Key points are not available for this paper at this time.
We previously reported that STAT3 plays a crucial role in transducing a signal for migration of keratinocytes (Sano, S., Itami, S., Takeda, K., Tarutani, M., Yamaguchi, Y., Miura, H., Yoshikawa, K., Akira, S., and Takeda, J. (1999) EMBO J. 18, 4657–4668). To clarify the role of STAT3 in signaling the migration, we studied the intracellular signaling pathway through an integrin receptor in STAT3-deficient keratinocytes. STAT3-deficient keratinocytes demonstrated increased adhesiveness and fast spreading on a collagen matrix. Staining with anti-phosphotyrosine antibody revealed that STAT3-deficient keratinocytes had an increased number of tyrosyl-hyperphosphorylated focal adhesions. Analyses with immunoprecipitation revealed that p130cas was constitutively hyperphosphorylated on tyrosine residues, while other focal adhesion molecules such as focal adhesion kinase and paxillin were not. Transfection of STAT3-deficient keratinocytes with an adenoviral vector encoding the wild-type Stat3 gene reversed not only impaired migration but also the increased tyrosine phosphorylation of p130cas. These results strongly suggest that STAT3 in keratinocytes plays a critical role in turnover of tyrosine phosphorylation of p130cas, modulating cell adhesiveness to the substratum leading to growth factor-dependent cell migration. We previously reported that STAT3 plays a crucial role in transducing a signal for migration of keratinocytes (Sano, S., Itami, S., Takeda, K., Tarutani, M., Yamaguchi, Y., Miura, H., Yoshikawa, K., Akira, S., and Takeda, J. (1999) EMBO J. 18, 4657–4668). To clarify the role of STAT3 in signaling the migration, we studied the intracellular signaling pathway through an integrin receptor in STAT3-deficient keratinocytes. STAT3-deficient keratinocytes demonstrated increased adhesiveness and fast spreading on a collagen matrix. Staining with anti-phosphotyrosine antibody revealed that STAT3-deficient keratinocytes had an increased number of tyrosyl-hyperphosphorylated focal adhesions. Analyses with immunoprecipitation revealed that p130cas was constitutively hyperphosphorylated on tyrosine residues, while other focal adhesion molecules such as focal adhesion kinase and paxillin were not. Transfection of STAT3-deficient keratinocytes with an adenoviral vector encoding the wild-type Stat3 gene reversed not only impaired migration but also the increased tyrosine phosphorylation of p130cas. These results strongly suggest that STAT3 in keratinocytes plays a critical role in turnover of tyrosine phosphorylation of p130cas, modulating cell adhesiveness to the substratum leading to growth factor-dependent cell migration. signal transducers and activators of transcription epidermal growth factor hepatocyte growth factor phosphate-buffered saline Src homology 2 and 3, respectively focal adhesion kinase protein-tyrosine phosphatase Signal transducers and activators of transcription (STATs)1 are a family of latent cytoplasmic transcription factors that are activated by many cytokines and growth factors (1.Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3330) Google Scholar, 2.Ihle J.N. Curr. Opin. Cell Biol. 2001; 13: 211-217Crossref PubMed Scopus (583) Google Scholar, 3.Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1636) Google Scholar). STATs are phosphorylated on tyrosine residues by activated kinases in receptor complexes, leading to formation of homo- or heterodimers and translocation to the nucleus in which they regulate transcription. STAT3 is activated by a variety of cytokines and growth factors such as interleukin-6, epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor, and granulocyte colony-stimulating factor. These cytokines and growth factors regulate the biological activities of keratinocytes (4.Bennett N.T. Schultz G.S. Am. J. Surg. 1993; 165: 728-737Abstract Full Text PDF PubMed Scopus (415) Google Scholar,5.Martin P. Science. 1997; 276: 75-81Crossref PubMed Scopus (3626) Google Scholar), suggesting that STAT3 plays a crucial role in keratinocytes. Because germ line STAT3 deletion leads to embryonic lethality (6.Takeda K. Noguchi K. Shi W. Tanaka T. Matsumoto M. Yoshida N. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3801-3804Crossref PubMed Scopus (1085) Google Scholar), to elucidate the biological roles of STAT3 in the skin, we previously generated keratinocyte-specific STAT3-deficient mice by conditional gene targeting using the Cre-loxP strategy (7.Sano S. Itami S. Takeda K. Tarutani M. Yamaguchi Y. Miura H. Yoshikawa K. Akira S. Takeda J. EMBO J. 1999; 18: 4657-4668Crossref PubMed Scopus (423) Google Scholar). The Stat3 gene was disrupted under the control of a keratin 5 promoter. The mutant mice were born with no apparent abnormalities, and their epidermis and hair follicle development was normal at birth. However, wound healing was markedly retarded, and the second hair cycle was impaired in keratinocyte-specific Stat3 gene knockout mice. An in vitro study with cultured keratinocytes revealed that this phenotype was attributed to impaired migration because of the failure of STAT3 activation. Cell migration is composed of several concerted steps (8.Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3226) Google Scholar, 9.Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1298) Google Scholar). Migration is initiated with membrane protrusion (filopodia and leading edge) and adhesion to the extracellular matrix, followed by cell traction and the release of adhesions at the rear portion of the cell. These events are regulated by multiple signaling mechanisms, such as tyrosine kinase and/or phosphatase signaling (10.Angers-Loustau A. Cote J.F. Charest A. Dowbenko D. Spencer S. Lasky L.A. Tremblay M.L. J. Cell Biol. 1999; 144: 1019-1031Crossref PubMed Scopus (247) Google Scholar, 11.Inagaki K. Noguchi T. Matozaki T. Horikawa T. Fukunaga K. Tsuda M. Ichihashi M. Kasuga M. Oncogene. 2000; 19: 75-84Crossref PubMed Scopus (79) Google Scholar, 12.Liu J. Huang C. Zhan X. Oncogene. 1999; 18: 6700-6706Crossref PubMed Scopus (72) Google Scholar, 13.Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (555) Google Scholar, 14.Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar), mitogen-activated protein kinase signaling (15.Klemke R.L. Leng J. Molander R. Brooks P.C. Vuori K. Cheresh D.A. J. Cell Biol. 1998; 140: 961-972Crossref PubMed Scopus (587) Google Scholar, 16.Nguyen D.H. Catling A.D. Webb D.J. Sankovic M. Walker L.A. Somlyo A.V. Weber M.J. Gonias S.L. J. Cell Biol. 1999; 146: 149-164Crossref PubMed Scopus (299) Google Scholar), small GTPase signaling (i.e. Rho and Rac) (17.Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5168) Google Scholar, 18.Rottner K. Hall A. Small J.V. Curr. Biol. 1999; 9: 640-648Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), and cytoskeletal reorganization (i.e. actin polymerization/depolymerizaton, actin/myosin motor) (19.Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar, 20.Welch M.D. Trends Cell Biol. 1999; 9: 423-427Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). However, it is still undetermined whether STAT3 is involved in these signaling events. In this report, we found that STAT3-deficient keratinocytes showed increased adhesiveness and forced spreading on a collagen matrix and that an increased number of hyperphosphorylated focal adhesions, in particular, an adaptor protein, p130cas, was constitutively hyperphosphorylated on tyrosine residues. These results strongly suggest that intracellular signaling of STAT3 in keratinocytes modulates tyrosine phosphorylation of p130cas and cell adhesiveness to the substratum, leading to cell migration in response to growth factors. Keratinocyte-specific STAT3-disrupted mice were generated by using the Cre -loxP strategy under the control of a keratin-5 promoter as previously described (7.Sano S. Itami S. Takeda K. Tarutani M. Yamaguchi Y. Miura H. Yoshikawa K. Akira S. Takeda J. EMBO J. 1999; 18: 4657-4668Crossref PubMed Scopus (423) Google Scholar). Briefly, mice carrying keratin-5 promoter-driven (K5)-Cre transgene and a STAT3-null allele (K5-Cre:STAT3+/−) were mated with STAT3flox/flox mice. Offspring carrying a floxed STAT3 allele and/or K5-Cre transgene (K5-Cre:STAT3flox/+,K5-Cre:STAT3flox/−, STAT3flox/+, STAT3flox/−) were examined for their genotypes by allele-specific PCR. Care of mice was in accordance with institutional guidelines. Primary cultures of mouse keratinocytes were established from newborn to 5-day-old mice. The total skin taken from mice was treated with 250 units/ml dispase (Godo Shusei, Tokyo, Japan) overnight at 4 °C, and the epidermis was peeled off from the dermis. Keratinocytes were collected upon trypsinization and washed in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were resuspended in MCDB153 medium containing 3% fetal calf serum and seeded onto type I collagen-coated dishes. Anti-p130cas, FAK, and paxillin were from Transduction Laboratories (Lexington, KY). Anti-c-Src (B-12) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phosphotyrosine monoclonal antibody (PY20) was from ICN Biomedicals, Inc. Anti-PTP-PEST monoclonal antibody was from Exalpha Biologicals, Inc. (Boston, MA). Horseradish peroxidase-conjugated goat anti-mouse secondary antibody was from Amersham Biosciences. Fluorescein isothiocyanate-conjugated anti-mouse antibody was from DAKO (Denmark). Keratinocytes were cultured in type I collagen-coated dishes until they reached confluence. After starvation for 24 h, they were treated with 10 μg/ml mitomycin C for 2 h to avoid a proliferative effect on the cells. A cell-free area was introduced by scraping the monolayer with a yellow pipette tip. Cell migration to the cell-free area was evaluated in the absence or presence of 10 ng/ml EGF (Upstate Biotechnology, Inc., Lake Placid, NY) or HGF (Collaborative Biomedical Products, Bedford, MA). After 48 h, photographs were taken using a phase-contrast microscope (DIA-PHOT 300; Nikon). The number of migrating keratinocytes was counted after taking photographs of four nonoverlapping fields. Values represent the mean ± S.D. of migrating cells per square millimeter beyond the front lines of the introduced wound edge. Student's t test was used for statistical analysis. Keratinocytes collected upon trypsinization of epidermal sheets were washed in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were resuspended in serum-free MCDB153 medium at 106 cells/ml and added to type I collagen-coated dishes. These dishes were incubated at 37 °C for the indicated times. Nonadherent cells were removed by washing with phosphate-buffered saline (PBS), and attached cells were counted. Values represent the mean ± S.D. and were analyzed statistically using Student's t test. Keratinocytes were seeded onto type I collagen-coated dishes at 5 × 105 cells/ml, and allowed to spread for the indicated times. After washing out the nonadherent cells, photographs were taken in the nonoverlapping fields under a phase-contrast microscope, and spread cells were counted. Spread cells were identified as opaque cells, exhibiting membranous protrusions. However, nonspread cells were rounded and phase-bright. Values represent the mean ± S.D. and were analyzed statistically using Student's t test. Keratinocytes (5 × 105 cells/ml) were plated onto type I collagen-coated coverslips for 24 h in MCDB153 medium containing 3% fetal calf serum. After serum starvation for 24 h, coverslips were washed twice with PBS and fixed for 5 min with 4% paraformaldehyde containing 0.5% Triton X-100 for permeabilization, followed by fixation with 4% paraformaldehyde for 15 min. After washing with PBS, fixed cells were blocked in PBS containing 1% bovine serum albumin. Cells were stained with a 1:100 diluted anti-phosphotyrosine antibody (PY20). Cells were further incubated with a fluorescein isothiocyanate-conjugated anti-mouse secondary antibody and washed with PBS. Images were analyzed using a confocal microscope (model LSM 410; Carl Zeiss). Keratinocytes were washed twice with PBS and lysed in a radioimmune precipitation buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mm EDTA, 1 mm sodium orthovanadate, 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). The protein concentration of the lysates was normalized before Western blotting or immunoprecipitation. For immunoprecipitation, equivalent lysates were incubated with precipitating antibodies bound to protein G-Sepharose beads (Amersham Biosciences) for 90 min at 4 °C. Immunoprecipitates were washed five times with the radioimmune precipitation buffer. The immunoprecipitates or whole lysates were separated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and reacted with primary antibodies. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL; Amersham Biosciences). A recombinant adenoviral vector containing the wild-type STAT3 expression cassette (Adeno-stat3) was constructed using the circular form of the adenoviral genome cloned in a cosmid and the Cre-loxPrecombination system according to Tashiro (21.Tashiro F. Niwa H. Miyazaki J. Hum. Gene Ther. 1999; 10: 1845-1852Crossref PubMed Scopus (38) Google Scholar). The pALC cosmid and wild-type STAT3 expression cassette were ligated. The resulting pALC-STAT3 cosmid vector was purified from E. coli DH5α and transfected into 293 cells with the pMC1-cre plasmid. The cosmid backbone was excised by transiently expressed Cre recombinase. Recombinant adenoviruses were purified by polyethylene glycol precipitation and cesium chloride ultracentrifugation. For reintroduction of wild-type STAT3, keratinocytes were infected with Adeno-stat3 during a serum starvation period for 24 h at a multiplicity of infection of 5. We previously demonstrated that keratinocyte-specific STAT3-disrupted mice showed retardation of skin wound healing and that migration of STAT3-deficient keratinocytes was impaired (7.Sano S. Itami S. Takeda K. Tarutani M. Yamaguchi Y. Miura H. Yoshikawa K. Akira S. Takeda J. EMBO J. 1999; 18: 4657-4668Crossref PubMed Scopus (423) Google Scholar). Cell migration is a coordinated and complex process, including cell adhesion to the extracellular matrix and organization of the actin cytoskeleton. Therefore, we hypothesized that STAT3 is involved in a specific point in these processes. First we compared cell attachment to the extracellular matrix. Freshly isolated keratinocytes were seeded onto type I collagen-coated dishes. Then nonadherent cells were removed by washing at the indicated time points, and attached cells were counted. As shown in Fig. 1a, STAT3-disrupted keratinocytes (white bars) showed significantly increased adhesiveness to the collagen matrix as compared with control cells (black bars) at 20 min, although no difference was observed after 1 or 3 h. The adhesion of keratinocytes to type I collagen is mediated through α2β1 and/or α3β1 integrins, and the STAT3 signaling pathway may modulate integrin expression (22.Wooten D.K. Xie X. Bartos D. Busche R.A. Longmore G.D. Watowich S.S. J. Biol. Chem. 2000; 275: 26566-26575Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) in some cells. However, no difference in integrin expression was found between control and STAT3-disrupted keratinocytes (Ref. 7.Sano S. Itami S. Takeda K. Tarutani M. Yamaguchi Y. Miura H. Yoshikawa K. Akira S. Takeda J. EMBO J. 1999; 18: 4657-4668Crossref PubMed Scopus (423) Google Scholar and data not shown). Therefore, increased adhesion of STAT3-disrupted keratinocytes was not attributed to increased integrin expression. Next, we compared cell spreading on the substratum of STAT3(−/−) keratinocytes with controls. Twenty min after seeding, an increased number of STAT3(−/−) keratinocytes were opaque with membranous protrusions (Fig. 1b, black arrows), which is a characteristic feature of spread cells, whereas control keratinocytes (STAT3(+/−)) were mostly round and phase-bright (nonspread) (Fig. 1b, upper panel). Quantitative evaluation revealed that the number of spread cells in STAT3-disrupted keratinocytes was 5-fold that at 20 min compared with controls, although no difference in cell spreading was observed between the two kinds of keratinocytes later than 1 h (Fig. 1c). Taken collectively, these results indicate that STAT3-disrupted cells were primed for attachment and spreading on the substratum, implying that STAT3 plays a critical role in regulating subsequent cell motility through integrin/focal adhesion signaling. In cultured cells, integrins and tyrosine-phosphorylated proteins are concentrated at focal adhesions, where actin cytoskeletons are connected with the extracellular matrix. Many lines of evidence have revealed that the number and/or tyrosine phosphorylation status of focal adhesions influenced adhesiveness, spreading, and cell migration (10.Angers-Loustau A. Cote J.F. Charest A. Dowbenko D. Spencer S. Lasky L.A. Tremblay M.L. J. Cell Biol. 1999; 144: 1019-1031Crossref PubMed Scopus (247) Google Scholar, 11.Inagaki K. Noguchi T. Matozaki T. Horikawa T. Fukunaga K. Tsuda M. Ichihashi M. Kasuga M. Oncogene. 2000; 19: 75-84Crossref PubMed Scopus (79) Google Scholar, 14.Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 23.Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1576) Google Scholar). Therefore, we next examined the formation and tyrosine phosphorylation status of focal adhesions, by immunostaining with an anti-phosphotyrosine monoclonal antibody. Strikingly, individual STAT3-disrupted keratinocytes exhibited coarse and strong signals on the surface (Fig. 2, right panel,red arrowheads) compared with control cells (Fig. 2, left panel), indicating that focal adhesions of STAT3-disrupted keratinocytes were hyperphosphorylated. To determine the focal adhesion molecule responsible for hyperphosphorylation in STAT3(−/−) keratinocytes, Western blot analysis was performed. Whole cell lysates (Fig. 3a, left panel) or PY20 immunoprecipitates (Fig. 3a,right panel) were subjected to Western blot analysis with PY20. Approximately 60–90- and 120–130-kDa proteins were hyperphosphorylated on tyrosine residues in STAT3-disrupted keratinocytes (Fig. 3a, black arrows). Previous studies showed that many tyrosine-phosphorylated proteins are associated with focal adhesions, including nonreceptor kinases, adaptor proteins, and cytoskeletal proteins (13.Parsons J.T. Martin K.H. Slack J.K. Taylor J.M. Weed S.A. Oncogene. 2000; 19: 5606-5613Crossref PubMed Scopus (555) Google Scholar, 24.Turner C.E. Nat. Cell Biol. 2000; 2: E231-E236Crossref PubMed Scopus (643) Google Scholar, 25.Vuori K. Hirai H. Aizawa S. Ruoslahti E. Mol. Cell. Biol. 1996; 16: 2606-2613Crossref PubMed Google Scholar). Among them, focal adhesion kinase (FAK;Mr ∼125,000), p130cas(Mr ∼130,000), paxillin (Mr ∼68,000), and Src (Mr ∼60,000) were candidates for the hyperphosphorylated proteins observed in Fig. 3a. Therefore, we analyzed the tyrosine phosphorylation state of these proteins. As shown in Fig. 3b, no differences were demonstrated in protein expression or tyrosine phosphorylation levels of FAK, Src, and paxillin between control and STAT3-disrupted keratinocytes. However, p130cas was constitutively hyperphosphorylated on tyrosine residues in STAT3(−/−) keratinocytes (Fig. 3b,black arrow). p130cas is thought to be an adaptor protein that contains an Src homology 3 (SH3) domain, a central substrate domain, and proline-rich motifs (26.Kirsch K.H. Georgescu M.M. Hanafusa H. J. Biol. Chem. 1998; 273: 25673-25679Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The central substrate domain has multiple phosphotyrosine motifs where Src homology 2 (SH2) domain-containing molecules recruit. The hyperphosphorylation of p130cas in STAT3-disrupted keratinocytes may result in abnormal assembly of interacting proteins, aberrant turnover of focal adhesions, increased cell adhesiveness, and faster cell spreading, leading to impaired migration. It is possible that the hyperphosphorylation of p130cas might be the result of down-regulation of protein-tyrosine phosphatases. Protein-tyrosine phosphatase, PTP-PEST, has been shown to interact with p130cas, and it has been demonstrated that PTP-PEST(−/−) fibroblasts have a phenotype similar to STAT3(−/−) keratinocytes (10.Angers-Loustau A. Cote J.F. Charest A. Dowbenko D. Spencer S. Lasky L.A. Tremblay M.L. J. Cell Biol. 1999; 144: 1019-1031Crossref PubMed Scopus (247) Google Scholar). Therefore, we examined expression levels of PTP-PEST. However, as shown in Fig. 3c, no difference was demonstrated in protein expression levels of PTP-PEST between control and STAT3(−/−) keratinocytes.
Kira et al. (Mon,) studied this question.