Requirement of Thyrotropin-Dependent Complex Formation of Protein Kinase A Catalytic Subunit with Inhibitor of B Proteins for Activation of
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内分泌学杂志 2005年第4期
Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan
Address all correspondence and requests for reprints to: Fukushi Kambe, M.D., Ph.D., Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Furo-cho, Chikusa-ku, Nagoya University, Nagoya 464-8601, Japan. E-mail: kambe@riem.nagoya-u.ac.jp.
Abstract
We previously demonstrated that TNF--dependent activation of p65 nuclear factor B in rat thyroid FRTL-5 cells requires TSH. In the present study, we investigated the mechanism of this TSH action. Western blot analysis revealed that, in both the presence and absence of TSH, degradation of a cytosolic B inhibitor (IB) occurred in response to TNF-, resulting in nuclear translocation of p65 in both conditions. However, no DNA binding of p65 was detected in the absence of TSH, suggesting that posttranslational modification of p65 by TSH is required for its binding. Treatment of the cells cultured in the presence of TSH with a protein kinase A (PKA) inhibitor, H89, markedly reduced p65 binding and its transcriptional activity. However, transient block of TSH/cAMP-dependent activation of PKA catalytic subunit (PKAc) by adenylate cyclase inhibitor, SQ22536, had no effects on the p65 activation. Interestingly, it was found that PKAc formed a complex with IB and ? only in the presence of TSH, and this PKAc could be activated by TNF-. TNF--dependent p65 activation was temporally associated with PKAc/IB complex formation. More than 3 h exposure of TSH was required for the complex formation and p65 activation. These results demonstrate that TSH induces the formation of PKAc/IB complex in FRTL-5 cells and that this PKAc bound with IB plays a critical role in TNF--dependent activation of p65.
Introduction
NUCLEAR FACTOR-B (NF-B) is a transcription factor composed of dimer of Rel family proteins such as p65 (RelA), p50, p52, c-Rel, and RelB (1). Typical NF-B dimer consists of p65 and p50 subunits. In unstimulated cells, NF-B is sequestered in cytoplasm bound to an inhibitor of B proteins (IB) that includes several isoforms such as IB and IB?. External stimuli such as TNF- induce activation of NF-B by promoting IB kinase-dependent phosphorylation of IB (2). The phosphorylated IB is then ubiquitinated and degraded by proteasome (3). After release from IB, NF-B translocates into nucleus, binds to regulatory element of target gene, and controls its transcription. Recently, it was demonstrated by Zhong et al. (4) that cAMP-dependent protein kinase [protein kinase A (PKA)] catalytic subunit (PKAc) contributes to p65 activation. They showed that IB binds with not only NF-B but also PKAc in cytoplasm. When IB is degraded in response to lipopolysaccharide (LPS), the liberated PKAc activates p65 via its phosphorylation in immune cells.
TSH is a prime regulator for proliferation and function of thyroid follicular cells. TSH exerts the effects by binding its cognate G protein-coupled receptor present in plasma membrane. Physiological and pathophysiological actions of TSH are largely mediated by activation of adenylate cyclase, followed by intracellular cAMP elevation and PKA activation. Inactive PKA is a tetramer composed of two regulatory (PKAr) and two catalytic (PKAc) subunits. Binding of the tetramer with cAMP dissociates the enzyme into a PKAr dimer and two free active PKAc. Most of effects of cAMP are mediated by PKAc through phosphorylation of serine or threonine residues of target proteins (5).
In a previous study, we demonstrated that the composition of NF-B subunits activated by TNF- in rat thyroid FRTL-5 cells is altered by the presence or absence of TSH in culture media (6). In the absence of TSH, p65-containing NF-B is not activated; only p50-containing NF-B is activated. In contrast, in the presence of TSH, p65/p50 heterodimer NF-B is activated in addition to p50-containing NF-B, and this p65/p50 activation is associated with an increase in the promoter activity driven by canonical B sites and in the expression of an endogenous NF-B target gene, IL-6. It was also demonstrated that an adenylate cyclase activator, forskolin, and thyroid-stimulating antibodies obtained from patients with Graves’ disease mimic this TSH action. However, how TSH is involved in the p65 activation remains to be elucidated. In the present study, we demonstrate that TSH/cAMP induces the complex formation of PKAc with IB and that this PKAc bound with IB contributes to TNF--dependent activation of p65.
Materials and Methods
Cell culture
FRTL-5 cells (CRL8305; American Type Culture Collection, Manassas, VA) were cultured as described previously (7). After 80–90% confluence, the cells were divided into two groups: one group was cultured in the presence of TSH (1 mU/ml; bovine TSH, 2 U/mg protein; Sigma, St. Louis, MO) and the other in its absence. After a 3- or 4-d culture, the cells were treated with 40 ng/ml recombinant human TNF- (Asahi Chemical, Tokyo, Japan) or 10 μg/ml LPS (Sigma) and harvested for EMSA or Western blot analysis. In some experiments, the cells were treated with 10 μM adenylate cyclase activator forskolin (Sigma), 30 μM adenylate cyclase inhibitor SQ22536 (Sigma), 50 μM PKA inhibitor H89 (Sigma), or 10 nM protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA; Sigma). Human thyroid cells in primary culture were prepared from surgical specimens of patients with Graves’ disease. Informed consents were obtained in accordance with guidelines of the Nagoya University School of Medicine (Nagoya, Japan).
EMSA
Preparation of nuclear extracts and procedures of EMSA were described previously (8, 9). The Bwt oligonucleotide probe (5'-TCGAGCAGAGGGGACTTTCCGAGAGTCGA-3') containing a canonical NF-B binding site (underlined) in mouse Ig enhancer (10) was used as a probe. Nuclear extracts (10 μg protein/lane) were used for EMSA. The gels were dried and subjected to BAS 2000 bioimage analyzer (Fuji Photo Film, Tokyo, Japan). In some experiments, nuclear extracts (10 μg) were incubated at 37 C for 60 min with 0.5 U bacterial alkaline phosphatase (BAP; Takara, Otsu, Shiga, Japan) in 20 μl EMSA binding buffer. They were then subjected to EMSA.
Western blot analysis
Preparation of nuclear and cytosolic extracts and procedures for Western blot analysis were described previously (9). In brief, the extracts (50 μg) were subjected to polyacrylamide gel electrophoresis and blotted onto a membrane (Hybond-C super; Amersham Biosciences Inc., Piscataway, NJ). The membrane was incubated with anti-p65, anti-IB, anti-IB?, or anti-PKAc antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or antiactin antibody (Sigma). It was then incubated with antirabbit-IgG goat IgG conjugated with alkaline phosphatase (Zymed, San Francisco, CA). After being washed, it was subjected to color development reaction using an nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate tablet (Roche Diagnostics, Mannheim, Germany).
Immunoprecipitation
Cells were lysed in a lysis buffer (10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.4% Nonidet P-40; 2 mM EDTA) with Complete Protease Inhibitor (Roche Diagnostics) on ice for 10 min. The cytosolic extracts were prepared by centrifugation of the lysates at 10,000 x g for 10 min at 4 C twice. After preclearing with protein G agarose beads (Amersham), the cytosolic extracts were incubated with anti-IB or anti-IB? antibodies overnight at 4 C with shaking, followed by incubation with protein G agarose beads for 60 min at 4 C. Immune complexes were washed three times in the lysis buffer, and the immunoprecipitated proteins were subjected to Western blot analysis using anti-PKAc antibody.
Reporter gene assay
Construction of NF-B-dependent reporter gene plasmid and procedures of reporter gene assay were described previously (9, 11). The plasmid named pGL3–3Bpro contains three NF-B sites in tandem. FRTL-5 cells were plated in six-well dishes (1 x 105 cells/well) and grown in the presence of TSH for 3 d. pGL3–3Bpro (each 2 μg/well) was transfected into the cells using a Lipofectamine reagent (Invitrogen, Carlsbad, CA). A ?-galactosidase-expressing plasmid (0.2 μg/well; pSV-?-gal; Promega, Madison, WI) was also transfected to monitor the efficiency of transfection. After 18 h incubation with the liposome-DNA solution, it was replaced with a fresh medium with TSH. After an additional 24-h incubation, the cells were pretreated with 50 μM H89 for 60 min and treated with 40 ng/ml TNF- for 8 h. The cells were then harvested, and the luciferase and ?-galactosidase activities in the cell lysates were determined by a luminometer.
PKA activity assay
The cells cultured in 6-cm dishes were lysed in 300 μl lysis buffer (20 mM 4-morpholinepropanesulfonic acid, 50 mM ?-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethanesulfonylfluoride, and 10 μg/ml leupeptin and aprotinin) and centrifuged at 13,000 rpm for 15 min. The supernatant, cytosolic fractions were used to determine kinase activity using a PKA kinase activity assay kit (Stressgen Biotechnologies Corp., Victoria, British Columbia, Canada). The assay is based on a solid-phase ELISA using a specific synthetic peptide as a substrate for PKA and antibody against the phosphorylated form of the substrate. Amounts of activated PKAc in the samples were determined referring to the standard curve of activities of active, purified PKA included in the kit and expressed as nanograms per microgram of total protein.
Statistical analysis
Statistical analysis was performed by one-way ANOVA followed by Student’s t test. P < 0.05 was considered significant.
Results
TSH is required for TNF-- and LPS-dependent p65/p50 heterodimer activation in thyroid cells
FRTL-5 cells cultured in the absence or presence of TSH were treated with TNF- or LPS. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe, which contains a canonical NF-B site. As shown in Fig. 1A, TNF- activated a single protein/DNA complex in the absence of TSH. In contrast, in the presence of TSH, TNF- induced an additional slow-migrating complex. Similar NF-B activation was observed in response to LPS. Supershift analysis using antibodies against various Rel family proteins revealed that a fast-migrating complex contains p50, but not p65, and that a slow-migrating complex represents p65/p50 heterodimer NF-B (data not shown), compatible with our previous report (6). The results demonstrated that TSH modifies the composition of NF-B subunits activated by TNF- or LPS in rat thyroid FRTL-5 cells.
FIG. 1. Effects of TSH on TNF-- and LPS-dependent activation of NF-B in thyroid follicular cells. FRTL-5 cells (A) and human thyroid follicular cells (B) cultured in the absence (–) or presence (+) of TSH for 3 d were treated with TNF- or LPS for 30 min. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe. Representative BAS 2000 images are shown. Fast- and slow-migrating complexes are indicated by white and black arrowheads, respectively.
Similar results were obtained from primary human thyroid follicular cells. As shown in Fig. 1B, TNF- mainly induced a fast-migrating complex in the absence of TSH. However, in the presence of TSH, TNF- induced a slow-migrating complex as well as a fast-migrating one, indicating that TSH is required for the p65 activation not only in rat but also in human thyroid cells.
Lack of TSH effect on TNF--dependent IB degradation and on p65 nuclear translocation
To address the mechanism of how TSH mediates the p65 activation, we first examined whether TSH modifies degradation of IB and -? in response to TNF- in FRTL-5 cells. Cytosolic extracts were subjected to Western blot analysis using anti-IB and -? antibodies. As shown in Fig. 2, in both the absence and presence of TSH, IB was degraded in response to TNF- with maximal degradation at 30 min and was recovered at 60 min. IB expression was shown to be positively regulated by NF-Bs, which include p65/p50 and p50-containing NF-B, through the NF-B-binding sites in the promoter (12). Therefore, the increase of IB at 60 min could be due to the NF-B activation by TNF-. In contrast, IB? was not degraded in the absence of TSH, whereas it was degraded in the presence of TSH. Actin levels were not altered by TNF-. Nuclear translocation of p65 occurred in both the absence and presence of TSH as evidenced by Western blot analysis using nuclear extracts. These results demonstrate that, even in the absence of TSH, TNF- induces nuclear translocation of p65, but this p65 has no DNA-binding ability. Thus, it is speculated that TSH-dependent posttranslational modification such as phosphorylation is required for DNA binding of p65.
FIG. 2. Effects of TSH on TNF--dependent degradation of IB and IB?, and nuclear translocation of p65. FRTL-5 cells cultured in the absence (–) or presence (+) of TSH for 3 d were treated with TNF- for the indicated time. Cytosolic extracts were subjected to Western blot analysis using anti-IB, anti-IB?, and antiactin antibodies, and nuclear extracts were subjected to the analysis using anti-p65 antibody.
H89 inhibits TNF--dependent p65/p50 activation
Thus, we examined possible involvement of phosphorylation in p65 binding. The nuclear extracts prepared from FRTL-5 cells cultured with TSH and TNF- were treated with BAP in vitro and subjected to EMSA. As shown in Fig. 3A, BAP treatment markedly attenuated DNA binding of p65/p50 heterodimer, suggesting that phosphorylation is required for the binding.
FIG. 3. Phosphorylation of p65 by PKA enhances its DNA-binding and transcriptional activities. A and B, Nuclear extracts were prepared from FRTL-5 cells cultured in the presence of TSH followed by treatment with TNF- for 30 min. They were then treated with or without 0.025 U/μl BAP at 37 C for 60 min and subjected to EMSA using Bwt oligonucleotide as a probe (A). FRTL-5 cells cultured in the presence of TSH were pretreated with 50 μM H89 for 60 min and treated with TNF- for 30 min. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe (B). Representative BAS 2000 images are shown. Fast- and slow-migrating complexes are indicated by white and black arrowheads, respectively. C, pGL3–3Bpro, a luciferase reporter plasmid driven by three canonical NF-B-binding sites, was transfected into FRTL-5 cells cultured in the presence of TSH. After 42 h incubation, the cells were pretreated with 50 μM H89 for 60 min and treated with TNF- for 8 h. The promoter activity was expressed as luciferase activity normalized by ?-galactosidase activity. The promoter activity of control (Cont) was arbitrarily expressed as 100. Mean ± SD (n = 3). *, P < 0.01 vs. control; #, P < 0.01 vs. TNF--treated cells.
Because most of TSH actions are mediated by cAMP-PKA pathway, we next investigated whether PKA is involved in p65 activation in FRTL5 cells by using a specific PKA inhibitor H89 that directly inhibits kinase activity of PKA in a competitive fashion against ATP (13). FRTL-5 cells cultured with TSH were pretreated with H89 and treated with TNF-. As shown in Fig. 3B, EMSA revealed that H89 markedly diminished the binding of p65/p50. Compatible with this result, luciferase reporter gene assay demonstrated that H89 markedly attenuated the transcriptional activity of p65/p50 in the presence of TSH (Fig. 3C). These results strongly suggested the involvement of PKA in phosphorylation of p65, which leads to TNF--dependent increase in DNA binding and transcriptional activities.
Adenylate cyclase inhibitor does not affect p65 activation in the presence of TSH
To further study the involvement of PKA in TNF--dependent activation of p65/p50, we next examined effects of an adenylate cyclase inhibitor, SQ22536, on the activation. As shown in Fig. 4A, the cells cultured in the presence of TSH were treated with SQ22536 followed by TNF-, and the nuclear extracts were subjected to EMSA. Unexpectedly, DNA binding of p65/p50 heterodimer was not diminished even in the presence of SQ22536 for 4 h. Because SQ22536 is effective in reducing TSH-dependent activation of PKAc (treatment of the cells with SQ22536 for 4 h significantly decreased the PKAc activity in the cytosol; Fig. 4B), these results suggested that PKA activated by TSH/cAMP may not directly contribute to the p65 phosphorylation.
FIG. 4. Effects of adenylate cyclase inhibitor on activation of PKAc and NF-B. A, FRTL-5 cells cultured in the presence of TSH were pretreated with 30 μM of an adenylate cyclase inhibitor, SQ22536 (SQ), for the indicated time (2–4 h). During the last 1 h, the cells were treated with TNF-. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe. Representative BAS 2000 image is shown. Fast- and slow-migrating complexes are indicated by white and black arrowheads, respectively. B, FRTL-5 cells cultured in the presence of TSH were treated for 4 h with 30 μM SQ22536 (SQ). During the last 1 h, the cells were treated with TNF-. Cytosolic fractions were used to determine PKAc activity. Amounts of activated PKAc were expressed as nanograms per microgram of total protein. Mean ± SD (n = 3). *, P < 0.01 vs. SQ (–) and TNF- (–); #, P < 0.01 vs. SQ (+) and TNF- (–).
On the other hand, it should be noted that the decrease in PKAc activity by SQ22536 was restored by the treatment with TNF- (Fig. 4B). This result suggested the presence of PKAc in thyroid cells, which can respond to TNF- and is not derived from a canonical heterotetramer complex of PKAc/PKAr. Because the presence of PKAc/IB complex that responds to LPS was demonstrated in immune cells (4), we questioned whether such a complex is present in FRTL-5 cells.
IB and -? form complexes with PKAc in the presence of TSH
Cytoplasmic extracts were prepared from the cells cultured with TSH and subjected to immunoprecipitation using anti-IB antibody or normal rabbit serum (NRS). The immunoprecipitates were then subjected to Western blot analysis using anti-PKAc antibody. As shown in Fig. 5A, when cytoplasmic extract without immunoprecipitation was subjected to Western blot analysis, a single band with a molecular weight of around 40,000 was detected. The size is compatible with the expected mass of PKAc. No PKAc band was detected, when NRS was used for immunoprecipitation. In contrast, when anti-IB antibody was used for immunoprecipitation, a single band corresponding to the PKAc band was detected, demonstrating that IB forms a complex with PKAc in FRTL-5 cells cultured with TSH.
FIG. 5. Effects of TSH on complex formation of PKAc with IB or IB?. A, Cytosolic extracts were prepared from FRTL-5 cells cultured in the presence of TSH. Immunoprecipitation (IP) was performed by NRS or anti-IB antibody. The precipitated samples were subjected to Western blot analysis (WB) using anti-PKAc antibody. Cyto, cytosolic extract without immunoprecipitation was used for Western blot analysis. The position of 35,000 molecular weight marker is indicated. B, FRTL-5 cells cultured in the absence of TSH for 4 d were treated with or without TSH for 3 d. Cytosolic extracts were subjected to immunoprecipitation (IP) using NRS and anti-IB and anti-IB? antibodies, followed by Western blot analysis (WB) using anti-PKAc antibody. Similar results were obtained from separate experiments.
We then examined whether IB? also forms a complex and whether TSH affects the complex formation. Cytoplasmic extracts prepared from FRTL-5 cells cultured with or without TSH were subjected to immunoprecipitation and Western blot analysis. As shown in Fig. 5B, IB and ? were not bound with PKAc in the absence of TSH. However, both IBs formed a complex with PKAc in the presence of TSH, raising a possibility that the PKAc bound with IBs would play an important role in p65 activation in the presence of TSH. To prove this possibility, we performed the following experiments.
p65 Activation is associated with formation of IB/PKAc complex
Duration of TSH exposure required for TNF--dependent p65 activation was investigated. FRTL-5 cells cultured with TSH for various lengths of time were treated with TNF-. As shown in Fig. 6A, EMSA revealed that a 3-h exposure to TSH was not sufficient for p65 activation. More than 3 h exposure was required for the activation. It should be noted that formation of IB/PKAc complex after TSH exposure occurred in a similar time course with the p65 activation (Fig. 6B). When amounts of PKAc bound with IB were analyzed by immunoprecipitation and Western blot analysis, more than 3 h exposure of TSH was required for the complex formation, demonstrating that TNF--dependent p65 activation was temporally associated with the formation of IB/PKAc complex. Total amounts of cytosolic PKAc were not altered by TSH (Fig. 6C). Taken together, these observations strongly suggest that PKAc, which is liberated from IB/PKAc complex in response to TNF- but not from PKAc/PKAr complex, is directly involved in p65 activation.
FIG. 6. Time courses of p65 activation in response to TNF- and of formation of PKAc/IB complex after TSH treatment. FRTL-5 cells cultured in the absence of TSH for 4 d were treated with TSH for various lengths of time. A, Cells were then treated with TNF- for 30 min, and the nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe. B, Cytosolic extracts were subjected to immunoprecipitation (IP) using anti-IB antibody, followed by Western blot analysis (WB) using anti-PKAc antibody. C, Cytosolic extracts were subjected to Western blot analysis (WB) using anti-PKAc antibody.
TSH-dependent IB/PKAc complex formation is mediated by the cAMP/PKA pathway
To define intracellular signaling pathway leading to the formation of IB/PKAc complex, effects of a PKC activator PMA and an adenylate cyclase activator forskolin on the complex formation were investigated. Because 6 h exposure to TSH was sufficient for the complex formation (Fig. 6B), FRTL-5 cells cultured in the absence of TSH were treated for 6 h with PMA, forskolin, TSH, SQ22536, and H89, alone or in combination. Cytosolic extracts were subjected to immunoprecipitation with anti-IB antibody, followed by Western blot analysis using anti-PKAc antibody. As shown in Fig. 7, no IB/PKAc complex was present in the cells cultured without TSH (lane 2). PMA did not induce the complex formation (lane 3). In contrast, forskolin and TSH induced the complex formation (lanes 4–6). SQ22536 (lane 7) and H89 (lane 8) prevented the TSH action. Forskolin-induced complex formation was also inhibited by H89 (data not shown). These results clearly demonstrate that the TSH-dependent IB/PKAc complex formation is mainly attributable to the cAMP/PKA-mediated TSH action but not to the PKC-mediated action.
FIG. 7. TSH-dependent IB/PKAc complex formation is mediated by cAMP/PKA pathway. FRTL-5 cells cultured in the absence of TSH for 4 d were treated for 6 h with 10 nM PMA, 10 μM forskolin (For), 0.25 mU/ml TSH, 1 mU/ml TSH, 1 mU/ml TSH plus 30 μM SQ22536 (SQ), or 1 mU/ml TSH plus 50 μM H89. The cytosolic extracts were subjected to immunoprecipitation (IP) with anti-IB antibody, followed by Western blot analysis (WB) using anti-PKAc antibody. Cyto, Cytosolic extract without immunoprecipitation was used for Western blot analysis.
Discussion
We previously demonstrated that TSH is required for TNF--dependent activation of p65 NF-B in rat thyroid FRTL-5 cells (6). The present study, to clarify the molecular mechanism of this TSH action, demonstrates that PKAc plays a pivotal role in p65 activation. PKAc does not affect nuclear translocation of p65 (Fig. 2) but increases its DNA-binding and transcriptional activities (Fig. 3). These observations are compatible with previous reports. Shirakawa and Mizel (14) first demonstrated that treatment of cytosolic extracts prepared from 70Z/3 murine pre-B cells with PKAc in vitro induced DNA-binding activity of NF-B. Naumann and Scheidereit (15) demonstrated that TNF--dependent phosphorylation of p65 is associated with its increased binding activity and that treatment of recombinant p65 with PKAc in vitro enhances its binding. It has been also shown, in various cells, that PKA inhibitors including H89 block the activation of NF-B, leading to decreased DNA-binding and transcriptional activities (16, 17, 18, 19). In addition, it was recently shown that IB-bound PKAc is involved in phosphorylation of p65 and, thereby, its transcriptional activity (4). However, some reports showed the opposite effects of PKA. Stimulation of PKA activity reduced p65 nuclear translocation and/or transcriptional activity (20, 21, 22). Therefore, it is suggested that PKA is involved in multiple steps of p65 activation process, and its effects could vary depending on cell types and culture conditions including external stimuli used.
Consistent with the report by Zhong et al. (4), our present study also demonstrated the interaction of endogenous PKAc with IB or -? in cytoplasm (Fig. 5), indicating that thyroid cells possess a pool of PKAc other than the well-established pool that consists of heterotetramer PKAc/PKAr complex. More importantly, it was demonstrated in this study that the formation of PKAc/IB complex is dependent on TSH; the complex was not detected in FRTL-5 cells cultured in the absence of TSH (Fig. 5). The absence of IB and/or PKAc proteins in cytoplasm is not the case because their expression in cytoplasm was detected by Western blot analysis even in the absence of TSH, and their contents were not affected by TSH (Figs. 2 and 6). Therefore, it is indicated that, even when both PKAc and IBs are present in cytoplasm, PKAc does not necessarily form a complex with IB.
Recently, it has been realized that PKA is localized at particular intracellular places through its association with A-kinase anchoring proteins that consist of more than 20 members, and this compartmentalization of PKA would account for site-, time-, and protein-specific phosphorylation by PKA (5, 23). For example, it was shown that AKAP79 can be associated with ?2-adrenergic receptor, one of G protein-coupled receptors, and with PKC as well as PKAc/PKAr, thereby forming a multicomponent signaling complex at the site of action (23, 24). On the other hand, it has been also shown that IB is a component of a large multiprotein complex so-called "signalsome" that is composed of IB kinases and interacting proteins (25, 26). Therefore, in thyroid follicular cells cultured in the absence of TSH, PKAc, and IB may be present in separate compartments in cytoplasm. When the cells are stimulated with TSH, formation of PKAc/IB complex is achieved, and this process requires more than 3 h stimulation with TSH. Such prolonged stimulation with TSH is likely to be required for the creation of new compartmentalization of PKAc/IB, the process of which may include de novo protein synthesis, intracellular protein trafficking, and/or complex formation.
Involvement of PKAc bound with IB in p65 activation was suggested by the experiment using adenylate cyclase inhibitor SQ22536 (Fig. 4). It was shown that 4 h exposure of this inhibitor did not affect TNF--dependent p65 activation in FRTL-5 cells cultured with TSH, suggesting that PKAr-bound PKAc does not directly contribute to the activation. Furthermore, it was shown that time course of TNF--dependent p65 activation after TSH exposure was well associated with that of the complex formation of PKAc/IB (Fig. 6). Therefore, these results strongly suggest the major role of IB-bound PKAc in p65 activation in FRTL-5 cells.
The present study showed that PKAc is involved in both DNA binding and transcriptional activities of p65. Recently, several phosphoserine residues in p65 have been identified. Ser276, Ser529, and Ser536 can be phosphorylated in cells by PKA, casein kinase II, and IB kinase, respectively (27, 28, 29, 30). Ser276 is also phosphorylated by mitogen- and stress-activated protein kinase-1 (31). When Ser276 is phosphorylated by PKA, it induces a conformational change of p65 molecule and creates a site for interaction of cAMP response element-binding protein/p300, thereby enhancing transcriptional activity of p65. On the other hand, phosphorylation of Ser276 as well as Ser529 and Ser536 has been shown to have little effect on DNA binding. However, more recently, Hou et al. (32) reported that phosphorylation of Ser337 in p50 (this residue corresponds to Ser276 in p65) and that of Ser276 in p65 augment the binding activity by inducing a conformational change in the hinge region of NF-B. It is, therefore, likely that PKA can regulate both transcriptional and binding activities of p65 via phosphorylation of Ser276.
In conclusion, the present study demonstrates that TSH induces the formation of PKAc/IB complex in cytoplasm of FRTL-5 thyroid cells and that this PKAc bound with IB plays a critical role in TNF--dependent activation of p65 in the cells cultured with TSH. These findings provide a novel aspect of biological effects of TSH on thyroid cells. Growing evidence has indicated that TNF- and NF-B are involved in pathogenesis of various autoimmune diseases. Recently, it is also shown that NF-B and its upstream kinase are key factors to link inflammation to cancer in some in vivo cancer models (33, 34). It is thus speculated that TSH might be implicated in tumorigenesis or promotion of thyroid cancers as well as pathogenesis of autoimmune thyroid diseases through the modulation of p65 activation.
References
Baldwin Jr AS 1996 The NF-B and IB proteins: new discoveries and insights. Annu Rev Immunol 14:649–683
Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M 1997 The IB kinase complex (IKK) contains two kinase subunits, IKK and IKK?, necessary for IB phosphorylation and NF-B activation. Cell 91:243–252
DiDonato J, Mercurio F, Rosette C, Wu-Li J, Suyang H, Ghosh S, Karin M, Roff M 1996 Mapping of the inducible IB phosphorylation sites that signal its ubiquitination and degradation: role of IB ubiquitination in signal-induced activation of NFB in vivo. Mol Cell Biol 16:1295–1304
Zhong H, SuYang H, Erdjument-Bromage H, Tempst P, Ghosh S 1997 The transcriptional activity of NF-B is regulated by the IB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89:413–424
Carnegie GK, Scott JD 2003 A-kinase anchoring proteins and neuronal signaling mechanisms. Genes Dev 17:1557–1568
Kikumori T, Kambe F, Nagaya T, Funahashi H, Seo H 2001 Thyrotropin modifies activation of nuclear factor B by tumour necrosis factor- in rat thyroid cell line. Biochem J 354:573–579
Kambe F, Seo H 1996 Mediation of the hormone- and serum-dependent regulation of thyroglobulin gene expression by thyroid-transcription factors in rat thyroid FRTL-5 cells. J Endocrinol 150:287–298
Kambe F, Nomura Y, Okamoto T, Seo H 1996 Redox regulation of thyroid-transcription factors, Pax-8 and TTF-1, is involved in their increased DNA-binding activities by thyrotropin in rat thyroid FRTL-5 cells. Mol Endocrinol 10:801–812
Kikumori T, Kambe F, Nagaya T, Imai T, Funahashi H, Seo H 1998 Activation of transcriptionally active nuclear factor-B by tumor necrosis factor- and its inhibition by antioxidants in rat thyroid FRTL-5 cells. Endocrinology 139:1715–1722
Baeuerle PA, Baltimore D 1988 IB: a specific inhibitor of the NF-B transcription factor. Science 242:540–546
Cao X, Kambe F, Ohmori S, Seo H 2002 Oxidoreductive modification of two cysteine residues in paired domain by Ref-1 regulates DNA-binding activity of Pax-8. Biochem Biophys Res Commun 297:288–293
Le Bail O, Schmidt-Ullrich R, Israel A 1993 Promoter analysis of the gene encoding the IB-/MAD3 inhibitor of NF-B: positive regulation by members of the rel/NF-B family. EMBO J 12:5043–5049
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272
Shirakawa F, Mizel SB 1989 In vitro activation and nuclear translocation of NF-B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol Cell Biol 9:2424–2430
Naumann M, Scheidereit C 1994 Activation of NF-B in vivo is regulated by multiple phosphorylations. EMBO J 13:4597–4607
Geng Y, Zhang B, Lotz M 1993 Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes. J Immunol 151:6692–6700
Lapointe R, Lemieux R, Olivier M, Darveau A 1996 Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes. Eur J Immunol 26:2376–2382
Um JH, Kang CD, Lee BG, Kim DW, Chung BS, Kim SH 2001 Increased and correlated nuclear factor-B and Ku autoantigen activities are associated with development of multidrug resistance. Oncogene 20:6048–6056
Bhat-Nakshatri P, Sweeney CJ, Nakshatri H 2002 Identification of signal transduction pathways involved in constitutive NF-B activation in breast cancer cells. Oncogene 21:2066–2078
Neumann M, Grieshammer T, Chuvpilo S, Kneitz B, Lohoff M, Schimpl A, Franza BJ, Serfling E 1995 RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A. EMBO J 14:1991–2004
Ollivier V, Parry GC, Cobb RR, de Prost D, Mackman N 1996 Elevated cyclic AMP inhibits NF-B-mediated transcription in human monocytic cells and endothelial cells. J Biol Chem 271:20828–20835
Takahashi N, Tetsuka T, Uranishi H, Okamoto T 2002 Inhibition of the NF-B transcriptional activity by protein kinase A. Eur J Biochem 269:4559–4565
Griffioen G, Thevelein JM 2002 Molecular mechanisms controlling the localisation of protein kinase A. Curr Genet 41:199–207
Fraser ID, Cong M, Kim J, Rollins EN, Daaka Y, Lefkowitz RJ, Scott JD 2000 Assembly of an A kinase-anchoring protein-?2-adrenergic receptor complex facilitates receptor phosphorylation and signaling. Curr Biol 10:409–412
Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J, Young DB, Barbosa M, Mann M, Manning A, Rao A 1997 IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-B activation. Science 278:860–866
Yamamoto Y, Kim DW, Kwak YT, Prajapati S, Verma U, Gaynor RB 2001 IKK /NEMO facilitates the recruitment of the IB proteins into the IB kinase complex. J Biol Chem 276:36327–36336
Zhong H, Voll RE, Ghosh S 1998 Phosphorylation of NF-B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1:661–671
Wang D, Baldwin Jr AS 1998 Activation of nuclear factor-B-dependent transcription by tumor necrosis factor- is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem 273:29411–29416
Wang D, Westerheide SD, Hanson JL, Baldwin Jr AS 2000 Tumor necrosis factor -induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem 275:32592–32597
Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W 1999 IB kinases phosphorylate NF-B p65 subunit on serine 536 in the transactivation domain. J Biol Chem 274:30353–30356
Vermeulen L, De Wilde G, Van Damme P, Vanden Berghe W, Haegeman G 2003 Transcriptional activation of the NF-B p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J 22:1313–1324
Hou S, Guan H, Ricciardi RP 2003 Phosphorylation of serine 337 of NF-B p50 is critical for DNA binding. J Biol Chem 278:45994–45998
Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E, Ben-Neriah Y 2004 NF-B functions as a tumour promoter in inflammation-associated cancer. Nature 431:461–466
Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, Kagnoff MF, Karin M 2004 IKK? links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 118:285–296(Xia Cao, Fukushi Kambe an)
Address all correspondence and requests for reprints to: Fukushi Kambe, M.D., Ph.D., Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Furo-cho, Chikusa-ku, Nagoya University, Nagoya 464-8601, Japan. E-mail: kambe@riem.nagoya-u.ac.jp.
Abstract
We previously demonstrated that TNF--dependent activation of p65 nuclear factor B in rat thyroid FRTL-5 cells requires TSH. In the present study, we investigated the mechanism of this TSH action. Western blot analysis revealed that, in both the presence and absence of TSH, degradation of a cytosolic B inhibitor (IB) occurred in response to TNF-, resulting in nuclear translocation of p65 in both conditions. However, no DNA binding of p65 was detected in the absence of TSH, suggesting that posttranslational modification of p65 by TSH is required for its binding. Treatment of the cells cultured in the presence of TSH with a protein kinase A (PKA) inhibitor, H89, markedly reduced p65 binding and its transcriptional activity. However, transient block of TSH/cAMP-dependent activation of PKA catalytic subunit (PKAc) by adenylate cyclase inhibitor, SQ22536, had no effects on the p65 activation. Interestingly, it was found that PKAc formed a complex with IB and ? only in the presence of TSH, and this PKAc could be activated by TNF-. TNF--dependent p65 activation was temporally associated with PKAc/IB complex formation. More than 3 h exposure of TSH was required for the complex formation and p65 activation. These results demonstrate that TSH induces the formation of PKAc/IB complex in FRTL-5 cells and that this PKAc bound with IB plays a critical role in TNF--dependent activation of p65.
Introduction
NUCLEAR FACTOR-B (NF-B) is a transcription factor composed of dimer of Rel family proteins such as p65 (RelA), p50, p52, c-Rel, and RelB (1). Typical NF-B dimer consists of p65 and p50 subunits. In unstimulated cells, NF-B is sequestered in cytoplasm bound to an inhibitor of B proteins (IB) that includes several isoforms such as IB and IB?. External stimuli such as TNF- induce activation of NF-B by promoting IB kinase-dependent phosphorylation of IB (2). The phosphorylated IB is then ubiquitinated and degraded by proteasome (3). After release from IB, NF-B translocates into nucleus, binds to regulatory element of target gene, and controls its transcription. Recently, it was demonstrated by Zhong et al. (4) that cAMP-dependent protein kinase [protein kinase A (PKA)] catalytic subunit (PKAc) contributes to p65 activation. They showed that IB binds with not only NF-B but also PKAc in cytoplasm. When IB is degraded in response to lipopolysaccharide (LPS), the liberated PKAc activates p65 via its phosphorylation in immune cells.
TSH is a prime regulator for proliferation and function of thyroid follicular cells. TSH exerts the effects by binding its cognate G protein-coupled receptor present in plasma membrane. Physiological and pathophysiological actions of TSH are largely mediated by activation of adenylate cyclase, followed by intracellular cAMP elevation and PKA activation. Inactive PKA is a tetramer composed of two regulatory (PKAr) and two catalytic (PKAc) subunits. Binding of the tetramer with cAMP dissociates the enzyme into a PKAr dimer and two free active PKAc. Most of effects of cAMP are mediated by PKAc through phosphorylation of serine or threonine residues of target proteins (5).
In a previous study, we demonstrated that the composition of NF-B subunits activated by TNF- in rat thyroid FRTL-5 cells is altered by the presence or absence of TSH in culture media (6). In the absence of TSH, p65-containing NF-B is not activated; only p50-containing NF-B is activated. In contrast, in the presence of TSH, p65/p50 heterodimer NF-B is activated in addition to p50-containing NF-B, and this p65/p50 activation is associated with an increase in the promoter activity driven by canonical B sites and in the expression of an endogenous NF-B target gene, IL-6. It was also demonstrated that an adenylate cyclase activator, forskolin, and thyroid-stimulating antibodies obtained from patients with Graves’ disease mimic this TSH action. However, how TSH is involved in the p65 activation remains to be elucidated. In the present study, we demonstrate that TSH/cAMP induces the complex formation of PKAc with IB and that this PKAc bound with IB contributes to TNF--dependent activation of p65.
Materials and Methods
Cell culture
FRTL-5 cells (CRL8305; American Type Culture Collection, Manassas, VA) were cultured as described previously (7). After 80–90% confluence, the cells were divided into two groups: one group was cultured in the presence of TSH (1 mU/ml; bovine TSH, 2 U/mg protein; Sigma, St. Louis, MO) and the other in its absence. After a 3- or 4-d culture, the cells were treated with 40 ng/ml recombinant human TNF- (Asahi Chemical, Tokyo, Japan) or 10 μg/ml LPS (Sigma) and harvested for EMSA or Western blot analysis. In some experiments, the cells were treated with 10 μM adenylate cyclase activator forskolin (Sigma), 30 μM adenylate cyclase inhibitor SQ22536 (Sigma), 50 μM PKA inhibitor H89 (Sigma), or 10 nM protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA; Sigma). Human thyroid cells in primary culture were prepared from surgical specimens of patients with Graves’ disease. Informed consents were obtained in accordance with guidelines of the Nagoya University School of Medicine (Nagoya, Japan).
EMSA
Preparation of nuclear extracts and procedures of EMSA were described previously (8, 9). The Bwt oligonucleotide probe (5'-TCGAGCAGAGGGGACTTTCCGAGAGTCGA-3') containing a canonical NF-B binding site (underlined) in mouse Ig enhancer (10) was used as a probe. Nuclear extracts (10 μg protein/lane) were used for EMSA. The gels were dried and subjected to BAS 2000 bioimage analyzer (Fuji Photo Film, Tokyo, Japan). In some experiments, nuclear extracts (10 μg) were incubated at 37 C for 60 min with 0.5 U bacterial alkaline phosphatase (BAP; Takara, Otsu, Shiga, Japan) in 20 μl EMSA binding buffer. They were then subjected to EMSA.
Western blot analysis
Preparation of nuclear and cytosolic extracts and procedures for Western blot analysis were described previously (9). In brief, the extracts (50 μg) were subjected to polyacrylamide gel electrophoresis and blotted onto a membrane (Hybond-C super; Amersham Biosciences Inc., Piscataway, NJ). The membrane was incubated with anti-p65, anti-IB, anti-IB?, or anti-PKAc antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or antiactin antibody (Sigma). It was then incubated with antirabbit-IgG goat IgG conjugated with alkaline phosphatase (Zymed, San Francisco, CA). After being washed, it was subjected to color development reaction using an nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate tablet (Roche Diagnostics, Mannheim, Germany).
Immunoprecipitation
Cells were lysed in a lysis buffer (10 mM HEPES, pH 7.9; 1.5 mM MgCl2; 10 mM KCl; 0.4% Nonidet P-40; 2 mM EDTA) with Complete Protease Inhibitor (Roche Diagnostics) on ice for 10 min. The cytosolic extracts were prepared by centrifugation of the lysates at 10,000 x g for 10 min at 4 C twice. After preclearing with protein G agarose beads (Amersham), the cytosolic extracts were incubated with anti-IB or anti-IB? antibodies overnight at 4 C with shaking, followed by incubation with protein G agarose beads for 60 min at 4 C. Immune complexes were washed three times in the lysis buffer, and the immunoprecipitated proteins were subjected to Western blot analysis using anti-PKAc antibody.
Reporter gene assay
Construction of NF-B-dependent reporter gene plasmid and procedures of reporter gene assay were described previously (9, 11). The plasmid named pGL3–3Bpro contains three NF-B sites in tandem. FRTL-5 cells were plated in six-well dishes (1 x 105 cells/well) and grown in the presence of TSH for 3 d. pGL3–3Bpro (each 2 μg/well) was transfected into the cells using a Lipofectamine reagent (Invitrogen, Carlsbad, CA). A ?-galactosidase-expressing plasmid (0.2 μg/well; pSV-?-gal; Promega, Madison, WI) was also transfected to monitor the efficiency of transfection. After 18 h incubation with the liposome-DNA solution, it was replaced with a fresh medium with TSH. After an additional 24-h incubation, the cells were pretreated with 50 μM H89 for 60 min and treated with 40 ng/ml TNF- for 8 h. The cells were then harvested, and the luciferase and ?-galactosidase activities in the cell lysates were determined by a luminometer.
PKA activity assay
The cells cultured in 6-cm dishes were lysed in 300 μl lysis buffer (20 mM 4-morpholinepropanesulfonic acid, 50 mM ?-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethanesulfonylfluoride, and 10 μg/ml leupeptin and aprotinin) and centrifuged at 13,000 rpm for 15 min. The supernatant, cytosolic fractions were used to determine kinase activity using a PKA kinase activity assay kit (Stressgen Biotechnologies Corp., Victoria, British Columbia, Canada). The assay is based on a solid-phase ELISA using a specific synthetic peptide as a substrate for PKA and antibody against the phosphorylated form of the substrate. Amounts of activated PKAc in the samples were determined referring to the standard curve of activities of active, purified PKA included in the kit and expressed as nanograms per microgram of total protein.
Statistical analysis
Statistical analysis was performed by one-way ANOVA followed by Student’s t test. P < 0.05 was considered significant.
Results
TSH is required for TNF-- and LPS-dependent p65/p50 heterodimer activation in thyroid cells
FRTL-5 cells cultured in the absence or presence of TSH were treated with TNF- or LPS. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe, which contains a canonical NF-B site. As shown in Fig. 1A, TNF- activated a single protein/DNA complex in the absence of TSH. In contrast, in the presence of TSH, TNF- induced an additional slow-migrating complex. Similar NF-B activation was observed in response to LPS. Supershift analysis using antibodies against various Rel family proteins revealed that a fast-migrating complex contains p50, but not p65, and that a slow-migrating complex represents p65/p50 heterodimer NF-B (data not shown), compatible with our previous report (6). The results demonstrated that TSH modifies the composition of NF-B subunits activated by TNF- or LPS in rat thyroid FRTL-5 cells.
FIG. 1. Effects of TSH on TNF-- and LPS-dependent activation of NF-B in thyroid follicular cells. FRTL-5 cells (A) and human thyroid follicular cells (B) cultured in the absence (–) or presence (+) of TSH for 3 d were treated with TNF- or LPS for 30 min. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe. Representative BAS 2000 images are shown. Fast- and slow-migrating complexes are indicated by white and black arrowheads, respectively.
Similar results were obtained from primary human thyroid follicular cells. As shown in Fig. 1B, TNF- mainly induced a fast-migrating complex in the absence of TSH. However, in the presence of TSH, TNF- induced a slow-migrating complex as well as a fast-migrating one, indicating that TSH is required for the p65 activation not only in rat but also in human thyroid cells.
Lack of TSH effect on TNF--dependent IB degradation and on p65 nuclear translocation
To address the mechanism of how TSH mediates the p65 activation, we first examined whether TSH modifies degradation of IB and -? in response to TNF- in FRTL-5 cells. Cytosolic extracts were subjected to Western blot analysis using anti-IB and -? antibodies. As shown in Fig. 2, in both the absence and presence of TSH, IB was degraded in response to TNF- with maximal degradation at 30 min and was recovered at 60 min. IB expression was shown to be positively regulated by NF-Bs, which include p65/p50 and p50-containing NF-B, through the NF-B-binding sites in the promoter (12). Therefore, the increase of IB at 60 min could be due to the NF-B activation by TNF-. In contrast, IB? was not degraded in the absence of TSH, whereas it was degraded in the presence of TSH. Actin levels were not altered by TNF-. Nuclear translocation of p65 occurred in both the absence and presence of TSH as evidenced by Western blot analysis using nuclear extracts. These results demonstrate that, even in the absence of TSH, TNF- induces nuclear translocation of p65, but this p65 has no DNA-binding ability. Thus, it is speculated that TSH-dependent posttranslational modification such as phosphorylation is required for DNA binding of p65.
FIG. 2. Effects of TSH on TNF--dependent degradation of IB and IB?, and nuclear translocation of p65. FRTL-5 cells cultured in the absence (–) or presence (+) of TSH for 3 d were treated with TNF- for the indicated time. Cytosolic extracts were subjected to Western blot analysis using anti-IB, anti-IB?, and antiactin antibodies, and nuclear extracts were subjected to the analysis using anti-p65 antibody.
H89 inhibits TNF--dependent p65/p50 activation
Thus, we examined possible involvement of phosphorylation in p65 binding. The nuclear extracts prepared from FRTL-5 cells cultured with TSH and TNF- were treated with BAP in vitro and subjected to EMSA. As shown in Fig. 3A, BAP treatment markedly attenuated DNA binding of p65/p50 heterodimer, suggesting that phosphorylation is required for the binding.
FIG. 3. Phosphorylation of p65 by PKA enhances its DNA-binding and transcriptional activities. A and B, Nuclear extracts were prepared from FRTL-5 cells cultured in the presence of TSH followed by treatment with TNF- for 30 min. They were then treated with or without 0.025 U/μl BAP at 37 C for 60 min and subjected to EMSA using Bwt oligonucleotide as a probe (A). FRTL-5 cells cultured in the presence of TSH were pretreated with 50 μM H89 for 60 min and treated with TNF- for 30 min. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe (B). Representative BAS 2000 images are shown. Fast- and slow-migrating complexes are indicated by white and black arrowheads, respectively. C, pGL3–3Bpro, a luciferase reporter plasmid driven by three canonical NF-B-binding sites, was transfected into FRTL-5 cells cultured in the presence of TSH. After 42 h incubation, the cells were pretreated with 50 μM H89 for 60 min and treated with TNF- for 8 h. The promoter activity was expressed as luciferase activity normalized by ?-galactosidase activity. The promoter activity of control (Cont) was arbitrarily expressed as 100. Mean ± SD (n = 3). *, P < 0.01 vs. control; #, P < 0.01 vs. TNF--treated cells.
Because most of TSH actions are mediated by cAMP-PKA pathway, we next investigated whether PKA is involved in p65 activation in FRTL5 cells by using a specific PKA inhibitor H89 that directly inhibits kinase activity of PKA in a competitive fashion against ATP (13). FRTL-5 cells cultured with TSH were pretreated with H89 and treated with TNF-. As shown in Fig. 3B, EMSA revealed that H89 markedly diminished the binding of p65/p50. Compatible with this result, luciferase reporter gene assay demonstrated that H89 markedly attenuated the transcriptional activity of p65/p50 in the presence of TSH (Fig. 3C). These results strongly suggested the involvement of PKA in phosphorylation of p65, which leads to TNF--dependent increase in DNA binding and transcriptional activities.
Adenylate cyclase inhibitor does not affect p65 activation in the presence of TSH
To further study the involvement of PKA in TNF--dependent activation of p65/p50, we next examined effects of an adenylate cyclase inhibitor, SQ22536, on the activation. As shown in Fig. 4A, the cells cultured in the presence of TSH were treated with SQ22536 followed by TNF-, and the nuclear extracts were subjected to EMSA. Unexpectedly, DNA binding of p65/p50 heterodimer was not diminished even in the presence of SQ22536 for 4 h. Because SQ22536 is effective in reducing TSH-dependent activation of PKAc (treatment of the cells with SQ22536 for 4 h significantly decreased the PKAc activity in the cytosol; Fig. 4B), these results suggested that PKA activated by TSH/cAMP may not directly contribute to the p65 phosphorylation.
FIG. 4. Effects of adenylate cyclase inhibitor on activation of PKAc and NF-B. A, FRTL-5 cells cultured in the presence of TSH were pretreated with 30 μM of an adenylate cyclase inhibitor, SQ22536 (SQ), for the indicated time (2–4 h). During the last 1 h, the cells were treated with TNF-. Nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe. Representative BAS 2000 image is shown. Fast- and slow-migrating complexes are indicated by white and black arrowheads, respectively. B, FRTL-5 cells cultured in the presence of TSH were treated for 4 h with 30 μM SQ22536 (SQ). During the last 1 h, the cells were treated with TNF-. Cytosolic fractions were used to determine PKAc activity. Amounts of activated PKAc were expressed as nanograms per microgram of total protein. Mean ± SD (n = 3). *, P < 0.01 vs. SQ (–) and TNF- (–); #, P < 0.01 vs. SQ (+) and TNF- (–).
On the other hand, it should be noted that the decrease in PKAc activity by SQ22536 was restored by the treatment with TNF- (Fig. 4B). This result suggested the presence of PKAc in thyroid cells, which can respond to TNF- and is not derived from a canonical heterotetramer complex of PKAc/PKAr. Because the presence of PKAc/IB complex that responds to LPS was demonstrated in immune cells (4), we questioned whether such a complex is present in FRTL-5 cells.
IB and -? form complexes with PKAc in the presence of TSH
Cytoplasmic extracts were prepared from the cells cultured with TSH and subjected to immunoprecipitation using anti-IB antibody or normal rabbit serum (NRS). The immunoprecipitates were then subjected to Western blot analysis using anti-PKAc antibody. As shown in Fig. 5A, when cytoplasmic extract without immunoprecipitation was subjected to Western blot analysis, a single band with a molecular weight of around 40,000 was detected. The size is compatible with the expected mass of PKAc. No PKAc band was detected, when NRS was used for immunoprecipitation. In contrast, when anti-IB antibody was used for immunoprecipitation, a single band corresponding to the PKAc band was detected, demonstrating that IB forms a complex with PKAc in FRTL-5 cells cultured with TSH.
FIG. 5. Effects of TSH on complex formation of PKAc with IB or IB?. A, Cytosolic extracts were prepared from FRTL-5 cells cultured in the presence of TSH. Immunoprecipitation (IP) was performed by NRS or anti-IB antibody. The precipitated samples were subjected to Western blot analysis (WB) using anti-PKAc antibody. Cyto, cytosolic extract without immunoprecipitation was used for Western blot analysis. The position of 35,000 molecular weight marker is indicated. B, FRTL-5 cells cultured in the absence of TSH for 4 d were treated with or without TSH for 3 d. Cytosolic extracts were subjected to immunoprecipitation (IP) using NRS and anti-IB and anti-IB? antibodies, followed by Western blot analysis (WB) using anti-PKAc antibody. Similar results were obtained from separate experiments.
We then examined whether IB? also forms a complex and whether TSH affects the complex formation. Cytoplasmic extracts prepared from FRTL-5 cells cultured with or without TSH were subjected to immunoprecipitation and Western blot analysis. As shown in Fig. 5B, IB and ? were not bound with PKAc in the absence of TSH. However, both IBs formed a complex with PKAc in the presence of TSH, raising a possibility that the PKAc bound with IBs would play an important role in p65 activation in the presence of TSH. To prove this possibility, we performed the following experiments.
p65 Activation is associated with formation of IB/PKAc complex
Duration of TSH exposure required for TNF--dependent p65 activation was investigated. FRTL-5 cells cultured with TSH for various lengths of time were treated with TNF-. As shown in Fig. 6A, EMSA revealed that a 3-h exposure to TSH was not sufficient for p65 activation. More than 3 h exposure was required for the activation. It should be noted that formation of IB/PKAc complex after TSH exposure occurred in a similar time course with the p65 activation (Fig. 6B). When amounts of PKAc bound with IB were analyzed by immunoprecipitation and Western blot analysis, more than 3 h exposure of TSH was required for the complex formation, demonstrating that TNF--dependent p65 activation was temporally associated with the formation of IB/PKAc complex. Total amounts of cytosolic PKAc were not altered by TSH (Fig. 6C). Taken together, these observations strongly suggest that PKAc, which is liberated from IB/PKAc complex in response to TNF- but not from PKAc/PKAr complex, is directly involved in p65 activation.
FIG. 6. Time courses of p65 activation in response to TNF- and of formation of PKAc/IB complex after TSH treatment. FRTL-5 cells cultured in the absence of TSH for 4 d were treated with TSH for various lengths of time. A, Cells were then treated with TNF- for 30 min, and the nuclear extracts were subjected to EMSA using Bwt oligonucleotide as a probe. B, Cytosolic extracts were subjected to immunoprecipitation (IP) using anti-IB antibody, followed by Western blot analysis (WB) using anti-PKAc antibody. C, Cytosolic extracts were subjected to Western blot analysis (WB) using anti-PKAc antibody.
TSH-dependent IB/PKAc complex formation is mediated by the cAMP/PKA pathway
To define intracellular signaling pathway leading to the formation of IB/PKAc complex, effects of a PKC activator PMA and an adenylate cyclase activator forskolin on the complex formation were investigated. Because 6 h exposure to TSH was sufficient for the complex formation (Fig. 6B), FRTL-5 cells cultured in the absence of TSH were treated for 6 h with PMA, forskolin, TSH, SQ22536, and H89, alone or in combination. Cytosolic extracts were subjected to immunoprecipitation with anti-IB antibody, followed by Western blot analysis using anti-PKAc antibody. As shown in Fig. 7, no IB/PKAc complex was present in the cells cultured without TSH (lane 2). PMA did not induce the complex formation (lane 3). In contrast, forskolin and TSH induced the complex formation (lanes 4–6). SQ22536 (lane 7) and H89 (lane 8) prevented the TSH action. Forskolin-induced complex formation was also inhibited by H89 (data not shown). These results clearly demonstrate that the TSH-dependent IB/PKAc complex formation is mainly attributable to the cAMP/PKA-mediated TSH action but not to the PKC-mediated action.
FIG. 7. TSH-dependent IB/PKAc complex formation is mediated by cAMP/PKA pathway. FRTL-5 cells cultured in the absence of TSH for 4 d were treated for 6 h with 10 nM PMA, 10 μM forskolin (For), 0.25 mU/ml TSH, 1 mU/ml TSH, 1 mU/ml TSH plus 30 μM SQ22536 (SQ), or 1 mU/ml TSH plus 50 μM H89. The cytosolic extracts were subjected to immunoprecipitation (IP) with anti-IB antibody, followed by Western blot analysis (WB) using anti-PKAc antibody. Cyto, Cytosolic extract without immunoprecipitation was used for Western blot analysis.
Discussion
We previously demonstrated that TSH is required for TNF--dependent activation of p65 NF-B in rat thyroid FRTL-5 cells (6). The present study, to clarify the molecular mechanism of this TSH action, demonstrates that PKAc plays a pivotal role in p65 activation. PKAc does not affect nuclear translocation of p65 (Fig. 2) but increases its DNA-binding and transcriptional activities (Fig. 3). These observations are compatible with previous reports. Shirakawa and Mizel (14) first demonstrated that treatment of cytosolic extracts prepared from 70Z/3 murine pre-B cells with PKAc in vitro induced DNA-binding activity of NF-B. Naumann and Scheidereit (15) demonstrated that TNF--dependent phosphorylation of p65 is associated with its increased binding activity and that treatment of recombinant p65 with PKAc in vitro enhances its binding. It has been also shown, in various cells, that PKA inhibitors including H89 block the activation of NF-B, leading to decreased DNA-binding and transcriptional activities (16, 17, 18, 19). In addition, it was recently shown that IB-bound PKAc is involved in phosphorylation of p65 and, thereby, its transcriptional activity (4). However, some reports showed the opposite effects of PKA. Stimulation of PKA activity reduced p65 nuclear translocation and/or transcriptional activity (20, 21, 22). Therefore, it is suggested that PKA is involved in multiple steps of p65 activation process, and its effects could vary depending on cell types and culture conditions including external stimuli used.
Consistent with the report by Zhong et al. (4), our present study also demonstrated the interaction of endogenous PKAc with IB or -? in cytoplasm (Fig. 5), indicating that thyroid cells possess a pool of PKAc other than the well-established pool that consists of heterotetramer PKAc/PKAr complex. More importantly, it was demonstrated in this study that the formation of PKAc/IB complex is dependent on TSH; the complex was not detected in FRTL-5 cells cultured in the absence of TSH (Fig. 5). The absence of IB and/or PKAc proteins in cytoplasm is not the case because their expression in cytoplasm was detected by Western blot analysis even in the absence of TSH, and their contents were not affected by TSH (Figs. 2 and 6). Therefore, it is indicated that, even when both PKAc and IBs are present in cytoplasm, PKAc does not necessarily form a complex with IB.
Recently, it has been realized that PKA is localized at particular intracellular places through its association with A-kinase anchoring proteins that consist of more than 20 members, and this compartmentalization of PKA would account for site-, time-, and protein-specific phosphorylation by PKA (5, 23). For example, it was shown that AKAP79 can be associated with ?2-adrenergic receptor, one of G protein-coupled receptors, and with PKC as well as PKAc/PKAr, thereby forming a multicomponent signaling complex at the site of action (23, 24). On the other hand, it has been also shown that IB is a component of a large multiprotein complex so-called "signalsome" that is composed of IB kinases and interacting proteins (25, 26). Therefore, in thyroid follicular cells cultured in the absence of TSH, PKAc, and IB may be present in separate compartments in cytoplasm. When the cells are stimulated with TSH, formation of PKAc/IB complex is achieved, and this process requires more than 3 h stimulation with TSH. Such prolonged stimulation with TSH is likely to be required for the creation of new compartmentalization of PKAc/IB, the process of which may include de novo protein synthesis, intracellular protein trafficking, and/or complex formation.
Involvement of PKAc bound with IB in p65 activation was suggested by the experiment using adenylate cyclase inhibitor SQ22536 (Fig. 4). It was shown that 4 h exposure of this inhibitor did not affect TNF--dependent p65 activation in FRTL-5 cells cultured with TSH, suggesting that PKAr-bound PKAc does not directly contribute to the activation. Furthermore, it was shown that time course of TNF--dependent p65 activation after TSH exposure was well associated with that of the complex formation of PKAc/IB (Fig. 6). Therefore, these results strongly suggest the major role of IB-bound PKAc in p65 activation in FRTL-5 cells.
The present study showed that PKAc is involved in both DNA binding and transcriptional activities of p65. Recently, several phosphoserine residues in p65 have been identified. Ser276, Ser529, and Ser536 can be phosphorylated in cells by PKA, casein kinase II, and IB kinase, respectively (27, 28, 29, 30). Ser276 is also phosphorylated by mitogen- and stress-activated protein kinase-1 (31). When Ser276 is phosphorylated by PKA, it induces a conformational change of p65 molecule and creates a site for interaction of cAMP response element-binding protein/p300, thereby enhancing transcriptional activity of p65. On the other hand, phosphorylation of Ser276 as well as Ser529 and Ser536 has been shown to have little effect on DNA binding. However, more recently, Hou et al. (32) reported that phosphorylation of Ser337 in p50 (this residue corresponds to Ser276 in p65) and that of Ser276 in p65 augment the binding activity by inducing a conformational change in the hinge region of NF-B. It is, therefore, likely that PKA can regulate both transcriptional and binding activities of p65 via phosphorylation of Ser276.
In conclusion, the present study demonstrates that TSH induces the formation of PKAc/IB complex in cytoplasm of FRTL-5 thyroid cells and that this PKAc bound with IB plays a critical role in TNF--dependent activation of p65 in the cells cultured with TSH. These findings provide a novel aspect of biological effects of TSH on thyroid cells. Growing evidence has indicated that TNF- and NF-B are involved in pathogenesis of various autoimmune diseases. Recently, it is also shown that NF-B and its upstream kinase are key factors to link inflammation to cancer in some in vivo cancer models (33, 34). It is thus speculated that TSH might be implicated in tumorigenesis or promotion of thyroid cancers as well as pathogenesis of autoimmune thyroid diseases through the modulation of p65 activation.
References
Baldwin Jr AS 1996 The NF-B and IB proteins: new discoveries and insights. Annu Rev Immunol 14:649–683
Zandi E, Rothwarf DM, Delhase M, Hayakawa M, Karin M 1997 The IB kinase complex (IKK) contains two kinase subunits, IKK and IKK?, necessary for IB phosphorylation and NF-B activation. Cell 91:243–252
DiDonato J, Mercurio F, Rosette C, Wu-Li J, Suyang H, Ghosh S, Karin M, Roff M 1996 Mapping of the inducible IB phosphorylation sites that signal its ubiquitination and degradation: role of IB ubiquitination in signal-induced activation of NFB in vivo. Mol Cell Biol 16:1295–1304
Zhong H, SuYang H, Erdjument-Bromage H, Tempst P, Ghosh S 1997 The transcriptional activity of NF-B is regulated by the IB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89:413–424
Carnegie GK, Scott JD 2003 A-kinase anchoring proteins and neuronal signaling mechanisms. Genes Dev 17:1557–1568
Kikumori T, Kambe F, Nagaya T, Funahashi H, Seo H 2001 Thyrotropin modifies activation of nuclear factor B by tumour necrosis factor- in rat thyroid cell line. Biochem J 354:573–579
Kambe F, Seo H 1996 Mediation of the hormone- and serum-dependent regulation of thyroglobulin gene expression by thyroid-transcription factors in rat thyroid FRTL-5 cells. J Endocrinol 150:287–298
Kambe F, Nomura Y, Okamoto T, Seo H 1996 Redox regulation of thyroid-transcription factors, Pax-8 and TTF-1, is involved in their increased DNA-binding activities by thyrotropin in rat thyroid FRTL-5 cells. Mol Endocrinol 10:801–812
Kikumori T, Kambe F, Nagaya T, Imai T, Funahashi H, Seo H 1998 Activation of transcriptionally active nuclear factor-B by tumor necrosis factor- and its inhibition by antioxidants in rat thyroid FRTL-5 cells. Endocrinology 139:1715–1722
Baeuerle PA, Baltimore D 1988 IB: a specific inhibitor of the NF-B transcription factor. Science 242:540–546
Cao X, Kambe F, Ohmori S, Seo H 2002 Oxidoreductive modification of two cysteine residues in paired domain by Ref-1 regulates DNA-binding activity of Pax-8. Biochem Biophys Res Commun 297:288–293
Le Bail O, Schmidt-Ullrich R, Israel A 1993 Promoter analysis of the gene encoding the IB-/MAD3 inhibitor of NF-B: positive regulation by members of the rel/NF-B family. EMBO J 12:5043–5049
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272
Shirakawa F, Mizel SB 1989 In vitro activation and nuclear translocation of NF-B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol Cell Biol 9:2424–2430
Naumann M, Scheidereit C 1994 Activation of NF-B in vivo is regulated by multiple phosphorylations. EMBO J 13:4597–4607
Geng Y, Zhang B, Lotz M 1993 Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes. J Immunol 151:6692–6700
Lapointe R, Lemieux R, Olivier M, Darveau A 1996 Tyrosine kinase and cAMP-dependent protein kinase activities in CD40-activated human B lymphocytes. Eur J Immunol 26:2376–2382
Um JH, Kang CD, Lee BG, Kim DW, Chung BS, Kim SH 2001 Increased and correlated nuclear factor-B and Ku autoantigen activities are associated with development of multidrug resistance. Oncogene 20:6048–6056
Bhat-Nakshatri P, Sweeney CJ, Nakshatri H 2002 Identification of signal transduction pathways involved in constitutive NF-B activation in breast cancer cells. Oncogene 21:2066–2078
Neumann M, Grieshammer T, Chuvpilo S, Kneitz B, Lohoff M, Schimpl A, Franza BJ, Serfling E 1995 RelA/p65 is a molecular target for the immunosuppressive action of protein kinase A. EMBO J 14:1991–2004
Ollivier V, Parry GC, Cobb RR, de Prost D, Mackman N 1996 Elevated cyclic AMP inhibits NF-B-mediated transcription in human monocytic cells and endothelial cells. J Biol Chem 271:20828–20835
Takahashi N, Tetsuka T, Uranishi H, Okamoto T 2002 Inhibition of the NF-B transcriptional activity by protein kinase A. Eur J Biochem 269:4559–4565
Griffioen G, Thevelein JM 2002 Molecular mechanisms controlling the localisation of protein kinase A. Curr Genet 41:199–207
Fraser ID, Cong M, Kim J, Rollins EN, Daaka Y, Lefkowitz RJ, Scott JD 2000 Assembly of an A kinase-anchoring protein-?2-adrenergic receptor complex facilitates receptor phosphorylation and signaling. Curr Biol 10:409–412
Mercurio F, Zhu H, Murray BW, Shevchenko A, Bennett BL, Li J, Young DB, Barbosa M, Mann M, Manning A, Rao A 1997 IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-B activation. Science 278:860–866
Yamamoto Y, Kim DW, Kwak YT, Prajapati S, Verma U, Gaynor RB 2001 IKK /NEMO facilitates the recruitment of the IB proteins into the IB kinase complex. J Biol Chem 276:36327–36336
Zhong H, Voll RE, Ghosh S 1998 Phosphorylation of NF-B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1:661–671
Wang D, Baldwin Jr AS 1998 Activation of nuclear factor-B-dependent transcription by tumor necrosis factor- is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem 273:29411–29416
Wang D, Westerheide SD, Hanson JL, Baldwin Jr AS 2000 Tumor necrosis factor -induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem 275:32592–32597
Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W 1999 IB kinases phosphorylate NF-B p65 subunit on serine 536 in the transactivation domain. J Biol Chem 274:30353–30356
Vermeulen L, De Wilde G, Van Damme P, Vanden Berghe W, Haegeman G 2003 Transcriptional activation of the NF-B p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J 22:1313–1324
Hou S, Guan H, Ricciardi RP 2003 Phosphorylation of serine 337 of NF-B p50 is critical for DNA binding. J Biol Chem 278:45994–45998
Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, Gutkovich-Pyest E, Urieli-Shoval S, Galun E, Ben-Neriah Y 2004 NF-B functions as a tumour promoter in inflammation-associated cancer. Nature 431:461–466
Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, Kagnoff MF, Karin M 2004 IKK? links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 118:285–296(Xia Cao, Fukushi Kambe an)