Lung-specific loss of α3 laminin worsens bleomycin-induced pulmonary fibrosis.

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-ß was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-ß. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.

Vimentin is sufficient and required for wound repair and remodeling in alveolar epithelial cells.

The physiological and pathophysiological implications of the expression of vimentin, a type III intermediate filament protein, in alveolar epithelial cells (AECs) are unknown. We provide data demonstrating that vimentin is regulated by TGFß1, a major cytokine released in response to acute lung injury and that vimentin is required for wound repair and remodeling of the alveolar epithelium. Quantitative real-time PCR shows a 16-fold induction of vimentin mRNA in TGFß1-treated transformed AECs. Luciferase assays identify a Smad-binding element in the 5' promoter of vimentin responsible for TGFß1-induced transcription. Notably, TGFß1 induces vimentin protein expression in AECs, which is associated with a 2.5-fold increase in cell motility, resulting in increased rates of migration and wound closure. These effects are independent of cell proliferation. TGFß1-mediated vimentin protein expression, cell migration, and wound closure are prevented by a pharmacological inhibitor of the Smad pathway and by expression of Ad-shRNA against vimentin. Conversely, overexpression of mEmerald-vimentin is sufficient for increased cell-migration and wound-closure rates. These results demonstrate that vimentin is required and sufficient for increased wound repair in an in vitro model of lung injury.

Vimentin regulates activation of the NLRP3 inflammasome.

Activation of the NLRP3 inflammasome and subsequent maturation of IL-1ß have been implicated in acute lung injury (ALI), resulting in inflammation and fibrosis. We investigated the role of vimentin, a type III intermediate filament, in this process using three well-characterized murine models of ALI known to require NLRP3 inflammasome activation. We demonstrate that central pathophysiologic events in ALI (inflammation, IL-1ß levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of Vim(-/-) mice challenged with LPS, bleomycin and asbestos. Bone marrow chimeric mice lacking vimentin have reduced IL-1ß levels and attenuated lung injury and fibrosis following bleomycin exposure. Furthermore, decreased active caspase-1 and IL-1ß levels are observed in vitro in Vim(-/-) and vimentin-knockdown macrophages. Importantly, we show direct protein-protein interaction between NLRP3 and vimentin. This study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the NLRP3 inflammasome.

The role of the ubiquitin proteasome pathway in keratin intermediate filament protein degradation.

Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation--causes, mechanisms, and consequences--will allow for a greater understanding of epithelial cell biology and lung pathology alike.