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.

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.

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.

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.

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.

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.

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.

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.

BTB/POZ domain of speckle-type POZ protein (SPOP) confers proapoptotic function in HeLa cells.

The proapoptotic function of SPOP protein was investigated in HeLa cells. HeLa cells underwent apoptosis by the overexpression of SPOP. Studies using SPOP deletion mutants suggest that BTB/POZ domain of SPOP protein is important for the induction of apoptosis in transfected cells. This study first proposes the proapoptotic aspect of the BTB/POZ domain of SPOP protein based on the finding that cells expressing the C-terminal fragment of SPOP containing the BTB/POZ domain underwent apoptosis.

Dose-dependent UV stabilization of p53 in cultured human cells undergoing apoptosis is mediated by poly(ADP-ribosyl)ation.

The effect of poly(ADP-ribosyl)ation on the stability of p53 in SK-HEP1 cells treated with UV light was examined. Intracellular levels of p53 increased in cells treated with a low dose of UV light (20 J/m2), whereas they increased but then declined after a higher dose of UV (100 J/m2). Intracellular levels of p53 in the UV treated SK-HEP1 cells were dependent on the UV dose. Use of proteasome inhibitors revealed that p53 is degraded by proteasomal proteolysis after high doses of UV light. We present evidence that, at low doses, poly(ADP-ribose)polymerase (PARP) poly(ADP-ribosyl)ates p53 and protects it from proteasomal degradation before caspase-3 is activated, whereas at high doses the cells undergo UV induced apoptosis and PARP is cleaved by caspase-3 before it can protect p53 from degradation. Destabilization of p53 by cleavage of PARP may be important in cell fate decision favoring apoptosis.

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.

Functional interaction between human papillomavirus type 18 E2 and poly(ADP-ribose) polymerase 1.

Human papillomavirus E2 protein is a transcription factor of viral gene expression and DNA replication. Here we show that PARP is a positive regulator of the E2 protein of human papillomavirus type 18 (HPV-18). PARP interacted with the COOH terminal region of HPV-18 E2 in vitro. The E2 interaction domain within PARP is located in the NH(2)-terminal zinc finger motif and the BRCT motif included in the automodification domain. Overexpression of either wild type or the NH(2)-terminal region of PARP containing zinc finger and BRCT stimulated E2-dependent transcription. Gel retardation assay indicates that PARP augments DNA binding activity of E2 in vitro. We also show that PARP-1 is recruited to E2-dependent promoter in vivo using ChIP assay. These results suggest that PARP serves a transcriptional co-activator in E2-dependent transcription by interacting directly with the HPV E2 protein.

Folate receptor mediated intracellular protein delivery using PLL-PEG-FOL conjugate.

To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins or other bioactive macromolecules into a specific cell, a di-block copolymer conjugate, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL), was synthesized. The PLL-PEG-FOL conjugate was physically complexed with fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) in an aqueous phase by ionic interactions. Cellular uptake of PLL-PEG-FOL/FITC-BSA complexes was greatly enhanced against a folate receptor over-expressing cell line (KB cells) compared to a folate receptor deficient cell line (A549 cells). The presence of an excess amount of free folate (1 mM) in the medium inhibited the intracellular delivery of PLL-PEG-FOL/FITC-BSA complexes. This suggests that the enhanced cellular uptake of FITC-BSA by KB cells in a specific manner was attributed to folate receptor-mediated endocytosis of the complexes having folate moieties on the surface. The PLL-PEG-FOL di-block copolymer could be potentially applied for intracellular delivery of a wide range of other biological active agents that have negative charges on the surface.

Folate receptor-mediated intracellular delivery of recombinant caspase-3 for inducing apoptosis.

Recombinant reversed caspase-3 (rev-caspase-3) is a pro-apoptotic gene capable of intracellular autocatalytic processing, which leads to programmed cell death. Folate receptor-specific intracellular delivery of the rev-caspase-3 gene into KB cells over-expressing folate receptors was explored by employing the folate-poly(ethylene glycol)-polyethylenimine (FOL-PEG-PEI) conjugate as a nonviral polymeric carrier. Using luciferase as a reporter gene, the conditions for formulation of DNA/polymer polyplexes were pre-optimized to attain the highest folate receptor-mediated gene transfection efficiency. FOL-PEG-PEI conjugate complexed with rev-caspase-3 plasmid in an optimized condition gave rise to a great increase in expression and activation of exogenous rev-caspase-3 in KB cells when pretreated with doxorubicin. The synthesized conjugate exhibited higher transfection efficiency than other commercially available transfection agents due to a unique mechanism of folate-receptor mediated endocytic gene transfer. The transfected cells showed a significant extent of apoptosis by rev-caspase-3. This study suggests the potential of using folate-receptor-mediated delivery of rev-caspase-3 gene for inducing tumor cell death in a target-specific manner.

Extracellular and intracellular glutathione protects astrocytes from Zn2+-induced cell death.

Free Zn(2+) is released in excess at excitatory synapses in pathological conditions including transient global and focal cerebral ischemia, which causes neuronal and glial cell death. In the current study, we explored the mechanism underlying Zn(2+)-induced cell death in primary cortical astroglial cultures. Chronic treatment with 30-35 microM Zn(2+) led to the death of 70-95% of astrocytes within 18 h, preceded by Zn(2+) influx. Extracellular glutathione (GSH; 100 microM) completely blocked the Zn(2+) influx and Zn(2+) toxicity. The Zn(2+) toxicity was also inhibited when intracellular GSH was increased. Conversely, it was aggravated when intracellular GSH was depleted by buthionine sulfoximine (BSO). Consistently, the level of cellular GSH was notably decreased with a concurrent increase in oxidized GSH in Zn(2+)-treated astrocytes. These results suggest that the disruption of proper maintenance of thiol homeostasis is a mechanism underlying Zn(2+) toxicity in primary cortical astrocytes.

Lactoferrin works as a new LPS-binding protein in inflammatory activation of macrophages.

Though lactoferrin (LF) is a glycoprotein that is involved in immunomodulation, its action mechanism has not been fully elucidated. Previous studies have suggested that lipopolysaccharide (LPS) activity is inhibited by direct binding between LPS and LF. However, here we show that when LPS and purified LF was mixed, and formed a complex (termed as LF-LPS), it was found to induce production of inflammatory mediators in macrophages to some extent, rather than inhibit LPS activity. Moreover, when macrophages were pretreated with LF-LPS, cells were rendered a tolerant state to LPS challenge. These macrophage-activating effects were mediated by Toll-like receptor 4 (TLR4)-NF-kappaB pathway. Comparative studies with C3H/HeN and C3H/HeJ mice demonstrated the strong dependency of the LF-LPS signal on TLR4. These findings suggest that the immunomodulatory properties of LF could be due, in part, to LPS binding.

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.