Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein.

Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.

Single-nucleotide polymorphisms in CCL2 gene are not associated with susceptibility to systemic sclerosis.

OBJECTIVE: To validate the reported association between CC chemokine ligand 2 (CCL2) -2518 G single nucleotide polymorphism and systemic sclerosis (SSc) in a much larger cohort of patients. We also performed subgroup analysis to test the hypothesis that CCL2 variants predispose to specific disease phenotypes. METHODS: Ninety-four Caucasian patients with SSc and 102 matched controls were genotyped by sequence-specific primers-polymerase chain reaction (SSP-PCR) methodology. RESULTS: Six biallelic single-nucleotide polymorphisms (SNP) were investigated (3 in the promoter region, 2 in the exon-coding sequence, and 1 in the 3x untranslated region), in addition to the known functional -2518 (A/G) variant. Six major haplotypes were constructed across all 7 SNP positions. No significant differences in genotype, allele, or haplotype frequency were observed between patients and controls or within disease subgroups. CONCLUSION: Genetic polymorphisms within CCL2 gene are associated with susceptibility neither to SSc nor to specific disease phenotypes.

Epithelial origin of myofibroblasts during fibrosis in the lung.

An understanding of the mechanisms underlying pulmonary fibrosis remains elusive. Once believed to result primarily from chronic inflammation, it is now clear that inflammation and chronic fibrosis, especially in diseases such as idiopathic pulmonary fibrosis/usual interstitial pneumonia, are often dissociated, and that inflammation is neither necessary nor sufficient to induce fibrosis. The origin of the primary effector cell of fibrosis in the lung, the myofibroblast, is not clearly established. Three potential sources have been hypothesized. Although conversion of resident fibroblasts and differentiation of circulating bone marrow-derived progenitors likely play a role, the possible contribution of alveolar epithelial cells (AECs), through a process termed "epithelial-mesenchymal transition" (EMT), has only recently received consideration. A process by which epithelial cells lose cell-cell attachment, polarity and epithelial-specific markers, undergo cytoskeletal remodeling, and gain a mesenchymal phenotype, EMT plays a prominent role in fibrogenesis in adult tissues such as the kidney. This review summarizes the evidence supporting a central role for EMT in the pathogenesis of lung fibrosis, the potential for EMT in AECs in vitro and in vivo and role of transforming growth factor-beta1 in this process, and the implications of epithelium-driven fibrosis on future research and treatment. Potential pathways involved in EMT are also discussed. It is hoped that a major shift in current paradigms regarding the genesis of pulmonary fibrosis and dissection of the relevant pathways may allow development of targeted interventions that could potentially reverse the process and ameliorate the debilitating effects of abnormal repair and progressive fibrosis.

Nuclear factor-kappaB activation in alveolar macrophages requires IkappaB kinase-beta, but not nuclear factor-kappaB inducing kinase.

Cytokine mediated activation of alveolar macrophages (AMs) is an important event in the pathogenesis of fibrosing alveolitis (FA). Through membrane-associated antigens, cytokines (e.g., tumor necrosis-factor-alpha and interleukin-1) are believed to activate a common kinase cascade that initiates the cytoplasmic degradation of IkappaB and nuclear translocation of "nuclear factor-kappaB" (NF-kappaB). In the nucleus, NF-kappaB promotes the transcription of genes encoding chemokines and cytokines involved in chronic inflammation. Preventing cytokine-mediated NF-kappaB activation is a potential strategy for attenuating the lung injury that occurs in FA. Previously, we have demonstrated that, unlike AMs from healthy volunteers, AMs from patients with inflammatory lung diseases express the coxsackie/adenovirus receptor and the alphav integrins required for adenovirus (Adv) infection. This property allows Adv-mediated transgene delivery to diseased, but not normal, AMs and analysis of molecular pathways involved in gene transcription. In this study, AMs were infected with Adv constructs expressing a defective beta subunit of IkappaB kinase (AdvIKKbetakd) and a defective NF-kappaB inducing kinase (AdvNIKkd) to investigate the contribution of these molecules to NF-kappaB activation. We observed that IKKbeta, but not NIK, was required for NF-kappaB activation. The results of this study identify IKKbeta, but not NIK, as a potential therapeutic target in diseases that involve NF-kappaB-dependent gene transcription.