Altered expression of p63 isoforms and expansion of p63- and club cell secretory protein-positive epithelial cells in the lung as novel features of aging

Abstract

Aging is a key contributor for subclinical progression of late-onset lung diseases. Basal, club, and type II alveolar epithelial cells (AECs) are lung epithelial progenitors whose capacities of differentiation are extensively studied. The timely transition of these cells in response to environmental changes helps maintain the intricate organization of lung structure. However, it remains unclear how aging affects their behavior. This paper demonstrates that the protein expression profiles of a type II AEC marker, prosurfactant protein C (pro-SPC), and a basal cell marker, p63, are altered in the lungs of 14-mo-old versus 7- to 9-wk-old mice. Expression of NH2-terminal-truncated forms of p63 (ΔNp63), a basal cell marker, and claudin-10, a club cell marker, in cytoplasmic extracts of lungs of 14-mo-old mice was upregulated. In contrast, nuclear expression of full-length forms of p63 (TAp63) decreases with age. These alterations in protein expression profiles coincide with dramatic changes in lung functions including compliance. Whole tissue lysates of middle-aged versus aged rhesus monkey lungs display similar age-associated alterations in pro-SPC expression. An age-associated decrease of TAp63 in nuclear lysates was observed in aged monkey group. Moreover, the lungs of 14-mo-old versus 7- to 9-wk-old mice display a wider spreading of ΔNp63-positive CCSP-positive bronchiolar epithelial cells. This expansion did not involve upregulation of Ki67, a representative proliferation marker. Collectively, it is postulated that 1) this expansion is secondary to a transition of progenitor cells committed to club cells from ΔNp63-negative to ΔNp63-positive status, and 2) high levels of cytoplasmic ΔNp63 expression trigger club cell migration.

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“…The lungs of the mice were perfused in ice‐cold saline and the left lung was excised and fixed in ice‐cold 4% PFA for 48h. After 48h, lung was immersed in 70% alcohol and processed by standard procedures 37,38 37 …”

“…First step in the process was a “Deep Inflation” perturbation to verify the proper insertion of the cannula without any obstruction or misplacement. The protocol followed a sequence of baseline measurement (SnapShot‐150, Quick Prime‐3) and was run thrice for each mouse as described 38,42 . The protocol was repeated three times and 9 measurements were recorded per mice.…”

“…After 48h, lung was immersed in 70% alcohol and processed by standard procedures. 37,38 Paraffin sections (5 µm) were cut and stained with hematoxylin and eosin and the extent of lung injury was assessed as previously described. 37 Paraffin-embedded lung sections were stained with Picrosirius red staining kit (24901-250) for the detection of the collagen depositions as per the manufacturer's instructions (Polysciences, Warrington, PA, USA).…”

“…The protocol followed a sequence of baseline measurement (SnapShot-150, Quick Prime-3) and was run thrice for each mouse as described. 38,42 The protocol was repeated three times and 9 measurements were recorded per mice. The tested parameters for adult mice (7-9 weeks) included inspiratory capacity (IC), respiratory system compliance (Crs), respiratory system elastance (Ers), tissue elastance (H), tissue damping (G), Newtonian resistance (Rn), and respiratory system resistance (RrS).…”

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“…δNp63 is known to have an anti-apoptotic effect, and TAp63 is a pro-apoptotic protein [120]. The expression of TAp63 decreases in the lungs of aging mice [121]. The specific functions of p63 isoforms are still unclear and require additional investigation.…”