`SLANTNNTTINNNUNNTNNUNNTNTNTTNT
`
`
`
`
`
`
`
`fitemuerubeTREB oo
`BPUAMEAMscnt
`
`me.ge
`6F
`
`
`
`
`
`at ot.
`
`whee
`
`NHS
`
`of sclerotic lesions (18, 19). Adenoviral gene transfer of
`soluble TGF-B1 receptorIl reduced the number of a-SMA
`(+) cells and ameliorated interstitial fibrosis in NTN (20). In
`our study, a-SMA was expressedinitially in the periglom-
`erular area on day 4; however, its periglomerular as well as
`mesangial expression was increased on day6, when the fi-
`bronectin expression wasalso increased. The periglomeru-
`lar o-SMA up-regulation can be attributed to myofibroblast
`formation. Activated myofibroblasts become hypertrophic
`and secrete extracellular matrix proteins, and this may lead
`to glomerulosclerosis, fibrous crescent formation and tubu-
`lointerstitial fibrosis (18).
`Phosphorylation and activation of p88 MAPK was noticed
`very early, only 5 hours after NTS induction, but most inter-
`estingly, its activation happened in a repeated way. P-p38
`MAPK expression was increased 5 hours after induction
`of NTN, reduced to normal levels during days 2-4 and re-
`lapsed on day 6. Transient inactivation of p-p38 MAPK may
`be due to its interaction with MAPK phosphatases (MKPs).
`MKPsare a family of protein phosphatases, which are re-
`sponsible for the dephosphorylation and inactivation of
`MAPKs. MKPsare activated simultaneously with MAPKs
`(20, 21), and they may be responsible for p-p38 MAPKin-
`activation.
`In the present study this inactivation seemed
`to be transient, because p38 MAPK wasreactivated later,
`probably as a result of cytokine and growth factor produc-
`tion. To our knowledge, this dual activation of p38 MAPK
`has not been described previously; however, more specific
`studies are necessary to investigate it further.
`There was only a small reduction in renal p-p38 MAPK ex-
`pression in IL-11-treated rats. These results suggest that
`glomerular expression of TGF-B1 and infiltration/transfor-
`mation of myofibroblasts may proceed independently of
`p-p38 MAPK,this is in accordance with a previous study,
`which showed that p-p38 MAPK was not the only down-
`stream signalling intermediate in the pathway from TGF-B1
`to a-SMA (22). Also, administration of a TGF-B1 receptor
`inhibitor (SD-208) resulted in the attenuation myofibroblast
`transformation of lung fibroblasts, an effect that could not
`be achieved by a p38 MAPKinhibitor(SD 282) (23).
`In our previous report, both high and low doses of IL-11
`reduced proteinuria and glomerular fibrinoid necrosis, but
`had a different effect on glomerular macrophages(10). Dai-
`ly treatment with a high dose (1,360 ug) of rhIL-11 reduced
`the numberofinfiltrating macrophages; a low dose (800
`yg) of rhIL-11 reduced only the numberof activated mac-
`rophages, not the total number of macrophages. Based on
`this reduction, the anti-TGF effect of IL-11 could be attrib-
`uted to its anti-inflammatory properties, a mechanism that
`has also been described for other agents (24). However,
`
`—
`
`weniels
`
`RH
`
`acceding
`
`Fig. 3 - Treatment with rhIL-11 in nephrotoxic nephritis (NTN).
`Treatment with high-dose IL-11 reduced expression of glom-
`erular TGF-81 (A), glomerular «&-SMA (C), periglomerular
`«-SMA (D), glomerular fibronectin (E) and periglomerular
`fibronectin (F) in comparison with vehicle-treated rats. The
`reduction in glomerular p-p38 MAPK(B) wasnotsignificant.
`
`
`
`In this study, we have shown that treatment with a high
`dose of IL-11 reduced glomerular expression of TGF-B1,
`a-SMA and fibronectin in NTN. To our knowledge, the
`present studyis the first demonstration that administration
`of rhIL-11 mayalleviate glomerular expression of TGF-B1
`activation of myofibroblasts and extracellular matrix depo-
`sition in experimental glomerulonephritis.
`In the kidney, myofibroblasts may derive from perivascular
`smooth muscle cells, pericytes and interstitial fibroblasts,
`after stimulation by cytokines such as TGF-$1 and IL-18
`(15-17). Myofibroblasts are implicated in the development
`
`© 2011 Societa Italiana di Nefrologia - SSN 1121-8428
`
`109
`
`Ex. 2001 - Page1544
`Ex. 2001 - Page1544
`
`
`
`Siangou et al: Anti-TGF effect of IL-11 in glomerulonephritis
`SLANTNNTTINNNUNNTNNUNNTNTNTTNT
`
`the fact that low-dose rhIL-11 was enough to reduceIL-
`1B production, and macrophageactivity, but not TGF-B1,
`Qa-SMA and fibronectin expression, makes the position
`more complicated. It seems that administration of rhlL-11
`has the potential to reduce inflammation, even when giv-
`en at lower doses, but this anti-inflammatory effect is not
`enough to justify its anti-TGF effect, which requires higher
`dosage, andis also independent from p38 MAPK dephos-
`phorylation and inactivation. One of the speculations is that
`the reduction in the number of glomerular macrophagesis
`needed to reduce the TGF-81 expression. Further work is
`needed to investigate this possibility.
`Findings of the present study suggest that IL-11 has a
`dose-dependenteffect in glomerular expression of TGF-B1,
`myofibroblast differentiation and extracellular matrix depo-
`sition in NTN. This finding may be of relevance to develop-
`ment of possible new applications of IL-11 and also novel
`treatment strategies in patients with glomerulonephritis.
`
`Financial support: This work was supported by a research project
`grant from Kidney Research UK. M.S. received a research project
`grant from the Greek Renal Association.
`
`Conflict of interest statement: James C. Keith Jr is an employee of
`Wyeth Research, Cambridge, MA, USA.
`
`Address for correspondence:
`Maria Stangou, MD
`Nephrology Department Aristotle University
`Hippokration Hospital
`50 Papanastasiou Street
`Thessaloniki, Greece
`mstangou@math.com
`
`
`
`8.
`
`1.
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`3.
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`4.
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`Trepicchio WL, Dorner AJ. Interleukin-11: a go130 cytokine.
`Ann N Y Acad Sci. 1998;856:12-21.
`2. Miuller-Newen G. The cytokine receptor gp130: faithfully pro-
`miscuous. Sci STKE. 2003;2003:PE40.
`Heinrich PC, Behrmann |, Haan S, Hermanns HM, Muller-
`Newen G, Schaper F. Principles of
`interleukin (IL)-6-
`type cytokine signalling and its regulation. Biochem J.
`2003;374:1-20.
`Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC,
`Turner KJ, Dorner AJ. Hematopoietic,
`immunomodula-
`tory and epithelial effects of
`interleukin-11. Leukemia.
`1999;13:1307-1315.
`Kurzrock R, Cortes J, Thomas DA,Jeha §S,Pilat S, Talpaz M.
`Pilot study of low-dose interleukin-11 in patients with bone
`marrow failure. J Clin Oncol. 2001;19:4165-4172.
` Ragni MV, Jankowitz RC, Chapman HL, et al. A phase Il
`prospective open-label escalating dose trial of recombinant
`interleukin-11 in mild von Willebrand disease. Haemophilia.
`2008;14:968-977.
`Schmitz B, Thiele J, Witte O, Kaufmann R, WickenhauserC,
`FischerR. Influence of cytokines(IL-1 alpha, IL-3, IL-11, GM-
`CSF) on megakaryocyte-fibroblast interactions in normal hu-
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`5.
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`6.
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`7.
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`man bone marrow. Eur J Haematol. 1995;55:24-32.
`Peterson RL, Wang L, Albert L, Keith JC Jr, Dorner AJ. Mo-
`lecular effects of recombinant human interleukin-11 in the
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`9.
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`10.
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`11.
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`HLA-B27 rat model of inflammatory bowel disease. Lab |n-
`vest. 1998;78:1503-1512.
`Tang W, Geba GP, Zheng T,et al. Targeted expression of IL-
`11 in the murine airway causes lymphocytic inflammation,
`bronchial remodeling, and airways obstruction. J Clin Invest.
`1996;98:2845-2853.
`Lai PC, Cook HT, Smith J, Keith JC Jr, Pusey CD, Tam FW.
`Interleukin-11 attenuates nephrotoxic nephritis in Wistar
`Kyoto rats. J Am Soc Nephrol. 2001;12:2310-2320.
`Lai PC, Smith J, Bhangal G, et al. Intedeukin-11 reduces
`renal
`injury and glomerular NF-kappa B activity in murine
`experimental glomerulonephritis. Nephron Exp Nephrol.
`2005;101:146-154.
`12. Tam FW, Smith J, Morel D, et al. Development of scarring
`and renal failure in a rat model of crescentic glomerulone-
`phritis. Nephrol Dial Transplant. 1999;14:1658-1666.
`13. Martin MM, BuckenbergerJA,Jiang J, et al. TGF-beta’ stim-
`ulates human AT1 receptor expression in lung fibroblasts by
`cross talk between the Smad, p38 MAPK, JNK, and PI3K
`signaling pathways. Am J Physiol Lung Cell Mol Physiol.
`2007;293:790-799.
`14. Kutz SM, Hordines J, McKeown-Longo PJ, Higgins PJ. TGF-
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`110
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`
`23 NEPHROL 2011; 24(01): 106-111
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`15.
`
`16.
`
`17.
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`18.
`
`19.
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`20.
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`B1-induced PAI-1 gene expression requires MEKactivity and
`cell-to-substrate achesion. J Cell Sci. 2001;114:3905-3914.
`Vesey DA, Cheung CW, Cuttle L, Endre ZA, Gobe G, Johnson
`DW.Interleukin-1 beta induces human proximaltubule cell in-
`jury, alpha-smooth muscle actin expression and fibronectin
`production. Kidney Int. 2002;62:31-40.
`Kondo S, Kagami S, Urushihara M, et al. Transforming growth
`factor-beta stimulates collagen matrix remodeling through
`increased adhesive and contractive potential by human renal
`fibroblasts. Biochim Biophys Acta. 2004;1693:91-100.
`Zeisberg EM, Potenta SE, Sugimoto H, Zeisberg M, Kalluri R.
`Fibroblasts in kidney fibrosis emerge via endothelial-to-mesen-
`chymaltransition. J Am Soc Nephrol. 2008;19:2282-2287.
`Goumenos D, Tsomi K, latrou C, et al. Myofibroblasts and
`the progression of crescentic glomerulanephritis. Nephrol
`Dial Transplant. 1998;13:1652-1661.
`Roberts IS, Burrows C, Shanks JH, Venning M, McWilliam
`LJ. Interstitial myofibroblasts: predictors of progression in
`membranous nephropathy. J Clin Pathol. 1997;50:123-127.
`Zhou A, Ueno H, Shimomura M, et al. Blockade of TGF-beta
`action ameliorates renal dysfunction and histologic progres-
`sion in anti-GBM nephritis. Kidney Int. 2003;64:92-101.
`
`21.
`
`22.
`
`23.
`
`24.
`
`Dickinson RJ, Keyse SM. Diverse physiological functions
`for dual-specificity MAP kinase phosphatases. J Cell Sci.
`2006;119:4607-4615.
`Ding Q, Gladson CL, Wu H, Hayasaka H, Olman MA. Fo-
`cal adhesion kinase (FAK)-related non-kinase inhibits
`myofibroblast differentiation through differential MAPK
`activation in a FAK-dependent manner. J Biol Chem.
`2008;283:26839-26849.
`Kapoun AM, Gaspar NJ, Wang Y, et al. Transforming growth
`factor-beta receptor type 1 (TGFbetaRI) kinase activity but
`not p38 activation is required for TGFbetaRl-induced myofi-
`broblast differentiation and profibrotic gene expression. Mol
`Pharmacol. 2006;70:51 8-531.
`Nagatoya K, Moriyama T, Kawada N, et al. Y-27632 prevents
`tubulointerstitial fiprosis in mouse kidneys with unilateral ure-
`teral obstruction. Kidney Int. 2002;61:1684-1695.
`
`Received: July 14, 2009
`Revised: February 22, 2010
`Accepted: March 07, 2070
`
`© 2011 Societa Italiana di Nefrologia - SSN 1121-8428
`
`111
`
`Ex. 2001 - Page1546
`Ex. 2001 - Page1546
`
`
`
`THE JOURNAL OF Bi
`
`
`© 1998cy The Americ bermistry and Molecular Biology, Ine
`
`3, L9DS
`Vol. 273, No. 1U, Iseue of March 6, pp. 550Printed ii
`
`ELSA,
`
`
`Transforming Growth Factor-—8 Stimulates Interleukin-11
`Transcription via Complex Activating Protein-1-dependent
`Pathways*
`
`(Received for publication, September 99, 1997, and in revised form, December 15, 1997}
`
`Weiliang Tangt, Liu Yang$, Yu-Chung Yangs, Shawn X. Lengt, and Jack A. Eliasi?
`Thus;
`
`Fromthe +
`ection ofPulmonary and Critical Care Medicine,
`Ya
`ty Schooi of Medicine, Department of Internal
`
`
`
`t 06520-8087 and SIndiana Unin
`sity School of Medicine, Departments of Medicine
`Medicine, New Haven, Connec
`(Hematology / Oncology), and Biochemistry /Molecular Biology, Walthe:
`7 Oncology Center, Indianapolis, Indiana 46202
`
`Studies were undertaken to characterize the mecha-
`nism by which transforming growth factor-§, (TGR-§,)
`stimulates epithelial cell interleukin (0L)-11 production.
`Nuclear run-on studies demonstrated that TGF-6, is a
`potent stimulator of DL-11 gene transcription. ToE-6,
`also stimulated the luciferase activity in cells trans-
`fected with reporter gene constructs containing mucle-
`otides -728 to +58 of the 0-11 promoter. Studies with
`progressive 5° deletion constructs and site-specific mu-
`tations domonstrated that this stimulation was depend-
`ent on 2 AP-.] sites between nucieotides —10G and —82 in
`the [L-11 promoter. Mobility shift assays demonstrated
`that TGR-8, stimulated AP-1 protein-DNA binding to
`both AP-1 sites. Supershifé analysis demonstrated that
`JunD was the major moiety contributing to AP-1-DNA
`binding im unstimulated cells and that ec-Jun-, Fra-1l-,
`and Fra-2-DNWA binding were increased whereas dhunD-
`DNA binding was decreased in TGF-6,-stimulated cells.
`The sequence in the UL-11 promoter that contains the
`AP-} sites also conferred TGE-8, responsiveness, in a
`position-independent fashion, om a heterologous mini-
`mal promoter. Thus, TGE-6, stimulates IL-11 gene tran-
`scription via a complex AP.l-dependent pathway that is
`dependent on 2 AP-1 motifs betweenmucleotides —106
`and -82 that fumetion as an enhancer in the IL-1}
`promoter.
`
`Interleukin-11 (L-11)' wasoriginaliy discovered as a solubie
`factor in supernatants from transformed stromal cells that
`stimulated plasmacytoma cell proliferation (1). It has subse-
`quentiy been shown to be a pleiotropic memberofthe I-6-type
`cytokine family that mediates
`its biologic activities via binding
`
`
`
`to a multimeric receptor complex that contains the gp13a0 mel-
`ecule (2-5). Among its many effects are the ability te regulate
`hematopoiesis, stimulate the production of acute phase pro-
`
`teins, induce thetis:
`ibitor of metalloproteinase-1, regu-
`
`
`* This work was supported by National Institutes of Health Grants
`
`FIL-36708, AT-34958, AL-54989, HL-56589
`(to F. A.B), DK-50576, and
`HAL-48819 (to Y.-C. ¥.). The casts of publication of this article were
`
`
`defrayed in part by the payment of page charges. Th
`rticle must
`
`therefore be hereby marked “advertisement” in accardance with 18
`
`U.S.C. Section 1734 solely to indicate this fact.
`{To whomcorrespondence should be addressed: Section of Pulme-
`
`
`nary and Critical Care Medicine, Yale University School of Medicine.
`
`
`
`Dept. of Internal Medicine, 353 Cedar St/105 LCI, New Haven, CT
`
`
`0652
`Tel: 208-
`5A183: Fax: 208-785-3826; E-mail:
`jack.
`chas@yals.edua.
`TOF-f,, transforming
`1 The abbreviations used are: UL, interleukin;
`
`
`
`
`
`prowth factor-8,; AP-1, activating protein-1; RSV, respiratory syncytial
`
`
`virus; DMEM, Dulbecco's modified Ragle’s medium; DTT, dithiathre-
`tho EMSA, electrophoretic mobility shift assays; bp, base pais
`
`thymidine kinase.
`
`late bone metabehsm, and alter epithelial proliferation (2,
`6—10). Studies from our laboratcries and others have also dem-
`onstrated that TL-11 can induce tissuefibrosis, regulate tissue
`myocyte and myofibroblast accumulation, alter airway physi-
`ology, and confer protection in the context of mucosal injury o!
`the respiratory and gastrointestinal tracts (11-14).
`tn keeping with the biologic importance of [L-11, a number of
`investigators have studied its sites of production and the reg-
`ulation of these responses. These studies demonstrated that
`
`IL-11 is produced by a variety of stromal cells in
`response to a
`varicty of stimuli, inchading cytokines, histarine, cosinephil
`AE
`
`major basic protein, and r piratory tropic viruses (7, 15-20). A
`
`prominent finding in our studies offibroblasts (18), epithelial
`eclls (19). and osteoblasts (20) and studies by others of chon-
`drocytes and synoviocytes (7) has been the importance of
`TGEF-8 moieties in the stimulation of IL-11 production. These
`studies also demonstrated that TGF-8, stimulation of IL-11
`
`protein production is associated with proportionate changes in
`IL-1] mRNA accumulation and,
`in our studies, IL-1] pene
`transcription (18).
`The IL-11 promoter has been cloned and the cis-elements
`and trans-acting factors that regulate the levels of basal TL-11
`production have been identified by cur laboratories (21). De-
`spite the demonstrated importance of gene transcription in the
`stimulation of IL-l) production, the cis-elements and trans-
`acting factors that mediate the transcriptional activation of
`TL-Li have not been investigated. To further our understand-
`ing of the regulation of IL-11, studies were undertaken to
`
`
`
`characterize the transcriptional elements utilized by TOP-6, in
`stimulation of IL-11. These studies identify twe activating
`n-1 (AP-1) motifs between —100 and —82 in the [1-11
`promoterthat are essential for TGF-g-induced IL-1] transcrip-
`
`tional activation. They also demonstrate that this stimulation
`is associated with camplex alterations in the composition of the
`AP-1 subunits that bind to these sites and that DNA which
`contains these AP-1 elements confers TGR-6, responsiveness
`on a heterologous promoter. Lastly they demonstrate that this
`mechanism is stimulus-specific since respiratory syncytial vi-
`rus (RSV) stimulates TL-11 transcription via a different
`mechanism.
`
`
`
`EXPERIMENTAL PROCEDURES
`
`Celi Culture and TGF-8, Stimulation
`
`A549 human alveolar epithelial-like cells were obtained from the
`American Type Oulture Collection (ATCC, Rockville, MD) and grownto
`
`
`confluence in Dulbecco’s modified Hagle’s medium (DMEM) supple-
`mented with 10% fetal hovine serum (22). Ai confluence, varying can-
`
`
`
`centralions of recombinant human TGP-6,
`(1-10 ng/ml) (Rh & D Sy
`
`
`tems, Minneapolis) or mediurs controls were
`added, and the«
`
`incubated for up to 48 bh. At the desired poinis in time, superr
`
`
`were removed and stered at —20°C, and nuc
`c harves
`further usage (see below).
`
`This paper is available on line at bitp://www.jbc.org
`
`Ex. 2001 - Page1547
`Ex. 2001 - Page1547
`
`
`
`deyWoypapeopumoc]
`
`
`
`
`
`
`
`SIOZ‘T]JOquisaaoNUoABOpOTg[2D[OFXISU]2
`
`
`
`t R
`
`
`
`
`
`TGE-8 Stumulates 4-11 Transcription
`eu
`The DNA fragments with the various 5’ deletions were then se
`
`by electrophoresis, electroeluted, and lpated into BaomHT/Xhol-
`
`linearized pXP2-luc vector. Clones from the subsequent transformation
`
`
`were sercened for insert
`siac, and DNA sequcneing was used to vorify
`junction sequences for all clones that were chosen for further
`utilization.
`
`5, a series of con-
`Through the combined efforts of bath approach
`
`728 to -8L. In
`structs were prepared whose 5’ ends extended from
`
`
`
`all cases, the 3' end was +58 relative to the tra
`ription initiation
`site.
`
`Site-directed Mutagenesis
`Mutation of the AP-1 sites in the parent [L-11 promoter was per-
`
`
`
`formed using the Muta-Gene M43 In Vitre Mutage
`Kat Bio-Rad,
`
`
`catalog number 170-3580) based an Kunkel’s method (29, 40). The
`$82-bp BomHVXhol fragment of Th-11 promoter was excised frors
`
`
`pXP2-1L-11-324 and subcloned into M13 phage. The recombinant pha
`
`BNA was then transformed into bacterial
`
`strain Escherichia coli CS236
`{dut-, ung—, thi-, and relA—) to generate uracil-containing single-
`stranded DNA. Such single-stranded DNA was allowed to anneal to
`
`mutagenic primer, and second strand DNA was synthesized with T7
`
`
`
`DNA palys
`and Ta DNA ligase. When transformedints bacterial
`strain MV1190(dut+, ung+), uracil-containiny single-stranded DNA
`
`
`template was degraded, and only newly synthesized mutation-bearing
`second strand DNAwould propagate. The wild type and mutated AP-1
`
`
`
`sequences are as follows: wild type &’ (distal) AP-1, 5 TGAGTC!
`:
`
`
`
`mutated 5’ (distal) AP-L, 5'-TGAcgaA-2'; wild type 3’ (pveximal) AP-1,
`
`5 -TGTGTCA-3'; mutated 3) (oraximal) AP-1, 5/-TGPepa d-8”. All ofthe
`AP-1 mutation constructs underwent DNA sequencing to verify the site
`and axtent of the induced alterations.
`
`
`Preparation of I.-C tikuc Constructs
`
`A 156-bp BamHT/Xhol fragment from the herp
`mplox virus thy-
`midine kinase (dk) minimal promoter/chioramphenicol acetyltrans-
`ferase reporter gene construct ptk~CAT (81) was obtained from Dr.
`
`Asuradha Ray (Yal
`siversity, New
`and subcloned inio the
`
`
`BamHl and Xhol
`of the pXP2-luc reporter pene construct
`to
`
`
`generate ptk-XP2.
`i
`cleotides (5'-GAT CCG AGG GTG AGT
`
`CAG GAT GTG TCA GGC CGA AGCTT-3' and 5'-GAT CAA GCT TCG
`
`
`
`
`GCC TGA CAC CTG ACT CAC CCT €G-3")
`were then synthesized and
`
`annealed to forra a 38-bp DNA duplex with sticky ends cormpatible witt
`the Bamlil site (' of herpes simplex virus th promoter) in ptk-XP2.
`This insert contaiis a 27-bp DNA sequence of the IL-1) promoter
`63
`
`
`ta -77 relative to ranseriptia start site) that contains both the 5’ and
`
` PS in the correct ((sense! (pILsisC++ the.XP) and reverse(ant
`Nor
`=<j
`35
`
`(pIL1(—)tk-XP2) directions. DNA sequencing was perforrsed to veri
`the sequence and orientation of DIVA insertion.
`
`Cell Transfection and Reporter Gene Assay
`
`A549 cells were seeded at, 40-05 confluence and incubated aver-
`
`
`nipht in DMEMwith
`high elucose and 10% fetal bovine serum. Trans-
`
`
`
`feetions were performed using the DEAE-dextran method as described
`proviously by our laboratory (22). Thecells were then incubated for 24h
`
`
`
`
`in
`serum-free DMEMalone or in DMEMsupyplemented with TGP-#, (10
`
`
`nefmb. In experiments where RSV was util
`d the A549 cells were
`
`incuhated for 90 mis with RSV Gnultiplic
`shiom = 8) or appra-
`priate medium control, washed, and then incubated for 24 h in serum-
`free DMEM. At the endof these incubations, cell lysates weve prepared,
`and luciferase activity was assessed on a Lumat luminometer using the
`
`Luciferase Assay System from Promega (Madison, WI
`
`
`tions the construct pOMY-&-gal (CLONTECH, Palo Alto, CA) was als
`included to contre! for transfection efficiency. The @-galactosidase ac-
`
`
`tivity in uostivulated and stimulated cell Iysaies was characterised
`
`
`
`using the CPRGmethod as descrihed previously in
`2)this laboratory (2%
`
`
` re
`e
`
`The G.galactosidase levels were then used te standardize the me
`ments of luciferase activity.
`
`Jc
`
`Rlectrophoretic Mobility Shift Assay (EMSA}
`
`
`Preparation of Nuclear Extracts--Nuciear extracts were prepared
`
`
`
`
`ing modifications of the
`techniques of Schreiberet ai. (82). Unstimu-
`
`lated, TOP-A,-stimulated, and RSV-infected A549 ces were prepared
`as noted above. At the desired points in time, the cells (107 per condi-
`tion) were mechanically detached, suspended in Tris-buffered saline
`
`freshly supplemented with protease inhibitors (1 uwe/ml leupeptin, 5
`ue/ml aprotinin, and 1 mM phenylracthylsulfonyl fluoride), pelicted at
`4°C, and resuspended, and swollen in solution A (10 mm HEPES, pH
`
`Ex. 2001 - Page1548
`Ex. 2001 - Page1548
`
`Nuclear Run-on Assay
`
`ve rates of genetranscription were assessed using mod
`
`
`
`
`
`xed by our laboratory (22-25).
`cations of protocols previously d
`A549 cells were incubated for 16 h under contral conditions, with
`TGF-g, (10
`ml), or after infection with respiratory syncyvial virus
`
`(RSV) at a multiplicity of infection of 8 as described previously (17-19).
`The eells (8 X 107 per condition) were then washed twice with ice-cold
`uspended in t
`uffer (16
`
`ram Tris-HC}, pH 24, 8 ron Meth, 2mm CaCh, 3 um dithbiothreite!
`
`
`(DPT), 300 mma sucrose, 0.5% Triton K-100). The nuclei were then
`harvested by centrifugation and resuspended tn 100 ul of storage buffer
`(50 mMTris-HCl pH 8.4, 5 wm MeCl, 0.1 mm EDTA, 40%slyceral) and
`stored at -80°C until farther utilized. Nylon membranes were pre-
`
`pared carrying 20 yg each of isolated cDNA fragments encoding Tl-11
`(a gift of De. Paul Schendel, GenetiesInstitute, Cambridge, MA) and
`pUCI1& without a cDNA i
`
`using a slot-blotting apparatus
`¢MINT.FEFOLDTr Schleic ser &Schue
`
`
`and baked in a vacuum oven (80°C for 2h). When ready,
`nuclei were
`
`thawed on ice and pelleted in a microcentrifuge at 4 °C
`30 s, and in
`vitro transeription and RNA labeling were earried out in transeription
`
`buffer (20 mm Tris-HCI, pH 8.3, 100 mM KCI, 4.5 mM MeCl,, 2mm DTT,
`and 400 um each of ATP, OTP, and CTP) in the presence of 200 pCi of
`la"@PLUTP (-3000 Ciummel, Amersham Corp.) and 20% ptyc
`
`
`t
`80 °C for 80 rain. TI
`ction was
`followed with a cold
`ch
`
`of 100 mv UTP for 10 rin at 86 °C. The reaction was then terminal
`
`by incubatingwith stop buffer (50 mm Tris-HICL pH §3, 500 mu Nac,
`5 mM EDTA) with 200 us/ml RNase-free DNase I and 750 unites
`RNasin (Bochringer Mansheim) at 20°C for 145 min. RNA was ox-
`
`
`tracted with phenol/chicreform, precipitated, and washed
`3 alcohol.
`
`Dried RNA pellets were dissolved in equal volurnes of TE buffer
`(0 mm
`Tris-HCl] and TimEDTA, pH 7.8), and radiaactivity was detarmined by
`
`
`
`the mean of duplicate countings of 1-1 aliquots.
`Hybridization was
`performed by incubating each membrane with equal numbers of counts
`of radiolabeled RNA. The membranes were then washed at high strin-
`
`gency, and binding was cvaluatcd using autoradiography.
`
`
`Primer Extension Analy,
`
`A549 cells were incubated for 16 h with TGF-f8, (10 ng/m). T
`supernatant
`e then removed, and poly(A)” RNA was
`isolated using
`
`
`
`ohge(dT) affinity based methodolngy as deserthed (26, 27). Primer ex-
`
`tension was ther
`ing a radiolabeled 20-h
`complemen-
`
`
`
`tary synthetic oligonucleotide corresponding to oligonucleotides -11 to
`
`
`+9 with respect to
`the tran
`ion start site. The 5’ end ofthe resulting
`
`TL-1 mRNA was defined using the Molency murine leukersia virus
`
`primer extension systen (Promega, Madison, WD as described by th
`manutacturer. In this system the 5’ end-labeled oligonucleotide hybrid-
`
`
`ized with the TL-11 mRNAand was utilized as a primer by the Moloney
`murine leukemia virus reverse transcriptase which, in the presence of
`deoxymuclestides, synthesized cDNA until the 5’ end of the mRNA was
`reached. The extended product was then resolved on an 8% uraal
`
`
`polyaerviamide sequencing gel along with a known DNA sequence
`ladder.
`
`Plasmid Construction
`
`A 786-bp Poul! fragment of the human IL-1] promoter was previ-
`ousty isolated and cloned in our lahoratovies (21). This promoter frag-
`ment contained
`uences between —728 and +58 relative to the
`
`transcription start site defined above. It was cloned into the Smalsite
`
`of the huciferase reporter gene vector pXP2-lue (ATCC) to generate the
`construct oXP2-IL-11-7%
`
`Preparation of 5’ Deletion Constructs
`
`
`
`Two techniques were used to ger
`ea series at 5’ deletions of the
`pxP2-1h-11-7528 parent construct. When appropriate restriction sites
`
`m mutants. This
`were present, they were util
`
`
`approach was utilized with the Aca
`the HinfT site ai
`
`- 96. In both cases, the — 728 to +88 fragment of the TL-11 premoter
`was subjected to enzyr
`the desired fragment was re-
`
`cloned inte the vector pRP2-luc using standard approaches. When ap-
`
`propriate restriction sites were net available Bal-31 exonuclease diges-
`
`tion was employed to introduce
`etiens. This technique takes
`advantage ofthe fact. that, Bal-21 degrades bath the 5’ and 8’ ends af
`
`double-stranded DNA without insertinginternal cleavages
`(28). Briefly,
`Bamlil-linearized parent construct pXP2-TL-11-324 was incubated
`with Bal-31 exonuclease for varying periods. BamHI linkers (New
`England Biolabs, cataloguc number 1071, Beverly, MA) were then
`added, and the DNA was subjected to BamHUXkhol double digestion
`
`9°
`
`xa8aQa dBB
`
`e
`
`
`
`
`
`SIOZ‘T]JOquisaaoNUoABOpOTg[2D[OFXISU]2
`
`
`
`5508
`
`incubation Conditions
`
`pUG
`
`}
`
`1-41
`
`TGF-68 Stimulates IL-Ii Transcription
`
`79,10 mu KCl, 0.l mm EDTA, 6.1 mm EGTA, 1 mv DTT with freshly
`added protease
`inhibitors as above)
`for 15 min on ice. Membrane lysis
`
`
`
`
`
`xccomplished by adding 25 ul of Nonidet P-40 followed by vigorous
`
`
`
`
`
`agitation. The nuclei were collected by eontrifugation, resuspended im
`59 yl of solution B (20 mM HEPES, pH 7.9, 400 mu NaCl, 1 mm EDTA,
`imM EGTA, 1 mm DTT and freshly added proteaseinhibiters as above),
`and agitated vigerously at 4°C for 15 min. The membrane debris was
`discarded, and the nuclear extracts were snap-frozen in small aliguots
`and stored at —80 °C.
`rotein concentrations of the nuclear extracts
`
`were determined using the DC Protein Assay System (Bio-Rad).
`i
`probes
`
`were used in these experiments. For the sake of simplicity, only the top
`
`strand DNA sequences are dlustrated here. Four olhgonucleatide probes
`
`were syoth
`ed using the oligonuclestide synt!
`liv at Yale
`
`
`
`University. They include the following: (i) wild type 6’ AP-1 sequence in
`the IL-1L promoter(5’ AP-1D (5'-GGGAGGGTGAGTCAGGATGTG-3’);
`
`(i) mutated 5 AP-1 (6-GGGAGGGTGAcgsAGGATGTG-3); Gi) wild
`type 3’ AP-1 sequence in the TL-11 promoter (6' AP-1) (5’-AGTCAGG-
`ATGTGTCAGGCCGGCCC-3'); and (iv) mutated 3’ AP-1 (5’-AGTCAG-
`
`GATGTcea AGGCCGGCCE.3)).
`Four other cligonucleotides ware obtained from conmercial sources
`(Stratagene, La Jolla, CA). They
`included the following: G) a classic
`
`
`AP-1 oligonucleotide (5'-CTAGTGATGAGTCAGCCGGATC-3"); GD an
`AP-2 oligonucleotide (5/-GATCGAACTGACCGCCCGCGGCCOCET-3).
`Gi) an AP-3 cligonuclectide (6'-CTAGTGGGACTTTCCACAGATC-3"),
`and Gv! as SP-1 oligonacleatide G' -GATCGATCQGGGCGGEGCGAT-
`C39.
`
`
`Ele
`straphoresis—EMSAs were performed using the techniques of
`
`
`y ef af.
`(32). Radiolabeled double-stranded cliponucleotide
`Se
`probes were prepared by annealing complementary oligonucleotides
`
`and ond-labcling using [y“*PIATP and T4 polynuclestide kinase (New
`England Biolabs). The laheled probes were purified by push-column
`chromatography, diluted with TE buffer (10 mMTris-TICl, pil 8.0, 1 mm
`EDTA) to the desired concentralion, and incubated with equal aliguots
`of nuclear extract (2-5 ug) and 2 ugof poly idl-dC}-polyidi-dC! in a total
`volume of 20 yl at room temperature for 1h. Resolution was accom-~
`
`plished by electropheresing 10 ul of the reaction solution on vertical 6%
`
`native polyacrylamide gels containing 2% elycerol using
`45% TRE
`
`buffer (28.5 mm Tris-HCl, 22.2 mmboric acid, (.25 mm EDTA, 5H 8.0).
`Binding was assessed via autoradiopraphy.
`Supershifi EMSA-—-Supershifi assays were used te determine which
`members of the AP-1 family were involved in TGF,-stimulation of
`
`TL-11 gene transcription. In tl
`studies EMSA were performed as
`desevibed above except tt
`sotype matched rabbit polyclonal antibod-
`
`
`ies against AP-1 proteins or contral preimmune antiserum were in-
`
`
`cluded during the 1-h radiclabeled probe-extract incubation period. All
`of the antibodies that were used were purchased from Sania Crox
`Biotechnology (Santa Cruz, CA). They included antibodies that react
`with all Jun family members (Pan-Jun) (catalogue number SC-44X),
`
`JTunB (catalopue oumber 46%), JunD (catalopue number SC-74X), c-Jun
`
` cr SC&22X): all Fos fa
`(catalogue nu
`2
`y members (pan-Pos) (cata-
`
`logue number SC-254X), ¢-Fos {catalogue number SC-52%), FosB (cat-
`
`alogue number 5U-48X), Pra-1
`(catalogue number 50-605X), and Pra-2
`(catalogue number SC604X).
`
`
`
`dyewoypepeojumog
`
`
`
`2a
`55ao\
`<22
`Oa
`m=
`2=
`
`=o
`
`2&g5 Z
`
`z°4
`2BaoOa=
`wOo=
`wn
`
`7y
`
`Fig. 1. Demonstration of the effects of TOP-2, aud RSV on
`IL-1 gene transeription. A549ce
`‘incubated in medium alone
`
`(Control), stinvalated with TGF-;
`PPf) (10 nefinl), ar infected with
`
`
`RSV. Twenty four hours later their nuclei were
`harvested, and gene
`transcription was assessed as deseribed under “Experimental Proce-
`dures
`ne fevels of TL-11 gene transcription are campared with tho
`
`hybridization noted with pUC18 @UC) without a cDNA insort which
`served as a negative control.
`
`Characterization of the Transcription Start Site in the 11-11
`
`
`
`Promoter-—Prior to initiating studies designed to define the
`stimulation-respensive cis-eclements in the HTL-11 promoter,
`
`primer extension analysis was used te characterize the tran-
`
`scription initiation site in TGP-8,-stimulated A549 cells. A
`
`single transcription initiation site was detected. This start site
`was 154 bp upstream of the ATG (data not shown) and is within
`i-2 bases of the start site previously described in unstimulated
`PU-24 primate bone marrow fibroblasts by our laboratories
`(20),
`TGFP-8, Stimulates IL-11 Promoter Activity—To begin to
`characterize the mechanism by which TGH-6, stimulates IL-1
`gene transcription, transient transfection assays were per-
`formed with a promoter-luciferase reporter gene construct con-
`
`taining IL-1L1 promoter elements between nuclectides
`7285
`
`
`and +58 (relative to the transcription start site), The levels of
`luciferase activity in A549 cells were evaluatedat base line and
`after TGFR-8, stimulation. As can be seen in Fig. 2, only a
`modest level of luciferase achivity was able te be detected in
`unstimulated A549 cells. In contrast, TGF-8, was an impres-
`sive, dose-dependent stimulator of the promoter activity of this
`construct (ig. 2). This demonstrates that the -728 to +58
`5
`&
`1
`oy
`fragment
`of the TL-11 promoter contains TGE-8,-responsive
`sequences.
`Previous studies from our laboratory dersonstrated that
`RSValso stimulates A549 cell IL-1L production and mRNA
`
`accumulation (17, 19). The studies noted above demonstrate
`that this stimulation is, at least in part, transeriptionally
`mediated. To determineif the cis-elements in the [L-11 pro-
`moter that respond to TGH-B, alsa respond to RSV, we com-
`
`pared the ability of these twostimuli to stimulate the —728 to
`+58 EL-1l1 promoter-lucifer