KLF4 Alleviates Hypertrophic Scar Fibrosis by Directly Activating BMP4 Transcription

Abstract

Background: Hypertrophic scars (HS) often occur after burns, surgery and extensive trauma. Krüppel-like factor 4 (KLF4) is a member of the Krüppel-like factor family, a group of conserved zinc finger transcription factors that regulate diverse cellular processes. KLF4 can participate in the regulation of fibrotic diseases in many organs, such as the lung, liver, and heart. However, the antifibrotic effect of KLF4 in skin HS remains elusive. Result: This study observed the inhibition of KLF4 on fibrosis in vivo and in vitro. Our results revealed that KLF4 expression was decreased in HS tissue and fibroblasts. The results of KLF4 transfection confirmed its ability to alleviate the transdifferentiation of fibroblasts into myofibroblasts both in vitro and in vivo, thereby inhibiting the development of fibrosis. In addition, ChIP assays showed that BMP4 was the target gene of KLF4 for inhibiting skin fibrosis. Conclusions: Collectively, this evidence indicates that KLF4 is associated with BMP4 and could play an important regulatory role in HS formation by downregulating myofibroblast transdifferentiation. Our study provides a new target for the prevention and treatment of hypertrophic scars.

Keywords: BMP4; Hypertrophic scar; Krüppel-like factor 4; Transcriptional activation.

Figure 1

KLF4 was dramatically downregulated in HS. (A) H&E and Masson staining of NS and HS originating from patients with HS. (B) IHC staining for KLF4 in NS and HS tissues from patients with HS. (C) Western blot and PCR for KLF4 in three paired NS and HS tissues. (D) Immunofluorescence double staining for KLF4 and α-SMA in NSFs and HSFs. *P < 0.05 and **P < 0.01 compared with the NS tissues.

Figure 2

KLF4 significantly dampened fibrosis-related molecules in HS fibroblasts. HSFs were infected with KLF4-overexpressing lentivirus or negative control vector, and NSFs were infected with the shKLF4 or control shRNA lentiviral vector. (A) KLF4 mRNA and protein levels were evaluated after infection in the indicated cells. (B) Fibrosis-related molecules, including α-SMA, Col1 and Col3, were analysed by qRT-PCR and western blot in the indicated cells. (C) Immunofluorescence staining for α-SMA in the indicated cells. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 compared with the negative control.

Figure 3

KLF4 significantly inhibited the proliferation of HS fibroblasts via cell cycle arrest. (A & C) CCK-8 assays were used to determine cell viability in the indicated HSFs infected with KLF4 or NC lentiviral vector and NSFs infected with shKLF4 or control shRNA lentiviral vector. (B & D) Cell cycle distributions were detected by flow cytometry. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the negative control.

Figure 4

KLF4 dramatically alleviated hypertrophic scar in vivo. (A) Bleomycin was persistently injected into BALB/c mice for 2 weeks, and then KLF4/NC adenovirus was injected into the region of bleomycin-induced dermal fibrosis in mice. (B) H&E and Masson staining were performed with HS tissues from the KLF4-overexpressing and NC groups. (C) Sirius Red staining was performed with HS tissues from the KLF4-overexpressing and NC groups. (D) Immunofluorescence double staining for KLF4 and α-SMA in HS skin tissues. (E) Immunofluorescence staining for Ki67 in HS skin tissue.

Figure 5

BMP4 was selected as a key downstream target of KLF4 via RNA sequencing. (A) Heatmap for the visualization of a clustering analysis of differentially expressed genes (DEGs). (B) Venn diagram of the overlap number of DEGs between comparisons. (C) DEGs, differentially expressed genes. (C) Volcano plots showing the upregulated (red) and downregulated (blue) DEGs for each comparison. BMP4 was clearly marked. (D) KEGG pathway network analysis of differentially expressed genes.

Figure 6

KLF4 positively regulated BMP4 transcription. (A) Schematic diagram of the BMP4 promotor. The blue region is the putative KLF4-binding site. (B) Amplification of the BMP4 promoter sequence from chromatin immunoprecipitation (ChIP) DNA was performed. Histone H3, IgG and input served as the positive, negative and internal controls, respectively. (C) qRT-PCR and western blot for BMP4 in HSFs infected with KLF4 lentiviral vector or NC vector and NSFs infected with shNC or shKLF4 lentivirus. (D) Downregulated BMP4 in HSFs inhibited proliferation and fibrosis-related molecule expression induced by KLF4 overexpression. A shBMP4-resistant reconstitution, BMP4Δ, was used to restore BMP4 expression, which reversed the fibrosis of HSFs. *P < 0.05, **P < 0.01, ***P < 0.001 and **** P < 0.0001 compared with the negative control.

Figure 7

Schematic diagram of the proposed mechanism.