TGF-β regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2

Cellular Signalling 18 (2006) 1417 – 1429 www.elsevier.com/locate/cellsig

TGF-β regulates differentially the proliferation of fetal and adult human skin fibroblasts via the activation of PKA and the autocrine action of FGF-2 Christina C. Giannouli, Dimitris Kletsas ⁎ Laboratory of Cell Proliferation and Ageing, Institute of Biology, National Centre for Scientific Research “Demokritos”, 153 10 Athens, Greece Received 14 July 2005; received in revised form 10 November 2005; accepted 10 November 2005 Available online 19 December 2005

Abstract Transforming growth factor-β (TGF-β) is a potent regulator of cell proliferation; interestingly its action is clearly cell type-dependent. In particular, it inhibits epithelial and endothelial cells' proliferation, while its action on many mesenchymal cells has been reported to be stimulatory. In this direction, we have recently shown that TGF-β regulates the proliferation of normal human skin fibroblasts according to their developmental origin: i.e. it inhibits fetal fibroblasts, while it stimulates the proliferation of adult ones. Here, we present evidence on the mechanisms underlying this differential action. Concerning fetal fibroblasts, we have found that TGF-β activates Protein Kinase A (PKA) and induces the expression of the cyclin-dependent kinase inhibitors (CKIs) p21CIP1/WAF1 and p15INK4B. Moreover, the specific PKA inhibitor H-89 blocks the induction of both CKIs and annuls the TGF-β-mediated inhibitory effect, indicating the central role of PKA in this process. In contrast, in adult cells no PKA activation is observed. Moreover, TGF-β stimulates cell proliferation by activating the MEK–ERK pathway, as the MEK inhibitor PD98059 blocks this effect. A specific neutralizing antibody against Fibroblast Growth Factor-2 (FGF-2) inhibits both ERK activation and the mitogenic activity of TGF-β, indicating that the latter establishes an autocrine loop, via FGF-2, leading to cell proliferation. This loop requires FGF receptor1 (FGFR-1), as its down-regulation by siRNA approach prevents TGF-β from stimulating ERK-1/2 activation and DNA synthesis. In conclusion, the differential proliferative response of fetal and adult normal human skin fibroblasts to TGF-β is regulated by distinct signaling pathways and furthermore it may provide information on the bimodal effect of this factor on cell proliferation, in general. © 2005 Elsevier Inc. All rights reserved. Keywords: TGF-β; Fetal; Adult; Skin; Fibroblasts; PKA; FGF-2; FGFR-1; Proliferation

1. Introduction Transforming growth factor-β (TGF-β) is the generic name of a large superfamily of highly conserved polypeptides that mediate a multiplicity of biological actions [1]. The function of all TGF-β isoforms (TGF-β1, -β2, and -β3, in humans) was found to be crucial for proper development and the maintenance of homeostasis, as they regulate a vast array of biological effects, such as proliferation, differentiation, immune response, apoptosis, extracellular matrix formation and tissue repair [2– 5]. In addition, subversion of TGF-β action is associated with developmental disorders and several diseases, like fibrosis, cancer or autoimmune diseases [4,6–8]. TGF-β exerts its biological actions through two transmembrane receptors, with serine/threonine kinase activities, termed ⁎ Corresponding author. Tel.: +30 210 6503565; fax: +30 210 6511767. E-mail address: [email protected] (D. Kletsas). 0898-6568/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2005.11.002

TGF-β receptor type I (TβR-I) and type II (TβR-II). Ligand binding to TβR-II leads to the formation of a receptor complex and the subsequent activation of TβR-I by phosphorylation, and initiates intracellular signaling [9]. Activated TβR-I serves as a docking site for receptor-associated Smad2 and Smad3 proteins which, after being phosphorylated, dissociate from the receptor, form a complex with the common Smad (Smad4) and translocate to the nucleus, where they regulate the transcription of target genes [10,11]. However, it has been reported that TGFβ can also activate several other signaling pathways, such as members of the Mitogen-Activated Protein Kinase (MAPK) family, phosphatidylinositol 3-kinase (PI-3K), Protein Kinase A and C (PKA and PKC), among others [12–15]. Even more, although the role of the Smad pathway is critical for many TGFβ-mediated biological effects, Smad-independent responses have also been well documented [16,17]. TGF-β is a pleiotropic factor and furthermore its action depends on the target cell type and the specific culture

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conditions [18–20]. Concerning cell proliferation, it is generally accepted that TGF-β inhibits epithelial, endothelial and hematopoietic cells, while it stimulates the growth of some mesenchymal cells, such as fibroblasts [8,21]. The inhibitory effect of TGF-β has been attributed to the upregulation of cyclin-dependent kinase inhibitors (CKIs), such as p21CIP1/WAF1 [22,23] and p15INK4B [24,25] or to the altered localization of p27Kip1 [25,26], while antiproliferative actions of TGF-β independent of CKIs have also been reported [27,28]. Loss of responsiveness to TGF-β, characterizing many types of cancer, is the result of mutational inactivation of its receptors or Smads [8,29]. On the other hand, the stimulatory action of TGFβ has been ascribed either to a down-regulation of p21CIP1/WAF1 [30,31] or to an indirect action via the autocrine secretion of other growth factors [20,32–35]. As mentioned above, TGF-β plays an important role in the tissue repair process. However, pronounced differences exist between fetal and adult wound healing, as the former is characterized by the absence of contracture and scar formation [36,37], thus implying the presence of different repair strategies. Furthermore, several lines of evidence indicate that these differences cannot be attributed only to the unique environment of the fetus, but they also represent intrinsic features of the fetal and the adult tissue [38]. In this context, we have studied the response of normal skin fibroblasts from these two developmental stages, to TGF-β, and have found that the latter can provoke opposing proliferative effects. In particular, we have shown that all three TGF-β isoforms strongly inhibit fetal human skin fibroblasts, while they stimulate the proliferation of fibroblasts from adult donors [39]. The aim of the present study was to investigate the mechanisms underlying the differential response of these normal cells to TGF-β. As the biological responses to TGF-β vary between different target cells (see above), this study could provide information not only on the differences between fetal and adult cells but also on the bimodal proliferative action of TGF-β, in general. Here, we report that TGF-β stimulates adult skin fibroblasts through the release of Fibroblast Growth Factor-2 (FGF-2) and, via its high affinity receptor FGFR-1, the ensuing activation of the MEK–ERK pathway. On the other hand, we show that TGF-β inhibits fetal fibroblasts by the activation of PKA, which is necessary for the subsequent up-regulation of the cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p15INK4B . 2. Materials and methods 2.1. Materials Human recombinant (h.r.) TGF-β1 and h.r. PDGF-BB, as well as rabbit antiTGF-β and goat anti-PDGF neutralizing antibodies were purchased from R&D Systems (Minneapolis, MN). Bovine pituitary FGF-2, calphostin C, U0126, indomethacin, caffeic acid, SB203580, NS-398, wortmannin, nimesulide, mouse anti-tubulin and rabbit anti-FGF-2 antibodies, as well as goat antimouse and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies were obtained from Sigma (St. Louis, MO). PD98059 was from Biomol Research Laboratories (Hamburg, Germany), while SP600125, NDGA and H-89 from Calbiochem (Scwalbach, Germany). The mouse neutralizing antibody against FGF-2 was obtained from Upstate Biotechnology (Dundee, UK). Rabbit phospho-ERK1/2 antibody that recognizes phosphorylated Thr202/

Tyr204 was obtained from Cell Signaling Technology (Hertfordshire, UK), mouse anti-pan-ERK and mouse anti-p21CIP1/WAF1 antibody from BD Transduction Laboratories (Bedford, MA); the rabbit polyclonal anti-p15INK4B and anti-FGFR-1 (C-15) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). [Methyl-3H]-Thymidine was from Amersham Biosciences (Buckinghamshire, UK) and [α-32P]-ATP was from Izotop (Budapest, Hungary). The rabbit antibodies against Smad2 and phospho-Smad2 (the latter recognizing phosphorylated Ser465/467), as well as the rabbit Smad3 antibody were generous gifts from Profs. A. Moustakas and D. Kardassis, respectively. The rabbit antibody against phospho-Smad3 which recognizes phosphorylated Ser433/435 was obtained from Cell Signaling Technology.

2.2. Cells and cell culture conditions The following normal human skin fibroblast strains have been used in this study: one strain from an 18-week fetus (Detroit 551, original source: ATCC, Rockville, USA), and one adult strain (AG01523c, Coriell Institute for Medical Research, Camden, USA). Both cell strains were routinely cultured in Eagle's Minimal Essential Medium (MEM), supplemented with non-essential amino acids, penicillin-streptomycin, glutamine (all from Biochrom AG, Berlin, Germany) and 10% Fetal Bovine Serum (FBS), from Gibco BRL (Invitrogen, Paisley, UK). Early passage cells were routinely subcultured when confluent by using a trypsin/citrate (0.25%/0.30% w/v) solution. The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cell counting, after trypsinization, was performed by using a Coulter counter (Beckman-Coulter, Fullerton, CA). Cells were tested periodically and found to be mycoplasma-free. In parallel experiments, the human immortalized epithelial HaCaT cell line, cultured in DMEM supplemented with 10% FBS, was also used.

2.3. DNA synthesis assay Cells were plated at a density of 2 × 104 cells/cm2, in MEM containing 10% FBS, and when grown to approximately 80% confluency the medium was changed to MEM containing 0.1% FBS for another 48 h. Fresh medium containing 0.1% FBS was then added to the quiescent cultures, along with the growth factors to be tested and [3H]-Thymidine (0.15 μCi/ml, 25 Ci/mmol). After 48 h of incubation, the culture medium was aspirated, the cells were washed with PBS, fixed with 10% ice-cold trichloroacetic acid (TCA), washed extensively under running tap water and air-dried. DNA was solubilized by the addition of 0.3 N NaOH/1% (w/v) SDS and the lysates were subjected to scintillation counting, as previously described [40]. When indicated, the cells were pre-incubated with the appropriate concentrations of kinase inhibitors 45 min before TGF-β treatment. Furthermore, when neutralizing antibodies were used, they were added in the culture medium 1 h before TGF-β treatment. In order to test the efficiency of these antibodies, these were pre-incubated with the respective growth factors at 37 °C for 1 h and then were added in the culture.

2.4. Cell cycle analysis Cell cycle analysis was performed by flow cytometry, as follows: cells were plated at a density of 104 cells/cm2 in MEM containing 10% FBS and after 24 h the medium was changed to one containing 1% FBS for another 24 h. Then, the cells were treated with TGF-β (2.5 ng/ml) and after a 24-h period they were trypsinized, washed with PBS, fixed in 50% (v/v) ethanol, and stained with propidium iodide (50 μg/ml), in the presence of MgCl2 (5 mM) and RNAse A (10 μg/ml) in Tris–HCl pH 7.5 (10 mM). DNA content was analyzed on a FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA) using the Modfit software.

2.5. Western analysis Human skin fibroblasts, treated with TGF-β (2.5 ng/ml) or FGF-2 (10 ng/ ml) for various time periods, were washed with ice-cold PBS and lysed as follows: The cells were scraped into 2 × hot SDS-PAGE sample buffer [125 mM Tris–HCl pH 6.8, 5% (w/v) SDS, 20% (v/v) glycerol, 125 mM βmercaptoethanol, 0.02% (w/v) bromophenol blue, supplemented with 2 mM PMSF, 2 mM NaF, and 2 mM Na3VO4], sonicated for 15 s, boiled for 5 min,

C.C. Giannouli, D. Kletsas / Cellular Signalling 18 (2006) 1417–1429 clarified by centrifugation, and stored at − 80 °C until use. The lysates were separated on SDS-PAGE and the proteins were transferred to PVDF membranes (Amersham Biosciences). The membranes were blocked with 5% (w/v) non-fat dried milk in 10 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20 (TTBS) buffer and incubated with the appropriate primary antibodies. After washing with TTBS, the membranes were incubated with the respective second antibody for 1 h, washed again with TTBS and the immunoreactive bands were visualized on Kodak-X-OMAT AR film by chemiluminescence (ECL kit) according to the manufacturer's (Amersham Biosciences) instructions. The intensity of the bands was quantified after capture with a CCD camera connected to a PC, using the BioProfil image analysis software (Vilber Lourmat, Torcy-Paris, France).

2.6. Preparation and transfection of small interfering RNAs targeting FGFR-1 Small interfering RNAs (siRNAs) targeting FGFR-1 were constructed from sense and antisense DNA oligonucleotides (Invitrogen), using the Silencer siRNA construction kit (Ambion Inc, USA). The sense sequence for FGFR-1 siRNA was 5′-AAGTCGGACGCAACAGAGAAA-3′, designed to eliminate all FGFR-1 isoforms containing intracellular kinase domains [41–43]. The scrambled siRNA, recommended for FGFR-1 (Control siRNA-A), was purchased form Santa Cruz Biotech. Human skin fibroblasts, when 90% confluent, were transfected, with 10 nM of either scrambled or FGFR-1 siRNA, in serum-free OPTI-MEM I Medium (Invitrogen), by using Lipofectamine 2000 (Invitrogen), according to manufacturer's instructions. Fresh medium containing FBS was added 6 h post-transfection [final concentration 10% (v/v)], and the cells were incubated for another 48 h. Then, the medium was changed to OPTIMEM I/1% FBS and 24 h later the cells were stimulated with either TGF-β (2.5 ng/ml) or FGF-2 (10 ng/ml). For DNA synthesis experiments, [3H]-Thymidine (0.2 μCi/ml) was added along with the growth factors for 48 h. DNA synthesis and western analysis experiments were performed in 48- and 12-well plates, respectively.

2.7. Northern analysis Fibroblasts were plated at a density of 104 cells/cm2 in MEM containing 10% FBS for 24 h. Then, the medium was changed to MEM containing 1% FBS and after 24 h the cells were stimulated with 2.5 ng/ml TGF-β for the indicated time points. Total RNA was isolated by using the Genelute Mammalian Total RNA Kit (Sigma). The RNA samples were electrophoresed (6 μg of total RNA per lane) in 1.3% (w/v) denaturing agarose gel containing 2.2 M formaldehyde and transferred to nitrocellulose membranes (Amersham Biosciences). After cross-linking RNA under ultraviolet light, the membranes were stained with methylene blue [44] and photographed, destained and pre-hybridized at 42 °C for 2 h, in a hybridization solution [5 × SSC, 0.1% (w/v) N-Laurosarcosine, 0.02% (w/v) SDS, 2 × Denhardt's solution, 50% (v/v) deionized formamide] [44]. Then, the denatured probes of human p21CIP1/WAF1or p15INK4B cDNAs were added (generous gifts from Profs. D. Kardassis and E. Kolettas, respectively), which have been previously labeled with [α-32P]ATP, by using Hexalabel DNA Labeling Kit (MBI Fermentas GMBH, Germany). After an overnight incubation, the membranes were washed twice in 2 × SSC, 0.1% SDS in 42 °C, twice in 0.1 × SSC, 0.1% SDS in room temperature and then they were exposed to an X-ray film (Kodak-X-OMAT AR) for at least 4 days at −80 °C. The intensity of the bands was quantified as above.

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2.9. In vitro kinase assay for PKA activity Human skin fibroblasts were plated at a density of 2 × 104 cells/cm2, in MEM containing 10% FBS, and when grown to approximately 80% confluency the medium was changed to MEM containing 0.1% FBS for another 48 h. Then, the cultures were treated with TGF-β (2.5 ng/ml) or forskolin (10 μM) for the indicated time periods, in the presence or absence of the PKA inhibitor H-89 (7 μM). Subsequently, the cells were washed with ice-cold TBS and kept on ice. Then, they were harvested by scrapping with a rubber policeman in an extraction buffer [25 mM Tris–HCl pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM PMSF and a cocktail of protease inhibitors (Sigma)] and then homogenized by sonication for 30 s. The protein content was determined and 6 μg of total protein was used in the PepTag Assay for a non-radioactive determination of PKA activity (Promega Corporation, Madison, WI). Briefly, the assay is based on the use of a fluorescent peptide substrate (Kemptide), specific for PKA. Phosphorylation of the PepTag peptide by PKA alters the net charge from +1 to − 1. This change in the charge of the substrate allows its phosphorylated and non-phosphorylated forms to be separated electrophoretically on an agarose gel (0.8% agarose in 50 mM Tris–HCl pH 8.0). The phosphorylated peptide migrates towards the negative electrode (cathode) while the non-phosphorylated one migrates toward the positive electrode (anode). The negative control lacks PKA enzyme and contains only buffer, while only the positive control contains the PKA catalytic subunit (final concentration 16 U/ml) supplied with the kit. The intensity of the bands was quantified as above.

2.10. RT-PCR analysis The expression of p21CIP1/WAF1 and p15INK4B genes after treatment with TGF-β in the presence of the PKA inhibitor H-89 was determined by semiquantitative RT-PCR, as follows: fetal skin fibroblasts were plated at a density of 104 cells/cm2 in MEM containing 10% FBS and after 24 h the medium was changed to MEM containing 1% FBS for another 24 h. Then, the cells (pre-incubated or not with 7 μM H-89 for 45 min) were stimulated with 2.5 ng/ml TGF-β and total RNA was isolated after 12 h, as described above. First strand cDNA was synthesized from 2 μg of total RNA, using oligo-dT12–18 and M-MLV reverse transcriptase following standard procedures indicated by the manufacturer (Invitrogen, Paisley, UK). The cDNA products were amplified in a buffer [10 mM Tris–HCl (pH 8.3), 2.5 mM MgCl2, and 50 mM KCl] containing 1 mM dATP, dCTP, dGTP, and dTTP, 2.5 units of Taq DNA polymerase (HyTest LTD, Turku, Finland), 0.2 μM of each set of specific primers: 5′-CCT CTT CGG CCC AGT GGA C-3′ and 5′-CCG TTT TCG ACC CTG AGA G-3′ (for p21CIP1/WAF1) [45] or 5′-TGG GGG CGG CAG CGA TGA G-3′ and 5′-AGG TGG GTG GGG GTG GGA AAT-3′ (for p15INK4B) [46]. The cDNAs were denatured at 94 °C for 30 s, amplified for 28 cycles for p21CIP1/WAF1 (annealing temperature 60 °C) and 30 cycles for p15INK4B (annealing temperature 56 °C), and extended at 72 °C for 45 s. The GAPDH gene was used for the normalization of the results by using the following primers: 5′-TGG TAT CGT GGA AGG ACT CAT GAC-3′ and 5′-ATG CCA GTG AGC TTC CCG TTC AGC-3′. The products were visualized on a 2% (w/v) agarose gel by using ethidium bromide staining. The intensity of the bands was quantified as described above.

3. Results

2.8. Determination of FGF-2 in conditioned media by ELISA

3.1. TGF-β inhibits fetal and stimulates adult human skin fibroblasts

Fetal and adult skin fibroblasts were plated in MEM containing 10% FBS, and when grown to approximately 80% confluency the medium was changed to MEM containing 0.1% FBS for another 48 h. Fresh medium containing 0.1% FBS was then added to the quiescent cultures and cells were stimulated with TGF-β (2.5 ng/ml) for the indicated time points. Then, the supernatants from treated and untreated cultures (in triplicates) were collected, centrifuged, aliquoted while on ice, frozen in liquid nitrogen and kept at − 80 °C until use. The amount of FGF-2 in the conditioned medium was determined by a human FGF-2 Duoset Elisa Kit, according to manufacturer's (R&D Systems) instructions.

TGF-β exerts a differential proliferative effect on human skin fibroblasts, depending on the developmental stage of the donor. In particular, while it strongly inhibits DNA synthesis in fetal fibroblasts, it stimulates adult cells (Fig. 1). The above results have also been confirmed in a series of fetal and adult skin fibroblast cell strains, either commercially available or in primary cultures developed in our laboratory [39]. This bimodal proliferative effect of TGF-β is also reflected on cell cycle

C.C. Giannouli, D. Kletsas / Cellular Signalling 18 (2006) 1417–1429 400

300

200

100

0

C

T

C

Fetal

T Adult

Fig. 1. Differential proliferative response of fetal and adult human skin fibroblasts to TGF-β. Fibroblast cultures were treated (T) or not (C) with 2.5 ng/ ml TGF-β, and after 48 h of incubation in the presence [3H]-Thymidine (0.15 μCi/ml; 25 Ci/mmol), DNA synthesis was estimated as described under Materials and methods. One representative experiment (performed in quadraplicate) out of multiple similar experiments is presented here. Results are expressed as mean values ± standard deviation. [Modified from [39], under permission.]

distribution, as deduced by flow cytometry. As can be seen in Table 1, in fetal fibroblasts treated with TGF-β for 24 h we observed a decline of the percentage of cells being in the S phase of the cell cycle with a concomitant increase of the G0/G1 phase. On the contrary, in adult cells TGF-β induces an increase in S phase coupled with a decrease of the percentage of cells being in the G0/G1 phase. Moreover, the percentage of cells being in G2/M remains practically unaltered in both fetal and adult fibroblasts. It must also be mentioned that TGF-β can inhibit or stimulate the proliferation of fetal and adult fibroblasts, respectively, independently of culture conditions, like cell density or serum-concentration: we observed that TGFβ exerts the same effect in confluent and subconfluent cultures, as well as in cells arrested by serum-deprivation or asynchronous proliferating in the presence of serum (not shown). 3.2. TGF-β stimulates human adult skin fibroblasts via autocrine FGF-2 action First, we studied the mechanism underlying the stimulatory effect of TGF-β on adult fibroblasts. It has been previously reported that TGF-β can stimulate the proliferation of many cell types indirectly, via an autocrine growth factor action. To test this hypothesis in our system, we performed DNA synthesis experiments by using specific neutralizing antibodies against certain growth factors. As expected, pre-incubation of TGF-β

with an anti-TGF-β neutralizing antibody annulled its action (Fig. 2). We also used a neutralizing antibody against PlateletDerived Growth Factor (PDGF), as previous reports implicate this factor in a TGF-β-induced autocrine loop leading to the stimulation of skin fibroblasts' proliferation [33]. In particular, we used an antibody that recognizes all three PDGF dimers (-AA, -BB and -AB), at the concentration of 10 μg/ml, that in preliminary experiments has been found to block completely the action of PDGF. When this antibody was added to the culture milieu it was unable to inhibit TGF-β-mediated induction of DNA synthesis (Fig. 2). In contrast, a monoclonal neutralizing antibody against Fibroblast Growth Factor-2 (FGF-2) was capable to block the stimulatory action of TGF-β at a great extent, comparable to the effect of the anti-TGF-β antibody (Fig. 2). Control (non-immune) IgGs–i.e. from the same species the neutralizing antibodies were raised–had no effect on the proliferative action of TGF-β in adult fibroblasts (not shown). The above results indicate that the indirect mitogenic effect of TGF-β is mediated via the autocrine action of FGF-2. Consequently, we studied the production and secretion of FGF-2 by adult fibroblasts after TGF-β treatment. To this end, lysates of TGF-β-treated cells were subjected to western analysis by using an anti-FGF-2 antibody (Fig. 3A). As can be seen, TGF-β stimulates the production of the 18 kDa FGF-2 isoform, i.e. the secreted FGF-2 isoform and with proven mitogenic activity [47]. This induction is rising gradually, reaching a maximum (approx. two-fold) after 24 h. In addition, we measured FGF-2 levels released from adult fibroblasts. Supernatants from TGF-β-treated or untreated cultures were tested by ELISA and, in accordance to the results presented in Fig. 3A, increased FGF-2 levels were found after the addition of TGF-β. This increase was observed 1–3 h after treatment and declined thereafter (Fig. 3B). On the other hand, in fetal fibroblasts, we have not observed any up-regulation of the 18

Adult Fibroblasts [3H]-Thymidine incorporation (% of control)

[3H]-Thymidine incorporation (% of control)

1420

400

300

200

100

0 N. anti-TGF-β

Control

Table 1 Cell cycle analysis, as estimated by flow cytometry, in fetal and adult human skin fibroblasts treated with TGF-β for 24 h

Fetal fibroblasts Adult fibroblasts

Control TGF-β-treated Control TGF-β-treated

G0/G1

S

G2/M

85.21% 89.50% 70.22% 63.97%

10.51% 5.19% 10.36% 19.57%

4.29% 5.32% 19.42% 16.45%

TGF-β

N. anti-FGF-2

FGF-2

N. anti-PDGF

PDGF

Fig. 2. Effect of neutralizing antibodies against TGF-β, PDGF and FGF-2 on the TGF-β-induced cell proliferation in adult skin fibroblasts. Adult fibroblasts were stimulated with TGF-β (2.5 ng/ml), FGF-2 (10 ng/ml) or PDGF (10 ng/ ml), in the presence or absence of neutralizing antibodies against TGF-β (N.antiTGF-β, 10 μg/ml), FGF-2 (N.anti-FGF-2, 5 μg/ml) or all PDGF isoforms (N. anti-PDGF, 10 μg/ml). After 48 h, in the presence of [3H]-Thymidine, DNA synthesis was estimated, as described under Materials and methods. One representative experiment (performed in quadraplicate) out of three similar experiments is presented here.

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A

Time (h)

0

0.5

1

2

3

6

12

24

TGF-β

-

+

+

+

+

+

+

+ FGF-2

Adult Tubulin FGF-2

Fetal

Tubulin

B Fetal Adult (% of control)

[FGF-2] in conditioned medium

200

150

100

1421

have studied the TGF-β-mediated ERK-1/2 activation by using an anti-FGF-2 neutralizing antibody at the time points of maximum ERK-1/2 stimulation, and have found that in the presence of this antibody, the delayed ERK-1/2 activation was completely inhibited (Fig. 4B). The TGF-β-mediated delayed ERK-1/2 activation was also blocked by using PD98059, a specific inhibitor of the MEK–ERK pathway (Fig. 4B). Similar results were obtained also with another MEK inhibitor, i.e. U0126 (not shown). Furthermore, we tested the role of the activation of the MEK–ERK pathway in the mitogenic action of TGF-β in adult fibroblasts. As can be seen in Fig. 4C and in Table 2, PD98059 can inhibit, in a dose-dependent manner, the stimulation of DNA synthesis provoked by TGF-β on adult cells. Similar results were also obtained with U0126 (Table 2). On the other hand, PD98059 and U0126 do not influence the inhibitory effect of TGF-β on fetal fibroblasts (Fig. 4D and Table 2). Notably, all other inhibitors used were unable to affect the mitogenic effect of TGF-β (Table 2). All the above indicate that TGF-β exerts an indirect mitogenic effect in adult human skin fibroblasts, via the autocrine action of FGF-2 and the subsequent activation of the MEK–ERK pathway.

50 0

1

2

3

4

5

6

Time after TGF-β stimulation (h) Fig. 3. Regulation of FGF-2 synthesis and secretion by human skin fibroblasts after TGF-β treatment. (A) Cultures of fetal and adult fibroblasts were stimulated with TGF-β (2.5 ng/ml). At the indicated time points cell lysates were collected and subjected to Western analysis, as described under Materials and methods, by using an anti-FGF-2 antibody. Tubulin was used as a loading control. (B) Fibroblast cultures were treated with TGF-β (2.5 ng/ml). At the indicated time points the supernatants were collected and subjected to ELISA for the identification of the secreted FGF-2 (see Materials and methods). In both (A) and (B) one representative experiment out of three similar ones is presented here.

kDa isoform of FGF-2 or of FGF-2 release in the culture milieu after treatment with TGF-β (Fig. 3A and B). 3.3. ERK-1/2 activation by TGF-β is necessary for the stimulation of adult skin fibroblasts Trying to identify the down-stream effectors that are responsible for the observed TGF-β-mediated stimulation of proliferation in adult fibroblasts we focused on the activation of the MEK–ERK pathway, known to be involved in the regulation of cell proliferation by several growth factors, FGF-2 included [48,49]. We observed, only in adult–and not in fetal fibroblasts (not shown)–a stimulation of ERK-1/2 activation by phosphorylation (Fig. 4A). However, in contrast to classical mitogens, such as FGF-2, that induces an immediate (within 10 min) ERK-1/2 activation (Fig. 4A), TGF-β treatment leads to a delayed activation, starting from 1 h after treatment and reaching a maximum after 3–6 h (Fig. 4A). This postponed activation is in agreement with the presence of an autocrine mechanism, and more specifically the early release of FGF-2 by these cells (Fig. 3B). To access further the involvement of FGF-2 in this phenomenon we

3.4. TGF-β stimulates ERK-1/2 activation and DNA synthesis through FGFR-1 The main receptor for FGF-2 in human skin fibroblasts is FGFR-1 [42,50]. In order to understand the role of this receptor in the TGF-β-induced stimulation of the ERK pathway and DNA synthesis, we have used siRNA to downregulate FGFR-1 levels. Western analysis revealed the expression of two bands of FGFR-1 of approx. 100 and 85 kDa respectively (Fig. 5A), as previously described in human skin fibroblasts [50]. As can be seen in Fig. 5A, treatment of cells with an FGFR-1 siRNA potently down-regulated its expression. Furthermore, we have shown that in cells with down-regulated FGFR-1 expression (after siRNA treatment) the mitogenic effect of FGF-2 is annulled (Fig. 5B), showing that indeed the effect of FGF-2 on DNA synthesis is mediated via FGFR-1. Even more interesting, these cells are also unable to respond to TGF-β (Fig. 5B). In contrast, scrambled siRNAtransfected cells responded normally to both growth factors (Fig. 5B). Finally, we have investigated the effect of FGFR-1 down-regulation in TGF-β-induced ERK activation. Accordingly, adult cells–control and transfected with either FGFR-1 siRNA or scrambled siRNA–were stimulated with TGF-β or FGF-2 for different time periods. Our data (Fig. 5C) indicate that in cells transfected with FGFR-1 siRNA FGF-2-induced ERK activation is diminished. Furthermore, the delayed ERK1/2 activation in response to TGF-β (4.5 h after treatment, see also Fig. 4A) was completely blocked. Once again, in the presence of scrambled siRNA the cells responded similarly to control, untransfected cells (Fig. 5C). These results confirm the above-mentioned data with the FGF-2 neutralizing antibody (Figs. 2 and 4) and further indicate that FGFR-1 is essential for ERK activation and stimulation of DNA synthesis by TGF-β in adult human fibroblasts.

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B

A 0

TGF-β (h)

0.5 1

2

3

4.5 6

8

FGF-2 (h) 0

0.15 1

2

TGF-β (h)

3

pERK-1/2

pERK-1/2

ERK-2

ERK-2

D

Adult Fibroblasts

[3H]-Thymidine incorporation (% of control)

[3H]-Thymidine incorporation (% of control)

C 400 300 200 100 0 0

10 M

15 M

20 M

25 M

0

+ PD98059 3 4.5 6

Fetal Fibroblasts 150

100

50

0 25 M

0

PD98059 Control

+ N. anti-FGF-2 0 3 4.5 6

PD98059

TGF-β

Control

TGF-β

Fig. 4. TGF-β stimulates the proliferation of adult skin fibroblasts via the MEK–ERK pathway. (A) Adult human skin fibroblasts were stimulated with TGF-β (2.5 ng/ml) or FGF-2 (10 ng/ml). At the indicated time points (h: hours) cell lysates were collected and subjected to Western analysis, as described under Materials and methods, by using an antibody against the phosphorylated forms of ERK-1/2 (pERK-1/2). For loading control, the membranes were reprobed with a pan-ERK antibody that recognizes mainly ERK-2. (B) Cultures of adult fibroblasts pre-incubated for 45 min with PD98059 (25 μM) or a neutralizing antibody against FGF-2 (5 μg/ml) were treated with TGF-β (2.5 ng/ml). Cell lysates were collected at the indicated time points and western analysis was performed as in (A). In (A) and (B) data are representative of three similar experiments. (C) Adult skin fibroblast cultures were pre-incubated for 45 min with increasing concentrations of PD98059. Then TGF-β (2.5 ng/ml) and [3H]-Thymidine were added and DNA synthesis was estimated after a 48-h incubation, as described under Materials and methods. One representative experiment (performed in quadraplicate) out of three similar experiments is presented here. (D) Fetal skin fibroblast cultures were pre-incubated with or without 25 μM PD98059. Forty-five minutes later, TGF-β (2.5 ng/ml) and [3H]-Thymidine were added, and DNA synthesis was estimated as in (C).

3.5. TGF-β-mediated inhibition of fetal skin fibroblasts is accompanied by the up-regulation of p21 CIP1/WAF1 and p15INK4B In several cell types of human origin, such as normal and transformed epithelial or endothelial cells, TGF-β inhibits their proliferation by arresting them in the late G1 phase of the cell cycle, via the induction of cyclin-dependent kinase inhibitors, like p21CIP1/WAF1 or p15INK4B , which inhibit the activities of the cyclin D-CDK4/6 and cyclin E-CDK2 complexes [23,24]. As we have shown that TGF-β can also inhibit normal human mesenchymal cells, and in particular human fetal skin fibroblasts by arresting them at the G0/G1 phase of the cell cycle (Table 1), we investigated the expression of these regulatory proteins in fetal, as well as in adult, fibroblasts after treatment with TGF-β. As can be seen in Fig. 6A, TGF-β can effectively stimulate the production of p21CIP1/WAF1 mRNA in fetal fibroblasts, as soon as 6 h after treatment, while a higher induction was observed at 12 h after growth factor addition. An intense up-regulation was observed also for the p15INK4B gene, starting from 6 h after treatment, as well. The above effect was accompanied by an up-regulation of p21CIP1/WAF1 and p15INK4B at the protein level, starting from 18 h after TGF-β treatment; a near two-fold induction was found for p21CIP1/WAF1 while the

stimulation of p15INK4B was more intense, i.e. more than fourfold compared to control levels (Fig. 6B). The expression of both p21CIP1/WAF1 and p15INK4B in the epithelial origin cell line HaCaT, in response to TGF-β, was used as a control (Fig. 6B). Table 2 Effect of inhibitors on the proliferative action of TGF-β on fetal and adult human skin fibroblasts Inhibitor

Target protein

Range of concentrations used

Effect on fetal fibroblasts

Effect on adult fibroblasts

H-89 Indomethacin Nimesulide NS-398 NDGA Caffeic Acid Wortmannin Calphostin C SB203580 SP600125 PD98059 UO126

PKA COX-1/COX-2 COX-2 COX-2 5-, 12-, 15-LOX 5-LOX PI-3K PKC p38 MAPK JNK MAPK MEK1,2 MEK1,2

0.5–10 μM 2.5–80 μM 100–200 μM 10–30 μM 0.2–1 nM 1–10 μM 50–200 nM 25–150 nM 1–10 μM 1–20 μM 5–25 μM 1–10 μM

+ − − − − − − − − − − −

− − − − − − − − − − + +

(+) denotes reversal of TGF-β (inhibitory or stimulatory) action; (−) denotes no effect on TGF-β action on skin fibroblasts' proliferation, determined by DNA synthesis assays.

C.C. Giannouli, D. Kletsas / Cellular Signalling 18 (2006) 1417–1429

A Scr siRNA

-

+

-

FGFR-1 siRNA

-

-

+

T

B [3H]-Thymidine incorporation (% of control)

250

200

150

100

50

0

1423

3.6. TGF-β inhibits the proliferation of fetal skin fibroblasts via PKA induction We have already shown that the inhibitory effect of TGF-β on fetal fibroblasts can be annulled by curcumin (diferyloylmethane), a plant polyphenol [39]. However, curcumin can affect several intracellular signaling pathways [51]. So, we studied the involvement of those pathways affected by curcumin by using specific inhibitors. In particular, we have used SP600125 (a JNK inhibitor), indomethacin [an inhibitor of cyclooxygenase (COX)-1 and -2], nimesulide and NS-398 (both inhibitors of COX-2), calphostin C (a specific inhibitor of PKC), caffeic acid [a 5-lipoxygenase (LOX) inhibitor] and NDGA (that inhibits 5-, 12-, and 15-lipoxygenase), as well as wortmannin (a PI-3K inhibitor), PD98059 and U0126 (both MEK inhibitors), and SB203580 (a p38 MAPK inhibitor). All these inhibitors, used at a range of non-cytotoxic concentrations, were unable to block the inhibitory effect of TGF-β (Table 2). However, as curcumin is known to inhibit also Protein Kinase A (PKA) [52], we used H-89, a specific PKA inhibitor. As can be seen in Fig. 7A, H-89 was found to annul the inhibitory effect of TGF-β on the proliferation of fetal cells,

A

Fetal

Time (h) TGF-β

6 -

Adult

12 +

-

6 +

-

12 +

-

+

C TGF-β

p21CIP1/WAF1

FGF-2 Time (h)

p15 INK4B pERK-1/2

18S rRNA ERK-2

Fig. 5. TGF-β stimulates proliferation through FGFR-1. (A) Lysates of adult skin fibroblasts, untransfected or transfected with 10 nM of either scrambled (Scr siRNA) or FGFR-1 siRNA, were subjected to western blot analysis by using an anti-FGFR-1 antibody. Tubulin expression was used as a loading control. (B) DNA synthesis after stimulation with TGF-β (2.5 ng/ml) or FGF-2 (10 ng/ml) in adult skin fibroblasts, untransfected or transfected with 10 nM of either scrambled (Scr siRNA) or FGFR-1 siRNA. DNA synthesis was estimated, as described under Materials and methods. One representative experiment (performed in quadraplicate) out of three similar experiments is presented here. (C) Western blot analysis of ERK-1/2 activation in adult skin fibroblasts, untransfected or transfected with 10 nM of either scrambled (Scr siRNA) or FGFR-1 siRNA, stimulated with TGF-β for 0.5 and 4.5 h or FGF-2 for 0.5 h. Western analysis was performed as in Fig. 4; one representative experiment out of two similar experiments is hereby presented.

In contrast, in adult cells we did not observe any significant upregulation of p21CIP1/WAF1 and p15INK4B mRNA: a small induction was found 6 h after TGF-β treatment, which is lost 6 h later (Fig. 6). However, we observed very low levels of p21CIP1/WAF1 and p15INK4B protein expression in adult cells and no further induction after the addition of TGF-β was found (not shown).

B Time (h) TGF-β

Fetal skin fibroblasts 18 -

20 +

-

HaCaT

24 +

-

+

-

22 +

p21CIP1/WAF1

p15 INK4B Tubulin Fig. 6. TGF-β induces p21CIP1/WAF1 and p15INK4B expression in fetal, and not in adult, human skin fibroblasts. Fetal and adult skin fibroblasts were treated with TGF-β (2.5 ng/ml) for the indicated time periods. In (A), total RNA was collected and subjected to Northern analysis by using [α-32P]-labeled cDNAs of p21CIP1/WAF1 or p15INK4B, as described under Materials and methods. 18S rRNA stained with methylene blue was used as a loading control. In (B), cell lysates were collected and Western analysis was performed with anti-p21CIP1/WAF1 or anti-p15INK4B antibodies. Lysates of HaCaT cells, treated with TGF-β (2.5 ng/ ml) for 22 h, were used as a positive control for p21CIP1/WAF1 or p15INK4B expression. The expression of tubulin served as a loading control. In both (A) and (B) similar results were obtained from at least three individual experiments.

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A

3.7. Activation of Smad2 and Smad3 proteins by TGF-β in fetal and adult human skin fibroblasts

[3H]-Thymidine incorporation (% of control)

Fetal Fibroblasts 150

100

50

0

0

2 µM

5 µM

7 µM

H-89 Control

TGF-β

B [3H]-Thymidine incorporation (% of control)

Adult Fibroblasts 200

150

100

50

0

7 µM

0

It has been recently shown in mink lung epithelial cells that the TGF-β-mediated PKA activation and the subsequent inhibition of proliferation depend on the activated, i.e. phosphorylated, Smad3 protein [54]. Accordingly, we studied in both fetal and adult human skin fibroblasts the activation of Smad proteins by TGF-β. In Fig. 9A, it can be seen that both Smad2 and Smad3 proteins are rapidly phosphorylated by TGF-β. Maximum activation was observed approx. 0.5 h after TGF-β treatment. A significant reduction of activated Smad2 and Smad3 was found 3 h after TGF-β addition (Fig. 9A), while Smad phosphorylation was completely annulled 24 h after stimulation (not shown); similar phosphorylation kinetics for Smad2 have been reported for mink lung epithelial cells [55]. Furthermore, these experiments showed a much more intense activation of Smad3 in fetal vs. adult cells. In order to ascertain these differences we performed western analysis on the same gel by loading the same amount of total protein from both cell types from cell lysates collected 0.5 and 3 h after TGF-β treatment, where we observed maximum and minimum activation, respectively (Fig. 9B). We have found that Smad2 is equally expressed and activated in both fetal and adult cells. In contrast, the basal and activated levels of Smad3 were nearly double in fetal

A

H-89 Control

Time (h)

0

TGF-β

-

0.25 0.5 +

+

1

2

3

+

+

+

TGF-β Non Phosphorylated

Non Phosphorylated

lin+ sko

sko For

For

lin

H-8

9

89 +H ed

-β+

-β

rea t

TGF

Unt

TGF

H89

rol ted

Unt rea

ont

Con at.

B

it. C

trol

Phosphorylated

Pos

with maximum effect seen at 5–7 μM. On the other hand, H-89 was unable to alter the stimulatory effect of TGF-β on adult fibroblasts (Fig. 7B and Table 2). Furthermore, in order to ascertain PKA involvement in TGFβ-mediated inhibition, we tested if TGF-β is indeed able to activate PKA in our cell system. So, we treated fetal and adult skin fibroblasts with TGF-β for several time periods and performed an in vitro kinase assay. To this end we have used a non-radioactive kinase assay (PepTag Assay) and as can be seen in Fig. 8A, as soon as 15 min after treatment, TGF-β stimulates PKA activation in fetal cells, more than 3-fold compared to the untreated control. This activation remains elevated for at least 3 h after TGF-β addition. In contrast, no PKA activation in response to TGF-β was observed in adult fibroblasts (Fig. 8A). Interestingly, the PKA activation induced by TGF-β was comparable to that provoked by forskolin (Fig. 8B), an established PKA activator [53]. Furthermore, the PKA activation, induced by either TGF-β or forskolin was completely blocked by H-89 (Fig. 8B).

Phosphorylated

Neg

Fig. 7. The role of PKA in the inhibition of skin fibroblasts' proliferation by TGF-β. (A) Fetal skin fibroblasts were pre-incubated with increasing concentrations of H-89 for 45 min and then they were treated with TGF-beta (2.5 ng/ml); [3H]-Thymidine was also included in the medium. 48 h later DNA synthesis was estimated, as described under Materials and methods. (B) Adult skin fibroblasts were pre-incubated with or without 7 μM H-89 and stimulated with TGF-β (2.5 ng/ml). DNA synthesis was estimated, as in (A) Data are representative of four separate experiments (performed in quadraplicate).

Non Phosphorylated Phosphorylated

Fig. 8. TGF-β induces the up-regulation of PKA activity in fetal but not in adult skin fibroblasts. (A) Fetal and adult skin fibroblasts were treated with TGF-beta (2.5 ng/ml). At the indicated time points cell lysates were collected and subjected to an in vitro PKA assay, as described under Materials and methods. (B) Fetal fibroblasts were treated for 3 h with TGF-β (2.5 ng/ml) or for 30 min with forskolin (10 μM), in the presence or absence of H-89 (7 μM); purified PKA catalytic subunit was used as a positive control (negative control contained only buffer), while all other samples contain cell lysates from treated and untreated cells. PKA activity was estimated as in (A). Similar results were obtained from three individual experiments.

C.C. Giannouli, D. Kletsas / Cellular Signalling 18 (2006) 1417–1429

A Adult TGF-β (h):

0 0.5 1

Fetal

2

3 4

0 0.5 1

2 3 4

p-Smad2

Smad2 Tubulin

p-Smad3

Smad3

1425

level. To this end, total RNA was collected from cells treated with TGF-β–pre-incubated or not with H-89–and then was subjected to RT-PCR. As can be seen in Fig. 10A, the induction of p21CIP1/WAF1 and p15INK4B mRNA by TGF-β was annulled when the cells were pre-incubated with H-89. This indicates that PKA activation is required for the transcriptional regulation of these two cyclin-dependent kinase inhibitors by TGF-β. Next, cell lysates from TGF-β-treated and untreated cells have been collected and analyzed by immunoblotting. We have found that at the protein level also H-89 blocks effectively the TGF-βmediated up-regulation of p21CIP1/WAF1 in a dose-dependent manner (Fig. 10B). Similarly, it blocks the induction of p15INK4B as well (Fig. 10C), showing that PKA activation is necessary for the stimulation of both proteins by TGF-β. These results collectively indicate that in human fetal skin fibroblasts

Tubulin

A B TGF-β (h):

Adult 0

0.5 3

H-89 TGF-β

Fetal 0

0.5

3

-

+

+ -

+ +

p21CIP1/WAF1

p-Smad2 GAPDH

Smad2 p15 INK4B

Tubulin GAPDH p-Smad3

B Smad3

7 M

H-89 TGF-β

Tubulin

Fig. 9. TGF-β stimulates more intensely Smad3, and not Smad2, in fetal compared to adult skin fibroblasts. (A) Kinetics of Smad2 and Smad3 phosphorylation by TGF-β. Fetal and adult skin fibroblasts were stimulated with 2.5 ng/ml TGF-β for the indicated time points. Cell lysates from fetal and adult were subjected to western blot analysis by using antibodies against the phosphorylated forms of Smad2 (p-Smad2) and Smad3 (p-Smad3). The membranes were reprobed with antibodies against total Smad2 or Smad3, and tubulin, for assessing equal loading. (B) Comparison of basal and phosphorylated (by TGF-β) levels of Smad2 and Smad3 in fetal and adult skin fibroblasts. The experimental procedure was as in (A), with samples of equal protein amount to be loaded on the same gel. Data are representative of three separate experiments.

cells compared to adult ones. Having in mind that phosphorylated Smad3, and not Smad2, is needed for PKA activation, these differences may be connected with the differential activation of PKA in fetal and adult fibroblasts (Fig. 8). 3.8. Up-regulation of p21CIP1/WAF1 and p15INK4B by TGF-β is dependent on PKA Finally, we investigated if PKA activation is responsible for the stimulation of p21CIP1/WAF1 and p15INK4B by TGF-β in fetal fibroblasts. First, this has been studied at the transcriptional

p21CIP1/WAF1

Tubulin

C H-89 TGF-β p15 INK4B

Tubulin

Fig. 10. TGF-β stimulates p21CIP1/WAF1 and p15INK4B expression in fetal fibroblasts through PKA activation. (A) The cells were pre-incubated for 45 min with H-89 (7 μM) and then treated with TGF-β (2.5 ng/ml) for 12 h. Total RNA was collected and p21CIP1/WAF1 and p15INK4B mRNA expression was estimated by semiquantitative RT-PCR, as described under Materials and methods. The expression of GAPDH was used as a loading control. (B) Parallel cultures of fetal fibroblasts were pre-treated for 45 min with or without H-89 (5 or 7 μM) and then were treated with TGF-β (2.5 ng/ml) for 20 h. Cells lysates were subjected to Western analysis with an anti-p21CIP1/WAF1 antibody. Tubulin was used as a loading control. (C) Cells pre-treated with or without H-89 (7 μM) and stimulated with TGF-β as in Fig. 8B, were subjected to Western analysis by using an anti-p15INK4B antibody. Tubulin was again used as a loading control. Similar results were obtained from four individual experiments.

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TGF-β-mediated PKA activation leads to the induction of cyclin-dependent kinase inhibitors p21CIP1/WAF1 and p15INK4B and consequently to growth arrest at the G1 phase of the cell cycle. 4. Discussion TGF-β plays an important role in many aspects of development and the regulation of tissue homeostasis in adults. Wound healing represents a major homeostatic mechanism, aiming at the maintenance of the structural and functional tissue integrity. However, profound differences have been observed between fetuses and adults in their wound repair strategies, as the former heal with a minimal inflammatory response and the absence of contraction and scar formation [36,37]. In agreement with the above, it has been shown that fetal fibroblasts express in vitro a unique phenotype [56–58]. More specifically, concerning the response of fibroblasts from these two developmental stages to TGF-β, it has been reported that fetal fibroblast contractility is inhibited by this factor, in contrast to adult cells [59]. Furthermore, marked differences have been observed in the secretion of extracellular matrix components in response to TGF-β [37,60]. In this context, we have added one more difference between fetal and adult skin fibroblasts concerning their response to this factor: all TGF-β isoforms stimulate the proliferation of adult skin fibroblasts, while they strongly inhibit cells of fetal origin [39]. Here we present evidence on the mechanism responsible for this differential proliferative effect and, in particular, the decisive roles of (a) the autocrine action of FGF-2 and (b) the activation of PKA, for the stimulatory and inhibitory action of TGF-β, respectively. The above-mentioned differential response probably indicates varying roles of TGF-β in the regulation of tissue homeostasis in different developmental stages. However, these findings are also important for understanding the regulatory role of TGF-β on cell proliferation. TGF-β inhibits many cell types, such as epithelial, endothelial or hematopoietic cells [8,21] and the fact that several cancer cell lines tend to become resistant to this anti-proliferative effect, attributes to this factor also a tumor suppressor role [3,29,61,62]. On the other hand, the action of TGF-β on cells of mesenchymal origin has, in general, been found to be stimulatory. Accordingly, the mechanisms underlying its stimulatory and inhibitory actions have been studied in normal and transformed/cancer cells of different origin and under various culture conditions. In contrast, the cellular model presented here, i.e. fetal vs. adult normal human skin fibroblasts, grown under identical conditions, represents a useful tool for the investigation of the pathways responsible for the proliferative and the anti-proliferative effect of TGF-β on the same cell type. Concerning the stimulatory effect of TGF-β on different cell types, several studies have attributed this to an indirect, autocrine action [20,32–35]. In this direction, it has been previously suggested that the action of TGF-β in mesenchymal cells, like mouse embryonic fibroblasts or human fibroblasts and smooth muscle cells, is mediated by the secretion of PDGF

or PDGF-like molecules [20,33,34]. However, in these studies when neutralizing antibodies raised against PDGF were used to test the autocrine proliferative effect, their inhibitory action was modest. Even more, the antibodies used were either nonspecific [63] or gave similar percentage of inhibition to the control cultures [20]. Here, by using an effective, pan-specific, anti-PDGF neutralizing antibody we have excluded this possibility in human adult skin fibroblasts, as this antibody was unable to block the mitogenic effect of TGF-β, when compared to their respective control, i.e. untreated cultures (Fig. 2). In contrast, we have shown that TGF-β establishes an FGF-2 autocrine loop leading to the stimulation of adult skin proliferation, the latter being in accordance with results from human fibroblasts from other tissues, such as kidney or cornea [32,64]. In particular, we have shown that in adult skin fibroblasts TGF-β induces the production of the secreted (18 kDa) isoform of FGF-2 (Fig. 3A) and the secretion of FGF-2 from the cells (Fig. 3B). Finally, the mitogenic effect of TGF-β is abrogated by a neutralizing antibody against FGF-2 (Fig. 2). It is interesting to note that FGF-2 secretion in the conditioned medium starts from 1 h and is observed until 4 h after TGF-β addition. It seems that this secretion is independent of novel protein synthesis, as in the presence of the protein synthesis inhibitor cyclohexamide the release of FGF-2 is not inhibited (data not shown). This is further substantiated by western analysis of whole cell lysates, showing that the increase of the levels of the 18 kDa FGF-2 starts from 3 h after TGF-β treatment (Fig. 3A), implying that the observed secretion (Fig. 3B) is due to pre-existing FGF-2. However, the mechanism of FGF-2 synthesis and release after stimulation with TGF-β in human fibroblasts obviously requires further investigation. In addition, it would be interesting to compare, in fetal vs. adult skin fibroblasts, the role of (a) the extracellular matrix and, specifically, the proteoglycans where FGF-2 is bound [48,65] and (b) of FGF-binding proteins (FGF-BPs) that facilitate its release from the extracellular matrix, protect it from degradation and enhance its activity [66,67]. After FGF-2 binding to its respective membrane receptors several signaling molecules and pathways can be activated, most prominent being the PLCγ-Protein Kinase C (PKC), and the Ras–Raf–MEK–ERK pathways [48,68]. Pre-treatment of adult fibroblasts with the specific PKC inhibitor calphostin C did not affect the stimulation of DNA synthesis by TGF-β (Table 2), indicating that PKC is not involved in the TGF-β– FGF-2 autocrine loop for the proliferation of these cells. Consequently, we have studied the MEK–ERK signaling pathway and specifically ERK-1/2 phosphorylation, and have found that TGF-β induces a delayed ERK-1/2 activation starting from 1 h and reaching a maximum between 3 and 6 h after stimulation (Fig. 4A). Having in mind that FGF-2 stimulates a rapid and sustained ERK-1/2 phosphorylation (Fig. 4A), as well as the kinetics of FGF-2 release (Fig. 3B), this delay is in accordance with the presence of an autocrine mechanism. This is further substantiated by the inhibition of this activation by an anti-FGF-2 neutralizing antibody (Fig. 4B). Finally, two specific MEK inhibitors PD98059 and U0126 can inhibit not only ERK-1/2 phosphorylation (Fig. 4B and data not

C.C. Giannouli, D. Kletsas / Cellular Signalling 18 (2006) 1417–1429

shown), but also the induction of DNA synthesis by TGF-β, in a dose-dependent manner (Fig. 4C and Table 2). All the above clearly show that TGF-β stimulates DNA synthesis in adult human skin fibroblasts by an autocrine induction of FGF-2 and the subsequent stimulation of the MEK–ERK pathway. In agreement to the above, it has been shown in human lung fibroblasts that TGF-β provokes the autocrine induction of FGF-2, and subsequently the activation of ERK-1/2 and the induction of the transcription factor activator protein-1 (AP-1) [69], although this was not correlated with any mitogenic activity. Having in mind that FGF-2 binds with high affinity to the fibroblast growth factor receptor-1 (FGFR-1) [42] and that human skin fibroblasts in culture express only this type of FGF receptor [50], we aimed at understanding its role in TGF-β proliferative effect. Accordingly, we used siRNA to inhibit FGFR-1 and we have found that in cells with downregulated FGFR-1 expression TGF-β is unable to activate the MEK–ERK pathway and to stimulate DNA synthesis (Fig. 5). These data are in agreement with the effect of FGF-2 neutralizing antibody (Figs. 2 and 4), and they further indicate that FGFR-1 is required for the mitogenic effect of TGF-β in adult human skin fibroblasts. In this vein, it has been recently reported that in human smooth muscle cells PDGF-BB induces the secretion of FGF-2 that via FGFR-1 activates ERK-1/2 and finally DNA synthesis [41]. Accordingly, it appears that the establishment of an FGF-2–FGFR-1–ERK activation circuit is a common theme in the indirect mitogenic effect induced by several growth factors and in different cell types. As shown in Fig. 1, human fetal skin fibroblasts, although of mesenchymal origin are strongly inhibited by TGF-β. This inhibition is accompanied by an increase of the percentage of cells being arrested in the G1 phase of the cell cycle (Table 1). In addition, TGF-β does not lead to an induction of FGF-2 production and the secretion of this factor to the culture milieu (Fig. 3), or the activation of the MEK–ERK pathway (not shown), indicating the presence of distinct regulatory mechanisms of cell proliferation in response to TGF-β in fetal and adult fibroblasts. As TGF-β is a potent inhibitor of epithelial, endothelial and hematopoietic cells, the majority of the data on this inhibitory mechanism stem from studies on these cell types. In brief, TGF-β binding to its transmembrane receptors leads to formation of active Smad2/Smad4 or Smad3/Smad4 complexes which interact physically with the transcription factor Sp1 and, in cooperation with the co-activators CBP and p300, regulate the expression of the cyclin-dependent kinase inhibitors (CKIs) p21CIP1/WAF1 and p15INK4B [10,22–25,70]. In accordance to the above, we report here for the first time that TGF-β stimulates the expression of p21CIP1/WAF1 and p15INK4B also in normal fetal skin fibroblasts, both at the mRNA and protein levels (Fig. 6). No other CKIs, such as p27KIP1 or p16INK4A , were found to be up-regulated by TGF-β (data not shown). On the other hand, in adult cells we observed a minor up-regulation of p21CIP1/WAF1 and p15INK4B mRNA only at early time points (Fig. 6). However, at the protein level no p21CIP1/WAF1 or p15INK4B induction was observed at any time

1427

point tested, indicating that a more intense or extended mRNA induction may be needed for the up-regulation of these proteins. We have already shown that the inhibitory effect of TGF-β on fetal fibroblasts is annulled in the presence of curcumin [39], which is an inhibitor of several signaling pathways [51]. Accordingly, we studied those pathways known by the literature to be suppressed by curcumin and also to be stimulated by TGFβ, in various cell types. To this end, we used a battery of specific inhibitors for these pathways, shown in Table 2. From all these compounds used, it came out that only the specific PKA inhibitor H-89 was able to cancel the inhibitory effect of TGF-β in a dose-dependent manner (Fig. 7). In agreement, H-89 blocked the expression of p21CIP1/WAF1 and p15INK4B by TGFβ, both at the transcriptional and the translational level, in fetal fibroblasts (Fig. 10). These results, with the use of the kinase inhibitor H-89, were verified by a specific PKA assay, showing that TGF-β can indeed activate PKA activity in fetal, and not in adult, fibroblasts (Fig. 8A). In general, PKA activation is usually mediated by a rise in cAMP levels [71], while a cAMP-independent activation has also been reported, i.e. by the degradation of IκB, a repressor of NF-κB [72]. However, recently it has been shown in immortalized mink lung epithelial cells that the TGF-βmediated activation of PKA is not associated with alterations in the cAMP levels or even IκB degradation [54]. In contrast, a novel mechanism has been proposed for these cells, where TGF-β treatment leads to the formation of an activated Smad3/ Smad4 complex that can bind the regulatory subunit of PKA, thus releasing the catalytic subunit and activating target genes, such as p21CIP1/WAF1 [54]. These results are obviously in agreement with our data presented here in normal human fibroblasts, as only in fetal cells–that are inhibited by TGF-β– the latter can activate PKA and subsequently up-regulate the expression of p21CIP1/WAF1. Furthermore, it seems that an additional target of this pathway is the activation of another CKI, i.e. p15INK4B. Since it is reported that PKA is activated by TGF-β through a Smad3-dependent mechanism, we tested the activation of the Smad pathway by this factor in fetal and adult fibroblasts. We report here for the first time that the basal levels and maximum activation of Smad2 by TGF-β are similar between normal fetal and adult cells, whereas Smad3 basal levels are double in fetal cells. In addition, we observed a significantly more intense phosphorylation and activation of Smad3 by TGF-β in fetal fibroblasts (Fig. 9). Having in mind that activated Smad3, and not Smad2, is necessary for PKA activation [54], our data imply the presence of a threshold of Smad3 phosphorylation for the activation of PKA and the subsequent inhibition of cell proliferation. Furthermore, the above are in agreement with the essential role of Smad3 in the TGF-β-mediated antiproliferative effect, as shown by experiments in fibroblasts from Smad3-null mice [73]. Finally, it would be interesting to investigate the differential contribution of other co-partners in this cascade in fetal and adult cells. One possible target could be the members of the A-kinase anchoring proteins (AKAPs) [74,75] that are necessary for the proper localization of PKA in

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the cell, since these proteins were found to participate in the Smad3-dependent PKA activation [54]. Furthermore, concerning PKA involvement in the regulation of cell proliferation, it has been shown that activation of this kinase by cAMP leads to the inactivation of the Ras–Raf– MEK–ERK pathway by the phosphorylation of the Raf-1 protein at the “gatekeeper” Ser259 [76,77]. In addition, it has been reported that, in mouse embryonic fibroblasts, TGF-β can stimulate the association of the catalytic subunit of PKA with Raf-1 (as shown by immunoprecipitation studies) and this has been proposed to result in a delay in the EGF-stimulated cellular proliferation [21]. Accordingly, we tested if TGF-β treatment leads to phosphorylation of Raf-1 at Ser259 in human fetal skin fibroblasts via PKA activation. However, this does not seem to be the case. Western analysis by using specific antibodies has not revealed any alterations in Raf-1 phosphorylation on Ser259 or ERK-1/2 activation, after stimulation of these cells with TGF-β, in the presence or absence of the PKA inhibitor H-89 (not shown). In conclusion, here we present data on the differential proliferative response of fetal and adult human skin fibroblasts to TGF-β as a result of the activation of distinct signaling cascades. In fetal fibroblasts, TGF-β up-regulates the activation of PKA and hence induces the expression of p21CIP1/WAF1 and p15INK4B , leading to growth arrest. In contrast, in adult cells, no PKA activation is observed, while TGF-β treatment leads to an autocrine loop, via the release of FGF-2 and, through FGFR-1, to the subsequent induction of the MEK–ERK pathway, and finally to growth stimulation. The comparison of the responses of fetal vs. adult normal skin fibroblasts could also represent a useful tool for the study of the different mechanisms that regulate the bimodal effect of TGF-β on cell proliferation. Acknowledgements We would like to thank Profs. A. Moustakas, D. Kardassis and E. Kolettas for generously providing antibodies and cDNAs, and Dr. S. Psarras for his help with the ELISA experiments. The continuous help of the members of our laboratory P. Handris and Dr. H. Pratsinis is also greatly acknowledged. This work was partially supported by the EURODISC project from the European Union (QLK6-CT-2002-02582). References [1] E. Piek, C.H. Heldin, P. ten Dijke, FASEB J. 13 (15) (1999) 2105. [2] P. ten Dijke, M.J. Goumans, F. Itoh, S. Itoh, J. Cell. Physiol. 191 (1) (2002) 1. [3] A. Moustakas, K. Pardali, A. Gaal, C.H. Heldin, Immunol. Lett. 82 (1–2) (2002) 85. [4] J.J. Letterio, A.B. Roberts, Annu. Rev. Immunol. 16 (1998) 137. [5] F. Verrecchia, A. Mauviel, J. Invest. Dermatol. 118 (2) (2002) 211. [6] G.C. Blobe, W.P. Schiemann, H.F. Lodish, N. Engl. J. Med. 342 (18) (2000) 1350. [7] A.B. Roberts, E. Piek, E.P. Bottinger, G. Ashcroft, J.B. Mitchell, K.C. Flanders, Chest 120 (1 Suppl.) (2001) 43S. [8] J. Massague, S.W. Blain, R.S. Lo, Cell 103 (2) (2000) 295. [9] C.H. Heldin, K. Miyazono, P. ten Dijke, Nature 390 (6659) (1997) 465.

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