Long-Term Exposure to Air Pollution Associates the Risk of Benign Brain Tumor: A Nationwide, Population-Based, Cohort Study in Taiwan.

Air pollutants as risk factors for benign brain tumor (BBT) remain unclear. Therefore, we conducted a nationwide retrospective cohort study by integrating the patients' clinical data and daily air quality data to assess the environmental risk factors of BBT in Taiwan.Daily air quality data were categorized into quartiles (Q1 to Q4). The adjusted hazard ratio (aHR) was evaluated by comparing the BBT incidence rate of the subjects in Q2-Q4 with that of the subjects in Q1 (the lowest concentration of air pollutants). A total of 161,213 subjects were enrolled in the study. Among the air pollutants tested, the aHR of BBT was significantly higher in the subjects who were exposed to the highest level (Q4) of CO (aHR 1.37, 95% CI 1.08-1.74), NO2 (aHR 1.40, 95% CI 1.09-1.78), and PM2.5 (aHR 1.30, 95% CI 1.02-1.65) than that in the subjects who were exposed to the lowest level (Q1). No significant risk association of BBT with SO2 and PM10 exposure was observed. The results revealed that long-term exposure to air pollutants, particularly CO, NO2, and PM2.5, is associated with the risk of BBT.

The intravenous administration of skin-derived mesenchymal stem cells ameliorates hearing loss and preserves cochlear hair cells in cisplatin-injected mice: SMSCs ameliorate hearing loss and preserve outer hair cells in mice.

Mesenchymal stem cells (MSCs) can be isolated from different tissue origins, such as the bone marrow, the placenta, the umbilical cord, adipose tissues, and skin tissues. MSCs can secrete anti-inflammatory molecules and growth factors for tissue repair and remodeling. However, the ability of skin-derived MSCs (SMSCs) to repair cochlear damage and ameliorate hearing loss remains unclear. Cisplatin is a commonly used chemotherapeutic agent that has the side effect of ototoxicity due to inflammation and oxidative stress. This study investigated the effects of SMSCs on cisplatin-induced hearing loss in mice. Two independent experiments were designed for modeling cisplatin-induced hearing loss in mice, one for chronic toxicity (4 mg/kg intraperitoneal [IP] injection once per day for 5 consecutive days) and the other for acute toxicity (25 mg/kg IP injection once on day one). Three days after cisplatin injection, 1 × 106 or 3 × 106 SMSCs were injected through the tail vein. Data on auditory brain responses suggested that SMSCs could significantly reduce the hearing threshold of cisplatin-injected mice. Furthermore, immunohistochemical staining data suggested that SMSCs could significantly ameliorate the loss of cochlear hair cells, TUNEL-positive cells and cleaved caspase 3-positive cells in cisplatin-injected mice. Neuropathological gene analyses revealed that SMSCs treatment could downregulate the expression of cochlear genes involved in apoptosis, autophagy, chromatin modification, disease association, matrix remodeling, oxidative stress, tissue integrity, transcription, and splicing and unfolded protein responses. Additionally, SMSCs treatment could upregulate the expression of cochlear genes affecting the axon and dendrite structures, cytokines, trophic factors, the neuronal skeleton and those involved in carbohydrate metabolism, growth factor signaling, myelination, neural connectivity, neural transmitter release, neural transmitter response and reuptake, neural transmitter synthesis and storage, and vesicle trafficking. Results from TUNEL and caspase 3 staining further confirmed that cisplatin-induced apoptosis in cochlear tissues of cisplatin-injected mice could be reduced by SMSCs treatment. In conclusion, the evidence of the effects of SMSCs in favor of ameliorating ototoxicity-induced hearing loss suggests a potential clinical application.

Umbilical Cord Mesenchymal Stromal Cell-Derived Exosomes Rescue the Loss of Outer Hair Cells and Repair Cochlear Damage in Cisplatin-Injected Mice.

Umbilical cord-derived mesenchymal stromal cells (UCMSCs) have potential applications in regenerative medicine. UCMSCs have been demonstrated to repair tissue damage in many inflammatory and degenerative diseases. We have previously shown that UCMSC exosomes reduce nerve injury-induced pain in rats. In this study, we characterized UCMSC exosomes using RNA sequencing and proteomic analyses and investigated their protective effects on cisplatin-induced hearing loss in mice. Two independent experiments were designed to investigate the protective effects on cisplatin-induced hearing loss in mice: (i) chronic intraperitoneal cisplatin administration (4 mg/kg) once per day for 5 consecutive days and intraperitoneal UCMSC exosome (1.2 µg/µL) injection at the same time point; and (ii) UCMSC exosome (1.2 µg/µL) injection through a round window niche 3 days after chronic cisplatin administration. Our data suggest that UCMSC exosomes exert protective effects in vivo. The post-traumatic administration of UCMSC exosomes significantly improved hearing loss and rescued the loss of cochlear hair cells in mice receiving chronic cisplatin injection. Neuropathological gene panel analyses further revealed the UCMSC exosomes treatment led to beneficial changes in the expression levels of many genes in the cochlear tissues of cisplatin-injected mice. In conclusion, UCMSC exosomes exerted protective effects in treating ototoxicity-induced hearing loss by promoting tissue remodeling and repair.

Targeting protein palmitoylation decreases palmitate­induced sphere formation of human liver cancer cells.

Although non­alcoholic fatty liver disease (NAFLD) is considered a benign disorder, hepatic steatosis has been proposed to be involved in the tumorigenesis of liver cancer. However, the underlying mechanism for carcinogenesis in fatty liver diseases remains unclear. Cancer stem cells (CSCs) have been hypothesized to serve a key role in tumorigenesis. Tumor formation begins with a subset of heterogeneous cells that share properties with stem cells, such as self­renewal and undifferentiated properties. Our previous study reported that the saturated fatty acid palmitate (PA) significantly enhanced the CSC properties of the HepG2 human liver cancer cell line; however, its underlying mechanisms are unknown. In the present study, a proteomic approach was used to investigate the palmitoylation of proteins in HepG2 CSCs. CSC behavior was induced in HepG2 cells via 200 µM PA. Proteomic analysis was performed to identify post­transcriptional modifications of proteins in HepG2 CSCs in response to PA treatment. The present study identified proteins modified by palmitoylation in HepG2 CSC spheres formed following PA treatment. It was therefore hypothesized that palmitoylation may be crucial for CSC sphere formation. Furthermore, the present study demonstrated that two palmitoylation inhibitors, tunicamycin (5, 10 and 25 µg/ml) and 2­bromohexadecanoic acid (25, 50 and 150 µM), significantly decreased CSC sphere formation without affecting cell viability. An association was identified between sphere formation capacity and tumor­initiating capacity of CSCs. The results of the present study demonstrated that protein palmitoylation may influence the PA­induced CSC tumor­initiating capacity, and that the inhibition of palmitoylation may be a suitable chemopreventive strategy for treating patients with NAFLD.

Increased risk of incident nasopharyngeal carcinoma with exposure to air pollution.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a race-specific malignancy. The nasal cavity is the main entry point for air pollutants or poisonous gases into the human body. However, the risk of NPC in populations exposed to air pollution remains unknown. METHODS: We combined data from the Taiwan Air Quality Monitoring Database (TAQMD) and the Longitudinal Health Insurance Database (LHID) to assess the risk of NPC in a population exposed to air pollution. RESULTS: Multivariate analysis revealed positive trends for the association between the risk of NPC and exposure to air pollution. After adjusting for potential covariates, the risk of developing NPC increased with the increase in nitrogen dioxide (NO2) and fine particulate matter (PM2.5) exposure concentrations from 1.39 to 2.28 and 2.01 to 1.97, respectively, compared to the risks at the lowest concentration levels. CONCLUSIONS: We identified a significant risk of NPC in a population exposed to air pollution. However, this study had several limitations. Moreover, additional experimental and clinical studies on the associations between environmental factors and NPC risk are warranted.

HBV infection increases the risk of macular degeneration: the roles of HBx-mediated sensitization of retinal pigment epithelial cells to UV and blue light irradiation.

BACKGROUND: Hepatitis B virus (HBV) infection is strongly associated with hepatocellular carcinoma due to the main pathogenic X protein of HBV (HBx). Whether HBV infection and the HBx protein could result in macular degeneration (MD) is not known. The aim of this study is to assess the association and underlying mechanisms between HBV infection and MD. METHODS: The National Health Research Institutes in Taiwan built a large database, the National Health Insurance Research Database (NHIRD), which includes the claims data from the Taiwan National Health Insurance (NHI) program. The Taiwan NHI is a single-payer, compulsory health insurance program for Taiwan citizens. The data for the present study were derived from the Longitudinal Health Insurance Database, which contains the claims data of 1 million insured people within the NHIRD, including beneficiary registration, inpatient and outpatient files, drug use, and other medical services. In this study, we first investigated the association of HBV infection and the risk of MD by a population-based cohorts study enrolling 39,796 HBV-infected patients and 159,184 non-HBV-infected patients. RESULTS: After adjustment of age, sex, and comorbidities, the risk of MD was significantly higher in the HBV-infected cohort than in the non-HBV-infected cohort (adjusted HR = 1.31; 95% CI = 1.17-1.46). In vitro, we provided evidence to demonstrate that overexpression of HBx in the human retinal pigment epithelial (RPE) cell line, ARPE19, significantly reduced cell viability and clonogenic survival upon UV and blue light irradiation. By gene microarray analysis, we further showed that almost all genes in DNA repair pathways including base excision repair, nucleotide excision repair, mismatch repair, and homologous recombination were significantly down-regulated in the UV-induced cell death of HBx-transfected ARPE19 cells. CONCLUSIONS: The HBx protein may sensitize RPE cells to UV and blue light irradiation and increase the risk of HBV-infection-associated MD through down-regulation of multiple DNA repair pathways.

Age-related hearing loss and dementia: a 10-year national population-based study.

Age-related hearing loss (ARHL) is postulated to affect dementia. Our study aims to investigate the relationship between ARHL and the prevalence, and 10-year incidence of dementia in the Taiwan National Health Insurance Research Database (NHIRD). We selected patients diagnosed with ARHL from the NHIRD. A comparison cohort comprising of patients without ARHL was frequency-matched by age, sex, and co-morbidities, and the occurrence of dementia was evaluated in both cohorts. The ARHL cohort consisted of 4108 patients with ARHL and the control cohort consisted of 4013 frequency-matched patients without ARHL. The incidence of dementia [hazard ratio (HR), 1.30; 95% confidence interval (CI 1.14-1.49); P = 0.002] was higher among ARHL patients. Cox models showed that being female (HR, 1.34; 95% CI 1.07-1.68), as well as having co-morbidities, including chronic liver disease and cirrhosis, rheumatoid arthritis, hypertension, diabetes mellitus, stroke, head injury, chronic kidney disease, coronary artery disease, alcohol abuse/dependence, and tobacco abuse/dependence (HR, 1.27; 95% CI 1.11-1.45), were independent risk factors for dementia in ARHL patients. We found ARHL may be one of the early characteristics of dementia, and patients with hearing loss were at a higher risk of subsequent dementia. Clinicians should be more sensitive to dementia symptoms within the first 2 years following ARHL diagnosis. Further clinical studies of the relationship between dementia and ARHL may be necessary.

Increased risk of fracture in patients with bipolar disorder: a nationwide cohort study.

OBJECTIVES: Bipolar disorder (BD) is a systemic inflammatory disease, and disrupted bone metabolism due to the inflammatory process can cause fracture. Despite evidence of an association between lower bone mineral density and an increased risk of fracture among patients with depression, schizophrenia, and anorexia nervosa, whether BD is a risk factor for subsequent fracture is unknown. To determine the association between BD and fracture and to examine the risk factors for fracture among patients with BD. METHODS: In this study, we enrolled patients diagnosed with BD from the Taiwan National Health Insurance Research Database. Patients newly diagnosed with BD (ICD-9-CM 296) from 2001 to 2008 were included in the BD cohort, and the date of the initial diagnosis of BD was used as the index date. The comparison cohort, comprising participants without BD, was frequency matched to the BD cohort by age, sex, and index year, and the occurrence of fracture was evaluated in both cohorts. RESULTS: The BD and comparison cohorts were comprised of 47,271 patients with BD and 1,89,084 frequency-matched participants without BD, respectively. The incidence of fracture was higher among patients with BD than among the controls. Cox models showed that BD was an independent risk factor for fracture irrespective of comorbidities [hazard ratio (HR) = 1.79, 95 % confidence interval (CI) = 1.73-1.84, p < .001]. CONCLUSION: Our study showed that patients with BD have a higher risk of subsequent fracture. Additional prospective clinical studies investigating the relationship between BD and fracture are warranted.

Activation of Aurora A kinase through the FGF1/FGFR signaling axis sustains the stem cell characteristics of glioblastoma cells.

UNLABELLED: Fibroblast growth factor 1 (FGF1) binds and activates FGF receptors, thereby regulating cell proliferation and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven SV40 T antigen has been shown to result in tumorigenesis in the brains of transgenic mice. FGF1B promoter (-540 to +31)-driven green fluorescent protein (F1BGFP) has also been used in isolating neural stem cells (NSCs) with self-renewal and multipotency from developing and adult mouse brains. In this study, we provide six lines of evidence to demonstrate that FGF1/FGFR signaling is implicated in the expression of Aurora A (AurA) and the activation of its kinase domain (Thr288 phosphorylation) in the maintenance of glioblastoma (GBM) cells and NSCs. First, treatment of FGF1 increases AurA expression in human GBM cell lines. Second, using fluorescence-activated cell sorting, we observed that F1BGFP reporter facilitates the isolation of F1BGFP(+) GBM cells with higher expression levels of FGFR and AurA. Third, both FGFR inhibitor (SU5402) and AurA inhibitor (VX680) could down-regulate F1BGFP-dependent AurA activity. Fourth, inhibition of AurA activity by two different AurA inhibitors (VX680 and valproic acid) not only reduced neurosphere formation but also induced neuronal differentiation of F1BGFP(+) GBM cells. Fifth, flow cytometric analyses demonstrated that F1BGFP(+) GBM cells possessed different NSC cell surface markers. Finally, inhibition of AurA by VX680 reduced the neurosphere formation of different types of NSCs. Our results show that activation of AurA kinase through FGF1/FGFR signaling axis sustains the stem cell characteristics of GBM cells. IMPLICATIONS: This study identified a novel mechanism for the malignancy of GBM, which could be a potential therapeutic target for GBM.

Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms in the regulation of mitochondrial metabolism of MSCs may ultimately improve therapeutic outcomes of stem cell therapy in the future.

Depression and the Risk of Peptic Ulcer Disease: A Nationwide Population-Based Study.

The risk of peptic ulcer disease (PUD) among patients with depression has raised concern. This study determined the association between depression and the subsequent development of PUD using claims data.Patients newly diagnosed with depression in 2000 to 2010 were identified as depression cohort from the Taiwan National Health Insurance Research Database. The comparison cohort was randomly selected from subjects without depression, frequency matched by age and gender and diagnosis date, with a size 2-fold of the size of the depression cohort. The incidence of PUD was evaluated for both cohorts by the end of 2011. We calculated the hazard ratios (HRs) and 95% confidence intervals (CIs) of PUD using the Cox proportional hazards regression model.The depression cohort consisted of 23,536 subjects (129,751 person-years), and the comparison cohort consisted of 47,069 subjects (285,592 person-years). The incidence of PUD was 2-fold higher in the depression cohort than in the comparison cohort (33.2 vs 16.8 per 1000 person-years) with an age adjusted HR of 1.97 (95% CI = 1.89-2.06) or a multivariable adjusted HR of 1.35 (95% CI = 1.29-1.42).Depression might increase the risk of developing PUD. Prospective clinical studies of the relationship between depression and PUD are warranted.

Association of Periodontitis and Subsequent Depression: A Nationwide Population-Based Study.

Periodontitis is a systemic and chronic inflammatory disease associated with multiple physical conditions. Distress and depression are other problems affecting the progression of periodontitis. However, the causal relationship between depression and periodontitis has not been adequately investigated. This aim of this study was to determine the association between periodontitis and the subsequent development of depression.We identified 12,708 patients with newly diagnosed periodontitis from 2000 to 2005 and 50,832 frequency-matched individuals without periodontitis. Both groups were followed until diagnosed with depression, withdrawal from the National Health Insurance program, or the end of 2011. The association between periodontitis and depressio was analyzed using Cox proportional hazard regression models.The incidence density rate of depression was higher in the periodontitis group than in the nonperiodontitis group, with an adjusted hazard ratio of 1.73 (95% confidence interval 1.58-1.89) when adjusting for sex, age, and comorbidity. Cox models revealed that periodontitis was an independent risk factor for depression in patients, except for comorbidities of diabetes mellitus (DM), alcohol abuse, and cancer.Periodontitis may increase the risk of subsequent depression and was suggested an independent risk factor regardless of sex, age, and most comorbidities. However, DM, alcohol abuse, and cancer may prevent the development of subsequent depression because of DM treatment, the paradoxical effect of alcohol, and emotional distress to cancer, respectively. Prospective studies on the relationship between periodontitis and depression are warranted.

Fluvastatin attenuates hepatic steatosis-induced fibrogenesis in rats through inhibiting paracrine effect of hepatocyte on hepatic stellate cells.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is associated with hepatic fibrogenesis. Despite well-known cholesterol-lowering action of statins, their mechanisms against NASH-mediated fibrogenesis remain unclear. This study aimed at investigating the in vitro and in vivo anti-fibrotic properties of fluvastatin (Flu). METHODS: Palmitate (PA)-induced changes in intracellular hydrogen peroxide levels in primary rat hepatocytes (PRHs) and human hepatoma cell line (HepG2) were quantified by dichlorofluorescein diacetate (DCF-DA) dye assay, whereas changes in expressions of NADPH oxidase gp91 (phox) subunit, α-smooth muscle actin (α-SMA), and NFκB p65 nuclear translocation were quantified with Western blotting. Quantitative real-time polymerase chain reaction (q-PCR) was used to investigate mRNA expressions of pro-inflammatory genes (ICAM-1, IL-6, TNF-α). Conditioned medium (CM) from PA-treated PRHs was applied to cultured rat hepatic stellate cell line, HSC-T6, with or without Flu-pretreatment for 2 h. Pro-fibrogenic gene expressions (COL1, TIMP-1, TGF-ß1, α-SMA) and protein expression of α-SMA were analyzed. In vivo study using choline-deficient L-amino acid defined (CDAA) diet-induced rat NASH model was performed by randomly assigning Wistar rats (n = 28) to normal controls (n = 4), CDAA diet with vehicles, and CDAA diet with Flu (5 mg/kg or 10 mg/kg) (n = 8 each) through gavage for 4 or 8 weeks. Livers were harvested for histological, Western blot (α-SMA), and q-PCR analyses for expressions of pro-inflammatory (IL-6, iNOS, ICAM-1) and pro-fibrogenic (Col1, α-SMA, TIMP-1) genes. RESULTS: In vitro, Flu (1-20 µM) inhibited PA-induced free-radical production, gp91 (phox) expression, and NFκB p65 translocation in HepG2 and PRHs, while CM-induced α-SMA protein expression and pro-fibrogenic gene expressions in HSC-T6 were suppressed in Flu-pretreated cells compared to those without pretreatment. Moreover, α-SMA protein expression was significantly decreased in HSC-T6 cultured with CM from PA-Flu-treated PRHs compared to those cultured with CM from PA-treated PRHs. Flu also reduced steatosis and fibrosis scores, α-SMA protein expression, mRNA expression of pro-inflammatory and pro-fibrogenic genes in livers of CDAA rats. CONCLUSIONS: We demonstrated PA-induced HSC activation through paracrine effect of hepatocyte in vitro that was significantly suppressed by pre-treating HSC with Flu. In vivo, Flu alleviated steatosis-induced HSC activation and hepatic fibrogenesis through mitigating inflammation and oxidative stress, suggesting possible therapeutic role of Flu against NASH.

Stem cell-based therapy in neural repair.

Cell-based therapy could aid in alleviating symptoms or even reversing the progression of neurodegenerative diseases and nerve injuries. Fibroblast growth factor 1 (FGF1) has been shown to maintain the survival of neurons and induce neurite outgrowth. Accumulating evidence suggests that combination of FGF1 and cell-based therapy is promising for future therapeutic application. Neural stem cells (NSCs), with the characteristics of self-renewal and multipotency, can be isolated from embryonic stem cells, embryonic ectoderm, and developing or adult brain tissues. For NSC clinical application, several critical problems remain to be resolved: (1) the source of NSCs should be personalized; (2) the isolation methods and protocols of human NSCs should be standardized; (3) the clinical efficacy of NSC transplants must be evaluated in more adequate animal models; and (4) the mechanism of intrinsic brain repair needs to be better characterized. In addition, the ideal imaging technique for tracking NSCs would be safe and yield high temporal and spatial resolution, good sensitivity and specificity. Here, we discuss recent progress and future development of cell-based therapy, such as NSCs, induced pluripotent stem cells, and induced neurons, in neurodegenerative diseases and peripheral nerve injuries.

The mood stabilizer valproate activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities.

Valproic acid (VPA) is the primary mood-stabilizing drug to exert neuroprotective effects and to treat bipolar disorder in clinic. Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) has been shown to recapitulate endogenous FGF1 gene expression and facilitates the isolation of neural stem/progenitor cells (NSPCs) from developing and adult mouse brains. In this study, we provide several lines of evidence to demonstrate the underlying mechanisms of VPA in activating FGF-1B promoter activity: (i) VPA significantly increased the FGF-1B mRNA expression and the percentage of F1BGFP(+) cells; (ii) the increase of F1BGFP expression by VPA involves changes of regulatory factor X (RFX) 1-3 transcriptional complexes and the increase of histone H3 acetylation on the 18-bp cis-element of FGF-1B promoter; (iii) treatments of other histone deacetylases (HDAC) inhibitors, sodium butyrate and trichostatin A, significantly increased the expression levels of FGF-1B, RFX2, and RFX3 transcripts; (iv) treatments of glycogen synthase kinase 3 (GSK-3) inhibitor, lithium, or GSK-3 siRNAs also significantly activated FGF-1B promoter; (v) VPA specifically enhanced neuronal differentiation in F1BGFP(+) embryonic stem cells and NSPCs rather than GFP(-) cells. This study suggested, for the first time, that VPA activates human FGF1 gene promoter through inhibiting HDAC and GSK-3 activities.

Leukemia inhibitory factor-induced Stat3 signaling suppresses fibroblast growth factor 1-induced Erk1/2 activation to inhibit the downstream differentiation in mouse embryonic stem cells.

In regular culture conditions with leukemia inhibitory factor (LIF), the majority of mouse embryonic stem cells (mESCs) are maintained in a self-renewal stage; very few mESCs have differentiated morphology. When LIF is withdrawn, mESCs tend to differentiate; this differentiation process can be enhanced by the introduction of exogenous fibroblast growth factor (FGF). Here, we show that even in the presence of exogenous FGF1, mESCs can maintain self-renewal and expression of pluripotency markers in the presence of LIF. To elucidate the mechanism in which LIF dominates over the FGF1, extracellular signal-regulated kinase 1/2 (Erk1/2) signaling of mESCs cultured in a medium containing FGF1 or LIF/FGF1 was examined. The results demonstrate that Erk1/2 was activated by FGF1 in the absence of LIF; however, the FGF1-induced Erk1/2 phosphorylation was suppressed when LIF was introduced. Moreover, FGF1-Erk1/2 downregulation was inhibited by a signal transducer and activator of the transcription 3 (Stat3) inhibitor WP1066, suggesting that LIF-induced Stat3 activation plays an important role in the FGF1-Erk1/2 inhibition in mESCs. We further demonstrate that the binding affinity of phospho-Erk1/2 and Sprouty2 was increased via Stat3 activation. Binding of phospho-Erk1/2 and Sprouty2 blocks the activation of Erk1/2 signaling, thus inhibiting the downstream differentiation process in mESCs. Our findings demonstrate, for the first time, that LIF-induced Stat3 phosphorylation plays an important role in promoting the binding of phospho-Erk1/2 and Sprouty2, and thus inhibiting FGF-induced differentiation.

Ciliogenic RFX transcription factors regulate FGF1 gene promoter.

Fibroblast growth factor 1 (FGF1) has been shown to regulate cell proliferation, cell division, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) was shown to recapitulate endogenous FGF1 gene expression. It can also be used to isolate neural stem/progenitor cells (NSPCs) and glioblastoma stem cells (GBM-SCs) from developing mouse brains and human glioblastoma tissues, respectively. However, the regulatory mechanisms of FGF-1B promoter and F1BGFP(+) cells are not clear. In this study, we present several lines of evidence to show the roles of ciliogenic RFX transcription factors in the regulation of FGF-1B gene promoter and F1BGFP(+) cells: (i) RFX1, RFX2, and RFX3 transcription factors could directly bind the 18-bp cis-element (-484 to -467), and contribute to the regulation of FGF1 promoter and neurosphere formation. (ii) We demonstrated RFX2/RFX3 complex could only be detected in the nuclear extract of FGF-1B positive cells, but not in FGF-1B negative cells. (iii) Protein kinase C inhibitors, staurosporine and rottlerin, could decrease the percentage of F1BGFP(+) cells and their neurosphere formation efficiency through reducing the RFX2/3 complex. (iv) RNA interference knockdown of RFX2 could significantly reduce the percentage of F1BGFP(+) cells and their neurosphere formation efficiency whereas overexpression of RFX2 resulted in the opposite effects. Taken together, this study suggests ciliogenic RFX transcription factors regulate FGF-1B promoter activity and the maintenance of F1BGFP(+) NSPCs and GBM-SCs.

Antifibrotic effects of triptolide on hepatic stellate cells and dimethylnitrosamine-intoxicated rats.

Triptolide (C38H42O6N2, TP, a diterpene triepoxide derived from Tripterygium wilfordii Hook F.), is a potent immunosuppresive and antiinflammatory agent. The present study investigated whether TP exerted antihepatofibrotic effects in vitro and in vivo. A cell line of rat hepatic stellate cells (HSC-T6) was stimulated with tumor necrosis factor-α (TNF-α) or transforming growth factor (TGF)-ß1. The inhibitory effects of TP on the nuclear factor-κB (NFκB) signaling cascade and fibrosis markers, including α-smooth muscle actin (α-SMA) and collagen, were assessed. An in vivo therapeutic study was conducted in dimethylnitrosamine (DMN)-treated rats. The rats were randomly assigned to one of three groups: control rats, DMN rats receiving vehicle only and DMN rats receiving TP (20 µg/kg). Treatment was given by gavage twice daily for 3 weeks starting 1 week after the start of DMN administration. TP (5-100 nM) concentration-dependently inhibited the NFκB transcriptional activity induced by TNF-α, lipopolysaccharide and phorbol 12-myristate 13-acetate in HSC-T6 cells. In addition, TP also suppressed TNF-α and TGF-ß1-induced collagen deposition and α-SMA secretion in HSC-T6 cells. In vivo, TP treatment significantly reduced hepatic fibrosis scores, collagen contents, IL-6 and TNF-α levels, and the number of α-SMA and NFκB-positive cells in DMN rats. The results showed that TP exerted antifibrotic effects in both HSC-T6 cells and DMN rats.

Regulation of FGF1 gene promoter through transcription factor RFX1.

Fibroblast growth factor 1 (FGF1) has been suggested to have an important role in cell growth, proliferation, and neurogenesis. Human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) has been shown to monitor endogenous FGF1 expression. F1BGFP could also be used to isolate neural stem/progenitor cells from embryonic, neonatal, and adult mouse brains or to isolate glioblastoma stem cells (GBM-SCs) from human glioblastoma tissues. Here, we present evidence that transcription factor RFX1 could bind the 18-bp cis-elements (-484 to -467) of the F1B promoter, modulate F1BGFP expression and endogenous FGF1 expression, and further regulate the maintenance of GBM-SCs. These observations were substantiated by using yeast one-hybrid assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, gain- and loss-of-function assays, and neurosphere assays. Overexpression of RFX1 was shown to down-regulate FGF-1B mRNA expression and neurosphere formation in human glioblastoma cells, whereas RNA interference knockdown of RFX1 demonstrated the opposite effects. Our findings provide insight into FGF1 gene regulation and suggest that the roles of FGF1 and RFX1 in the maintenance of GBM-SCs. RFX1 may negatively regulate the self-renewal of GBM-SCs through modulating FGF-1B and FGF1 expression levels by binding the 18-bp cis-elements of the F1B promoter.

Brain-specific 1B promoter of FGF1 gene facilitates the isolation of neural stem/progenitor cells with self-renewal and multipotent capacities.

Fibroblast growth factor 1 (FGF1) has been shown to maintain proliferation and self-renewal capacities of neural stem/progenitor cells (NSPCs) in vitro. We have previously identified FGF1B as the major transcript of FGF1 gene expressed exclusively in brain areas that are known to be abundant for NSPCs in vivo. The 540-bp (-540 to +31) sequence upstream of the 1B transcription start site (F1B) is sufficient to drive the expression of a heterologous luciferase reporter in cultured cells. In this study, we report a direct genetic and functional approach to isolate F1B(+) NSPCs using green fluorescent protein (GFP) reporter gene under the control of human F1B promoter. The F1B-GFP reporter could facilitate the isolation of NSPCs with self-renewal and multipotent capacities from human glioblastoma tissues, developing or adult mouse brains by fluorescence-activated cell sorting. Future work elucidating the mechanisms that control FGF1B expression will help to identify new NSPC-related genes.