Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in human keratinocytes is regulated by EGF and TGFβ1

Download Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in human keratinocytes is regulated by EGF and TGFβ1

Post on 06-Jun-2016




1 download

Embed Size (px)


<ul><li><p>Expression of Insulin-Like Growth FactorBinding Protein-3 (IGFBP-3) in Human</p><p>Keratinocytes Is Regulated byEGF and TGFb1</p><p>STEPHANIE R. EDMONDSON,1 MARI M. MURASHITA,2 VINCENZO C. RUSSO,1</p><p>CHRISTOPHER J. WRAIGHT,1 AND GEORGE A. WERTHER1*1Centre for Hormone Research, Royal Childrens Hospital, Victoria, Australia</p><p>2Department of Paediatrics, Hokkaido University School of Medicine, Sapporo, Japan</p><p>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. 179:201207, 1999. 1999 Wiley-Liss, Inc.</p><p>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.</p><p>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</p><p>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-</p><p>Contract grant sponsor: National Health and Medical ResearchCouncil of Australia.</p><p>*Correspondence to: George A Werther, Centre for Hormone Re-search, Royal Childrens Hospital, Flemington Road, Parkville, Vic-toria 3052, Australia. E-mail:</p><p>Received 24 June 1998; Accepted 16 November 1998</p><p>JOURNAL OF CELLULAR PHYSIOLOGY 179:201207 (1999)</p><p> 1999 WILEY-LISS, INC.</p></li><li><p>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).</p><p>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.</p><p>MATERIALS AND METHODSMaterials</p><p>Keratinocyte serum-free medium (SFM), bovine pi-tuitary extract (BPE), recombinant epidermal growthfactor (rEGF), 13Hanks balanced salt solution(HBSS), Gentamicin (10 mg/ml), 0.25% Trypsin1 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 (1422 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 Childrens Hos-pital, Melbourne, Australia). Human placental totalRNA was a gift from Ms. Katy Freed (Perinatal Unit,Royal Womens 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).</p><p>Keratinocyte cultureHuman prepubertal foreskins were obtained from</p><p>routine circumcision and placed into Gibcos 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 4C. The epidermal layer was then lifted fromthe dermis and digested for 1520 min at 37C in 2 mlof 0.025% Trypsin1 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 7090% confluent.</p><p>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).</p><p>Western ligand blottingKeratinocyte CM (250 ml) was concentrated, and</p><p>samples were electrophoresed as previously described(Murashita et al, 1995) and according to the method ofLaemmli (1970). CM from cells cultured in growth fac-torfree 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 106cpm) of 125I-IGF-I/125I-IGF-II, as described by Hossen-lopp et al. (1986) and were then exposed to x-ray filmfor 310 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</p><p>202 EDMONDSON ET AL.</p></li><li><p>of the control, untreated sample and statistically ana-lyzed by one-way ANOVA using a Dunnetts MultipleComparison post test (Prism Graph Pad Software, SanDiego, CA).</p><p>RNA isolation and Northern analysisKeratinocytes (in 90 mm petri dishes) were grown to</p><p>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 50C 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 42C (5 h) and hybridizedwith labelled cDNA separately at 48C overnight in50% (v/v) formamide, 0.05% (w/v) sodium pyrophos-phate, 2.53 SSC, 53 Denhardts 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 at50C. Membranes were then exposed to X-ray film for312 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 Students t-test (Prism Graph PadSoftware).</p><p>RESULTSIGFBP-3 abundance is reduced</p><p>by EGF and TGFb1In this study, we have investigated the effect of EGF,</p><p>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.</p><p>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-</p><p>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.</p><p>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).</p><p>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).</p><p>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. 3ad).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 whe...</p></li></ul>


View more >