Elsevier

Matrix Biology

Volume 25, Issue 2, March 2006, Pages 118-129
Matrix Biology

Tandem Sp1/Sp3 sites together with an Ets-1 site cooperate to mediate α11 integrin chain expression in mesenchymal cells

https://doi.org/10.1016/j.matbio.2005.10.002Get rights and content

Abstract

α11β1 integrin is a collagen receptor, which is expressed in a highly regulated manner in a specific subset of ectomesenchymally and mesodermally derived cells. We previously established that a 3 kb region upstream of the transcription start site of the ITGA11 gene efficiently induced α11 transcription in a cell-type specific manner. Using the human fibrosarcoma cell line HT1080 and human skin fibroblasts, we now report that the majority of the activity in the proximal promoter resides in a region spanning nt + 25 to nt − 176. Mutation and deletion analyses using luciferase reporter assays showed that tandem low affinity Sp1/Sp3 binding sites, together with an Ets-1-like binding site, were needed for the proximal promoter activity in mesenchymal cells. EMSAs and supershift assays showed that Sp1 and Sp3 both bind to the Sp1/Sp3 binding sites, whereas occupation of the Ets-1 binding site appears to be Sp3-dependent. Chromatin immunoprecipitation assays verified that Sp1, Sp3 and Ets-1 can bind the promoter in vivo. In heterologous Drosophila SL2 cells, Sp1, Sp3 and Ets-1 all transactivated the α11 promoter, with Sp1 being the most efficient activator. The lack of any synergistic effect of Sp1/Sp3 and Ets-1 in SL2 cells indicates that an Ets family member other than Ets-1 might be involved in regulating α11 transcription in mesenchymal cells. The central role of Sp1 in regulating α11 RNA transcription was further verified by the ability of the Sp1 inhibitor mithramycin A to efficiently attenuate α11 RNA and protein levels in primary fibroblasts. The proximal promoter itself was able to confer cell-type specific transcription on HT1080 cells and embryonic fibroblasts but not on U2OS and JAR cells. We speculate that the “mesenchymal signature” of α11 integrin gene expression is controlled by the activity of Sp1/Sp3, fibroblast-specific combinations of Ets family members and yet unidentified enhancer-binding transcription factors.

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.

References (61)

  • S. Gruner et al.

    Multiple integrin–ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo

    Blood

    (2003)
  • D. Gullberg et al.

    Collagen-binding I domain integrins — what do they do?

    Prog. Histochem. Cytochem.

    (2002)
  • B. Han et al.

    MRG1 expression in fibroblasts is regulated by Sp1/Sp3 and an Ets transcription factor

    J. Biol. Chem.

    (2001)
  • L. He et al.

    The contributions of the α 2β1 integrin to vascular thrombosis in vivo

    Blood

    (2003)
  • O. Holtkotter et al.

    Integrin α2-deficient mice develop normally, are fertile, but display partially defective platelet interaction with collagen

    J. Biol. Chem.

    (2002)
  • R.O. Hynes

    Integrins: bidirectional, allosteric signaling machines

    Cell

    (2002)
  • B. Jacquelin et al.

    Characterization of inherited differences in transcription of the human integrin α2 Gene

    J. Biol. Chem.

    (2001)
  • C.G. Knight et al.

    The collagen-binding A-domains of integrins α1β1 and α2β1 recognize the same specific amino acid sequence, GFOGER, in native (triple-helical) collagens

    J. Biol. Chem.

    (2000)
  • T.T. Li et al.

    Genetic variation responsible for mouse strain differences in integrin α2 expression is associated with altered platelet responses to collagen

    Blood

    (2004)
  • M. Marin et al.

    Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation

    Cell

    (1997)
  • W. Nishida et al.

    A triad of serum response factor and the GATA and NK families governs the transcription of smooth and cardiac muscle genes

    J. Biol. Chem.

    (2002)
  • S.N. Popova et al.

    The mesenchymal α11β1 integrin attenuates PDGF-BB-stimulated chemotaxis of embryonic fibroblasts on collagens

    Dev. Biol.

    (2004)
  • G. Suske

    The Sp-family of transcription factors

    Gene

    (1999)
  • A. Tajima et al.

    Mouse integrin av promoter is regulated by transcriptional factors Ets and Sp1 in melanoma cells

    Biochim. Biophys. Acta

    (2000)
  • Y. Takagi et al.

    Conserved neuron promoting activity in Drosophila and vertebrate laminin α1

    J. Biol. Chem

    (1996)
  • C.-F. Tiger et al.

    α11β1 integrin is a receptor for interstitial collagens involved in cell migration and collagen reorganization on mesenchymal non-muscle cells

    Dev. Biol.

    (2001)
  • M. Tulla et al.

    Selective binding of collagen subtypes by integrin α 1I, β 2I, and α 10I domains

    J. Biol. Chem.

    (2001)
  • T. Velling et al.

    cDNA cloning and chromosomal localization of human α11 integrin. A collagen-binding, I domain-containing, β1-associated integrin α-chain present in muscle tissues

    J. Biol. Chem.

    (1999)
  • T. Velling et al.

    Polymerization of type I and III collagens is dependent on fibronectin and enhanced by integrins α11β1 and α2β1

    J. Biol. Chem.

    (2002)
  • F. Verrecchia et al.

    Blocking sp1 transcription factor broadly inhibits extracellular matrix gene expression in vitro and in vivo: implications for the treatment of tissue fibrosis

    J. Invest. Dermatol.

    (2001)
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