Deletion of the PDGFR-� Gene Affects Key Fibroblast FunctionsImportant for Wound Healing*

Received for publication, November 19, 2004Published, JBC Papers in Press, December 6, 2004, DOI 10.1074/jbc.M413081200

Zhiyang Gao‡, Toshiyasu Sasaoka§, Toshihiko Fujimori¶, Takeshi Oya‡, Yoko Ishii‡,Hemragul Sabit‡, Makoto Kawaguchi�, Yoko Kurotaki¶, Maiko Naito§, Tsutomu Wada**,Shin Ishizawa‡, Masashi Kobayashi**, Yo-Ichi Nabeshima¶, and Masakiyo Sasahara‡ ‡‡§§

From the ‡Department of Pathology, the §Department of Clinical Pharmacology and the **First Department of InternalMedicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, the ¶Department ofPathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, the �Department ofPathology, Niigata Rosai Hospital, Labor Health and Welfare Organization, 1-7-12 Tooun-cho, Jhoetsu, Niigata 942-8502,and ‡‡Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho,Kawaguchi-shi, Saitama 332-0012, Japan

This study provides new perspectives of the unique as-pects of platelet-derived growth factor �-receptor(PDGFR-�) signaling and biological responses through theestablishment of a mutant mouse strain in which two loxPsequences were inserted into the introns of PDGFR-� ge-nome sequences. Isolation of skin fibroblasts from the mu-tant mice and Cre recombinase transfection in vitro in-duced PDGFR-� gene deletion (PDGFR-��/�). The resultantdepletion of the PDGFR-� protein significantly attenuatedplatelet-derived growth factor (PDGF)-BB-induced cell mi-gration, proliferation, and protection from H2O2-inducedapoptosis of the cultured PDGFR-��/� dermal fibroblasts.PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration inPDGFR-��/� fibroblasts. Concerning the PDGF signaling,PDGF-BB-induced phosphorylation of Akt, ERK1/2, andJNK, but not p38, decreased in PDGFR-��/� fibroblasts, butPDGF-AA-induced signaling was not altered. Overexpres-sion of the phospholipid phosphatases, SHIP2 and/orPTEN, inhibited PDGF-BB-induced phosphorylation of Aktand ERK1/2 in PDGFR-��/� fibroblasts but did not affectthat of JNK and p38. These results indicate that disruptionof distinct PDGFR-� signaling pathways in PDGFR-��/�

dermal fibroblasts impaired their proliferation and sur-vival, but completely inhibits migratory response, and thatPDGF-BB-induced phosphorylation of Akt and ERK1/2 pos-sibly mediated by PDGFR-� is regulated, at least in part, bythe lipid phosphatases SHIP2 and/or PTEN. Thus, thePDGFR-� function on dermal fibroblasts appears to be crit-ical in PDGF-BB action for skin wound healing and isclearly distinctive from that of PDGFR-� in the ligand-induced biological responses and the underlying propertiesof cellular signaling.

Platelet-derived growth factors (PDGFs)1 are major mito-gens for connective tissue cells that are involved in diverse

biological processes including physiological development, tis-sue repair, tumorigenesis, and atherosclerosis (1). PDGF fam-ily members, PDGF-A, -B, -C, and -D, are assembled as disul-fide-linked homo- or heterodimers and exert their activity bybinding to and activating specific high affinity cell surfacereceptors. Two receptor subtypes with protein-tyrosine kinaseactivity have been identified that can form homo- and het-erodimeric receptor complexes: the �-subunit, which can bindto the A-, B-, and C-chains of PDGF, and the �-subunit, specificfor the B-, C- and D-chains (1–4). Dermal fibroblasts are one ofthe major target cells of PDGF in the initiation and propaga-tion of wound healing in the skin (5). Levels of PDGF receptor(PDGFR)-� expression are high in fibroblasts during early em-bryogenesis, and disruption of PDGFR-� results in a reductionin fibroblasts throughout the embryo (6, 7). In contrast, tar-geted deletion of PDGFR-� and analysis of blastocyst chimerasdemonstrated no effect of PDGFR-� on fibroblast development(8, 9). However, in mice prepared from the blastocyst chimerasthat contain a combination of wild-type and PDGFR-��/� cells,analysis of granulation tissue formation following the subcuta-neous implantation of sponges demonstrated that PDGFR-��/� cells were depleted in the granulation tissue (10). Thesereports suggest that the two subtypes of PDGFR play distinctroles in development and that PDGFR-� is important in woundhealing by dermal fibroblasts. Analysis of mutant mice inwhich the cytoplasmic signaling domain of the PDGFR-� wasused to replace the PDGFR-� cytoplasmic domain showed noobvious defects in any of the PDGFR-�-dependent cell types(11). On the other hand, when the PDGFR-� was dependentupon PDGFR-� cytoplasmic domain, multiple abnormalitiesoccurred in vascular smooth muscle cell development (11).These data suggest that PDGFR-� has unique signaling capac-ities compared with PDGFR-�.

PDGF binding to the receptors activates a variety of intra-cellular signaling molecules (12). One of these is phosphatidyl-inositol 3-kinase (PI3K), which results in the local accumula-tion of PI(3,4,5)P3 at the plasma membrane. Synthesized

* This study was supported in part by Grants-in-aid for ScientificResearch 16390114 and 12470053 from the Ministry of Education,Science, and Culture of Japan. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

§§ To whom correspondence should be addressed: Dept. of Pathology,Toyama Medical and Pharmaceutical University, 2630 Sugitani,Toyama 930-0194, Japan. Tel.: 81-76-434-7238; Fax: 81-76-434-5016;E-mail: sasahara@ms.toyama-mpu.ac.jp.

1 The abbreviations used are: PDGF, platelet-derived growth factor;

BrdUrd, 5-bromo-2�-deoxyuridine; DMEM, Dulbecco’s modified Eagle’smedium; ERK, extracellular signal-related kinase; ES cells, embryonicstem cells; FBS, fetal bovine serum; JNK, c-Jun NH2-terminal kinase;MAPK(s), mitogen-activated protein kinase(s); m.o.i., multiplicity ofinfection; neo-TK, neomycin-thymidine kinase; PDGFR, platelet-de-rived growth factor receptor; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PI3K, phosphatidylinositol 3-kinase; PTEN, phospha-tase and tensin homolog deleted on chromosome 10; SHIP2, Srchomology domain 2-containing inositol phosphatase 2; pfu, plaque-forming units.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 10, Issue of March 11, pp. 9375–9389, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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PI(3,4,5)P3 recruits the pleckstrin homology domain-contain-ing signaling molecule Akt/PKB to the membrane (12, 13).PDGFs are also known to activate the mitogen-activated pro-tein kinase (MAPK) families, including the extracellular sig-nal-regulated kinase (ERK1/2), c-Jun NH2-terminal kinase(JNK), and p38 MAPK. ERK1/2 is predominantly activated byreceptor tyrosine kinases of growth factors, whereas JNK andp38 are preferentially activated by stress-inducing stimuli suchas UV light, heat shock, pro-inflammatory cytokines, and bymitogenic stimuli as well (14). The PI3K/Akt and MAPKs path-ways play important roles in the regulation of cell growth,migration, and survival during the wound healing process (13,15, 16), although the relative importance of these kinases invarious cellular phenomena differ depending on the cell type(14). SHIP2 and PTEN are recently identified phospholipidphosphatases that are thought to negatively regulate thePDGF activation of the PI3K/Akt pathway by removing 5�- and3�-phosphates, respectively, from PI(3,4,5)P3. In addition,PTEN and SHIP2 also appear to inhibit PDGF activation ofERK1/2 (17, 18). However, the specific role of PDGFR-� in theactivation of Akt and MAPKs and the implication of the phos-pholipid phosphatases in PDGFR-�-mediated signaling arestill poorly understood.

To elucidate the biological properties and signaling path-ways of PDGFR-�, it is critical to analyze its function in non-transformed cells that physiologically express the receptor.Because homozygous disruption of PDGF-B or PDGFR-� re-sults in perinatal death in mouse embryos (8, 19), it has beendifficult to evaluate the role of the PDGF-B/PDGFR-� systemin repair of adult tissues, such as wound healing. To addressthis problem, we established a new mouse strain in which thePDGFR-� exons are flanked by two loxP sequences. The Cre-mediated recombination by adenovirus-gene transfer markedlyreduced the expression of PDGFR-� in the dermal fibroblastsderived from the mutant mice without affecting the expressionof PDGFR-�. By using these dermal fibroblasts, we studied thePDGF-induced biological effects, including proliferation, mi-gration, and apoptosis, which are central processes in woundhealing. In addition, we examined the involvement ofPDGFR-� in the activation of Akt and MAPKs to clarify theintracellular signals activated by the PDGFs. Finally, we ex-amined the effect of the phospholipid phosphatases, SHIP2 andPTEN, in the regulation of PDGF-BB signaling in dermal fi-broblasts lacking PDGFR-�.

EXPERIMENTAL PROCEDURES

Materials—Human recombinant PDGF-AA was provided by Strath-mann Biotech AG (Hamburg, Germany), and human recombinantPDGF-BB was purchased from Invitrogen. Polyclonal anti-PDGFR-�antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). Poly-clonal anti-PDGFR-� antibody, polyclonal anti-Tyr857 phosphospecificPDGFR-� antibody, monoclonal anti-phosphotyrosine antibody (PY99),monoclonal anti-Akt1 antibody, and monoclonal anti-PTEN antibodywere from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonalanti-Thr308 phosphospecific Akt antibody, polyclonal ERK1/2 antibody,polyclonal anti-Thr202/Tyr204 phosphospecific ERK1/2 antibody, poly-clonal anti-JNK antibody, polyclonal anti-Thr183/Tyr185 phosphospecificJNK antibody, polyclonal anti-p38 antibody, and polyclonal anti-Thr180/Tyr182 phosphospecific p38 antibody were from Cell Signaling Technol-ogy, Inc. (Beverly, MA). Polyclonal anti-SHIP2 antibody was generatedas described previously (20). Fetal bovine serum (FBS) was obtainedfrom BioWhittaker A Cambrex Inc. (Walkersville, MD). Dulbecco’s mod-ified Eagle’s medium (DMEM) was from Nissui Pharmaceutical Co.,Ltd. (Tokyo, Japan). All other reagents were of analytical grade andwere purchased from Sigma or Wako Pure Chemical Industries Ltd.(Osaka, Japan).

Generation of PDGFR-�flox/flox Mutant Mice—For the construction ofthe targeting vector, a 13.5-kb BamHI/SpeI genomic fragment of thePDGFR-� gene from the 129X1/SvJ mouse was cloned into pBluescriptSK� (Stratagene, La Jolla, CA). We inserted a neomycin-thymidine

kinase (neo-TK) selection cassette and one-loxP sequence at the 5�- and3�-ends, respectively, of the 3.3-kb FspI/HpaI genomic fragment thatincludes exons 4–7 encoding the extracellular domain of the PDGFR-�(Fig. 1A). The neo-TK selection cassette consisted of MC1-neo andMC1-TK selection cassette flanked by two loxP sites. A diphtheria toxinselection cassette (DT-A) was inserted at the 3�-end of the vector (Fig.1A). After linearization, the vector was electroporated into 129SVmouse embryonic stem (ES) cells. The obtained recombinant ES cloneswere transfected with the pCre-Pac plasmid by electroporation to deletethe neo-TK selection genes. The resulting ES clones in which exons 4–7of the PDGFR-� gene were flanked by two loxP sequences (PDGFR-�flox/�) were then injected into blastocysts to obtain chimeric mice. Thechimeras were bred with C57BL/6 mice (Sankyo Laboratory, Tokyo,Japan) to obtain the F1 progenies containing the recombinant allele(PDGFR-�flox/�). These F1 progenies were cross-bred to obtain PDGFR-�flox/flox homozygous mice. Genotyping was performed by both Southernblotting and PCR-based analyses. The probes for Southern blotting areshown in Fig. 1A. The primers for genomic PCR were as follows: primer1, 5�-TAGCCATGGAGTCATCTCTTCAGCCCTAAA-3�; primer 2, 5�-C-CTGCATCAAGTAGCTCACAACTGCCTGTA-3�; primer 3, 5�-TTCTTG-TCTGAGAGCCTGTTGTGTGATGGA-3�; primer 4, 5�-TGTCTGCAGA-TCTCTAGCCTTGGGGAAATC-3�; and primer 5, 5�-AGCAAGGTCGC-GCAAGGGATAACAGC-3�.

Adenovirus—We used adenovirus expression vectors with an E1-deleted replication deficiency. Two adenoviral vectors expressing Crerecombinase and LacZ tagged with a nuclear localization signal underthe control of the CAG promoter (21) were kindly provided by Dr. IzumuSaito (Tokyo University, Tokyo, Japan) (22). The recombinant adenovi-rus expressing PTEN (23) was a generous gift of Dr. Tomoichiro Asano(Tokyo University, Tokyo, Japan). The SHIP2-expressing adenovirusvector was constructed as described previously (18).

Cell Culture and Infection with Adenovirus—Mouse dermal fibro-blast cells were prepared from 8- to 12-week-old male control C57BL/6mice and PDGFR-�flox/flox mice. Briefly, mice were anesthetized withpentobarbital (50 mg/kg body weight), and a full thickness of the backskin was cut out by scissors. The skin tissues were cut into small piecesand were implanted into plastic tissue culture dishes containing DMEMwith 10% FBS. The fibroblast cultures were used after three to sevenpassages. Cre recombinase, PTEN, and SHIP2 were transiently ex-pressed in cultured cells by adenovirus-mediated gene transfer. Cellswere infected in DMEM containing 5% FBS at a multiplicity of infection(m.o.i.) of 10 pfu/cell. After 16 h, the virus was removed by replacing themedium. Experiments were conducted 24–48 h after the initial addi-tion of the virus.

DNA Synthesis Assay—The cultured fibroblasts obtained from wild-type or PDGFR-�flox/flox mice were infected with Cre-adenovirus asdescribed above. The cells were cultured in DMEM containing 10% FBSfor an additional 24 h. The cells were then plated into 96-well plates at1.5 � 104 cells/well in DMEM containing 10% FBS. After 24 h, the cellswere serum-starved for 24 h and then were incubated for 18 h with 1 nM

PDGF-AA, 1 nM PDGF-BB, or 10% FBS in DMEM containing 10 mM

5-bromo-2�-deoxyuridine (BrdUrd). BrdUrd incorporation into DNAwas determined with a 5-bromo-2�-deoxyuridine labeling and detectionkit (Roche Applied Science) according to the manufacturer’s instruc-tions and detected with an enzyme-linked immunosorbent assay platereader (Nippon InterMed. Ltd., Tokyo, Japan).

Wound Scratch Assay—Confluent cultured fibroblasts obtained fromwild-type or PDGFR-�flox/flox mice were grown in 6-well plates and wereinfected with Cre-adenovirus as described above. The cells were cul-tured in DMEM containing 10% FBS for an additional 24 h. After thecells were serum-starved for 24 h in DMEM, they were scratched witha 1-ml plastic pipette tip and washed twice with PBS to remove thefloating cells. The cells were then cultured in DMEM supplementedwith 1 nM PDGF-AA, 1 nM PDGF-BB, or 10% FBS. After 48 h, the cellswere viewed with an inverted phase contrast microscope (OlympusCK30, Tokyo, Japan) and photographed.

Apoptosis Assay—Apoptosis was detected using an APOPercentageTM

kit (Biocolor Ltd., Belfast, Northern Ireland). Briefly, confluent wild-type or PDGFR-� flox/flox fibroblasts grown in 6-well plates were infectedwith the Cre-adenovirus as described above. After infection, the cellswere replated into 12-well plates and cultured for an additional 24 h inDMEM containing 10% FBS. Apoptosis was induced by 1 �M H2O2 in 1ml of DMEM containing 1 nM PDGF-AA, 1 nM PDGF-BB, or 10% FBS,and 50 �l of APOPercentage Dye. After 60 min, the medium was drainedfrom the wells, and the cells were washed twice with PBS. The cellswere then observed with an inverted phase contrast microscope.

Immunoprecipitation and Western Blotting—Confluent wild-typeand PDGFR-�flox/flox fibroblasts grown in 6-well plates were infected for

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16 h with the Cre-adenovirus at an m.o.i. of 10 pfu/cell. The cells werethen incubated for 24 h in DMEM containing 10% FBS. Next, the cellswere serum-starved for 48 h and then treated with various concentra-tions of PDGF-AA or PDGF-BB at 37 °C for the indicated times. Thecells were lysed for 15 min at 4 °C in a buffer consisting of 50 mM Tris,150 mM NaCl, 10 mM EDTA, 0.5% sodium deoxycholate, 1% TritonX-100, 2 mM Na3VO4, 150 mM sodium fluoride, 2 mM phenylmethylsul-fonyl fluoride, 10 �g/ml aprotinin, and 10 �g/ml leupeptin (pH 7.4).Lysates obtained from the same number of cells were centrifuged toremove insoluble materials. The supernatants were incubated with theindicated antibodies for 4 h at 4 °C, and then immune complexes weremixed with glutathione-Sepharose for 2 h at 4 °C. The immune com-plexes were precipitated by centrifugation. Immune complexes or wholecell lysates were separated by SDS-PAGE and then electrophoreticallytransferred to polyvinylidene difluoride membranes. The membraneswere incubated for 1 h at 20 °C in a buffer containing 50 mM Tris, 150mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk. The membraneswere then probed with specified antibodies for 16 h at 4 °C. After themembranes had been washed in a buffer containing 50 mM Tris, 150 mM

NaCl, and 0.1% Tween 20, the blots were incubated with an appropriatehorseradish peroxidase-conjugated secondary antibody. Immunoreac-tive bands were detected using enhanced chemiluminescence reagents(Amersham Biosciences) according to the manufacturer’s instructions.

Immunofluorescence—Cultured wild-type or PDGFR-�flox/flox fibro-blasts were infected with the Cre-adenovirus as described above andwere replated in chamber slides. All staining procedures were per-formed at room temperature. After growth for 24 h, the cells were fixedwith 1% formalin in PBS for 10 min. Nonspecific immunoreactions wereblocked by incubation for 20 min with 10% goat serum in PBS. Cellswere then incubated for 2 h with 1:100 anti-PDGFR-�, washed threetimes for 5 min with PBS, and then incubated for 1 h with 1:1,000 AlexaFluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene,OR). The cells were mounted with Vectashield mounting medium con-taining 4,6-diamidino-2-phenylindole (Vector Laboratories, Burl-ingame, CA) and observed with an Olympus AX80 microscope.

Statistical Analysis—All data were presented as means � S.D. Stu-dent’s t test was used to determine the p values, and p � 0.05 wasconsidered statistically significant.

RESULTS

PDGFR-�flox/flox Mutant Mice Are Healthy and Show NoGross Abnormalities—After transfection of the targeting vector(Fig. 1A) and selection with neomycin, we obtained four ES cellclones with homologous recombination out of the 319 clonesexamined. In addition to a 12.8-kb fragment, clones that hadundergone recombination generated a 7.3-kb fragment inSouthern blotting using a 5�-probe after digestion of thegenomic DNA with XbaI (Fig. 1B). Generation of a 5.7-kbfragment after SacI digestion and an 8.8-kb fragment afterXbaI digestion was detected by Southern blotting using a 3�-probe and neo probe, respectively (Fig. 1B).

ES cell clones that had undergone homologous recombina-tion were transfected with Cre-expressing plasmid (pCre-Pac;gift from Dr. Takeshi Yagi, Osaka University, Japan) to deletethe neo-TK selection cassette. Deletion of the neo-TK selectiongene was confirmed by Southern blotting with an internalprobe (data not shown). The resulting ES clones (PDGFR-�flox/�) were used for the generation of chimeric mice.

The PDGFR-�flox/� ES clones were also transfected with thepCre-Pac plasmid to confirm that the Cre recombinase caninduce recombination and eliminate the floxed PDGFR-� al-lele. After transfection with the pCre-Pac plasmid, genomicPCR with primers 1, 2, and 5 generated bands of 271, 329, and410 bp, which corresponded to the PDGFR-� genes for wild-type, floxed, and deleted alleles (PDGFR-��), respectively (Fig.1C). These results confirmed that the Cre recombinase causedPDGFR-� gene deletion in the PDGFR-�flox/� ES cells.

The chimeras generated from PDGFR-�flox/� ES cells werebred with C57BL/6 mice, generating F1 progenies containingthe recombinant allele (PDGFR-�flox/�). These mice werecrossed with each other to obtain homozygous PDGFR-�flox/flox

mice. The PDGFR-�flox/flox mice were healthy, and no gross

abnormalities were detected. The genotype of the mice wasconfirmed by PCR with primers 1 and 2 (Fig. 1D) and primers3 and 4 (data not shown). PCR with primers 1 and 2 generateda 271-bp band from the wild-type PDGFR-� gene and a 329-bpband from the floxed allele.

Adenovirus-mediated Cre Expression Eliminates the Expres-sion of PDGFR-� in PDGFR-�flox/flox Dermal Fibroblasts—Wefirst investigated whether Cre-adenovirus infection could ab-rogate the expression of PDGFR-� in PDGFR-�flox/flox dermalfibroblasts. Similar levels of PDGFR-� were expressed in bothwild-type and PDGFR-�flox/flox fibroblasts in the absence ofCre-adenovirus infection (Fig. 2A). Treatment with 1 nM

PDGF-BB induced similar levels of tyrosine phosphorylation ofPDGFR-� in both types of fibroblast (Fig. 2A). Infection withthe Cre-adenovirus caused a marked reduction in the expres-sion and tyrosine phosphorylation of PDGFR-� in PDGFR-�flox/flox but not wild-type fibroblasts. However, Cre transfec-tion had no effect on the expression of PDGFR-� in wild-type orPDGFR-�flox/flox fibroblasts (Fig. 2A). Immunofluorescentstudies revealed that the cytoplasmic immunoreactivity for thePDGFR-� was eliminated in greater than 90% of PDGFR-�flox/flox fibroblasts after Cre transfection (PDGFR-��/�)(Fig. 2B).

The effects of Cre transfection were further assessed byexamining PDGF-AA- and PDGF-BB-induced tyrosine phos-phorylation of PDGFR-� and PDGFR-�. Stimulation by eitherPDGF-AA or PDGF-BB induced equivalent and dose-depend-ent tyrosine phosphorylation of PDGFR-� in both Cre-trans-fected wild-type and PDGFR-�flox/flox fibroblasts (Fig. 2C, aand b). In contrast, PDGF-BB-induced tyrosine phosphoryla-tion of PDGFR-� was not detected in PDGFR-��/� fibroblasts,although it was clearly observed in wild-type fibroblasts follow-ing Cre transfection (Fig. 2C, c). Thus, Cre transfection ofPDGFR-�flox/flox fibroblasts selectively abrogated the expres-sion and function of PDGFR-� but not PDGFR-�.

PDGF-BB- and Fetal Bovine Serum-induced BrdUrd Incor-poration Is Decreased after PDGFR-� Gene Deletion—PDGFRis known to play an important role in cell proliferation (12). Wetherefore examined the effect of Cre transfection on PDGF-AA-,PDGF-BB-, and fetal bovine serum (FBS)-induced BrdUrd in-corporation in wild-type and PDGFR-�flox/flox fibroblasts. Asshown in Fig. 3, 1 nM PDGF-AA induced similar levels ofBrdUrd incorporation in wild-type and PDGFR-��/� cells. Incontrast, 1 nM PDGF-BB- and 10% FBS-induced BrdUrd incor-poration were significantly decreased in PDGFR-��/� fibro-blasts compared with wild-type fibroblasts, 48.4 � 1.2 and34.2 � 1.9%, respectively. These results demonstrate a signif-icant contribution of PDGFR-� to cell proliferation in responseto PDGF-BB and FBS, but not PDGF-AA.

In Vitro Repair of a Scratch Wound Is Inhibited afterPDGFR-� Gene Deletion—Because PDGFR is also known to beimplicated in cell migration (12), we examined the effect of thedeletion of the PDGFR-� gene on the combined migratory andproliferative response to scratch wounding that mimics aspectsof wound healing. A wound was formed in confluent cell mono-layers by scratching with a plastic pipette tip. In wild-typefibroblasts cultured for 48 h with 1 nM PDGF-BB, the woundwas completely closed (Fig. 4, A and B). Similarly, substantialnumbers of wild-type fibroblasts had filled in the scratchedarea following treatment with 10% FBS for 48 h (Fig. 4, E andF). In contrast, the scratched wound remained unclosed at 48 hafter culture with 1 nM PDGF-BB in PDGFR-��/� fibroblasts(Fig. 4, C and D). Wound closure was mostly suppressed inPDGFR-��/� cells after culture with 10% FBS (Fig. 4, G and H).These results indicate that the PDGF-BB/PDGFR-� system isessential for the combined migration and proliferation required

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FIG. 1. Construction of PDGFR-� gene targeting vector and genotyping. A, diagram of the targeting vector. Three loxP sequences andneo-TK and diphtheria toxin selection cassettes were inserted into the PDGFR-� locus, and it was subcloned into the pBluescript SK� plasmid.XbaI and SacI sites were artificially introduced in the indicated positions. WT, wild type. B, Southern blotting of ES cells after homologousrecombination (RE) using three different probes. Genomic DNA prepared from isolated ES cells was digested with XbaI or SacI. C, Cre-mediatedgene depletion in ES cells. PDGFR-�flox/� ES cells were treated with Cre recombinase, and genomic PCR was performed. The 410-bp bandcorresponds to the PDGFR-�-deleted allele (PDGFR-��) after Cre treatment. D, PCR-based genotyping of mice. Genomic PCR products obtainedfrom mouse tail DNA show distinct band patterns in PDGFR-�flox/flox, PDGFR-��/�, and PDGFR-�flox/� mice.

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FIG. 2. Depletion of PDGFR-� protein expression after Cre-adenovirus infection. A, wild-type (WT) and PDGFR-�flox/flox fibroblasts wereinfected with the Cre-adenovirus at an m.o.i. of 10 pfu/cell and were stimulated with 1 nM PDGF-BB for 5 min. Whole cell lysates were immunoblotted withanti-PDGFR-�, anti-PDGFR-�, or anti-Tyr857 phosphospecific PDGFR-� antibody. B, the Cre-adenovirus-infected wild-type or PDGFR-�flox/flox fibroblastswere immunostained with anti-PDGFR-� antibody (green). Representative immunofluorescent staining for PDGF-� is shown. Fibroblasts were unambig-uously identified by 4,6-diamidino-2-phenylindole staining of nuclei (blue). C, PDGF-induced phosphorylation of PDGFR after deletion of the PDGFR-� gene.Wild-type and PDGFR-�flox/flox fibroblasts were infected with the Cre-adenovirus at an m.o.i. of 10 pfu/cell and serum-starved for 24 h. a, the cells werestimulated with various concentrations of PDGF-AA for 5 min. The cell lysates were immunoprecipitated with anti-PDGFR-� antibody and immunoblottedwith anti-phosphotyrosine antibody (PY99) or anti-PDGFR-� antibody. b, cells were stimulated with various concentrations of PDGF-BB for 5 min. Proteinswere immunoprecipitated from cell lysates using anti-PDGFR-� antibody and then immunoblotting was performed with PY99 or anti-PDGFR-� antibody.c, cells were stimulated with various concentrations of PDGF-BB for 5 min. Proteins were immunoprecipitated from cell lysates using anti-PDGFR-�antibody, and immunoblotting was performed with PY99 or anti-PDGFR-� antibody.

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to fill in a scratch wound with PDGF-BB- and 10% FBS asstimulants and suggest that the role of PDGF-� is minimal.This concept was further supported by the fact that PDGF-AAdid not induce apparent wound closure in either wild-type orPDGFR-��/� cells (Fig. 4, I–L).

Apoptosis Induced by H2O2 Is Enhanced by Elimination ofthe PDGFR-� Gene—We next examined the effect of the dele-tion of the PDGFR-� gene on H2O2-induced apoptosis in dermalfibroblasts. To detect apoptosis, we utilized the APOPercent-ageTM kit, which stains apoptotic cells but not necrotic cellswith a purple-red color (24, 25). Few apoptotic cells were ob-served in wild-type or PDGFR-��/� fibroblasts when the cellswere cultured with 10% FBS (data not shown). In wild-typecells cultured with DMEM supplemented only with 0.2% bo-vine serum albumin, the addition of H2O2 increased the num-ber of apoptotic cells by 78.3 � 3.2%. The addition of 1 nM

PDGF-BB protected wild-type cells from apoptosis. However,PDGFR-��/� fibroblasts showed an increase in the number ofapoptotic cells from 23.3 � 3.7% in wild-type to 56.9 � 6.5% inPDGFR-��/� fibroblasts treated with 1 nM PDGF-BB (Fig. 5, A,B, and G). These data indicate that PDGF-BB mediates pro-tection from H2O2-induced apoptosis through PDGFR-�. Incontrast, H2O2 induced only a low level of apoptosis in wild-type and PDGFR-��/� fibroblasts cultured in the presence of10% FBS or 1 nM PDGF-AA, and there was no significantdifference in the degree of apoptosis among the four treatments(apoptosis for wild-type cells in 10% FBS was 23.8 � 3.6%; forwild-type cells in PDGF-AA, 21.3 � 2.9%; for PDGFR-��/� cellsin 10% FBS, 24.3 � 1.9%; and for PDGFR-��/� cells in PDGF-AA, 23.2 � 1.6%; Fig. 5, C–G). These data provide further datathat PDGFR-� signaling by PDGF-AA is not perturbed byconditional deletion of PDGFR-� and that it is sufficient toinhibit H2O2-induced apoptosis in fibroblasts.

PDGF-BB-induced Phosphorylation of Akt Is Decreased afterPDGFR-� Gene Deletion—Because various biological actionswere impaired in PDGFR-��/� fibroblasts, we next investigatedthe effect of PDGFR-� deletion on PDGF-induced intracellularsignaling. Akt is one of the important signaling moleculesinvolved in the biological effects of PDGF (12). As shown in Fig.6A, stimulation with PDGF-BB for 5 min dose-dependentlyincreased the phosphorylation of Akt in wild-type fibroblasts,and the levels of phosphorylation reached a maximum at 1 nM

PDGF-BB. Compared with the wild-type cells, the levels ofphosphorylation following treatment with PDGF-BB were sig-nificantly lower in PDGFR-��/� fibroblasts. At 0.2 nM PDGF-BB, Akt phosphorylation was decreased by 50.8 � 2.1% inPDGFR-��/� cells compared with wild-type cells.

We further conducted time course studies of PDGF-BB-in-duced phosphorylation of Akt. In wild-type cells, PDGF-BBinduced the phosphorylation of Akt in a time-dependent man-ner for up to 60 min, and the phosphorylation was sustained athigh levels until 120 min after stimulation (Fig. 6C). Akt phos-phorylation was significantly reduced at all time points inPDGFR-��/� fibroblasts, whereas the dose (Fig. 6B) and timedependence (Fig. 6D) of PDGF-AA-induced phosphorylation ofAkt were comparable in wild-type and PDGFR-��/� fibroblasts.

PDGF-BB-induced Phosphorylation of ERK1/2 and JNK,but Not p38, Are Decreased after Deletion of the PDGFR-�Gene—In addition to Akt, MAPKs, including ERK1/2, JNK,and p38, are known to be key molecules in the biological actionsof PDGF (26). Therefore, we investigated the effect of PDGFR-�gene deletion on PDGF-induced phosphorylation of ERK1/2,JNK, and p38. In wild-type fibroblasts, PDGF-BB dose-depend-ently induced the phosphorylation of ERK1/2 at up to 1 nM (Fig.7A). In the presence of 1 nM PDGF-BB, the levels of phospho-rylation reached a maximum after 5 min with a gradual de-crease thereafter (Fig. 7C). PDGF-BB-induced ERK1/2 phos-phorylation was significantly decreased at all doses and timepoints in PDGFR-��/� cells (Fig. 7, A and C). Phosphorylationof JNK was induced by PDGF-BB in a similar dose- and time-dependent manner as that observed in ERK1/2. JNK phospho-rylation was also decreased in PDGFR-��/� cells comparedwith wild-type cells (Fig. 8, A and C). At 0.4 nM PDGF-BB, thelevels of ERK1/2 and JNK phosphorylation were reduced by42.7 � 3.8 and 53.2 � 2.7%, respectively, in PDGFR-��/� cellscompared with wild-type cells. The phosphorylation of ERK1/2and JNK induced by PDGF-AA was essentially identical be-tween wild-type and PDGFR-��/� cells at all doses and timepoints examined (Figs. 7, B and D, and Fig. 8, B and D).

PDGF-BB and PDGF-AA dose-dependently induced thephosphorylation of p38 up to a concentration of 2 nM (Fig. 9, Aand B). In addition, both 1 nM PDGF-BB and 1 nM PDGF-AAcaused a transient phosphorylation that lasted for 15 min,decreasing thereafter and returning to a basal level at 120 min(Fig. 9, C and D). Most interestingly, both PDGF-BB- andPDGF-AA-induced phosphorylation of p38 was nearly identicalat all dose and time points in wild-type and PDGFR-��/� cells.

Effect of PTEN and SHIP2 Expression on the Phosphoryla-tion of Akt and MAPKs in Wild-type and PDGFR-��/� Fibro-blasts—PTEN and SHIP2 are phospholipid phosphatases thatmay act as negative regulators of PDGF signaling (17, 18). Tounderstand their roles in PDGF-BB-induced phosphorylationof Akt and MAPKs, we overexpressed PTEN and/or SHIP2 inwild-type and PDGFR-��/� fibroblasts. As shown in Fig. 10A,we were able to enhance the expression of PTEN and SHIP2 by10-fold of endogenous expression. PDGF-BB-induced phospho-rylation of Akt was only modestly decreased by the expressionof either PTEN or SHIP2 but was significantly inhibited whenboth phosphatases were overexpressed in wild-type cells. Inagreement with the results in Fig. 6, the levels of the Aktphosphorylation were decreased by the depletion of PDGFR-�,and expression of either PTEN or SHIP2 effectively inhibitedPDGF-BB-induced phosphorylation of Akt in PDGFR-��/� cells(64.2 � 4.1% and 65.5 � 3.1% reduction, respectively;Fig. 10B).

In wild-type cells, PDGF-BB-induced phosphorylation of theERK1/2 did not appear to be affected by the overexpression ofSHIP2 or PTEN. However, in PDGFR-��/� cells, overexpres-

FIG. 3. Effect of PDGFR-� depletion on BrdUrd incorporation.Wild-type (WT) and PDGFR-�flox/flox fibroblasts were infected withCre-adenovirus at an m.o.i. of 10 pfu/cell. The cells were serum-starvedfor 24 h and then treated with 1 nM PDGF-AA, 1 nM PDGF-BB, or 10%FBS. After 18 h, BrdUrd incorporation was assessed. Results representmeans � S.D. of six separate experiments. *, p � 0.05 versus BrdUrdincorporation in wild-type fibroblasts.

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FIG. 4. Effect of PDGFR-� deletionon wound scratch assay. Confluentwild-type (WT) and PDGFR-�flox/flox fibro-blasts were infected with the Cre-adeno-virus. The cells were subjected to injuryby scratching with a plastic pipette tip.The cells were then treated for 48 h with1 nM PDGF-BB (A–D), 10% FBS (E–H), or1 nM PDGF-AA (I–L). Large black dotsmarked the bottom surface of the culturedish to specify the site of observation.Representative images are shown fromthree separate experiments.

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sion of SHIP2 but not PTEN inhibited PDGF-BB-induced phos-phorylation of ERK1/2 (30.6 � 1.2% reduction; Fig. 10C). Incontrast to the results with Akt and ERK1/2, expression ofPTEN, SHIP2, or both did not affect PDGF-BB-induced phos-phorylation of JNK (Fig. 10D) or p38 (Fig. 10E) in eitherwild-type or PDGFR-��/� fibroblasts.

DISCUSSION

In the current studies, we developed a new mouse line inwhich a portion of the PDGFR-� exons were flanked by twoloxP sequences that did not affect development of the mutantmouse. The expression of PDGFR-� and -� and the level ofligand-induced phosphorylation were comparable between der-mal fibroblasts isolated from wild-type and mutant mice with-out Cre transfection. The adenovirus-mediated Cre transfec-tion in PDGFR-�flox/flox cells efficiently abrogated PDGFR-�expression without affecting PDGFR-� expression and its sig-naling pathways. Therefore, this is a powerful approach forunambiguously determining the role of the two PDGFR sub-types in the biological effects of and signaling pathways stim-ulated by PDGF.

In wild-type fibroblasts, PDGF-AA and PDGF-BB inducedthe incorporation of BrdUrd, but PDGF-BB- and 10% FBS-induced BrdUrd incorporation were significantly lower inPDGFR-��/� fibroblasts than in wild-type fibroblasts. Theseresults indicate that PDGFR-� plays an important role in me-diating PDGF-BB- and 10% FBS-induced DNA synthesis. How-ever, because the depletion of PDGFR-� caused only a partialdecrease of BrdUrd incorporation, PDGFR-� appears to partlymediate the effects of PDGF-BB in dermal fibroblasts. In ad-dition, PDGF-AA-induced DNA synthesis was unaffected bydepletion of PDGFR-�, consistent with all of the effects ofPDGF-AA being mediated by PDGFR-�.

In the wound scratch assay, PDGF-BB and 10% FBS, butnot PDGF-AA, induced wound closure in wild-type fibro-blasts. The wound closure induced by PDGF-BB and 10%FBS was significantly inhibited by depletion of the PDGFR-�.Wound closure is known to be dependent on both cell motilityand proliferation. Because PDGF-AA clearly induced cell pro-liferation in both wild-type and PDGFR-��/� cells, the lack ofwound closure by PDGF-AA suggests that PDGFR-� signal-ing is insufficient to stimulate cell motility. Similarly, studieswith aortic endothelial cells transfected with equal numbersof PDGFR-� or -� showed that chemotactic migration wasmediated only by PDGFR-� (27). Also, in dermal fibroblasts,PDGF-BB is known to be the major inducer of chemotaxis ontype I collagen, although the subtype of PDGFR that wasinvolved was not determined (28). Furthermore, the contri-bution of PDGFR-� �/� fibroblasts to granulation formationwas reduced by 85% after subcutaneous implantation ofsponges in the blastocyst chimeric mice expressing mixturesof wild-type and PDGFR-� �/� cells (10). Taken together, ourresults clearly indicate that in dermal fibroblasts PDGFR-�,but not PDGFR-�, plays a crucial role particularly in themediation of PDGF-induced cellular motility.

PDGF-AA, PDGF-BB, and 10% FBS protected wild-type fi-broblasts from H2O2-induced apoptosis to similar extents. InPDGFR-��/� fibroblasts, the effect of PDGF-BB was mostlylost, whereas the protective effects of PDGF-AA and 10% FBSwere essentially unchanged. These results indicate that thePDGFR-� plays an important role in the ability of PDGF-BB toprotect dermal fibroblasts from H2O2-induced apoptosis andthat the remaining PDGFR-� appears to be sufficient for theprotection by PDGF-AA and 10% FBS.

Based on these results, the functional importance ofPDGFR-� and PDGFR-� appears to differ, depending on thespecific biological effects triggered by PDGF-AA, PDGF-BB,and 10% FBS. Our studies have further clarified the functionalsignificance of PDGFR-� signaling in mediating its biologicaleffects by examining the activation of two of its downstreampathways, Akt and MAPKs. Compared with wild-type fibro-blasts, there was a decrease in PDGF-BB-induced phosphoryl-ation of Akt, ERK1/2, and JNK in PDGFR-��/� fibroblasts.

FIG. 5. Effect of PDGFR-� deletion on H2O2-induced apoptosis.Wild-type (WT) and PDGFR-�flox/flox fibroblasts were infected with theCre-adenovirus at an m.o.i. of 10 pfu/cell and then were cultured in12-well plates. After 24 h, 1 �M H2O2 and dye from the APOPercent-ageTM kit were added along with 1 nM PDGF-BB (A and B), 10% FBS (Cand D), or 1 nM PDGF-AA (E and F). After 1 h, the cells were washedtwice with PBS and then photographed. G, results represent themeans � S.D. of three separate experiments. *, p � 0.05 versusPDGF-BB incubation in wild-type fibroblasts.

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FIG. 6. Effect of PDGFR-� depletion on PDGF-induced phosphorylation of Akt. Wild-type (WT) and PDGFR-�flox/flox fibroblasts wereinfected with Cre-adenovirus at an m.o.i. of 10 pfu/cell, and the cells were serum-starved for 48 h. The cells were then treated at 37 °C for 5 minwith the indicated concentrations of PDGF-BB (A) or PDGF-AA (B), or they were treated at 37 °C for the indicated times with 1 nM PDGF-BB (C)or 1 nM PDGF-AA (D). Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Thr308 phosphospecific Akt antibody oranti-Akt1 antibody. The relative amount of phosphorylated Akt was determined by densitometry. Results represent means � S.D. of three separateexperiments. *, p � 0.05 versus Akt phosphorylation in wild-type fibroblasts.

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FIG. 7. Effect of PDGFR-� depletion on PDGF-induced phosphorylation of ERK1/2. Wild-type (WT) and PDGFR-�flox/flox fibroblasts wereinfected with the Cre-adenovirus at an m.o.i. of 10 pfu/cell, and the cells were serum-starved for 48 h. The cells were treated at 37 °C for 5 minwith the indicated concentrations of PDGF-BB (A) or PDGF-AA (B), or they were treated at 37 °C for the indicated times with 1 nM PDGF-BB (C)or 1 nM PDGF-AA (D). Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Thr202/Tyr204 phosphospecific ERK1/2antibody or anti-ERK1/2 antibody. The relative amount of phosphorylated ERK1/2 was determined by densitometry. Results represent themeans � S.D. of three separate experiments. *, p � 0.05 versus ERK1/2 phosphorylation in wild-type fibroblasts.

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FIG. 8. Effect of PDGFR-� depletion on PDGF-induced phosphorylation of JNK. Wild-type (WT) and PDGFR-�flox/flox fibroblasts wereinfected with the Cre-adenovirus at an m.o.i. of 10 pfu/cell, and the cells were serum-starved for 48 h. The cells were treated at 37 °C for 5 minwith the indicated concentrations of PDGF-BB (A) or PDGF-AA (B), or they were treated at 37 °C for the indicated times with 1 nM PDGF-BB (C)or 1 nM PDGF-AA (D). Cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Thr183/Tyr185 phosphospecific JNK antibodyor anti-JNK antibody. The relative amount of phosphorylated JNK was determined by densitometry. Results represent means � S.D. of threeseparate experiments. *, p � 0.05 versus JNK phosphorylation in wild-type fibroblasts.

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FIG. 9. Effect of PDGFR-� depletion on PDGF-induced phosphorylation of p38. Wild-type (WT) and PDGFR-�flox/flox fibroblasts were infectedwith the Cre-adenovirus at an m.o.i. of 10 pfu/cell, and the cells were serum-starved for 48 h. The cells were treated at 37 °C for 5 min with the indicatedconcentrations of PDGF-BB (A) or PDGF-AA (B), or they were treated at 37 °C for the indicated times with 1 nM PDGF-BB (C) or 1 nM PDGF-AA (D).The cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Thr180/Tyr182 phosphospecific p38 antibody or anti-p38 antibody. Therelative amount of phosphorylated p38 was determined by densitometry. Results represent means � S.D. of three separate experiments.

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FIG. 10. Effect of co-infection with PTEN and SHIP2 on PDGF-induced phosphorylation of Akt, ERK1/2, JNK, and p38 in wild-typeand PDGFR-��/� fibroblasts. Wild-type (WT) and PDGFR-�flox/flox fibroblasts were co-infected with the Cre-adenovirus, and LacZ-, PTEN-, orSHIP2-adenovirus at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 48 h and then treated at 37 °C for 5 min with 1 nM PDGF-BB. Thecell lysates were separated by 7.5% SDS-PAGE and immunoblotted with PTEN or SHIP2 antibody (A), anti-Thr308 phosphospecific Akt andanti-Akt1 antibodies (B), anti-Thr202/Tyr204 phosphospecific ERK1/2 and anti-ERK1/2 antibodies (C), anti-Thr183/Tyr185 phosphospecific JNK andanti-JNK antibodies (D), or anti-Thr180/Tyr182 phosphospecific p38 and anti-p38 antibodies (E). The relative amounts of phosphorylated Akt,ERK1/2, JNK, and p38 were determined by densitometry. Results represent means � S.D. of three separate experiments. *, p � 0.05 versus Aktphosphorylation of Cre- and LacZ-infected wild-type fibroblasts in B and versus ERK1/2 phosphorylation of Cre- and LacZ-infected PDGFR-�flox/flox

fibroblasts in C. �, p � 0.05 versus Akt phosphorylation in Cre- and LacZ-infected PDGFR-�flox/flox fibroblasts in B.

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Most interestingly, PDGF-BB-induced phosphorylation of p38MAPK was unaffected by depletion of PDGFR-�. On the otherhand, PDGF-AA-induced phosphorylation of Akt, ERK1/2,JNK, and p38 was comparable between wild-type and PDGFR-��/� fibroblasts. These results indicate that PDGFR-� is in-volved in PDGF-BB-induced phosphorylation of Akt, ERK1/2,and JNK. Because the inhibition of these pathways was par-tial, we presume that PDGFR-� mediates part of the signaltriggered by PDGF-BB. In addition, our data suggest thatPDGFR-� mediates PDGF-AA-induced phosphorylation of Akt,ERK1/2, JNK, and p38, and, unexpectedly, PDGF-BB-inducedphosphorylation of p38 as shown in our dose- and time-depend-ent course studies.

In NIH3T3 fibroblasts, PDGFR-�, but not PDGFR-�, medi-ates PDGF signals leading to the activation of JNK, althoughboth PDGFR-� and PDGFR-� activate ERK1/2 (29). This isdifferent from our current finding that ERK1/2 and JNK arepossible downstream signaling molecules for both PDGFR-�and PDGFR-�. The apparent discrepancy may be due to thespecific cell types examined and/or the experimental proce-dures employed. A recent study (30) reported that mouse em-bryonic fibroblasts lacking both JNK1 and JNK2 exhibit muchslower proliferation than wild-type fibroblasts. In addition,studies with pharmacological inhibitors, antisense oligonucleo-tides, and c-Jun�/� fibroblasts suggest that JNK plays a keyrole in cell cycle progression (31). Furthermore, by using dom-inant-negative mutants, Kawano et al. (26) showed that ERKand JNK, but not p38, are involved in PDGF-BB-induced me-sangial cell proliferation. In the current studies, we found thatPDGFR-��/� cells have reduced PDGF-BB-induced cell prolif-eration and decreased activation of ERK1/2 and JNK, but therewas no effect on the activation of p38. Taken together, thesefindings support the idea that PDGFR-� mediates PDGF-BB-induced proliferation through ERK1/2 and JNK, but notthrough p38, in dermal fibroblasts.

The involvement of p38 and JNK in cell migration is contro-versial, possibly because of different findings in various celltypes and different methods to evaluate cell migration. Ras-mediated activation of p38, but not JNK, has been reported tobe crucial for the migration of PDGFR-transfected porcine aor-tic endothelial cells (32). In contrast, Javelaud et al. (33)showed that transforming growth factor-� stimulates cell mo-tility via the activation of JNK and c-Jun in JNK�/� mouseembryonic fibroblasts and human dermal fibroblasts. Consist-ent with these findings, our data showed that, in PDGFR-��/�

cells, the phosphorylation of JNK was decreased, whereas thatof p38 was unaffected. Collectively, our results suggest that thedecrease in PDGF-BB-induced phosphorylation of JNK inPDGFR-��/� fibroblasts may contribute to the lack of cell move-ment and that normal activation of p38 is not sufficient toinduce cell motility.

Ligand binding- or stress-induced activation of PDGFR-� isknown to mediate Akt activation resulting in the protection ofcells from apoptosis (34, 35). After PDGF-BB stimulation, as-sociation of the activated Akt with I�B appears to be a mech-anism for protection of human skin fibroblasts from apoptosis(36). In PDGF-BB-stimulated wild-type cells, the phosphoryl-ation of Akt sustained at high levels until 2 h after stimulation,whereas the phosphorylation of MAPKs showed transientpeaks and tended to decrease within 15–30 min after stimula-tion. The decrease of phosphorylation of Akt was marked until2 h in PDGF-BB-stimulated PDGFR-��/� fibroblasts. Thesefeatures of Akt activation may imply that the Akt, rather thanMAPKs, contributed to the protection of PDGF-BB-stimulatedwild-type cells from H2O2-induced apoptosis.

The phospholipid phosphatases SHIP2 and PTEN have beenreported to be negative regulators of PDGF signaling. Thesephosphatases lower the level of PI(3,4,5)P3, which results in areduction in Akt activation (17, 18, 37). We found that theoverexpression of either SHIP2 or PTEN effectively inhibitedPDGF-BB-induced phosphorylation of Akt in PDGFR-��/� fi-broblasts, whereas Akt phosphorylation was only partially in-hibited by expression of both SHIP2 and PTEN in wild-typefibroblasts. In addition, SHIP2 is known to competitively in-hibit Shc/Grb2 binding, which is required for the activation ofp21 Ras and ERK1/2 (20). Consistent with this, PDGF-BB-induced phosphorylation of ERK1/2 was inhibited by the ex-pression of SHIP2 but not PTEN in PDGFR-��/� fibroblasts.These results indicate that the PDGF-induced phosphorylationof Akt and ERK1/2 mediated by PDGFR-�, and not PDGFR-�,is preferentially regulated by phospholipid phosphatases. How-ever, we cannot rule out the possibility that SHIP2 or PTENalone are insufficient for the negative regulation of PDGF-BB-induced Akt phosphorylation when it is mediated by bothPDGFR-� and PDGFR-�. In contrast to the involvement ofSHIP2 and/or PTEN in Akt and ERK1/2 activation, expressionof SHIP2 and PTEN did not affect PDGF-BB-induced phospho-rylation of JNK or p38 MAPK in either wild-type or PDGFR-��/� fibroblasts. These results indicate that the phospholipidphosphatases are a negative regulator of certain, but not all,PDGF-BB-induced intracellular signaling events. Furtherstudies are needed to determine whether phospholipid phos-phatases play a direct role in the negative regulation ofPDGFR-�- or PDGFR-�-mediated biological effects.

In summary, we examined the biological effects and signal-ing stimulated by PDGF in dermal fibroblasts in whichPDGFR-� was depleted by the adenovirus-mediated Cre/loxPsystem. This system allowed us to assess the functional impor-tance of PDGFR-� and to examine the role of its specific intra-cellular signaling pathways. Our results indicate the following.1) PDGFR-� plays a crucial role in PDGF-BB-induced cellproliferation, migration, and protection from apoptosis. 2)PDGFR-� activation cannot mediate fibroblast migratory re-sponse to fill in a scratch wound, although it supports a prolif-erative response. 3) PDGF-BB-induced phosphorylation of Akt,ERK1/2, and JNK, but not p38, is involved in the biologicaleffects of PDGF-BB. 4) Although PDGFR-� alone can mediatePDGF-BB-induced activation of p38, it is not enough to medi-ate the cellular responses in fibroblasts, including cell prolifer-ation, migration, and protection from apoptosis. 5) SHIP2, andnot PTEN, preferentially plays a role as a negative regulator ofPDGFR-�-mediated phosphorylation of Akt and ERK1/2, butnot of JNK or p38.

Acknowledgments—We thank Dr. Kazuhito Fukui, Dr. Hiroshi Tsuneki,Yoichi Kurashige, Takako Matsushima, and Sayaka Takakuwa (ToyamaMedical and Pharmaceutical University, Japan) for their technical assist-ance. We also thank Prof. Elaine W. Raines (University of Washington) forcritical reading of the manuscript. We are also grateful to Dr. Izumu Saito(Tokyo University, Japan) for providing AxCANCre (Cre) and AxCALNLZ(LacZ) adenovirus vectors, Dr. Junichi Miyazaki (Osaka University Gradu-ate School of Medicine, Japan) for the CAG promoter, and Dr. TomoichiroAsano (Tokyo University, Japan) for providing the recombinant adenovirusexpressing PTEN.

REFERENCES

1. Heldin, C. H., and Westermark, B. (1999) Physiol. Rev. 79, 1283–13162. Li, X., Ponten, A., Aase, K., Karlsson, L., Abramsson, A., and Uutela, M. (2000)

Nat. Cell Biol. 2, 302–3093. Bergsten, E., Uutela, M., Li, X., Pietras, K., Ostman, A., and Heldin, C. H.

(2001) Nat. Cell Biol. 3, 512–5164. LaRochelle, W. J., Jeffers, M., McDonald, W. F., Chillakuru, R. A., Giese, N. A.,

and Lokker, N. A. (2001) Nat. Cell Biol. 3, 517–5215. Singer, A. J., and Clark, R. A. (1999) N. Engl. J. Med. 341, 738–7466. Orr-Urtreger, A., and Lonai, P. (1992) Development (Camb.) 115, 1045–10587. Schatteman, G. C., Morrison-Graham, K., van Koppen, A., Weston, J. A., and

Bowen-Pope, D. F. (1992) Development (Camb.) 115, 123–131

Roles of PDGFR-� in Dermal Fibroblasts9388

by guest on July 14, 2018http://w

ww

.jbc.org/D

ownloaded from

8. Soriano, P. (1994) Genes Dev. 8, 1888–18969. Crosby, J. R., Seifert, R. A., Soriano, P., and Bowen-Pope, D. F. (1998) Nat.

Genet. 18, 385–38810. Crosby, J. R., Tappan, K. A., Seifert, R. A., and Bowen-Pope, D. F. (1999)

Am. J. Pathol. 154, 1315–132111. Klinghoffer, R. A., Mueting-Nelsen, P. F., Faerman, A., Shani, M., and Sori-

ano, P. (2001) Mol. Cell 7, 343–35412. Heldin, C. H., Ostman, A., and Ronnstrand, L. (1998) Biochim. Biophys. Acta

1378, F79–F11313. Cantley, L. C. (2002) Science 296, 1655–165714. Widmann, C., Gibson, S., Jarpe, M. B., and Johnson, G. L. (1999) Physiol. Rev.

79, 143–18015. Yates, S., and Rayner, T. E. (2002) Wound Repair Regen. 10, 5–1516. Chang, L., and Karin, M. (2001) Nature 410, 37–4017. Mahimainathan, L., and Choudhury, G. G. (2004) J. Biol. Chem. 279,

15258–1526818. Sasaoka, T., Kikuchi, K., Wada, T., Sato, A., Hori, H., Murakami, S., Fukui, K.,

Ishihara, H., Aota, R., Kimura, I., and Kobayashi, M. (2003) Endocrinology144, 4204–4214

19. Leveen, P., Pekny, M., Gebre-Medhin, S., Swolin, B., Larsson, E., and Bet-sholtz, C. (1994) Genes Dev. 8, 1875–1887

20. Ishihara, H., Sasaoka, T., Hori, H., Wada, T., Hirai, H., Haruta, T., Langlois,W. J., and Kobayashi, M. (1999) Biochem. Biophys. Res. Commun. 260,265–272

21. Niwa, H., Yamamura, K., and Miyazaki, J. (1991) Gene (Amst.) 108, 193–19922. Kanegae, Y., Lee, G., Sato, Y., Tanaka, M., Nakai, M., Sakaki, T., Sugano, S.,

and Saito, I. (1995) Nucleic Acids Res. 23, 3816–382123. Ono, H., Katagiri, H., Funaki, M., Anai, M., Inukai, K., Fukushima, Y.,

Sakoda, H., Ogihara, T., Onishi, Y., Fujishiro, M., Kikuchi, M., Oka, Y., and

Asano, T. (2001) Mol. Endocrinol. 15, 1411–142224. Charalampopoulos, I., Tsatsanis, C., Dermitzaki, E., Alexaki, V. I., Castanas,

E., Margioris, A. N., and Gravanis, A. (2004) Proc. Natl. Acad. Sci. U. S. A.101, 8209–8214

25. Sun, Y., Mochizuki, Y., and Majerus, P. W. (2003) J. Biol. Chem. 278,43645–43653

26. Kawano, H., Kim, S., Ohta, K., Nakao, T., Miyazaki, H., Nakatani, T., andIwao, H. (2003) J. Am. Soc. Nephrol. 14, 584–592

27. Eriksson, A., Siegbahn, A., Westermark, B., Heldin, C. H., and Claesson-Welsh, L. (1992) EMBO J. 11, 543–550

28. Li, W., Fan, J., Chen, M., Guan, S., Sawcer, D., Bokoch, G. M., and Woodley,D. T. (2004) Mol. Biol. Cell 15, 294–309

29. Yu, J., Deuel, T. F., and Kim, H. R. (2000) J. Biol. Chem. 275, 19076–1908230. Tournier, C., Hess, P., Yang, D. D., Xu, J., Turner, T. K., Nimnual, A.,

Bar-Sagi, D., Jones, S. N., Flavell, R. A., and Davis, R. J. (2000) Science 288,870–874

31. Du, L., Lyle, C. S., Obey, T. B., Gaarde, W. A., Muir, J. A., Bennett, B. L., andChambers, T. C. (2004) J. Biol. Chem. 279, 11957–11966

32. Matsumoto, T., Yokote, K., Tamura, K., Takemoto, M., Ueno, H., Saito, Y., andMori, S. (1999) J. Biol. Chem. 274, 13954–13960

33. Javelaud, D., Laboureau, J., Gabison, E., Verrecchia, F., and Mauviel, A.(2003) J. Biol. Chem. 278, 24624–24628

34. Kauffmann-Zeh, A., Rodriguez-Viciana, P., Ulrich, E., Gilbert, C., Coffer, P.,Downward, J., and Evan, G. (1997) Nature 385, 544–548

35. Chen, K., Albano, A., Ho, A., and Keaney, J. F., Jr. (2003) J. Biol. Chem. 278,39527–39533

36. Romashkova, J. A., and Makarov, S. S. (1999) Nature 401, 86–9037. Sly, L. M., Rauh, M. J., Kalesnikoff, J., Buchse, T., and Krystal, G. (2003) Exp.

Hematol. 31, 1170–1181

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Ishizawa, Masashi Kobayashi, Yo-Ichi Nabeshima and Masakiyo SasaharaHemragul Sabit, Makoto Kawaguchi, Yoko Kurotaki, Maiko Naito, Tsutomu Wada, Shin

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Gene Affects Key Fibroblast Functions Important forβPDGFR-Deletion of the

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