1Department of Internal Medicine, Scott and White Clinic, Texas A&M University System Health Science Center College of Medicine, Temple, Texas; and 2Department of Immunology, The Scripps Research Institute, La Jolla, California
Submitted 24 February 2005 ; accepted in final form 19 May 2005
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ABSTRACT |
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cytokines; intracellular kinases; cancer
Interleukin-6 (IL-6) is a multifunctional cytokine that mediates diverse tissue responses in response to environmental stimuli (21). The expression of IL-6 is increased during infection, trauma, or cellular stresses. In addition to a major role in host cell defense and the regulation of inflammatory responses, IL-6 signaling pathways have been shown to contribute to tumor progression in epithelial (e.g., cholangiocarcinoma) as well as hematopoeitic (e.g., multiple myeloma) human tumors (41). These may involve IL-6 effects on either cell proliferation or cell survival. Cell survival resulting from inhibition of apoptosis by IL-6 has also been described in several settings (6, 17, 29). Although signaling pathways mediating proliferative responses by IL-6 are well characterized, signaling for cell survival remains poorly understood (20, 21). The activation of cell survival signaling can promote tumor growth by providing neoplastic cells with the ability to survive under adverse environmental conditions such as limited growth factor availability or during treatment with cytotoxic drugs.
The p38 mitogen-activated protein kinase (MAPK), like SGK, acts as a critical intracellular regulator of environmental changes. The p38 MAPK pathway can be activated by heat shock, bacterial lipopolysaccharide, inflammatory cytokines, UV radiation, or hormones and is an important signal transduction mechanism mediating cellular responses to environmental changes. Activation of p38 MAPK has diverse downstream signaling effects, including cell cycle progression, apoptosis, differentiation, and cellular inflammatory responses (33). Several lines of evidence support a critical role of p38 MAPK signaling in IL-6-mediated signaling during growth of human tumors. IL-6 is an autocrine factor involved in human cholangiocarcinoma growth (34). We have shown that IL-6 stimulation activates the p38 MAPK in malignant but not in nonmalignant biliary tract epithelia (35, 37). Furthermore, we have also demonstrated that inhibition of p38 MAPK signaling reduces anchorage-independent growth in vitro and decreases xenograft growth in immunodeficient mice (42, 49). In addition, p38 MAPK signaling activates a dominant cell survival pathway in response to double-stranded RNA, a potent inducer of IL-6 expression (43). These observations suggest that IL-6 may promote cell survival by p38 MAPK-dependent pathways. However, the downstream mediators of survival signaling by p38 MAPK are poorly characterized. Our results show that IL-6 can regulate SGK by multiple p38 MAPK-dependent mechanisms, including regulation of transcriptional expression as well as phosphorylation of SGK at the Ser78 residue. Furthermore, regulation of SGK involves constitutive p38 MAPK activity. Several distinct isoforms of p38 MAPK, p38-, -
, -
, and -
have been identified in mammalian cells. Although these isoforms have 6070% identity, there is considerable overlap in their substrate specificities in vitro. Our studies show that SGK regulation by IL-6 specifically involves the p38
isoforms. In addition, chemotherapeutic drug toxicity in malignant biliary epithelial cells is increased in cells in which SGK expression is reduced. Taken together, these results suggest that survival signaling by IL-6 may involve transcriptional as well as posttranslational regulation of the survival kinase SGK via a p38 MAPK-dependent pathway.
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MATERIALS AND METHODS |
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Cell lines and cultures. Malignant human cholangiocarcinoma cell lines KMCH-1 and Mz-ChA-1 were obtained as previously described (42, 43). KMCH-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, and Mz-ChA-1 cells were cultured in CMRL 1066 medium with 10% fetal bovine serum, 1% L-glutamine, and 1% antimycotic antibiotic mix. Cell culture media and supplements were obtained from Invitrogen (St. Louis, MO) unless otherwise noted. Cells were cultured in 100-mm plates and used when 7580% confluent. Cell lines overexpressing IL-6 for xenograft studies were obtained from Mz-ChA-1 cells stably transfected with the pTarget (Promega, Madison, WI) expression plasmid containing full-length IL-6 under the control of a cytomegalovirus (CMV) promoter and selected by growth in medium containing G418. These cell lines were designated as Mz-IL-6, and empty vector controls cells were designated as Mz-1, respectively, and comprised a mixed population of stable transfectants without isolation of specific clones. For studies involving IL-6, cells were serum starved for 12 h before being stimulated with IL-6. For inhibitor studies, cells were preincubated with inhibitors 1 h before the addition of IL-6, except for studies with phosphatidylinositol 3-kinase (PI3-kinase) inhibitors, which were preincubated for 2 h.
Transient transfections. Cells (2 x 106) were plated in 100-mm dishes in culture media for 24 h. The media were then replaced, and plasmids were transfected in serum-free media using 1518 µg of plasmid DNA per dish and TransIT-LT1 transfection reagent (Mirus, Madison, WI). After 46 h, the media were replaced with regular media containing 10% serum and the cells were incubated for 48 h before study.
Construction of recombinant adenoviruses encoding wild-type and mutant p38.
The recombinant adenoviruses encoding the wild-type and mutant p38
were produced according to the method of He et al. (19) with some modifications. Briefly, the FLAG-tagged wild-type and mutant human p38
cDNAs were cloned into the shuttle vector pAdTrack-CMV, linearized, and cotransformed into Escherichia coli BJ5183 cells along with the adenoviral backbone vector pAdEasy-1. Recombinants were selected for kanamycin resistance and confirmed with the use of restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into an adenovirus packaging cell line: human embryonic kidney-293 cells. Recombinant adenoviruses were collected 1014 days after infection and were concentrated using a CsCl gradient. The shuttle vector pAdTrack-CMV also encodes green fluorescent protein (GFP) driven by a separate CMV promoter, and thus the titers of the viral stocks were estimated by counting GFP-expressing cells. An adenovirus-expressing GFP tag (AdGFP) under the control of a separate CMV promoter, which was a gift from Dr. Kim Heidenreich (Dept. of Pharmacology, University of Colorado HSC, Denver, CO), was used as a control.
Preparation of nuclear and cytoplasmic extracts. Nuclear and cytoplasmic fractions were obtained using the NE-PER extraction kit (Pierce, Rockford, IL) according to the manufacturer's instructions. Protein concentrations in nuclear and cytoplasmic fractions were determined using the Bradford method, and reagents were obtained from Bio-Rad (Hercules, CA).
Immunoprecipitation and in vitro kinase assay.
KMCH cells were stimulated with IL-6 in the presence or absence of p38 MAPK inhibitors. Cells were placed on ice and extracted with lysis buffer containing 50 mM -glycerophosphate, pH 7.3, 1.5 mM EDTA, 1 mM EGTA, 1 mM DTT, and phosphatase inhibitor cocktails I and II (Sigma). Lysates were centrifuged for 15 min at 12,000 g, and SGK was immunoprecipitated from 150 µg of cell extract using anti-SGK monoclonal antibody (Cell Signaling, Beverly, MA) and the Seize immunoprecipitation kit (Pierce, Rockford, IL). In vitro kinase assays were then performed using the SGK kinase assay kit (StressGen, Vancouver, BC, Canada).
Western blot analysis. After treatment, confluent cell monolayers in 100-mm dishes were washed twice with ice-cold phosphate-buffered saline and lysed by incubation for 20 min in 1 ml of ice-cold cell lysis buffer (1% Nonidet P-40, 50 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, and 1x protease mixture) and stored at 70°C. Protein concentrations were measured using a Bradford protein assay kit (Bio-Rad). Equivalent amounts of protein were resolved and mixed with 6x SDS-PAGE sample buffer and then subjected to SDS-PAGE in a 420% linear gradient Tris·HCl-ready gel (Bio-Rad). The resolved proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk (except for phospho-SGK and p38 Western blots, in which 5% bovine serum albumin was used) in Tris-buffered saline, pH 7.4, containing 0.05% Tween 20, and were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies according to the manufacturer's instructions. The protein of interest was visualized with enhanced chemiluminescence (Amersham Biosciences) and a charge-coupled device camera-based imager (ChemiImager 4000, Alpha Innotech, San Leandro, CA) and quantitated using NIH Image software.
Isolation of RNA and real-time PCR quantification.
RNA was isolated using an RNA isolation kit (Bio-Rad), and reverse transcription was performed using 1 µg of total RNA and the reverse transcription kit (Invitrogen) as described by the manufacturer. To measure the mRNA levels of SGK, quantitative real-time PCR was performed on a MX 3000P real-time PCR Instrument (Stratagene, San Diego, CA) using the resulting total cDNA. The mRNA level of -actin was used as a control. For the quantitative SYBR Green real-time PCR, 100 ng of cDNA was used per reaction. Each 20-µl SYBR Green reaction consisted of 2 µl of cDNA (50 ng/µl), 10 µl of 2x Universal SYBR Green PCR Master Mix (Sigma), and 2 µl of 20 nM forward and reverse primers. Optimization was performed for each gene-specific primer prior to the experiment to confirm that primer concentrations and reaction conditions did not produce artificial amplification signals in the no-template control tubes. Primer sequences were designed using Primer Express software (PerkinElmer Life Sciences) and were as follows: SGK forward, CCT TGT GGA TAT GCT GTG TGA ACC G; SGK reverse, 5'-TGG GGC ATT GGT CCA TAA AAA CC-3'; IL-6 forward, 5'-GCA GAA TGA GAT GAG TTG TC-3'; IL-6 reverse, 5'-GCC TTC GGT CCA GTT GCC TT-3';
-actin forward, 5'-CCA AGG CCA ACC GCG AGA AGA TGA C-3'; and
-actin reverse, 5'-AGG GTA CAT GGT GGT GCC GCC AGA C-3'. PCR parameters were as follows: 10 min at 95°C and then 40 cycles of 30 s at 95°C, 1 min at 68°C, and 1 min at 72°C. Specificity of the produced amplification product was confirmed by melting curve analysis of the reaction products using SYBR Green as well as by visualization on ethidium bromide-stained 1.8% agarose gels to confirm a single band of the expected size. Each sample was tested in triplicate with quantitative real-time PCR, and samples obtained from at least three independent experiments were used for calculation. Threshold values were determined for each sample-primer pair, and means ± SD values were calculated. SGK or IL-6 mRNA expression was normalized against
-actin expression.
Nude mouse xenograft model. Eight-week-old male athymic nu/nu mice were obtained from Charles River Laboratories (Wilmington, MA) and fed food and water ad libitum. The mice were housed 4 per cage, and fluorescent light was controlled to provide alternate light and dark cycles of 12 h each. The animals received a subcutaneous injection of either Mz-1 or Mz-IL-6 cells (3 x 106 viable cells suspended on 0.5 ml of extracellular matrix gel) on their right flanks. Tumor volume was estimated by serial measurements obtained twice per week. The xenografts were excised. Tissue was divided and homogenized to obtain cell lysates or used for extraction of nuclear proteins or mRNA isolation. Animal protocols were approved by the Institutional Animal Care and Use Committee.
RNA interference. RNA interference for gene silencing was performed using small interfering 21-nucleotide double-stranded RNA (siRNA) molecules. SiRNA specific for SGK and control siRNA were obtained from Ambion (Austin, TX). KMCH cells were transfected as previously described (49). Briefly, 0.1 µg of siRNA was mixed with 6 µl of transfection agent (TransIt TKO, Mirus, Madison, WI), and the mixture was incubated in 1 ml of medium at room temperature for 1520 min before being added to cultured cells grown to 5060% confluence for 48 h. The efficacy of gene silencing was assessed by immunoblot analysis.
Cytotoxicity assay. Transfected cells were seeded into 96-well plates (10,000 viable cells/well) and incubated with gemcitabine, 5-fluorouracil, or appropriate diluent controls in a final volume of 200-µl medium. After 24 h, cell viability was assessed using a commercially available tetrazolium bioreduction assay for viable cells (CellTiter 96 AQ; Promega, Madison, WI), and cytotoxicity was assessed as previously described (43).
Statistical analysis. Data are expressed as the means ± SE from at least three separate experiments performed in triplicate, unless otherwise noted. The differences between groups were analyzed using a double-sided Student's t-test when only two groups were present. Statistical significance was considered to be P < 0.05. Statistical analyses were performed with the GB-STAT statistical software program (Dynamic Microsystems, Silver Spring, MD).
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RESULTS |
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IL-6-dependent SGK Ser78 phosphorylation is PI3-kinase independent. IL-6 receptor ligation results in the activation of several intracellular signaling pathways, such as JAK-STAT, ERK, p38 MAPK, and PI3-kinase pathways (20). Our studies with pharmacological inhibitors indicated that PI3-kinase or p38 MAPK pathways can mediate the survival effect of IL-6 (data not shown). Thus SGK activation by IL-6 may involve signaling by activation of PI3-kinase signaling. The mechanism of SGK activation by PI3-kinase signaling involves reversible phosphorylation at the Thr256 site in the activation loop of SGK (36). Thr256 resides in the action loop of the catalytic domain and is a major site for activation of SGK by phosphorylation by the 3-phosphoinositide-dependent kinases. Although considered to be the major site for the activation of SGK, the presence of additional active phosphorylation sites is suggested by studies showing that mutagenesis of Thr256 does not abrogate total cellular phosphorylation of SGK and is supported by the identification of Ser78 as a phosphorylation- dependent site for SGK activation by the BMK-1 (18). Thus we evaluated phosphorylation of SGK at both Thr256 and Ser78 using phosphorylation site-specific antibodies. As expected, phosphorylation at both sites occurred following stimulation by IL-6. However, incubation with the PI3-kinase inhibitor wortmannin inhibited IL-6-induced SGK phosphorylation at Thr256 but not Ser78 (Fig. 3). Furthermore, the increased translocation of SGK to the nucleus after IL-6 treatment was not substantially reduced by either wortmannin or by LY-294002, two specific inhibitors of PI3-kinase, even when used at relatively high concentrations (Fig. 3B). Similarly, translocation of p38 MAPK to the nucleus and reduction of SGK levels in the cytoplasm was not affected by wortmannin (data not shown). Taken together, these observations place SGK phosphorylation downstream of p38 MAPK following IL-6 stimulation in a pathway distinct from PI3-kinase signaling.
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Upregulation of SGK mRNA expression by IL-6 and/or p38 MAPK stimulation.
To further examine the effects of p38
overexpression on total SGK protein expression, we performed real-time PCR to quantitate alterations in SGK transcript levels. Overexpression of p38
caused a concentration-dependent increase in SGK mRNA, supporting a direct transcriptional rather than posttranslational mechanism for increased SGK protein expression after overexpression of p38
(Fig. 7, A and B). On the basis of these observations, we postulated that transcriptional upregulation of SGK may represent an additional mechanism involved in survival signaling by IL-6. To further examine the mechanism, we quantitated alterations in transcript levels with IL-6 stimulation. Basal levels of SGK mRNA were detectable by quantitative real-time PCR and increased in a concentration-dependent manner after treatment with IL-6 (Fig. 7C). A profound induction of mRNA was observed, with a remarkable >4-fold induction with IL-6 (10 ng/ml) after 6 h. The mRNA levels of
-actin were nearly identical in all of these samples, indicating the specificity of the response. These quantitative real-time PCR data demonstrate, for the first time, concentration-dependent transcriptional regulation of SGK by IL-6 stimulation as well as by p38
overexpression. Furthermore, the increased SGK mRNA expression correlated well with total SGK protein expression assessed by immunoblot analysis (Fig. 7D). The rapidity of the transcriptional response suggests that SGK expression may mediate the survival effects of IL-6. To establish whether IL-6 induces SGK mRNA by altering transcription or by altering mRNA stability, the expression of SGK was assessed in the presence of the transcriptional inhibitor actinomycin D. First, we assessed the effect of preincubation with actinomycin D on SGK mRNA expression induced by IL-6 or by p38
overexpression. The increase in SGK mRNA in response to either IL-6 stimulation or enforced expression of p38
was blocked in the presence of actinomycin D. Next, we assessed the time course of SGK mRNA expression in the presence of actinomycin D and did not observe any significant effect on the rate of loss of SGK mRNA levels. Thus IL-6 increases SGK mRNA by a transcriptional mechanism and does not alter SGK mRNA stability.
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DISCUSSION |
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Although it was initially identified as being under acute transcriptional control by serum and glucocorticoids, SGK has subsequently been shown to be stimulated by diverse stimuli such as osmotic and oxidative stress, growth factors, or hormones (1, 5, 28, 30, 32, 46). In contrast to that of most protein kinases, the regulation of SGK is complex and can occur at multiple levels involving transcriptional control, subcellular localization, and posttranslational modifications involving kinase phosphorylation/dephosphorylation (see Ref. 13 for review). These mechanisms are differentially regulated in a cell-type and stimulus-specific manner. The presence of multiple regulatory mechanisms for SGK activation suggests that this kinase acts as a common downstream integrator of the response to diverse stimuli and signaling cascades. The activation of SGK by IL-6 and p38 MAPK activation involves phosphorylation at the Ser78 residue in preference to classical SGK phosphorylation sites at Thr256 or Ser422 (36). Although Ser78 is not located within the catalytic domain, SGK activation has been shown to occur following phosphorylation at this site by BMK1, a member of the MAP kinase family (18). Unlike Thr256, the Ser78 site is not phosphorylated by members of the 3-phosphoinositide-dependent kinase family. The presence of PI3-kinase-independent mechanisms has been described previously for SGK activation under specific conditions such as by cell detachment, but the precise nature was not defined (40). Although p38 MAPK dependent phosphorylation at Ser78 residue occurs independently of the PI3-kinase pathway, our studies do not exclude an additional contribution of this or other p38 MAPK-independent signaling pathways during SGK activation by IL-6. Indeed, our studies indicate that IL-6 can phosphorylate the Thr256 site independent of p38 MAPK activation. Taken together, these observations indicate that SGK activation by IL-6 can occur via temporally separated mechanisms involving activation by different signaling cascades, phosphorylation at different sites, and transcriptional regulation. Concomitant activation of stimulus-dependent signaling cascades can activate different intracellular pathways that act in concert and converge onto SGK. Upstream regulators of SGK can thereby provide the mechanism for stimulus specificity by which SGK coordinates specific cellular responses. Thus SGK is well positioned to act as a central integration point of the cellular survival response to diverse physiological or pathophysiological stimuli mediated by either the p38 MAPK or the PI3-kinase pathways.
These studies identify SGK as a downstream target kinase for p38. Many substrates of p38 MAPK are themselves serine/threonine kinases. These include the MAPK-activated protein kinases (MK) such as MK2 and MK3, the p38-regulated and -activated protein kinases or MK5, the MAPK kinase-interacting kinases 1 and 2 (MNK-1 and MNK-2), and the mitogen- and stress-activated kinases 1 and 2. Downstream kinases can mediate physiologically relevant effects of p38 MAPK signaling as demonstrated in MK2-knockout mice, which are able to resist stress and survive endotoxin-induced septic shock (26). Transcriptional regulation of SGK expression by p38 MAPK provides an additional level of regulation of SGK by p38 MAPK. Several transcription factors have been identified as direct targets of the p38 MAPK signaling. These include activating transcription factor 2, CHOP/GADD153, and myocyte enhancer factor 2. Additional studies will be required to identify specific transcriptional factors involved in regulation of SGK expression by p38 MAPK. Because of the common responses to environmental stresses described for both p38 MAPK and SGK as well as the multiple mechanisms by which p38 MAPK can regulate SGK expression, it seems probable that the p38 MAPK/SGK axis is a critical mediator of the cellular response to perturbations in the extracellular environment.
Unlike most other kinases involved in p38 MAPK signaling, SGK is regulated transcriptionally as well as posttranslationally, raising the intriguing possibility that SGK may serve to amplify specific stress responses after p38 MAPK activation. Recently, the mitogen-activated protein kinase kinase kinase 3 (MEKK3) has been shown to be a target substrate for SGK (7). MEKK3 lies upstream of mitogen-activated protein kinase kinase (MKK) 3 and 6, which are activators of p38 MAPK signaling. This raises the possibility of feedback loops involving the p38 MAPK-SGK axis with the potential for self- limiting the activation cascade independent of phosphatase activity by inhibition of an upstream activator.
Considerable similarity exists in the mechanisms of activation and downstream effects of SGK and Akt. Both Akt and SGK can be activated by growth factors in a PI3-kinase-dependent manner. The preponderance of available evidence shows that Akt and SGK constitute major cell survival pathways. Furthermore, several identical downstream targets of Akt, such as Bad and FOXO-3, are also downstream substrates for SGK. These observations suggest that SGK and Akt may act in an analogous fashion to promote cellular survival. While unique functions for SGK have been reported, these may not directly mediate cell survival (39). However, the cell and stimulus specificity of activation of survival signaling by Akt and SGK are undefined. We have observed a lack of Akt activation by IL-6 and the inability of dominant negative Akt to ameliorate the survival effects of IL-6 in cholangiocarcinoma cells (data not shown). These observations support the primary involvement of SGK during survival signaling by IL-6.
IL-6 increases resistance of cholangiocarcinoma cells to undergo apoptosis in response to chemotherapeutic agents, such as gemcitabine or 5-fluorouracil (48). Indeed, production of IL-6 has been correlated with induction of drug resistance in several tumors such as myeloma, lung, breast, ovarian, prostate, and colorectal cancer (3, 9, 11, 14, 25, 41). High levels of IL-6 are produced by multidrug-resistant cancer cells, whereas some cancer cells that are sensitive to drug treatment do not express IL-6 (9). In human hepatoma cells, endogenous production of IL-6 can confer resistance to chemotherapy through induction of multidrug-resistant protein (27). Furthermore, anti-IL-6 has been shown to restore the ability of hematopoietic K562 mutant cells to undergo chemotherapy-induced apoptosis (12). It is therefore noteworthy that manipulation of SGK expression by siRNA can also modulate cellular responses to chemotherapeutic agents. Thus our observations identify SGK as a functionally relevant intermediate in survival signaling by IL-6 and implicate the p38 MAPK-SGK signaling axis in IL-6- induced chemoresistance.
IL-6 is an autocrine factor important in the growth of several tumors such as cholangiocarcinoma and selectively activates p38 MAPK signaling in malignant cholangiocarcinoma cells. We propose that dysregulation of p38 MAPK/SGK-mediated signaling in tumor cells permits the survival of transformed cells under otherwise adverse environmental conditions, such as exposure to chemotherapeutic drugs or to hypoxia or under growth-factor limited conditions. Additional studies to assess the contribution of SGK in biliary tract tumorigenesis and transformed cell behavior are warranted.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Biondi RM, Kieloch A, Currie RA, Deak M, and Alessi DR. The PIF-binding pocket in PDK1 is essential for activation of S6K and SGK, but not PKB. EMBO J 20: 43804390, 2001.
3. Borsellino N, Bonavida B, Ciliberto G, Toniatti C, Travali S, and D'Alessandro N. Blocking signaling through the Gp130 receptor chain by interleukin-6 and oncostatin M inhibits PC-3 cell growth and sensitizes the tumor cells to etoposide and cisplatin-mediated cytotoxicity. Cancer 85: 134144, 1999.[CrossRef][ISI][Medline]
4. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, and Greenberg ME. Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol 21: 952965, 2001.
5. Buse P, Tran SH, Luther E, Phu PT, Aponte GW, and Firestone GL. Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum- and glucocorticoid-inducible protein kinase, Sgk, in mammary tumor cells. A novel convergence point of anti-proliferative and proliferative cell signaling pathways. J Biol Chem 274: 72537263, 1999.
6. Chauhan D, Kharbanda S, Ogata A, Urashima M, Teoh G, Robertson M, Kufe DW, and Anderson KC. Interleukin-6 inhibits Fas-induced apoptosis and stress-activated protein kinase activation in multiple myeloma cells. Blood 89: 227234, 1997.
7. Chun J, Kwon T, Kim DJ, Park I, Chung G, Lee EJ, Hong SK, Chang SI, Kim HY, and Kang SS. Inhibition of mitogen-activated kinase kinase kinase 3 activity through phosphorylation by the serum- and glucocorticoid-induced kinase 1. J Biochem (Tokyo) 133: 103108, 2003.
8. Collins BJ, Deak M, Arthur JS, Armit LJ, and Alessi DR. In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation. EMBO J 22: 42024211, 2003.
9. Conze D, Weiss L, Regen PS, Bhushan A, Weaver D, Johnson P, and Rincon M. Autocrine production of interleukin 6 causes multidrug resistance in breast cancer cells. Cancer Res 61: 88518858, 2001.
10. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95105, 2000.[CrossRef][ISI][Medline]
11. De Vita F, Orditura M, Auriemma A, Infusino S, Roscigno A, and Catalano G. Serum levels of interleukin-6 as a prognostic factor in advanced non-small cell lung cancer. Oncol Rep 5: 649652, 1998.[ISI][Medline]
12. Dedoussis GV, Mouzaki A, Theodoropoulou M, Menounos P, Kyrtsonis MC, Karameris A, and Maniatis A. Endogenous interleukin 6 conveys resistance to cis-diamminedichloroplatinum-mediated apoptosis of the K562 human leukemic cell line. Exp Cell Res 249: 269278, 1999.[CrossRef][ISI][Medline]
13. Firestone GL, Giampaolo JR, and O'Keeffe BA. Stimulus-dependent regulation of serum and glucocorticoid inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity. Cell Physiol Biochem 13: 112, 2003.[CrossRef][ISI][Medline]
14. Frassanito MA, Cusmai A, Iodice G, and Dammacco F. Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis. Blood 97: 483489, 2001.
15. Han J, Lee JD, Bibbs L, and Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808811, 1994.[ISI][Medline]
16. Han J, Lee JD, Jiang Y, Li Z, Feng L, and Ulevitch RJ. Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J Biol Chem 271: 28862891, 1996.
17. Hardin J, MacLeod S, Grigorieva I, Chang R, Barlogie B, Xiao H, and Epstein J. Interleukin-6 prevents dexamethasone-induced myeloma cell death. Blood 84: 30633070, 1994.
18. Hayashi M, Tapping RI, Chao TH, Lo JF, King CC, Yang Y, and Lee JD. BMK1 mediates growth factor-induced cell proliferation through direct cellular activation of serum and glucocorticoid-inducible kinase. J Biol Chem 276: 86318634, 2001.
19. He XS, Rivkina M, and Robinson WS. Construction of adenoviral and retroviral vectors coexpressing the genes encoding the hepatitis B surface antigen and B7-1 protein. Gene 175: 121125, 1996.[CrossRef][ISI][Medline]
20. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, and Schaper F. Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374: 120, 2003.[CrossRef][ISI][Medline]
21. Hirano T. Interleukin 6 and its receptor: ten years later. Int Rev Immunol 16: 249284, 1998.[Medline]
22. Jiang Y, Chen C, Li Z, Guo W, Gegner JA, Lin S, and Han J. Characterization of the structure and function of a new mitogen-activated protein kinase (p38). J Biol Chem 271: 1792017926, 1996.
23. Jiang Y, Gram H, Zhao M, New L, Gu J, Feng L, Di Padova F, Ulevitch RJ, and Han J. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38delta. J Biol Chem 272: 3012230128, 1997.
24. Kobayashi T, Deak M, Morrice N, and Cohen P. Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase. Biochem J 344: 189197, 1999.[CrossRef][ISI][Medline]
25. Komoda H, Tanaka Y, Honda M, Matsuo Y, Hazama K, and Takao T. Interleukin-6 levels in colorectal cancer tissues. World J Surg 22: 895898, 1998.[CrossRef][ISI][Medline]
26. Kotlyarov A, Neininger A, Schubert C, Eckert R, Birchmeier C, Volk HD, and Gaestel M. MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nat Cell Biol 1: 9497, 1999.[CrossRef][ISI][Medline]
27. Lee G and Piquette-Miller M. Influence of IL-6 on MDR and MRP-mediated multidrug resistance in human hepatoma cells. Can J Physiol Pharmacol 79: 876884, 2001.[CrossRef][ISI][Medline]
28. Leong ML, Maiyar AC, Kim B, O'Keeffe BA, and Firestone GL. Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells. J Biol Chem 278: 58715882, 2003.
29. Lichtenstein A, Tu Y, Fady C, Vescio R, and Berenson J. Interleukin-6 inhibits apoptosis of malignant plasma cells. Cell Immunol 162: 248255, 1995.[CrossRef][ISI][Medline]
30. Mizuno H and Nishida E. The ERK MAP kinase pathway mediates induction of SGK (serum- and glucocorticoid-inducible kinase) by growth factors. Genes Cells 6: 261268, 2001.
31. Mora A, Komander D, van Aalten DM, and Alessi DR. PDK1, the master regulator of AGC kinase signal transduction. Semin Cell Dev Biol 15: 161170, 2004.[CrossRef][ISI][Medline]
32. Naray-Fejes-Toth A, Fejes-Toth G, Volk KA, and Stokes JB. SGK is a primary glucocorticoid-induced gene in the human. J Steroid Biochem Mol Biol 75: 5156, 2000.[CrossRef][ISI][Medline]
33. Nebreda AR and Porras A. p38 MAP kinases: beyond the stress response. Trends Biochem Sci 25: 257260, 2000.[CrossRef][ISI][Medline]
34. Okada K, Shimizu Y, Nambu S, Higuchi K, and Watanabe A. Interleukin-6 functions as an autocrine growth factor in a cholangiocarcinoma cell line. J Gastroenterol Hepatol 9: 462467, 1994.[ISI][Medline]
35. Park J, Gores GJ, and Patel T. Lipopolysaccharide induces cholangiocyte proliferation via an interleukin-6-mediated activation of p44/p42 mitogen-activated protein kinase. Hepatology 29: 10371043, 1999.[CrossRef][ISI][Medline]
36. Park J, Leong ML, Buse P, Maiyar AC, Firestone GL, and Hemmings BA. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J 18: 30243033, 1999.
37. Park J, Tadlock L, Gores GJ, and Patel T. Inhibition of interleukin 6-mediated mitogen-activated protein kinase activation attenuates growth of a cholangiocarcinoma cell line. Hepatology 30: 11281133, 1999.[CrossRef][ISI][Medline]
38. Pramanik R, Qi X, Borowicz S, Choubey D, Schultz RM, Han J, and Chen G. p38 isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun. The determinant roles of the isoforms in the p38 MAPK signal specificity. J Biol Chem 278: 48314839, 2003.
39. Sakoda H, Gotoh Y, Katagiri H, Kurokawa M, Ono H, Onishi Y, Anai M, Ogihara T, Fujishiro M, Fukushima Y, Abe M, Shojima N, Kikuchi M, Oka Y, Hirai H, and Asano T. Differing roles of Akt and serum- and glucocorticoid-regulated kinase in glucose metabolism, DNA synthesis, and oncogenic activity. J Biol Chem 278: 2580225807, 2003.
40. Shelly C and Herrera R. Activation of SGK1 by HGF, Rac1 and integrin-mediated cell adhesion in MDCK cells: PI-3K-dependent and -independent pathways. J Cell Sci 115: 19851993, 2002.
41. Szczepek AJ, Belch AR, and Pilarski LM. Expression of IL-6 and IL-6 receptors by circulating clonotypic B cells in multiple myeloma: potential for autocrine and paracrine networks. Exp Hematol 29: 10761081, 2001.[CrossRef][ISI][Medline]
42. Tadlock L and Patel T. Involvement of p38 mitogen-activated protein kinase signaling in transformed growth of a cholangiocarcinoma cell line. Hepatology 33: 4351, 2001.[CrossRef][ISI][Medline]
43. Tadlock L, Yamagiwa Y, Marienfeld C, and Patel T. Double-stranded RNA activates a p38 MAPK-dependent cell survival program in biliary epithelia. Am J Physiol Gastrointest Liver Physiol 284: G924G932, 2003.
44. Waldegger S, Barth P, Raber G, and Lang F. Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 94: 44404445, 1997.
45. Waldegger S, Gabrysch S, Barth P, Fillon S, and Lang F. h-sgk serine-threonine protein kinase as transcriptional target of p38/MAP kinase pathway in HepG2 human hepatoma cells. Cell Physiol Biochem 10: 203208, 2000.[CrossRef][ISI][Medline]
46. Webster MK, Goya L, Ge Y, Maiyar AC, and Firestone GL. Characterization of sgk, a novel member of the serine/threonine protein kinase gene family, which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13: 20312040, 1993.[Abstract]
47. Wu W, Chaudhuri S, Brickley DR, Pang D, Karrison T, and Conzen SD. Microarray analysis reveals glucocorticoid-regulated survival genes that are associated with inhibition of apoptosis in breast epithelial cells. Cancer Res 64: 17571764, 2004.
48. Yamagiwa Y, Marienfeld C, Meng F, Holcik M, and Patel T. Translational regulation of XIAP by interleukin-6: a novel mechanism of tumor cell survival. Cancer Res 64: 12931298, 2004.
49. Yamagiwa Y, Marienfeld C, Tadlock L, and Patel T. Translational regulation by p38 mitogen-activated protein kinase signaling during human cholangiocarcinoma growth. Hepatology 38: 158166, 2003.[ISI][Medline]