Mechanical ventilation enhances Acinetobacter baumannii-induced lung injury through JNK pathways.

BACKGROUND: Patients in intensive care units (ICUs) often received broad-spectrum antibiotic treatment and Acinetobacter baumannii (A.b.) and Pseudomonas aeruginosa (P.a.) were the most common pathogens causing ventilator-associated pneumonia (VAP). This study aimed to examine the effects and mechanism of mechanical ventilation (MV) on A.b.-induced lung injury and the involvement of alveolar macrophages (AMs). METHODS: C57BL/6 wild-type (WT) and c-Jun N-terminal kinase knockout (JNK1-/-) mice received MV for 3 h at 2 days after nasal instillation of A.b., P.a. (1 × 106 colony-forming unit, CFU), or normal saline. RESULTS: Intranasal instillation of 106 CFU A.b. in C57BL/6 mice induced a significant increase in total cells and protein levels in the bronchoalveolar lavage fluid (BALF) and neutrophil infiltration in the lungs. MV after A.b. instillation increases neutrophil infiltration, interleukin (IL)-6 and vascular cell adhesion molecule (VCAM) mRNA expression in the lungs and total cells, IL-6 levels, and nitrite levels in the BALF. The killing activity of AMs against A.b. was lower than against P.a. The diminished killing activity was parallel with decreased tumor necrosis factor-α production by AMs compared with A.b. Inducible nitric oxide synthase inhibitor, S-methylisothiourea, decreased the total cell number in BALF on mice receiving A.b. instillation and ventilation. Moreover, MV decreased the A.b. and P.a. killing activity of AMs. MV after A.b. instillation induced less total cells in the BALF and nitrite production in the serum of JNK1-/- mice than those of WT mice. CONCLUSION: A.b. is potent in inducing neutrophil infiltration in the lungs and total protein in the BALF. MV enhances A.b.-induced lung injury through an increase in the expression of VCAM and IL-6 levels in the BALF and a decrease in the bacteria-killing activity of AMs. A lower inflammation level in JNK1-/- mice indicates that A.b.-induced VAP causes lung injury through JNK signaling pathway in the lungs.

Inhibition of nitric oxide production reverses diabetes-induced Kupffer cell activation and Klebsiella pneumonia liver translocation.

Klebsiella pneumoniae (KP) is the most common pathogen of pyogenic liver abscess in East and Southeast Asia and diabetes mellitus (DM) is a major risk factor. The effect and mechanism of diabetes on KP liver abscess was examined in streptozotocin-induced diabetic mice and Akita mice (C57BL/6J-Ins2Akita). KP translocation to liver and plasma alaine transaminase levels were increased and liver clearance of KP was decreased in DM mice. Diabetic mice exhibited overgrowth of Enterococcus as well as E.coli and decreased lactobacilli/bifidas growth in intestine, increased intestinal iNOS protein and nitrite levels in portal vein, and increased IL-1ß and TNF-α expression of Kupffer cells. Fructooligosaccharides (FOS) or dead L. salivarius (dLac) supplementation reversed diabetes-induced enteric dysbiosis, NO levels in portal vein, and KP translocation to liver. L-NAME treatment decreased intestinal iNOS protein expression as well as Kupffer cell activation and increased liver clearance of KP in DM mice. Dead E.coli (2×108 CFU/ml) feeding for one week induced iNOS and TLR4 expression of intestine in germ-free (GF) mice. Dead bacteria feeding induced IL-1ß and TNF-α expression of Kupffer cells in GF mice but not in GF TLR4-/- mice. In conclusion, balance of intestinal microflora is important for preventing intestinal iNOS expression, Kupffer cell activation, and KP liver translocation in diabetes. Reversal of diabetes-induced enteric dysbiosis with FOS or dead L. salivarius decreases diabetes-induced intestinal iNOS expression and KP liver translocation. Diabetes induces Kupffer cell activation and KP liver translocation through enteric dysbiosis and nitric oxide production.

Pseudomonas aeruginosa Ventilator-Associated Pneumonia Induces Lung Injury through TNF-α/c-Jun NH2-Terminal Kinase Pathways.

Ventilator-associated pneumonia (VAP) is a common nosocomial infection among intensive care unit (ICU) patients. Pseudomonas aeruginosa (PA) is the most common multidrug-resistant Gram-negative pathogen and VAP caused by PA carries a high rate of morbidity and mortality. This study examined the molecular mechanism of PA VAP-induced lung injury. C57BL/6 wild-type (WT) mice and JNK1 knockout (JNK1-/-) mice received mechanical ventilation (MV) for 3 h at 2 days after receiving nasal instillation of PA. The WT and JNK1-/- mice also received MV after the induction of lung injury by instillation of supernatants from PA-stimulated alveolar macrophages (AMs). AMs isolated from WT, IκB-kinase (IKK)ßΔMye (IKKß was selectively deleted in macrophages), and JNK1-/- mice were ex vivo stimulated with live PA and supernatants were collected for cytokine assay. Intranasal instillation of 106 PA enhanced MV-induced NF-κB DNA binding activity in the lungs and nitrite levels in BALF. MV after PA instillation significantly increased the expression of ICAM and VCAM in the lungs and TNF-α, IL-1ß, and IL-6 levels in bronchoalveolar lavage fluid (BALF) of WT mice, but not in JNK1-/- mice. MV after supernatant instillation induced more total protein concentration in BALF and neutrophil sequestration in the lungs in WT mice than JNK1-/- mice and cytokine assay of supernatants indicated that TNF-α is a critical regulator of PA VAP-induced lung injury. Ex vivo PA stimulation induced TNF-α production by AMs from WT as well as JNK1-/- mice but not IKKßΔMye mice. In summary, PA colonization plays an important role in PA VAP-induced lung injury through the induction of JNK1-mediated inflammation. These results suggest that the pathogenesis mechanism of PA VAP involves production of TNF-α through activation of IKK/NF-κB pathways in AMs and JNK signaling pathway in the lungs.

Pseudomonas aeruginosa colonization enhances ventilator-associated pneumonia-induced lung injury.

BACKGROUND: Pseudomonas aeruginosa (PA) is the single-most common pathogen of ventilator-associated pneumonia (VAP). Large quantities of PA in the trachea of ventilated patients are associated with an increased risk of death. However, the role of PA colonization in PA VAP-induced lung injury remains elusive. This study examined the effect and mechanism of PA colonization in VAP-induced lung injury. METHODS: C57BL/6 wild-type (WT) and c-Jun N-terminal kinase knockout (JNK1(-/-)) mice received mechanical ventilation for 3 h at 2 days after receiving nasal instillation of PA (1 × 10(6) colony forming unit) or normal saline. RESULTS: Intranasal instillation of PA or mechanical ventilation induced the expression of interleukin-6 (IL-6) in the lungs. Phospho-JNK protein expression in the lungs was significantly increased in mice receiving mechanical ventilation after PA instillation as compared with those receiving ventilation alone. Mechanical ventilation after PA instillation significantly increased the expression of tumor necrosis factor-α (TNF-α), IL-1ß, and macrophage inflammatory protein-2 (MIP-2) proteins; neutrophil sequestration; and TNF-α, IL-1ß, and IL-6 levels in the lungs of WT mice, but not in JNK1(-/-) mice. CONCLUSION: PA colonization plays an important role in PA VAP-induced lung injury through the induction of JNK1-mediated inflammation. PA-induced VAP causes lung injury through JNK signaling pathway in the lungs. JNK inhibition in ICU patients with higher percentages of PA colonization may reduce VAP-induced lung injury and mortality.

AIBp regulates mitotic entry and mitotic spindle assembly by controlling activation of both Aurora-A and Plk1.

We previously reported that Aurora-A and the hNinein binding protein AIBp facilitate centrosomal structure maintenance and contribute to spindle formation. Here, we report that AIBp also interacts with Plk1, raising the possibility of functional similarity to Bora, which subsequently promotes Aurora-A-mediated Plk1 activation at Thr210 as well as Aurora-A activation at Thr288. In kinase assays, AIBp acts not only as a substrate but also as a positive regulator of both Aurora-A and Plk1. However, AIBp functions as a negative regulator to block phosphorylation of hNinein mediated by Aurora-A and Plk1. These findings suggest a novel AIBp-dependent regulatory machinery that controls mitotic entry. Additionally, knockdown of hNinein caused failure of AIBp to target the centrosome, whereas depletion of AIBp did not affect the localization of hNinein and microtubule nucleation. Notably, knockdown of AIBp in HeLa cells impaired both Aurora-A and Plk1 kinase, resulting in phenotypes with multiple spindle pole formation and chromosome misalignment. Our data show that depletion of AIBp results in the mis-localization of TACC3 and ch-TOG, but not CEP192 and CEP215, suggesting that loss of AIBp dominantly affects the Aurora-A substrate to cause mitotic aberrations. Collectively, our data demonstrate that AIBp contributes to mitotic entry and bipolar spindle assembly and may partially control localization, phosphorylation, and activation of both Aurora-A and Plk1 via hNinein during mitotic progression.

Differential roles of Bcl2L12 and its short variant in breast cancer lymph node metastasis.

Bcl2L12 plays a role in post-mitochondrial apoptosis through multiple mechanisms involving p53, αB-crystallin, caspase-3 and -7 in glioblastoma. Bcl2L12 is reported to be a good prognostic marker in breast cancer and correlated with ER and Bcl2 expression status. However, the mechanisms by which Bcl2L12 regulates apoptosis in breast cancer (BCa) remain unknown. Recent studies have shown that Bcl2L12 expression is a useful biomarker in other types of cancer. Thus, we examined whether Bcl2L12 and Bcl2L12A mRNA were associated with breast cancer progression or a specific subtype. In total, 106 paraffin-embedded, different stage breast cancer specimens were prepared and quantified for Bcl2L12 and Bcl2L12A expression by PCR. The correlation between Bcl2L12 and Bcl2L12A mRNA levels and clinicopathological characteristics was statistically analyzed. The results showed that Bcl2L12 and Bcl2L12A mRNA expression was not significantly different across the different stage, grade and TNM classification groups (P>0.005). Using linear regression, Bcl2L12 mRNA was associated with Bcl2L12A mRNA, grade 3 tumor and the triple-negative breast cancer (TNBC) subtype. In non-TNBC specimens, Bcl2L12 mRNA was only correlated with Bcl2L12A mRNA. Bcl2L12A mRNA was positively associated with Bcl2L12 mRNA and the number of lymph node metastases, but negatively correlated with staging in the non-TNBC group. Specifically, Bcl2L12, but not Bcl2L12A, mRNA was significantly higher in TNBC and grade 3 tumors, respectively. In non-TNBC, Bcl2L12A mRNA was significantly highly expressed in tumors with ≥ 12 metastatic lymph nodes. Bcl2L12 and its variant mRNA were highly expressed in carcinoma in situ (CIS) samples. In addition, they were estimated to be correlated with the total sample and non-TNBC, but not the TNBC group. In summary, a high Bcl2L12 mRNA expression was associated with the high-grade BCa and TNBC subtype. In addition, the interplay between Bcl2L12 and its variant may be associated with high lymph node metastasis in non-TNBC tumors.

GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

GSK3ß binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3ß mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3ß binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3ß but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3ß are required in this novel PKA/GSKIP/GSK3ß/Drp1 complex. Moreover, overexpressed kinase-dead GSK3ß-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3ß recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3ß function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation.

NF-κB activation in myeloid cells mediates ventilator-induced lung injury.

BACKGROUND: Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated. METHODS: To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKß(Δmye)), IL-6(-/-) to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI. RESULTS: Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1ß, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKß(Δmye) mice as well as in IL6(-/-) to WT chimeric mice. CONCLUSION: Given that IKKß(Δmye) mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB-IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.

Blocking TNF-α enhances Pseudomonas aeruginosa-induced mortality in burn mice through induction of IL-1ß.

PURPOSE: Tumor necrosis factor (TNFα) is a proinflammatory cytokine and has been a target for intervention in human sepsis. However, inhibition of TNF-α with a high dose of a TNF-receptor fusion protein in patients with septic shock worsened patient survival. This study was designed to investigate whether blocking TNF-α enhances mortality in infected burn mice through the induction of IL-1ß. METHODS: WT or Tnfrsf1a(-/-) mice received Pseudomonas aeruginosa injection in the back at 8h after burn injury. The animals were sacrificed at 24h after burn and lung tissues were harvested and examined for determining myeloperoxidase (MPO) activity, pulmonary microvascular dysfunction, NF-κB DNA binding activity, and IL-1ß expression. Also, the lung and blood were harvested for bacterial count assay. RESULT: Thermal injury alone induced NF-κB DNA binding activity and neutrophil infiltration in the lung in WT but not in Tnfrsf1a(-/-) mice. A 50% total body surface area (TBSA) burn induced a significant increase of mortality in WT compared with Tnfrsf1a(-/-) mice. In contrast, P. aeruginosa injection with a 30% TBSA burn pretreatment enhanced IL-1ß expression, bacterial counts in lung and blood, pulmonary microvascular dysfunction, and mortality in Tnfrsf1a(-/-) mice compared with WT mice. Injection of the IL-1 receptor antagonist, Anakinra, reduced P. aeruginosa infection with burn pretreatment-induced blood bacterial counts, IL-1ß levels as well as permeability of lung, and mortality in Tnfrsf1a(-/-) mice. CONCLUSIONS: Our findings suggest that thermal injury induces lung NF-κB activation and neutrophil sequestration through TNFα signaling. However, blocking TNF-α enhances P. aeruginosa infection-induced lung damage in burn mice via induction of IL-1ß. Using an IL-1 receptor antagonist combined with the neutralization of TNF-α could be a useful strategy for decreasing P. aeruginosa infection-induced mortality in burn patients.

TNF-alpha decreases infection-induced lung injury in burn through negative regulation of TLR4/iNOS.

BACKGROUND: Sepsis is an infectious process-induced generalized inflammatory response that mediates the excessive production of cytokines. However, anti-tumor necrosis factor (TNF)-α therapy has failed in decreasing mortality of sepsis patients due to undefined mechanisms. This study was designed to investigate whether absence of TNF receptor enhanced lung damage and mortality through toll-like receptors (TLRs) and inducible nitric oxide synthase (iNOS). MATERIALS AND METHODS: We injected Pseudomonas aeruginosa or lipopolysaccharide in the backs of wild-type, Tnfrsf1a(-/-) (deficient of TNF-α receptor 1), and TLR4(-/-) mice at 8 h after 30% total body surface area burn. The animals were sacrificed at 16 h after burn and lung tissues were harvested and examined for determining pulmonary microvascular dysfunction and interleukin (IL)-1ß, iNOS, and TLR4 expression. The blood of animals was harvested for bacterial count assay. The effect of S-methylisothiourea, an iNOS inhibitor, on P aeruginosa infection with thermal injury pretreatment-induced lung damage was also examined. RESULTS: P aeruginosa or lipopolysaccharide injection with thermal injury pretreatment enhanced TLR4, iNOS, and IL-1ß expression and pulmonary microvascular dysfunction in Tnfrsf1a(-/-) mice compared with wild-type mice. P aeruginosa infection with thermal injury pretreatment did not induce IL-1ß or iNOS expression and mortality in TLR4(-/-) mice. S-methylisothiourea treatment significantly decreased P aeruginosa infection with thermal injury pretreatment-induced lung injury, blood bacterial counts, pulmonary IL-1ß expression, and mortality in Tnfrsf1a(-/-) mice. CONCLUSIONS: Given that absence of the TNF-α receptor 1 is associated with increased lung permeability, we conclude that TNF-α decreases P aeruginosa infection-induced lung damage in burn mice through negative regulation of TLR4 as well as iNOS expression, and iNOS inhibitor might be useful in reversing anti-TNF-α therapy-induced lung injury in burn.

PTEN overexpression attenuates angiogenic processes of endothelial cells by blockade of endothelin-1/endothelin B receptor signaling.

Arteriovenous (AV) graft is frequently used as vascular access in hemodialysis patients. However, clotting or thrombosis of AV grafts often occurs and requires surgical removal. At present, the molecular pathogenesis underlying thrombosis of AV graft is not clear. The PTEN/Akt signaling has been implicated in the pathogenesis of vascular diseases. In this study, elevated PTEN expression and concomitant Akt inactivation was observed in endothelium of atherosclerotic brachial arteries from hemodialysis patients. To investigate whether PTEN upregulation affects endothelial function, adenovirus-mediated PTEN (Ad-PTEN) overexpression was performed in aorta rings and cultured endothelial cells. It was found that PTEN overexpression potently inhibited the microvessel sprouting in aorta rings and the angiogenic activities of endothelial cells including migration and tube formation. On the contrary, PTEN knockdown by RNA interference promoted the endothelial migration and reversed the Ad-PTEN-induced inhibition of endothelial migration. Expression analysis showed that PTEN overexpression attenuated the expression of endothelin-1 (ET-1) and endothelin B receptor (ETBR) in endothelial cells at transcriptional levels. However, exogenous ET-1 supply only partially reversed the PTEN-induced inhibition of migration and tube formation. This was delineated due to that PTEN overexpression also perturbed endothelial nitric oxide synthase (eNOS) activation and vascular endothelial growth factor (VEGF) release. In summary, PTEN upregulation induces endothelial dysfunction by attenuating the availability and signaling of multiple angiogenic pathways in endothelial cells, thereby may contribute to thrombosis of AV graft.

GSK3ß regulates Bcl2L12 and Bcl2L12A anti-apoptosis signaling in glioblastoma and is inhibited by LiCl.

BCL2L12 has been reported to be involved in post-mitochondrial apoptotic events in glioblastoma, but the role of BCL2L12A, a splicing variant of BCL2L12, remains unknown. In this study, we showed that BCL2L12 and BCL2L12A were overexpressed in glioblastoma multiforme (GBM). Large-scale yeast two-hybrid screening showed that BCL2L12 was a GSK3b binding partner in a testis cDNA library. Our data demonstrated that GSK3b interacts with BCL2L12 but not BCL2L12A, whose C terminus lacks a binding region. We found that a BCL2L12(153-191) fragment located outside of the C-terminal BH2 motif is responsible for GSK3b binding. In contrast, no interaction was detected between BCL2L12A and GSK3b. In vitro kinase and l-phosphatase assays showed that GSK3b phosphorylates BCL2L12 at S156, while this site is absent on BCL2L12A. Moreover, our data also showed that the BCL2L12(153-191) fragment directly interrupted GSK3bmediated Tau phosphorylation in a dose-dependent manner. Ectopic expression of GFP-fused BCL2L12 or BCL2L12A in U87MG cells leads to repression of apoptotic markers and protects against staurosporine (STS) insults, indicating an antiapoptotic role for both BCL2L12 and BCL2L12A. In contrast, no anti-apoptotic ability was seen in BCL2L12(S156A). When BCL2L12-expressing U87MG cells were co-administrated with STS and LiCl, cells underwent apoptosis. This effect could be reversed by LiCl. In short, we established a model to demonstrate that GSK3b interacts with and phosphorylates BCL2L12 and might also affect BCL2L12A to modulate the apoptosis signaling pathway in glioblastoma. These findings suggest that LiCl may be a prospective therapeutic agent against GBM.

Gut flora enhance bacterial clearance in lung through toll-like receptors 4.

BACKGROUND: The influence of the gut flora on lung inflammatory reaction against bacterial challenge remains undefined. This study was designed to investigate whether gut flora enhances lung defense against E.coli pneumonia through TLR4 signaling. METHODS: C3H/HeN (WT) mice and C3H/HeJ (TLR4 deficient) mice were treated with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in intestinal tract. At the end of 4th week, E.coli was injected to trachea to induce E.coli pneumonia. RESULTS: We found that commensal depletion by antibiotic pretreatment before E.coli pneumonia challenge induced a 30% decrease of MPO activity in the lung, a significant decrease of bacterial killing activity of alveolar macrophage, and bacterial counts in C3H/HeN mice but not in C3H/HeJ (TLR4 deficient) mice. LPS, a TLR4 ligand, supplementation during antibiotic pretreatment reversed these effects and decreased E.coli pneumonia-induced mortality in C3H/HeN mice. Furthermore, commensal depletion induced a suppression of NF-κB DNA binding activity and an increase of KC, MIP-2, IL-1ß expression in the lung in C3H/HeN mice but not in C3H/HeJ mice. CONCLUSIONS: Taken together with that commensal depletion increased E.coli pneumonia-induced mortality and LPS supplementation decreased it, we conclude that gut flora enhances bacterial clearance against E.coli pneumonia through TLR4.

Commensal microflora contribute to host defense against Escherichia coli pneumonia through Toll-like receptors.

The influence of the gut-lung axis on the lung immunity, although appreciated, remains undefined mechanically. This study was designed to investigate whether commensal microflora in gut increase host defense against subsequent pneumonia through toll-like receptor (TLR) signaling and if oral TLR4 ligand supplementation enhances lung defense against bacterial challenge. We found that commensal gut depletion by antibiotic pretreatment before Escherichia coli pneumonia challenge induced a 15-fold and a 3-fold increase in bacterial counts in blood and lung, respectively, and a 30% increase of mortality when compared with the E. coli group. Commensal depletion also induced a suppression of cytokines expression as well as nuclear factor κB activity in intestine. Furthermore, LPS supplementation during antibiotic pretreatment reversed these effects. Commensal depletion also decreased bacterial killing activity of alveolar macrophages and increased IL-6 as well as IL-1ß levels and keratinocyte-derived chemokine, macrophage inflammatory protein 2, and IL-1ß expression of lung, and LPS supplementation reversed them. In conclusion, commensal gut microflora in the intestinal tract appear to be critical in inducing TLR4 expression as well as nuclear factor κB activation of intestine and lung innate defense against E. coli pneumonia.

Differential expression of hedgehog signaling components and Snail/E-cadherin in human brain tumors.

The hedgehog (Hh) transcription factor Gli induces transformation of epithelial cells via induction of Snail, a repressor of E-cadherin. Epithelial-mesenchymal transition is also a determinant of the progression of tumorigenesis, following down-regulation of E-cadherin. However, the role of Hh signaling components and Snail/E-cadherin in brain tumors is not yet fully understood. We analyzed the expression of Hh signaling components and Snail/E-cadherin in 69 brain tumors by reverse transcription-polymerase chain reaction (RT-PCR). The data showed that overexpression of Smo (35/69), Ptch (50/69), Gli1 (56/69), Gli2 (29/69) and N-myc (39/69) might contribute to brain tumorigenesis. Our results also indicated that Snail and E-cadherin showed opposing expression in malignant tumors (high grade astrocytoma and metastasis). Snail and E-cadherin showed less correlation in benign brain tumors. We further investigated mutations of Gli2 and Snail by RT-PCR and direct sequencing. No mutation was observed on Gli2 but several sporadic mutations on Snail were found, including S96G, S111L, S111L/S119Y and one nonsense mutation at codon 158 (Y158*). An in vitro E-cadherin promoter assay showed that S96G, S111L, S111L/S119Y Snail mutants were decreased by 15, 25 and 50%, respectively, whereas Y158* was increased by 40% compared to wild-type. Furthermore, our data showed that wild-type Snail and S96G, S111L, S111L/S119Y translocated to the nucleus, while the Y158* mutant failed to translocate to the nucleus. Taken together, our results demonstrate that Hh signaling components, the expression and mutations of Snail and the expression of E-cadherin may play an important role in human brain tumorigenesis.

Functional characterization of AIBp, a novel Aurora-A binding protein in centrosome structure and spindle formation.

Aurora-A is involved in chromosome alignment, centrosome maturation, mitotic spindle assembly and regards to an oncogene. Aurora-A is also known to bind to several other proteins affecting its up-regulation or down-regulation and localization. However, how these different binding signals work together to regulate Aurora-A is not properly known. To explore more Aurora-A interacting proteins, the low-copy yeast two-hybrid screening using Aurora-A as bait protein was performed. One novel gene, AIBp, was demonstrated to associate with Aurora-A by the yeast two-hybrid method and in vitro GST pull-down assay. Molecular characterization showed that AIBp possessed a binding site at the C-terminal with Aurora-A (kinase domain). Interestingly, AIBp also interacts with hNinein at the N-terminal, which overlaps with a previously reported hNinein and GSK3beta binding site. Using a kinase assay, AIBp interacts with the Aurora-A kinase domain functions as a positive regulator, whereas AIBp binding to hNinein appears to block the phosphorylation of hNinein by both Aurora-A and GSK3beta. siRNA-mediated elimination of AIBp from HeLa cells, results in a doughnut-like shape, asymmetrical spindle pole and multiple spindle pole formation. We also demonstrated that both AIBp and Aurora-A are co-overexpressed in various brain tumors. These studies demonstrate that AIBp may not only be required for the dynamic movement of Aurora-A at the centrosomes and spindle apparatus during the cell cycle, but may also be important during brain tumorigenesis.

Commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4.

BACKGROUND: Major burn is associated with decreased gut barrier function and increased bacterial translocation (BT). This study is to investigate whether commensal microflora induce host defense and decrease BT in burn mice. METHODS: First, we treated Wild type (WT) mice with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in gut. The intestinal permeability, glutathione level, NF-kappaB DNA-binding activity, TLR4 expression of intestinal mucosa, BT to mesenteric lymph nodes (MLNs), and bacterial killing activity of peritoneal cells were measured after thermal injury. Second, lung of animals were harvested for MPO activity and TNFalpha mRNA expression assay. Third, WT animals were treated with oral antibiotics with or without LPS supplement after burn. At 48 hr after burn, TLR4 expression of intestinal mucosa and bacterial killing activity of cells were examined. Finally, bacterial killing activity and BT to MLNs after thermal injury in C3H/HeJ (TLR4 mutant) mice were measured. RESULTS: Burn induced BT to MLNs in WT mice. Commensal depletion decreased TLR4 expression as well as NF-kappaB activation of intestine, myeloperoxidase (MPO) activity as well as TNFalpha expression of lung, and bacterial killing activity of peritoneal cells. Oral LPS supplement markedly reduced 81% of burn-induced BT and increased TLR4 expression, MPO activity of lung, as well as bacterial killing activity of peritoneal cells. LPS supplement did not change BT or bacterial killing activity in C3H/HeJ mice. CONCLUSIONS: Collectively, commensal microflora induce TLR4 expression of intestine and bacterial killing activity of inflammatory cells in burn. TLR4 ligand increases bacterial killing activity and decreases burn-induced BT. Taken together with the abolition of LPS effect in TLR4 mutant mice, we conclude that commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4.

Peritonitis-induced peroxynitrite and lung damage depends on c-Jun NH2-terminal kinase signaling of hematopoietic cells.

OBJECTIVES: Abdominal sepsis is a common, life-threatening condition in critically ill patients, and pseudomonas peritonitis remains a serious clinical complication of peritoneal dialysis. This study was performed to determine whether peritonitis induces lung damage through the c-Jun NH2-terminal kinase. DESIGN: : Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Peritonitis models in the mice. INTERVENTIONS: Wild-type, c-Jun NH2-terminal kinase1, and c-Jun NH2-terminal kinase1 mice were subjected to peritonitis. A c-Jun NH2-terminal kinase inhibitor, SP600125 or leflunomide, was administered to mice immediately after peritonitis. MEASUREMENTS AND MAIN RESULTS: The changes of plasma dihydrorhodamine 123 oxidation level, the myeloperoxidase activity, and extravasations of Evans blue dye of lung in wild-type mice with or without c-Jun NH2-terminal kinase inhibitor; c-Jun NH2-terminal kinase1 mice and c-Jun NH2-terminal kinase1 mice; and chimeric mice (wild-type --> wild-type, c-Jun NH2-terminal kinase1 --> wild-type) with Pseudomonas aeruginosa-induced peritonitis were determined to evaluate the role of c-Jun NH2-terminal kinase signaling of the hematopoietic cells in peritonitis-induced lung damage. Our results showed that peritonitis induced dihydrorhodamine 123 oxidation, myeloperoxidase activity, activator protein-1 (AP-1) DNA binding activity, phosphorylated-c-Jun NH2-terminal kinase and inducible nitric oxide synthase expression, and Evans blue dye extravasations in lungs, and administration of specific c-Jun NH2-terminal kinase inhibitor decreased the peritonitis-induced dihydrorhodamine 123 oxidation and lung damage. Also, both c-Jun NH2-terminal kinase1 and c-Jun NH2-terminal kinase1 mice showed a decreased dihydrorhodamine 123 oxidation and lung damage after peritonitis. Finally, dihydrorhodamine 123 oxidation, reactive oxygen species, inducible nitric oxide synthase expression, and lung damage were decreased in c-Jun NH2-terminal kinase1 --> wild-type but not in wild-type --> c-Jun NH2-terminal kinase1 chimeric mice. CONCLUSIONS: Collectively, our data suggest that peritonitis-induced inducible nitric oxide synthase expression, peroxynitrite production, and lung damage depend on the c-Jun NH2-terminal kinase signaling of the hematopoietic cells.

GSKIP, an inhibitor of GSK3beta, mediates the N-cadherin/beta-catenin pool in the differentiation of SH-SY5Y cells.

Emerging evidence has shown that GSK3beta plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3beta interaction protein (GSKIP) able to negatively regulate GSK3beta in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH-SY5Y cells treated with retinoic acid (RA) to differentiate to neuron-like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH-SY5Y cells. GSKIP may affect GSK3beta activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases beta-catenin in the nucleus and raises the level of cyclin D1 to promote cell-cycle progression in SH-SY5Y cells. Additionally, overexpression of GSKIP downregulates N-cadherin expression, resulting in decreased recruitment of beta-catenin. Moreover, depletion of beta-catenin by small interfering RNA, neurite outgrowth is blocked in SH-SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3beta/beta-catenin, beta-catenin/cyclin D1, and beta-catenin/N-cadherin pool during RA signaling in SH-SY5Y cells.

Nod2 mutation enhances NF-kappa B activity and bacterial killing activity of macrophages.

NOD2, an intracellular sensor of bacteria, are linked to increased susceptibility to bacteria in Crohn's disease (CD). The NOD2 protein is expressed mainly by macrophages and dendritic cells. This study is to examine the role of NOD2 in the innate response of macrophages to bacterial challenge. First, peritoneal macrophages and alveolar macrophages were harvested from WT, Nod2(2939iC), as well as TLR4(-/-) mice and incubated with E. coli or P. aeruginosa. Bacterial killing activity; IL-1beta and TLR4 protein expression; NF-kappaB DNA binding activity assay; as well as IL-1beta, TNFalpha, TLR2, TLR4 and TLR9 mRNA expression of macrophages were examined. We found that alveolar macrophages and peritoneal macrophages of Nod2(2939iC) mice but not WT mice or TLR4(-/-) mice demonstrated a significant increase of E. coli killing activity. Bacterial challenge also induced a significant increase of pro-IL-1beta protein expression; NF-kappaB DNA binding activity; as well as IL-1beta and TNFalpha mRNA expression of the peritoneal macrophages in Nod2(2939iC) mice. Collectively, the increase of bacterial killing activity, IL-1beta expression, and NF-kappaB DNA binding activity of macrophages in Nod2(2939iC) mice suggests that NOD2 is a positive regulator of NF-kappaB/IL-1beta-mediated innate response to bacteria challenge in Crohn's disease.