©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of Inducible Nitric-oxide Synthase by the Heterotrimeric G Protein G(*)

(Received for publication, November 28, 1995)

Kenichiro Kitamura (1)(§) William D. Singer (3) Robert A. Star (2) Shmuel Muallem (4) R. Tyler Miller(¶) (1)

From the  (1)Departments of Medicine and Pharmacology, University of Florida, Gainesville, Florida 32610 and the Departments of (2)Medicine, (3)Pharmacology, and (4)Physiology, University of Texas, Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

While the functions of several G protein alpha subunits such as alpha(s) and alpha(q) are relatively well understood, the action of others such as alpha remain largely undefined. Because of recent interest in regulation of nitric-oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha, we studied the effect of alpha on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha or its GTPase-deficient mutant, alpha, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha) or were minimally increased in cells expressing activated alpha (alpha) and alpha(q) (alpha), members of the three other G protein alpha chain families. Transient co-expression of alpha or alpha increased the activity of an iNOS promoter-CAT construct demonstrating that alpha increases iNOS expression through transcription. Consequently, alpha induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents.


INTRODUCTION

The G protein family of heterotrimeric signaling proteins couples receptors on the outside of cells to effector molecules in cell membranes or inside cells. The alpha subunits have primary responsibility for determining the specificity of receptor and effector interactions. Individual alpha chains couple to multiple effectors (enzymes and ion channels), and the effects of multiple G proteins converge on single effector systems at multiple levels. These characteristics create signaling networks and allow for integration and modulation of signals in cells(1, 2) . The functions of some alpha subunits such as alpha(s), that stimulates adenylyl cyclase, and alpha(q), that regulates phospholipase Cbeta isoforms, are relatively well defined in biochemical and physiologic terms (1, 2, 3) .

The functions of other alpha chains such as those of the alpha/alpha family remain largely undefined. The alpha and alpha subunits are widely expressed in mammalian tissues(4, 5) . We have demonstrated that stable expression of alpha in epithelial cells increases calcium influx(6) . Transient or stable overexpression of alpha increases Na/H exchange (NHE-1) activity, although the mechanism is not fully defined(6, 7, 8) . Expression of an activated form of alpha can transform cells in tissue culture and increase expression of immediate early genes and activate Jun/stress-activated kinases(9, 10, 11, 12) .

Receptors for thrombin and thromboxane, both ligands that participate in inflammatory reactions, stimulate guanine nucleotide exchange by alpha in platelet membranes(13) . This finding suggests that alpha may be involved in signaling by proinflammatory substances and could regulate cell processes that are involved in inflammatory responses, including intracellular calcium (Ca)(^1)(13) . Recently, considerable interest has been expressed in regulation of NO production by G protein-coupled signaling systems. To explore involvement of alpha in inflammation and provide direct evidence for regulation of NO production by heterotrimeric G proteins, we studied the effect of stable expression of alpha and members of the three other G protein alpha chain families (alpha(s), alpha(i), and alpha) on NO production.


EXPERIMENTAL PROCEDURES

Materials

Restriction and DNA modifying enzymes were obtained from Promega and radionucleotides from DuPont NEN. Tissue culture medium, serum, and G418 were from Life Technologies, Inc., and radiochemicals were from DuPont NEN or Amersham. Tissue culture plasticware was from Falcon. Calphostin-C was obtained from LC Laboratories. Other chemicals were from Sigma or molecular biology grade from Fisher.

Cell Culture

MCT cells, an SV40-transformed mouse proximal tubule cell line were a gift from Eric Neilson(14) . Cultures were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 (50:50) plus 5% fetal bovine serum. Experiments were performed when cells were approximately 90% confluent. Serum and G418 were removed 18-24 h before experiments.

Construction of cDNAs and Expression in Mammalian Cells

The cDNA constructs, vectors, and stable expression of alpha, alpha, alpha, and alpha were described previously(6, 15) . alpha was subcloned into Zem-228, and stable, neomycin-resistant pooled cells expressing it were selected as described(6) . The cDNAs were expressed in MCT cells using Zem-228 that contains a mouse metallothionein promoter. All studies were performed without heavy metal induction of the alpha chains, because we found adequate expression without induction. A clonal cell line expressing alpha and neomycin-resistant pooled cells expressing alpha or alpha were used for studies. The expressed alpha chain protein was increased 2-4-fold in membrane fractions over that in untransfected cells. (^2)Pooled neomycin-resistant MCT cells expressing Zem-228 without an insert were used as controls (neomycin-resistant control cells)(6) .

We obtained the iNOS promoter-CAT (p1 iNOS-CAT) from Carl Nathan and Qiao-wen Xie (Cornell) and alpha from Henry Bourne (University of California, San Francisco)(8, 16) . alpha and alpha were expressed in Zem-228 and pCMV5 vectors (17, 18) . Truncation mutants of p1 iNOS-CAT were made using convenient restriction sites, blunt-ending the overhangs with polymerase, and religating the plasmids. cDNAs were expressed transiently using a modification of the technique of Martin et al.(19) . MCT cells were grown to near confluence, suspended, and aliquots of approximately 10^6 cells incubated with 4 µg of DNA (2 µg of p1 iNOS-CAT, 1 µg of alpha construct, and 1 µg of SV40 beta-galactosidase) in the presence of DEAE-dextran (M(r) 5 times 10^5) and 5% serum. The control sample contained 1 µg of pCMV5 in place of the alpha chain construct.

CAT Assays

Cells were harvested 48 h after transfection, extracts prepared, and normalized for protein(20) . beta-Galactosidase activity correlated with protein. Aliquots of cell extract (35 µg of protein) were assayed for chloramphenicol acetyltransferase (CAT) activity using [^14C]chloramphenicol as a substrate for 4 h at 37 °C(19, 20) . Acetylated and nonacetylated [^14C]chloramphenicol were separated by thin layer chromatography and the plates exposed to x-ray film.

Immunoblotting

30 µg of post-nuclear cell extract protein was size-fractionated on a 7% SDS-polyacrylamide gel. The antibody to the macrophage-inducible form of NOS (iNOS) (from Transduction Laboratories, N32030, Louisville, KY.), was used at a 1:500 dilution in Tris-buffered saline with 0.1% Tween 20. The filters were washed and incubated with goat anti-mouse coupled to horseradish peroxidase in 1.0% Tween 20 at a 1:10,000 dilution. The blots were developed with the Amersham ECL system.

RNA Preparation and Analysis

Poly(A) RNA, prepared by the guanidinium thiocyanate method and oligo(dT)-cellulose chromatography, was size-fractionated, transferred to nylon filters (GeneScreen Plus, DuPont NEN), and hybridized as described previously (6) . Radiolabeled cDNA probes were made by random primer using beta-actin and iNOS cDNAs(6, 21) .

Measurement of NOS Activity

NOS activity was measured in cell extracts as conversion of [^3H]arginine to [^3H]citrulline by a modification of the method of Bredt and Snyder(22) . Cells were scraped in buffer containing 50 mM HEPES, pH 8.0, and assorted protease inhibitors, homogenized, and cytosolic extracts prepared by centrifugation. Each reaction contained 50 µg of cell extract, 20 mM HEPES, pH 7.4, flavin adenine dinucleotide (10M), flavin mononucleotide (10M), tetrahydrobiopterin (2 times 10M), beta-NADPH (2 times 10M), EDTA (5 times 10M), CaCl(2) (5 times 10M), arginine (2 times 10M), and [^3H]arginine (approximately 180,000 cpm). The reactions were carried out for 15 min at 37 °C and stopped by the addition of 400 µl of stop solution containing 20 mM HEPES, pH 5.5, 2 mM EDTA, and 2 mM EGTA and placement on ice. [^3H]Citrulline was separated by column chromatography. Reactions were performed in triplicate.

Statistical Analysis

Experimental groups were compared by analysis of variance using the InStat biostatistics program from GraphPad Software (San Diego, CA). Multiple comparisons among groups were made using the Student-Newman-Keuls test. p values < 0.05 were considered significant.


RESULTS

Our approach to studying regulation of NOS activity by heterotrimeric G protein alpha chains was to stably express members of each of the four G protein alpha chain families, alpha(s), alpha(i), alpha, and alpha, in MCT cells(23) . The expressed alpha chains (alpha, alpha, alpha, and alpha) contained mutations, substitution of leucine for glutamine at a conserved site in the guanine nucleotide binding domain that blocks GTPase activity and produces an activated protein(24) . The GTPase-deficient forms of the alpha chains were studied because they provide the strongest intracellular signals.

NOS Activity in Cells Expressing G Protein alpha Chains

Fig. 1shows NOS activity in extracts from neomycin-resistant control cells and MCT cells that stably express alpha, alpha, alpha, alpha, and alpha. Stable expression of the two forms of alpha, alpha, and alpha increase NOS activity to a much greater degree than alpha chains from other families. N-nitro-L-arginine (solid bars, Fig. 1) blocked the majority of the conversion of [^3H]arginine to [^3H]citrulline. Similar results were obtained when NO(2) production in whole cells was measured.^2


Figure 1: NOS activity in cell extracts from neomycin-resistant control cells and cells expressing alpha, alpha, alpha, alpha, and alpha. Open bars represent total citrulline conversion activity, and closed bars represent NOS activity in the presence of 1 mMN-nitro-L-arginine. Values represent the means of multiple experiments expressed as multiples of the control neomycin-resistant control cells value ± S.E. (neomycin-resistant control cells (Neo), n = 9; alpha, n = 4; alpha, n = 4; alpha, n = 9;, alpha, n = 4; alpha, n = 4).



Expression of NOS in Cells Expressing G Protein alpha Chains

The question we proceeded to ask was whether the increased NOS activity seen in the cells expressing the two forms of alpha was due to increased activity of existing protein, increased protein expression, and what form of NOS was involved. To address these questions, we performed immunoblots with the cell extracts using antibodies to iNOS (2A), bNOS (2B), and eNOS (2C). Only iNOS was detected in any of the cell types. RNA blots with cDNA probes to the three NOS isoforms were performed at reduced stringency (30% formamide, hybridization at 37 °C), but again, only iNOS was detected.^2Fig. 2A shows that iNOS antigen expression correlated with NOS activity. The cells expressing alpha and alpha contained the most iNOS protein (approximately 15-fold increased over neomycin-resistant control cells), while the increases in the cells expressing alpha and alpha were much more modest, only 3-4-fold over neomycin-resistant control cells.


Figure 2: Immunoblot of cell extracts from neomycin-resistant control cells (Neo) and cells expressing alpha, alpha, alpha, alpha, and alpha using antibodies to iNOS (A), bNOS (B), and eNOS (C). The first lane contains 400 ng of cell extract enriched in iNOS (A), bNOS (B), or eNOS protein. The blot shown is representative of four separate blots.



The next question addressed was whether the increase in iNOS protein was due to increased mRNA levels. We performed Northern blots with a cDNA probe for iNOS. We found that expression of alpha substantially increased iNOS mRNA abundance (approximately 10-fold) as shown in Fig. 3. For comparison, the increase in iNOS mRNA in the cells expressing alpha, the cells with the next highest increase in NOS activity and protein expression was relatively small, approximately 2-fold.


Figure 3: RNA blot using 8 µg of poly(A) RNA from neomycin-resistant control cells (Neo) and cells expressing alpha and alpha. In the top panel, the blot was probed for mouse iNOS, and in the bottom panel the blot was probed for beta-actin to normalize for loading. The position of the ribosomes is shown at the right.



Induction of iNOS by alpha

iNOS mRNA can be regulated by a variety of mechanisms, including mRNA stabilization, degradation, and transcription(25) . In view of the robust increase in iNOS mRNA, protein, and activity in the cells expressing alpha, we determined if alpha caused an increase in iNOS mRNA through increased transcription using transient co-expression of a CAT reporter construct (p1 iNOS-CAT) that contains -1588 to +161 of the mouse iNOS 5`-flanking regulatory region with alpha or its activated mutant, alpha(8, 19) . Fig. 4A demonstrates that alpha increased the activity of p1 iNOS-CAT and, therefore, transcription of the iNOS promoter reporter gene construct. alpha stimulated p1 iNOS-CAT activity more than alpha. When the wild type and mutant alpha were expressed with pCMV5, a vector with a strong cytomegalovirus promoter, the relative stimulation of p1 iNOS-CAT was greater than that obtained with pZem-228, a vector with a weaker metallothionein promoter. These results indicate that activity of the iNOS promoter is related to both the amount of alpha expressed and its activity.


Figure 4: A, regulation of iNOS promoter activity by alpha and alpha in MCT cells. MCT cells were transiently co-transfected with p1 iNOS-CAT and pCMV5 without insert (control) or one of the alpha constructions (alpha or alpha) in the vectors indicated (Zem-228 or pCMV5). Cells were grown for 48 h, extracts prepared, and equal amounts of cell extract (normalized for protein) were assayed for CAT activity. The experiment shown is representative of four separate experiments. B summarizes the effect of protein kinase C inhibition on alpha and alpha-stimulated p1 iNOS-CAT activity (left panel: open columns, control; , filled columns; shaded columns, calphostin C) and activation of adenylyl cyclase on alpha and alpha-stimulated P1 iNOS-CAT activity (right panel: open columns, control; filled columns, forskolin + isobutylmethylxanthine) relative to the alpha control. Cells were transiently transfected with the alpha chains (in pCMV5 vectors) and p1 iNOS-CAT as indicated. After 24 h, the cells were treated with phorbol 12-myristate 13-acetate (400 nM), calphostin C (10 nM), or isobutylmethylxanthine and forskolin (50 µM and 100 nM, respectively) for 24 h as indicated. Cells were harvested and assayed for CAT activity as described above. The experiments shown are representative of at least three experiments.



Galpha had a marked stimulatory effect on iNOS expression, while alpha and alpha had smaller effects. In order to demonstrate the specificity of the alpha effect and that the other alpha chains act by different mechanisms, we studied the effects of chemical activators or inhibitors of several signaling pathways on expression of p1 iNOS-CAT co-expressed with alpha, alpha, or alpha. Other investigators have demonstrated that inhibition of protein kinase C blocks the ability of alpha(q), but not alpha to stimulate Na/H exchanger activity(7) . Consequently, in cells transiently expressing p1 iNOS-CAT and alpha or alpha, we inhibited protein kinase C by down-regulating it with a high dose of phorbol 12-myristate 13-acetate, and by treatment with calphostin C. As shown in Fig. 4B (left panel), inhibition of protein kinase C reduced stimulation of p1 iNOS-CAT by alpha, but had no effect on its expression in response to alpha. In similar experiments (Fig. 4B (right panel)), isobutylmethylxanthine and forskolin prevented stimulation of p1 iNOS-CAT by alpha, but increased its expression (rather than decreased it) to a small degree in the cells with alpha. These results demonstrate that alpha stimulates iNOS expression by a mechanism that is distinct from those of alpha(q) and alpha.

The iNOS promoter-CAT reporter construct contains 161 bases of the 5`-untranslated region(16) , so in principle, alpha could regulate expression of this construct through stabilization of the mRNA. In order to determine what region of p1 iNOS-CAT responds to alpha, we made a series of truncation mutations using convenient restriction sites, -724 to +161 (P3 iNOS-CAT), -333 to +161 (p4 iNOS-CAT), and -48 to +161 (p6 iNOS-CAT)(16) . Fig. 5shows that stimulation of iNOS-CAT activity by alpha requires the 285-base region between -333 and -48. Consequently, the principal effect of alpha cannot be through stabilization of mRNA, but must be through increased transcription. The exact sequence responsible for the effect of alpha on iNOS expression is currently under investigation.


Figure 5: Effect of successive truncations of p1 iNOS-CAT on alpha-stimulated iNOS-CAT activity. A shows a map of p1 iNOS-CAT and the truncations made. B shows CAT activity of p1 iNOS-CAT and the truncation mutants with pCMV5 vector alone (top) and pCMV5-alpha (bottom). The cells were harvested and assayed for CAT activity as described above.




DISCUSSION

The novel and central finding of these studies is that iNOS is regulated at the transcriptional level by a specific G protein alpha chain, Galpha. Induction of iNOS is usually associated with inflammatory reactions initiated by a variety of stimuli, particularly bacterial products and/or cytokines, not by G protein-dependent signaling systems(25) . Consequently, induction of iNOS by alpha and the receptors that couple to it represents a new mechanism for regulation of iNOS and possibly other proteins that are induced during inflammatory reactions. Regulation of iNOS expression by alpha is not limited to epithelial cells. In experiments similar to those shown in Fig. 4, alpha and alpha increased iNOS promoter activity in RAW 264.7 cells, a macrophage-like cell line.^2 These studies indicate that substances that act through receptors coupled to alpha such as thrombin and thromboxane (13) could also initiate cellular responses similar to those initiated by cytokines and bacterial products such as lipopolysaccharide.

Both the wild type and mutant forms of alpha increase iNOS expression and activity. In the stable pooled cells the level of iNOS expression and activity was similar in response to alpha and alpha. One would expect that expression of the mutant alpha chain might result in higher levels of iNOS expression. However, higher levels of NOS could be toxic to the cells and result in similar levels of expression in cells containing mutant and wild-type alpha proteins. In the transient system where the total number of cells and efficiency of expression is lower than the stable system, alpha is more effective than alpha.

alpha induces iNOS by a specific mechanism. alpha chains from other families either do not induce iNOS (alpha) or do so to a much smaller degree and by a different mechanism (alpha and alpha). We do not believe that alpha or alpha(q) acts primarily through increased Ca(i), because the resting Ca(i) levels and calcium pool sizes are similar in neomycin-resistant control cells and the cells expressing the alpha chains(6, 26) . Cells expressing alpha have substantially elevated basal phospholipase C activity and presumably protein kinase C activity. alpha probably induces iNOS through protein kinase C because inhibition of protein kinase C blocks stimulation of iNOS by alpha. The small increase in iNOS activity and protein in the cells that express alpha is due to inhibition of adenylyl cyclase by overexpression of this alpha subunit, because the low level of iNOS induction is blocked by activators of the adenylyl cyclase system.

alpha may signal by a mechanism that has not been described previously for G protein alpha chains, because activated representatives of the other G protein alpha chain families either do not stimulate iNOS expression (alpha) or cause only a minor increase in its expression (alpha and alpha through their respective second messengers and kinases). Regulation of iNOS expression is relatively well characterized in macrophages and other tissues where it is induced by cytokines and lipopolysaccharide via interferon response elements and NF-kappaB(25, 27) . Interferon- signals through pathways involving JAK/STAT proteins, and lipopolysaccharide activates a complex signaling pathway involving NF-kappaB and mitogen-activated protein kinases. alpha appears not to require the upstream GAS or ISRE sequences in the iNOS gene(19) , but may couple receptors for substances like thrombin and thromboxane to regulation of iNOS expression through activation of other sequences shared by cytokines or lipopolysaccharide.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants R29DK41726, DK39298, F32-GM15359, and GM31954 with funds from the American Heart Association, Florida Affiliate (to R. T. M.), a training grant from the National Kidney Foundation (to K. K.), and by an Established Investigator Award from the American Heart Association (to R. A. S.). 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.

§
Current address: Third Dept. of Internal Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan.

To whom correspondence should be addressed: Nephrology, Hypertension, and Transplantation, University of Florida, Box 100224 JHMHC, 1600 S.W. Archer Rd., Gainesville, FL 32610. Tel.: 352-392-4008; Fax: 352-392-3581.

(^1)
The abbreviations used are: Ca, intracellular calcium; CAT, chloramphenicol acetyltransferase; bNOS, brain nitric-oxide synthase or NOS I; eNOS, endothelial nitric-oxide synthase or NOS III; iNOS, inducible nitric-oxide synthase or NOS II; NO, nitric oxide.

(^2)
K. Kitamura and R. T. Miller, unpublished observations.


ACKNOWLEDGEMENTS

We thank Paul Sternweis, Elliott Ross, Al Gilman, and Kimio Tomita for helpful discussions; Paul McLeroy for expert technical assistance; Carl Nathan and Qiao-wen Xie for p1 iNOS-CAT; Henry Bourne for alpha; and John Drake for persistant encouragement.


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