The Phytopathogenic Fungus Pallidocercospora crystallina-Caused Localized Subcutaneous Phaeohyphomycosis in a Patient with a Homozygous Missense CARD9 Mutation.

PURPOSE: In the past decade, an increasing number of otherwise healthy individuals suffered from invasive fungal infections due to inherited CARD9 mutations. Herein, we present a patient with a homozygous CARD9 mutation who was suffering from localized subcutaneous phaeohyphomycosis caused by the phytopathogenic fungus Pallidocercospora crystallina which has not been reported to cause infections in humans. METHODS: The medical history of our patient was collected. P. crystallina was isolated from the biopsied tissue. To characterize this novel pathogen, the morphology was analyzed, whole-genome sequencing was performed, and the in vivo immune response was explored in mice. Whole-exome sequencing was carried out with samples from the patient's family. Finally, the expression and function of mutated CARD9 were investigated. RESULTS: A dark red plaque was on the patient's left cheek for 16 years and was diagnosed as phaeohyphomycosis due to a P. crystallina infection. Whole-genome sequencing suggested that that this strain had a lower pathogenicity. The in vivo immune response in immunocompetent or immunocompromised mice indicated that P. crystallina could be eradicated within a few weeks. Whole-exome sequencing revealed ahomozygous missense mutation in CARD9 (c.1118G>C p.R373P). The mRNA and protein expression levels were similar among cells carrying homozygous (C/C), heterozygous (G/C), and wild-type (G/G) CARD9 alleles. Compared to PBMCs or neutrophils with heterozygous or wild-type CARD9 alleles, however, PBMCs or neutrophils with homozygous CARD9 alleles showed impaired anti-P. crystallina effects. CONCLUSION: Localized subcutaneous phaeohyphomycosis caused by P. crystallina was reported in a patient with a homozygous CARD9 mutation. Physicians should be aware of the possibility of a CARD9 mutation in seemingly healthy patients with unexplainable phaeohyphomycosis.

Hydroa vacciniforme-like lymphoproliferative disorder: Clinicopathologic study of 41 cases.

BACKGROUND: Hydroa vacciniforme-like lymphoproliferative disorder (HVLLPD) is a rare Epstein-Barr virus (EBV)-related disease that is usually found in East Asians and Latin Americans. OBJECTIVE: To report the characteristics of HVLLPD in Chinese patients. METHODS: Retrospective analysis of patients with HVLLPD from a single institute. RESULTS: A total of 41 patients were enrolled. All patients presented with papulovesicular lesions, mainly distributed on sun-exposed areas, with 26 patients showing systemic symptoms. Follow-up data were available for 20 patients, 16 patients were alive, and 4 patients died. Of the 4 deceased patients, 3 had taken a serum EBV DNA test that showed high viral loads. These 3 patients also received chemotherapy. Histopathology was characterized by dense proliferation of lymphocytes in the dermis. Angiotropism or angiodestruction was found in the majority of patients, whereas prominent cellular polymorphism was noticed in only 4 patients. All patients were positive for CD3, TIA1 cytotoxic granule associated RNA binding protein, and EBV-encoded RNA in situ hybridization. LIMITATIONS: This was a retrospective study. CONCLUSIONS: HVLLPD in Chinese patients showed indolent behavior in the majority of cases, which differed from the characteristics of HVLLPD in Latin Americans. Patients with high serum EBV DNA loads had an increased risk of their disease evolving into aggressive disease. Chemotherapy should not be considered as first-line treatment for most Chinese patients.

The MEK/ERK signalling cascade is required for sonic hedgehog signalling pathway-mediated enhancement of proliferation and inhibition of apoptosis in normal keratinocytes.

Keratinocytes (KCs) play a critical role in maintaining the cutaneous structure and are involved in various physiological and pathologic processes of the skin. Many inflammatory skin diseases and skin cancers result from excessive proliferation and insufficient apoptosis of KCs. Recent data suggested that the sonic hedgehog (Shh) signalling pathway plays an essential role in the proliferation and apoptosis of normal KCs. However, the mechanism remains poorly defined. Here, we provide evidence that Shh signalling induces proliferation and inhibits apoptosis in normal KCs via cyclin D1 and Bcl2 in an extracellular signal-regulatedkinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent manner. In addition, the effect is independent of phosphoinositide-3 kinase (PI3K)/AKT or Janus kinase/signal transducer and activator of transcription (JAK/STAT) 1/3 pathways. Furthermore, we observed that epidermal growth factor receptor (EGFR) signalling modulates the activity of Shh signalling pathway; besides, Shh and EGFR signalling act additively to induce the ERK activation and the increases in cyclin D1 and Bcl2 thereby affecting proliferation and apoptosis in KCs in vitro. The present study suggests that the MEK/ERK1/2 activation is part of the mechanism of Shh signal-mediated proliferation and apoptosis in normal KCs. Our results may help to elucidate the regulatory mechanisms of the Shh pathway in normal KCs and the pathogenesis of related skin disorders.

Comparison of the 308-nm excimer laser with the 308-nm excimer lamp in the treatment of vitiligo--a randomized bilateral comparison study.

BACKGROUND: Vitiligo is an acquired pigment disorder characterized by areas of depigmented skin resulting from the loss of epidermal melanocytes. Recently, several investigations have documented the benefits of excimer phototherapy (e.g., using the 308-nm excimer laser or the 308-nm excimer lamp) for the treatment of vitiligo. AIM: To compare the effectiveness of the 308-nm excimer laser with the 308-nm excimer lamp in the treatment of vitiligo patients. METHODS: This intervention study was designed as a randomized self-control trial. Fourteen subjects with 48 symmetrical vitiligo lesions were enrolled in this study. One lesion was treated with the 308-nm excimer laser, and its counterpart was treated with the 308-nm excimer lamp. Lesions were treated three times a week with the same dose on both sides for a total of 20 sessions. RESULTS: All of the patients completed the study, and 48 lesions were treated. The two treatments exhibited similar results in terms of repigmentation. CONCLUSIONS: The 308-nm excimer lamp and the 308-nm excimer laser exhibited similar efficacies in treating vitiligo.

WITHDRAWN: Comparision between 308-nm excimer laser and 308-nm excimer lamp in the treatment of vitiligo - a randomized self control study.

Ahead of Print article withdrawn by publisher.

[Association of matrix metalloproteinase-3 gene polymorphisms with subtypes of ischemic stroke].

OBJECTIVE: To assess the association between matrix metalloproteinase-3 (MM-3) gene polymorphisms and subtypes of ischemic stroke (IS) in northern Han Chinese population. METHODS: A total of 289 patients with acute IS (within 3 days after the onset, including 185 with large artery atherosclerosis (LAA) and 104 for small artery occlusion (SAO)) and 175 matched healthy controls were recruited for this case-control study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or sequenc-based typing (SBT) was carried out to analyze 3 SNPs of the MMP-3 gene. RESULTS: An incomplete linkage disequilibrium (LD) block was constructed with the 3 SNPs, and the distribution of genotypes of the 3 SNPs differed between the LAA group and controls in a dominant model: Carriers of 5A allele (5A5A+5A6A) of the rs3025058 locus were 1.72 times more susceptible to LAA stroke compared with carriers of 6A6A alleles (P=0.017, OR=1.72, 95% CI: 1.10-2.69), carriers of G alleles (GG+AG) of the rs522616 locus were 0.52 times more susceptible to LAA stroke compared with carriers of AA alleles (P=0.005, OR=0.52, 95% CI: 0.33-0.82), whilst carriers of A allele of the rs679620 locus were 1.55 times more susceptible to LAA stroke compared with carriers of GG alleles (P=0.042, OR=1.55, 95% CI: 1.01-2.37). However, no significant difference has been found between particular genotypes of such SNPs between SAO patients and controls (P> 0.05). Furthermore, 5A-A-A and 6A-A-A haplotypes were significantly more common in LAA group than the controls (P< 0.05), whilst 6A-G-G haplotype has been the opposite (P< 0.01). CONCLUSION: Our study has demonstrated that serum MMP-3 level is significantly increased at acute stage of LAA as well as SAO type strokes. There may be an association of rs3025058, rs522616 and rs679620 of MMP-3 gene with susceptibility to LAA stoke in northern Han Chinese population.

A Social Emotional Learning Training Programme in a Poor Rural Primary School in Central China: A Pre-Post Intervention Study.

INTRODUCTION: Many universal school-based social and emotional learning (SEL) programmes in the U.S. and Europe have been found to improve social skills and reduce emotional distress and behaviour problems. The aim of this study is to determine whether an adapted version of the SEL can reduce social, emotional, and behavioural difficulties in children in mainland China, using a pre-post intervention design. METHODS: The study was conducted in a primary school in an economically-disadvantaged rural area in Henan province in central China. The intervention consisted of 16 weekly 90-minunte classroom sessions involving all 190 children in the school. Social and emotional problems were assessed pre- and post- intervention using the Chinese version of the Strengths and Difficulties Questionnaire (SDQ). The results suggest that: (1) the programme can reduce children's peer relationship problems, and that the reduction was sustainable at the two post-intervention assessments; (2) the intervention effects on emotional symptoms or total difficulties in the overall population are very few, but children identified as high risk in the initial assessment benefited from the programme. CONCLUSIONS: This is the first published report on the effectiveness of a school-based SEL programme in mainland China. Although the improvement are limited, the programme does benefit some children.

Effect of Edaravone Combined with Anticoagulant Therapy on the Serum hs-CRP, IL-6, and TNF-α Levels and Activity of Daily Living in Patients with Acute Cerebral Infarction.

Objective: To explore the effect of edaravone combined with anticoagulant therapy on the serum hs-CRP, IL-6, and TNF-α levels and the activity of daily living (ADL) in patients with acute cerebral infarction (ACI). Methods: The clinical data of 84 ACI patients treated in our hospital from August 2020 to August 2021 were retrospectively analyzed, and they were divided into the routine group (n = 42) and the combined group (n = 42) according to the order of admission. Both groups were treated with routine clinical treatment, and the combined group was additionally treated with edaravone combined with anticoagulant therapy. Serum samples were collected from both groups after treatment. ELISA was used to detect the serum inflammatory factor levels, and the modified Barthel index score was used to evaluate the ADL of patients. Results: Compared with the routine group, the combined group achieved obviously lower levels of PMA, CD62p, and serum inflammatory factors after treatment (P < 0.001), higher modified Barthel score after treatment (P < 0.001), lower plasma viscosity, platelet aggregation rate, and plasma fibrinogen level after treatment (P < 0.001), and higher clinical overall efficacy (P < 0.05). Conclusion: Edaravone combined with anticoagulant therapy is a reliable method to enhance ADL and reduce the inflammatory response of ACI patients. This strategy greatly reduces the platelet-activating factor levels of patients and improves the comprehensive clinical efficacy, and its further research will help to establish a better solution for these patients.

Teachers' Perceptions of Student Mental Health in Eastern China: A Qualitative Study.

In China, primary and secondary school teachers, known as ban zhu ren, have pastoral responsibility for the students in their class. The aim of this preliminary study is to identify how ban zhu ren perceive the mental health of their students, and how they have acted on these perceptions. Content analysis was used to organize the data and distinguish categories or themes derived from in-depth semi-structured interviews conducted with 27 ban zhu ren from Zhejiang and Anhui provinces. Frequencies of informant responses were used to identify the areas of agreement and disagreement across identified categories and themes among the informants. The results illustrate that the informants consider issues, such as not paying attention in class (n = 14), not getting along well with classmates (n = 12), and excessive gaming (n = 11) to be indicative of mental illness, although these would commonly be considered normal adolescent behaviors. Fifteen informants admitted that they found it difficult to work with student mental health issues, and 18 felt they had inadequate or non-existent training. However, all informants stated that they had intervened with what they perceived to be students' mental health issues, although only 9 informants had referred students for professional help. The informants reported that they were reluctant to provide referrals, due to the stigmatization they believed students would experience if given a diagnosis of mental illness. We conclude that among our informants there is a lack of agreement on what behavioral and mental health issues are, and that informants may be confusing what are, in actuality, non-conformist or non-compliant (yet often normal), adolescent behaviors with mental illness due to insufficient mental health training.

A retrospective study: Clinicopathological and immunohistochemical analysis of 54 cases of tufted angioma.

BACKGROUND: Tufted angioma is a rare benign lesion with vascular proliferation. AIM: To retrospectively analyze the clinicopathological manifestations and immunohistochemical features of tufted angioma. METHODS: Clinical and histopathological features of tufted angioma (n = 54) were evaluated and analyzed retrospectively in the Department of Dermatology, Xijing Hospital from 2003 to 2014. RESULTS: Clinically, tufted angioma usually presented as erythematous plaques and papules on the head and neck (n = 11), trunk (n = 21) and extremities (n = 22), mainly in children (n = 48), without gender difference (24 males and 30 females). A total of 45 cases showed solitary lesions and nine cases showed multiple lesions. Common symptoms included pain (n = 11), tenderness (n = 7), itching (n = 1), hypertrichosis (n = 7), hyperhidrosis (n = 6) and Kasabach-Merritt phenomenon (n = 1). Histopathologically, typical tufted angioma (n = 37) showed proliferation of endothelial cells in a so-called cannonball pattern, while in the early (n = 4) and regressed (n = 13) stages the tufted appearance was not prominent. The proliferated endothelial cells were diffusely positive for CD31 and Wilms tumor 1, focally positive for D2-40 and Prox1, and negative for Glut-1. LIMITATIONS: Our research was confined to patients of Chinese origin and our sample size was limited. CONCLUSIONS: Tufted angioma is a rare vascular neoplasm with diverse clinical manifestations and unique pathological features. It should be recognized as a vascular tumor with lymphatic differentiation. We emphasize the importance of considering tufted angioma in the differential diagnoses of any congenital or acquired vascular tumor.

Associations of serum C-peptide and insulin-like growth factor binding proteins-3 with breast cancer deaths.

C-peptide is usually considered as a marker of insulin secretion and has no physiological function. This study aimed to assess the association between serum C-peptide level as independent risk factor and breast cancer and explored the possible underlying mechanisms. This was a population-based cohort study. All the data was collected according to a standard protocol. The C-peptide and insulin-like growth factor binding proteins-3(IGFBP-3) concentrations were measured in blood. The breast cancer deaths were confirmed by National Death Index records. Cox proportional hazard regression analysis was conducted to determine the hazard ratio of serum C-peptide level for breast cancer deaths. Analysis of covariance was used to assess the association between serum C-peptide and IGFBP-3 level, and the linear trend was tested by using a linear model. A total of 8,373 women 17 years of age or older were included in the study, and 57 breast cancer deaths were observed over the study period. The result of survival analysis showed that breast cancer deaths increased with increasing levels of serum C-peptide. The hazard ratio was 1.69 (95% confidence interval, 1.17-2.45). The levels of circulating IGFBP-3 were positively associated with changes in serum C-peptide levels and showed a strong linear trend in the covariance analysis. Serum C-peptide level was associated with increased risk of breast cancer death. Our results suggest that the increased risk of breast cancer death can be via a pathway that serum C-peptide level positive associated with the change in serum IGFBP-3 level.

Gut Microbial Dysbiosis and Plasma Metabolic Profile in Individuals With Vitiligo.

Autoimmune diseases are increasingly linked to aberrant gut microbiome and relevant metabolites. However, the association between vitiligo and the gut microbiome remains to be elucidated. Thus, we conducted a case-control study through 16S rRNA sequencing and serum untargeted-metabolomic profiling based on 30 vitiligo patients and 30 matched healthy controls. In vitiligo patients, the microbial composition was distinct from that of healthy controls according to the analysis on α- and ß-diversity (P < 0.05), with a characteristic decreased Bacteroidetes: Firmicutes ratio. Meanwhile, the levels of 23 serum metabolites (including taurochenodeoxycholate and L-NG-monomethyl-arginine) in the vitiligo patients were different from those in the healthy individuals and showed significant correlations with some microbial markers. We found that Corynebacterium 1, Ruminococcus 2, Jeotgalibaca and Psychrobacter were correlated significantly with disease duration and serum IL-1ß level in vitiligo patients. And Psychrobacter was identified as the most predictive features for vitiligo by machine learning analysis ("importance" = 0.0236). Finally, combining multi-omics data and joint prediction models with accuracies up to 0.929 were established with dominant contribution of Corynebacterium 1 and Psychrobacter. Our findings replenished the previously unknown relationship between gut dysbiosis and vitiligo circulating metabolome and enrolled the gut-skin axis into the understanding of vitiligo pathogenesis.

Overexpression of glycogen synthase kinase 3beta sensitizes neuronal cells to ethanol toxicity.

The developing central nervous system (CNS) is particularly susceptible to ethanol toxicity. The loss of neurons underlies many of the behavioral deficits observed in fetal alcohol spectrum disorders (FASD). The mechanisms of ethanol-induced neuronal loss, however, remain incompletely elucidated. We demonstrated that glycogen synthase kinase 3beta (GSK3beta), a multifunctional serine/threonine kinase, was involved in ethanol neurotoxicity. The activity of GSK3beta is negatively regulated by its phosphorylation at serine 9 (Ser9). Ethanol induced dephosphorylation of GSK3beta at Ser9 and the activation of Bax as well as caspase-3 in the developing mouse brain. These ethanol-induced alterations were ameliorated by the pretreatment of a GSK3beta inhibitor, lithium. To determine the role of GSK3beta in ethanol neurotoxicity, we overexpressed wild-type (WT), S9A mutant or dominant-negative (DN) mutant GSK3beta in a neuronal cell line (SK-N-MC). Ethanol only modestly reduced the viability of parental SK-N-MC cells but drastically induced caspase-3 activation and apoptosis in cells overexpressing WT or S9A GSK3beta, indicating that the high levels of GSK3beta or the active form of GSK3beta increased cellular sensitivity to ethanol. Contrarily, overexpression of DN GSK3beta conferred resistance to ethanol toxicity. Lithium and other specific GSK3beta inhibitors abolished the hypersensitivity to ethanol caused by WT or S9A overexpression. Bax, a proapoptotic protein, is a substrate of GSK3beta. Cells overexpressing WT or S9A GSK3beta were much more sensitive to ethanol-induced Bax activation than parental SK-N-MC cells. Our results indicate that GSK3beta may be a mediator of ethanol neurotoxicity, and its expression status in a cell may determine ethanol vulnerability.

The role of glycogen synthase kinase 3beta in the transformation of epidermal cells.

Glycogen synthase kinase 3beta (GSK3beta) is a multifunctional serine/threonine kinase. We showed that the expression of GSK3beta was drastically down-regulated in human cutaneous squamous cell carcinomas and basal cell carcinomas. Due to its negative regulation of many oncogenic proteins, we hypothesized that GSK3beta may function as a tumor suppressor during the neoplastic transformation of epidermal cells. We tested this hypothesis using an in vitro model system, JB6 mouse epidermal cells. In response to epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), the promotion-sensitive JB6 P+ cells initiate neoplastic transformation, whereas the promotion-resistant JB6 P- cells do not. JB6 P- cells expressed much higher levels of GSK3beta than JB6 P+ cells; JB7 cells, the transformed derivatives of JB6, had the least amount of GSK3beta. The activity of GSK3beta is negatively regulated by its phosphorylation at Ser9. EGF and TPA induced strong Ser9 phoshorylation in JB6 P+ cells, but phosphorylation was seen at a much lesser extent in JB6 P- cells. EGF and TPA-stimulated Ser9 phosphorylation was mediated by phosphoinositide-3-kinase (PI3K)/Akt and protein kinase C (PKC) pathways. Inhibition of GSK3beta activation significantly stimulated activator protein-1 (AP-1) activity. Overexpression of wild-type (WT) and S9A mutant GSK3beta in JB6 P+ cells suppressed EGF and TPA-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient (K85R) GSK3beta, in contrast, potentiated anchorage-independent growth and drastically enhanced in vivo tumorigenicity. Together, these results indicate that GSK3beta plays an important role in skin tumorigenesis.

Ethanol promotes endoplasmic reticulum stress-induced neuronal death: involvement of oxidative stress.

One of the most devastating effects of ethanol exposure during development is the loss of neurons in selected brain areas. The underlying cellular/molecular mechanisms remain unclear. The endoplasmic reticulum (ER) is involved in posttranslational protein processing and transport. The accumulation of unfolded or misfolded proteins in the ER lumen triggers ER stress, which is characterized by translational attenuation, synthesis of ER chaperone proteins such as GRP78, and activation of transcription factors such as ATF4, ATF6, and CHOP. Sustained ER stress ultimately leads to cell death. ER stress response can be induced experimentally by treatment with tunicamycin and thapsigargin. Using SH-SY5Y neuroblastoma cells and primary cerebellar granule neurons as in vitro models, we demonstrated that exposure to ethanol alone had little effect on the expression of markers for ER stress; however, ethanol drastically enhanced the expression of GRP78, CHOP, ATF4, ATF6, and phosphorylated PERK and eIF2 alpha when induced by tunicamycin and thapsigargin. Consistently, ethanol promoted tunicamycin- and thapsigargin-induced cell death. Ethanol rapidly caused oxidative stress in cultured neuronal cells; antioxidants blocked ethanol's potentiation of ER stress and cell death, suggesting that the ethanol-promoted ER stress response is mediated by oxidative stress. CHOP is a proapoptotic transcription factor. We further demonstrated that CHOP played an important role in ethanol-promoted cell death. Thus, the effect of ethanol may be mediated by the interaction between oxidative stress and ER stress.

Differential requirement of EGF receptor and its tyrosine kinase for AP-1 transactivation induced by EGF and TPA.

The transcription factor activator protein-1 (AP-1) has been implicated in a large variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. It is thought that the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 activity is because of the activation of the PKC/MAPK/AP-1 pathway, although the detailed molecular mechanism has not been fully characterized. The tyrosine kinases of epidermal growth factor receptor (EGFR) lie at the head of a complex of signal transduction cascade that modulates cell proliferation, survival, adhesion, migration and differentiation. Currently, little is known about whether EGFR or its tyrosine kinase is necessary for TPA-induced AP-1 activation. In the present study, we investigated this issue using a well-characterized mouse fibroblast B82 cell line, which is devoid of the EGFR, and its stable transfectants with either wild-type EGFR (B82L) or tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721). We demonstrated that the TPA or epidermal growth factor (EGF) induced AP-1 activation in the B82L cells that express wild-type EGFR, but not in the B82 cell, whereas autophosphorylation at tyrosine(1173) of EGFR in B82L cells was only induced by EGF, but not TPA. The expression of tyrosine kinase-deficient EGFR (mutation at Lys-721) (B82M721) resulted in deficiency of AP-1 induction in cellular response to EGF, while TPA treatment led to fully AP-1 activation. Furthermore, the mutation at Lys-721 of EGFR resulted in impairing of EGFR autophosphorylation at tyrosine(1173) induced by EGF. Based on these results, we conclude that TPA-induced AP-1 activation requires the basal level-EGFR protein, but not EGFR tyrosine kinase and EGFR autophosphorylation at tyrosine(1173), whereas both EGFR tyrosine kinase and EGFR autophosphorylation at Y(1173) play a critical role in EGF-induced AP-1 activation.

Overexpression of ErbB2 enhances ethanol-stimulated intracellular signaling and invasion of human mammary epithelial and breast cancer cells in vitro.

Both epidemiological and experimental studies indicate that ethanol is a tumor promoter and may promote metastasis of breast cancer. However, the molecular mechanisms underlying ethanol-mediated tumor promotion remain unknown. Overexpression of ErbB proteins in breast cancer patients is generally associated with poor prognosis. The ErbB proteins are a family of receptor kinases that include four closely related members: epidermal growth factor receptor (EGFR/ErbB1), ErbB2/neu, ErbB3, and ErbB4. Particularly, ErbB2 plays a pivotal role in ErbB-mediated activities. Here we demonstrated that amplification of ErbB2 expression sensitized a specific cellular response to ethanol. Human breast cancer cells or mammary epithelial cells with a high expression of ErbB2 exhibited an enhanced response to ethanol-stimulated cell invasion in vitro. Ethanol also stimulated cell proliferation; however, this stimulation was independent of ErbB2 levels. Ethanol triggered divergent intracellular signaling among cells expressing different ErbB2 levels. In the cells overexpressing ErbB2, ethanol was more effective in the activation of c-Jun NH2 terminal protein kinases (JNKs) and p38 mitogen-activated protein kinase (p38 MAPK) as well as the induction of reactive oxygen species (ROS) than the cells with normal ErbB2 expression. Blockage of either JNKs or p38 MAPK activation eliminated ethanol-mediated cell invasion. In contrast, the reduction of hydrogen peroxide concentration by catalase exposure had little effect on ethanol-induced cell invasion. These results indicated that ethanol-induced cell invasion was primarily mediated by JNKs and p38 MAPK, whereas the involvement of ROS formation might be minimal. Our study suggests that overexpression of ErbB2 may augment ethanol-elicited signaling and promote ethanol-stimulated tumor metastasis.

The role of epidermal growth factor receptor in ethanol-mediated inhibition of activator protein-1 transactivation.

A potential mechanism underlying ethanol-induced alterations in gene expression is the disruption of transcription factor activity. Growth factor receptors, particularly receptor tyrosine kinases, play an important role in modulating many biological effects of ethanol. We demonstrated here that the expression of epidermal growth factor receptor (EGFR) mediated the effect of ethanol on the activity of transcription factor activator protein-1 (AP-1). Ethanol had little effect on AP-1 activity in the fibroblast cells devoid of EGFR (B82); however, it significantly suppressed AP-1 activity in B82 cells that were stably transfected with either a wild-type EGFR (B82L) or a kinase-deficient receptor (B82M721) in a concentration-dependent manner. EGF activated AP-1 only in B82L cells; the activation was mediated primarily by Akt and ERK. Ethanol inhibited EGF-induced EGFR autophosphorylation, phosphorylation of ERK as well as Akt and its substrate GSK-3beta, and subsequently blocked EGF-stimulated AP-1 activation in B82L cells. On the other hand, ethanol had little effect on EGF-stimulated JNK activation. Phorbol ester 12-O-teradecanoyl-phorbol-13-acetate (TPA) activated AP-1 in B82L and B82M721 cells, but not B82 cells. TPA-induced activation of ERK and PKCdelta was dependent on the expression of EGFR although the intrinsic kinase activity of EGFR was not required. In contrast, TPA-induced phosphorylation of p38 MAPK, JNKs and other PKC isoforms was independent of EGFR. Ethanol selectively inhibited TPA-induced phosphorylation of ERK and PKCdelta, and modestly suppressed TPA-stimulated AP-1 activation in B82L and B82M721 cells. Thus, EGFR plays a critical role in the interaction between ethanol and AP-1.

Glycogen synthase kinase 3beta (GSK3beta) mediates 6-hydroxydopamine-induced neuronal death.

The causes of sporadic Parkinson's disease (PD) are poorly understood. 6-Hydroxydopamine (6-OHDA), a PD mimetic, is widely used to model this neurodegenerative disorder in vitro and in vivo; however, the underlying mechanisms remain incompletely elucidated. We demonstrate here that 6-OHDA evoked endoplasmic reticulum (ER) stress, which was characterized by an up-regulation in the expression of GRP78 and GADD153 (Chop), cleavage of procaspase-12, and phosphorylation of eukaryotic initiation factor-2 alpha in a human dopaminergic neuronal cell line (SH-SY5Y) and cultured rat cerebellar granule neurons (CGNs). Glycogen synthase kinase-3 beta (GSK3beta) responds to ER stress, and its activity is regulated by phosphorylation. 6-OHDA significantly inhibited phosphorylation of GSK3beta at Ser9, whereas it induced hyperphosphorylation of Tyr216 with little effect on GSK3beta expression in SH-SY5Y cells and PC12 cells (a rat dopamine cell line), as well as CGNs. Furthermore, 6-OHDA decreased the expression of cyclin D1, a substrate of GSK3beta, and dephosphorylated Akt, the upstream signaling component of GSK3beta. Protein phosphatase 2A (PP2A), an ER stress-responsive phosphatase, was involved in 6-OHDA-induced GSK3beta dephosphorylation (Ser9). Blocking GSK3beta activity by selective inhibitors (lithium, TDZD-8, and L803-mts) prevented 6-OHDA-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), DNA fragmentations and cell death. With a tetracycline (Tet)-controlled TrkB inducible system, we demonstrated that activation of TrkB in SH-SY5Y cells alleviated 6-OHDA-induced GSK3beta dephosphorylation (Ser9) and ameliorated 6-OHDA neurotoxicity. TrkB activation also protected CGNs against 6-OHDA-induced damage. Although antioxidants also offered neuroprotection, they had little effect on 6-OHDA-induced GSK3beta activation. These results suggest that GSK3beta is a critical intermediate in pro-apoptotic signaling cascades that are associated with neurodegenerative diseases, thus providing a potential target site amenable to pharmacological intervention.

GSK3ß mediates the carcinogenic effect of HPV16 in cervical cancer.

Cervical cancer is one of the most prevalent and fatal cancers among women and infection of the human papillomavirus (HPV) is the most important risk factor. This study investigated how HPV16 regulated GSK3ß expression and function to promote cervical cancers. The expression of GSK3ß was analyzed by quantitative PCR and western blot. The proliferation, invasion, and clonogenic survival of cells with different E6/E7 levels were measured by MTT, transwell invasion assays, and soft agar colony-forming assays, respectively. The levels of GSK3ß were correlated with the copy numbers and expression levels of HPV16 E6/E7 genes. HPV16 E6/E7 genes regulated GSK3ß transcription through an element located in the promoter 85 and 250 base pairs upstream of the transcription start site. The abilities of cell proliferation, invasion, and clonogenic survival were increased in C33A cells by ectopic HPV16 E6/E7 and decreased in CaSki cells by knocking down HPV16 E6/E7 levels. Meanwhile, LiCl increased GSK3ß transcript levels and the proliferation of CaSki cells in a HPV16-dependent manner. These data indicated that GSK3ß may participated in HPV16 mediated deregulation of wnt/ß-catenin and other signaling pathways promoting the progression and invasion of cervical cancers.