spr (-/-) mice display variable 'open-field' behaviors, impaired motor functions on the 'rotating rod', and dystonic 'hind-limb clasping'. In this study, we report that these aberrant motor deficits displayed by Spr (-/-) mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr (-/-) mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA) and its metabolites in Spr (-/-) mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr (-/-) mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency.

Absence of a human DnaJ protein hTid-1S correlates with aberrant actin cytoskeleton organization in lesional psoriatic skin.

The biochemical mechanism by which the human tumorous imaginal disc1(S) (hTid-1(S)) interferes with actin cytoskeleton organization in keratinocytes of human skin epidermis was investigated. We found that hTid-1, specifically hTid-1(S), interacts with MK5, a p38-regulated/activated protein kinase, and inhibits the protein kinase activity of MK5 that phosphorylates heat shock protein HSP27 in cultured HeLa cells. Thus, hTid-1(S) expression inhibits the phosphorylation of HSP27 known to play important roles in F-actin polymerization and actin cytoskeleton organization. The interplay between MK5/HSP27 signaling and hTid-1(S) expression was supported by the inhibition of HSP27 phosphorylation and MK5 activity in HeLa cells in response to hypoxia during which hTid-1(S) expression was down-regulated. We also found that overexpression of hTid-1(S) results in the inhibition of HSP27 phosphorylation, F-actin polymerization, and actin cytoskeleton organization in transduced HaCaT keratinocytes. This study further proposes that the loss of hTid-1(S) expression in the basal layer of skin epidermis correlates with the enhanced HSP27 phosphorylation, keratinocyte hyperproliferation, and excess actin cytoskeleton organization in lesional psoriatic skin.

Pten coordinates retinal neurogenesis by regulating Notch signalling.

Development of nervous tissue is a coordinated process of neural progenitor cell (NPC) proliferation and neuronal differentiation. Intracellular signalling events that regulate the balance between NPC proliferation and neuronal differentiation, therefore, determine the size and composition of nervous tissues. Here, we demonstrate that negative regulation of phosphoinosite 3-kinase (PI3K)-Akt signalling by phosphatase tensin homologue (Pten) is essential for maintaining NPC population in mouse retina. We found that mouse retinal progenitor cells (RPCs) lacking the Pten gene complete neurogenesis earlier than their normal developmental schedule, resulting in their premature depletion in the mature retina. We further discover that Notch intracellular domain (NICD) fails to form transcription activator complex in Pten-deficient RPCs, and thereby unable to support RPC maintenance. Taken together, our results suggest that Pten plays a pivotal role in retinal neurogenesis by supporting Notch-driven RPC maintenance against neurogenic PI3K-Akt signalling.

Hypermethylation of repetitive DNA elements in livers of mice fed an atherogenic diet.

OBJECTIVE: DNA methylation status was examined in C57BL/6J obese mice fed an atherogenic diet (AD) to establish the correlation between epigenetic alterations and obesity-related abnormalities. METHODS: Six-week-old male C57BL/6J mice were fed a normal diet (ND) or AD for 8 wk. Methylation levels of global DNA and repetitive DNA elements in livers of ND-fed mice and AD-fed mice were examined. RESULTS: The total amounts of 5-MeC genomic contents in livers of AD-fed mice were increased as compared with those of ND-fed mice. Hypermethylation of repetitive DNA elements was observed in livers of AD-fed mice. CONCLUSION: Hypermethylation of repetitive DNA elements in livers of AD-fed mice proposes epigenetic changes by nutritional intervention.

Transcriptional changes of secreted Wnt antagonists in hindlimb skeletal muscle during the lifetime of the C57BL/6J mouse.

The canonical Wnt pathway plays a critical role in myogenesis and age-related inefficient muscle regeneration. To gain insights into changes in Wnt signaling in muscle during the lifetime of a mouse, mRNA levels of secreted Wnt antagonists were investigated. Among 13 analyzed antagonists, seven genes were found to be down-regulated in skeletal muscles of adult and old mice. Epigenetic modifications at the promoter regions of these seven Wnt antagonists were then examined to understand how these correlate with this transcriptional repression. DNA methylation was stably maintained, while chromatin modifications changed to transcriptionally inactive states over the course of a lifetime. Similar patterns of changes in chromatin modifications were observed at the promoters of all of the studied genes. The observations indicated that an upstream factor might regulate the chromatin states and the transcriptional repression of Wnt antagonists. Several bioinformatic analyses revealed that a FOXD3 binding motif is present within promoter regions of the seven antagonists. Furthermore, age-dependent differential FOXD3 binding is observed at the motifs of the seven gene promoters. Our results suggest that FOXD3 as a potential epigenetic regulator may mediate the transcriptional repression of the seven antagonists, possibly through regulation of histone modifications.

Autophagy induction by tetrahydrobiopterin deficiency.

Tetrahydrobiopterin (BH4) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH4 deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH4-deficient Spr(-/-) mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH4 synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr(-/-) mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr(-/-) mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr(-/-) mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pah(enu2) mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH4 deficiency.

Transcriptional repression of repeat-derived transcripts correlates with histone hypoacetylation at repetitive DNA elements in aged mice brain.

In order to better characterize epigenetic alterations at repetitive DNA elements with aging, DNA methylation and histone marks at various repeat classes were investigated. Repetitive DNA elements were hypermethylated in the brains of old mice. Histone hypoacetylation and altered histone trimethylation at repetitive sequences were detected in brain tissues during aging. The expression of repeat-derived transcripts (RDTs) was then measured to explore any correlations with the observed epigenetic alterations. Large numbers of RDTs investigated were down-regulated along with age. Bisulfite sequencing revealed that CpG dinucleotide methylation patterns at the repeats of the RDT promoter region were mostly well maintained during aging. ChIP assay showed that histones were deacetylated at the promoter region of RDTs in aged mice brain. The observations indicate that the transcriptional repression of RDTs appears to be related to histone hypoacetylation, but not to DNA hypermethylation at repeat DNA elements in the brains of aged mice.

Alkaline stress-induced autophagy is mediated by mTORC1 inactivation.

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.

Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways.

Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

Retrotransposon-specific DNA hypomethylation and two-step loss-of-imprinting during WW45 haploinsufficiency-induced hepatocarcinogenesis.

Liver cancer development follows a multistep process that includes epigenetic changes beginning at the initiation stage, changes that have been studied for their potential diagnostic value. Here, we examined long-term, cancer-associated epigenetic changes during carcinogenesis using a mouse model of liver cancer. WW45-haploinsufficient (WW45(+/-)) mice developed liver cancer after 12 months due to dysregulation of the Hippo pathway and consequent Yap overexpression. There was no pathological sign of neoplastic regions in the livers of 10-month-old WW45(+/-) mice but whole-gene expression patterns statistically proved the resemblance between 10-month-old livers and hepatomas from WW45(+/-) mice. We found epigenetic features in the livers of 10-month-old WW45(+/-) mice which were already distinctive from the wild-type counterparts prior to tumorigenesises. H19 ICR showed loss-of-imprinting in two steps and allelic histone marker signature during tumorigenesis showed similarity with ES cells. Progressive cancer pathognomonic global hypomethylation was a characteristic post-10-month feature and was well reflected in retrotransposons. Heterochromatic histone modifications also decreased in retrotransposons after 10 months in the liver of WW45(+/-) mice. This study showed potential epigenetic features for cancer prognostic use and supported the epigenetic progenitor model of cancer.

Notch signal activates hypoxia pathway through HES1-dependent SRC/signal transducers and activators of transcription 3 pathway.

We report a Notch signal-induced pathway that leads to transcriptional activation of HIF1-alpha gene. HeLa/rtTAA/TRE-N1-IC cell line capable of doxycycline-induced expression of human Notch1-IC was established. The induction of Notch signaling activates HIF1-alpha and its target gene expression in HeLa/rtTAA/TRE-N1-IC cells. Notch signaling enhanced signal transducers and activators of transcription 3 (STAT3) phosphorylation required for HIF1-alpha expression. SRC kinase was found to be responsible for the enhanced STAT3 phosphorylation in response to Notch signaling. Activation of SRC/STAT3 pathway by Notch signaling was dependent on the expression of Notch effector HES1 transcription factor. The induction of HES1 enhanced STAT3 phosphorylation at Tyr 705 as well as SRC phosphorylation at Tyr 416 in inducible HeLa/rtTAA/TRE-HES1 cells, which express HES1 in response to doxycycline treatment. However, the treatment of Trichostatin A that interferes with HES1 transcriptional regulation did not affect STAT3 phosphorylation, and the expression of dominant negative HES1 failed to interfere with HES1-dependent SRC/STAT3 pathway. These observations have led us to the conclusion that HES1-dependent activation of SRC/STAT3 pathway is independent of HES1 transcription regulation. This study first reports HES1-dependent SRC/STAT3 pathway that provides a functional link between Notch signaling and hypoxia pathway.

Akt stabilizes estrogen receptor alpha with the concomitant reduction in its transcriptional activity.

We have investigated the effect of Akt on estrogen receptor (ER) alpha protein level and its transcriptional activity. Transient transfection studies revealed that constitutively active Akt1 up-regulated ERalpha at the post-transcriptional level. Studies using Akt inhibitor and dominant-negative Akt1 showed that Akt1 kinase activity is required for the up-regulation of ERalpha. Cycloheximide decay assays and studies with proteasome inhibitor indicated that Akt1-mediated up-regulation of ERalpha was maintained by inhibiting proteasome-mediated degradation of ERalpha. When Akt consensus phosphorylation site mutant, ERalphaS167A was tested for Akt1-mediated up-regulation, increase of ERalphaS167A by Akt1 was significantly impaired as compared to wild type ERalpha. In addition, dominant-negative glycogen synthase kinase (GSK) 3beta and LiCl could also partially up-regulate ERalpha protein level, suggesting that concerted action of Akt1-mediated phosphorylation on S167 and kinase activity of Akt-downstream GSK3beta could affect ERalpha protein level. Paradoxically, co-expression of Akt1 could down-regulate transcriptional activity of ERalpha. The inhibitory effect of Akt1 on ERalpha transcriptional activity was not attributable to changes in subcellular distribution of ERalpha. Transfection studies using increasing amount of Akt1 and ERalpha indicated that the transcriptional activity of ERalpha was negatively regulated by ERalpha protein quantities at higher ERalpha concentrations. Chromatin immunoprecipitation assays revealed that at Akt1 concentration high enough to induce up-regulation of ERalpha, association of ERalpha to promoter region of ERalpha target pS2 gene was impaired. Taken together, these data suggest that Akt1 could increase ERalpha protein level with simultaneous reduction in its transcriptional activity, possibly by modulating association of ERalpha to the target gene promoters.

Intracellular delivery enhancement of poly(amino acid) drug carriers by oligoarginine conjugation.

Poly(2-hydroxytethyl aspartamide) (PHEA) was effectively translocated in both fixed and unfixed HeLa cells, when oligoarginine (Arg(8)) known as one of the cell-penetrating peptides was conjugated via a thioether linkage. The internalization of PHEA-Arg(8) into cells was a temperature-dependent process, and the studies at endocytosis inhibition conditions suggested that an endocytosis was a key mechanism. The fluorescence spectra of PHEA-Arg(8) in liposome solutions showed that PHEA-Arg(8) was collectively adsorbed in the negative liposome membrane due to the high cationic property of a conjugated Arg(8), representing that a surface adsorption was a first step in the internalization of PHEA-Arg(8). The membrane leakage activity of PHEA-Arg(8) was much lower than that of Arg(8) own, meaning that PHEA-Arg(8) does not effectively disrupt the cell membrane integrity. The uptake of polymer conjugates increased with the incubation time and reached saturation after several hours. The increase in the number of peptide conjugated to one polymer chain could increase the collective adsorption of polymer conjugates and enhance the cellular uptake. Thus, it is believed that PHEA-Arg(8) could be internalized by an adsorptive-endocytosis. A model conjugate of PHEA-Arg(8) with methotrexate (PHEA-MTX-Arg(8)) inhibited the cell proliferation about several orders of magnitude more active than PHEA-MTX.

14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization.

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.

Integrin-linked kinase controls Notch1 signaling by down-regulation of protein stability through Fbw7 ubiquitin ligase.

Integrin-linked kinase (ILK) is a scaffold and protein kinase that acts as a pivotal effector in integrin signaling for various cellular functions. In this study, we found that ILK remarkably reduced the protein stability of Notch1 through Fbw7. The kinase activity of ILK was essential for the inhibition of Notch1 signaling. Notably, the protein level and transcriptional activity of the endogenous Notch1 intracellular domain (Notch1-IC) were higher in ILK-null cells than in ILK wild-type cells, and the level of endogenous Notch1-IC was increased by the blocking of the proteasome, suggesting that ILK enhances the proteasomal degradation of Notch1-IC. ILK directly bound and phosphorylated Notch1-IC, thereby facilitating proteasomal protein degradation through Fbw7. Furthermore, we found down-regulation of Notch1-IC and up-regulation of ILK in basal cell carcinoma and melanoma patients but not in squamous cell carcinoma patients. These results suggest that ILK down-regulated the protein stability of Notch1-IC through the ubiquitin-proteasome pathway by means of Fbw7.

Inhibition of NF-kappaB acetylation and its transcriptional activity by Daxx.

We propose a biochemical mechanism by which Daxx modulates NF-kappaB transcriptional activity. Both chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) have confirmed Daxx-mediated repression of transcriptional competence of NF-kappaB in HeLa cells. Overexpression of Daxx repressed the expression of NF-kappaB-regulated genes such as I kappa B alpha and IL8. Co-immunoprecipitation assay revealed the existence of intermolecular association between endogenous Daxx and p65 subunit of NF-kappaB stimulated by TNFalpha. Here, we suggest that Daxx-mediated repression of NF-kappaB transactivation correlates with the inhibition of p65 acetylation by Daxx. Based on the finding that the Daxx binding N-terminal side of p65 includes the major sites of acetylation mediated by p300/CBP, we further propose that the physical interaction between Daxx and p65 provides a functional framework for the inhibition of p65 acetylation by p300/CBP and subsequent repression of NF-kappaB transcriptional activity.

Interaction of HCV core protein with 14-3-3epsilon protein releases Bax to activate apoptosis.

Through protein-protein binding assays, we found that HCV core protein interacted with 14-3-3epsilon protein. Interestingly, the expression of HCV core protein induced apoptosis in 293T cells. The apoptosis induced by core expression is accompanied by translocation of Bax from cytosol to mitochondria, disruption of mitochondrial membrane potential, cytochrome c release, and activation of caspase-9 and caspase-3. Furthermore, over-expression of 14-3-3epsilon inhibited the core-induced apoptosis and Bax translocation to mitochondria. These results indicate that HCV core protein induces the Bax-mediated apoptosis by interacting with 14-3-3epsilon protein in 293T cells. As a mechanism of apoptosis induction by HCV core, we propose that the interaction of HCV core with 14-3-3epsilon causes the dissociation of Bax from the Bax/14-3-3epsilon complex in cytosol, and the free Bax protein provokes activation of the mitochondrial apoptotic pathway.