MRCK as a Potential Target for Claudin-Low Subtype of Breast Cancer.

To find new molecular targets for triple negative breast cancer (TNBC), we analyzed a large-scale drug screening dataset based on breast cancer subtypes. We discovered that BDP-9066, a specific MRCK inhibitor (MRCKi), may be an effective drug against TNBC. After confirming the efficacy and specificity of BDP-9066 against TNBC in vitro and in vivo, we further analyzed the underlying mechanism of specific activity of BDP-9066 against TNBC. Comparing the transcriptome of BDP-9066-sensitive and -resistant cells, the activation of the focal adhesion and YAP/TAZ pathway were found to play an important role in the sensitive cells. Furthermore, YAP/TAZ is indeed repressed by BDP-9066 in the sensitive cells, and active form of YAP suppresses the effects of BDP-9066. YAP/TAZ expression and activity are high in TNBC, especially the Claudin-low subtype, consistent with the expression of focal adhesion-related genes. Interestingly, NF-κB functions downstream of YAP/TAZ in TNBC cells and is suppressed by BDP-9066. Furthermore, the PI3 kinase pathway adversely affected the effects of BDP-9066 and that alpelisib, a PI3 kinase inhibitor, synergistically increased the effects of BDP-9066, in PIK3CA mutant TNBC cells. Taken together, we have shown for the first time that MRCKi can be new drugs against TNBC, particularly the Claudin-low subtype.

Development of triazole-based PKC-inhibitors to overcome resistance to EGFR inhibitors in EGFR-mutant lung cancers.

Protein kinase C delta (PKCδ) is prominently expressed in the nuclei of EGFR-mutant lung cancer cells, and its presence correlates with poor survival of the patients undergoing EGFR inhibitor treatment. The inhibition of PKCδ has emerged as a viable approach to overcoming resistance to EGFR inhibitors. However, clinical-grade PKCδ inhibitors are not available, highlighting the urgent needs for the development of effective drugs that target PKCδ. In this study, we designed and synthesized a series of inhibitors based on the chemical structure of a pan PKC inhibitor sotrastaurin. This was achieved by incorporating a triazole ring group into the original sotrastaurin configuration. Our findings revealed that the sotrastaurin derivative CMU-0101 exhibited an elevated affinity for binding to the ATP-binding site of PKCδ and effectively suppressed nuclear PKCδ in resistant cells in comparison to sotrastaurin. Furthermore, we demonstrated that CMU-0101 synergistically enhanced EGFR TKI gefitinib sensitivity in resistant cells. Altogether, our study provides a promising strategy for designing and synthesizing PKCδ inhibitors with improved efficacy, and suggests CMU-0101 as a potential lead compound to inhibit PKCδ and overcome TKI resistance in lung cancers.

Brassinosteroid recruits FERONIA to safeguard cell expansion in Arabidopsis.

Plant cell expansion is driven by turgor pressure and regulated by hormones. How plant cells avoid cell wall rupture during hormone-induced cell expansion remains a mystery. Here we show that brassinosteroid (BR), while stimulating cell elongation, promotes the plasma membrane (PM) accumulation of the receptor kinase FERONIA (FER), which monitors cell wall damage and in turn attenuates BR-induced cell elongation to prevent cell rupture. The GSK3-like kinase BIN2 phosphorylates FER, resulting in reduced FER accumulation and translocation from endoplasmic reticulum to PM. By inactivating BIN2, BR signaling promotes dephosphorylation and increases PM accumulation of FER, thereby enhancing the surveillance of cell wall integrity. Our study reveals a vital signaling circuit that coordinates hormone signaling with mechanical sensing to prevent cell bursting during hormone-induced cell expansion.

Mapping the signaling network of BIN2 kinase using TurboID-mediated biotin labeling and phosphoproteomics.

Elucidating enzyme-substrate relationships in posttranslational modification (PTM) networks is crucial for understanding signal transduction pathways but is technically difficult because enzyme-substrate interactions tend to be transient. Here, we demonstrate that TurboID-based proximity labeling (TbPL) effectively and specifically captures the substrates of kinases and phosphatases. TbPL-mass spectrometry (TbPL-MS) identified over 400 proximal proteins of Arabidopsis thaliana BRASSINOSTEROID-INSENSITIVE2 (BIN2), a member of the GLYCOGEN SYNTHASE KINASE 3 (GSK3) family that integrates signaling pathways controlling diverse developmental and acclimation processes. A large portion of the BIN2-proximal proteins showed BIN2-dependent phosphorylation in vivo or in vitro, suggesting that these are BIN2 substrates. Protein-protein interaction network analysis showed that the BIN2-proximal proteins include interactors of BIN2 substrates, revealing a high level of interactions among the BIN2-proximal proteins. Our proteomic analysis establishes the BIN2 signaling network and uncovers BIN2 functions in regulating key cellular processes such as transcription, RNA processing, translation initiation, vesicle trafficking, and cytoskeleton organization. We further discovered significant overlap between the GSK3 phosphorylome and the O-GlcNAcylome, suggesting an evolutionarily ancient relationship between GSK3 and the nutrient-sensing O-glycosylation pathway. Our work presents a powerful method for mapping PTM networks, a large dataset of GSK3 kinase substrates, and important insights into the signaling network that controls key cellular functions underlying plant growth and acclimation.

ezGeno: an automatic model selection package for genomic data analysis.

MOTIVATION: To facilitate the process of tailor-making a deep neural network for exploring the dynamics of genomic DNA, we have developed a hands-on package called ezGeno. ezGeno automates the search process of various parameters and network structures and can be applied to any kind of 1D genomic data. Combinations of multiple abovementioned 1D features are also applicable. RESULTS: For the task of predicting TF binding using genomic sequences as the input, ezGeno can consistently return the best performing set of parameters and network structure, as well as highlight the important segments within the original sequences. For the task of predicting tissue-specific enhancer activity using both sequence and DNase feature data as the input, ezGeno also regularly outperforms the hand-designed models. Furthermore, we demonstrate that ezGeno is superior in efficiency and accuracy compared to the one-layer DeepBind model and AutoKeras, an open-source AutoML package. AVAILABILITY AND IMPLEMENTATION: The ezGeno package can be freely accessed at https://github.com/ailabstw/ezGeno. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Growth arrest-specific 6 protein in HIV-infected patients: Determination of plasma level and different antiretroviral regimens.

BACKGROUND/PURPOSE: Growth arrest-specific 6 (Gas6) protein is involved in cell proliferation, differentiation, adhesion, migration in response to inflammatory processes. Human immunodeficiency virus (HIV) infection induces a chronic inflammatory condition and combination of antiretroviral therapy improves immune function and decreases the inflammatory state. The aim of this study was to assess the implications of Gas6 in chronic inflammation status of HIV-infected patients undergoing different third regimens of antiretroviral therapy. The Gas6 may be a marker of chronic inflammation of HIV-infected patients. METHODS: A total of 356 adult males, including 258 HIV-infected patients and 98 healthy controls, were recruited. The demographic and clinical characteristics of the patients were collected. Laboratory assessment included hemogram, CD4 count, plasma HIV RNA load (PVL), hepatitis B and C viruses, and serum biochemistry. Plasma Gas6 concentrations were determined. RESULTS: The values of Gas6 were lower in HIV patients compared to healthy subjects (14.3 ± 6.4 vs 21.5 ± 15.2, p = 0.01). HIV patients that received antiviral regimen with abacavir had similar Gas6 level than those who received antiviral regimens with tenofovir (14.3 ± 6.5 vs 13.8 ± 5.9, p = 0.99). HIV patients that received antiviral regimen with protease inhibitors (PIs) had lower Gas6 level (13.1 ± 3.5 vs 14.2 ± 6.6 vs 14.6 ± 6.5, p = 0.03) than those who received antiviral regimens with non-nucleoside reverse transcriptase inhibitors (nNRTIs) and integrase inhibitors (INSTIs), respectively. CONCLUSIONS: Decreased plasma Gas6 concentrations were observed in HIV patients. Gas6 levels are associated with different third regimen of highly active antiretroviral therapy. Gas6 may represent a unique marker for assessing the chronic inflammation state difference among cART regimens in HIV patients.

PKCδ-mediated SGLT1 upregulation confers the acquired resistance of NSCLC to EGFR TKIs.

The tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) have been widely used for non-small cell lung cancer (NSCLC) patients, but the development of acquired resistance remains a therapeutic hurdle. The reduction of glucose uptake has been implicated in the anti-tumor activity of EGFR TKIs. In this study, the upregulation of the active sodium/glucose co-transporter 1 (SGLT1) was found to confer the development of acquired EGFR TKI resistance and was correlated with the poorer clinical outcome of the NSCLC patients who received EGFR TKI treatment. Blockade of SGLT1 overcame this resistance in vitro and in vivo by reducing glucose uptake in NSCLC cells. Mechanistically, SGLT1 protein was stabilized through the interaction with PKCδ-phosphorylated (Thr678) EGFR in the TKI-resistant cells. Our findings revealed that PKCδ/EGFR axis-dependent SGLT1 upregulation was a critical mechanism underlying the acquired resistance to EGFR TKIs. We suggest co-targeting PKCδ/SGLT1 as a potential strategy to improve the therapeutic efficacy of EGFR TKIs in NSCLC patients.

DockCoV2: a drug database against SARS-CoV-2.

The current state of the COVID-19 pandemic is a global health crisis. To fight the novel coronavirus, one of the best-known ways is to block enzymes essential for virus replication. Currently, we know that the SARS-CoV-2 virus encodes about 29 proteins such as spike protein, 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), Papain-like protease (PLpro), and nucleocapsid (N) protein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) for viral entry and transmembrane serine protease family member II (TMPRSS2) for spike protein priming. Thus in order to speed up the discovery of potential drugs, we develop DockCoV2, a drug database for SARS-CoV-2. DockCoV2 focuses on predicting the binding affinity of FDA-approved and Taiwan National Health Insurance (NHI) drugs with the seven proteins mentioned above. This database contains a total of 3,109 drugs. DockCoV2 is easy to use and search against, is well cross-linked to external databases, and provides the state-of-the-art prediction results in one site. Users can download their drug-protein docking data of interest and examine additional drug-related information on DockCoV2. Furthermore, DockCoV2 provides experimental information to help users understand which drugs have already been reported to be effective against MERS or SARS-CoV. DockCoV2 is available at https://covirus.cc/drugs/.

Cigarette smoke-induced LKB1/AMPK pathway deficiency reduces EGFR TKI sensitivity in NSCLC.

Smoker patients with non-small cell lung cancer (NSCLC) have poorer prognosis and survival than those without smoking history. However, the mechanisms underlying the low response rate of those patients to EGFR tyrosine kinase inhibitors (TKIs) are not well understood. Here we report that exposure to cigarette smoke extract enhances glycolysis and attenuates AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR; this in turn reduces the sensitivity of NSCLC cells with wild-type EGFR (EGFRWT) to EGFR TKI by repressing expression of liver kinase B1 (LKB1), a master kinase of the AMPK subfamily, via CpG island methylation. In addition, LKB1 expression is correlated positively with sensitivity to TKI in patients with NSCLC. Moreover, combined treatment of EGFR TKI with AMPK activators synergistically increases EGFR TKI sensitivity. Collectively, the current study suggests that LKB1 may serve as a marker to predict EGFR TKI sensitivity in smokers with NSCLC carrying EGFRWT and that the combination of EGFR TKI and AMPK activator may be a potentially effective therapeutic strategy against NSCLC with EGFRWT.

The Roles of Peptide Hormones and Their Receptors during Plant Root Development.

Peptide hormones play pivotal roles in many physiological processes through coordinating developmental and environmental cues among different cells. Peptide hormones are recognized by their receptors that convey signals to downstream targets and interact with multiple pathways to fine-tune plant growth. Extensive research has illustrated the mechanisms of peptides in shoots but functional studies of peptides in roots are scarce. Reactive oxygen species (ROS) are known to be involved in stress-related events. However, recent studies have shown that they are also associated with many processes that regulate plant development. Here, we focus on recent advances in understanding the relationships between peptide hormones and their receptors during root growth including outlines of how ROS are integrated with these networks.

Monitoring early developed low bone mineral density in HIV-infected patients by intact parathyroid hormone and circulating fibroblast growth factor 23.

BACKGROUND/PURPOSE: HIV-infected patients have a high prevalence of low bone mineral density (BMD), but BMD changes remain unclear. This cross-sectional retrospective observational study aimed to characterize the prevalence and associated factors of low BMD in HIV patients. METHODS: Between 1 January 2015 and 31 December 2016, all patients aged 20 years or greater who sought for HIV care were included. BMD was measured by dual-energy X-ray absorptiometry. Multivariable analyses of the association with HIV disease status, treatment and anthropometric parameters were performed. Circulating fibroblast growth factor 23 and intact parathyroid hormone were measured. RESULTS: A total of 137 patients was included; their median age was 39 years old; 97.8% were treated with combination antiretroviral therapy (cART); Body mass index (BMI) was 21.97 kg/m2. Sixty-one patients (44.5%) showed low BMD (osteopenia and osteoporosis) based on the WHO criteria. The median BMD was -0.80 g/cm2 (IQR, -1.5 to -0.2). The prevalence rate of low BMD was 37% in those who were aged 20-29 years, 45.2% in those who were aged 30-39 years, 45.2% in those who were aged 40-49 years, 45.8% in those who were aged 50-59 years, and 53.8% in those who were aged ≧60 years. More than half of patients (50.4%, 69/137) were younger than 40 years. Compared with normal BMD group, the low BMD group has a higher proportion of secondary hyperparathyroidism (18.0% vs 5.3%, p: 0.026) and a lower median C-terminal FGF23 level (48.92 vs 62.61 pg/ml, p: 0.008). Univariate and multivariate analyses of the factors associated with low BMD. We found that only serum intact-parathyroid hormone (iPTH) > 69 pg/ml (OR, 3.86; 95% CI, 1.14-13.09) was statistically significant associated with low BMD in multivariate analysis. CONCLUSIONS: This cohort-based survey showed a high prevalence of low BMD among HIV-infected adults which included young-age patient in an university hospital. Secondary hyperparathyroidism was significantly associated with low BMD. There was no association between FGF23 and low BMD.

Automatic cell fusion via optically-induced dielectrophoresis and optically-induced locally-enhanced electric field on a microfluidic chip.

Cell fusion technology has been exploited in a wide variety of biomedical applications, and physical, chemical, and biological approaches can all be used to fuse two different types of cells; however, no current technique is adept at inducing both cell pairing and fusion at high efficiencies and yields. Hence, we developed a new method featuring the use of optically induced dielectrophoresis (ODEP) in conjunction with an optically induced, locally enhanced electric field for accurate and automatic cell pairing and fusion on a microfluidic device. After pairing cells via ODEP, a locally enhanced electric field generated by "virtual electrodes" by projecting light patterns was enacted to induce a proper transmembrane potential at the cell contact area such that cell fusion could be triggered by white light exposure. As a fusion yield of 9.67% was achieved between Pan1 and A549 cells, we believe that this may be a promising technique for automatically fusing different cell types.

Cigarette smoke enhances oncogene addiction to c-MET and desensitizes EGFR-expressing non-small cell lung cancer to EGFR TKIs.

Cigarette smoking is one of the leading risks for lung cancer and is associated with the insensitivity of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, it remains undetermined whether and how cigarette smoke affects the therapeutic efficacy of EGFR TKIs. In this study, our data showed that chronic exposure to cigarette smoke extract (CSE) or tobacco smoke-derived carcinogen benzo[α]pyrene, B[α]P, but not nicotine-derived nitrosamine ketone (NNK), reduced the sensitivity of wild-type EGFR-expressing NSCLC cells to EGFR TKIs. Treatment with TKIs almost abolished EGFR tyrosine kinase activity but did not show an inhibitory effect on downstream Akt and ERK pathways in B[α]P-treated NSCLC cells. CSE and B[α]P transcriptionally upregulate c-MET and activate its downstream Akt pathway, which is not inhibited by EGFR TKIs. Silencing of c-MET reduces B[α]P-induced Akt activation. The CSE-treated NSCLC cells are sensitive to the c-MET inhibitor crizotinib. These findings suggest that cigarette smoke augments oncogene addiction to c-MET in NSCLC cells and that MET inhibitors may show clinical benefits for lung cancer patients with a smoking history.

A WD40 protein, AtGHS40, negatively modulates abscisic acid degrading and signaling genes during seedling growth under high glucose conditions.

The Arabidopsis thaliana T-DNA insertion mutant glucose hypersensitive (ghs) 40-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The ghs40-1 mutant displayed severely impaired cotyledon greening and expansion and showed enhanced reduction in hypocotyl elongation of dark-grown seedlings when grown in Glc concentrations higher than 3 %. The Glc-hypersensitivity of ghs40-1 was correlated with the hyposensitive phenotype of 35S::AtGHS40 seedlings. The phenotypes of ghs40-1 were recovered by complementation with 35S::AtGHS40. The AtGHS40 (At5g11240) gene encodes a WD40 protein localized primarily in the nucleus and nucleolus using transient expression of AtGHS40-mRFP in onion cells and of AtGHS40-EGFP and EGFP-AtGHS40 in Arabidopsis protoplasts. The ABA biosynthesis inhibitor fluridone extensively rescued Glc-mediated growth arrest. Quantitative real time-PCR analysis showed that AtGHS40 was involved in the control of Glc-responsive genes. AtGHS40 acts downstream of HXK1 and is activated by ABI4 while ABI4 expression is negatively modulated by AtGHS40 in the Glc signaling network. However, AtGHS40 may not affect ABI1 and SnRK2.6 gene expression. Given that AtGHS40 inhibited ABA degrading and signaling gene expression levels under high Glc conditions, a new circuit of fine-tuning modulation by which ABA and ABA signaling gene expression are modulated in balance, occurred in plants. Thus, AtGHS40 may play a role in ABA-mediated Glc signaling during early seedling development. The biochemical function of AtGHS40 is also discussed.

Fluorodeoxyglucose Uptake in Advanced Non-small Cell Lung Cancer With and Without Pulmonary Lymphangitic Carcinomatosis.

AIM: To assess the correlation between advanced non-small cell lung cancer (NSCLC) with or without pulmonary lymphangitic carcinomatosis (PLC) and fluorodeoxyglucose (FDG) uptake and its effect on survival outcomes. PATIENTS AND METHODS: We retrospectively reviewed 157 patients with NSCLC. The mean and maximum standardized uptake values (SUVmean and SUVmax, respectively), metabolic tumor volume (MTV) and total lesion glycolysis (TLG) were evaluated for their effect on overall survival (OS) and progression-free survival (PFS). RESULTS: The PLC group included 55 patients and the non-PLC group included 102 patients. The SUVmean, SUVmax, MTV and TLG values were lower in the non-PLC group. In the PLC group, primary lung tumor TLG was a significant predictor of PFS, while whole-body TLG was found to be a significant predictor in non-PLC patients. CONCLUSION: Primary lung tumor TLG was a good predictor in PLC patients. Whole-body TLG could be a useful predictor only in patients without PLC.

Lapatinib increases motility of triple-negative breast cancer cells by decreasing miRNA-7 and inducing Raf-1/MAPK-dependent interleukin-6.

Lapatinib, a dual epidermal growth factor receptor (EGFR) and HER2 tyrosine kinase inhibitor (TKI), has been approved for HER2-positive breast cancer patients. Nevertheless, its inhibitory effect on EGFR did not deliver clinical benefits for triple-negative breast cancer (TNBC) patients even EGFR overexpression was frequently found in this disease. Moreover, lapatinib was unexpectedly found to enhance metastasis of TNBC cells, but the underlying mechanisms are not fully understood. In this study, we explored that the level of interleukin-6 (IL-6) was elevated in lapatinib-treated TNBC cells. Treatment with IL-6 antibody abolished the lapatinib-induced migration. Mechanistically, the signaling axis of Raf-1/mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinases (JNKs), p38 MAPK, and activator protein 1 (AP-1) was activated in response to lapatinib treatment to induce IL-6 expression. Furthermore, our data showed that microRNA-7 directly binds and inhibits Raf-1 3'UTR activity, and that down-regulation of miR-7 by lapatinib contributes to the activation of Raf-1 signaling pathway and the induction of IL-6 expression. Our results not only revealed IL-6 as a key regulator of lapatinib-induced metastasis, but also explored the requirement of miR7/Raf-1/MAPK/AP-1 axis in lapatinib-induced IL-6 expression.

Influence of thyroid transcription factor-1 on fluorodeoxyglucose uptake and prognosis of non-small cell lung cancer.

AIM: To assess the correlation between thyroid transcription factor-1 (TTF1) protein expression in primary tumors from patients with non-small cell lung cancer (NSCLC) and fluorodeoxyglucose (FDG) uptake, and to determine its effect on survival outcomes. PATIENTS AND METHODS: We categorized 112 patients with NSCLC according to TTF1 expression (TTF1(+): n=59, TTF1(-): n=53), and retrospectively determined whether positron-emission topography measurements, including standardized uptake values [mean (SUVm) and maximum (SUVmax)], metabolic tumor volume (MTV), total lesion glycolysis (TLG), and other clinical factors could predict progression-free (PFS) or overall (OS) survival of these patients. RESULTS: The SUVm, SUVmax, MTV, and TLG values were lower in the TTF1(+) group; their survival outcomes were also better. The SUVm, SUVmax, and TLG values were good prognostic indicators for OS and PFS in this group. CONCLUSION: Primary NSCLC tumors expressing TTF1 had lower FDG uptake than those that did not and this was a good prognostic indicator.

AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.

TNF-α induces cytosolic phospholipase A2 expression in human lung epithelial cells via JNK1/2- and p38 MAPK-dependent AP-1 activation.

BACKGROUND: Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. PRINCIPAL FINDINGS: We demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, p42, p38, JNK2, c-Jun, c-Fos, or ATF2. We showed that TNF-α markedly stimulated p42/p44 MAPK, p38 MAPK, and JNK1/2 phosphorylation which were attenuated by their respective inhibitors. In addition, TNF-α also stimulated c-Jun and ATF2 phosphorylation which were inhibited by pretreatment with SP600125 and SB202190, respectively, but not PD98059. Furthermore, TNF-α-induced cPLA2 promoter activity was abrogated by transfection with the point-mutated AP-1 cPLA2 construct. Finally, we showed that TNF-α time-dependently induced p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs. On the other hand, TNF-α induced in vivo binding of c-Jun, c-Fos, ATF2, and p300 to the cPLA2 promoter in these cells. In an in vivo study, we found that TNF-α induced leukocyte count in BAL fluid of mice and cPLA2 mRNA levels in lung tissues via MAPKs and AP-1. SIGNIFICANCE: Taken together, these results demonstrated that TNF-α-induced cPLA2 expression was mediated through p38 MAPK- and JNK1/2-dependent p300/c-Fos/c-Jun/ATF2 complex formation in HPAEpiCs.