Scleraxis and NFATc Regulate the Expression of the Pro-α1(I) Collagen Gene in Tendon Fibroblasts

Veéronique Leéjard,Gaëlle Brideau,Freédeéric Blais,Ruchanee Salingcarnboriboon,Gerhard Wagner,Michael H.A Roehrl,Masaki Noda,Delphine Duprez,Pascal Houillier,Jerome Rossert

doi:10.1074/jbc.m610113200

Abstract

The combinatorial action of separate cis-acting elements controls the cell-specific expression of type I collagen genes. In particular, we have shown that two short elements located between -3.2 and -2.3 kb and named TSE1 and TSE2 are needed for expression of the mouse COL1a1 gene in tendon fibroblasts. In this study, we analyzed the trans-acting factors binding to TSE1 and TSE2. Gel shift experiments showed that scleraxis (SCX), which is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts, binds TSE2, preferentially as a SCX/E47 heterodimer. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring either four copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Analysis of TSE1 showed that it contains a consensus binding site for NFATc transcription factors. This led us to show that the NFATc4 gene is expressed in tendons of developing mouse limbs and in TT-D6 cells, a cell line that has characteristics of tendon fibroblasts. In gel shift assays, TSE1 bound NFATc proteins present in nuclear extracts from TT-D6 cells. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring four copies of TSE1 cloned upstream of the COL1a1 minimal promoter. By contrast, inhibition of the nuclear translocation of NFATc proteins in TT-D6 cells strongly inhibited the expression of the COL1a1 gene. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene specifically in tendon fibroblasts.

Highlights

DNA Constructs—For transfection experiments, various segments of the COL1a1 proximal promoter were cloned upstream of the lacZ gene in the placH reporter plasmid [4]. pJ320 contains a segment of the COL1a1 promoter extending from Ϫ3,150 to ϩ110 bp. p4TSE1mp contains four copies of TSE1 cloned upstream of the COL1a1 minimal promoter that extends from Ϫ220 to ϩ110 bp. p4TSE2mp contains four copies of TSE2 cloned upstream of the COL1a1 minimal promoter. pJ320-MTSE2 is identical to pJ320, except that the E-box in TSE2 was mutated (CACGTG 3 GGATCC) by site-directed mutagenesis using the QuickChange site-directed mutagenesis kit (Stratagene) and following the manufacturer’s instructions
This complex was competed by an excess of unlabeled TSE1 probe (Fig. 4, lane 2) but not by a mutated probe deleted of the GGAAA motif (Fig. 4, lane 3). It was competed by a double-stranded oligonucleotide corresponding to a well described NFATc-binding site in the mouse IL-4 promoter but not by an oligonucleotide in which this binding site had been mutated (Fig. 4, lanes 4 –9). These results indicate that TSE1 binds NFATc proteins that are present in tendon fibroblasts
By generating transgenic mice harboring various segments of the COL1a1 proximal promoter cloned upstream of the lacZ reporter gene, we have previously shown that two short cis-acting elements, named TSE1 and TSE2, are necessary for activation of this promoter in tendon fibroblasts [8]

Summary

Introduction

The segment of the mouse pro-␣1(I) collagen (COL1a1) promoter located downstream of Ϫ2.3 kb induces high level expression of reporter genes in osteoblasts of transgenic mice, whereas the segment of COL1a1 located between Ϫ3.2 and Ϫ2.3 kb drives high level expression of the lacZ reporter gene in tendon, ligament, and fascia fibroblasts (4, 6 – 8) Analysis of this latter segment has shown that two short cisacting elements, named TSE14 and TSE2, are necessary for activation of the COL1a1 promoter in tendon fibroblasts [8]. It has been shown that a biologically active variant of Smad promotes the differentiation of C3H10T1/2 mesenchymal cells into tendon fibroblasts in the presence of bone morphogenetic protein 2, indicating that this Smad pathway is involved in the differentiation of mesenchymal cells into tendon fibroblasts [19] It induced the expression of the SCX, EphA4, and COL1a1 genes. These data suggest that SCX and NFATc activate the COL1a1 gene in tendon fibroblasts, and are part of a regulatory network involved in differentiation of mesenchymal cells into tendon fibroblasts

Methods

Results

Conclusion