Cyb5R3 in Alveolar Epithelial Cells Confers Protection Against Lung Fibrosis Through Suppression of TGF‐β Signaling
Authors: Bueno Marta , Alvarez Diana , Calyeca Jazmin , Baker Christian , Shulkowski Stephanie , Nasser Amro , Mathien August , Sembrat John , Miller Megan , Hahn Scott , Rojas Mauricio , Straub Adam , Mora Ana 
Published: April 2020

BackgoundAlterations in the redox homeostasis play a role in the pathogenesis of age‐related diseases such idiopathic pulmonary fibrosis (IPF). The most accepted theory in the pathogenesis of IPF is the injury and activation of alveolar epithelial type II cells (AECII) triggering excessive and chronic wound‐healing responses, mediated by growth factors such as TGF‐β. A mitochondrial membrane‐bound NADH‐dependent redox enzyme that contributes to stress protection and associates with healthspan and aging is cytochrome b5 reductase isoform 3 (Cyb5R3). Cyb5R3 is a flavoprotein recognized for its ability to reduce heme iron, a major target of reactive oxygen species. However, the link between Cyb5R3 expression levels in AECII with the modulation of the TGF‐β signaling pathways and the age‐related vulnerability to injury and fibrosis in the lung is unknown.MethodsLungs from young, old and IPF donors were lysated to test Cyb5R3 levels. Cyb5R3 flox mice (Cyb5R3 fl/fl or WT) and mice carrying a doxycycline‐inducible Cre‐recombinase under the control of a regulatory sequence of the SP‐C gene were bred. To promote recombination and generate a homozygous conditional type II lung epithelial cells (AECII) CYB5R3 knockout mice (AECII Cyb5R3 KO), mice were fed doxycycline chow (625 mg/kg, Envigo) for 2 weeks before injury or AECII isolation. Mice were infected intranasally with gammaherpesvirus 68 (MHV68) to evaluate inflammatory and fibrotic responses in the lung. Mouse alveolar epithelial cell line MLE12 was used to knockdown Cyb5R3 using adenovirus carrying scrambleshRNA or Cyb5R3shRNA constructs.ResultsAged donor and IPF total lung lysates show lower levels of Cyb5R3. In addition, isolated murine primary AECII showed that Cyb5R3 is significantly diminished in aging AECIIs (24 mo vs. 4 mo) associated with cellular oxidative stress and a senescence phenotype. Conditional deletion of Cyb5R3 in AECII show increased susceptibility to lung fibrosis. AECII Cyb5R3 KO mice showed higher collagen deposition with significant higher transcript levels of TIMP1 (tissue inhibitor of the MMPs) and osteopontin than Cyb5R3 WT mice. Since TGF‐β is a key modulator of the net balance between MMPs and TIMPs, we analyzed the role of Cyb5R3 in TGF‐β signaling. In mouse epithelial cells, deficiency of Cyb5R3 enhances TGF‐β signaling with upregulation of profibrotic and senescence genes by modulation of cGMP‐PKG and cAMPPKA pathways.ConclusionsAltogether, these data suggest a possible role for Cyb5R3 in the fragility of AECIIs and the age related susceptibility to lung fibrosis. New therapies designed to modulate Cyb5R3 expression and activity might lessen severity of pulmonary fibrosis, especially in the context of the aging lung.Support or Funding InformationThis work was funded by the Aging Institute & Vascular Medicine Institute at the University of Pittsburgh