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发表于 2009-3-6 09:33 |只看该作者 |倒序浏览 |打印

1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, UK

2 Celltech plc, Slough SL1 4EN, UK6 f! Q: y, Z$ \+ a3 d

Abstract! |$ v$ ~- z! G) W1 c$ N

SCC4 human keratinocytes are derived from a squamous cell carcinoma of the tongue and undergo very little spontaneous differentiation. Introduction of a wild-type ß1 integrin subunit into SCC4 cells stimulates differentiation, suggesting either that the cells have a defect in the integrin signaling pathways that control differentiation or that the ß1 subunit itself is defective. Here we describe a heterozygous mutation in the SCC4 ß1 subunit. The mutation, T188I, maps to the I-like domain. It results in constitutive activation of ligand binding, irrespective of the partner {alpha}  subunit, in solid phase assays with recombinant protein and in living cells. The mutation promotes cell spreading, but not proliferation, motility, or invasiveness. It results in sustained activation of Erk MAPK independent of cell spreading. When introduced into SCC4 keratinocytes, the wild-type ß1 integrin stimulates differentiation, whereas the mutant is inactive. Activation of ß1 integrins in normal keratinocytes also suppresses differentiation. These results establish, for the first time, mutation as a mechanism by which integrins can contribute to neoplasia, because the degree of differentiation in epithelial cancers is inversely correlated with prognosis. They also provide new insights into how integrins regulate keratinocyte differentiation.

Key Words: integrin; keratinocyte; differentiation; adhesion; tumor
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Integrin extracellular matrix receptors regulate growth and differentiation of many cell types, including epidermal keratinocytes . Integrin loss or overexpression contributes to the pathogenesis of benign epidermal disorders, such as psoriasis, and influences the incidence and prognosis of squamous cell carcinomas and other tumors . Integrin mutations underlie several heritable disorders, including the epidermal blistering disease epidermolysis bullosa , and there is growing evidence that integrin polymorphisms predispose to certain diseases . However, to date, no integrin mutations in tumors of any origin have been reported.
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When cultured epidermal keratinocytes are deprived of contact with an adhesive substratum, they initiate terminal differentiation. This can be partly inhibited by ligation of ß1 integrins with antibodies or high concentrations of extracellular matrix proteins . To investigate the mechanism by which integrins regulate differentiation, we have previously introduced a series of wild-type and mutant chick ß1 integrin subunits into primary human keratinocytes and tested the ability of chick-specific anti-integrin antibodies to block suspension-induced terminal differentiation . Those studies established that the differentiation signal can still be transduced by ß1 cytoplasmic domain mutants that fail to localize to focal adhesions. A point mutation that abolishes ß1 ligand binding is inactive in regulating suspension-induced differentiation and this led us to conclude that the differentiation signal is "do not differentiate," transduced by ligand-occupied receptors, rather than "do differentiate," transduced by unoccupied receptors.4 Y1 v+ \( r; k- H

Introduction of the wild-type chick ß1 subunit into primary human keratinocytes has no effect on the proportion of cells that undergo spontaneous terminal differentiation; this is also true for several lines of human keratinocytes derived from squamous cell carcinomas. However, transduction of one such line, SCC4, does have a strong positive effect on differentiation . SCC4 are derived from a squamous cell carcinoma of the tongue . They are poorly differentiated, with only 1% of cells expressing the terminal differentiation marker, involucrin, in culture . Introduction of the wild-type ß1 subunit into SCC4 cells increases the percentage of involucrin-positive cells to 10%, which is similar to the percentage in preconfluent primary keratinocytes) q, T  B0 v6 m, d1 q$ `# N
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Because poorly differentiated squamous cell carcinomas have a worse prognosis than tumors exhibiting a moderate or high degree of differentiation , the nature of the differentiation defect in SCC4 cells could have broad significance for our understanding of how integrins influence the course of the disease. The failure of SCC4 cells to differentiate does not reflect integrin loss or overexpression, because the level and range of integrins (predominantly 2ß1, 3ß1, 5ß1, 6ß4, and ß5) expressed are similar to primary human keratinocytes . To test the hypothesis that the SCC4 ß1 integrin subunit is inherently defective in differentiation control, we sequenced the complete ß1 cDNA. Here we describe a point mutation in the ß1 subunit and report on its functional consequences.- e# s( K- z9 r. x& L+ F

Results
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Identification of the mutation  {/ f! S% ~& p
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SCC4 keratinocytes were heterozygous for a point mutation within the I-like domain of the ß1 subunit, T188I . The equivalent region was sequenced from 18 normal individuals. Because none of these contained the mutation, we excluded the possibility of a polymorphism. We also sequenced the ß1 subunit from three squamous cell carcinoma lines previously shown not to differentiate when transduced with the wild-type chick ß1 subunit  and found that they were all homozygous for the wild-type human ß1 sequence. Threonine 188 lies on the upper surface of the I-like domain on a loop that projects from the surface containing the MIDAS (metal ion–dependent adhesion site) and ADMIDAS (adjacent to MIDAS) sites  . The loop is a small cysteine-bridged structure present in all ß subunits except ß4 . This region, the "specificity loop," is involved in ligand binding and, when exchanged between ß1 and ß3, determines the ligand specificity of the corresponding v heterodimers . In vß3, the specificity loop is displaced over 1.5 Å on ligand binding .9 s  \; G+ ?+ d- x9 q# N" \# c5 Z6 G

fig.ommtted

Location of the T188I mutation. (a) The predicted structure of the ß1 I-like domain, based on comparison with the ß3 subunit using the SWISS-MODEL server and SwissPDB Viewer , is shown in two orientations. The threonine 188 residue (arrowhead) lies within the specificity loop, above the surface containing the MIDAS (metal ion–dependent adhesion site) and ADMIDAS (adjacent to MIDAS) sites. ß sheets are shown in yellow and  helices in red. The ADMIDAS cation is shown in blue. (b) Position and sequence comparison of the T188I-containing loop in ß1 and other ß subunits.

The T188I mutation increases the ligand binding ability of recombinant ß1 integrins2 j) L1 t7 B6 d# P
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To study the functional significance of the mutation, we expressed it as a soluble Fc fusion protein in combination with the 5 and v integrin subunits . The Fc chimeric integrins contained the extracellular domain of each subunit fused to the human 1 immunoglobulin Fc domain and were recovered from the medium of transiently transfected mammalian cells . The binding of wild-type and mutant {alpha} 5ß1 and {alpha} vß1 to 2 µg/ml fibronectin in the presence of 1 mM magnesium ions was examined.

fig.ommtted
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Increased ligand binding by T188I mutant relative to wild-type recombinant ß1 integrins. Dose response curves of binding to 2 µg/ml fibronectin by soluble -Fc/ß-Fc fusion proteins in the presence of 1 mM Mg2 . (a) 5ß1; (b) vß1. Data are means and SEM of duplicate wells.
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At all concentrations tested, the heterodimers containing the T188I mutant ß1 subunit showed increased binding to fibronectin relative to heterodimers containing the wild-type ß1 subunit. In the case of 5ß1 , the curves approached a plateau at the highest concentration tested; this allowed us to obtain an estimate of the relative affinity of the mutant and wild-type heterodimers for fibronectin. The concentration of wild-type 5ß1 that gave 50% binding was 175.9 ng/ml, compared with 37.9 ng/ml in the case of T188I 5ß1. This suggests that the mutant increased the affinity of {alpha} 5ß1 for fibronectin 4.6-fold relative to the wild-type integrin., V- M# ?6 P, k; [8 ^0 ^
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The affinity of recombinant vß1 for fibronectin is lower than that of 5ß1, and the transfection efficiency of the ß1 Fc chimeras was also lower ( b; unpublished data). Hence, it was not possible to obtain an indication of the relative affinity of mutant and wild-type vß1 for fibronectin. Nevertheless, over a broad range of concentrations, T188I vß1 showed increased binding to fibronectin when compared with wild-type vß1 . The solid phase assays thus demonstrate that T188I increased the binding of two different ß1 integrin heterodimers to fibronectin.( ~: ]2 L/ i, o3 H/ Y, o1 b
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The T188I mutation promotes ß1 integrin–mediated adhesion to a greater extent than the wild-type subunit
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Threonine 188 is conserved between human and chick ß1, and to compare the T188I mutation with the wild-type subunit in intact cells, we engineered the mutation in the chick ß1 subunit. We used the chick subunit to allow detection with species-specific antibodies in both human and mouse cells. The wild-type chick ß1 subunit forms functional heterodimers with human 2, 3, and 5 in primary human keratinocytes and behaves in the same way as human ß1 in assays of keratinocyte differentiation, extracellular matrix adhesion, and Erk MAPK activation* ?/ y7 r+ e/ \# d, j
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A ß1-null mouse embryo fibroblast cell line, GD25 , was retrovirally transduced with either the wild-type  or mutant  integrin. The use of high titre retroviral packaging cells enabled us to study mixed populations, avoiding clonal selection . The level of cell surface expression of wild-type and T188I ß1 was the same (, a and b). Parental GD25 cells express only the vß3 integrin (vitronectin receptor); when the ß1 subunit is introduced, it is expressed as heterodimers with 3 and 6 (laminin receptors) and with {alpha} 5 (fibronectin receptor)
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Increased ligand binding by T188I mutant relative to wild-type ß1 integrins in whole cell adhesion assays. (a and b) Flow cytometry of parental GD25 cells (gray lines) or GD25 cells transduced with (a) wild-type (WT) or (b) mutant (T188I) chick ß1 integrin subunits (black lines) stained with antibody to chick ß1 (JG22). (c and d) Adhesion of ß1-null parental GD25 cells or GD25 transduced with wild-type (GD25cß1wt) or mutant (GD25cß1T188I) ß1 integrin subunits to (c) 10 µg/ml fibronectin (Fn) or (d) 10 µg/ml laminin (Ln) in TBS (black bars), 10 mM Mg2  (white bars), 1 mM Mn2  (horizontal striped bars), or 1 mM Ca2  (diagonal striped bars). Error bars are the SD of triplicate wells. Asterisks indicate conditions under which T188I- expressing cells were more adhesive than cells expressing wild-type ß1 (P

The parental cells showed little adhesion to fibronectin or laminin in the absence of divalent cations or in the presence of magnesium, manganese, or calcium ions (). The wild-type subunit conferred adhesiveness to both ligands, but cells expressing the T188I mutant had a greater ability to bind fibronectin than cells expressing the wild-type subunit in the presence of each cation (P  increased adhesion to fibronectin was observed at all concentrations tested between 2.5 and 20 µg/ml; on 2.5 µg/ml fibronectin, there was no attachment of GD25 cells expressing the wild-type integrin, whereas there was one-third maximal attachment of cells expressing the mutant (unpublished data). Maximal attachment of cells expressing wild-type or mutant ß1 occurred at ">=" 5 µg/ml fibronectin (unpublished data). In magnesium or calcium ions, the wild-type and mutant ß1 subunits promoted laminin adhesion equally; however, in manganese, T188I promoted adhesion to a greater extent (P  The results support the conclusion from the solid phase assays that T188I increases the ligand binding ability of ß1 integrins.$ m  D$ ]9 k, }  [: d" S- J- S; S3 f+ G

Enhanced ligand binding and ligand-independent ß1 activation in SCC4 cells compared with keratinocytes homozygous for wild-type ß1 integrin subunit

In the solid phase and GD25 assays, the T188I mutation promoted ligand binding when tested in the absence of wild-type ß1. Because SCC4 cells are heterozygous for the mutation, we examined whether the mutant also promoted adhesion when coexpressed with the wild-type subunit. We compared the adhesion of SCC4 cells with other squamous cell carcinoma–derived keratinocytes (SCC13) that are homozygous for wild-type ß1. In the presence of magnesium or manganese ions, the proportion of SCC4 cells that adhered to fibronectin or collagen was significantly greater than the proportion of SCC13 cells (P  the sole form of ß1  or when coexpressed with wild-type ß1 .
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Integrin activation and increased adhesiveness of SCC4 keratinocytes. (a and b) Adhesion of SCC4 and SCC13 keratinocytes to (a) 10 µg/ml fibronectin (Fn) or (b) 10 µg/ml collagen (Col) in TBS (black bars), 10 mM Mg2  (white bars), or 1 mM Mn2  (horizontal striped bars). Error bars are the SD of triplicate wells. Asterisks indicate conditions under which SCC4 were more adhesive than SCC13 cells (P 2  (gray lines) or 1 mM Mn2  (black lines).5 G: Y# c7 N7 a

As a further measure of the activation status of ß1 integrins containing the T188I mutation, we performed flow cytometry of primary keratinocytes and SCC4 cells labeled with HUTS-21, an antibody that detects a high-affinity ß1 conformation MOLT4 cells, a lymphoblast cell line, express high levels of the HUTS-21 epitope in the presence of manganese ions and therefore served as a positive control. The epitope was undetectable in any strain of normal keratinocytes (cells from five individuals tested) and in three SCC lines expressing wild-type ß1 subunits ( c; unpublished data). However, SCC4 cells bound HUTS-21 in the presence of manganese. Thus, keratinocytes heterozygous for T188I expressed a ß1 activation epitope in the absence of ligand, whereas keratinocytes homozygous for wild-type ß1 did not8 }& e' I  O1 k) D6 B
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Effects of the T188I mutation on cell behavior* }) \% T3 ^& x7 a2 ?
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To study the effects of the T188I mutation on cell behavior, we monitored the spreading, motility, and invasiveness of parental GD25 cells and cells transduced with the wild-type or mutant ß1 subunit . Both the wild-type and mutant integrins localized to focal adhesions, but the mutant promoted more rapid cell spreading than the wild type Both forms of the ß1 subunit promoted the random motility of GD25 cells on fibronectin equally (P = 0.34; , a and b). The wild-type subunit promoted invasiveness through Matrigel to a greater extent than the mutant , probably reflecting the inverse relationship between integrin affinity and the optimal ligand concentration for cell movement

fig.ommtted

Effects of wild-type and T188I mutant ß1 subunits on spreading and Erk MAPK activation of GD25 cells. (a) Both the T188I and the wild-type (WT) ß1 subunits localized to focal adhesions. Cells were stained with V2E9 (anti–chick ß1 integrin, green), TRITC–phalloidin (red), and Hoescht nuclear stain (blue). Bars, 10 µm. (b) Rate of spreading on 10 µg/ml fibronectin. 50% of cells expressing the T188I subunit (triangles) began to spread within 10 min of plating, whereas cells expressing the wild-type ß1 subunit (squares) took >20 min to achieve the same degree of spreading. By 50 min, : N2 g! Q8 Q% A; p2 b- k+ v/ S

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Motility and invasiveness of GD25 cells expressing wild-type or T188I mutant ß1 integrins. (a and b) Random cell migration within 24 h of plating on 10 µg/ml fibronectin. (a) Starting (green) and final (red) positions of individual cells. (b) Mean speeds of 40 cells. Error bars are the SD. (c) Number of cells that migrated through Matrigel 2 d after plating. Parental GD25 cells were compared with cells expressing the wild-type (GD25ß1WT) or T188I mutant (GD25ß1T188I) subunit. Error bars are the SD of triplicate wells.9 h' U) u7 t, y$ y! G
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To determine whether the mutation had any effect on integrin signaling, we examined activation of Erk MAPK in GD25 cells expressing mutant or wild-type ß1 . ß1 signaling through MAPK is known to suppress the initiation of terminal differentiation in normal human keratinocytes . When GD25 cells were plated on fibronectin in the presence of EGF, Erk MAPK was activated for longer in cells expressing the mutant than in cells expressing the wild-type ß1 subunit. This was independent of cell spreading, because it was evident at 40 min, after maximal spreading of cells expressing the mutant had occurred. At the 40-min time point, the level of phosphorylated Erk relative to total Erk was 2.4-fold higher in cells expressing mutant ß1 than in cells expressing wild-type ß1. When GD25 cells were plated on fibronectin in the absence of EGF, the initial peak of Erk MAPK activation (7 min) was 1.9-fold greater in cells expressing T188I than in those expressing wild-type ß1 ; again this was independent of cell spreading, because it occurred at a time when neither population of cells had spread appreciably (unpublished data). We conclude that the mutant integrin is more effective at activating Erk MAPK than the wild-type integrin, a property that is independent of its ability to promote cell spreading.

The T188I mutation is inactive in regulating keratinocyte differentiation
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To examine whether the T188I mutation was responsible for the failure of SCC4 keratinocytes to undergo terminal differentiation , SCC4 cells were transduced with the wild-type or T188I mutant chick ß1 integrin. The level of expression of each chick integrin subunit, determined using species-specific antibodies, was equivalent and corresponded to that of the endogenous subunits . Neither subunit had any major effect on the growth rate of SCC4 cells . However, whereas the wild-type subunit stimulated terminal differentiation (as measured by the proportion of cells expressing involucrin or cornifin), the mutant subunit had no effect . To test whether activation of ß1 integrins also inhibited differentiation of primary keratinocytes, we incubated preconfluent adherent cultures for 48 h with the activating antibody TS2/16 or control anti-ß1 antibodies. At the end of the incubation period, the proportion of differentiated keratinocytes was ~ 13% in the presence of control antibodies, but was only 7% in TS2/16-treated cultures (P  d). Thus antibody-induced activation of the wild-type ß1 subunit can suppress keratinocyte terminal differentiation. This is consistent with the observation that high concentrations of fibronectin can inhibit suspension-induced terminal differentiation of human keratinocytes
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fig.ommtted

Effect of integrin activation on growth and differentiation of SCC4 cells and normal keratinocytes. (a) Flow cytometry of parental SCC4 cells (gray lines) or SCC4 transduced with wild-type (WT) or mutant (T188I) chick ß1 integrin subunits (black lines) stained with antibody to chick ß1 (JG22). (b) Growth curves of parental SCC4 cells transduced with empty retroviral vector (diamonds), SCC4 expressing the T188I mutant ß1 subunit (triangles), and SCC4 expressing the wild-type ß1 subunit (squares). 103 cells were seeded per well in 24-well dishes and harvested at the time points shown. Error bars are the SD of triplicate wells. (c) Percent of SCC4 cells expressing the terminal differentiation markers involucrin and cornifin in preconfluent cultures. Parental SCC4 cells were compared with cells expressing the empty retroviral vector (SCC4 pBp) or the mutant (SCC4 cß1 T188I) or wild-type (SCC4 cß1 wt) ß1 integrin subunits. Error bars are the SD of triplicate wells. (d) Primary human epidermal keratinocytes were seeded at 4 x 103/cm2 on a 3T3 feeder layer. 24 h later, the cells were incubated with 20 µg/ml of the following anti–human ß1 antibodies: TS2/16 (adhesion activating), P5D2, or A2B2 (inhibitory anti–human ß1 antibodies, which did not interfere with adhesion under these assay conditions). Control cells were incubated with no antibody (Km) or with W1B10 (chick-specific anti-ß1 antibody). 48 h later, the cells were harvested and the proportion of cells expressing involucrin or cornifin was determined. Error bars are the SD of triplicate wells. Asterisks indicate a significant difference between treated and control cells (P $ }2 j* q0 u0 }8 _5 ?5 \3 b

Discussion
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The T188I mutation we have identified is remarkable for three reasons. It is the first integrin mutation to be found in a human tumor. It is, unlike the majority of integrin disease mutations , a gain of function mutation, increasing the affinity of ß1 integrins for a variety of ligands. It impairs the ability of keratinocytes to undergo terminal differentiation and thus can contribute to the neoplastic phenotype.

The adhesion-promoting effect of the mutation was observed when T188I was expressed as 5ß1 (, vß1  3ß1 or 6ß1 , and 2ß1 (collagen receptor;  b) heterodimers. Nevertheless, the extent to which adhesion was activated did appear to depend on the  partner and the assay conditions . By modeling the ß1 I-like domain on the crystal structures of vß3 , we can speculate about how the T188I mutation might increase ligand binding. The threonine may interact with other residues to stabilize a low-affinity form of the receptor; replacing the threonine with isoleucine might prevent this interaction. Alternatively, the presence of isoleucine may increase ligand binding by altering the normal movement of the loop that occurs on activation." `' g* u. d( @& ?! S9 O1 _

Consistent with our observations, replacement of the ß2 specificity loop with the ß3 loop activates binding of {alpha} Lß2 to ICAM1 . Swapping the specificity loops of the ß1 and ß3 subunits determines the ability of each subunit to regulate Rho GTPases, demonstrating its importance in translating information from ligand binding into intracellular signaling events . It is possible that in addition to, or as a consequence of, increasing ligand binding, the T188I mutation might alter the interactions of ß1 integrins with other membrane proteins or affect organization of the extracellular matrix ; however, this remains to be investigated.. _2 K8 U3 ?5 V8 _3 S
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The evidence that T188I is indeed responsible for the failure of SCC4 to differentiate rests on the observation that transduction of SCC4 cells with wild-type ß1 increases differentiation, whereas transduction with T188I does not . Furthermore, antibody-mediated activation of the wild-type ß1 subunit, to mimic the effect of the T188I mutation, suppresses differentiation of normal keratinocytes. We have previously shown that the mechanism by which ß1 integrins regulate keratinocyte differentiation is via occupied receptors transducing a "do not differentiate" signal . The properties of the T188I mutation support this conclusion, in that by strongly promoting ligand binding, cells expressing the mutant integrin receive a strong "do not differentiate" message. The enhanced activation of Erk MAPK by T188I independent of cell spreading adds weight to the view that activation of this pathway downstream of ß1 integrins plays a role in suppressing keratinocyte differentiation . It is interesting that the decision to differentiate appears to be an all or nothing response to relatively modest changes in integrin-mediated adhesion, both in the case of the T188I mutation and when a dominant negative integrin mutation is introduced into keratinocytes.

Changes in integrin expression, whether loss, de novo expression, or overexpression, are all reported to influence the prognosis of epithelial tumors , the best-documented mechanism being by promoting invasion . In contrast, the T188I mutation is not correlated with altered integrin expression levels  and contributes to the neoplastic phenotype by inhibiting integrin-regulated differentiation. It is now of interest to determine the overall frequency of integrin mutations in tumors, to uncover the extent to which such mutations affect the onset and outcome of the disease.
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Materials and methods

cDNA and genomic sequencing

RNA was isolated from cultured SCC4 cells using the easyRNA protocol (QIAGEN). A first strand cDNA was synthesized using random hexamer primers with the Superscript2 reverse polymerase (GIBCO BRL). Three overlapping 1-kb fragments of the coding region of the human ß1 integrin cDNA were synthesized using the PWO proofreading enzyme (Boehringer) and cloned into the pCR-Blunt vector (Invitrogen). Competent cells were then transformed, and 10 clones were picked for each fragment and sequenced multiple times in both the forward and reverse orientation. The entire ß1 cDNA was sequenced from six other sources of cultured human keratinocytes: the squamous cell carcinoma lines SCC12F2, SCC12B2, and SCC13  and three strains of primary keratinocytes. A small fragment containing the site of the mutation (corresponding to residues 673–832 of the ß1 cDNA) was amplified from genomic DNA of 15 normal individuals and also sequenced.: i. q5 m8 L. {% @( Z& Y4 w
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Solid phase assays with recombinant integrins
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Integrin cDNAs were cloned into the vector pEE12.2h, which contains the human 1 Fc domain as a Sal1–EcoR1 genomic fragment. Separate vectors encoding the extracellular regions of the human 5, v, wild-type ß1, and T188I ß1 integrin subunits were constructed, essentially as described previously . The appropriate  and ß integrin vectors were transiently coexpressed in CHOL761h cells, and integrin chimeras were captured from the culture supernatant with goat anti–human-Fc antiserum. Solid phase binding to fibronectin-coated plates was performed as follows. 96-well plates (Nunc) were coated with the 50-kD fragment of fibronectin at 2 µg/ml in 0.1 M sodium bicarbonate, pH 8.5. Plates were blocked with 5% (wt/vol) BSA, 1% (vol/vol) Tween-20 in PBS. Supernatants containing soluble integrins were pretreated with 25 mM EDTA and then extensively dialyzed against 20 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, pH 7.5. Titrations of integrins were performed in 1% (wt/vol) BSA, 20 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, pH 7.5, and incubated on the fibronectin for 2 h at room temperature. Bound integrins were detected with an HRP-labeled goat anti–human IgG-Fc serum (Jackson ImmunoResearch Laboratories).
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Cell culture and retroviral infection8 L0 O8 Y/ o& |& ~$ c. \; f
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Primary human keratinocytes and SCC4, SCC13, and GD25 cells were cultured as described previously . The pBabe-puro ß1 T188I construct was made using the pBabe-puro chick ß1 wild-type vector as a template via the Quikchange mutagenesis protocol (Stratagene). Retroviral packaging cell lines were generated as described previously  or by transfecting Phoenix packaging cells (G. Nolan, Stanford University, Stanford, CA) and then using the virus-containing supernatant either to directly infect GD25 cells or to make stable amphotropic virus AM12 producers . The AM12 producer lines were used to infect SCC4 cells as described previously 4 C' f$ O8 A% ~' n% A8 u6 T: F

Cell attachment, spreading, motility, and invasion# @$ ~  J' Y& i; C  ?& r) N

96-well flat-bottomed assay plates (Dynex) were coated with fibronectin as described previously . Preconfluent 75-cm2 flasks of cells were washed twice in serum-free medium and incubated with 5 ml of serum-free medium containing 5 µM CellTracker green dye (Molecular Probes) for 20 min at 37°C. The cells were washed twice, placed in serum-containing medium for a further 20 min, and then harvested. Cells were resuspended at 3 x 105 cells/ml in TBS containing Mg2  (10 mM), Mn2  (1 mM), or Ca2  (1 mM). 100 µl of cell suspension was added per well to triplicate wells and incubated at 37°C for 15 min. The plate was then washed twice with TBS. The fluorescence value from each well was measured and compared with a standard curve prepared from serial dilutions of the same cell suspension.4 c! K* w7 A: ~! j0 j
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To quantitate cell motility and spreading, subconfluent cultures were harvested and plated onto dishes coated in 10 µg/ml fibronectin, at a density that allowed tracking of individual cells. The cells were filmed for 25 h. Individual cells were tracked using Kinetic Imaging Tracking software and the results analyzed using Mathematica (Wolfram Research). Cell spreading was measured during the first 3 h of filming: the number of unspread cells and the total number of cells in every third frame were recorded, and the percentage of spread cells was calculated. Any cells that failed to spread over the period of the film (24 h) were discounted as nonviable.2 C/ O, R" H9 Q8 J  Y: @
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Invasion assays were performed as described previously . GD25 cells were mixed with Matrigel (Becton Dickinson) to give a final concentration of 3 x 105 cells/ml in a 1:2 dilution of Matrigel. 100 µl of the mixture was placed in 0.8-µm pore size 24-well cell culture inserts (Becton Dickinson) and left to gel for 1 h at 37°C. 100 µl serum-free medium was placed above the gel and 750 µl medium containing 10% donor calf serum was placed in the lower compartment. After 2 d, the inserts were removed from their wells, the Matrigel was removed, and the membranes were fixed in methanol and stained with crystal violet. The center of each membrane was photographed, and the number of cells was counted using NIH Image analysis software.% k: L, z9 I. e. A0 e- G

Measurement of Erk MAPK activation

GD25 cells expressing wild-type or mutant ß1 integrins were plated on 10 µg/ml fibronectin in the presence or absence of 10 ng/ml EGF for various times and then lysed and immunoblotted as described previously . The antibody specific for phosphorylated Erk1/2 was purchased from New England Biolabs, Inc., and the antibody to total Erk was from Santa Cruz Biotechnology, Inc. Protein bands were visualized with HRP-conjugated secondary antibodies using ECL (Amersham Biosciences). The level of Erk phosphorylation was quantitated using the Scion Image package, dividing the value for each phosphoErk band with the value for the corresponding total Erk band.

Cell differentiation assays4 c8 ^2 H& @& v8 N0 h- [
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The proportion of terminally differentiated cells was determined essentially as described previously . Involucrin was detected with SY-5 or DH-1 antibodies  and cornifin with SQ37C, a generous gift of A. Jetten (National Institute of Environmental Health Sciences, Research Triangle Park, NC) .
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Acknowledgments

We are deeply grateful to Simon Broad for extensive practical assistance throughout the project.% `( e/ G2 x7 c3 C6 N

F.M. Watt and R.D. Evans are supported by Cancer Research UK.: W/ L! f. S% G  w
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Submitted: 3 September 2002

Revised: 6 December 2002

Accepted: 30 December 2002
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References

Adams, J.C., and F.M. Watt. 1989. Fibronectin inhibits theterminal differentiation of human keratinocytes. Nature. 340:307–309.6 U9 w5 B. Y% V2 w, m8 S0 c
- t1 ]% f" n9 w2 Z0 W
Bagutti, C., P.M. Speight, and F.M. Watt. 1998. Comparison of integrin, cadherin, and catenin expression in squamous cell carcinomas of the oral cavity. J. Pathol. 186:8–16.  c. w' N' H* d" P0 v! o, a
$ h( q9 {( d# e5 b1 Z4 @, D! d8 k
Bishop, L.A., W.J. Kee, A.J. Zhu, and F.M. Watt. 1998. Lack of intrinsic polarity in the ligand-binding ability of keratinocyte ß1 integrins. Exp. Dermatol. 7:350–361.( s. b3 D6 ^) X! `# R  a) k

Fujimoto, W., G. Nakanishi, J. Arata, and A.M. Jetten. 1997. Differential expression of human cornifin {alpha}  and ß in squamous differentiating epithelial tissues and several skin lesions. J. Invest. Dermatol. 108:200–204.
$ w9 @0 _% _: J% x9 F
Giancotti, F.G., and E. Ruoslahti. 1999. Integrin signaling. Science. 285:1028–1032.( k$ Y" O/ o6 ?, x# p$ k6 X( i
8 K5 [- Q% ~) `# d/ n4 j) O
Haase, I., R.M. Hobbs, M.R. Romero, S. Broad, and F.M. Watt. 2001. A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis. J. Clin. Invest. 108:527–536.3 |( M( V% y; e: i
9 V/ V8 W1 s2 o! E
Hogg, N., and P.A. Bates. 2000. Genetic analysis of integrin function in man: LAD-1 and other syndromes. Matrix Biol. 19:211–222.+ U* G" d1 p2 K2 B* i% x

Jones, J., F.M. Watt, and P.M. Speight. 1997. Changes in the expression of {alpha} v integrins in oral squamous cell carcinomas. J. Oral Pathol. Med. 26:63–68.+ T& Q# X% P" ?8 X
; v$ k7 ~* D. @# l; |
Kamata, T., K.K. Tieu, T. Tarui, W. Puzon-McLaughlin, N. Hogg, and Y. Takada. 2002. The role of the CPNKEKEC sequence in the ß(2) subunit I domain in regulation of integrin {alpha} (L)ß(2) (LFA-1). J. Immunol. 168:2296–2301.

Kunicki, T.J. 2001. The role of platelet collagen receptor (glycoprotein Ia/IIa; integrin {alpha} 2ß1) polymorphisms in thrombotic disease. Curr. Opin. Hematol. 8:277–285./ L/ v! G, U  h  T5 A+ [/ P
' x0 G& f# R# W2 J- u- ~
Lacy, P.D., E.L. Spitznagel, Jr., and J.F. Piccirillo. 1999. Development of a new staging system for recurrent oral cavity and oropharyngeal squamous cell carcinoma. Cancer. 86:1387–1395.

Leitinger, B., and N. Hogg. 2002. The involvement of lipid rafts in the regulation of integrin function. J. Cell Sci. 115(Pt. 5):963–972.
2 E7 a3 |" s! a' m
Levy, L., S. Broad, A.J. Zhu, J.M. Carroll, I. Khazaal, B. Péault, and F.M. Watt. 1998. Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice. Gene Ther. 5:913–922.
- c( z# C  V0 K' O
Levy, L., S. Broad, D. Diekmann, R.D. Evans and F.M. Watt. 2000. ß1 integrins regulate keratinocyte adhesion and differentiation by distinct mechanisms. Mol. Biol. Cell. 11:453–466.; [; t3 E: S; \! o" l4 S$ F

Lochter, A., M. Navre, Z. Werb, and M.J. Bissell. 1999. {alpha} 1 and {alpha} 2 integrins mediate invasive activity of mouse mammary carcinoma cells through regulation of stromelysin-1 expression. Mol. Biol. Cell. 10:271–282.

Luque, A., M. G車mez, W. Puzon, Y. Takada, F. S芍nchez-Madrid, and C. Cabañas. 1996. Activated conformations of very late activation integrins detected by a group of antibodies (HUTS) specific for a novel regulatory region (355-425) of the common ß1 chain. J. Biol. Chem. 271:11067–11075.
6 f$ J' n, y7 l: [) K9 ]& T$ f
Mercurio, A.M., and I. Rabinovitz. 2001. Towards a mechanistic understanding of tumor invasion–lessons from the {alpha} 6ß4 integrin. Semin. Cancer Biol. 11:129–141.+ a* J- ^5 ?4 n, O
# M) C) H' ~+ K) O& I. i
Miao, H., S. Li, Y.L. Hu, S. Yuan, Y. Zhao, B.P. Chen, W. Puzon-McLaughlin, T. Tarui, J.Y. Shyy, Y. Takada, et al. 2002. Differential regulation of Rho GTPases by ß1 and ß3 integrins: the role of an extracellular domain of integrin in intracellular signaling. J. Cell Sci. 115:2199–2206.6 u8 [3 [; Z4 l2 k- d0 r& c
$ f( ?1 {* N& p0 p0 b" m
Palecek, S.P., J.C. Loftus, M.H. Ginsberg, D.A. Lauffenburger, and A.F. Horwitz. 1997. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature. 385:537–540.' v3 }: [' y3 r. B( e- O" U

Petter, G., and U.F. Haustein. 2000. Histologic subtyping and malignancy assessment of cutaneous squamous cell carcinoma. Dermatol. Surg. 26:521–530.) m# X0 ~) l9 N' t: Z% _; E
2 ]/ `8 D/ Y$ W! M) k: _
Rheinwald, J.G., and M.A. Beckett. 1981. Tumorigenic keratinocyte lines requiring anchorage and fibroblast support cultured from human squamous cell carcinomas. Cancer Res. 41:1657–1663.
6 z3 p. G! l3 g9 j: s3 C; A. {
Schwartz, M.A. 2001. Integrin signaling revisited. Trends Cell Biol. 11:466–470.
- e8 l# j3 N- O' q% c
Stephens, P.E., S. Ortlepp, V.C. Perkins, M.K. Robinson, and H. Kirby. 2000. Expression of a soluble functional form of the integrin {alpha} 4ß1 in mammalian cells. Cell Adhes. Commun. 7:377–390.
9 r0 f! p9 g9 T2 a
Takada, Y., and W. Puzon. 1993. Identification of a regulatory region of integrin ß1 subunit using activating and inhibiting antibodies. J. Biol. Chem. 268:17597–17601., N: w+ Y+ S) @* B- Q3 x

Takagi, J., T. Kamata, J. Meredith, W. Puzon-McLaughlin, and Y. Takada. 1997. Changing ligand specificities of {alpha} vß1 and {alpha} vß3 integrins by swapping a short diverse sequence of the ß subunit. J. Biol. Chem. 272:19794–19800.# B  w7 a% y6 j& t* C

Thomas, G.J., M.P. Lewis, I.R. Hart, J.F. Marshall, and P.M. Speight. 2001. {alpha} vß6 integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9. Int. J. Cancer. 92:641–650.
  q0 K2 ?- m5 |$ H% P
van der Flier, A., and A. Sonnenberg. 2001. Function and interactions of integrins. Cell Tissue Res. 305:285–298.8 g4 x3 ^9 K) W9 d

van Waes, C., K.F. Kozarsky, A.B. Warren, L. Kidd, D. Paugh, M. Liebert, and T.E. Carey. 1991. The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin {alpha} 6ß4. Cancer Res. 51:2395–2402.
/ g1 X# R- c( F0 m9 w& h/ N
Watt, F.M. 2002. Role of integrins in regulating epidermal adhesion, growth and differentiation. EMBO J. 21:3919–3926.

Wennerberg, K., L. Lohikangas, D. Gullberg, M. Pfaff, S. Johansson, and R. Fässler. 1996. ß1 integrin-dependent and -independent polymerization of fibronectin. J. Cell Biol. 132:227–238." {8 _! D! t3 o! @8 P. i/ h5 A% W
* z$ ?0 T9 L1 E+ j  o
Xiong, J.P., T. Stehle, B. Diefenbach, R. Zhang, R. Dunker, D.L. Scott, A. Joachimiak, S.L. Goodman, and M.A. Arnaout. 2001. Crystal structure of the extracellular segment of integrin {alpha} vß3. Science. 294:339–345.8 O2 q1 w8 D3 {: e$ V7 A
6 o8 Z9 i- Z2 B- x
Xiong, J.P., T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S.L. Goodman, and M.A. Arnaout. 2002. Crystal structure of the extracellular segment of integrin {alpha} vß3 in complex with an Arg-Gly-Asp ligand. Science. 296:151–155.
; q* ~- `5 `  k6 j; W
Zhu, A.J., I. Haase, and F.M. Watt. 1999. Signaling via ß1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. Proc. Natl. Acad. Sci. USA. 96:6728–6733.3 T  P+ C: U# W$ |  y
) U: B: l; W7 Q+ J6 e; o% |

! o8 m# ~. f* P. b

(Richard D. Evans Vivienne C. Perkins Alistair Henry Paul E. Stephens Martyn K. Robinson and Fiona M.)

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