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

The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin α5β1. In this work, we determined whether the activity directed by the α5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/α5 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 α5 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 α5β1 integrin activates a signal transduction pathway that results in the transcriptional activation of the α5 gene likely through altering the phosphorylation state of Sp1. The accumulation of fibronectin (FN) in response to corneal epithelium injury has been postulated to turn on expression of the FN-binding integrin α5β1. In this work, we determined whether the activity directed by the α5 gene promoter can be modulated by FN in rabbit corneal epithelial cells (RCEC). The activity driven by chloramphenicol acetyltransferase/α5 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 α5 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 α5β1 integrin activates a signal transduction pathway that results in the transcriptional activation of the α5 gene likely through altering the phosphorylation state of Sp1. extracellular matrix fibronectin rabbit corneal epithelial cells fibronectin-responsive element electrophoretic mobility shift assays chloramphenicol acetyltransferase mitogen-activated protein kinases extracellular signal-regulated kinase MAPK/ERK kinase human growth hormone base pair oncostatin M low density lipoprotein receptor vascular endothelial growth factor acetylcholine receptor fetal bovine serum 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 (1Reim M. Kottek A. Schrage N. Prog. Retin. Eye Res. 1997; 16: 183-225Crossref Scopus (47) Google Scholar). 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. 2Humphries M.J. Obara M. Olden K. Yamada K.M. Cancer Invest. 1989; 7: 373-393Crossref Scopus Google Scopus Google Scopus Google Scholar). In the FN is by the of the corneal epithelium basement membrane Google Invest. Google A. Scopus Google Scholar). corneal the cells that the and stromal of FN Google K. K. Eye Res. Scopus Google M. Invest. Google A. A. Eye Res. Scopus Google M. N. Eye Res. 1997; Scopus Google Scholar). FN corneal cell migration in Scopus Google K. Yamada K.M. Invest. Google and in Invest. Google by as a extracellular matrix to corneal epithelial cells as the K. Yamada K.M. Invest. Google M. Scopus Google Scholar). the wound is the of FN Google Invest. Google Cell Scopus Google N. Scopus Google Scholar). The in FN expression that has been to corneal wound healing was postulated to be with the expression of its integrin receptor α5β1 Google as has also been shown for and and integrin receptor and Invest. Google Invest. Google 1997; Scopus Google Scholar). the integrin α5β1 was shown to be corneal wound healing Invest. Scholar). evidence that FN can α5β1 integrin expression the protein and has been provided through FN expression in the human cell M. 1997; Scopus Google as as in Scopus Google Scholar). evidence expression of α5β1 to that of FN has also Google A. Google Scholar). a is that through its with on the of differentiation, and by through a of signal transduction the transcription of are to these of ECM such as with integrin the activation of such as adhesion mitogen-activated protein kinases and (for a review see Scopus Google Scholar). of the signal transduction pathway is of to transcriptional of that are for cell growth and The results provided evidence by on α5 gene such a of signal transduction cell adhesion properties as The of that are activation of proteins through receptor kinases MAPK/ERK kinase and Scopus Google Scholar). of through phosphorylation to the been to and transcription such as and 16: Scopus Google Cell Scopus Google Scopus Google as as of the as M. Google Scholar). In the we that FN can the transcription of the α5 integrin gene the promoter a FN responsiveness was shown to be determined by the binding of the transcription factor Sp1 to a that is of a perfect inverted repeat by can confer FN responsiveness to an otherwise unresponsive heterologous promoter. of the signal transduction pathway to require activation of the Sp1 DNA binding as a the FN responsiveness of the α5 promoter found to be by activation with the MEK kinase inhibitor these results the that the through activation of the can its synthesis in a that is on the extracellular of corneal epithelial cells the of rabbit as M. Eye Res. Scopus Google and grown to low of the or cell density for in with and human FN as Google or ECM membrane was for on the culture with and with bovine serum in and grown as of was by RCEC in the of of the inhibitor PD98059 for cells cells with and The plasmids and all bear the chloramphenicol acetyltransferase gene to DNA the human α5 gene sequence to and all a 3′ been Google Scholar). The plasmids or of the α5 FRE or its by the the promoter of the gene the that has been into its designated M. Scopus Google The Sp1 expression was provided by the expression was The in the a They the DNA sequence the human and and designated the α5 FRE or its the DNA binding for human in Scopus Google the affinity binding for the transcription factor Sp1 Scopus Google or the identified in the promoter the gene M. Scopus Google Scholar). RCEC low cells culture cells culture or cells culture cell density transfected the as by the of the and of the human growth hormone K. Scopus Google Scholar). cells transfected to the M. Scopus Google M. DNA Cell Scopus Google a density of cells culture of activity for all transfected cells determined as M. DNA Cell Scopus Google and to the of into the culture and a for of the expression of the proved to be transfected cells to the of by the and with the of the of and of In the with the Sp1 expression the was for of The for transfected to the of in be to be the by the to chloramphenicol is also provided for transfected RCEC grown on or FN-coated culture and as Google that a of was to all the in to in the α5 FRE or the affinity Sp1 as DNA was with proteins in the RCEC grown on or FN-coated culture in the of of in human cells also in as a for for by through as Res. Scopus Google Scholar). and to the of the in of proteins RCEC grown in the of FN as that and of the DNA sequence of the α5 the for human in Scopus Google the affinity binding for the transcription factor Sp1 Scopus Google or the the gene M. Scopus Google to the binding to on the in by in the of proteins RCEC grown with or in the of of with or to of a rabbit the transcription factor Sp1 in was and was for on and as of was proteins cells as a or RCEC grown on culture or with FN as was by the and of of was to of proteins on a and a of protein was also as a to protein The was in and in and a of a rabbit the transcription factor Sp1 was to the and for The was in and an in a of a The membrane was in and and by M. 1997; Scopus Google and Scopus Google provided evidence that the of expression for the the α5 integrin was modulated by the of the extracellular matrix of RCEC plasmids the gene to the human α5 gene promoter Google in to whether such an in α5 be determined by the α5 gene this a the to the gene was transfected into RCEC on or FN-coated culture cell RCEC on FN-coated the activity driven by the when transfected low cell density of the However, and for cell the activity of the was found to be and when cells are grown on FN-coated culture on of the serum into the culture to cell on FN-coated culture on the activity directed by The of the α5 promoter FN responsiveness was by RCEC an cell density on culture with or of FN to shown on the activity directed by the increased to the of FN on the culture a in was FN that the activity of the human α5 promoter can be drastically increased when RCEC are grown on FN-coated culture and that such a is cell are to many of the that are through signal transduction by binding proteins with the α5 promoter sequence required to confer FN plasmids of the α5 promoter transfected into RCEC grown density on and FN-coated culture dishes. the of the α5 promoter to the in α5 promoter activity when RCEC are grown on FN-coated However, the of the α5 to abolished the FN responsiveness of the of this sequence the of a perfect inverted repeat of the FN responsiveness of the α5 promoter to be determined by a of DNA sequence and to the α5 proteins such as and are also commonly found in the extracellular whether the ECM FN can also the expression directed by the α5 promoter in this RCEC grown to cell density on or culture transfected with the The ECM membrane and shown on the activity driven by is increased by only when RCEC are grown on The activity directed by the α5 promoter was to when FN and the ECM are on the culture dishes. However, promoter activation was when FN was on the culture of RCEC on culture with the plasmids the of the α5 promoter identified the element and that the ECM FN only a on the α5 promoter activity and that is through a element that FN responsiveness in α5 FRE can confer FN responsiveness to the promoter of the heterologous a the α5 inverted repeat to was in or the promoter of the gene M. Scopus Google Scholar). The transfected into RCEC grown on culture with or FN activity was and to the directed by the the of of the α5 FRE inverted in the 3′ or the half-site the α5 or in the promoter in The activity directed by was in RCEC grown on culture or with FN as in A. the of α5 FRE inverted are indicated by a activity was as in A. of the as a in was in by the with proteins RCEC grown on FN-coated in the of and the DNA sequence of the FRE or that of of its The of the is with that of the binding of Sp1 to the α5 FRE as by in and the protein a to Sp1. of proteins RCEC grown with or FN with the FRE or in the of of the Sp1 of the was by the as The of the to and the as is with that of the as the FRE was in the of of a serum or of the Sp1 with or proteins and of was as of the of Sp1 in proteins RCEC grown on culture with or fibronectin and in the Sp1 as a of a cells also to RCEC on the The shown to and The of the Sp1 is indicated responsiveness directed by of the α5 promoter in activity directed by plasmids of the α5 promoter in RCEC grown with or RCEC an cell density cells on or FN-coated culture with plasmids to and to the α5 the human α5 gene promoter. activity was and as the of FN is provided for of a perfect inverted repeat in the that FN responsiveness. The DNA sequence of a perfect inverted repeat that has been designated as the α5 FRE and identified and the human α5 gene promoter is indicated the of α5 FRE promoter activity in RCEC grown with or cell density of the α5 promoter FN responsiveness. RCEC low cells cells and cell density cells on or FN-coated culture dishes. transfected with the and and to as is as the of the activity RCEC grown on FN-coated that of RCEC grown on is provided for activation of the α5 promoter FN responsiveness in RCEC on culture that been with or of FN RCEC transfected with the and activity was determined and as is as by ECM and FN on α5 promoter promoter activity in RCEC grown on or FN dishes. RCEC grown to on or on FN or ECM and culture to with the α5 cells and activity was determined and as is as in the to activity directed by plasmids of the α5 promoter in RCEC grown with or RCEC an cell density cells for on or culture transfected with the the α5 promoter activity was and as the of ECM is provided for In to whether the FN responsiveness by the α5 promoter also the to inverted repeat that has been designated as the fibronectin-responsive element on its by transcription this the the α5 FRE was and with of proteins and RCEC grown on or FN-coated culture shown in and a the most and of proteins also in proved to be The signal to a and was found to be in the RCEC grown on FN-coated culture dishes. for the of these was by in as for transcription of and be by a of that of was a abolished a of a and be by an the sequence for in However, that of when a of the was suggesting that binding of a of the of transcription likely accounts for the of this of an the affinity binding for the transcription factor Sp1 for of a and as as the FRE a sufficient to in evidence that Sp1 or of this M. Res. Scopus Google is the transcription factor binding the α5 a the sequence for Sp1 that we identified in the promoter the gene designated M. Scopus Google was also as Sp1 the Sp1 by the of the is by a in the It is also with the 3′ half-site of the α5 FRE of the identified as for of the element by Sp1 are also in the α5 FRE M. Scopus Google shown in the element as as the FRE for the of and in results that of a and likely results the of the α5 FRE by of the Sp1 of transcription and that the of that by a of the of the DNA sequence the α5 FRE the of a perfect for see and that has been to this transcription factor Google Cell 1997; Scopus Google Scholar). in to whether Sp1 was binding the FRE to in The was protein and of in the of or to of an human Sp1. of a that of was found to be when the RCEC grown on FN-coated was or suggesting that Sp1 expression its DNA binding is increased when RCEC are on FN-coated dishes. The addition of the Sp1 in a of and a with a electrophoretic mobility the of by the Sp1 The proportion of the signal by the Sp1 was in the RCEC grown on FN-coated culture and of with RCEC grown on evidence that Sp1 or its DNA binding is increased in cells grown on FN-coated culture dishes. be when the Sp1 was with the is as a in such the Sp1 as the of that RCEC the of Sp1 of whether are on or FN-coated culture evidence that an DNA binding a in the of expression of likely accounts for the increased binding of Sp1 to the α5 FRE when RCEC are on FN-coated dishes. However, proteins RCEC required in to an Sp1 signal of to that proteins human cells as a for Sp1 substantial a affinity of the Sp1 directed rabbit Sp1 in the affinity Sp1 as also a signal when RCEC are that of proteins RCEC and cells the Sp1 binding in RCEC likely that Sp1 is a in RCEC in Sp1 a affinity for the Sp1 for its in the α5 only a of the affinity Sp1 is sufficient to binding of Sp1 to the a of the α5 FRE was required to of this in whether the inverted repeat the α5 FRE is sufficient to confer FN responsiveness to a heterologous a the α5 sequence to as was the promoter of the The gene a inhibitor expression is to the the and the M. Scopus Google Scholar). shown that the promoter the to in is to in most transfected cell M. Scopus Google Google Scholar). However, to by the Sp1 identified in the of the the FRE was in a that into the Sp1 M. Scopus Google transfected into only a was in the activity directed by the when FN was on the culture However, of or of an the α5 FRE the promoter in and in in the half-site of the inverted repeat on the FRE on the activity when cells are grown on or on the FN responsiveness when are on FN-coated culture when with the directed by the the that of the 3′ half-site of the FRE and most of its sequence on the promoter activity abolished FN responsiveness when RCEC on FN-coated dishes. the and the α5 FRE the as the 3′ half-site that the of FN responsiveness the 3′ half-site of the α5 FRE was the of Sp1 with its sequence in the in proteins RCEC grown on FN-coated culture and with the in the of of the sequence the FRE or of its shown in of the with proteins RCEC the a in and as as a of FRE of this a of the FRE as the FRE for the of the Sp1 evidence that these with the of the by Sp1. the of the FRE in the 3′ half-site the 3′ half-site or in with the in of the complex, when a evidence that are to Sp1. that the inverted repeat identified in the promoter of the α5 gene can confer FN responsiveness to an otherwise unresponsive heterologous promoter and that only the 3′ repeat of the with its is required for this to evidence that FN responsiveness by the α5 FRE was determined through its by cells been to be in this transcription as as many in an for gene expression or transcription factor (for a review see Google Scholar). the and the plasmids into cells or with a a the Sp1 the of gene promoter and of Sp1 expression in promoter activity when transfected in However, when with a in promoter activity was with the with the The promoter transfected or with into cells The substantial of in only to the of Sp1. However, the addition of the α5 FRE in in a in the activity directed by with the in RCEC in the half-site of the FRE only a on the activation of the promoter the activation be the 3′ half-site or the 3′ and of the FRE the plasmids and results are with of the shown in and evidence that Sp1 to the 3′ half-site of the FRE in to the activity of its promoter this the α5 or the or on with FN or with a the sequence has been shown to in the activation of mitogen-activated protein kinases K. Google such as extracellular signal-regulated kinases been shown to be to the ECM A. Yamada K.M. Cell Scopus Google Scholar). Sp1 has been as of the transcription by kinases Scopus Google M. A. Cell Scopus Google Scholar). shown that its to with the α5 FRE is increased activation of the signal whether the FN responsiveness directed by the α5 FRE was due to the activation of the pathway Scopus Google RCEC grown to cell density on culture or with FN transfected with the plasmids or and with or of the kinase inhibitor the activity directed by the transfected was increased when RCEC on FN-coated However, the addition of as as of the PD98059 inhibitor of this inhibitor Scopus Google M. A. Cell Scopus Google abolished this FN the of activity to the results also with the of the α5 FRE the promoter of the gene and the in the α5 FN responsiveness of the promoter was abolished when cells in the of the inhibitor RCEC grown on or FN-coated culture in the of or and in with the α5 a Sp1 signal that increased when RCEC grown on FN be with RCEC that been to the inhibitor and in However, RCEC in the of the inhibitor abolished of the when cells grown on that activation of the pathway through the of the with its ECM FN accounts for the in α5 promoter activity when RCEC are grown on FN-coated culture and that this is most likely on the phosphorylation state of Sp1 by of the corneal epithelium is to wound healing by migration and of the corneal epithelial cells that the and the cells the corneal of the migration is to be by the of FN by the stromal and the cells that the Google K. K. Eye Res. Scopus Google M. Invest. Google A. A. Eye Res. Scopus Google M. N. Eye Res. 1997; Scopus Google Scopus Google K. Yamada K.M. Invest. Google Invest. Google M. Scopus Google Scholar). provided evidence that the of expression for the the α5 integrin was modulated by the of such FN M. 1997; Scopus Google Scopus Google Scholar). The was in to whether through its α5β1 signal transduction can the transcriptional activity directed by the promoter of the α5 integrin provided evidence that FN can the transcriptional activity of the α5 promoter by altering the DNA binding affinity of the transcription factor Sp1 for a and that has been designated as the α5 FRE. The α5 FRE a perfect inverted repeat of the However, site-directed mutagenesis provided evidence that only the 3′ half-site with its 3′ base was required for FN responsiveness to of sequence the α5 promoter was found to be to a sequence the acetylcholine receptor gene that was to be required for expression of 1989; Scopus Google is and was postulated as the sequence for the transcription factor 1989; Scopus Google Scholar). However, has been as a protein that be to phosphorylation by protein The substantial we in the of Sp1 to the α5 FRE in RCEC grown with or FN alteration in the binding affinity of Sp1 or in the of Sp1 protein by RCEC culture However, to in the of Sp1 RCEC grown with or FN in the results are with by Scopus Google see in Sp1 protein an increased binding of Sp1 to the factor response element the promoter of the acetylcholine receptor with in the α5 promoter activity be by the affinity of Sp1 for the FRE by altering its state of phosphorylation through proteins that to the such as and of the state of phosphorylation for the transcription factor Sp1 has been to or its properties in M. A. Cell Scopus Google 1997; Scopus Google Scholar). Scopus Google that Sp1 also be a for identified a sequence that responsiveness of the human low density lipoprotein receptor to oncostatin M through a signal transduction pathway that phosphorylation of and by the kinases and The sequence the repeat also an of the the identified in the α5 FRE of also the sequence of the 3′ repeat that proved to be required for the FN responsiveness directed by the α5 FRE. only Sp1 and the protein been shown to repeat Scopus Google Scholar). However, these been to in Sp1 binding by or in the of Sp1 by Scopus Google Scholar). to also in Sp1 binding to the α5 FRE be RCEC grown with or FN when only of proteins However, the of proteins to or a binding of Sp1 to the FRE when RCEC are grown on FN-coated culture is also by a of the of this is likely to be and these M.J. Res. 1997; Google in Sp1 by Scopus Google also identified Sp1 as a of the pathway in the activation of the vascular endothelial growth factor However, transcriptional activation of through this transduction pathway only requires binding of Sp1 to a the promoter and also that of with the to the response Scopus Google Scholar). the Sp1 and binding been shown by to be increased when the pathway is However, evidence has been provided as to whether this is on an state of phosphorylation of or by an in the of M. A. Cell by the pathway with the kinase inhibitor that phosphorylation of Sp1 through is required in to Sp1 binding to the gene promoter. and as also by the DNA binding of Sp1 as a its properties can be likely through phosphorylation by all are to by the of the to the has been postulated as for transcriptional activation of the gene M.J. Res. 1997; Google Scholar). repeat also an that is for transcriptional is by the of the Sp1 repeat M.J. Res. 1997; Google Scholar). M.J. Res. 1997; Google postulated that the expression of an that Sp1 to a to the transcriptional transduction through activation of also to phosphorylation of Sp1 and account for transcriptional activation M.J. Res. 1997; Google has been shown to for the gene promoter M. A. Cell Scholar). the α5 promoter has has been shown to an that is likely sufficient to the transcriptional Google Scholar). The α5 FRE is the a to that for the Sp1 and the sequence the most M.J. Res. 1997; Google Google Scholar). for the repeat the affinity identified in the α5 promoter and to the responsiveness of the α5 promoter be on the on the results shown in the we that phosphorylation of Sp1 and of its to the transcriptional are required to the signal by extracellular the for an is required to the response to be of integrin α5 has also been to be by the binding of Sp1 to promoter the transcriptional activity directed by the integrin gene promoter was to be on its by Sp1 and DNA Cell 1997; 16: Scopus Google Scholar). directed by the promoter of the and the integrin has been to be through the of Sp1 and of the of transcription such as M. Google M. Scopus Google Scholar). also identified in the promoter of the integrin gene 1997; Google Scholar). phosphorylation of Sp1 to be required for of the Sp1 in of an element such as the α5 FRE is a the of the activation of the signal transduction pathway by FN results a the extracellular signal transduction and the to expression of the α5 integrin of also to the pathway by the α5β1 integrin and for a was Cell Scopus Google Scholar). M. of of for the of the α5

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.