Inhibition of Thyrotropin-Stimulated DNA Synthesis by - Molecular and ...

MOLECULAR

AND

CELLULAR BIOLOGY, Aug. 1993,

p.

Vol. 13, No. 8

4477-4484

0270-7306/93/084477-08$02.00/0 Copyright X 1993, American Society for Microbiology

Inhibition of Thyrotropin-Stimulated DNA Synthesis by Microinjection of Inhibitors of Cellular Ras and Cyclic AMP-Dependent Protein Kinase ERIK KUPPERMAN,' WEI WEN,2 AND JUDY L. MEINKOTHl 3* Departments of Medicine' and Chemistry2 and Cancer Center,3 University of California at San Diego, La Jolla, California 92093 Received 16 November 1992/Returned for modification 16 January 1993/Accepted 1 May 1993

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSHJ)-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMPdependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSHstimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase. model system for studying Ras involvement in G.-coupled mitogenic pathways. TSH stimulates DNA synthesis in the absence of other growth factors and serum (30). The TSH receptor is a seven-transmembrane-spanning receptor which couples to G. (11). Injection of a highly specific antibody raised against the carboxy terminus of Ga. abolished TSHstimulated morphological changes and changes in gene expression and DNA synthesis in WRT cells (30). TSH treatment activates adenylyl cyclase activity, resulting in increased levels of intracellular cyclic AMP (cAMP) and activation of the cAMP-dependent protein kinase (cAPK). This pathway is thought to be the primary signaling pathway induced by TSH in most thyroid cells. Thyroid follicular cells also respond to alterations in Ras activity. Several investigators have reported high frequencies of ras mutations in human follicular neoplasms (38). The high frequency of ras point mutations in both benign and malignant tumors suggests that ras activation is an early event in thyroid tumorigenesis (32). Consistent with a putative role in growth control, overexpression of H-Ras results in the partial transformation of human follicular cells (24), and microinjection of oncogenic H-Ras protein stimulates DNA synthesis in WRT cells (29). To assess the role of cellular Ras protein in TSH-initiated signal transduction, a dominant interfering Ras protein (N17 Ras) was injected into living cells. N17 Ras contains a serine-to-asparagine substitution at position 17 (15), and recent evidence suggests that N17 Ras exerts its inhibitory effect through competition with cellular Ras for a guanine nucleotide-releasing factor (13). Without access to guanine nucleotide-releasing factors, cellular Ras cannot exchange bound GDP for GTP and remains inactive. Microinjection of N17 Ras caused a significant reduction in TSH-induced DNA synthesis. Inhibition of cAPK through microinjection of the heat-stable protein kinase inhibitor (PKI) also decreased TSH-stimulated DNA synthesis. When N17 Ras and PKI were coinjected, TSH-stimulated DNA synthesis was almost abolished. The results of these experiments demonstrate that both Ras and cAPK function in growth pathways

The ras gene family is one member of a nucleotide-binding superfamily which participates in a diverse range of cellular activities, including growth, differentiation, and protein trafficking. Experimental evidence has shown that Ras is involved in growth factor-stimulated proliferation in many cell types. Sequencing and crystallographic data revealed that Ras shares structural homology with heterotrimeric G proteins. Ras and heterotrimeric G proteins are also functionally related; both contain intrinsic GTPase activity and alternate between active and inactive states, depending on whether GTP or GDP is bound. Oncogenic forms of the Ras protein are generated by mutations which reduce the protein's normal GTPase activity or eliminate the enhancement of its GTPase activity by GTPase-activating protein (GAP) (43). One result of these mutations is a constitutively active signaling molecule. Oncogenic Ras stimulates proliferation in fibroblasts and differentiation in PC12 cells (4, 14, 35). Conversely, disruption of Ras function with anti-Ras antibodies or a dominant interfering mutant of Ras (N17 Ras) blocks mitogenic responses to platelet-derived growth factor, epidermal growth factor, insulin, and serum in fibroblasts (6, 7, 31) and neuronal differentiation induced by nerve growth factor in PC12 cells (39). One common feature of Ras-regulated signaling has been its role in downstream events initiated by receptor tyrosine kinases. Although many growth factors stimulate proliferation through receptors containing (or associated with) tyrosine kinase activity, there are a large number of mitogenic peptides and hormones which stimulate proliferation through G protein-coupled receptors (10, 17, 25, 28) (for a review, see reference 5). To assess whether Ras is a component of signaling pathways emanating from G protein-coupled receptors, the role of cellular Ras in thyrotropin (thyroid-stimulating hormone [TSH])-induced DNA synthesis in cultured thyroid cells was examined. Wistar rat thyroid (WRT) follicular cells provide a useful

*

Corresponding author. 4477

4478

KUPPERMAN ET AL.

stimulated by TSH. Furthermore, these findings suggest that Ras may have a generalized role in growth factor signal transduction pathways, including those mediated by Got subunits. MATERIALS AND METHODS Reagents. Two types of bovine TSH (bTSH) were used in these experiments: crude bTSH (1 IU/mg) from Sigma and purified bTSH (30 IU/mg) kindly provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. Cells and cell culture. WRT cells were kindly provided by Roberto Toccafondi (Florence, Italy). The methods used to culture WRT cells have been described elsewhere (30). Briefly, cells were cultured in a six-hormone-containing medium (6H) consisting of Coon's modified Ham's F12 medium supplemented with TSH, insulin, somatostatin, glycyl-L-histidine acetate, hydrocortisone, transferrin (3), 100 U of penicillin, and 100 ,ug of streptomycin sulfate per ml. WRT CRE (30) cells were grown in the same medium further supplemented with 150 ,ug of Geneticin (G418) per ml. For starvation, subconfluent cells were incubated in basal medium (Coon's modified Ham's F12 medium containing 0.3% bovine serum albumin [BSA]) for at least 24 h prior to injection or hormone treatment. All cells were grown at 37°C in an atmosphere of 5% CO2. Measurements of DNA synthesis. DNA synthesis was assessed through incorporation of the thymidine analog bromodeoxyuridine (BrdU) and its subsequent detection by indirect immunofluorescence (23, 30). Following incubation in medium containing BrdU, cells were fixed in 95% ethanol-5% acetic acid for 30 min at room temperature. Prior to antibody staining, cells were permeabilized in phosphatebuffered saline (PBS) containing 0.1% Tween for 2 min at room temperature. BrdU incorporation was detected by staining with a mouse monoclonal anti-BrdU antibody (Amersham) followed by a biotinylated anti-mouse antibody (Vector Laboratories) and Texas red-coupled streptavidin (Amersham). Injected cells were detected with a fluorescein isothiocyanate (FITC)-coupled anti-rabbit antibody (Jackson Laboratories). The biotinylated and FITC antibodies were diluted (1:400 and 1:200, respectively) in PBS containing homologous cell extract and 0.1 mg of BSA per ml. Texas red-coupled streptavidin was diluted 1:200 in PBS. Detection of ,8-Gal activity. WRT CRE cells were starved in basal medium for at least 24 h prior to microinjection or TSH stimulation. For TSH stimulation, the cells were induced with basal medium containing crude bTSH at 10 mU/ml for 5 to 6 h. Following stimulation, the cells were fixed in PBS containing 3.7% formaldehyde for 5 min at room temperature and subsequently stained in 5 mM K3Fe(CN)6-5 mM K4Fe(CN)6. 3Hj0-2 mM MgCl2-1 mg of

5-chloro-4-bromo-3-indolyl-1-D-galactopyranoside (X-Gal)

per ml in PBS for 16 h at 37°C. Cells which express 3-Gal contain a dark blue precipitate and can be readily distinguished from nonexpressing colorless cells. Injected cells were detected after X-Gal staining by permeabilizing the cells as described above and staining them with an FITCcoupled anti-rabbit antibody (Jackson Laboratories). Microinjection. All microinjection experiments were performed by using an automatic micromanipulator (Eppendorf, Fremont, Calif.). Microinjection needles were pulled on a vertical pipette puller (Kopf, Tujunga, Calif.). Cells were plated on glass coverslips, grown to 60 to 80% confluency, and placed in basal medium for at least 24 h prior to

MOL. CELL. BIOL.

injection. Cells were injected into the cytoplasm as described previously (30). Affinity-purified rabbit immunoglobulin G (IgG) was coinjected (at a final concentration of 4 to 5 mg/ml) in all experiments in order to unambiguously identify the injected cells. Cellular Ras and N17 Ras proteins (injected at a final concentration of 3.2 mg/ml) and T24 Ras protein (injected at 0.5 mg/ml) were injected in 20 mM Tris (pH 7.4)-2 mM MgCl2-0.1 mM EDTA-1 mM ,3-mercaptoethanol-20 mM NaCl. Cellular Ras, T24 Ras, and N17 Ras were purified from Escherichia coli as previously described (16) and were >95% pure as estimated by polyacrylamide gel electrophoresis analysis. Recombinant PKI was purified as described previously (41) and quantitated by titration against the cAPK catalytic subunit. RESULTS Microinjection of cellular Ras protein stimulates DNA synthesis in WRT cells. Since Ras gene activation occurs at high frequency in thyroid follicular cells (38), the ability of overexpressed cellular Ras protein to stimulate DNA synthesis was examined. Quiescent WRT cells were injected with cellular Ras protein (3.2 mg/ml) and maintained in basal medium containing BrdU for 48 h. This time was chosen since previous experiments demonstrated that DNA synthesis occurs between 30 and 48 h following hormone treatment. After 48 h, the cells were fixed and stained for the presence of both BrdU and IgG coinjected with the Ras protein. Microinjection of cellular Ras protein stimulated DNA synthesis in the absence of all other growth factors and serum, as shown by the brightly stained nuclei in Fig. lb. Approximately 37% of the cells injected with cellular Ras protein synthesized DNA, while only 6.5% of the cells maintained in basal medium underwent DNA replication (Fig. lc). As previously reported (29), similar results were obtained with mutationally activated T24 Ras protein (Fig. lc). Control injections of an affinity-purified rabbit IgG had no effect on DNA synthesis (0% labeled nuclei, n = 40). Furthermore, injection of N17 Ras protein, which differs from cellular Ras by a single amino acid substitution, did not stimulate DNA synthesis (Table 1), demonstrating that the effects of cellular and T24 Ras were specific for Ras proteins active in growth signaling. Microinjection of dominant interfering N17 Ras protein reduces TSH-stimulated DNA synthesis. To determine whether endogenous Ras protein is required for TSH-stimulated DNA synthesis, dominant interfering N17 Ras protein was injected into quiescent cells, which were then stimulated with TSH for 48 h. Injection of N17 Ras consistently reduced both TSH (38 to 52%)- and 6H (33%)-stimulated DNA synthesis. In contrast, injection of rabbit IgG had no inhibitory effect on DNA synthesis. Examples of injected cells are shown in Fig. 2a and b, and the results are summarized in Table 1. If quiescent cells were stimulated with TSH for 19 to 25 h prior to injection of N17 Ras, no significant reduction in DNA synthesis was observed. These results indicate that N17 Ras must be present during the time in which the cells become committed to undergo DNA synthesis in order to exert an inhibitory effect. Neither cellular nor T24 Ras protein exerted an inhibitory effect on TSH-stimulated DNA synthesis (data not shown), although both proteins are purified like N17 Ras. These results indicate that the inhibition observed following N17 Ras injection is not a nonspecific effect due to a contaminant in the protein preparation or a nonspecific inhibition of BrdU incorporation. Several experiments were performed to determine

VOL. 13, 1993

TSH-STIMULATED DNA SYNTHESIS REQUIRES Ras AND cAPK

4479

% DNA synthiesis

C 100

90 s0 70 60

40 30

10 0

i wt ras

T24

basal

FIG. 1. Induction of DNA synthesis in WRT cells by microinjection of cellular or T24 Ras. WRT cells were plated on glass coverslips and starved in basal medium 24 h prior to injection. T24 Ras was injected at 0.5 mg/ml, and cellular Ras was injected at 3.2 mg/ml. Forty-eight hours after injection, cells were fixed and stained as described in Materials and Methods. (a and b) Representative examples of injected cells. (a) Fluorescent photomicrograph depicting injected cells; (b) the same field of cells stained for BrdU incorporation. (c) Quantitation of DNA synthesis induced by cellular (wild-type [wt]) or T24 Ras. For cellular Ras protein, the results from three independent experiments (330 total injected cells) are summarized. For T24 Ras protein, the results represent two independent experiments (87 total injected cells). Over 1,300 cells were counted for the basal control, which represents uninjected cells maintained in basal medium.

whether N17 Ras was maximally effective in inhibiting TSH-stimulated DNA synthesis under the conditions used. First, the time between injection and TSH stimulation was increased from 30 min to 5 h to allow more time for the proper cellular localization of the injected Ras protein. With both times, DNA synthesis was reduced by 52% (204 and 923 injected cells were examined for the 30-min and 5-h time points, respectively). Dose-response experiments indicated that similar reductions were observed in response to injection of N17 Ras at 3.2 (used above) and 2.2 mg/ml, but less inhibition was observed at 1.5 mg/ml, consistent with maximal inhibition occurring at concentrations of N17 Ras greater than 2.2 mg/ml. Finally, the ability of N17 Ras to abolish cellular Ras-stimulated DNA synthesis was examined. Coinjection of cellular Ras (2 mg/ml) with N17 Ras (4 mg/ml) reduced Ras-mediated DNA synthesis by >80% (la). Since the concentration of injected Ras should be in excess over that of cellular Ras, the amount of injected N17 Ras should have been sufficient to abolish TSH-induced DNA synthesis if all of it proceeded through Ras. Inhibition of both cellular Ras and cAPK abolishes TSHstimulated DNA synthesis. Since TSH elevates intracellular cAMP levels, experiments were performed to assess the effect of inhibitors of cAPK on TSH-stimulated DNA synthesis. PKI is a highly specific and potent inhibitor of the cAPK catalytic subunit (for a recent review, see reference 47). Microinjection of PKI into quiescent cells stably trans-

fected with a cAMP response element (CRE)-regulated lacZ (30) abolished both TSH- and 8-bromo-cAMP (8Brc AMP)-stimulated changes in gene expression (Table 2). In contrast, PKI only partially reduced DNA synthesis in response to either agent. These results suggest either that PKI is insufficiently stable to abolish DNA synthesis or that different thresholds are required for the inhibition of gene expression and DNA synthesis. However, the reduction in TSH-stimulated DNA synthesis by PKI indicates that, as expected, at least part of the mitogenic action of TSH is transmitted tlirough cAPK. Since TSH-stimulated DNA synthesis also requires cellular Ras, the effect of coinjection of PKI and N17 Ras was examined. Quiescent WRT cells were injected with PKI, N17 Ras, or both inhibitors together and stimulated with TSH. As seen before, injection of N17 Ras or PKI resulted in partial reductions in DNA synthesis (Tables 1 and 3). In contrast, coinjection of the two proteins resulted in a much greater reduction in DNA synthesis (compare Fig. 2e and f with Fig. 2a and b or Fig. 2c and d). In over 800 injected cells, coinjection of both inhibitors resulted in levels of DNA synthesis similar to those observed in cells maintained in basal medium (summarized in Table 3). These results suggest that both cellular Ras and cAPK are required for maximal levels of TSH-stimulated DNA synthegene

SIS.

cAPK is not required for Ras-stimulated DNA synthesis. To and cAPK were components of the same

assess whether Ras

TABLE 1. N17 Ras injections into WRT cells Injected

Treatment

No. of injected cells analyzed

% BrdU incorporated in injected cells

No. of uninjected cells analyzed

% BrdU incorporated in uninjected cells

% Reduction

1 nM TSHb 10 nM TSHb 10 mU of TSHd/ml

529 1,127 800 565 474 168

16 (13-19)C 26 (23-29) 23 (20-26) 58 (54-62) 67 (62-70) 3 (0-6)

633 3,078 1,390 602 788 525

26 (23-29) 54 (52-56) 45 (42-48) 87 (84-90) 65 (62-68) 3 (2-4)

38 52 49 33 0 0

material

N17 Rasa

6He Rabbit IgGf N17 Ras

10 nM TSH None

a Injected at 3.2 mg/ml. Purified bTSH (30 IU/mg). c The 95% confidence interval, given in parentheses, was calculated by using the standard d Crude bTSH (1 IU/mg). ' Fully supplemented growth medium containing crude TSH. f Injected at 4 to 5 mg/ml. b

error of proportion.

4480

KUPPERMAN ET AL.

MOL. CELL. BIOL.

FIG. 2. Inhibition of DNA synthesis by microinjection of N17 Ras, PKI, or both inhibitors. WRT cells were prepared for injection as described in the legend to Fig. 1. (a and b) Cells injected with N17 Ras (3.2 mg/ml); (c and d) cells injected with PKI (1.9 mg/ml); (e and f) cells injected with both inhibitors together at the same concentration of either inhibitor alone. In all cases, quiescent cells were injected and subsequently stimulated with TSH (10 mU/ml) for 48 h. Panels a, c, and e are fluorescent micrographs depicting injected cells; panels b, d, and f are the same fields of cells stained for BrdU incorporation.

or different signaling pathways, Ras-induced DNA synthesis was measured in the presence or absence of PKI. Coinjec-

tion of PKI had no significant effect on cellular Ras-stimulated DNA synthesis (Table 4). The injected PKI was biologically active, as it reduced TSH-stimulated DNA synthesis by 51% in the same experiment. These results suggest that cAPK activity is not required for the stimulation of DNA synthesis by cellular Ras and suggest that Ras and

cAPK signaling pathways may be distinct, at least in the early cytoplasmic steps of signal transduction. Alterations in Ras activity have no effect on CRE-regulated gene expression. To assess whether Ras signaling pathways converged on CRE-regulated gene expression, quiescent WRT CRE cells were injected with N17 Ras and stimulated with TSH. As reported previously (30), TSH treatment stimulated high levels of CRE-regulated gene expression


VOL. 13, 1993

4481

TABLE 2. Effects of PKI on TSH- and 8BrcAMP-stimulated gene expressiona and DNA synthesisb No. of

Injected

Treatment

material

PKIc Rabbit IgGf PKI Rabbit IgG

cells injected analyzed

10 mU of TSHd/ml 1 mM 8BrcAMP 10 mU of TSH/ml 1 mM 8BrcAMP 10 mU of TSH/ml 1 mM 8BrcAMP 10 mU of TSHIml 1 mM 8BrcAMP

% 13-Gal

expression in

UIJ~L~Li ijcecls

536 502 596 472

6

No. of % 13-Gal % BrdU expression in incorporated in uninjected cells uninjected

injected cells inctdcells

(4-8)e

12 (9-15) 53 (49-57) 78 (74-82)

1,572 938

30 (28-32) 30 (27-33) 78 (75-81) 48 (43-53)

647 377

% BrdU rated in unecdcls uinjcted cls

Reduction

analyzed

cells

1,333

57 (54-60)

89

1,400 1,451 1,237 2,422 1,135 695 484

84 (82-86) 60 (57-63) 83 (81-85)

86 12 6 52 40 0 0

63 50 73 48

(61-65) (47-53) (70-76) (44-52)

a Assessed in WRT CRE cells. b Assessed in WRT cells. c Injected at 1.9 mg/ml. d Crude bTSH (1 IU/mg). The 95% confidence interval, given in parentheses, was calculated by using the standard error of proportion. f Injected at 4 to 5 mg/ml.

(Table 2). A reduction in ,B-Gal expression was observed following injection of N17 Ras, but this was quantitatively similar to the effects observed following control injections with rabbit IgG and is consistently observed in the WRT CRE cell line (Table 5). In related experiments, cellular Ras protein was injected into quiescent WRT CRE cells to determine whether overexpression of Ras would stimulate CRE-regulated gene expression. Injection of cellular Ras protein at the same concentration which stimulated DNA synthesis in WRT cells did not stimulate CRE-dependent 1-Gal expression (Table 6). Similar results were obtained following injection of T24 Ras protein (data not shown). Together, these experiments suggest that cellular Ras protein influences TSH-stimulated DNA synthesis but does not affect CRE-containing promoters. DISCUSSION The frequent mutational activation of the Ras gene in human tumors suggests that Ras functions in pathways regulating cellular proliferation. Experimental evidence has revealed that Ras functions in both growth and differentiative signaling pathways in a wide variety of cell types. Ras appears to serve as one of the downstream effectors of tyrosine kinase-containing receptors, including those for platelet-derived growth factor, epidermal growth factor, insulin, and nerve growth factor (6, 7, 31, 39). Similarly, Ras functions in signal transmission where cell surface receptors lack endogenous tyrosine kinase activity but associate with cytoplasmic tyrosine kinases (8). A number of peptides, including thrombin, bombesin, and bradykinin, stimulate

proliferation in fibroblasts through seven-transmembranespanning receptors which couple through G proteins (17, 34, 46). Many hormones, such as TSH, follicle-stimulating hormone, luteinizing hormone, and parathyroid hormone, stimulate both differentiated functions and proliferation through G protein-coupled receptors in their respective cell types (1, 9, 30, 33). The possibility that Ras function is required for growth stimulation from G protein-coupled receptors was tested. In thyroid follicular cells, TSH stimulates proliferation through a seven-transmembrane-spanning receptor coupled to Gs. In WRT cells, virtually all of the effects of TSH were abolished following microinjection of an antibody to Gas (30), suggesting that all of TSH signaling proceeds through Gs in this cell type. Unlike the case for human or rat FRTL-5 thyroid cells (12), TSH does not increase intracellular calcium levels in WRT cells (28a), suggesting that the TSH receptor does not activate a second G protein coupled to phosphoinositide turnover in this cell type. At least part of the mitogenic action of TSH is mediated through cAMP, since elevations in intracellular cAMP are mitogenic in thyroid cells (reviewed in reference 12). As expected, microinjection of PKI reduced TSH-stimulated DNA synthesis (Tables 2 and 3). However, since PKI only partially inhibited DNA synthesis in response to 8BrcAMP, it was not possible to determine whether all or only part of TSH action utilized cAPK. Interestingly, microinjection of N17 Ras protein reduced TSH-stimulated DNA synthesis (Table 1), suggesting that both cAPK and cellular Ras are required for the full mitogenic action of TSH. Consistent with this idea, coinjec-

TABLE 3. PKI and N17 Ras injections into WRT cells Injected material

Treatment


% BrdU incorporated in injected cells

pKIa N17 Rasd PKI + N17 Ras None

10 mU of TSHb/ml 10 mU of TSH/ml 10 mU of TSH/ml None

193 148 816 NAC

24 (18-30)C 14 (9-19) 7 (5-9) NA


331 261

1,402 3,228

a Injected at 1.9 mg/ml. b Crude bTSH (1 IU/mg). c The 95% confidence interval, given in parentheses, was calculated by using the standard error of proportion. d Injected at 3.2 mg/ml. I NA, not applicable.


48 40 48 7

(43-53) (34-46) (46-50) (6-8)

% Reduction

50 65 85 NA

4482

MOL. CELL. BIOL.

KUPPERMAN ET AL.

TABLE 4. Cellular Ras and PKI injections into WRT cells Injected material

Treatment

No. of injected

cells analyzed

in injected cells



Cellular Rasa Cellular Ras + PKIb PKI

None None 1 nM TSHC

43 572 107

33 30 20

213 963 231

14 11.5 41

% BrdU

incorporated

%

Stimulation

58 62 od

a Injected at 3.2 mg/ml. b Injected at 1.9 mg/ml. Purified bTSH (30 IU/mg). d There was a 51% reduction in BrdU incorporated in injected cells.

tion of PKI and N17 Ras reduced TSH-stimulated DNA synthesis to near background levels (Table 3). A similar requirement for Ras has been found in phenylephrine-induced hypertrophy in neonatal cardiocytes (42) and in thrombin-stimulated DNA synthesis in human astrocytes (22a). All of these systems utilize Ga subunits coupled to receptors of the seven-transmembrane type. Taken together, these results suggest that Ras function may be crucial for many G protein-coupled signaling pathways in addition to its established role in tyrosine kinase-mediated pathways. It will be important to assess how Ras is coupled to these serpentine receptors and whether heterotrimeric G proteins are distal, proximal, or totally distinct from Ras. The relationship of Ras to the other components of the TSH signal transduction cascade remains to be defined. Ras may lie distal to the TSH receptor or in a parallel and independent pathway which is somehow activated by TSH stimulation. Under the conditions used in these experiments, basal levels of DNA synthesis were quite low. Thus, WRT cells do not contain a constitutively activated growth signaling pathway. Furthermore, since N17 Ras is much less effective as an inhibitor of activated Ras (36), it is unlikely that WRT cells contain an activating Ras mutation. The abolition of TSH-stimulated DNA synthesis following injection of a specific Gas antibody (30) or coinjection of PKI and N17 Ras is consistent with a model in which Ras is distal to Gs. However, it remains formally possible that Ras is activated through another G protein or some other putative adaptor molecule. It seems unlikely that both Ras and cAPK are components of one linear pathway distal to the TSH receptor. Unlike TSH (Table 2) or purified cAPK catalytic subunit (not shown), microinjection of cellular (Table 6) or activated Ras failed to stimulate the expression of a CREregulated marker gene. Similarly, microinjection of N17 Ras did not reduce TSH-stimulated marker gene expression (Table 5). It should be noted that a number of cAMPinducible genes lack obvious consensus CREs in their promoters (2). For example, thyroglobulin gene expression is stimulated by TSH through an unknown sequence motif not likely to be a CRE (22). Also, activation of the cAPK pathway stimulates other promoters, for example, those

containing AP-2 elements (21), and some of these promoters may be targets for Ras. However, Ras-stimulated DNA synthesis was insensitive to PKI (Table 4), suggesting that cAPK is not required for Ras-mediated DNA synthesis. Although Ras and cAPK-mediated signaling pathways appear to be distinct, these pathways may interact. In a small number of cells, microinjection of N17 Ras prevented 8BrcAMP-stimulated DNA synthesis (not shown). Similar results were obtained with a mutationally altered cAPK catalytic subunit, which stimulates DNA synthesis in WRT cells (47a). In Saccharomyces cerevisiae, both Ras and cAPK are components of nutrient-stimulated growth pathways. Therefore, Ras expression may also affect cAPKmediated signaling pathways in mammalian cells which are mitogenically responsive to cAMP. If Ras is one of the components of G protein-coupled signaling pathways, several predictions can be made. First, regulators of Ras function should also affect signal transduction from these receptors. Two predominant classes of Ras regulators have been identified, nucleotide exchange factors and GAPs. Guanine nucleotide exchange factors (49) stimulate GDP-to-GTP exchange on Ras and activate Ras as a signaling molecule. Thrombin stimulation results in increased levels of GTP-bound Ras (22a, 45). GAP or NF-1 (27, 43, 50) accelerates GTP hydrolysis and converts Ras to an inactive form, although GAP may also function as an effector of Ras action (51). Consistent with the effects of N17, microinjection of type I GAP reduced TSH-stimulated DNA synthesis (la). Second, some mechanism must exist for signal transmission from G protein-coupled receptors to Ras regulators. For receptor tyrosine kinases, tyrosine phosphorylation acts to recruit a variety of SH2 and SH3 domaincontaining proteins to the receptor, which act as downstream effectors in signal transduction. Interestingly, activation of G protein-coupled receptors is also associated with increased tyrosine phosphorylation (18, 40), and Gas subunits are substrates for tyrosine phosphorylation by pp6Oc-s?

(19).

Since WRT cells are mitogenically responsive to both Ras and cAMP, this cell type provides a model system for understanding how these signaling pathways may interact.

TABLE 5. N17 Ras injections into WRT CRE cells Injected

Treatment


% 3-Gal expression in injected cells


% 3-Gal expression in uninjected cells

% Reduction

N17 Rasa Rabbit IgGd

10 mU of TSHb 10 mU of TSH

999 205

51 (48-54)C 47 (40-54)

2,168 434

71 (69-73) 60 (55-65)

28 22

material

a Injected at 3.2 mg/ml. b Crude bTSH (1 IU/mg). c The 95% confidence interval, given in parentheses, was calculated by using the standard d Injected at 4 to 5 mg/ml.

error of proportion.

VOL. 13, 1993


TABLE 6. Cellular Ras injections into WRT CRE cells Injected material

Cellular Rasa Rabbit IgGc

No. of % 1-Gal No. of % 13-Gal injected expression uninjected expression in % cells in injected cells uninjected Stimulation cells analyzed analyzed cells

337 188

0 0

769 363

1 (0-_)b

1 (0-1.7)

0 0

a Injected at 3.2 mg/ml. Values in parentheses are 95% confidence limits. I Injected at 4-5 mg/ml.

b

Mutations in both Ras and Gs have been identified in thyroid While mutations in either molecule alone appear restricted to benign neoplastic growth (32, 37), mutations in both might be sufficient to induce malignant transformation. Chronic TSH stimulation results in hyperthyroidism in humans, which can be associated with an increased incidence of cancer. In animal models, chronic TSH stimulation results in carcinogenesis (20). Since TSHinduced signaling appears to be regulated by both Ras and Gs, it will be important to determine whether activating mutations in both Ras and G5 (or cAPK) are sufficient to induce transformation. The insulin-like growth factors IGF-1 and IGF-2 are also potent mitogens for thyroid cells, and overexpression of IGF-1 or its receptor occurs during neoplastic conversion (48). In cultured cells, TSH and IGF-1 together exert synergistic effects upon DNA synthesis (44). It will be interesting to assess whether the synergism between TSH and IGF-1 involves pathways regulated by Ras, cAMP, or both molecules. tumors (26, 37).

ACKNOWLEDGMENTS We thank Susan Taylor for kindly providing PKI, Gerard Burrow for support and encouragement, and Joe Dela Cruz for expert technical assistance. We thank Jim Feramisco and Andrew Thorburn for critical reviews of the manuscript. This research was supported by funds provided to J.L.M. by the Cigarette and Tobacco Surtax Fund of the State of California through the Tobacco-Related Disease Research Program of the University of California, grant 2KT0030. This material is based on work supported under a National Science Foundation graduate research fellowship awarded to E.K., a member of the Biomedical Sciences program. W.W. is a member of the Chemistry graduate program.

REFERENCES 1. Abney, T., and L. Carswall. 1986. Regulation of Leydig cell DNA synthesis. Mol. Cell. Endocrinol. 45:157-165. la.AI-Alawi, N. Unpublished data. 2. Albanese, C., T. Kay, N. Troccoli, and J. Jameson. 1991. Novel cyclic adenosine 3',5'-monophosphate response element in human chorionic gonadotropin 1-subunit gene. Mol. Endocrinol. 5:693-702. 3. Ambesi-Impiombato, F., L. Parks, and H. Coon. 1980. Culture of hormone dependent epithelial cells from rat thyroids. Proc. Natl. Acad. Sci. USA 77:3455-3459. 4. Bar-Sagi, D., and J. Feramisco. 1985. Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell 42:841-848. 5. Bourne, H., D. Sanders, and F. McCormick 1990. The GTPase superfamily: a conserved switch for diverse cell functions. Nature (London) 348:125-132. 6. Burgering, B., R. Medema, J. Maassen, M. Van de Wetering, A. Van der Eb, F. McCormick, and J. Bos. 1991. Insulin stimulation of gene expression mediated by p2lras activation. EMBO J. 10:1103-1109.

4483

7. Cai, H., J. Szeberenyi, and G. Cooper. 1990. Effect of a dominant inhibitory Ha-Ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol. Cell. Biol. 10:5314-5323. 8. Campbell, M., and B. Sefton. 1992. Association between B-lymphocyte membrane immunoglobulin and multiple members of the src family of protein tyrosine kinases. Mol. Cell. Biol. 12:2315-2321. 9. Canalis, E., M. Centrella, W. Burch, and T. McCarthy. 1989. Insulin-like growth factor I mediates selective anabolic effects of parathyroid hormone in bone cultures. J. Clin. Invest. 83:60-65. 10. Chambard, J. C., S. Paris, G. L'Allemain, and J. Pouyssegur. 1987. Two growth factor signaling pathways in fibroblasts distinguished by pertussis toxin. Nature (London) 326:800-803. 11. Dib, K., B. Saunier, B. Delemer, C. Correze, and C. Jacquemin. 1991. Thyrotropin and forskolin induce changes in the expression of guanine nucleotide regulatory proteins (G proteins) in cultured thyroid follicles, p. 529-531. In A. Gordon, J. Gross, and G. Hennemann (ed.), Progress in thyroid research. Balkema, Rotterdam, The Netherlands. 12. Dumont, J., F. Lamy, P. Roger, and C. Maenhaut. 1992. Physiological and pathological regulation of thyroid cell proliferation and differentiation by thyrotropin and other factors. Physiol. Rev. 72:667-697. 13. Farnsworth, C., and L. Feig. 1991. Dominant inhibitory mutations in the Mg2+-binding site of RasH prevent its activation by GTP. Mol. Cell. Biol. 11:4822-4829. 14. Feramisco, J., M. Gross, T. Kamata, M. Rosenberg, and R. Sweet. 1984. Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells. Cell 38:109-117. 15. Fieg, L., and G. Cooper. 1988. Inhibition of NIH3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol. Cell. Biol. 8:3235-3243. 16. Gross, M., R. W. Sweet, G. Sathe, S. Yolcoyama, 0. Fasano, M. Goldfarb, M. Wigler, and M. Rosenberg. 1985. Purification and characterization of human H-Ras proteins expressed in Escherichia coli. Mol. Cell. Biol. 5:1015-1024. 17. Gupta, S., C. Gallego, and G. Johnson. 1992. Mitogenic pathways regulated by G protein oncogenes. Mol. Biol. Cell. 3:123128. 18. Gutkind, J. S., and K. C. Robbins. 1992. Activation of transforming G protein-coupled receptors induces rapid tyrosine phosphorylation of cellular proteins, including p125FAK and the p130 v-src substrate. Biochem. Biophys. Res. Commun. 188:155-161. 19. Hausdorff, W. P., J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz. 1992. Tyrosine phosphorylation of G protein alpha subunits by pp60csrc. Proc. Natl. Acad. Sci. USA 89:5720-5724. 20. Hill, R. N., L. S. Erdreich, and 0. E. Paynter. 1989. Thyroid follicular cell carcinogenesis. Fundam. Appl. Toxicol. 12:629697. 21. Imagawa, M., R. Chiu, and M. Karin. 1987. Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51:251-260. 22. Javaux, F., A. Donda, G. Vassart, and D. Christophe. 1991. Cloning and sequence analysis of TFE, a helix-loop-helix transcription factor able to recognize the thyroglobulin gene promoter in vitro. Nucleic Acids Res. 19:1121-1127. 22a.LaMorte, V. J. Biol. Chem., in press. 23. LaMorte, V., P. Goldsmith, A. Spiegel, J. Meinkoth, and J. Feramisco. 1992. Inhibition of DNA synthesis in living cells by microinjection of Gi2 antibodies. J. Biol. Chem. 267:691-694. 24. Lemoine, N. R., S. Staddon, J. Bond, F. S. Wyllie, J. J. Shaw, and D. Wynford-Thomas. 1990. Partial transformation of human thyroid epithelial cells by mutant Ha-ras oncogene. Oncogene 5:1833-1837. 25. Letterio, J., S. Coughlin, and L. Williams. 1986. Pertussis toxin-sensitive pathway in the stimulation of c-myc expression and DNA synthesis by Bombesin. Science 234:1117-1119. 26. Lyons, J., C. A. Landis, G. Harsh, L. Vallar, K. Grfinewald, H. Feichtinger, Q. Y. Dub, 0. H. Clark, E. Kawasaki, H. R.

4484

KUPPERMAN ET AL.

Bourne, and F. McCormicL 1990. Two G protein oncogenes in human endocrine tumors. Science 249:655-659. 27. Martin, G. A., D. Viskochil, G. Bollag McCabe, P. C., W. J. Crosier, H. Haubruck, L. Conroy, R. Clark, P. O'Connell, R. M. Cawthon, M. A. Innis, and F. McCormick. 1990. The gap-related domain of the neurofibromatosis type I gene product interacts with ras p21. Cell 63:843-849. 28. McCarthy, T., M. Centrella, and E. Canalis. 1989. Parathyroid hormone enhances the transcript and polypeptide levels of insulin-like growth factor I in osteoblast-enriched cultures from fetal rat bone. Endocrinology 124:1247-1253. 28a.Meinkoth, J. Unpublished data. 29. Meinkoth, J., J. Dela Cruz, and G. Burrow. 1991. TSH, IGF-1 and activated ras protein induce DNA synthesis in cultured thyroid cells. Thyroidology 3:103-107. 30. Meinkoth, J., P. Goldsmith, A. Spiegel, J. Feramisco, and G. Burrow. 1992. Inhibition of thyrotropin-induced DNA synthesis in thyroid follicular cells by microinjection of an antibody to the stimulatory G protein of adenylate cyclase, Gs. J. Biol. Chem. 267:13239-13245. 31. Mulcahy, L. S., M. R. Smith, and D. W. Stacy. 1985. Requirement for Ras protooncogene function during serum-stimulated growth of NIH3T3 cells. Nature (London) 312:241-243. 32. Namba, H., S. A. Rubin, and J. A. Fagin. 1990. Point mutations of ras oncogenes are an early event in thyroid tumorigenesis. Mol. Endocrinol. 4:1474-1479. 33. Roy, S., and G. Greenwald. 1991. Mediation of follicle-stimulating hormone action on follicular deoxyribonucleic acid synthesis by epidermal growth factor. Endocrinology 129:1903-1908. 34. Shenker, A., P. Goldsmith, C. Unson, and A. Spiegel. 1991. The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family. J. Biol. Chem. 266:9309-9313. 35. Stacey, D., and H. Kung. 1984. Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein. Nature (London) 310:508-511. 36. Stacey, D. W., L. A. Feig, and J. B. Gibbs. 1991. Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras. Mol. Cell. Biol. 11:4053-4064. 37. Suarez, H., J. du Vilard, B. Caillou, M. Schlumberger, C. Parmentier, and R. Monier. 1991. Gsp mutations in human thyroid tumors. Oncogene 6:677-679. 38. Suarez, H. G., J. du Villard, M. Severino, B. Caillou, M. Schlumberger, M. Tubiana, C. Parmentier, and R. Monier. 1990. Presence of mutations in all three ras genes in human thyroid tumors. Oncogene 5:565-570. 39. Szeberenyi, J., H. Cai, and G. Cooper. 1990. Effect of a

MOL. CELL. BIOL.

dominant inhibitory Ha-ras mutation on neuronal differentiation of PC12 cells. Mol. Cell. Biol. 10:5324-5332. 40. Takahashi, S., M. Conti, C. Prokop, J. J. Van Wyk, and H. S. Earp 3d. 1991. Thyrotropin and insulin-like growth factor I regulation of tyrosine phosphorylation in FRTL-5 cells. Interaction between cAMP-dependent and growth factor-dependent signal transduction. J. Biol. Chem. 266:7834-7841. 41. Thomas, J., S. M. Van Patten, P. Howard, K. H. Day, R. D. Mitchell, T. Sosnick, J. Trewhella, D. A. Walsh, and R. A. Maurer. 1991. Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase. J. Biol. Chem. 266:10906-10911. 42. Thorburn, A., J. Thorburn, S.-Y. Chen, S. Powers, H. E. Shubeita, J. R. Feramisco, and K. R. Chien. 1993. Hras dependent pathway for cardiac muscle hypertrophy. J. Biol. Chem. 268:2244-2249. 43. Trahey, M., and F. McCormick. 1987. A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. Science 238:542-545. 44. Tramontano, D., G. W. Cushing, A. C. Moses, and S. H. Ingbar. 1986. Insulin-like growth factor-I stimulates the growth of rat thyroid cells in culture and synergizes the stimulation of DNA synthesis induced by TSH and Graves'-IgG. Endocrinology 119:940-942. 45. Van Corven, E. J., P. L. Hordik, R. H. Medema, J. L. Bos, and W. H. Moolenaar. 1993. Pertussis toxin-sensitive activation of p23ras by G protein-coupled receptor agonists in fibroblasts. Proc. Natl. Acad. Sci. USA 90:1257-1261. 46. Vincentini, L., and M. V'illereal. 1984. Serum, bradykinin and vasopressin stimulate release of inositol phosphates from human fibroblasts. Biochem. Biophys. Res. Commun. 123:663670. 47. Walsh, D., K. Angelos, S. Van Patten, D. Glass, and L. Garetto. 1990. The inhibitor of the cAMP-dependent protein kinase, p. 43-84. In B. Kemp (ed.), CRC critical reviews: peptides and protein phosphorylation. CRC Press, Boca Raton, Fla. 47a.Wen, W. Unpublished data. 48. Williams, D. W., E. D. Williams, and D. Wynford-Thomas. 1989. Evidence for autocrine production of IGF-1 in human thyroid adenomas. Mol. Cell. Endocrinol. 61:139-143. 49. Wolfman, A., and I. G. Macara. 1990. A cytosolic protein catalyzes the release of GDP from p2lras. Science 248:67-69. 50. Xu, G. F., B. Un, K. Tanaka, D. Dunn, D. Wood, R. Gesteland, R. White, R. Weiss, and F. Tamanoi. 1990. The catalytic domain of the neurofibromatosis type I gene product stimulates ras GTPase and complements IRA mutants of S. cerevisiae. Cell 63:835-841.