JOURNAL OF CELLULAR PHYSIOLOGY 148:133-138 (19911
Prolactin Induces Proliferation of Vascular Smooth Muscle Cells Through a Protein Kinase C-Dependent Mechanism MARIE D. SAURO* AND N A N C Y E. ZORN Department of Pharmacology and l'herapeutics, University of South Florida College of Medicine, and H. lee Moffitt Cancer Center and Research Institute, Tampa, Florida 336 12 The effects of prolactin (PRL) on A10 (aortic smooth muscle) cell proliferation were examined by measuring both [jHIthymidine incorporation and increases in cell number. PRL induced a significant proliferative response from l o - ' ' to l o p 7 M, with optimal activity at lo-'' M. PRL also enhanced platelet-derived growth factor (PDCF)-induced proliferation. The possibility that PRL induces proliferation through a protein kinase C (PKC)-mediated mechanism was also examined. PRL caused activation of PKC from 1Op'* to l o - * M. Antiserum to PRL, a monoclonal antibody directed against the PRL receptor and the immunosuppressive agent cyclosporine A, were able to inhibit PRL-induced proliferation and activation of PKC. The PKC inhibitors, staurosporine, sphingosine, and 1-(-5-iso-quinolinesulfonyl)-2-methylpiperazine (H-7) also antagonized both proliferation and PKC activation. These data strongly suggest that PRL-induced A1 0 cell proliferation is mediated through the PKC pathway and that this may play a role in vascular smooth muscle cell hyperplasia, characteristic of the pathogenesis of cardiovascular diseases such as hypertension and atherosclerosis.
Cardiovascular diseases such as hypertension and atherosclerosis are characterized by structural and functional alterations in the vasculature. In hypertension, structural changes include hypertrophy and hyperplasia of the vascular media (Owens, 19891, rarefication of resistance vessels, and develo ment of intimal lesions, resulting in increased perip era1 resistance (reviewed by Winquist et al., 1982). Hypertension is thought t o be a major risk factor for atherosclerosis development, although the underlying mechanisms have not been elucidated. In atherosclerosis, following endothelial cell damage, vascular smooth muscle cells (VSMCs) migrate from the medial layer to the intima, where they proliferate and together with infiltrated leukocytes and li id droplets form atherosclerotic plaques (reviewed y Ross, 1986). The mechanisms responsible for abnormal cell growth involve a multitude of factors, many of which are not well understood. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) have been extensively studied and shown to be mitogenic for VSMC. Other hormones may play a role as well. Prolactin (PRL)is a polypeptide hormone best known for its lactogenic properties. More recently, PRL has been im licated in a variety of physiological functions, one of w ich is growth promotion/mitogenesis (Buckley et al., 1986). Receptors for PRL have been identified in a number of tissues, including the vasculature (Russell et al., 198413; Buckley et al., 1984). PRL has been implicated as a factor in blood pressure regulation; however, these findings are controversial (Mills et al., 1981,1982; Manku et al., 1979).It has also been shown that mononuclear cells, which are in intimate contact
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0 1991 WILEY-LISS, INC.
with the vasculature, secrete a PRL-like molecule, although the stimulus for release is not known (Montgomery et al., 1987). Because of these cell-cell interactions, it would seem logical that the vasculature is a target tissue for PRL. Although there are numerous examples demonstrating a role for PRL in blood pressure regulation, the receptor-mediated intracellular mechanism of action is not well characterized. Signal transduction pathways such as CAMP,cGMP, phosphoinositides, and tyrosine kinases do not appear to be involved in PRL intracellular signallin Buckley et al. (1986, 1987) have demonstrated t at PRL may operate via a protein kinase C (PKCI-mediated mechanism. Because of its mitogenic pro erties and its implicated role in blood pressure regu ation, we investigated the possibilities that PRL induces VSMC hyperplasia and that the response is mediated through PKC.
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MATERIALS AND METHODS Materials A10 cells were purchased from the American Type Culture Collection (Rockville, MD). Dulbecco's modified Eagle's media (DMEM), penicillinistreptomycin, and heat-inactivated fetal calf serum (FCS) were purchased from Gibco (Grand Island, NY). PDGF, sphingosine sulfate, l-(-5-isoquinolinesulfonyl)-2-methylReceived October 17, 1990; accepted March 14,1991. *To whom reprint requestsicorrespondence should be addressed at: Masonic Medical Research Laboratory, 2150 Bleecker St., Utica. NY 13501.
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piperazine (H-7), phosphatidyl serine, 1,2-diolein, leupeptin, CAMP-dependent protein kinase inhibitor, histone (type IIIs), trypsin-EDTA (10x), and phorbol 12,13-dibutyrate (PDBu) were purchased from Sigma Chemical Co. (St. Louis, MO). Ovine PRL-18 (oPRL) and anti-PRL antiserum were obtained from NIDDK (Bethesda, MD). The anti-PRL receptor monoclonal antibody (Tl) was a generous gift from Dr. Paul Kelly. Cyclosporine A (CsA) was obtained from Sandoz Pharmaceuticals (Newark, NJ). Staurosporine was purchased from Kamiya (Thousand Oaks, CA). L3H1thymidine and [32P]ATP were purchased from Dupont (Wilmington, DE). All other chemicals were reagent grade and were purchased from Sigma. Cell culture Aortic smooth muscle cells (A10 cells), derived from thoracic aorta of embryonic rats were used in this study. Cells were grown in DMEM containing 10%FCS and 1%penicillinistreptomycin in 175 ml dis osable tissue culture flasks, maintained at 37°C in a umidified atmos here of 5% co2/95%air and were subcultured week y. At confluency, monolayers were washed with 10 ml Hank’s balanced salt solution (HBSS) without MgC12, treated with 5 ml trypsin-EDTA (diluted 1:lO with HBSS) for 3 min, and centrifuged at 1,200 rpm for 10 min. Cells were resuspended in 5-10 ml DMEM with 10% FCS and 1%penicillin/ streptomycin and seeded at a ratio of 1 to 4. Experiments were performed at passages 20-35; it has been reported by others that DNA synthesis in these cells remains stable at passages 16-150 (Cascieri et al., 1986). DNA synthesis Confluent cultures were washed and treated with trypsin-EDTA (diluted 1 : l O with HBSS) for 3 min, centrifuged at 1,200 rpm for 10 min, and resuspended in DMEM containing 10% FCS and 1%penicillin/ streptomycin. Cells were plated at an initial concentration of 5,000 cellsiwell in 96-well culture plates in a total volume of 200 pl and were allowed to adhere overnight. Quiescence was induced by a 24-hr serum deprivation (Cascieri et al., 1986) and was verified by flow cytometry. Quiescent cells were washed with HBSS and then treated with various pharmacological agents, 25 p1 per well, in DMEM plus or minus 0.1% FCS. Direct effects of each agent were tested in the absence of PRL. Cells were incubated for 24 hr, which was shown to give optimal [3H]thymidine incorporation, at 37°C in 5% C02i95%air, and ulsed for the last 4 hr with [3H]thymidine, 0.5 pCiiwe 1 (6.7 Ci/mmole). Media were aspirated, and cells were washed with HBSS, trypsinized for 3 min (trypsin-EDTA diluted 1 : l O with deionized water), and collected on filter paper disks using a Skatron cell harvester. Wells and filters were washed five times with deionized water. [3Hlthymidine incorporation was counted using liquid scintillation spectroscopy. Cells remained viable after 48 hr as determined by trypan blue exclusion. None of the agents tested resulted in significant loss of cell viability at the concentrations used, also determined by trypan blue exclusion. Results are expressed as cpm L3H1thymidine incorporated into cells.
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Determination of cell number Cells were plated at an initial concentration of 5,000 cellsiwell in six-well culture plates in a total volume of 2 ml and were allowed to adhere overnight. As in previously described studies, quiescence was induced by a 24 hr serum deprivation. Quiescent cells were treated with various pharmacological agents in DMEM plus or minus FCS and were allowed to incubate for 24 hr at 37°C. After 24 hr, media were aspirated, and cells were washed with HBSS, trypsinized for 3 min, and centrifuged at 1,200 rpm for 10 min. Cells were resuspended in 0.1-0.2 ml DMEM, and 50 pl of the cell suspension was added to 50 p1 of trypan blue. The suspension was mixed well, and cells were counted using a hemocytometer. PKC determination Confluent cells in 175-ml flasks were serum-deprived for 24 hr. Media were aspirated, and cells were allowed to equilibrate in warmed HBSS at 37°C for 30 min. HBSS was changed two times; the last time it was replaced with HBSS containing 0.75 mM CaC12,which was shown to ive optimal PKC activation. Cells were then incubate with various agents at 37°C for 15 min, which again allowed for optimal PKC activation. The reaction was terminated by aspirating solution and adding ice-cold homogenization buffer [20 mM Tris, pH 7.4, 0.3 M sucrose, 5 mM EDTA, 2 mM EGTA, 5 mM dithiothreitol (DTT), 25 pgiml leupeptin, and 1 mM pheny lmethylsulfonylf luoride (PMSF)]. Cells were scraped and processed using a slight modification of the method of Sauro and Fitzpatrick (1990). Briefly, cells suspended in homogenization buffer were sonicated for 5 sec five times, and homogenates were centrifuged at 106,OOOg for 30 min at 4°C. The supernatant from this centrifugation is the crude cytosolic fraction. Pellets were resuspended in homogenization buffer containing 0.1% Triton X-100, sonicated for 5 sec five times, and gently rocked for 45 min at 4°C. The homogenate was centrifuged at 106,OOOgfor 20 min at 4°C. The supernatant from this centrifugation is the crude particulate fraction. It was found that there was no appreciable PKC activity in the pellet from this second centrifugation. PKC was then partially purified using affinity chromatography (DEAE cellulose columns) and eluted with 100 mM NaC1. PKC activity was assayed at 37°C for 5 min in a total volume of 250 pl of reaction buffer [20 mM Tris, pH 7.4, 10 mM Mg(C2H3O2I2,200 pgirnl histone, 100 pM ATP (containing [32PlATPwith 4-5 x lo6 CPM), 3.5 mM CAMP-dependent protein kinase inhibitor, 50 p1 sample (containing 10-40 kg proteiniml, well within the linear range for PKC measurement), and either 5 mM EGTA or 1 mM CaC12 and liposomes (30 pgiml phosphatidylserine and 3 pg/ml1,2-dioleinl. Reaction buffer minus the sample was preincubated for 5 min at 37°C. The reaction was initiated by addition of sample and terminated after 5 min by addition of 3 ml ice-cold 10% trichloroacetic acid (TCA).Samples were allowed to sit on ice for 30 rnin to ensure complete protein precipitation, collected on filter paper disks using a Brandel cell harvester, and counted in a Beckman scintillation counter. Protein concentration was determined using a
%
135
PRL-INDUCED A10 CELL PROLIFERATION THROUGH PKC
Biorad protein assay kit. Results are expressed as pmol phosphateiminimg protein. Statistical analysis For all experiments, statistical significance was established using an analysis of variance or Student's t test, with P < 0.05 considered to be significant.
RESULTS Figure 1illustrates the res onsiveness of A10 cells to increasing concentrations ofF! CS. The inset shows the concentration response over a range of 0-1% FCS; 0.1% FCS was chosen for experiments because of its ability to stimulate proliferation, yet not possess levels of PRL and other growth factors that could interfere with data interpretation. Figure 2 illustrates the effects of oPRL (10p12-10-7 M) on DNA synthesis in A10 cells as determined by L3H1thymidineuptake. When cells were incubated with oPRL in DMEM minus FCS, PRL induced a significant stimulation of proliferation from 10p11-10-7 M ( P < 0.05). An oPRL concentration of lo-'' M gave an optimal response (33% increase in [3Hlthymidine uptake). When cells were incubated with oPRL in DMEM plus 0.1% FCS, 10-1'-10-9 M oPRL induced a significant proliferative response ( P < 0.05), again with lop1' M giving optimal activity (25%increase in proliferation). Table 1 shows the effects of various agents on oPRLinduced A10 cell proliferation. Concentrations of'inhibitors used were those that were found to have minimal effects on basal proliferative activity, but could significantly inhibit oPRL-induced proliferation. PDBu (lo-' M) was used as a ositive control. Alone, PDBu significantly stimulate proliferation but antagonized oPRLinduced proliferation. Anti-PRL antiserum and antiPRL receptor antibody were able to antagonize the PRL response, as was the immunosuppressive agent CsA. The PKC inhibitors H-7, staurosporine, and sphingosine also inhibited oPRL-induced L3H]thymidine incorporation. Each PKC inhibitor was tested for its effects on cell viability using trypan blue exclusion and was found to have minimal or no effect at the concentrations used. Also studied was whether oPRL-induced VHlthymidine incorporation was a true measure of proliferation or merely increasing the intracellular pool of thymidine. Table 2 shows that oPRL induces an increase in cell number, which could be blocked using PRL antiserum, anti-PRL receptor antibody as well as PKC inhibitors. We then examined the possibility of an interaction between PRL and PDGF, a known VSMC mitogen. Ovine PRL (lop1' M) significantly enhanced PDGFinduced A10 cell proliferation ( P < 0.05) (Fig. 3A,B). This was true for cells cultured in both 0% FCS and 0.1% FCS. PRL induced significant activation of PKC in A10 cells with optimal articulate activity occurring at lop1' M ( P < 0.05) (fig. 4).In the particulate fraction, a "bell-shaped,' concentration response was seen, which is characteristic for PRL. The effects of various agents on oPRL-induced PKC activation in the particulate fraction are shown in Table 3. Anti-PRL antiserum and
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[Fetal Calf Serum] (%) Fig. 1. Effects of fetal calf serum (FCS) concentration on A10 cell proliferation. Cells were prepared as described in Materials and Methods and incubated in DMEM containing various concentrations of FCS for 24 hr a t 37°C. Results are expressed as mean value i SEM from three experiments. Inset shows the concentration response from 0-1% FCS.
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LOG [oPRL] (M) Fig. 2. Effects of oPRL on A10 cell proliferation. Cells were prepared as described in Materials and Methods and incubated in DMEM (plus or minus FCS) and various concentrations of oPRL for 24 hr at 37°C. Results are expressed as mean value ISEM from seven to ten experiments. In 0% FCS, oPRL significantly stimulated proliferation from lo-'' to M ( P < 0.05). In 0.1% FCS, oPRL significantly stimulated proliferation from lo-" to W9M ( P < 0.05). Abbreviations: FCS, fetal calf serum; oPRL, ovine prolactin.
anti-PRL receptor antibody inhibited PRL-induced PKC activation as did the PKC inhibitors, H-7, staurosporine, and sphingosine. Finally, the interaction between oPRL and other PKC activators was examined. Table 4 illustrates that, whereas oPRL, PDBu, and PDGF by themselves induced significant activation of particulate PKC, PDGF inhibited PRL-induced activity when incubated together, although activity was not totally blocked. PDBu also had an inhibitory effect on PRL-induced PKC activity. DISCUSSION In both hypertension and atherosclerosis, structural alterations in the vasculature occur, resulting in a
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TABLE 1. Effects of various agents on oPRL-induced proliferation in A10 cells' cpm [3H]thymidine incorporated -oPRT, +oPRL 0% FCS CTR Antiserum (11750) AntirecAb (10 pg/ml) CsA M)
PDBu (lo-' M) Staurosporine (1 nM) H-7 (6 pM) Sphingosine (10 pM) 0.1% FCS CTR Antiserum (1/750) AntirecAb (10 pg/ml) CsA M) PDBu M) Staurosporine (1 nM) H-7 (6 pM) Sphingosine (10 pM)
3.250 F 123 3,022 f 373 3,305 i- 253 3,084 f 165 4,944 f 162* 3,012 F 321 3,659 f 139 4,322 f 900
4,071 k 252* 3,160 F 43** 3,028 f 232** 2,970 f 164** 3,163 F 81** 2,892 f 67** 3,498 f 66** 2,988 f 189**
4,420 F 138 4,741 F 188 4,801 f 218 4,760 i 300 6,209 f 111* 4,120 f 553 5,015 f 255 4,984 f 657
5,331 f 163* 4,526 f 149** 4,676 f 182** 4,660 f 84** 4,504 i 43** 4,263 f 65** 4,354 f 375** 2,779 f 474**
'Cells were prepared a s described in Materials and Methods and incubated in DMEM plus or minus FCS with each agent alone or with oPRL (lO-"M) plus each agent. Abbreviations: FCS, fetal calf serum; oPRL, ovine prolactin; CTR, control; antiserum, prolactin antiserum: antirecAb, antiprolactin receptor antibody; CsA, cyclosporine A PDBu, phorbol 12, 13-dibutyrate;H-7, 1-(-5-isoquinolinesuIfonyl)-2methylpiperazine. n = 6-10. *Significant stimulation over basal (P< 0.05). **Significant inhibition of PRL-induced proliferation (P< 0.05).
pathological response (Win uist et al., 1982). Characteristic of both diseases is V MC hyperplasia; however, the mechanisms by which this occurs are not well understood (ROSS,1986; Owens, 1989). In this study, we have illustrated that PRL is mitogenic for VSMC and that its proliferative response may be mediated through a PKC-dependent mechanism. We have also demonstrated that PRL is able to enhance PDGFinduced VSMC proliferation. oPRL caused a concentration-dependent proliferative response in A10 cells when incubated in either 0% FCS or 0.1% FCS with an optimal response occurring at 10-l' M (33% increase in L3H1thymidineuptake). When incubated with 0.1% FCS, higher concentrations of oPRL did not stimulate an increase in [3Hlthymidine uptake. This could possibly be due to the fact that serum contains PRL, and, when cells are treated with high PRL concentrations, rece tor down-regulation may occur; it has been shown t at internalized PRL persists in cells for long periods of time and binds almost irreversibly to its receptor (Kelly et al., 1979). To ensure that PRL was not merely increasing the intracellular pool of thymidine, we looked at the ability of oPRL to increase cell number and found that PRL did indeed induce proliferation. Anti-PRL antiserum and an anti-PRL receptor antibody antagonized the proliferative response t o PRL. Furthermore, the immunosuppressive agent CsA, which antagonizes many actions of PRL by competitively binding to the PRL receptor (Russell et al., 1984a,b),also attenuated oPRL-induced L3HJthymidine uptake. These data strongly suggest that PRL causes proliferation in these cells through a receptor-mediated mechanism. PRL has been previously shown to be mitogenic in a
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TABLE 2. Effects of PRL and inhibitors on A10 cell number' Aeent CTR oPRL (lo-'* M) oPRL (lo-" M) oPRL (lo-" M) + Antiserum (1/750) AntirecAb (10 pg/ml) Staurosporine (1 nM) H-7 (6 pM) Sphingosine (10 pM)
Cell number ( N O 3 ) 0% FCS 0.1% FCS 5.2 6.8 8.4
5.9 7.1 9.1
5.4 4.9 5.3 5.1 5.8
5.1 6.2 4.9 6.3 6.1
'Cells were prepared a s described in Materials and Methods and incubated in DMEM plus or minus FCS with each agent. This experiment was repeated three times with similar results. Variation in cell numberwas
Prolactin Induces Proliferation of Vascular Smooth Muscle Cells Through a Protein Kinase C-Dependent Mechanism MARIE D. SAURO* AND N A N C Y E. ZORN Department of Pharmacology and l'herapeutics, University of South Florida College of Medicine, and H. lee Moffitt Cancer Center and Research Institute, Tampa, Florida 336 12 The effects of prolactin (PRL) on A10 (aortic smooth muscle) cell proliferation were examined by measuring both [jHIthymidine incorporation and increases in cell number. PRL induced a significant proliferative response from l o - ' ' to l o p 7 M, with optimal activity at lo-'' M. PRL also enhanced platelet-derived growth factor (PDCF)-induced proliferation. The possibility that PRL induces proliferation through a protein kinase C (PKC)-mediated mechanism was also examined. PRL caused activation of PKC from 1Op'* to l o - * M. Antiserum to PRL, a monoclonal antibody directed against the PRL receptor and the immunosuppressive agent cyclosporine A, were able to inhibit PRL-induced proliferation and activation of PKC. The PKC inhibitors, staurosporine, sphingosine, and 1-(-5-iso-quinolinesulfonyl)-2-methylpiperazine (H-7) also antagonized both proliferation and PKC activation. These data strongly suggest that PRL-induced A1 0 cell proliferation is mediated through the PKC pathway and that this may play a role in vascular smooth muscle cell hyperplasia, characteristic of the pathogenesis of cardiovascular diseases such as hypertension and atherosclerosis.
Cardiovascular diseases such as hypertension and atherosclerosis are characterized by structural and functional alterations in the vasculature. In hypertension, structural changes include hypertrophy and hyperplasia of the vascular media (Owens, 19891, rarefication of resistance vessels, and develo ment of intimal lesions, resulting in increased perip era1 resistance (reviewed by Winquist et al., 1982). Hypertension is thought t o be a major risk factor for atherosclerosis development, although the underlying mechanisms have not been elucidated. In atherosclerosis, following endothelial cell damage, vascular smooth muscle cells (VSMCs) migrate from the medial layer to the intima, where they proliferate and together with infiltrated leukocytes and li id droplets form atherosclerotic plaques (reviewed y Ross, 1986). The mechanisms responsible for abnormal cell growth involve a multitude of factors, many of which are not well understood. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) have been extensively studied and shown to be mitogenic for VSMC. Other hormones may play a role as well. Prolactin (PRL)is a polypeptide hormone best known for its lactogenic properties. More recently, PRL has been im licated in a variety of physiological functions, one of w ich is growth promotion/mitogenesis (Buckley et al., 1986). Receptors for PRL have been identified in a number of tissues, including the vasculature (Russell et al., 198413; Buckley et al., 1984). PRL has been implicated as a factor in blood pressure regulation; however, these findings are controversial (Mills et al., 1981,1982; Manku et al., 1979).It has also been shown that mononuclear cells, which are in intimate contact
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0 1991 WILEY-LISS, INC.
with the vasculature, secrete a PRL-like molecule, although the stimulus for release is not known (Montgomery et al., 1987). Because of these cell-cell interactions, it would seem logical that the vasculature is a target tissue for PRL. Although there are numerous examples demonstrating a role for PRL in blood pressure regulation, the receptor-mediated intracellular mechanism of action is not well characterized. Signal transduction pathways such as CAMP,cGMP, phosphoinositides, and tyrosine kinases do not appear to be involved in PRL intracellular signallin Buckley et al. (1986, 1987) have demonstrated t at PRL may operate via a protein kinase C (PKCI-mediated mechanism. Because of its mitogenic pro erties and its implicated role in blood pressure regu ation, we investigated the possibilities that PRL induces VSMC hyperplasia and that the response is mediated through PKC.
%
f
MATERIALS AND METHODS Materials A10 cells were purchased from the American Type Culture Collection (Rockville, MD). Dulbecco's modified Eagle's media (DMEM), penicillinistreptomycin, and heat-inactivated fetal calf serum (FCS) were purchased from Gibco (Grand Island, NY). PDGF, sphingosine sulfate, l-(-5-isoquinolinesulfonyl)-2-methylReceived October 17, 1990; accepted March 14,1991. *To whom reprint requestsicorrespondence should be addressed at: Masonic Medical Research Laboratory, 2150 Bleecker St., Utica. NY 13501.
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piperazine (H-7), phosphatidyl serine, 1,2-diolein, leupeptin, CAMP-dependent protein kinase inhibitor, histone (type IIIs), trypsin-EDTA (10x), and phorbol 12,13-dibutyrate (PDBu) were purchased from Sigma Chemical Co. (St. Louis, MO). Ovine PRL-18 (oPRL) and anti-PRL antiserum were obtained from NIDDK (Bethesda, MD). The anti-PRL receptor monoclonal antibody (Tl) was a generous gift from Dr. Paul Kelly. Cyclosporine A (CsA) was obtained from Sandoz Pharmaceuticals (Newark, NJ). Staurosporine was purchased from Kamiya (Thousand Oaks, CA). L3H1thymidine and [32P]ATP were purchased from Dupont (Wilmington, DE). All other chemicals were reagent grade and were purchased from Sigma. Cell culture Aortic smooth muscle cells (A10 cells), derived from thoracic aorta of embryonic rats were used in this study. Cells were grown in DMEM containing 10%FCS and 1%penicillinistreptomycin in 175 ml dis osable tissue culture flasks, maintained at 37°C in a umidified atmos here of 5% co2/95%air and were subcultured week y. At confluency, monolayers were washed with 10 ml Hank’s balanced salt solution (HBSS) without MgC12, treated with 5 ml trypsin-EDTA (diluted 1:lO with HBSS) for 3 min, and centrifuged at 1,200 rpm for 10 min. Cells were resuspended in 5-10 ml DMEM with 10% FCS and 1%penicillin/ streptomycin and seeded at a ratio of 1 to 4. Experiments were performed at passages 20-35; it has been reported by others that DNA synthesis in these cells remains stable at passages 16-150 (Cascieri et al., 1986). DNA synthesis Confluent cultures were washed and treated with trypsin-EDTA (diluted 1 : l O with HBSS) for 3 min, centrifuged at 1,200 rpm for 10 min, and resuspended in DMEM containing 10% FCS and 1%penicillin/ streptomycin. Cells were plated at an initial concentration of 5,000 cellsiwell in 96-well culture plates in a total volume of 200 pl and were allowed to adhere overnight. Quiescence was induced by a 24-hr serum deprivation (Cascieri et al., 1986) and was verified by flow cytometry. Quiescent cells were washed with HBSS and then treated with various pharmacological agents, 25 p1 per well, in DMEM plus or minus 0.1% FCS. Direct effects of each agent were tested in the absence of PRL. Cells were incubated for 24 hr, which was shown to give optimal [3H]thymidine incorporation, at 37°C in 5% C02i95%air, and ulsed for the last 4 hr with [3H]thymidine, 0.5 pCiiwe 1 (6.7 Ci/mmole). Media were aspirated, and cells were washed with HBSS, trypsinized for 3 min (trypsin-EDTA diluted 1 : l O with deionized water), and collected on filter paper disks using a Skatron cell harvester. Wells and filters were washed five times with deionized water. [3Hlthymidine incorporation was counted using liquid scintillation spectroscopy. Cells remained viable after 48 hr as determined by trypan blue exclusion. None of the agents tested resulted in significant loss of cell viability at the concentrations used, also determined by trypan blue exclusion. Results are expressed as cpm L3H1thymidine incorporated into cells.
K
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Determination of cell number Cells were plated at an initial concentration of 5,000 cellsiwell in six-well culture plates in a total volume of 2 ml and were allowed to adhere overnight. As in previously described studies, quiescence was induced by a 24 hr serum deprivation. Quiescent cells were treated with various pharmacological agents in DMEM plus or minus FCS and were allowed to incubate for 24 hr at 37°C. After 24 hr, media were aspirated, and cells were washed with HBSS, trypsinized for 3 min, and centrifuged at 1,200 rpm for 10 min. Cells were resuspended in 0.1-0.2 ml DMEM, and 50 pl of the cell suspension was added to 50 p1 of trypan blue. The suspension was mixed well, and cells were counted using a hemocytometer. PKC determination Confluent cells in 175-ml flasks were serum-deprived for 24 hr. Media were aspirated, and cells were allowed to equilibrate in warmed HBSS at 37°C for 30 min. HBSS was changed two times; the last time it was replaced with HBSS containing 0.75 mM CaC12,which was shown to ive optimal PKC activation. Cells were then incubate with various agents at 37°C for 15 min, which again allowed for optimal PKC activation. The reaction was terminated by aspirating solution and adding ice-cold homogenization buffer [20 mM Tris, pH 7.4, 0.3 M sucrose, 5 mM EDTA, 2 mM EGTA, 5 mM dithiothreitol (DTT), 25 pgiml leupeptin, and 1 mM pheny lmethylsulfonylf luoride (PMSF)]. Cells were scraped and processed using a slight modification of the method of Sauro and Fitzpatrick (1990). Briefly, cells suspended in homogenization buffer were sonicated for 5 sec five times, and homogenates were centrifuged at 106,OOOg for 30 min at 4°C. The supernatant from this centrifugation is the crude cytosolic fraction. Pellets were resuspended in homogenization buffer containing 0.1% Triton X-100, sonicated for 5 sec five times, and gently rocked for 45 min at 4°C. The homogenate was centrifuged at 106,OOOgfor 20 min at 4°C. The supernatant from this centrifugation is the crude particulate fraction. It was found that there was no appreciable PKC activity in the pellet from this second centrifugation. PKC was then partially purified using affinity chromatography (DEAE cellulose columns) and eluted with 100 mM NaC1. PKC activity was assayed at 37°C for 5 min in a total volume of 250 pl of reaction buffer [20 mM Tris, pH 7.4, 10 mM Mg(C2H3O2I2,200 pgirnl histone, 100 pM ATP (containing [32PlATPwith 4-5 x lo6 CPM), 3.5 mM CAMP-dependent protein kinase inhibitor, 50 p1 sample (containing 10-40 kg proteiniml, well within the linear range for PKC measurement), and either 5 mM EGTA or 1 mM CaC12 and liposomes (30 pgiml phosphatidylserine and 3 pg/ml1,2-dioleinl. Reaction buffer minus the sample was preincubated for 5 min at 37°C. The reaction was initiated by addition of sample and terminated after 5 min by addition of 3 ml ice-cold 10% trichloroacetic acid (TCA).Samples were allowed to sit on ice for 30 rnin to ensure complete protein precipitation, collected on filter paper disks using a Brandel cell harvester, and counted in a Beckman scintillation counter. Protein concentration was determined using a
%
135
PRL-INDUCED A10 CELL PROLIFERATION THROUGH PKC
Biorad protein assay kit. Results are expressed as pmol phosphateiminimg protein. Statistical analysis For all experiments, statistical significance was established using an analysis of variance or Student's t test, with P < 0.05 considered to be significant.
RESULTS Figure 1illustrates the res onsiveness of A10 cells to increasing concentrations ofF! CS. The inset shows the concentration response over a range of 0-1% FCS; 0.1% FCS was chosen for experiments because of its ability to stimulate proliferation, yet not possess levels of PRL and other growth factors that could interfere with data interpretation. Figure 2 illustrates the effects of oPRL (10p12-10-7 M) on DNA synthesis in A10 cells as determined by L3H1thymidineuptake. When cells were incubated with oPRL in DMEM minus FCS, PRL induced a significant stimulation of proliferation from 10p11-10-7 M ( P < 0.05). An oPRL concentration of lo-'' M gave an optimal response (33% increase in [3Hlthymidine uptake). When cells were incubated with oPRL in DMEM plus 0.1% FCS, 10-1'-10-9 M oPRL induced a significant proliferative response ( P < 0.05), again with lop1' M giving optimal activity (25%increase in proliferation). Table 1 shows the effects of various agents on oPRLinduced A10 cell proliferation. Concentrations of'inhibitors used were those that were found to have minimal effects on basal proliferative activity, but could significantly inhibit oPRL-induced proliferation. PDBu (lo-' M) was used as a ositive control. Alone, PDBu significantly stimulate proliferation but antagonized oPRLinduced proliferation. Anti-PRL antiserum and antiPRL receptor antibody were able to antagonize the PRL response, as was the immunosuppressive agent CsA. The PKC inhibitors H-7, staurosporine, and sphingosine also inhibited oPRL-induced L3H]thymidine incorporation. Each PKC inhibitor was tested for its effects on cell viability using trypan blue exclusion and was found to have minimal or no effect at the concentrations used. Also studied was whether oPRL-induced VHlthymidine incorporation was a true measure of proliferation or merely increasing the intracellular pool of thymidine. Table 2 shows that oPRL induces an increase in cell number, which could be blocked using PRL antiserum, anti-PRL receptor antibody as well as PKC inhibitors. We then examined the possibility of an interaction between PRL and PDGF, a known VSMC mitogen. Ovine PRL (lop1' M) significantly enhanced PDGFinduced A10 cell proliferation ( P < 0.05) (Fig. 3A,B). This was true for cells cultured in both 0% FCS and 0.1% FCS. PRL induced significant activation of PKC in A10 cells with optimal articulate activity occurring at lop1' M ( P < 0.05) (fig. 4).In the particulate fraction, a "bell-shaped,' concentration response was seen, which is characteristic for PRL. The effects of various agents on oPRL-induced PKC activation in the particulate fraction are shown in Table 3. Anti-PRL antiserum and
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[Fetal Calf Serum] (%) Fig. 1. Effects of fetal calf serum (FCS) concentration on A10 cell proliferation. Cells were prepared as described in Materials and Methods and incubated in DMEM containing various concentrations of FCS for 24 hr a t 37°C. Results are expressed as mean value i SEM from three experiments. Inset shows the concentration response from 0-1% FCS.
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LOG [oPRL] (M) Fig. 2. Effects of oPRL on A10 cell proliferation. Cells were prepared as described in Materials and Methods and incubated in DMEM (plus or minus FCS) and various concentrations of oPRL for 24 hr at 37°C. Results are expressed as mean value ISEM from seven to ten experiments. In 0% FCS, oPRL significantly stimulated proliferation from lo-'' to M ( P < 0.05). In 0.1% FCS, oPRL significantly stimulated proliferation from lo-" to W9M ( P < 0.05). Abbreviations: FCS, fetal calf serum; oPRL, ovine prolactin.
anti-PRL receptor antibody inhibited PRL-induced PKC activation as did the PKC inhibitors, H-7, staurosporine, and sphingosine. Finally, the interaction between oPRL and other PKC activators was examined. Table 4 illustrates that, whereas oPRL, PDBu, and PDGF by themselves induced significant activation of particulate PKC, PDGF inhibited PRL-induced activity when incubated together, although activity was not totally blocked. PDBu also had an inhibitory effect on PRL-induced PKC activity. DISCUSSION In both hypertension and atherosclerosis, structural alterations in the vasculature occur, resulting in a
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SAURO AND ZORN
TABLE 1. Effects of various agents on oPRL-induced proliferation in A10 cells' cpm [3H]thymidine incorporated -oPRT, +oPRL 0% FCS CTR Antiserum (11750) AntirecAb (10 pg/ml) CsA M)
PDBu (lo-' M) Staurosporine (1 nM) H-7 (6 pM) Sphingosine (10 pM) 0.1% FCS CTR Antiserum (1/750) AntirecAb (10 pg/ml) CsA M) PDBu M) Staurosporine (1 nM) H-7 (6 pM) Sphingosine (10 pM)
3.250 F 123 3,022 f 373 3,305 i- 253 3,084 f 165 4,944 f 162* 3,012 F 321 3,659 f 139 4,322 f 900
4,071 k 252* 3,160 F 43** 3,028 f 232** 2,970 f 164** 3,163 F 81** 2,892 f 67** 3,498 f 66** 2,988 f 189**
4,420 F 138 4,741 F 188 4,801 f 218 4,760 i 300 6,209 f 111* 4,120 f 553 5,015 f 255 4,984 f 657
5,331 f 163* 4,526 f 149** 4,676 f 182** 4,660 f 84** 4,504 i 43** 4,263 f 65** 4,354 f 375** 2,779 f 474**
'Cells were prepared a s described in Materials and Methods and incubated in DMEM plus or minus FCS with each agent alone or with oPRL (lO-"M) plus each agent. Abbreviations: FCS, fetal calf serum; oPRL, ovine prolactin; CTR, control; antiserum, prolactin antiserum: antirecAb, antiprolactin receptor antibody; CsA, cyclosporine A PDBu, phorbol 12, 13-dibutyrate;H-7, 1-(-5-isoquinolinesuIfonyl)-2methylpiperazine. n = 6-10. *Significant stimulation over basal (P< 0.05). **Significant inhibition of PRL-induced proliferation (P< 0.05).
pathological response (Win uist et al., 1982). Characteristic of both diseases is V MC hyperplasia; however, the mechanisms by which this occurs are not well understood (ROSS,1986; Owens, 1989). In this study, we have illustrated that PRL is mitogenic for VSMC and that its proliferative response may be mediated through a PKC-dependent mechanism. We have also demonstrated that PRL is able to enhance PDGFinduced VSMC proliferation. oPRL caused a concentration-dependent proliferative response in A10 cells when incubated in either 0% FCS or 0.1% FCS with an optimal response occurring at 10-l' M (33% increase in L3H1thymidineuptake). When incubated with 0.1% FCS, higher concentrations of oPRL did not stimulate an increase in [3Hlthymidine uptake. This could possibly be due to the fact that serum contains PRL, and, when cells are treated with high PRL concentrations, rece tor down-regulation may occur; it has been shown t at internalized PRL persists in cells for long periods of time and binds almost irreversibly to its receptor (Kelly et al., 1979). To ensure that PRL was not merely increasing the intracellular pool of thymidine, we looked at the ability of oPRL to increase cell number and found that PRL did indeed induce proliferation. Anti-PRL antiserum and an anti-PRL receptor antibody antagonized the proliferative response t o PRL. Furthermore, the immunosuppressive agent CsA, which antagonizes many actions of PRL by competitively binding to the PRL receptor (Russell et al., 1984a,b),also attenuated oPRL-induced L3HJthymidine uptake. These data strongly suggest that PRL causes proliferation in these cells through a receptor-mediated mechanism. PRL has been previously shown to be mitogenic in a
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TABLE 2. Effects of PRL and inhibitors on A10 cell number' Aeent CTR oPRL (lo-'* M) oPRL (lo-" M) oPRL (lo-" M) + Antiserum (1/750) AntirecAb (10 pg/ml) Staurosporine (1 nM) H-7 (6 pM) Sphingosine (10 pM)
Cell number ( N O 3 ) 0% FCS 0.1% FCS 5.2 6.8 8.4
5.9 7.1 9.1
5.4 4.9 5.3 5.1 5.8
5.1 6.2 4.9 6.3 6.1
'Cells were prepared a s described in Materials and Methods and incubated in DMEM plus or minus FCS with each agent. This experiment was repeated three times with similar results. Variation in cell numberwas
