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(Hypertension. 1997;29:356.)
© 1997 American Heart Association, Inc.

Arthur C. Corcoran Memorial Lecture

-Thrombin Stimulates sis-Inducing Factor-A DNA Binding Activity in Rat Aortic Smooth Muscle Cells

G. Jayarama Bhat; S. Thomas Abraham; Harold A. Singer; Kenneth M. Baker

From the Weis Center for Research, Geisinger Clinic, Danville, Pa.

Correspondence to G. Jayarama Bhat, PhD, Weis Center for Research, 100 N Academy Ave, Danville, PA 17822

	Abstract

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Exposure of rat aortic vascular smooth muscle cells to -thrombin resulted in the appearance of sis-inducing factor-A (SIF-A)-like DNA binding activity. This response to -thrombin was delayed (detectable at 1 hour) compared with the rapid activation (15 to 30 minutes) by platelet-derived growth factor and the cytokine interleukin-6. -Thrombin-induced SIF-A was sensitive to treatment with the tyrosine kinase inhibitor genistein. The thrombin inhibitor hirudin prevented the -thrombin-mediated SIF-A induction. Cycloheximide had no effect on the ability of -thrombin to induce SIF-A, suggesting that induction does not require new protein synthesis. -Thrombin-induced SIF-A could be resolved into two additional subcomplexes termed SIF-A_F and SIF-A_S. Antibodies against Stat3 reacted with -thrombin-induced SIF-A_F, suggesting that Stat3 or a related protein is present in this subcomplex. Induction of SIF-A DNA binding activity may contribute to -thrombin-mediated cellular responses, including wound healing, cell proliferation, and inflammation in the vasculature.

Key Words: thrombin • transcription factors • muscle, smooth, vascular • signal transduction

Abbreviations: Ang II = angiotensin II • IL-6 = interleukin-6 • PDGF = platelet-derived growth factor • SIE = sis-inducing element • SIF-A = sis-inducing factor A • STAT = signal transducers and activators of transcription • VSM = vascular smooth muscle

	Introduction

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The multifunctional serine protease -thrombin, generated at sites of vascular injury, has central functions in hemostasis but also promotes a wide range of cellular responses (reviewed in References 1 and 2). In addition to its potent stimulatory effect on platelet aggregation,³ it acts as a mitogen in lymphocytes⁴ and aortic VSM cells^5,6 and stimulates the induction of the growth-associated proto-oncogene c-fos.^7,8 Acting through a seven-transmembrane, G protein-coupled receptor, -thrombin activates phospholipase C and subsequent generation of inositol trisphosphate, which results in an increase in intracellular Ca²⁺.^9,10 It also activates a series of protein kinase cascades, including protein kinase C, p42/p44 mitogen-activated protein (MAP) kinases, p38 MAP kinase, Src family kinases, and Janus kinases (JAK).^6,11–15 Stimulation of these signal transduction pathways may collectively contribute to the ability of -thrombin to cause multiple biological responses.

In recent reports, we and others demonstrated that Ang II, coupling through the G protein-coupled angiotensin type 1 (AT₁) receptor, stimulated the STAT pathway.^16–18 Six STAT family members (Stat1 through Stat6) have been identified, with additional subtypes.¹⁹ Upon tyrosine phosphorylation, STAT proteins form homodimers or heterodimers and translocate to the nucleus.^19,20 Binding of STAT proteins (Stat1 and Stat3) to the regulatory DNA element SIE can result in the formation of DNA-protein complexes referred to as SIF.¹⁹ For example, the c-fos gene promoter contains an SIE DNA responsive to the STAT pathway.¹⁹ Depending on the ligand, SIF appears in three different forms: complex A, B, and C (SIF-A, SIF-B, and SIF-C). SIF-A contains Stat3-Stat3 homodimer, SIF-B contains Stat1-Stat3 heterodimer, and SIF-C contains Stat1-Stat1 homodimer.¹⁹ PDGF induces all the three complexes; IL-6 induces mainly complex A, and interferon gamma mainly complex C.^21,22 In the present study, we determined whether -thrombin was capable of stimulating the STAT pathway in rat aortic VSM cells. Using electrophoretic mobility shift assays, which measure the binding of activated STAT proteins to [³²P]-labeled SIE DNA in vitro,^16,17 we showed that -thrombin stimulated the formation of DNA-protein complex SIF-A. The characteristics of -thrombin-induced SIF-A formation observed in the present study were similar to the stimulation of SIF-A by Ang II in rat cardiac fibroblasts and Chinese hamster ovary K1 (CHO-K1) cells expressing AT₁ receptors.^16,17

	Methods

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Materials
Cell culture media, fetal bovine serum, antibiotics, tissue culture flasks, and murine recombinant IL-6 were purchased from Life Technologies. -Thrombin and r-hirudin were purchased from Sigma Chemical Co. [-³²P]ATP was purchased from DuPont-NEN; polyclonal antibodies to Stat1, Stat2, Stat3, and Stat4 were purchased from Santa Cruz Biotechnology; PDGF was purchased from Upstate Biotechnology.

Cell Culture and Treatment With Agonists
VSM cells were isolated from the thoracic aorta of rats (150 to 180 g) by enzymatic dissociation as previously described.²³ VSM cells were grown in medium (50% Dulbecco's modified Eagle's medium and 50% F-12) containing 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamate, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells between passages 5 and 10 were seeded for 24 hours, serum-starved for 24 to 48 hours, and stimulated with agonists as indicated.

Preparation of Nuclear Extracts
Nuclear extracts were prepared as previously described.^16,17 After -thrombin or other treatments, VSM cells were rinsed with phosphate-buffered saline at 0°C and scraped into the same buffer. Harvested cells were resuspended in 10 vol hypotonic buffer (10 mmol/L HEPES [pH 7.9], 10 mmol/L KCl, 1.5 mmol/L MgCl₂, 0.5 mmol/L dithiothreitol), supplemented with protease and phosphatase inhibitors, incubated for 10 minutes on ice, and sedimented. Cells were resuspended with 0.6 mL of the same buffer, dounce-homogenized, and sedimented at 1000g for 10 minutes, and the pellet (nuclei) was collected. The pelleted nuclei were incubated for 30 minutes at 4°C in high salt buffer (20 mmol/L HEPES [pH 7.9], 25% glycerol, 400 mmol/L NaCl, 1 mmol/L EDTA) supplemented with protease and phosphatase inhibitors and sedimented at 12 000 rpm in a microcentrifuge for 15 minutes at 4°C. This supernatant (nuclear extract) was dialyzed against low salt buffer (same as the high salt buffer except that NaCl was 50 mmol/L) for 4 hours, protein concentration was determined, and the extract was stored at 80°C.

Electrophoretic Mobility Shift Assay
Mobility shift assays were performed as previously described.^16,17 Five micrograms of nuclear extract was incubated with 1 µg poly(dI-dC) in 20 µL of 10 mmol/L HEPES (pH 7.9), 50 mmol/L NaCl, 1 mmol/L EDTA, and 10% glycerol for 20 minutes at 25°C. The samples were incubated with 1 or 2 fmol radiolabeled probes (approximately 5000 cpm) for 10 minutes at 25°C. The sequence of the probes used in the present study has been previously described.¹⁶ Binding reactions were resolved on a 4% native polyacrylamide gel containing 0.5x Tris borate-EDTA buffer (25 mmol/L Tris, 25 mmol/L boric acid, 0.5 mmol/L EDTA). Gels were run at 200 V for 2 or 5 hours in a cold room (4°C to 8°C) and exposed to x-ray film.

	Results

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Effect of -Thrombin on Induction of SIF Activity
For determination of whether -thrombin was capable of inducing SIF activity, rat aortic VSM cells were treated with -thrombin for differing periods of time, and nuclear extracts were prepared and subjected to electrophoretic mobility shift assays (2 hours, 200 V) using [³²P]-labeled SIE. As a positive control, cells were treated with the growth factor PDGF or the cytokine IL-6. Fig 1 demonstrates the formation of nuclear SIF complex induced by -thrombin, IL-6, and PDGF. IL-6- and PDGF-induced activation was rapid and maximal at 15 to 30 minutes. In contrast, induction of SIF by -thrombin was delayed, with initial activity being detected at 1 hour and a maximal response at 3 to 6 hours. The positive control PDGF induced three different SIF complexes (complexes A, B, and C), and IL-6 induced mainly one (complex A), in agreement with published reports.^16,21,22 When -thrombin-, IL-6-, and PDGF-induced samples were run on the same gel, the -thrombin-induced SIF comigrated with IL-6-induced SIF-A (data not shown). When the gels were run for longer periods of time (5 hours at 200 V), the -thrombin-induced SIF complex could be further resolved into two distinct subcomplexes (see Fig 3). These results indicate that -thrombin and PDGF/IL-6 exhibit distinct kinetics of SIF formation. The delayed appearance of SIF by -thrombin is similar to the time course of SIF induction by Ang II,^16,17 indicating that a common mechanism may govern the activation of SIF by -thrombin and Ang II.

View larger version (73K): [in this window] [in a new window]   FIG 1. Time course for -thrombin-, IL- 6-, and PDGF-stimulated SIF-A activity in VSM cells. Nuclear extracts were prepared from untreated cells and cells treated with -thrombin (3.4 nmol/L; 0.4 U/mL), IL-6 (20 ng/mL), and PDGF (10 ng/mL) for indicated times, incubated with [32P]-labeled SIE, and analyzed by electrophoretic mobility shift assay. Positions of the three SIF complexes (A, B, and C) are indicated by arrows.

View larger version (37K): [in this window] [in a new window]   FIG 3. -Thrombin-induced SIF-A complex is recognized by antibody to Stat3. A, Nuclear extracts were prepared from -thrombin (Th)-treated cells (2 hours) and incubated with [32P]-labeled SIE for 10 minutes. Two micrograms of anti-Stat3 was added and the incubation continued for an additional 1 hour on ice. Complexes were resolved by electrophoretic mobility shift assay. Lane 4 represents -thrombin-treated nuclear extract incubated with [32P]-labeled SIE to which a 100-fold excess of unlabeled SIE-DNA complex was added as a competitor. B, Nuclear extracts from -thrombin-treated cells (2 hours) were incubated with [32P]-labeled SIE for 10 minutes. Two micrograms of anti-Stat1 (lane 3), anti-Stat2 (lane 4), anti-Stat3 (lane 5), or anti-Stat4 (lane 6) were added and supershift assays performed as described for Fig 3A. The SIF and supershifted (SS) complexes are indicated by arrows.

Characterization of -Thrombin-Induced SIF Response
To determine whether the -thrombin-induced SIF is mediated through an -thrombin-specific receptor, we pretreated the cells with hirudin for 15 minutes and subsequently exposed them to -thrombin for 1 hour. Hirudin acts as a specific inhibitor of -thrombin actions and has been used to block thrombin-mediated proliferation of smooth muscle cells.⁵ Nuclear extracts were prepared and subjected to electrophoretic mobility shift assay. As shown in Fig 2A, lane 3, pretreatment of cells with hirudin completely inhibited -thrombin-induced SIF formation. To determine whether the SIF response by -thrombin required the action of tyrosine kinases, we pretreated the cells with the tyrosine kinase inhibitor genistein before the addition of -thrombin. As shown in Fig 2A, genistein prevented -thrombin-induced formation of SIF (lane 4). These data indicate that the -thrombin-induced signaling pathway involves the action of tyrosine kinases. We also observed that induction of SIF by -thrombin was insensitive to cycloheximide treatment, suggesting that the induction is a posttranslational event (data not shown). SIF induction by -thrombin was detected at concentrations of -thrombin as low as 0.3 nmol/L (Fig 2B).

View larger version (40K): [in this window] [in a new window]   FIG 2. Characterization of -thrombin-induced SIF complex. A, Effect of the thrombin inhibitor hirudin and tyrosine kinase inhibitor genistein on -thrombin-induced SIF. Serum-starved cells were untreated (lane 1) or treated with -thrombin (Th, 3.4 nmol/L) for 1 hour (lane 2). Alternatively, cells were pretreated with hirudin (0.4 U/mL) (lane 3) or genistein (100 µg/mL) (lane 4) for 15 minutes and then treated with -thrombin (0.4 U/mL=3.4 nmol/L) for 1 hour. Nuclear extracts were prepared and subjected to electrophoretic mobility shift assay using [32P]-labeled SIE. B, Effect of different concentrations of -thrombin on SIF induction. Serum-starved cells were treated with -thrombin at the indicated concentration for 1 hour, and nuclear extracts were prepared and analyzed using [32P]-labeled SIE.

-Thrombin-Induced SIF Complex Contains Stat3 or a Related STAT Protein
PDGF- or IL-6-induced SIF-A has been shown to contain Stat3 protein.^17,21,22 Since the -thrombin-induced SIF complex comigrated with PDGF- or IL-6-induced complex A, we determined whether the -thrombin-induced SIF contained Stat3 as a component protein. Nuclear extracts prepared from -thrombin-stimulated cells were incubated with anti-Stat3, and the resulting DNA-protein complexes were analyzed in a gel mobility shift assay. As shown in Fig 3A, the -thrombin-induced SIF could be resolved into two distinct complexes. We refer to the slower migrating complex as SIF-A_S and the faster migrating complex as SIF-A_F. Addition of anti-Stat3 super-shifted mainly SIF-A_F, indicating that Stat3 or a related protein is a component of -thrombin-induced SIF-A_F (lane 3). Addition of unlabeled SIE-DNA (100-fold excess) competed away the -thrombin-induced SIF and supershifted complexes (lane 4). These results suggested that -thrombin induced specific SIF complexes. Addition of anti-Stat1, anti-Stat2, and anti-Stat4 did not supershift the -thrombin-induced SIF-A (Fig 3B, lanes 3, 4, and 6, respectively). It is currently not known whether the SIF-A_S contains other known members of the STAT family (Stat5 and Stat6) or novel transcription factors.

	Discussion

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Using the c-fos regulatory element SIE, we demonstrated in the present study that -thrombin stimulates the formation of SIF-A complex in rat aortic smooth muscle cells. We also demonstrated that the -thrombin-induced SIF-A complex can be further resolved into a faster and slower migrating SIF, termed SIF-A_F and SIF-A_S, respectively. Stat3 or a related protein was present in SIF-A_F; however, the identity of the STAT member in SIF-A_S is not known. As shown earlier for cytokines,²⁴ treatment of cells with genistein inhibited the -thrombin-induced SIF-A, suggesting that tyrosine kinases were involved in the signal transduction pathway leading to the DNA binding activity. Cycloheximide treatment did not affect the ability of -thrombin to stimulate the SIF-A, indicating that the induction did not require new protein synthesis. We also demonstrate evidence that IL-6 induces SIF-A activity in VSM cells.

With respect to the time course, -thrombin-induced SIF-A significantly differed from that elicited by PDGF or IL-6. In contrast to the rapid induction of SIF by PDGF and IL-6 (maximal within 30 minutes), -thrombin induced a delayed formation of SIF, with initial activity detectable at 1 hour and maximal at 3 to 6 hours. This delay by -thrombin appears similar to the Ang II-induced delayed activation of SIF-A observed previously in cardiac fibroblasts,¹⁶ CHO-K1 cells expressing AT_1A receptors,¹⁷ and CHO-K1 cells expressing endothelin receptors.²⁵ These data suggest that SIF-A activation by all three different agonists (Ang II, endothelin, and -thrombin) may involve a common mechanism.

In cultured VSM and endothelial cells, -thrombin was shown to induce c-fos mRNA in a transient fashion, with induction detected at 15 to 30 minutes but maximal at 60 minutes after exposure.^7,8 The 5' promoter region of the c-fos gene contains three main regulatory elements: (1) the SIE, controlled by the STAT pathway; (2) the serum-response element, which is activated by the MAP kinase pathway; and (3) the cAMP/Ca²⁺-response element, which is responsive to protein kinase A.^26,27 Since -thrombin is a potent inducer of the MAP kinase cascade (within 5 minutes)⁶ and increases intracellular Ca²⁺,^7,8 it is likely that these two pathways contribute to the early phase in the induction of c-fos mRNA. Since SIF-A induction by -thrombin is delayed (1 hour), the STAT pathway may contribute to c-fos induction at 1 hour.

It is not known at present whether JAK kinases are involved in -thrombin-stimulated tyrosine phosphorylation of Stat3. The reports of activation of JAK kinases by -thrombin in human platelets¹⁵ suggest a role for these kinases in Stat3 tyrosine phosphorylation. It is notable that the human -thrombin receptor sequence contains a potential Stat3 binding site, YXXQ (amino acid YPMQ; 206– 209),⁹ which is demonstrated to be important for the interaction of Stat3 with cytokine receptors.²⁸ Whether Stat3 binds to the YPMQ motif in the -thrombin receptor requires further study.

It is also important to point out that the -thrombin-, PDGF-, and IL-6-induced SIF responses were variable between cultures. This probably reflects, at least in part, differences in the level of receptors expressed in these primary cultures. Although studies in primary cultures are important for establishing the physiological relevance of thrombin-induced SIF activation, future studies on the mechanism of SIF induction (activation of JAK kinases and Stat3 tyrosine phosphorylation) may be better addressed in model cell lines transfected with -thrombin receptors.

We show in this study that Stat3 is a component of -thrombin-induced SIF-A_F. Stat3 was originally identified as an IL-6-dependent transcription factor that bound to the acute phase response element in a variety of promoters of genes encoding acute phase response proteins.²⁹ The significance of Stat3/SIF-A activation by -thrombin and its influence on gene expression in VSM cells is not clear at this stage. However, it may have implications in -thrombin-mediated cellular responses, such as wound healing, and in pathological disorders involving the vascular or perivascular wall, such as atherosclerosis.

	Acknowledgments

 
This work was supported in part by grants from the American Heart Association (Pennsylvania Affiliate and National) (G.J.B.) and RO1 HL-40992 (H.A.S.) and the Geisinger Clinic Foundation. K.M.B. is an Established Investigator of the American Heart Association. The technical assistance of David Cooney and Kathy M. Conrad is gratefully acknowledged.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bhat, G. J. Articles by Baker, K. M. Search for Related Content PubMed PubMed Citation Articles by Bhat, G. J. Articles by Baker, K. M.