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Myoglobin is an oxygen storage molecule that is selectively expressed in cardiac and slow-twitch skeletal muscles that have a high oxygen demand. Numerous studies have implicated hypoxia in the regulation of myoglobin expression as an adaptive response to hypoxic stress. However, the details of this relationship remain undefined. In the present study, adult mice exposed to 10% oxygen for periods up to 3 wk exhibited increased myoglobin expression only in the working heart, whereas myoglobin was either diminished or unchanged in skeletal muscle groups. In vitro and in vivo studies revealed that hypoxia in the presence or absence of exercise-induced stimuli reprograms calcium signaling and modulates myoglobin gene expression. Hypoxia alone significantly altered calcium influx in response to cell depolarization or depletion of endoplasmic reticulum calcium stores, which inhibited the expression of myoglobin. In contrast, our whole animal and transcriptional studies indicate that hypoxia in combination with exercise enhanced the release of calcium from the sarcoplasmic reticulum via the ryanodine receptors triggered by caffeine, which increased the translocation of nuclear factor of activated T-cells into the nucleus to transcriptionally activate myoglobin expression. The present study unveils a previously unrecognized mechanism where the hypoxia-mediated regulation of calcium transients from different intracellular pools modulates myoglobin gene expression. In addition, we observed that changes in myoglobin expression, in response to hypoxia, are not dependent on hypoxia-inducible factor-1 or changes in skeletal muscle fiber type. These studies enhance our understanding of hypoxia-mediated gene regulation and will have broad applications for the treatment of myopathic diseases. nuclear factor of activated T cells; calcineurin; skeletal muscle

DEP domain containing 1(DEPDC1) is involved in the tumorigenesis of a variety of cancers. But its role in tumorigenesis of lung adenocarcinoma (LUAD) is not fully understood. Here, we investigated the role and the underlying mechanisms of DEPDC1 in the development of LUAD. The expression and prognostic values of DEPDC1 in LUAD were analysed by using the data from public databases. Gene enrichment in TCGA LUAD was analysed using GSEA software with the pre‐defined gene sets. Cell proliferation, migration and invasion of A549 cells were examined with colony formation, Transwell and wound healing assays. The function of DEPDC1 in autophagy and RAS‐ERK1/2 signalling was determined with Western blot assay upon DEPDC1 knockdown and/or overexpression in A549, HCC827 and H1993 cells. The results demonstrated that DEPDC1 expression was up‐regulated in LUAD tissues, and its high expression was correlated with unfavourable prognosis. The data also showed that DEPDC1 knockdown impaired proliferation, migration and invasion of A549 cells. Most notably, the results showed that DEPDC1 up‐regulated RAS expression and thus enhanced ERK1/2 activity, through which DEPDC1 could inhibit autophagy. In conclusion, our study revealed that DEPDC1 is up‐regulated in LUAD tissues and plays an oncogenic role in LUAD, and that DEPDC1 inhibits autophagy through the RAS‐ERK1/2 signalling in A549, HCC827 and H1993 cells. [ABSTRACT FROM AUTHOR]

Cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is a newly identified cytosolic DNA sensor, but its function in lung epithelial cells is relatively unknown. In the present study, the effects of lipopolysaccharide (LPS) on the expression and function of cGAS in the A549 lung epithelial cell line was investigated. The cells were treated with LPS at different concentrations (e.g., 100, 200, 400 and 800 ng/ml), and the cGAS expression levels were examined via western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The cells were pretreated with LPS, followed by E. coli DNA transfection using Lipofectamine® 3000. After 24 h, interferon (IFN)-β production was measured using ELISA and the expression of the autophagic markers, microtubule-associated proteins 1A/1B light chain 3 and sequestosome-1, were determined using western blot analysis. The cells were also pretreated with either a toll-like receptor (TLR) 4 inhibitor, a serine/threonine-protein kinase TBK1 (TBK1) inhibitor or an nuclear factor (NF)-κB inhibitor, followed by LPS treatment, and the cGAS expression levels were examined via western blot analysis and RT-qPCR. The result showed that LPS treatment upregulated cGAS expression in a dose-dependent manner. E. coli DNA treatment could induce IFN-β production and autophagy via cGAS, which was enhanced by LPS pretreatment. The effect of LPS on cGAS expression was suppressed by treatment with a TLR4 inhibitor, a TBK1 inhibitor and an NF-κB inhibitor. In conclusion, LPS enhances DNA-induced IFN-β production and autophagy by upregulating cGAS expression through the myeloid differentiation primary response protein MyD88-independent TLR4 signaling pathway in A549 cells. [ABSTRACT FROM AUTHOR]

A previous study revealed that DEP domain containing 1 (DEPDC1) is involved in the carcinogenesis of bladder cancer via forming a complex with zinc finger protein 224 (ZNF224) to suppress A20 expression, resulting in the activation of the nuclear factor (NF)-κB signaling pathway; however, the role of DEPDC1 in liver cancer remains unclear. Hep G2 cells were treated with 11R-DEP: 611–628, a peptide capable of disrupting the DEPDC1-ZNF224 complex. Cell proliferation was examined using an MTT assay and apoptosis was analyzed via detection of the apoptotic marker caspase-3 using western blot analysis. A20 expression was examined via reverse transcription-quantitative polymerase chain reaction and NF-κB subcellular localization was determined via immunofluorescence staining. microRNA (miR)-130a was overexpressed in HepG2 cells and its effects on proliferation and apoptosis were examined. The results demonstrated that 11R-DEP: 611–628 (3 µM) and miR-130a inhibited cell proliferation and promoted apoptosis in HepG2 cells by activating A20 expression, which blocks the nuclear transportation of NF-κB. In addition, the results demonstrated that the 11R-DEP: 611–628 (3 µM) treatment resulted in downregulation of DEPDC1 expression, indicating that DEPDC1 expression is regulated by the DEPDC1-ZNF224 complex. In conclusion, the data indicated that DEPDC1 suppresses apoptosis to promote cell proliferation through the NF-κB signaling pathway in HepG2 cells and that DEPDC1 is a potential target for the treatment of liver cancer. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a chronic, fatal and progressive fibro-proliferative lung disease, and fibroblast-to-myofibroblast differentiation is a crucial process in the development of IPF. Elongation factor-2 kinase (eEF2K) has been reported to play an important role in various disease types, but the role of eEF2K in IPF is unknown. In this study, we investigated the role of eEF2K in normal lung fibroblast (NHLF) proliferation, differentiation, apoptosis, and autophagy as well as the interaction between eEF2K and p38 MAPK signaling through in vitro experiments. We found that the inhibition of eEF2K markedly augmented cell proliferation and differentiation, suppressed apoptosis and autophagy, and reversed the anti-fibrotic effects of a p38 MAPK inhibitor. Together, our results indicate that eEF2K might inhibit TGF-β1-induced NHLF proliferation and differentiation and activate NHLF cell apoptosis and autophagy through p38 MAPK signaling, which might ameliorate lung fibroblast-to-myofibroblast differentiation. [ABSTRACT FROM AUTHOR]

The ectopic expression of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) has been demonstrated to facilitate tumorigenesis and induce proliferation in a various types of cancer. However, the role of IGF2BP2 in esophageal squamous cell carcinoma (ESCC) has yet been fully elucidated. In this regard, the current study assessed the expression patterns and clinical significance of IGF2BP2 in 94 Chinese patients diagnosed with ESCC. Immunohistochemistry and reverse transcription-quantitative PCR assays were employed to assess IGF2BP2 expression in ESCC tissues compared with adjacent healthy tissues. The results revealed that the protein expression of IGF2BP2 was substantially upregulated in ESCC tissues compared with adjacent ESCC tissues. More specifically, higher IGF2BP2 expression strongly associated with tumor node metastasis stage, lymphatic infiltration and lymph node metastasis. Using two ESCC cell lines (TE-1 and TE-10), the inhibition of IGF2BP2 expression by small interfering RNA was proven to induce apoptosis and suppress proliferation, migration and cell cycle progression in vitro. Collectively, the present findings indicated that IGF2BP2 may serve a major role in the development of ESCC carcinogenesis. The present study may be helpful in the design of potential drug targets in the treatment of ESCC. [ABSTRACT FROM AUTHOR]

Abstract: Homers are scaffolding proteins that bind Ca2+ signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca2+ signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca2+ signaling complex in an isoform-specific manner. Homer2 increases the GAP activity of RGS proteins and PLCβ that accelerate the GTPase activity of Gα subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP3Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca2+ channels Cav1.2 and Cav1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Cav1.2 to regulate E–C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca2+ signaling and create a negative bias that can be reversed by the immediate early gene form of Homer1. Hence, the Homers should be viewed as the buffers of Ca2+ signaling that ensure a high spatial and temporal fidelity of the Ca2+ signaling and activation of downstream effects. [Copyright &y& Elsevier]

The NICE-3 protein serves an oncogenic role in hepatocellular carcinoma, but its role in lung adenocarcinoma (LUAD) remains unknown. The aim of the present study was to investigate the potential role and underlying mechanisms of NICE-3 in LUAD. In the present study, NICE-3 expression in LUAD tissues and its association with patient prognosis were analyzed using datasets from The Cancer Genome Atlas and Gene Express Omnibus. After NICE-3-knockdown with small interfering RNA in LUAD cells, cell proliferation was measured by cell counting, cell cycle was examined by flow cytometry, cell invasion and migration were detected by Transwell assays and autophagic markers LC3 and p62, as well as phosphorylation of S6K and AKT, were determined by western blotting. The results of public database analysis demonstrated that compared with normal lung tissues, NICE-3 expression was increased in LUAD tissues, where high expression levels were associated with a poor prognosis. The results of in vitro experimentation in LUAD cells indicated that NICE-3-knockdown inhibited proliferation, cell cycle, migration and invasion, but enhanced autophagy. Notably, NICE-3-knockdown inhibited AKT/mTORC1 signaling. The present results suggested that NICE-3 may serve an oncogenic role in LUAD via the AKT/mTORC1 signaling pathway and may therefore be a potential therapeutic target for LUAD. [ABSTRACT FROM AUTHOR]

Melanocyte proliferating gene 1 (MYG1) is an exonuclease that participates in RNA processing and is required for normal mitochondrial function. However, its role in tumorigenesis remains unknown. The present study aimed to investigate the role of MYG1 and its underlying mechanisms in human lung adenocarcinoma (LUAD). The expression levels of MYG1 in tumor tissues of patients with LUAD were obtained from public cancer databases and analyzed using the UALCAN online software. The association between MYG1 expression levels and the prognosis of patients with LUAD was analyzed using the Kaplan-Meier plotter. In addition, the role of MYG1 in the LUAD A549 and H1993 cell lines was determined by knocking down MYG1 expression with a specific small interfering RNA or by overexpressing it with a MYG1-containing plasmid. The results demonstrated that MYG1 expression levels were upregulated in LUAD tissues compared with those in normal lung tissues from healthy subjects, and high MYG1 expression levels were associated with an unfavorable prognosis. MYG1 promoted the proliferation, migration and invasion of A549 and H1993 cells. In addition, MYG1 inhibited autophagy via the AMP-activated protein kinase/mTOR complex 1 signaling pathway. Collectively, the present results suggested that MYG1 may serve an oncogenic role in LUAD and may be a potential therapeutic target for LUAD. [ABSTRACT FROM AUTHOR]

Methyltransferase-like 1 (METTL1) is a transfer RNA and microRNA modifying enzyme. However, its role in lung adenocarcinoma (LUAD) remains unknown. The present study aimed to investigate the effect of METTL1 in LUAD and determine the association between METTL1 expression and prognosis of patients with LUAD. The expression profile of METTL1 in LUAD tissues was downloaded from public cancer databases and analyzed using the Gene Expression Profiling Interactive Analysis database and UALCAN online software. In addition, the association between METTL1 expression and prognosis of patients with LUAD was assessed using the Kaplan-Meier Plotter software. The effect of METTL1 in the A549 cell line was determined in vitro via overexpression and knockdown experiments. The results demonstrated that METTL1 was upregulated in LUAD tissues, and its increased expression was associated with unfavorable prognosis. Furthermore, METTL1 promoted proliferation and colony formation of A549 cells, and inhibited autophagy via the AKT/mechanistic target of rapamycin complex 1 signaling pathway. Taken together, the results of the present study suggest that METTL1 acts as an oncogene in LUAD, thus may be a potential prognostic predictor and therapeutic target for LUAD. [ABSTRACT FROM AUTHOR]

Cigarette smoke (CS) is the primary risk factor for chronic obstructive pulmonary disease (COPD) and dampens antiviral response, which increases viral infections and leads to COPD acute exacerbation (AECOPD). Adenovirus, a nonenveloped DNA virus, is linked with AECOPD, whose DNAs trigger innate immune response via interacting with pattern recognition receptors (PRRs). Stimulator of interferon genes (STING), as a cytosolic DNA sensor, participates in adenovirus-induced interferon β (IFNβ)-dependent antiviral response. STING is involved in various pulmonary diseases, but role of STING in pathogenesis of AECOPD is not well documented. In the present study, we explored relationship between STING and AECOPD induced by recombinant adenovirus vectors (rAdVs) and CS in wild type (WT) and STING-/- mice; and also characterized the inhibition of STING- IFNβ pathway in pulmonary epithelium exposed to cigarette smoke extract (CSE). We found that CS or CSE exposure alone dramatically inhibited STING expression, but not significantly effected IFNβ production. Moreover, CS or CSE-exposed significantly suppressed activation of STING-IFNβ pathway induced by rAdVs and suppressed clearance of rAdVs DNA. Inflammation, fibrosis and emphysema of lung tissues were exaggerated when treated with CS plus rAdVs, which further deteriorate in absences of STING. In A549 cells with knockdown of STING, we also observed enhancing apoptosis related to emphysema, especially CSE and adenovirus vectors in combination. Therefore, STING may play a protective role in preventing the progress of COPD. • CS damped STING-IFNβ pathway activated by rAdVs in vitro and vivo. • CS alleviated rAdVs DNA clearance in lung which partially depended on suppression of STING. • STING played a protective role in the pathophysiology of AECOPD model induced by CS plus rAdVs. [ABSTRACT FROM AUTHOR]

Background: Lung adenocarcinoma (LUAD) is a major type of lung cancer with poor prognosis and low 5-year survival rate, which urgently needs further investigation in order to elucidate its mechanisms completely and discover novel therapeutic targets. C1orf74 is a novel protein with unknown function either in normal cells or cancer cells. The aim of this study is to investigate the expression and function of C1orf74 in LUAD cells., Methods: The expression of C1orf74 in LUAD was analyzed using the LUAD datasets from public databases. The prognostic value of C1orf74 in LUAD was analyzed using Kaplan-Meier Plotter. C1orf74 expression in LUAD cell line A549, H1993 and HCC827 was silenced using small interfering RNA, and then the effects of C1orf74 knockdown on proliferation, migration and invasion of LUAD cells were detected by colony formation assay and Transwell assay, the role of C1orf74 in EGFR/AKT/mTORC1 signaling pathway was examined by Western blot, and the function of C1orf74 in cell cycle was detected by flow cytometry., Results: The results of LUAD clinical data showed that C1orf74 was upregulated in LUAD tissues, and its high expression was associated with poor prognosis. The results from cultured LUAD cells demonstrated that C1orf74 knockdown inhibited cell proliferation, migration and invasion, but induced cell cycle arrest and autophagy. Moreover, C1orf74 knockdown suppressed EGFR/AKT/mTORC1 signaling in LUAD cells. In conclusion, the present study revealed that C1orf74 is upregulated in LUAD tissues and plays an oncogenic role in LUAD, and that C1orf74 positively regulates cell proliferation and mobility through the EGFR/AKT/mTORC1 signaling pathway in LUAD cells., Competing Interests: The authors declare that they have no competing interests., (© 2022 Guo et al.)

Anticancer agents that present nonapoptotic cell death pathways are required for treating apoptosis-resistant pancreatic cancer. Here, we synthesized three fluorescent dithiocarbazate-copper complexes, {[Cu II (L)(Cl)] 1 , [Cu II 2 (L) 2 (NO 3 ) 2 ] 2 , and [Cu II 2 Cu I (L) 2 (Br) 3 ] 3 }, to assess their antipancreatic cancer activities. Complexes 1-3 showed significantly greater cytotoxicity toward several pancreatic cancer cell lines with better IC 50 than those of the HL ligand and cisplatin. Confocal fluorescence imaging showed that complex 3 was primarily localized in the mitochondria. Primarily, compound 3 also can be applied to in vivo imaging. Further studies revealed that complex 3 kills pancreatic cancer cells by triggering multiple mechanisms, including ferroptosis. Complex 3 is the first copper complex to evoke cellular events consistent with ferroptosis in cancer cells. Finally, it significantly retarded the ASPC-1 cells' growth in a mouse xenograft model.

Ectopic expression of DEP domain containing 1 (DEPDC1) in lung adenocarcinomas is associated with poor prognosis, but its role and the underlying mechanism remain unknown. In this study, DEPDC1 expression in lung cancer cell lines was examined with Western blot assay, and DEPDC1-positive cell A549 was selected for further experiments. DEPDC1 inhibitor miR-130a was overexpressed in A549 cells, and the proliferation and apoptosis of these cells were analyzed with cell counting and flow cytometry assay. Interfering peptide 11R-DEP:611-628 and JNK inhibitor SP600125 were used alone or in combination to treat A549 cells, and the cell proliferation and apoptosis were assessed by flow cytometry assay; caspase 3 and cleaved caspase 3, phosphor-JNK, and total JNK were detected by Western blotting; and nuclear factor kappa B (NF-κB) localization was determined by immunofluorescence staining. We found that miR-130a and 11R-DEP:611-628 peptides (5 μM) both inhibited A549 proliferation and induced apoptosis. We observed that 11R-DEP:611-628 peptide treatment resulted in elevated A20 expression, dramatically reduced nuclear NF-κB, and increased phosphor-JNK. These findings indicate that DEPDC1 inhibits apoptosis of A549 cell by suppressing A20 expression to regulate NF-κB activity, and that JNK plays a protective role upon 11R-DEP:611-628 peptide treatment. In conclusion, DEPDC1 might be a novel therapeutic target for lung cancer, and the 11R-DEP:611-628 peptide is a potent apoptosis inducer in A549 cells., Competing Interests: Disclosure The authors report no conflicts of interests in this work.

The aim of the present study was to investigate the role of the Toll‑like receptor (TLR)4 signaling pathway in cellular response to lipopolysaccharide (LPS) in rat pulmonary artery smooth muscle cells (PASMCs). Chronic obstructive pulmonary disease (COPD) rats were established with passive inhaling cigarette smoke plus injection of LPS. The TLR4 protein in lung tissues was determined with immunohistochemical staining and protein levels of the components of the TLR4 pathway in PASMCs were analyzed with western blotting. The production of interferon (IFN)‑γ upon LPS stimulation in PASMCs was measured with ELISA. TLR4 expression in lung tissue from COPD rats was increased obviously compared with that in normal group. LPS enhances TLR4 expression in rat PASMCs and induced production of IFN‑γ dramatically. LPS treatment resulted in increased phosphor‑interleukin‑1 receptor‑associated kinase (IRAK), IκB and IκB kinase, as well as the total protein of nuclear factor (NF)‑κB p65. TLR4 inhibitor TAK‑242, IRAK1/4 inhibitor and NF‑κB inhibitor Bay 117082 were capable of suppressing the effects of LPS. TLR4 signaling pathway is functional in PASMCs, and may be involved in the inflammatory response during the pathogenesis of COPD.

The present study aimed to determine the underlying molecular mechanism of the antifibrotic effect of artesunate in pulmonary fibrosis (PF). Primary lung fibroblasts were isolated from the lung tissues of rats, and treated with artesunate (8 µg/ml) and transforming growth factor (TGF)-β1 (5 ng/ml). For in vivo experiments, the rats were administered bleomycin intratracheally, followed by daily intraperitoneal artesunate injections for 27 days. Western blotting, and immunohistochemical and immunofluorescent staining were used to assess the expression of key components of the Notch signaling pathway, including α-smooth muscle actin (α-SMA) and type IV collagen. Artesunate (8 µg/ml) was identified to inhibit TGF-β1-induced α-SMA and collagen protein expression, and repress the Notch signaling pathway, in primary lung fibroblasts. Downregulation of α-SMA and collagen by artesunate was associated with inhibition of the Notch signaling pathway. The daily intraperitoneal injection of artesunate (1 mg/kg) in rats was determined to inhibit bleomycin-induced overexpression of α-SMA and type IV collagen proteins, and inhibit the Notch signaling pathway, in lung tissues. In conclusion, the results of the current study indicate that artesunate inhibits the TGF-β1-induced differentiation of rat primary lung fibroblasts into myofibroblasts and ameliorates bleomycin-induced PF. In addition, the results of the present study suggest that the underlying molecular mechanism for these effects of artesunate is repression of the Notch signaling pathway.

Lung cancer is the most frequent cancer worldwide, in terms of incidence and mortality. Due to challenges in the diagnosis of the disease, the 5-year overall survival rate is only ~16%. Previous studies have suggested that malignant transformations originate from adult stem cells, and malignant lesions may therefore express stem-cell-associated markers. The purpose of the present study is to investigate the expression and clinical significance of the stem cell-associated markers Sal-like protein 4 (SALL4) and leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) in lung cancer, and to provide novel diagnostic markers and targets for the treatment of lung cancer. The expression of the stem cell-associated markers SALL4 and LGR5 was analyzed by immunohistochemistry performed on 135 human lung cancer tissue specimens and 10 non-cancer lung tissue specimens. The clinical significance of the expression of these markers and correlation between their expression and clinical parameters was also assessed. SALL4 expression was highly upregulated in lung cancer tissues, but was not present in non-cancerous lung tissues, and the sensitivity and specificity of SALL4 reached 88% and 100%, respectively. By contrast, LGR5 demonstrated 97% sensitivity, but the specificity was poor. Therefore, SALL4 may be an extremely useful diagnostic marker for lung cancer, but LGR5 is not as useful.

In the present study, paclitaxel (PTX) were encapsulated with polyethylene glycol (PEG)-polylactide (PLA)/D-α tocopheryl polyethylene glycol 1000 succinate (TPGS) (PEG-PLA/TPGS) and the enhanced anti-tumor activity of this PTX mixed micelles (PTX-MM) was evaluated in lung cancer cells. The PTX-MM prepared by a solvent evaporation method was demonstrated to have high drug-loading efficiency (23.2%), high encapsulation efficiency (76.4%), and small size (59 nm). In vitro release assay showed the slow release behavior of PTX-MM, suggesting the good stability of the PTX-MM essential for long circulation time. In vitro kinetics assay demonstrated that PTX-MM could promote absorption and increase relative bioavailability. The anti-cancer efficiency of PTX-MM was also examined by both in vitro and in vivo studies. PTX-MM exhibits obvious cytotoxicity against lung cancer cells with much lower IC50 value when compared with commercial formulated PTX or PTX + TPGS. The xenograft tumor model studies on nude mice indicated that PTX-MM inhibits tumor growth more effectively than other formulations. It was also found that most of mixed micelles were integral in tumor site to exhibit anti-cancer activity. Our results suggested that the use of PTX-MM as an anti-cancer drug may be an effective approach to treat lung cancer., (© The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.)

High-risk human papillomavirus (HPV) is a possible cause of esophageal cancer. However, the molecular pathogenesis of HPV-infected esophageal cancer remains unclear. The expression levels of some microRNAs including miR-125b have been negatively correlated with HPV infection, and miR-125b downregulation is associated with tumorigenesis. In addition, Wnt/β-catenin signaling pathway has been suggested to play an important role in esophageal cancer (EC). We examined miR-125b and Wnt/β-catenin signaling pathway in HPV-16 E6 promoted tumor progression in EC. HPV-16 E6 transfection decreased markedly the expression levels of miR-125b and promoted the colony formation in the Eca 109 and Kyse 150 cell lines, and restoration of miR-125b expression level antagonized the increased colony formation in HPV-16 E6 transfected cell lines. We also demonstrated that overexpression of E6 upregulated the Wnt/β-catenin signaling activity via modulating the multiple regulators including TLE1, GSK3β, and sFRP4. Overexpression of miR-125b restored the expression levels of these proteins. Expression of miR-125b was lower in HPV-16 E6 positive esophageal cancer tissues, and was negatively correlated with E6 mRNA levels. Our results indicate that HPV-16 E6 promotes tumorigenesis in EC via down-regulation of miR-125b, and this underlying mechanism may be involved in the activation of the Wnt/β-catenin signaling pathway.

Our previous study revealed that serum deprivation upregulated human alkaline ceramidase 2 (haCER2) activity and mRNA in HeLa cells, but the mechanism remains unknown. In the present study, serum deprivation also upregulated haCER2 activity in HepG2 human hepatoma cell line cells due to an increase in haCER2 mRNA, in which mRNA transcription, not mRNA stability, is involved. Furthermore, p38 mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) signaling pathway is involved in haCER2 mRNA upregulation by serum deprivation, and this mechanism may explain why haCER2 is upregulated in human liver cancer. In conclusion, p38 MAPK, AP-1 or haCER2 may be used as targets in liver cancer therapy.

This study aimed to investigate KIF18A expression in hepatocellular carcinoma (HCC) and to determine the possibility of KIF18A expression being a biomarker in HCC diagnosis or being an independent predictor of disease-free survival (DFS) and overall survival (OS) in HCC patients underwent surgical resection. KIF18AmRNA was detected in 216 cases of HCC tissues by quantitative real-time PCR (qRT-PCR) and in 20 cases of HCC tissues by semi-quantitative RT-PCR. KIF18A protein was determined in 32 cases of HCC tissues by immunohistochemistry (IHC). The survival probability was analyzed by Kaplan-Meier method, and survival curves between groups were obtained by using the log-rank test. Independent predictors associated with DFS were analyzed with Stepwise Cox proportional hazard models. High KIF18A mRNA level was detected in 154 out of 216 (71.3%) cases of HCC. The positive rate of KIF18A expression was significantly higher in liver cancer tissues than that in adjacent normal liver tissues (ANLT) from HCC patients [65.6% (21 of 32) vs. 25.0% (8 of 32), P=0.001]. The KIF18A expression level had positive relevance to the alpha-fetoprotein (AFP) (≥ 200 ng/ml), tumor size (≥ 5 cm), clinical tumor-node-metastasis (TNM) stage and portal vein tumor thrombus (PVTT) in HCC (all P <0.05). A survival analysis indicated that HCC patients with higher KIF18A expression had a significantly shorter DFS and OS after resection. A multivariate analysis suggested that KIF18A upregualtion was an independent factor for DFS [hazard risk (HR)=1.602; 95% confidence interval (CI), 1.029-2.579; P=0.031] and OS (HR=1.682; 95% CI, 1.089-2.600; P=0.019). KIF18A might be a biomarker for HCC diagnosis and an independent predictor of DFS and OS after surgical resection.

Homers are scaffolding proteins that bind Ca(2+) signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca(2+) signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca(2+) signaling complex in an isoform-specific manner. Homer2 increases the GAP activity of RGS proteins and PLCbeta that accelerate the GTPase activity of Galpha subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP(3)Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca(2+) channels Ca(v)1.2 and Ca(v)1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Ca(v)1.2 to regulate E-C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca(2+) signaling and create a negative bias that can be reversed by the immediate early gene form of Homer1. Hence, the Homers should be viewed as the buffers of Ca(2+) signaling that ensure a high spatial and temporal fidelity of the Ca(2+) signaling and activation of downstream effects.

Excitation-contraction (E-C) coupling and Ca(2+)-induced Ca(2+) release in smooth and cardiac muscles is mediated by the L-type Ca(2+) channel isoform Ca(v)1.2 and the ryanodine receptor isoform RyR2. Although physical coupling between Ca(v)1.1 and RyR1 in skeletal muscle is well established, it is generally assumed that Ca(v)1.2 and RyR2 do not directly communicate either passively or dynamically during E-C coupling. In the present work, we re-examined this assumption by studying E-C coupling in the detrusor muscle of wild type and Homer1(-/-) mice and by demonstrating a Homer1-mediated dynamic interaction between Ca(v)1.2 and RyR2 using the split green fluorescent protein technique. Deletion of Homer1 in mice (but not of Homer2 or Homer3) resulted in impaired urinary bladder function, which was associated with higher sensitivity of the detrusor muscle to muscarinic stimulation and membrane depolarization. This was not due to an altered expression or function of RyR2 and Ca(v)1.2. Most notably, expression of Ca(v)1.2 and RyR2 tagged with the complementary C- and N-terminal halves of green fluorescent protein and in the presence and absence of Homer1 isoforms revealed that H1a and H1b/c reciprocally modulates a dynamic interaction between Ca(v)1.2 and RyR2 to regulate the intensity of Ca(2+)-induced Ca(2+) release and its dependence on membrane depolarization. These findings define the molecular basis of a "two-state" model of E-C coupling by Ca(v)1.2 and RyR2. In one state, Ca(v)1.2 couples to RyR2 by H1b/c, which results in reduced responsiveness to membrane depolarization and in the other state H1a uncouples Ca(v)1.2 and RyR2 to enhance responsiveness to membrane depolarization. These findings reveal an unexpected and novel mode of interaction and communication between Ca(v)1.2 and RyR2 with important implications for the regulation of smooth and possibly cardiac muscle E-C coupling.

Disruption of protein synthesis and folding results in ER stress, which is associated with the pathophysiology of diverse diseases affecting secretory and muscle cells. Cells are protected against ER stress by activation of the unfolded protein response (UPR) that is regulated by the protein kinase PERK, which phosphorylates the translation initiation factor 2 eIF2alpha to attenuate protein synthesis. PERK-/- cells are unable to modulate ER protein load and experience high levels of ER stress. In addition to its role in protein synthesis, the ER also orchestrates many signaling events essential for cell survival, prominent among which is Ca2+ signaling. It is not known, however, whether there is a relationship between ER stress and the function of the Ca2+-signaling pathway in muscle and non-muscle cells. To directly address this question we characterized Ca2+ signaling in the secretory pancreatic and parotid acinar cells and in urinary bladder smooth muscle (UBSM) cells obtained from PERK-/- and wild-type mice. Deletion of PERK that results in high levels of ER stress, and distention and fragmentation of the ER slowed the rate of agonist-mediated Ca2+ release from the ER and reduced Ca2+-induced Ca2+ release, although IP3 production, localization of the IP3 receptors, IP3-mediated Ca2+ release, Ca(v)1.2 current and RyRs activity remained unaltered. On the other hand, ER stress disrupted the integrity of the Ca2+-signaling complexes in both secretory and UBSM cells, as revealed by markedly reduced co-immunoprecipitation of plasma membrane- and ER-resident Ca2+-signaling proteins. These findings establish a relationship between the unfolding protein response, ER stress and Ca2+ signaling and highlight the importance of communication within the terminal ER-plasma membrane microdomain for propagation of the Ca2+ signal from the plasma membrane into the cell.

Regulators of G protein signaling (RGS) proteins accelerate the GTPase activity of Galpha subunits to determine the duration of the stimulated state and control G protein-coupled receptor-mediated cell signaling. RGS2 is an RGS protein that shows preference toward Galpha(q). To better understand the role of RGS2 in Ca(2+) signaling and Ca(2+) oscillations, we characterized Ca(2+) signaling in cells derived from RGS2(-/-) mice. Deletion of RGS2 modified the kinetic of inositol 1,4,5-trisphosphate (IP(3)) production without affecting the peak level of IP(3), but rather increased the steady-state level of IP(3) at all agonist concentrations. The increased steady-state level of IP(3) led to an increased frequency of [Ca(2+)](i) oscillations. The cells were adapted to deletion of RGS2 by reducing Ca(2+) signaling excitability. Reduced excitability was achieved by adaptation of all transporters to reduce Ca(2+) influx into the cytosol. Thus, IP(3) receptor 1 was down-regulated and IP(3) receptor 3 was up-regulated in RGS2(-/-) cells to reduce the sensitivity for IP(3) to release Ca(2+) from the endoplasmic reticulum to the cytosol. Sarco/endoplasmic reticulum Ca(2+) ATPase 2b was up-regulated to more rapidly remove Ca(2+) from the cytosol of RGS2(-/-) cells. Agonist-stimulated Ca(2+) influx was reduced, and Ca(2+) efflux by plasma membrane Ca(2+) was up-regulated in RGS2(-/-) cells. The result of these adaptive mechanisms was the reduced excitability of Ca(2+) signaling, as reflected by the markedly reduced response of RGS2(-/-) cells to changes in the endoplasmic reticulum Ca(2+) load and to an increase in extracellular Ca(2+). These findings highlight the central role of RGS proteins in [Ca(2+)](i) oscillations and reveal a prominent plasticity and adaptability of the Ca(2+) signaling apparatus.