Larouche et al. 1 Expression of the α5 integrin subunit gene ...

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Expression of the 5 integrin subunit gene promoter is positively regulated by the

extracellular matrix component fibronectin through the transcription factor Sp1 in

corneal epithelial cells in vitro.

Larouche, K., Leclerc, S., Salesse, C. and Gurin, S.L.*

Oncology and Molecular Endocrinology Research Center, and Ophthalmology

Research Unit, CHUL/CHUQ and Laval University, Qubec G1V 4G2, Canada.

Address correspondence to: Dr. Sylvain L. Gurin

Oncology and Molecular Endocrinology Research

Center

CHUL Research Center

2705 Laurier Blvd., Ste-Foy (Qubec)

Canada, G1V 4G2

Tel.: (418) 654-2296 FAX: (418) 654-2761

Email:[email protected]

Running title: FN-responsiveness of the 5 integrin gene promoter

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 19, 2000 as Manuscript M002945200 by guest on A

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SUMMARY

The accumulation of fibronectin (FN) in response to corneal epithelium injury has been

postulated to turn on expression of the FN-binding integrin 51. 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 CAT/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 EMSA 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 51

integrin activates a signal transduction pathway that results in the transcriptional

activation of the 5 gene likely through altering the phosphorylation state of Sp1.

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INTRODUCTION

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) 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 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 (5,8-11).

FN promotes corneal cell migration both in vivo (12,13) and in vitro (14) by acting as a


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temporary extracellular matrix to which corneal epithelial cells attach as they migrate

over the wounded area (13,15). Once the wound is reepithelialized, the subepithelial

immunohistological staining of FN progressively decreases (5,16-18).

The increase in FN expression that has been reported to occur during corneal wound

healing was postulated to be coordinated with the expression of its major integrin

receptor 51 (5), as has also been shown for laminin and tenascin and their

corresponding integrin receptor subunits 6 and 9, respectively (19-21). For instance,

the integrin 51 was shown to be present during corneal wound healing after radial

keratectomy (22). Direct evidence that FN can positively alter 51 integrin expression

at both the protein and mRNA levels have been provided through FN antisense

expression studies performed in the epithelium-derived human colon carcinoma cell

line Moser (23) as well as in murine AKR-2B fibroblasts (24). Other indirect evidences

linking expression of 51 to that of FN have also emerged from recent studies (25,26).

As a consequence, it is not surprising that EM, through its interactions with membrane

bound integrins, exerts profound influences on the major cellular programmes of

growth, differentiation and apoptosis by altering, through a number of signal

transduction pathways, the transcription of genes whose specific functions are linked to

these cellular functions. Binding of ECM components, such as FN, with their

corresponding integrin receptors will trigger the activation of intracellular signalling

mediators such as focal adhesion kinase (FAK), mitogen-activated protein kinases


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(MAPKs), and Rho-family GTPases (for a review, see 27). Activation of the MAPK

signal transduction pathway is of particular interest since it links integrin-mediated

signaling to transcriptional regulation of genes that are crucial for cell growth and

differentiation. The results presented hereby provided evidence that, by acting on 5

gene expression, such a route of signal transduction might alter cell adhesion properties

as well. The downstream cascade of family members that are activated following

transient activation of Ras GTP-binding proteins through receptor tyrosine kinases

include MAPK/ERK kinase (designated MEK or MAPKK) and ERK1 (p44)/ERK2 (p42)

(28). Activation of ERK1/ERK2 through phosphorylation causes their translocation to

the nucleus, where they have been reported to phosphorylate and activate distinct

transcription factors, such as ELK, c-Jun, and c-Myc (29-31), as well as members of the

ETS family (such as PEA3) (32).

In the present study, we demonstrated that FN can alter the transcription of the 5

integrin subunit gene at the promoter level. Such a FN-responsiveness was shown to be

determined by the binding of the transcription factor Sp1 to a target site that is part of a

perfect inverted repeat which, by itself, can confer FN-responsiveness to an otherwise

unresponsive heterologous promoter. Most of all, the FN-activated, integrin-mediated

signal transduction pathway appears to require activation of ERK1/ERK2 since the Sp1

DNA binding affinity, and, as a consequence, the FN-responsiveness of the 5

promoter, were both found to be diminished by blocking their activation with the MEK

kinase inhibitor PD98059. Together, these results demonstrate the novel finding that the


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5 integrin subunit, through activation of the MAPK pathway, can autoregulate its own

synthesis in a manner that is dependent on the extracellular concentration of FN.


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EXPERIMENTAL PROCEDURES

Cell culture and media

Rabbit corneal epithelial cells (RCECs) were obtained from the central area of freshly

dissected rabbit corneas as described previously (33) and then grown to low (near 15%

coverage of the plates), intermediate (near 75% coverage), or high cell density (100%

coverage for more than 48h) under 5% CO2 in SHEM medium supplemented with 5%

FBS and 20g/ml gentamycin. When indicated, human plasma FN (obtained as

previously described (34)) or ECM gel (basement membrane matrice from Engelbert

Holm Swarm Mouse Sarcoma, Fisher) was coated for 18 hrs at 37oC on the culture

dishes at varying concentrations (FN: 1 to 16 g per cm2; ECM: 10 g per cm2). Coated

petris were washed twice with PBS and blocked at 37oC with 2% BSA in PBS. Cells were

then seeded and grown as above. Inhibition of ERK1/ERK2 was performed by

culturing subconfluent RCEC in the presence of 10M of the MEK/kinase inhibitor

PD98059 (Sigma, Oakville, On, Canada) for 48h before cells were harvested. Drosophila

Schneider cells (ATCC CRL-1963) were cultured at 28oC without CO2 in Schneider

medium (Sigma, Oakville, On, Canada) supplemented with 10% FBS and 20g/ml

gentamycin.

Plasmids and oligonucleotides

The plasmids 5-41, 5-92, 5-178, and 5-954, which all bear the chloramphenicol

acetyl transferase (CAT) reporter gene fused to DNA fragments from the human 5

gene upstream regulatory sequence extending up to 5' positions -41, -92, -178, and -954,


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respectively, but all sharing a common 3' end located at position +23, have been

previously described (35). The recombinant plasmids bearing one or two sense copies of

either the 5 FRE or its mutant derivatives were created by inserting the corresponding

double-stranded oligomers upstream the basal promoter of the mouse p12 gene (into

the unique BamHI site) that has been previously mutated into its Sp1 binding site (and

designated p12.108/M (36)). The Sp1 expression vector pPacSp1 was generously

provided by Dr. Guntram Suske (Institute fr Molecularbiology und Tumorforschung,

Philipps Universitt Marburg, Germany) whereas the LacZ expression plasmid

pAC5/V5-His/LacZ was obtained from InVitro Gene (Carlsbad, CA).

The double-stranded oligonucleotides used in the present study were chemically

synthesized using a Biosearch 8700 apparatus (Millipore). They contained the DNA

sequence from the human 5 promoter comprised between positions -82 to -56 and

designated the 5 FRE (5'-GATCAGCCGGGAGTTTGGCAAACTCCTCCCC-3'), or its

mutated derivatives (5FRE/m5': 5'-GATCAGCCGAAAAATTGGCAAACTCCT-

CCCC-3'; 5FRE/m3': 5'-GATCAGCCGGGAGTTTGGCAAACTAAAAAAC-3';

5FRE/m5'+3': 5'-GATCAGCCGAAAAATTGGCAAACTAAAAAAC-3'), the DNA

binding site for human HeLa CTF/NF-I in adenovirus type 2 (Ad2) (5'-GATCTTAT-

TTTGGATTGAAGCCAATATGAG-3') (37), the high-affinity binding site for the

positive transcription factor Sp1 (5'-GATCATATCTGCGGGGCGGGGCAGACACAG-

3') (38), or the Sp1 binding site (designated p12.A) identified in the basal promoter from

the mouse p12 gene (5'-GATCCAGTGGGTGGAGCCTG-3') (36).


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Transient transfection and CAT assay

RCEC plated at either low (5x104 cells per 35mm tissue-culture plates), intermediate

(5x105 cells per 35mm tissue-culture plates), or high (1,5x106 cells per 35 mm tissue-

culture plates) cell density were transiently transfected using the polycationic detergent

Lipofectamine (Gibco BRL, Burlington, Ontario) as recommended by the manufacturer.

Each Lipofectamine-transfected plate received 1.5g of the test plasmid and 0.5 g of

the human growth hormone (hGH)-encoding plasmid pXGH5 (39). Drosophila

Schneider cells were transfected according to the calcium phosphate precipitation

procedure (36,40) at a density of 1x106 cells per 60mm culture plate.

Levels of CAT activity for all transfected cells were determined as described (40) and

normalized to the amount of hGH secreted into the culture media and assayed using a

kit for quantitative measurement of hGH (Immunocorp, Montral, Qubec). Because the

metallothionein-I promoter which direct expression of hGH from the pXGH5 plasmid

proved to be highly inefficient in Drosophila cells, CAT activities from transfected

Schneider cells were normalized to the amount of -gal encoded by the plasmid

pAC5/V5-His/LacZ and cotransfected along with the CAT recombinant constructs.

Each cell-containing plate therefore received 15g of the test plasmid, 4g of pAC5/V5-

His/LacZ and 1g of pPAC (empty vector)). In the cotransfection experiments

performed with the Sp1 expression plasmid, the empty pPAC was substituted for 1g

pPacSp1. The value presented for each individual test plasmid transfected corresponds


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to the mean of at least three separate transfections done in triplicate. To be considered

significant, each individual value needed to be at least three times over the background

level caused by the reaction buffer used (usually corresponding to 0.15%

chloramphenicol conversion). Standard deviation is also provided for each transfected

CAT plasmid.

Nuclear extract preparation

Crude nuclear extracts were prepared from RCEC grown solely on plastic or FN-coated

culture dishes and dialyzed against DNaseI buffer (50 mM KCl, 4 mM MgCl2, 20 mM

K3PO4 (pH 7.4), 1 mM -mercaptoethanol, 20% glycerol) as described (41) except that a

combination of protease inhibitors (pepstatin A (0.5 g/ml), leupeptin (5 g/ml),

chymostatin (5 g/ml), antipain (5 g/ml), aprotinin (5 g/ml), benzamidine (5 mM))

(all reagents from Sigma Co.) was added to all the buffers used in order to restrict

proteolysis. Extracts were kept frozen in small aliquots at -80C until use.

Electrophoretic mobility shift assays (EMSA) and supershift experiments

EMSAs were carried out using either the 27-bp 5 FRE or the high affinity Sp1 oligomer

as 5'-end labeled probes. Approximately 2x104 cpm labeled DNA was incubated with

crude nuclear proteins (as specified in the figure legends) from RCEC grown on either

untreated or FN-coated culture dishes in the presence of 500 ng poly(dI-dC).poly(dI-

dC) (Pharmacia-LKB) in buffer D (5 mM N-2-hydroxyethylpipera-zine-N-2'-ethane-

sulfonic acid (HEPES) (pH7.9), 10% glycerol (v/v), 25 mM KCl, 0.05 mM EDTA, 0.5 mM


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dithiothreitol, 0.125 mM PhMeSO2F). Occasionnally, crude nuclear extracts from human

HeLa cells were also used in EMSA as a positive control for comparison purposes.

Incubation proceeded at room temperature for 10 min upon which time DNA-protein

complexes were separated by gel electrophoresis through 6% native polyacrylamide

gels run against Tris-glycin buffer as described (42). Gels were dried and

autoradiographed at -80C to reveal the position of the shifted DNA-protein complexes

generated. Competitions in EMSA were performed using 10g of crude nuclear

proteins from RCEC grown in the presence of FN at 8g per cm2 as above except that

molar excesses (100- and 500-fold) of synthetic double-stranded oligonucleotides

bearing the DNA sequence of the 5 FRE, the DNA binding site for human HeLa

CTF/NF-I in adenovirus type 2 (Ad2) (37), the high-affinity binding site for the positive

transcription factor Sp1 (38), or the p12.A Sp1 binding site from the mouse p12 gene (36)

were added to the binding reaction prior to loading on the gel. Supershift experiments

in EMSA were conducted by first incubating varying amounts (as specified in the figure

legends) of crude nuclear proteins from RCEC grown either with (8g per cm2) or

without FN, in the presence of 250ng poly(dI-dC).poly(dI-dC), with either no or 1l

(corresponding to 1g) of a commercially engineered, rabbit antiserum raised against

the transcription factor Sp1 (Santa Cruz Biotechnology , Inc.) in buffer D. Then, 2x104

cpm FRE labeled probe was added and incubation was extended for another 15 min at

room temperature. Samples were finally loaded on high-ionic strength, 6% native

polyacrylamide gels and run at 4C against Tris-glycine buffer as above. Formation of

DNA-protein complexes was revealed following autoradiography at -70C.


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SDS-PAGE and Western blot

Crude nuclear proteins were obtained from either HeLa cells (used as a positive control)

of from RCEC grown on culture dishes coated or not with FN (8g per cm2) as detailed

above. Protein concentration was evaluated by the Bradford procedure and further

validated following Coomassie blue staining of SDS-polyacrylamide fractionated

nuclear proteins. One volume of sample buffer (6 M urea, 63 mM Tris (pH6.8), 10%

(v/v) glycerol, 1% SDS, 0,00125% (w/v) bromophenol blue, 300 mM -

mercaptoethanol) was added to 20g proteins before they were size-fractionated on a

10% SDS-polyacrylamide minigel and transferred onto a nitrocellulose filter. A full set

of protein molecular mass markers (Gibco BRL, Burlington, On, Canada) was also

loaded as a control to evaluate protein sizes. The blot was then washed once in TS

buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.4)) and 4-times (5 min each at 22C) in

TSM buffer (TS buffer plus 5% (w/v) fat free Carnation milk and 0.1% Tween 20). Then,

a 1:500 dilution of a rabbit monoclonal antibody raised against the transcription factor

Sp1 (Santa Cruz Biotechnology, Inc.) was added to the membrane-containing TSM

buffer and incubation proceeded further for 4 h at 22C. The blot was then washed in

TSM buffer and incubated an additional 1 h at 22C in a 1:1000 dilution of a peroxidase-

conjugated goat antimouse immunoglobulin G (Jackson Immunoresearch Lab.). The

membrane was successively washed in TSM (4-times, 5 min each) and TS (twice, 5 min

each) buffers before immunoreactive complexes were revealed using Western blot

chemiluminescence reagents (Renaissance, NEN Dupont) and autoradiographed.


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RESULTS

The activity directed by the 5 integrin subunit gene promoter is positively regulated

by fibronectin.

Studies conducted by Rajagopal et al. (23) and Huang et al. (24) both provided evidence

that the level of expression for the mRNA encoding the 5 integrin subunit was

positively modulated by the presence of the extracellular matrix component fibronectin.

We therefore exploited transient transfection of primary cultured RCEC using

recombinant plasmids bearing the CAT reporter gene fused to various segments from

the human 5 gene promoter (35) in order to evaluate whether such an FN-dependent

increase in 5 mRNA could be determined by discrete cis-acting elements from the 5

gene upstream regulatory region. For this purpose, a recombinant plasmid bearing the

5 promoter up to position -954 (5-954) inserted upstream from the CAT reporter gene

was transfected into RCEC plated either on plastic or FN-coated culture dishes

(2g/cm2) at varying cell densities. As Figure 1A indicates, culturing RCEC on FN-

coated petris did not alter the activity driven by the 5-954 plasmid when transfected at

low cell density (near 15% coverage of the plates). However, at both intermediate (near

75% coverage) and high (100% coverage for more than 48h) cell density, the activity of

the 5 promoter was found to be respectively 6.1- and 6.4-fold higher when cells are

grown on FN-coated culture plates rather than solely on plastic. Witdrawal of the

serum contained into the culture medium (which normally contains 5% FBS) prior to

cell seeding on FN-coated culture dishes had no statistical effect on the CAT activity

directed by 5-954 (results not presented).


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The dose-dependency of the 5 promoter FN-responsiveness was next evaluated by

transfecting RCEC plated at an intermediate cell density on culture dishes coated with

either no or increasing concentrations of FN (from 1 to 16g/cm2). As shown on Figure

1B, the activity directed by the 5-954 plasmid increased proportionnally to the amount

of FN coated on the culture dishes, reaching a drastic 18-fold stimulation at 16g/cm2

FN. No further increase in 5 promoter function was observed at FN concentrations

above 16g/cm2 (results not presented). We therefore conclude that the activity of the

human 5 promoter can be drastically increased when RCEC are grown on FN-coated

culture dishes and that such a positive influence is obviously cell-density dependent.

A distinct cis-acting element from the basal promoter of the human 5 gene mediates

FN-responsiveness in RCEC.

Discrete cis-acting regulatory elements are known to mediate many of the regulatory

effects that are triggered through signal transduction pathways by binding trans-acting

nuclear proteins with distinctive regulatory properties. To more precisely determine

the minimal 5 promoter sequence required to confer FN-responsiveness, CAT

recombinant plasmids bearing various 5' deletions of the 5 promoter were transfected

into RCEC grown at intermediate density on both plastic and FN-coated (2g/cm2)

culture dishes. Neither the deletion of the 5 promoter down to position -178 nor -92

could prevent the average 5-fold increase in 5 promoter activity observed when RCEC

are grown on FN-coated plates. However, the further deletion of the 5 sequences


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down to position -41 almost totally abolished the FN-responsiveness of the 5

promoter. A detailed examination of this 41 bp sequence revealed the presence of a

perfect inverted repeat of the following sequence: 5'-GGAGTTTG-3' (Figure 2B).

Therefore, FN-responsiveness of the 5 promoter appears to be determined by a short

strech of DNA sequence contained between positions -41 to -92 relative to the 5 mRNA

start site.

Influence of ECM components other than FN on the activity of the 5 promoter.

Apart from FN, proteins such as collagen IV, vitronectin, entactin and laminin are also

commonly found in the extracellular matrix. We examined whether components from

the ECM other than FN can also alter the expression directed by the 5 promoter in

RCEC. For this purpose, RCEC were grown to intermediate cell density on either

untreated or ECM-coated culture dishes before they were transiently transfected with

the 5-954 plasmid. The ECM gel (basement membrane matrice from Engelbert Holm

Swarm Mouse Sarcoma, Fisher) contains laminin, collagen IV, entactin, and heparin

sulfate proteoglycans but no FN. As shown on Figure 3A, the CAT activity driven by

5-954 is increased by only 2.5-fold when RCEC are grown on ECM-coated dishes

(10g/cm2). The CAT activity directed by the 5 promoter was raised to 4-fold when

both FN (2g/cm2) and the ECM gel (10g/cm2) are coated together on the culture

dishes. However, optimal promoter activation was obtained when FN (2g/cm2) was

coated alone on the culture plates (7.6-fold activation). Transient transfection of RCEC

plated on ECM-coated culture dishes with the recombinant plasmids bearing the


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various 5' deletions of the 5 promoter identified the ECM-responsive element

somewhere between positions -178 and -954 (Figure 3B). We conclude that components

from the ECM other than FN had only a moderate effect on the 5 promoter activity

and that their action is mediated through a cis-acting element distinct from that which

determine FN-responsiveness in RCEC.

The transcription factor Sp1 intereacts specifically with the 5 FRE in vitro.

In order to determine whether the FN-responsiveness mediated by the ---41/-92 5

promoter segment (which also bear the ---82 to ---56 inverted repeat that has been

designated as the Fibronectin Responsive Element ; FRE) depends on its recognition by

nuclear transcription factors, EMSAs were performed. For this purpose, the synthetic

oligomer bearing the 5 FRE was 5' end-labeled and incubated with increasing amounts

of crude nuclear proteins (2, 5, 10, and 20 g) from RCEC grown either on plastic or FN-

coated culture flasks (8g/cm2). As shown on Figure 4A, three distinct DNA-protein

complexes (designated a, b, and c) were observed upon autoradiography, complex a

being the most abundant at 5, 10, and 20g proteins (a few other fast-migrating

complexes were also occasionnally observed in EMSA but their formation proved to be

highly inconsistent). The signal corresponding to both complexes a and c was usually

found to be much stronger in the crude extract prepared from RCEC grown on FN-

coated culture dishes. Specificity for the formation of these complexes was then

evaluated by competition experiments in EMSA using, as unlabeled competitors,

various double-stranded oligonucleotides bearing target sequences for known


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transcription factors. Formation of both complexes a and b could easily be competed off

by a 100-fold molar excess of unlabeled FRE whereas that of complex c was partly

prevented at a 100-fold molar excess but nearly completely abolished at a 500-fold

excess (Figure 4B). Formation of both complexes a and b could not be prevented by an

unrelated oligomer bearing the target sequence for HeLa CTF/NF-I in adenovirus type

2. However, that of complex c was efficiently prevented when a 500-fold molar excess of

the NF1 oligomer was used suggesting that binding of a member of the NF1 family of

transcription factors likely accounts for the formation of this complex. Most of all, an

oligomer bearing the high affinity binding site for the positive transcription factor Sp1

could compete for formation of complexes a and b even as efficiently as the FRE itself, a

100-fold molar excess being sufficient to almost totally prevent their formation in EMSA

(Figure 4B). As a further evidence that Sp1 or any other member of this family (43) is the

major transcription factor binding the 5 FRE, a synthetic oligomer bearing the target

sequence for Sp1 that we identified in the basal promoter from the mouse p12 gene (and

designated p12.A) (36) was also used as unlabeled competitor. This Sp1 site diverges

from the Sp1 consensus by the lack of the central C residue (Figure 2B) which is

substituted by a T in the p12.A element. It is also relatively well preserved with the 3'

half-site of the 5 FRE (9 out of 12 residues) since the five G residues identified as

critical for recognition of the p12.A element by Sp1 are also preserved in the 5 FRE (36)

(see Figure 10). As shown on Figure 4B, the p12.A element competed nearly as well as

the FRE for the formation of both complexes a and b in EMSA. These results suggest

that formation of complexes a and b likely results from the recognition of the labeled 5


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FRE by distinct members of the Sp1 family of transcription factors and that complex c

might result from the recognition of that same probe by a member of the NF1 family. A

detailed examination of the DNA sequence from the 5 FRE indeed revealed the

presence of a perfect half-palyndromic site for NF1 (TGGCA; see Figures 2B and 10) that

has been previously reported to bind this transcription factor (44,45).

We next performed supershift experiments in EMSA to establish clearly whether Sp1

was truly binding the FRE to yield complexes a and b in EMSA. The experiment was

conducted at three different protein concentrations (5, 10, and 20g crude nuclear

proteins) in the presence of either no or 1l (corresponding to 1g) of an antiserum

raised against human Sp1. Again, formation of complex a but not that of complex c,

was found to be much stronger when the extract from RCEC grown on FN-coated petris

was used at either 10 or 20g (but not at 5g; Figure 5A) suggesting that Sp1 expression

(or its corresponding DNA binding affinity) is increased when RCEC are cultured on

FN-coated dishes. The further addition of the Sp1 antiserum resulted in a strong

reduction of complex a formation and yielded a new complex (a/Sp1Ab) with a lower

electrophoretic mobility resulting from the recognition of complex a by the Sp1

antibody. The proportion of the signal supershifted by the Sp1 antibody was much

stronger in the extract from RCEC grown on FN-coated culture dishes (at both 10g and

20g but not at 5g proteins) than with RCEC grown solely on plastic providing a

further evidence that either Sp1 expression, or its DNA binding affinity, is indeed

increased in cells grown on FN-coated culture dishes. As Figure 5B indicates, no


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supershifted complex could be obtained when the Sp1 antiserum was substituted with

the non-immune serum, which is used as a negative control in such experiments.

Western blot analysis using the Sp1 antiserum as the source of primary antibody

revealed that RCEC express nearly the same amount of Sp1 irrespective of whether they

are cultured on plastic or FN-coated culture dishes (Figure 5C) therefore providing

evidence that an improved DNA binding affinity, rather than a variation in the level of

expression of Sp1, likely account for the increased binding of Sp1 to the 5 FRE when

RCEC are cultured on FN-coated dishes. However, nearly five times more proteins

from RCEC were required in order to detect an Sp1 signal of equal strenght to that

obtained using proteins from human HeLa cells (often used as a positive control for Sp1

expression). This substantial difference did not arise from a reduced affinity of the anti-

human Sp1 antibody directed against rabbit Sp1 in our experiments since EMSAs

performed using the high affinity Sp1 oligomer as labeled probe (Figure 6A) also

revealed a much weaker shifted signal when crude nuclear extracts from RCEC are

selected, despite that equal amounts of proteins from both RCEC and HeLa cells were

used. Therefore, the reduced Sp1 binding observed in nuclear extracts from RCEC likely

suggests that Sp1 is expressed at a much lower level in RCEC than in HeLa cells.

Furthermore, Sp1 clearly possesses a higher affinity for the Sp1 oligomer than for its

target site in the 5 FRE since only a 100-fold molar excess of the high affinity Sp1

oligomer is sufficient to totally prevent binding of Sp1 to the Sp1 labeled probe,

whereas a 500-fold excess of the 5 FRE was required to almost totally prevent

formation of this complex in EMSA (Figure 6B).


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The inverted repeat from the 5 basal promoter mediates Sp1-dependent FN-

responsiveness.

To answer whether the inverted repeat from the 5 FRE (Figure 2B) is sufficient to

confer FN-responsiveness to an heterologous promoter, a synthetic, double-stranded

oligonucleotide bearing the 5 sequence from -82 to -56 (designated as the fibronectin

responsive element, FRE) was inserted upstream the basal promoter of the mouse p12

gene. The p12 gene encodes a 12kDa secretory protease inhibitor whose expression is

mainly restricted to the ventral prostate, the coagulating gland and the seminal vesicule

(46). We have previously shown that the basal promoter from the p12 gene, which

extends from position -108 to +7 in plasmid p12.108, is constitutively expressed to

relatively high levels in most transfected cell types (36,47). However, to avoid any

interference by the Sp1 site identified in the middle of the p12 basal promoter, the FRE

was inserted in a derivative from p12.108 that bears mutations into the p12 Sp1 target

site (p12.108/M (36)) (Figure 7A). When transfected into mid-confluent RCEC, only a

weak difference (1.6-fold activation) was observed in the CAT activity directed by the

parental plasmid p12.108/M when 8g/cm2 FN was coated on the culture plates

(Figure 7A). However, insertion of either one (in plasmid p12/FRE) or two sense copies

(in plasmid p12/2xFRE) of an oligomer bearing the -82/-56 5 FRE immediately

upstream from the p12 basal promoter resulted in 6.1- and 8.8-fold increase in CAT

activity, respectively. Mutations introduced in the 5 half-site of the inverted repeat

contained on the FRE (refer to the Materials and Methods section) had no statistically


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significant effect on either the basal p12 promoter-driven activity when cells are grown

on plastic or on the FN-responsiveness when they are cultured on FN-coated culture

dishes (32% reduction when compared to the level directed by the wild-type p12/FRE)

(Figure 7B). On the other hand, mutations that altered part of the 3 half-site of the FRE

and most of its downstream GC-rich sequence had no affect on the unstimulated, p12

promoter basal activity but totally abolished FN-responsiveness when RCEC were

cultured on FN-coated dishes. As expected, mutating both the 3 and 5 half-sites from

the 5 FRE had the same effect as mutating the 3 half-site alone. To confirm that the

lack of FN-responsiveness resulting from mutating the 3 half-site of the 5 FRE was the

consequence of preventing Sp1 from properly interacting with its target sequence in the

FRE, competitions experiments in EMSA were performed. Crude nuclear proteins were

prepared from mid-confluent RCEC grown on FN-coated culture dishes and incubated

with the 5 FRE labeled probe in the presence of varying concentrations of unlabeled

oligonucleotides bearing the sequence from either the wild-type FRE, or any of its

mutated derivatives. As shown on Figure 7C, incubation of the labeled probe with

nuclear proteins from RCEC yielded the typical Sp1/FRE complex observed above (also

denoted complex a in both Figures 4 and 5). As expected, as little as a 100-fold molar

excess of unlabeled wild-type FRE totally prevented formation of this complex.

Similarly, a 100-fold molar excess of the 5 half-site mutated FRE competed as well the

unmutated FRE for the formation of the Sp1 complex providing evidence that these

mutated positions did not interfer with the recognition of the oligomer by Sp1. On the

other hand, derivatives of the FRE bearing mutations in the 3 half-site (altering either


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the 3 half-site alone or in combination with the 5 half-site) were totally inefficient in

preventing formation of the Sp1/FRE complex, even when used at a 500-fold molar

excess, therefore providing evidence that both mutated oligomers are unable to bind

Sp1. We therefore conclude that the inverted repeat identified in the basal promoter of

the 5 gene can confer Sp1-dependent FN responsiveness to an otherwise unresponsive

heterologous promoter and that only the 3 repeat of the FRE, along with its

downstream GC-rich sequence, is required for this effect to occur.

As a further evidence that FN-responsiveness mediated by the 5 FRE was determined

through its recognition by Sp1, co-transfection experiments were therefore conducted

into Drosophila Schneider cells. These cells have been reported to be deficient in

producing this transcription factor, as well as many others expressed in higher

eukaryotes, which make them an ideal system for studying gene expression or

transcription factors funtion (for a review, see 48). Both the FRE-bearing 5-92 and the

FRE-depleted 5-42 plasmids were co-transfected into Schneider cells either alone or

with a recombinant plasmid (pPacSp1, a generous gift from Dr. Guntram Suske,

Institute fr Molecularbiology und Tumorforschung, Philipps Universitt Marburg,

Germany) containing the Sp1 cDNA under the control of the Drosophila actin gene

promoter and therefore ensuring high levels of Sp1 expression in Schneider cells.

Neither 5-41 nor 5-92 could determine high basal promoter activity when

individually transfected in Schneider cells. However, when co-transfected along with

pPacSp1, a dramatic 75-fold increase in promoter activity was observed with the FRE-


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containing plasmid 5-92 but not with the FRE-deleted plasmid 5-41 (Figure 8A). The

recombinant 5FRE/p12 promoter constructs were then transfected either alone or with

pPacSp1 into Schneider cells (Figure 8B). The parental plasmid p12.108/M, although

encoding substantial amounts of CAT in Schneider cells, responded only weakly (3.5-

fold activation) to the presence of Sp1. However, the further addition of the 5 FRE in

p12/FRE resulted in a strong increase (52-fold) in the CAT activity normally directed by

p12.108/M. As with the transfection experiments conducted in RCEC (see Figure 7A

and 7B), mutations introduced in the 5 half-site of the FRE (in plasmid p12/FREm5)

had only a modest effect on the Sp1-mediated activation of the p12 promoter (Figure

8B). On the other hand, no Sp1-mediated activation could be observed upon mutating

either the 3 half-site alone or both the 3 and 5 half-sites of the FRE (in the plasmids

p12/FREm3 and p12/FRE3+5, respectively). These results are consistent with those

of the competition experiment shown in Figure 7C and provide clear evidence that Sp1

does bind to the 3 half-site of the FRE in order to positively influence the activity of its

downstream promoter (in this case, either the 5 or the p12 promoter).

Activation of ERK1/ERK2 mediates the FN-responsiveness of the 5 promoter

Culturing murine Swiss 3T3 or rat REF52 fibroblasts on substrata coated with either FN

or with a synthetic peptide containing the RGD sequence have been shown to result in

the activation of mitogen activated protein kinases (MAPK) (49), such as extracellular-

signal-regulated kinases (ERKs), which have been shown to be recruited to the ECM

ligand/integrin binding site (50). Sp1 has been recently recognized as one of the few


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target transcription factors phosphorylated by ERK kinases (51-53). We have shown

above that its ability to interact with the 5 FRE is strongly increased upon activation of

the FN/51 integrin-mediated signal transduction. To determine whether the Sp1-

mediated FN-responsiveness directed by the 5 FRE was due to the activation of the

ras-Erk signaling pathway (27), RCEC grown to intermediate cell density on culture

dishes coated (8 g/cm2) or not with FN were transiently transfected with either the

recombinant plasmids 5-92 or p12/FRE and cultured with either no or 10M of the

Mek 1 kinase inhibitor PD98059. As expected, the CAT activity directed by the

transfected plasmid 5-92 was strongly increased (9.9-fold increase) when RCEC were

cultured on FN-coated dishes (Figure 9A). However, the addition of as little as 10M of

the PD98059 inhibitor (many studies have used doses 5- to 10-fold higher of this

inhibitor (51-53)) totally abolished this FN-responsiveness, the level of CAT activity

returning to the unstimulated level. Identical results were also obtained with the

recombinant plasmid p12/FRE, which bears one sense copy of the 5 FRE inserted

upstream the basal promoter of the p12 gene (see Figure 7A and B). Again, the near 3-

fold increase in the 5 FRE-mediated FN-responsiveness of the p12 promoter was

totally abolished when cells were cultured in the presence of the inhibitor (Figure 9B).

Crude nuclear extracts were prepared from RCEC grown either on plastic or FN-coated

culture dishes in the presence of either no or 10M PD98059, and then used in EMSAs.

Upon incubation with the 5 FRE labeled probe, a clear Sp1 signal that increased

several fold when RCEC were grown on FN could be observed with nuclear extracts

from RCEC that have not been exposed to the Mek-1 inhibitor (compare the 1st lane with


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the 3r lane in Figure 9C). However, culturing RCEC in the presence of the inhibitor

totally abolished formation of the Sp1/5FRE complex (Figure 9C) even when cells

were grown solely on plastic. We therefore conclude that activation of the ras-Erk

signaling pathway through the interaction of the 51 integrin with its ECM ligand FN

accounts for the increase in 5 promoter activity when RCEC are grown on FN-coated

culture dishes, and that this effect is most likely dependent on the altered

phosphorylation state of Sp1 by activated ERK1/ERK2.


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DISCUSSION

Debridement of the corneal epithelium is known to promote wound healing by

stimulating migration and differentiation of both the basal corneal epithelial cells that

border the injured area and the precursor cells from the corneal limbus. Induction of the

migration process is known to be influenced by the massive production of FN by both

the stromal keratocytes and the basal cells that border the injured area (5,8-15).

Moreover, recent studies provided evidence that the level of expression for the mRNA

encoding the 5 integrin subunit was positively modulated by the presence of such FN

(23,24). The present study was therefore conducted in order to investigate whether FN,

through its 51 receptor-mediated signal transduction pathway, can alter the

transcriptional activity directed by the promoter of the 5 integrin subunit gene. We

provided clear evidence that FN can indeed alter the transcriptional activity of the 5

promoter by altering the DNA binding affinity of the positive transcription factor Sp1

for a short 5 promoter segment located between positions -77 and -61 that has been

designated as the 5 FRE. The 5 FRE bears a perfect inverted repeat of the following

sequence: 5'-GGAGTTTG-3'. However, site-directed mutagenesis provided evidence

that only the 3 half-site (along with its nearby 3 GC-rich basepairs (TCCCC)) was

required for FN-responsiveness to occur. This short stretch of sequence from the 5

promoter was found to be highly homologous to a 12 bp sequence from the murine

acetylcholine receptor (AChR) -subunit gene that was reported to be absolutely

required for muscle-specific expression of AChR- (54) (see Fig. 10). This cis-acting

element, which is comprised between positions -106 to -95, was postulated as being the


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target sequence for the transcription factor myogenin (54). However, myogenin has not

been reported as a target protein that might be subjected to differential phosphorylation

by protein kinases.

The substantial variations we have observed in the ability of Sp1 to bind the 5 FRE in

RCEC grown with or without FN might either result from alteration in the binding

affinity of Sp1 or from modification in the amount of Sp1 protein produced by RCEC

under both culture conditions. However, our inability to detect any significant variation

in the absolute amount of Sp1 between RCEC grown with or without FN in Western

blot analyses rather favors the former hypothesis. Our results are consistent with those

recently reported by Alroy et al. (55) who could not see any variation in Sp1 protein

levels despite an increased binding of Sp1 to the NDF-response element (NRE) from the

promoter of the acetylcholine receptor upon stimulation with Neu differentiation

factors (NDFs). Variations in the 5 promoter activity might then be triggered by

modifying the affinity of Sp1 for the FRE target site by altering its state of

phosphorylation through nuclear proteins that belong to the MAPkinase family, such as

ERK-1 (p44) and ERK-2 (p42). Alteration of the state of phosphorylation for the

transcription factor Sp1 has been reported to alter, either positively or negatively, its

DNA binding properties in vitro (52,55,56). Li et al. (51) recently reported that Sp1

might also be a target for ERK proteins. Indeed, they identified a 16bp sequence (repeat

3) that mediates responsiveness of the human low density lipoprotein receptor (LDLR)

to oncostatin M (OM) through a signal transduction pathway that involves


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phosphorylation of ERK-1 and ERK-2 by the upstream kinases MEK1 and MEK2. The

sequence from the LDLR repeat 3 also bears an intact copy of the GGAGTTT motif (on

the non-coding strand) identified in the 5 FRE (see Figure 9). Most of all, it also

contains the GC-rich sequence (TCCCC) located downstream of the 3 repeat that

proved to be required for the FN responsiveness directed by the 5 FRE. Interestingly,

only Sp1 and the Sp1-related protein Sp3, have been shown to bind repeat 3 (51).

However, these authors have been unable to detect any OM-induced alterations in Sp1

binding by EMSA or in the ratio of hyper versus hypophosphorylated Sp1 by Western

blot analyses (51). As figure 5A reveals (and to some extend, also Figure 4A), no

significant changes in Sp1 binding to the 5 FRE could be observed between nuclear

extracts obtained from RCEC grown with or without FN when only 5g crude nuclear

proteins were used. However, raising the amount of proteins to either 10 or 20g clearly

revealed a much stronger binding of Sp1 to the FRE when RCEC are grown on FN-

coated culture dishes which is also supported by a more intense supershift of the

Sp1/FRE DNA-protein complex. Formation of this DNA-protein complex is therefore

likely to be concentration-dependent and might explain why these authors could not

detect any alterations in Sp1 binding (57). A recent study conducted by Milanini et al.

(53) also identified Sp1 as a target of the p42/p44 MAP kinase pathway in the activation

of the vascular endothelial growth factor (VEGF) gene. However, transcriptional

activation of VEGF through this p42/p44 transduction pathway not only requires

binding of Sp1 to a GC-rich region from the VEGF promoter located between positions -

88 to -66, but also that of AP-2, which synergizes with the former to ensure the proper


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regulatory response (53). Both the Sp1 and AP-2 binding activities have been shown by

EMSA to be increased when the p42/p44 MAP kinase pathway is activated. However,

no clear evidence has been provided yet as to whether this effect is dependent on an

altered state of phosphorylation of both factors or by an increase in the amount of both

proteins. Recently, Merchant et al. (52) have shown, by blocking the EGF-induced ras-

Erk pathway with the Mek 1 kinase inhibitor PD98059, that phosphorylation of Sp1

through Erk2 is indeed required in order to induce Sp1 binding to the gastrin gene

promoter. Therefore, and as also supported by our results, the DNA binding ability of

Sp1 and, as a consequence, its transactivation properties, can be enhanced likely

through phosphorylation by activated ERK1/ERK2.

Not all Sp1/Sp3 binding sites are subjected to regulation by the MAP kinase pathway.

Proper positioning of the Sp1/Sp3 target site relative to the TATA box has been

postulated as being particularly critical for OM-mediated transcriptional activation of

the LDLR gene (57). Indeed, although LDLR repeat 1 also bears an Sp1 binding site that

is critical for basal transcriptional activity, it is not affected by the presence of OM,

unlike the more proximal Sp1 site from repeat 3 (57). Liu et al. postulated that OM may

induce the expression of an Sp1-dependent coactivator that would bridge Sp1 bound to

a proper positioned target site, to the general transcriptional machinery (57).

Alternatively, OM-mediated transduction pathway, through activation of ERK1/ERK2,

might also lead to post-translational phosphorylation of Sp1 and account for LDLR

transcriptional activation (57), which has been recently shown to occur for the gastrin


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gene promoter (52). Although the 5 promoter has no TATA box, it has been shown to

contain an initiator site located at position -45 that is likely sufficient to bind the general

transcriptional machinery (35). The 5 FRE is therefore located approximately 15bp

upstream from the putative initiator site, a positioning nearly identical to that observed

for the LDLR Repeat 3 Sp1 site and the LDLR TATA-like sequence (17bp from the most

5 located TATA-like sequence) (57,58). As for the LDLR repeat 1, the putative high

affinity Sp1 binding sites identified in the 5 promoter between positions -178 and -92

did not contribute much to the FN-mediated responsiveness of the 5 promoter since

they could be deleted without any significant effect on the CAT activity. Based on the

results shown in the present study, we suggest that both phosphorylation of Sp1 and

proper positioning of its target site relative to the general transcriptional machinery are

required to properly transduce the signal triggered by extracellular FN. Whether the

need for a Sp1-dependent coactivator is required to produce the proper response

remains to be demonstrated.

Expression of integrin subunit genes other than 5 has also been reported to be

positively influenced by the binding of Sp1 to their promoter sequence. Indeed, the

transcriptional activity directed by the 6 integrin subunit gene promoter was reported

to be dependent on its recognition by both Sp1 and AP2 (59). Transcription directed by

the promoter of both the 2/CD18 and the IIb integrin subunits has recently been

reported to be positively regulated through the synergistic action of both Sp1 and

members of the Ets family of transcription factors, such as GABP (60-61). Two tandemly


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repeated Sp1 binding sites were also identified in the promoter of the 2 integrin

subunit gene (62). Interestingly, phosphorylation of Sp1 appears to be required for

formation of the Sp1 DNA-protein complex in vitro.

Characterization of an FN-responsive element such as the 5 FRE is a first step toward

the understanding of the nuclear events taking place upon activation the signal

transduction pathway normally triggered by membrane-bound FN-integrins. Our

results provide a link between the extracellular ligand/integrin mediated signal

transduction and the nuclear events leading to expression of the 5 integrin subunit

gene. Most of all, it also provides further support to the major signaling pathway

activated by the 51 integrin and for which a model was recently proposed (63).


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ACKNOWLEDGEMENTS

We would like to thank Dr. Thomas M. Birkenmeir (Department of Internal Medicine,

Washington University School of Medicine, St-Louis, Missouri) for his generous gift of

the 5 promoter-CAT constructs. K. Larouche is supported by a Scholarship from the

Natural Sciences and Engineering Research Council of Canada (NSERC). S. Gurin and

C. Salesse are Scholars from the Fonds de la Recherche en Sant du Qubec (FRSQ).

This work was supported by grants from the Medical Research Council (MRC), the

Fondation sur les Maladies de l'Oeil and the FRSQ Vision network.


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FIGURE LEGENDS

Fig. 1. 5 promoter activity in RCEC grown with or without FN. (A) Cell-density

dependency of the 5 promoter FN-responsiveness. RCEC were plated at low

(1x105 cells per petri; L), intermediate (5x105 cells per petri; I) and high cell density

(1.5x106 cells per petri; H) on either uncoated or FN-coated (2g/cm2) tissue

culture dishes. Cells were transiently transfected 24 h later with the 5-954

recombinant plasmid and harvested 48 hrs post-transfection. CAT activities were

measured and normalized to hGH as described in Materials and Methods. Each

value is expressed as the ratio of the CAT activity from RCEC grown on FN-coated

petris over that of RCEC grown solely on plastic. Standard deviation is provided

for each individual value. (B) Dose-dependent activation of the 5 promoter FN-

responsiveness in RCEC. RCEC (3x105 cells/petri) were plated on culture dishes

that have been coated with either no or increasing concentrations of FN (1 to 16

g/cm2). RCEC were then transfected with the 5-954 recombinant construct and

harvested 48 hrs later. CAT activity was determined and normalized as described

in the Materials and Methods section. Each value is expressed as detailed in panel

A.

Fig. 2. FN-responsiveness directed by recombinant constructs bearing 5' deletions of

the 5 promoter in RCEC. (A) CAT activity directed by recombinant plasmids

bearing 5-deletions of the 5 promoter in RCEC grown with or without FN.

RCEC plated at an intermediate cell density (5x105 cells per petri) on either regular


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or FN-coated (2g/cm2) culture dishes were transiently transfected, 24 hrs later,

with CAT recombinant plasmids bearing various lengths (up to positions -954,

-178, -92, and -41 relative to the 5 mRNA start site) from the human 5 gene

promoter. CAT activity was measured and expressed as the ratio of CAT+FN over

CAT-FN. Standard deviation is provided for each value. (B) Identification of a

perfect inverted repeat in the 5 promoter segment that mediates FN-

responsiveness. The DNA sequence of a perfect inverted repeat that has been

designated as the 5 FN responsive element (FRE) and identified between position

-77 to -61 from the human 5 gene promoter is indicated in bold capitel letters.

Arrows indicate the position of each 5 FRE half-sites.

Fig. 3. Influence exerted by both ECM and FN on 5 promoter function. (A) 5

promoter activity in RCEC grown on FN-, ECM-, or both FN+ECM-coated dishes.

RCEC were grown to mid-confluency either solely on plastic (-) or on coated (ECM

(10g/cm2), FN (2 g/cm2) or both ECM+FN; (10g/cm2 and 2 g/cm2,

respectively) culture dishes prior to their transfection with the 5 recombinant

plasmid 5-954. Transfected cells were harvested 48 hrs later and CAT activity

determined and normalized as described in the Materials and Methods section.

Each value is expressed as detailed in the legend to Fig. 1. (B) CAT activity

directed by recombinant plasmids bearing 5-deletions of the 5 promoter in

RCEC grown with or without ECM. RCEC plated at an intermediate cell density

(5x105 cells per petri) for 24 hrs on either regular or ECM-coated (10g/cm2)


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culture dishes were transiently transfected with the various 5' deletion constructs

from the 5 promoter (see Fig. 2A). CAT activity was measured and expressed as

the ratio of CAT+ECM over CAT-ECM. Standard deviation is provided for each

value.

Fig. 4. Binding of nuclear proteins from RCEC to the 5 FRE in vitro. (A) EMSA

analysis of the nuclear proteins from RCEC interacting with the 5 FRE. The

double-stranded oligonucleotide bearing the 5 FRE was 5 end-labeled and

incubated with varying concentrations (2 to 20g) of crude nuclear proteins from

RCEC grown on either no (FN-) or FN-coated (FN+) culture dishes (8g/cm2). The

position of three DNA-protein complexes is shown (a, b, and c) along with that of

the free probe (U). P: labeled probe alone. (B) Competitions in EMSA. The 5' end-

labeled 5 FRE was incubated with 10g crude nuclear proteins from RCEC

grown on FN-coated culture dishes in the presence of either 100- or 500-fold molar

excess of various unlabeled double-stranded oligonucleotide competitors (FRE,

Sp1, NF1, and p12.A). U: free labeled probe; P: labeled probe alone; C: labeled

probe with nuclear proteins but without unlabeled competitor.

Fig. 5. In vitro binding of Sp1 to the 5 FRE as revealed by supershift analyses in

EMSA and Western blots. (A) The nuclear protein yielding complex a

corresponds to Sp1. Varying amounts (5 to 20g) of crude nuclear proteins from

RCEC grown with (8g/cm2) or without FN were incubated with the labeled FRE


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either alone or in the presence of 1l of the Sp1 antiserum. Formation of the shifted

DNA-protein complexes was examined by the EMSA as above. The position of

both the Sp1/FRE (corresponding to complex a) and the supershifted

Sp1Ab/Sp1/FRE (identified as a/Sp1Ab) complexes is provided, along with that

of the free probe (U). (B) As controls, the FRE labeled probe was incubated in the

presence of either 1l of a non-immune serum (NIS) or 1l of the Sp1 antiserum

(Sp1Ab) with or without crude nuclear proteins (NP) and formation of DNA-

protein complexes was evaluated as above. U: unbound fraction of the labeled

probe. (C) Western blot analysis of Sp1 in RCEC. Crude nuclear proteins were

obtained from RCEC grown on culture dishes coated with (+FN: 8g/cm2) or

without (-FN) fibronectin and tested in Western blot analyses using the Sp1

antiserum as detailed in Materials and Methods. As a positive control, increasing

concentrations (5 to 20g) of a crude nuclear extract from HeLa cells were also

loaded next to those from RCEC on the SDS-gel. The molecular mass marker

shown correspond to myosin (200 kDa) and phosphorylase B (97.4 kDa). The

position of the endogenous Sp1 is indicated (Sp1 (95/106)).

Fig. 6. EMSA analyses of Sp1 in RCEC. (A) Equal amounts (1, 2, and 5g) of nuclear

proteins from either RCEC (grown on culture dishes coated with 8g/cm2 FN) or

HeLa cells were incubated in the presence of an oligomer bearing the Sp1 high

affinity binding site Sp1 (5'-GATCATATCTGCGGGGCGGGGCAGAC-ACAG-3')

and formation of the Sp1 complex was evaluated by EMSA as described in


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42

Materials and Methods. The position of the Sp1 complex is indicated, as well as

that of three other complexes of which two likely correspond to complexes b and c

from figure 5, whereas the third (ns) was shown in panel B as being non-specific.

(B) Approximately 5g crude nuclear proteins from RCEC grown on FN-coated

dishes (8g/cm2) were incubated with the high affinity Sp1 labeled probe with

either no or increasing concentrations (100- and 500-fold molar excess) of the

unlabeled Sp1, FRE, or NF1 oligomers. Formation of DNA-protein complexes was

then monitored by EMSA as in panel A. The position of the specific Sp1 complex is

indicated along with that of a non-specific complex (ns). U: unbound fraction of

the labeled probe.

Fig. 7. The 5 FRE can confer FN-responsiveness to the basal promoter of the

heterologous p12 gene. (A) A synthetic oligonucleotide bearing the 5 inverted

repeat from position -82 to -56 (5 FRE) was inserted in either one (in plasmid

p12/FRE) or two sense copies (in plasmid p12/2xFRE) upstream the basal

promoter of the mouse p12 gene (p12.108/M) (36). The recombinant constructs

were transiently transfected into midconfluent RCEC grown on tissue culture

dishes coated with either no (FN-) or 8 g/cm2 FN (FN+). CAT activity was

measured and expressed relative to the unstimulated level directed by the parental

plasmid p12.108/M. Arrows indicate the position of each of the wild-type,

unmutated 5 FRE inverted repeats. (B) Double-stranded oligonucleotides bearing

mutations in either the 3' (in plasmid p12/FREm3'), or the 5' (in plasmid


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43

p12/FREm5') half-site from the 5 FRE, or both (in plasmid p12/FREm5'+3'), were

inserted in single-sense copies upstream the p12 promoter in p12.108/M. The CAT

activity directed by each recombinant construct was assessed following transient

transfections in mid-confluent RCEC grown on culture dishes coated or not with

FN (8 g/cm2) as in panel A. Arrows indicate the position of each unmutated 5

FRE inverted repeat whereas mutation-bearing repeats are indicated by an X. CAT

activity was measured as in panel A. (C) Formation of the Sp1/5FRE

DNA/protein complex (identified as complex a in Fig. 5) was evaluated in EMSA

by incubating the 5 FRE labeled probe with crude nuclear proteins from

midconfluent RCEC grown on FN-coated dishes ((8g/cm2), in the presence of

unlabeled double-stranded oligonucleotides (both 100- and 500-fold molar excess)

bearing the DNA sequence of either the wild-type, unmutated FRE or that of any

of its mutated derivatives (FREm3; FREm5; FREm3+5). The position of both the

Sp1/FRE and NF1/FRE complexes is provided, along with that of the free

probe(U). P: probe without nuclear proteins.

Fig. 8. Transient transfection experiments in Sp1-deficient Drosophila Schneider

cells. (A) Both the recombinant plasmids 5-41 and 5-92 were transiently

transfected either alone or in combination with the Sp1 expression plasmid

pPacSp1 into Drosophila Schneider cells. Cells were harvested 48 hrs later and

CAT activity determined and normalized as detailed in Materials and Methods.

(B) Same as in panel A except that the recombinant plasmids p12.108/M and


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44

p12/FRE, or its mutated derivatives p12/FREm3, p12/FREm5, and

p12/FREm3+m5 (refer to Materials and Methods for details), were selected for

the co-transfection experiments.

Fig. 9. The integrin-mediated FN-responsiveness is abolished by the MEK/kinase

inhibitor PD98059. (A) The recombinant plasmid 5-92 was transiently transfected

into RCEC grown on plastic (-FN) or FN-coated (+FN; 8 g/cm2) culture dishes

with either no or 10M of the MEK/kinase inhibitor PD98059. Cells were

harvested 48 hrs later and CAT activity determined and normalized as detailed in

Materials and Methods. (B) Same as in panel A except that the recombinant

plasmid p12/FRE (see Figure 7) was substituted to 5-92 for the transfection

experiments. (C) The double-stranded oligonucleotide bearing the 5 FRE was 5

end-labeled and incubated with crude nuclear proteins (5g) from RCEC grown

on either no (-) or FN-coated (+) culture dishes (8g/cm2), in the presence of either

no or 10M PD98059. Formation of the Sp1/FRE complex was then monitored by

EMSA as detailed in Figure 4 except that the concentration of the polyacrylamide

gel was lowered to 4%. The position of three Sp1/FRE complex is shown (Sp1)

along with that of the free probe (U). P: labeled probe alone.

Fig. 10. Sequence homology between the 5 FRE and other gene regulatory target

sequences. The DNA sequence from the human 5 FRE is aligned with the Sp1

binding site identified in the promoter of the mouse p12 gene (p12.A), the 16bp


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45

repeat 3 element from the human low density lipoprotein receptor (LDLR), and

sequences from the murine acetylcholine receptor (AChR) -subunit gene

promoter that also bear a binding site for the transcription factor myogenin

(underlined). Arrows indicate the position of each 5 FRE half inverted repeats

whereas black dots indicate the position of those G residues whose methylation by

DMS interfere with the recognition of the p12.A element by Sp1. The DNA

sequences which show homology to both the NF1 and Sp1 target sites are

indicated.


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Kathy Larouche, Steeve Leclerc, Christian Salesse and Sylvain L. Guerincorneal epithelial cells in vitro

extracellular matrix component fibronectin through the transcription factor Sp1 in 5 integrin subunit gene promoter is positively regulated by theExpression of the

published online September 19, 2000J. Biol. Chem.

10.1074/jbc.M002945200Access the most updated version of this article at doi:

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