Human chorionic gonadotropin suppresses activation of nuclear transcription factor-kappa B and activator protein-1 induced by tumor necrosis factor.

Human chorionic gonadotropin (hCG) suppresses cell-mediated allogeneic reactions, viral replication, tumorigenesis, and metastasis, most of which require activation of nuclear transcription factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). In the present report, we investigated the effect of hCG on NF-kappaB and AP-1 activated by tumor necrosis factor (TNF). Treatment of the CaCOV3 human ovarian cell line with hCG blocked TNF-induced activation of NF-kappaB, IkappaBalpha degradation, and NF-kappaB-dependent reporter gene transcription. hCG also blocked NF-kappaB activation induced by ceramide. The effect of hCG on NF-kappaB was mediated through inhibition of phosphorylation of IkappaBalpha. Because hCG also blocked TNF receptor-associated factor-2 and NF-kappaB-inducing kinase reporter gene expression, hCG must act at a step that causes phosphorylation of IkappaBalpha. AP-1 activation induced by TNF and ceramide was also suppressed by hCG. hCG abrogated the TNF-induced activation of mitogen-activated protein kinase kinase and c-Jun N-terminal kinase required for NF-kappaB and AP-1, respectively. Dideoxyadenosine and H-8 reversed the effect, and dibutyryl cAMP mimicked the effect, suggesting that hCG suppresses the transcription factors through cAMP-induced protein kinase A pathway. Overall, our results indicate that hCG inhibits the activation of NF-kappaB and AP-1, which may be the molecular basis by which hCG suppresses viral replication, cell proliferation, tumorigenesis, and metastasis.

Human chorionic gonadotropin (hCG) 1 is a heterodimeric placental glycoprotein required to maintain pregnancy (1). This hormone exerts its effects through lutropin receptors by stimulating progesterone production (2). Several reports suggest that hCG regulates the immune system by an unknown mech-anism (3). For instance, hCG suppresses the cell-mediated allogeneic reaction involving maternal lymphocytes and fetal implants (4) and down-regulates interleukin-2 and oncostatin M secretion from phytohemagglutinin-stimulated peripheral blood mononuclear cells (5,6). This gonadotropin inhibits the production of human immunodeficiency virus (HIV)-1 (7). It has been shown to block tumorigenesis and metastasis in Kaposi's sarcoma xenografts in mice (8) and to suppress the wasting syndrome frequently associated with HIV-1 infection (9).
The protective effects of pregnancy against breast cancer have been supported by epidemiological studies in humans and experimental data in animals (10 -12). Suppression of mammary carcinogenesis similar to that induced by pregnancy has been achieved by treating young virgin rats with hCG (13). hCG also inhibits the development of 9,10-dimethyl-1,2-benz(a)anthracene-induced mammary carcinoma in a dosedependent manner (14). In vitro, hCG has been shown to block the proliferation of breast tumor cells (15)(16)(17).
How hCG exerts this broad spectrum of effects is not understood. Because various genes that are required for carcinogenesis, immune modulation, cell proliferation, and HIV-1 replication are regulated by the nuclear transcription factor-B (NF-B) and activator protein-1 (AP-1), we hypothesized that many of hCG's effects are mediated through suppression of NF-B and AP-1 activation. Numerous lines of evidence suggest this possibility. For example, various agents that promote tumorigenesis activate NF-B (18), including phorbol ester, okadaic acid, and tumor necrosis factor (TNF). In addition, several genes that are involved in tumorigenesis, metastasis, and inflammation are regulated by NF-B (18). Recent reports indicate that NF-B protects cells from undergoing apoptosis and thus promotes proliferation (19,20), and most activators of NF-B also induce apoptosis (18).
Our hypothesis suggests that hCG would affect the TNFinduced activation of NF-B and AP-1. The activation of NF-B by TNF has been shown to involve interaction of TNF receptor-1 through its death domain with TNF-receptor-associated death domain (TRADD), which then binds to TNF receptorassociated factor (TRAF)-2. TRAF-2 then binds to NF-B-inducing kinase (NIK), which activates IB␤ kinase (IKK-␤) to result in phosphorylation of IB␣, which in turn leads to ubiquitination, degradation, and finally NF-B activation (18). The activation of NF-B is regulated by several other kinases, including those that belong to the mitogen-activated protein (MAP) kinase family (21). Various agents that activate NF-B also activate AP-1, another transcription factor (22). The activation of AP-1 is mediated through the activation of a stressactivated protein kinase called c-Jun N-terminal kinase (JNK) (22,23).
Because hCG inhibits various cellular responses that require NF-B and AP-1 activation, we tested the hypothesis that these effects are mediated through its modulation of activation of NF-B, AP-1, and members of the MAP kinase family. We found that hCG is a potent inhibitor of NF-B and AP-1 activation, and inhibits TNF-induced JNK and MAP kinase kinase (MEK) activation.

EXPERIMENTAL PROCEDURES
Materials-Highly purified urinary hCG (lot CR-127) with a specific activity of 14900 IU/mg protein, recombinant hCG (lot AFP8456A), and human lutropin (lot AFP4395A) were kindly supplied by Dr. A. F. Parlow under the National Hormone and Pituitary Program (supported by the National Institute of Child Health and Human Development and the United States Department of Agriculture, Rockville, MD). An antibiotic-antimycotic mixture (containing penicillin, streptomycin, and amphotericin B), RPMI 1640 medium, and fetal bovine serum were obtained from Life Technologies, Inc. Glycine, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), ceramide, NaCl, calpain inhibitor I (ALLN, N-acetyl leucine leucine norleucinal), and bovine serum albumin were obtained from Sigma. Bacteria-derived recombinant human TNF, purified to homogeneity with a specific activity of 5 ϫ 10 7 units/mg, was kindly provided by Genentech, Inc. (South San Francisco, CA). Antibodies against IB␣ and double-stranded oligonucleotide having the AP-1 consensus sequence were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-IB␣ (Ser-32) antibody was purchased from New England Biolabs (Beverly, MA). Carrier-free Na 125 I was purchased from Amersham Pharmacia Biotech; PD-10 (prepacked Sephadex G-25 medium) columns were from Amersham Pharmacia Biotech; IODO-GEN and gelatin were from Sigma. Expression plasmids encoding FLAG-tagged NIK (24) were kindly provided by Dr. David Wallach (Weizmann Institute of Science, Rehovot, Israel). The expression plasmid encoding myc-tagged TRAF-2 has been previously described (25).
Cell Lines-CaCOV3 (human ovarian) cells, U937 (human histiocytic lymphoma), Jurkat (T cells), and HeLa (epithelial cells) were obtained from the American Type Culture Collection (Manassas, VA). Most of the studies were performed with human ovarian cells, because the hCG receptors on these cells have been well characterized (2). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and a 1ϫ antibiotic-antimycotic mixture. Cells were free from mycoplasma as determined using the Gen-Probe Mycoplasma Rapid Detection Kit (Fisher Scientific, Pittsburgh, PA).
NF-B Activation Assay-To assay NF-B activation, electrophoretic mobility shift assays (EMSAs) were carried out as described elsewhere (26). Briefly, nuclear extracts prepared from TNF-treated cells (2 ϫ 10 6 cells/ml) were incubated with 32 P-end-labeled, 45-mer, double-stranded NF-B oligonucleotide (6 g of protein with 16 fmol of DNA) from the HIV long terminal repeat, 5Ј-TTGTTACAAGGGACTTTCCGCTGGG-GACTTTCCAGGGAGGCGTGG-3Ј (underlining indicates NF-B binding sites) for 15 min at 37°C, and the DNA-protein complex formed was resolved from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5Ј-TTGTTACAACTCACTT-TCCGCTGCTCACTTTCCAGGGAGGCGTGG-3Ј, was used to examine the specificity of binding of NF-B to the DNA. The specificity of binding was also examined using competition assay with the unlabeled oligonucleotide. The gels were dried, and the radioactive bands were visualized and quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software.
AP-1 Activation Assay-To assay AP-1 activation, 6 g of nuclear extract, prepared as indicated above, was incubated with 16 fmol of the 32 P-end-labeled AP-1 consensus oligonucleotide 5Ј-CGCTTGATGACT-CAGCCGGAA-3Ј (underlining indicates the AP-1 binding site) for 15 min at 37°C and analyzed on 6% native polyacrylamide gel. The specificity of binding was examined by competition assay with unlabeled oligonucleotide. The radioactive bands were visualized and quantified as indicated above.
c-Jun Kinase Assay-The c-Jun kinase assay was performed as described elsewhere (28) with modifications. Briefly, after cells (3 ϫ 10 6 / ml) were treated with TNF for 10 min, cell extracts were prepared by lysing the cells in buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 1% Nonidet P-40, 2 g/ml leupeptin, 2 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml benzamidine, and 1 mM dithiothreitol (DTT). Cell extracts (150 g/sample) were immunoprecipitated with 0.3 g of anti-JNK antibody for 60 min at 4°C. Immune complexes were collected by incubation with protein A/G sepharose beads for 45 min at 4°C. The beads were washed four times with 400 L of lysis buffer and twice with 400 L of kinase buffer (20 mM HEPES, pH 7.4, 1 mM DTT, and 25 mM NaCl). Kinase assays were performed for 15 min at 30°C with GST-Jun (1-79) as a substrate (2 g/sample) in 20 mM HEPES (pH 7.4), 10 mM MgCl 2 , 1 mM DTT, and 10 Ci of [␥ 32 P]ATP. Reactions were stopped with the addition of 15 L of 2 ϫ SDS sample buffer, boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis (9%). GST-Jun (1-79) was visualized by staining with Coomassie Blue, and the dried gel was analyzed with a PhosphorImager (Molecular Dynamics).
NF-B-dependent Reporter Gene Transcription-The effect of hCG on TNF-, TRAF-2-, NIK-, and p65 (transactivation subunit of NF-B)induced NF-B-dependent reporter gene transcription was measured as described previously (29). Briefly, HeLa cells (0.1 ϫ 10 6 cells/well) were plated in 6-well plates, treated with 50 ng/ml hCG for 12 h, and then transfected using the calcium phosphate method. Cells were transfected with medium (1 ml) containing plasmid DNAs for TRAF2, NIK, or p65 (1 g each) along with 0.5 g of NF-B promoter DNA linked to the heat-stable secretory alkaline phosphatase (SEAP) gene. The total final amount of DNA was maintained at 3 g by the addition of the control plasmid pCMVFLAG1 DNA.
To examine TNF-induced reporter gene expression, we transfected cells with the SEAP expression plasmid for 10 h before treating them with TNF (1 nM). In all transfections, treatment with hCG was continued during the transfection reaction. Cell culture-conditioned medium was harvested 24 h after transfection and analyzed (25 L) for alkaline phosphatase activity as described by the manufacturer (CLONTECH, Palo Alto, CA). SEAP activity was assayed on a 96-well fluorescent plate reader (Fluoroscan II, Lab Systems) with the excitation wavelength set at 360 nm and the emission wavelength at 460 nm. This reporter system was specific, because TNF-induced NF-B SEAP activity was inhibited by overexpression of either an IB␣ mutant lacking Ser-32/36, a kinase inactive NIK, or a dominant-negative TRAF-2 mutant (25,29).

RESULTS
hCG Inhibits TNF-induced NF-B Activation-CaCOV3 cells were treated with hCG for 24 h and then stimulated with 1 nM TNF for 30 min, and nuclear extracts were prepared and assayed for NF-B by EMSA. hCG alone did not activate NF-B (Fig. 1A). Neither the concentration of hCG nor the duration of treatment had any effect on the viability of CaCOV3 cells (data not shown). TNF induced NF-B activation by 7-fold; hCG pretreatment completely suppressed it in a dose-dependent manner with maximum inhibition at 50 ng/ml (Fig. 1A). We next tested the incubation period required for hCG to block TNF-induced NF-B activation by incubating cells with hCG for 6, 12, 18, or 24 h before adding TNF and then treating cells with TNF for 30 min. Only when the cells were pretreated for 24 h with hCG was maximum inhibition of NF-B activation observed (Fig. 1B). Co-treatment with hCG did not inhibit NF-B activation.
Activated NF-B That Is Inhibited by hCG Consists of p50 and p65 Subunits-Various combinations of Rel/NF-B proteins can constitute an active NF-B heterodimer that binds to specific sequences in DNA (19). To show that the retarded band visualized by EMSA in TNF-treated cells was indeed NF-B, we incubated nuclear extracts from TNF-activated cells with antibody to either the p50 (NF-BI) or p65 (Rel A) subunits and then conducted EMSA. Antibodies to either subunit of NF-B shifted the band to a higher molecular weight (Fig. 1C), thus suggesting that the TNF-activated complex consisted of p50 and p65 subunits. Neither preimmune serum nor such irrelevant antibodies as anti-cRel or anti-cyclin D1 affected the mobility of NF-B. Excess unlabeled NF-B (100-fold) caused complete disappearance of the band but mutant oligo did not, indicating the specificity of NF-B.
Inhibition of NF-B Activation by hCG Is Not Cell Typespecific-All of the effects of hCG described here were observed hCG Blocks Activation of NF-B and AP-1 in human ovarian CaCOV3 cells. Recent reports indicate, however, that the NF-B activation pathway may differ in different cell types (30). The receptors for hCG have been demonstrated on lymphoid cells and other cell types (2,17,31). Therefore, we also studied whether hCG could block TNF-induced NF-B activation in myeloid (U-937) cells, epithelial (HeLa) cells, and T (Jurkat) cells. In these experiments (Fig. 2) hCG inhibited TNF-induced NF-B activation in all cell types, suggesting that this effect is not restricted to ovarian cells.
hCG Also Blocks Ceramide-induced Activation of NF-B-NF-B is also activated by a variety of other agents besides TNF, including phorbol ester, LPS, okadaic acid, and ceramide (19). However, the signal transduction pathways induced by these agents differ. We therefore examined the effect of hCG on the activation of NF-B by these agents as well as by TNF. hCG completely blocked the activation of NF-B induced by ceramide but not that by H 2 O 2 , LPS, okadaic acid, or phorbol ester (Fig. 3). These results suggest that TNF and ceramide activate NF-B by a pathway that is different from that of the other agents and that hCG may act at a step where both of these agents converge in the signal transduction pathway that leads to NF-B activation.
Lutropin Also Inhibits TNF-induced NF-B Activation-As noted earlier (Fig. 1A), TNF-induced NF-B activation was completely suppressed by hCG. Because hCG shares a common receptor with a pituitary hormone, lutropin (2), we also examined the effect of leutinizing hormone on TNF-induced NF-B FIG. 1. hCG inhibits TNF-dependent NF-B activation. Ca-COV3 cells (2 ϫ 10 6 /ml) were incubated at 37°C for 24 h with different concentrations (0 -50 ng/ml) of hCG followed by 30-min incubation at 37°C with 1 nM TNF. After these treatments, nuclear extracts were prepared and then assayed for NF-B as described under "Experimental Procedures" (A). Cells were preincubated at 37°C with 50 ng/ml hCG for the indicated times and then tested for NF-B activation after incubation at 37°C for 30 min either with or without 1 nM TNF. After these treatments, nuclear extracts were prepared and then assayed for NF-B (B). Nuclear extracts were prepared from untreated or TNFtreated (1 nM) cells (2 ϫ 10 6 /ml), incubated for 15 min with different antibodies and unlabeled NF-B probe, and then assayed for NF-B as described under "Experimental Procedures" (C). activation. As shown in Fig. 4A, like hCG, pretreatment of cells with leutinizing hormone for 24 h suppressed TNF-induced NF-B activation in a dose-dependent manner, with maximum suppression occurring at 100 ng/ml. Although we used highly purified natural hCG in our studies, it is possible that the suppressive effects on NF-B activation were due to a contaminant in the hCG preparation (32). To explore this possibility, we used synthetic hCG prepared by recombinant DNA methods. As shown in Fig. 4B, this hormone also inhibited TNF-induced NF-B activation in a dose-dependant manner, indicating that the activity was not due to a contaminant in the hCG preparation.
hCG Inhibits TNF-dependent Phosphorylation and Degradation of IB␣-The translocation of NF-B to the nucleus is preceded by the phosphorylation and proteolytic degradation of IB␣ (23). To determine whether the inhibitory action of hCG was due to an effect on IB␣ degradation, the cytoplasmic levels of IB␣ proteins were examined by Western blot analysis. IB␣ was maximally degraded 15 min after TNF treatment of cells and resynthesis began thereafter. The pretreatment of cells with hCG completely abolished the TNF-induced degradation of IB␣ (Fig. 5A).
To determine if inhibition of TNF-induced IB␣ degradation by hCG was due to suppression of IB␣ phosphorylation, we treated the cells with the proteosome inhibitor ALLN (33) for 1 h and detected a hyperphosphorylated form of IB␣ by using antibodies that recognize only the serine-phosphorylated form. The hyperphosphorylated form of IB␣ appears as a slowmigrating band on SDS-polyacrylamide gel electrophoresis (Fig. 5B). TNF clearly induced the phosphorylation of IB␣ and hCG suppressed it (compare lane 4 with lane 8). The lack of a slow-migrating band in hCG-treated cells further demonstrates that hCG blocked TNF-induced IB␣ phosphorylation (see lower panel of Fig. 5B).
hCG Represses TNF-induced NF-B-dependent Reporter Gene Expression-So far we have shown that hCG blocks the DNA binding of NF-B protein to its consensus sequence. DNA binding alone did not always correlate with NF-B-dependent gene transcription, suggesting that additional regulatory steps have a role (34). To determine the effect of hCG on TNFinduced NF-B-dependent reporter gene expression, the hCGpretreated or untreated HeLa cells were transiently transfected with the SEAP reporter construct and then stimulated with TNF. Although SEAP activity increased 5-fold over the vector control after stimulation with TNF, TNF-induced SEAP activity was almost completely abolished by pretreatment with hCG (Fig. 6). These results demonstrate that hCG also represses NF-B-dependent reporter gene expression induced by TNF.
TNF-induced NF-B activation is mediated through sequential interaction of the TNF receptor with TRADD, TRAF2, NIK, and IKK-␤, resulting in phosphorylation of IB␣. To delineate the site of action of hCG in the TNF-signaling pathway leading to NF-B activation, cells were transfected with TRAF2, NIK, and p65 plasmids, and then NF-B-dependent SEAP expression was monitored in hCG-untreated and -treated cells. As shown in Fig. 6, TRAF2, NIK, and p65 plasmids induced gene expression and hCG suppressed TRAF-2 and NIK-induced but not p65-induced NF-B reporter expression. Thus hCG must act at a step downstream from IKK-␤. Because NIK is known to activate IKK-␤, which in turn phosphorylates IB␣, it appears that hCG must block the activity of IKK-␤.
hCG Inhibits TNF-induced AP-1 Activation-We next tested the effect of hCG on TNF-induced activation of AP-1 (35). TNF induced AP-1 expression by 5-fold in ovarian cells at a concentration of 1 nM. The activation of AP-1 was completely inhibited by hCG in a dose-dependent manner, with maximum suppression occurring at 50 ng/ml (Fig. 7A). Supershift analysis with specific antibodies against c-fos and c-jun indicate that TNFinduced AP-1 consisted of c-fos and c-jun (Fig. 7B). Lack of supershift by unrelated antibodies and disappearance of the AP-1 band by competition with unlabeled oligo shows the specificity.
Most agents that activate NF-B also activate AP-1 (35); therefore, we also investigated the effect of hCG on AP-1 activation by PMA, LPS, H 2 O 2 , okadaic acid, and ceramide. Like NF-B, all these agents activated AP-1, but only ceramideinduced AP-1 was blocked by hCG. Thus hCG blocked both NF-B and AP-1 induced by TNF and ceramide.
hCG Inhibits TNF-induced JNK and MEK Activation-TNF is a potent activator of JNK and MEK. Activation of AP-1 and NF-B are known to require the activation of JNK and MEK, respectively. To determine whether these kinases are modu-lated by hCG, we pretreated CaCOV3 cells with different concentrations of hCG for 24 h and then stimulated the cells with TNF (1 nM) for 10 min; JNK activation was then measured. TNF activated JNK by about 4-fold, an activation that gradually decreased with increasing concentrations of hCG. A 50 ng/ml concentration of hCG inhibited most of the JNK induced by TNF (Fig. 8A). We found that hCG also abolished TNFinduced MEK activation (Fig. 8B).
hCG Blocks TNF-induced NF-B Activation through the cAMP Pathway-hCG is known to be a potent inducer of cAMP (1). To investigate whether hCG-induced cAMP plays a role in suppression of NF-B activation, cells were treated with the hormone in the presence of different concentrations of dideoxyadenosine, which is known to block adenylate cyclase (36), the enzyme responsible for generation of cAMP. As shown in Fig. 9A, dideoxyadenosine had no effect by itself on TNFinduced NF-B activation, but it abolished the suppressive effect of hCG on TNF-induced NF-B activation in a dose-dependent manner achieving a complete block at 500 M. These results suggest that the effects of hCG are caused by cAMP. To further confirm this, we treated cells with a cell-permeable FIG. 6. hCG inhibits the NF-B-dependent reporter gene expression induced by TNF, TRAF-2, and NIK. HeLa cells were either pretreated or treated with hCG (50 ng/ml) overnight and then transiently transfected with indicated plasmids along with NF-B-containing plasmid linked to the SEAP gene. Where indicated, cells were exposed to 1 nM TNF for 2 h. Cells were assayed for secreted alkaline phosphatase activity as described under "Experimental Procedures." Results are expressed as fold activity over the nontransfected control.
hCG Blocks Activation of NF-B and AP-1 analogue of cAMP, dibutyryl cAMP. Bt 2 cAMP induced a gradual decrease in TNF-induced NF-B activation with increases in concentration (Fig. 9B) just as hCG did.
Since cAMP activates PKA, we also examined the effect of the PKA inhibitor H-8 on hCG-induced inhibition of NF-B activation. Cells were either exposed to 2 M H-8 for 1 h at 37°C and then treated with hCG for 24 h or exposed simultaneously to 2 M H-8 and hCG for 24 h or treated with 2 M H-8 after the hormone exposure. The cells were then examined for TNF-induced NF-B activation. The PKA inhibitor blocked the inhibitory effects of hCG when the cells were pretreated or co-treated with the hormone but post-treatment with H-8 produced almost no protection against hCG-mediated inhibition of NF-B activation (Fig. 9, panel C). These results indicate that the effects of hCG are mediated through activation of PKA.

DISCUSSION
Because hCG exhibits anticarcinogenic, growth regulatory, and immunomodulatory effects, we hypothesized that these effects of the hormone are mediated through suppression of NF-B activation, an early mediator of the pleiotropic effects of TNF. Our results clearly demonstrate that hCG is a potent inhibitor of NF-B activation induced by TNF. The inhibition of NF-B activation by hCG correlated with suppression of IB␣ phosphorylation and degradation and NF-B-dependent reporter gene transcription. hCG also suppressed the TNF-induced activation of AP-1, MEK, and JNK. We show that the suppressive effects of hCG are likely mediated through cAMP.
There are several possibilities for how hCG might inhibit TNF-induced NF-B activation. NF-B activation requires sequential phosphorylation, ubiquitination, and degradation of IB␣, and in our experiments hCG blocked IB␣ phosphorylation and degradation, as indicated by the following: Treatment of cells with hCG did not produce the retarded IB␣ band typical of the phosphorylated molecule (33,37). Similarly, antibodies against the phosphorylated form of IB␣ revealed di-minished phosphorylation of IB␣ in hCG-treated cells. The phosphorylation of IB␣ is regulated by a large number of kinases, including IKK-␣, IKK-␤, IKK-␥, NIK, transforming growth factor-␤-activated kinase-1, and MEKK1 (21, 23, 24, 38 -41). MEKK2 and MEKK3 have also been implicated in NF-B activation, whereas MEKK4 activates JNK (42). Given that MEKK induces the phosphorylation of MEK and that hCG inhibits the activation of MEK, it is possible that hCG inhibited IB␣ phosphorylation by inhibiting the activity of MEKK1 or other kinases.
Extensive research in the last few years has revealed that the induction of NF-B by TNF involves sequential activation of the TNF receptor, TRADD, TRAF2, NIK, and IKK-␤, which in turn results in phosphorylation of IB␣ (21). Because hCG blocked NF-B reporter gene expression induced by overexpression of TRAF2 and NIK plasmids but not that induced by NF-B transactivating subunit p65, hCG must inhibit IKK-␤, which is consistent with suppression of TNF-induced IB␣ phosphorylation by hCG.
We found that hCG also blocked ceramide-induced NF-B activation but not okadaic acid-, LPS-, PMA-, and H 2 O 2 -induced activation. This suggests that these activators activate NF-B by different pathways (43). This is consistent with a recent report demonstrating that ionizing radiation and shortwavelength UV activate NF-B through two distinct mechanisms (44). hCG blocked NF-B activation induced by both TNF and ceramide. Because TNF is known to induce ceramide production (45), it is possible that hCG blocks TNF-induced NF-B activation through suppression of ceramide production. Ceramide, however, is known not to play a major role in TNFinduced NF-B activation (46 and references therein).
Our results also demonstrate that hCG blocks TNF-induced NF-B activation on a wide variety of cells. hCG receptors have been demonstrated on epithelial, lymphoid, and myeloid cells (2,(5)(6)(7)17). hCG inhibits the proliferation of breast epithelial cells (15)(16)(17) and suppresses secretion of interleukin-2 from peripheral blood mononuclear cells (5). Because NF-B is a survival factor in most cell types (19,20) and is constitutively expressed in breast cancer cells (47), hCG may inhibit the growth of these cells through suppression of NF-B. Our results indicate that hCG also inhibits the activation of AP-1, another transcription factor known to play a role in cell proliferation (22). Suppression of AP-1 activation may also contribute to the growth inhibitory effects of hCG.
The effects of hCG we observed were not due to a contaminant of the hormone preparation because synthetic recombinant hCG also inhibited NF-B activation. In addition, the pituitary-derived hormone lutropin, which shares a common receptor with hCG (2), also suppressed the TNF-induced NF-B activation. How hCG suppresses NF-B activation is not clear, but it appears to be related to its ability to produce cAMP and activate protein kinase A (PKA). We clearly show that treatment of cells with Bt 2 cAMP blocks TNF-induced NF-B activation. Another pituitary-derived hormone, melanocyte-stimulating hormone, also blocks TNF-induced NF-B activation via cAMP and PKA activation (48).
We also investigated how cAMP generated by activation of cells with hCG inhibits NF-B activation. That inhibitors of PKA reversed the suppressive effect of hCG suggests that cAMP may activate this kinase. This possibility is intriguing, because it has been shown that PKA-mediated phosphorylation of NF-B is involved in inducible and constitutive activation of NF-B (49 -51). It was also shown that the catalytic subunit of PKA associates with IB␣, the inhibitory subunit of NF-B, in the cytoplasm (51). On stimulation of cells with either LPS or IL-1, PKA is activated, leading to phosphorylation of the p65

FIG. 8. hCG inhibits TNF-induced JNK (A) and MEK (B) activation.
A, ovarian cells were pretreated with different concentrations of hCG as indicated and then stimulated with 1 nM TNF at 37°C for 10 min. Then the cells were washed, and pellets were extracted and assayed for JNK as described under "Experimental Procedures." B, ovarian cells were pretreated with hCG (50 ng/ml) for 24 h and then stimulated with different concentrations of TNF at 37°C for 30 min. Cells were then washed, and pellets were extracted and assayed for MEK as described under "Experimental Procedures." subunit of NF-B and in turn to NF-B's translocation to the nucleus (51). Because PKA activation observed in these studies was cAMP-independent, it is unlikely that this is a mechanism of suppression of NF-B activation by hCG. Besides, in our studies, PKA activation did not result in activation of NF-B, as shown by Zhong et al. (51), but rather in its suppression.
Our results are consistent with other reports that show that elevation of cAMP reduces NF-B activity (52)(53)(54), but the mechanism is controversial. Ollivier et al. (54) found inhibition of NF-B mediated transcription by elevated cAMP or by overexpression of PKA without any inhibition of IB␣ degradation or nuclear translocation of p65. In contrast, Chen and Rothenberg (52) and Neumann et al. (53) reported that the effects of cAMP are mediated through stabilization of IB␣ and impairment of the nuclear transport of p65. Similarly, we found that elevation of intracellular cAMP induced by hCG inhibited both IB␣ degradation and gene transcription. It is possible that cAMP inhibits NF-B activation by inhibiting the MAP kinase kinase (MEK)-c-Jun N-terminal kinase (JNK) pathway, because overexpression of MEKK reversed the inhibitory effects of cAMP on NF-B activation (55).
We found that hCG blocked NF-B-dependent reporter gene expression. Several genes are involved in tumor promotion that are regulated by NF-B. This includes growth factors, cyclooxygenase-2, metalloproteases, and cell surface adhesion molecules (56). It is possible that the anticarcinogenic effects of hCG (10 -14) are mediated through the suppression of NF-B-de-pendent gene expression. Because NF-B-regulated genes also play a critical role in inflammation, hCG may also exhibit anti-inflammatory effects. TNF is one of the NF-B-regulated genes involved in tumor promotion, inflammation, and the wasting syndrome (for references see Ref. 57). hCG suppresses HIV-1-associated wasting syndrome, perhaps by suppressing TNF production. Because replication of certain viruses such as HIV-1 is also dependent on NF-B (21), hCG may also abolish viral replication. The suppression of HIV-1 replication by hCG has indeed been demonstrated (7). The concentration of hCG used in our studies is pharmacologically achievable and is quite safe, as indicated by the presence of 5000 mIU/ml to over 100,000 mIU/ml in normal maternal sera during the second trimester (58). During pregnancy, hCG may play a role in suppression of cell-mediated allogeneic reactions. Our results suggest that hCG may also have applications for various diseases, including cancer, inflammation, and AIDS. Its lack of toxicity even in large doses broadens its potential for therapeutic use. These possibilities require further investigation in detail.