Human collagen Krox up-regulates type I collagen expression in normal and scleroderma fibroblasts through interaction with Sp1 and Sp3 transcription factors.

Despite several investigations, the transcriptional mechanisms that regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a -112/-61-bp sequence in FF, ANF, and SF. Knockdown of hc-Krox by short interfering RNA and decoy strategies confirmed the transactivating effect of hc-Krox and decreased substantially COL1A1 transcription levels in all fibro-blast types. The -112/-61-bp sequence bound specifically hc-Krox but also Sp1 and CBF. Attempts to elucidate the potential interactions between hc-Krox, Sp1, and Sp3 revealed that all of them co-immunoprecipitate from FF cellular extracts when a c-Krox antibody was used and bind to the COL1A1 promoter in chromatin immunoprecipitation assays. Moreover, hc-Krox DNA binding activity to its COL1A1-responsive element is increased in SF, cells producing higher amounts of type I collagen compared with ANF and FF. These data suggest that the regulation of COL1A1 gene transcription in human dermal fibroblasts involves a complex machinery that implicates at least three transcription proteins, hc-Krox, Sp1, and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox

Effets de C-krox, Sp1/Sp3 sur la régulation de l'expression du collagène de type I dans des fibroblastes dermiques humains (relation avec la sclérodermie)

CAEN-BU Sciences et STAPS (141182103) / SudocSudocFranceF

NFκB signaling in <i>a/a ma ft/ma ft/J</i> mice.

<p><b>A.</b> P5 WT and <i>a/a ma ft/ma ft/J</i> mouse skin cryosections were immunostained with an anti-p50 antibody. Fluorescence was visualized with a 20x lens. Scale bar = 50 µm. (<b>B</b>). Epidermal protein extracts from P5 WT and <i>a/a ma ft/ma ft/J (ft/ft)</i>mice were submitted to WB, performed with anti-p50, anti-p-p65 and anti-β-actin antibodies, and detected by chemiluminescence. Quantification of p50 and p-p65 expression normalized to β-actin is represented in histograms. (<b>C</b>). Total RNAs were extracted from WT and <i>a/a ma ft/ma ft/J (ft/ft)</i> mice dorsal skin, and real-time PCRs were performed using primers specific for <i>Vcam</i>, <i>Icam</i>, and <i>Il6</i>. Ct values were normalized to <i>hprt</i>. The experiment was realized in groups of 10 mice and statistically significant differences were calculated with the student T-test (**p<0.1 and ***p<0.001).</p

Acanthosis and inflammation in 5-days-old <i>a/a ma ft/ma ft/J</i> skin.

<p><b>A.</b> Dorsal skin of P5 C57blk6/J wild type (WT) and flaky tail (<i>a/a ma ft/ma ft/J</i>) mice was collected, fixed in PFA 4% and embedded in paraffin. H&E staining was performed and sections were visualized with a 20x lens. Thickening of the epidermis, hypogranulosis (white arrowheads), lymphocytic exocytosis (arrows) and inflammatory infiltrates (black arrowheads) are observed. Magnification of two representative fields is presented in order to distinguish inflammatory infiltrates in the <i>a/a ma ft/ma ft/J</i> mouse skin. (<b>B</b> and <b>C</b>)<b>.</b> Dorsal skin biopsies were embedded in OCT and cryosections were prepared. Immunofluorescence was performed using antibodies against keratin 5 (KRT5) <b>(B)</b> and keratin 6 (KRT6) <b>(C)</b>. Fluorescence was visualized with a 20x lens (LSM 700 Zeiss confocal microscope (B) or Zeiss Axiovision microscope (C)). Scale bars = 50 µm.</p

Increased Sprr2 expression in <i>a/a ma ft/ma ft/J</i> mouse epidermis.

<p>A. cDNA was prepared from P5 WT and <i>a/a ma ft/ma ft/J (ft/ft)</i> mice, real-time PCRs were performed using primers specific for <i>Sprr2a</i> and <i>Sprr2d</i>. Ct values were normalized with the <i>hprt</i> house-keeping gene. The experiment was realized in groups of 10 mice and statistically significant differences were calculated with the student T-test (***p<0.001). B. P5 WT and <i>a/a ma ft/ma ft/J</i> mouse skin cryosections were immunostained with an anti-SPRR2 antibody. Fluorescence was visualized with a 20x lens. Scale bar = 50 µm.</p

Real time PCR analysis of gene expression in K14-TSLP tg mouse skin.

<p>cDNA was prepared from P5 WT and <i>K14-TSLP tg</i> mice, and real-time PCRs were performed using primers specific for (A) <i>Il13</i>, (B) <i>Il6</i>, (C) <i>Sprr2a</i> and (D) <i>Sprr2d</i>. The experiment was realized in groups of 5 WT and 5 <i>K14-TSLP</i> P5 mice and statistically significant differences were calculated with the student T-test (*p<0.05 and **p<0.01).</p

Characterization of a Non-fibrillar-Related Collagen in the Mollusc Haliotis tuberculata and its Biological Activity on Human Dermal Fibroblasts

International audienc

SOX9 Exerts a Bifunctional Effect on Type II Collagen Gene (COL2A1) Expression in Chondrocytes Depending on the Differentiation State

International audienc

Chondroitin sulphate decreases collagen synthesis in normal and scleroderma fibroblasts through a Smad-independent TGF-β pathway - implication of C-Krox and Sp1

International audienceDespite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. In this study, we determined the effects of both native ichtyan chondroïtin sulphate (CS) and its derived hydrolytic fragments (CSf) on human normal (NF) and scleroderma (SF) fibroblasts. Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF. These glycosaminoglycan molecules repress COL1A1 gene transcription through a -112/-61 bp sequence upstream the start site of transcription and imply hc-Krox and Sp1 transcription factors. In addition, CS and CSf induced a down-regulation of TbetaRI expression. As a conclusion, our findings highlight a possible new role for CS and CSf as anti-fibrotic molecules and could help in elucidating the mechanisms of action by which CS and CSf exert their inhibitory effect on type I collagen synthesis