Báo cáo khoa học: Thrombin-mediated impairment of fibroblast growth factor-2 activity doc

Thrombin-mediated impairment of fibroblast growth factor-2 activity Pierangela Totta 1, *, Raimondo De Cristofaro 2, *, Claudia Giampietri 3 , Maria S. Aguzzi 1 , Debora Faraone 1 , Maurizio C. Capogrossi 1 and Antonio Facchiano 1 1 Laboratorio di Patologia Vascolare, IDI-IRCCS, Istituto Dermopatico dell’Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy 2 Institute of Internal Medicine, Haemostasis Research Center, Catholic University School of Medicine, Rome, Italy 3 Department of Histology and Medical Embriology, University of Rome ‘Sapienza’, Italy Fibroblast growth factor (FGF)-2 belongs to the 23-member family of FGFs [1]. It is known as one of the most potent angiogenic factors controlling embry- onic development [2], tissue remodeling [3], stem cell physiology [4] and tumor growth [5]. FGF-2 activity is finely modulated at several levels, and recent evidence shows that different FGF-2 concentrations may exert opposing effects [6]. Studies have shown that the interaction of several molecules with this growth factor [7–9] or with its receptors [10,11] participates in the control of FGF-2 activity. A few studies have examined FGFs proteolytic degradation [12–15]. For example, FGF-2 degradation by the zinc-endopro- tease neprilysin has been recently demonstrated. This Keywords cell proliferation; digestion; fibroblast growth factor-2; maturation; thrombin Correspondence A. Facchiano, Laboratorio di Patologia Vascolare, Istituto Dermopatico dell’Immacolata, IDI-IRCCS, Via Monti di Creta 104, 00167 Rome, Italy Fax: +39 06 6646 2430 Tel: +39 06 6646 2431 E-mail: a.facchiano@idi.it *These authors contributed equally to this work (Received 6 October 2008, revised 19 March 2009, accepted 6 April 2009) doi:10.1111/j.1742-4658.2009.07042.x Thrombin generation increases in several pathological conditions, including cancer, thromboembolism, diabetes and myeloproliferative syndromes. During tumor development, thrombin levels increase along with several other molecules, including cytokines and angiogenic factors. Under such conditions, it is reasonable to predict that thrombin may recognize new low-affinity substrates that usually are not recognized under low-expression levels conditions. In the present study, we hypothesized that fibroblast growth factor (FGF)-2 may be cleaved by thrombin and that such action may lead to an impairment of its biological activity. The evidence collected in the present study indicates that FGF-2-induced proliferation and chemo- taxis ⁄ invasion of SK-MEL-110 human melanoma cells were significantly reduced when FGF-2 was pre-incubated with active thrombin. The inhibi- tion of proliferation was not influenced by heparin. Phe-Pro-Arg-chlorom- ethyl ketone, a specific inhibitor of the enzymatic activity of thrombin, abolished the thrombin-induced observed effects. Accordingly, both FGF-2-binding to cell membranes as well as FGF-2-induced extracellular signal-regulated kinase phosphorylation were decreased in the presence of thrombin. Finally, HPLC analyses demonstrated that FGF-2 is cleaved by thrombin at the peptide bond between residues Arg42 and Ile43 of the mature human FGF-2 sequence. The apparent k cat ⁄ K m of FGF-2 hydroly- sis was 1.1 · 10 4 m )1 Æs )1 , which is comparable to other known low-affinity thrombin substrates. Taken together, these results demonstrate that throm- bin digests FGF-2 at the site Arg42-Ile43 and impairs FGF-2 activity in vitro, indicating that FGF-2 is a novel thrombin substrate. Abbreviations ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; HMW, high molecular weight; HUVEC, human umbilical vein endothelial cell line; LMW, low molecular weight; PAR, protease-activated receptor; PPACK, Phe-Pro-Arg-chloromethyl ketone; TRAP, thrombin receptor-activating peptide. FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS 3277 metalloprotease was found to cleave the Leu135- Gly136 peptide bond of FGF-2, severely inhibiting its angiogenic activity, as demonstrated in a murine cor- neal pocket angiogenesis model [12]. In addition, more recently, high molecular weight (HMW) FGF-2 was shown to be cleaved by thrombin, and its degradation product stimulated endothelial cell migration and pro- liferation in a similar manner to low molecular weight (LMW) FGF-2 [13]. Moreover, fibrinogen and fibrin, by a direct interaction, are known to protect FGF-2 from in vitro proteolytic degradation induced by tryp- sin and chymotrypsin [16,17]. Finally, different FGF-2 fragments inhibit FGF-2 [18,19], further suggesting that proteolytic processing of FGF-2 may represent an endogenous way to modulate its activity. Thrombin is a serine protease generated from its zymogen precursor prothrombin after endothelial cell damage and induction of the coagulation cascade [20]. Its activation is known to be increased in thrombo- embolism [21], diabetes [22] and cancer [23]. Thrombin pro-coagulant activity converts fibrinogen to fibrin monomer, which then polymerizes to form the fibrous matrix of blood clots [24]. Moreover, thrombin cleaves protease-activated receptor (PAR)-1 and PAR-4, which are expressed on human platelet membranes, activating their hemostatic properties [25]. In addition to these clot-promoting activities, thrombin down-regulates its own generation through activation of the protein C pathway. Activated protein C inactivates cofactors Va and VIIIa, thereby blunting further thrombin genera- tion [26]. Thrombin also participates directly in its final inhibition and clearance from the circulation by specifically recognizing the serine protease inhibitors (serpins) antithrombin and heparin cofactor II [27]. Thrombin interaction with PAR-1 and PAR-4 does not activate only hemostatic functions. Indeed, these receptors are expressed on the membrane of different cell types, including fibroblast [27], endothelial [28] and cancer cells [29], and their activation enhances cytokine release, cell permeability and cell growth. Furthermore, thrombin recognizes several other membrane receptors and substrates, such as platelet glycoprotein Ib and glycoprotein V [30], and additional noncanonical thrombin substrates, even in the intracellular compart- ment, have been identified [31,32]. Taken together, these data indicate that thrombin recognizes a complex substrates network with several biological functions, in addition to the classical clot- promoting effects, and prompted us to investigate additional substrates not directly involved in blood coagulation. In the present study, we show, for the first time, that thrombin digests the 18 kDa LMW isoform of human FGF-2, modulating its biological activities such as in vitro cell proliferation and chemo- taxis induction. In addition, we also identified the cleavage site on FGF-2. Results FGF-2-induced proliferation of SK-MEL-110 is inhibited by thrombin Human metastatic melanoma cell line SK-MEL-110 was chosen as a model to test the FGF-2 mitogenic and chemotactic activity in vitro. Figure 1A shows that 1 h of thrombin pre-incubation significantly reduces FGF- 2-induced cell growth as a function of concentration in 48 h proliferation assays. The inhibitory action reached a plateau at 0.1 nm thrombin; therefore, all the experi- ments were carried out at this thrombin concentration, which corresponds to 0.01 UÆmL )1 . Figure 1A shows that thrombin activity was blocked with the irreversible selective thrombin inhibitor Phe-Pro-Arg-chloromethyl ketone (PPACK). As a control, thrombin and PPACK, alone or mixed, do not affect cell proliferation under these experimental conditions (Fig. 1A, inset). Time-course proliferation experiments were then car- ried out. Pre-treating FGF-2 with thrombin for 1 h was sufficient to completely inhibit the mitogenic effect of FGF-2 at 48 and 72 h of proliferation (Fig. 1B). The experiments shown in Fig. 1 were performed by pre- incubating FGF-2 with active thrombin; next, before exposing cells to this mixture, PPACK was added to block the enzyme. This protocol was chosen to exclude the possibility that thrombin enzymatic action may interfere with cell growth by cleaving and activating PARs receptors, which are known to be expressed in SK-MEL-110 cells (data not shown) and in other mela- noma cells [29]. To further rule out this possibility, we exposed SK-MEL-110 cells to the action of specific PARs agonists, namely thrombin receptor-activating peptide (TRAP)-1 and TRAP-4, to show that specifi- cally activating thrombin-receptors does not itself determine any inhibition of cell-proliferation. Fig- ure 2A shows that the specific agonists TRAP-1 alone and TRAP-4 alone induced some proliferation of mela- noma cells in the absence of other mitogenic stimuli, whereas they did not affect the FGF-2-induced prolifer- ation (Fig. 2B). These data allowed us to exclude the possibility that thrombin agonism may per se inhibit SK-MEL-110 proliferation in vitro. Therefore, to mimic more closely the conditions occurring under in vivo conditions, cells were directly exposed to the mixture containing FGF-2 and active thrombin. Figure 3 shows that, under these conditions, SK-MEL-110 proliferate significantly less than cells exposed to FGF-2 alone and FGF-2 is a thrombin substrate P. Totta et al. 3278 FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS that such inhibition was absent in the presence of inac- tive thrombin (i.e. thrombin pre-incubated with PPACK), further confirming that the enzymatic activity of thrombin inhibits the mitogenic action of FGF-2. To evaluate the influence of heparan sulfate proteogly- cans on thrombin susceptibility of FGF-2, the effect of heparin was investigated in proliferation assays. Hepa- rin alone (at 10 and 50 nm) did not influence the spon- taneous proliferation of SK-MEL-110 (Fig. 4A). Thus, the effect of heparin (at 50 nm) was tested in the pres- ence of FGF-2 and thrombin. Figure 4B shows that heparin reduces the mitogenic activity of FGF-2; how- ever, the inhibitory effect induced by thrombin was maintained both in the absence and in the presence of heparin, indicating that, at these doses, heparin does not influence the observed thrombin–FGF-2 interplay. As a specificity control, similar experiments were car- ried out on a different cellular model. Figure 5 shows that, similar to melanoma cells, the mitogenic effect of FGF-2 was inhibited by thrombin (0.1 nm) in the primary human umbilical vein endothelial cell line (HUVEC). Taken together, these findings indicate that the mitogenic activity of FGF-2 is controlled by throm- bin and that FGF-2 may be a thrombin substrate. FGF-2-induced SK-MEL-110 invasion/migration on different matrices We then investigated whether thrombin modulates the ability of FGF-2 to induce cell invasion through differ- ent matrices. Invasion assays were carried out in vitro, in modified Boyden chambers, on filters coated either with vitronectin, collagen IV or fibronectin. Migration 0 h 48 h 25 50 75 100 A B Cell number (%) + + + – – – FGF-2 Thrombin (n M ) 0.1 PPACK + 0.1 – – – – – – – – – 0 25 50 75 100 Cell number (%) – FGF-2 0.001 Thrombin (n M) 0.01 1– +++++ 0.1– – PPACK +++++ * * ** ** Cell number (%) 0 100 300 500 700 900 1100 1300 0 24 48 72 Time ( h ) * ** BSA BSA + PPACK Thrombin Thrombin + PPACK FGF2 + PPACK FGF2 + Thrombin + PPACK Fig. 1. Dose–response and time-course proliferation assays. (A) Dose–response proliferation assay: SK-MEL-110 (4 · 10 4 ) cells were seeded in six-well plates and grown for 24 h at 37 °C, 5% CO 2 in complete medium. Medium was then replaced and cells were starved overnight with incomplete medium. Subsequently, cells were stimulated for 48 h with medium 0.1% BSA, FGF-2 (10 ngÆmL )1 , 0.6 nM) or thrombin (0.1 nM) pre-incubated for 1 h at 37 °C. Other cells were stimulated with medium 0.1% BSA con- taining FGF-2 alone (10 ngÆmL )1 , 0.6 nM), thrombin alone (0.1 nM) or FGF-2 (10 ngÆmL )1 , 0.6 nM) with different thrombin concentra- tions (0.001, 0.01, 0.1 and 1 n M), pre-incubated for 1 h at 37 °C and then supplemented with PPACK (50 n M) to block the enzymatic activity of thrombin. The mitogenic effect of FGF-2 is significantly reduced in the presence of different concentrations of thrombin (*P < 0.005; **P < 0.001). Neither thrombin alone nor thrombin with PPCAK nor PPACK alone influenced SK-MEL-110 growth with respect to control (A, inset). Data are expressed as a percentage of cell number versus cells treated with FGF-2 alone (100% corre- sponds to 1.9 · 10 5 cells). Data reported are the mean ± SEM of five independent experiments carried out in duplicate. (B) Time- course proliferation assay: SK-MEL-110 (4 · 10 4 ) cells were seeded and grown for 24 h in complete medium. Medium was then replaced and cells were starved overnight with incomplete med- ium. Subsequently, cells were stimulated for 24, 48 and 72 h with the indicated stimuli, with doses as described in (A). FGF-2-induced SK-MEL-110 proliferation is inhibited when FGF-2 is pre-incubated with thrombin alone for 1 h and then thrombin is blocked with PPACK (*P < 0.0005; **P < 0.05). Both thrombin alone and throm- bin + PPACK do not influence SK-MEL-110 cell proliferation com- pared to BSA and BSA + PPACK. Data are expressed as a percentage of cell number versus cell number at t 0 (100% corre- sponds to 1.9 · 10 5 cells). Data reported are the mean ± SEM of five independent experiments carried out in duplicate. P. Totta et al. FGF-2 is a thrombin substrate FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS 3279 assays were also carried out using gelatin-coated filters. The invasion ⁄ chemoattractant properties of FGF-2 were markedly and significantly reduced in the presence of thrombin (0.1 nm, corresponding to 0.01 UÆmL )1 ) on all tested matrices (Fig. 6A–D), with different potency. We observed approximately 50% inhibition on vitronectin (Fig. 6A), approximately 30% inhibition on collagen IV and fibronectin (Fig. 6B,C) and approx- imately 40% inhibition on gelatin (Fig. 6D). These data indicate that thrombin strongly inhibits the chemotactic and invasion properties of FGF-2. FGF-2-dependent extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase phosphorylation is reduced by thrombin Mitogen-activated protein kinases and ERK1 ⁄ 2 are important transducers of mitogenic and differentiation signals induced by FGF-2 and are often altered in mel- anoma progression [33]. We therefore investigated whether FGF-2 pre-incubation with thrombin alters the ability to activate ERK1 ⁄ 2 in our experimental model. Figure 7A shows that ERK1 ⁄ 2 phosporylation induced by FGF-2 is reduced when FGF-2 is incu- bated with thrombin. The lower band corresponding to the 42 kDa form (i.e. ERK2) was found to be reduced, as also revealed in densitometry analysis (Fig. 7B), suggesting that thrombin incubation impairs the ability of FGF-2 to signal toward one of the key 0 h 48 h – 0 50 100 150 200 AB Cell number (%) 0 50 100 150 200 Cell number (%) FGF-2 TRAP-1 TRAP-4 – + – – – – – – – + – * + – + – – – + – – + + – – – – FGF-2 TRAP-1 TRAP-4 0 h 48 h Fig. 2. TRAP-treatment of SK-MEL-110 cells. SK-MEL-110 (4 · 10 4 ) cells were seeded and grown for 24 h in complete medium as described in Fig. 1. Medium was then replaced and cells were starved overnight with incomplete medium. Then, cells were directly exposed (48 h) to FGF-2 (10 ngÆmL )1 ) in the presence or the absence of 5.7 lM TRAP-1 or TRAP-4. (A) TRAP-1 and TRAP-4 induced some prolifera- tion as compared to control (*P < 0.05). The mitogenic effect of FGF-2 was not influenced by TRAP-1 or TRAP-4 (B). Data are expressed as a percentage of cell number versus cells treated with FGF-2 (100% corresponds to 1.9 · 10 4 cells). The data reported are the mean ± SEM of five independent experiments carried out in duplicate. 0 20 40 60 80 100 Cell number (%) FGF-2 Thrombin PPACK 0 h 48 h – + ++ + + – + + + – +– + – + – – – – – * Fig. 3. Influence of PPACK-thrombin in SK-MEL-110 proliferation. SK-MEL-110 (4 · 10 4 ) cells were seeded and grown for 24 h in complete medium. Medium was then replaced and cells were starved overnight with incomplete medium. Then, cells were directly exposed for 48 h to FGF-2 (10 ngÆmL )1 ) in the presence or absence of thrombin (0.1 n M) pre-incubated or not with PPACK (50 n M) or PPACK alone (50 nM). As a control, cells were stimulated with thrombin alone (0.1 n M) or PPACK alone (50 nM). FGF-2- induced proliferation was measured after 48 h stimulation with thrombin in the presence or in the absence of PPACK. PPACK blocks thrombin enzymatic activity and reverts the thrombin anti- mitogenic effect (*P < 0.01), suggesting that FGF-2 is degraded by thrombin protease function. Data are expressed as a percentage of cell number versus FGF-2 (100% corresponds to 1.9 · 10 5 cells). The data reported are the mean ± SEM of five independent experi- ments carried out in duplicate. FGF-2 is a thrombin substrate P. Totta et al. 3280 FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS intracellular signal pathways mediating the biological activity of FGF-2 [34–36]. Proteolytic degradation of FGF-2 induced by thrombin According to data reported above, we hypothesized that FGF-2 may be directly cleaved by thrombin. Its degradation was then investigated by HPLC analysis (Fig. 8A) and by Western blotting (see, Fig. S1). Fig- ure 8A shows the elution chromatogram of FGF-2 alone and FGF-2 incubated with thrombin for 30 min. Incubation of FGF-2 with thrombin lowered the peak corresponding to the full length FGF-2, eluting at 23.5 min, and induced the appearance of the peak eluting at approximately 13.6 min, corresponding to a proteolytic fragment of FGF-2. Area values corre- sponding to the full length FGF-2 were then fitted according to Eqn (1) (see Experimental procedures), which allows calculation of the kinetic rate constant of the peak’s area decay, equal to 4.8 ± 0.3 · 10 2 min )1 . This value, under our experimental conditions, reflects an apparent k cat ⁄ K m of FGF-2 hydrolysis equal to 1.1 · 10 4 m )1 Æs )1 , which is comparable to the value of other low-affinity thrombin substrates, such as zymo- gen protein C or thrombin-activatable fibrinolysis inhibitor [37,38]. As shown in the (Fig. S1), FGF-2 incubated with PPACK appears as a main unique band (arrowhead, 18 kDa), whereas one immunoreac- tive additional band appears in the FGF-2 incubated with thrombin for 1 h (1 : 1 molar ratio) with an apparent molecular weight of 15 kDa. FGF-2 incu- bated with inactive thrombin (i.e. thrombin pre-incu- bated with PPACK) shows no additional bands compared to basal conditions. We then confirmed the observed biochemical degradation of FGF-2 induced by thrombin with a biological assay. Structural degra- dation and loss of function were then investigated as a 0 h 24 h FGF-2 Thrombin * 0 25 50 75 100 Cell number (%) – – + + + +– –– – Fig. 5. HUVEC proliferation assay. HUVEC (8 · 10 4 ) were seeded and grown for 24 h in complete medium. Medium was then replaced and cells were starved overnight with incomplete med- ium. HUVEC growth was examined after 24 h of stimulation with FGF-2 (10 ngÆmL )1 ) in the presence or in the absence of thrombin (0.1 n M). The mitogenic effect of FGF-2 is significantly reduced in the presence of thrombin and thrombin alone has no effect on HUVEC proliferation. (*P < 0.01). Data are expressed as a percent- age of cell number versus FGF-2 (100% corresponds to 12.7 · 10 4 cells) and are the mean ± SEM of five independent experiments carried out in duplicate. 0 25 50 75 100 Cell number (%) –Thrombin +Thrombin FGF-2 Heparin (n g ·mL –1 ) – – – + 50 + * * 48 h 0 h 10 50 – 0 25 50 75 100A B FGF-2 Heparin (ng·mL –1 ) – – – – – Fig. 4. Heparin does not influence the effect of thrombin on FGF-2. SK-MEL-110 (4 · 10 4 ) cells were seeded and grown for 24 h in complete medium. Medium was then replaced and cells were starved overnight with incomplete medium. Cells were stimulated for 48 h with two different heparin concentrations (10 and 50 ngÆmL )1 ). At either dose, heparin alone does not influence spon- taneous SK-MEL-110 growth (A); FGF-2-induced proliferation is par- tially affected by 50 n M heparin; however, heparin does not influence the inhibitory effect of thrombin. Indeed, thrombin pre- incubation inhibits FGF-2 activity similarly both in the absence and in the presence of heparin (B) (*P < 0.05). Data are expressed as a percentage of cell number versus FGF-2 (100% corresponds to 1.9 · 10 5 cells) and are the mean ± SEM of five independent experiments carried out in duplicate. P. Totta et al. FGF-2 is a thrombin substrate FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS 3281 function of time of exposure to thrombin. Figure 8B shows that increasing times of FGF-2 ⁄ thrombin incu- bation strongly reduced both the integrity of FGF-2 (expressed as a percentage of peak area on the chro- matogram) as well as its mitogenic action (expressed as a percentage of proliferation). After 60 min of incuba- tion, both the integrity and function of FGF-2 were markedly reduced to an approximately residual 20% of that at t 0 , with an almost complete loss of signal after 120 min of incubation. The strong loss of func- tion and loss of integrity observed after 60 min of incubation agree with the almost complete inhibition of mitogenic activity of FGF (Fig. 1B) carried out under similar experimental conditions (1 h of incuba- tion of FGF-2 and thrombin). FGF-2 binding to cells was then investigated in a cytofluorimetric assay. Figure 8C shows that FGF-2 binding to cells (detected via a primary antibody recognizing FGF-2 and a secondary fluorescent anti- body) is lowered to control levels when FGF-2 is pre- treated with thrombin for 1 h. These data indicate that FGF-2 pre-treated with thrombin diminishes binding to cell membranes, explaining, at least in part, the observed impairment of biological activity. Cleavage site determination Additional investigations were then carried out to iden- tify the FGF-2 site cleaved by thrombin. The fragment eluting at 13.6 min (Fig. 8A) was sequenced and revealed a N-terminal sequence of I-H-P-D-G-R-V-D, corresponding to the fragment Ile43 to Asp50 of mature FGF-2. As expected, the N-terminal sequence of undigested FGF-2 was sequenced as M-A-A-G-S-I- T, corresponding to the reported N-terminal sequence of mature human FGF-2 (accession number P09038). These experiments show that thrombin cleaves FGF-2 at the peptide bond between Arg42 and Ile43, releasing the N-terminal segment from Met1 to Arg42 and the remaining C-terminal fragment from the intact mole- cule. We then investigated whether FGF-2 shares sequence homology with known thrombin-recognized cleavage sites. The sequence alignment reported in Table 1 shows structural similarities of several known thrombin cleavage sites to that of the site found on FGF-2 in the present study. Beside the invariant argi- nine (R) residue at the P1 position, common residues are present at any position, specifically the aromatic residues phenylalanine (F), tyrosine (Y) and tryptophan VitronectinAB CD 0 20 40 60 80 100 Cells/field (%) FGF-2 Thrombin Collagen IV 0 20 40 60 80 100 Gelatin 0 20 40 60 80 100 Fibronectin 0 20 40 60 80 100 Cell/field (%) FGF-2 Thrombin – – + + + +– –– – + + + +– – – – + + + +– – – – + + + +– – Fig. 6. FGF-2-induced SK-MEL-110 inva- sion ⁄ migration in modified Boyden cham- bers. The invasion ⁄ migration properties of FGF-2 is reduced in the presence of throm- bin on different protein matrices, namely vitronectin (A), collagen IV (B), fibronectin (C) and gelatin (D). Data are expressed as a percentage of cell number ⁄ field versus cells exposed to FGF-2, (100% corresponds to 49 cells per field for vitronectin, 22 cells per field for collagen IV, 30 cells per field for fibronectin and 41 cells per field for gelatin). The data reported are the mean ± SEM of three independent experiments carried out in duplicate. FGF-2 is a thrombin substrate P. Totta et al. 3282 FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS (W) at P3; the leucine (L) and isoleucine (I) residues at P2; and proline (P) residues at the P3¢ position. Discussion FGF-2 is a multi-function factor with a key role in cell proliferation and tissue differentiation. It is mainly bound to low-affinity receptors (heparan sulfates) and stored on the membranes and the extracellular matrix; low levels of FGF-2 are also found circulating in the blood [39]. Thrombin cleaves the coagulation cascade substrates and binds and cleaves PARs receptors [25,29], as well as other membrane-bound substrates such as platelet glycoprotein V [30]. It circulates either in inactive form (prothrombin) or at low concentration in active form, subsequent to vascular damage and activation of the coagulation cascade. In particular, within 45 s to 5 min after venipuncture, active thrombin appears ($3ngÆmL )1 , 0.08 nm) and, after 2–10 min, further thrombin generation occurs, result- ing in clotting after 15–27 min at a thrombin concen- tration of 40–50 ngÆmL )1 (1–2.4 nm) [40]. In the present study, we hypothesized that FGF-2 may be cleaved by thrombin when either thrombin or FGF-2 expression levels strongly increase. This hypothesis stems from the observation that thrombin levels increase and coagulation is activated in several physio- pathological conditions [41,42]. During cancer, levels of thrombin, FGF-2 and several other factors increase [43,44]; therefore, we hypothesized that, under such conditions, low-affinity substrates, which usually are only marginally hydrolyzed, may be recognized and digested as a part of a feedback control mechanism. FGF-2 is present in four isoforms: three HMW forms (22, 22.5 and 24 kDa), predominantly localized in the nucleus, and one LMW form (18 kDa), mainly present in the cytoplasm and on the membranes. A recent study showed that thrombin is able to cleave HMW FGF-2, generating a fragment somewhat simi- lar to the LMW FGF-2. Indeed, similar to LMW, the cleaved form stimulates endothelial cell migration and proliferation [13]. By contrast, in the present study, we assessed whether the real LMW FGF-2 is recognized by thrombin; indeed, the real LMW FGF-2 is the cir- culating form and therefore the form naturally exposed to thrombin [39]. Thrombin at a dose of 0.1 nm (i.e. the level reached in vivo after vascular damage [45]) strongly reduced the biological functions of FGF-2 and such inhibitory effects were abolished by blocking the enzymatic activity of thrombin with its selective inhibitor PPACK, indicating that the enzymatic action of trhombin was involved. The presence of heparin did not change the observed effects. Thrombin also decreased binding of FGF-2 to cell membranes, as well as FGF-2-dependent ERK2 phosphorylation, one of the main signal-pathways mediating FGF-2 activity. Finally, HPLC analysis indicated a thrombin-depen- dent digestion, with kinetics matching the functional impairment of FGF-2. The inhibitory effects of throm- bin on FGF-2, as observed in the present study on the human metastatic melanoma cell line SK-MEL-110, were also confirmed in additional proliferation assays carried out using human endothelial cells (HUVEC). The k cat ⁄ K m value of FGF-2 hydrolysis revealed a relatively low specificity for thrombin; however, the high concentrations of active thrombin attained in vivo during the activation of coagulation [45] may be suffi- cient to sustain significant FGF-2 cleavage. Although conclusive in vivo evidence is lacking, these data sug- gest that thrombin-dependent FGF-2 cleavage may occur under physiological ⁄ pathological conditions with enhanced thrombin generation, such as in athero- thrombosis, or with elevated levels of FGF-2 and thrombin, such as in cancer. The cleavage site identi- fied between Arg42 and Ile43 of the mature FGF-2 1.5 p-ERK1/2 A B Total ERK1/2 FGF-2 Thrombin – + + – + + – – 0 0.5 1.0 p-ERK2 / total ERK2 Fig. 7. FGF-2-induced SK-MEL-110 ERK ⁄ MAPK phosphorylation in the presence of active thrombin. SK-MEL-110 (4 · 10 4 ) cells were seeded and grown for 24 h in complete medium. Medium was then replaced and cells were starved overnight with incomplete medium. These cells were stimulated for 10 min with FGF-2 (10 ngÆmL )1 ), thrombin (0.1 nM) or FGF-2 with thrombin pre-incu- bated for 1 h at 37 °C and then added PPACK (50 n M) to block thrombin enzymatic activities. Next, cells were lysated and Western blotting analysis was performed to evaluate ERK1 ⁄ 2 phosporylation. (A) Active thrombin in the presence of FGF-2 (lane FGF-2 + thrombin) significantly reduces ERK-2 (42 kDa) phosphory- lation (one representative experiment). (B) Densitometry analysis of three separate experiments. P. Totta et al. FGF-2 is a thrombin substrate FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS 3283 sequence is close to the Asp57 residue, initiating the FREG fragment of FGF-2, which was recently demon- strated to strongly modulate FGF-2 activity [19]. We hypothesize that proteolytic degradation as well as the release of active fragments may represent, at least in part, a feedback regulatory mechanism for modulating the angiogenic properties of FGF-2 when FGF-2 levels increase. The reported proteolytic effect of thrombin on human matrix proteins, such as vitronectin, fibronectin Fig. 8. Thrombin-induced FGF-2 structural and functional impair- ment. (A) FGF-2 degradation investigated by HPLC. The lower chro- matogram reports the elution profile of FGF-2 alone; the upper chromatogram reports the elution profile of FGF-2 incubated with thrombin for 30 min. In the presence of thrombin, the peak eluting at 23.5 min is reduced and an additional peak appears after 13.6 min of elution, corresponding to the degradation fragment. (B) The structural impairment parallels the functional impairment of FGF-2 in the presence of different time points of thrombin incuba- tion. Structural impairment is expressed as time-dependent decrease of the peak eluting at 23.5 min, whereas functional impairment is expressed as the time-dependent loss of a mitogenic effect on SK-MEL-110 cells. Structural and functional impairment are approximately 50% at 30 min, approximately 80% at 60 min and almost complete after 120 min of incubation. (C) FGF-2 binding to SK-Mel-110 cells. SK-MEL-110 (2 · 10 5 ) cells were seeded and grown for 24 h in complete medium. Medium was then replaced and cells were starved overnight with incomplete medium. These cells were exposed to FGF-2 (50 ngÆmL )1 ) or to thrombin (0.1 nM) or to FGF-2 previously incubated with thrombin for 1 h at 37 °C (50 ngÆmL )1 and 0.1 nM respectively) FGF-2 binding to cells, detected with a primary antibody and a secondary fluorescent anti- body, was then measured by flow cytometry. The peak on the right, corresponding to the peak at higher fluorescence, is increased and enlarged toward the right-hand side (i.e. higher fluo- rescence) in cells exposed to FGF-alone compared to control cells, whereas it is lowered to the control level in cells exposed to FGF-2 pre-incubated with thrombin. One representative graph from three experiments is reported. 80 100 80 100 FGF-2 peak area (%) 60 40 60 40 Cell number (%) Time (min) Absorbance 214 nm 5 1015202530 Time (min) 30 20 0 20 0 60 90 120 FGF-2 FGF-2 + Thrombin No FGF-2 Counts FL1 log 10 0 10 1 10 2 0 A B C Table 1. P3–P3¢ sequence of the most common thrombin substrates. The structural similarities of FGF-2 to other known thrombin sub- strates are indicated in bold. Substrate Position Residue no. Swiss-Prot database entryP3 P2 P1 P1¢ P2¢ P3¢ FGF-2 FLRI HP 40–45 P09038 Fibrinogen S A R G H R 12–17 P02675 PAR-4 A P R GYP 45–50 Q96RI0 Factor V (1) G IRS F R 737–742 Q15430 Factor V (2) YL RS N N 1571–1576 Q15430 Factor VIII Q IRS V A 389–394 P00451 Platelet glycoproteinV G P R GPP 474–479 P40197 Carboxypeptidase B2 (TAFI) S P RA S A 112–117 Q96IY4 Vitronectin W G R T S A 302–307 P04004 FGF-2 is a thrombin substrate P. Totta et al. 3284 FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS and murine collagen IV [46,47], might indicate dual effects on cell migration ⁄ invasion. Indeed, on the one hand, thrombin digests FGF-2, reducing its angiogenic and invasive action; on the other hand, thrombin degrades matrix proteins, facilitating cell invasion. The net effect measured in vitro in the present study was a strong reduction of FGF-2-induced cell migra- tion ⁄ invasion. Preliminary results indicate that, at simi- lar concentrations, thrombin recognizes and degrades at least one other angiogenic growth factor, namely platelet-derived growth factor-BB, suggesting that increased thrombin concentrations may modulate angiogenesis and cell chemotaxis ⁄ invasion at different levels. A number of studies indicate that coagulation proteases play significant roles in cancer biology [28]. For example, melanoma is a highly metastatic cancer, and evidence exists indicating that thrombin contrib- utes to this aggressive pattern. Furthermore, previous studies show that the assembly and regulation of the prothrombinase complex on the murine melanoma cell line B16F10 is accelerated with enhanced thrombin formation [48]. These conditions, along with PAR-1 expression in melanoma cell lines [29], likely play a rel- evant role in the metastatic potential of these cancer cells. The additional effects of thrombin on growth factors and matrix proteins reported in the present study may further contribute to these effects, indicat- ing that pathological conditions with increased thrombin levels may lead to proteolytic matura- tion ⁄ degradation of both growth factors and extracel- lular matrix proteins, affecting cell mitogenic ⁄ invasion features at different levels. Cancer can activate the coagulation cascade, as also demonstrated by the enhanced rate of thromboembolic complications and the beneficial effect of anticoagulant therapies in the prevention of these disorders in cancer patients [49]. However, it is less well known whether activation of the coagulation system may also support or inhibit tumor progression. The findings reported in the pres- ent study outline the functional link between thrombin activity and the mitogenic ⁄ invasive properties of mela- noma cells. This observation may contribute to explaining why paradoxical pro-apoptotic effects of a high thrombin concentration on different cell types recently were reported [49]. Two reports available in the literature indicate that HMW FGF-2 and FGF-1 are cleaved by thrombin, whereas they also report that LMW FGF-2 is not cleaved by thrombin [13,50]. In this respect, it should be highlighted that the experimental conditions (i.e. the amount of thrombin and FGF-2, as well as the incubation times) are markedly different. Indeed, we used 0.1 nm = 0.01 UÆmL )1 thrombin, whereas both Lobb [50] and Yu et al. [13] used at least 100-fold more thrombin. The source of thrombin was also dif- ferent; both Lobb [50] and Yu et al. [13] used commer- cial human thrombin, whereas we used in-house highly-purified human thrombin [51]. Lobb [50] used in-house purified bovine brain-derived FGF-2, whereas both Yu et al. [13] and ourselves used commercial human recombinant FGF-2. The incubation times were also markedly different: Lobb [50] reports 6–20 h of incubation of brain-derived bovine FGF-2 with thrombin, whereas Yu et al. [13] used human recombi- nant FGF-2 at unspecified doses incubated with thrombin for 5–15 min. In the present study, specified doses of human recombinant FGF-2 were incubated for significantly longer times (60–120 min). The experi- mental conditions employed in the present study were designed to mimic in vitro, as much as possible, the conditions occurring under pathological states that show increased thrombin levels and increased FGF-2 levels, and were confirmed, in different cell models and in the presence of heparin, to support the possible in vivo relevance of the observed effects. In conclusion, in the present study, we show that thrombin is able to cleave human LMW FGF-2 in vitro, further unravelling the complex linkage between coagu- lation activation and cancer progression. Experimental procedures Cell culture and proliferation Human metastatic melanoma cell line SK-MEL-110 [52] (4 · 10 4 ) cells were seeded in six-well plates and grown for 24 h at 37 °C, 5% CO 2 in DMEM supplemented with 1% penicillin–streptomycin, 1% l-glutamine and 10% charcoal stripped fetal bovine serum (Gibco, Carlsbad, CA, USA). Medium was then replaced and cells were starved overnight with DMEM supplemented with 1% penicillin–streptomy- cin and 1% l-glutamine. Subsequently, dose–response and time-course proliferation assays were performed using dif- ferent stimuli and different time points. For dose–response and for time-course assays (Fig. 1), human recombinant FGF-2 (10 ngÆmL )1 , 0.6 nm) (Pierce Endogen, Rockford, IL, USA) and human a-thrombin (thrombin) [51] (0.001, 0.01, 0.1 and 1 nm or 0.1 nm) were pre-incubated for 1 h at 37 °C; enzymatic activity was then blocked with PPACK (50 nm) (Calbiochem, BIOMOL International LP, Exeter, UK) and cells were stimulated with these mixtures for 48 or 24 h and 48 and 72 h, respectively. In other experiments (Figs 2–4), SK-MEL-110 cells were directly exposed for 48 h to FGF-2 (10 ngÆmL )1 , 0.6 nm)in the presence or the absence of 5.7 lm TRAP-1 or TRAP-4 [53] (PRIMM Srl, Milan, Italy), or different mixtures of P. Totta et al. FGF-2 is a thrombin substrate FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS 3285 PPACK (50 nm) or heparin 25 000 IU per 5 mL (10 and 50 ngÆmL )1 under the assumption that 170 IU corresponds to 1 mg) (Epsoclar from Mayne Pharma Srl, Napoli, Italia) [54] and ⁄ or thrombin (0.1 nm, 0.01 UÆmL )1 ). Finally, in the experiments depicted in Fig. 8B, FGF-2 (10 ngÆmL )1 , 0.6 nm) was pre-incubated at different time points with thrombin (0.1 nm, 0.01 UÆmL )1 ); the enzymatic reaction was then blocked with PPACK (50 nm) and SK-MEL-110 proliferation was stimulated for 48 h. Stimuli were resus- pended in all cases in DMEM with 0.1% BSA (Sigma- Aldrich, St Louis, MO, USA). Primary HUVEC (Cambrex, Walkersville, MD, USA) were also used. Cells were grown at 37 °C, 5% CO 2 . HUVEC (8 · 10 4 ) were seeded in six-well plates and grown for 24 h in endothelial cell basal medium 2 (EBM-2; Cambrex) supple- mented with endothelial growth medium 2 (Cambrex), 1% penicillin–streptomycin and 1% l-glutamine. Medium was then replaced and cells were starved overnight with EBM-2 supplemented with 1% penicillin–streptomycin and 1% l-glutamine. Subsequently, HUVECs were stimulated with 10 ngÆmL )1 FGF-2 resuspended in EBM-2 with 0.1% BSA in the presence or the absence of thrombin 0.1 nm, for 24 h. Time-course experiments were also carried out. After treatment, cells were harvested with trypsin (Gibco), stained with trypan blue solution (Sigma-Aldrich) and counted in a hemocytometer (improved Neubauer chamber) in quadruplicate. Cell invasion assay The SK-MEL-110 invasion assay was carried out in mod- ified Boyden chambers as previously described [55]. Briefly, 8 lm pore-size polycarbonate filters (Costar, Cam- bridge, MA, USA) were coated with human plasma vitro- nectin (Calbiochem), murine collagen type IV (Becton Dickinson, Bedford, MA, USA), human plasma fibronec- tin (Invitrogen, Carlsbad, CA, USA) or type A gelatin from porcine skin (10 lgÆmL )1 ) (Sigma-Aldrich). SK-MEL-110 cells were grown in DMEM supplemented with 1% penicillin–streptomycin, 1% l-glutamine and 10% fetal bovine serum and then replaced with DMEM supplemented with 1% penicillin–streptomycin, 1% l-glu- tamine overnight. Cells were then harvested by trypsiniza- tion, resuspended in DMEM supplemented with 1% penicillin–streptomycin, 1% l-glutamine and 0.1% BSA, and 800 lL were added to the upper portion of the Boy- den chambers containing 1 · 10 6 cellsÆmL )1 ; the lower portion of the chambers contained either DMEM supple- mented with 0.1% BSA or 10 ngÆmL )1 (0.6 nm) FGF-2 with 0.1% BSA, alone or mixed with 0.1 nm thrombin. Invasion was allowed to proceed for 4 h at 37 °Cin5% CO 2 ; then cells were fixed in 95% ethanol and stained with 0.5% toluidine blue in NaCl ⁄ Pi (Gibco), pH 7.4, for 10 min. Migrated cells were counted by evaluation of 15 fields at · 400 magnification. Phosphorylation and degradation analyses by Western blotting ERK1 ⁄ 2 phosphorylation analysis (Fig. 6): SK-MEL-110 cells were grown in DMEM supplemented with 1% penicil- lin–streptomycin, 1% l-glutamine and 10% fetal bovine serum, replaced with DMEM supplemented with 1% peni- cillin–streptomycin, 1% l-glutamine overnight. FGF-2 (10 ngÆmL )1 , 0.6 nm), human a-thrombin (thrombin) (0.1 nm, 0.01 UÆmL )1 ) and FGF-2 with human a-thrombin (thrombin) (0.1 nm, 0.01 UÆmL )1 ) were pre-incubated for 1 h at 37 °C and then PPACK (50 nm) was added to block, where necessary, thrombin enzymatic activities. Cells were treated with these mixtures for 10 min, rinsed with ice-cold NaCl ⁄ Pi and lysed for 15 min with 1% triton, 10% glyc- erol, 100 mm NaCl, 5 mm EDTA, 20 mm Hepes (pH 7.4), 10 mm NaF, 2 mm phenylmethanesulfonyl fluoride, 10 lm NaVO3 and 1% protease inhibitor cocktail (Sigma- Aldrich). Lysates were then boiled for 7 min. After determi- nation of protein concentration (Bio-Rad Laboratories, Hercules, CA, USA), 30 lg of total proteins were resolved in SDS ⁄ PAGE with NuPAGE Ò pre-cast gels for protein electrophoresis (10%) and NuPAGE Ò SDS running buffer (Mops buffer) (Invitrogen), transferred to nitrocellulose membrane and blocked with 5% milk in T-NaCl ⁄ Pi (0.1% Tween 20 in NaCl ⁄ Pi, pH 7.4). After three washes with T-NaCl ⁄ Pi, membranes were incubated with monoclonal anti-pERK or anti-ERK sera (Cell Signaling Technology, Beverly, MA, USA) for 1 h. For detection, secondary anti- mouse serum (1 : 5000) (Pierce Endogen) was used followed by chemiluminescence (ECL; Amersham, Little Chalfont, UK) and autoradiography. FGF-2 degradation analysis (see, Fig. S1) FGF-2 was dissolved in a 50 mm Tris–HCl containing 150 mm NaCl, pH 8, and incubated for 1 h alone, with PPACK, with thrombin, or with inactivated-thrombin (thrombin pre-incubated with PPACK, PPACK-thrombin) (1 : 1 molar ratio, 100 lgÆmL )1 )at37°C. After incubation, active-thrombin was blocked by PPACK (1 : 100 molar ratio). FGF-2 (500 ng) was then resolved in SDS ⁄ PAGE with NuPAGE Ò pre-cast gels for protein electrophoresis (12%) and NuPAGE Ò SDS running buffer (Mes buffer; Invitrogen), transferred to nitrocellulose membrane and blocked with 5% milk in T-NaCl ⁄ Pi. After three washes with T-NaCl ⁄ Pi, membranes were incubated with poly- clonal anti-FGF-2 (0.3 lgÆmL )1 ) serum (Oncogene Research Products, Darmstad, Germany) for 1 h. For detection, secondary anti-goat serum (1 : 5000) (Pierce Endogen) was used followed by chemiluminescence (ECL; Amersham) and autoradiography. Bands were quantified using a calibrated imaging densi- tometer (GS 710; Bio-Rad Laboratories) and analyzed using quantity one software (Bio-Rad Laboratories). FGF-2 is a thrombin substrate P. Totta et al. 3286 FEBS Journal 276 (2009) 3277–3289 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... 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Fluorescence of 10 000 cells per sample was acquired in logarithmic mode to quantify the binding of the relevant molecules summit 4.3 software (Beckman Coulter) was used for data elaboration Acknowledgements The present study was supported by the Italian Ministry of University and Research (‘PRIN-2005’) to R.D.C.; Progetto Oncoproteomica Italia-USA (no 527B ⁄ 2A ⁄ 5) to A.F.; and Ministry of Health MS-RO2006,... 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Edwards-Ingram LC, Holladay A & Saffell JL (2006) Ligand concentration is a driver of divergent signaling and pleiotropic cellular responses to FGF J Cell Physiol 206, 386–393 7 De Marchis F, Ribatti D, Giampietri C, Lentini A, Faraone D, Scoccianti M, Caporossi MC & Facchiano A (2002) A platelet-derived growth factor inhibits basic fibroblast growth factor angiogenic properties in vitro and in vivo through its alpha... rate of 1 mLÆmin)1 The peaks were detected routinely at 214 nm Under these conditions, the observed velocity of the reaction did not reach saturation, such that pseudo-first order conditions were met (concentration < Km of the reaction) Hence, the FGF-2 peak area remaining after cleavage by thrombin was fitted to the equation: Pt (%) ¼ 100  exp ðÀkobs tÞ ð1Þ where kobs is the pseudo-first order rate of . Thrombin-mediated impairment of fibroblast growth factor-2 activity Pierangela Totta 1, *, Raimondo De Cristofaro 2, *, Claudia Giampietri 3 ,. strong loss of func- tion and loss of integrity observed after 60 min of incubation agree with the almost complete inhibition of mitogenic activity of FGF (Fig.