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. 2022 Aug 22;7(16):e160688.
doi: 10.1172/jci.insight.160688.

KLF4 is a therapeutically tractable brake on fibroblast activation that promotes resolution of pulmonary fibrosis

Loka R Penke, Jennifer M Speth, Steven K Huang, Sean M Fortier, Jared Baas, Marc Peters-Golden

KLF4 is a therapeutically tractable brake on fibroblast activation that promotes resolution of pulmonary fibrosis

Loka R Penke et al. JCI Insight. .

Abstract

There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibited fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a therapeutic approach in fibrosis of the lung and perhaps other organs.

Keywords: Fibrosis; Pulmonology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. KLF4 expression is diminished in fibrotic fibroblasts.
Baseline KLF4 expression in fibroblasts outgrown from lungs of patients with IPF and control nonfibrotic (normal) lungs by real-time quantitative PCR (qPCR) analysis (A) and protein densitometry of Western blots (B). (C) Correlation analysis between the baseline KLF4 and FOXM1 mRNA expression in normal (blue dots) and IPF (red dots) fibroblasts; the cell lines displayed in C are an unselected subset of those displayed in A. Baseline KLF4 expression in fibroblasts outgrown from mouse lungs on day 21 after bleomycin and saline by qPCR analysis (D) and protein densitometry of Western blots (E). KLF4 expression in CCL210 cells after 48 hours of TGF-β (2 ng/mL) stimulation analyzed by qPCR (F) and Western blot (G). Expression of KLF4 protein in CCL210 cells after 3 hours of PGE2 (0.5 M) treatment analyzed by Western blot (results depicted from 1 experiment representative of 3 experiments (H). In A–C, each symbol represents a single patient lung-derived fibroblast line, and in D and E, each symbol represents an individual murine lung-derived fibroblast line. In A, B, and D–F, mRNA and protein values are normalized to GAPDH and expressed relative to the control (normal human or saline-treated mouse). Data in A–F are shown as mean ± SEM. *P < 0.05, 2-way ANOVA.
Figure 2
Figure 2. KLF4 expression regulates fibroblast differentiation and proliferation.
(A) KLF4 protein expression in MRC5 cells 48 hours after lentiviral transduction of UBC promoter-driven GFP (UBC-GFP) or human KLF4 (UBC-KLF4). (B and C) Effect of UBC-KLF4 or -GFP on TGF-β–induced expression of α-SMA and COL1α2 mRNA at 24 hours by qPCR (B) and protein at 48 hours by Western blot (C). (D–F) Effect of UBC-KLF4 or -GFP on FGF-2–induced fibroblast proliferation as determined at 72 hours by the CyQuant NF DNA binding assay (D) and proliferation-associated expression of FOXM1 and cyclin B1 (CYCB1) mRNA by qPCR (E) and protein by Western blot (F) at 48 hours. (G) CRISPR-mediated knockdown of KLF4 protein in MRC5 cells as determined by Western blot. (H and I) Effect of KLF4 knockdown on basal proliferation of fibroblasts as determined by the CyQuant NF DNA binding assay (H) and basal expression of FOXM1 and CYCB1 mRNA by qPCR (I). (J and K) Effect of KLF4 knockdown on TGF-β–induced expression of α-SMA analyzed at 48 hours by qPCR (J) and Western blot (left) and its protein densitometry (right) (K). (L) KLF4 protein induction in fibroblasts after treatment with APTO-253 (250 nM) for 36 hours. (M) Effect of APTO-253 on baseline and FGF-2–induced proliferation of fibroblasts as determined by the CyQuant NF DNA binding assay. (N and O) Effect of APTO-253 on TGF-β–induced expression of α-SMA and COL1α2 analyzed at 48 hours by qPCR (N) and Western blot (O). GAPDH mRNA and protein were used to normalize α-SMA, COL1α2, FOXM1, and CYCB1 expression by qPCR and Western blot, respectively. In A, C, F, G, K, L and O, representative Western blot of 2–3 experiments is shown. Data in B, E, I, J and N are expressed relative to control values and are shown as mean ± SEM from 3 independent experiments. *P < 0.05, 2-way ANOVA.
Figure 3
Figure 3. Restoration of KLF4 in elicited or IPF myofibroblasts promotes their dedifferentiation and restores apoptosis susceptibility.
(A) Scheme illustrating the experimental layout for molecular restoration of KLF4 in myofibroblasts to evaluate their differentiation status and functional response to Fas-mediated apoptosis. (B–G) MRC5 cells stably transduced with DOX-KLF4 or DOX-GFP were treated for 48 hours with TGF-β to promote a myofibroblast phenotype. Cells were then treated with/without doxycycline (1 μg/mL) for 16 hours, and the expression of α-SMA (B) and COL1α2 (C) was analyzed by qPCR at 24 hours; or cells were further stimulated with Fas Ab and assessed for relative annexin V binding at 24 hours (D), caspase-3/7 activity at 16 hours (E), and expression of APAF1 (F) and BIRC5 (G) measured by qPCR at 24 hours. (H) Scheme illustrating the experimental layout for treatment of elicited myofibroblasts or IPF fibroblasts with the pharmacologic KLF4 inducer APTO-253 and evaluation of differentiation status and functional response to Fas-mediated apoptosis. (I–L) MRC5 cells were treated for 48 hours with TGF-β to elicit myofibroblast differentiation. They were then treated with/without APTO-253 (250 nM) for 36 hours and assessed for the induction of KLF4 by qPCR (I) and α-SMA protein by densitometric analysis of Western blot (J) or further incubated with Fas Ab and assessed for relative annexin V binding at 24 hours (K), with expression of APAF1 and BIRC5 measured by qPCR at 24 hours (L). (M–O) IPF fibroblasts were treated with/without APTO-253 (250 nM) for 36 hours and assessed for the induction of KLF4 by qPCR (M) or analyzed for the expression of α-SMA by qPCR (N) and protein densitometry (O). GAPDH mRNA and protein were used to normalize α-SMA and COL1α2 expression by qPCR and Western blot, respectively. (M–O) Each symbol represents a single patient lung-derived fibroblast line. mRNA values are expressed relative to control. (B and C) The dashed line represents the value of fibroblasts not treated with TGF-β. All data represent mean values (± SE) from 3–4 independent experiments. *P < 0.05, 2-way ANOVA.
Figure 4
Figure 4. Conditional deletion of fibroblast KLF4 exacerbates peak lung fibrosis after bleomycin challenge.
(A) Schematic illustrating the timelines for in vivo administration of bleomycin and tamoxifen and the determination of experimental endpoints at day 21. (B–F) Effect of conditional deletion of fibroblast KLF4 (cKLF4 KO) in mice treated with and without bleomycin, as reflected by Masson’s trichrome staining for collagen deposition (stains blue) (B), changes in the lung hydroxyproline content (C), and the expression of fibrotic markers Tgf-β1 in whole lung tissue (D) and α-Sma and Col1α1 in fibroblasts outgrown from lung tissue (E and F). Images in B are at original magnification of 40×; scale bar = 300 μm. In C–F, each symbol represents an individual mouse. In D–F, mRNA values are expressed relative to the WT saline control. Results are expressed as mean ± SEM, n = 5–8 mice per group. *P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test.
Figure 5
Figure 5. Conditional deletion of fibroblast KLF4 impairs spontaneous resolution in a transient model of lung fibrosis.
(A) Schematic illustrating the timelines for in vivo administration of bleomycin and tamoxifen and the determination of experimental endpoints; tamoxifen administration was initiated at peak fibrosis (day 21) and continued until assessment at resolution (day 42). (B–F) Comparison of cKLF4-KO and WT mice at day 42, as reflected by Masson’s trichrome staining for collagen deposition (stains blue) (B), changes in the lung hydroxyproline content (C), and expression of fibrotic markers Col1α1, Ctgf, and Tgf-β1 mRNA in whole lung tissue (D–F). Images in B are at original magnification of 40×, scale bar = 300 μm. In C–F, each symbol represents an individual mouse. Results are expressed as mean ± SEM, n = 6–8 mice per group. *P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test.
Figure 6
Figure 6. Pharmacologic restoration of KLF4 attenuates peak fibrosis and promotes resolution in a model of persistent pulmonary fibrosis.
(A) Schematic illustrating the timelines for in vivo administration of single-dose bleomycin and APTO-253 in a transient model of lung fibrosis and the determination of experimental endpoints at peak fibrosis (day 21). (B and C) Effect of APTO-253 administration in mice treated with and without bleomycin, as reflected by Masson’s trichrome staining for collagen deposition (stains blue) (B) and changes in the lung hydroxyproline content (C). (D) Schematic illustrating the timelines for in vivo administration of bleomycin in a 3-dose model characterized by persistent fibrosis and administration of APTO-253 during the fibrotic phase of this model beginning at day 42 and continuing until harvest at day 63. Effect of APTO-253 administration in mice treated with and without repeated bleomycin challenge, as reflected by changes in body weight (E), lung hydroxyproline content (F), Masson’s trichrome staining for collagen deposition (stains blue) (G), and expression of fibrotic markers Ctgf in whole lung tissue (H) and α-Sma in fibroblasts outgrown from lung tissue (I). Images in B and G are at original magnification of 40×, scale bar = 300 μm. In C, E, F, and H and I, each symbol represents an individual mouse. Results are expressed as mean ± SEM, n = 5–8 mice per group. *P < 0.05, 2-way ANOVA with Tukey’s multiple comparisons test.
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