Spatiotemporal roles of AMPK in PARP-1- and autophagy-dependent retinal pigment epithelial cell death caused by UVA.

BACKGROUND: Although stimulating autophagy caused by UV has been widely demonstrated in skin cells to exert cell protection, it remains unknown the cellular events in UVA-treated retinal pigment epithelial (RPE) cells. METHODS: Human ARPE-19 cells were used to measure cell viability, mitochondrial reactive oxygen species (ROS), mitochondrial membrane potential (MMP), mitochondrial mass and lysosomal mass by flow cytometry. Mitochondrial oxygen consumption rate (OCR) was recorded using Seahorse XF flux analyzer. Confocal microscopic images were performed to indicate the mitochondrial dynamics, LC3 level, and AMPK translocation after UVA irradiation. RESULTS: We confirmed mitochondrial ROS production and DNA damage are two major features caused by UVA. We found the cell death is prevented by autophagy inhibitor 3-methyladenine and gene silencing of ATG5, and UVA induces ROS-dependent LC3II expression, LC3 punctate and TFEB expression, suggesting the autophagic death in the UVA-stressed RPE cells. Although PARP-1 inhibitor olaparib increases DNA damage, ROS production, and cell death, it also blocks AMPK activation caused by UVA. Interestingly we found a dramatic nuclear export of AMPK upon UVA irradiation which is blocked by N-acetylcysteine and olaparib. In addition, UVA exposure gradually decreases lysosomal mass and inhibits cathepsin B activity at late phase due to lysosomal dysfunction. Nevertheless, cathepsin B inhibitor, CA-074Me, reverses the death extent, suggesting the contribution of cathepsin B in the death pathway. When examining the role of EGFR in cellular events caused by UVA, we found that UVA can rapidly transactivate EGFR, and treatment with EGFR TKIs (gefitinib and afatinib) enhances the cell death accompanied by the increased LC3II formation, ROS production, loss of MMP and mass of mitochondria and lysosomes. Although AMPK activation by ROS-PARP-1 mediates autophagic cell death, we surprisingly found that pretreatment of cells with AMPK activators (A769662 and metformin) reverses cell death. Concomitantly, both agents block UVA-induced mitochondrial ROS production, autophagic flux, and mitochondrial fission without changing the inhibition of cathepsin B. CONCLUSION: UVA exposure rapidly induces ROS-PARP-1-AMPK-autophagic flux and late lysosomal dysfunction. Pre-inducing AMPK activation can prevent cellular events caused by UVA and provide a new protective strategy in photo-oxidative stress and photo-retinopathy.

Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

Diabetic retinopathy (DR) is a major cause of blindness in adult, and the accumulation of advanced glycation end products (AGEs) is a major pathologic event in DR. Methylglyoxal (MGO), a highly reactive dicarbonyl compound, is a precursor of AGEs. Although the therapeutic potential of metformin for retinopathy disorders has recently been elucidated, possibly through AMPK activation, it remains unknown how metformin directly affects the MGO-induced stress response in retinal pigment epithelial cells. Therefore, in this study, we compared the effects of metformin and the AMPK activator A769662 on MGO-induced DR in mice, as well as evaluated cytotoxicity, mitochondrial dynamic changes and dysfunction in ARPE-19 cells. We found MGO can induce mitochondrial ROS production and mitochondrial membrane potential loss, but reduce cytosolic ROS level in ARPE-19 cells. Although these effects of MGO can be reversed by both metformin and A769662, we demonstrated that reduction of mitochondrial ROS production rather than restoration of cytosolic ROS level contributes to cell protective effects of metformin and A769662. Moreover, MGO inhibits AMPK activity, reduces LC3II accumulation, and suppresses protein and gene expressions of MFN1, PGC-1α and TFAM, leading to mitochondrial fission, inhibition of mitochondrial biogenesis and autophagy. In contrast, these events of MGO were reversed by metformin in an AMPK-dependent manner as evidenced by the effects of compound C and AMPK silencing. In addition, we observed an AMPK-dependent upregulation of glyoxalase 1, a ubiquitous cellular enzyme that participates in the detoxification of MGO. In intravitreal drug-treated mice, we found that AMPK activators can reverse the MGO-induced cotton wool spots, macular edema and retinal damage. Functional, histological and optical coherence tomography analysis support the protective actions of both agents against MGO-elicited retinal damage. Metformin and A769662 via AMPK activation exert a strong protection against MGO-induced retinal pigment epithelial cell death and retinopathy. Therefore, metformin and AMPK activator can be therapeutic agents for DR.

Epigenetic upregulation of spleen tyrosine kinase in cancer cells through p53-dependent downregulation of DNA methyltransferase.

Syk is a tumor suppressor gene in some solid tumors. Currently, it remains unknown how Syk gene hypermethylation is controlled by DNA methyltransferase (DNMT) and p53. In colorectal cancer HCT116 cells, we found that protein and mRNA levels of Syk were much higher in WT than in p53-/- cells. Both p53 inhibitor PFT-α and p53 silencing can reduce the protein and mRNA expression of Syk in WT cells, while DNMT inhibitor 5-Aza-2'-dC can increase Syk expression in p53-/- cells. Interestingly, the DNMT expression in p53-/- HCT116 cells was higher than that in WT cells. PFT-α can not only enhance Syk gene methylation but also increase DNMT1 protein and mRNA levels in WT HCT116 cells. In metastatic lung cancer cell lines A549 and PC9, which express WT p53 and gain function of p53, respectively, PFT-α can also downregulate Syk mRNA and protein expression. However, the Syk methylation level was increased by PFT-α in A549 but not in PC9 cells. Likewise, 5-Aza-2'-dC transcriptionally increased Syk gene expression in A549 cells, but not in PC9 cells. In summary methylation of Syk promoter requires DNMT1, and p53 can upregulate Syk expression via downregulation of DNMT1 at the transcriptional level.

P2X7 Is Involved in the Mouse Retinal Degeneration via the Coordinated Actions in Different Retinal Cell Types.

Adenosine triphosphate (ATP) released from dying cells with high concentrations is sensed as a danger signal by the P2X7 receptor. Sodium iodate (NaIO3) is an oxidative toxic agent, and its retinal toxicity has been used as the model of dry age-related macular degeneration (AMD). In this study, we used NaIO3-treated mice and cultured retinal cells, including BV-2 microglia, 661W photoreceptors, rMC1 Müller cells and ARPE-19 retinal epithelial cells, to understand the pathological action of P2X7 in retinal degeneration. We found that NaIO3 can significantly decrease the photoreceptor function by reducing a-wave and b-wave amplitudes in electroretinogram (ERG) analysis. Optical coherence tomography (OCT) analysis revealed the degeneration of retinal epithelium and ganglion cell layers. Interestingly, P2X7-/- mice were protected from the NaIO3-induced retinopathy and inflammatory NLRP3, IL-1ß and IL-6 gene expression in the retina. Hematoxylin and eosin staining indicated that the retinal epithelium was less deteriorated in P2X7-/- mice compared to the WT group. Although P2X7 was barely detected in 661W, rMC1 and ARPE-19 cells, its gene and protein levels can be increased after NaIO3 treatment, leading to a synergistic cytotoxicity of BzATP [2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-triphosphate tri(triethyleneammonium)salt] and NaIO3 administration in ARPE-19 cells. In conclusion, the paracrine action of the ATP/P2X7 axis via cell-cell communication is involved in NaIO3-induced retinal injury. Our results show that P2X7 antagonist might be a potential therapy in inflammation-related retinal degeneration.

Decoy receptor 3 is involved in epidermal keratinocyte commitment to terminal differentiation via EGFR and PKC activation.

Decoy receptor 3 (DcR3) is a soluble receptor for Fas ligand, LIGHT and TL1A, but it also exerts effector functions. Previously, we found that DcR3 is upregulated in the serum and lesional skin of patients with psoriasis and is upregulated by EGFR activation in proliferating primary human epidermal keratinocytes. However, the functional role of intracellular DcR3 in keratinocyte differentiation is still incompletely defined. Herein, primary cultured human epidermal keratinocytes were differentiated by phorbol 12-myristate 13-acetate (PMA) treatment, calcium treatment and cell confluence, which are three standard in vitro differentiation models. We found that the constitutive expression of the DcR3 gene and protein was progressively suppressed during terminal differentiation of keratinocytes. These changes were correlated with downregulation of EGFR activation during keratinocyte differentiation. EGFR inhibition by gefitinib further decreased confluence-induced suppression of DcR3 mRNA expression, and, vice versa, knocking down DcR3 expression attenuated EGFR and EGFR ligand expression as well as EGFR activation. Under conditions without a change in cell growth, DcR3 silencing reduced the expression of involucrin and transglutaminase 1 but enhanced the induction of the terminal differentiation markers keratin 10 and loricrin. Of note, DcR3 interacted with PKCα and PKCδ and enhanced PKC activity. In keratinocytes with PKCα and PKCδ silencing, differentiation markers were differentially affected. In conclusion, DcR3 expression in keratinocytes is regulated by EGFR and forms a positive feedback loop to orchestrate constitutive EGFR and PKC activity. During differentiation, DcR3 is downregulated and involved in modulating the pattern of terminal differentiation.

Blimp-1 Upregulation by Multiple Ligands via EGFR Transactivation Inhibits Cell Migration in Keratinocytes and Squamous Cell Carcinoma.

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of Blimp-1 expression and cellular functions in keratinocytes and cancer cells. Previously we demonstrated that EGF can upregulate Blimp-1 gene expression in keratinocytes, playing a negative role in regulation of cell migration and inflammation. Because it remains unclear if Blimp-1 can be regulated by other stimuli beyond EGF, here we further investigated multiple stimuli for their regulation of Blimp-1 expression in keratinocytes and squamous cell carcinoma (SCC). We found that PMA, TNF-α, LPS, polyIC, H2O2 and UVB can upregulate the protein and/or mRNA levels of Blimp-1 in HaCaT and SCC cells. Concomitant EGFR activation was observed by these stimuli, and EGFR inhibitor gefitinib and Syk inhibitor can block Blimp-1 gene expression caused by PMA. Reporter assay of Blimp-1 promoter activity further indicated the involvement of AP-1 in PMA-, TNF-α-, LPS- and EGF-elicited Blimp-1 mRNA expression. Confocal microscopic data indicated the nuclear loclization of Blimp-1, and such localization was not changed by stimuli. Moreover, Blimp-1 silencing enhanced SCC cell migration. Taken together, Blimp-1 can be transcriptionally upregulated by several stimuli in keratinocytes and SCC via EGFR transactivation and AP-1 pathway. These include growth factor PMA, cytokine TNF-α, TLR ligands (LPS and polyIC), and ROS insults (H2O2 and UVB). The function of Blimp-1 as a negative regulator of cell migration in SCC can provide a new therapeutic target in SCC.

PARP-1 regulates inflammasome activity by poly-ADP-ribosylation of NLRP3 and interaction with TXNIP in primary macrophages.

Poly(ADP-ribose) polymerase-1 (PARP-1) plays an essential role in DNA repair by catalyzing the polymerization of ADP-ribose unit to target proteins. Several studies have shown that PARP-1 can regulate inflammatory responses in various disease models. The intracellular Nod-like receptor NLRP3 has emerged as the most crucial innate immune receptor because of its broad specificity in mediating immune response to pathogen invasion and danger signals associated with cellular damage. In our study, we found NLRP3 stimuli-induced caspase-1 maturation and IL-1ß production were impaired by PARP-1 knockout or PARP-1 inhibition in bone marrow-derived macrophages (BMDM). The step 1 signal of NLRP3 inflammasome activation was not affected by PARP-1 deficiency. Moreover, ATP-induced cytosolic ROS production was lower in Parp-1-/- BMDM, resulting in the decreased inflammasome complex assembly. PARP-1 can translocate to cytosol upon ATP stimulation and trigger the PARylation modification on NLRP3, leading to NLRP3 inflammasome assembly. PARP-1 was also a bridge between NLRP3 and thioredoxin-interacting protein (TXNIP) and participated in NLRP3/TXNIP complex formation for inflammasome activation. Overall, PARP-1 positively regulates NLRP3 inflammasome activation via increasing ROS production and interaction with TXNIP and NLRP3, leading to PARylation of NLRP3. Our data demonstrate a novel regulatory mechanism for NLRP3 inflammasome activation by PARP-1. Therefore, PARP-1 can serve as a potential target in the treatment of IL-1ß associated inflammatory diseases.

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages.

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNß, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Incorporating Post-Cessation Weight-Control Coaching into Smoking Cessation Therapy to Reduce Type 2 Diabetes Risk.

Post-cessation weight gain (PCWG) facilitates short-term type 2 diabetes (T2D) risk in prediabetic smokers in the absence of complementary measures. In this shared decision-making-based non-randomized controlled trial, prediabetic smokers joined the Fight Tobacco and Stay Fit (FIT2) program or received usual care. The 16-week FIT2 program combined smoking cessation therapy with individualized coaching in diet and physical activity strategies for PCWG restriction (NCT01926041 at ClinicalTrials.gov). During a mean follow-up period of 1316 days, 217 participants (36.8%) developed T2D, and 68 (11.5%) regressed to normoglycemia. In the intention-to-treat analysis (n = 589), the FIT2 program was associated with a reduced T2D risk (HR, 0.58; 95% CI, 0.40-0.84) and a higher probability of regression to normoglycemia (HR, 1.91; 95% CI, 1.04-3.53) compared with usual care. The post-program quitters were at lower T2D risk (HR, 0.63; 95% CI, 0.44-0.92) and were more likely to regress to normoglycemia (HR, 1.83; 95% CI, 1.01-3.30) compared with the controls in the time-varying analysis (n = 532). We demonstrated that the FIT2 program was negatively associated with long-term T2D risk and positively associated with the probability of regression to normoglycemia compared with usual care. To prevent T2D development, we recommend simultaneously promoting smoking abstinence and lifestyle coaching for PCWG restriction.

Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

Gout is a common inflammatory arthritis caused by the deposition of monosodium urate (MSU) crystals in the joints. This activates the macrophages into a proinflammatory state by inducing NLRP3-dependent interleukin-1ß (IL-1ß) secretion, resulting in neutrophil recruitment. Soluble decoy receptor 3 (DcR3) is an immune modulator and can exert biological functions via decoy and non-decoy actions. Previously, we showed that DcR3 suppresses lipopolysaccharides (LPS)- and virus-induced inflammatory responses in the macrophages and promotes the macrophages into the M2 phenotype. In this study, we clarified the actions of DcR3 and its non-decoy action motif heparin sulfate proteoglycan (HSPG) binding domain (HBD) in the MSU crystal-induced NLRP3 inflammasome activation in the macrophages and in mice. In bone marrow-derived macrophages, THP-1 and U937 cells, we found that the MSU crystal-induced secretion of IL-1ß and activation of NLRP3 were suppressed by both DcR3.Fc and HBD.Fc. The suppression of the MSU-induced NLRP3 inflammasome activation is accompanied by the inhibition of lysosomal rupture, mitochondrial production of the reactive oxygen species (ROS), expression of cathepsins, and activity of cathepsin B, without affecting the crystal uptake and the expression of NLRP3 or pro-IL-1ß. In the air pouch mice model of gout, MSU induced less amounts of IL-1ß and chemokines secretion, an increased M2/M1 macrophage ratio, and a reduction of neutrophil recruitment in DcR3-transgenic mice, which expresses DcR3 in myeloid cells. Similarly, the mice intravenously treated with DcR3.Fc or HBD.Fc displayed less inflammation response. These findings indicate that HBD of DcR3 can reduce MSU crystal-induced NLRP3 inflammasome activation via modulation of mitochondrial and lysosomal functions. Therefore, we, for the first time, demonstrate a new therapeutic potential of DcR3 for the treatment of gout.

PARP-1 involves in UVB-induced inflammatory response in keratinocytes and skin injury via regulation of ROS-dependent EGFR transactivation and p38 signaling.

UV irradiation can injure the epidermis, resulting in sunburn, inflammation, and cutaneous tissue disorders. Previous studies demonstrate that EGFR in keratinocytes can be activated by UVB and contributes to inflammation. Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme and plays an essential role in DNA repair under moderate stress. In this study, we set out to understand how PARP-1 regulates UVB irradiation-induced skin injury and interplays with EGFR to mediate the inflammation response. We found that PARP-1 deficiency exacerbated the UVB-induced inflammation, water loss, and back skin damage in mice. In human primary keratinocytes, UVB can activate PARP-1 and enhance DNA damage upon PARP-1 gene silencing. Moreover, PARP-1 silencing and PARP inhibitor olaparib can suppress UVB-induced COX-2 and MMP-1 expression, but enhance TNF-α and IL-8 expression. In addition, EGFR silencing or EGFR inhibition by gefitinib can decrease UVB-induced COX-2, TNF-α, and IL-8 expression, suggesting EGFR activation via paracrine action can mediate UVB-induced inflammation responses. Immunoblotting data revealed that PARP-1 inhibition decreases UVB-induced EGFR and p38 activation. Pharmacological inhibition of p38 also dramatically led to the attenuation of UVB-induced inflammatory gene expression. Of note, genetic ablation of PARP-1 or EGFR can attenuate UVB-induced ROS production, and antioxidant NAC can attenuate UVB-induced EGFR-p38 signaling axis and PARP-1 activation. These data suggest the regulatory loops among EGFR, PARP-1, and ROS upon UVB stress. PARP-1 not only serves DNA repair function but also orchestrates interactions to EGFR transactivation and ROS production, leading to p38 signaling for inflammatory gene expression in keratinocytes.

Multiresolution Imaging Using Bioluminescence Resonance Energy Transfer Identifies Distinct Biodistribution Profiles of Extracellular Vesicles and Exomeres with Redirected Tropism.

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP-specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.

Galectin-3 regulates UVB-induced inflammation in skin.

BACKGROUND: Galectin-3 is widely expressed in many immunocytes and epithelial cells including skin keratinocytes. Galectin-3 can regulate immunological or inflammatory processes and plays a proinflammatory role in some disease models. Galectin-3 has a role in disorders related to ultraviolet (UV) photodamage such as apoptosis, skin squamous cell carcinoma and basal cell carcinoma. However, the evidence of galectin-3 in UVB-induced skin inflammation is still limited and the underlying molecular mechanism remains elusive. OBJECTIVE: We aimed to investigate the effects of galectin-3 in human epidermal keratinocytes and in mice after UVB irradiation. METHODS: Primary human epidermal keratinocytes with galectin-3 knockdown were used as the in vitro model. ELISA, QPCR, and western blotting were applied to evaluate the released cytokine, mRNA and protein expression. Histologic analysis, measurement of erythema and transepidermal water loss (TEWL) were applied to evaluate UVB-induced skin damage in galectin-3 knockout mice. RESULTS: In UVB-irradiated human keratinocytes, galectin-3 knockdown downregulated the UVB-induced ASC crosslinking, cleavage of caspase-1, and formation of active IL-1ß. Galectin-3 knockdown also decreased UVB-induced production of reactive oxygen species, p38 phosphorylation, and COX2 expression in human keratinocytes. After four days of UVB irradiation, galectin-3 knockout mice showed reduced gross erythema, histologic features of tissue inflammation, quantified levels of erythema and TEWL compared to wild type mice. The skin tissue lysate also showed less expression of active IL-1ß and COX2 in galectin-3 knockout mice. CONCLUSION: Galectin-3 may play a positive regulatory role in UVB-induced skin inflammation.

Synergistic Anti-Tumour Effect of Syk Inhibitor and Olaparib in Squamous Cell Carcinoma: Roles of Syk in EGFR Signalling and PARP1 Activation.

Syk is a non-receptor tyrosine kinase involved in the signalling of immunoreceptors and growth factor receptors. Previously, we reported that Syk mediates epidermal growth factor receptor (EGFR) signalling and plays a negative role in the terminal differentiation of keratinocytes. To understand whether Syk is a potential therapeutic target of cancer cells, we further elucidated the role of Syk in disease progression of squamous cell carcinoma (SCC), which is highly associated with EGFR overactivation, and determined the combined effects of Syk and PARP1 inhibitors on SCC viability. We found that pharmacological inhibition of Syk could attenuate the EGF-induced phosphorylation of EGFR, JNK, p38 MAPK, STAT1, and STAT3 in A431, CAL27 and SAS cells. In addition, EGF could induce a Syk-dependent IL-8 gene and protein expression in SCC. Confocal microscopic data demonstrated the ability of the Syk inhibitor to change the subcellular distribution patterns of EGFR after EGF treatment in A431 and SAS cells. Moreover, according to Kaplan-Meier survival curve analysis, higher Syk expression is correlated with poorer patient survival rate and prognosis. Notably, both Syk and EGFR inhibitors could induce PARP activation, and synergistic cytotoxic actions were observed in SCC cells upon the combined treatment of the PARP1 inhibitor olaparib with Syk or the EGFR inhibitor. Collectively, we reported Syk as an important signalling molecule downstream of EGFR that plays crucial roles in SCC development. Combining Syk and PARP inhibition may represent an alternative therapeutic strategy for treating SCC.

Chronic Viral Hepatitis Signifies the Association of Premixed Insulin Analogues with Liver Cancer Risks: A Nationwide Population-Based Study.

This study sought to determine whether chronic hepatitis B or C would modify the association between insulin analogues and hepatocellular carcinoma (HCC) risks. We conducted a nationwide nested case-control study for HCC cases and matched controls from 2003 to 2013 among newly diagnosed type 2 diabetes patients on any antidiabetic agents in Taiwan before and after exclusion of chronic viral hepatitis, respectively. A total of 5832 and 1237 HCC cases were identified before and after exclusion of chronic viral hepatitis, respectively. Incident HCC risks were positively associated with any use of premixed insulin analogues (adjusted odds ratio (OR), 1.27; 95% CI 1.04 to 1.55) among total participants, especially among current users (adjusted OR, 1.45; 95% CI 1.12 to 1.89). However, the association between HCC occurrence and premixed insulin analogues diminished among participants without chronic viral hepatitis (adjusted OR, 1.35; 95% CI 0.92 to 1.98). We also observed a significant multiplicative interaction between chronic viral hepatitis and premixed insulin analogues on HCC risks (P = 0.010). Conclusions: Chronic viral hepatitis signifies the role of premixed insulin analogues in HCC oncogenesis. We recommend a closer liver surveillance among patients prescribed premixed insulin analogues with concomitant chronic viral hepatitis.

Anti-obesity action of INDUS810, a natural compound from Trigonella foenum-graecum: AMPK-dependent lipolysis effect in adipocytes.

OBJECTIVE: We investigated the anti-obesity effect and underlying action mechanism of INDUS810 isolated from Trigonella foenum-graecum L. (Fabaceae), an annual herb commonly known as fenugreek and reported to have hypocholesterolemic, antidiabetic, anticancer and gastroprotective properties. METHODS: For obese animal study, 4-week old mice were fed with normal diet or high-fat diet together with or without intraperitoneal injection of INDUS810 (200mg/kg) twice per week for 15weeks. 3T3-L1 cells were used to study action mechanism of INDUS810 in adipocyte differentiation and lipid metabolism. RESULTS: We found that INDUS810 can reduce high-fat diet-induced weight increase in epididymal white adipose tissue, interscapular brown adipose tissue and liver, as well as serum levels of total cholesterol and low-density lipoprotein cholesterol. Moreover, the insulin sensitivity was significantly improved in INDUS810-treated obese mice. In 3T3-L1 adipocytes, we found that INDUS810 could inhibit lipid accumulation at either differentiating or mature stages, and increase lipolysis activity in mature adipocytes. Additionally, INDUS810 has no effects on cell viability nor the expressions of adipocyte differentiation markers like fatty acid synthase, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α. In contrast, INDUS810 can increase protein levels of peroxisome proliferator-activated receptor α, peroxisome-proliferator-activated receptor-γ co-activator 1ß, sirtuin 1 and sirtuin 3. Of note, INDUS810 can activate adenosine monophosphate-activated protein kinase, which leads to the reduction of lipid contents in adipocytes. CONCLUSION: Our in vitro and in vivo studies suggest that INDUS810 is a potential anti-obesity agent, and this action depends on activate adenosine monophosphate-activated protein kinase activation.

BLIMP1 transcriptionally induced by EGFR activation and post-translationally regulated by proteasome and lysosome is involved in keratinocyte differentiation, migration and inflammation.

BACKGROUND: B lymphocyte-induced maturation protein-1 (BLIMP1) is a transcriptional repressor, and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of BLIMP1 expression in keratinocytes and crosstalk between EGFR and BLIMP1 in skin homeostasis. OBJECTIVE: The aim of the study was to investigate the regulation and functional link between EGFR and BLIMP1 in human epidermal keratinocytes. METHODS: Immunoblotting and Q-PCR were used to determine the molecular mechanism of BLIMP1 expression induced by EGFR in primary human epidermal keratinocytes (NHEK) and HaCaT cells. In functional assay, effects of BLIMP1 knockdown on EGFR-mediated cytokine production, differentiation, and migration in NHEK were evaluated by Q-PCR, ELISA, immunoblotting, and/or wound-healing assay. RESULTS: EGFR activation by EGFR ligands could upregulate the protein and mRNA levels of BLIMP1 in NHEK and HaCaT cells. This effect was dependent on PKC, p38, and ERK activation. Additionally, the stability of BLIMP1 protein was under the control of the proteasome and lysosome degradation systems. EGF could also upregulate BLIMP1 expression in skin squamous cell carcinomas. In addition, BLIMP1 knockdown enhanced the EGFR-mediated IL8, CXCL5 and IL6 gene expression and keratinocyte migration, but reduced the EGFR-mediated suppression of differentiation marker K10. CONCLUSIONS: Our findings shed new insights into the regulation of BLIMP1 expression by EGFR-mediated gene transcription and proteasome/lysosome-mediated degradation in keratinocytes. Functionally, BLIMP1 is a negative regulator of EGF-induced inflammation and migration in keratinocytes, and exerts a gene-specific regulation on keratinocyte differentiation.

Coordinate effects of P2X7 and extracellular acidification in microglial cells.

Extracellular adenosine 5'-triphosphate (ATP) is a damage-associated molecular pattern and contributes to inflammation associated diseases including cancer. Extracellular acidosis is a novel danger signal in the inflammatory sites, where it can modulate inflammation, immunity and tumor growth. Extracellular acidification was shown to inhibit P2X7-mediated channel currents, while it remains unknown how acidification and P2X7 together affect cellular responses. Here, we treated BV-2 microglial cells with ATP in a short period (<15 min) or a sustained acidified condition. For short acidification we compared the actions of neutralized ATP and acidic ATP in a condition with pH buffering. For sustained acidification, we treated cells with neutralized ATP in acidic medium or acidic ATP in medium without pH buffering. In the short acidified condition, neutralized ATP induced higher responses than acidic ATP to increase intracellular calcium and reactive oxygen species, decrease intracellular potassium and induce cell death. In contrast, these cellular responses and mitochondrial fission caused by neutralized ATP were enhanced by pH 6.0 and pH 4.5 media. P2X7 activation can also rapidly block mitochondrial ATP turnover and respiration capacity, both of which were mimicked by nigericin and enhanced by acidity. Taken together P2X7-mediated ionic fluxes and reactive oxygen species production are attenuated under short acidification, while sustained acidification itself can induce mitochondrial toxicity which deteriorates the mitochondrial function under P2X7 activation.

Decoy receptor 3: an endogenous immunomodulator in cancer growth and inflammatory reactions.

Decoy receptor 3 (DcR3), also known as tumor necrosis factor receptor (TNFR) superfamily member 6b (TNFRSF6B), is a soluble decoy receptor which can neutralize the biological functions of three members of tumor necrosis factor superfamily (TNFSF): Fas ligand (FasL), LIGHT, and TL1A. In addition to 'decoy' function, recombinant DcR3.Fc is able to modulate the activation and differentiation of dendritic cells (DCs) and macrophages via 'non-decoy' action. DcR3-treated DCs skew T cell differentiation into Th2 phenotype, while DcR3-treated macrophages behave M2 phenotype. DcR3 is upregulated in various cancer cells and several inflammatory tissues, and is regarded as a potential biomarker to predict inflammatory disease progression and cancer metastasis. However, whether DcR3 is a pathogenic factor or a suppressor to attenuate inflammatory reactions, has not been discussed comprehensively yet. Because mouse genome does not have DcR3, it is not feasible to investigate its physiological functions by gene-knockout approach. However, DcR3-mediated effects in vitro are determined via overexpressing DcR3 or addition of recombinant DcR3.Fc fusion protein. Moreover, CD68-driven DcR3 transgenic mice are used to investigate DcR3-mediated systemic effects in vivo. Upregulation of DcR3 during inflammatory reactions exerts negative-feedback to suppress inflammation, while tumor cells hijack DcR3 to prevent apoptosis and promote tumor growth and invasion. Thus, 'switch-on' of DcR3 expression may be feasible for the treatment of inflammatory diseases and enhance tissue repairing, while 'switch-off' of DcR3 expression can enhance tumor apoptosis and suppress tumor growth in vivo.

Spleen tyrosine kinase mediates the actions of EPO and GM-CSF and coordinates with TGF-ß in erythropoiesis.

Erythropoietin (EPO) and GM-CSF are involved in erythropoiesis, while TGF-ß inhibits proliferation but potentiates differentiation of erythroblasts. Since Syk inhibitor may induce anemia side effect in clinic, here we investigated the role of Syk in the biological actions of EPO and GM-CSF in erythropoiesis. In human erythroleukemia cell line TF-1, Syk inhibitor R406 exerts an enhancement effect with TGF-ß to decrease cell viability, either in the absence or presence of EPO or GM-CSF. Such effect of R406 results from the reduced cell cycle progression and increased cell apoptosis. Notably, unlike Syk, Src family kinases are not involved in the viability control of TF-1 cells. Signaling studies showed that Syk is required for STAT5 and ERK activation induced by EPO, and Akt and ERK activation induced by GM-CSF. Nevertheless, R406 does not change the Smad2/3 signal caused by TGF-ß, and TGF-ß neither affects above signal pathways of EPO and GM-CSF. Of note, Syk is constitutively associated with EPOR in plasma membrane and can bind to STAT5 at active status upon EPO stimulation. Furthermore, EPO-induced hemoglobin γ expression was reduced by R406. In BFU-E and CFU-E colony formation assays in Syk-deficient erythroid progenitor cells, we confirmed the essential role of Syk in erythropoiesis mediated by EPO. Taken together, Syk is a novel upstream signaling molecule of EPOR, and contributes to erythroblast proliferation, survival and differentiation.