Heparin-binding Epidermal Growth Factor-like Growth Factor Is an ...

6 downloads 151 Views 4MB Size Report
Aug 5, 2006 - regulin were kindly provided by Dr. Judith A. Abraham (Scios Nova Inc. ...... Wille, J., Jr., Pittelkow, M. R., Shipley, G. D., and Scott, R. E. (1984) J. Cell ... Shoyab, M., McDonald, V. L., Bradley, J. G., and Todaro, G. J. (1988) Proc ...
THE JOURNALOF BIOLCGICAL CHEMISTRY 0 1994 by The American Societyfor Biochemistry and Molecular Biolom, Inc.

Vol. 269, No. 31, Issue of August 5, pp. 20060-20066, 1994 Printed in U.S.A.

Heparin-binding Epidermal GrowthFactor-like Growth Factor Is an Autocrine GrowthFactor for Human Keratinocytes" (Received for publication, May 4, 1994)

Koji HashimotoS, Shigeki HigashiyamaSlfl,Hideo Asada, Etsuro Hashimura, Teruaki Kobayashi, Kaori Sudo, Takatoshi Nakagawas, Deborah Dammll, Kunihiko Yoshikawa, and Naoyuki TaniguchiO From the Departments of Dermatology and $Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan and llscios Nova Znc., Mountain View, California 94043

Since heparin-binding epidermal growth factor (HBEGF) is a member of the EGF family and binds to EGF receptor, we tested recombinant HB-EGF for its ability to stimulate human keratinocyte proliferation. The effect of HB-EGFon human keratinocytes was dependent on the cell density. HB-EGF optimally increased the cell number by 1.8-fold at 1.0 ng/ml for a 4-day incubation period under subconfluent culture. In contrast, under confluent culture, 10 ng/ml HB-EGF optimally increased the DNA synthesis 2.1-fold. To examine the production of HB-EGF byhuman keratinocytes, the analysis of human keratinocyte-conditionedmedium wasundertaken by a combination of heparin affinity column chromatography, EGF receptor-stimulating assay, immunoblotting, and neutralization. Heparin column chromatography fractionated three activities, peaks 1, 2, and 3, which contained immunoreactive 30- and 27-, 19-,and 14.5-kDa bands, respectively. The anti-HB-EGF-blocking antibody neutralized the activities of peaks 2 and 3 by 38and 22%, respectively, but did not neutralize the activity of peak 1 at all. Theantibody reduced the cell growth by 37%for a 4-day incubation period. Northern blot analysis detected a 2.5-kilobase transcript of HB-EGF.The addition of 1ng/ml HB-EGFoptimally increased the levels of HBEGF mRNA SA-foldat 1h and TGF-a mRNA 3.1-fold at 3 h. Interestingly, the addition of TGF-a at 1 ngml to keratinocyte cultures enhanced the level ofHB-EGF mRNA 10.2-fold at 6 h. 1ng/ml EGF also increased HBEGF mRNA levels 10.9-fold at 1h. These results suggest that HB-EGF is an autocrine growth factor for human keratinocytes, and HB-EGF and TGF-a act not only by an autoinductive mechanism but also by mutual amplification.

sequence of human HB-EGF cDNA predicts a precursor protein of 208 amino acidscomposed of a signal peptide, a hydrophilic domain, an EGF-like domain, and a transmembrane domain. The precursor proteinwould then be cleaved to yield a mature protein containing at least 86 amino acids and comprising an EGF-like domain and a basic region responsible for heparin receptor and is binding (2). This growth factorbinds to the EGF a potent mitogen for smooth musclecells and Balb/c 3T3 fibroblasts (1).HB-EGF is also transcribed and regulated in human vascular endothelial cells (3) and in human (4) and rat aortic smooth muscle cells (5). Keratinocytes are the main component cells in the human epidermis, and their growth is stimulated by a variety of growth factors. Among these, the most important are members of the EGF family of growth factors, including transforming growth factor-a (TGF-a)(6) and amphiregulin (7). TGF-a and amphiregulin are produced and secreted by human keratinocytes, which suggests that they are autocrine growth factors. (8). HB-EGF and amphiregulin share structural characteristics Amphiregulin binds to heparin and is composed of 84 amino acids, close to the number in HB-EGF. In the present work, we demonstrate for the first time that human keratinocytes produce HB-EGF, which stimulates the growth of these cells in culture. Furthermore, anti-HB-EGF antibody reduced the growth of human keratinocytes. We also show that the addition of recombinant HB-EGF or TGF-a increasesthetranscription of both HB-EGF andTGF-a mRNAs. These data suggest that HB-EGF is an autocrine growth factor for human keratinocytes and that endogenous keratinocyte EGF-like growth factors share propertiesof mutual amplification. EXPERIMENTAL.PROCEDURES

Heparin-binding EGF-like growth factor (HB-EGF)' is a new member of the EGF family initially purified from conditioned medium of the U-937 macrophage-like cell line (1). The coding

* This work was supported by Grant-in-aid for Scientific Research 03454274 from the Ministry of Education, Science, and Cultureof Japan (toK. Y.) and Grant-in-aidfor Cancer Research 05151047(to S. H. and N. T.). The costsof publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked "advertisement"in accordancewith18U.S.C.Section1734 solely to indicate this fact. t Contributed equally to this work and should be considered first authors. fl To whom correspondence should be addressed. Tel.: 81-6-879-3421; Fax: 81-6-879-3429. The abbreviations used are: HB-EGF, heparin-bindingEGF-like growthfactor;EGF,epidermalgrowthfactor;TGF-a, transforming growth factor-a; DIG, digoxigenin; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PBS, phosphate-bufferedsaline; BrdUrd, bromodeoxyuridine.

Materials RecombinanthumanEGF and TGF-aweregenerousgiftsfrom Otsuka PharmaceuticalCo., Ltd. (Tokyo, Japan). HB-EGF and amphiby Dr. Judith A. Abraham (Scios Nova Inc., regulin were kindly provided Mountain View, CA) and Dr. Michael Klagsbrun (Children's Hospital, Harvard Medical School, Boston, MA), respectively. p-cellulin was a kind gift from Dr. Reiko Sasada (Takeda Chemical Industries, Osaka, Japan). pSP65 plasmid containing a fragment of human TGF-ol cDNA (1.27 kilobases) was a generous gift from Dr. Rik Derynck (University of California, San Francisco, CAI. Cell Culture Human keratinocytes were cultured as previously described (9, 10). Briefly, normal human skin, obtained during plastic surgery, was cut into 3-5-mm piecesand floated on dispase solution(500unitdml) overnight at 4 "C. Afterthe separation of epidermis from dermis by forceps, the epidermal sheets were rinsed with Ca2+-and Me-free PBS, incubated in a 0.25% trypsin solution for 10 minat 37 "C, and teasedwith forceps. Epidermal cells were suspended in optimized nutrient medium MCDB153 (Kyokuto Co.) supplemented with insulin (5 pdml), hydro-

20060

Factor HB-EGF Is an Autocrine Growth for cortisone (5 x

M), ethanolamine (0.1 mM), phosphoethanolamine(0.1

m ~ ) and , bovine hypothalamic extract (150 pg/ml). The stock solution

consisted of MCDB153 supplemented with 0.1 mM Ca2+containing elevated concentrations of amino acids (0.75 mM isoleucine, 0.24m~ histidine, 0.09 m~ methionine, 0.09 m~ phenylalanine, 0.045 mM tryptophan, 0.075 m~ tyrosine) and antibiotics (100 unitdml penicillin and 100 pg/ml streptomycin). Second-passage cells were used in these experiments. Unless indicated otherwise, keratinocytes were plated in 6-well plates (Falcon 3046, Becton Dickinson, Bedford, MA). Since no serum was present in the medium, the collected medium was centrifuged, and the supernatant was frozen at -20“Cfor storage. The EP170.7 cell line, which was provided by Dr. Jackie Pierce (U), was grown in RPMI 1640 medium supplemented with 10% fetal calf serum (11)and containing penicillin and 5% WEHI-3 cell-conditioned medium (100 unitdml) and streptomycin (100 pg/ml). This cell undergoes the DNA synthesis through binding of the ligands to the EGF receptors on the cell surface. Measurement of Bromodeoxyuridine (BrdUrd) Uptake Keratinocytes were seeded on 24-well plates at the density of 8 x 104/well. Afterreaching confluency, the cells were refed with the medium lacking bovine hypothalamic extract. The following day, the cells were refed once again with the same medium containing various concentrations of HB-EGF and incubated overnight. The cells were then incubated with the medium containing BrdUrd for 2 h. Incorporated BrdUrd was measured using a cell proliferation assay kit (Amersham Corp.) accordingto the manufacturer’s instructions. The absorbance at 410 nm was measured using a model 450 MicroplateReader (Bio-Rad). Growth Factor Assay EP170.7 cells were washed with RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 pg/ml). Cells (2 x lo4) were plated in 96-well plates in a total volume of 200 pl. 36h later, 1 pCi of 1,HIthymidine wasadded in 10 pl of PBS. After4 h, cells were harvested, and incorporated L3H1thymidine was determined using a 1205 Betaplate system (Pharmacia Biotech Inc.). Recombinant HB-EGF was used as a standard to estimate the HB-EGF content of samples. Column Chromatographies Conditioned media were directly applied to a Bio-Rex 70 column (20 ml) (Bio-Rad) equilibrated with 20 mM Tris-HC1, pH 7.2. The column was extensively washed with the equilibration buffer, and the bound protein was eluted with 1M NaC1, 20 m~ Tris-HC1, pH 7.2. The protein fraction was diluted four times with the equilibration buffer and applied to a TSK-heparin 5PW column (8 x 75 mm, Tosoh, Tokyo, Japan) equilibrated with 0.02 M NaC1, 0.01 M Tris-HC1, pH 7.4, using a fast protein liquid chromatography system (Pharmacia). The column was washed with 20 ml of equilibration buffer, and bound protein was eluted with a 40-ml linear gradient of 0.2-2 M NaCl in 0.01 M Tris-HC1, pH 7.4, at a flow rate of 1 mumin. 1-ml fractions were collected, and 10 p1of each fraction were tested for mitogenicactivity on EP170.7 cells as described above. Measurement of TGF-a in CultureMedium TGF-a in culture medium was concentrated by Sep-Pak C,, (Waters Associates) and then eluted with acetonitryl and lyophilized. Samples were solubilizedin 0.5 ml of PBS and quantitated by an enzyme-linked immunosorbent assay kit (OtsukaPharmaceutical Co., Ltd.) according to the manufacturer’s instructions (12). Immunological Methods WesternBlotting-The fractions containing mitogenic activity obtained by heparin affinity chromatography were pooled, diluted twice with 0.01 M Tris-HC1,pH7.4, and concentrated by a mini-heparinSepharose column (50pl). Samples were fractionated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis under reducing conditions as previously described (8). Proteins in the gels were transferred to a nitrocellulose membrane (Schleicher& Schuell)in 150 m~ CAPS buffer, pH 11, containing 20% methanol a t 200 mA for 3 h. The nitrocellulose membrane was incubated with 5% skim milk in PBS for 2 h at 4 “C to block nonspecific adsorption. HB-EGF was detected by incubating the nitrocellulose first with rabbit anti-HB-EGF antibody 2998 (4) or rabbit anti-amphiregulin antibody -1, which was raised against synthetic peptide corresponding to the 1-16 sequence, then with alkaline phosphatase-conjugated goat anti-rabbit IgG (Promega), and finally with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate

Human Keratinocytes

20061

(Promega). 50 ng of highly purified U-937 cell-conditioned mediumderived “Peak-1” HB-EGF(8) was used as a standard. Neutralization by Anti-HB-EGFAntibody-A neutralizing anti-HBEGFpolyclonalantibody,197, was raised in goats directed against recombinant 77-amino acid human HB-EGF. To neutralize HB-EGF, increasing concentrations of 197 antibody and 1 ng of recombinant HB-EGF or 10-pl samples were added directly to EP170.7 cells.To test the specificity of 197 antibody, 1 ng of EGF, TGF-a, amphiregulin, or p-cellulin was used instead of HB-EGF. Probe Preparation Digoxigenin(DIG)-labeled single-stranded RNA probeswere prepared using a DIGRNA labeling kit (Boehringer Mannheim GmbH Biochemica) according tothe manufacturer’s instructions (13). For the generation of human HB-EGF probe,a fragment of human cDNA (315969) (1)was subcloned into the Bluescript pKS(-) plasmid. This plasmid was linearized by BanHI and transcribed with T3 RNA polymerase to generate a 0.65-kilobase-longantisense (tRNA) probe. pSP65 plasmid was linearized byHind111 and transcribed with SP6 polymerase to generate a 1.27-kilobase-longantisense probe of TGF-a. Northern Blot Analysis Total RNA from cultured human keratinocytes was prepared by the acid guanidinium thiocyanate/phenoVchloroformmethod (14). 20 pg of total RNMane was fractionated on a 1.8% formaldehyde-agarosegel and transferred to Genescreen (DuPont NEN) in 20 x SSC (150 mM sodium chloride,15 mM sodium citrate) according to standard procedure (15). Ultraviolet cross-linking was performed by ultraviolet treatment in a Stratalinker (Stratagene). Prehybridization was carried out in sealed plastic bags for 6 h a t 65 “Cin a hybridization buffer (5 x SSC, 10 x Denhardt’s solution, 10 mM NaHPO,,pH6.5, 0.5% SDS, 50% formamide, 0.1 mg/ml sonicated herring sperm DNA). Hybridization was performed using hybridization buffer supplemented by 1000 ng of DIG-labeled RNA probe/ml of buffer at 65 “C for 16 h. The hybridized filters were then washed in 2 x SSC, 0.1% SDS once, and 0.2 x SSC, 0.1% SDS twicea t 65 “C. DIG-labeled nucleic acids were detected as follows (16, 17). All the steps were carried out at room temperature. Reagents were part of the Boehringer DIG DNAdetection kit. The filter was brieflyrinsed in DIG buffer 1(0.1M maleic acid, 0.15 M NaC1, pH 7.5) twice,and subsequently nonspecific binding sites were blockedby incubating for 60 minin DIG buffer 1containing 1.5%(w/v)Boehringer blocking reagent, a specially purified fraction of dried milk powder. Anti-DIG Fab fragment conjugated to alkaline phosphatase diluted 1:10,000 in DIG buffer 1 containing 0.2% Tween 20was applied for 30min. Excess antibody was washed off in DIG buffer 1containing 0.2% Tween20 for 20 min twice, and this buffer was then exchanged forDIG buffer 3 (0.1 M Tris-HC1,O.lM NaC1, 50 mM MgCl,, pH 9.5) for 3 min. The filter was washed with the assay buffer (100 mM diethanolamine, 2 m~ MgCl,, 0.02% NaN,) for 10 min twice and incubated with 3-(2’-spiroadamantane)-4-methoxy-4-(3”phosphoryloxy)-pheny1-1,2-dioxetanesolution (0.1 mg/ml in the assay buffer) in a plastic bag as previously described for 15 min. The filter was then transferred and sealed in a new plastic bag and exposed to Kodak X-Omat AR film at room temperature from 30 min to 2 h. For all of the Northern blots, rRNA (28 and 18 S) in gels was stained with ethidium bromide and photographed before transferring t o a nylon membrane to confirm the presence of equivalent amounts of RNA in each lane. The relative signal intensities of mRNA levels were quantitated by densitometric scanning using a dual wavelength flying spot scanner (CS-9000, Shimadzu, Tokyo, Japan). RESULTS Recombinant HB-EGF Stimulates Proliferation of Normal H u m a n Keratinocytes-First, we tested recombinant HB-EGF

for its ability to stimulate human keratinocyte growth in the absence of exogenous TGF-a and EGF under subconfluent conditions. HB-EGF stimulated keratinocyte growth in a dosedependent manner. Optimal stimulation occurred at 1.0 ng/ml (approximately a 1.8-fold increase in cell number) (Fig. lA). It showed a tendency to inhibit cell growth at higher concentrations. The effective doserange of HB-EGF for keratinocytes is almost the same as that of BalbIc-3T3 cells and bovine aortic smooth muscle cells (1).Since keratinocytes are at confluency i n vivo, we also studied the effect of HB-EGF on the growth of human keratinocytes under confluent conditions. DNA synthe-

HB-EGF Is an Autocrine Growth Factor for HumanKeratinocytes

20062 A.

*

*

I

6-

T

4-

28s +

2-

18S+

0 - L Lr 0

HB-EGF (ng/ml)

0.3

1 FIG.2. Expression of HB-EGF mRNA in normal human keratinocytes. Human keratinocytesa t subconfluency were harvested, and total RNA was extracted.RNA blot analysis was performed with 20 pg of RNAllane.

Production and Secretion of HB-EGF and Related Proteins by Human Keratinocytes-To examine the production and secretion of HB-EGF by human keratinocytes, we partially purified it from the conditioned medium of cultured human keratinocytes by heparin affinity column chromatography. An EP170.7 cell DNA synthesis assay wasused to detectmitogenic activity. Three mitogenic peaks, peaks 1, 2, and 3, were detected after HB-EGF (ng/rnl) fractionation by the heparincolumn, as shown in Fig. 3A, and FIG.1. Effect of HB-EGF onthe proliferation of human kera- subjected to Western blotting analyses. As shown in Fig. 3B, tinocytes.A, effect of HB-EGF on subconfluent keratinocytes. Human keratinocytes were plated a t a density of 1 x lo4 celldwell in 6-well the three peaks contained a total of four peptides that crossreacted with anti-HB-EGF antibody 2998. The first peak conplates. The next day, cells were refed with fresh medium containing various dosesof recombinant HB-EGF. After 4 days, cell numbers were tained two bands of 30 and 27 kDa. The second peak revealed countedusing a hemocytometer. B , effect of HB-EGF on confluent a 19-kDa band comigrating with highly purified HB-EGF from keratinocytes. DNA synthesis was examinedby measuring BrdUrd indemoncorporation a s described under “Experimental Procedures.” Recombi- U-937 cell-conditioned medium (8). The third peak nant HB-EGF was addedat the concentrationsof 0.1,0.5,1, 5, 10, and strated a lower molecular mass band of 14.5 kDa as well as a 20 ng/ml. Asterisks denote a significant difference ( p < 0.05) from the faint 19-kDa band. On the other hand, anti-amphiregulin ancorresponding control (no addition).p values were calculatedby a two- tibody detected an 18.5-kDa band in peaks2 and 3,which was sample t test. Results are representativeof three independent experidifferent from the 19-kDa band detected by 2998 antibody in ments. peaks 2 and 3 and which seemed to be identical to amphiregulin in human keratinocyte-conditionedmedium, as previously sis was examined by measuring bromodeoxyuridine incorpora- reported (7). A batch of conditioned medium was fractionated tion. In contrast to subconfluent keratinocytes, HB-EGF stimu- by heparin column chromatography and subjected to neutrallated cell growth in a dose-dependent manner up to20 ng/ml. izing studies. The chromatographic pattern was almost the Optimum stimulation wasobserved at 10 ng/ml(a.l-fold)(Fig. same as that shown in Fig. 3 A , and fractions 11, 17, and 22 represented peaks 1 , 2 a n d3, respectively (data not shown). 50 lB), at which the keratinocyte growth was inhibited under subconfluent conditions. These results indicate that the effect pg/ml anti-HB-EGF-blocking antibody 197, which is specific for of HB-EGF on human keratinocytegrowth is dependent on the HB-EGF and does not neutralize EGF, TGF-a, amphiregulin, cell density, and theeffective dose is higher inconfluent kera- or P-cellulin (Fig. 4A),neutralized theactivities of fractions 17 tinocytes. (peak 2) and 22 (peak 3) by 38 and 22%, respectively, whereas Expression of HB-EGF mRNA in Normal HumanKeratino- it did not affect the activity of fraction 11 (peak 1)a t all (Fig. cytes-Digoxigenin-labeled antisense HB-EGF-specific RNA 4B). Antibody 197 neutralized36% of the total activity pooled probe hybridized to a single 2.5-kilobase transcript of HB-EGF from fractions 9 to 25 (Fig. 4B).These results indicate that fractionated from the total RNA of normal human keratino- cultured humankeratinocytes secrete heparin-bindingEGF recytes (Fig. 2). The size of the transcript is consistent with thatceptor ligands, including HB-EGF and amphiregulin. in human macrophages (11, the U-937 cell line (11, rat aortic Growth Inhibition of Human Keratinocytes by Anti-HB-EGFsmooth muscle cells (5),and humanumbilical vein endothelial blocking Antibody-To characterize the autocrine nature of cells (3). This result of Northern blot analysis demonstrated HB-EGF in human keratinocyte culture,we examined the efthat the HB-EGF gene is expressed in cultured human kera- fect of anti-HB-EGF-blocking antibody 197 on human keratinocytes. tinocyte growth. The antibody 197 (45pg/ml) reduced the cell

HB-EGF Is an Autocrine Growth Factor for Human Keratinocytes

A

20063

A I

3

\ 3 Q

1

30

20

10

40

Fraction Number

B

Antibody (pg/mi)

- HB-EGF-

-AR-

kDa

B

kDa 30

3

1201

21.5

19-

14.3

E

1

2

3 1

2

3

W I

03 X

FIG.3. TSK-heparin column chromatographyof keratinocyteJ conditioned medium and Westernblotting analyses.A,heparini i i ”. 1 1 10 100 affinity column analysis. Keratinocyte-conditionedmedium (4 liters) Antibody (pg/mi) was concentrated by a Bio-Rex 70column and applied to a TSK-heparin column. Fractions were eluted with a 0.2-2 M NaCl gradient andexamFIG. 4. Neutralization of heparin-binding mitogens of kerained for their ability to stimulate DNAsynthesis in EP170.7 cells. These results were reproducible in keratinocyte-conditioned medium from tinocyte-conditionedmedium. A, specificity of anti-HB-EGF antifour different cultures. B , Western blot analysis. Fractions 10-12, 16- body 197. 1ng of recombinant human EGF (O), TGF-a (W), amphiregu18, and 20-23 were separately pooled and concentrated on heparin- lin (0).P-cellulin (A),or HB-EGF (0)and anappropriate concentration Sepharose minicolumns (50 pl). The concentrated samples were divided of antibody 197 were simultaneously added to a 96-well plate of and fractionated by SDS-polyacrylamide gel electrophoresis under re- EP170.7 cells. L3H1Thymidineincorporation was measured according to ducing conditions. Proteins were transferred to a nitrocellulose mem- the method described under “Experimental Procedures.” B,neutralizabrane and detected with rabbit antibodies to HB-EGF and amphiregu- tion of the mitogenic activities of the conditioned medium fractionated 22 . 1in.Lune 1,peak 1 inA;lane2,peak2inA;lane3,peak3inA;HB-EGF,by heparin column chromatography. 10 pl of fraction 11 (A), 17 (0) 50 ng of highly purified U-937 cell-conditioned medium-derived HB- (O),or pooled fractions 9-25 (A)or 1ng of recombinant human HB-EGF EGF. The left panel was detected by anti-HB-EGF antibody 2998 (HB- ( 0 )and an appropriate concentration of antibody 197 were simultaneously added to a 96-well plate of EP170.7 cells. c3H1Thymidine incorEGF) and the right panel by anti-amphiregulin antibody ( A R ) . poration was measured.

growth to 66% compared with the pooled normal goat IgG from four individuals at thesame concentration after a 4-day incubation period (Fig. 5). The cells cultured in the presence or absence of the normal goat IgG (45 pg/ml) showed almost similar growth. This result also indicates the autocrine nature of HB-EGF in human keratinocytes. Autoinduction of HB-EGF mRNAinHumanKeratinocytes-As TGF-a induces production of its own mRNA in an autocrine manner (6),we examined whether the addition of exogenous HB-EGF to culture medium could enhance the level of HB-EGF mRNA in keratinocytes. Since HB-EGF significantly stimulated the keratinocyte growth a t 1 ng/ml under both subconfluent and confluent conditions as shown above, we tested whetherthis concentration of HB-EGF induces HB-EGF mRNA. Cells were harvested a t 0, 1,3,6,12, and24 h after the addition of HB-EGF. HB-EGF a t 1 ng/ml optimally increased the level of HB-EGF mRNA 5.4-fold a t 1h (Fig. 6A). Furthermore, the sameconcentration of HB-EGF enhanced the level of TGF-a mRNA 3.1-fold a t 3 h (Fig. 6B). Since TGF-a and EGF are structurallyrelated to HB-EGF and share thesame recep-

tor, we tested TGF-a and EGF for their ability to enhance the level of HB-EGF mRNA expression. 1ng/ml TGF-a was added, at which concentration TGF-a stimulates the growth of human keratinocytes (18).TGF-a optimally increased the level of HBEGF mRNA 10.2-fold at 6 h (Fig. 7A), and EGF (1ng/ml) also increased the HB-EGF mRNA level 10.9-fold at 1h (Fig. 7B). The finding that 1 ng/mlHB-EGF not only stimulated the growth of human keratinocytes but also induced its mRNA expression under both subconfluent and confluent conditions demonstrated the autocrine loop of HB-EGF productionin human keratinocyte culture. Furthermore, HB-EGF and TGF-a mutually increased their respective mRNA expressions. This indicates that HB-EGF and TGF-a interact with each other by a mutual amplification mechanism as well as by autocrine induction. Interestingly, HB-EGF and EGF optimally enhanced HB-EGF mRNA as early as at1h. In contrast, TGF-a optimally induced HB-EGF mRNA a t 6 h. Enhancement of TGF-a Production by HB-EGF-The addition of HB-EGF increased the amount of TGF-a mRNA in cul-

HB-EGF Is a n Autocrine Growth Factor for Human Keratinocytes

20064 125

0

A

1

We-

3

6 12 24 (hr) ,

~

,

T

100

28s + 75

18s-

50

B 25

28s "*

0

18s-

FIG.7.Time course of HB-EGF mRNAinduction by TGF-a and FIG.5. Growth inhibition of human keratinocytes by anti-HB- EGF. Confluent keratinocytes were incubated with1 ng/ml TGF-a (A) EGF-blocking antibody.Human keratinocytes were plateda t a den- and 1 ng/ml EGF (B). Total RNA was extracted a t t h eindicated times. sity of 1 x 10' cells/well in 6-well plates. The next day, cells were refed Time point0 h represents the control. RNA blot analysis wasperformed with fresh medium containing anti-HB-EGF antibody IgG (45 pg/ml) or with 10 pg of RNNlane. After electrophoresis, the RNA was transferred pooled normal goat(NG) I& (45 pg/ml). The cells were also cultured into nylon filters, which were hybridized DIG-labeled to human HB-EGF the absence of IgG. After 4 days, cell numbers were counted using a probe. hemocytometer. The cell growth was expressed a s a percentage compared with control culture. The cell number of control culture was 36.2 x 10". Asterisk denotes a significant difference ( p < 0.05) from the corresponding controls (no IgG or normal goat I&). p values were calculated by a two-sample t test. The experiment was performed in duplicate. A

0

1

3

6 12 24 (hr)

n

18S+

so

100

1sn

200

TGF-a (ng/ml)

FIG.8. TGF-a protein production induced byHB-EGF and EGF. Confluent keratinocytes were cultured in fresh medium containB

0

1

3

6 12 24 (hr)

28s "* 18s-

ing 20 ng/ml HB-EGF or 10 ng/ml EGF, and conditioned medium was harvested after 24 h. Conditioned medium was concentrated by SepPak C,, and eluted with acetonitrile. After lyophilization andsolubilization, the amount of TGF-a was measured by enzyme-linked immunosorbent assay. Asterisks denote a significant difference ( p < 0.001) from the corresponding control. p values were calculated by a twosample t-test.

HB-EGF increased TGF-a protein production 4.6-fold, while EGF enhanced it to aneven greater extent (Fig. 8). DISCUSSION

Human keratinocyte growth is under coordinated regulation

FIG. 6. Time course of HB-EGFmRNA and TGF-a mRNA induc- by positive and negative mediators. As positive growth mediation by HB-EGF. Confluent keratinocytes were incubated with 1ng/ml tors, EGF, TGF-a, amphiregulin, fibroblast growth factors,and HB-EGF, and total RNA was extracted at the indicated times. Time point 0 h represents the control. RNA blot analysis was performed with interleukin-6have beenreported (7, 19-22). Among these, 10 pg of RNNlane.Each lane containedapproximatelythesame members of the EGF family are thought tobe central because amount of total RNAas ascertained by ethidium bromide staining. After anti-EGF receptor antibody abrogates thegrowth of keratinoelectrophoresis, the RNA was transferred to nylon filters, which were hybridized to DIG-labeled human HB-EGF and TGF-aprobes. A, HB- cytes (23). Although the growth factor-independent proliferation of human keratinocytes was reported, now it isunderstood EGF mRNAB,TGF-a mRNA.

tured keratinocytes as described above. EGF has the abilityto enhance TGF-a protein production in cultured keratinocytes (6). Therefore, we examined the effect of HB-EGF on TGF-a protein production. Human keratinocytes were incubated in the presence of 20 ng/ml HB-EGF or 10 ng/ml EGF. Conditioned medium was harvested after a 24-h incubation period.

as being endogenous growth factor-dependent growth induced by TGF-a and amphiregulin, members of the EGF family (7, 24). In other words, TGF-a and amphiregulin regulate human keratinocyte growth in an autocrine manner (6, 7). This is the phenomenon by which a cell produces and secretes a growth factor(s) that, in turn, stimulates/inhibits the proliferation of that same cell (25). We demonstrated in this study that HB-

HB-EGF Is an Autocrine Growth Factor for

Human Keratinocytes

20065

heparin column with about 1.0 M NaCl (peak 2) and comigrated with highly purified HB-EGF U-937 cell-conditioned medium. This form of HB-EGF, consisting of 86 aminoacids, is generally considered the dominantform (1).Anti-HB-EGF antibody 2998 cross-reacted with three other bands with30- and 27-kDa molecular masses in peak l and a 14.5-kDa molecular mass in peak 3. Based on the results that anti-HB-EGF-blocking antibody 197 did not neutralize the activity of peak 1, the immunoreactive 30- and 27-kDa bands in peak 1would be different from HB-EGF. I t is also unknown whether the immunoreactive 30- and 27-kDa bands would be mitogens for EP170.7 cells. However, the possibility cannot be excluded at the present time that they maybe new EGF-like growth factorscross-reactive to KERATINOCYTE the anti-HB-EGF antibody 2998. The lower molecular mass FIG.9. Schematic representation of autocrine stimulation in keratinocytesby TGF-a, amphiregulin, andHB-EGF. Exposure of form of HB-EGF has been found in wound fluid and possibly keratinocytes to EGF, TGF-a, amphiregulin, andHB-EGF results in the generated by deglycosylation or proteolytic degradation (34). binding of these growth factors to EGF receptors and stimulation of the Partial neutralizationof the activity in peak3 also supports the same cell growth, which is followed by an increase in theproduction of possible explanation that the 14.5-kDa band might be a deglyTGF-a, amphiregulin, andHB-EGF by the same cells. Secreted TGF-a, cosylated or proteolytic product of HB-EGF. amphiregulin, and HB-EGF bind to EGF receptors and furtherenhance Anti-HB-EGF-blockingantibody 197neutralized at least cell growth and their own synthesis. 36% of the total activity obtained from heparin column chrofactor for EGF is also an autocrine growth factor for human keratino- matography.Since TGF-a is an autocrine growth cytes because it is both produced by them and stimulates their human keratinocytes and hasno affinity for heparin, theratio growth. Further evidence for the autocrine nature of HB-EGF of HB-EGF in EGF receptor ligands found in the conditioned in human keratinocyte culture was provided by the ability of medium is estimated to be under 36%. However, the blocking antibody 197 reduced the cell growth by 37% for a 4-day incuanti-HB-EGF-blockingantibody to reduce theirgrowth,although the inhibition was less than half. This indicates that bation period, suggesting that not only secreted HB-EGF but HB-EGF may be one of the crucial factors for the autocrine also membrane-anchored HB-EGF might be involved in thecell growth of human keratinocytes. It is of great interest that growth control. Namely, a jwtacrine loop of HB-EGF might HB-EGF and TGF-a act notonly by autoinduction but alsoby work in keratinocyte growthas well as the autocrine loop. HB-EGF has been found in wound fluid (34). Hence, our a mutual amplification mechanism, asshown in this study. We possibility thatkeratinocytesare one expect that amphiregulin would also be involved in this mecha- findingsuggeststhe nism. Fig. 9 shows a schematic model of how HB-EGF, TGF-a, source of HB-EGF in wound fluid. and amphiregulin might regulate human keratinocyte growth The present study demonstrates new aspects of the role of HB-EGF in the regulation of epidermal growth. This study, and by an autoinduction and a mutual amplification mechanism. The reason keratinocytesneed three EGFfamily growth fac- others, will lead to clarification of the complex but coordinated tors remained unclarified. The presence of TGF-a and HB- mechanism of keratinocyte proliferation and differentiation by EGF2 in normal epidermis was observed (4, 26). However, am- autocrine growth factors. phiregulin mRNA was not detected in normal human Acknowledgments-We thank Dr. Judith A. Abraham and Brett Garepidermis, although it markedly increased in epidermis of psorick (Scios Nova Inc.) for assistance with the neutralizing antibody and riasis, a hyperproliferative skin disease (27). Correlation of Dr. Peter Royce (Terumo Corporation Research and Development CenTGF-a to keratinocyte differentiation was reported (28, 29). ter) for critical review of the manuscript. We are indebted to Sumi These observations may suggest possibility the that theywould Hayakawa for excellent technical assistance. be involved in different stagesof proliferation and differentiaREFERENCES tion in human keratinocytes. 1. Higashiyama, S., Abraham, J. A., Miller, J., Fiddes, J. C., and Klagsbrun, M. In addition to its distinct character as an autocrine growth (1991) Science 261,936-939 2. Thompson, 5. A,, Higashiyama, S., Wood, K., Pollitt, N. S., Damm, D., McEnfactor forkeratinocytes, we would like to emphasizea different roe, G., Garrick, B., Ashton, N., Lau, K., Hancock, N., Klagsbrun, M., and aspect of HB-EGF, namely a possible role as a paracrine growth Abraham, J. A. (1994) J . B i d . Chem. 269, 2541-2549 factor for fibroblasts. Epithelia-mesenchyme interaction plays 3. Yoshizumi, M., Kourembanas, S., Temizer, D. H., Cambria, R. P., Quertermous, T., and Lee, M. E. (1992) J. Biol. Chem. 267,9467-9469 an important role in the regulation of both keratinocyte and 4. Dluz, S., Higashiyama, S., Damm, D., Abraham, J. A., and Klagsbrun, M. fibroblast proliferation under normal and pathological condi(1993) J. Biol. Chem. 268, 18330-18334 5. Temizer, D. H., Yoshizumi, M., Perrella, M. A,, Susanni,E. E., Quertermous, T., tions. Wound healing isone of the situations in which epitheliaand Lee, M. E. (1992) J. Biol. Chem. 267,24892-24896 dermis interaction is involved. Recently, it has been demon6. Coffey, R., Jr., Derynck, R., Wilcox, J. N., Bringman, T. S., Goustin, A. S., by keratinocytes strated that proteins synthesized and secreted Moses, H. L., and Pittelkow, M. R. (1987) Nature 326,817420 in vivo may traverse the basement membrane into the dermis 7. Cook, P.W., Mattox, P.A., Keeble, W.W., Pittelkow, M. R., Plowman, G. D., Shoyab, M., Adelman, J. P., and Shipley, G. D. (1991) Mol. Cell. Biol. 11, where they may ultimately be systemically detected (30).Three 2547-2557 (31), 8. Higashiyama, S., Lau, K., Besner, G. E., Abraham, J. A,, and Klagsbrun, M. factors that areproduced by human keratinocytes, TGF-a amphiregulin (321, and interleukin-1 (33), have already been 9. (1992) J. B i d . Chem. 267,6205-6212 Matsumoto, K., Hashimoto, K., Nishida, Y., Hashiro, M., and Yoshikawa, K. As shown t o act as growth factorsfor human dermal fibroblasts. (1990) Biochem. Biophys. Res. Commun. 166,916-923 10. Wille, J., Jr., Pittelkow, M. R., Shipley, G. D., and Scott, R. E. (1984) J. Cell. HB-EGF is mitogenic for fibroblasts (l), the present study inPhysiol. 121, 31-44 dicates that it may also be a fourth paracrine growthfactor for 11. Pierce, J. H., Ruggiero, M., Fleming, T.P., Di-Fiore, P. P., Greenberger, J. S., dermal fibroblasts. Varticovski, L., Schlessinger, J., Rovera, G., and Aaronson, S.A. (1988) The present study showed that human keratinocytes synthe- 12. Science 239, 628-631 Higashiyama, M., Matsumoto, K., Hashimoto, K., andyoshikawa, K. (1991) J . size the 19-kDa form of HB-EGF, which was eluted from a Dermatol. (Tokyo) 18, 117-119 S.,Ito, A., Morii, E., Wanaka, A., 'lbhyama, M., Kitamura, Y., and Nomura, S. (1992) Mol. Brain Res. 16, 47-54 14. Chomczynski, P., and Sacchi, N. (1987)AnaL Biochem. 162, 15g159 15. Sambrook, J., Fritsch, E. F.,and Maniatis, T. (1989) Molecular Cloning: A 13. Hirota,

M. Higashiyama, K. Hashimoto, and S. Higashiyama, unpublished observations.

20066 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

HB-EGF Is an Autocrine Growth Factor for Human Keratinocytes

Laboratory Manual,2nd Ed., pp. 7.3-7.52, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY During, K. (1991) Anal. Biochem. 196, 433-438 Lanzillo, J. J. (1991) Anal. Biochem. 194, 45-53 Pittelkow, M. R., Lindquist, P. B., Abraham, R. T., Graves-Deal, R., Derynck, R., and Coffey, R., Jr. (1989) J. B i d . Chem. 264, 5164-5171 Barrandon, Y., and Green, H. (1987) Cell 50, 1131-1137 Elder, J. T.,Sartor, C. I., Boman, D.K., Benrazavi, S., Fisher, G. J., and Pittelkow, M.R. (1992) Arch. Dermatol. Res. 284,324-332 OKeefe, E. J., Chiu, M. L., and Payne, R., Jr. (1988) J. Inuest. Dermatol. 90, 767-769 Shipley, G. D., and Pittelkow, M. R. (1987) Arch. Dermatol. 123,1541a-1544a Hashimoto, IC, and Yoshikawa, K. (1992)J. Dermatol. (Tokyo) 19,648-651 Cook, P.W., Pittelkow, M. R., and Shipley, G . D. (1991) J . Cell. Physiol. 146, 277-289 Sporn, M. B., and Todaro, G. J. (1980) N. Engl. J . Med. 303,878480 Elder, J. T., Fisher, G. J., Lindquist, P. B., Bennett, G. L., Pittelkow, M. R.,

27. 28. 29. 30. 31. 32. 33. 34.

Coffey, R., Jr., Ellingsworth, L., Derynck, R., and Voorhees, J. J. (1989) Science 243,811-814 Cook, P. W., Pittelkow, M. R., Keeble, W. W., Graves-Deal, R., Coffey, R., Jr., and Shipley, G. D. (1992) Cancer Res. 52, 3224-3227 Finzi, E., Harkins, R., and Horn, T. (1991) J . Inuest. Dermatol. 96, 328-332 Finzi, E., Ho, T.,Anhalt, G., Hawkins, W., Harkins, R., and Horn,T. (1992)Am. J . Pathol. 141, 643-653 Fenjves, E. S., Gurdon, D. A,, Pershing, L. K., Williams, D. L., and Taichman, L. B. (1989)Proc. Natl. Acad. Sci. U.S. A. 86,88034807 Pittelkow, M. R. (1992) Adu. Dermatol. 7, 55-81 Shoyab, M., McDonald, V. L., Bradley, J. G., and Todaro, G. J. (1988)Proc. Natl. Acad Sci. U.S. A. 85,6528-6532 Blanton, R. A,, Kuppe,T. S., MeDougall, J. K., and Dower, S. (1989)Proc. Natl. Acad. Sci. U. S. A. 86, 1273-1277 Marikovsky, M., Breuing, K., Liu, P. Y., Eriksson, E., Higashiyama, S., Farber, P. A,, Abraham, J. A., and Klagsbrun, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,38893893