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Transcriptional Stimulation of the Surfactant Protein B Gene by STAT3 in Respiratory Epithelial Cells

2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês

10.1074/jbc.m109986200

ISSN

1083-351X

Autores

Cong Yan, Angela Naltner, Michelle Martin, Michael Naltner, Jessica M. Fangman, Okyanus Gurel,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

The function of the lung is dependent upon differentiation and proliferation of respiratory epithelial cells and the synthesis/secretion of surfactant lipids and proteins into air space. During the respiratory inflammatory response, cytokines produced by macrophages and epithelial cells in the respiratory system have significant influence on surfactant protein homeostasis. We report here that among family members of Janus family tyrosine kinase (JAK) and signal transducers and activators of transcription (STAT), only JAK 1 and STAT3 stimulated the −500 to +41 promoter activity of the surfactant protein B (SP-B) gene in respiratory epithelial cells. JAK1 and STAT3 were co-localized in alveolar type II epithelial cells where SP-B is synthesized and secreted. Interleukin 6 and interleukin 11, known to activate STAT3 synergistically, stimulated the SP-B promoter activity with retinoic acid, which is at least partially mediated through interactions between STAT3 and retinoid nuclear receptor enhanceosome proteins in pulmonary epithelial cells. The function of the lung is dependent upon differentiation and proliferation of respiratory epithelial cells and the synthesis/secretion of surfactant lipids and proteins into air space. During the respiratory inflammatory response, cytokines produced by macrophages and epithelial cells in the respiratory system have significant influence on surfactant protein homeostasis. We report here that among family members of Janus family tyrosine kinase (JAK) and signal transducers and activators of transcription (STAT), only JAK 1 and STAT3 stimulated the −500 to +41 promoter activity of the surfactant protein B (SP-B) gene in respiratory epithelial cells. JAK1 and STAT3 were co-localized in alveolar type II epithelial cells where SP-B is synthesized and secreted. Interleukin 6 and interleukin 11, known to activate STAT3 synergistically, stimulated the SP-B promoter activity with retinoic acid, which is at least partially mediated through interactions between STAT3 and retinoid nuclear receptor enhanceosome proteins in pulmonary epithelial cells. The lung has the largest epithelial surface area of the body in order to facilitate air exchange. The structure of alveoli is protected by surfactant membrane. Pulmonary surfactant is a complex mixture of lipids and proteins that form an insoluble film to reduce surface tension at the air/liquid interface in the alveoli. The reduction of surface tension at the alveolar surface promotes lung expansion on inspiration and prevents lung collapse on expiration. Deficiency of pulmonary surfactant is responsible for increased surface tension along the alveolar epithelium and brings about alveolar collapse and epithelial cell lysis, resulting in respiratory distress syndrome, a major cause of morbidity and mortality in preterm infants. Pulmonary surfactant is composed of 90–95% lipids and 5–10% proteins. Among surfactant proteins, SP-B 1The abbreviations used are: SP-Bsurfactant protein BRAretinoic acidRAREretinoic acid responsive elementRARretinoid nuclear receptorACTRactivator of thyroid and retinoic acid receptorADactivation domainBDbinding domainCREBcAMP-response element-binding proteinCBPCREB-binding proteinILinterleukinJAKJanus family tyrosine kinaseSTATsignal transducers and activators of transcriptionTTF-1thyroid transcription factor 1hhuman 1The abbreviations used are: SP-Bsurfactant protein BRAretinoic acidRAREretinoic acid responsive elementRARretinoid nuclear receptorACTRactivator of thyroid and retinoic acid receptorADactivation domainBDbinding domainCREBcAMP-response element-binding proteinCBPCREB-binding proteinILinterleukinJAKJanus family tyrosine kinaseSTATsignal transducers and activators of transcriptionTTF-1thyroid transcription factor 1hhuman is a 79-amino acid amphipathic peptide produced by the proteolytic cleavage of proSP-B in alveolar type II and Clara epithelial cells. The SP-B peptide is stored in lamellar bodies and secreted with phospholipids into the airway lumen. It facilitates the stability and rapid spreading of surfactant phospholipids during respiratory cycles (1.Whitsett J.A. Nogee L.M. Weaver T.E. Horowitz A.D. Physiol. Rev. 1995; 75: 749-757Crossref PubMed Scopus (160) Google Scholar). SP-B plays a critical role in postnatal lung function. Null mutations in the SP-B gene cause lethal respiratory distress in newborn infants and in SP-B-deficient mice produced by gene targeting (2.Clark J.C. Wert S.E. Bachurski C.J. Stahlman M.T. Stripp B.R. Weaver T.E. Whitsett J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7794-7798Crossref PubMed Scopus (554) Google Scholar, 3.Nogee L.M. Garnier G. Dietz H.C. Singer L. Murphy A.M. deMello D.E. Colten H.R. J. Clin. Invest. 1994; 93: 1860-1863Crossref PubMed Scopus (463) Google Scholar).During respiratory cycles, respiratory epithelium of conducting airways and peripheral alveoli are repeatedly exposed to airborne particles and microorganisms from the external environment. Innate defenses and the acquired immune system protect the lung from microorganisms in part mediated and regulated by the production of proinflammatory cytokines, chemokines, and growth factors (4.Zhang P. Summer W.R. Bagby G.J. Nelson S. Immunol. Rev. 2000; 173: 39-51Crossref PubMed Scopus (336) Google Scholar). Airway macrophages and epithelial cells express numerous pleiotropic cytokines that play a pivotal role in inflammatory remodeling responses and the pathogenesis of airway disorders (5.Holgate S.T. Clin. Exp. Allergy. 2000; 30 Suppl. 1: 37-41Crossref PubMed Google Scholar, 6.Takizawa H. Int. J. Mol. Med. 1998; 1: 367-378PubMed Google Scholar). It is poorly understood how homeostasis of surfactant proteins is affected by cytokines and their downstream signaling molecules during pulmonary inflammation.The JAKs/STATs pathway was originally discovered through the study of interferon-induced intracellular signal transduction (7.Yan C. Tamm I. J. Biol. Chem. 1990; 265: 20188-20194Abstract Full Text PDF PubMed Google Scholar, 8.Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar, 9.Fu X.Y. Schindler C. Improta T. Aebersold R. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7840-7843Crossref PubMed Scopus (447) Google Scholar). Subsequent studies showed that they are activated in response to many different cytokines and growth factors (10.Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1460) Google Scholar). Upon ligand binding, JAKs activate members of the STAT family through phosphorylation of a single tyrosine. Activated STATs form dimers, translocate to the nucleus, and bind to specific response elements in promoters of target genes. Four mammalian JAK members (JAK1, JAK2, JAK3, and TYK2) and seven STAT members (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) have been identified to date. A given JAK or STAT family member is only activated by specific cytokines (10.Leonard W.J. O'Shea J.J. Annu. Rev. Immunol. 1998; 16: 293-322Crossref PubMed Scopus (1460) Google Scholar, 11.Ihle J.N. Curr. Opin. Cell Biol. 2001; 13: 211-217Crossref PubMed Scopus (585) Google Scholar). In the lung, the functions of JAKs and STATs on lung development and surfactant protein gene expression remain unclear.Expression of the SP-B gene is highly tissue-specific, restricted to respiratory alveolar type II epithelial cells and non-ciliated bronchiolar epithelial cells (Clara cells). In vitro, expression of the SP-B gene is also highly cell line-specific. Many non-lung cell lines do not support SP-Bgene expression, including HeLa cells (12.Di Bohinski R.J. Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar). Human pulmonary adenocarcinoma H441 cells have been used extensively in the characterization of SP-B gene regulation (12.Di Bohinski R.J. Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar, 13.Yan C. Sever Z. Whitsett J.A. J. Biol. Chem. 1995; 270: 24852-24857Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 14.Yan C. Whitsett J.A. J. Biol. Chem. 1997; 272: 17327-17332Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16.Yan C. Ghaffari M. Whitsett J.A. Zeng X. Sever Z. Lin S. Am. J. Physiol. 1998; 275: L239-L246Crossref PubMed Google Scholar, 17.Yan C. Naltner A. Conkright J. Ghaffari M. J. Biol. Chem. 2001; 276: 21686-21691Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), which shares many characteristics of Clara cells. There are two regions (−33 to −150 and −331 to −500) highly conserved in the promoters of both human and mouse SP-B genes (18.Bruno M.A. Bohinski R.J. Carter J.E. Foss K.A. Whitsett J.A. Am. J. Physiol. 1995; 268: L381-L390PubMed Google Scholar). The region located between −150 and −33 bp contains functional binding sites for thyroid transcription factor 1 (TTF-1) and hepatocyte nuclear factor 3 (HNF3) (12.Di Bohinski R.J. Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar). The region located between −500 and −331 bp contains TTF-1 and retinoic acid response element (RARE) sites mediating retinoic acid (RA) action (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19.Naltner A. Wert S. Whitsett J.A. Yan C. Am. J. Physiol. 2000; 279: L1066-L1074Crossref PubMed Google Scholar). RA receptors (RARs) and nuclear receptor co-activators (including p160 co-activators and CBP/p300) strongly stimulated expression of the hSP-B 500 luciferase reporter gene through RARE in the −500 to −331 bp region (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19.Naltner A. Wert S. Whitsett J.A. Yan C. Am. J. Physiol. 2000; 279: L1066-L1074Crossref PubMed Google Scholar). More importantly, RARE sites are overlapping with clustered TTF-1 binding sites. Mutations at clustered TTF-1 DNA binding sites abolished the RAR/nuclear receptor co-activator-mediated RA action (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). TTF-1 is a homeodomain containing a tissue-specific transcription factor of Nkx2 family members that strongly stimulated hSP-B transcription (12.Di Bohinski R.J. Lauro R. Whitsett J.A. Mol. Cell. Biol. 1994; 14: 5671-5681Crossref PubMed Scopus (484) Google Scholar, 13.Yan C. Sever Z. Whitsett J.A. J. Biol. Chem. 1995; 270: 24852-24857Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). TTF-1 was only expressed in the lung, thyroid, and part of forebrain (20.Lazzaro D. Price M. de Felice M. Di Lauro R. Development. 1991; 113: 1093-1104Crossref PubMed Google Scholar, 21.Kimura S. Hara Y. Pineau T. Fernandez-Salguero P. Fox C.H. Ward J.M. Gonzalez F.J.. Genes Dev. 1996; 10: 60-69Crossref PubMed Scopus (989) Google Scholar). In the lung, TTF-1 was present at the earliest stages of differentiation of epithelium and was later confined to conducting airway epithelial cells and type II epithelial cells in alveoli (19.Naltner A. Wert S. Whitsett J.A. Yan C. Am. J. Physiol. 2000; 279: L1066-L1074Crossref PubMed Google Scholar,20.Lazzaro D. Price M. de Felice M. Di Lauro R. Development. 1991; 113: 1093-1104Crossref PubMed Google Scholar, 22.Zhou L. Lim L. Costa R.H. Whitsett J.A. J. Histochem. Cytochem. 1996; 44: 1183-1193Crossref PubMed Scopus (243) Google Scholar, 23.Stahlman M.T. Gray M.E. Whitsett J.A. J. Histochem. Cytochem. 1996; 44: 673-678Crossref PubMed Scopus (190) Google Scholar). Studies of the mammalian two-hybrid and glutathione S-transferase pull-down systems indicated a direct interaction between RAR and TTF-1 (17.Yan C. Naltner A. Conkright J. Ghaffari M. J. Biol. Chem. 2001; 276: 21686-21691Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Together, RAR, TTF-1, and nuclear receptor co-activators (p160 co-activators and CBP/p300) formed an enhanceosome in the hSP-B −500 to −331 bp region (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17.Yan C. Naltner A. Conkright J. Ghaffari M. J. Biol. Chem. 2001; 276: 21686-21691Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar,19.Naltner A. Wert S. Whitsett J.A. Yan C. Am. J. Physiol. 2000; 279: L1066-L1074Crossref PubMed Google Scholar).In this report, stimulation of hSP-B 500 transcription by JAK1 and STAT3 and their upstream stimuli, interleukin 6 (IL-6) and interleukin 11 (IL-11), was observed. In addition, a synergistic stimulatory effect between RA and IL-6/IL-11 was observed in H441 cells. This was mediated at least by interaction between STAT3 and hSP-B enhanceosome proteins, including TTF-1, RARα, and ACTR (activator of thyroid and retinoic acid receptor).MATERIALS AND METHODSConstruction of PlasmidsThe plasmid constructs of hSP-B 500 bp, pG5LUC, pM RARα BD, and pVP16 TTF-1 AD were made previously (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17.Yan C. Naltner A. Conkright J. Ghaffari M. J. Biol. Chem. 2001; 276: 21686-21691Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). pM STAT3 BD was made by subcloning the STAT3 cDNA into the pM BD vector (CLONTECH, Palo Alto, CA) at the EcoRI/XbaI sites following a procedure as described previously (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). pVP16 STAT3 AD was made by subcloning STAT3 cDNA into the pVP-16 vector (CLONTECH) at the EcoRI/XbaI sites. Plasmid pM-53 BD and pVP16-CP AD were purchased from CLONTECH..Cell CultureHuman pulmonary adenocarcinoma cells (H441) were cultured in RPMI supplemented with 10% fetal calf serum, glutamine, and penicillin/streptomycin. Cells were maintained and passaged weekly at 37 °C in 5% CO2/air.Transfection and Reporter Gene AssaysH441 cells were seeded at densities of 2 × 105 cells per well in 6-well plates. The hSP-B 500 luciferase reporter construct (0.25 μg) or the hSP-B (−500/−331)/SV40 promoter luciferase reporter construct (0.25 μg) and pCMV-βgal plasmid (0.5 μg) were transfected with various concentrations of JAKs, STATs, IL-6 receptor subunits, and IL-11 receptor subunits into H441 cells by FuGENE 6 (Roche Molecular Biochemicals) as specified in the figure legends. Treatment of H441 cells with IL-6, IL-11 (Sigma), and/or all -trans RA (CalBiochem) was on the following day. After 2 days of incubation, cells were lysed, and luciferase activities were performed using the luciferase assay system (Promega). The light units were assayed by luminometry (Monolight 3010, Analytical Luminescence Laboratory, San Diego, CA). In each transfection, β-galactosidase activities were determined for normalization of transfection efficiency.Mammalian Two-hybrid System AssayTransient transfections of the mammalian two-hybrid constructs were performed as described previously (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17.Yan C. Naltner A. Conkright J. Ghaffari M. J. Biol. Chem. 2001; 276: 21686-21691Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). H441 cells were seeded at a density of 3 × 105 cells per well in a 6-well plate. The paired AD/BD constructs were transfected along with the pCMV/β-gal vector (0.5 μg) and the pG5Luciferase reporter vector (0.5 μg) (15)using FuGENE 6 (Roche/Roche Molecular Biochemicals) as specified in the figure legends. The cells were harvested 48 h after transfection and assayed by luminometry. β-Galactosidase activities were determined for normalization.ImmunohistochemistryThe lung from a wild type FVB/N adult mouse was infused with a fixative solution (4% paraformaldehyde, 1 × phosphate-buffered saline) and was dissected out and stored in fixative at 4 °C for ∼24 h. After fixation and embedding in paraffin, lung tissue sections were cut to 5 μm thick. The adult lung slides were baked at 60 °C for 2 h and washed in a series of xylene and ethanol to remove paraffin from the tissues followed by antigen retrieval procedure. Endogenous peroxidase activity was removed from the adult mouse lung tissues by incubating the tissue slides in methanol and hydrogen peroxide for 15 min. Tissue slides were incubated overnight at 4 °C with primary JAK1 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), STAT3 (1:1,000, Santa Cruz Biotechnology), or TTF-1 (1:10,000) antibodies. No primary antibody was added in the negative control. The tissues were washed and treated with secondary conjugated antibodies 24 h later. The interactions were amplified with Vectastain's Elite ABC kit to visualize the signals.Purification of mRNA and Reverse Transcription-PCRFor the in vivo assay, 8-week-old Swiss black mice received 600 units of IL-6 or 0.3 μg of IL-11. The control animals received phosphate-buffered saline. After 24 h, whole lungs were dissected out from animals and were placed on dry ice immediately. Total RNAs were purified, and RNA concentrations were determined. One μg of total RNA from each sample was subject to reverse transcription-PCR assay using a pair of primers corresponding to the SP-B coding regions described previously (16.Yan C. Ghaffari M. Whitsett J.A. Zeng X. Sever Z. Lin S. Am. J. Physiol. 1998; 275: L239-L246Crossref PubMed Google Scholar). After gel electrophoresis, DNAs were transferred onto a nitrocellular membrane and were hybridized with the radiolabeled SP-B cDNA probe to confirm authentic PCR bands.RESULTSJAK and STAT Stimulation of hSP-B TranscriptionTo understand the mechanism whereby cytokines regulate SP-B gene expression in lung epithelial cells, the human SP-B 500 luciferase reporter gene vector was co-transfected with various JAK and STAT family members into H441 cells. In a dose-dependent study of JAK family members, only JAK1 modestly stimulated expression of the hSP-B 500 luciferase reporter gene (Fig. 1A). JAK2 and JAK3 showed a small inhibitory effect. The effect is statistically significant by an ANOVA (analysis of variance) analysis (p < 0.05). In another dose-dependent study of STAT family members, only STAT3 stimulated expression of the hSP-B 500 luciferase reporter gene (Fig. 1B). These studies demonstrated that the hSP-B 500 promoter was selectively activated by JAK1 and STAT3, implying that only a subset of cytokines may influence h SP-B gene expression through the hSP-B −500 to +41 bp region in respiratory epithelial cells.STAT3C Stimulation and STAT3β Suppression of hSP-B TranscriptionIt has been reported previously (24.Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2472) Google Scholar) that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of STAT3 resulted in a constitutively active form, STAT3C. STAT3C dimerized spontaneously to bind to DNA and activated transcription (24.Bromberg J.F. Wrzeszczynska M.H. Devgan G. Zhao Y. Pestell R.G. Albanese C. Darnell Jr., J.E. Cell. 1999; 98: 295-303Abstract Full Text Full Text PDF PubMed Scopus (2472) Google Scholar). When STAT3C was co-transfected with the hSP-B 500 luciferase reporter gene into H441 cells, it was a much more potent stimulator to the hSP-B promoter than wild type STAT3 in a dose-dependent study (Fig. 2A). On the other hand, a dominant negative form, STAT3β, was also identified (31.Caldenhoven E. van Dijk T.B. Solari R. Armstrong J. Raaijmakers J.A. Lammers J.W. Koenderman L. de Groot R.P. J. Biol. Chem. 1996; 271: 13221-13227Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Co-transfection of STAT3β significantly suppressed hSP-B500 luciferase reporter gene activity in a dose-dependent study (Fig. 2B). These results further support the functional role of STAT3 in hSP-B transcription.Figure 2Transcriptional regulation of hSP-B 500 by STAT3C and STAT3β in H441 cells. Various concentrations of STAT3C (A) and STAT3β (B) expression vectors were co-transfected with the hSP-B 500 bp luciferase reporter gene vector (0.25 μg) and with 0.5 μg of pCMV-βgal vector into H441 cells. After 2 days of incubation, cells were lysed, and luciferase activities were performed using the luciferase assay system (Promega). The light units were assayed by luminometry. In each transfection, β-galactosidase activities were determined for normalization of transfection efficiency. Values are means ±S.D., n = 3. wt, wild type;Conc, concentration.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Localization of JAKs and STATs in Lung Epithelial CellsTo detect whether lung epithelial cells express JAK1 and STAT3 proteins, immunohistochemistry was performed using antibodies against JAK1 and STAT3 in lung tissue sections. TTF-1 was used as a positive control in this study, which was expressed restrictively in respiratory epithelial cells. Similar to TTF-1, immunohistochemical staining detected expression of STAT3 in alveolar type II epithelial cells where SP-B is synthesized and processed (Fig. 3A). On the other hand, JAK1 expression was detected in more cell types. In addition, both JAK1 and STAT3 were detected in epithelial cells of conducting airways (Fig. 3B). No staining was observed in a negative control. Expression of STAT3 in the lung was also observed by Northern blot analysis (25.Akira S. Nishio Y. Inoue M. Wang X.J. Wei S. Matsusaka T. Yoshida K. Sudo T. Naruto M. Kishimoto T. Cell. 1994; 77: 63-71Abstract Full Text PDF PubMed Scopus (864) Google Scholar). Taken together, JAK1 and STAT3 were co-localized with SP-B, TTF-1, RAR, and nuclear receptor co-activators in the same respiratory epithelial cells of the lung (15.Naltner A. Ghaffari M. Whitsett J.A. Yan C. J. Biol. Chem. 2000; 275: 56-62Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19.Naltner A. Wert S. Whitsett J.A. Yan C. Am. J. Physiol. 2000; 279: L1066-L1074Crossref PubMed Google Scholar).Figure 3JAK1 and STAT3 expression in lung epithelial cells. The adult mouse lung was stained with TTF-1, JAK1, and STAT3 antibodies as described under "Materials and Methods." Original magnification was at ×460.View Large Image Figure ViewerDownload Hi-res image Download (PPT)IL-6 and IL-11 Stimulation of hSP-B Transcription in H441 CellsSTAT3 is known to be activated by IL-6 family cytokines (26.Zhong Z. Wen Z. Darnell Jr., J.E. Science. 1994; 264: 95-98Crossref PubMed Scopus (1693) Google Scholar, 27.Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1286) Google Scholar). Since STAT3 stimulated transcription of the hSP-Bpromoter, stimulation of the hSP-B 500 luciferase reporter gene by IL-6 and IL-11 was assessed. The hSP-B 500 luciferase reporter gene vector was co-transfected with IL-6 receptor (IL-6R/gp130) or IL-11 receptor (IL-11R/gp130) subunit expression vectors into H441 cells. Cells were then treated with 5 units/ml IL-6 or 5 ng/ml IL-11. Without receptor co-transfection, the hSP-B 500 luciferase reporter gene did not respond to the stimulation of IL-6 or IL-11 treatment, indicating that H441 cells did not have enough receptors to mediate IL-6 or IL-11 signaling. With cotransfection of IL-6R/gp130 subunits or IL-11R/gp130 subunits, significant stimulation (∼4-fold) of the hSP-B 500 luciferase reporter gene was observed (Fig. 4, A and B). This is probably due to the effect of pre-existing cytokines in tissue culture medium. Treatment of IL-6 or IL-11 in the presence of receptors further stimulated expression of the hSP-B 500 luciferase reporter gene to 8–10-fold (Fig. 4, A and B).Figure 4Transcriptional activation of hSP-B 500 by IL-6 and IL-11 in H441 cells and in the mouse lung. In one group, the hSP-B 500 bp luciferase reporter gene vector (0.25 μg) and 0.5 μg of the pCMV-βgal vector were transfected into cells (A). In a second group, IL-6R (1.0 μg) and hgp130 (1.0 μg) subunit expression vectors were co-transfected with the hSP-B 500 bp luciferase reporter gene vector (0.25 μg) and with the pCMV-βgal vector (0.5 μg) into H441 cells. On the next day, both groups of cells were treated with 5 units/ml of IL-6 (Sigma). In controls, no cytokine was added. After 2 days of incubation, cells were lysed, and luciferase activities were performed using the luciferase assay system (Promega). In each transfection, β-galactosidase activities were determined for normalization of transfection efficiency. Values are means +S.D., n = 3. In panel B, the experiment was performed essentially the same as outlined in panel A except that IL-11R (1.0 μg) and hgp130 (1.0 μg) subunit expression vectors were used for co-transfection and treated with 5 ng/ml of IL-11.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SP-B mRNA Elevation by IL-6 and IL-11 in MiceThe in vivo effect of IL-6 and IL-11 on SP-B gene expression was also assessed in mice. Adult Swiss black mice were intratracheally administered IL-6 (600 units/mouse) or IL-11 (0.3 μg/mouse). After 24 h, lungs were dissected out, and total mRNAs were purified. Semiquantitative reverse transcription-PCR was used to study regulation of SP-B mRNA expression by IL-6 and IL-11. SP-B mRNAs in IL-6- and IL-11-treated adult animal lungs were significantly elevated in comparison with control (Fig. 5). This seems to indicate that Clara cells or alveolar type II epithelial cells express IL-6 or IL-11 receptors to mediate SP-B expression in vivo.Figure 5Increase of SP-B mRNA by IL-6 and IL-11 treatment in mice. Lungs from phosphate-buffered saline-, IL-6- (600 units/animal), or IL-11- (0.3 μg/animal) treated animals were dissected out, and total mRNAs were purified. SP-B mRNA was semiquantitatively determined by reverse transcription-PCR using a pair of primers corresponding to the SP-B coding regions. No significant change of glyceraldehyde-3-phosphate dehydrogenase mRNAs was observed in IL-6- or IL-11-treated animals (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Synergistic Stimulation between IL-6/IL-11 and RA on the hSP-B (−500/−331)/SV40 Promoter Luciferase Reporter GeneSince both RA and IL-6 or IL-11 stimulated the hSP-B 500 luciferase reporter gene, interactions between the two signaling pathways were investigated. H441 cells were co-transfected with IL-6R/gp130 or IL-11R/gp130 subunits and the hSP-B (−500/−331)/SV40 promoter luciferase reporter gene that contains hSP-B −500 to −331 bp region and mediates RA/RAR and nuclear receptor co-activator stimulation. H441 cells were treated with all-trans RA, IL-6, or IL-11. RA, IL-6, and IL-11 stimulated expression of the chimeric reporter gene individually (Fig. 6). When H441 cells were co-treated with RA and IL-6 (or IL-11), a synergistic stimulation was observed (Fig. 6), suggesting that RA and IL-6 (or IL-11) signaling pathways can cross-talk in the hSP-B −500 to −331 bp region. No stimulation was observed when the SV40 promoter luciferase reporter gene vector was used in a similar study (data not shown). It is noticed that, unlike the hSP-B 500 luciferase reporter gene, the hSP-B(−500/−331)/SV40 promoter luciferase reporter gene was not stimulated by IL-6R/gp130 or IL-11R/gp130 subunit transfection in the absence of IL-6 or IL-11, implying that other cis-elements outside the hSP-B −500 to −331 bp region may also play a role in mediating the stimulatory effect of IL-6 family cytokines.Figure 6Interaction between RA and IL-6 family cytokines in H441 cells. IL-6R/gp130 subunit expression vectors or IL-11/gp130 subunit expression vectors were co-transfected with the hSP-B (−500/−331)/SV40 promoter luciferase reporter construct (0.25 μg) and with 0.5 μg of the pCMV-βgal vector into H441 cells. On the next day, H441 cells were treated with all-trans RA (10−5m), 5 units/ml of IL-6, 5 ng/ml of IL-11, a combination of RA/IL-6, or a combination of RA/IL-11. After 2 days of incubation, cells were lysed, and luciferase activities were performed using the luciferase assay system (Promega). In each transfection, β-galactosidase activities were determined for normalization of transfection efficiency. Values are means +S. D., n = 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interactions between STAT3 and RARα Enhanceosome ProteinsTo see whether this synergistic effect between IL-6 family cytokines and RA might be a result of downstream transcription factor interactions, the mammalian two-hybrid system was utilized to characterize interaction

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