Expression of Insulin-Like Growth FactorBinding Protein-3 (IGFBP-3) in Human

Keratinocytes Is Regulated byEGF and TGFb1

STEPHANIE R. EDMONDSON,1 MARI M. MURASHITA,2 VINCENZO C. RUSSO,1

CHRISTOPHER J. WRAIGHT,1 AND GEORGE A. WERTHER1*1Centre for Hormone Research, Royal Children’s Hospital, Victoria, Australia

2Department of Paediatrics, Hokkaido University School of Medicine, Sapporo, Japan

Insulin-like growth factor-I (IGF-I) is essential for normal epidermal homeostasis;however, the role of IGF binding proteins (IGFBPs), regulators of IGF action,remains unclear. Here we examine the regulation of human keratinocyte-pro-duced IGFBPs by epidermal growth factor (EGF), transforming growth factor beta1 (TGFb1), and IGF-I, growth factors known to be active in skin. In the absenceof added growth factors, IGFBP-3 was the major binding protein secreted into themedium by primary keratinocytes. Addition of EGF or TGFb1 to keratinocytecultures resulted in a significant decrease in IGFBP-3 abundance in conditionedmedium when compared with control, untreated cells. Specifically, EGF (50ng/ml) and TGFb1 (50 ng/ml) reduced IGFBP-3 abundance to 15 6 6% and 22 69%, respectively. Using Northern blot analysis, we found EGF and TGFb1 (50ng/ml) to reduce IGFBP-3 mRNA levels in keratinocytes to 51 6 12% and 50 638%, respectively, when compared with control, untreated cells. Treatment withIGF-I or its analogue des(1-3)IGF-I did not lead to any consistent change inIGFBP-3 abundance. However, both IGF-I and des(1-3)IGF-I at 100 ng/ml led toa modest increase in IGFBP-3 mRNA levels in keratinocytes, suggesting posttran-scriptional regulation of IGFBP-3 abundance. We propose that local modulationof IGFBP-3 abundance may represent another level of regulation of growth factoraction in the epidermis, where EGF and TGFb1 and possibly other local growthfactors specifically regulate the availability of IGF-I to its keratinocytereceptors. J. Cell. Physiol. 179:201–207, 1999. © 1999 Wiley-Liss, Inc.

Insulin-like growth factor-I (IGF-I) action on kera-tinocytes appears to be essential for the development ofa normal epidermis since transgenic mice lacking theIGF-I receptor have abnormally thin, translucent epi-dermis with no spinous layer (Liu et al., 1993). IGF-I inthe skin is produced by dermal fibroblasts and epider-mal melanocytes (Tavakkol et al., 1992), and dermallyderived IGF-I has been shown to stimulate keratino-cytes in vitro (Barreca et al., 1992). Keratinocytes donot synthesize IGF-I but are highly responsive to IGF-Ithrough their IGF-I receptors, which are most abun-dant in the basal layer (Krane et al., 1992). As withmost tissues studied so far, IGF-I action in the epider-mis occurs in the presence of IGF-I binding proteins(IGFBPs), whose roles remain unclear.

IGFBPs, of which at least six have been cloned andcharacterized in many cell systems, serve as carrierproteins for IGFs and thus modulate their bioactivity(Jones and Clemmons, 1995). Although IGFBPs sharesequence and structural homology, they each possessunique characteristics, such as the capacity to associ-ate with components of the extracellular matrix, whichcan affect the molecular mechanisms by which they

augment or inhibit IGF action (Jones and Clemmons,1995). Recent studies (Oh et al., 1993a,b; Mohseni-Zadeh and Binoux, 1997) suggest IGFBPs may alsohave biological activity which is independent of IGFs.Thus, IGFBPs add a further level of complexity in thebiological action of IGFs. We previously demonstratedthat in the absence of added growth factors IGFBP-3 isthe major IGFBP produced by cultured human kera-tinocytes, that IGFBP-3 is produced by basal keratin-ocytes in vivo, and that it is the major IGFBP detectedin the epidermis (Batch et al., 1994; Murashita et al.,1995; Wraight et al., 1997). Our analysis of an immor-talized basal keratinocyte cell line (HaCaT) revealedthat the mitogenic effects of IGF-I are blunted by IG-FBP synthesis (Wraight et al., 1994). IGFBP expres-

Contract grant sponsor: National Health and Medical ResearchCouncil of Australia.

*Correspondence to: George A Werther, Centre for Hormone Re-search, Royal Children’s Hospital, Flemington Road, Parkville, Vic-toria 3052, Australia. E-mail: werther@cryptic.rch.unimelb.edu.au

Received 24 June 1998; Accepted 16 November 1998

JOURNAL OF CELLULAR PHYSIOLOGY 179:201–207 (1999)

© 1999 WILEY-LISS, INC.

sion is highly regulated by growth factors in manytissues (Jones and Clemmons, 1995). In cultured der-mal fibroblasts, locally produced growth factors such asepidermal growth factor (EGF) and transforminggrowth factor beta 1 (TGFb1) regulate IGFBP produc-tion and specifically alter the IGF-I response (Yatemanet al., 1993).

In the present study, we have investigated whethergrowth factors regulate the expression of IGFBP-3 bycultured human primary keratinocytes and therebypotentially influence IGF-I action in the epidermis. Weshow that while IGF-I and its analogue des(1-3)IGF-I,which has reduced affinity for IGFBPs, do not signifi-cantly regulate IGFBP-3 production by keratinocytesin culture, two other important skin growth factors,EGF and TGFb1, significantly decrease secreted IG-FBP-3 abundance.

MATERIALS AND METHODSMaterials

Keratinocyte serum-free medium (SFM), bovine pi-tuitary extract (BPE), recombinant epidermal growthfactor (rEGF), 13Hank’s balanced salt solution(HBSS), Gentamicin (10 mg/ml), 0.25% Trypsin–1 mMEDTA were all obtained from GIBCO (Grand Island,NY). Keratinocyte basal medium (KBM) withoutgrowth factors and calcium were purchased from Clo-netics (San Diego, CA). Dispase was obtained fromCollaborative Biomedical Products (Bedford, MA).Phosphate buffered saline (PBS) (calcium- and magne-sium-free) and fetal calf serum (FCS) were from TraceAmerica (Miami, FL). Falcon 3024 plastic tissue cul-ture flasks were obtained from Becton Dickinson (Lin-coln Park, NJ). Nitrocellulose filters (0.45 mM) wereobtained from Schleicher and Schuell (Dassel, Germany).X-ray films (X-Omat AR) were purchased from Kodak(Rochester, NY). RIA grade bovine serum albumin(BSA) was purchased from Sigma Chemical Co (St.Louis, MO). 125I-IGF-I (2,000 Ci/mmol), 14C proteinmolecular weight (14–22 kDa) markers, 32P-dCTP(3,000 Ci/mmol), 32P-ATP (3,000 Ci/mmol), and Hy-bond-N nylon membranes were purchased from Amer-sham (Buckinghamshire, UK). 125I-IGF-II (1,500 Ci/mmol) was a generous gift from Dr. L.A. Bach(Department of Medicine, University of Melbourne,Austin, and Repatriation Medical Centre, Heidelberg,Australia). The cDNA for human IGFBP-3 was kindlyprovided by Dr. S. Shimasaki (Whittier Institute, LaJolla, CA [Shimasaki and Ling, 1991]). Prepubertal ratkidney (PPRK) total RNA was a gift from Dr. S. Ymer(Centre for Hormone Research, Royal Children’s Hos-pital, Melbourne, Australia). Human placental totalRNA was a gift from Ms. Katy Freed (Perinatal Unit,Royal Women’s Hospital, Melbourne, Australia). Re-combinant human IGF-I was a gift from Dr. A Skottner(KabiPharmacia, Peptide Hormones, Uppsala, Swe-den). Recombinant human EGF was purchased fromAmersham (Sydney, Australia). The cDNA randompriming kit was purchased from Boehringer Mannheim(Sydney, Australia). TGFb1 and des(1-3)IGF-I weregifts from Dr. C. Williams (Auckland University, NewZealand).

Keratinocyte cultureHuman prepubertal foreskins were obtained from

routine circumcision and placed into Gibco’s Keratino-cyte serum-free medium (SFM) containing Gentamicin(5 mg/ml). After washing with 70% ethanol, foreskinswere rinsed with PBS, and tissues were cut into smallpieces (approximately 3 3 3 mm). Tissues were thenimmersed in HBSS containing dispase (25.0 caseino-lytic U/ml)/Gentamicin (5 mg/ml) and incubated for18 h at 4°C. The epidermal layer was then lifted fromthe dermis and digested for 15–20 min at 37°C in 2 mlof 0.025% Trypsin–1 mM EDTA. Following incubation,0.5 ml of FCS was added, and the cell suspension wasfiltered though gauze. Matrix-free cells were spun at1,000 rpm for 10 min at room temperature. The kera-tinocyte cell pellet was resuspended in complete kera-tinocyte medium containing 50 mg/ml BPE and 5 ng/mlEGF, counted by haemocytometer, and seeded into 75cm2 flasks at a cell density 4 3 104 cells/cm2. Mediumwas changed every 3 days, and cells were passagedwhen 70–90% confluent.

For regulation studies, cells (n 5 4; at passage 3)were seeded into 2 cm2 wells and fed every 3 days withkeratinocyte complete medium. At 1 day postconflu-ence, cells were washed three times with PBS, and themedium was changed to keratinocyte basal medium(KBM) containing 0.1 mM CaCl2. After 48 h, KBM wasreplaced with KBM/0.02% BSA containing the appro-priate dilution of either IGF-I (0-100 ng/ml), des (1-3)IGF-I (0-100 ng/ml), EGF (0-50 ng/ml), or TGFb1 (0-50ng/ml) (triplicate wells). After 24 h, the medium, withor without growth factor, was replaced with identicalmedium, and at 48 h the conditioned medium (CM) wascollected for analysis of IGFBPs by Western ligandblot. Cells from each well were trypsinized and countedin a Coulter Industrial C Counter (Coulter, Bedford-shire, UK).

Western ligand blottingKeratinocyte CM (250 ml) was concentrated, and

samples were electrophoresed as previously described(Murashita et al, 1995) and according to the method ofLaemmli (1970). CM from cells cultured in growth fac-tor–free KBM/0.02% BSA were used as controls, and14C molecular weight markers and human serum (5 ml)were used as molecular weight markers. Separatedproteins were then electrophoretically transferred tonitrocellulose filters which were stained with Ponceaured to verify even loading and transfer of samples.Inspection of the filters indicated that the growth fac-tor treatments had no effect on overall protein abun-dance and profile in keratinocyte CM (data not shown).Filters were probed with equal proportions (1 3 106

cpm) of 125I-IGF-I/125I-IGF-II, as described by Hossen-lopp et al. (1986) and were then exposed to x-ray filmfor 3–10 days. X-ray autoradiographs of Western li-gand blots (WLBs) were densitometrically scanned andanalyzed using a Bio-Rad, GS-670 imaging densitome-ter (Bio-Rad, North Ryde, Australia) and MolecularAnalyst software (Bio-Rad). For statistical analysis,triplicate IGFBP-3 band densities were corrected forcell number, averaged, and then compared to the aver-age band density of IGFBP-3 in the control, untreatedsample. Combined data was expressed as a percentage

202 EDMONDSON ET AL.

of the control, untreated sample and statistically ana-lyzed by one-way ANOVA using a Dunnett’s MultipleComparison post test (Prism Graph Pad Software, SanDiego, CA).

RNA isolation and Northern analysisKeratinocytes (in 90 mm petri dishes) were grown to

1 day postconfluence and washed three times withPBS, and the medium was then changed to KBM/0.1mM CaCl2. After 48 h, KBM was replaced with KBM/0.02% BSA containing either no growth factors, IGF-I(100 ng/ml), des(1-3)IGF-I (100 ng/ml), EGF (50 ng/ml),or TGFb1 (50 ng/ml). After 24 h, total RNA was iso-lated using the guanidium-thiocyanate method (Chom-czynski and Sacchi, 1987) and quantified spectropho-tometrically at 260 nm. RNA integrity was confirmedby 1% agarose/TAE electrophoresis. Total RNA wasthen denatured in 1 M glyoxal, 54% DMSO, 10 mMsodium phosphate buffer (pH 7.0) at 50°C for 1 h,fractionated on a 1% agarose gel with recirculatingbuffer (10 mM sodium phosphate, pH 7.0), transferredto Hybond-N membrane in 203 SSC (3 M NaCl, 0.3 Mtrisodium citrate dihydrate, pH 7.0), and UV cross-linked (2.5 J/cm2). A cDNA for human IGFBP-3 waslabelled with 32P-dCTP using a random priming kit toan average specific activity of 1 3 109 dpm/mg. Mem-branes were prehybridized at 42°C (5 h) and hybridizedwith labelled cDNA separately at 48°C overnight in50% (v/v) formamide, 0.05% (w/v) sodium pyrophos-phate, 2.53 SSC, 53 Denhardt’s solution, 25 mM so-dium phosphate, 0.5% (w/v) SDS, and 100 mg/ml boiledherring sperm DNA. Membranes were then washedtwice in 23 SSC, 0.1% SDS for 30 min at room tem-perature and once in 0.13 SSC, 0.1% SDS for 1 h at50°C. Membranes were then exposed to X-ray film for3–12 days. X-ray autoradiographs were densitometri-cally scanned and analyzed using a Bio-Rad GS-670imaging densitometer and Molecular Analyst software.Corrections for loading of total RNA were based oncomparisons with the intensity of the corresponding18S rRNA band in each lane. Data (duplicates) fromthree (TGFb1, EGF) or four (IGF-I, des(1-3)IGF-I) sep-arate cell cultures were expressed as IGFBP-3 mRNAband intensity from cells treated with specific growthfactors as a percentage of the band intensity for control,untreated cells. Data were then statistically analyzedusing an unpaired Student’s t-test (Prism Graph PadSoftware).

RESULTSIGFBP-3 abundance is reduced

by EGF and TGFb1In this study, we have investigated the effect of EGF,

TGFb1, IGF-I, or des(1-3)IGF-I on the appearance ofIGFBPs in medium conditioned by confluent humanprimary keratinocytes. Des(1-3)IGF-I, an analogue ofIGF-I which has minimal affinity for the IGFBPs butthe same affinity for the IGF-I receptor (Ballard et al.,1989), was used in order to determine if any potentialeffects of IGF-I on IGFBP abundance in conditionedmedium required interaction with IGFBPs.

In the primary cell lines used in this study (n 5 4),IGFBP-3 was the only consistently detected and regu-lated IGFBP found in keratinocyte-conditioned me-dium. In some WLBs of keratinocyte-conditioned me-

dia, extremely low levels of a 24 kDa binding protein,previously identified by us as IGFBP-4 (Murashita etal., 1995), were also detected. When present, IGFBP-4levels were not significantly affected by the addition ofgrowth factors. Figures 1a, 2a, and 3a,c are represen-tative autoradiographs of WLBs of keratinocyte-condi-tioned medium from one primary keratinocyte cell linegrown in the presence of specific growth factors. Fig-ures 1b, 2b, and 3b,d are graphical representations ofcombined densitometric analyses of IGFBP-3 bands inWLBs from the cell cultures used in this study. IG-FBP-3 abundance (mean 6 SEM) is expressed as apercentage of the control, untreated cells.

As shown in Figure 1, EGF had an inhibitory effecton IGFBP-3 (Fig. 1a), decreasing the IGFBP-3 level inconditioned medium to 34 6 12% and 15 6 6% of thatfrom control, untreated cells at 5 ng/ml (P , 0.05) and50 ng/ml (P , 0.01), respectively (Fig. 1b).

As shown in Figure 2, TGFb1 also had an inhibitoryeffect on IGFBP-3 (Fig. 2a), decreasing the IGFBP-3levels in conditioned medium to 55 6 29%, 21 6 7%,and 22 6 9% of that from control, untreated cells at 0.5ng/ml (P , 0.05), 5 ng/ml (P , 0.01), and 50 ng/ml (P ,0.01), respectively (Fig. 2b).

Addition of IGF-I or des(1-3)IGF-I to basal mediumdid not lead to consistent change in IGFBP-3 levels inconditioned medium when compared to IGFBP-3 levelsin control, untreated conditioned medium (Fig. 3a–d).For example, Figure 3a shows a WLB in which thereappears to be a slight decrease in IGFBP-3 abundancein the conditioned medium from one primary keratin-ocyte cell line when cultured in the presence of highconcentrations of IGF-I (100 ng/ml). Nonetheless, thecombined densitometric analysis of WLBs of condi-tioned medium from all cell line studies, when cor-rected for cell numbers, reveals that the overall effectof IGF-I on IGFBP-3 levels was negligible (Fig. 3b).Similar results were obtained when the effects of des(1-3)IGF-I on IGFBP-3 abundance in keratinocyte condi-tioned medium were assessed (Fig. 3c,d).

IGFBP-3 mRNA abundance is reducedby EGF and TGFb1

To determine whether the observed regulation ofIGFBP-3 levels in keratinocyte-conditioned mediumcorrelated with regulation of IGFBP-3 mRNA levels,we treated keratinocytes with growth factors at maxi-mal concentration; then total RNA was analyzed byNorthern blotting.

Fig. 4a is a Northern blot autoradiograph of 30 mg oftotal RNA from one primary keratinocyte cell line andshows that the predicted 2.6 kb IGFBP-3 mRNA band(Spratt et al., 1990) was clearly identified in untreatedkeratinocytes (Fig. 4a, control lane). Figure 4b is agraphical representation of the combined densitomet-ric analysis of IGFBP-3 mRNA bands in Northern blotsfrom all cell cultures used in this study. In order tocorrect for variation in total RNA loading, we standard-ized IGFBP-3 mRNA band intensities against 18SrRNA bands from the same sample. IGFBP-3 mRNAlevels are expressed as a percentage of the control,untreated cells. As shown in Figure 4a,b both EGF andTGFb1 caused a significant reduction in IGFBP-3mRNA (P , 0.05) when compared to control, untreatedkeratinocytes. Specifically, EGF (50 ng/ml) reduced IG-

203EGF AND TGFb REGULATE KERATINOCYTE IGFBP-3

FBP-3 mRNA levels to 51 6 12%, and TGFb1 (50ng/ml) reduced IGFBP-3 mRNA levels to 50 6 38% ofcontrol, untreated keratinocytes. Treatment withIGF-I or des(1-3)IGF-I (100 ng/ml) resulted in a mod-est, variable increase in IGFBP-3 mRNA abundance(P 5 0.054 and P 5 0.045, respectively) (Fig. 4b).

DISCUSSIONIn this study, we have assessed the regulation of

IGFBP-3, the major IGF binding protein produced bycultured human primary keratinocytes under basalconditions, by three growth factors known to be impor-tant in epidermal growth and skin homeostasis: EGF,TGFb1, and IGF-I. We previously showed that, in theabsence of any added growth factor, cultured humankeratinocytes secrete IGFBP-3 in abundance (Mu-rashita et al., 1995). Furthermore, our in situ hybrid-ization and immunohistochemical studies revealedthat IGFBP-3 is produced by basal keratinocytes andappears to be the predominant species in the epidermis(Batch et al., 1994; Wraight et al., 1997); thus, theproduction of IGFBP-3 by cultured human keratino-cytes is likely to be physiologically relevant.

In the present study, EGF had a potent inhibitoryeffect on the production of IGFBP-3 by cultured human

primary keratinocytes. In fact, IGFBP-3 levels werereduced to almost undetectable by EGF, an observationconsistent with the fact that IGFBP-3 is difficult todetect in conditioned media when cells are grown inmedium containing EGF (Murashita et al., 1995). Thisinhibitory effect appears to be mediated via a decreasein the steady-state level of IGFBP-3 mRNA and isconsistent with our studies in the cultured human ker-atinocyte cell line, HaCaT (Wraight et al., 1995). Syn-ergism between the EGF and IGF-I systems has beendescribed for many cell culture systems (Coppola et al.,1994; Burgaud et al., 1996; Ristow, 1996), includingkeratinocytes where IGF-I via transmodulation of theEGF receptor is able to stimulate keratinocyte growth(Krane et al., 1991). Hence, it is possible that an addi-tional level of synergism exists in keratinocytes whereEGF can suppress the synthesis of IGFBP-3, thereforemodulating the level of free IGF-I and consequentlypotentiating IGF-I action.

TGFb1, which is normally an anti-proliferative andprodifferentiation stimulus for keratinocytes (Shipleyet al., 1986), had an inhibitory effect on IGFBP-3. Sim-ilar to the EGF effect, the concomitant reduction inIGFBP-3 mRNA levels suggested that the effect onIGFBP-3 was via alteration in steady-state mRNA lev-els. These observations of the effect of TGFb1 on IG-

Fig. 1. EGF reduces IGFBP-3 abundance in human keratinocyte–conditioned medium. Postconfluent keratinocytes were treated withtwo daily additions of EGF at the concentrations indicated. Cell-conditioned media were collected and IGFBP abundance measured byWLB as in Material and Methods. a: A representative WLB autora-diograph of keratinocyte-conditioned medium from one primary hu-man keratinocyte cell culture. HS, human serum; M, 14C molecularweight markers. b: Combined scanning densitometric analysis of IG-FBP-3 abundance detected in keratinocyte-conditioned medium byWLB. Data are expressed as a percentage of the control, untreatedsample and are shown as the mean 6 SEM of experiments performedon three primary human keratinocyte cell cultures (with each condi-tion in triplicate for each experiment). *P , 0.01. **P , 0.05.

Fig. 2. TGFb1 reduces IGFBP-3 abundance in human keratinocyteconditioned medium. Postconfluent keratinocytes were treated withtwo daily additions of TGFb1 at the concentrations indicated. Cell-conditioned media were collected and IGFBP abundance measured byWLB as in Material and Methods. a: A representative WLB autora-diograph of keratinocyte-conditioned medium from one primary hu-man keratinocyte cell culture. HS, human serum; M, 14C molecularweight markers. b: Combined scanning densitometric analysis of IG-FBP-3 abundance detected in keratinocyte-conditioned medium byWLB. Data are expressed as a percentage of the control, untreatedsample and are shown as the mean 6 SEM of experiments performedon three primary human keratinocyte cell cultures (with each condi-tion in triplicate for each experiment). *P , 0.01. **P , 0.05.

204 EDMONDSON ET AL.

FBP-3 levels are consistent with recent findings inendothelial cells (Erondu et al., 1996) but contrast withrecent findings in other cell systems, such as dermalfibroblasts (Martin and Baxter, 1993; Yateman et al.,1993) and breast cancer cells (Oh et al., 1995), whereTGFb1 was a stimulator of IGFBP-3 production. Al-though TGF is detected throughout the epidermis(Schmid et al., 1993), the TGFb1 receptor is expressedwith increasing abundance through the upper differen-tiated layers of the epidermis and not at all in the basallayer. A possible explanation for the lack of IGFBP-3expression in suprabasal layers (Batch et al., 1994;Wraight et al., 1997) is therefore inhibition of its ex-pression by TGFb1.

In the current study, IGF-I treatment of human ker-atinocytes did not consistently regulate IGFBP-3.Des(1-3)IGF-I, which has a greatly reduced affinity forIGF binding proteins, also did not consistently regulateIGFBP abundance. IGF-I or des(1-3)IGF-I both causeda modest increase in IGFBP-3 mRNA levels when com-pared with IGFBP-3 mRNA levels of control, untreatedkeratinocytes; however, this increase did not translateinto a consistent increase in protein abundance. The

possibility of a degree of posttranscriptional regulationof IGFBP-3 abundance cannot therefore be ruled out.

The lack of IGFBP-3 regulation by IGF-I is relativelynovel since in most cultured cells IGF-I increases IG-FBP-3 abundance (Martin and Baxter, 1992; Ramag-nolo et al., 1994; Grimes and Hammond et al., 1994;Lalou et al., 1994; Leighton et al., 1994; Price et al.,1995). Furthermore, previous studies revealed thatIGF-I increased IGFBP-3 abundance in a human epi-dermal squamous cell carcinoma line (SCL-1) (Neelyand Rosenfeld, 1992). In contrast, a recent study of ratmesenchephalic cells revealed IGF-I alone did not reg-ulate IGFBP-3 abundance but, when added togetherwith basic fibroblast growth factor (bFGF), was able toaugment the increase in IGFBP-3 abundance producedby bFGF alone (Kummer et al., 1996). In addition,IGF-I did not affect IGFBP-3 abundance in rat hepato-cyte cultures, but, when added in the presence of co-cultures of hepatocytes and Kupffer cells, IGFBP-3abundance was increased (Scharf et al., 1996). Boththese studies indicate that under certain conditionsIGF-I may exert its effects on IGFBP-3 abundance onlyin the presence of additional growth factors. Although

Fig. 3. IGF-I and des(1-3)IGF-I alone are unable to regulate IG-FBP-3 abundance in human keratinocyte-conditioned medium. Post-confluent keratinocytes were treated with two daily additions of IGF-Ior des(1-3)IGF-I at the concentrations indicated. Cell-conditioned me-dia were collected and IGFBP abundance measured by WLB as inMaterial and Methods. a,c: Representative WLB autoradiographs ofkeratinocyte-conditioned medium from one primary human keratin-ocyte cell culture after treatment with either (a) IGF-I or (c) des(1-

3)IGF-I, as indicated. HS, human serum; M, 14C molecular weightmarkers. b,d: Combined scanning densitometry analysis of IGFBP-3abundance in keratinocyte-conditioned medium following exposure toIGF-I (b) or des(1-3)IGF-I (d) and detection in keratinocyte-condi-tioned medium by WLB. Data are expressed as a percentage of thecontrol, untreated sample and are shown as the mean 6 SEM ofexperiments performed on four primary human keratinocyte cell cul-tures (with each condition in triplicate for each experiment).

205EGF AND TGFb REGULATE KERATINOCYTE IGFBP-3

this study was designed to examine the effects of indi-vidual growth factors, it remains to be determinedwhether IGF-I in combination with growth factorspresent in the epidermis may also regulate keratino-cyte-derived IGFBP abundance in a similar manner.

From our results, it is clear that IGFBP-3 productionby cultured human primary keratinocytes can be reg-ulated by two cytokines known to be active in thecontrol of epidermal growth. Cytokine regulation ofIGFBP production in fibroblasts has been shown toaffect IGF-I responsiveness (Yateman et al., 1993).This may occur by regulating the sensitivity of cells tolocal “free” IGF-I or by altering IGFBP-mediated tar-geting of IGFs to their receptors. IGFBPs may eitherinhibit or enhance IGF-I cellular responses depending

on their individual properties and on whether they arecell- or matrix-associated (Jones and Clemmons, 1995).Furthermore, increasing evidence suggests that IG-FBP-3 can exert IGF-I–independent effects on culturedcells (Cohen et al., 1993; Oh et al., 1993a,b; Valentiniset al., 1995; Lalou et al., 1996; Mosheni-Zadeh andBinoux, 1997). It therefore remains possible that kera-tinocyte-derived IGFBP-3 may exert both IGF-I andIGF-I–independent effects on epidermal keratinocytesin vivo. Thus, it is likely that in keratinocytes themodulation of IGFBP-3 abundance by specific growthfactors represents an additional level of regulation ofIGF-I action in the skin in both the healthy state anddiseased states.

LITERATURE CITEDBallard FJ, Francis GL, Bagley CJ, Szabo L, Wallace JC. 1989. Effects

of insulin-like growth factors on protein metabolism: why are somemolecular variants more potent? Biochem Soc Symp 55:91–104.

Barreca A, deLuca M, delMonte P, Bondanza S, Damonte G, CariolaG, diMarco E, Giordano G, Cancedda R, Minuto F. 1992. In vitroparacrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors. J Cell Physiol 151:262–268.

Batch JA, Mercuri FA, Edmondson SR, Werther GA. 1994. Localiza-tion of messenger ribonucleic acid for insulin-like growth factorbinding proteins in human skin by in situ hybridization. J ClinEndocrinol Metab 79:1444–1449.

Burgaud JL, Baserga R. 1996. Intracellular transactivation of theinsulin-like growth factor-I receptor by an epidermal growth factorreceptor. Exp Cell Res 223:412–419.

Chomczynski P, Sacchi N. 1987. Single-step method of RNA isolationby acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem 162:156–159.

Cohen P, Lamson G, Okajima T, Rosenfeld RG. 1993. Transfection ofthe human insulin-like growth factor binding protein-3 gene intoBalb/c fibroblasts inhibits cellular growth. Mol Endocrinol 7:380–386.

Coppola D, Ferber A, Miura M, Sell C, D’Ambrosio C, Rubin R,Baserga R. 1994. A functional insulin-like growth factor receptor isrequired for the mitogenic and transforming activities of the epi-dermal growth factor receptor. Mol Cell Biol 14:4588–4595.

Erondu NE, Dake BL, Moser DR, Lin M, Boes M, Bar RS. 1996.Regulation of endothelial IGFBP-3 synthesis and secretion by IGF-Iand TGF-beta. Growth Regulation 6:1–9.

Grimes RW, Hammond JM. 1994. Proteolytic degradation of insulin-like growth factor (IGF)–binding protein-3 by porcine granulosacells in culture: regulation by IGF-I. Endocrinology 134:337–343.

Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M.1986. Analysis of serum insulin-like growth factor binding proteinsusing Western blotting: use of the method for titration of the bind-ing proteins and competitive binding studies. Anal Biochem 154:138–143.

Jones J, Clemmons D. 1995. Insulin-like growth factors and theirbinding proteins: biological actions. Endocr Rev 16:3–33.

Krane JF, Murphy DP, Carter DM, Krueger JG. 1991. Synergisticeffects of epidermal growth factor (EGF) and insulin-like growthfactor I/somatomedin C (IGF-I) on keratinocyte proliferation may bemodulated by IGF-I transmodulation of the EGF receptor. J InvestDermatol 96:419–424.

Krane JF, Gottlieb AB, Carter DM, Krueger JG. 1992. The insulin-like growth factor I receptor is over-expressed in psoriatic epider-mis, but is differentially regulated from the epidermal growth factorreceptor. J Exp Med 175:1081–1090.

Kummer JL, Zawada WM, Freed CR, Chernausek SD, HeidenreichKA. 1996. Insulin-like growth factor binding proteins in fetal ratmesenchephalic culture: regulation by fibroblast growth factor andinsulin-like growth factor-I. Endocrinology 137:3551–3556.

Laemmli UK. 1970. Cleavage of structural proteins during the assem-bly of the head of bacteriophage T4. Nature 227:680–685.

Lalou C, Silve C, Rosato R, Segovia B, Binoux M. 1994. Interactionsbetween insulin-like growth factor-I (IGF-I) and the system of plas-minogen activators and their inhibitors in the control of IGF-bind-ing protein-3 production and proteolysis in human osteosarcomacells. Endocrinology 135:2318–2326.

Lalou C, Lassarre C, Binoux M. 1996. A proteolytic fragment ofinsulin-like growth factor (IGF) binding protein-3 that fails to bind

Fig. 4. EGF and TGFb1 reduce IGFBP-3 mRNA abundance in hu-man keratinocytes. Thirty micrograms of total RNA from keratino-cytes exposed to EGF (50 ng/ml), TGFb1 (50 ng/ml), IGF-I (100 ng/ml),des(1-3)IGF-I (100 ng/ml), or no growth factors (control) was subjectedto Northern analysis as described in Materials and Methods. a: Rep-resentative IGFBP-3 mRNA Northern blot of total RNA extractedfrom keratinocytes following exposure to the indicated growth factors.C, control; E, EGF; T, TGFb1; I, IGF-I; I9, des(1-3)IGF-I. b: Combineddensitometric analysis of IGFBP-3 mRNA abundance detected byNorthern blot. Data are expressed as a percentage of the control,untreated sample and are shown as the mean 6 SEM of four exper-iments. *P , 0.05. **P 5 0.054.

206 EDMONDSON ET AL.

IGFs inhibits the mitogenic effects of IGF-I and insulin. Endocri-nology 137:3206–3212.

Leighton JK, Grimes RW, Canning SF, Hammond JM. 1994. IGF-binding proteins are differentially regulated in an ovarian granu-losa cell line. Mol Cell Endocrinol 106:75–80.

Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. 1993.Mice carrying null mutations of the genes encoding insulin-likegrowth factor I (IGF-I) and type 1 IGF receptor (IGF1r). Cell 75:59–72.

Martin JL, Baxter RC. 1992. Transforming growth factor-b stimu-lates production of insulin-like growth factor–binding protein-3 byhuman skin fibroblasts. Endocrinology 128:1425–1433.

Martin JL, Baxter RC. 1993. Release of fibroblast IGFBPs by TGF-beta and IGF-I occurs through distinct mechanisms. Growth Reg-ulation 3:62–65.

Mosheni-Zadeh SM, Binoux M. 1997. The 16 kDa proteolytic fragmentof insulin-like growth factor (IGF) binding protein-3 inhibits themitogenic action of fibroblast growth factor on mouse fibroblastswith a targeted disruption of the type I IGF receptor gene. Endo-crinology 138:3069–3072.

Murashita MM, Russo VC, Edmondson SR, Wraight CJ, Werther GA.1995. Identification of insulin-like growth factor binding proteinsfrom cultured human epidermal keratinocytes. J Cell Physiol 163:339–345.

Neely EK, Rosenfeld RG. 1992. Insulin-like growth factors (IGFs)reduce IGF-binding protein-4 (IGFBP-4) concentration and stimu-late IGFBP-3 independently of IGF receptors in human fibroblastsand epidermal cells. Endocrinology 130:985–993.

Oh Y, Muller HL, Lamson G, Rosenfeld RG. 1993a. Insulin-likegrowth factor (IGF)–independent action of IGF-binding protein-3 inHs578T human breast cancer cells. J Biol Chem 268:14964–14971.

Oh Y, Muller HL, Pham H, Rosenfeld RG. 1993b. Demonstration ofreceptors for insulin-like growth factor binding protein-3 on Hs578Thuman breast cancer cells. J Biol Chem 268:26045–26048.

Oh Y, Muller HL, Ng L, Rosenfeld RG. 1995. Transforming growthfactor-b–induced cell growth inhibition in human breast cancercells is mediated through insulin-like growth factor–binding pro-tein-3 action. J Biol Chem 270:13589–13592.

Price WA, Moats-Staats BM, Stiles AD. 1995. Insulin-like growthfactor-I (IGF-I) regulates IGFBP-3 and IGFBP-4 by multiple mech-anisms in A549 human adenocarcinoma cells. Am J Respir Cell MolBiol 13:466–476.

Ramagnolo D, Akers RM, Byatt JC, Wong EA, Turner JD. 1994.IGF-I–induced IGFBP-3 potentiates the mitogenic actions of IGF-Iin mammary epithelial MD-IGF-I cells. Mol Cell Endocrinol 102:131–139.

Ristow HJ. 1996. Studies on stimulation of DNA synthesis with epi-

dermal growth factor and insulin-like growth factor-I in culturedhuman keratinocytes. Growth Regulation 6:96–109.

Scharf J, Ramadori G, Braulke T, Hartmann H. 1996. Synthesis ofinsulin-like growth factor binding proteins and of the acid-labilesubunit in primary cultures of rat hepatocytes, of Kupffer cell, andin cocultures: regulation by insulin, insulin-like growth factor, andgrowth hormone. Hepatology 23:818–827.

Schmid P, Cox D, Bilbe G, McMaster G, Morrison C, Stahelin H,Luscher N, Seiler W. 1993. TGF-betas and TGF-beta type II recep-tor in human epidermis: differential expression in acute and chronicskin wounds. J Pathol 171:191–197.

Shimasaki S, Ling N. 1991. Identification and molecular character-ization of insulin-like growth factor binding proteins (IGFBP-1, -2,-3, -4, -5 and -6). Prog Growth Factor Res 3:243–266.

Shipley GD, Pittelkow MR, Wille JJ, Scott RE, Moses HL. 1986.Reversible inhibition of normal human prokeratinocyte prolifera-tion by type beta transforming growth factor-growth inhibitor inserum free medium. Cancer Res 46:2068–2071.

Spratt SK, Tatsuno GP, Yamanaka MK, Ark BC, Detmer J, Mas-carenhas D, Flynn J, Talkington-Verser C, Spencer EM. 1990. Clon-ing and expression of human insulin-like growth factor bindingprotein 3. Growth Factors 3:63–72.

Tavakkol A, Elder JT, Griffiths CEM, Cooper KD, Talwar H, FisherGJ, Keane KM, Foltin SK, Voorhees JJ. 1992. Expression of growthhormone receptor, insulin-like growth factor I (IGF-I) and IGF-Ireceptor mRNA and proteins in human skin. J Invest Dermatol99:343–349.

Valentinis B, Bhala A, DeAngelis T, Baserga R, Cohen P. 1995. Thehuman insulin-like growth factor (IGF) binding protein-3 inhibitsthe growth of fibroblasts with a targeted disruption of the IGF-I-receptor gene. Mol Endocrinol 9:361–367.

Wraight CJ, Murashita MM, Russo VC, Werther GA. 1994. A kera-tinocyte cell line synthesizes a predominant insulin-like growthfactor-binding protein (IGFBP-3) which modulates insulin-likegrowth factor-I action. J Invest Dermatol 103:627–631.

Wraight CJ, Werther GA. 1995. Insulin-like growth factor-I and epi-dermal growth factor regulate insulin-like growth factor bindingprotein-3 (IGFBP-3) in the human keratinocyte cell line HaCaT.J Invest Dermatol 105:602–607.

Wraight CJ, Edmondson SR, Fortune DW, Varigos G, Werther GA.1997. Expression of insulin-like growth factor binding protein-3(IGFBP-3) in the psoriatic lesion. J Invest Dermatol 108:452–456.

Yateman ME, Claffey DC, Cwyfan-Hughes SC, Frost VJ, Wass JA,Holly JM. 1993. Cytokines modulate the sensitivity of human fibro-blasts to stimulation with insulin-like growth factor-I (IGF-I) byaltering endogenous IGF-binding protein production. J Endocrinol137:151–159.

207EGF AND TGFb REGULATE KERATINOCYTE IGFBP-3