Fig 1.
Lung staining for SA-β-galactosidase activity.
(A and B) 5 μM sections of lung from of patients with IPF, (C) normal donor controls, (D) scleroderma, or (E) hypersensitivity pneumonitis were stained for SA-βgal activity. Note the blue staining epithelial cells in IPF lung (A and B, arrows) but not normal individuals, or patients with scleroderma or hypersensitivity pneumonitis. Images are representative of staining of tissue from 8 patients with IPF, 14 control subjects, 5 patients with scleroderma, and 8 patients with hypersensitivity pneumonitis.
Fig 2.
Immunohistochemistry of IPF and normal lung with p16, p21, or p53 antibodies.
5 μM sections of normal lung immunostained for (A) p16, (B) p21, (C) p53 show an absence of staining. In contrast, IPF lung immunostained for (D) p16, (E) p21, or (F) p53 show immunoreactive cells lining airspaces consistent with type II AECs. (G-I) Isotype control for p16, p21, or p53 are shown. Images are representative of staining of tissue from 12 patients with IPF and 10 control subjects.
Fig 3.
Expression of senescence markers in isolated type II AECs.
(A) Immunoblot for p16, p21 and p53 shows higher levels of these proteins in type II AECs isolated from IPF patients relative to those from control subjects. Relative densitometric ratios of p16, p21, and p53 to β-actin are presented by bar graphs (* p < 0.05). (B) SA-βgal activity in type II AECs isolated from IPF lungs was detected in an average of 23.1 ± 13% of cells relative to type II AECs isolated from disease controls (1.2 ± 1.3%), control subjects (0.8 ± 0.6%) and unstatined type II AECs (1.5 ± 0.7%). (C) Scatter plots of age and SA-βgal activity of patients whose type II AECs were analyzed by flow cytometry.
Fig 4.
qRT-PCR for miRNAs isolated from type II AECs.
Box plots of normalized relative log2 expression of miRNAs in type II AECs isolated from IPF patients (n = 15) and control subjects (n = 15). The levels of miR-34a (A), miR-34b (B), and miR-34c (C) were significantly higher in type II AECs isolated from IPF patients compared to those from control subjects (* p< 0.001 by Mann-Whitney U test). The levels of (D) miR-20a, (E) miR-29c, and (F) miR-let-7f were not significantly different in IPF patients relative to control subjects. The boxes are drawn extending from the 75th percentile to the 25th percentile. The horizontal line inside the box represents median values. The whiskers above and below the box delineate the maximum and minimum.
Fig 5.
SA-βgal activity detected by flow cytometry in A549 cells overexpressing miR-34a, miR-34b, and miR-34c.
The relative percentage of cells with detectable SA-gal activity was measured in A549 cells overexpressing (A) miR-34a, (B) miR-34b, (C) miR-34c, or (D) mCherry control. Note the higher percentage of cells with SA-βgal activity in cells overexpressing miR-34a, miR-34b, or miR-34c compared to treated control cells.
Fig 6.
Targets of miR-34 family in isolated type II AECs and A549 cells overexpressing miR-34 family miRNAs.
(A-F) Box plots of normalized relative log2 expression of SIRT1, E2F1, c-Myc, CDK4, CDK6, and CCNE2 in type II AECs isolated from IPF patients (n = 15) and control subjects (n = 15). The expression levels of (B) E2F1, (C) c-Myc, and (F) CCNE2 were significantly lower in type II AECs isolated from IPF patients compared to those from control subjects (* p < 0.05 by Mann-Whitney U test). The boxes are drawn extending from the 75th percentile to the 25th percentile. The horizontal line inside the box represents median values. The whiskers above and below the box delineate the maximum and minimum. (G) Expression levels of candidate targets of miR-34 family were also measured in A549 overexpressing miR-34a, miR-34b, or miR-34c by qRT-PCR.
Fig 7.
Proposed relationship between miR-34 miRNAs and their targets as mediators of cellular senescence in IPF type II AECs.
p53 activation leads to up-regulation of miR-34 family of miRNAs in IPF type II AECs. Elevated miR-34a, miR-34b, or miR-34c levels lead to down-regulation of key target genes, including E2F1, c-Myc, and CCNE2, involved in the cell cycle, leading to cellular senescence.