Tandem Sp1/Sp3 sites together with an Ets-1 site cooperate to mediate α11 integrin chain expression in mesenchymal cells
Introduction
Integrins are heterodimeric cell adhesion receptors composed of non-covalently associated α and β chains. Analyses of genomic sequences in a number of vertebrate species have revealed that the integrin gene family is composed of 18 α subunits and 8 β subunits, which can combine into 24 heterodimers. Integrins fulfill a dual role as mechanical links and signalling receptors involved in a variety of biological processes (Hynes, 2002).
A subgroup of integrins composed of α1β1, α2β1, α10β1 and α11β1 act as collagen receptors (Gullberg and Lundgren-Åkerlund, 2002). Although these collagen-binding integrins also display a lower affinity for a restricted number of other ligands, most notably laminins (Pfaff et al., 1994), they show the highest affinity towards collagens. Available data on gene-deficient mice support the view that these integrins exert their biological role when binding collagens (Pozzi et al., 1998, Chen et al., 2002, Holtkotter et al., 2002). At the molecular level, the cell-collagen interactions involve residues in the MIDAS site of the integrin αI domain and GFOGER-like sequences in collagens (Knight et al., 2000, Zhang et al., 2003). α1- and α10-I domains show a preference for the network-forming collagens IV and VI (Tuckwell et al., 1995, Dickeson and Santoro, 1998, Kern and Marcantonio, 1998), whereas α2-I and α11-I domains appear to prefer the fibrillar collagens (Tulla et al., 2001, Zhang et al., 2003). In addition to the different collagen specificities of collagen-binding integrins, they vary with respect to the mode by which they generate intracellular signals.
At the cellular level, collagen-binding integrins exert their biological roles by functioning in cell attachment, cell migration, collagen reorganization and cell proliferation (Gullberg and Lundgren-Åkerlund, 2002), and also by taking part in matrix assembly and matrix remodeling (Velling et al., 2002). Collagen-binding integrins have been shown to control collagen turnover by regulating collagen synthesis, matrix metalloproteinase (MMP) synthesis (White et al., 2004) and collagen phagocytosis (Lee et al., 1996, Segal et al., 2001).
Collagen-binding integrins show tissue-specific expression patterns and have unique non-redundant functions. α1β1 integrin is prominently expressed in smooth muscle and capillary endothelium, where it appears to interact with the basement membrane collagen IV (Duband et al., 1992, Gardner et al., 1996), whereas α1β1 on fibroblasts interacts with fibrillar collagens. Mice deficient in α1 display a dermal defect due to dysregulated collagen turnover and a proliferation defect in fibroblasts (Gardner et al., 1999). α2β1 integrin is expressed in a variety of cell types including platelets, epithelia, capillary endothelium and fibroblasts (Wu and Santoro, 1994). Mice lacking the α2 chain display a platelet adhesion defect with respect to collagen in vitro (Chen et al., 2002, Holtkotter et al., 2002) and in vivo (Gruner et al., 2003, He et al., 2003). An important role of α1β1 and α2β1 integrins in tumor angiogenesis has been suggested by antibody studies (Senger et al., 1997), and tumors grown in α1-defective mice are characterized by reduced angiogenesis (Pozzi et al., 2000).
Due to their recent discovery, less is known about the function of the α10 (Camper et al., 1998) and α11 (Velling et al., 1999) integrin chains. α10 is expressed in cartilage (Camper et al., 1998, Camper et al., 2001) and α10-deficient mice display a mild cartilage phenotype (Bengtsson et al., 2005). α11 is expressed in a subset of non-muscle mesodermal cells in the perichondrium, periosteum and the ectomesenchyme in the head (Tiger et al., 2001, Popova et al., 2004).
In an effort to characterize the molecular basis for the restricted ectomesenchymal and mesodermal expression pattern of α11, we have set out to characterize the transcriptional regulation of α11. In the current report we extend our previous analysis of the core ITGA11 promoter (Zhang et al., 2002) and identify a proximal promoter driving high-level transcription in mesenchymal cells. We identify tandem low affinity Sp1/Sp3 binding sites and an Ets-1 binding site as being able to mediate the regulation of α11 expression and suggest that the binding of specific combinations of trans-activating factors is necessary for the mesenchymal α11 expression pattern observed in vivo.
Section snippets
Identification of the ITGA11 proximal promoter
We have previously used luciferase constructs to analyze the region upstream of the major transcription start site in the human integrin α11 gene (ITGA11) for promoter activity. In this study we constructed a panel of 12 additional luciferase constructs to map the 3 kb promoter region in more detail. We routinely used the easily transfectable cell line HT1080 for the promoter analyses (low α11 expression), but confirmed the obtained results in primary fibroblasts (high α11 expression).
Discussion
We have shown previously that 3 kb of the sequence upstream of the transcription start site in the human α11 integrin promoter could drive transcription in vitro (Zhang et al., 2002). We now map the sites conferring proximal promoter activity on two Sp1/Sp3 binding sites and an Ets-1-like binding site and show that the proximal promoter itself is able to direct a certain cell-type specific expression.
A growing number of integrin genes have been found to be regulated by various combinations of
Cells and reagents
Primary human foreskin fibroblasts AG1518 (Genetic Mutant Cell Repository, Camden, NJ), human fibroblasts BJ (ATCC, VA), primary mouse embryonic fibroblasts (MEF) (Popova et al., 2004), human fibrosarcoma cell line HT1080 (provided by S. Johansson, Uppsala), human osteosarcoma cell line U2OS (provided by C. Svensson, Uppsala) and human chorioncarcinoma cell line JAR (provided by L. Sorokin, Lund) were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum. The Drosophila
Acknowledgements
This research was supported by grants from Svenska Vetenskapsrådet NT-K (DG), the Wenner-Gren Foundation (WMZ), Konung Gustaf V:s minnesfond (DG) and Meltzerfondet (DG ). We are grateful for the generous gift of Sp1/Sp3 vectors from G. Suske, the Ets-1 vector from P. Marsden and the β-gal vector from Y. Engström.
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