Induction of cytosolic phospholipase A2 by lipopolysaccharide in canine tracheal smooth muscle cells: involvement of MAPKs and NF-kappaB pathways

Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandins (PG) synthesis induced by bacterial lipopolysaccharide (LPS) and cytokines. However, the intracellular signaling pathways mediating LPS-induced cPLA2 expression and PGE2 synthesis in canine tracheal smooth muscle cells (TSMCs) remains unknown. LPS-induced expression of cPLA2 and release of PGE2 was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (D609), phosphatidylinositol-phospholipase C (U73122), PKC (GF109203X and staurosporine), removal of Ca2+ by BAPTA/AM plus EDTA, MEK1/2 (PD98059), p38 (SB202190), JNK (SP600125), and phosphatidylinositol 3-kinase (PI3-K; LY294002 and wortmannin). The involvement of MPAKs in LPS-induced responses was further confirmed by transfection of TSMCs with dominant negative mutants of ERK2 and p38. LPS-induced cPLA2 expression and PGE2 synthesis was inhibited by a selective NF-kappaB inhibitor (helenalin) and transfection with dominant negative mutants of NF-kappaB inducing kinase (NIK), IkappaB kinase (IKK)-alpha, and IKK-beta, consistent with that LPS-stimulated both IkappaB-alpha degradation and NF-kappaB translocation into nucleus in these cells. LPS-stimulated cPLA2 phosphorylation was inhibited by PD98059, GF109203X, and staurosporine, indicating the regulation by p42/p44 MAPK and PKC. Moreover, LPS-induced up-regulation of cPLA2 and COX-2 linked to PGE2 synthesis was inhibited by AACOCF3 (a selective cPLA2 inhibitor), implying the involvement of cPLA2 in these responses. These findings suggest that phosphorylation and expression of cPLA2 correlates with the release of PGE2 from LPS-challenged TSMCs, at least in part, mediated through MAPKs and NF-kappaB signaling pathways. LPS-mediated responses were modulated by PLC, Ca2+, PKC, tyrosine kinase, and PI3-K in TSMCs.     

Artocarpin induces cell apoptosis in human osteosarcoma cells through endoplasmic reticulum stress and reactive oxygen species

Osteosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. However, because of side effects and drug resistance in chemotherapy and the insufficiency of an effective adjuvant therapy for osteosarcoma, it is necessary to research novel treatments. This study was the first to investigate the anticancer effects of the flavonoid derivative artocarpin in osteosarcoma. Artocarpin induced cell apoptosis in three human osteosarcoma cell lines—U2OS, MG63, and HOS. Artocarpin was also associated with increased intracellular reactive oxygen species (ROS). Mitochondrial dysfunction was followed by the release of cytochrome c from mitochondria and accompanied by decreased antiapoptotic Bcl-2 and Bcl-xL and increased proapoptotic protein Bak and Bax. Artocarpin triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels and increased glucose-regulated protein 78 and 94 expressions, and also increased calpains expression and activity. Animal studies revealed a dramatic 40% reduction in tumor volume after 18 days of treatment. This study demonstrated a novel anticancer activity of artocarpin against human osteosarcoma cells and in murine tumor models. In summary, artocarpin significantly induced cell apoptosis through ROS, ER stress, mitochondria, and the caspase pathway, and may thus be a novel anticancer treatment for osteosarcoma.     

Induction of COX-2/PGE2/IL-6 is crucial for cigarette smoke extract-induced airway inflammation: Role of TLR4-dependent NADPH oxidase activation

Exposure to cigarette smoke extract (CSE) leads to airway and lung inflammation through an oxidant-antioxidant imbalance. Cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂) have been shown to play critical roles in respiratory inflammation. Here, we show that COX-2/PGE₂/IL-6 induction is dependent on Toll-like receptor 4 (TLR4)/NADPH oxidase signaling in human tracheal smooth muscle cells (HTSMCs). CSE induced COX-2 expression in vitro in HTSMCs and in vivo in the airways of mice. CSE also directly caused an increase in TLR4. Moreover, CSE-regulated COX-2, PGE₂, and IL-6 generation was inhibited by pretreatment with TLR4 Ab; inhibitors of c-Src (PP1), NADPH oxidase (diphenylene iodonium chloride and apocynin), p38 MAPK (SB202190), MEK1/2 (U0126), JNK1/2 (SP600125), and NF-κB (helenalin); a ROS scavenger (N-acetyl-l-cysteine); and transfection with siRNA of TLR4, MyD88, TRAF6, Src, p47^phox, p38, p42, JNK2, or p65. CSE-induced leukocyte numbers in BAL fluid were also reduced by pretreatment with these inhibitors. Furthermore, CSE induced p47^phox translocation and TLR4/MyD88/TRAF6 and c-Src/p47^phox complex formation. We found that PGE₂ enhanced IL-6 production in HTSMCs and leukocyte count in BAL fluid. In addition, treatment with nicotine could induce COX-2, PGE₂, and IL-6 generation in in vivo and in vitro studies. These results demonstrate that CSE-induced ROS generation was mediated through the TLR4/MyD88/TRAF6/c-Src/NADPH oxidase pathway, in turn initiated the activation of MAPKs and NF-κB, and ultimately induced COX-2/PGE₂/IL-6-dependent airway inflammation.     

Induction of cyclooxygenase-2 expression in human tracheal smooth muscle cells by interleukin-1β: Involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-κB

Interleukin-1 (IL-1) has been recognized as a potent stimulus for the synthesis of prostaglandin (PG), which has been implicated in inflammatory responses of the airways. However, the mechanisms underlying IL-1-induced cyclooxygenase (COX) expression and PGE₂ synthesis via activation of p42/p44 and p38 mitogen-activated protein kinases (MAPKs) in human tracheal smooth muscle cells (HTSMCs) are not completely understood. We found that IL-1 increased COX-2 expression and PGE₂ synthesis in time- and concentration-dependent manners. Both specific phosphatidylcholine-phospholipase C inhibitor (D609) and protein kinase C inhibitor (GF109203X) attenuated IL-1-induced responses in HTSMCs. IL-1-induced COX-2 expression and PGE₂ synthesis were also inhibited by an inhibitor of MEK1/2 (PD98059) and inhibitors of p38 MAPK (SB203580 and SB202190), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by the transient activation of p42/p44 and p38 MAPKs induced by IL-1. Furthermore, IL-1-induced activation of nuclear factor-B (NF-B) was inversely correlated with the degradation of IB- in HTSMCs. IL-1-induced COX-2 expression and PGE₂ synthesis were inhibited by the NF-B inhibitor pyrrolidinedithiocarbamate. These findings suggest that the expression of COX-2 is correlated with the release of PGE₂ from IL-1-challenged HTSMCs, which is mediated, at least in part, through p42/p44 and p38 MAPKs and NF-B signaling pathways in HTSMCs.     

Lipoteichoic acid-stimulated p42/p44 MAPK activation via Toll-like receptor 2 in tracheal smooth muscle cells

Lipoteichoic acid (LTA), the principal component of the cell wall of gram-positive bacteria, triggers several inflammatory responses. However, the mechanisms underlying its action on human tracheal smooth muscle cells (HTSMCs) were largely unknown. This study was to investigate the mechanisms underlying LTA-stimulated p42/p44 mitogen-activated protein kinase (MAPK) using Western blotting assay. LTA stimulated phosphorylation of p42/p44 MAPK via a Toll-like receptor 2 (TLR2). Pretreatment with pertussis toxin attenuated the LTA-induced responses. LTA-stimulated phosphorylation of p42/p44 MAPK was attenuated by inhibitors of tyrosine kinase (genistein), phosphatidylcholine-phospholipase C (PLC; D609), phosphatidylinositol (PI)-PLC (U-73122), PKC (staurosporine, G?-6976, rottlerin, or Ro-318220), MEK1/2 (U-0126), PI 3-kinase (LY-294002 and wortmannin), and an intracellular Ca(2+) chelator (BAPTA-AM). LTA directly evoked initial transient peak of [Ca(2+)](i), supporting the involvement of Ca(2+) mobilization in LTA-induced responses. These results suggest that in HTSMCs, LTA-stimulated p42/p44 MAPK phosphorylation is mediated through a TLR2 receptor and involves tyrosine kinase, PLC, PKC, Ca(2+), MEK, and PI 3-kinase.     

Tumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells: involvement of p42/p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaB

This study was to determine the mechanism of tumor necrosis factor-alpha (TNF-alpha)-enhanced cyclooxygenase (COX)-2 expression associated with prostaglandin E2 (PGE2) synthesis in human tracheal smooth muscle cells (HTSMCs). TNF-alpha markedly increased COX-2 expression and PGE2 synthesis in a time- and concentration-dependent manner, whereas COX-1 remained unaltered. Tyrosine kinase inhibitor (genistein), phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D-609) and PKC inhibitor (GF109203X) attenuated TNF-alpha-induced COX-2 expression and PGE2 synthesis in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis were also inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 and SB202190 (inhibitors of p38 MAPK), respectively, suggesting the involvement of p42/p44 and p38 MAPKs in these responses. This hypothesis was further supported by that TNF-alpha induced a transient activation of p42/p44 and p38 MAPKs in a time-and concentration-dependent manner. Furthermore, TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) reversely correlated with the degradation of IkappaB-alpha in HTSMCs. TNF-alpha-induced COX-2 expression and PGE2 synthesis was also inhibited by NF-kappaB inhibitor pyrrolidinedithiocarbamate (PDTC). These findings suggest that the increased expression of COX-2 correlates with the release of PGE2 from TNF-alpha-challenged HTSMCs, at least in part, mediated through p42/p44 and p38 MAPKs as well as NF-kappaB signaling pathways in HTSMCs.     

miR-26a attenuates cardiac apoptosis and fibrosis by targeting ataxia–telangiectasia mutated in myocardial infarction

Apoptosis and fibrosis play a vital role in myocardial infarction (MI) induced tissue injury. Although microRNAs have been the focus of many studies on cardiac apoptosis and fibrosis in MI, the detailed effects of miR-26a is needed to further understood. The present study demonstrated that miR-26a was downregulated in ST-elevation MI (STEMI) patients and oxygen-glucose deprivation (OGD)-treated H9c2 cells. Downregulation of miR-26a was closely correlated with the increased expression of creatine kinase, creatine kinase-MB and troponin I in STEMI patients. Further analysis identified that ataxia–telangiectasia mutated (ATM) was a target gene for miR-26a based on a bioinformatics analysis. miR-26a overexpression effectively reduced ATM expression, apoptosis, and apoptosis-related proteins in OGD-treated H9c2 cells. In a mouse model of MI, the expression of miR-26a was significantly decreased in the infarct zone of the heart, whereas apoptosis and ATM expression were increased. miR-26a overexpression effectively reduced ATM expression and cardiac apoptosis at Day 1 after MI. Furthermore, we demonstrated that overexpression of miR-26a improved cardiac function and reduced cardiac fibrosis by the reduced expression of collagen type I and connective tissue growth factor (CTGF) in mice at Day 14 after MI. Overexpression of miR-26a or ATM knockdown decreased collagen I and CTGF expression in cultured OGD-treated cardiomyocytes. Taken together, these data demonstrate a prominent role for miR-26a in linking ATM expression to ischemia-induced apoptosis and fibrosis, key features of MI progression. miR-26a reduced MI development by affecting ATM expression and could be targeted in the treatment of MI.     

Polymicrobial Bloodstream Infection in Neonates: Microbiology,Clinical Characteristics,and Risk Factors

Background

Polymicrobial bloodstream infections (PBSIs) have been associated with complex underlying medical conditions and a high incidence of specific microorganisms in several settings, but the relevant data are scarce in neonates.

Methods

Positive blood cultures from January 2004 to December 2011 in the neonatal intensive care unit (NICU) of Chang Gung Memorial Hospital (CGMH) were reviewed. Each neonate with PBSI (case episode) was matched to two episodes of monomicrobial BSI (control episode) by birth weight, gestational age and gender. Records were reviewed to compare their underlying medical conditions, organisms isolated, adequacy of therapy, clinical characteristics and outcomes.

Results

Forty-five episodes of PBSI (4.4% of all neonatal BSIs) were identified in 43 neonates. Gram-negative organisms constituted 59.8% of all PBSI pathogens, and 33 (73.3%) of PBSIs were caused by at least one Gram-negative organism. PBSIs were significantly more likely to be the recurrent episode and have endotracheal tube in place. No significant difference was found between PBSIs and controls in terms of demographics and most chronic conditions. PBSIs were significantly associated with a higher severity of illness, a longer duration of septic symptoms, and a higher rate of modification of antimicrobial regimens than monomicrobial BSIs. However, the sepsis-attributable mortality rates were comparable between these two groups.

Conclusions

In the NICU, PBSIs were more often caused by Gram-negative bacilli, and often occurred in neonates without any chronic conditions. The clinical significance of PBSIs included a more severe illness, longer duration of septic symptoms and a higher rate of modification of antimicrobial regimens.