J Am Soc Nephrol 12: 891–899, 2001 Activated Coagulation : A Novel Mitogenic Stimulus for Human Mesangial Cells

RAFFAELLA MONNO,* GIUSEPPE GRANDALIANO,* ROBERTA FACCIO,† ELENA RANIERI,* CARMELA MARTINO,* LORETO GESUALDO,* and FRANCESCO P. SCHENA* *Division of Nephrology, Department of Emergency and Transplantation, and †Institute of Human Anatomy, University of Bari, Bari, Italy.

Abstract. Intraglomerular activation of the coagulation cascade of leupeptin, a specific inhibitor, and neutral- is a common feature of mesangioproliferative glomerulone- izing anti-PDGF antibody. To investigate the role of tyrosine phritis. Besides , very little is known about the cellu- kinase (TK) activation on FXa mitogenic effect, FXa-stimu- lar effects of other components of the coagulation system. This lated thymidine uptake was evaluated in the presence of study investigated the effect of activated factor X (FXa) on genistein and herbimycin A, two powerful and specific TK cultured human mesangial cells. This serine protease induced a inhibitors. FXa-elicited DNA synthesis was also examined significant and dose-dependent increase in DNA synthesis. In after protein kinase C (PKC) downregulation by prolonged addition to its mitogenic effect, FXa caused a striking upregu- incubation with phorbol-12-myristate-13-acetate to study the lation of platelet-derived growth factor (PDGF) A and B chain influence of the phospholipase C–PKC axis. The proliferative gene expression. Next, the intracellular mitogenic signaling effect of FXa required its proteolytic activity, and the activa- pathways activated by FXa were investigated. FXa induced a tion of TK was only partially dependent on PKC activation rapid spike in cytosolic calcium concentration followed by a while it was PDGF independent. Finally, it was shown by sustained plateau. This response was not influenced by the reverse transcription-PCR that mesangial cells do not express downregulation of thrombin receptors. In addition, FXa stim- the signaling splicing variant of the putative FXa receptor, ulated a significant upregulation of different tyrosine-phospho- effector protease receptor-1. In conclusion, the present study rylated proteins. One of these phosphorylated cellular proteins demonstrated that FXa is a powerful mitogenic factor for was represented by the c-jun N-terminal kinase, a member of human mesangial cells, and it induces its cellular effect not the mitogen-activated protein kinase family. To evaluate the through effector protease receptor-1, but most likely by bind- role of FXa enzymatic activity and of PDGF autocrine secre- ing a protease-activated receptor and activating phospholipase tion, FXa-induced DNA synthesis was studied in the presence C–PKC and TK signaling pathways.

The local activation of the coagulation cascade with conse- receptor (8), growing interest has been focused on the cell- quent intraglomerular thrombosis and fibrin deposition is a activating properties of proteases, and in the last few years common feature of a variety of experimental and human me- three more protease-activated receptors (PAR) have been sangioproliferative glomerulonephritides (1–3). Among the cloned and characterized (9–11). The presence of different components of the coagulation system, thrombin recently has PAR with similar properties suggests that other coagulation been suggested to play a pivotal role in the abnormal activation proteases also could share with thrombin the ability to modu- of glomerular cells, constantly observed in glomerulonephrit- late the mechanisms of cell activation. Interestingly, earlier ides (4). Indeed, this serine protease can induce mitogenesis works suggested that activated factor X (FXa), the enzymati- and growth factor and cytokine secretion in resident glomerular cally active constituent of the prothrombinase complex respon- cells interacting with a specific cell surface receptor (4–7). The sible for the conversion of prothrombin to thrombin, triggers unusual proteolytic activation, induced by thrombin itself, of complex pathways that are involved in the regulation of cel- this transmembrane protein is responsible for different intra- lular growth. Binding of FXa to vascular endothelial cells cellular signaling events (8). Since the cloning of thrombin induces the release of platelet-derived growth factor (PDGF)- like molecules, thus providing a paracrine mechanism of cell proliferation (12). Moreover, FXa directly stimulates prolifer- Received August 10, 1998. Accepted October 9, 2000. ation of rat aortic smooth muscle cells (SMC) independently Correspondence to Prof. Francesco P. Schena, Division of Nephrology, De- from thrombin formation (13). An FXa-specific receptor, ef- partment of Emergency and Transplantation, University of Bari, Polyclinic, fector protease receptor-1 (EPR-1), has been cloned in leuko- Piazza Giulio Cesare, 11, 70124 Bari, Italy. Phone: 39-80-5592237; Fax: 39-80-5575710; E-mail: [email protected] cytes and has been shown to be expressed also by endothelial 1046-6673/1205-0891 and SMC, although very little is known about the signaling Journal of the American Society of Nephrology cascade activated by this receptor, which eventually leads to an Copyright © 2001 by the American Society of Nephrology FXa mitogenic effect. 892 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 891–899, 2001

The aim of this study was to investigate the effect of FXa on PMA for 48 h, with genistein (25 ␮M) for 18 h, or with leupeptin (1 mesangial cell proliferation and to evaluate the potential sig- ␮g/ml) or neutralizing polyclonal rabbit anti human PDGF AB anti- naling pathways that are activated by this coagulation factor. body (50 ␮g/ml) for 30 min before adding FXa for 24 h. At the end of the incubation period, cells were pulsed for 4 h with 1.0 ␮Ci/ml Our data demonstrate that FXa induces DNA synthesis and 3 PDGF gene expression in human mesangial cells. Interestingly, H-thymidine. The medium then was removed, and the cells were washed twice in ice-cold 5% TCA and incubated in 5% TCA for 5 the FXa mitogenic action is PDGF independent and most likely min. The cell layers were solubilized by adding 0.75 ml of 0.25 N involves its binding to a protease-activated receptor with con- 2ϩ NaOH in 0.1% sodium dodecyl sulfate (SDS). Half milliliter aliquots sequent increase in cytosolic calcium ([Ca ]i) and in tyrosine were then neutralized and counted in scintillation fluid using a ␤ phosphorylation of cytoplasmic proteins. counter. Thus, we can propose FXa as a novel growth factor for human mesangial cells (HMC) and support the hypothesis of a pathogenic involvement of this coagulation factor in mesan- RNA Isolation and Northern Blot Analysis gioproliferative glomerulonephritides. For each experiment, 2 ϫ 106 cells were plated in 75-mm2 flasks and cultured as detailed above. After reaching confluence, HMC were Materials and Methods serum-starved for 48 h and then incubated for the indicated time Reagents periods in serum-free insulin-free RPMI 1640 containing 10 nM FXa. At the end of incubation, cells were lysed with 4 M guanidium RPMI 1640 medium, trypsin, penicillin, and streptomycin were isothiocyanate containing 25 mM sodium citrate (pH 7.0), 0.5% obtained from Mascia Brunelli (Milan, Italy). Fetal bovine serum, sarcosyl, and 0.1 mM 2-␤-mercaptoethanol. Total RNA was isolated L-glutamine, sodium pyruvate, nonessential amino acids, , in- by the single-step method, using phenol and chloroform/isoamylalco- sulin, transferrin, and selenium were from Sigma Cell Culture (Milan, hol (15). Italy). FXa was purchased from Calbiochem (La Jolla, CA). This PDGF A chain gene expression was studied by Northern blotting as preparation was homogeneous and was guaranteed by the manufac- described previously (6). Briefly, electrophoresis of 20 ␮g of total turer to contain no detectable thrombin contamination. Bovine throm- RNA from each experimental condition was carried out in 1% agarose bin, phorbol-12-myristate-13-acetate (PMA), genistein, leupeptin, gel with 2.2 M formaldehyde. The gel was transferred overnight to a fura 2 acetoxymethylester, and ionomycin were from Sigma Chemical nylon membrane (Schleicher & Schuell, Dassel, Germany). The mem- Co. (Milan, Italy). Neutralizing polyclonal rabbit anti-human PDGF brane was stained with ethidium bromide to evaluate the 28-S and AB antibody was from Genzyme (Cambridge, MA). The monoclonal 18-S bands and then prehybridized at 42°C for2hin50%Formamide, antiphosphotyrosine antibody Py20 was obtained from Upstate Bio- 0.5% SDS, 5x SSC, and 0.1 mg/ml salmon sperm DNA. The cDNA technology Inc. (Lake Placid, NY). The monoclonal anti-phospho jun probe used was a 1.3-kb fragment encoding the human PDGF A chain N-terminal kinase (JNK) was purchased from USB (Cleveland, OH). isolated from pGEM1 plasmid with EcoRI. The probe was labeled by The horseradish-peroxidase-conjugated sheep anti-mouse antibody random priming using a commercially available kit (Amersham) and was supplied from ECL (Amersham, UK). [32P]dCTP and [methyl- [32P]dCTP (specific activity, 3000 Ci/mmol). The 32P-labeled probe 3H]-thymidine were purchased from Amersham. All other chemicals (106 cpm/ml) was added to 10 ml of prehybridization solution, and the were reagent grade. blots were hybridized for 16 h at 42°C. The membranes were then washed once in 2x SSC, 0.1% SDS at room temperature for 5 min, Cell Isolation and Culture once in the same buffer at 55°C for 30 min, and in 1x SSC, 0.1% SDS Normal-appearing portions of human kidneys that were surgically at 55°C for another 30 min. After drying, membranes were exposed to removed for renal carcinoma were used to culture mesangial cells a Kodak X-OMAT film with intensifying screens at Ϫ70°C. from outgrowths of collagenase-treated glomeruli. HMC were estab- lished and characterized as previously reported (14). Cells were grown until confluent in RPMI 1640 medium supplemented with 17% heat- Reverse Transcription-PCR ␮ inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 g/ml HMC express extremely low levels of PDGF B chain-specific streptomycin, 2 mM L-glutamine, 2 mM sodium pyruvate, 1% (vol/ transcript, which do not allow for conventional Northern blot analysis. ␮ ␮ vol) nonessential amino acids, 5 g/ml insulin, 5 g/ml transferrin, Therefore, in preliminary experiments, we tried to analyze the target and 5 ng/ml selenium. For passage, confluent cells were washed with gene expression using a highly sensitive and specific RNase protec- PBS, removed with 0.025% trypsin/0.5 mM ethylenediaminetetraac- tion assay performed exactly as described previously (16). Unfortu- etate (EDTA) in PBS and plated in RPMI. Experiments included in nately, this technical approach also failed to identify measurable this study were performed on cells between the 5th and 10th passages. amounts of PDGF B chain mRNA in 100 ␮g of total RNA extracted from unstimulated cells. Thus, we resolved to address this issue by Culture Condition and Cell Growth adopting a semiquantitative reverse transcription-PCR (RT-PCR), DNA synthesis was measured as the amount of [methyl-3H]-thy- which allowed us to compare the relative amounts of target gene midine incorporated into trichloroacetic acid (TCA)-precipitable ma- transcripts in the different experimental conditions selected. terial as described previously (14). Briefly, cells were plated in One ␮g of total RNA from cultured HMC was used in an RT 24-well dishes at a density of 4 ϫ 104/well, grown to confluence, and reaction. Twenty ␮l of the reaction mixture containing 1 ␮g of total made quiescent by placing them in serum-free medium for 48 h. RNA, PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl), 5 mM

Before the serum-free medium was added, the cells were washed three MgCl2, 1 mM dNTPs, 20 U of RNase inhibitor, 2.5 mM oligo (dT), times with PBS to avoid any possible contamination with thrombin and 100 U of Moloney murine leukemia virus reverse transcriptase and/or prothrombin present in the FBS. Then cells were incubated were incubated at 42°C for 30 min and then heated to 95°C for 5 min with FXa at the indicated concentrations for 24 h at 37°C. In a to inactivate the enzyme activity and to denature RNA-cDNA hybrids. separate set of experiments, cells were preincubated with 10Ϫ7M All samples were reverse transcribed in the same set of experiments, J Am Soc Nephrol 12: 891–899, 2001 FXa and Mesangial Cell Proliferation 893

and the efficiency of the reaction was checked by glyceraldehyde supernatant first was incubated with antiphosphotyrosine antibodies phosphate dehydrogenase (GAPDH) amplification. for2honarocking platform at 4°C and then with agarose-linked PCR was performed with two separate sets of oligonucleotide protein A for 2 h at 4°C. The immunobeads were washed twice with primers, specific for human PDGF B chain and GAPDH, respectively: RIPA buffer and twice with 0.5 mM LiCl, 0.1 mM Tris-HCl (pH 7.5), PDGF B chain: 5'-ATG CTG AGT GAC CAC TCG-3' upstream, and 1 mM sodium orthovanadate. The beads were then resuspended in 5'-CCT GAA TTT CCG GTG CTT GCC-3' downstream; GAPDH: sample buffer and boiled. The immunoprecipitated proteins were 5'-TGG TAT CGT GGA AGG ACT CAT GAC-3' upstream, 5'-ATG separated by electrophoresis on a 10% polyacrylamide gel and trans- CCA GTG AGC TTC CCG TTC AGC-3' downstream. ferred onto a nitrocellulose membrane. The membrane was blocked as PDGF B chain or GAPDH cDNA amplification was performed in described and incubated with mouse anti-phospho JNK antibody two separate sets of reactions at a final concentration of 1x PCR (1:500) for4hatroom temperature, washed, and incubated with ␮ ␮ buffer, 1.5 mM MgCl2, 200 M dNTPs, 0.15 M PDGF B primers or horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody 0.15 ␮M GAPDH primers, and 1.25 U of AmpliTaq DNA polymerase (1:1500). The ECL enhanced chemiluminescence system was used for (Perkin Elmer Cetus, Norwalk, CT), in a total volume of 50 ␮l. The detection. amplification profile involved denaturation at 95°C for 1 min, primer annealing at 61°C for PDGF B or at 55°C for GAPDH for 1 min, and 2ϩ Measurements of [Ca ]i extension at 72°C for 1 min. PCR products were electrophoresed in 2ϩ [Ca ]i was measured at 37°C by dual-wavelength fluorescence ␮ ϩ 1.6% agarose gel in Tris borate/EDTA buffer, loading 10 l of either microscopy in groups of 7 to 10 cells loaded with the Ca2 -sensitive PDGF B or GAPDH PCR products for each sample. The expected size indicator fura 2, as described previously (17). Briefly, cells seeded at of the amplified fragments was 588 bp and 450 bp for PDGF B chain a density of 2 ϫ 104/24 mm diameter round glass coverslips were and GAPDH, respectively. loaded with 3 to 10 ␮m of fura 2 acetoxymethylester in serum-free In a separate set of experiments, total RNA from growing HMC medium at 37°C and 5% CO2 for 60 min. Coverslips were rinsed was reverse transcribed as described and amplified using the follow- twice and then incubated subsequently with FXa and thrombin at ␤ ing sets of primers specific for human EPR-1 and -actin: EPR-1: 37°C in Krebs-Ringer-HEPES buffer (125 mm NaCl, 5 mm KCl, 1.2 5'-TTACGCCAGACTTCAGCCTG-3' upstream, 5'-TGGGTAACA- mm KH PO , 1.2 mm MgSO , 2 mm CaCl , 265 mm HEPES, 6 mm ␤ 2 4 4 2 GTGGCTGCTTC-3' downstream; -actin: 5'-GGCATCGTGATG- glucose). Cells were not washed between the two different stimuli. GACTCCG-3' upstream, 5'-GCTGGAAGGTGGACAGCGA-3' 2ϩ [Ca ]i-dependent fluorescence was measured with a microfluorom- downstream. The expected size of the fragments was 295 bp and 746 eter (Cleveland Bioinstrumentation, Cleveland, OH) connected to a ␤ bp for the two EPR-1 transcripts and 700 bp for -actin. Zeiss IM35 inverted microscope equipped with a Nikon CF X40 fluor objective. Recordings were performed at 340- and 380-nm excitation Western Blot Analysis wavelengths (bandwidth, 0.5 nm). Emission was collected by a pho- HMC were grown in 60-mm2 Petri dishes to confluence in RPMI tomultiplier carrying a 505-nm cutoff filter. Emission from 340 and containing 17% FBS. The cell monolayer was incubated 48 h in 380 nm and real-time 340- to 380-nm ratios were recorded by a serum-free medium and then exposed to 10 nM FXa for the indicated specific software (Labview, National Instruments Corporation, Aus- time periods. At the end of the treatment, the cell monolayer was tin, TX). In each experiment, the maximal fluorescence ratio was 2ϩ rinsed rapidly twice with ice-cold PBS and lysed in 100 ␮l of RIPA determined by addition of Ca ionophore ionomycin, and the min- buffer (1 mM phenylmethylsulphonyl fluoride, 5 mM EDTA, 1 mM imal fluorescence ratio was determined by addition of 7.5 mM eth- sodium orthovanadate, 150 mM sodium chloride, 8 ␮g/ml leupeptin, yleneglycol-bis(␤-aminoethyl ether)-N,N'-tetraacetic acid and 60 mM 1.5% nonidet P-40, 20 mM tris-HCl [pH 7.4]). The lysates were set on Tris (hydroxymethyl) aminomethane HCl (pH 10.5). Fura 2 dissoci- 2ϩ ice for 30 min and centrifuged at 12,000 ϫ g at 4°C for 5 min, and the ation constant was assumed to be 224 nM. [Ca ]i was calculated supernatants were collected and stored at Ϫ80°C until used. Aliquots using previously described formulas (18). containing 7.5 ␮g of proteins from each lysate were subjected to SDS-polyacrylamide gel electrophoresis on 7.5% gels under reducing Statistical Analyses conditions and then electrotransferred onto nitrocellulose membranes Data are presented as mean Ϯ SD. Data were compared using (Hybond C; Amersham). After blocking nonspecific binding through ANOVA or two-tailed unpaired t test, as appropriate. P Ͻ 0.05 was incubation with 2% bovine serum albumin and 0.1% Tween-20 in considered significant. PBS (TBS) overnight at room temperature, the membranes were incubated with monoclonal antiphosphotyrosine antibody Py20 at Results room temperature for 4 h. The membranes were washed twice in TBS Mitogenic Effect and then incubated with horseradish-peroxidase-conjugated sheep an- We first evaluated whether FXa had an effect on HMC ti-mouse antibody at 1:1500 dilution in 0.1% tween-20 in PBS for 2 h at room temperature. The membranes were washed three times at proliferation. Twenty-four h of incubation with this serine room temperature in TBS and then once with 0.1% SDS in PBS. The protease induced a dose-dependent increase in DNA synthesis, 3 ECL enhanced chemiluminescence system was used for detection. as shown by [ H]-thymidine incorporation (Figure 1). A sig- nificant increase over the basal counts occurred with concen- Immunoprecipitation and Anti-Phospho trations of 2.5 nM and peaked at 20 nM. Higher concentrations JNK Immunoblotting of FXa did not cause any further upregulation of DNA syn- thesis (data not shown). Confluent HMC in 60-mm2 culture dishes were placed in serum- free medium for 48 h. FXa was then added for the indicated time 2ϩ periods. Cells were washed twice with ice-cold PBS and lysed with Effect on [Ca ]i Mobilization RIPA buffer for 30 min at 4°C. The cell lysate was centrifuged at Activation of the four known PAR results in the induction of 10,000 ϫ g for 30 min at 4°C. A total of 100 ␮g of protein from the phospholipase C (PLC) activity with the subsequent increase in 894 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 891–899, 2001

shown to induce tyrosine phosphorylation of several cellular proteins (19). Therefore, the ability of FXa to induce the activation of intracellular TK was evaluated by immunoblot- ting using a specific monoclonal antiphosphotyrosine antibody. FXa (10 nM) caused a time-dependent increase in tyrosine phosphorylation of a panel of cellular proteins. The apparent molecular weight of the most prominent tyrosine-phosphory- lated proteins was 140, 100, 80, 60, and 44 kD (Figure 3). FXa has been shown to activate p44 mitogen-activated pro- tein kinase in different cell types. In an attempt to identify one of the TK substrates phosphorylated in response to FXa, we blotted the antiphosphotyrosine immunoprecipitate with a spe- cific anti-phospho JNK antibody, recognizing the activated JNK, another member of the MAP kinase family. As shown in Figure 4, FXa caused a rapid and significant increased in phosphorylated JNK in the antiphosphotyrosine immunopre- cipitate, strongly suggesting a TK-dependent activation of this signaling enzyme induced by FXa.

Effect of FXa on PDGF mRNA Expression Figure 1. Activated factor X (FXa)-induced [3H] thymidine incorpo- PDGF is one of the most powerful growth factors for HMC, ration into human mesangial cell (HMC) DNA. Mesangial cells were and most of the other mitogens, including thrombin, induce the made quiescent by incubation in serum-free, insulin-free medium for expression of its two subunits (4,20). We sought to determine 48 h, exposed to FXa for a total of 24 h and then pulsed with [3H] whether FXa was able to stimulate PDGF A and B chain gene thymidine for 4 h. DNA synthesis, measured as described in the Materials and Methods section, was expressed in cpm (count per expression in quiescent HMC. Figure 5 demonstrates the time min)/well. Data represent means Ϯ SD (n ϭ 3, each point in quadru- course of changes that occurred in PDGF A chain mRNA plicate). *, P Ͻ 0.001 versus basal. expression, studied by Northern blotting, in HMC exposed to FXa (10 nM). When compared with control cells, FXa-stimu- lated HMC exhibited a marked increase of PDGF A chain intracellular calcium concentration (8–11). To investigate the mRNA starting at 3 h, with a peak at 6 h, returning to near intracellular mitogenic signaling pathways elicited by FXa, we basal levels by 12 h and increasing again at 24 h without studied the activation of PLC indirectly by measuring the reaching the maximum expression found at 6 h. Then, we cytosolic calcium levels in groups of 7 to 10 human mesangial determined the effect of FXa on PDGF B chain mRNA. As 2ϩ cells stimulated with FXa (10 nM). Increases in [Ca ]i rapidly shown in Figure 6, FXa also induced PDGF B chain mRNA occurred, peaked within a few minutes, and declined slowly to expression with a peak at 6 h and a subsequent reduction to reach a plateau at a slightly elevated steady-state level, which basal level after 12 h. was maintained for several minutes (Figure 2A). After the early response to FXa was exhausted, the same cells were Regulation of FXa-Induced Mitogenesis stimulated with thrombin (10 U/ml) to evaluate the activation To determine whether the enzymatic activity of FXa was and consequent downregulation of thrombin receptors by FXa necessary to elicit the mitogenic effect observed, we evaluated in HMC. The FXa-stimulated cells were still responsive to the effect of a specific serine-protease inhibitor, leupeptin, on 2ϩ thrombin, which induced an increase in [Ca ]i following a DNA synthesis stimulated by FXa. Leupeptin at the concen- kinetic similar to that previously described (4). The response tration of 1 ␮g/ml completely abolished the increase in thymi- mirrored the pattern described for FXa stimulation but with a dine uptake induced by FXa (Figure 7) without affecting the higher peak and a more sustained plateau. To confirm the basal level (data not shown). To investigate whether FXa 2ϩ ability of FXa to elicit a [Ca ]i spike despite the downregu- mitogenic effect was dependent on thrombin release, we used lation of thrombin receptors, we repeated the same experiments a specific thrombin inhibitor, hirudin. This mol- exposing the cells first to thrombin and then to FXa. The ecule at the concentration of 20 UI/ml did not affect FXa- downregulation of thrombin receptors did not influence the induced DNA synthesis (Figure 7). cells’ responsiveness to FXa (Figure 2B). Conversely, both Because FXa induced both PDGF A and B chain mRNA FXa and thrombin completely downregulated their own re- expression in human mesangial cells, we investigated whether sponse in HMC (Figure 2, C and D). its proliferative effect was dependent on the autocrine effect of PDGF. For this purpose, we studied FXa-induced DNA syn- Effect of FXa on Intracellular Tyrosine Kinases thesis in HMC in the presence of a polyclonal neutralizing Tyrosine phosphorylation is a key event in the intracellular anti-human PDGF antibody (50 ␮g/ml) coincubated with FXa signaling of different mitogens (19). Recently, non-tyrosine (10 nM). No significant reduction was observed in FXa-trig- kinase (non-TK) receptor agonists, including thrombin, were gered thymidine incorporation (Figure 6), whereas the mito- J Am Soc Nephrol 12: 891–899, 2001 FXa and Mesangial Cell Proliferation 895

Figure 2. Stimulation of cytosolic-free calcium ([Ca2ϩ]) by FXa (10 nM) and thrombin (5 U/ml) (A) and vice versa (B), in groups of 7 to 10 cultured HMC. In each set of experiments (n ϭ 6), the different stimuli were added at the indicated time (FXa, arrow; thrombin, arrowhead), without washing cells between the addition of the two proteases. Note that FXa stimulates a quick increase in [Ca2ϩ] both before and after thrombin stimulation (A and B). (C and D) FXa and thrombin, respectively, were added twice to the cells at the indicated time. genic response to PDGF AB (10 ng/ml) was completely abol- receptor. We then investigated by RT-PCR whether HMC ished (control, 555 Ϯ 50; PDGF AB, 2400 Ϯ 320; PDGF AB express EPR-1. HepG2, a hepatocarcinoma cell line, express ϩ neutralizing antibody, 625 Ϯ 115). both EPR-1 isoforms, whereas in human mesangial cells we To understand the role of the increased calcium mobilization observed only the expression, at very low levels, of the tran- and protein tyrosine phosphorylation in FXa-induced mitogen- script encoding the nuclear protein (Figure 8). esis in HMC, we examined the effect of Ca2ϩ-dependent protein kinase C (PKC) downregulation and TK inhibition on Discussion DNA synthesis stimulated by FXa. The PKC downregulation, The property of thrombin to influence pleiotropic cell func- obtained as previously demonstrated (21), by a prolonged tions (22) through the interaction with its proteolytically acti- preincubation with PMA, significantly, although not com- vated receptor (23) and the recent discovery of other PAR pletely, reduced thymidine uptake stimulated by FXa (Figure (9–11) have directed us toward the hypothesis that other co- 7). Finally, genistein, an isoflavone compound that specifically agulation proteases may regulate the mechanisms of cell acti- inhibits TK, only partially reduced FXa-induced DNA synthe- vation. The increasing interest in clotting proteases has driven sis (Figure 7), whereas a more pronounced and significant us to consider these enzymes as growth factors, leading to the inhibition was observed with herbimycin A, a more powerful demonstration of an FXa-proliferative effect on aortic SMC TK inhibitor. (13,24) and factor XII mitogenic action on several target cells (25). EPR-1 Expression in HMC The availability of these factors in the glomerular microen- Several reports support the hypothesis that the transmem- vironment is indicated by the presence of prominent fibrin brane protein EPR-1 is the potential FXa receptor. Interest- deposits during several forms of glomerular diseases (3). De- ingly, EPR-1 gene transcript undergoes an alternative splicing spite this observation, the possible role of the coagulation with the production of two mature mRNA, the longer one proteases in the activation of glomerular cells is still largely encoding a nuclear protein and the shorter the potential FXa unknown. Mesangial cells are specialized glomerular pericytes 896 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 891–899, 2001

Figure 5. FXa-induced platelet-derived growth factor (PDGF) A chain gene expression in HMC. Quiescent mesangial cells were incubated Figure 3. Effect of FXa on protein-tyrosine phosphorylation (A and with FXa (10 nM) and harvested after 0, 3, 6, 12, and 24 h, and then B). Confluent quiescent HMC were stimulated with FXa (10 nM) for total RNA was extracted. PDGF A chain mRNA expression was 5, 15, 30, and 60 min and then lysed. Proteins (7.5 ␮g) from cell lysate studied by Northern blot analysis (upper panel). 28-S and 18-S ribo- were separated by sodium dodecyl sulfate-polyacrylamide gel elec- somal RNA bands on ethidium bromide-stained gel were used to trophoresis (SDS-PAGE). Proteins were electrotransferred onto a control the RNA loading (lower panel). nitrocellulose filter and probed with mouse monoclonal antiphospho- tyrosine antibody, as described in the Materials and Methods section. Molecular mass markers are on the left (in kD). Arrows, on the right, indicate the most prominent tyrosine phosphorylated proteins whose apparent molecular weight were 140, 100, 80, 60, and 44 kD.

Figure 6. FXa -induced PDGF B chain gene expression in HMC. Quiescent mesangial cells were incubated with FXa (10 nM) and Figure 4. FXa-induced jun N-terminal kinase (JNK) phosphorylation harvested after 0, 3, 6, 12, and 24 h, and then total RNA was extracted. in HMC. Confluent quiescent HMC were stimulated with FXa (10 PDGF B chain mRNA was detected by reverse transcription-PCR nM) for the indicated time periods and then lysed. Equal amounts of (RT-PCR; upper panel), and signals revealed were normalized to the protein from each cell lysate were immunoprecipitated with antiphos- expression of glyceraldehyde phosphate dehydrogenase (GAPDH; photyrosine antibody, separated by SDS-PAGE, transferred onto ni- lower panel). Representative of three experiments. trocellulose filters, and probed with anti-phospho JNK antibody as described in the Materials and Methods section. Molecular markers are shown on the left. Representative of three experiments. progression of glomerular injury toward sclerosis. The data reported in this study demonstrate, for the first time, that FXa activates HMC, inducing mitogenesis and PDGF A and B that are intimately involved in the hemodynamic regulation of chain gene expression. Interestingly, FXa exerts its prolifera- the glomerular microvascular bed. Proliferation of these cells is tive effect at doses even lower than its physiologic plasma a histopathologic hallmark of a variety of experimental and concentrations (15 to 75 nM), with a significant increase in human glomerular diseases, and it has been implicated in the thymidine uptake starting at 2.5 nM. Therefore, we can hy- J Am Soc Nephrol 12: 891–899, 2001 FXa and Mesangial Cell Proliferation 897

expression of PDGF B chain and PDGF ␤ receptor has been demonstrated in experimental and human mesangioprolifera- tive glomerulonephritis (27–30). Although earlier data (12), confirmed by recent findings (24), showed that FXa stimulates the release of PDGF from endothelial and vascular SMC, the effect of this serine-protease on PDGF A and B chain gene expression has not yet been investigated. Ko et al. (24) sug- gested that all of the effects induced by FXa in rat SMC were due to the release of PDGF, because neutralizing anti-PDGF antibodies were able to abolish any cell response to this factor. We excluded that FXa-induced HMC proliferation was caused by an autocrine effect of PDGF de novo synthesized or released by intracellular storage. Although FXa-induced thymidine up- take in the presence of a polyclonal anti-human PDGF anti- body was not reduced significantly, we could not exclude an Figure 7. Regulation of FXa-induced DNA synthesis in HMC. Qui- intracytoplasmic autocrine loop. However, our data on FXa- escent confluent mesangial cells were respectively co-incubated with induced tyrosine phosphorylation do not reveal the presence of ␮ leupeptin (leu, 1 g/ml) and with a polyclonal neutralizing anti- a phosphorylated protein of 170 kD corresponding to PDGF human PDGF antibody (PDGF ab, 50 ␮g/ml) and preincubated for receptors. In addition, we found that anti-PDGF antibody does 18 h with genistein (gen, 25 ␮M) and 48 h with phorbol-12-myristate- 13-acetate (PMA) (10Ϫ7 M), before the addition of FXa (10 nM). not inhibit FXa-induced tyrosine phosphorylation, further sup- DNA synthesis was measured as the amount of [3H] thymidine porting the hypothesis that PDGF molecules eventually avail- incorporation in trichloroacetic acid –insoluble precipitate as de- able are not responsible for cellular response mediated by scribed in the Materials and Methods section. Data represent means Ϯ tyrosine phosphorylation. Conversely, we cannot rule out de- SD (n ϭ 3, each point in quadruplicate). *, P Ͻ 0.001 versus basal; finitively a role for other growth factors that potentially are **, P Ͼ 0.001 versus FXa. secreted in response to FXa, including bFGF. However, the signaling events observed within a few minutes or seconds after the addition of FXa to the culture medium strongly suggest that this protease may stand on its own as a mitogenic factor. Once a direct FXa mitogenic effect on HMC was demon- strated, the next question to answer concerned the receptor used and the intracellular signaling pathways elicited by this serine protease. Recently, Altieri (31) cloned in a leukemia cell line a cell surface receptor, called EPR-1, that specifically binds FXa. This receptor presents a single transmembrane domain and a short cytoplasmic tail with different potential serine-phosphorylation sites. Herbert et al. (32) demonstrated in human aortic SMC that a blocking anti–EPR-1 antibody is able to inhibit FXa-induced rabbit SMC growth. However, our cells do not express EPR-1 as demonstrated by RT-PCR. This Figure 8. Effector protease receptor-1 (EPR-1) expression in HMC. finding, together with the observation that a nonspecific pro- Growing mesangial cells and HepG2, a hepatocarcinoma cell line tease inhibitor completely blocks the mitogenic effect of FXa, (positive control), were harvested and total RNA was extracted as suggests the potential involvement of a protease-activated re- described in the Materials and Methods section. EPR-1 chain mRNA ceptor. There is now a growing interest in PAR that, for their was detected by RT-PCR. HMC express very low levels of the longer structure and functions, belong to the G protein-coupled recep- transcript (lane 1), whereas HepG2 present both of the EPR-1 tran- tor superfamily. Besides the first thrombin receptor, in the last ␤ scripts (lane 2). In the lanes 4 (mesangial) and 5 (HepG2), the -actin few years, three more PAR have been cloned and characterized expression is observed in the same samples. The molecular marker is (9–11). Two of these transmembrane proteins can interact in lane 3. Molecular weights of the amplified bands are indicated. Representative of three experiments. specifically with thrombin, although they are not expressed at the renal level. These receptors, activated through limited proteolysis of the amino-terminal sequence, are able to induce 2ϩ pothesize that in vivo, the small amounts of FXa locally pro- an increase in [Ca ]i (4–6), the activation of PKC (21), the duced by activating the coagulation system during glomerular modulation of gene expression, and the synthesis of DNA (22). damage could be sufficient to induce mitogenesis in HMC. We investigated whether the proteolytic activity of FXa was PDGF is the most powerful mitogen for HMC (26), and its necessary to elicit the mitogenic effect on HMC. The complete synthesis always has been associated in vitro as well as in vivo abolition of FXa-induced DNA synthesis by leupeptin, a spe- with the proliferation of this cell line (20). Indeed, an increased cific serine protease inhibitor, indicates that this enzyme re- 898 Journal of the American Society of Nephrology J Am Soc Nephrol 12: 891–899, 2001 quires its enzymatic activity to trigger cellular response. This unaffected by TK inhibition (36). However, it has been re- observation suggests the interaction with a membrane receptor ported that an increase in intracellular calcium, caused by whose activation would occur by a proteolytic cleavage. Acti- angiotensin II or by a calcium ionophore, stimulates the ty- vation of all four known PAR results in the induction of PLC rosine phosphorylation of several cellular proteins (37). Be- activity with subsequent intracellular calcium mobilization (8– cause FXa-induced tyrosine phosphorylation does not occur 11). We demonstrated for the first time that FXa stimulates an rapidly but has slow kinetics, it is conceivable that a cytoplas- 2ϩ 2ϩ increase in [Ca ]i at physiologic concentrations in HMC. The mic TK could be activated by an increase in [Ca ]i concen- kinetics observed mirrored the one described previously for tration through a calcium-binding regulatory protein. thrombin (4). FXa induced, within 1 min, a peak followed by Although the precise identity of most tyrosine phosphory- a plateau at a slightly elevated steady-state level. This suggests lated proteins remains to be determined, the 44- to 46-kD an early release from intracellular stores and a later influx of protein most likely may represent one of the MAP kinase extracellular calcium. Interestingly, we observed that HMC, isoforms. Indeed, extracellular signal-regulated kinase-1 acti- soon after stimulation with FXa at doses that maximally in- vation by FXa was demonstrated in rat vascular SMC mito- duced DNA synthesis, maintain their responsiveness to throm- genesis (24). Interestingly, FXa in HMC induces the phosphor- bin. On the basis of this observation, it is conceivable that FXa ylation of another kinase belonging to the MAP kinase does not occupy the same thrombin receptor(s), which there- superfamily, JNK. JNK is a downstream target of several fore remain available to interact with this serine protease and to cytoplasmic TK and can activate, through jun phosphorylation, 2ϩ induce a spike of [Ca ]i. As expected, stimulation of HMC the transcription factor activator protein-1 that, in turn, can first with thrombin and then with FXa showed the same pattern modulate the expression of several genes (38). 2ϩ of [Ca ]i release observed using the opposite experimental In conclusion, we demonstrated for the first time that FXa is conditions. This finding strongly supports the involvement of a powerful mitogenic stimulus for HMC and induces PDGF A two different classes of receptors in the cell interaction with and B chain gene expression. Its proliferative effect, requiring these serine proteases. Indeed, Vu et al. (8) demonstrated that its proteolytic activity, may be mediated by a protease-acti- thrombin receptor, PAR-1, PAR-3, and PAR-4 are highly vated receptor through the activation of the PLC-PKC and TK specific for thrombin and are poorly activated by other pro- signaling pathways. Our findings suggest that FXa, through the teases. Nystedt et al. (9) found that PAR-2 is specifically biologic effects described here, could be responsible for the activated by trypsin. Thus, another unknown PAR could be activation of mesangial cells during glomerular injury. involved in the FXa-induced cellular effects. The activation of PLC in FXa-stimulated HMC demon- Acknowledgments 2ϩ strated indirectly by the [Ca ]i mobilization leads to PKC This study was presented at the 30th Annual Congress of The ϩ activation. To evaluate the involvement of Ca2 -dependent American Society of Nephrology, held in San Antonio, TX, Novem- PKC in HMC proliferation induced by FXa, we downregulated ber 2 to 5, 1997. It was supported partly by the Baxter Extramural it by a prolonged preincubation with PMA (21), finding a grant (eight round, 1996 to 1998), the Associazione per il Progresso significant but not complete reduction of mitogenesis. This Scientifico in Nefrologia e Trapianto (APSNT), the Consiglio Nazio- observation suggests an involvement of Ca2ϩ-dependent PKC nale delle Ricerche (CNR) (96.3476), the CNR target project on but does not exclude that other PKC isozymes, not affected by Biotechnology, and by grants from the Ministero dell’ Universita’ e della Ricerca Scientifica e Tecnologica (40%: 96.746, 96.7404). We PMA preincubation, such as PKC␨ (21), could be responsible thank the skillful technical experience of Michele Ursi. of the residual mitogen effect. Interestingly, downregulation of PKC in the presence of PMA did not result in any significant effect on thrombin-induced DNA synthesis (21), suggesting a References 1. Kincaid Smith P: Coagulation and renal disease. Kidney Int 2: divergence in the intracellular pathways elicited by these two 183–190, 1972 serine proteases. 2. 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