Regulation of fibronectin gene expression in cardiac fibroblasts by scleraxis.
The glycoprotein fibronectin is a key component of the extracellular matrix. By interacting with numerous matrix and cell surface proteins, fibronectin plays important roles in cell adhesion, migration and intracellular signaling. Up-regulation of fibronectin occurs in tissue fibrosis, and previous studies have identified the pro-fibrotic factor TGFβ as an inducer of fibronectin expression, although the mechanism responsible remains unknown. We have previously shown that a key downstream effector of TGFβ signaling in cardiac fibroblasts is the transcription factor scleraxis, which in turn regulates the expression of a wide variety of extracellular matrix genes. We noted that fibronectin expression tracked closely with scleraxis expression, but it was unclear whether scleraxis directly regulated the fibronectin gene. Here, we report that scleraxis acts via two E-box binding sites in the proximal human fibronectin promoter to govern fibronectin expression, with the second E-box being both sufficient and necessary for scleraxis-mediated fibronectin expression to occur. A combination of electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that scleraxis interacted to a greater degree with the second E-box. Over-expression or knockdown of scleraxis resulted in increased or decreased fibronectin expression, respectively, and scleraxis null mice presented with dramatically decreased immunolabeling for fibronectin in cardiac tissue sections compared to wild-type controls. Furthermore, scleraxis was required for TGFβ-induced fibronectin expression: TGFβ lost its ability to induce fibronectin expression following scleraxis knockdown. Together, these results demonstrate a novel and required role for scleraxis in the regulation of cardiac fibroblast fibronectin gene expression basally or in response to TGFβ.
Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre, 351 Tache Avenue, Winnipeg, MB, R2H 2A6, Canada.|Department of Physiology and Pathophysiology, College of Medicine, University of Manitoba, Winnipeg, MB, Canada.|Division of Cardiology, School of Medicine, University of Colorado Denver, Anschutz Medical Campus, RC2- Room 8450, Aurora, CO, 80045, USA.
Rushita A
RA
Bagchi
Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre, 351 Tache Avenue, Winnipeg, MB, R2H 2A6, Canada.
Justin
J
Lin
Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre, 351 Tache Avenue, Winnipeg, MB, R2H 2A6, Canada.
Ryan
R
Wang
Institute of Cardiovascular Sciences, St. Boniface Hospital Albrechtsen Research Centre, 351 Tache Avenue, Winnipeg, MB, R2H 2A6, Canada. mczubryt@sbrc.ca.|Department of Physiology and Pathophysiology, College of Medicine, University of Manitoba, Winnipeg, MB, Canada. mczubryt@sbrc.ca.
Michael P
MP
Czubryt
D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D024721:E-Box Elements / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology*; D058628:Myofibroblasts / Q000378:metabolism; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D017207:Rats, Sprague-Dawley; D015533:Transcriptional Activation / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D005353:Fibronectins; D016212:Transforming Growth Factor beta; C503815:Scx protein, mouse
Extracellular matrix; Gene expression; Gene regulation; Promoter; Transcription