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J Biol Chem, Vol. 274, Issue 39, 27562-27566, September 24, 1999


Galpha 13 Stimulates Rho-dependent Activation of the Cyclooxygenase-2 Promoter*

Lee W. SliceDagger , John H. Walsh, and Enrique Rozengurt

From the Division of Digestive Diseases, Department of Medicine, CURE, Digestive Diseases Research Center, and Molecular Biology Institute, University of California, Los Angeles, California 90095

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND CONCLUSIONS
REFERENCES

Cyclooxygenase-2 (COX-2) gene expression is rapidly increased by cytokines, tumor promoters, and growth factors and is markedly enhanced in various cancer cells. Here, we examine the regulation of COX-2 promoter activity by alpha  subunits of heterotrimeric G proteins in NIH 3T3 cells. Using a transient transfection assay with a reporter vector in which the murine COX-2 promoter drives the production of luciferase and expression vectors encoding for alpha  subunits of G-proteins, we show that overexpression of wild type and constitutively active Galpha 13 and Galpha q induced transcription from the COX-2 promoter. The highest level of induced luciferase activity (5.8-fold) occurred in cells expressing the constitutively active Galpha 13(Q226L). We also show that expression of a constitutively active mutant of Rho (RhoQ63L) also induced transcription from the COX-2 promoter. Co-expression of Clostridium botulinum C3 toxin specifically blocked induction of the COX-2 promoter by either Galpha 13Q226L or RhoQ63L but did not prevent the activation of this promoter by Ras, Rac, v-src, or forskolin. We conclude that Galpha 13 signals through a Rho-dependent pathway leading to activation of the COX-2 promoter. This pathway is not inhibited by either cytochalasin D, which disrupts actin filament organization, or genistein, a broad spectrum tyrosine kinase inhibitor, indicating a bifurcation of the signaling pathway used by Galpha 13/Rho to induce COX-2 expression from that used to induce stress fiber formation and tyrosine phosphorylation of focal adhesion proteins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND CONCLUSIONS
REFERENCES

Prostaglandins play a pivotal role in a broad range of physiological and pathological processes including inflammation, pain transmission, maintenance of gastrointestinal integrity, and progression of colorectal cancer (1-3). The rate-limiting enzymes for production of prostaglandins are cyclooxygenases (COX)1 type 1 and 2 (4-6). COX-1 is constitutively expressed in nearly all cells, whereas COX-2 expression is induced as an immediate-early gene in response to pro-inflammatory cytokines, tumor promoters, and growth factors (7-10). COX-2 is overexpressed in cancers of the colon, stomach, and breast (11-14), and chronic inhibition of COX activity has been associated with chemopreventive effects on colon cancer (15). Consequently, the identification of the pathways and regulatory elements that lead to COX-2 expression are the subject of major interest.

An increase in the rate of COX-2 gene transcription is mediated by several cis-acting promoter elements that respond to multiple signal transduction pathways (8, 16-21). Platelet-derived growth factor, serum, and v-src promote COX-2 expression through Ras-mediated increases in c-Jun NH2-terminal kinase and extracellular signal-related kinase pathways in NIH 3T3 cells (22-24). Agonists that signal through cAMP induce COX-2 expression through the cAMP response element binding transcription factor (25). These pathways converge onto a common regulatory region, the ATF/cAMP response element located between -56 and -48 of the murine COX-2 promoter (22). Recent studies have shown that COX-2 expression also is induced by activation of the seven transmembrane domain receptor for bombesin (26), which interacts with both of the Gq and G12 families of heterotrimeric G-proteins (27). Given that protein kinase C activation induces COX-2 expression (28), it is likely that Galpha q, which couples to phospholipase C and thereby to the production of inositol 1,4,5-trisphosphate, which mobilizes Ca2+ from internal stores, and diacylglycerol, which activates protein kinase C (29), contributes to COX-2 transcriptional activation. In contrast, the role of Galpha 12/13 in COX-2 expression was unknown.

The ubiquitously expressed Galpha 12/13 (30) has been implicated in cell migration, proliferation, and transformation (31-33). Expression of the constitutively activated mutant of Galpha 13Q226L transforms NIH 3T3 cells and induces Egr-1 and c-Fos expression from serum response elements through the transcriptional activation of serum response factor (34). Expression of constitutively activated Galpha 12/13 also stimulates stress fiber formation, focal adhesion assembly, and cytochalasin-sensitive tyrosine phosphorylation of the nonreceptor tyrosine kinase p125FAK and the adapter proteins p130CAS and paxillin (35, 36). The small G-protein Rho plays a critical role in many biological responses induced by Galpha 12/13 (37, 38), and recent studies have shown that the Rho-activating guanine nucleotide exchange factor, pGEF115, is linked to Galpha 13 (39, 40), thus providing a defined signaling pathway from Galpha 13 to Rho.

Here, we examined whether the Galpha 13/Rho pathway leads to transcriptional activation of COX-2. Our results show that mutationally activated or aluminum fluoride-stimulated Galpha 13 induces activation of the COX-2 promoter through a Rho-dependent pathway. COX-2 expression by Galpha 13/Rho can be dissociated from stress fiber formation, focal adhesion assembly, and tyrosine phosphorylation of focal adhesion proteins, indicating a bifurcation of the signaling pathway used by Galpha 13/Rho to induce COX-2 expression from that used to induce actin cytoskeleton reorganization.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND CONCLUSIONS
REFERENCES

Cell Line and Expression Vectors-- NIH 3T3 cells were obtained from the American Type Tissue Collection (ATTC, Manassas, VA). All tissue culture reagents were purchased from Life Technologies, Inc. The reporter plasmid, pTIS-10s encodes the luciferase cDNA under the control of the murine COX-2 promoter (-371/+70). The murine Galpha 12 and Galpha 13 cDNAs including the constitutively active mutants Galpha 12Q229L and Galpha 13Q226L in pcDNA-1 (Invitrogen, San Diego, CA) were gifts from H. Bourne (University of California, San Francisco, CA). The murine Galpha q cDNA and mutant Galpha qQ209L in pcDNA-1 were obtained from ATTC. The rat gastrin- releasing peptide receptor expression vector, prGRPR, is described in Slice et al. (41). Expression vectors for Rho, RhoQ63L, Ras, dominant negative Ras, RasV12, Rac, dominant negative Rac, and RacV12 in pcDNA-3 were provided by D. Chang (University of California, Los Angeles, CA). The expression vector for v-src was provided by H. Herschman (University of California, Los Angeles, CA). pcDNA-3 was purchased from Invitrogen. R. Treisman (Imperial Cancer Research Fund, London) provided the plasmids pEF-LacZ and pEF-C3.

Cell Culture, Transfection, and Luciferase Assay-- NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 10 mM Hepes, glutamine, penicillin, and streptomycin. The cells were plated onto 60-mm dishes (Nunc, Napierville, IL) at a density of 105/plate. The next day, a transfection mixture was prepared by adding 1.5 µg of the reporter plasmid into 150 µl of Dulbecco's modified Eagle's medium with a combination of pcDNA-3 and the appropriate expression vector (totaling 1.0 µg). After a 20-min incubation after the addition of SuperFect (5 µl, Qiagen, Chatsworth, CA), the transfection mixture was combined with 1.0 ml of fresh medium and placed onto the cells in 60-mm plates. The cells were incubated for 2 days, and then the media was replaced with low serum media (Dulbecco's modified Eagle's medium with 0.1% calf serum and 0.1% bovine serum albumin). The next day, the cells were harvested with or without treatment with inhibitor (5 h), which in preliminary experiments was determined to be the optimal time for detection of changes in luciferase expression.

Cells were washed with phosphate-buffered saline and then lysed with luciferase assay buffer (400 µl, Promega, Madison, WI). The cell particulate was removed by brief centrifugation, and the protein concentration was measured using a protein assay Kit (Bio-Rad). Luciferase assays were performed using a Turner TD20/20 luminometer, with 90 µl of luciferase reaction mix (Promega, Madison, WI) mixed with 20 µl of protein extract. The relative light units (RLU) were normalized for the amount of protein in each extract, and the results were reported as relative changes in luciferase activity with a relative fold increase of 1.0, signifying no change. The data was reported as the mean value ± S.E. from two or more sets of transfections that were done in triplicate.

Agonists and Inhibitors-- Cytochalasin D, forskolin, and genistein were purchased from Calbiochem. AlCl3 and NaF were purchased from Sigma. Gastrin-releasing peptide was purchased from Bachem (Torrance, CA), and epidermal growth factor was purchased from Amersham Pharmacia Biotech.

Phalloidin Staining and Immunoblotting Assays-- NIH 3T3 cells were grown on poly-L-lysine-coated glass coverslips. The cells were incubated with and without cytochalasin D (2 µM, 30 min) before exposure to media containing 20% calf serum. After 10 min, the cells were fixed using 4% paraformaldehyde in phosphate-buffered saline at room temperature for 10 min. The cells were treated with rhodamine-phalloidin (Molecular Probes, Eugene, OR) according to manufacturer's protocol. The fluorescent image was acquired by a Spot-2 CCD camera attached to a Zeiss Axioskop microscope using a rhodamine filter set (Chroma #41002c).

NIH cells were serum-starved overnight before a 1-h incubation with or without genistein (50 µM). The cells were harvested 10 min after exposure to epidermal growth factor (50 ng/ml). Proteins were separated by size using SDS-polyacrylamide gel electrophoresis and then electrotransferred onto nitrocellulose. The immobilized proteins were screened using anti-phosphotyrosine antibodies (4G10, Upstate Biotechnology, Lake Placid, NY) and ECL (Amersham Pharmacia Biotech).

    RESULTS AND CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND CONCLUSIONS
REFERENCES

Galpha 13 Induces COX-2 Promoter Activity-- To determine whether Galpha 13 can regulate COX-2 expression, NIH 3T3 cells were co-transfected with a reporter plasmid in which the production of luciferase is driven by the COX-2 promoter (-371 to +70) and expression vectors encoding wild type Galpha 13 or constitutively activated Galpha 13(Galpha 13Q226L). As shown in Fig. 1A, expression of Galpha 13Q226L induced a marked concentration-dependent increase in COX-2 promoter activity. The maximal increase in relative luciferase activity was 5.8-fold in cells transfected with 0.25 µg of plasmid compared with control cells transfected with pcDNA-3. Expression of wild type Galpha 13 also induced higher levels of luciferase activity with a maximal relative increase of 2.9-fold that was achieved in cells transfected with 0.25 µg of plasmid. The relative high activity of the wild type Galpha 13 could be due to the low level of GTPase activity of this Galpha subunit (42, 43). The results presented in Fig. 1A show for the first time that Galpha 13 signaling leads to activation of the COX-2 promoter.


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  		Fig. 1.   Galpha 13 induces luciferase expression in transfected NIH 3T3 cells. A, cells were co-transfected with reporter (1.5 µg) and increasing amounts of expression vectors encoding wild type or Q226L Galpha 13 (QL mutant) with pcDNA-3 (total DNA was 2.5 µg in all transfections). After 2 days, the cells were serum-starved overnight before harvesting. B, cells were co-transfected with reporter vector, pcDNA-3, and either the expression vector (0.25 µg) encoding the wild type cDNA for alpha q or the constitutively active alpha qQ209L (total DNA was 2.5 µg for each transfection). The cells were serum-starved overnight before harvesting.

Signaling by Galpha q activates protein kinase C via phospholipase Cbeta , and it is known that direct protein kinase C stimulation by phorbol esters induces COX-2 expression in NIH 3T3 cells (26, 28). Consequently, we tested the effects of wild type and constitutively activated Galpha q on COX-2 promoter-mediated gene transcription. Using the same conditions (0.25 µg of expression vector) that were employed for maximal Galpha 13-induced transcriptional activation of COX-2, expression of Galpha qQ209L resulted in a 3.2-fold relative increase in luciferase activity (Fig. 1B).

To determine whether the effects of Galpha 13Q226L arose from long term activation of signaling pathways leading to secretion of autocrine factors or altered expression of Galpha 13 regulators, we examined Galpha 13 signaling in an acutely regulated system. Aluminum fluoride activates heterotrimeric G-proteins by its ability to mimic the gamma -phosphoryl group of GTP when associated with GDP-bound alpha -subunits (44, 45). We co-transfected NIH cells with wild type Galpha 13 and with the reporter plasmid, and after 72 h, the cells were exposed to 2.5 µM aluminum fluoride for 5 h (Fig. 2A). Treatment with aluminum fluoride significantly increased luciferase activity in cells overexpressing wild type Galpha 13. In contrast, aluminum fluoride induced luciferase activity only slightly in cells transfected with the COX-2 promoter reporter vector and a control vector (i.e. in the absence of Galpha 13 overexpression). Furthermore, conditioned media collected from cells overexpressing either Galpha 13 (wild type or QL mutant) or Galpha q (wild type or QL mutant) did not induce any significant increase in luciferase activity (Fig. 2B) when added to cells transfected with the COX-2 promoter reporter vector. The results shown in Fig. 2 indicate that the activation of the COX-2 promoter by the alpha  subunit of heterotrimeric G proteins is not caused by the release of autocrine factors during the course of the experiments.


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  		Fig. 2.   Direct induction of luciferase by Galpha 13 in transfected NIH 3T3 cells. A, cells were co-transfected with reporter vector and pcDNA-3 or the expression vector encoding Galpha 13 (0.125 µg). This lower amount of expression vector for Galpha 13 was chosen to decrease the basal level of luciferase activity. The cells were serum-starved overnight and then stimulated with AlF4- (2.5 µM, 5 h). The cells were washed and lysed, and luciferase activity measured. B, cells were transfected with reporter vector (1.5 µg), pcDNA-3 (control), and expression vectors (0.25 µg) encoding Galpha q (wild type and Q206L) or Galpha 13 (wild type and Q226L) (total DNA was 2.5 µg). The conditioned media was collected after 3 days. New NIH 3T3 cells were transfected with reporter vector and pcDNA-3. The cells were serum-starved overnight 2 days after transfection and then incubated with the conditioned media for 5 h.

Transcriptional Activation of the COX-2 Promoter by Galpha 13 Is Rho-dependent-- Recent evidence has shown that the small G-protein Rho mediates some of the biological effects induced by Galpha 13 (37, 38). To test whether activation of the COX-2 promoter involves a Rho-signaling pathway, cells were co-transfected with the COX-2 promoter and either pcDNA-3 (control) or expression vectors encoding wild type or constitutively active Rho (RhoQ63L). The results presented in Fig. 3A show for the first time that cells overexpressing RhoQ63L exhibit a 3-fold relative increase in luciferase activity compared with either control cells or cells overexpressing wild type Rho. The results indicate that Rho activation is a potential pathway leading to COX-2 transcriptional activation.


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  		Fig. 3.   Galpha 13 induces luciferase expression in transfected NIH 3T3 cells by a Rho-dependent pathway. A, cells were co-transfected with reporter vector (1.5 µg) and expression vectors (0.25 µg) encoding wild type Rho or constitutively active RhoQ63L (Rho QL). Control cells were co-transfected with pcDNA-3. The cells were serum-starved overnight before harvesting. B, NIH 3T3 cells were co-transfected with reporter vector (1.5 µg), pcDNA-3 (control), expression vectors encoding RhoQ63L (0.25 µg) or Galpha 13Q226L (0.125 µg), pEF-LacZ (0.25 µg, untreated), and pEF-C3 (0.25 µg, C3 toxin). The total amount of DNA in each transfection was 2.5 µg. The cells that were treated with forskolin were co-transfected with reporter vector and pcDNA-3. Cells were serum-starved overnight, and the appropriate cells were stimulated with forskolin (10 µM, 5 h). All the cells were harvested 72 h after transfection.

The Clostridium botulinum C3 toxin, which specifically ADP-ribosylates Rho at residue 41 and impairs its function (46), was used to test whether activation of the COX-2 promoter by Galpha 13 proceeds through a Rho-dependent pathway. Expression of C3 toxin blocked the increase in the level of luciferase activity produced in cells expressing either the RhoQ63L or Galpha 13Q226L (Fig. 3B). In contrast, C3 toxin did not prevent the increase in luciferase activity induced by treatment with forskolin, which stimulates COX-2 promoter activity via cAMP. These results indicate that Rho mediates COX-2 promoter activation by Galpha 13.

Transcriptional Activation of the COX-2 Promoter by Galpha 13 Is Ras- and Rac-Independent-- Previous studies have shown that the COX-2 promoter is induced by constitutively active Ras and Rac (22-24). Furthermore, it has been reported that Galpha 13Q226L requires functional Rac to stimulate Na+/H+ antiport activity (47). Therefore, we examined whether Ras or Rac are required for COX-2 promoter activation by Galpha 13 or Rho. Expression of Ras and Rac or dominant negative forms of Ras, RasDN, and Rac, RacDN did not affect the level of activation of the COX-2 promoter by Galpha 13Q226L (Fig. 4A). As a positive control, we confirmed that expression of RasDN and RacDN significantly reduced v-src-dependent activation of the COX-2 promoter (Fig. 4B). These results indicate that induction of the COX-2 promoter by Galpha 13Q226L is not dependent on Ras or Rac signaling pathways.


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  		Fig. 4.   Galpha 13 induces luciferase expression in transfected NIH 3T3 cells through Ras/Rac-independent pathways. A, cells were co-transfected with reporter vector (1.5 µg), expression vector (0.125 µg) encoding Galpha 13Q226L, and expression vectors (0.25 µg) encoding wild type (wt) Ras and Rac or dominant negative (DN) mutants of Ras and Rac. Control cells were co-transfected with pcDNA-3. B, cells were co-transfected with reporter vector (1.5 µg), pcDNA-3 (control), expression vectors encoding v-src (0.125 µg), or dominant negative mutants of Ras and Rac (0.25 µg). C, cells were co-transfected with reporter vector (1.5 µg), pcDNA-3 (control), expression vectors encoding v-src (0.125 µg), constitutively active mutants (V12) of Ras and Rac (0.25 µg), and C3 toxin (0.25 µg). D, cells were co-transfected with reporter vector (1.5 µg), expression vector (0.5 µg) encoding GRP receptor, pEF-C3 (0.25 µg), and pcDNA-3. All the cells were transfected with a total of 2.5 µg of DNA. All the cells were serum-starved overnight before harvesting 72 h after transfection.

We next wanted to determine the specificity of C3 toxin effects on small GTPase-dependent activation of the COX-2 promoter. We confirmed that in our experimental system, the constitutive active V12 forms of both Ras and Rac induced the COX-2 promoter (Fig. 4C). In contrast to RhoQ63L, expression of C3 toxin did not inhibit the relative luciferase induction by RasV12, RacV12, or v-src. This demonstrates selective inhibition of Rho by C3 toxin. There was a slight increase in the relative luciferase induction by RasV12, RacV12, v-src, and forskolin in cells expressing C3 toxin. This could be attributed to a lower basal level of luciferase activity in cells expressing C3 toxin. This supports an earlier proposal that NIH 3T3 cells have an elevated basal level of Rho activity (48).

Next, we determined whether agonist binding to the GRP receptor, which induces COX-2 mRNA and COX-2 (26), leads to activation of the COX-2 promoter via the Galpha 13/Rho pathway. NIH 3T3 cells were co-transfected with the reporter plasmid and an expression vector containing the GRP receptor cDNA. The cells were challenged with GRP for 5 h and then assayed for luciferase activity. GRP induced luciferase activity over 4-fold compared with unchallenged cells (Fig. 4D). Cells not transfected with the GRP receptor expression vector did not respond to GRP (data not shown). Expression of C3 toxin strikingly inhibited GRP-dependent induction of luciferase activity. These results indicate that GRP, through the GRP receptor, induces COX-2 promoter activity primarily through a Galpha 13/Rho-dependent pathway.

Dissociation of Rho-dependent Stress Fiber Formation, Tyrosine Phosphorylation, and COX-2 Transcriptional Activation-- Rho mediates multiple signaling pathways including gene expression, tyrosine phosphorylation, and stress fiber formation (49). Expression of either Galpha 13Q226L or RhoQ63L stimulates the formation of actin stress fibers and focal adhesion contacts leading to the tyrosine phosphorylation of the nonreceptor tyrosine kinase p125FAK and of the adapter proteins paxillin and p130CAS (35, 50-53). We examined if activation of the COX-2 promoter by Galpha 13 or Rho is dependent on actin cytoskeleton remodeling and/or tyrosine phosphorylation of focal adhesion proteins. Treatment of NIH 3T3 cells with cytochalasin D under conditions that disrupted actin stress fibers, as shown by phalloidin staining (Fig. 5C), did not prevent the activation of the COX-2 promoter by Galpha 13Q226L (Fig. 5A). To determine whether activation of the COX-2 promoter by Galpha 13 was through a tyrosine kinase-dependent pathway, cells were treated with increasing concentrations of the broad-spectrum tyrosine kinase inhibitor, genistein. Inhibition of tyrosine kinase activity did not prevent activation of the COX-2 promoter by Galpha 13Q226L (Fig. 5B). In fact, the relative luciferase activity levels increased in the cultures exposed to higher doses of genistein (50 µM). This high dose of genistein inhibited epidermal growth factor-stimulated tyrosine phosphorylation (Fig. 5D). The results obtained with both genistein and cytochalasin D suggest that activation of the COX-2 promoter by Galpha 13 is not dependent on tyrosine phosphorylation of focal adhesion proteins.


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  		Fig. 5.   Galpha 13 induction of luciferase is not inhibited by cytochalasin D or genistein. A, NIH 3T3 cells were co-transfected with reporter vector (1.5 µg) and either the expression vector encoding Galpha 13(Q226L (QL)) (0.125 µg) or pcDNA-3 (control) (total DNA was 2.5 µg). The cells were serum-starved overnight and then incubated for 5 h with cytochalasin D (2.0 µM). The cells were washed and lysed, and the luciferase activity was measured. B, NIH 3T3 cells were co-transfected with reporter vector (1.5 µg), and either the expression vector encoding Galpha 13(Q226L) (0.125 µg) or pcDNA-3 (control) (total DNA was 2.5 µg). The cells were serum-starved overnight and then treated with increasing concentrations of genistein for 5 h before harvesting. C, NIH 3T3 cells were grown on coverslips and serum-starved overnight. The appropriate cells were treated with cytochalasin D (2 µM) 30 min before induction with media containing 20% calf serum. After 10 min, the cells were fixed and stained with rhodamine-phalloidin. D, serum-starved NIH 3T3 cells were incubated with and without genistein (50 µM) for 1 h. The indicated cells were treated with epidermal growth factor (EGF, 50 ng/ml, 10 min). A Western blot was done on the cellular proteins using an anti-phosphotyrosine antibody.

Concluding Remarks-- COX-2 is a prostaglandin endoperoxide synthase that is dramatically induced by multiple extracellular stimuli and oncogene products (7-10). Given the pivotal role of inducible prostaglandin production in many normal and abnormal processes, the transcriptional regulation of COX-2 expression is the subject of intense interest. Our results demonstrate for the first time that mutationally activated or aluminum fluoride-stimulated Galpha 13 induces the activation of the COX-2 promoter. These findings identify a novel regulatory mechanism leading to COX-2 promoter activation and raise the possibility that COX-2 induction is a downstream event in Galpha 13 signaling.

Activated Galpha 13 induces a variety of biological responses including stimulation of Na+/H+ antiport activity, stress fiber and focal adhesion formation, and tyrosine phosphorylation of focal adhesion proteins through Rho (35, 47, 50-53). This is consistent with the recent observation that expression of constitutively active Galpha 13 increases the level of active Rho (Rho-GTP) (54). Here, we report for the first time that expression of constitutively activated Rho induces activation of the COX-2 promoter. Furthermore, our results also show that inactivation of Rho by C. botulinum C3 toxin selectively blocks induction of COX-2 promoter activity by Galpha 13, Rho, and by the GRP receptor. Neither Ras or Rac is downstream of Galpha 13 nor is Rho downstream of Ras or Rac in the pathway(s) leading to COX-2 promoter activation. In addition, signaling to the COX-2 promoter by Galpha 13/Rho can be dissociated from both cytoskeletal reorganization and tyrosine phosphorylation pathways. Taken together, the findings presented here indicate that the Galpha 13-Rho pathway provides a novel mechanism leading to COX-2 promoter activation.

Increasing evidence indicates that overexpression of COX-2 contributes to the development of a variety of human cancers (1). It is also known that constitutively activated Galpha 13 can act as a potent oncogene (31-33) and that Rho function is necessary for transformation (55). In addition, constitutive or autocrine stimulation of heptahelical receptors that couple to Galpha 13 and Rho are also implicated in tumorogenesis (56, 57). Our results identifying a novel link between Galpha 13, Rho, and COX-2 suggest that this pathway may contribute to tumor development in at least some cell types, a proposition that warrants further experimental work.

    ACKNOWLEDGEMENTS

We thank Dr. Henry R. Bourne for the kind gifts of the expression constructs for G12-Q229L and G13-Q226L, Dr. David Chang for the Rho, Rac, and Ras expression vectors, and Dr. Richard Treisman for the kind gifts of the expression constructs for C3 and beta -galactosidase.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK35740, DK17294 (to J. H. W.), and DK55003 (to E. R.) and Veterans Administration Merit Review and CURE Center grants.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Please address correspondence to: Dept. of Medicine, UCLA School of Medicine, Warren Hall 11-151, 900 Veteran Ave., Los Angeles, CA 90095-1786. Tel.: 310-206-0909; Fax: 310-794-5332; E-mail: lslice@med1.medsch.ucla.edu.

    ABBREVIATIONS

The abbreviations used are: COX, cyclooxygenase; G-protein, heterotrimeric guanine nucleotide-binding protein; GRP, gastrin-releasing peptide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND CONCLUSIONS
REFERENCES

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