Additional file 1: of YAP/TAZ regulates TGF-β/Smad3 signaling by induction of Smad7 via AP-1 in human skin dermal fibroblasts

Figure S1. Smad7, YAP, and TAZ mRNA expression in human skin dermis in vivo and primary dermal fibroblasts in vitro. (A) Human skin dermis was prepared by cutting off epidermis at a depth of 1 mm by cryostat. Dermal total RNA was prepared using a commerical kit (RNeasy midikit, Qiagen, Chatsworth, CA). N = 8. (B) Total RNA from primary dermal dibroblasts was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). N = 3 mRNA levels for Smad7, YAP, and TAZ were determined by real-time RT-PCR. mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. Data are expressed as mean + SEM, *p < 0.05 vs Smad7. Figure S2. Full-length Western blots for Fig. 1a. Figure S3. Full-length Western blots for Fig. 2b. Figure S4. Full-length Western blots for Fig. 3b. Figure S5 A. Full-length Western blots for Fig. 5d. B. Full-length Western blots for Fig. 5f. C. Full-length Western blots for Fig. 5g. Figure S6 YAP/TAZ knockdown did not alter AP-1 family transcription factors mRNA expression. Primary dermal fibroblasts were transfected with non-specific control siRNA or YAP/TAZ siRNAs (400 nM) for 48 h. AP-1 family transcription factors mRNA levels were quantified by real-time RT-PCR. mRNA levels were normalized to mRNA for 36B4, a ribsomal protein used as an internal control for quantitation. N = 4, data are expressed as mean + SEM. Figure S7. Smad3 antibody specificity testing by Smad3. Primary dermal fibroblasts were transfected with with non-specific control siRNA or Smad3 siRNA (400 nM). 48 h after transfection, cells were treated with TGF-β1 (ng/ml) for one hour and whole cell extract was prepared. Protein levels of phospho-Smad3 were determined by Capillary electrophoresis immunoassay and normalized to β-actin (loading control). Band intensities were quantified by Compass software. Bands show representative digital images. (PDF 175 kb

CCN2 and FN are primarily expressed in the dermis of normal human skin, stromal tissues of skin SCC, and wounded human skin.

<p>(A, B) Epidermis and dermis were captured by LCM. Total RNA was extracted from captured tissue, and mRNA levels were quantified by real-time RT-PCR. CCN2 (A) and FN (B) mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Data are relative levels to 36B4 (mean±SEM), N = 6, *p<0.05. (C) Double immunostaining for CCN2 and FN in normal human skin. OCT-embedded normal human skin sections (7μm) were co-immunofluorescence stained with CCN2 and FN. Representative of six individuals. Bar = 50μm. (D) Expression of CCN2 and FN in the stromal tissues of SCC was determined by immunohistology. Arrow heads indicate tumor islands. Representative of five SCC. Bar = 100μm. (E) Double immunostaining for CCN2 and FN. Representative of six individuals. Bar = 50μm. (F) Partial thickness wounds were made in forearm skin of healthy adult individuals by CO₂ laser (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173191#sec002" target="_blank">Methods</a> for details). Skin samples were obtained at indicated times, and mRNA levels were quantified by real-time RT-PCR. CCN2 and FN mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Mean±SEM, N = 6, *p<0.05 vs control.</p

CCN2 functions primary mediator in Smad3-dependent expression of FN in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with the indicated siRNAs (20nM) and vectors (1μg). Total RNA and whole cell extract were prepared 48 hours after transfection. mRNA and protein levels were quantified by real-time RT-PCR and Western analysis, respectively. mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative Western blots. (A, C) CCN2 and FN mRNA levels. (B, D) CCN2, FN, Smad2, and Smad3 protein levels. (E) The ability of CCN2 to regulate FN expression is dependent on intact TGF-β signaling. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. Data are expressed as mean±SEM, N = 3, *p<0.05 vs control.</p

FN expression is regulated by CCN2 in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with non-specific control siRNA or CCN2 siRNAs (20nM) (A, B, C), or control vector (pCDNA3.1, 2μg) or increasing amounts of CCN2 vector (0.5, 1, and 2μg) (D, E). Total RNA and whole cell extract were prepared 48 hours after transfection. (A) CCN2 mRNA levels. (B) FN mRNA levels. (C) CCN and FN Protein levels. (D) FN mRNA levels. (E) CCN and FN protein levels. mRNA levels were quantified by real-time RT-PCR. Protein levels were determined by ProteinSimple capillary electrophoresis immunoassay (C) and Western analysis (E). mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative digital images (C) and Western blots (E). Data are expressed as mean±SEM, N = 3–5, *p<0.05.</p

CCN2 and FN expression are primarily regulated by Smad3 in primary dermal fibroblasts.

<p>Primary dermal fibroblasts (1 × 10⁶) were transfected with the indicated siRNAs (20nM). Total RNA and whole cell extract were prepared 48 hours after transfection. mRNA levels were quantified by real-time RT-PCR. Protein levels were determined by ProteinSimple capillary electrophoresis immunoassay (B, C) and Western blots (E). mRNA levels were normalized to mRNA for 36B4, a ribosomal protein used as an internal control for quantitation. Protein levels were normalized by β-actin (loading control). Insets show representative digital images (B, C) and Western blots (E). (A) Smad2 and Smad3 mRNA levels. (B) CCN2, FN, and Smad3 protein levels. (C) CCN2, FN, and Smad2 protein levels. (D) CCN2 and FN mRNA levels. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. (E) CCN2, FN, Smad2, and Smad3 protein levels. 32 hours after transfection, cell were treated with TGF-β1 (5 ng/ml) for 16 hours. Data are expressed as mean±SEM, N = 3, *p<0.05 vs control.</p

Oxidative exposure induces CCN1 expression and inhibits type I procollagen in adult human primary dermal fibroblasts.

<p>Fibroblasts were exposed to vehicle (CTRL) or H₂O₂ on two successive days (ROS), as described in Methods. (a) Oxidative exposure induces endogenous ROS. RedoxSensor Red CC-1, an indicator of ROS level, was visualized and quantified by fluorescence microscopy, as described in Methods. Oxidative exposure induces CCN1 (b) mRNA and (c) protein. Oxidative exposure reduces Type I procollagen (COL-1) (d) mRNA and (e) protein. mRNA levels were quantified by real-time RT-PCR and normalized to an internal control housekeeping gene (36B4). Protein levels were determined by Western blot and normalized to an internal control (β-actin). Inset shows representative blot. Data are means±SEM, N = 3, *<i>p</i><0.05 vs. CTRL.</p

CCN1 induction by oxidative exposure mediates type I procollagen reduction in human dermal fibroblasts.

<p>Fibroblasts were exposed to vehicle (CTRL) or H₂O₂ on two successive days (ROS), as described in Methods. Cells were transfected with control siRNA (CTRL si) or CCN1 siRNA (CCN1 si), and harvested two days later. (a) CCN1 siRNA reduces oxidant-induced CCN1 protein. Knockdown of oxidant-induced CCN1 prevents reduction of type I procollagen (COL-1) (b) mRNA and (c) protein. mRNA levels were quantified by real-time RT-PCR normalized to internal control housekeeping gene (36B4). COL-1 protein levels were determined by Western blot normalized to an internal control (β-actin). Inset shows representative blot. Data are means±SEM, N = 3, *<i>p</i><0.05 vs. CTRL, **<i>p</i><0.05 vs. oxidative exposure with CTRL si.</p

Oxidant exposure activates AP-1 binding site-dependent CCN1 promoter activity in human dermal fibroblasts.

<p>Fibroblasts were exposed to vehicle (CTRL) or H₂O₂ on two successive days (ROS), as described in Methods. Fibroblasts were transfected with CCN1 promoter/luciferase reporter constructs: 1, wild-type (−883/+1); 2, AP-1 mutation (−883/+1); and 3, AP-1 deletion (605/+1). Luciferase activities were determined two days after transfection. (a) Mutation or deletion of AP-1 binding site abolishes activation of CCN1 promoter by oxidative exposure. Data are means±SEM, N = 3, *<i>p</i><0.05 vs. CTRL. (b) Nucleotide sequence of the CCN1 proximal promoter. AP-1-binding sequences are marked in box. Heavy underline denotes electrophoretic mobility shift assay (EMSA) probe. Arrow indicates transcription start site. (c) Oxidant-exposure increases protein-binding to AP-1 site in CCN1 promoter. DNA/protein complex was analyzed by EMSA. Arrows indicate specific retarded complexes. Closed triangle indicates free probes. Results are representative of at least three independent experiments.</p

c-Jun mediates oxidative exposure induction of CCN1 and reduction of type I procollagen in human dermal fibroblasts.

<p>Fibroblasts were exposed to vehicle (CTRL) or H₂O₂ on two successive days (ROS), as described in Methods. (a) Oxidative exposure induces c-Jun mRNA and (b) total and phospho c-Jun proteins. mRNA levels were quantified by real-time RT-PCR normalized to an internal control housekeeping gene (36B4). Protein levels were determined by Western blot normalized to internal control (β-actin). Inset shows representative blot. (c) Dominant negative c-Jun (DN c-Jun) reduces oxidant activation of CCN1 promoter. Fibroblasts were co-transfected with CCN1 promoter/luciferase reporter construct (−883/+1) and control (CTRL) or DN c-Jun expression vector, and analyzed two days later. (d–f) DN c-Jun reduces oxidant induction of (d) CCN1 mRNA and (e) CCN1 protein. Fibroblasts were transfected with CTRL or DN c-Jun expression vectors, and analyzed two days later. (f) DN c-Jun mitigates loss of type I procollagen (COL-1) by oxidative exposure. Fibroblasts were transfected with CTRL or DN c-Jun alone or together with CCN1 expression vectors, and analyzed two days later. Data are means±SEM, N = 3, *<i>p</i><0.05 vs. CTRL, **<i>p</i><0.05 vs. CTRL with oxidative stress, ***<i>p</i><0.05 vs DN-c-Jun alone.</p

Oxidant Exposure Induces Cysteine-Rich Protein 61 (CCN1) via c-Jun/AP-1 to Reduce Collagen Expression in Human Dermal Fibroblasts

<div><p>Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin <i>in vivo</i>. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin.</p></div