Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

Abstract

Epithelial-mesenchymal cell interactions and factors that control normal lung development are key players in lung injury, repair, and fibrosis. A number of studies have investigated the roles and sources of epithelial progenitors during lung regeneration; such information, however, is limited in lung fibroblasts. Thus, understanding the origin, phenotype, and roles of fibroblast progenitors in lung development, repair, and regeneration helps address these limitations. Using a combination of platelet-derived growth factor receptor α-green fluorescent protein (PDGFRα-GFP) reporter mice, microarray, real-time polymerase chain reaction, flow cytometry, and immunofluorescence, we characterized two distinct interstitial resident fibroblasts, myo- and matrix fibroblasts, and identified a role for PDGFRα kinase activity in regulating their activation during lung regeneration. Transcriptional profiling of the two populations revealed a myo- and matrix fibroblast gene signature. Differences in proliferation, smooth muscle actin induction, and lipid content in the two subpopulations of PDGFRα-expressing fibroblasts during alveolar regeneration were observed. Although CD140α(+)CD29(+) cells behaved as myofibroblasts, CD140α(+)CD34(+) appeared as matrix and/or lipofibroblasts. Gain or loss of PDGFRα kinase activity using the inhibitor nilotinib and a dominant-active PDGFRα-D842V mutation revealed that PDGFRα was important for matrix fibroblast differentiation. We demonstrated that PDGFRα signaling promotes alveolar septation by regulating fibroblast activation and matrix fibroblast differentiation, whereas myofibroblast differentiation was largely PDGFRα independent. These studies provide evidence for the phenotypic and functional diversity as well as the extent of specificity of interstitial resident fibroblasts differentiation during regeneration after partial pneumonectomy.

Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development

“…In contrast, expression of CD29 (β1 integrin) identified proliferative myofibroblasts. Our previously published realveolarization data suggested that PDGFRα + /CD29 + /CD34 + fibroblasts contribute to the transient re-emergence of αSMA + myofibroblasts as they lose CD34 expression (Green et al, 2016). We performed flow cytometry to determine whether similar functional differences within PDGFRα + (CD140a + ) fibroblasts are associated with CD29 and CD34 during lung development.…”

“…PDGFRα + lung fibroblasts are essential for alveolarization and realveolarization (Green et al, 2016; Bostrom et al, 2002, 1996; Lindahl et al, 1997; Chen et al, 2012; Perl and Gale, 2009). As early as E12.5, a population of PDGFRα-expressing, SHH-responsive Gli1+ mesenchymal cells can be traced throughout postnatal lung development and contribute to nearly all of the alveolar myofibroblasts required for septation (Li et al, 2015; Liu et al, 2013).…”

“…During the initial phase of septal eruption (~P4-7) there is an abrupt increase in αSMA expression organized along the developing septal crest (Green et al, 2016; Chen et al, 2012; Branchfield et al, 2016; McGowan et al, 2008; Ntokou et al, 2015; Mariani et al, 2002). Following eruption, there is a phase of septal elongation and maturation in which αSMA expression is dramatically reduced, elastin expression and organization proceeds, and there is thinning of the alveolar interstitium (Branchfield et al, 2016; McGowan and McCoy, 2011; Mariani et al, 2002).…”

“…Over the last few decades, investigation of the pulmonary mesenchyme has been challenged by the remarkable complexity and diversity of fibroblast and other mesenchymal stem cell populations (Hogan et al, 2014; Bertoncello and McQualter, 2013; Volckaert et al, 2011; McQualter et al, 2009, 2013; Green et al, 2016). Mesenchymal subpopulations can be grouped by their anatomical location: tracheal, peribronchiolar, perivascular, alveolar and subpleural.…”

“…Using a Pdgfra GFP reporter mouse, we have previously established different functional roles, cellular markers and transcriptional profiles of PDGFRα + lung fibroblasts during adult lung regeneration following partial pneumonectomy. With PDGFRα kinase gain- or loss-of-function and in combination with manipulation of FGFR and/or PPAR-gamma signaling pathways, we identified and characterized temporally dynamic and functionally diverse subpopulations of matrix-, myo-, and lipofibroblasts that are required for injury-induced repair and regeneration (Green et al, 2016; Chen et al, 2012; Perl and Gale, 2009). Although others have reported on the temporal-spatial localization of PDGFRα + lung fibroblasts (Branchfield et al, 2016; Kimani et al, 2009; McGowan et al, 2008; Ntokou et al, 2015), a more thorough analysis that couples spatial-temporal localization with functional analysis and gene expression data may improve our understanding of the role of PDGFRα fibroblast populations during distal lung development.…”