KLF4 is a therapeutically tractable brake on fibroblast activation that promotes resolution of pulmonary fibrosis
Loka R. Penke, … , Jared Baas, Marc Peters-Golden
Loka R. Penke, … , Jared Baas, Marc Peters-Golden
Published July 19, 2022
Citation Information: JCI Insight. 2022;7(16):e160688. https://doi.org/10.1172/jci.insight.160688.
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Research Article Pulmonology

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

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.

Authors

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

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Figure 2

KLF4 expression regulates fibroblast differentiation and proliferation.

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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.