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Drug resistance is a key factor underlying the failure of tumor chemotherapy. It enhances the stem‑like cell properties of cancer cells, tumor metastasis and relapse. Luteolin is a natural flavonoid with strong anti‑tumor effects. However, the mechanism(s) by which luteolin protects against paclitaxel (PTX)‑resistant cancer cell remains to be elucidated. The inhibitory effect of luteolin on the proliferation of EC1/PTX and EC1 cells was detected by cell counting kit‑8 assay. Colony formation and flow cytometry assays were used to assess clonogenic capacity, cell cycle and apoptosis. Wound healing and Transwell invasion tests were used to investigate the effects of luteolin on the migration and invasion of EC1/PTX cells. Western blotting was used to detect the protein levels of EMT‑related proteins and stem cell markers after sphere formation. Parental cells and drug‑resistant cells were screened by high‑throughput sequencing to detect the differential expression of RNA and differential genes. ELISA and western blotting were used to verify the screened PI3K/Akt signaling pathway, key proteins of which were explored by molecular docking. Hematoxylin and eosin staining and TUNEL staining were used to observe tumor xenografts on morphology and apoptosis in nude mice. The present study found that luteolin inhibited tumor resistance (inhibited proliferation, induced cell cycle arrest and apoptosis and hindered migration invasion, EMT and stem cell spherification) in vitro in PTX‑resistant esophageal squamous cell carcinoma (ESCC) cells. In addition, luteolin enhanced drug sensitivity and promoted the apoptosis of drug‑resistant ESCC cells in combination with PTX. Mechanistically, luteolin may inhibit the PI3K/AKT signaling pathway by binding to the active sites of focal adhesion kinase (FAK), Src and AKT. Notably, luteolin lowered the tumorigenic potential of PTX‑resistant ESCC cells but did not show significant toxicity in vivo . Luteolin enhanced drug chemosensitivity by downregulating the FAK/PI3K/AKT pathway in PTX‑resistant ESCC and could be a promising agent for the treatment of PTX‑resistant ESCC cancers.

Consumers are more inclined to choose beef with a high intramuscular fat content (IMF), which regulated by lots of factors. It is very significant to find a miRNA that plays a key role in the accumulation of IMF. In our study, we found that bta-miR-330 was highly expressed in Japanese black cattle and differentially expressed at intramuscular pre-adipocytes differentiation processes. Furthermore, we transfected the bta-miR-330 mimic & inhibitor in intramuscular pre-adipocytes. The results showed that bta-miR-330 inhibits the proliferation but promotes the adipogenesis of intramuscular pre-adipocytes. Subsequently, our study showed that bta-miR-330 binds to SESN3, which inhibits the adipogenesis of intramuscular pre-adipocytes. Moreover, we established the mechanism that bta-miR-330 promotes the adipogenesis of intramuscular pre-adipocytes by targeting SESN3 to activate the Akt-mTOR signaling pathway. Overall, our results revealed that bta-miR-330-SESN3-Akt-mTOR axis plays an important role in adipogenesis of intramuscular pre-adipocytes, which provides a molecular basis for increasing IMF content in beef cattle., Competing Interests: Declaration of competing interest We declare that we have no interest in any business or organization. The submitted manuscripts have not been published elsewhere and are not considered for publication elsewhere. All the authors approved the manuscript we submitted., (Copyright © 2024. Published by Elsevier B.V.)

Nitrate (NO 3 ) is a significant inorganic nitrogen source in soil, playing a crucial role in influencing crop productivity. As sessile organisms, plants have evolved complex mechanisms for nitrate uptake and response to varying soil levels. Recent advancements have enhanced our understanding of nitrate uptake and signaling pathways. This mini-review offers a comparative analysis of nitrate uptake mechanisms in Arabidopsis and rice. It also examines nitrate signal transduction, highlighting the roles of AtNRT1.1 and AtNLP7 as nitrate receptors and elucidating the OsNRT1.1B-OsSPX4-OsNLP3 cascade. Additionally, it investigates nuclear transcriptional networks that regulate nitrate-responsive genes, controlled by various transcription factors (TFs) crucial for plant development. By integrating these findings, we highlight mechanisms that may help to enhance crop nitrogen utilization.- ) is a significant inorganic nitrogen source in soil, playing a crucial role in influencing crop productivity. As sessile organisms, plants have evolved complex mechanisms for nitrate uptake and response to varying soil levels. Recent advancements have enhanced our understanding of nitrate uptake and signaling pathways. This mini-review offers a comparative analysis of nitrate uptake mechanisms in Arabidopsis and rice. It also examines nitrate signal transduction, highlighting the roles of AtNRT1.1 and AtNLP7 as nitrate receptors and elucidating the OsNRT1.1B-OsSPX4-OsNLP3 cascade. Additionally, it investigates nuclear transcriptional networks that regulate nitrate-responsive genes, controlled by various transcription factors (TFs) crucial for plant development. By integrating these findings, we highlight mechanisms that may help to enhance crop nitrogen utilization., (© 2024 Scandinavian Plant Physiology Society.)

This study aimed to elucidate the opioid mechanisms underlying dexamethasone-induced pain antihypersensitive effects in neuropathic rats. Dexamethasone (subcutaneous and intrathecal) and membrane-impermeable Dex-BSA (intrathecal) administration dose-dependently inhibited mechanical allodynia and thermal hyperalgesia in neuropathic rats. Dexamethasone and Dex-BSA treatments increased expression of dynorphin A in the spinal cords and primary cultured microglia. Dexamethasone specifically enhanced dynorphin A expression in microglia but not astrocytes or neurons. Intrathecal injection of the microglial metabolic inhibitor minocycline blocked dexamethasone-stimulated spinal dynorphin A expression; intrathecal minocycline, the glucocorticoid receptor antagonist Dex-21-mesylate, dynorphin A antiserum, and κ-opioid receptor antagonist GNTI completely blocked dexamethasone-induced mechanical antiallodynia and thermal antihyperalgesia. Additionally, dexamethasone elevated spinal intracellular cAMP levels, leading to enhanced phosphorylation of PKA, p38 MAPK and CREB. The specific adenylate cyclase inhibitor DDA, PKA inhibitor H89, p38 MAPK inhibitor SB203580 and CREB inhibitor KG-501 completely blocked dexamethasone-induced anti-neuropathic pain and increased microglial dynorphin A exprression. In conclusion, this study reveal that dexamethasone mitigateds neuropathic pain through upregulation of dynorphin A in spinal microglia, likely involving the membrane glucocorticoid receptor/cAMP/PKA/p38 MAPK/CREB signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Diabetic retinopathy (DR) is established as the primary cause of visual impairment and preventable blindness, posing significant social and economic burdens on healthcare systems worldwide. Oxidative stress has been identified as a major contributor to DR, yet the precise role of the transmembrane glycoprotein CD200R in this context remains elusive. We studied human retinal pigment epithelia ARPE-19 cells to investigate the role of CD200R in high-glucose (HG) induced oxidative stress. Under HG conditions, we found a significant increase in CD200R expression in a time-dependent pattern. Conversely, knockdown of CD200R effectively alleviated oxidative stress and restored cell viability in HG-treated ARPE-19 cells, a phenomenon corroborated by the addition of a reactive oxygen species (ROS) scavenger. Exploration of the AKT/mTOR signaling pathway confirmed its mediating role regarding CD200R knockdown suppression of the expression of key proteins induced by HG conditions. Additionally, we found that the inhibition of mTOR signaling with Rapamycin effectively countered HG-induced oxidative stress in ARPE-19 cells, suggesting a promising therapeutic target against oxidative stress in the context of DR. This study establishes the crucial role of CD200R in HG-induced oxidative stress and identifies potential therapeutic avenues for the treatment of DR., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interests exist., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Pathological cardiac hypertrophy (referred to as cardiac hypertrophy) is a maladaptive response of the heart to a variety of pathological stimuli, and cardiac hypertrophy is an independent risk factor for heart failure and sudden death. Currently, the treatments for cardiac hypertrophy are limited to improving symptoms and have little effect. Elucidation of the developmental process of cardiac hypertrophy at the molecular level and the identification of new targets for the treatment of cardiac hypertrophy are crucial. In this review, we summarize the research on multiple active substances related to the pathogenesis of cardiac hypertrophy and the signaling pathways involved and focus on the role of transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling in the development of cardiac hypertrophy and the identification of potential targets for molecular intervention. We aim to identify important signaling molecules with clinical value and hope to help promote the precise treatment of cardiac hypertrophy and thus improve patient outcomes.

Cytokine storms are an important mechanism of sepsis. TNF-α is an important cytokine. As a regulator of TNF superfamily receptors, RIPK1 not only serves as the basis of the scaffold structure in complex I to promote the activation of the NF-κB and MAPK pathways but also represents an important protein in complex II to promote programmed cell death. Ubiquitination of RIPK1 is an important regulatory function that determines the activation of cellular inflammatory pathways or the activation of death pathways. In this paper, we introduce the regulation of RIPK1, RIPK1 PANoptosome's role in Inflammatory and sepsis, and perspectives., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Tang, Chen, Gao, Zhang and Xiao.)

The use of antimicrobial peptide (AMP), found in all forms of life and playing a pivotal role in the innate immune system, has been developed as a new strategy for maintaining intestinal health and reducing antibiotic usage due to its ability to resist pathogens and commensal microbes. The current study investigated the effects of l-threonine on β-defensin expression, the intestinal mucosal barrier and inflammatory cytokine expression in porcine intestinal epithelial cell lines (IPEC-J2). The results revealed that in IPEC-J2 cells, l-threonine significantly increased the expression of β-defensin (including pBD-1, pBD-2, and pBD-3) in a dose- and time-dependent manner (P < 0.05). By using different concentrations and treatment times of l-threonine, the results showed that the expression of β-defensin was upregulated to the greatest extent in IPEC-J2 cells cultured with 1 mM l-threonine for 24 h. Although the mRNA expression levels of β-defensins were markedly increased (P < 0.05), there was relatively little inducible pBD-1, pBD-2 and pBD-3 mRNA expression at the sub-confluent and confluent densities in comparison with post-confluent densities. Furthermore, we found that l-threonine enhanced the β-defensin expression by suppressing the expression of SIRT1, which increased acetylated p65 expression, and activating the NF-κB signaling pathway, which induced the translocation of p65 from the cytoplasm to the nucleus. In addition, l-threonine significantly prevented LPS-induced intestinal mucosal barrier damage by attenuating the decreasing tendency of the mRNA expression of Mucin1 and Mucin2 (P < 0.05). Simultaneously, l-threonine enhanced the expression of β-defensins upon LPS challenge in IPEC-J2 cells (P < 0.05). l-Threonine obviously decreased the mRNA expression of inflammatory cytokines compared to that in untreated cells (P < 0.05). In conclusion, l-threonine can upregulate β-defensin expression and reduce inflammatory cytokine expression in IPEC-J2 cells; meanwhile, l-threonine alleviates LPS-induced intestinal mucosal barrier damage in porcine intestinal epithelial cells. The l-threonine-mediated modulation of endogenous defensin expression may be a promising approach to reduce antibiotic use, enhance disease resistance and intestinal health in animals.

Purpose: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection., Methods: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans-infected corneas. B6 CD11c-DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively., Results: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1β levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1β, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas., Conclusions: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection-induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.

Psoriasis is one of the most common chronic inflammatory skin diseases that affects approximately 3% of the world's population. Hyper proliferation, infiltration of inflammatory cells and aberrant differentiation of keratinocytes are the three most important characteristics of psoriasis. Previous reports showed that NF-κBis the crucial mediator linking psoriatic keratinocytes and immune cell states through its effects on chemokine and cytokine production. To identify the role of NF-κB in psoriasis, we conducted ELISA assay to detect the activity of NF-κB in lesional skin and nonlesional skin of patients with psoriasis. Mounting evidence suggests that the interaction between long noncoding RNAs (lncRNAs) and microRNAs plays important role in the regulation of the initiation and development of various diseases. In this article, we identified that lncRNA UCA1 was down-regulated in lesional skin of patients with psoriasis. Further studies showed that lncRNA UCA1 could promote the expression of A20 by inhibitingmiR125a, and up-regulated A20 decreased the activity of NF-κB through its ubiquitin editing function. Taken together, we identified and demonstrated that lncRNA UCA1 negatively regulated NF-κB activity in psoriasis through the miR125a-A20 axis., (© 2021 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Triptolide, a component of the Chinese herb Tripterygium wilfordii Hook F, has been proved to be effective in the treatment of rheumatoid arthritis (RA). However, its underlying mechanisms on RA have not yet been well established. We observed the inhibitory effect of triptolide on the expression of inflammatory cytokines and proliferation of fibroblast-like synoviocytes (FLS) induced by the complex of interleukin-6 (IL-6) and the soluble form of the IL-6 receptor (sIL-6R). Furthermore, to clarify the underlying mechanisms, we treated FLS with the Janus-activated kinase 2 (JAK2) inhibitor/signal transducer and activator of transcription 3 (STAT3) activation blocker AZD1480. In this study, immunohistochemical staining was used to identify vimentin (+) and CD68 (-) in FLS. The FLS proliferation was measured by cell proliferation assay, and the cell cycles were analyzed by flow cytometry. Furthermore, ELISA was used to detect the expression of the inflammatory factors in culture solution. The expression levels of p-JAK2, JAK2, p-STAT3 and STAT3 were investigated through Western blotting analysis. The results showed that IL-6/sIL-6R significantly increased the cell proliferation and expression of inflammatory cytokines, including IL-6, interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF). Triptolide or AZD1480 inhibited the cell proliferation and inflammatory cytokine expression in IL-6/sIL-6R-stimulated FLS by suppressing JAK2/STAT3. The study suggested that the physiological effects of triptolide on RA were due to its contribution to the inhibition of the inflammatory cytokine expression and FLS proliferation by suppressing the JAK2/STAT3 signaling pathway. It may provide an innovative insight into the effect of triptolide in preventing RA pathogenesis.

Doxorubicin is a commonly used anthracycline chemotherapeutic agent; however, its application is limited owing to its cardiotoxicity. Current clinical treatments cannot efficiently or fully prevent doxorubicin-induced toxicity, primarily because its pathogenesis and mechanisms of action remain unknown. In this study, we established a rat model of chronic doxorubicin-induced cardiotoxicity, in which the severity of cardiac fibrosis and hydroxyproline levels increased in a time-dependent manner. Doxorubicin damaged the mitochondria and blood vessels and induced autophagy. Cells undergoing endothelial-to-mesenchymal transition (EndoMT)and those expressing endothelial cell and myofibroblast markers were simultaneously observed in vitro and in rats treated with doxorubicin. The NF-κB pathway was activated during EndoMT, andp65 and p-p65 were strongly expressed in the nucleus of endothelial cells in vitro. Taken together, these results suggest that vascular injury and cardiac fibrosis are characteristic symptoms of doxorubicin-induced cardiotoxicity. The NF-κB pathway-associated EndoMT may influence the pathogenesis of doxorubicin-induced cardiotoxicity, and the constituents of this pathway may be potential therapeutic targets to prevent the development of this condition., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Purpose: Autophagy plays an important role in balancing the inflammatory response to restore homeostasis. The aim of this study was to explore the mechanism by which trehalose suppresses inflammatory cytokines via autophagy activation in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress., Methods: An in vitro dry eye model was used in which HCECs were cultured in hyperosmolar medium with the addition of sodium chloride (NaCl). Trehalose was applied in different concentrations. The levels of TNF-α, IL-1β, IL-6, and IL-8 were detected using RT-qPCR and ELISA. Cell viability assays, immunofluorescent staining of LC3B, and western blots of Beclin1, Atg5, Atg7, LC3B, and P62 were conducted. The key factors in upstream signaling pathways of autophagy activation were measured: P-Akt, Akt, and transcription factor EB (TFEB)., Results: Trehalose reduced the proinflammatory mediators TNF-α, IL-1β, IL-6, and IL-8 in primary HCECs at 450 mOsM. This effect was osmolarity dependent, and a level of 1.0% trehalose showed the most suppression. Trehalose promoted autophagosome formation and autophagic flux, as evidenced by increased production of Beclin1, Atg5, and Atg7, as well as higher LC3B I protein turnover to LC3B II, with decreased protein levels of P62/SQSTM1. The addition of 3-methyladenine blocked autophagy activation and increased the release of proinflammatory cytokines. Trehalose further activated TFEB, with translocation from cytoplasm to the nucleus, but diminished Akt activity., Conclusions: Our findings demonstrate that trehalose, functioning as an autophagy enhancer, suppresses the inflammatory response by promoting autophagic flux via TFEB activation in primary HCECs exposed to hyperosmotic stress, a process that is beneficial to dry eye.

We aimed to identify key genes and pathways associated with different immune statuses of hepatitis B virus (HBV) infection. The gene expression and DNA methylation profiles were analysed in different immune statuses of HBV infection. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were identified, followed by their functional and integrative analyses. The differential expression of IgG Fc receptors (FcγRs) in chronic HBV-infected patients and immune cells during different stages of HBV infection was investigated. Toll-like receptor (TLR) signalling pathway (including TLR6) and leucocyte transendothelial migration pathway (including integrin subunit beta 1) were enriched during acute infection. Key DEGs, such as FcγR Ib and FcγR Ia, and interferon-alpha inducible protein 27 showed correlation with alanine aminotransferase levels, and they were differentially expressed between acute and immune-tolerant phases and between immune-tolerant and immune-clearance phases. The integrative analysis of DNA methylation profile showed that lowly methylated and highly expressed genes, including cytotoxic T lymphocyte-associated protein 4 and mitogen-activated protein kinase 3 were enriched in T cell receptor signalling pathway during acute infection. Highly methylated and lowly expressed genes, such as Ras association domain family member 1 and cyclin-dependent kinase inhibitor 2A were identified in chronic infection. Furthermore, differentially expressed FcγR Ia, FcγR IIa and FcγR IIb, CD3 - CD56 + CD16 + natural killer cells and CD14 high CD16 + monocytes were identified between immune-tolerant and immune-clearance phases by experimental validation. The above genes and pathways may be used to distinguish different immune statuses of HBV infection., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

This study was to explore the role and mechanism of macrophages in pollen-triggered allergic inflammation. A murine model of short ragweed (SRW) pollen-induced experimental allergic conjunctivitis (EAC), and bone marrow (BM)-macrophages cultures were used. Typical allergic manifestations and TSLP-stimulated Th2 hyperresponse were observed in ocular surface of EAC model in wild-type (WT) mice induced by SRW. The M2 phenotype markers, Arg1, Ym1 and FIZZ1, were highly expressed by conjunctiva and draining cervical lymph nodes (CLNs) of WT-EAC mice when compared with controls, as evaluated by RT-qPCR and Immunofluorescent double staining with macrophage marker F4/80. The stimulated expression of TSLPR and OX40L by macrophage was detected in conjunctiva and CLNs by RT-qPCR, double staining, and flow cytometry. M2 macrophages were found to produce TARC and MDC. In contrast, EAC model with TSLPR -/- mice did not show allergic signs and any increase of Th2 cytokines (IL-4, IL-5 and IL-13) and M2 markers. In vitro cultures confirmed that SRW extract stimulates expression of TSLPR, OX40L, TARC, MDC, and three M2 markers by BM-macrophages from WT mice, but not from TSLPR -/- mice. These findings demonstrate that SRW pollen primes macrophage polarization toward to M2 phenotype via TSLP/TSLPR/OX40L signaling to amplify allergic inflammation.

Lung cancer belongs to a leading popular and malignant cancer around the world. However, the root mechanism underlying lung cancer progression remains unclear. Recently, long noncoding RNA (lncRNA) has been identified as important for tumorigenesis. LncRNA MNX1-AS1 is proven to regulate colon adenocarcinoma, cervical cancer, glioblastoma, and ovarian cancer. Whether MNX1-AS1 participates in lung cancer needs investigation. In our research, we found that MNX1-AS1 was dramatically upregulated in lung cancer. MNX1-AS1 upregulation indicated poor prognosis in lung cancer patients. Functionally, MNX1-AS1 promoted lung cancer progression through regulating proliferation, migration, and invasion. Mechanistically, MNX1-AS1 was found to locate in the cytoplasm and interact with miR-527. Through inhibiting miR-527 availability, MNX1-AS1 facilitated BRF2 expression. Restoration of BRF2 rescued defects of proliferation, migration, and invasion caused by MNX1-AS1 knockdown. Taken together, our study found a novel signaling pathway, namely MNX1-AS1/miR-527/BRF2 axis, involved in lung cancer progression., (© 2019 Wiley Periodicals, Inc.)

Background: While most studies focus on pro-allergic cytokines, the protective role of immunosuppressive cytokines in allergic inflammation is not well elucidated. This study was to explore a novel anti-inflammatory role and cellular/molecular mechanism of IL-27 in allergic inflammation., Methods: A murine model of experimental allergic conjunctivitis (EAC) was induced in BALB/c, C57BL/6 or IL-27Rα-deficient (WSX-1 -/- ) mice by short ragweed pollen, with untreated or PBS-treated mice as controls. The serum, eyeballs, conjunctiva, cervical lymph nodes (CLNs) were used for study. Gene expression was determined by RT-qPCR, and protein production and activation were evaluated by immunostaining, ELISA and Western blotting., Results: Typical allergic manifestations and stimulated thymic stromal lymphopoietin (TSLP) signaling and Th2 responses were observed in ocular surface of EAC models in BALB/c and C57BL/6 mice. The decrease of IL-27 at mRNA (IL-27/EBI3) and protein levels were detected in serum, conjunctiva and CLN, as evaluated by RT-qPCR, immunofluorescent staining, ELISA and Western blotting. EAC induced in WSX-1 -/- mice showed aggravated allergic signs with higher TSLP-driven Th2-dominant inflammation, accompanied by stimulated Th17 responses, including IL-17A, IL-17F, and transcription factor RORγt. In contrast, Th1 cytokine IFNγ and Treg marker IL-10, with their respective transcription factors T-bet and foxp3, were largely suppressed. Interestingly, imbalanced activation between reduced phosphor (P)-STAT1 and stimulated P-STAT6 were revealed in EAC, especially WSX-1 -/- -EAC mice., Conclusion: These findings demonstrated a natural protective mechanism by IL-27, of which signaling deficiency develops a Th17-type hyperresponse that further aggravates Th2-dominant allergic inflammation., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Low-level inorganic arsenic (iAs) in drinking water is a risk factor for β cells dysfunction. Taurine (Tau) is a kind of semi-essential β amino acid, and beneficial for β cell function. However, the effects of Tau on arsenic trioxide (As 2 O 3 ) induced β cells dysfunction and related mechanisms are still uncertain. Here, we found that Tau relieved As 2 O 3 -induced endoplasmic reticulum (ER) stress, inflammation and pyroptosis in rat pancreas. In INS-1 cells, with NOD-like receptor family pyrin domain-containing 3 (NLRP3) inhibitor pretreatment, As 2 O 3 -induced activation of pyroptosis was decreased; with tumor necrosis factor-α (TNF-α) inhibitor pretreatment, As 2 O 3 -induced activation of NLRP3 inflammasome and pyroptosis were decreased; further, with the inositol-requiring enzyme 1 alpha (IRE1α) inhibitor, As 2 O 3 -induced induction of TNF-α was decreased. Tau markedly protected As 2 O 3 -induced β cells dysfunction by reducing the phosphorylation of IRE1α, production of TNF-α, activation of NLRP3 inflammasome and pyroptosis. Our results revealed that ER stress dependent inflammation and pyroptosis are critical pathogenic components of As 2 O 3 -induced β cell dysfunction. Moreover, TNF-α was a critical signaling node that linked As 2 O 3 -induced ER stress and pyroptosis. Tau was an effective supplement against As 2 O 3 -induced β cells dysfunction through the pathway as mentioned above., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Anticancer activities of flavonoids derived from Tephroseris kirilowii (Turcz.) Holub. were evaluated in human cancer cells. We isolated and identified, for the first time, eight flavonoids from T. kirilowii and found that three of them (IH: isorhamnetin, GN: genkwanin, and Aca: acacetin) inhibited cell proliferation in a variety of human cancer cell lines. These active flavonoids caused cell cycle arrest at G2/M phase and induced apoptosis and autophagy in human breast cancer cells. Molecular docking revealed that these flavonoids dock in the ATP binding pocket of PI3Kγ. Importantly, treatment with these flavonoids decreased the levels of PI3Kγ-p110, phospho-PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6K, and phospho-ULK. Pretreatment with PI3Kγ specific inhibitor AS605240 potentiated flavonoids-mediated inactivation of AKT, mTOR, p70S6K, ULK, and apoptosis. Taken together, these findings represent a novel mechanism by which downregulation of PI3Kγ-p110 and consequent interruption of PI3K/AKT/mTOR/p70S6K/ULK signaling pathway might play a critical functional role in these flavonoids-induced cell cycle arrest at G2/M phase, apoptosis, and autophagy. Our studies provide novel insights into the anticancer activities of selected flavonoids and their potential uses in anticancer therapy.

The effects of MC-3129, a synthetic cyclohexene derivative, on cell viability and apoptosis have been investigated in human leukemia cells. Exposure of leukemia cells to MC-3129 led to the inhibition of cell viability and induction of apoptosis through the dephosphorylation and mitochondrial translocation of cofilin. A mechanistic study revealed that interruption of the RhoA/ROCK1/PTEN/PI3K/Akt signaling pathway plays a crucial role in the MC-3129-mediated dephosphorylation and mitochondrial translocation of cofilin and induction of apoptosis. Our in vivo study also showed that the MC-3129-mediated inhibition of the tumor growth in a mouse leukemia xenograft model is associated with the interruption of ROCK1/PTEN/PI3K/Akt signaling and apoptosis. Molecular docking suggested that MC-3129 might activate the RhoA/ROCK1 pathway by targeting LPAR2. Collectively, these findings suggest a hierarchical model, in which the induction of apoptosis by MC-3129 primarily results from the activation of RhoA/ROCK1/PTEN and inactivation of PI3K/Akt, leading to the dephosphorylation and mitochondrial translocation of cofilin, and culminating in cytochrome c release, caspase activation, and apoptosis. Our study reveals a novel role for RhoA/ROCK1/PTEN/PI3K/Akt signaling in the regulation of mitochondrial translocation of cofilin and apoptosis and suggests MC-3129 as a potential drug for the treatment of human leukemia.

Hirsutine extracted from Uncaria rhynchophylla has been shown to exhibit anti-cancer activity. However, the molecular mechanism by which hirsutine exhibits anti-lung cancer activity remains unclear. In the present study, we showed that hirsutine induces apoptosis in human lung cancer cells via loss of mitochondrial membrane potential (∆ψm), adenosine triphosphate (ATP) depletion, ROS production, as well as cytochrome c release. Dephosphorylation of GSK3β is involved in hirsutine-mediated mitochondrial permeability transition pore (mPTP) opening through ANT1/CypD interaction. Mechanistic study revealed that interruption of ROCK1/PTEN/PI3K/Akt signaling pathway plays a critical role in hirsutine-mediated GSK3β dephosphorylation and mitochondrial apoptosis. Our in vivo study also showed that hirsutine effectively inhibits tumor growth in a A549 xenograft mouse model through ROCK1/PTEN/PI3K/Akt signaling-mediated GSK3β dephosphorylation and apoptosis. Collectively, these findings suggest a hierarchical model in which induction of apoptosis by hirsutine stems primarily from activation of ROCK1 and PTEN, inactivation of PI3K/Akt, leading in turn to GSK3β dephosphorylation and mPTP opening, and culminating in caspase-3 activation and apoptosis. These findings could provide a novel mechanistic basis for the application of hirsutine in the treatment of human lung cancer.

Key Points: Smooth muscle myosin regulatory light chain (RLC) is phosphorylated by Ca 2+ /calmodulin-dependent myosin light chain kinase and dephosphorylated by myosin light chain phosphatase (MLCP). Tracheal smooth muscle contains significant amounts of myosin binding subunit 85 (MBS85), another myosin phosphatase targeting subunit (MYPT) family member, in addition to MLCP regulatory subunit MYPT1. Concentration/temporal responses to carbachol demonstrated similar sensitivities for bovine tracheal force development and phosphorylation of RLC, MYPT1, MBS85 and paxillin. Electrical field stimulation releases ACh from nerves to increase RLC phosphorylation but not MYPT1 or MBS85 phosphorylation. Thus, nerve-mediated muscarinic responses in signalling modules acting on RLC phosphorylation are different from pharmacological responses with bath added agonist. The conditional knockout of MYPT1 or the knock-in mutation T853A in mice had no effect on muscarinic force responses in isolated tracheal tissues. MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction., Abstract: Ca 2+ /calmodulin activation of myosin light chain kinase (MLCK) initiates myosin regulatory light chain (RLC) phosphorylation for smooth muscle contraction with subsequent dephosphorylation for relaxation by myosin light chain phosphatase (MLCP) containing regulatory (MYPT1) and catalytic (PP1cδ) subunits. RLC phosphorylation-dependent force development is regulated by distinct signalling modules involving protein phosphorylations. We investigated responses to cholinergic agonist treatment vs. neurostimulation by electric field stimulation (EFS) in bovine tracheal smooth muscle. Concentration/temporal responses to carbachol demonstrated tight coupling between force development and RLC phosphorylation but sensitivity differences in MLCK, MYPT1 T853, MYPT1 T696, myosin binding subunit 85 (MBS85), paxillin and CPI-17 (PKC-potentiated protein phosphatase 1 inhibitor protein of 17 kDa) phosphorylations. EFS increased force and phosphorylation of RLC, CPI-17 and MLCK. In the presence of the cholinesterase inhibitor neostigmine, EFS led to an additional increase in phosphorylation of MYPT1 T853, MYPT1 T696, MBS85 and paxillin. Thus, there were distinct pharmacological vs. physiological responses in signalling modules acting on RLC phosphorylation and force responses, probably related to degenerate G protein signalling networks. Studies with genetically modified mice were performed. Expression of another MYPT1 family member, MBS85, was enriched in mouse, as well as bovine tracheal smooth muscle. Carbachol concentration/temporal-force responses were similar in trachea from MYPT1 SM+/+ , MYPT1 SM-/- and the knock-in mutant mice containing nonphosphorylatable MYPT1 T853A with no differences in RLC phosphorylation. Thus, MYPT1 T853 phosphorylation was not necessary for regulation of RLC phosphorylation in tonic airway smooth muscle. Furthermore, MLCP activity may arise from functionally shared roles between MYPT1 and MBS85, resulting in minimal effects of MYPT1 knockout on contraction., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Main Conclusion: Fackel (FK) is involved in the flowering of Arabidopsis mainly via the gibberellin pathway and vernalization pathway. This new function of FK is partially dependent on the FLOWERING LOCUS C ( FLC ). A common transitional process from vegetative stage to reproductive stage exists in higher plants during their life cycle. The initiation of flower bud differentiation, which plays a key role in the reproductive phase, is affected by both external environmental and internal regulatory factors. In this study, we showed that the Arabidopsis weak mutant allele fk-J3158, impaired in the FACKEL (FK) gene, which encodes a C-14 reductase involved in sterol biosynthesis, had a long life cycle and delayed flowering time in different photoperiods. In addition, FK overexpression lines displayed an earlier flowering phenotype than that of the wild type. These processes might be independent of the downstream brassinosteroid (BR) pathway and the autonomous pathway. However, the fk-J3158 plants were more sensitive than wild type in reducing the bolting days and total leaf number under gibberellic acid (GA) treatment. Further studies suggested that FK mutation led to an absence of endogenous GAs in fk-J3158 and FK gene expression was also affected under GA and paclobutrazol (PAC) treatment. Moreover, the delayed flowering time of fk-J3158 could be rescued by a 3-week vernalization treatment, and the expression of FLOWERING LOCUS C (FLC) was accordingly down-regulated in fk-J3158. We also demonstrated that flowering time of fk-J3158 flc double mutant was significantly earlier than that of fk-J3158 under the long-day (LD) conditions. All these results indicated that FK may affect the flowering in Arabidopsis mainly via GA pathway and vernalization pathway. And these effects are partially dependent on the FLOWERING LOCUS C (FLC).

Ethnopharmacological Relevance: Xinqin, a polyherbal medicine, is an important traditional Chinese herbal formula used in traditional oriental medicine for treatment of allergic rhinitis (AR). The formula is based on the Chinese Pharmacopoeia AIM OF THE STUDY: Previously, Xinqin exhibited potent anti-allergic effect in a guinea pig model of AR. In this study, we explored the molecular mechanism of the anti-allergic effect mediated by Xinqin., Materials and Methods: AR was induced in guinea pigs (Hartley) with toluene-2, 4-diisocyanate (TDI) in vivo and in HMC-1 mast cells with A23187/phorbol 12-myristate-13-acetate (PMA) in vitro. The releases of allergic inflammatory mediators such as histamine, leukotriene (LT) D4, immunoglobulin (Ig) E, TNF-α, and IL-6 were analyzed for allergy. The mast cell degranulation was displayed in HMC-1 mast cells. The activities of janus protein kinase 2 (JAK2), signal transduction and activator of transcription 5 (STAT5) and suppressor of cytokine signaling 3 (SOCS3) were evaluated by Western blot., Results: Treatment with Xinqin resulted in AR symptoms and decreases in levels of histamine, LTD4, IgE, TNF-α, and IL-6 in serum of guinea pig model of AR and in A23187/PMA-stimulated HMC-1 mast cells. Treatment with Xinqin also inhibited cell degranulation in A23187/PMA-stimulated HMC-1 mast cells. The JAK2/STAT5 signaling pathway could play an important role in the anti-allergic activity mediated by Xinqin., Conclusions: Xinqin exerts the anti-allergic effect by modulating mast cell-mediated allergic responses by down-regulating JAK2/STAT5 signaling pathway. Results from this study provide a mechanistic basis for the application of Xinqin in the treatment of AR., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Clinical studies have shown that probiotics influence gastrointestinal motility. However, the molecular mechanisms by which probiotic Lactobacillus modulates intestinal motility in traumatic brain injury (TBI) mouse model have not been explored. In the present study, we provided evidence showing that treatment of TBI mice with Lactobacillus acidophilus significantly improved the terminal ileum villus morphology, restored the impaired interstitial cells of Cajal (ICC) and the disrupted ICC networks after TBI, and prevented TBI-mediated inhibition of contractile activity in intestinal smooth muscle. Mechanistically, the decreased concentration of MLCK, phospho-MLC20 and phospho-MYPT1 and increased concentration of MLCP and PKC were observed after TBI, and these events mediated by TBI were efficiently prevented by Lactobacillus acidophilus application. These findings may provide a novel mechanistic basis for the application of Lactobacillus acidophilus in the treatment of TBI.

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb prescribed as an immunostimulant, hepatoprotective, antiperspirant, a diuretic or a tonic as documented in Chinese Materia Medica. In the present study, we employed a high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS to investigate the possible mechanism of action involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. Differential proteins were identified, among which 13 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (vimentin and Gap43) were randomly selected, and their expression levels were further confirmed by western blots analysis. The results matched well with those of proteomics. Furthermore, network analysis of protein-protein interactions (PPI) and pathways enrichment with AGS-IV associated proteins were carried out to illustrate its underlying molecular mechanism. Proteins associated with signal transduction, immune system, signaling molecules and interaction, and energy metabolism play important roles in neuroprotective effect of AGS-IV and Raf-MEK-ERK pathway was involved in the neuroprotective effect of AGS-IV against glutamate-induced neurotoxicity in PC12 cells. This study demonstrates that comparative proteomics based on shotgun approach is a valuable tool for molecular mechanism studies, since it allows the simultaneously evaluate the global proteins alterations.

Hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) play important roles in angiogenesis and tumor growth. Tanshinone IIA (T2A) is a novel antiangiogenic agent with promising antitumor effects; however, the molecular mechanism underlying the antiangiogenic effects of T2A remains unclear. In the present study, we provided evidence showing that T2A inhibited angiogenesis and breast cancer growth by down-regulating VEGF expression. Specifically, T2A repressed HIF-1α expression at the translational level and inhibited the transcriptional activity of HIF-1α, which led to the down-regulation of VEGF expression. Suppression of HIF-1α synthesis by T2A correlated with strong dephosphorylation of mammalian target of rapamycin (mTOR) and its effectors ribosomal protein S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), a pathway regulating HIF-1α expression at the translational level. In addition, we also found that T2A inhibited the angiogenesis and growth of human breast cancer xenografts in nude mice through suppression of HIF-1α and VEGF. Our study provides novel perspectives and potential targets for the treatment of human breast cancer.

Numerous evidences have indicated that a signal system is composed by signal pathways, each pathway is composed by sub-pathways, and the sub-pathway is composed by the original signal terminals initiated with a protein/gene. We infer the terminal signals merged signal transduction system as "signal basin". In this article, we discussed the composition and regulation of signal basins, and the relationship between the signal basin control and triple W of spatiotemporal cell biology. Finally, we evaluated the importance of the systemic regulation to gene expression by signal basins under triple W. We hope our discussion will be the beginning to cause the attention for this area from the scientists of life science., (© 2013 Wiley Periodicals, Inc.)

R-(-)-gossypol acetic acid (AT-101) is a natural cottonseed product that exhibits anticancer activity. However, the molecular mechanism behind the antileukemic activity of AT-101 has not been well characterized. In this study, we investigated how AT-101 induces apoptosis in human leukemia cells. Exposure to AT-101 significantly increased apoptosis in both human leukemia cell lines and primary human leukemia cells. This increase was accompanied by the activation of caspases, cytochrome c release, Bcl2-associated X protein (Bax) translocation, myeloid cell leukemia-1 (Mcl-1) downregulation, Bcl-2-associated death promoter (Bad) dephosphorylation, Akt inactivation, and RhoA/Rho-associated coiled-coil containing protein kinase 1/phosphatase and tensin homolog (RhoA/ROCK1/PTEN) activation. RhoA, rather than caspase-3 cleavage, mediated the cleavage/activation of ROCK1 that AT-101 induced. Inhibiting RhoA and ROCK1 activation by C3 exoenzyme (C3) and Y27632, respectively, attenuated the ROCK1 cleavage/activation, PTEN activity, Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and apoptosis mediated by AT-101. Knocking down ROCK1 expression using a ROCK1-specific siRNA also significantly abrogated AT-101-mediated apoptosis. Constitutively active Akt prevented the AT-101-induced Mcl-1 downregulation, Bad dephosphorylation, and apoptosis. Conversely, AT-101 lethality was potentiated by the phosphatidylinositol 3-kinase inhibitor LY294002. In vivo, the tumor growth inhibition caused by AT-101 was also associated with RhoA/ROCK1/PTEN activation and Akt inactivation in a mouse leukemia xenograft model. Collectively, these findings suggest that AT-101 may preferentially induce apoptosis in leukemia cells by interrupting the RhoA/ROCK1/PTEN pathway, leading to Akt inactivation, Mcl-1 downregulation, Bad dephosphorylation, and Bax translocation, which culminate in mitochondrial injury and apoptosis.

Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates smooth muscle myosin regulatory light chain (RLC) to initiate contraction. We used a tamoxifen-activated, smooth muscle-specific inactivation of MLCK expression in adult mice to determine whether MLCK was differentially limiting in distinct smooth muscles. A 50% decrease in MLCK in urinary bladder smooth muscle had no effect on RLC phosphorylation or on contractile responses, whereas an 80% decrease resulted in only a 20% decrease in RLC phosphorylation and contractile responses to the muscarinic agonist carbachol. Phosphorylation of the myosin light chain phosphatase regulatory subunit MYPT1 at Thr-696 and Thr-853 and the inhibitor protein CPI-17 were also stimulated with carbachol. These results are consistent with the previous findings that activation of a small fraction of MLCK by limiting amounts of free Ca(2+)/calmodulin combined with myosin light chain phosphatase inhibition is sufficient for robust RLC phosphorylation and contractile responses in bladder smooth muscle. In contrast, a 50% decrease in MLCK in aortic smooth muscle resulted in 40% inhibition of RLC phosphorylation and aorta contractile responses, whereas a 90% decrease profoundly inhibited both responses. Thus, MLCK content is limiting for contraction in aortic smooth muscle. Phosphorylation of CPI-17 and MYPT1 at Thr-696 and Thr-853 were also stimulated with phenylephrine but significantly less than in bladder tissue. These results indicate differential contributions of MLCK to signaling. Limiting MLCK activity combined with modest Ca(2+) sensitization responses provide insights into how haploinsufficiency of MLCK may result in contractile dysfunction in vivo, leading to dissections of human thoracic aorta.

Effects of phenethyl isothiocyanate (PEITC) have been investigated in human leukemia cells (U937, Jurkat, and HL-60) as well as in primary human acute myeloid leukemia (AML) cells in relation to apoptosis and cell signaling events. Exposure of cells to PEITC resulted in pronounced increase in the activation of caspase-3, -8, -9, cleavage/degradation of PARP, and apoptosis in dose- and time-dependent manners. These events were accompanied by the caspase-independent downregulation of Mcl-1, inactivation of Akt, as well as activation of Jun N-terminal kinase (JNK). Inhibition of PI3K/Akt by LY294002 significantly enhanced PEITC-induced apoptosis. Conversely, enforced activation of Akt by a constitutively active Akt construct markedly abrogated PEITC-mediated JNK activation, Mcl-1 downregulation, caspase activation, and apoptosis, and also interruption of the JNK pathway by pharmacological or genetically (e.g., siRNA) attenuated PEITC-induced apoptosis. Finally, administration of PEITC markedly inhibited tumor growth and induced apoptosis in U937 xenograft model in association with inactivation of Akt, activation of JNK, as well as downregulation of Mcl-1. Taken together, these findings represent a novel mechanism by which agents targeting Akt/JNK/Mcl-1 pathway potentiate PEITC lethality in transformed and primary human leukemia cells and inhibitory activity of tumor growth of U937 xenograft model.

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-kappaB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-kappaB activity. In this study, we have demonstrated the effect of PKA on NF-kappaB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-kappaB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-kappaB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-kappaB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-kappaB activation; PKA-activating agents enhanced NF-kappaB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-kappaB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-kappaB-induced breast cancer cell proliferation and multiple NF-kappaB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-kappaB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

Background: The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs)., Methodology/principal Findings: Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-kappaB activation, but were potent in eliciting EGFR signaling in a TGF-alpha-related pathway in HCECs. Concomitant with the lack of NF-kappaB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling., Conclusions/significance: Our data suggest that inflammatory response mediated by NF-kappaB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues.

We demonstrate that conditional ablation of the homeobox transcription factor Cdx2 from early endoderm results in the replacement of the posterior intestinal epithelium with keratinocytes, a dramatic cell fate conversion caused by ectopic activation of the foregut/esophageal differentiation program. This anterior homeotic transformation of the intestine was first apparent in the early embryonic Cdx2-deficient gut by a caudal extension of the expression domains of several key foregut endoderm regulators. While the intestinal transcriptome was severely affected, Cdx2 deficiency only transiently modified selected posterior Hox genes and the primary enteric Hox code was maintained. Further, we demonstrate that Cdx2-directed intestinal cell fate adoption plays an important role in the establishment of normal epithelial-mesenchymal interactions, as multiple signaling pathways involved in this process were severely affected. We conclude that Cdx2 controls important aspects of intestinal identity and development, and that this function is largely independent of the enteric Hox code.

Cytoskeleton is a stereo wire frame system of filament, which is in a dynamic state. It can assemble or disassemble continuously with the change of physiological condition, and be regulated by many factors interior and exterior of the cell. Insulin is an important hormone involving in lots of physiological functions such as transport of glucose, gene expression and DNA synthesis. Normal secretion of insulin is the significant premise for which to educe its functions. Many studies indicate that cytoskeleton plays an important role in the process of insulin action and secretion, with a mechanism referring to insulin related signal transduction pathways tightly. The change of cytoskeleton influencing insulin related signal transduction pathways will lead to incidence of insulin resistance.

Ovarian cancer is one of the most common cancers among women. Recent studies demonstrated that the gene encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K) is frequently amplified in ovarian cancer cells. PI3K is involved in multiple cellular functions, including proliferation, differentiation, antiapoptosis, tumorigenesis, and angiogenesis. In this study, we demonstrate that the inhibition of PI3K activity by LY-294002 inhibited ovarian cancer cell proliferation and induced G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins, including cyclin D1, cyclin-dependent kinase (CDK) 4, CDC25A, and retinoblastoma phosphorylation at Ser(780), Ser(795), and Ser(807/811). Expression of CDK6 and beta-actin was not affected by LY-294002. Expression of the cyclin kinase inhibitor p16(INK4a) was induced by the PI3K inhibitor, whereas steady-state levels of p21(CIP1/WAF1) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation of AKT and p70S6K1, but not extracellular regulated kinase 1/2. The G(1) cell cycle arrest induced by LY-294002 was restored by the expression of active forms of AKT and p70S6K1 in the cells. Our study shows that PI3K transmits a mitogenic signal through AKT and mammalian target of rapamycin (mTOR) to p70S6K1. The mTOR inhibitor rapamycin had similar inhibitory effects on G(1) cell cycle progression and on the expression of cyclin D1, CDK4, CDC25A, and retinoblastoma phosphorylation. These results indicate that PI3K mediates G(1) progression and cyclin expression through activation of an AKT/mTOR/p70S6K1 signaling pathway in the ovarian cancer cells.

Intracellular recording methods with "sharp" microelectrodes were used to study signal transduction mechanisms underlying the excitatory action of bradykinin (BK) in morphologically identified neurons in the small intestinal submucosal plexus. Exposure to BK evoked slowly activating membrane depolarization and enhanced excitability associated with increased input resistance in AH-type and decreased input resistance in S-type neurons. Preincubation with pertussis toxin did not affect the BK-evoked responses. Pretreatment with the cyclooxygenase inhibitors indomethacin or piroxicam suppressed or abolished the BK-evoked responses. Application of prostaglandin (PG) E(2) or PG analogs evoked BK-like depolarizing responses in the submucosal plexus with a potency order of PGE(2) > PGE(1) > 17-phenyl trinor-PGE(2) > PGI(2) > sulprostone > PGF(2alpha). Depolarizing responses to bradykinin or PGE(2) in S-type neurons were suppressed in the presence of the phospholipase C inhibitor U73122 [(1-6-[([17beta]-3-methoxyestra-1,3,5[10]-tren-17-71)amino]hexyl)-1H-pyrrole-2,5-dione)], but not the inactive analog U73343 [(1-6-[([17beta]-3-methoxyestra-1,3,5[10]trien-17yl)amino]hexyl)-2,5-pyrrolidinedione)]. The inositol-1,4,5-trisphosphate receptor antagonist 2-aminoethoxy-diphenylborane and the calmodulin inhibitor W-7, but not ryanodine, suppressed both bradykinin- and PGE(2)-evoked responses. KN-62, an inhibitor of calmodulin kinases, or GF109203X, a specific protein kinase C inhibitor, suppressed both BK- and PGE(2)-evoked depolarizing responses. Selective protein kinase A inhibitors did not alter BK- or PGE(2)-evoked depolarizing responses in S neurons. The results suggest that BK stimulates synthesis and release of PGE(2), which acts at EP(1) receptors to evoke depolarizing responses in submucosal neurons. The postreceptor transduction cascade includes activation of phospholipase C, inositol-1,4,5-trisphosphate production, intraneuronal Ca2+ mobilization, activation of protein kinase C and/or calmodulin kinases, and phosphorylation of cationic channels.

Previous studies have shown that a constitutively active isoform of Ras is able to produce superoxide radical (O2(-)). The present study investigate the mechanisms by which O2(-) radical mediates signals from Ras protein to the nucleus, leading to cellular responses such as apoptosis in Cr(VI)-stimulated cells. Two human prostate tumor cell lines, Ras(+), which overexpresses Ras, and Ras(-), which has a normal Ras level, were utilized. Compared to Ras(-) cells, Ras(+) cells exhibited higher susceptibility to apoptosis induced by Cr(VI). Catalase, sodium formate, and deferoxamine inhibited Cr(VI)-induced apoptosis. Similar differences were observed in both cellular DNA damage and the activation of p53 protein. The differences in Cr(VI)-induced cell responses in Ras(+) and Ras(-) cells were due to differences in the generation of free radicals between these two cells. ESR spin trapping measurements showed that Ras(+) cells generated more hydroxyl radical ((.)OH), O2(-) radical, and Cr(V) than Ras(-) cells following Cr(VI) stimulation. The generation of the reactive oxygen species (ROS) can be abolished by the addition of superoxide dismutase (SOD) or if the experiment were carried out in an argon atmosphere. Catalase inhibited spin adduct signals but was much less potent than SOD. The mechanism of ROS generation in Cr(VI)-stimulated Ras(+) cells involves the reduction of molecular oxygen to O2(-) radical by a flavoenzyme-containing NADPH oxidase complex as shown by oxygen consumption and diphenylene iodonium (DPI) inhibition. Results shown above support the following conclusions: (a) Ras protein mediates O2(-) radical generation through reduction of molecular oxygen by NADPH oxidase in Cr(VI)-stimulated cells. (b) The O2(-) radical and Cr(VI) produce other reactive species, including H2O2, OH radical, and Cr(V) through O2(-) dismutation and Haber-Weiss type of reactions. (c) Among these reactive species, (.)OH radical is responsible for the further transduction of signals from Ras to the nucleus, leading to various cell responses.

Arsenite is ubiquitous in the environment, particularly in the form of contaminated water. Although this metal is a known human carcinogen, its exact mechanism of action remains unclear. P70S6K1 phosphorylates the ribosomal 40S protein leading to increased protein translation, and is an important regulator of cell growth and proliferation. Hypoxia inducible factor-1 (HIF-1) is a basic helix-loop-helix transcription factor composed of two subunits, HIF-1alpha and HIF-1beta. HIF-1 activates the transcription of a number of genes that mediate angiogenesis and tumor formation. In this study we demonstrated that arsenite treatment increased levels of p70S6K1 phosphorylation and p70S6K1 activity in a PI3K and mTOR sensitive manner. We have also shown that arsenite specifically induces HIF-1alpha, but not HIF-1beta, protein levels in prostate cancer cells in a mTOR-dependent manner.

Prostate cancer is one of the most common cancers among men. Recent studies demonstrated that PI3K signaling is an important intracellular mediator which is involved in multiple cellular functions including proliferation, differentiation, anti-apoptosis, tumorigenesis, and angiogenesis. In the present study, we demonstrate that the inhibition of PI3K activity by LY294002, inhibited prostate cancer cell proliferation and induced the G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins including cyclin D1, CDK4, and Rb phosphorylation at Ser780, Ser795, and Ser807/811, whereas expression of CDK6 and beta-actin was not affected by LY294002. The expression of cyclin kinase inhibitor, p21(CIP1/WAF1), was induced by LY294002, while levels of p16(INK4) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation and p70(S6K), but not MAPK. PI3K regulates cell cycle through AKT, mTOR to p70(S6K). The mTOR inhibitor rapamycin has similar inhibitory effects on G(1) cell cycle progression and expression of cyclin D1, CDK4, and Rb phosphorylation. These results suggest that PI3K mediates G(1) cell cycle progression and cyclin expression through the activation of AKT/mTOR/p70(S6K) signaling pathway in the prostate cancer cells.

Chromium(VI) (Cr(VI)) is widely used in industry and is a potent inducer of tumors in animals. The present study demonstrates that Cr(VI) induces hypoxia-inducible factor 1 (HIF-1) activity through the specific expression of HIF-1alpha but not HIF-1beta subunit and increases the level of vascular endothelial growth factor (VEGF) expression in DU145 human prostate carcinoma cells. To dissect the signaling pathways involved in Cr(VI)-induced HIF-1 expression, we found that p38 mitogen-activated protein kinase signaling was required for HIF-1alpha expression induced by Cr(VI). Neither phosphatidylinositol 3-kinase nor extracellular signal-regulated kinase activity was required for Cr(VI)-induced HIF-1 expression. Cr(VI) induced expression of HIF-1 and VEGF through the production of reactive oxygen species in DU145 cells. The major species of reactive oxygen species responsible for the induction of HIF-1 and VEGF expression is H(2)O(2). These results suggest that the expression of HIF-1 and VEGF induced by Cr(VI) may be an important signaling pathway in the Cr(VI)-induced carcinogenesis.