Journal: Journal of Clinical Investigation

Article Title: IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/jci128678

Figure Lengend Snippet: Figure 2. KLF4 binds to the IL-36γ promoter and regulates IL-36γ transcriptional activity in response to EGFR inhibition. (A) Luciferase reporter assay of human IL-36γ transcriptional activity in PHKs transfected with IL-36γ-pGL3 (1630 bp) reporter plasmid, followed by exposure to erlotinib and C. acnes for 16 hours. Renilla luciferase activity was measured to determine transfection efficiency. Data represent mean ± SEM (n = 4). E.V., empty vector. (B and D) Schematic of 5′-deletion and mutant constructs of the human IL-36γ promoter. Site-directed mutagenesis was performed to introduce the indicated mutation at the EGFR inhibitor–responsive site (EiRS) and p65 binding site. (C and E) 5′-Deletion and mutation study of the human IL-36γ promoter activi ty. PHKs were transfected with indicated plasmids, followed by exposure to erlotinib and C. acnes for 16 hours. Data represent mean ± SEM (n = 4). (F) Quantitative PCR was performed to evaluate the gene expression of transcription factor candidates binding to the EiRS. PHKs were exposed to erlotinib for 6 hours. Data represent mean ± SEM (n = 3). (G) Gel shift, competition, and supershift EMSA analysis using a Cy5-labeled oligonucleotide probe for the EiRS-containing region and HEK293T cell lysate containing KLF4 protein. (H) PHKs were exposed to erlotinib and C. acnes for 24 hours. (I) DNA pull-down assay using biotinylated wild-type– or mutant-oligonucleotide probe of the EiRS-containing region. These probes were incubated with extracts from PHKs exposed to erlotinib for 24 hours. DNA-associated proteins were visualized by Western blotting. (J) Ex vivo skin explants from healthy controls were exposed to erlotinib for 24 hours and KLF4 expression was assessed by Western blotting. The blot shown is representative of 2 different skin donors. Data were analyzed with 1-way ANOVA followed by Dunnett’s multiple-comparisons test (A and E) or with 2-tailed unpaired Mann-Whitney U (C) or t test (F). *P < 0.05; **P < 0.01; ***P < 0.001. All blots were run contemporaneously with the same protein samples.

Article Snippet: The following cDNAs were subcloned into pcDNA3.1 (Invitrogen) or pMXs-IP (gift from Toshio Kitamura, University of Tokyo, Japan): mouse Klf4 (Addgene, 15920), human ERK1 (Addgene, 23509), human ERK2 (Addgene, 23498), and human ubiquitin (Addgene, 31815).

Techniques: Activity Assay, Inhibition, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Mutagenesis, Construct, Introduce, Binding Assay, Real-time Polymerase Chain Reaction, Gene Expression, Gel Shift, Labeling, Pull Down Assay, Incubation, Western Blot, Ex Vivo, Expressing, MANN-WHITNEY

Journal: Journal of Clinical Investigation

Article Title: IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/jci128678

Figure Lengend Snippet: Figure 3. Lack of KLF4 binding site results in loss of synergistic IL-36γ production in mice. (A) PMKs were exposed to erlotinib (1 μM) and murine IL-36γ (100 ng/mL) for 6 hours; isolated RNA was analyzed by quantitative PCR. Data represent mean ± SEM (n = 3). (B) PMKs from wild-type or KLF4-knock out mouse were exposed to murine IL-36γ (100 ng/mL) for 6 hours. Data represent mean ± SEM (n = 3). PMK cell lysates were analyzed by SDS-PAGE and immunoblotting. Blots were run contemporaneously with the same protein samples. (C) Schematic of the human and murine IL-36γ promoter with predicted KLF4 binding site and p65 binding site by JASPAR. (D) Gel shift and competition EMSA analysis using a Cy5-labeled oligonucleotide probe for human and mouse KLF4 binding sites and HEK293T cell lysate containing murine KLF4 protein. Sequence-specific binding of human probe to murine KLF4 was demonstrated as a positive control. Gel shift reflecting formation of protein-DNA complexes with the murine probe, and KLF4 was not observed. Data are representative of 3 independent experiments.

Article Snippet: The following cDNAs were subcloned into pcDNA3.1 (Invitrogen) or pMXs-IP (gift from Toshio Kitamura, University of Tokyo, Japan): mouse Klf4 (Addgene, 15920), human ERK1 (Addgene, 23509), human ERK2 (Addgene, 23498), and human ubiquitin (Addgene, 31815).

Techniques: Binding Assay, Isolation, Real-time Polymerase Chain Reaction, Knock-Out, SDS Page, Western Blot, Gel Shift, Labeling, Sequencing, Positive Control

Journal: Journal of Clinical Investigation

Article Title: IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/jci128678

Figure Lengend Snippet: Figure 4. Blockade of the EGFR/MEK/ERK pathway increases keratinocyte expression of KLF4. (A) Quantitative PCR was performed to evaluate gene expression in RNA isolated from biopsies of 4 patients with acneiform eruption and 10 healthy control (HC) skin biopsies. Data represent mean ± SD. (B) PHKs were pre-exposed to the BRAF inhibitor vemurafenib (1 μg/mL) for 30 minutes and exposed to the MEK inhibitor trametinib (2 μg/mL) and C. acnes (MOI of 10) for 6 hours. Data represent mean ± SEM (n = 3). Data were analyzed with 2-tailed unpaired t test (A) or 1-way ANOVA followed by Tukey’s mul tiple-comparisons test (B). *P < 0.05; **P < 0.01; ***P < 0.001. (C) PHKs were exposed to erlotinib (1 μM) or trametinib (2 μg/mL) for 24 hours and total cell lysates were collected for Western blotting with antibodies against KLF4 and β-actin. (D) ERK1 and ERK2 were silenced by siRNA in PHKs and cell lysates were analyzed by SDS-PAGE and immunoblotting with indicated antibodies. (E) HEK293T cells were transfected with FLAG-tagged KLF4 and Myc-tagged ERK1 and ERK2 for 24 hours. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with the indicated antibod ies. (F) HEK293T cells were transfected with FLAG-tagged KLF4, HA-tagged ubiquitin, and constitutively active ERK (CA-ERK) for 24 hours. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. All blots were run contemporaneously with the same protein samples. Data are representative of 3 independent experiments.

Article Snippet: The following cDNAs were subcloned into pcDNA3.1 (Invitrogen) or pMXs-IP (gift from Toshio Kitamura, University of Tokyo, Japan): mouse Klf4 (Addgene, 15920), human ERK1 (Addgene, 23509), human ERK2 (Addgene, 23498), and human ubiquitin (Addgene, 31815).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression, Isolation, Control, Western Blot, SDS Page, Transfection, Immunoprecipitation, Ubiquitin Proteomics

Journal: Journal of Clinical Investigation

Article Title: IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/jci128678

Figure Lengend Snippet: Figure 5. KLF4 is critical for IL-36γ transcrip- tional activity upon EGFR/MEK inhibition. (A) KLF4-overexpressing primary keratinocytes were exposed to C. acnes for 24 hours. (B) Flag- tagged wild-type (WT) and dominant-negative (DN) KLF4 were overexpressed in response to doxycycline using a Tet-on system for 24 hours, followed by exposure to Pam3CSK4 for another 24 hours. The cell lysates were collected for Western blotting and quantitative PCR (qPCR). Data represent mean ± SEM (n = 3). (C) KLF4 siRNA–treated PHKs were exposed to erlotinib and C. acnes for 6 hours and IL36G levels were analyzed by qPCR. Data represent mean ± SEM (n = 3). (D) Keratinocyte cell lines in which KLF4 was knocked out by CRISPR/Cas9 were exposed to trametinib (2 μg/mL) for 24 hours and total cell lysates were collected for Western blotting with antibodies against KLF4 and β-actin. The cells were exposed to trametinib for 24 hours and isolated RNA was analyzed by qPCR. Data represent mean ± SEM (n = 3). All blots were run contemporaneously with the same protein samples. Data are representative of 3 independent experiments. (E) Mutations generated by CRISPR/Cas9 in the KLF4 binding site. Red nucleotides are the PAM sequence and blue nucleotides hybridize to the sgRNA. KLF4 binding site–mutant cells were exposed to trametinib and Pam3CSK4 for 24 hours. Total RNA was analyzed by qPCR. Data repre sent mean ± SEM (n = 3). Data were analyzed with 1-way ANOVA followed by Dunnett’s (B and E) or Tukey’s multiple-comparisons test (C) or with 2-tailed unpaired t test (D). **P < 0.01; ***P < 0.001.

Article Snippet: The following cDNAs were subcloned into pcDNA3.1 (Invitrogen) or pMXs-IP (gift from Toshio Kitamura, University of Tokyo, Japan): mouse Klf4 (Addgene, 15920), human ERK1 (Addgene, 23509), human ERK2 (Addgene, 23498), and human ubiquitin (Addgene, 31815).

Techniques: Activity Assay, Inhibition, Dominant Negative Mutation, Western Blot, Real-time Polymerase Chain Reaction, CRISPR, Isolation, Generated, Binding Assay, Sequencing, Mutagenesis

Journal: Journal of Clinical Investigation

Article Title: IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/jci128678

Figure Lengend Snippet: Figure 6. Increased KLF4 levels in vitro and in vivo upon EGFR inhibition. (A) Representative images of KLF4 expression (green) in PHKs after erlotinib or control DMSO exposure for 24 hours. Nuclei were stained with DAPI. Scale bars: 10 μm. Data are representative of 3 independent experiments. (B and C) Immunofluorescent staining with mouse anti-KLF4 (green) and rabbit anti–IL-36γ (red) antibodies of formalin-fixed, paraf fin-embedded skin sections of acneiform eruption patients and healthy controls (HC). Nuclei were stained with DAPI. The white-boxed regions in B were zoomed separately in C. Scale bars: 50 μm (B) and 10 μm (C). Pictures are representative of 5 patients and 3 healthy individuals.

Article Snippet: The following cDNAs were subcloned into pcDNA3.1 (Invitrogen) or pMXs-IP (gift from Toshio Kitamura, University of Tokyo, Japan): mouse Klf4 (Addgene, 15920), human ERK1 (Addgene, 23509), human ERK2 (Addgene, 23498), and human ubiquitin (Addgene, 31815).

Techniques: In Vitro, In Vivo, Inhibition, Expressing, Control, Staining