Epithelial NF-κB signaling promotes EGFR-driven lung carcinogenesis via macrophage recruitment.

Several studies have demonstrated that NF-κB activation is common in lung cancer; however, the mechanistic links between NF-κB signaling and tumorigenesis remain to be fully elucidated. We investigated the function of NF-κB signaling in epidermal growth factor receptor (EGFR)-mutant lung tumors using a transgenic mouse model with doxycycline (dox)-inducible expression of oncogenic EGFR in the lung epithelium with or without a dominant inhibitor of NF-κB signaling. NF-κB inhibition resulted in a significant reduction in tumor burden in both EGFR tyrosine kinase inhibitor (TKI)-sensitive and resistant tumors. However, NF-κB inhibition did not alter epithelial cell survival in vitro or in vivo, and no changes were detected in activation of EGFR downstream signaling pathways. Instead, we observed an influx of inflammatory cells (macrophages and neutrophils) in the lungs of mice with oncogenic EGFR expression that was blocked in the setting of NF-κB inhibition. To investigate whether inflammatory cells play a role in promoting EGFR-mutant lung tumors, we depleted macrophages and neutrophils during tumorigenesis and found that neutrophil depletion had no effect on tumor formation, but macrophage depletion caused a significant reduction in tumor burden. Together, these data suggest that epithelial NF-κB signaling supports carcinogenesis in a non-cell autonomous manner in EGFR-mutant tumors through recruitment of pro-tumorigenic macrophages.

Neutrophil-Derived IL-1ß Impairs the Efficacy of NF-κB Inhibitors against Lung Cancer.

Although epithelial NF-κB signaling is important for lung carcinogenesis, NF-κB inhibitors are ineffective for cancer treatment. To explain this paradox, we studied mice with genetic deletion of IKKß in myeloid cells and found enhanced tumorigenesis in Kras(G12D) and urethane models of lung cancer. Myeloid-specific inhibition of NF-κB augmented pro-IL-1ß processing by cathepsin G in neutrophils, leading to increased IL-1ß and enhanced epithelial cell proliferation. Combined treatment with bortezomib, a proteasome inhibitor that blocks NF-κB activation, and IL-1 receptor antagonist reduced tumor formation and growth in vivo. In lung cancer patients, plasma IL-1ß levels correlated with poor prognosis, and IL-1ß increased following bortezomib treatment. Together, our studies elucidate an important role for neutrophils and IL-1ß in lung carcinogenesis and resistance to NF-κB inhibitors.

Extensive phenotyping of individuals at risk for familial interstitial pneumonia reveals clues to the pathogenesis of interstitial lung disease.

RATIONALE: Asymptomatic relatives of patients with familial interstitial pneumonia (FIP), the inherited form of idiopathic interstitial pneumonia, carry increased risk for developing interstitial lung disease. OBJECTIVES: Studying these at-risk individuals provides a unique opportunity to investigate early stages of FIP pathogenesis and develop predictive models of disease onset. METHODS: Seventy-five asymptomatic first-degree relatives of FIP patients (mean age, 50.8 yr) underwent blood sampling and high-resolution chest computed tomography (HRCT) scanning in an ongoing cohort study; 72 consented to bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsies. Twenty-seven healthy individuals were used as control subjects. MEASUREMENTS AND MAIN RESULTS: Eleven of 75 at-risk subjects (14%) had evidence of interstitial changes by HRCT, whereas 35.2% had abnormalities on transbronchial biopsies. No differences were noted in inflammatory cells in BAL between at-risk individuals and control subjects. At-risk subjects had increased herpesvirus DNA in cell-free BAL and evidence of herpesvirus antigen expression in alveolar epithelial cells (AECs), which correlated with expression of endoplasmic reticulum stress markers in AECs. Peripheral blood mononuclear cell and AEC telomere length were shorter in at-risk individuals than healthy control subjects. The minor allele frequency of the Muc5B rs35705950 promoter polymorphism was increased in at-risk subjects. Levels of several plasma biomarkers differed between at-risk subjects and control subjects, and correlated with abnormal HRCT scans. CONCLUSIONS: Evidence of lung parenchymal remodeling and epithelial dysfunction was identified in asymptomatic individuals at risk for FIP. Together, these findings offer new insights into the early pathogenesis of idiopathic interstitial pneumonia and provide an ongoing opportunity to characterize presymptomatic abnormalities that predict progression to clinical disease.

ß-catenin in the alveolar epithelium protects from lung fibrosis after intratracheal bleomycin.

RATIONALE: Alveolar epithelial cells (AECs) play central roles in the response to lung injury and the pathogenesis of pulmonary fibrosis. OBJECTIVES: We aimed to determine the role of ß-catenin in alveolar epithelium during bleomycin-induced lung fibrosis. METHODS: Genetically modified mice were developed to selectively delete ß-catenin in AECs and were crossed to cell fate reporter mice that express ß-galactosidase (ßgal) in cells of AEC lineage. Mice were given intratracheal bleomycin (0.04 units) and assessed for AEC death, inflammation, lung injury, and fibrotic remodeling. Mouse lung epithelial cells (MLE12) with small interfering RNA knockdown of ß-catenin underwent evaluation for wound closure, proliferation, and bleomycin-induced cytotoxicity. MEASUREMENTS AND MAIN RESULTS: Increased ß-catenin expression was noted in lung parenchyma after bleomycin. Mice with selective deletion of ß-catenin in AECs had greater AEC death at 1 week after bleomycin, followed by increased numbers of fibroblasts and enhanced lung fibrosis as determined by semiquantitative histological scoring and total collagen content. However, no differences in lung inflammation or protein levels in bronchoalveolar lavage were noted. In vitro, ß-catenin-deficient AECs showed increased bleomycin-induced cytotoxicity as well as reduced proliferation and impaired wound closure. Consistent with these findings, mice with AEC ß-catenin deficiency showed delayed recovery after bleomycin. CONCLUSIONS: ß-Catenin in the alveolar epithelium protects against bleomycin-induced fibrosis. Our studies suggest that AEC survival and wound healing are enhanced through ß-catenin-dependent mechanisms. Activation of the developmentally important ß-catenin pathway in AECs appears to contribute to epithelial repair after epithelial injury.

Opposing effects of bortezomib-induced nuclear factor-κB inhibition on chemical lung carcinogenesis.

Since recent evidence indicates a requirement for epithelial nuclear factor (NF)-κB signaling in lung tumorigenesis, we investigated the impact of the NF-κB inhibitor bortezomib on lung tumor promotion and growth. We used an experimental model in which wild-type mice or mice expressing an NF-κB reporter received intraperitoneal urethane (1 g/kg) followed by twice weekly bortezomib (1 mg/kg) during distinct periods of tumor initiation/progression. Mice were serially assessed for lung NF-κB activation, inflammation and carcinogenesis. Short-term proteasome inhibition with bortezomib did not impact tumor formation but retarded the growth of established lung tumors in mice via effects on cell proliferation. In contrast, long-term treatment with bortezomib resulted in significantly increased lung tumor number and size. This tumor-promoting effect of prolonged bortezomib treatment was associated with perpetuation of urethane-induced inflammation and chronic upregulation of interleukin-1ß and proinflammatory C-X-C motif chemokine ligands (CXCL) 1 and 2 in the lungs. In addition to airway epithelium, bortezomib inhibited NF-κB in pulmonary macrophages in vivo, presenting a possible mechanism of tumor amplification. In this regard, RAW264.7 macrophages exposed to bortezomib showed increased expression of interleukin-1ß, CXCL1 and CXCL2. In conclusion, although short-term bortezomib may exert some beneficial effects, prolonged NF-κB inhibition accelerates chemical lung carcinogenesis by perpetuating carcinogen-induced inflammation. Inhibition of NF-κB in pulmonary macrophages appears to play an important role in this adverse process.

Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress.

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, ß-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs.

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection.

We investigated whether nuclear factor kappa B (NF-kappaB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re-modeling. We generated a doxycycline inducible transgenic mouse model to activate NF-kappaB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF-kappaB signaling cascade. We found that activation of NF-kappaB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase-3, an indicator of apoptosis. We also found that activation of NF-kappaB during lactation with no additional involution signals had a similar effect. The observation that NF-kappaB is a key regulator of milk production led us to investigate the role of NF-kappaB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF-kappaB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF-kappaB in mammary epithelium. Together, our data reveal that activation of NF-kappaB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF-kappaB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact.

Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection.

Recent evidence suggests that dysfunctional type II alveolar epithelial cells (AECs) contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Based on the hypothesis that disease-causing mutations in surfactant protein C (SFTPC) provide an important paradigm for studying IPF, we investigated a potential mechanism of AEC dysfunction suggested to result from mutant SFTPC expression: induction of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We evaluated biopsies from 23 IPF patients (including 3 family members with L188Q SFTPC mutations, 10 individuals with familial interstitial pneumonia without SFTPC mutations, and 10 individuals with sporadic IPF) and sections from 10 control lungs. After demonstrating UPR activation in cultured A549 cells expressing mutant SFTPC, we identified prominent expression of UPR markers in AECs in the lungs of patients with SFTPC mutation-associated fibrosis. In individuals with familial interstitial pneumonia without SFTPC mutations and patients with sporadic IPF, we also found UPR activation selectively in AECs lining areas of fibrotic remodeling. Because herpesviruses are found frequently in IPF lungs and can induce ER stress, we investigated expression of viral proteins in lung biopsies. Herpesvirus protein expression was found in AECs from 15/23 IPF patients and colocalized with UPR markers in AECs from these patients. ER stress and UPR activation are found in the alveolar epithelium in patients with IPF and could contribute to disease progression. Activation of these pathways may result from altered surfactant protein processing or chronic herpesvirus infection.

Epithelial NF-kappaB activation promotes urethane-induced lung carcinogenesis.

Chronic inflammation is linked to carcinogenesis in several organ systems. In the lungs, NF-kappaB, a central effector of inflammatory responses, is frequently activated in non-small-cell lung cancer, but its role in tumor promotion has not been studied. Several lines of evidence indicate that ethyl carbamate (urethane)-induced lung tumor formation, a prototypical mouse model of multistage lung carcinogenesis, is potentiated by inflammation. We found that mouse strains susceptible to lung tumor formation (FVB, BALB/c) exhibited early NF-kappaB activation and inflammation in the lungs after urethane treatment. However, a resistant strain (C57B6) failed to activate NF-kappaB or induce lung inflammation. In FVB mice, we identified urethane-induced NF-kappaB activation in airway epithelium, as well as type II alveolar epithelial cells and macrophages. Using an inducible transgenic mouse model (FVB strain) to express a dominant inhibitor of NF-kappaB specifically in airway epithelial cells, we found that urethane-induced lung inflammation was blocked and tumor formation was reduced by >50%. Selective NF-kappaB inhibition resulted in increased apoptosis of airway epithelial cells at 2 weeks after urethane treatment in association with a marked reduction of Bcl-2 expression. These studies indicate that NF-kappaB signaling in airway epithelium is integral to tumorigenesis in the urethane model and identify the NF-kappaB pathway as a potential target for chemoprevention of lung cancer.

Targeted immunomodulation of the NF-kappaB pathway in airway epithelium impacts host defense against Pseudomonas aeruginosa.

We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-kappaB activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-kappaB activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-kappaB before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-kappaB activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-kappaB. The TNF-alpha concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-kappaB up-regulation were abrogated in TNF-alpha-deficient mice. In contrast, NF-kappaB inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-kappaB pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.

Activation of proteinase-activated receptor-2 in mesothelial cells induces pleural inflammation.

Pleural inflammation underlies many pleural diseases, but its pathogenesis remains unclear. Proteinase-activated receptor-2 (PAR(2)) is a novel seven-transmembrane receptor with immunoregulatory roles. We hypothesized that PAR(2) is present on mesothelial cells and can induce pleural inflammation. PAR(2) was detected by immunohistochemistry in all (19 parietal and 11 visceral) human pleural biopsies examined. In cultured murine mesothelial cells, a specific PAR(2)-activating peptide (SLIGRL-NH(2)) at 10, 100, and 1,000 muM stimulated a 3-, 42-, and 1,330-fold increase of macrophage inflammatory protein (MIP)-2 release relative to medium control, respectively (P < 0.05 all) and a 2-, 32-, and 75-fold rise over the control peptide (LSIGRL-NH(2), P < 0.05 all). A similar pattern was seen for TNF-alpha release. Known physiological activators of PAR(2), tryptase, trypsin, and coagulation factor Xa, also stimulated dose-dependent MIP-2 release from mesothelial cells in vitro. Dexamethasone inhibited the PAR(2)-mediated MIP-2 release in a dose-dependent manner. In vivo, pleural fluid MIP-2 levels in C57BL/6 mice injected intrapleurally with SLIGRL-NH(2) (10 mg/kg) were significantly higher than in mice injected with LSIGRL-NH(2) or PBS (2,710 +/- 165 vs. 880 +/- 357 vs. 88 +/- 46 pg/ml, respectively; P < 0.001). Pleural fluid neutrophil counts were higher in SLIGRL-NH(2) group than in the LSIGRL-NH(2) and PBS groups (by 40- and 26-fold, respectively; P < 0.05). This study establishes that activation of mesothelial cell PAR(2) potently induces the release of inflammatory cytokines in vitro and neutrophil recruitment into the pleural cavity in vivo.

p47phox deficiency impairs NF-kappa B activation and host defense in Pseudomonas pneumonia.

We examined the role of redox signaling generated by NADPH oxidase in activation of NF-kappaB and host defense against Pseudomonas aeruginosa pneumonia. Using mice with an NF-kappaB-driven luciferase reporter construct (HIV-LTR/luciferase (HLL)), we found that intratracheal administration of P. aeruginosa resulted in a dose-dependent neutrophilic influx and activation of NF-kappaB. To determine the effects of reactive oxygen species generated by the NADPH oxidase system on activation of NF-kappaB, we crossbred mice deficient in p47(phox) with NF-kappaB reporter mice (p47(phox-/-)HLL). These p47(phox-/-)HLL mice were unable to activate NF-kappaB to the same degree as HLL mice with intact NADPH oxidase following P. aeruginosa infection. In addition, lung TNF-alpha levels were significantly lower in p47(phox-/-)HLL mice compared with HLL mice. Bacterial clearance was impaired in p47(phox-/-)HLL mice. In vitro studies using bone marrow-derived macrophages showed that Toll-like receptor 4 was necessary for NF-kappaB activation following treatment with P. aeruginosa. Additional studies with macrophages from p47(phox-/-) mice confirmed that redox signaling was necessary for maximal Toll-like receptor 4-dependent NF-kappaB activation in this model. These data indicate that the NADPH oxidase-dependent respiratory burst stimulated by Pseudomonas infection contributes to host defense by modulating redox-dependent signaling through the NF-kappaB pathway.

Modulation of endotoxin-induced NF-kappa B activation in lung and liver through TNF type 1 and IL-1 receptors.

We investigated the requirement for tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 receptors in the pathogenesis of the pulmonary and hepatic responses to Escherichia coli lipopolysaccharide (LPS) by studying wild-type mice and mice deficient in TNF type 1 receptor [TNFR1 knockout (KO)] or both TNF type 1 and IL-1 receptors (TNFR1/IL-1R KO). In lung tissue, NF-kappaB activation was similar among the groups after exposure to aerosolized LPS. After intraperitoneal injection of LPS, NF-kappaB activation in liver was attenuated in TNFR1 KO mice and further diminished in TNFR1/IL-1R KO mice; however, in lung tissue, no impairment in NF-kappaB activation was found in TNFR1 KO mice and only a modest decrease was found in TNFR1/IL-1R KO mice. Lung concentrations of KC and macrophage-inflammatory peptide 2 were lower in TNFR1 KO and TNFR1/IL-1R KO mice after aerosolized and intraperitoneal LPS. We conclude that LPS-induced NF-kappaB activation in liver is mediated through TNF-alpha- and IL-1 receptor-dependent pathways, but, in the lung, LPS-induced NF-kappaB activation is largely independent of these receptors.

Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy.

FK506 binding proteins 12 and 12.6 (FKBP12 and FKBP12.6) are intracellular receptors for the immunosuppressant drug FK506 (ref. 1). The skeletal muscle ryanodine receptor (RyR1) is isolated as a hetero-oligomer with FKBP12 (ref. 2), whereas the cardiac ryanodine receptor (RyR2) more selectively associates with FKBP12.6 (refs 3, 4, 5). FKBP12 modulates Ca2+ release from the sarcoplasmic reticulum in skeletal muscle and developmental cardiac defects have been reported in FKBP12-deficient mice, but the role of FKBP12.6 in cardiac excitation-contraction coupling remains unclear. Here we show that disruption of the FKBP12.6 gene in mice results in cardiac hypertrophy in male mice, but not in females. Female hearts are normal, despite the fact that male and female knockout mice display similar dysregulation of Ca2+ release, seen as increases in the amplitude and duration of Ca2+ sparks and calcium-induced calcium release gain. Female FKBP12.6-null mice treated with tamoxifen, an oestrogen receptor antagonist, develop cardiac hypertrophy similar to that of male mice. We conclude that FKBP12.6 modulates cardiac excitation-contraction coupling and that oestrogen plays a protective role in the hypertrophic response of the heart to Ca2+ dysregulation.