Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP-mediated transcription

Journal of Cellular Biochemistry 83:414±425 (2001)

Retinoblastoma Protein Complexes With C/EBP Proteinsand Activates C/EBP-Mediated Transcription

Amos Charles,1 Xiaoren Tang,1 Erika Crouch,3 Jerome S. Brody,1 and Zhi-Xiong Jim Xiao1,2*1The Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts2Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts3Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri

Abstract The retinoblastoma protein (RB) recruits histone deacetylase (HDAC) to repress E2F-mediatedtransactivation that plays a critical role in cell cycle regulation. RB is also involved in activation of expression of anumber of tissue speci®c- and differentiation-related genes. In this study, we examined the mechanism by which RBstimulated the expression of a differentiation-related gene, the surfactant protein D (SP-D), which plays important rolesin innate host defense and the regulation of surfactant homeostasis. We demonstrated that RB speci®cally stimulated theactivity of human SP-D gene promoter. The RB family member, p107 but not p130, also increased SP-D promoteractivity. Activation by RB was mediated through a NF-IL6 (C/EBPb) binding motif in the human SP-D promoter, and thissequence speci®cally bound to C/EBPa, C/EBPb, and C/EBPd. RB formed stable complexes with all three C/EBP familymembers. RB small pocket (amino acid residues 379±792), but not the C-pocket (amino acid residues 792±928), wasnecessary and suf®cient for its interaction with C/EBP proteins. Furthermore, we demonstrated that the complexescontaining RB and C/EBP proteins directly interacted with C-EBP binding site on DNA. These ®ndings indicate that RBplays a positive, selective, and direct role in the C/EBP-dependent transcriptional regulation of human SP-D expression.J. Cell. Biochem. 83: 414±425, 2001. ß 2001 Wiley-Liss, Inc.

Key words: retinoblastoma protein; C/EBP; transcription; differentiation; surfactant protein D

Inactivation of retinoblastoma protein (RB)has been documented in many human tumorsand cancers [Weinberg, 1995; Dyson, 1998]. RBplays a critical role in cell cycle regulation byphysical association with the members of theE2F transcription factors to form active repres-sor complexes [Weintraub et al., 1995; Zhanget al., 1999]. Loss of RB-mediated transactiva-tion repression is thought to be critical intumorigenesis [for review see Harbour andDean, 2000].

RB not only negatively regulates cell prolif-eration but also is important in cell differentia-tion and development. It is not clear, however,whether RB-mediated transrepression of E2Factivity accounts for the RB function in celldifferentiation and development. RB has beenimplicated in activating transcription of anumber of genes associated with cell differen-tiation. For instance, RB interacts with thehuman bromoprotein (hBrm) to potentiateglucocorticoid receptor±mediated transactiva-tion [Singh et al., 1995]. In addition, RB posit-ively regulates adipocyte differentiation bybinding to C/EBPa and activates transcriptionof the UCP-1 promoter in preadipocytes [Chenet al., 1996a]. Differentiation of monocytes isassociated with RB binding to NF-IL6 which isalso called C/EBPb [Chen et al., 1996b]. Fur-thermore, RB associates directly with the tran-scription factor MyoD in promoting muscle celldifferentiation [Halevy et al., 1995; Skapeket al., 1995].

The RB family consists of RB and the relatedproteins p107 and p130. It has been shown that

ß 2001 Wiley-Liss, Inc.DOI 10.1002/jcb.1239

Abbreviations used: RB, retinoblastoma protein; SP-D,surfactant protein D; HDAC, histone deacetylase; DMEM,Dulbecco's modi®ed Eagle medium; CAT, chloramphenicolacetyltransferase; PBS, phosphate-buffered saline.

Amos Charles and Xiaoren Tang are co-®rst authors.

Grant sponsor: NIH (to J.S.B.); Grant number: P01HL474049; Grant sponsor: NIH (to E.C.); Grant number:HL44015.

*Correspondence to: Zhi-Xiong Jim Xiao, Ph.D., BostonUniversity School of Medicine, 88 East Newton Street,Evans 605, Boston, MA 02118. E-mail: [email protected]

Received 17 May 2001; Accepted 15 June 2001

the expression of RB and the closely relatedproteins p107 and p130 is developmentally, butnot necessarily coordinately regulated [Cobri-nik et al., 1996; Jiang et al., 1997] and that theseproteins are expressed in a tissue and cell-speci®c fashion [Dyson, 1998].

RB has been implicated in lung cell prolifera-tion and differentiation. It has been shown thatRB expression is induced during fetal lungmaturation or terminal growth arrest of aconditionally immortalized fetal rat lungepithelial cell line [Levine et al., 1998]. It is welldocumented that loss of RB expression is closelyassociated with the development of small celllung carcinoma [ Yokota et al., 1988; Ookawaet al., 1993; Dean et al., 1994].

The surfactant-associated proteins includeSP-A, SP-B, SP-C, and SP-D that are expressedin epithelial cells of the peripheral lung in adevelopmentally regulated fashion [Xu et al.,1995]. The surfactant-associated proteins areassociated with lung epithelial type II celldifferentiation and are important in lungalveolar function and immune response [forreviews see Crouch et al., 2000; Lawson andReid, 2000]. SP-A and SP-D are highly homo-logous collagenous lectins. Both proteins arebelieved to participate in the pulmonaryresponse to lung injury, and the production ofboth proteins can be increased in response tolung injury and infection. Although the SP-Aand SP-D genes are expressed late in gestationin the same distal lung epithelial cells, theyappear to be differentially regulated [Mendel-son et al., 1991; McCormick and Mendelson,1994; Smith et al., 1995; Rust et al., 1996].Importantly, surfactant protein expression isclosely correlated with the expression of themembers of the CAAT-enhancing binding pro-teins (C/EBP) [Li et al., 1995].

C/EBP (CCAAT/enhancer-binding protein) isa family of bZIP (basic region-leucine zipper)proteins consisting of three subfamilies, a, b(also called NF-IL6), and d. Because of theirhigh similarity in the basic region, all threemembers, C/EBPa, C/EBPb, and C/EBPd havebeen shown to interact with virtually identicalDNA sequences [Cao et al., 1991; Williams et al.,1991; Osada et al., 1996]. Furthermore, the C/EBP members can readily form heterodimerswith one another that are capable of binding toC/EBP-biding motif. C/EBP factors play impor-tant roles in the cell-speci®c gene expressionand in cell differentiation, most notably in liver,

fat, and monocytes [Wu et al., 1995; Darlingtonet al., 1998; Diehl, 1998; Lekstrom-Himes andXanthopoulos, 1998; Poli, 1998].

A body of evidence suggests that C/EBPproteins are involved in lung cell differentia-tion. C/EBPa is expressed in lung alveolar typeII cells and, to a less extent, in bronchiolar Claracells [Li et al., 1995]. In the embryonic rat lung,C/EBPa expression closely correlates with theexpression of SP-A in the alveolar epithelialcells and the expression of CCSP in thebronchiolar epithelium [Li et al., 1995]. Inter-estingly, both SP-A and SP-D gene promotersequences contain C/EBP (NF-IL6) bindingsites [Crouchetal.,2000].Furthermore,C/EBPaknockout mice exhibit immature lung [Wanget al., 1995] as well as alveolar abnormalit-ies with hyperproliferation of epithelial cells[Flodby et al., 1996]. In addition, other membersof C/EBP proteins may play important rolesin the lung development, as well. For example,C/EBPd appears to be most abundant in themouse lung [Cao et al., 1991; Sugahara et al.,1999]. C/EBPd is localized primarily to alveolarepithelial cells and is rapidly induced duringdifferentiation of human fetal lung in culture[Breed et al., 1997].

In this study, we demonstrated that RBfunctions as a co-activator in upregulation oftissue speci®c SP-D gene expression. RB andp107, but not p130, speci®cally stimulatedhuman SP-D gene reporter activity. In addition,we demonstrated that RB directly interactswith C/EBPa, C/EBPb as well as C/EBPd andthat RB-C/EBP complexes can directly bind toDNA containing C/EBP binding site.

MATERIALS AND METHODS

Cell Culture and Transfection

MLE-15 cells are embryonic lung epithelialcells immortalized by SV40 T with expression ofendogenous surfactant proteins A, B, and C[Wikenheiser et al., 1993]. HeLa and MLE-15cells were maintained in Dulbecco's Modi®edEagle Medium (DMEM) containing 4.5 g/L glu-cose and L-glutamine and 10% heat-inactivatedfetal bovine serum (FetalClone I, Hyclone,Logan, Utah), penicillin G (100 U/ml),and streptomycin (100 mg/ml) at 378C in ahumidi®ed 5% CO2 incubator. Cells grown to40±60% con¯uence were used in transienttransfection by a calcium phosphate protocol.Brie¯y, cells were cotransfected with a DNA

RB Activates C/EBP-Mediated Transcription 415

mixture containing 2 mg of the pCMV-b-galactosidase plasmid, 7.5 mg of either a ratSP-A/CAT or a human SP-D/CAT reporterplasmid in the absence or presence of anindicated amount of pCMV-pRB. Thirty-sixhours after transfection, cells were collectedand lysed in Promega lysis buffer (Promega,Madison, WI). Aliquots of cell lysates wereassayed for CAT and b-galactosidase activities,respectively.

Cell Extracts and Western Blotting Analysis

Nuclear extract of HeLa and MLE-15 cellswere essentially performed according to theprotocol of Rana and Farmer [Rana et al., 1995].Brie¯y, cells were washed with ice-cold PBS andlysed with NP-40 lysis buffer (10 mM Tris-HCl,pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5%Nonidet P-40) on ice for 15 min. Cell pellets werecollected by centrifugation at 1,000g for 5 min at48C, washed with the NP-40 lysis buffer, andresuspended in a nuclear extraction buffer(20 mM HEPES pH.7.9, 0.2 mM EDTA, 1.5 mMMgCl2, 350 mM NaCl, 25% glycerol, 0.5 mMDTT, 2 mg/ml leupeptin, 2 mg/ml aprotinin, and10 mg/ml PMSF). After incubation on ice for20 min and centrifugation at 14000 rpm for15 min at 48C, the supernatants were collectedand stored at ÿ808C.

For Western blot analyses, proteins wereseparated by electrophoresis on 10% SDS poly-acrylamide gels, transferred to nitrocellulosemembranes, and blocked with 4% non-fat drymilk in TBST (10 mM Tris-HCl pH.8.0, 165 mMNaCl, 0.25% Tween 20). Antibodies used inthis study, C/EBPa (14AA), C/EBPb (C-19), orC/EBPd (M-17), were purchased from SantaCruz Biotechnology, Santa Cruz, CA. Proteinswere visualized with an enhanced chemolumi-nescence system (ECL, Amersham, Piscataway,NJ).

Electrophoresis Mobility Shift Assay (EMSA)

A reaction mixture contained 10 mg of nu-clear extract proteins, 1 ml of radiolabeleddouble stranded DNA oligo, 3 mg of poly(dI/dC), 5 mg of bovine serum albumin, and 4 ml ofbinding buffer (20 mM Tris-HCl pH7.5, 5 mMMgCl2, 40 mM KCl, 2 mM DTT, 125 mM MnCl2,and 10% glycerol) in a total volume of 20 ml. Thereaction mixture was incubated at room tem-perature for 30 min prior to electrophoresis onnon-denaturing 6% polyacrylamide gels in Tris-borate-EDTA buffer (90 mM Tris-borate, 2 mM

EDTA HEPES [pH 8.0]). In supershift studies,nuclear extracts were pre-incubated for 10 minat room temperature with a speci®c GST-RBfusion protein (2 mg) prior to the addition of the32P-radiolabeled probe. Unlabeled wild type ormutant oligo DNA was used at 50-fold molarexcess for binding competition of the 32P-labeledoligo. A wild-type 40 base-pair oligonucleotide,SP-D(WT), contains the DNA sequences fromÿ355 to ÿ316 in the human SP-D promoter(50GCAGTTGTGAGTTCCTTTTGCAATCGC-TGTAGGTCATTGT30) [Rust et al., 1996]. Thisoligonucleotide contains the consensus NF-IL6binding site (TT/GNNGNAAT/G). A mutantoligonucleotide, SP-D(mt) with the NF-IL6binding site scrambled (50GCAGTTGTGA-GTTCCGTATATTCTCGCTGTAGGTCATTG-T30), was used in parallel.

The double stranded DNA oligo was radiola-beled with [g-32P]dATP (New England BioLab)using T4 polynucleotide kinase.

RESULTS

RB Activates SP-D Gene

Since the human SP-D gene contains anumber of putative RB-responsive elementsincluding a NF-IL6 site (ÿ340/ÿ323) and twohalf GRE sites (Fig. 1) [Rust et al., 1996], wewondered whether RB can function as a tran-scription co-activator for human SP-D gene.Using a CAT-reporter construct in which theCAT expression is under the control of the SP-D50 promoter segment (SP-D698), we examinedthe effect of RB on the SP-D698 reporter activityin the lung epithelial MLE-15 cells [Wikenhei-ser et al., 1993]. As shown in Figure 2A, when co-transfected to MLE-15 cells, RB stimulated SP-D698/CAT reporter activity more than threefold, whereas RB had only marginal effect onSP-A236/CAT reporter activity despite thepresence of a consensus NF-IL6 binding site(TTCTGCAAT) n the SP-A promoter region(ÿ184/ÿ176) [Lacaze-Masmonteil et al., 1992].The positive effect of RB on the SP-D698/CATreporter was also observed in HeLa cells (datanot shown).

Because p107 and p130 are members of theRB family, we examined whether p107 or p130have similar effects. As shown in Figure 2B, co-transfection of p107 led to a reproduciblesimulation on the SP-D698/CAT reporter activ-ity. However, there was no stimulation by p130despite the expression of p130 in the cells was

416 Charles et al.

comparable with that of RB and p107 (Fig. 2B,inserted).

Dose-Dependent RB Activation of SP-D Requiresthe Presence of a C/EBP Binding Motif

We next examined whether the RB stimula-tory effect on human SP-D promoter is dose-dependent. MLE-15 cells were co-transfectedwith a ®xed amount of the SP-D698/CATreporter and an increasing amount of pCMV-RB (0±10 mg). RB-mediated stimulation wasevidently dose-dependent (Fig. 3A). In contrast,despite of an increasing amount of input RBplasmid, RB had no signi®cant effect on the SP-D161/CAT reporter construct (Fig. 3A). SinceSP-D161/CAT is a deletion construct that con-tains only 161 base pairs upstream of thetranscriptional start site and lacks the putativeNF-IL6 (C/EBPb)-binding site [Rust et al.,1996], the data suggested that the NF-IL6 siteis an important RB-responsive element. Tofurther examine this possibility, we constru-cted a targeted deletion mutant (SP-D698/CATD) which lacks the 9 bp (TTTTGCAAT) C/EBP-binding site in the SP-D698 promoterregion. When tested for RB effect, SP-D698D/

CAT apparently lost the RB-responsiveness(Fig. 3B).

C/EBP Proteins Speci®cally Bind to the C/EBP-Binding Site in the Human SP-D Promoter

Since it is likely that RB functions as atranscription co-activator for SP-D gene, wesought to identify speci®c proteins that maymediate the RB stimulatory effect. We analyzedthe expression of C/EBP proteins in both MLE-15 and HeLa cells. As indicated in Figure 4A,both MLE-15 (M) and HeLa (H) cells expressedC/EBPb including two major protein species (38and 18 kDa) and one minor (42 kDa) proteinspecies. Expression of C/EBPdwas also detectedin both cell lines. Two C/EBPa protein species(42 and 30 kDa) were readily detected in MLE-15 cells but no immunoreactive C/EBPa wasfound in HeLa cells indicating that HeLa cellslack the detectable expression of C/EBPa.

Next, we performed EMSA to examinewhether C/EBP proteins bind to the C/EBP-binding motif in the human SP-D promoter.Using a 32P-labled 40 bp double-stranded DNAoligo (ÿ355/ÿ316 in the human SP-D promoter)as probe, we detected at least three major

Fig. 1. DNA sequence and several putative regulatory elements in 50 untranslated region of the human SP-D gene. A putative TATA sequence is indicated. The transcriptional start site is indicated by an arrow [Rustet al., 1996].


speci®c protein-DNA complexes from MLE-15nuclear extracts (Fig. 4B, arrowheads). Incontrast, only two major protein-DNA com-plexes were observed in HeLa nuclear extracts(Fig. 5A), consistent with the observation thatHeLa cells lack C/EBPa expression. To demon-strate the speci®city of the protein-DNA com-plexes, we performed competition analysis. Thewild-type oligo, SP-D(wt), completely elimi-nated all three major proteins-DNA complexes(Fig. 4B, lane 3) whereas the mutant oligo, SP-D(mt) that contains a scrammed sequence in the9 bp C/EBP-binding site, was unable to do so(Fig. 4B, lane 4). Furthermore, addition of a34 bp DNA oligo derived from the G-CSFpromoter sequence (ÿ183 to ÿ150), whichcontains a functional NF-IL6 (C/EBP) site[Natsuka et al., 1992; Tanaka et al., 1995], alsoexhibited effective competition (Fig. 4B, lane 2).Similar competition was observed using HeLa

cell lysate (Fig. 5A, lanes 2±4). Thus, weconclude that the human SP-D promoter seg-ment (ÿ355/ÿ316) contains a functional NF-IL6(C/EBP) binding site.

Next, we performed antibody supershiftassays to further examine the speci®city of theprotein-DNA complexes. Addition of a controlrabbit IgG antibody in the reaction mixture hadnoeffect ontheprotein-DNAcomplexes (Fig.4C,lane 2). However, a C/EBPa-speci®c rabbit poly-clonal antibody caused the complete disappear-ance of the second protein-DNA complex, adecrease in the fastest migrated DNA-proteincomplex, and the appearance of a slower migr-ating band (Fig. 4C, lane 3). A C/EBPb-speci®cantibody supershifted the fastest migratingprotein-DNA complex (Fig. 4C, lane 4), andan antibody speci®c for C/EBPd caused thedisappearance of the upper band (Fig. 4C,lane 5). These data indicate that the C/EBP

Fig. 2. RB speci®cally upregulates SP-D/CAT reporter activity.A CAT reporter containing either a 698 bps of the human SP-D(SP-D698) promoter (see Fig. 1) or a 236 bps of the rat SP-Apromoter (SP-A236) was used in cotransfection experiments.A: Exponentially growing lung epithelial MLE-15 cells weretransfected with a DNA mixture containing 2 mg of pCMV-b-galactosidase plasmid, 7.5 mg of either the SP-A236/CAT or theSP-D698/CAT reporter plasmid DNA in the absence or presenceof 5 mg of pCMV-RB plasmid per 100 mm plate. In the mockedtransfections, 5 mg of pCMV plasmid was used instead. B: MLE-15 cells were co-transfected with DNA mixtures containing 2 mg

of pCMV-b-galactosidase, 7.5 mg of SP-D698/CAT reporter inthe presence of either 5 mg of pCMV-RB, pCMV-p107 or pCMV-130. Aliquots of cell lysates were assayed for chloramphenicolacetyltransferase (CAT) and b-galactosidase activities. Thecorrected CAT activities are presented as fold activation. Datashown is the mean from at least three independent experimentsin duplicate with standard derivations. Protein expression in thetransient transfection experiments were con®rmed (inserted inpanel B) by Western blot analysis using antibodies speci®c forRB, p107 and p130, respectively (purchased from Santa Cruz).

418 Charles et al.

proteins can speci®cally bind the human SP-Dpromoter.

Similar experiments were performed usingHeLa cell extracts. Because HeLa cells expressC/EBPb and C/EBPd, but not C/EBPa (Fig. 4A),we could only detect two protein-DNA proteincomplexes that were supershifted with antibo-dies to C/EBPb and C/EBPd, respectively (datanot shown).

C/EBP Proteins Speci®cally Interact With the RBSmall Pocket and the C/EBP-RB Protein

Complexes Directly Bind to DNA

Because RB stimulatory effect on SP-Dpromoter requires the presence of the NF-IL6site, we examined the interactions between RBand the C/EBP proteins by EMSAs. As expected,two speci®c protein-DNA complexes wereidenti®ed from HeLa cell lysate (Fig. 5A, lane1). Addition of the GST-RB large pocket [GST-RB(379±928)] or GST-RB small pocket [GST-RB(379±792)], led to a supershift of these twoprotein-DNA complexes, respectively (Fig. 5A,lanes 5 and 6). In contrast, addition of acomparable amount of RB C-pocket [GST-

RB(792±928)], or GST alone, had no effect onthese two protein-DNA complexes (Fig. 5A,lanes 7 and 8). Similarly, GST-RB(379±928)led to supershifts of both C/EBPa-DNA and C/EBPb-DNA complexes and the loss of the C/EBPd-DNA complex in MLE-15 cells (Fig. 5B,lane 4), whereas GST alone (Fig. 5B, lane 2) orGST-RBC(792-928) (Fig. 5B, lane 3) had noeffect on the protein-DNA complexes. Additionof an antibody speci®c for C/EBPa (Fig. 5B,compares lanes 5 and 6) or C/EBPb (Fig. 5B,compare lanes 7 and 8) further supershifted C/EBP-RB-DNA complexes (indicated by openarrows). These data indicate that RB interactswith C/EBP proteins and the RB-C/EBP proteincomplexes can directly bind to DNA.

Because RB interacts with C/EBP-DNA com-plexes in nuclear extracts, we next examinedwhether RB can form stable complexes with C/EBP proteins within intact epithelial cells.Since RB expression in MLE-15 cells is low(data not shown), we could not detect reprodu-cible RB-C/EBP-DNA complexes using antibodysupershift assay. We, therefore, transientlytransfected MLE-15 cells with pCMV-RB,

Fig. 3. RB-mediated upregulation of SP-D is dose-dependentand requires presence of the NF-IL6 site (ÿ340/ÿ332). A: MLE-15 cells were transfected with a DNA-mixture containing 2 mg ofpCMV-b-galactosidase, 7.5 mg of either SP-D698/CAT reporteror SP-D161/CAT and an increasing amount of pCMV-RB, asindicated. B: MLE-15 cells were transfected with 2 mg of pCMV-

b-galactosidase, 7.5 mg SP-D698 or a deletion mutant (SP-D698D) which lacks the NF-IL6 site (TTTTGCAAT), in theabsence or presence of 5 mg of pCMV-RB. The corrected CATactivities are presented as fold activation. Data shown is themean with standard derivations from at least three independentexperiments in duplicate.


Fig. 4. C/EBP proteins interact with the NF-IL6 binding site inthe human SP-D promoter. A: Expression of C/EBP proteins inHeLa and MLE-15 cells. Equal amounts (50 mg) of whole celllysates from HeLa cells (H) or MLE-15 cells (M) cells wereseparated on a 10% SDS±PAGE and Western blot analyses wereperformed using antibodies speci®c for C/EBPa (14AA), C/EBPb(C-19), or C/EBPd (M-17), respectively. All antibodies werepurchased from Santa Cruz Biotech, Inc. B: At least threecellular proteins interact with the NF-IL6 site in the SP-Dpromoter. Aliquots of nuclear extract (6 mg) derived from MLE-15 cells were used for EMSA, using 32P-labeled, double-stranded DNA oligo (SP-D) that contains the NF-IL6 site(ÿ355/ÿ315) as probe. Fifty fold molar access of cold

DNA oligos were used in competition assays. The mutant SP-D DNA oligos (SP-Dmt) contain a scrambled sequence at theNF-IL6 site (ÿ340/ÿ332). The G-CSF oligo contains a NF-IL6binding site, which has been documented previously [Natsukaet al., 1992]. C: C/EBP proteins interact with the NF-ILsite. Aliquots of MLE-15 nuclear extracts were incubatedwith 1 ml of either rabbit IgG, or polyclonal antibody speci®cfor C/EBPa, C/EBPb, or C/EBPd prior to the addition of theprobe. The reaction mixtures were separated on a 4% non-denaturing acrylamide gel and visualized by autoradiography.Speci®c DNA-protein-antibody complexes are indicated byarrows.

420 Charles et al.

which encodes the wild-type human RB. RBprotein expression in the transfected cells wascon®rmed by Western blot analysis (data notshown). Nuclear extracts from pCMV-RB trans-fected MLE-15 cells were used in EMSA. Toidentify speci®c RB-C/EBP protein complexes,the puri®ed GST-RB(379±928) fusion proteinwas used in EMSA (duplicated in Fig. 6, lanes 2and 3), which led to the formation of two speci®cRB-C/EBP protein complexes (Fig. 6, comparelane 1 with lanes 2 and 3). Expression of humanRB proteins in MLE-15 cells led to two addi-tional DNA-protein complexes (Fig. 6, openarrows) in addition to the three C/EBP-DNAprotein complexes (solid arrows). These data

indicate that RB-C/EBP protein complexes candirectly bind to DNA.

DISCUSSION

RB has been documented to function as atumor suppressor protein by negatively regu-lating cell cycle G1 exit [Weinberg, 1995]. RBassociates E2Fs and recruits histone deacety-lase (HDAC), resulting in transcriptional re-pression [Weintraub et al., 1995; Brehm et al.,1998; Luo et al., 1998; Magnaghi-Jaulin et al.,1998]. Thus, the primary function of RB in cellcycle regulation is to act as a transcriptional co-repressor. On the other hand, RB can function

Fig. 5. RB-C/EBP protein complexes directly interact withDNA. Equal amounts (6 mg) of whole cell extracts from HeLacells (panel A) or nuclear extracts from MLE-15 cells (panel B)were used in EMSA using 32P-labeled human SP-D (WT) (ÿ355/ÿ315) as probe. Fifty fold molar access of the cold SP-D (WT),SP-D(mt) or the G-CSF oligo was used in the competition assay(panel A, lanes 2±4). For assessing RB-C/EBP protein interac-tion, 1mg of the puri®ed GST-RBLP (panel A, lane 5), GST-RBSP(panel A, lane 6), GST-RBC (panel A, lane 7), or GST protein(panel A, lane 8) was added in the reaction mixture prior

to the addition of the 32P-labeled probe. In antibody-supershift-ing experiments, 1 ml of polyclonal antibody speci®c for C/EBPa,C/EBPb, or C/EBPd was added to the reaction mixture andincubated for 2 h prior to addition of the 32P-labeled probe.The reaction mixtures were separated on a 4% non-denatur-ing acrylamide gel and visualized by autoradiography. Speci®cC/EBP protein-DNA complexes are indicated by arrows withsolid heads. The RB-C/EBP-DNA complexes are indicated byarrows with hollow heads.


as transcription co-activator through its physi-cal interaction with selective transcriptionalfactors, such as hBrm and C/EBP [Singh et al.,1995; Chen et al., 1996a; Chen et al., 1996b].Recently, it has been shown that RB interactswith Jun to activate Jun-mediated transcrip-tion through an AP-1 consensus sequence[Nishitani et al., 1999]. RB can also interactwith AP2 to activate transcription [Batscheet al., 1998]. In this study, we demonstratedthat RB performs a positive regulatory functionin regulating differentiation-speci®c geneexpression through direct interaction with C/EBP-DNA complexes, and that p107 but notp130, likely functions in a similar fashion.

RB-Mediated Transcriptional Activation is notCell Type-Speci®c

RB-mediated stimulation of human SP-Dgene expression is observed in both non-lungcells (HeLa) and lung epithelial cells (MLE-15).This is consistent with recent studies indicat-ing that SP-D is not a lung speci®c protein[Crouch et al., 2000]. Overexpression of RB canstimulate gene transcription in both cell lines,suggesting that the components involved in RB-mediated transcriptional activation are com-mon in both cell types and, therefore, unlikely tobe tissue speci®c.

Like RB, p107 but not p130 stimulates SP-Dpromoter activity. It is known that RB, p107,and p130 have overlapping but different func-tions at different cell cycle stages by targetingdifferent E2F family members [Hurford et al.,1997; Dyson, 1998]. Importantly, the RB familygenes are differentially regulated during devel-opment. It has been noted that the E2F-5knockout mice display a speci®c developmentalabnormality in the choroid plexus that is notpresent in either RB null mice nor in p107 nullmice [Lindeman et al., 1998]. This suggests thatp130, a primary partner of E2F-5, could havefunctions in development and cell differentia-tion that differ from RB and p107.

RB Selectively Activates Promoter ActivityThrough the C/EBP Element at ÿ340

RB activation of SP-D gene expression requ-ires the NF-IL6 binding motif (TTTTGCAAT)located in the 50 promoter region of SP-D.Deletion of the sequence totally eliminatesthe RB effect on reporter activity (Fig. 4).Interestingly, the rat SP-A promoter, whichcontains a perfect NF-IL6 consensus sequence

Fig. 6. RB forms complexes with C/EBP proteins in vivo. Pre-con¯uent (40±60%) MLE-15 cells were transfected with 5 mg ofpCMV-RB plasmid using calcium phosphate procedure. Thirty-six hours after transfection, nuclear extracts were prepared. Sixmicrograms of proteins from mock transfected (lanes 1±3) orRB-transfected MLE-15 cell lysate (lane 4) was used in a reactionmixture, using 32P-labeled human SP-D (WT) (ÿ355/ÿ315) asprobe. As controls for RB-C/EBP-DNA complexes, 1 mg ofpuri®ed GST-RB protein (duplicated in lanes 2 and 3) was addedin the reaction mixture prior to the addition of the 32P-labeledprobe. The reaction mixtures were separated on a 4% non-denaturing acrylamide gel and visualized by autoradiography.Speci®c C/EBP-DNA complexes are indicated by arrows withsolid heads. The newly formed RB-C/EBP-DNA complexes inRB-transfected cell lysate are indicated by arrows with hollowheads

422 Charles et al.

(TTCTGCAAT) at position ÿ184 to ÿ176 in thepromoter region [Smith et al., 1995], is notactivated by RB. One possibility for this selec-tivity of RB-mediated transactivation is that RBfunctionally interacts not only the C/EBP sitebut also the adjacent DNA elements. Alterna-tively, RB may also target on other regulatoryproteins that functionally interact with certaincorresponding elements adjacent to the C/EBPsite.

RB-C/EBP Complexes Bind Directly to DNA

It has been reported that that RB directlyinteracts with NF-IL6 (i.e., C/EBPb) [Chenet al., 1996a; Chen et al., 1996b]. This interac-tion between RB and NF-IL6 occurs when U937cells differentiate along the monocyte/macro-phage lineage. Consequently, RB enhances C/EBP proteins to bind to its cognate DNA sequ-ences and thus to increase C/EBP-mediatedtranscription [Chen et al., 1996b]. Here, wehave demonstrated that RB speci®cally inter-acts with three major C/EBP isoforms and thatRB-C/EBP protein complexes can directly inter-act with DNA. This particular ®nding implies,although does not prove, that RB might bedirectly involved in the transcriptional machin-ery that activates transcription.

It is interesting to note that RB activates C/EBP-mediated increases in SP-D promoteractivity in the absence of C/EBPa in HeLa cells,suggesting that C/EBPb and C/EBPd, throughtheir interaction with RB, can activate tran-scription. This possibility is supported by the C/EBP-speci®c antibody supershifts of nuclearextracts that bind to the NF-IL6 (C/EBPb) inRB-transfected cells. Interestingly, it appearsthat C/EBPa and C/EBPd are reciprocallyexpressed in lung alveolar type II cell prolifera-tion and in acute lung injury [Sugahara et al.,1999]. Thus, RB may function in interactionwith different C/EBP factors in regulating cellproliferation and differentiation.

Physiological Relevance of RB-MediatedActivation on SP-D Gene

The surfactant proteins A and D (SP-A andSP-D) are primarily found in the surfactant onthe pulmonary epithelial cells. They can also befound in cells lining the gastrointestinal tract.There is accumulating evidence that SP-D, likeSP-A, is an important component of the innateimmune response to microbial pathogens. SP-Dinteracts directly with glycoconjugates and/or

lipid moieties on the surface of microorganisms,which in turn initiates a variety of biologicalreactions. Additionally, SP-A and SP-D are alsoimplicated in in¯ammation as immunomodula-tors [Crouch et al., 2000; Lawson and Reid,2000]. It has been shown that a recombinant SP-D protein inhibits human T lymphocyte prolif-eration and IL-2 production, suggesting thatSP-D may contribute to the suppression ofstimulated T cell proliferation [Borron et al.,1998; Wang et al., 1998]. Mice lacking SP-Dexhibit chronic in¯ammation, emphysem, and®brosis on the lung, suggesting that SP-D may,directly or indirectly, in¯uence the state ofactivation of alveolar macrophages [Wert et al.,2000]. The observation that RB can activate SP-D gene expression raises an interesting possi-bility that RB might be involved in non-acquiredhost responses to infection. Co-activation by RBand C/EBPs on the expression of SP-D, but notSP-A, suggests a mechanism for differentialregulation on surfactant genes.

ACKNOWLEDGMENTS

We thank Dr. Sheldon Feinstein for the rat-SP-A construct and for the valuable input fromDr. Steve Farmers and Dr. Qiang Yu. This workis supported by NIH grant P01HL474049 toJ.S.B. and HL440 to EC.

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Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP-mediated transcription