Effect of Janus Kinase 3 Inhibitor on Sebaceous Gland Regeneration during Skin Wound Healing.

BACKGROUND: Janus kinase (Jak) 3 has recently been shown as a beneficial target for the treatment of chronic inflammatory diseases, such as psoriasis and alopecia areata. The role of Jak3 in tissue repair and remodeling is emerging. OBJECTIVE: This study aimed to investigate the role of Jak3 signaling in the remodeling of the sebaceous gland (SG) during skin wound repair, and the development of in vitro SGs. METHODS: Mouse skin tissue (ICR mouse) was obtained from the recovered skin eight days after a 4 mm biopsy punch wound. To observe the role of Jak3, the selective inhibitors WHI-p131 and PF06651600 was administered. Formation of in vitro SG was examined using primary sebocyte cultures obtained postnatally from 3-day-old mice. RESULTS: The data showed that SGs showed highly positive signals with anti-isolectin B4, which also used for detection of angiogenetic vessels and the basal epidermis. Isolectin B4 could be a good indicator of SGs. The Jak3 inhibitors significantly reduced the area and volume of SG remodeling with reduced expression of p-Jak3. In addition, the area of cultured intact SG in vitro was significantly decreased in a concentration-dependent manner by Jak3 inhibition. CONCLUSION: These data showed that Jak3 signaling is a potent regulator to develop SGs. Jak3 inhibition did not decrease the number of sebocytes in SGs but decreased the area and volume of SG remodeling. Therefore, Jak3 inhibition may be a potential target for the treatment of SG hyperplasia and associated skin diseases.

Silica-coated magnetic-nanoparticle-induced cytotoxicity is reduced in microglia by glutathione and citrate identified using integrated omics.

BACKGROUND: Nanoparticles have been utilized in brain research and therapeutics, including imaging, diagnosis, and drug delivery, owing to their versatile properties compared to bulk materials. However, exposure to nanoparticles leads to their accumulation in the brain, but drug development to counteract this nanotoxicity remains challenging. To date, concerns have risen about the potential toxicity to the brain associated with nanoparticles exposure via penetration of the brain blood barrier to address this issue. METHODS: Here the effect of silica-coated-magnetic nanoparticles containing the rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)] were assessed on microglia through toxicological investigation, including biological analysis and integration of transcriptomics, proteomics, and metabolomics. MNPs@SiO2(RITC)-induced biological changes, such as morphology, generation of reactive oxygen species, intracellular accumulation of MNPs@SiO2(RITC) using transmission electron microscopy, and glucose uptake efficiency, were analyzed in BV2 murine microglial cells. Each omics data was collected via RNA-sequencing-based transcriptome analysis, liquid chromatography-tandem mass spectrometry-based proteome analysis, and gas chromatography- tandem mass spectrometry-based metabolome analysis. The three omics datasets were integrated and generated as a single network using a machine learning algorithm. Nineteen compounds were screened and predicted their effects on nanotoxicity within the triple-omics network. RESULTS: Intracellular reactive oxygen species production, an inflammatory response, and morphological activation of cells were greater, but glucose uptake was lower in MNPs@SiO2(RITC)-treated BV2 microglia and primary rat microglia in a dose-dependent manner. Expression of 121 genes (from 41,214 identified genes), and levels of 45 proteins (from 5918 identified proteins) and 17 metabolites (from 47 identified metabolites) related to the above phenomena changed in MNPs@SiO2(RITC)-treated microglia. A combination of glutathione and citrate attenuated nanotoxicity induced by MNPs@SiO2(RITC) and ten other nanoparticles in vitro and in the murine brain, protecting mostly the hippocampus and thalamus. CONCLUSIONS: Combination of glutathione and citrate can be one of the candidates for nanotoxicity alleviating drug against MNPs@SiO2(RITC) induced detrimental effect, including elevation of intracellular reactive oxygen species level, activation of microglia, and reduction in glucose uptake efficiency. In addition, our findings indicate that an integrated triple omics approach provides useful and sensitive toxicological assessment for nanoparticles and screening of drug for nanotoxicity.

Glutamate Dehydrogenase as a Neuroprotective Target Against Neurodegeneration.

Regulation of glutamate metabolism via glutamate dehydrogenase (GDH) might be the promising therapeutic approach for treating neurodegenerative disorders. In the central nervous system, glutamate functions both as a major excitatory neurotransmitter and as a key intermediate metabolite for neurons. GDH converts glutamate to α-ketoglutarate, which serves as a TCA cycle intermediate. Dysregulated GDH activity in the central nervous system is highly correlated with neurological disorders. Indeed, studies conducted with mutant mice and allosteric drugs have shown that deficient or overexpressed GDH activity in the brain can regulate whole body energy metabolism and affect early onset of Parkinson's disease, Alzheimer's disease, temporal lobe epilepsy, and spinocerebellar atrophy. Moreover, in strokes with excitotoxicity as the main pathophysiology, mice that overexpressed GDH exhibited smaller ischemic lesion than mice with normal GDH expression. In additions, GDH activators improve lesions in vivo by increasing α-ketoglutarate levels. In neurons exposed to an insult in vitro, enhanced GDH activity increases ATP levels. Thus, in an energy crisis, neuronal mitochondrial activity is improved and excitotoxic risk is reduced. Consequently, modulating GDH activity in energy-depleted conditions could be a sound strategy for maintaining the mitochondrial factory in neurons, and thus, protect against metabolic failure.

Jak kinase 3 signaling in microgliogenesis from the spinal nestin+ progenitors in both development and response to injury.

During spinal cord development, endogenous progenitors expressing nestin can migrate into the target and differentiate into neurons and other glial cells. Microglial cells can also be derived from nestin progenitor cells, even in the adult brain. Knockdown of Jak kinase 3 (Jak3) signaling can increase neurogenesis with longer neurite outgrowth in cortical progenitor cells. This study investigated the effect of Jak3 signaling on differentiation from nestin progenitor cells using E13.5 spinal progenitor cell cultures. In growth factors-enriched culture, developing neurons could not survive after several days and also a significant proportion of nestin-expressing cells transformed into ameboid Iba1 microglial cells, which increased exponentially after 5 days. This microgliogenesis was predominantly regulated by Jak3 signaling, which was confirmed by transcription factors responsible for microgliogenesis, and microglial migration and phagocytosis, such as Pu.1, Irf8, and Runx1. Jak3 inhibition also significantly increased the Tuj1 growing neurites with little microglial activation. These results indicated that neuronal and microglial cell differentiation was regulated primarily by Jak3 signaling and the developing neurons and neurite outgrowth might also be regulated by Jak3-dependent microglial activity.

Glutamate dehydrogenase as a neuroprotective target against brain ischemia and reperfusion.

Deregulation of glutamate homeostasis is associated with degenerative neurological disorders. Glutamate dehydrogenase (GDH) is important for glutamate metabolism and plays a central role in expanding the pool of tricarboxylic acid (TCA) cycle intermediate alpha-ketoglutarate (α-KG), which improves overall bioenergetics. Under high energy demand, maintenance of ATP production results in functionally active mitochondria. Here, we tested whether the modulation of GDH activity can rescue ischemia/reperfusion-induced neuronal death in an in vivo mouse model of middle artery occlusion and an in vitro oxygen/glucose depletion model. Iodoacetate, an inhibitor of glycolysis, was also used in a model of energy failure, remarkably depleting ATP and α-KG. To stimulate GDH activity, the GDH activator 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid and potential activator beta-lapachone were used. The GDH activators restored α-KG and ATP levels in the injury models and provided potent neuroprotection. We also found that beta-lapachone increased glutamate utilization, accompanied by a reduction in extracellular glutamate. Thus, our hypothesis that mitochondrial GDH activators increase α-KG production as an alternative energy source for use in the TCA cycle under energy-depleted conditions was confirmed. Our results suggest that increasing GDH-mediated glutamate oxidation represents a new therapeutic intervention for neurodegenerative disorders, including stoke.

Effect of Continuous-Wave Low-Intensity Ultrasound in Inflammatory Resolution of Arthritis-Associated Synovitis.

BACKGROUND: Low-intensity ultrasound (LIUS) can reduce pain and improve function in arthritic joints. Neutrophils are first-line actors in host defense that recruit macrophages. Dead neutrophils are removed during resolution of inflammation. Delayed neutrophil clearance can lead to extended inflammation or even chronic autoimmune disease. Although neutrophil extracellular traps (NETs) in arthritic tissue are involved in the pathogenesis of arthritis, their functional role has not been clarified. OBJECTIVES: This study aimed to investigate the effect of LIUS on synovial inflammation and its resolution via neutrophil clearance. METHODS: Synovitis was induced by intra-articular injection of complete Freund's adjuvant (CFA) into the left knee joint of 58 adult male Sprague-Dawley rats. Low-intensity ultrasound (1 MHz, 200 mW/cm(2)) was applied for 10 minutes daily. Neutrophil clearance was assessed with the expression of myeloperoxidase (MPO). In addition, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and NET formation in the synovium were observed. In neutrophil and macrophage cultures from peripheral blood, the effect of NET clearance by LIUS was investigated. RESULTS: In CFA-induced synovitis, MPO-positive neutrophils peaked after 2 to 3 days, filling the inflammatory core. Monocytes and macrophages in the periphery later infiltrated the core and were reduced thereafter. Low-intensity ultrasound reduced synovial hyperplasia and induced earlier MPO clearance. Neutrophils in the core of the inflamed synovium exhibited NET formation, which LIUS increased. Low-intensity also induced NETs in peripheral polymorphonuclear cells in an intensity-dependent manner and potentiated phorbol myristate acetate (PMA)-induced NETosis. The PMA-induced NETs were cleared by macrophages; clearance was enhanced by LIUS. LIMITATIONS: The effect of LIUS on CFA-induced inflammation was observed only during the acute phase. Although the effect of LIUS on NETosis in the in vitro neutrophil culture system was clear, the in vivo NETosis cannot be quantified. CONCLUSIONS: Neutrophil extracellular traps act in inflammatory synovitis, and LIUS enhanced the NETs and resulted in neutrophil clearance by enhancing the phagocytosis of macrophages, which might be a factor underlying the therapeutic effect of LIUS in arthritic synovium.

Fructose-1,6-bisphosphate suppresses lipopolysaccharide-induced expression of ICAM-1 through modulation of toll-like receptor-4 signaling in brain endothelial cells.

Fructose-1,6-bisphosphate (FBP) is a glycolytic intermediate with salutary effects in various brain injury models, but its neuroprotective mechanism is incompletely understood. In this study, we examined the effects of FBP on the expression of adhesion molecules in cerebrovascular endothelial cells and explored the possible mechanisms therein involved. FBP significantly down-regulated lipopolysaccharide (LPS)-induced expression of adhesion molecules and leukocyte adhesion to brain endothelial cells and inhibited NF-κB activity, which is implicated in the expression of adhesion molecules. FBP abrogated ICAM-1 expression and NF-κB activation induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or overexpression of MyD88 or TRAF6. FBP suppressed TRAF6-induced phosphorylation of TAK1, IKKß and IκBα, but fail to affect NF-κB activity induced by ectopic expression of IKKß. In addition, LPS-induced IRAK-1 phosphorylation was inhibited by FBP, suggesting the presence of multiple molecular targets of FBP in MyD88-dependent signaling pathway. FBP significantly attenuated ICAM-1 expression and NF-κB activity induced by poly[I:C] or overexpression of TRIF or TBK1. FBP significantly repressed the expression of interferon-ß (IFN-ß) and the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C] or overexpression of TRIF or TBK1, but fail to affect IRF3 activity induced by ectopic expression of constitutively active IRF3. Overall, our results demonstrate that FBP modulates both MyD88- and TRIF-dependent signaling pathways of TLR4 and subsequent inflammatory responses in brain endothelial cells, providing insight into its neuroprotective mechanism in brain injury associated with inflammation.

Isobavachalcone attenuates lipopolysaccharide-induced ICAM-1 expression in brain endothelial cells through blockade of toll-like receptor 4 signaling pathways.

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte-endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-ß, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.

Effects of low-intensity ultrasound on gramicidin D-induced erythrocyte edema.

OBJECTIVES: To determine whether low-intensity ultrasound (US) can reduce red blood cell (RBC) edema and, if so, whether the US activity is associated with aquaporin 1 (AQP-1), a water channel in the cell membrane. METHODS: Red blood cell edema was induced by gramicidin D treatment at 40 ng/mL for 20 minutes and evaluated by a hematocrit assay. Low-intensity continuous wave US at 1 MHz was applied to RBCs for the last 10 minutes of gramicidin D treatment. To determine whether US activity was associated with AQP-1, RBCs were treated with 40 µM mercuric chloride (HgCl(2)), an AQP-1 inhibitor, for 20 minutes at the time of gramicidin D treatment. Posttreatment morphologic changes in RBCs were observed by actin staining with phalloidin. RESULTS: Red blood cell edema increased significantly with gramicidin D at 20 (1.8%), 40 (6.7%), 60 (16.7%), and 80 (11.3%) ng/mL, reaching a peak at 60 ng/mL, compared to the control group (20 ng/mL, P = .019; 40, 60, and 80 ng/mL, P < .001). No significant RBC hemolysis was observed in any group. Edema induced by gramicidin D at 40 ng/mL was significantly reduced by US at 30 (3.4%; P = .003), 70 (4.4%; P = .001), and 100 (2.9%; P = .001) mW/cm(2). Subsequent experiments showed that edema reduction by US ranged from 7% to 10%. Cotreatment with HgCl(2) partially reversed the US effect and showed a significantly different level of edema compared to gramicidin D-alone and US-cotreated groups (P = .001). These results were confirmed by microscopic observation of RBC morphologic changes. CONCLUSIONS: Low-intensity US could reduce gramicidin D-induced RBC edema, and its effect appeared to at least partly involve regulation of AQP-1 activity. These results suggest that low-intensity US can be used as an alternative treatment to control edema and related disorders.

JAK-STAT pathway and myogenic differentiation.

Myogenic differentiation plays an important role in muscle regeneration and is regulated by two transcription factor families, MRFs and MEF2, which induce differentiation of myoblasts through expression of the muscle-specific gene, myogenin. In addition, many intracellular signaling pathways are also involved in myogenic differentiation, including p38 MAPK, ERK/MAPK and PI3K/AKT. The JAK-STAT pathway is activated by various cytokines and positively or negatively regulates the differentiation of myoblasts. JAK1 plays a notable role in proliferation; whereas, JAK2 and JAK3 function mainly in differentiation. The STATs, molecules downstream of JAK, regulate myogenesis. With JAK1, STAT1 promotes proliferation, while STAT3 has a dual effect on proliferation and differentiation. The JAK-STAT negative regulator, SOCS, is also associated with myogenesis; although, its role is controversial. In this review, we will discuss the role of the JAK-STAT pathway on myogenic differentiation.

Seizure susceptibility in immature brain due to lack of COX-2-induced PGF2α.

The immature brain is prone to seizure; however, the mechanism underlying this vulnerability has not been clarified. Febrile seizure is common in young children, and the use of non-steroidal anti-inflammatory drugs for febrile seizure is not recommended. In previous studies, we established that prostaglandin (PG) F2α, a product of cyclooxygenase (COX), acts as an endogenous anticonvulsant in the adult mouse. Therefore, we assumed that COX-2 activity was involved with seizure susceptibility in early life. In the present study, immature mice (postnatal day 9) were far more prone to kainic acid (KA)-induced seizures than mature mice (after postnatal day 35). Seizure activity began later in immature mice, but was more severe and was unaffected by a potent COX inhibitor, indomethacin; in contrast, indomethacin aggravated seizure activity in mature mice. Immature mouse brains exhibited little basal COX-2 expression and little KA-induced COX-2 induction, while KA-induced COX-2 expression and PGF2α release were prominent in mature brains. During brain development, COX expression was increased and glycosylated in an age-dependent manner, which was necessary for COX enzyme activity. Intracisternal PGF2α administration also reduced KA-induced seizure activity and mortality. Taken together, low COX activity and the resulting deficiency of PGF2α may be an essential cause of increased seizure susceptibility in the immature brain.

Fructose-1,6-bisphosphate ameliorates lipopolysaccharide-induced dysfunction of blood-brain barrier.

Fructose-1,6-bisphosphate (FBP), a glycolytic intermediate, has neuroprotective effects in various brain injury models. However, its effects on blood-brain barrier (BBB) are largely unknown. In this study, we investigated the effects of FBP on lipopolysaccharide (LPS)-induced BBB dysfunction in in vitro BBB model comprising co-culture of mouse brain endothelial cell line, bEnd.3 and mouse primary astrocyte and explored its action mechanism therein involved. LPS induced the impairment of endothelial permeability and transendothelial electrical resistance (TEER). The functional changes were confirmed by alterations in immunostaining for junctional proteins occludin, ZO-1 and VE-cadherin, such as the loss of cortical staining pattern and appearance of intercellular gaps in endothelial cells. Co-administration of FBP alleviated the deleterious effects of LPS on BBB permeability and TEER in a dose dependent manner. And also FBP inhibited the LPS-induced changes in the distribution of endothelial junctional proteins, resulting in the better preservation of monolayer integrity. FBP suppressed the production of reactive oxygen species (ROS) but did not affect cyclooxygenase-2 expression and prostaglandin E2 production in endothelial cells stimulated with LPS. Taken together, these data suggest that FBP could ameliorate LPS-induced BBB dysfunction through the maintenance of junctional integrity, which might be mediated by downregulation of ROS production.

Genomic copy number alterations with transcriptional deregulation at 6p identify an aggressive HCC phenotype.

Genomic analyses have revealed the enormous heterogeneity in essentially all cancer types. However, the identification of precise subtypes, which are biologically informative and clinically useful, remains a challenge. The application of integrative analysis of multilayered genomic profiles to define the chromosomal regions of genomic copy number alterations with concomitant transcriptional deregulation is posited to provide a promising strategy to identify driver targets. In this study, we performed an integrative analysis of the DNA copy numbers and gene expression profiles of hepatocellular carcinoma (HCC). By comparing DNA copy numbers between HCC subtypes based on gene expression pattern, we revealed the DNA copy number alteration with concordant gene expression changes at 6p21-p24 particularly in the HCC subtype of aggressive phenotype without expressing stemness genes. Among the genes at 6p21-p24, we identified IER3 as a potential driver. The clinical utility of IER3 copy numbers was demonstrated by validating its clinical correlation with independent cohorts. In addition, short hairpin RNA-mediated knock-down experiment revealed the functional relevance of IER3 in liver cancer progression. In conclusion, our results suggest that genomic copy number alterations with transcriptional deregulation at 6p21-p24 identify an aggressive HCC phenotype and a novel functional biomarker.

Prostaglandin E2-induced intercellular adhesion molecule-1 expression is mediated by cAMP/Epac signalling modules in bEnd.3 brain endothelial cells.

BACKGROUND AND PURPOSE: Prostaglandin E2 (PGE2) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells. EXPERIMENTAL APPROACH: bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE2 with or without agonists/antagonists of PGE2 receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry. KEY RESULTS: PGE2 significantly up-regulated the expression of ICAM-1, which was blocked by EP4 antagonist (ONO-AE2-227) and knock-down of EP4. PGE2 effects were mimicked by forskolin, dibutyryl cAMP (dbcAMP) and an exchange protein directly activated by cAMP (Epac) activator (8-Cpt-cAMP) but not a protein kinase A activator (N6-Bnz-cAMP). PGE2-induced ICAM-1 expression was reduced by knock-down of Epac1. A PI3K specific inhibitor (LY294002), Akt inhibitor VIII (Akti) and NF-κB inhibitors (Bay-11-7082 and MG-132) attenuated the induction of ICAM-1 by PGE2. PGE2, dbcAMP and 8-Cpt-cAMP induced the phosphorylation of Akt, IκB kinase and IκBα and the translocation of p65 to the nucleus and increased NF-κB dependent reporter gene activity, which was diminished by Akti. CONCLUSION AND IMPLICATIONS: Our findings suggest that PGE2 induces ICAM-1 expression via EP4 receptor and Epac/Akt/NF-κB signalling pathway in bEnd.3 brain endothelial cells, supporting its pathophysiological role in brain inflammation.

Role of JAK3 in myogenic differentiation.

Skeletal muscle differentiation is regulated by transcription factors, including members of the myogenic regulatory factor (MRF) family and many signaling pathways. The JAK1 and JAK2 pathways are known to each have different effects on myoblast proliferation and differentiation; however, the role of JAK3 in myoblast differentiation remains unclear. In this study, we investigated the effect of JAK3 inhibition on myogenic differentiation in the C2C12 mouse myoblast cell line. During myogenic differentiation, treatment with the JAK3 inhibitor WHIp154 significantly increased the number of MHC-positive multinucleated myotubes and the expressions of myosin heavy chain (MHC), myogenin (MGN), MyoD, and myogenic enhancer factor 2 (MEF2). Knockdown of the JAK3 gene using siJAK3 also significantly increased MHC, MGN and MyoD mRNA expressions as well as insulin-like growth factor-II (IGF-II) gene expression. During differentiation, JAK3 was initially activated and later decreased. Differentiation decreased STAT1, which was further decreased by WHIp154. In contrast, STAT3 gradually was elevated during differentiation, and was increased by JAK3 inhibition. Moreover, we found that up-regulation of AKT activity and down-regulation of ERK activity cooperated to accelerate myogenic differentiation. Taken together, these data indicate that JAK3 inhibition potently facilitates myoblast differentiation through antagonistic STAT1/STAT3 activities. Additionally, JAK3 inhibition induced precocious differentiation and played important roles for terminal differentiation, including fusion, which is involved with regulation of AKT and ERK pathways.

Differential regulation of proliferation and differentiation in neural precursor cells by the Jak pathway.

Neuronal precursor cells (NPCs) are temporally regulated and have the ability to proliferate and differentiate into mature neurons, oligodendrocytes, and astrocytes in the presence of growth factors (GFs). In the present study, the role of the Jak pathway in brain development was investigated in NPCs derived from neurosphere cultures using Jak2 and Jak3 small interfering RNAs and specific inhibitors. Jak2 inhibition profoundly decreased NPC proliferation, preventing further differentiation into neurons and glial cells. However, Jak3 inhibition induced neuronal differentiation accompanied by neurite growth. This phenomenon was due to the Jak3 inhibition-mediated induction of neurogenin (Ngn)2 and NeuroD in NPCs. Jak3 inhibition induced NPCs to differentiate into scattered neurons and increased the expression of Tuj1, microtubule associated protein 2 (MAP2), Olig2, and neuroglial protein (NG)2, but decreased glial fibrillary acidic protein (GFAP) expression, with predominant neurogenesis/polydendrogenesis compared with astrogliogenesis. Therefore, Jak2 may be important for NPC proliferation and maintenance, whereas knocking-down of Jak3 signaling is essential for NPC differentiation into neurons and oligodendrocytes but does not lead to astrocyte differentiation. These results suggest that NPC proliferation and differentiation are differentially regulated by the Jak pathway.

Gadd45beta is a novel mediator of cardiomyocyte apoptosis induced by ischaemia/hypoxia.

AIMS: Because apoptotic death plays a critical role in cardiomyocyte loss during ischaemic heart injury, a detailed understanding of the mechanisms involved is likely to have a substantial impact on the optimization and development of treatment strategies. The goal of this study was to assess gene profiling during ischaemia/hypoxia and to evaluate the functions of ischaemia/hypoxia-responsive genes in in vivo and in vitro ischaemia/hypoxia-induced cardiomyocyte apoptosis models. METHODS AND RESULTS: DNA microarray analysis and real-time polymerase chain reaction were performed on hearts obtained from an in vivo rat transient ischaemia model and on neonatal rat cardiomyocytes from an in vitro hypoxia model. Three genes, namely Ddit4, Gadd45beta and Atf3, were found to be up-regulated in vivo and in vitro. Using loss-of-function and gain-of-function techniques, the functions of these ischaemia/hypoxia-responsive genes were evaluated. Ischaemia/hypoxia-induced cardiomyocyte apoptosis was remarkably attenuated by the small interfering RNA-mediated down-regulation of Gadd45beta in vivo and in vitro, whereas ectopic Gadd45beta expression significantly aggravated hypoxia-induced apoptosis in vitro. CONCLUSION: These results suggest that Gadd45beta is a key player in ischaemia/hypoxia-induced apoptotic cardiomyocyte death, and that strategies based on its inhibition might be of benefit in the treatment of acute ischaemic heart disease.

Role of PKCbetaII and PKCdelta in blood-brain barrier permeability during aglycemic hypoxia.

Blood-brain barrier (BBB) dysfunction contributes to the pathophysiology of cerebrovascular diseases such as stroke. In the present study, we investigated the role of PKC isoforms in aglycemic hypoxia-induced hyperpermeability using an in vitro model of the BBB consisting of mouse bEnd.3 cells. PKCbetaII and PKCdelta isoforms were activated during aglycemic hypoxia. CGP53353, a specific PKCbetaII inhibitor, significantly attenuated aglycemic hypoxia-induced BBB hyperpermeability and disruption of occludin and zonula occludens-1 (ZO-1), indicating a deleterious role of PKCbetaII in the regulation of BBB permeability during aglycemic hypoxia. Conversely, rottlerin, a specific PKCdelta inhibitor, exacerbated BBB hyperpermeability and tight junction (TJ) disruption during aglycemic hypoxia, indicating a protective role of PKCdelta against aglycemic hypoxia-induced BBB hyperpermeability. Furthermore, disruption of TJ proteins during aglycemic hypoxia was attenuated by PKCbetaII DN and PKCdelta WT overexpression, and aggravated by PKCbetaII WT and PKCdelta DN overexpression. These results suggest that PKCbetaII and PKCdelta counter-regulate BBB permeability during aglycemic hypoxia.

Cadmium stimulates the expression of vascular cell adhesion molecule-1 (VCAM-1) via p38 mitogen-activated protein kinase (MAPK) and JNK activation in cerebrovascular endothelial cells.

In this study, we examined the effect of Cd on the expression of vascular cell adhesion molecule-1 (VCAM-1) and its mechanisms in bEnd.3 cells. The treatment with Cd increased protein and mRNA expressions of VCAM-1 and increased the phosphorylations of p38, JNK, and ERK. The Cd-induced VCAM-1 expression was significantly suppressed by either a specific p38 mitogen-activated protein kinase (MAPK) inhibitor (SB202190) or a JNK inhibitor (SP600125), but not by an ERK inhibitor (U0126). These results suggest that Cd induces the expression of VCAM-1, at least in part, via p38 and JNK pathways in bEnd.3 cells.

Calpain-mediated N-cadherin proteolytic processing in brain injury.

Neural-cadherin (N-cadherin), a member of the classical cadherin family of transmembrane glycoproteins, mediates cellular recognition and cell-cell adhesion through calcium-dependent homophilic interactions and plays important roles in the development and maintenance of the nervous system. Metalloproteinase is known to cleave N-cadherin, which is further cleaved by gamma-secretase. The intracellular domain of N-cadherin interacts with beta-catenin, and beta-catenin stability is critical for cell-cell adhesion and cell survival. In the present study, we showed that N-cadherin is cleaved specifically by calpain, resulting in the generation of a novel 110 kDa fragment. The cleavage occurred in ischemic brain lesions and in vitro neural cells in the presence of NMDA and ionomycin, and was restored by calpain inhibitors but not matrix metalloproteinase or gamma-secretase inhibitors. Calpain directly cleaved N-cadherin in in vitro calpain assays, and calpain inhibitors prevented its cleavage in a dose-dependent manner. Using N-cadherin deletion mutants, we found that calpain cleavage sites exist in at least four regions of the cytoplasmic domain. Treatment with NMDA induced neuronal death, and it suppressed the expression of surface N-cadherin and the N-cadherin/beta-catenin interaction, effects that were prevented by calpain inhibitor. Furthermore, calpain-mediated N-cadherin cleavage significantly affected cell-cell adhesion, AKT signaling, the N-cadherin/beta-catenin interaction and the Wnt target gene expressions through the accumulation of nuclear beta-catenin.