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Tissue barrier function is directly mediated by tight junction transmembrane proteins known as claudins. Cells that form tight junctions typically express multiple claudin isoforms which suggests that heterotypic (head-to-head) binding between different claudin isoforms may play a role in regulating paracellular permeability. However, little is known about motifs that control heterotypic claudin compatibility. We found that although claudin-3 and claudin-4 were heteromerically compatible when expressed in the same cell, they did not heterotypically interact despite having extracellular loop (EL) domains that are highly conserved at the amino acid level. Claudin-1 and -5, which were heterotypically compatible with claudin-3, did not heterotypically bind to claudin-4. In contrast, claudin-4 chimeras containing either the first EL domain or the second EL domain of claudin-3 were able to heterotypically bind to claudin-1, claudin-3, and claudin-5. Moreover, a single point mutation in the first extracellular loop domain of claudin-3 to convert Asn 44 to the corresponding amino acid in claudin-4 (Thr) produced a claudin capable of heterotypic binding to claudin-4 while still retaining the ability to bind to claudin-1 and -5. Thus, control of heterotypic claudin-claudin interactions is sensitive to small changes in the EL domains.

Introduction Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease with an average survival of only 3 to 5 years. The mechanisms underlying the initiation and progression of IPF are poorly understood, and treatments available have only modest effect on disease progression. Interestingly, the incidence of IPF is approximately 60 times more common in individuals aged 75 years and older, but the mechanism by which aging promotes fibrosis is unclear. The authors hypothesized that aged lungs have a profibrotic phenotype that render it susceptible to disrepair after injury. Methods Young and old mice were treated with bleomycin to examine disrepair in the aged lung. In addition, uninjured young and old mouse lungs were analyzed for transforming growth factor-beta 1 (TGF-β1) production, extracellular matrix composition and lung fibroblast phenotype. Lung fibroblasts were treated with a DNA methyltransferase inhibitor to examine the potential epigenetic mechanisms involved in age-associated phenotypic alterations. Results The lungs of old mice showed worse fibrosis after bleomycin-induced injury compared with the lungs from young mice. At baseline, aged lungs expressed a profibrotic phenotype characterized by increased mRNA expression for fibronectin extracellular domain A (Fn-EDA) and the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Old lungs also expressed higher levels of TGF-β receptor 1 and TGF-β1 mRNA, protein and activity as determined by increased Smad3 expression, protein phosphorylation and DNA binding. Lung fibroblasts harvested from aged lungs showed reduced expression of the surface molecule Thy-1, a finding also implicated in lung fibrosis; the latter did not seem related to Thy-1 gene methylation. Conclusion Altogether, aged lungs manifest a profibrotic phenotype characterized by enhanced fibronectin extracellular domain A and MMP expression and increased TGF-β1 expression and signaling and are populated by Thy-1–negative fibroblasts, all implicated in the pathogenesis of lung fibrosis.

Although it is well recognized that alcohol abuse impairs alveolar macrophage immune function and renders patients susceptible to pneumonia, the mechanisms are incompletely understood. Alveolar macrophage maturation and function requires priming by GM-CSF, which is produced and secreted into the alveolar space by the alveolar epithelium. In this study, we determined that although chronic ethanol ingestion (6 wk) in rats had no effect on GM-CSF expression within the alveolar space, it significantly decreased membrane expression of the GM-CSF receptor in alveolar macrophages. In parallel, ethanol ingestion decreased cellular expression and nuclear binding of PU.1, the master transcription factor that activates GM-CSF-dependent macrophage functions. Furthermore, treatment of ethanol-fed rats in vivo with rGM-CSF via the upper airway restored GM-CSF receptor membrane expression as well as PU.1 protein expression and nuclear binding in alveolar macrophages. Importantly, GM-CSF treatment also restored alveolar macrophage function in ethanol-fed rats, as reflected by endotoxin-stimulated release of TNF-α and bacterial phagocytosis. We conclude that ethanol ingestion dampens alveolar macrophage immune function by decreasing GM-CSF receptor expression and downstream PU.1 nuclear binding and that these chronic defects can be reversed relatively quickly with rGM-CSF treatment in vivo.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disorder of unknown cause. Several studies suggest an association between Epstein-Barr virus pulmonary infection and the development of IPF. Objectives: To determine whether reduction of ␥-herpesvirus reactivation from latency would alter progressive lung fibrogenesis in an animal model of virus-induced pulmonary fibrosis. Methods: IFN-␥ receptor-deficient (IFN-␥R؊/؊ ) mice infected intranasally with murine ␥-herpesvirus 68 (MHV68) develop lung fibrosis that progresses for up to at least 180 days after initial infection. Viral replication during the chronic phase of infection was controlled by two methods: the administration of cidofovir, an antiviral drug effective at clearing lytic but not latent virus, and by using a mutant ␥-herpesvirus defective in virus reactivation from latency. Measurements and Main Results: Ten percent of the asymptomatic MHV68-infected animals that received antiviral treatment beginning on Day 45 postinfection had severe pulmonary fibrosis compared with 40% of the control saline-treated animals. Absence of severe fibrosis was also observed in IFN-␥R؊/؊ mice infected with the defective reactivation mutant MHV68 v-cyclin stop. Decreased fibrosis was associated with lower levels of transforming growth factor-␤, vascular endothelial growth factor, and markers of macrophage alternative activation. When antiviral treatment was administered on Day 60 in symptomatic animals, survival improved from 20 to 80% compared with untreated symptomatic animals, but lung fibrosis persisted in 60% of the mice. Conclusions: MHV68-induced fibrosis is a result of viral lytic replication during chronic lung herpesvirus infection in mice. We speculate that antiviral therapy might help to control lung fibrosis in humans with IPF and associated herpesvirus infection.

Tobacco-related lung diseases are associated with alterations in tissue remodeling and are characterized by increased matrix deposition. Among the matrix molecules found to be highly expressed in tobacco-related lung diseases is fibronectin, a cell adhesive glycoprotein implicated in tissue injury and repair. We hypothesize that nicotine, a component of tobacco, stimulates the expression of fibronectin in lung fibroblasts via the activation of intracellular signals that lead to increased fibronectin gene transcription. In support of this, we found that nicotine stimulated the expression of fibronectin in lung fibroblasts and that its stimulatory effect was associated with activation of protein kinase C and mitogen-activated protein kinases, increased levels of intracellular cAMP, and phosphorylation and DNA binding of the transcription factor CREB. Increased transcription of the gene was dependent on cAMP-response elements (CREs) present on the 5' end of its gene promoter. The stimulatory effect of nicotine on fibronectin expression was abolished by alpha-bungarotoxin, an inhibitor of alpha7 nicotinic acetylcholine receptors (alpha7 AChRs). Of note, nicotine increased the expression of alpha7 nAChRs on fibroblasts. Our data suggest that nicotine induces lung fibroblasts to produce fibronectin by stimulating alpha7 nAChR-dependent signals that regulate the transcription of the fibronectin gene.

Oxidant stress has been implicated in the pathogenesis of chronic lung disorders like idiopathic pulmonary fibrosis. However, mechanisms that link oxidant stress to fibrogenesis remain partially elucidated. Emerging data suggest an important role for the extracellular thiol/disulfide redox environment. The cysteine (Cys)/cystine (CySS) redox couple represents the predominant low-molecular-weight thiol/disulfide pool found in plasma and is sensitive to aging, smoking, and other host factors. We hypothesized that an oxidized extracellular Cys/CySS redox potential (E(h) Cys/CySS) affects lung fibroblasts by inducing intracellular signals that stimulate proliferation and matrix expression. We tested this hypothesis in primary murine lung fibroblasts and found that an oxidized E(h) Cys/CySS (-46 mV) stimulated lung fibroblast proliferation. Furthermore, it stimulated their expression of fibronectin, a matrix glycoprotein highly expressed in fibrotic lung diseases and implicated in lung injury. This stimulatory effect was dependent on protein kinase C activation. Oxidant stress also increased the phosphorylation of cAMP response element binding protein, a transcription factor known for its ability to stimulate fibronectin expression, and increased the expression of mRNAs and proteins coding for the transcription factors nuclear factor (NF)-kappaB and mothers against decapentaplegic homolog 3. Fibroblasts cultured in normal (-80 mV) or reduced (-131 mV) E(h) Cys/CySS showed less induction. Furthermore, fibronectin expression in response to an oxidized E(h) Cys/CySS was associated with expression of transforming growth factor-beta1 (TGF-beta1) and was inhibited by an anti-TGF-beta1 antibody and SB-431542, a TGF-beta1 receptor inhibitor. These studies suggest that extracellular oxidant stress activates redox-sensitive pathways that stimulate lung fibroblast proliferation and matrix expression through upregulation of TGF-beta1.

Connexin43 (Cx43) is a gap junction protein that forms multimeric channels that enable intercellular communication through the direct transfer of signals and metabolites. Although most multimeric protein complexes form in the endoplasmic reticulum (ER), Cx43 seems to exit from the ER as monomers and subsequently oligomerizes in the Golgi complex. This suggests that one or more protein chaperones inhibit premature Cx43 oligomerization in the ER. Here, we provide evidence that an ER-localized, 29-kDa thioredoxin-family protein (ERp29) regulates Cx43 trafficking and function. Interfering with ERp29 function destabilized monomeric Cx43 oligomerization in the ER, caused increased Cx43 accumulation in the Golgi apparatus, reduced transport of Cx43 to the plasma membrane, and inhibited gap junctional communication. ERp29 also formed a specific complex with monomeric Cx43. Together, this supports a new role for ERp29 as a chaperone that helps stabilize monomeric Cx43 to enable oligomerization to occur in the Golgi apparatus. INTRODUCTIONConnexins form gap junction channels that mediate intercellular communication by allowing the direct transfer of ions and small aqueous molecules between neighboring cells or by serving as hemichannels at the plasma membrane (Harris, 2001;Goodenough and Paul, 2003;Saez et al., 2003;Laird, 2006). There are Ͼ20 human connexin genes and one of the most ubiquitously expressed is connexin43 (Cx43). Several diseases have been linked to mutations in different connexin genes, including Cx43 (Kelsell et al., 2001;Laird, 2008). Many of these mutant connexins are not efficiently processed and lack the ability to form functional gap junction channels. Determining how mutant connexins are mistargeted requires a more complete understanding of how normal connexins are processed by the cell.A gap junction hemichannel consists of six connexins, which oligomerize before delivery to the plasma membrane (Martin and Evans, 2004;Segretain and Falk, 2004;Koval, 2006). Unlike most multimeric membrane channels, Cx43 is unusual in that it does not oligomerize in the endoplasmic reticulum (ER) (Musil and Goodenough, 1993), which is more commonly a prerequisite to further transport along the secretory pathway (Ellgaard and Helenius, 2003;Anelli and Sitia, 2008). Instead, Cx43 is transported out of the ER as an apparent monomer that then oligomerizes in the Golgi complex (Musil and Goodenough, 1993;Koval et al., 1997). Although other connexins, such as Cx46 (Koval et al., 1997), also oligomerize in the Golgi apparatus, this is not a universal pathway for connexin oligomerization because other gap junction proteins, such as Cx32, preferentially oligomerize in the ER (Martin and Evans, 2004;Koval, 2006). Understanding the molecular basis for this difference, as well as the ability of monomeric Cx43 to be transported from the ER to the Golgi apparatus has proven difficult, because little is known about chaperones that regulate connexin folding. In particular, it seemed likely that one or more chaperones would be requir...