JOURNAL OF CELLULAR PHYSIOLOGY 150:475-483 (1992)

Thrombin Signal Transduction Mechanisms in Human Glomerular Epithelial Cells C I - J I A N G HE,* M.N. PERALDI, C. A D I D A , J.M.REBIBOU, Q. MEULDERS, J.D. SRAER, AND E. R O N D E A U lNSERM U 64, Hepita/ Tenon, 75020 Paris, France We have previously shown that a-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-I). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ( [Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 n M nicardipine, a dihydroperidine agent, or by 4 m M EGTA, indicating that increase in [CaL']i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 'H thymidine incorporation was inhibited by 300 n M nicardipine, and restored by addition of l o p 8 M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 pM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and y-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 'H thymidine incorporation but stimulated U-PA, t-PA, and PAL1 synthesis although to a lesser extent than a-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by a-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAIL1 synthesis but not mitogenesis.

Besides its central effect in blood coagulation, a-thrombin (a-THR) plays a n important role in cell activation (Fenton, 1986; Fenton and Bing, 1986). Such a n action has been demonstrated for many cell types, including platelets (Berndt and Philipps, 1981), fibroblasts (Carney et al., 1985; Paris e t al., 1988), smooth muscle cells (Berk et al., 1990; Bar-Shavit e t al., 19901, epithelial cells (He et al., 1991), and endothelial cells (Awbrey et al., 1979; Gelehrter and Szycer-Laszuk, 1986; Dupuy et al., 1989; Wotja e t al., 1989). All these cells possess specific membrane receptors for thrombin (Carney and Cunningham, 1978). However, the mechanism of cell activation by thrombin and the response of the cells seem to be highly dependent on cell type. In fibroblasts, it has been shown that both membrane binding of thrombin and catalytic activity of the enzyme were necessary for the cell to be activated (Gordon 8 1992 WILEY-LISS. INC.

and Carney, 1986; Frost et al., 1987). This is the twosignal hypothesis which was initially proposed to explain why diisopropylfluorophosphate-inactivated thrombin (DFP-THR), which binds thrombin receptor,

Received June 13,1991; accepted September 20, 1991. *To whom reprint requestsicorrespondence should be addressed. Abbreviations: a-THR, a-thrombin; y-THR, y-thrombin; DFPTHR, diisopropylfluorophosphate-inactivated thrombin; u-PA, urokinase type plasminogen activator; t-PA, tissue type plasminogen activator; PAI-1, plasminogen activator inhibitor 1;PIPB, phosphatidylinositol-bisphosphate; PC, phosphatidylcholine; PKC, protein kinase C; DAG, diacylglycerol; IP3, inositoltrisphosphate; [Ca2+]i,intracellular calcium concentration.

476

HE ET AL

does not induce cell activation. Moreover, y-thrombin (y-THR), a degraded form of thrombin which retains some enzymatic activity of a-THR but which does not bind the thrombin receptor, has no cellular effect (Gordon and Carney, 1986). Addition of DFP-THR, or a monoclonal antithrombin receptor antibody, with y-THR can, however, reproduce the effects of a-THR (Frost et al., 1987). Some evidence has been provided that the enzymatic activity of thrombin was required to induce a n activation of intracellular protein kinase C (PKC) (Carney et al., 1985; Gordon and Carney, 1986; Leach e t al., 1991; Berk et al., 1990); and recently cloning of a functional thrombin receptor revealed that thrombin itself might partially cleave its receptor and thereby created a new receptor aminoterminus that functions as a tethered ligand and activates the receptor (Vu et al., 1991). It has been shown that inositol trisphosphate (IP3)and diacylglycerol (DAG) were generated by phospholipase C activation after thrombin addition (Paris et al., 1988; Berk et al., 19901, with a subsequent raise in cytosolic calcium concentration and PKC activation. In endothelial cells and in fibroblasts, activation of these pathways were shown to stimulate protein synthesis and cell proliferation (Gelehrter and Szycer-Laszuk, 1986; Carney et al., 1985; Paris et al., 1988). However, in other cell types, different mechanisms of action seem to be involved in the signal transduction pathway of thrombin. The two signals are not required for smooth muscle cell isolated from the bovine aorta since in these latter DFP-THR stimulates 3H-thymidine incorporation and proliferation similar to native a-THR (BarShavit e t al., 1990). On the other hand, a-THR does not stimulate proliferation of adult rat vascular smooth muscle cells (Berk et al., 1990). In both these cell types, i t does stimulate protein synthesis through phospholipase C and PKC pathways. Furthermore, these events may occur in the absence of increase in intracellular calcium concentration ([Ca2+]i),suggesting that phosphatidylcholine (PC) rather than phosphatidyl-inositolbisphosphate (PIP2) may be the source for DAG generation and further PKC activation (Berk e t al., 1990). We recently demonstrate that human kidney glomerular epithelial cells in culture have two classes of high affinity receptors for a-THR and that in these cells a-thrombin stimulates the synthesis of specific proteins of the fibrinolytic system (urokinase-type plasminogen activator, u-PA; tissue-type plasminogen activator, t-PA; plasminogen activator inhibitor 1, PAI-l), which regulate the conversion of plasminogen into plasmin, the main fibrinolytic enzyme (Collen and Lijnen, 1986). In glomerular epithelial cells, a-THR is also able to stimulate cell proliferation (He et al., 1991). Binding and enzymatic activity of a-THR were required to obtain these effects. In the present study, we investigate the signal transduction pathways of thrombin in glomerular epithelial cells. Thrombin-induced cell proliferation appears to be dependent on extracellular calcium entry and PKC activation and to be blunted by calcium channel blockers. Conversely, thrombin effect on the synthesis of fibrinolytic components requires PKC activation but is not inhibited by calcium channel blockers and may be observed without change in [Ca2+]i.

MATERIALS AND METHODS 1. Materials The following materials were obtained as indicated: purified human a-THR (3000 U/mg, 1U/ml = lo-’ M), collagenase type IV (Sigma, St. Louis); nicardipine (Sandoz, Basel, Switzerland); human urokinase reference standard grade from Choay (Paris, France); human t-PA, rabbit polyclonal anti-human t-PA, and anti-human u-PA antibodies (Biopool, Sweden); ionomycin, fura-2-acetoxymetylester from Calbiochem (San Diego, CA); diisopropylfluorophosphate (Fluka, AG, Chem Fabrik Buchs). DFP-THR has less than 1%of the procoagulant activity of native thrombin as measured by the coagulation time of a 2 mg/ml fibrinogen solution. y-THR (3,000 U/mg) was a generous gift from Professor Guillain, Laboratoire d’Hemostase, HBpital Bichat, Paris. 2. Cell cultures The glomerular epithelial cells were cultured from normal human kidneys (n = 6) judged to be unsuitable for transplantation. Glomerular epithelial cells were obtained by collagenase digestion of the isolated glomeruli, as previously described with slight modifications (Rondeau et al., 1989). Briefly, glomeruli were isolated from renal cortex by differential sieving through a 180 pm sieve to exclude vessels and tubules and were then washed over a 105 pm sieve which allowed cellular debris to pass through but retained glomeruli. Isolated glomeruli were then suspended in RPMI medium (Flow Laboratories, Irvine, UK) containing 500 U/ml collagenase for 30 min a t 37°C. The suspension was passed over a 25 pm sieve which retained partially digested glomeruli. Epithelial cells in the filtrate were cultured a t 37°C in RPMI medium containing 20% fetal calf serum in a humidified atmosphere of 5% CO, in air. They were purified using cloning rings in order to isolate groups of homogenous cells and identified by their morphologic features a s previously reported (Stricker et al., 1980; Rondeau et al., 1989). Epithelial cells are polyhedral a t confluency. By immunofluorescence, the epithelial origin of these cells was confirmed by positive staining with anti-cytokeratin antibodies. All the cells were positively stained by anti-urokinase antibodies but not by anti-myosin nor anti-factor VIII-related antigen antibodies, excluding mesangial and endothelial contamination. These cells were also positively stained with a n anti-CALLA monoclonal antibody (10 T5 from Immunotechnology) which binds to podocytes and to the brush border of normal human kidney. Finally, these cells were stained by a monoclonal anti-human GECs antibody (PHM5 from Artarmon, Australia). By electron microscopy, GECs grown on plastic appeared flat with rare villi. Weibel-Palade bodies were not observed.

3. Incubation procedures Incubations were performed on the third subculture of glomerular epithelial cells. For the third passage the cells were grown in multidish wells (Nunclon, Nunc, Roskilde, Denmark) in hormonally defined medium (DMEM 50%, HAM F12 50%, HEPES 10 mM, glu-

THROMBIN ACTION ON GLOMERULAR EPITHELIAL CELLS

tamine 2 mM, insuline 5 pg/ml, dexamethasone 5.10p8M, transferrine 5 pg/ml, selenium 3.10p8M,penicilline 10 pg/ml, streptomycine 100 pg/ml) with 1% fetal calf serum. Before cells reached confluence, each well was washed three times with serum-free defined medium to remove serum-derived plasminogen activators and plasminogen activator inhibitors and incubated 24 hours in minimum defined medium (DMEM 50%,HAM F12 50%, HEPES 10 mM, glutamine 2 mM). Adherent cells were then incubated with the compound to be tested or with vehicle alone a t 37°C in 1 ml of minimum defined medium in 5% CO, atmosphere. At the end of incubation, the culture media were collected separately for determination of fibrinolytic components. Cells were counted after detachment with trypsin. Care was taken that the viability of the cells did not differ between experimental and control conditions: in all cases more than 95% of the cells excluded trypan blue dye.

477

utes. After centrifugation, the supernatants were titrated to pH 7.5 with 10N KOH, IP3 was further purified using Amprep SAX minicolumns in order to eliminate phosphatidylinositol monophosphate and bisphosphate. Eluted IP3 fractions were tested by the D-myo-inositol-l,4,5-triphosphate 3H assay system according to the standard conditions recommended by the manufacturer. A standard curve was constructed by addition of different amounts of IP3 in the binding assay to fixed amounts of 3H IP3 and of bovine adrenal binding protein preparation. The bound IP3 was then separated from free ligant by centrifugation and the radioactivity in the pellet was counted in a p-scintillation counter.

6. Measurement of 3H thymidine incorporation and proliferation of GECs After a 24 hour incubation in minimum defined medium, resting cells were challenged with the compounds to be tested or their vehicle for 24 to 48 hours. 4. Cytosolic free calcium concentration [Ca2+]i Six hours before cell count 1 ~ c i / ~thymidine H was determination added to each well. At the end of incubation cells were [Ca2'li in human glomerular epithelial cells was washed, incubated 30 minutes in minimum defined memeasured according to Mene et al. (1987) with minor dium containing 1 mg/ml non-labeled thymidine, and modifications. Twenty-four hours before the experi- then harvested by trypsinization. Cells were filtered on ments, the cells were incubated in a serum-free mini- 0.45 pm Millipore filter and the radioactivity of the mum defined medium. At the end of the incubation, the filter was counted in a f3-scintillation counter. Aliquots cells were washed with 10 mM HEPES buffer (pH 7.4), of detached cells were counted in Malassez plates and containing 135 mM NaC1,5 mM KC1, 1mM NaH P04, control of cell viability assessed by Trypan blue dye 1.8 mM CaC12, 0.5 mM MgS04, 10 mM glucose, and 1 exclusion. Each experimental condition was made in mg/ml BSA. Then the cells were loaded a t 37°C with triplicate. fura-2-acetyloxymethylester (4 pmol/L) in the same buffer for 40 minutes. Loaded cells were successively 7. Enzyme linked immunosorbent assay (ELISA) for t-PA, u-PA and PAI-1 washed twice with the above buffer, incubated at 37°C Determination of u-PA, t-PA, and PAI-1 antigen with 3 ml EDTA (0.02%) and 1ml trypsin (0.05%)for 5 minutes in order to be dissociated, collected, and centri- were performed by ELISA a s previously described (Lafuged for 3 min a t 1,OOOg; washed twice with 5 ml above cave et al., 1989). ELISA for PAI-1 was slightly modibuffer containing 0.25 mg/ml BSA, and finally resus- fied: microtiters polyvinylchloride plates were coated pended in 2 ml of the same buffer in a quartz cuvette a t with specific goat polyclonal antihuman PAI-1 IgG 37°C under constant stirring. Fluorescence (F) was (Biopool, Sweden) and free and complexed PAI-1 were monitored continuously in a Perkin-Elmer model LS-5 detected with a monoclonal anti-PAI-1 antibody (250 spectrofluorometer before and after addition of the ngiml) which binds to both free PAI-1 and complexed agents tested using 339 nm excitation/500 nm emission PAI-1 (MA1 12, Biopool, Sweden). The assay was then wavelengths with 5 mm slit widths. Calibration of the continued as described previously (Lacave et al., 1989). Ca2+-dependentfluorescence for each experiment was The detection limits for PAI-1, u-PA, and t-PA were 0.4 obtained by addition of 20 pM ionomycin (maximum ng/ml, 0.9 ng/ml, and 0.14 ngiml, respectively. fluorescence, F max) followed by 4 mM EGTA in 60 mM 8. Cellular cyclic AMP generation Tris-HC1 buffer, pH 10.5 (minimal fluorescence, F m i d . Thrombin (0.1 U/ml to 10 U/ml) was added Cells were pretreated with serum-free minimum dealone or after exposure to nicardipine or EGTA. [Ca2+1i fined medium containing lop4 M 1-methyl-3 isobutyl was calculated using the following formula: [Ca2+]i= xanthine (Sigma, St. Louis), then washed 3 times and (F - F min) x Kd/(F max - F) with Kd = 224 nM. incubated in the same medium with or without a-THR at 37°C for 5 minutes. Reaction was stopped by addition 5. Determination of inositol 1,4,5 triphosphate of 500 pl extraction medium (85%ethanol, 15% formic UP31 release acid, vol/vol). Cyclic AMP was measured by radioimGeneration of IP3 was measured by the D-myo-inosi- munoassay after acetylation of the samples and of the tol-1,4,5-triphosphate L3H1 assay system (Amersham, standards according to a previously published techUK) (Aducci and Marra, 1990). The cells were incu- nique (Friedlander et al., 1983). bated in a serum-free-medium 24 hours before experiRESULTS ments and then thrombin (0.1 to 10 U/ml) was tested for 1. Intracellular free calcium concentration a short time (10 to 300 seconds). The reaction was As shown in Figure lA, a-THR induced a rapid and stopped by addition of 20% ice-cold perchloric acid, then the cells were dissociated and kept on ice for 20 min- transient increase in [Ca2'li level which was dose-de-

HE ET AL

0

2

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6

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Thrombin (U/ml)

Fig, 1. Variations of Ca2+ intracellular concentrations. A: Dose response effect of a-thrombin ( 5 to 10 Uiml). The [Ca"li increased from 109 f 11nM in basal conditions to 218 ? 48 nM in the presence of 5 to 10 Uiml a-thrombin (P < 0,001, n = 6). B: Effects of a-thrombin ( 5 Uiml) DFP-thrombin and y-thrombin (5 Uiml) alone or in association.

Nicardipine (Nic) (300 nM) and EGTA (4 mM) significantly inhibited a-thrombin effect. Mean 2 SEM of percent of increase from basal value are shown (*P < 0.01 and **P < 0.001 compared to a-thrombin alone).

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Fig. 2. Inositol triphosphate generation induced by a-thrombin. A Time course effect of 1Uiml a-thrombin (MI, 1 Uiml y-thrombin (O), 1 Uiml DFP-thrombin ( A ) , or combination of y-thrombin and DFPthrombin (A).B: Dose-response effect of a-thrombin measured after 10 seconds of incubation. *P < 0.01; **P < 0,001.

pendent. A plateau was reached with 1-2 U/ml a-THR. DFP-THR or y T H R (2.5 Ulml) had no effect when used alone or in association, indicating that simultaneous binding and intact proteolytic activity a t the cell surface was required to induce [Ca2+1iincrease. Furthermore, receptor occupancy by DFP-THR induced a halfinhibition of a-THR effect. An inhibition in a-THR-

stimulated [Ca2'li increase was observed when cells were tested in the presence of nicardipine or after pretreatment by 4 mM EGTA, suggesting that external Ca2+ influx into the cells was the main source of increase in [Ca2+li(Fig. lB), the remaining elevation of Ca2+ being due probably to release from intracellular stores. Maximal inhibition (60 to 70%) of a-THR-stimu-

THROMBIN ACTION ON GLOMERULAR EPITHELIAL CELLS

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lated ICa2+li was obtained with 300 nM (Fig. 1B). A similar inhibition was observed with 3 pM nicardipine (not shown).

2. IP3 generation As shown in Figure 2A, a-THR induced a rapid and transient increase in IP3 level in glomerular epithelial cells, which reached a fivefold increase after 10 seconds of incubation and then progressively decreased to control value. This effect was not observed when cells were incubated with DFP-THR or y-THR whether they were added alone or in combination. The dose-effect of a-THR is shown in Figure 2B. The maximal effect was reached a t 1U/ml a-THR. 3. Effects of nicardipine on thrombin actions The mitogenic effect of a-THR was inhibited in a dose-dependent manner by nicardipine, a calcium channel blocker, but in contrast thrombin-stimulated protein synthesis was not modified by this drug (Fig. 3). Verapamil, which also inhibits L-type voltage-operated

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calcium channels, also had a n inhibitory effect on a-THR-stimulated thymidine incorporation (not shown). Cell count was not modified significantly by nicardipine, excluding a nonspecific toxic effect due to cell detachment. To control that inhibition of cell growth was related to inhibition of calcium entry by nicardipine, we looked at the effect of ionomycin, which is known to increase calcium entry. As shown in Figure 4, lo-* M ionomycin was able to restore the growth promoting effect of a-THR inhibited by nicardipine. 4. Separate control of cell growth and protein

synthesis We have previously shown that a-THR stimulated DNA synthesis and u-PA, t-PA, and PAI-1 synthesis in human glomerular epithelial cells (He et al., 1991). DFP-THR or y-THR, when used alone, were ineffective on both cell growth and protein synthesis (Fig. 5). However, when added simultaneously to the cells, they stimulated significantly synthesis of u-PA, t-PA, and

HE ET AL

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Fig. 4. Incorporation of 3H-thymidine induced by 1Uiml a-thrombin (THR),with or without 0.3 pM nicardipine (Nic) and M ionomycin (Ion).Mean i SEM of 2 experiments made in triplicate are shown. **P < 0.01 compared to control value; *P < 0.05 compared to a-thrombin alone, +P < 0.05 compared to a-thrombin + nicardipine.

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PAI-1 but had no mitogenic activity (Fig. 5). Addition of M) to DFP-THR and y-THR did ionomycin (lop9to not modify the 3H thymidine incorporation nor protein synthesis (not shown). a-THR induced a more than threefold increase of thymidine incorporation and protein synthesis, whereas combination of DFP-THR and y-THR induced only a twofold stimulation of protein synthesis (Tables 1-3). All these results indicated that protein synthesis could be stimulated without [Ca2+]i Fig. 5. Effect of 1U/ml a-thrombin (a-THR), 1Uiml DFP-thrombin increase but that maximal effect of a-THR on cell (DFP-THR), 1 Uiml y-thrombin (y-THR), alone or in combination on growth and protein synthesis required a n increase of 'H thymidine incorporation (A), and on synthesis of u-PA (B), t-PA (C), and PAI-1 (D).Mean f SEM of 3 to 5 experiments made in tripli[Ca2 li. cate are shown. *P < 0.01; **P < 0.001; +P < 0.05 compared to re+

5. Role of protein kinase C activation

As shown in Tables 1 and 2, both DNA and protein synthesis by human glomerular epithelial cells were stimulated by 1U/ml a-THR. These effects were significantly inhibited by H7 (Hidaka et al., 1984), a n inhibitor of protein kinases (Table l ) , and by pretreatment of the cells for 24 hours by phorbol myristate acetate (PMA) (Table 2). PMA pretreatment has been shown to down-regulate PKC activity (Nishizuka, 1984). In separate experiments, PMA pretreatment for 24 hours was found to decrease the a-THR stimulated [Ca2+li increase from 236 16 to 160 rt 10 nM (P < 0.01, n = 3). The synthesis of fibrinolytic-related proteins induced by DFP-THR combined with y-THR was also inhibited by H7 and PMA pretreatment (Tables 1 and 2). Taken together these results suggest that PKC activity is stimulated by a-THR or DFP-THR combined with y-THR in these cells. It mediates protein synthesis and is necessary but not sufficient for cell growth activation. THR did not stimulate CAMPgeneration in these cells (not shown) and addition of 8 bromocyclic AMP, to induce protein kinase A activation, had a n inhibitory effect on cellular actions of a-THR (Table 3). Similarly, forskolin, which induces a maximal adenylate cyclase activation, inhibited a-THR effects on thymidine incorporation and protein synthesis (Table 3).

*

spective control values.

DISCUSSION We have previously shown that a-THR stimulated the proliferation of human glomerular epithelial cells in culture and that it increased the synthesis of t-PA, u-PA, and PAI-1 by these cells (He et al., 1991). We reported that DFP-THR alone had no effect, nor had y-THR alone. This indicates that both receptor occupancy and enzymatic activity of thrombin are required to induce these cellular effects, and is consistent with previous observations reported for fibroblasts and for endothelial cells (Carney et al., 1985; Paris e t al., 1988; Gelehrter and Szycer-Laszuk, 1986). We now demonstrate that cell proliferation and synthesis of specific proteins are stimulated by a-THR through different pathways in human glomerular epithelial cells. First, simultaneous addition of DFP-THR and y-THR does not stimulate cell growth and does not induce a n increase in [Ca2+1i but still stimulates the synthesis of proteins of the fibrinolytic system. Second, addition of nicardipine to thrombin-stimulated cells inhibits about 70% of the [Ca2'li increase and suppresses the thrombin-stimulated thymidine incorporation. Conversely nicardipine had no effect on the thrombin-

THROMBIN ACTION ON GLOMERULAR EPITHELIAL CELLS

481

TABLE 1. Effect of H7 on cell proliferation and synthesis of fibrinolytic components induced by a-thrombin or DFP-thrombin combined with y-thrombin'

3H thymidine

Culture treatment

incorporation (cpm/1o5 cells)

Control a-thrombin(lU/ml) DFP-thrombin( 1U/ml) y-thrombin(lU/ml) H7 (10pM) H7 a-thrombin H7 DFP-thrombin y-thrombin

2,042 i 220 7,113 f 670** 2,811 f 435

80 f 12 242 f 18** 168 f 17*

2,059 f 416 2,309 f 505**** 1,858 f 231

75 f 11 115 f 15**** 102 f 21***

+ +

+ +

Fibrinolytic components synthesis (ng/105 cells) u-PA t-PA PAI-1 2.8 f 0.4 12.3 f 1.2** 7.8 f 1.1*

2.5 f 0.7 3.9 f 1.6**** 3.7 f 0.4***

252 f 22 590 i 51** 427 35* 231 f 43 287 f 32**** 267 k 39***

'Cells were incubated in serum-free minimum defined medium with the indicated compounds for 24 hours. 'H-thymidine incorporation was measured during thelast 6 hours. The concentrations of fibrinolytic components were measured in the 24 hour-conditioned medium. Mean k SEM of 3 experiments made in triplicate are given. *P< 0.01; *+P< 0.001 a s compared to control. ***P< 0.01; ****P< 0.001 a s compared to a-thrombin or DFP-thrombin -,-thrombin.

+

TABLE 2. Effect of PMA pretreatment on cell proliferation and synthesis of fibrinolytic components induced by a-thrombin or DFP-thrombin combined with y-thrombin' Culture treatment Control a-thrombin(lU/ml) DFP-thrombin(lU/ml) y-thrombin(lU/ml) PMA pretreatment (16 nM) PMA pretreatment a-thrombin PMA pretreatment DFP-thrombin y-thrombin

+

+ + +

3H thymidine incorporation (cum/105 cells)

1,560 f 244 4,618 f 390** 2,110 f 372

Fibrinolytic components synthesis (ng/105 cells) u-PA t-PA PAI-1 12 f 18 251 i 32** 157 i 25*

2.6 f 0.6 12.5 f 1.4** 6.2 f 0.9*

271 i 42 632 f 61** 526 f 68*

1,284 f 295

58 f 10

2.0 f 0.6

234 f 31

1,634 f 278****

85 f 12****

3.3 f 0.7****

289 f 54****

1,478 i 267

77 f 17***

2.7 f 0.4***

262 f 48***

'Cells were incubated in serum-free minimum defined medium with the indicated compounds for 24 hours. %thymidine incorporation was measured during the last 6 hours.Theconcentrations of fibrinolytic components weremeasured in the 24 hour-conditioned medium. Mean k SEM of 3 experiments made in triplicate are given. *P< 0.01; **P< 0.001 a s compared to control. ***P< 0.01; ****P< 0.001 a s compared to a-thrombin or DFP-thrombin t 7-thrombin.

TABLE 3. Effect of 8 bromo cAMP and forskolin on a-thrombin-induced cell proliferation and fibrinolytic components synthesis of GECs' Culture treatment Control @-thrombin(1U/ml) 8 bromo cAMP (~o-~M) Forskolin M) a-thrombin 8 bromo cAMP a-thrombin fornkolin

+ +

3Hthymidine incorporation ( c ~ r n / 1 0cells) ~ 2,042 f 220 7,113 f 670** 1,570 i 61*

Fibrinolytic components synthesis (ng/105 cells) u-PA t-PA PAI-1 61 f 4 160 f 12** 45 i 8*

1,207 i 110* 3,044 f 289****

58 f 11 43 f 7****

1,843 f 121****

72

* 15***

3.6 f 0.8 10.4 i 1.4** 2.0 f 0.4*

192 i 31 444 f 52** 165 f 23

3.1 f 0.6 2.1 f OX****

176 f 18 171 f 24****

3.9 f 0.9***

191 f 27***

'Cells were incubated in serum-free minimum defined medium with the indicated compounds for 24 hours. "H-thymidine incorporation was measured during the last 6 hours. The concentrations of fibrinolytic components were measured in the 24 hour conditioned medium. Mean f SEM of at least 3 different experiments made in triplicate are given. *P< 0.05; **P< 0.001 a s compared to contol. ***P< 0.01; ****P< 0.001 a s compared to thrombin alone.

stimulated synthesis of u-PA, t-PA, and PAI-1. Third, a-THR does not induce cAMP generation as previously suggested (Gordon et al., 1984; Shah, 1989) and cAMP or forskolin addition had rather an inhibitory effect on a-THR actions. 1. Extracellular calcium entry is induced by a-THR and is required for cell growth but not for u-PA, t-PA, and PAI-1 synthesis stimulation.

Nicardipine is a dihydroperidine-derivativeand is an inhibitor of L-type voltage-dependent calcium channels (Catteral, 1988). Our results suggest that a-THR stimulates calcium influx through voltage-operated channels. This is also supported by the reduction in [Ca2+]i increase induced by EGTA and is in accordance with previous reports of calcium influx in human mesangial cells stimulated by a-THR (Shultz and Raij, 1990).Sim-

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ilar inhibitory effects were observed with another inhibitor of L-type calcium channels, verapamil (not shown). Nicardipine does not prevent 1251a-THR binding and does not inhibit to a similar extent the proliferative response to EGF (not shown). We cannot, however, exclude that nicardipine also prevents calcium entry mediated by inositol 1,3,4,5 tetrakisphosphate (Irvine and Moore, 1986) or mobilization of intracellular Ca2+ by binding to receptor sites on the sarcoplasmic reticulum (Oeken et al., 1986). However, increase in intracellular free calcium induced by a-THR seems to be required for cell growth stimulation. This is further supported by the effect of ionomycin, which restores the proliferative response induced by a-THR despite the presence of nicardipine (Fig. 4).It has to be noted that calcium influx alone induced by ionomycin does not stimulate DNA synthesis indicating that other intracellular signals generated by a-THR are also required. The mechanism of activation of L-type calcium channels by a-THR is not well understood. Intact enzymatic activity of a-THR seems to be required since neither DFP-THR nor y-THR alone or in combination was able to stimulate [Ca2+li increase. Furthermore, the combination of ionomycin DFP-THR and y-THR did not reproduce the effects of a-THR on cell proliferation and protein synthesis. This may suggest that another signal is induced by the intact enzymatic activity of a-THR. It is intriguing to observe that a-THR is mitogenic for some cell types but not for others (Berk, 1990), and that among thrombin-sensitive cells some respond to active a-THR, whereas other proliferate in the presence of DFP-THR (Bar-Shavit et al., 1990). It has recently been shown that a-THR could cleave a membrane protein which after autoactivation may transduce intracellular signals (Vu et al., 1991). In cell types which did not require the enzymatic activity of a-THR, it is possible that this membrane protein is endogenously cleaved and activated by binding of DFPTHR. Further studies are needed to answer this question. Calcium influx was not required for stimulation of u-PA, t-PA, and PAI-1 synthesis by a-THR, since nicardipine did not inhibit this effect. To our knowledge, this is the first report showing that proliferation and protein synthesis stimulated by a-THR can be inhibited separately. 2. Protein kinase C activation is required for thrombin to induce cell growth and synthesis of u-PA, t-PA, and PAI-1 by glomerular epithelial cells. IP3 generation was stimulated by a-THR and associated with a free [Ca2'li increase which was not inhibited by nicardipine or EGTA. These results are very consistent with a phospholipase C activation, PIP2 breakdown, and generation of IP3 and DAG, a s described in fibroblasts (Berk et al., 1990) and in endothelial cells (Dupuy et al., 1989). Although we did not measure directly PKC activity, it is likely that this effect induces PKC activation for the following reasons: 1)PMA has been shown to stimulate u-PA synthesis in glomerular epithelial cells (Rondeau et al., 1989) and t-PA and PAI-1 synthesis in other cell types (Levin et al., 1989); 2) the effects of a-THR on glomerular epithelial cells were inhibited by H7 and by pretreatment of the cells by PMA to induce PKC desensitization. The role of PKC is not well defined since PMA

pretreatment was found to inhibit also the a-THR-induced [Ca2+]iincrease. Whether this is due to a decrease in a-THR-receptors or to a direct role of PKC on calcium channels remains to be determined. Similar mechanisms may explain the inhibitory effect of CAMP on a-THR actions. Interestingly, stimulation of both cell growth and protein synthesis by a-THR require PKC activation. PIP2 breakdown may not be the only mechanism of PKC activation by a-THR since the combination of DFP-THR and yTHR also induces a n increased synthesis of u-PA, t-PA and PAI-1 without IP3 nor [Ca2+]i increase. This effect of THR-DFP and y-THR was also inhibited by H7 and PMA pretreatment, suggesting a role for PKC activation. It has recently been shown that DAG generation and PKC activation may also occur through PC hydrolysis in rat vascular smooth muscle cells (Berk et al., 1990). Moreover, it has been shown in IIC9 cells, a subclone of Chinese hamster embryo fibroblasts, that at high concentrations (0.1 to 1 U/ml), a-THR can stimulate both PC and PIP2 hydrolysis, whereas at lower concentrations it increases only PC hydrolysis. In these cells, PC hydrolysis alone was not associated with PKC activation, although there was DAG generation (Leach et al., 1991). It is likely that a-THR also stimulates PIP2 and PC hydrolysis in glomerular epithelial cells and that the combination of DFPTHR and y-THR only stimulates PC hydrolysis. In contrast to what was observed in IIC9 cells, PKC activation was promoted in the absence of PIP2 breakdown, suggesting that PKC activation following PC hydrolysis may be cell or tissue-dependent. However, we found a lower stimulation of u-PA, t-PA, and PAI-1 synthesis by DFP-THR and y-THR than by a-THR and further studies are required to determine the respective roles of PC and PIP2 hydrolysis. In conclusion, we describe different signalling pathways stimulated by a-THR in human lomerular epithelial cells. a-THR stimulates free [Ca8+]i increase by activation of external Ca2+ influx and by IP3 generation, which stimulates release of Ca2+ from internal stores; thrombin also stimulates PIP2 breakdown through phospholipase C activation and presumably PC breakdown, which results in PKC activation. Thrombin-stimulated cell growth is highly dependent on both Ca2+ influx and PKC activation, whereas thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1 does not depend on Ca2+influx but requires PKC activation.

ACKNOWLEDGMENTS This work was supported by grants from the Association Claude Bernard, Paris, France, and the Institut National de la Sant6 et de la Recherche Medicale. We are indebted to Miss Mina Mallet for excellent secretarial assistance. LITERATURE CITED Aducci, P., and Marra, M. (1990) IP3 levels and modulation by fusicoccin measured by a novel [H31IP3 binding assay. Biochem. Biophys. Res. Commun., I68:1041-1046. Awbrey, B.J., Hoak, J.C., and Owen, W.G. (1979) Binding of thrombin to cultured human endothelial cells. J . Biol. Chem., 254:4092-4095. Bar-Shavit, R., Benezra, M., Eldor, A., Hy-Am, E., Fenton, J.W., 11, Wilner, G.D., and Vlodavsky, I. (1990) Thrombin immobilized to

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