Expression of the α5 Integrin Subunit Gene Promoter Is Positively Regulated by the Extracellular Matrix Component Fibronectin through the Transcription Factor Sp1 in Corneal Epithelial Cells in Vitro

Abstract

The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin ␣ 5 ␤ 1 . In this work, we determined whether the activity directed by the ␣ 5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/␣ 5 promoter-bearing plasmids was drastically increased when transfected into RCEC grown on FN-coated culture dishes. The promoter sequence mediating FN responsiveness was shown to bear a perfect inverted repeat that we designated the fibronectin-responsive element (FRE). Analyses in electrophoretic mobility shift assays provided evidence that Sp1 is the predominant transcription factor binding the FRE. Its DNA binding affinity was found to be increased when RCEC are grown on FN-coated dishes. The addition of the MEK kinase inhibitor PD98059 abolished FN responsiveness suggesting that alteration in the state of phosphorylation of Sp1 likely accounts for its increased binding to the ␣ 5 FRE. The FRE also proved sufficient to confer FN responsiveness to an otherwise unresponsive heterologous promoter. However, site-directed mutagenesis indicated that only the 3 half-site of the FRE was required to direct FN responsiveness. Collectively, binding of FN to its ␣ 5 ␤ 1 integrin activates a signal transduction pathway that results in the transcriptional activation of the ␣ 5 gene likely through altering the phosphorylation state of Sp1.Corneal wounds account for a substantial proportion of all visual disabilities and medical consultations for ocular problems in North America. They can be superficial with damage limited to the epithelium or associated with a deeper involvement of the epithelial basement membrane and of the stromal lamella. Severe recurrent and persistent corneal wounds are most commonly secondary to ocular diseases and damage such as recurrent erosion, mild chemical burns, superficial herpetic infections, neuroparalytic cornea, autoimmune diseases, and stromal ulcerations due to viral or bacterial infections or to severe burns (1). Despite currently available treatments, many of these corneal wounds persist for weeks and months or else recur frequently and can progress to corneal perforation.Tissue repair requires cell migration, proliferation, and adhesion. Cell adhesion and migration in turn require extracellular matrix (ECM) 1 synthesis and assembly. ECM is a complex, cross-linked structure of proteins and polysaccharides. It organizes the geometry of normal tissues. Fibronectin (FN) is an ECM adhesion protein identified as a potential wound healing agent because of its cell attachment, migration, differentiation, and orientation properties (for a review see Refs. 2-4). In the unwounded rat eye, FN is observed by immunohistological staining at the level of the corneal epithelium basement membrane (5-7). Shortly after corneal injury, the basal cells that border the injured area and stromal keratocytes start producing massive amounts of FN (5,[8]...

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

“…Interestingly, in silico analysis of the mouse and human ␤3 integrin gene promoters (30, 31) (GenBank TM accession numbers AF026510 and AF02055, respectively) revealed considerable sequence homology across a 1.3-kb region upstream of the transcription start site and several conserved binding elements for Sp1 but not for Smad proteins. Recently, it has been demonstrated that cooperation between Sp1 and Smad proteins may represent a general regulatory mechanism for conferring the TGF␤1 inducibility of several genes, including ␤5, ␣5, and ␣11 integrins (13,20,32) and different types of collagen (33,34). In these studies the antibiotic mithramycin A selectively and efficiently reduced TGF␤-induced collagen and ␣11 integrin expression in mesenchymal cells via inhibition of Sp1 binding to the gene promoter.…”

“…Cooperation between the Smad proteins and the transcription factor Sp1 may represent a general mechanism for conferring TGF␤1 inducibility of several genes, including integrins (13,20). We sought to determine whether Sp1 was involved in the up-regulation of ␣v␤3 integrin induced by TGF␤1.…”

“…7, A and B). To further validate the role of p38 MAPK, we transiently overexpressed a kinase mutant p38 MAPK (p38-KM) construct in fibroblasts (20). As expected, an increased level of p38 MAPK protein was seen in p38-KMtransfected cells but not in cells transfected with empty pcDNA3 vector (supplemental Fig.…”

Angiotensin II–Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1

“…Larouche et al identified 2 residues in Sp1, namely Thr453 and Thr739, that are phosphorylated by ERK. 8 We recently demonstrated that both these residues in Sp1 are required for ERK-dependent fibroblast growth factor-2 repression of PDGF receptor-␣ transcription in SMCs. 54 Moreover, these residues are required for ERK-dependent galectin-1 regulation of p27 transcription in carcinoma cells.…”

“…For example, Sp1 phosphorylation has been linked with shear stress induction of the tissue factor promoter, 4 hepatocyte growth factor-inducible vascular endothelial growth factor/ vascular permeability factor (VEGF/VPF) transcription, 5 epidermal growth factor-inducible apolipoprotein A-I expression, 6 and T-cell receptor-inducible interleukin-21 receptor gene expression. 7 Protein kinases able to phosphorylate Sp1 include extracellular signal-regulated kinase, 8,9 MEK1, 10 casein kinase II, 11 HIV type 1 Tat, 12 and DNA-dependent protein kinase. 3 We have shown previously that Sp1 phosphorylation and expression of the extrinsic apoptotic ligand, FasL are processes critically dependent on the integrity of the atypical protein kinase C, PKC-, as demonstrated by the blockade of both of these processes with dominant-negative (kinase dead) PKC-.…”