©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of NFAT-mediated Transcription by G-coupled Receptors in Lymphoid and Non-lymphoid Cells (*)

(Received for publication, February 27, 1996)

Valerie Boss Deepa J. Talpade Thomas J. Murphy (§)

From the Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The nuclear factor of activated T cells (NFAT) was discovered as an inducible transcription factor activated by antigen stimulation of the T cell receptor in lymphocytes. Stimulation of NFAT-mediated transcription is now reported in both lymphoid and non-lymphoid cells following activation of a neurotransmitter receptor. Carbachol induces robust luciferase responses in Jurkat and pheochromocytoma PC12 cells expressing an NFAT-luciferase reporter construct and a G(q)-coupled m3 muscarinic receptor. Cyclosporin blocks this response in PC12 cells, as in Jurkat cells. In PC12 cells expressing a G(i)-coupled m2 muscarinic receptor, carbachol induces NFAT-mediated luciferase activity that is strictly dependent upon co-expression of a chimeric Galpha(q)/alpha(i) subunit, which confers G(q)-effector coupling on G(i)-linked receptors. These findings suggest that neurotransmitters, autacoids, or hormones acting on G(q)-protein-coupled receptors may serve as physiological stimulators of NFAT in lymphoid and non-lymphoid cells.


INTRODUCTION

The nuclear factor of activated T cells (NFAT), (^1)a transcription factor activated by antigen stimulation of the T cell receptor (TCR), is known mainly for its role in the co-ordinate induction of interleukin and cytokine genes during immune cell activation(1) . Stimulation of the TCR and subsequent protein-tyrosine phosphorylation initiates several downstream events, including the activation of isoforms of phospholipase C (PLC). PLC, like other PLC isoforms, catalyzes the breakdown of phosphoinositides. The resultant co-activation of Ca and protein kinase C (PKC) signaling pathways is required for induction of NFAT-mediated transcription(2, 3, 4) .

Stimulation of receptors coupled to the G(q) subfamily of heterotrimeric G-proteins activates beta isoforms of PLC (PLCbeta), and thus the possibility exists that such receptors may participate in induction of NFAT-dependent gene expression. Previous experiments have shown that stimulation of an ectopically expressed muscarinic receptor in T cells induces phosphoinositide hydrolysis(5) , interleukin 2 (IL-2) production(6) , and lymphoid NFAT activity(7) . G(q)-protein-coupled receptors are expressed in many cell types, and NFAT or NFAT-like activities have recently been discovered in a variety of lymphoid and non-lymphoid cells and tissues(1, 8, 9, 10, 11, 12, 13, 14, 15) . The present study demonstrates that NFAT can act as a downstream effector of G(q)-protein-coupled receptors in the non-lymphoid PC12 cell line, as well as in lymphoid cells.


EXPERIMENTAL PROCEDURES

Cell Lines and Plasmids

A plasmid containing multimers of the NFAT binding site located upstream from the minimal IL-2 promoter and the firefly luciferase reporter gene (NFAT-luciferase) (16) was a gift from Rick Bram (St. Jude Children's Research Hospital, Memphis, TN). Plasmids coding for human muscarinic receptors m2 and m3 (17) were obtained from Allan Levey (Emory University, Atlanta, GA). Plasmids encoding G-protein alpha subunits (wild type Galpha(q), and a chimera, Galpha(q)/alpha(i), in which the last five residues from alpha(i) are replaced with corresponding amino acids from Galpha(q)(18) ) were gifts from Bruce Conklin (Gladstone Cardiovascular Center, San Francisco, CA). Jurkat cells and PC12 cells (subcolony originated by Simon Haleguoa, SUNY, Stonybrook, NY) were obtained from Jeremy Boss and Ken Minneman, respectively (Emory University).

Transfections

Cells (0.8 ml) were electroporated in a 0.4-cm gap cuvette using a Bio-Rad Gene Pulser II at 950 microfarads and 300 V (for Jurkat cells) or 360 V (for PC12 cells), at cell densities of 1 times 10^7 cells/ml (for Jurkat cells) or 3-7 times 10^6 cells/ml (for PC12 cells). NFAT-luciferase plasmid DNA (10 µg) and pRSV-Tag plasmid DNA (5 µg), as well as 5 µg of each additional plasmid DNA, were included in every transfection, and all transfections were performed in duplicate in RPMI 1640 medium. Transfected cells were aliquoted in multiwell plates in RPMI 1640 with 10-20% FBS (for Jurkat) or Dulbecco's modified Eagle's medium with 10% FBS (for PC12) with penicillin/streptomycin and maintained at 37 °C (5% CO(2)).

Assays

For luciferase assays, receptor agonists and other drugs were added to Jurkat or PC12 cells between 46 and 50 h after transfection. After an additional 8-12 h, cells were harvested, and luciferase activity was determined as described previously(19) . For phosphoinositide hydrolysis assays, fresh growth medium containing 1 µCi/ml myo-[^3H]inositol (Amersham Corp.) was added at 36 h after transfection. After an additional 18 h of incubation, agonists and additional drugs were added to fresh medium containing 5 mM LiCl, and the incubation was continued for 60 min. Cells were lysed and [^3H]inositol phosphates were measured as described previously(20) .

Materials

RPMI 1640 and bicarbonate-buffered Dulbecco's modified Eagle's medium used for growth media were made from powder obtained from Life Technologies, Inc. The RPMI 1640 (liquid) used for transfections, FBS, and calf serum were purchased from Cellgro Mediatech (Herndon, VA), and horse serum was from Atlanta Biologicals (Norcross, GA). Carbachol, ionomycin, phorbol 12-myristate 13-acetate (PMA), and chemicals for buffers were purchased from Sigma. Firefly luciferin was obtained from Boehringer Mannheim. Cyclosporin A (CsA) was a gift from Sandoz Pharmaceuticals (Basel, Switzerland).


RESULTS AND DISCUSSION

Transcriptional responses to Ca and PKC establish the salient features of NFAT-mediated gene expression in lymphoid (Jurkat) and non-lymphoid (PC12) cells. These were transfected with a well characterized NFAT-inducible luciferase reporter plasmid containing a concatemer of the distal IL-2 gene NFAT-responsive element on a minimal IL-2 gene promoter(16, 21) , as well as a plasmid encoding the SV40 large T antigen (pRSV-Tag) to ensure plasmid replication. In Jurkat cells (n = 4 experiments) and in PC12 cells (n = 3 experiments), the respective basal luciferase responses in the absence of added drugs (0.04 ± 0.01 and 0.06 ± 0.02 Turner light units; mean ± S.E.) are near the lower limit of detectability. The PKC activator PMA does not significantly stimulate luciferase activity in either cell line. In Jurkat cells, the Ca ionophore ionomycin induces a modest increase in luciferase activity (5.5 ± 1.6-fold over basal). Consistent with previous reports(1, 7, 16, 22) , ionomycin and PMA together elicit synergistic stimulation of luciferase activity (550 ± 170-fold over basal) (Fig. 1a). Similarly, in PC12 cells, modest luciferase responses elicited by ionomycin alone (5.3 ± 1.4-fold) are potentiated by PMA (13 ± 3-fold in PC12 cells) (Fig. 1b). NFAT-mediated responses induced by ionomycin in PC12 cells exhibit dose-related increases at concentrations up to 1 µM, which attenuate at higher concentrations (Fig. 1c). Virtually identical results have been obtained using ionomycin in Jurkat cells (data not shown)(22) . These data suggest that PC12 cells, like Jurkat cells, express a functional NFAT capable of mediating transcriptional events in response to Ca and PKC signaling.


Figure 1: Ionomycin and PMA synergistically increase NFAT-mediated responses in Jurkat and PC12 cells. a, in Jurkat cells transfected with NFAT-luciferase and pRSV-Tag plasmids, (n = 4 experiments, each performed in duplicate), ionomycin (iono; 0.25 µM), but not PMA (10 nM), induces significant (p < 0.01) luciferase activity compared with basal. Ionomycin and PMA together induce a luciferase response that is significantly greater than basal (p < 0.005) or than responses elicited by ionomycin (p < 0.005) or PMA (p < 0.005) alone. b, in similarly transfected PC12 cells (n = 3), ionomycin (0.5 µM), but not PMA (10 nM), induces a significant (p < 0.001) increase in luciferase activity compared with basal. The response elicited by ionomycin and PMA together is significantly greater than basal (p < 0.001) or responses elicited by ionomycin (p < 0.005) or PMA (p < 0.005). c, in transfected PC12 cells, ionomycin alone (open squares) induces a dose-dependent stimulation of luciferase activity, which is potentiated (closed squares) by 10 nM PMA. Student's t test was used for data analysis in these and all subsequent experiments.



TCR activation of PLC defines the major known pathway for induction of NFAT-mediated transcription in lymphocytes. Recent studies suggest that lymphoid NFAT may also be induced through stimulation of receptors that activate PLCbeta through coupling to heterotrimeric G(q)-proteins(6, 7) . Carbachol (1 mM) elevates luciferase activity (410 ± 44-fold increase; n = 5 experiments) in Jurkat cells expressing a muscarinic m3 receptor but not in cells transfected with empty vector as control (Fig. 2a). Similarly, carbachol elicits a dose-dependent increase in luciferase activity that is 34 ± 7-fold over basal at the highest dose applied (1 mM carbachol) in PC12 cells expressing the m3 receptor but not in control cells transfected with empty vector (n = 5 experiments) (Fig. 2b). Carbachol-induced phosphoinositide hydrolysis and luciferase responses in PC12 cells were compared in side-by-side experiments. Both responses were elicited in cells expressing the m3 receptor but not in those transfected with empty vector instead (Table 1), although luciferase measurements clearly serve as a more sensitive indicator of receptor stimulation than inositol phosphate responses.


Figure 2: Stimulation of G(q)-coupled receptors induces NFAT-mediated luciferase activity in Jurkat and PC12 cells. Cells were transfected with NFAT-luciferase and pRSV-Tag plasmids, as well as either a plasmid encoding the m3 muscarinic receptor or an empty vector as control. a, significantly greater (p < 0.005) stimulation of NFAT-dependent luciferase activity is elicited by 1 mM carbachol (+) than by vehicle(-) in Jurkat cells transfected with the m3 receptor plasmid but not in cells transfected with empty vector instead (n = 5). b, carbachol elicits a dose-dependent increase in luciferase activity in PC12 cells transfected with the m3 receptor (closed squares) but not in those transfected with empty vector instead (open squares) (n = 5). c, PC12 cells were transfected with NFAT-luciferase and pRSV-Tag plasmids, as well as either a plasmid coding for the m2 receptor or an empty vector as control. Transfections also included either a plasmid coding for the chimeric Galpha(q)/alpha(i) subunit or wild type Galpha(q). Carbachol elicits a dose-dependent increase in luciferase activity in PC12 cells transfected with an m2 receptor plasmid (filled circles) or empty vector (filled circles) in addition to a plasmid coding for the chimeric Galpha(q)/alpha(i) subunit. Carbachol does not increase luciferase activity in cells transfected with an m2 receptor plasmid (filled squares) or empty vector (open squares) in addition to a plasmid coding for a wild type Galpha(q) subunit (n = 6). Student's t tests were used as statistical analyses.





A chimeric Galpha(q)/alpha(i) subunit that switches the coupling of G(i)-linked receptors from inhibition of adenylate cyclase to stimulation of PLC (18) was used to establish that heterotrimeric G-proteins are involved in muscarinic receptor-induced NFAT responses. PC12 cells were transfected with reporter plasmids, and either an empty vector, a plasmid coding for a wild type Galpha(q), or a plasmid coding for Galpha(q)/alpha(i) was added. A dose-related increase in carbachol-stimulated luciferase activity is elicited in PC12 cells expressing Galpha(q)/alpha(i) alone but not in control cells expressing wild type Galpha(q) (Fig. 2c) or transfected with empty vector (data not shown). This response is likely mediated by a muscarinic m4 receptor endogenous to PC12 cells, which is known to couple negatively to adenylate cyclase(23) . In parallel experiments, larger dose-related increases in carbachol-stimulated luciferase activity are elicited in cells expressing a G(i)-coupled m2 receptor, and these responses are strictly dependent upon the presence of Galpha(q)/alpha(i) (Fig. 2c). Carbachol elicits both phosphoinositide hydrolysis and NFAT-luciferase responses in side-by-side assays in PC12 cells expressing the m2 receptor in the presence, but not the absence, of the chimeric Galpha(q)/alpha(i) subunit (Table 1). Since the chimeric G-protein complements a response otherwise refractory in the cells, the simplest interpretation of these data is that G(q)-protein coupling is both necessary and sufficient for induction of NFAT-mediated transcription by muscarinic receptors.

The immunosuppressant CsA forms a complex with a cyclophilin that inhibits calcineurin, a Ca-dependent phosphatase essential for the nuclear translocation of NFAT and subsequent NFAT-dependent transcription(22, 24, 25, 26) . As in Jurkat cells (data not shown) (22) Ca- and PKC-dependent NFAT activation in PC12 cells is inhibited by CsA at concentrations consistent with its effects on immune cells(21, 22, 24, 26, 27, 28) . CsA also inhibits carbachol-induced luciferase expression in cells expressing the m3 receptor (Fig. 3). Co-dependence of NFAT activation upon elevations of intracellular Ca and PKC activity and its sensitivity to CsA are principal characteristics of NFAT activity (for reviews, see (2, 3, 4) ). Therefore, the finding that m3 receptor-stimulated luciferase responses are blocked by CsA in PC12 cells provides further evidence that G(q)-protein-coupled receptors can mediate NFAT induction in non-immune cells.


Figure 3: CsA inhibits NFAT-mediated luciferase activity in PC12 cells. Luciferase responses elicited by co-application of 1 µM ionomycin and 10 nM PMA (filled squares) or by 1 mM carbachol (filled circles) or buffer (open squares) in PC12 cells transfected with NFAT-luciferase, pRSV-Tag, and m3 receptor plasmids are inhibited by increasing doses of CsA but not by vehicle containing 0.1% ethanol and 0.05% Tween 80 (no CsA). Data are presented as mean ± S.E. of 4 experiments, each performed in duplicate.



This study establishes that NFAT transcription factors can serve as downstream effectors for G(q)-protein-coupled receptors that activate PLC and that such receptors can induce NFAT activity in both lymphoid and non-lymphoid cells. NFAT proteins or NFAT-like DNA binding and/or transcriptional activities and NFAT isoform mRNA have been identified in a variety of tissues (8, 14) and cell types, including lymphoid(1, 9, 10, 12) , endothelial(13) , neuronal(11) , and mast cells(15) . Receptors coupled to G(q)-proteins are represented in numerous cell types and respond to a diverse array of hormones, autacoids, and neurotransmitters. The present studies indicate that these agents are capable of acting as physiological activators of NFAT and that NFAT-mediated transcription represents a heretofore unappreciated mechanism by which these receptor agonists can modulate cellular physiology. Furthermore, immunosuppressant therapy with CsA is limited by its toxicity to neural, hepatic, bone, and renal systems. The present findings raise the possibility that some of these side effects might arise in part through disruption of normal cellular processes that are modulated by virtue of G(q)-protein-coupled receptor regulation of NFAT-mediated transcriptional activities.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL48252 and HL52810, during the tenure of a grant-in-aid from the American Heart Association and Sanofi-Winthrop. 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. Tel.: 404-727-2466; Fax: 404-727-0365.

(^1)
The abbreviations used are: NFAT, nuclear factor of activated T cells; TCR, T cell receptor; PLC, phospholipase C; PKC, protein kinase C; IL-2, interleukin 2; FBS, fetal bovine serum; NFAT-luciferase, firefly luciferase reporter plasmid; pRSV-Tag, plasmid encoding the SV40 large T antigen; CsA, cyclosporin A; PMA, phorbol 12-myristate 13-acetate.


ACKNOWLEDGEMENTS

We are grateful to Drs. Jeremy Boss, Melissa Brown, Ray Dingledine, and Ken Minneman for thoughtful comments and to Drs. Rick Bram, Bruce Conklin, and Allan Levey for supplying plasmids.


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©1996 by The American Society for Biochemistry and Molecular Biology, Inc.