©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the STAT Pathway by Angiotensin II in T3CHO/AT Cells
CROSS-TALK BETWEEN ANGIOTENSIN II AND INTERLEUKIN-6 NUCLEAR SIGNALING (*)

(Received for publication, March 29, 1995; and in revised form, June 12, 1995)

G. Jayarama Bhat (§) Thomas J. Thekkumkara (¶) Walter G. Thomas (**) Kathleen M. Conrad Kenneth M. Baker (§§)

From theWeis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We recently reported that angiotensin II (AII), acting through the STAT (Signal Transducers and Activators of Transcription) pathway, stimulated a delayed SIF (sis-inducing factor)-like DNA binding activity (maximal at 2-3 h) (Bhat, G. J., Thekkumkara, T. J., Thomas, W. G., Conrad, K. M., and Baker, K. M.(1994) J. Biol. Chem. 269, 31443-31449). Using a cell line transfected with the AT receptor (T3CHO/AT), we further characterized the AII-induced SIF response and explored the possible reasons for the delay in stimulated SIF activity. In cells transfected with a chloramphenicol acetyltransferase reporter plasmid, under the control of a SIE (sis-inducing element), AII markedly stimulated chloramphenicol acetyltransferase activity. The delayed SIF activation by AII was not due to a requirement for the release of other SIF inducing factors into the medium and contrasts with the rapid (5 min) induction elicited by the cytokine, interleukin-6 (IL-6). Interestingly, both agents stimulated tyrosine phosphorylation of Stat92 and predominantly the formation of SIF complex A. We tested the hypothesis that AII initially activated an inhibitory pathway, which was responsible for delaying the maximal SIF stimulation until 2 h. Pretreatment of cells for 15 min with AII resulted in significant inhibition of the IL-6-induced nuclear SIF response (10 min) and Stat92 tyrosine phosphorylation, which was blocked by EXP3174, an AT(1) receptor antagonist. This inhibition was transient with return of the IL-6-induced SIF response at 2 h, suggesting that the delayed maximal activation of SIF by AII occurs following an initial transient inhibitory phase. Pretreatment of cells with phorbol 12-myristate 13-acetate for 15 min, to activate protein kinase C, resulted in inhibition of the IL-6-induced SIF response (10 min). However, down-regulation of protein kinase C activity prevented phorbol 12-myristate 13-acetate, but not AII mediated inhibition of the IL-6-induced SIF response. Although the mechanism is not clear, the results presented in this paper raise the interesting possibility that the activation of SIF/Stat92 by AII is characterized by an initial inhibitory phase, followed by the induction process. The observation that AII and IL-6 utilize similar components of the STAT pathway and that AII can cross-talk with IL-6 signaling through inhibition of IL-6-induced SIF/Stat92, implies a modulatory role for AII in cellular responses to cytokines.


INTRODUCTION

Angiotensin II (AII) (^1)stimulates a variety of physiological responses related to the regulation of blood pressure, salt, and fluid homeostasis(1, 2) . In addition, AII promotes growth responses in many cells, including cardiomyocytes, cardiac fibroblasts, and vascular smooth muscle cells(3, 4, 5, 6, 7, 8) . Angiotensin II exerts effects through specific G-protein coupled receptors, predominantly the AT(1) receptor subtype. AT(1) receptors couple to intracellular calcium mobilization, activation of tyrosine kinases such as p125, p46, and p54, and induction of serine/threonine kinases, including protein kinase C (PKC) and mitogen-activated protein kinases (2, 9, 10, 11, 12, 13, 14) . Angiotensin II may act directly through these signaling pathways or indirectly via the release of growth factors such as PDGF and TGF-beta, as demonstrated for rat vascular smooth muscle cells(4, 7) . Like other growth factors, AII induces a rapid increase of the growth associated nuclear proto-oncogenes c-myc, c-fos, and c-jun and several cellular genes including tenascin, fibronectin, and collagen(4, 15, 16, 17, 18, 19) . These studies indicate that AII can induce rapid changes in gene expression and function, that may ultimately lead to increased cell growth(20) .

Growth factors and cytokines transduce signaling through a common pathway (STAT pathway) from receptor to the nucleus(21) . The c-fos regulatory element SIE (sis-inducing element) has been used extensively to study the activation of STAT pathways by many ligands, including PDGF, epidermal growth factor, IFN-, and IL-6(22, 23, 24, 25, 26, 27, 28) . Binding of the ligand to the receptor activates tyrosine kinases, which phosphorylate monomeric STAT proteins in the cytoplasm, leading to dimerization and formation of complexes referred to as SIF (sis-inducing factor)(29) . SIF subsequently translocates to the nucleus and interacts with SIE, or SIE-like elements, in the promoter of genes to induce expression. Depending upon the ligand, SIF appears in three different forms: complex A, B, and C(22, 24) . PDGF and epidermal growth factor induce all three complexes; IL-6 induces mainly complex A and IFN- mainly complex C.

We have recently shown (30) that AII stimulates the STAT pathway and induces predominantly SIF complex A in both neonatal rat cardiac fibroblasts and CHO-K1 cells expressing AT receptors (T3CHO/AT). Like growth factors, activation of SIF by AII was post-translational and required the actions of tyrosine kinases. Angiotensin II-induced SIF complexes contained tyrosine phosphorylated Stat91, with activation occurring in the cytoplasm followed by translocation to the nucleus. However, with respect to the time course of SIF activation, the AII-induced response significantly differed from that of growth factor/cytokine responses. Unlike the rapid induction of SIF by cytokines and growth factors (maximal in less than 30 min), AII-induced activity although detectable at 30 min, was maximal at 2-3 h(30) . In the present study, using T3CHO/AT cells, we explored the possible reasons for the delayed maximal SIF activation by AII. Our data indicate that AII directly activates SIF activity and that the delayed maximal activation of SIF is not due to the secondary release of other SIF inducing factors into the medium. We also provide evidence that AII evokes a PKC independent, transient inhibitory effect on the rapid SIF induction by IL-6. We propose that similar transient inhibitory mechanisms may be responsible for delaying the maximal SIF activation by AII (2 h).


EXPERIMENTAL PROCEDURES

Materials

Cell culture media, fetal bovine serum, antibiotics, Geneticin, tissue culture flasks, IL-6, and TGF-beta, were purchased from Life Technologies, Inc.; AII was purchased from U. S. Biochemical Corp; Nitrocellulose membranes were purchased from Amersham Corp; [-P]ATP was from DuPont NEN; polyclonal antibodies to Stat92 and protein A/G-agarose were purchased from Santa Cruz Biotechnology; monoclonal antibodies to phosphotyrosine and PDGF were purchased from Upstate Biotechnology; goat anti-rabbit IgG and rabbit anti-mouse IgG were purchased from Bio-Rad; and other chemicals were purchased from Sigma.

Cell Culture and Treatment with Agonists

T3CHO/AT cells (31) were grown in alpha-minimum essential medium containing 10% fetal bovine serum and 200 µg/ml Geneticin antibiotic for 12-24 h, serum starved for 12 h, and treated with one or more growth factors or cytokines at the following concentrations: AII, 100 nM; PDGF, 10 ng/ml; TGF-beta, 5 ng/ml; IL-6, 10 ng/ml. Where indicated, the non-peptide AT(1) receptor antagonist EXP3174 was added to a final concentration of 100 µM. For PKC down-regulation, cells were exposed to PMA (250 nM) for 24 h, prior to stimulation with different agonists. At the time of experiments, cultures were subconfluent.

Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay

Nuclear extracts were prepared and gel mobility shift assays were performed as described previously(30) , using P-labeled, double stranded oligonucleotides representing the SIE-DNA (top strand: 5`-CAGTTCCCGTCAATC-3`).

Immunoprecipitation and Western Blots

Immunoprecipitations were performed according to previously described methods(30) , on protein extracts obtained from the nuclei of cells untreated or treated with AII or IL-6. One hundred µg of protein from each sample was diluted with an equal volume of immunoprecipitation buffer (1 = 10 mM Tris (pH 7.4), 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate) and immunoprecipitated with 1 µg of anti-Stat92 antibody and protein A/G-agarose. Immunocomplexes were harvested by centrifugation, washed 3 times with immunoprecipitation buffer, proteins resolved by 8% polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane (Amersham), and incubated with antiphosphotyrosine antibody(30) . Immunoreactive bands were visualized using a chemiluminescence Western blotting system (Amersham) according to the manufacturer's instructions. The blots were stripped (Amersham) and reprobed with polyclonal anti-Stat92 antibody as described previously(30) .

Construction of Reporter Plasmids

The parental plasmid pBLCAT2 was obtained from B. Luckow and G. Schutz(32) . This plasmid has a bacterial chloramphenicol acetyltransferase gene under the control of the thymidine kinase (tk) promoter. DNA representing three tandem repeats of SIE (see above) and mutant SIE (5`-CAGCCACCGTCAATC-3`) were inserted at the HindIII-BamHI site of the pBLCAT2 vector to generate SIE/pBLCAT2 and m.SIE/pBLCAT2, respectively.

Transfections and Chloramphenicol Acetyltransferase (CAT) Assay

T3CHO/AT cells were grown in 60-mm tissue culture plates to 50-60% confluence until the day of transfection. Cells were transfected with 2 µg of pBLCAT2 (vector), SIE/pBLCAT2, or m.SIE/pBLCAT2 using Lipofectin according to the manufacturer's (Life Technologies, Inc.) instructions. To measure the transfection efficiency, cells were co-transfected with pSV-beta-galactosidase plasmid (Promega). Twenty-four hours after transfections, the cells were serum starved for 3 h and AII was added for 24 h. Cells were collected and extracts prepared by three cycles of freezing and thawing. Extracts were heated to 60 °C for 10 min, sedimented at 14,000 rpm in a microcentrifuge, and protein concentrations determined (33) . CAT assay was performed as described previously with minor modifications(34) . The reaction mixture contained 140 mM Tris-HCl (pH 7.8), 0.2 µCi of [^14C]chloramphenicol, 4 mM acetyl-coenzyme A, and 40 µg of cell extract in a final volume of 150 µl. The mixture was incubated at 37 °C, for 1 h and then extracted with ethyl acetate. The organic phase was transferred, dried, and the pellet redissolved in 15 µl of ethyl acetate. The labeled chloramphenicol and acetylated derivatives were separated by ascending thin layer chromatography using chloroform/methanol (95:5, v/v) and the chromatograms subjected to autoradiography at -70 °C.


RESULTS

Effect of Different Agents on SIF Induction in T3CHO/ATCells

We reported previously (30) that AII induces maximal SIF activity between 2 and 3 h in T3CHO/AT cells. This delay in maximal response to AII, compared to the rapid activation (30 min) by cytokines and growth factors, is not due to a requirement for new protein synthesis. Given the observation that, in rat aortic smooth muscle cells, AII causes an increase in the expression/secretion of growth factors such as PDGF and TGF-beta(4, 7) , we first considered the possibility that the delayed SIF induction resulted from secondary release of growth factors. We tested the ability of different agents (PDGF, TGF-beta, and IL-6) to stimulate SIF activity in T3CHO/AT cells. Nuclear extracts were made from treated (PDGF, TGF-beta, IL-6, 30 min; AII, 2 h) and untreated cells and analyzed in an gel mobility shift assay. As shown in Fig.1, PDGF and TGF-beta failed to induce SIF activity (lanes 3 and 5), while induction was observed with AII (positive control; lane 2) and IL-6 (lane 4). These results suggest that AII induced SIF activity is not due to the release of PDGF and TGF-beta in these cells.


Figure 1: Effect of different agents on the induction of SIF activity in T3CHO/AT cells. Cells were treated with AII (100 nM) for 2 h (lane 2); PDGF (10 ng/ml) for 30 min (lane 3); IL-6 (10 ng/ml) for 30 min (lane 4); and TGF-beta (5 ng/ml) for 30 min (lane 5). Nuclear extracts were prepared and analyzed in an electrophoretic mobility shift assay using P-labeled SIE. The positions of SIF complexes A, B, and C are indicated. These data are representative of three separate experiments. Ctl, untreated control (lane 1).



The AII-induced SIF Activity Is Specifically Mediated by the ATReceptor

To investigate the possibility that AII stimulation causes the release of SIF inducing factors, we collected conditioned medium from T3CHO/AT cells treated with AII for 2 h, the time of maximal stimulation. This conditioned media was tested for the ability to induce SIF activity at early (30 min) and delayed (2 h) time points. As shown in Fig.2, conditioned medium from cells exposed to AII induced significant levels of SIF, only at 2 h (lane 6), but not at 30 min (lane 4), as would be expected if the response was mediated by a released factor. However, the activity at 2 h was completely blocked by pretreatment with the AT(1) receptor antagonist, EXP3174 (lane 7). These results indicate that AII acts directly through the AT receptor to induce SIF activity and that the delayed activity is not secondary to the release of other SIF inducing factors.


Figure 2: Angiotensin II directly induces SIF activity through the AT receptor. Cells were treated for 2 h with AII (100 nM, lane 2), conditioned medium was removed and added to quiescent cells. The incubation was continued for 30 min or 2 h in the presence (lanes 5 and 7) or absence (lanes 4 and 6) of a 100-fold excess of EXP3174, an AT(1) receptor antagonist. Nuclear extracts were prepared and analyzed in an electrophoretic mobility shift assay using P-labeled SIE. Lane 3 represents cells pretreated with EXP3174 (EXP) and stimulated with AII for 2 h, as a positive control for the specificity of AII action. This experiment was repeated three times. Ctl, untreated control (lane 1).



SIF Activation by AII Requires Continuous Exposure

In order to determine whether continuous receptor occupancy is required for AII-mediated SIF induction, we first treated the cells with AII (100 nM) for varying periods (5, 15, and 35 min and 1 h), and then with 100-fold excess of the AT(1) receptor antagonist EXP3174 until 2 h. Nuclear extracts were made from these cells and analyzed in a gel mobility shift assay. As shown in Fig.3A, cells exposed to AII for 5 min and 15 min before the addition of EXP3174 showed no or very little SIF induction (lanes 3 and 4). Cells exposed to AII for 35 min and 1 h before the addition of EXP3174 showed significant SIF induction (lanes 5 and 6). These results indicate that a 15-35 min continuous exposure to AII is required for SIF induction.


Figure 3: SIF activation by AII requires continuous exposure where as activation by IL-6 is rapid. A, time course of activation of SIF by AII: as a positive control for AII induced SIF, cells were stimulated with AII (100 nM) for 2 h (lane 2). Alternatively, cells were exposed to AII for varying time periods, 5 min (lane 3), 15 min (lane 4), 35 min (lane 5), 1 h (lane 6), followed by the addition of a 100-fold excess of EXP3174 for a total incubation period of 2 h. Nuclear extracts were prepared and analyzed in a gel mobility shift assay using P-labeled SIE. This experiment was repeated three times. Ctl, untreated control (lane 1). B, time course of activation of SIF by IL-6: cells were treated with IL-6 (10 ng/ml) for the indicated times, nuclear extracts were prepared and analyzed in a gel mobility shift assay using P-labeled SIE (lanes 2-6). This representative experiment was reproduced four times. Ctl, untreated control (lane 1).



Differing Time Courses of SIF Activation by AII and IL-6

To determine whether T3CHO/AT cells respond appropriately to a known rapid SIF inducer (IL-6)(24) , we compared the time course of SIF activation by AII, to that of IL-6. For these experiments, we treated serum-starved T3CHO/AT cells with AII and IL-6 for varying periods of time, up to 6 h. Nuclear extracts were prepared and analyzed in a gel mobility shift assay. As we have reported previously(30) , in this cell line and in neonatal cardiac fibroblasts, AII stimulates predominantly a delayed SIF-A, with maximal activity at 2 h. In contrast, IL-6 stimulated high levels of SIF activity (complex A) as early as 5 min (Fig.3B), consistent with the findings of others using different cell types(24) . These results suggest that the delayed SIF response is not an aberration of this cell line, but rather a characteristic of AII stimulation.

The AII-induced SIF Complex A Contains Stat92 or a Related Protein

Since IL-6 induced SIF (complex A) has been demonstrated to contain Stat92 (Stat3, also referred to as acute phase response factor)(35, 36, 37) , we tested the relatedness of the AII and IL-6-induced complexes using anti-Stat92 antibody in a gel mobility supershift assay. Nuclear extracts were prepared from AII and IL-6-stimulated T3CHO/AT cells and incubated with Stat92 antibody. As shown in Fig.4, the anti-Stat92 antibodies recognized SIF complex A in both AII and IL-6-treated nuclear extracts, as demonstrated by the complete disappearance of SIF complex A and the formation of supershift complexes (lanes 3 and 7). When AII and IL-6-induced nuclear extracts were incubated with IgG from a nonimmunized rabbit, supershifted complexes were not observed demonstrating the specificity of protein-antibody interactions (lanes 5 and 9). We conclude from these results that Stat92 or an antigenically related protein is likely to be part of AII-induced SIF complex A.


Figure 4: Angiotensin II-induced SIF complex A contains Stat92 or a related protein. Nuclear extracts from AII (100 nM, 2 h) or IL-6 (10 ng/ml, 10 min) treated cells were incubated with P-labeled SIE. For supershift assays, nuclear extracts from similarly treated cells were incubated with 2 µg of anti-Stat92 antibody (lanes 3 and 7), incubated on ice for 1 h, and complexes resolved in a gel mobility shift assay. The SIF complex A (SIF A) and supershifted complexes (SS) are indicated. Supershifted complexes were competed with a 100-fold excess of unlabeled (competitor) SIE (lanes 4 and 8). This blot is representative of three experiments. Ctl, untreated control (lane 1); SS, supershifted complex; NRA, non-related antibody; com. SIE, competitor SIE.



Angiotensin II Pretreatment Inhibits the IL-6-induced Nuclear SIF Response

Our previous report on AII (30) and the results obtained in Fig.3B on IL-6, demonstrate that AII and IL-6 induce maximal nuclear SIF activation at different time points with AII at 2-3 h and IL-6 at 5-30 min. The longer time required for maximal SIF activation by AII is different from the rapid activation by most cytokines and growth factors, where the maximal response is detected in less than 30 min(22, 23, 24) . We speculated as to whether AII could activate an inhibitory pathway, in the initial phase following AII exposure, which was responsible for delaying the maximal SIF stimulation until 2 h. Since both AII and IL-6 appear to utilize Stat92-related protein for SIF-A formation (see Fig.4), if AII promotes an inhibitory pathway, pretreatment of T3CHO/AT cells with AII would be expected to affect the rapid induction of SIF by IL-6. To test this hypothesis, T3CHO/AT cells were pretreated with AII for 15 min, following which IL-6 was added for an additional 10 min. As a positive control for stimulation of SIF activity, cells were treated with AII (25 min and 2 h) or IL-6 alone (10 min). Nuclear extracts were prepared and subjected to electrophoretic mobility shift assays and immunoprecipitations. As shown in Fig.5, pretreatment of T3CHO/AT cells with AII for 15 min resulted in significant inhibition of the IL-6-induced SIF response at 10 min (lane 5). Pre-exposure of cells to EXP3174, an AT(1) receptor antagonist, prevented the inhibitory action of AII (lane 6), demonstrating that this phenomena is mediated through the transfected AT receptor.


Figure 5: Angiotensin II pretreatment inhibits the rapid induction of SIF by IL-6. Cells were treated with AII alone (100 nM) for 25 min (lane 2) and 2 h (lane 3), and IL-6 alone (10 ng/ml) for 10 min (lane 4). Alternatively, cells were pretreated with AII (100 nM) for 15 min followed by IL-6 (10 ng/ml) for 10 min (lane 5), or pretreated with EXP3174 (1 10^5M) for 30 min and then sequentially with AII (100 nM) for 15 min and IL-6 (10 ng/ml) for 10 min (lane 6). Nuclear extracts were prepared and analyzed in a mobility shift assay using P-labeled SIE. This blot is representative of three experiments. Ctl, untreated control (lane 1); EXP, EXP3174.



Angiotensin II Pretreatment Markedly Reduced the IL-6-induced Stat92 Phosphorylation

Since both AII and IL-6-induced SIF complexes contain Stat92/or a related protein (see Fig.4), we determined whether the inhibition of the IL-6-induced SIF response by AII was reflected in the degree of tyrosine phosphorylation of Stat92. The nuclear extracts corresponding to the samples described in Fig.5were immunoprecipitated with anti-Stat92 antibody, separated by SDS-polyacrylamide gel electrophoresis, blotted, and probed with antiphosphotyrosine antibodies. As shown in Fig.6A, AII caused the tyrosine phosphorylation of a single 92-kDa protein at 2 h (lane 3). In contrast, IL-6 induced the tyrosine phosphorylation of two proteins, of molecular mass 92 and 89 kDa (lane 4), consistent with the finding of others(36) . A recent report suggests that the two forms of Stat92 results from differential phosphorylation(38) . In untreated nuclei, both forms of tyrosine-phosphorylated proteins were not observed (lane 1). Interestingly, pretreatment with AII markedly reduced the IL-6-induced tyrosine phosphorylation of both forms of Stat92 (lane 5). The AT(1) receptor antagonist EXP3174 prevented the inhibitory action of AII on IL-6-induced tyrosine phosphorylation (lane 6). To determine whether Stat92 related proteins were present in the unphosphorylated form in the nucleus, the blot in Fig.6A was stripped and reprobed with anti-Stat92 antibody. Fig.6B shows that Stat92-related proteins are present mainly in the nucleus of AII (2 h) and IL-6-treated cells (lanes 3 and 4), consistent with the pattern observed by others, in which only tyrosine-phosphorylated STAT proteins translocate to the nucleus (21, 22, 23, 24) . Most importantly, samples treated sequentially with AII and IL-6 had little or no Stat92-related proteins in the nucleus (lane 5), suggesting that inhibition of the Stat92 activation occurs in the cytoplasm. The AT(1) receptor antagonist EXP3174 prevented the inhibitory action of AII on IL-6 induced appearance of Stat92-related proteins (lane 6). We have confirmed that this inhibition occurs outside the nucleus based on an attenuation of the tyrosine phosphorylation of Stat92-related proteins from cytoplasmic fractions of AII/IL-6-treated samples (data not shown).


Figure 6: Angiotensin II pretreatment inhibits IL-6- induced Stat92 tyrosine phosphorylation. A, phosphotyrosine blots of immunoprecipitated Stat92. Samples corresponding to those described in the legend to Fig.6were immunoprecipitated with anti-Stat92 antibody (1 µg) for 12 h at 4 °C and the resulting immunoprecipitates resolved on an 8% polyacrylamide-SDS gel. Proteins were transferred to nitrocellulose and probed with antiphosphotyrosine antibody and the presence of tyrosine-phosphorylated proteins detected by chemiluminescence. Migration of differentially phosphorylated Stat92 protein is indicated by an arrow. B, the same blot in A was stripped and reprobed with Stat92 antibody. The major band seen below the 55-kDa marker corresponds to immunoglobulin heavy chain (IgG). This experiment was repeated three times.



Inhibition of IL-6-induced SIF by AII Is Transient

We next addressed the question of whether inhibition of the IL-6-induced SIF response by AII was transient and if this response would reappear at 2 h, the usual time of maximal SIF activation by AII. To test this hypothesis, cells were pretreated with AII for 15 min and IL-6 was added for 10 min and 2 h. As positive controls, we also treated the cells with AII, or IL-6 alone, for 10 min and 2 h. Nuclear extracts were prepared and analyzed in a gel mobility shift assay. As shown in Fig.7, lane 6, in AII-pretreated cells (15 min), we observed a complete inhibition of the IL-6-induced SIF response (10 min), confirming that an inhibitory pathway is activated by AII to suppress IL-6 induced SIF activity at 10 min. If the inhibitory pathway activated by AII is transient, SIF activity would be expected to return at 2 h. Following a 15-min pretreatment with AII, when cells were challenged with IL-6 for 2 h, significant levels of SIF activity were observed (lane 7). The reappearance of this nuclear SIF activity at 2, h in cells sequentially treated with AII (15 min) and IL-6 (2 h), suggests that the initial AII induced inhibitory phase is reversed, allowing a stimulatory response. However, the presence of SIF activity at 2 h, could be due to either AII or IL-6 or both. To determine whether IL-6 contributes to the SIF response at 2 h, we pretreated the cells with AII for 15 min, followed by addition of the AT(1) receptor antagonist (EXP3174) to block subsequent stimulatory actions of AII. As detailed in Fig.3A, under these experimental conditions, the addition of EXP3174 effectively blocked the stimulatory actions of AII at 2 h. In the presence of EXP3174, we detected abundant amounts of SIF activity (Fig.7, lane 8), suggesting that the observed induction at 2 h was due to IL-6 stimulation. These results indicate that AII mediated inhibition of the IL-6- induced SIF response is transient.


Figure 7: Inhibition of IL-6 induced SIF by AII is transient. Cells were treated with AII for 2 h (100 nM, lanes 2 and 3), IL-6 (10 ng/ml, lanes 4 and 5), or sequentially with AII and IL-6 (lanes 6 and 7), or sequentially with AII, EXP3174, and IL-6 (lane 8) for the indicated times. Nuclear extracts were prepared and analyzed in a gel mobility shift assay using P-labeled SIE. This experiment was performed four times. Ctl, untreated control (lane 1).



Inhibition of IL-6 SIF Induction by AII Is Mimicked by PMA

To investigate the mechanism of rapid inhibition of IL-6-induced SIF by AII, we determined the role of protein kinase C (PKC), which has been shown to be activated by AII in a number of cell types(9, 11, 13, 16) . To establish a role for PKC in the inhibitory effects on IL-6-induced SIF, the cells were pretreated with AII or phorbol 12-myristate 13-acetate (PMA), prior to IL-6 addition. Gel shift assays were performed on nuclear extracts prepared from cells untreated, treated with IL-6 (10 min) alone, or treated sequentially with PMA (15 min) and IL-6 (10 min) or with AII (15 min) and IL-6 (10 min). Fig.8demonstrates that PMA (lane 3) mimics the inhibitory actions of AII (lane 4), abolishing the IL-6-mediated induction of SIF. To further determine whether AII mediates the inhibition of IL-6-SIF induction through the activation of PKC, we down-regulated cellular PKC activity by treating the cells with PMA for 24 h. Gel shift assays were performed on nuclear extracts prepared from cells untreated, treated with IL-6 (10 min), or sequentially treated with PMA (15 min) and IL-6 (10 min) or with AII (15 min) and IL-6 (10 min). As shown in Fig.8, in PKC down-regulated cells, PMA failed to inhibit IL-6-stimulated SIF induction by IL-6 (lane 7), while the capacity of AII to inhibit IL-6-stimulated SIF was unaffected (lane 8).


Figure 8: Inhibition of IL-6-induced SIF by AII is mimicked by PMA. Cells were treated with IL-6 (10 ng/ml) for 10 min (lane 2), or sequentially with PMA (100 nM) for 15 min and then with IL-6 (10 ng/ml) for 10 min (lane 3), or sequentially with AII (100 nM) for 15 min and then with IL-6 (10 ng/ml) for 10 min (lane 4). Nuclear extracts were prepared and analyzed in a gel mobility shift assay using P-labeled SIE. Lanes 5-8 represent experiments performed using PKC down-regulated cells, with identical treatment conditions as described for lanes 1-4. For PKC down-regulation, the cells were exposed for 24 h to PMA (250 nM) before treating with agonists. This experiment was repeated three times. Ctl, untreated control (lanes 1 and 5).



Angiotensin II Stimulates CAT Activity in Cells Transfected with SIE/CAT Reporter Plasmid

We have shown that AII induces SIF activity at 2 h, thus the importance of this induction on gene expression was assessed using a CAT reporter assay. Two constructs, one carrying wild type SIE (SIE/pBLCAT2) and the other mutant SIE (m.SIE/pBLCAT2) placed in front of the tk promoter, were used in these experiments. As shown in Fig.9, addition of AII to T3CHO/AT cells transfected with SIE/pBLCAT2 showed enhanced CAT activity (lane 2) which was completely blocked by the AT(1) receptor antagonist EXP3174 (lane 3). Cells transfected with mutant m.SIE/pBLCAT2 (lane 4) or the parental vector (pBLCAT2) showed no CAT activity (lane 5). Similar results were obtained when we transiently co-transfected pBLCAT2 vectors carrying wild type SIE or mutant SIE with an expression vector carrying cDNA for the AT receptor (data not shown). These results indicate that the SIF complexes formed in response to AII are capable of stimulating gene expression.


Figure 9: Gene transcription is activated by AII-induced SIF. T3CHO/AT cells were transfected with CAT reporter plasmid pBLCAT2, or this plasmid carrying three copies of wild type SIE (SIE/pBLCAT2) or three copies of mutant SIE (m.SIE/pBLCAT2). Twenty-four hours after transfections, cells were serum starved for 3 h and treated with AII (100 nM) or EXP3174 (100 µM) as indicated, for 12 h. Cell extracts were prepared and CAT activity measured using thin layer chromatography. This experiment was repeated three times. EXP, EXP3174.




DISCUSSION

SIF complexes (A, B, and C) contain tyrosine-phosphorylated STAT proteins. Complex A contains dimerized Stat92, complex B is a heterodimer of Stat92 and Stat91, and complex C is a homodimer of Stat91. We have previously demonstrated that stimulation of the AII G-protein coupled receptor (AT) induces SIF activity (mainly complex A) and activation of Stat91(30) . We now identify a more significant contribution of Stat92 to AII stimulated SIF formation consistent with the predominance of complex A. Activation of Stat92 by AII was delayed compared to its rapid induction by IL-6 which parallels our initial observation (30) that nuclear SIF activity stimulated by AII was delayed (maximal at 2-3 h). Angiotensin II directly activated SIF activity through the AT receptor and the delayed activation was not due to a requirement for the release of other SIF inducing factors. We hypothesized that AII signaling was bifunctional, capable of activating an inhibitory pathway that prevented/suppressed SIF/Stat92 activation during the initial phase (0-30 min) after AII exposure, followed by a stimulatory pathway that induced significant levels of SIF/Stat92 activity at 2 h. In agreement with this speculation, detectable levels of SIF activity were only observed after a 30-min exposure to AII (Fig.3A). To test this hypothesis, we determined the capacity of AII to modulate the rapid induction of SIF/Stat92 by IL-6. We demonstrated that AII transiently inhibits SIF activation by IL-6 in T3CHO/AT cells, suggesting that similar transient/reversible inhibitory mechanisms may be responsible for the delayed maximal SIF activation by AII. The delayed SIF activation by AII is potentially an important observation suggesting that: 1) the mechanism of the AII-induced Stat92 activation and SIF complex formation is unique and probably involves additional steps not utilized by growth factors and cytokines; and that 2) AII activates the transcription of a subset of genes containing SIE or SIE-like elements at 2 h, which may differ from those stimulated (30 min) by growth factors and cytokines. Our observation that AII can cross-talk with a cytokine signaling pathway raises the possibility that this AII induced inhibitory pathway is utilized by cells to modulate/inhibit IL-6-related cytokine responses.

Other mechanisms for the delayed SIF/Stat92 activation by AII can be envisaged. A recent study suggested that the interactions of a specific Src homology 2 (SH2) domain on STATs, with particular phosphotyrosine-containing motifs within the cytoplasmic domains of activated receptors, was the crucial determinant by which STAT or STATs were activated by JAK kinases in response to ligands(39, 40) . While it is unclear how the AT receptor couples to these activation events, AII stimulation has been shown to phosphorylate the AT(1) receptor on both serine and tyrosine residues(41) . Although the identity of the phosphorylated tyrosine residues are not yet determined, tyrosine residues (Tyr-312, Tyr-319, and Tyr-339) within the cytoplasmic tail of the AT receptor are potential candidates and may be accessible to JAKs. A recent study reported that AII activates JAK2/tyk2 kinases(42) , suggesting that members of the JAK kinase family may be involved in the activation of STAT proteins by this peptide. Interestingly, the early activation of JAK2 and tyk2 (within 5 min) coincides with the rapid tyrosine phosphorylation of Stat91 (within 5 min). However, despite this early activation of JAK2/tyk2, tyrosine phosphorylation of Stat92 was not detected until 60 min following exposure to AII(42) . We have shown that Stat92 is the principal component of AII-stimulated SIF, the time course of activation of which is confirmed in the preceding report (42) . Thus, the mechanism for AII-induced Stat92 activation may require other kinases or may involve processes distinct from those described for growth factors and cytokines. Alternatively, in context with what is known about STAT signaling, the delayed activation of Stat92 by AII may be explained if the SH2 domain of Stat92 has low affinity for the phosphorylated AII receptor.

Our observation that AII can inhibit IL-6 stimulated SIF/Stat92 activation is novel and may occur at multiple levels. It is possible that the interference occurs proximally at the level of the IL-6 receptor or the signal transducer protein gp130. Since the activation of the STAT pathway by IL-6 involves tyrosine phosphorylation and dimerization of gp130(43) , the possibility that AII activates a tyrosine phosphatase which prevents these events, requires further investigation. In support of this hypothesis, there is evidence that a G-protein coupled receptor (the somatostatin receptor) can activate and recruit a tyrosine phosphatase(44) . Similarly, other downstream events such as the tyrosine phosphorylation and activation of JAK kinases and Stat92 may be transiently suppressed by a tyrosine phosphatase.

It has been demonstrated, in many cell types, that AII rapidly increases intracellular calcium and cellular PKC activity(9, 11, 14) . Additionally, AII can modulate levels of cAMP(9, 45, 46) . To determine the possible role of cAMP, calcium, and PKC in the AII mediated inhibition of SIF induction by IL-6, we tested the ability of forskolin, ionomycin, and PMA (which activate cAMP, calcium, and PKC, respectively) to modulate the SIF induction by IL-6. Pretreatment of cells with forskolin and ionomycin for 15 min had no effect on the IL-6-induced SIF response. (^2)Only PMA inhibited the rapid induction of SIF by IL-6. Although, PMA mimicked the actions of AII, the mechanism by which AII inhibited the IL-6-induced SIF/Stat92 activation appeared to be independent of PMA-sensitive isoforms of PKC. It is possible, however, that a PMA-insensitive isoform of PKC is involved or that the inhibition may involve other kinases.

Thus, we have shown that the AT receptor mediates the activation of SIF complex A and Stat92 or a related protein. Stat92 appears to play a general role, integrating diverse signals from receptor tyrosine kinases (PDGF and EGF), from JAK kinase-dependent receptors (IL-6, IFN-alpha, IFN-, and growth hormone) (36, 37) and from G-protein coupled receptors (AT), as we have demonstrated. Although Stat92 is a common major component for both AII and IL-6-induced SIF, the timing of activation, along with its interactions with other members of the STAT family may result in differential gene transcription. We have demonstrated that AII and IL-6 display distinct kinetics of Stat92 activation, providing a basis for cross-talk between AII and cytokine-mediated cellular processes. Such cross-talk may occur in cardiac tissues given the presence of receptors for AII (3, 4, 5) and a variety of IL-6-related cytokines(47) . The interplay between signaling pathways for AII and cytokines opens a new area of investigation.


FOOTNOTES

*
This work was supported in part by Grants from Pennsylvania Affiliate of the American Heart Association (to G. J. B. and K. M. B.), from the American Heart Association (94013470 to T. J. T. and 91003020 to K. M. B.), from the National Institutes of Health (HL-44883 to K. M. B.), and by the Geisinger Clinic Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Weis Center for Research, 100 North Academy Ave., Danville, PA 17822. Tel.: 717-271-6815; Fax: 717-271-6668.

Present address: C281, Dept. of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, 80262.

**
Recipient of a C. J. Martin fellowship from the National Health and Medical Research Council of Australia.

§§
Established Investigator of the American Heart Association.

^1
The abbreviations used are: AII, angiotensin II; G-protein, guanyl nucleotide-binding protein; AT, angiotensin II receptor subtype 1A; CHO-K1, Chinese hamster ovary cells; PDGF, platelet-derived growth factor; IFN-, interferon-; IL-6, interleukin-6; SIE, sis-inducing element; SIF, sis-inducing factor; JAK, Janus kinase; TGF-beta; transforming growth factor-beta; STAT, signal transducers and activators of transcription; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; EXP, EXP3174; CAT, chloramphenicol acetyltransferase.

^2
G. J. Bhat and K. Baker, unpublished results.


ACKNOWLEDGEMENTS

We thank Drs. H. Singer and T. Abraham for valuable discussions; Drs. H. Morgan and H. Singer for critical reading of the manuscript; Dr. D. Dostal for suggestions and expert assistance in the preparation of figures; Dr. S. Saha for providing plasmids; T. Motel for part of the tissue culture work; and Dupont Merck Pharmaceutical Co. for providing the AII receptor antagonist, EXP3174.


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