Lung-specific loss of the laminin α3 subunit confers resistance to mechanical injury

Abstract

SummaryLaminins are heterotrimeric glycoproteins of the extracellular matrix that are secreted by epithelial cells and which are crucial for the normal structure and function of the basement membrane. We have generated a mouse harboring a conditional knockout of a3 laminin (Lama3 fl/fl ), one of the main laminin subunits in the lung basement membrane. At 60 days after intratracheal treatment of adult Lama3 fl/fl mice with an adenovirus encoding Cre recombinase (Ad-Cre), the protein abundance of a3 laminin in whole lung homogenates was more than 50% lower than that in control-treated mice, suggesting a relatively long half-life for the protein in the lung. Upon exposure to an injurious ventilation strategy (tidal volume of 35 ml per kg of body weight for 2 hours), the mice with a knockdown of the a3 laminin subunit had less severe injury, as shown by lung mechanics, histology, alveolar capillary permeability and survival when compared with Ad-Null-treated mice. Knockdown of the a3 laminin subunit resulted in evidence of lung inflammation. However, this did not account for their resistance to mechanical ventilation. Rather, the loss of a3 laminin was associated with a significant increase in the collagen content of the lungs. We conclude that the loss of a3 laminin in the alveolar epithelium results in an increase in lung collagen, which confers resistance to mechanical injury.

Lung-Specific Loss of α3 Laminin Worsens Bleomycin-Induced Pulmonary Fibrosis

“…Here, we show that a3 laminin is expressed uniformly throughout the alveolar basement membrane in normal human lungs. Despite the widespread expression of a3 laminin protein in the normal lung, a3 laminin mRNA was nearly undetectable in lung homogenates, suggesting that a3 laminin may have a very long half-life in the human lung, as we have previously observed in mice (3 mo) (12). In patients with lung fibrosis, a normal basement membrane pattern of laminin staining was observed in areas of the lung without fibrosis.…”

“…Global deletion of the laminin a3 gene in mice results in a similar blistering skin disease, which is the cause of death of the animals shortly after birth, before lung development is complete (25). Thus, we developed a mouse line lacking the a3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the SPC promoter (SPC-Cre) with mice expressing floxed alleles encoding the a3 laminin gene (Lama3 fl/fl ), which we have previously described (12). Expression of the SPC gene in early progenitors of the lung epithelium during development results in Cre-mediated gene recombination in the entire airway epithelium (13,26).…”

“…Unless indicated otherwise, we used mice that were 8-12 weeks of age (20-25 g). Mice harboring floxed alleles in the gene encoding a3 laminin (Lama3 fl/fl ) were created by generating a vector that targets exon 42 of the mouse a3 laminin allele flanked by loxP (locus of x-ing over bacteriophage P) sequences and introduced this into embryonic stem cells, as we previously described (12). To generate a line of mice deficient in a3 laminin in the lung epithelium, we crossed Lama3 fl/fl animals with transgenic mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) (a kind gift of Dr. B. Hogan, Duke University, Durham, NC) (13).…”

Epithelial β1 integrin is required for lung branching morphogenesis and alveolarization

“…In the mammary gland, epithelial β1 integrin deletion results in epithelial detachment from the basement membrane without inflammation Naylor et al, 2005). In contrast, increased numbers of macrophages were observed in the lungs of mice with laminin α3 chain mutations (Urich et al, 2011) and in the lungs of humans with integrin α3 mutations (Nicolaou et al, 2012), suggesting that β1 integrin-mediated regulation of inflammation is specific to the lung epithelium. Whereas β1 integrin deficiency results in increased ROS production and MCP-1 secretion from alveolar epithelial cells, the molecular mechanisms accounting for these findings will require further study.…”

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell–cell trans-dimerization of Na,K-ATPase β1 subunits

“…A total of 1-ml of PBS was instilled into the lungs and then aspirated three times. BALF was centrifuged to remove cells and used to determine proteins (Urich et al, 2011). RNA was isolated from lungs using an RNeasy kit (QIAGEN, Valencia, CA) and reverse transcribed using qScript cDNA synthesis (Quanta Biosciences, Gaithersburg, MD, USA).…”