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The Kindler Syndrome Protein Is Regulated by Transforming Growth Factor-β and Involved in Integrin-mediated Adhesion

2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês

10.1074/jbc.m307978200

ISSN

1083-351X

Autores

Susanne Kloeker, Michael B. Major, David Calderwood, Mark H. Ginsberg, David A. Jones, Mary C. Beckerle,

Tópico(s)

Cell Adhesion Molecules Research

Resumo

Transforming growth factor-β1 (TGF-β1) contributes to tumor invasion and cancer progression by increasing the motility of tumor cells. To identify genes involved in TGF-β-mediated cell migration, the transcriptional profiles of human mammary epithelial cells (HMEC) treated with TGF-β were compared with untreated cells by cDNA microarray analysis. One gene up-regulated by TGF-β was recently named kindlerin (Jobard, F., Bouadjar, B., Caux, F., Hadj-Rabia, S., Has, C., Matsuda, F., Weissenbach, J., Lathrop, M., Prud'homme, J. F., and Fischer, J. (2003) Hum. Mol. Genet. 12, 925–935). This gene is significantly overexpressed in some cancers (Weinstein, E. J., Bourner, M., Head, R., Zakeri, H., Bauer, C., and Mazzarella, R. (2003) Biochim. Biophys. Acta 1637, 207–216), and mutations in this gene lead to Kindler syndrome, an autosomal-recessive genodermatosis. TGF-β stimulation of HMEC resulted in a marked induction of kindlerin RNA, and Western blotting demonstrated a corresponding increase in protein abundance. Kindlerin displays a putative FERM (four point one ezrin radixin moesin) domain that is closely related to the sequences in talin that interact with integrin β subunit cytoplasmic domains. The critical residues in the talin FERM domain that mediate integrin binding show a high degree of conservation in kindlerin. Furthermore, kindlerin is recruited into a molecular complex with the β1A and β3 integrin cytoplasmic domains. Consistent with these biochemical findings, kindlerin is present at focal adhesions, sites of integrin-rich, membrane-substratum adhesion. Additionally, kindlerin is required for normal cell spreading. Taken together, these data suggest a role for kindlerin in mediating cell processes that depend on integrins. Transforming growth factor-β1 (TGF-β1) contributes to tumor invasion and cancer progression by increasing the motility of tumor cells. To identify genes involved in TGF-β-mediated cell migration, the transcriptional profiles of human mammary epithelial cells (HMEC) treated with TGF-β were compared with untreated cells by cDNA microarray analysis. One gene up-regulated by TGF-β was recently named kindlerin (Jobard, F., Bouadjar, B., Caux, F., Hadj-Rabia, S., Has, C., Matsuda, F., Weissenbach, J., Lathrop, M., Prud'homme, J. F., and Fischer, J. (2003) Hum. Mol. Genet. 12, 925–935). This gene is significantly overexpressed in some cancers (Weinstein, E. J., Bourner, M., Head, R., Zakeri, H., Bauer, C., and Mazzarella, R. (2003) Biochim. Biophys. Acta 1637, 207–216), and mutations in this gene lead to Kindler syndrome, an autosomal-recessive genodermatosis. TGF-β stimulation of HMEC resulted in a marked induction of kindlerin RNA, and Western blotting demonstrated a corresponding increase in protein abundance. Kindlerin displays a putative FERM (four point one ezrin radixin moesin) domain that is closely related to the sequences in talin that interact with integrin β subunit cytoplasmic domains. The critical residues in the talin FERM domain that mediate integrin binding show a high degree of conservation in kindlerin. Furthermore, kindlerin is recruited into a molecular complex with the β1A and β3 integrin cytoplasmic domains. Consistent with these biochemical findings, kindlerin is present at focal adhesions, sites of integrin-rich, membrane-substratum adhesion. Additionally, kindlerin is required for normal cell spreading. Taken together, these data suggest a role for kindlerin in mediating cell processes that depend on integrins. The survival of cancer patients with solid tumors decreases dramatically when tumors are invasive and have an increased likelihood of metastasizing to distal sites. The enhanced invasiveness of tumor cells is attributed to epithelial to mesenchymal transition (EMT), 1The abbreviations used are: EMT, epithelial to mesenchymal transition; TGF-β1, transforming growth factor β; HMEC, human mammary epithelial cells; Mig-2, mitogen inducible gene-2; FERM, four point one ezrin radixin moesin; PH, pleckstrin homology; siRNA, small interfering siRNA; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PH, pleckstrin homology. 1The abbreviations used are: EMT, epithelial to mesenchymal transition; TGF-β1, transforming growth factor β; HMEC, human mammary epithelial cells; Mig-2, mitogen inducible gene-2; FERM, four point one ezrin radixin moesin; PH, pleckstrin homology; siRNA, small interfering siRNA; PBS, phosphate-buffered saline; MES, 4-morpholineethanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PH, pleckstrin homology. (5Savagner P. BioEssays. 2001; 23: 912-923Crossref PubMed Scopus (605) Google Scholar, 6Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5408) Google Scholar, 7Akhurst R.J. Derynck R. Trends Cell Biol. 2001; 11: S44-S51PubMed Scopus (727) Google Scholar, 8Derynck R. Akhurst R.J. Balmain A. Nat. Genet. 2001; 29: 117-129Crossref PubMed Scopus (1939) Google Scholar) a normal biological process that is critical for wound healing and development. EMT is characterized by a change in cell shape from a polarized epithelial cell to a flattened fibroblast-like morphology, a decrease in cell-cell junctions concurrent with a decrease in E-cadherin expression, and an increase in cell motility (5Savagner P. BioEssays. 2001; 23: 912-923Crossref PubMed Scopus (605) Google Scholar, 6Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5408) Google Scholar). Transforming growth factor-β1 (TGF-β) is a major contributor to tumor progression and metastasis (9Oft M. Heider K.H. Beug H. Curr. Biol. 1998; 8: 1243-1252Abstract Full Text Full Text PDF PubMed Google Scholar, 10McEarchern J.A. Kobie J.J. Mack V. Wu R.S. Meade-Tollin L. Arteaga C.L. Dumont N. Besselsen D. Seftor E. Hendrix M.J. Katsanis E. Akporiaye E.T. Int. J. Cancer. 2001; 91: 76-82Crossref PubMed Scopus (127) Google Scholar, 11Janda E. Lehmann K. Killisch I. Jechlinger M. Herzig M. Downward J. Beug H. Grunert S. J. Cell Biol. 2002; 156: 299-314Crossref PubMed Scopus (603) Google Scholar), and several studies have shown that TGF-β promotes tumor cell invasiveness by promoting EMT (9Oft M. Heider K.H. Beug H. Curr. Biol. 1998; 8: 1243-1252Abstract Full Text Full Text PDF PubMed Google Scholar, 12Boland S. Boisvieux-Ulrich E. Houcine O. Baeza-Squiban A. Pouchelet M. Schoevaert D. Marano F. J. Cell Sci. 1996; 109: 2207-2219Crossref PubMed Google Scholar, 13Bakin A.V. Tomlinson A.K. Bhowmick N.A. Moses H.L. Arteaga C.L. J. Biol. Chem. 2000; 275: 36803-36810Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar, 14Ellenrieder V. Hendler S.F. Boeck W. Seufferlein T. Menke A. Ruhland C. Adler G. Gress T.M. Cancer Res. 2001; 61: 4222-4228PubMed Google Scholar, 15Bhowmick N.A. Ghiassi M. Bakin A. Aakre M. Lundquist C.A. Engel M.E. Arteaga C.L. Moses H.L. Mol. Biol. Cell. 2001; 12: 27-36Crossref PubMed Scopus (849) Google Scholar). Despite the key role established for TGF-β in stimulating EMT and tumor progression, the molecular mechanisms by which TGF-β promotes EMT have not been fully elucidated.Microarray analysis of a TGF-β-responsive cell line, human mammary epithelial cells (HMEC), led us to identify kindlerin as a TGF-β-inducible gene. Kindlerin is mutated in Kindler syndrome, a rare autosomal-recessive genodermatosis (1Jobard F. Bouadjar B. Caux F. Hadj-Rabia S. Has C. Matsuda F. Weissenbach J. Lathrop M. Prud'homme J.F. Fischer J. Hum. Mol. Genet. 2003; 12: 925-935Crossref PubMed Scopus (194) Google Scholar, 3Siegel D.H. Ashton G.H. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. South A.P. Smith F.J. Prescott A.R. Wessagowit V. Oyama N. Akiyama M. Al Aboud D. Al Aboud K. Al Githami A. Al Hawsawi K. Al Ismaily A. Al-Suwaid R. Atherton D.J. Caputo R. Fine J.D. Frieden I.J. Fuchs E. Haber R.M. Harada T. Kitajima Y. Mallory S.B. Ogawa H. Sahin S. Shimizu H. Suga Y. Tadini G. Tsuchiya K. Wiebe C.B. Wojnarowska F. Zaghloul A.B. Hamada T. Mallipeddi R. Eady R.A. McLean W.H. McGrath J.A. Epstein E.H. Am. J. Hum. Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Early in life patients with Kindler syndrome endure blistering of the skin and photosensitivity, which progresses to diffuse poikiloderma followed by cutaneous atrophy (16Kindler T. Br. J. Dermatol. 1954; 66: 104-111Crossref PubMed Scopus (164) Google Scholar, 17Forman A.B. Prendiville J.S. Esterly N.B. Hebert A.A. Duvic M. Horiguchi Y. Fine J.D. Pediatr. Dermatol. 1989; 6: 91-101Crossref PubMed Scopus (58) Google Scholar). The clinical presentation of this disease is similar to patients with junctional epidermolysis bullosa harboring mutations in α6 and β4 integrin genes (18Vidal F. Aberdam D. Miquel C. Christiano A.M. Pulkkinen L. Uitto J. Ortonne J.P. Meneguzzi G. Nat. Genet. 1995; 10: 229-234Crossref PubMed Scopus (323) Google Scholar, 19Pulkkinen L. Kimonis V.E. Xu Y. Spanou E.N. McLean W.H. Uitto J. Hum. Mol. Genet. 1997; 6: 669-674Crossref PubMed Scopus (123) Google Scholar).Kindlerin (also known as URP1 for UNC-112 related protein 1 or kindlin) is a member of a newly recognized protein family, which also includes Mig-2 and URP2 (2Weinstein E.J. Bourner M. Head R. Zakeri H. Bauer C. Mazzarella R. Biochim. Biophys. Acta. 2003; 1637: 207-216Crossref PubMed Scopus (67) Google Scholar, 3Siegel D.H. Ashton G.H. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. South A.P. Smith F.J. Prescott A.R. Wessagowit V. Oyama N. Akiyama M. Al Aboud D. Al Aboud K. Al Githami A. Al Hawsawi K. Al Ismaily A. Al-Suwaid R. Atherton D.J. Caputo R. Fine J.D. Frieden I.J. Fuchs E. Haber R.M. Harada T. Kitajima Y. Mallory S.B. Ogawa H. Sahin S. Shimizu H. Suga Y. Tadini G. Tsuchiya K. Wiebe C.B. Wojnarowska F. Zaghloul A.B. Hamada T. Mallipeddi R. Eady R.A. McLean W.H. McGrath J.A. Epstein E.H. Am. J. Hum. Genet. 2003; 73: 174-187Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). An apparent kindlerin orthologue, UNC-112, has been studied in Caenorhabditis elegans (20Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (164) Google Scholar). The unc-112 gene is essential for embryogenesis, and it displays a genetic interaction with integrins (20Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (164) Google Scholar). Because integrins play a critical role in mammalian cell adhesion and migration, we postulated that kindlerin may be involved in TGF-β-stimulated EMT through interactions with integrins.Here we report that kindlerin expression is responsive to TGF-β levels, that kindlerin localizes in focal adhesions (integrin-rich signaling centers that integrate extracellular matrix attachment and cytoskeletal organization), and that kindlerin forms complexes with integrin β subunit cytoplasmic domains. Furthermore, cell spreading is perturbed upon reduction of kindlerin protein. Taken together with the observation that kindlerin is overexpressed in colon and lung carcinomas (2Weinstein E.J. Bourner M. Head R. Zakeri H. Bauer C. Mazzarella R. Biochim. Biophys. Acta. 2003; 1637: 207-216Crossref PubMed Scopus (67) Google Scholar), our data support a role for kindlerin in mammalian cell adhesion and suggest that kindlerin may mediate TGF-β signaling in tumor progression via contributions to integrin-dependent cellular functions.EXPERIMENTAL PROCEDURESCell Culture and Drug Treatments—HaCaT cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and were split every 3rd day or at 80% confluency. The HaCaT immortalized keratinocyte cell line was a gift from D. Grossman. HMEC were obtained from BioWhittaker and cultured in complete mammary epithelial growth media. HMEC were seeded at passages 7 or 8 and harvested at no greater than 80% confluency for all experiments.cDNA Microarray Data Analysis—The construction of the microarrays, generation of the microarray probes, microarray hybridization, and scanning were performed as described previously (21Karpf A.R. Peterson P.W. Rawlins J.T. Dalley B.K. Yang Q. Albertsen H. Jones D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14007-14012Crossref PubMed Scopus (172) Google Scholar). First-strand cDNAs were generated by reverse transcription from the mRNA samples in the presence of Cy-3dCTP or Cy-5dCTP. The resulting labeled cDNAs were combined and simultaneously hybridized to the microarray slide displaying 4608 randomly selected and minimally redundant cDNAs from the unigene set (22Miller G.S. Fuchs R. Comput. Appl. Biosci. 1997; 13: 81-87PubMed Google Scholar). Each of the 4608 minimally redundant genes was present in duplicate on the microarray, and the comparisons were completed three separate times resulting in a total of six measurements for each gene. In each case, mRNA from TGF-β-treated cells was directly compared with mRNA from vehicle-treated cells. The GeneSpring software program (version 5.1; Silicon Genetics) was utilized for all steps in the data analysis. To normalize the microarray data, a Lowess curve was fit to the log intensity versus log ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. We selected TGF-β-responsive genes based on a statistical analysis using the gene expression program GeneSpring (version 5.1). We applied the inclusion criteria of 1.5-fold induction, and a p value of less than 0.05 (Student's t test) in defining TGF-β up-regulated genes. The list of genes shown in Table I was selected from the total normalized data set as having a fold induction greater than 1.5 in at least 4 of 6 data points (see the experimental description within Table I). Genes with a p value (Students t test) greater than 0.05 were not included. DNA sequencing and BLAST analysis confirmed the identity of each microarray clone corresponding to those genes listed in Table I.Table IGenes Induced by TGF-β in HMEC Human mammary epithelial cells were treated with TGF-β (10 ng/ml) for 3 h; mRNA harvested from treated and non-treated HMEC samples (3 independent replicates) was reverse-transcribed into cDNA in the presence of either a green or red fluorescent tag. Competitive hybridization of the labeled cDNAs on glass slides containing 4608 cDNAs spotted in duplicate revealed the relative increase and decrease of specific mRNAs. Stringent data analysis (greater than 1.5-fold in 4 of the 6 microarray ratios; p value less than 0.05) facilitated the identification of 17 TGF-β up-regulated transcripts.Gene nameFold changep valueap values were calculated using the Student's t test from 6 independent microarray expression ratiosPlasminogen activator inhibitor, type I (PAI1)3.077.76E-07Integrin α22.554.46E-04Kindlerin2.305.36E-05Transglutaminase2.175.07E-03TGF-β induced, 68-kDa1.964.92E-03Connective tissue growth factor1.944.32E-05PMEPA11.895.56E-03Sox-41.881.66E-02Tubulin α-4 chain1.762.13E-02Tis11D/ERF-21.703.22E-04Gadd45β1.697.24E-04EST (accession no. Al828370)1.666.81E-03DNA G/T mismatch-binding protein1.636.79E-05Thrombospondin 11.621.68E-02TGF-β inducible early protein (TIEG)1.614.04E-05Early growth response (EGR-1)1.583.63E-02Fibronectin1.588.22E-05a p values were calculated using the Student's t test from 6 independent microarray expression ratios Open table in a new tab Northern Blotting—For treatments with TGF-β1 (PeproTech), HMEC cells were not serum-starved prior to the addition of growth factor. The vehicle control for TGF-β1 was composed of 4 mm HCl, 1 mg/ml bovine serum albumin. Cycloheximide (Calbiochem) was used at 10 μg/ml and was added to the cells 15 min prior to the addition of TGF-β. Total RNA was isolated using Trizol (Invitrogen) followed by poly(A) RNA selection using a Poly(AT) Tract mRNA Isolation kit (Promega). Poly(A) RNA was fractionated through formaldehyde-containing agarose gels and transferred onto nylon membranes (Amersham Biosciences). Probes were generated using the Rediprime II random prime labeling system (Amersham Biosciences) supplemented with [32P]dCTP. Hybridizations with 32P-labeled probes were carried out using ULTRAhyb buffer (Ambion) as recommended by the manufacturer. The kindlerin probe was generated by amplification of the insert of a cDNA image clone (accession number 299593) using T7 and T3 primers.Construction of FLAG-tagged Kindlerin cDNA—In order to manufacture the longest piece of the 5′ end of kindlerin cDNA, the EcoRI/SwaI fragment of AA158566 was subcloned into the EcoRI/SwaI sites of AI147142 to make clone A. Clone A was used as a template for PCR to add an EcoRI site directly 5′ of the ATG (sense oligonucleotide 5, 5′-ACG AAT TCA ATG CTG TCA TCC ACT GAC TTT AC-3′) and ended at the native PstI site (antisense oligonucleotide 6, 5′-CGA GGA TGC TGC AGT TTT GTT CC-3′). The EcoRI/PstI insert from clone A was excised and replaced with the PCR product digested with EcoRI/PstI in order to remove the 5′-untranslated region to generate clone B. The 3′ end of kindlerin cDNA was isolated by reverse transcriptase-PCR using RNA isolated from HMEC treated with TGF-β. The sense oligonucleotide 7 (5′-ATA GGT ACC TCA ATC CTG ACC GCC GGT CAA-3′) began at the HaeII site, and the antisense oligonucleotide 8 (5′-GAA GCG GCG CTT TCT AAT TTG GA-3′) added a KpnI site directly 3′ of the stop codon. The complete FLAG-kindlerin cDNA was assembled by three-piece ligation containing clone B digested with EcoRI and HaeII, the reverse transcriptase-PCR product digested with HaeII and KpnI, and FLAGpCMV2 digested with EcoRI and KpnI.Kindlerin Antibody Generation—A construct expressing the C-terminal third of kindlerin (amino acid residues 500 to end) with a His tag was engineered as follows: a PCR product was generated using sense oligonucleotide 11 (5′-GCT CCA TAT GAT TCT TTC CTT TCT GAA GAT GCA GCA T-3′) and antisense oligonucleotide 12 (5′-ATG CGG CCG CTC ACA CCC AAC CAC TGG TAA GTT T-3′) using the FLAG-kindlerin cDNA as template and cloned into NdeI and NotI sites of the pET28a vector (Novagen). The construct was verified by DNA sequencing and utilized to transform competent BL21 cells (Novagen). The recombinant His-tagged kindlerin fragment was purified under denaturing conditions in 6 m urea. Fractions containing purified protein were pooled and concentrated using a Centricon-30 (Millipore). Concentrated protein was loaded onto SDS-PAGE, briefly stained with BioSafe Coomassie Blue (Bio-Rad), and excised from the gel. Gel pieces containing kindlerin were used to inject rabbits for polyclonal antibody production (Harlan Bioproducts for Science).Western Blotting—For the TGF-β time course, HMEC or HaCaT were treated with vehicle or TGF-β (2 ng/ml) for the indicated times. Protein lysates were harvested from HaCaT or HMEC in a buffer containing 25 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm CaCl2, 1% Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm benzamidine, 1 mg/ml pepstatin A, and 1 mg/ml phenanthroline. The lysates were centrifuged at 20,800 × g for 10 min at 4 °C. The supernatant was transferred to a clean tube and assayed for protein concentration using the DC Protein Assay (Bio-Rad). For antibody characterization (Fig. 3A), lysates were heated at 70 °C for 10 min in LDS sample buffer (Invitrogen) and then separated by Tris-glycine 10% SDS-PAGE and transferred to nitrocellulose (Millipore). The blots were incubated with indicated kindlerin, vinculin (Sigma), or E-cadherin (Transduction Laboratories) antibodies, followed by incubation with donkey anti-rabbit horseradish peroxidase or sheep anti-mouse horseradish peroxidase (Amersham Biosciences). Immune complexes were visualized with Western Lightning Chemiluminescence Reagent (PerkinElmer Life Sciences). For the time course experiments, lysates were harvested and analyzed as indicated above, with the exception that BisTris 4–12% NuPAGE using MES running buffer (Invitrogen) was utilized to separate the lysates.Phase Contrast Imaging—HMEC were allowed to adhere overnight and were subsequently treated with vehicle or TGF-β for 48 h. Cell morphology was observed using a Nikon microscope and recorded with a digital camera.Affinity Purification of Kindlerin Antibody and Indirect Immunofluorescence—Kindlerin antiserum was affinity-purified by using a method described previously (23Beckerle M.C. J. Cell Biol. 1986; 103: 1679-1687Crossref PubMed Scopus (103) Google Scholar). Briefly, antiserum was incubated with recombinant kindlerin immobilized on nitrocellulose. After extensive washing of the membrane, the bound antibodies were eluted with 100 mm glycine, pH 2.5, and immediately combined with 1 m Tris-HCl, pH 9.0, sufficient to neutralize the glycine-HCl. The eluted material was then concentrated in a Centricon-10 device (Amicon). The control antibody was absorbed to nitrocellulose without kindlerin protein. Indirect immunofluorescence was performed by plating HaCaT cells on glass coverslips coated with 10 μg/ml fibronectin (Sigma) in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). To examine kindlerin localization in the presence of TGF-β, cells were treated with vehicle or TGF-β (2 ng/ml) for 48 h. Coverslips were washed in PBS, fixed for 15 min with 4% formaldehyde, 5 μm CaCl2, 0.5 μm MgCl2 in PBS, and permeabilized for 4 min with 0.2% Triton X-100 in PBS. After washing in PBS, the coverslips were incubated with primary antibodies at 37 °C for 120 min, washed in PBS for 10 min, and subsequently incubated with Alexa-488- or Alexa-594-conjugated secondary antibodies (Molecular Probes). The coverslips were mounted with ProLong (Molecular Probes) after washing for 10 min in PBS. Primary antibodies used in this study include affinity-purified rabbit polyclonal kindlerin antibody R-2230 (1:100), control antibody (1:100), rabbit polyclonal talin antibody B-11 (1:800), and vinculin monoclonal antibody (1:300 Sigma). Actin filaments were visualized using Alexa-488-conjugated phalloidin (1:100 Molecular Probes). Cell images were visualized on a Zeiss Axiophot microscope and recorded with a digital camera.Kindlerin and Talin Amino Acid Alignment and Modeling—The primary sequences of kindlerin and talin were aligned by blast searching (www.ncbi.nlm.nih.gov/blast/) and refined using structure-based alignments performed by Swiss-PdbViewer (us.expasy.org/spdbv/) using the structure of talin subdomains F2 and F3 (4Garcia-Alvarez B. de Pereda J.M. Calderwood D.A. Ulmer T.S. Critchley D. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar) (Protein Data Bank code 1MK7) as template. Based on the sequence alignment, the putative kindlerin F3 subdomain (residues 566–655) was modeled on the talin F3 subdomain (residues 309–400 from Protein Data Bank code 1MK7). Modeling was performed using Swiss model (24Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9472) Google Scholar, 25Peitsch M.C. Biochem. Soc. Trans. 1996; 24: 274-279Crossref PubMed Scopus (898) Google Scholar, 26Peitsch M.C. Bio/Technology. 1995; 13: 658-660Crossref Scopus (113) Google Scholar), and the quality of the resulting model was assessed using WHAT_CHECK and WHAT-IF version 19970813-1517 (27Vriend G. J. Mol. Graphics. 1990; 8: 52-56Crossref PubMed Scopus (3353) Google Scholar, 28Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1794) Google Scholar).Integrin β Tail Binding Assays—Affinity chromatography was performed by using recombinant models of integrin cytoplasmic tails as described previously (29Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar). Briefly, Chinese hamster ovary cells transfected with FLAG-kindlerinpCMV2 were lysed as described previously (29Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar), and the indicated amount of lysate was mixed with 50 μl of His-Bind resin coated with recombinant integrin tails. Beads were washed and bound proteins fractionated by SDS-PAGE, and kindlerin was detected by Western blotting with FLAG antibodies. Loading of recombinant integrin tails onto the beads was assessed by Coomassie Blue staining.siRNA Transfection—HaCaT cells were transfected using siPORT Amine (Ambion) with a 21-nucleotide irrelevant RNA (control) or with a 21-nucleotide RNA targeting the kindlerin sequence (only sense sequence is shown): 5′-GAA GUU ACU ACC AAA AGC UTT. Cells were harvested ∼44 h after adding siRNA duplexes and examined for kindlerin expression by Western blot analysis. To show equivalent loading of protein, the membrane was probed with a talin monoclonal antibody (Sigma).Cell Spreading—Approximately 44 h after addition of the siRNA duplexes, cells were trypsinized and washed 2 times in Opti-MEM (Invitrogen). Cells were plated on fibronectin (10 μg/ml, Sigma)-coated coverslips and were allowed to spread for 30 min at 37 °C, 5% CO2. Cells were stained by indirect immunofluorescence as described above using talin and vinculin antibodies. A minimum of four randomly selected fields (∼180 cells total) was recorded with a digital camera. The area of cell spreading visualized by talin immunofluorescence was measured using Openlab software.RESULTSTGF-β Treatment Increases Kindlerin Expression—TGF-β elicits its biological effects by activation of a SMAD-dependent transcriptional program (reviewed in Refs. 30Massague J. Annu. Rev. Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 31Christian J.L. Nakayama T. BioEssays. 1999; 21: 382-390Crossref PubMed Scopus (50) Google Scholar, 32Verrecchia F. Mauviel A. J. Investig. Dermatol. 2002; 118: 211-215Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 33Derynck R. Zhang Y.E. Nature. 2003; 425: 577-584Crossref PubMed Scopus (4201) Google Scholar). To identify potential downstream targets of TGF-β signaling that are involved in EMT and cell migration, we used microarray analysis to compare the transcriptional profiles of HMEC treated with 10 ng/ml TGF-β to those of vehicle-treated cells. Seventeen genes exhibited a greater than 1.5-fold induction and a p value of less than 0.05 (Table I). Induced genes included a number of known TGF-β-responsive genes with roles in cell motility and adhesion such as plasminogen activator inhibitor type I (34Gerwin B.I. Keski-Oja J. Seddon M. Lechner J.F. Harris C.C. Am. J. Physiol. 1990; 259: L262-L269PubMed Google Scholar, 35Stampfer M.R. Yaswen P. Alhadeff M. Hosoda J. J. Cell. Physiol. 1993; 155: 210-221Crossref PubMed Scopus (55) Google Scholar), transglutaminase (36Akimov S.S. Belkin A.M. J. Cell Sci. 2001; 114: 2989-3000Crossref PubMed Google Scholar), thrombospondin 1 (37Claisse D. Martiny I. Chaqour B. Wegrowski Y. Petitfrere E. Schneider C. Haye B. Bellon G. J. Cell Sci. 1999; 112: 1405-1416Crossref PubMed Google Scholar), and fibronectin (35Stampfer M.R. Yaswen P. Alhadeff M. Hosoda J. J. Cell. Physiol. 1993; 155: 210-221Crossref PubMed Scopus (55) Google Scholar). We examined Table I for novel genes with predicted roles in cell adhesion and migration, and we recognized that the third ranking gene encoded kindlerin, a protein required for stable attachment of epithelial cells to the lamina densa (1Jobard F. Bouadjar B. Caux F. Hadj-Rabia S. Has C. Matsuda F. Weissenbach J. Lathrop M. Prud'homme J.F. Fischer J. Hum. Mol. Genet. 2003; 12: 925-935Crossref PubMed Scopus (194) Google Scholar). Kindlerin displays similarity throughout its sequence to two other human proteins, Mig-2 (65% identity) and URP2 (57% identity), as shown in Fig. 1 and appears to be related to the C. elegans protein, UNC-112 (44% identity). UNC-112 is required for the organization of dense bodies and attachment of muscle cells to the hypodermis, and UNC-112 interacts genetically with integrins (20Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (164) Google Scholar). Thus we focused on kindlerin as a possible mediator of TGF-β effects on integrin function.Fig. 1Comparison of the amino acid sequences of kindlerin paralogues and UNC-112. Amino acid alignment of kindlerin, Mig-2, URP2, and UNC-112. Identical amino acids are highlighted in black, and dashes indicate gaps in the alignment. The amino acid sequences were aligned using the Clustal method.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to verify the induction of the kindlerin gene by TGF-β, mRNA was isolated every2hupto12h after treatment with TGF-β and examined by Northern blot analysis for kindlerin expression (Fig. 2A). A 4.6-kb kindlerin mRNA transcript was induced ∼10-fold 6 h after addition of TGF-β. The level of kindlerin transcript remained elevated after exposure to TGF-β for 96 h (data not shown). To examine the role of protein synthesis in regulation of kindlerin mRNA, we examined the effect of cycloheximide on TGF-β stimulated HMEC. Kindlerin mRNA abundance was markedly increased by the addition of TGF-β (Fig. 2B). Addition of cycloheximide significantly reduced the effect of TGF-β on kindlerin mRNA, indicating that protein synthesis is required for the TGF-β stimulation of kindlerin transcription. Thus, kindlerin is a TGF-β-inducible gene.Fig. 2TGF-

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