Keratinocyte Growth Factor/Fibroblast GrowthFactor-7-regulated Cell Migration and Invasionthrough Activation of NF-�B Transcription Factors*
Received for publication, July 19, 2006, and in revised form, December 13, 2006 Published, JBC Papers in Press, January 1, 2007, DOI 10.1074/jbc.M606878200
Jiangong Niu‡1,2, Zhe Chang‡§1, Bailu Peng‡, Qianghua Xia‡§, Weiqin Lu§¶, Peng Huang§¶, Ming-Sound Tsao�,and Paul J. Chiao‡§**3
From the Departments of ‡Surgical Oncology, ¶Molecular Pathology, and **Molecular and Cellular Oncology, The University ofTexas M. D. Anderson Cancer Center, Houston, Texas 77030, the §Program of Cancer Biology, Graduate School of BiomedicalSciences, The University of Texas-Houston Health Science Center, Houston, Texas 77030, and the �Departments of LaboratoryMedicine and Pathobiology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network,University of Toronto, Toronto, Ontario M5G 2M9, Canada
Keratinocyte growth factor (KGF)/fibroblast growth factor-7(FGF-7) is a paracrine- and epithelium-specific growth factor pro-duced by cells of mesenchymal origin. It acts exclusively throughFGF-7 receptor (FGFR2/IIIb), which is expressed predominantlyby epithelial cells, but not by fibroblasts, suggesting that it mightfunction as a paracrinemediator ofmesenchymal-epithelial inter-actions.KGF/FGF-7plays an essential role in the growthof epithe-lial cells and is frequently overexpressed in cancers of epithelialorigin such as pancreatic cancer, switching paracrine stimulationofKGF/FGF-7 to an autocrine loop. Less is known, however, aboutthe signaling pathways by which KGF/FGF-7 regulates theresponse of epithelial cells. To delineate the signaling pathwaysactivated by KGF/FGF-7 and examine cellular response to KGF/FGF-7 stimulation, we performed functional analysis of KGF/FGF-7 action. In this report, we show that KGF/FGF-7 activatednuclear factor �B (NF-�B), which in turn induced expression ofVEGF, MMP-9, and urokinase-type plasminogen activator andincreased migration and invasion of KGF/FGF-7-stimulatedhuman pancreatic ductal epithelial cells. Expression of phospho-rylation-defective I�B� (I�B�S32A,S36A), which blocked NF-�Bactivation, inhibitedKGF/FGF-7-inducedgeneexpressionandcellmigration and invasion. Our results demonstrate for the first timethat KGF/FGF-7 induces NF-�B activation and that NF-�B playsan essential role in regulation of KGF/FGF-7-inducible geneexpression and KGF/FGF-7-initiated cellular responses. Thus,these findings identify one signaling pathway for KGF/FGF-7-reg-ulated cell migration and invasion and suggest that paracrinesourcesofKGF/FGF-7areoneof themalignancy-contributing fac-tors from tumor stroma.
Keratinocyte growth factor (KGF)4/fibroblast growth fac-tor-7 (FGF-7) is a paracrine-actingmitogenproducedby cells ofmesenchymal origin in response to pro-inflammatory cyto-kines and steroid hormones (1–4). KGF/FGF-7 is a heparin-binding growth factor that acts exclusively through a splicingvariant of FGF receptor 2, FGFR2-IIIb, which is expressed pre-dominantly by epithelial cells (5–7). Functional assays in organand cell cultures and in vivo analysis show that KGF/FGF-7 ismitogenically active only on epithelial cells derived from a vari-ety of tissues (8).Many studies show that KGF/FGF-7 is up-reg-ulated after epithelial injury and plays an important role in tis-sue repair (9). For example, recombinant human KGF/FGF-7induces proliferation of pancreatic ductal epithelial cells inadult rats after daily systemic administration for 1 to 2 weeks,indicating that KGF/FGF-7 is a potent in vivomitogen for pan-creatic ductal epithelial cells (10). These effects of KGF/FGF-7are attributed in part to the mechanisms that act collectively tostimulate cell proliferation, migration, and survival; DNArepair; and induction of enzymes involved in the detoxificationof reactive oxygen species (11).The mitogenic property of KGF also has been implicated in
the growth of cancer cells. For instance, KGF/FGF-7 andFGFR2-IIIb have been shown to be overexpressed in both pan-creatic cancer cells and the acinar and ductal cells adjacent tocancer cells (12, 13). These findings suggest that KGF/FGF-7acts as a unique stromal mediator of epithelial cell proliferationin a paracrine manner to stimulate pancreatic cancer cellgrowth in vivo (12, 13). Tumor-associated fibroblasts may playimportant roles in tumor progression through paracrinemech-anisms. KGF may be one of the molecules secreted by thesefibroblasts, which could stimulate the adjacent tumor cells. Anumber of studies show that pro-inflammatory cytokinestumornecrosis factor-� (TNF�), interleukin-6 (IL-6), and espe-* This work was supported in part by NCI, National Institutes of Health Grants
CA097159 and CA109405 and by grants from the Lockton Fund for Pancre-atic Cancer Research. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.
1 Both authors contributed equally to this work.2 An Odyssey fellow supported by the Odyssey Program and The H-E-B Award
for Scientific Achievement at M. D. Anderson Cancer Center.3 To whom correspondence should be addressed: Dept. of Surgical Oncolo-
gy/Molecular and Cellular Oncology, Unit 107, The University of Texas M. D.Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.:713-794-1030; Fax: 713-794-4830; E-mail: [email protected].
cially IL-1 are potent inducers of KGF expression, suggestingthat infiltrating inflammatory cells such asmonocytes and neu-trophils produce inflammatory cytokines, including IL-1,which induce KGF/FGF-7 expression from local mesenchymalcells to promote epithelial proliferation (3, 4). KGF/FGF-7 isone of the AP-1-regulated genes induced by the pro-inflamma-tory cytokines (14). However, KGF/FGF-7-mediated down-stream signaling pathways are still unclear.Nuclear factor �B (NF-�B) is a family of pleiotropic tran-
scription factors that control the expression of numerous genesinvolved in growth, tumorigenesis, tumor metastasis, differen-tiation, embryonic development, apoptosis, and inflammation(15–18). Interaction of c-Rel, RelA, and RelB with their inhibi-tors, the I�Bs, results in inactive complexes in the cytoplasm bymasking the nuclear localization signal (19, 20). In most celltypes, NF-�B proteins are sequestered in the cytoplasm by theinhibitor I�B in an inactive form (16, 19, 20). On stimulation,I�B is phosphorylated by I�B kinase (IKK) and polyubiquiti-nated, which triggers its rapid degradation by proteasome (21–23). Consequently, NF-�B proteins are released and translo-cated into the nucleus, where they activate the expression oftarget genes (15–17, 24). One of the key target genes regulatedby NF-�B is its inhibitor I�B�. A feedback inhibition path-way for control of I�B� gene transcription and down-regu-lation of transient activation of NF-�B activity has beendescribed previously (25–27).Members of the NF-�B family are involved in the develop-
ment of cancer.Many tumors have acquired genetic alterationsin the signaling pathways that regulateNF-�B activation. In onestudy, for example, defective I�B� led to constitutive nuclearNF-�B activity, which in turn conferred a growth advantage toHodgkin disease tumor cells (28). Elevated IKK activities alsowere found in some of the tumor cells, suggesting that IKK isactivated by as yet unidentified aberrant upstream signalingcascades (28). We previously reported that RelA, the p65 sub-unit of the NF-�B transcription factor, is constitutively acti-vated in most pancreatic cancer tissues and human pancreaticcancer cell lines but not in normal pancreatic tissues or immor-talized pancreatic ductal epithelial cells (29, 30). ConstitutiveRelA activity plays a key role in pancreatic cancer metastasisand tumor progression (31, 32). We recently showed that IL-1autocrine stimulation is involved in constitutive NF-�B activityin pancreatic cancer. Key features of pancreatic cancer aremarked proliferation of stromal fibroblasts and deposition ofextracellular matrix components such as matrix metallopro-teinases (MMP) and collagens, suggesting that microenviron-mental cellular interactions are important in the pathogenesisof this disease. The role of tumor-associated stromal fibroblastsin pancreatic cancer pathogenesis remains unclear.In the study reported here, we show that NF-�B activation
and NF-�B-regulated genes are induced in response to stimu-lation by epidermal growth factor (EGF) and KGF/FGF-7 inimmortalized/nontumorigenic human pancreatic ductal epi-thelial (HPDE) cells (33) and that Ras, phosphatidylinositol3-kinase, and Akt are involved in these pathways. Inhibitionof NF-�B activation by a phosphorylation-defective I�B�(I�B�S32A,S36A) blocked KGF/FGF-7-induced gene expres-sion and cell migration and invasion. Our findings suggest that
NF-�B plays an essential role in the regulation of KGF/FGF-7-inducible gene expression and KGF/FGF-7-initiated cellularresponses, and that paracrine stimulation of KGF/FGF-7mightbe one of the factors contributing to growth of malignanciesfrom tumor stroma.
EXPERIMENTAL PROCEDURES
Cell Lines and Reagents—Human papillomavirus type 16early gene 6 and 7-immortalized/nontumorigenic HPDE cellshave been described elsewhere (33). Mouse embryonic fibro-blasts (MEFs) were grown in Dulbecco’s modified Eagle’smedium, which contained 4.5 g/liter glucose, glutamine, andnonessential amino acids, and was supplemented with 10%heat-inactivated fetal bovine serum. Human KGFR cDNA wasobtained fromDr. T. Miki (NCI, National Institutes of Health).EGF and KGF were obtained from R&D Systems. HPDE cellswere cultured in keratinocyte-SFM (serum-free medium,Invitrogen). Anti- phospho-I�B� (Ser-32), anti-phospho-Akt, and anti-I�B� antibodies were obtained from Cell Sig-naling Technology. Anti-Ras antibody was obtained fromCalbiochem. Anti-Paxillin antibody was obtained from Neo-Markers, Inc. Anti-�-actin antibody was obtained fromSigma. Anti-uPA antibody was obtained from American Diag-nostica Inc. Anti-human MMP9 was obtained from BindingSite Inc., and anti-vascular endothelial growth factor (VEGF),anti-p65/NF-�B, anti-Akt, and anti-MEKK3 antibodies wereobtained from Santa Cruz Biotechnology (Santa Cruz, CA).Growth Curve and Cell Cycle Analysis—HPDE cells were
kept in keratinocyte-SFM without growth factors for 48 h andwere stimulated or not stimulated with KGF/FGF-7 or EGF forvarious periods. Cells were counted every 24 h in triplicates,and cell cycle profiles of HPDE cells unstimulated or stimulatedwith KGF (100 ng/ml) or EGF (100 ng/ml) at different timeintervals were analyzed by flow cytometry. Each experimentwas performed independently at least three times with similarresults each time. Results are expressed as the means � S.E. ofthree independent experiments. All of the statistical analyseswere performed using StatView 5.0 (Abacus Concepts Inc.,Berkeley, CA).Nuclear Extract Preparation and Electrophoretic Mobility
Shift Assay—HPDE cells were starved for 48 h, then stimulatedwith EGF (100 ng/ml) or KGF (100 ng/ml) for different timeintervals or TNF� (10 ng/ml) for 30 min as control. Electro-phoretic mobility shift assay (EMSA) was performed as previ-ously described by Schmidt et al. (34). Nuclear extracts wereprepared according to the method of Andrews and Faller (35).DNA binding assays for NF-�B proteins were performed withnuclear extracts (10 �g) as described by Wang et al. (29). 32P-Labeled double-stranded oligonucleotides (5�-CTCAACAGA-GGGGACTTTCCGAGAGGCCAT-3�) containing the �B sitefound in theHIV long terminal repeat were used as probes. Themutant �B site for HIV long terminal repeat (5�-CTCAACAG-AGTTGACTTTTCGAGAGGCCAT-3�) was used for compe-tition studies. The competition was performed with a 50-foldexcess of unlabeled wild-type or mutant �B oligonucleotides.The supershift experiments were performed with anti-RelAantibody (Santa Cruz Biotechnology). The reactions were ana-
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lyzed on 4% polyacrylamide gels containing 0.25�Tris/borate/EDTA buffer.Western Blot Analysis—HPDE cells were starved for 48 h,
then treated with EGF or KGF (100 ng/ml) for various timeperiods, or with TNF� (10 ng/ml) for 30 min as a positive con-trol. Cell cytoplasmic extracts were used for detection of phos-phor-I�B�, I�B�, Paxillin, and �-actin protein levels. Cellnuclear extracts were used to detect RelA/NF-�B and Paxillinprotein levels. For detection of uPA,MMP9, andVEGF levels inconditioned medium, the conditioned media from unstimu-lated and growth factor-stimulated HPDE cells were harvestedat the same ratio of medium volume to cell number and sub-jected to dialysis for 24 h; dialyzed medium (6 ml) was thendried to a volume of 40–80 �l. The concentrated medium or100 �g of protein extracts was resolved by SDS-PAGE, trans-ferred to nylon membranes (Immobilon-P, Millipore), andprobed with antibodies. The subsequent Western blot analysiswas carried out with Lumi-lightWestern blot substrate (RocheApplied Science).Transfection and Luciferase Reporter Assays—HPDE cells
and wild-type MEFs at 70% confluence in growth factor-freemediumwere transfected with either 1.0�g of �B-Luc promot-er-reporter construct alone or co-transfected with variousexpression vectors using FuGENE 6 transfection reagent(Roche Applied Science); pRL-CMV Renilla luciferase was co-transfected as a control reporter vector. After 24–48 h, the cellswere treated or not treated with EGF or KGF (100 ng/ml) for1–4 h. At the end of treatment, reporter activity was deter-mined using a dual-luciferase reporter gene assay according tothe manufacturer’s instructions (Promega, Madison, WI). Theresults are shown as the means � S.E. of three independentexperiments.Reverse Transcription-PCR—Total RNA was extracted using
TRIzol reagent according to the protocol of the manufacturer(Invitrogen). The RNAwas then subjected to reverse transcrip-tion into cDNA. The primers used for PCR amplification ofthe KGFR were 5�-CTCAAGCACTCGGGGATAAA-3� and5�-CTGTTTTGGCAGGACAGTGA-3�; the 150-bp productcorresponded to nucleotides 1352–1501 of human KGFRcDNA, which are located in the IgIIIb exon of human KGFRcDNA (29). The PCR conditions were as follows: 94 °C for 5min, then 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for30 s, and finally extension at 72 °C for 7 min. Glyceraldehyde-3-phosphate dehydrogenase was used as a positive control.Cell Migration and Invasion Assay—Invasion assay was per-
formed in 24-well plates by using a BD Biocoat growth factor-reduced Matrigel invasion chamber (BD Biosciences) with an8.0-�m pore size PET membrane. Each membrane had a thinlayer of GFR Matrigel Basement Membrane Matrix, whichserves as a reconstituted basement membrane in vitro. Theinserts were rehydrated by adding 0.5 ml of warm culturemedium at 37 °C to the inserts for 2 h. Invasion of KGF/FGF-7-stimulatedHPDE/I�B�Mcells was determined, andHPDE/pu-romycin (HPDE/CTL) cells were used as the control. TheseHPDE cells, kept in SFM without growth factors for 24 h, wereseeded (5� 104 cells in 0.5ml of SFM) to the invasion chamberswith orwithout KGF (100 ng/ml) and heparin (29.4�g/ml), and750 �l of complete keratinocyte-SFM was added to the lower
well of the invasion plate. Cells were incubated at 37 °C in ahumidified atmosphere of 95% air and 5% CO2 for 48 h. Non-invading cells from the interior of the inserts were removed byusing cotton-tipped swabs. Invading cells on the bottom side ofthemembrane were fixed by 10% formaldehyde for 10min, andthen stained with 1% crystal violet, and at least three randomfields per insertwere counted under anOlympusmicroscope.Arepresentative field of each experiment was photographed with4� lens and 2.5� magnification. Results are expressed as themeans � S.E. of three independent experiments.Migration assay was performed in 24-well plates by using
Falcon cell culture inserts, which have a PET membrane with1 � 105 8.0-�m pores per cm2 (BD Biosciences Labware).Before the assay was run, HPDE cells were starved for 24 h, and700 �l of complete keratinocyte-SFM was added to the lowerwell of themigration plate. The cells (5� 104 cells/0.3ml) wereseeded to the insert in keratinocyte-SFM with or without KGF(100 ng/ml) and heparin (29 �g/ml). The incubation, staining,counting, and photographing procedures were the same as forthe invasion assay. The representative fields of each experimentare shown. Results are expressed as the means � S.E. of threeindependent experiments.
RESULTS
KGF/FGF-7 Induces Proliferation, Invasion, andMigration inImmortalized HPDE Cells—KGF/FGF-7 is a potent mitogenicfactor for epithelial cells derived from a variety of tissues andplays an important role in tissue repair (8, 9). To determinewhether KGF/FGF-7 has any effects on immortalized/nontu-morigenic HPDE cells, we stimulated HPDE cells with KGF/FGF-7 using EGF-treated cells as a positive control. Our resultsshow that KGF/FGF-7 induced the HPDE cells to proliferate asdetermined by cell counting and flow cytometry analysis for cellcycle profile (Fig. 1,A andB). Cellmigration and invasion assayswere performed as described in “Experimental Procedures.”The representative fields of cell migration and invasion exper-iment are shown (Fig. 1, C and E). Results are expressed as themeans � S.E. of three independent experiments (Fig. 1, D andF). These results show that KGF/FGF-7 stimulated the invasionand migration of the HPDE cells (Fig. 1, C–F), suggesting thatexogenous KGF/FGF-7 mimics paracrine stimulation.KGF/FGF-7 Induces NF-�B Activation in Immortalized
HPDE Cells—To test whether KGF/FGF-7-mediated pathwayscould activate NF-�B, HPDE cells were kept in growth factor-free medium for 48 h, and then stimulated with KGF/FGF-7(100 ng/ml) or EGF (100 ng/ml, as a positive control) at varioustime intervals. Our results show that bothKGF/FGF-7 and EGFinduced activation of NF-�B, and a comparable amount ofnuclear extracts was used as determined by Oct-1 EMSA forloading controls (Fig. 2A). KGF/FGF-7-induced NF-�B activa-tion was time-dependent, and the activity peaked at 2 h (Fig.2A). As shown in Fig. 2B, to confirm the specificity of the KGF/FGF-7- and EGF-induced �B DNA binding activity, competi-tion and supershift assays were performed by using the samenuclear extracts for NF-�B EMSA in Fig. 2A. The unlabeledoligonucleotides containing wild-type �B binding sites com-pletely blockedNF-�BDNAbinding activity, whereas the unla-beled oligonucleotides with mutant �B binding sites had no
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effect on NF-�B DNA binding activity (Fig. 2B). In supershiftexperiments, the mobility of the NF-�B binding activities wasfurther retarded after incubation with p65 antibody as indi-
cated by an arrow (Fig. 2B). Theseresults suggest that the NF-�BDNAbinding activity was specific andthat one of the subunits of the DNAbinding complex was p65 (RelA). Asshown in Fig. 2C, I�B� phosphoryl-ation and degradation were inducedby KGF/FGF-7 and EGF in a time-dependent manner as determinedby Western blot analysis using thecytoplasmic extracts, isolated forthe experiments described in Fig.2A, with anti-phospho-I�B� andanti-I�B� antibodies. Because I�B�phosphorylation by IKK, polyubiq-uitination, and proteasome-medi-ated degradation are the necessarysteps for NF-�B activation (16),these results further indicate thatKGF/FGF-7 and EGF inducedNF-�B activation. Interestingly, thedegradation patterns of I�B� variedslightly between EGF and KGF/FGF-7 stimulation (Fig. 2C, lanes 4and 7). Because I�B� is one of thegenes transcriptionally regulated byNF-�B, the expression levels ofI�B� may oscillate depending onthe nature of the stimulation (25,26). To further demonstrate thatKGF/FGF-7 induces NF-�B activa-tion as observed in NF-�B EMSAand I�B� immunoblots, the levels ofnuclear RelA/NF-�B were deter-mined in the nuclear extracts fromtheHPDE cells stimulatedwith EGFand KGF/FGF-7 at various timeintervals. The results show that thelevels of RelA/NF-�B wereincreased in the nuclear fractions inthe EGF and KGF/FGF-7 timecourse stimulation (Fig. 2C). Therelative protein loading was shownby the use of anti-�-actin antibody,and the quality of the nuclearextracts was determined by probingboth cytoplasmic and nuclearextracts with an antibody againstPaxillin, a cytoplasmic protein. Asshown in Fig. 2C, the findings showthat NF-�B activation was inducedby KGF/FGF-7. To determine theexpression of NF-�B-regulatedgenes that are induced by KGF/FGF-7, the NF-�B reporter gene
assays were performed in HPDE cells. The results showed thatKGF/FGF-7 induced NF-�B-dependent promoter activity sim-ilar to that induced by EGF and TNF�, further indicating that
FIGURE 1. KGF and EGF promote the growth, migration, and invasion of HPDE cells. A, HPDE cells (2 � 105)were cultured in keratinocyte-SFM, either without growth factors or with KGF (100 ng/ml) or EGF (100 ng/ml) asa positive control. Cells were counted in triplicate at different time intervals as indicated. Results are expressedas the means � S.E. of three independent experiments. B, cell cycle profiles of HPDE cells unstimulated andstimulated with KGF (100 ng/ml) or EGF (100 ng/ml) at different time intervals were analyzed by flow cytom-etry. C and D, cell migration assay. To examine KGF-induced HPDE cell migration, HPDE cells were added to thetop compartment of a Boyden chamber in the presence and absence of KGF (100 ng/ml) in the lower wells.After incubation for 48 h, cells that migrated through filters were stained, counted, and photographed. Therepresentative fields are shown in C (10� magnification). Migrated cells were counted in at least three ran-domized fields per insert; results are shown in D as the means � S.E. of three independent experiments. Themigration of HPDE cells with and without KGF stimulation was compared and analyzed statistically. E and F, cellinvasion assay. To examine KGF-induced HPDE cell invasion, HPDE cells were added to the top compartment ofa Boyden chamber coated with Biocoat growth factor-reduced Matrigel basement in the presence or absenceof KGF (100 ng/ml) in the lower wells. After incubation for 48 h, cells that traversed the Matrigel-coated filterswere stained, counted, and photographed. The representative fields are shown in E (10� magnification). Thenumber of KGF-stimulated HPDE cells that traversed Matrigel-covered filters was determined by counting atleast three randomized fields per insert; results in F are expressed as the means � S.E. of three independentexperiments.
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KGF/FGF-7 induced NF-�B activation (Fig. 2D). Takentogether, these results show for the first time that KGF/FGF-7activates NF-�B.KGF/FGF-7 Induces Expression of NF-�B Downstream Tar-
get Genes—To determine whether expression of NF-�B down-stream target genes was induced by KGF/FGF-7, levels of uPA,MMP9, and VEGF expression in HPDE cells were examined(Fig. 3). The HPDE cells were kept in growth factor-freemedium for 48 h, and then stimulated with KGF/FGF-7 (50ng/ml) for various time intervals (Fig. 3). The conditionedmedia from these cells were harvested at the same volume/cellnumber ratios at each time point and were subjected to dialysisand concentration prior to Western blot analysis. The loadingcontrol for the concentrated conditioned media was shown bythe use of Coomassie Blue staining of the identical gels. Theresults show that KGF/FGF-7 induced the expression of NF-�Bdownstream target genes uPA, MMP9, and VEGF and thatthese inductions were dose- and time-dependent (Fig. 3A).However, the patterns of uPA, MMP9, and VEGF expressioninduced by KGF/FGF-7 were different. At 24- and 48-h timepoints, only uPA expressionwas increasedwithout KGF/FGF-7stimulation and possibly by other stimuli in the media.To further demonstrate the expression of uPA, MMP9 and
VEGF were induced by KGF/FGF-7 through NF-�B activation,time- and dose-dependent phosphorylation, and degradationof I�B�, and the levels of nuclear RelA/NF-�Bwere determinedin cytoplasmic and nuclear extracts by immunoblotting. Asshown in Fig. 3B, I�B� phosphorylation and degradation wereinduced by KGF/FGF-7 in a time- and dose-dependentmanneras determined by Western blot analysis using anti-phospho-I�B� and anti-I�B� antibodies. At 24- and 48-h time points,both I�B� phosphorylation and degradation appeared to berather constant in the cells with and without KGF/FGF-7 stim-ulation, suggesting the I�B� re-synthesis and phosphorylation/degradation through the I�B� autoregulation loop reached thesteady state (25, 26). The levels of nuclear RelA/NF-�B wereincreased in the nuclear fractions in the KGF/FGF-7 timecourse stimulation (Fig. 3B). The quality of the nuclear extractswas determined by probing both cytoplasmic and nuclearextracts with an antibody against Paxillin, and relative proteinloadingwas shown by the use of anti-�-actin antibody as shownin Fig. 3B. Although the increased level of nuclear RelA/NF-�Bin the KGF/FGF-7-stimulated cells was consistent with I�B�phosphorylation and degradation, it should be mentioned that
FIGURE 2. EGF and KGF activate NF-�B. A, HPDE cells were cultured in keratino-cyte-SFM without growth factors for 48 h, and then were stimulated with 100ng/ml KGF or EGF for the indicated time and fractionated into cytoplasmic and
nuclear extracts. Nuclear extracts (15 �g) were used in this analysis with anHIV �B probe. An Oct-1 probe was used as a loading control for quality andquantity of cell nuclear extracts. B, competition and supershift assays wereperformed using 15 �g of the same nuclear extracts from KGF- or EGF-stimulated cells for determining the specificity of inducible RelA/p50NF-�B DNA binding activity. The supershifted NF-�B band was indicatedby an arrow. C, for Western blot analysis for I�B� phosphorylation and deg-radation, cytoplasmic protein extracted as described for A was probed withanti-phosphorylated I�B� (p-I�B�), anti-I�B�, and anti-Paxillin antibodies.For nuclear p65/NF-�B immunoblots, the same nuclear extracts used in NF-�BEMSA in A were probed with an anti-p65/NF-�B and anti-Paxillin antibodies.Relative protein loading was shown by using anti-�-actin antibody. D, the �Breporter gene assay was performed with wild-type and mutant �B-luciferasereporter gene constructs as well as TK Renilla for control. After transfectionwith the reporter gene constructs, HPDE cells were cultured in growth factor-free medium for 48 h and then stimulated with KGF, EGF, or TNF� for 2 h, andthe dual luciferase assay was performed.
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the nuclear RelA/NF-�B detected inWestern blot reflected thetotal RelA/NF-�B proteins, which include those from an inac-tive complex with I�B� unable to bind to �B enhancer in thenucleus, as part of the feedback regulation mechanism, andthose that are capable of binding to �B enhancer as determinedby NF-�B EMSA.KGF/FGF-7-inducedNF-�BActivation Is Inhibited byMouse
I�B� (S32A,S36A) PhosphorylationMutant I�B�M—Todeter-mine the role of NF-�B in KGF/FGF-7-mediated signaling
pathways, we used retroviral infection to generate stable clones(HPDE/I�B�M) that expressed I�B�M (I�B�S32A,S36A) withmutated PEST domain for increase of its stability (36). Asshown in Fig. 4A, the expression of I�B�M appeared as a fastermigration band, which was largely due to 3 amino acid residuesshorter in length, and the lack of phosphorylation on PESTregion in this mutated mouse I�B� was confirmed in pooledpuromycin-resistant HPDE cell clones by Western blot usinganti-I�B� antibody. In the presence of I�B�M in HPDE cells,the endogenous I�B� protein was reduced to a minimum level(Fig. 4A), and the same finding in pancreatic cancer cell linewasreported (31). The reduction of endogenous I�B� protein sug-gest the inhibition of NF-�B-mediated basal transcription ofI�B� by I�B�M and the faster turnover rate for endogenousI�B�. TNF�- and KGF/FGF-7-induced NF-�B activation wasinhibited completely by I�B�Mas determined by EMSA (Fig. 4,B and C). To further demonstrate that TNF�- and KGF/FGF-7-induced NF-�B activation is suppressed by I�B�M, inhibi-tion of TNF�- and KGF/FGF-7-regulated I�B� phosphoryla-tion and degradation and level of nuclear RelA/NF-�B weredetermined in HPDE/CTL (infected with a retroviral vectoronly expressing a puromycin-resistant gene) and HPDE/I�B�M cells by Western blot analysis (Fig. 4, B and C). Theendogenous I�B� level was very low, and phosphorylation ofendogenous I�B� induced by KGF/FGF-7, a much weakerNF-�B inducer as compared with TNF-�, cannot be readilydetected inHPDE/I�B�Mcells, but endogenous I�B� degrada-tion was detected and I�B�Mwas not degraded (Fig. 4C). Thequality of the nuclear extracts and relative protein loading weredetermined by Western blot analysis described above. Takentogether these data demonstrate that the TNF�- and KGF/FGF-7-induced I�B� phosphorylation and degradation andincrease of nuclear RelA/NF-�B protein were inhibited byexpression of I�B�M.KGF/FGF-7-induced Expression of NF-�B-regulated Genes Is
Inhibited byMouse I�B� (S32A,S36A) PhosphorylationMutantI�B�M—Todeterminewhether KGF/FGF-7-inducible expres-sion of NF-�B downstream target genes was inhibited byI�B�M, levels of uPA, MMP9, and VEGF in the conditionedmedia from HPDE/CTL and HPDE/I�B�M cells were exam-ined as already described above. The loading control for theconcentrated conditioned media was shown by the use of Coo-massie Blue staining of the identical gels (Fig. 5A). The results ofWestern blot analysis show that KGF/FGF-7-induced expres-sion of the NF-�B downstream target genes uPA and MMP9was inhibited by I�B�M, further suggesting that NF-�B playsan essential role in regulation of KGF/FGF-7-inducible geneexpression (Fig. 5A). The presence of a low level of uPA inHPDE/I�B�Mcells at 12 h with KGF/FGF-7 stimulationmightbe due to the KGF/FGF-7-induced activation of NF-�B fromdegradation of endogenous I�B�. The disappearance of uPA inHPDE/I�B�M cells at 24 and 48 h of KGF/FGF-7 stimulationsuggest that the residual NF-�B activity induced from endoge-nous I�B� complex was suppressed by I�B�M expressed athigher level with greater stability. Interestingly, theKGF/FGF-7induced expression of VEGF is not inhibited by I�B�M, sug-gesting VEGF may be regulated by other transcription factorsmediated by KGF/FGF-7.
FIGURE 3. A, expression of the NF-�B downstream target genes uPA, MMP9,and VEGF was induced by KGF. Western blot analysis was performed withanti-uPA, anti-MMP9, and anti-VEGF antibodies to determine the levels ofuPA, MMP9, and VEGF expression in the conditioned media from HPDE cellsstimulated or not stimulated with KGF (50 ng/ml) for the indicated times. Theconditioned media were collected in the same volume/cell number ratios andsubjected to dialysis and concentration prior to the analysis. The equal load-ing of the concentrated conditioned media was shown by the use of Coomas-sie Blue staining of the identical gels. B, KGF/FGF-7-induced NF-�B activation.HPDE cells were cultured in keratinocyte-SFM without growth factors for 48 h,and then were stimulated with 50 ng/ml KGF/FGF-7 for the indicated timeand fractionated into cytoplasmic and nuclear extracts. Western blot analysiswas performed as described for probing with anti-phosphorylated I�B�(p-I�B�), anti-I�B�, RelA/NF-�B (p65), and Paxillin. Relative protein loading isshown by using anti-�-actin antibody.
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To further demonstrate KGF/FGF-7-induced expression ofuPA, MMP9 and VEGF were suppressed by I�B�M-mediatedinhibition of NF-�B activation, time-dependent phosphoryla-tion, and degradation of I�B�, and increased nuclear RelA/NF-�B protein levels were determined in cytoplasmic andnuclear extracts by immunoblotting. As shown in Fig. 5B, I�B�phosphorylation and degradation were induced by KGF/FGF-7in a time-dependent manner as determined by Western blot
FIGURE 4. Phosphorylation-defective I�B� mutant (I�B�M) inhibitedKGF-induced NF-�B activation. A, expression of I�B�M in I�B�M retrovirus-infected HPDE cells was determined by Western blot analysis using anti-I�B�antibody. Relative protein loading was shown by using anti-�-actin antibody.B and C, EMSA were performed to examine TNF�- and KGF-induced NF-�BDNA binding activity in HPDE/CTL and HPDE/I�B�M cells as described. TheHPDE/CTL and HPDE/I�B�M cells were cultured in keratinocyte-SFM withoutgrowth factors for 48 h, then stimulated with TNF� for 30 min or with 100ng/ml KGF for the indicated times; the cells were then fractionated into cyto-plasmic and nuclear extracts. Nuclear extracts (15 �g) were used in this anal-ysis with an HIV �B probe. The cytoplasmic extracts (50 �g) were subjected toWestern blot using anti-phosphorylated I�B� (p-I�B�), anti-I�B�, and Paxillinantibodies, and the nuclear extracts were probed with an anti-p65/NF-�B andanti-Paxillin antibodies. Relative protein loading is shown by using anti-�-actin antibody.
FIGURE 5. KGF/FGF-7-induced expression of NF-�B downstream targetgenes uPA, MMP9, and VEGF was inhibited by I�B�M. A, Western blotanalysis was performed with anti-uPA, anti-MMP9, and anti-VEGF antibodiesto determine the levels of uPA, MMP9, and VEGF in the conditioned mediafrom HPDE/CTL and HPDE/I�B�M cells stimulated or not stimulated with KGF(50 ng/ml) for the indicated times. The conditioned media were collected inthe same volume/cell number ratio and subjected to dialysis and concentra-tion prior to the analysis. The equal loading of the concentrated conditionedmedia is shown by the use of Coomassie Blue staining of the identical gels.B, I�B�M inhibited KGF/FGF-7-induced NF-�B activation. HPDE cells were cul-tured in keratinocyte-SFM without growth factors for 48 h and then werestimulated with 50 ng/ml KGF/FGF-7 for the indicated time and fractionatedinto cytoplasmic and nuclear extracts. Western blot analysis was performedas described for probing with anti-phosphorylated I�B� (p-I�B�), anti-I�B�,RelA/NF-�B (p65), and Paxillin. Relative protein loading is shown by usinganti-�-actin antibody.
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analysis using anti-phospho-I�B� and anti-I�B� antibodies. Inthe KGF/FGF-7 time-course stimulation, the levels of nuclearRelA/NF-�B were increased in the nuclear fractions of HPDE/CTL cells but not in HPDE/I�B�M cells (Fig. 5B). The nuclearextracts showed very little contaminants with cytoplasmic pro-teins as determined by the level of Paxillin, and protein loadingsin each lane were comparable as determined by the use of anti-�-actin antibody (Fig. 5B). These results suggest that I�B�Minhibited the KGF/FGF-7-induced expression of the NF-�B-regulated downstream target genes uPA and MMP9.KGF/FGF-7-induced Cell Migration and Invasion Are
Suppressed by I�B�M-mediated Inhibition of NF-�B—Todemonstrate whether NF-�B activity plays a key role in KGF/FGF-7-induced cellular responses, cell migration and inva-sion assays were performed as described under “Experimen-tal Procedures” using HPDE/I�B�M cells and HPDE/CTLcells as a control. The representative fields of the cell migrationexperiment are shown (Fig. 6A) as are the representative fields
of the cell invasion experiment (Fig.6C). Results are expressed as themeans � S.E. of three independentexperiments (Fig. 6, B and D). Theresults in Fig. 6 showed that KGF/FGF-7 significantly enhancedHPDE/CTL cellmigration and inva-sion, whereas its effects on migra-tion and invasion of HPDE/I�B�Mcells were suppressed by I�B�M-mediated inhibition of NF-�B activ-ity. Taken together, these resultssuggest that NF-�B plays an essen-tial role in regulation of KGF/FGF-7-inducible gene expression andKGF/FGF-7-mediated cell migra-tion and invasion.KGF Receptor-mediated Signal-
ing Cascades Induce NF-�B Acti-vation—To determine whetherKGFR, a splicing variant of FGFR-2,FGFR2-IIIb, relays KGF/FGF-7 sig-nals for induction of NF-�B activa-tion, and to generate a specific cellline for identifying the signalingcomponents in KGF/FGF-7-in-duced NF-�B activation, an expres-sion vector for a human KGFR wasconstructed. Wild-type MEFs cells,which do not express mouse KGFR,were transfected with this expres-sion vector to obtain stable clones.As shown in Fig. 7A, human KGFRtranscript was detected by reversetranscription-PCR in these G418-resistant clones after transfection ofKGFR expression plasmid, indicat-ing that human KGFR wasexpressed. NF-�B was activated byKGF/FGF-7 in the MEFs/KGFR but
not in the parental MEFs (Fig. 7B, lanes 2 and 5). A low level ofNF-�B activity was detected in MEFs/KGFR without exoge-nous KGF/FGF-7, suggesting that expression of human KGFRin MEFs partially connected the KGF/FGF-7 autocrine stimu-lation loop (Fig. 7B, lane 4). The expression of KGF/FGF-7 inMEFs cells was detected by Western blot (data not shown).EGF-inducedNF-�B activation served as a positive control (Fig.7B). NF-�B reporter gene assays showed that KGF/FGF-7-in-duced NF-�B activity was dependent on expression of KGFR(Fig. 7C). Furthermore, the high level of reporter gene activityin the cell transfected with KGFR without exogenous KGF/FGF-7 stimulation is consistent with the results from theNF-�B EMSA in Fig. 7B, suggesting KGF/FGF-7 that autocrinestimulation is at work. In Fig. 7 (D and E), both KGF/FGF-7-and EGF-inducedNF-�B reporter gene activity was observed inMEFs/KGFR cells only transfected with NF-�B-luciferasereporter gene. In the MEFs/KGFR cells co-transfected withNF-�B-luciferase reporter gene and phosphorylation-defective
FIGURE 6. I�B�M-mediated inhibition of NF-�B suppressed KGF/FGF-7-induced cell migration and inva-sion. A and B, cell migration assay. HPDE/CTL and HPDE/I�B�M cells were added to the top compartment of aBoyden chamber in the presence or absence of KGF (100 ng/ml) in the lower wells. At various time points, cellsthat migrated through filters were stained and counted, and the representative fields were photographed andare shown in A (10� magnification). The number of migrated cells (i.e. KGF-stimulated HPDE/CTL and HPDE/I�B�M cells that traversed the filters) was determined by counting at least three randomized fields per insert intriplicate. The results are expressed as the means � S.E. of three independent experiments and are shown in B.C and D, cell invasion assay. To examine KGF-induced HPDE/CTL and HPDE/I�B�M cells invasion, HPDE/CTLand HPDE/I�B�M cells were added to the top compartment of a Boyden chamber coated with Biocoat growthfactor-reduced Matrigel basement in the presence or absence of KGF (100 ng/ml) in the lower wells. Afterincubation for 48 h, cells that traversed the Matrigel-coated filters were stained and counted, and the repre-sentative fields were photographed and are shown in C (10� magnification). The number of KGF-stimulatedHPDE/CTL and HPDE/I�B�M cells that traversed Matrigel-covered filters was determined by counting at leastthree randomized fields per insert. The results are expressed as the means � S.E. of three independent exper-iments and are shown in D.
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I�B� (S32A,S36A), kinase-dead IKK1/� (��), or IKK2/�(��), NF-�B reporter gene activity was significantly inhibitedby phosphorylation-defective I�B� and these kinase-deadmutants (Fig. 7,D and E). Taken together, these results suggestthat KGF/FGF-7 activates NF-�B through KGFR- and IKK-de-pendent mechanisms.Activation of Akt Plays a Key Role in KGF/FGF-7-induced
NF-�BActivation—The reports by Romashkova et al. andOzeset al. (37, 38) show that NF-�B has a role in growth factor sig-naling and define an anti-apoptotic Ras/phosphatidylinositol3-kinase/Akt/IKK/NF-�B pathway (37, 38). To identify the sig-nalingmolecules involved in growth factor-mediated activationof IKK andNF-�B, �B-luciferase reporter gene assays were per-formed using MEFs/KGFR co-transfected with various Aktmutants. The results show that a myristoylated active form ofAkt (myr-Akt) greatly enhanced KGF/FGF-7- and EGF-in-duced NF-�B reporter gene activity, and constitutively acti-vated Akt mutant (AktDD) induced NF-�B reporter geneactivity without exogenous growth factors, whereas activa-tion-defective (AktAA) and kinase-dead (AktK179M) AKTmutants inhibited KGF/FGF-7- and EGF-induced NF-�Breporter gene activity (Fig. 8A). The expression levels of varioustransfectedAktmutants are comparable as determined by anti-Akt immunoblot using the same extracts for the NF-�Breporter gene assay (Fig. 8A). Although kinase-dead MEKK3(MEKK3KM) only reduced KGF/FGF-7- and EGF-inducedNF-�B activation by �3-fold, Ras (RsN17) mutant, Akt (Akt-K179M) mutants, and LY294002, a specific inhibitor of phos-phatidylinositol 3-kinase, the upstream kinase of Akt, com-pletely inhibited KGF/FGF-7- and EGF-induced NF-�Breporter gene activity (Fig. 8B). The expression levels of trans-fected kinase-dead MEKK3KM, RsN17, and Akt-K179Mmutants were very similar as determined by the immunoblotusing the same extracts for the NF-�B reporter gene assay (Fig.8B). As shown in Fig. 8C, phosphorylation of Akt and KGF/FGF-7- and EGF-induced NF-�B DNA binding activity alsowere inhibited by LY294002 in HPDE cells. Taken together,these results show that the signaling pathways mediated byKGF/FGF-7 involve activation of Akt and NF-�B.
DISCUSSION
We have demonstrated for the first time that KGF/FGF-7, aparacrine and epithelium-specific growth factor produced bycells of mesenchymal origin, is an inducer of NF-�B activationand that NF-�B plays an essential role in regulation of KGF/FGF-7-inducible gene expression. In this report, we providecrucial data supporting this novel function of KGF/FGF-7, asfollows: 1) KGF/FGF-7 stimulated growth,migration, and inva-sion of immortalized HPDE cells (Fig. 1); 2) KGF/FGF-7inducedNF-�B activation as demonstrated by EMSA (Fig. 2); 3)KGF/FGF-7 induced expression of NF-�B downstream targetgenes (Fig. 3); 4) KGF/FGF-7-induced NF-�B activation wasinhibited by I�B�M, a phosphorylation mutant of I�B�(S32A,S36A), andNF-�B plays an essential role in regulation ofKGF/FGF-7-inducible uPA and MMP9 expression, but KGF/FGF-7-induced VEGF expression was not regulated by NF-�B(Figs. 4 and 5); 5) KGF/FGF-7-induced cell migration and inva-sion were suppressed by I�B�M-mediated inhibition of NF-�B
FIGURE 7. Expression of FGFR2/IIIb receptor (KGFR) in MEFs activated NF-�B.A, expression of KGFR and glyceraldehyde-3-phosphate dehydrogenase(GAPDH, control) in MEFs was determined by reverse transcription-PCR afterFGFR2/IIIb transfection. B, KGF-induced NF-�B DNA binding activity in MEFs andMEFs/KGFR was determined by EMSA as described. MEFs and MEFs/KGFR werecultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum for 48 h and then fractionated into cytoplasmic andnuclear extracts. Nuclear extracts (10 �g) were used in this analysis with an HIV �Bprobe. C–E, dual luciferase assays were performed to determine the activation ofNF-�B reporter gene. C, the �B luciferase and TK Renilla reporter genes wereco-transfected into MEFs with KGFR; the cells were cultured in serum-freemedium for 48 h and then stimulated or not stimulated with KGF (100 ng/ml) for2 h. D and E, the �B luciferase and TK Renilla reporter genes were co-transfectedinto MEFs with phosphorylation-defective I�B� mutant and kinase-dead IKK1/�and IKK2/�. These transfectants were cultured in SFM for 48 h and then stimu-lated or not stimulated with EGF (100 ng/ml) or KGF (100 ng/ml) for 2 h, andprotein extracts were isolated for dual luciferase assay.
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(Fig. 6); 6) KGF/FGF-7 receptor-mediated signaling cascadesinduced NF-�B activation (Fig. 7); and 7) activation of Aktplayed a key role in KGF/FGF-7-inducedNF-�B activation (Fig.8). These results suggest that NF-�B plays an essential role inregulation of KGF/FGF-7 signaling cascades and KGF/FGF-7-induced cellular responses.The tumor microenvironment is composed of fibroblasts,
various inflammatory cells, and vascular structures. Interactionbetween tumor cells and their microenvironment is a highlydynamic process, with presumed effects on tumor progression.The interplay is based on three basic processes: production ofautocrine andparacrine factors, cell-matrix contact, and signal-ing by direct cell-cell interactions (39, 40). To establish cellculture models for the study of the tumorigenesis of humanpancreatic cancer in vitro, we have developed immortalizedcultures of HPDE cells derived from normal epithelium (41).The aim of these studies was to investigate the immortaliza-tion-transformation sequence of these cells to understand bet-ter the implication of various mutations, including the role ofparacrine factors such as KGF/FGF-7, a potent mitogenic para-crinemediator of epithelial cells, in the development of pancre-atic cancer.We investigated the transforming potential of KGFon pancreatic epithelial cells, because one of the key features ofpancreatic epithelial cancer is amarked proliferation of stromalfibroblasts. We tested exogenous effects of KGF/FGF-7 onhuman pancreatic epithelial cells by mimicking paracrine orautocrine KGF/FGF-7 stimulation. KGF/FGF-7 stimulatedHPDE cells and enhanced cell growth, migration, and invasion,suggesting the transforming potential of KGF/FGF-7 onhumanpancreatic epithelial cells.We previously showed that NF-�B is constitutively activated
in most human pancreatic cancer tissues and cell lines but notin normal pancreatic tissues and immortalized pancreatic duc-tal epithelial cells (29, 42). A number of recent studies haveshown that phosphorylation mutant I�B� (S32A,S36A)(I�B�M)-mediated inhibition of constitutive NF-�B activity inhuman pancreatic cancer cells suppresses tumorigenesis andlivermetastasis in an orthotopic nudemousemodel, suggestingthat constitutive NF-�B activation plays an important role inpancreatic tumor progression and metastasis (31, 32). We pre-viously found that an IL-1 autocrine mechanism accounts forthe constitutive activation of NF-�B in metastatic human pan-creatic cancer cell lines (42). Our results also demonstrate thatregulation of IL-1 expression is primarily dependent on growth
FIGURE 8. Akt plays a key role in regulation of KGF/FGF-7-induced NF-�Bactivation. A, the �B luciferase reporter genes and TK Renilla were trans-fected into MEFs/KGFR with control expression vector or with various AKTmutants (Myrkt, AktDD, AktAA, and Akt-K179M) as indicated. These MEFs were
cultured in SFM for 48 h and then stimulated with either 100 ng/ml EGF or 100ng/ml KGF/FGF-7 for 2 h, and protein extracts were isolated from these cellsfor dual luciferase assays and Western blots to determine the expression ofAkt mutants and �-actin as indicated. B, MEFs/KGFR cells were transfectedwith control expression vector, kinase-dead MEKK3 and Akt, or a mutant Ras(RsN17), cultured in Dulbecco’s modified Eagle’s medium without growthfactors for 48 h and then either stimulated with EGF (100 ng/ml) or KGF (100ng/ml) for 2 h or treated or not treated with LY294002 (30 �M) for 2 h. Dualluciferase assay and Western blots for determining the expression of Akt,MEKK3, Ras mutants, and B-actin were performed as indicated. C, HPDE cellswere starved for 48 h, and then treated with 30 �M LY294002 for 30 min. Thenthe cells were treated with 100 ng/ml EGF or KGF/FGF-7 for 1 h. The nuclearand cytoplasmic extracts were isolated; nuclear extracts were used to deter-mine NF-�B activities by EMSA and cytoplasmic extracts to determine phos-phorylation of Akt by Western blot analysis using an anti-phospho-Aktantibody.
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factor-regulated AP-1 activity (43). These findings suggest apossible mechanism by which constitutive activation of NF-�Bin metastatic human pancreatic cancer cells is initially inducedby KGF/FGF-7 and further enhanced by IL-1 autocrine stimu-lation, because IL-1 is one of the downstream target genes reg-ulated by NF-�B. Interestingly, Chedid et al. (3) reported thatthe pro-inflammatory cytokine IL-1 strongly induces theexpression of KGF/FGF-7 in fibroblasts from multiple sources(3). Furthermore, it has been shown that, in a cell co-culturesystem, expression ofKGF/FGF-7 is strongly enhanced in fibro-blasts, and expression of IL-1 is greater in co-cultured kerati-nocytes than in monocultures (44). Thus it was postulated thatIL-1, which had no effect on keratinocyte proliferation, inducesthe expression of growth factors to stimulate keratinocyte pro-liferation, such as KGF/FGF-7 in fibroblasts. This is consistentwith our finding that KGF/FGF-7 produced from fibroblastsinduced NF-�B activation and stimulated the expression ofNF-�B-regulated genes such as IL-1, which in turn inducedexpression of KGF/FGF-7 in fibroblasts, resulting in a doubleparacrine stimulation loop for a dynamic and reciprocal mod-ulation of cytokine and growth factor production in epithelialmesenchymal interactions. Themutually induced signaling cir-cuits for growth regulation may have in vivo functional signifi-cance, because marked proliferation of stromal fibroblasts isone of the key features of pancreatic cancer. Furthermore, thesignificance of this reciprocal modulation of the growth of epi-thelial cells is regulated by a double paracrine mechanismthrough release of pro-inflammatory cytokine IL-1 from epi-thelial cells, which elicit enhanced expression of growth factors,particularly KGF/FGF-7, in fibroblasts; thus IL-1, in addition toits pro-inflammatory function, may play an essential role inregulating fibroblast proliferation.
Acknowledgments—We are grateful to Dr. Toru Miki at NCI,National Institutes of Health for providing human KGFR plasmid.We also thank Kathryn Hale for editorial assistance.
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KGF/FGF-7 Induces NF-�B Activation
MARCH 2, 2007 • VOLUME 282 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 6011
