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Research Article

Smads oppose Hox transcriptional activities

Xuelin Li a, Shuyi Nieb, Chenbei Changb, Tao Qiua, Xu Caoa,⁎aDepartment of Pathology, University of Alabama at Birmingham, 1670 University Blvd., VH G003, Birmingham, AL 35294-0019, USAbDepartment of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA

A R T I C L E I N F O R M A T I O N

YFP; yellow fluorescence protein

⁎ Corresponding author. Fax: +1 205 934 1775.E-mail address: cao@path.uab.edu (X. Cao

0014-4827/$ – see front matter © 2005 Elsevdoi:10.1016/j.yexcr.2005.12.002

A B S T R A C T

Article Chronology:Received 10 August 2005Revised version received28 November 2005Accepted 1 December 2005Available online 10 January 2006

BMPs and Hox proteins play crucial roles in developmental processes. Beyond their mutualregulation of gene expression, little is known about the relations between their mechanismsof actions. Previously, we have shown that Hoxc8 acts as a downstream repressor in the BMPsignaling pathway. Smad1 and Smad6 interact with Hoxc8 and regulate its repressionactivities. The Hox family contains 39 genes divided into 13 paralogs. In this report, wesystemically examined the potential functions of all the paralogous Hox proteins as BMPdownstream transcription factors. Representative Hox proteins from each paralog weretested. In the gel-shift assay, we found that Smad1, Smad4, and Smad6 interactedwithmostof the Hox proteins in ways similar to their interactions with Hoxc8. The interactions wereconfirmed in mammalian cells. We also examined the effects of Smads on Hox-inducedtransactivation. Particularly, we determined that for Hoxd10 as a transcriptional activator,both Smad1 and Smad6 opposed its activity. In addition, Smad6 also inhibited Hoxc8- andHoxb7-induced osteoprotegerin (OPG) transactivation. Furthermore, Smad1 inhibitedHoxb4-mediated target gene Irx5 expression during early Xenopus development. Ourfindings suggest that Hox proteins act as general downstream DNA-binding proteins inBMP signaling cascade and their transcriptional activities are regulated by Smads.

© 2005 Elsevier Inc. All rights reserved.

Keywords:BMPSmadHoxTranscriptionXenopusDevelopment

Abbreviations:Abd-B; Abdominal-BBMPs; bone morphogenetic proteinsGST; glutathione S-transferaseIrx5; Xenopus Iroquois 5OPG; osteoprotegerinOPN; osteopontinPCA; protein-fragmentcomplementation assayTGF-β; transforming growth factor β

Introduction

Bone morphogenetic proteins (BMPs) are members of thetransforming growth factor β (TGF-β) superfamily of signalingmolecules that regulate diverse biological events, including

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cell growth, differentiation, and apoptosis [1,2]. In particular,these signal molecules regulate a broad range of morphoge-netic events during embryonic development, such as neuraldifferentiation, tissue patterning, skeletogenesis, etc. [2]. BMPstrigger cell responses mainly through the Smad pathway,

d.

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which requires two types of serine/threonine transmembranereceptor kinases and phosphorylation of receptor-regulatedSmad proteins (R-Smads, Smad1, 5, and 8) [1,3–7]. Uponphosphorylation, the R-Smads form complexes with thecommon partner, Smad4, and translocate into the nucleuswhere the complexes recruit distinct transcription factors andmediate gene transcription in a tissue-specific manner [6,7].Inhibitory Smads, Smad6 and Smad7, represent anothersubclass of Smads which block phosphorylation of R-Smads,indicating their negative feedback functions in the signaling[8,9]. While Smad7 is a general inhibitor for the TGF-βsuperfamily, Smad6 is more specific for the BMP pathway[1,3]. Although the BMP pathway has been well-established,less is known about the downstream transcription factorsrecruited by BMP signaling [4,6].

Hox transcription factors are key regulators of skeletalpatterning during embryonic development [10,11]. The Hoxgene family has distinct features such that all the membersshare a well-conserved homeobox sequence and the genefamily has characteristic organization along the genome. Inmice and humans, 39 Hox genes have been identified whichare arranged into four genomic clusters (Hoxa-d). According tothe sequence similarities and positions along the clusters, the39 genes are divided into 13 paralogs [12–14]. Hox genemutations lead to structure malformations. For example,homeotic transformations of vertebrae as well as limb skeletalmalformations were observed in Hoxa11 mutant mice [15];mutation of Hoxa13 was found to be the molecular basis fordigit arch malformation in Hypodactyly (Hd) mice [16]. Hoxproteins and BMPs participate in many common developmen-tal processes during normal embryogenesis, playing eitheropposite roles or similar roles. First, BMPs are primary factorspromoting cartilage and bone formation [2,17–19]. On theother hand, several Hox proteins have been shown to inhibitskeletogenesis. Hoxa2 inhibits cartilage condensation andbone formation in the second branchial arch, likely viaexcluding Sox9 expression and down-regulating Cbfa1 expres-sion [20]. Hoxd11 and Hoxd13 arrest cartilage growth of tibiaand fibula, resulting in decreased bone length [21]. Hoxa13inhibits the cell proliferation and differentiation of zeugopodcartilage rudiments [22]. Second, BMP-2 [23] as well as 5′ Hoxd[24] genes had been shown to be involved in limb anterior–posterior axis patterning. Third, both BMP-4 and Abd-B-like(paralog 9–13) Hox proteins act as downstream factors of sonichedgehog signaling and control regionalization of hindgut[25]. All the evidence implies that the functions of BMPs andHox proteins during development are closely related. Accu-mulating evidence shows that BMPs can regulate Hox geneexpression in many developmental contexts [26–28]. There isalso evidence showing that Hox proteins regulate Bmp-4promoter activities [29]. However, beyond these mutual generegulations, little is known as to whether their transcriptionalmechanisms are intrinsically linked.

We have previously identified Hoxc8 as a downstreamtranscription repressor in BMP-mediated osteopontin (OPN)and osteoprotegerin (OPG) gene expression [30–33]. Otherstudies further support that osteopontin is a direct target ofHoxc8 in vivo [34]. In response to BMP, Smad1 dislodges Hoxc8from the promoter and activates both osteopontin andosteoprotegerin gene transcription [30,33]. On the other hand,

Smad6 acts as a co-repressor for Hoxc8 and enhances itsrepressor activity [31]. We also provided evidence that Hoxa9has similar interactionswithSmad1andSmad6 [30,31]. Similarto our findings, group 13Hoxproteinshave beendemonstratedto interact with the R-Smads [35]. Also, homeoprotein DLX-1was found to interact with Smad4 through its homeodomainand inhibit TGF-β/BMP/Activin-induced transactivation [36].

Given that all the Hox proteins share the well-conservedDNA-binding domain known as homeodomain (HD), wewanted to know how universal these Smad–Hox interactionsare and whether Smad proteins mediate Hox transcriptionalactivities as a general rule. For this purpose, we tested 12 Hoxproteins, each from a different paralog (except for paralog 8),and demonstrated that most of the Hox proteins tested hadsimilar interactions with Smad1, Smad4, and Smad6. Inaddition to what we found before for Hoxc8 as a repressor,we determined that when Hoxd10 acts as an activator, bothSmad1 and Smad6 opposed its activity. In addition, Smad6inhibited both Hoxc8- and Hoxb7-induced OPG transactiva-tion. Furthermore, we demonstrated that Smad1 inhibitedHoxb4-induced gene transcription during early Xenopus de-velopment. Our data suggest that Hox proteins act as generaldownstream DNA-binding proteins in BMP signaling pathwayand their transcriptional activities are regulated by Smads.

Materials and methods

Plasmid construction and cell culture

Hoxa1 cDNA was a gift from Dr. V. Zappavigna (University ofModena and Reggio Emilia, Modena, Italy), Hoxa2 cDNA was agift from Dr. M. Mallo (Max-Planck Institute of Immunobiol-ogy, Freiburg, Germany), Hoxb3 and b4 cDNAs were gifts fromDr. K. R. Humphries (British Columbia Cancer Agency, BC,Canada), Hoxa5 cDNA was amplified from human breastcancer cDNA library and confirmed by sequencing, Hoxd10cDNA was a gift from Dr. M. Featherstone (McGill University,Quebec, Canada), and Hoxa11 cDNA was a gift from Dr. S. S.Potter (Children's Hospital Research Foundation, Cincinnati,OH). Hoxb6, b7, a9, d12, and d13 cDNAs were gifts from Dr. C.Largman (University of California, San Francisco, CA). Smad1and Smad4 cDNAs were gifts from Dr. R. Derynck (Universityof California, San Francisco, CA). Smad6 cDNA was a gift fromDr. A. Hemmati–Brivanlou (Rockefeller University, New York,NY). To get GST-fusion constructs, Hoxa1, a2, b3, b4, a5, b7,d10, a11, and d13 cDNAswere cloned into pGEX-5X-1 vector bystandard PCR. Hoxb6 and d12 cDNAs were cloned into pGEX-KG vector. The generation of GST-Hoxa9 and GST-Smadconstructs was described previously [30,31]. The plasmidsexpressing YFP1 (a.a. 1–158) and YFP2 (a.a. 159–239) were giftsfrom Dr. S. W. Michnick (University of Montreal, Quebec,Canada) and Odyssey Thera, Inc. (San Ramon, CA). To get Hox–YFP2 constructs, Hoxa1, b4, and d10 cDNAs were cloned in-frame to the N-terminus of YFP2. To get YFP1–Smad con-structs, Smad1 and Smad6 cDNAs were cloned in-frame to theC-terminus of YFP1 by standard PCR. Expression plasmids forFLAG-tagged Smad1/2/4/6 and HA-tagged Hoxc8 were de-scribed previously [30,31]. To get HA-tagged Hoxb7 expressionplasmid, Hoxb7 cDNAwas cloned into the pCDNA3 vector with

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HA at carboxyl terminal. pSG5-Hoxd10 expression plasmidwas obtained from Dr. M. Featherstone. OPG-luciferase (OPG-luc) reporter plasmid containing two Hoxc8 binding sites wasconstructed previously [33]. pTHCR reporter plasmid contain-ing a Hoxd10-responsive element [37] was obtained fromDr. V.Zappavigna. 293T cells, COS-1 cells, and mouse fibroblastNIH3T3 cells were incubated in Dulbecco's modified Eagle'smedium supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin at 37°C in 5% CO2. Human prostatecancer LNCaP cells were cultured in RPMI-1640 medium with10% fetal bovine serum and 1% penicillin/streptomycin at 37°Cin 5% CO2.

Gel-shift assay

Gel-shift assays were performed as previously described [38].The GST-fusion plasmids were transformed into BL21 Escher-ichia coli. The purification of the GST-fusion proteins wasperformed as described [39]. The amounts of GST fusionproteins used in the gel-shift assay were as follows: Hoxa1 0.3μg, Hoxa2 0.1 μg, Hoxb3 50 ng, Hoxb4 0.1 μg, Hoxa5 50 ng,Hoxb6 50 ng, Hoxb7 0.4 μg, Hoxa9 0.1 μg, Hoxd10 0.3 μg, Hoxa110.2 μg, Hoxd12 0.5 μg, Hoxd13 0.3 μg, Smad1 3 μg, Smad4 2 μg,and Smad6 3 μg. The osteopontin promoter element contain-ing the Hox binding site (OPN5, 5′-GACATCGTTCATCAG-TAATGCTTG-3′) and the Hoxd13 binding element (5′-CTGCGATGATTTATGACCGC-3′) were 32P-labeled as probes.

YFP protein-fragment complementation assay (PCA)

293T cells (in 6-well plate, 80% confluence) were transfected withHox–YFP2 and YFP1–Smad expression plasmids as indicated inFig. 2A using Tfx-50 reagent according to the manufacturer'sinstructions (Promega). 48 h after transfection, cells were viewedfor green fluorescence under an OLYMPUS IX70 InvertedResearch Microscope with objective lens of Hoffman ModulationContrast®, HMC 20 LWD PL FL, 0.4 NA/1 OPTICS INC at roomtemperature. Digital pictures were taken with an Olympus1XTRINOC camera and processed with MagnaFire® SP imagingsoftware (Optronics). To visualize the subcellular localization,COS-1 cells were transfected with the indicated YFP-fusionplasmids (Fig. 2B) using the same method, except using theobjective lens of HMC 40 LWD PL FL, 0.6 NA/1.

Transient transfection, luciferase assay and immunoblotting

To examine the effects of Smads on Hox protein-induced genetranscription, two reporter plasmids (OPG-luc and pTHCR)were used in the transient transfection assays. For the OPG-lucactivity assay, 50 ng OPG-luc and different expressionplasmids were transfected into LNCaP cells (in 12-well cultureplate, 50% confluence) as indicated in Fig. 3 usingLipofectamine as instructed (Invitrogen). 5 ng pRL-SV40plasmid expressing Renilla luciferase (Promega) was co-transfected in each well as internal control. Total DNA waskept constant by adding pcDNA3 plasmid. For pTHCR activityassay, NIH3T3 cells (in 24-well culture plate, 50% confluence)were transfected with 25 ng pTHCR reporter plasmid anddifferent expression plasmids as indicated (Fig. 4) using thesamemethod. 5 ng pRL-SV40 plasmid was added in each well.

Cellswere harvested 48 h after transfection. Luciferase activitywas measured and normalized with Renilla luciferase activityusing Dual-luciferase assay kit according to the manufac-turer's instructions (Promega). Luciferase values shown in thefigures are representative of transfection experiments per-formed in triplicate in three independent experiments. Todetermine the expression levels of HA-tagged Hoxb7, c8, andFLAG-tagged Smad6 in Fig. 3, the same transfection wasperformed in 6-well plate, with the exception that theamounts of the plasmids were doubled. LNCaP cells werelysed in the NP-40 buffer (137 mM NaCl, 2 mM EDTA, 10%glycerol, 1% Nonidet P-40, 20 mM Tris–HCl pH 8.0) 48 h aftertransfection. Supernatants (50 μg) were boiled and separatedby 8.5% SDS-PAGE. After electrophoresis, proteins weretransferred to nitrocellulose membranes and immunoblottedby 1:1000 anti-FLAGM2monoclonal antibody (Sigma) or 1:1000anti-HA monoclonal antibody (Babco). The membranes werewashed three times with phosphate-buffered saline contain-ing 0.05% Tween-20 and then incubated with 1:10,000 goatanti-mouse antibody (Bio-Rad). The blots were visualized bythe enhanced chemiluminescence (ECL) kit (Amersham).

Embryos, RNA injections, and animal cap assay

Xenopus embryos were obtained and staged as described [40].Hoxb4 mRNA for embryo injection was synthesized in vitrousing T7 mMessage mMachine kit (Ambion). The template forHoxb4 transcription was generated by linearizing the pCI-Hoxb4 expression plasmid with ClaI. Smad1 and Smad2mRNAs were synthesized as previously described [41]. ThemRNAs were injected into the animal poles of the two-cellstage embryos. Animal caps were removed at late blastulastages and cultured until control embryos reached neurulastages, then total RNA was extracted and subjected to RT-PCRanalysis.

RT-PCR assay

RT-PCR assay was performed as described previously [42]. TotalRNA was extracted from animal caps or whole embryos.Approximately 1 μg total RNA was used for reverse transcriptionwithTaqmankit (Roche). 2 μl of total 30 μl cDNAwere used in PCR.Primers used for EF1α and Irx5 amplification were as follows:EF1α-U: 5′-CAGATTGGTGCTGGATATGC-3′ and EF1α-D: 5′-ACTGCCTTGATGACTCCTAG-3′; Irx5-U: 5′-ACTCTGGTCCTTGG-CAGAGA-3′ and Irx5-D: 5′-AGGGTAAAAGGGGATGCTGT-3′. ThePCR conditionswere as follows: 25 cycles of 95°C, 30 s; 55°C, 1min;72°C, 30 s for EF1αamplificationand27 cycles of 94°C, 30 s; 60°C, 30s; 72°C, 1min for Irx5 amplification. 5 μl of total 25 μl PCR productswere subjected to 5% PAGE and visualized by autoradiography.

Results

Smad1, Smad4, and Smad6 modulate the DNA-bindingactivities of different paralogous Hox proteins

Previously, we demonstrated distinct Smad–Hoxc8 interac-tions in BMP-mediated gene transcription. Smad1 interactswith Hoxc8 homeodomain (HD) and inhibits its DNA binding

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activity [30,32], while Smad6 forms a heterodimer with Hoxc8and enhances its binding to DNA [31]. Other studies also showthat homeoprotein DLX-1 interacts with Smad4 through itshomeodomain [36]. The fact that the homeodomain thatmediates Hoxc8–Smad1 interaction is well conserved in theHox family raises the question as to whether the Smad–Hoxinteractions are universal for most, if not all, Hox proteins. Invertebrate, the 39 Hox genes are divided into 13 paralogs sothat genes within each paralog share maximum sequencehomology. To examine our hypothesis, we first chose 12 Hoxproteins, each from a different paralog group (except group 8,which has been characterized), to test their interactions withSmads using a gel-shift assay with purified Hox and Smadproteins. As shown in Fig. 1A, 11 Hox proteins bound to theosteopontin promoter element (OPN5), which contains a coreTAAT sequence [30], with different affinities. Smad1, Smad4,or Smad6 alone did not bind to the Hox consensus element.When coexpressed, Smad1 inhibited most of the Hox bindingto DNA, while Smad4 inhibited all of the Hox binding. Smad6,on the other hand, formed complexes with most of the Hoxproteins on the DNA element. These results were similar towhat we found previously for Hoxc8 and suggested thatSmad–Hox interactions may be a general theme for most Hoxparalogs.

Since Hoxd13 did not bind OPN5 element (data not shown),we used an alternative DNA target that contains a core TTAT

Fig. 1 – Smad1, Smad4, and Smad6 alter the DNA-binding activitThe osteopontin Hox binding element (OPN5: 5′-TAGTTAATGACThe proteins usedwere purified GST-fusion proteins. 0.3μg Hoxa0.05 μg Hoxb6, 0.4 μg Hoxb7, 0.1 μg Hoxa9, 0.3 μg Hoxd10, 0.2 μg(lanes 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46), together with 3μμg Smad4 (lanes 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48), or 3 μSmad1 and Smad4 inhibit Hoxd13 binding to the DNA target. Hoxused as probe and incubated with 0.3 μg GST–Hoxd13 alone (lan

sequence and had been demonstrated to be a selective targetfor Abd-B-like Hox proteins [43]. Similar to other Hox proteins,Smad1 and Smad4 inhibited Hoxd13 binding to the DNA target(Fig. 1B, lanes 7 and 8), but unlike other Hox proteins, Smad6did not form a complex with Hoxd13 (Fig. 1B, lane 9). It ispossible that complex formation between Smad6 and Hoxproteins is dependent on the identity of the DNA target. In ourprevious studies, theMH1 domain of Smad6was found to bindon OPN5, and it may contribute to the complex formation ofSmad6 and Hoxc8 on OPN5 [44]. So far, we do not haveevidence as to whether Smad6 domains bind to a TTATsequence. Taken together, our results suggest that Smad4mayinteract with all, while Smad1 and Smad6 may interact withmost of the paralogous Hox proteins and they may potentiallyact as general cofactors for Hox proteins by modulating theirDNA-binding activities.

Smad1 and Smad6 interact with representative Hox proteinsin mammalian cells

Our gel-shift assays demonstrated that Smads modulated theDNA binding of most of the Hox proteins. To confirm theinteractions, we next analyzed the Smad–Hox interactions in293T cells, using protein-fragment complementation assay(PCA). It had been shown that when the N-terminal and C-terminal YFP fragments were fused with two proteins

ies of representative Hox proteins from different paralogs. (A)ATCGTTCATCAG-3′) was 32P-labeled and used as the probe.1, 0.1μg Hoxa2, 0.05μg Hoxb3, 0.1μg Hoxb4, 0.05μg Hoxa5,Hoxa11, 0.5 μg Hoxd12 were incubated with the probe alone

g Smad1 (lanes 7, , 11, 15, 19, 23, 27, 31, 35, 39, 43, and 47), or 2g Smad6 (lanes 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, and 49). (B)d13 binding element (5′-CTGCGATGATTTATGACCGC-3′) wase 6) or together with Smads (same amounts as A, lanes 7–9).

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respectively, no fluorescent signals could be detected unlessthe two proteins interacted to bring the two YFP fragmentstogether to fold in active form and give green signal [45]. Inour assays, we chose three distal, nonparalogous Hoxproteins (Hoxa1, Hoxb4, and Hoxd10) for their interactionswith Smad1 and Smad6 for the following reasons. First,different paralogous Hox proteins define the identities ofdifferent domains along the anterior–posterior body axis,and functional redundancy normally occurs between para-logous as well as neighboring nonparalogous Hox proteins,but rarely between distal nonparalogous Hox proteins. Thethree distal Hox proteins we chose therefore should notshare redundant activities. Second, Smad1 inhibited theDNA-binding activities of these three Hox proteins atdifferent levels. The binding of Hoxa1 was remarkablydecreased, Hoxd10 binding was moderately decreased, andthere was no obvious inhibition of Hoxb4 binding by Smad1(Fig. 1A, lanes 7, 19, and 39). To test the interactions, wemade two sets of expression plasmids. One set of theplasmids express YFP1–Smad (Smad1 and Smad6, respec-

Fig. 2 – Smad1 and Smad6 interact with representative Hox pro293T cells in a 6-well plate were transfected with Hox–YFP2 andexpression plasmids used for transfection were 1.5 μg each. 48under fluorescence microscope at room temperature. (B) COS-1 ch after transfection, the subcellular protein localization was visua

tively), which contain the N-terminal YFP (a.a. 1–158) and thefull-length Smad1 or Smad6 separated by a small linkerregion. Another set of the plasmids express Hox–YFP2 fusionproteins which contain the full-length Hox proteins (Hoxa1,Hoxb4, and Hoxd10, respectively) and the C-terminal YFP (a.a. 159–239). While cells transfected with either YFP1–Smad1or Hox–YFP2 alone did not have any detectable signal, co-transfection of YFP1–Smad1 and Hox–YFP2 resulted ingeneration of green fluorescence (Fig. 2A). The data indicatethat Smad1 interacts with the three Hox proteins in thecells. Combined with previous results that Smad1 interactswith Hoxc8 and Hoxa9, we propose that Smad1 may interactwith most of the Hox proteins directly, though it is alsopossible that cofactors may exist for a subset of Hox proteinsto interact with Smad1. In similar experiments, Smad6 alsointeracted with the three Hox proteins in 293T cells (Fig. 2A).These results confirmed the Smad–Hox interactions inmammalian cells.

To further visualize the subcellular localization of theinteractions, we transfected YFP1–Smad and Hox–YFP2

teins in protein-fragment complementation assays (PCA). (A)/or YFP1–Smad plasmids as indicated. The amounts of theh after transfection, cells were checked for fluorescent signalells were transfected with indicated expression plasmids. 48lized with higher magnification (see Materials andmethods).

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plasmids into COS-1 cells and viewed the signals with anobjective lens of highermagnification. Both Smad1 and Smad6interacted with Hox proteins in the nucleus as well as in thecytoplasm, although the nuclear colocalization was predom-inant (Fig. 2B). It indicates that Hox proteins may undergoposttranslational modifications in the cytoplasm. One suchexample is Hoxb6, which is phosphorylated by casein kinase IIand PKA [46,47] and is predominantly localized in thecytoplasm of epidermal keratinocytes [47,48].

Smads oppose Hox transcriptional activities

Interactions of Smads with most paralogous Hox proteinssuggest that Smads may potentially regulate Hox transcrip-tional activities. We had shown before that Smad1 antag-onizes the repressor function of Hoxc8 and activates genetranscription, while Smad6 acts as a co-repressor for Hoxc8 tofurther silence gene transcription [30–32]. Different Hoxproteins act as either transcriptional activators or repressors.Furthermore, the same Hox protein possesses either activa-tion or repression functions, depending on the biologicalcontexts as well as the genes it regulates. One such example isHoxc8, which mediates a broad range of target gene transcrip-tion [34]. Our results indicate that different Hox proteinsinteract with Smads in similar patterns (Fig. 1). Here wewanted to examine how Smads modulate the activatorfunction of Hox proteins.

Fig. 3 – Smad6 blocks Hoxc8- and Hoxb7-induced OPG activationHA-Hoxc8 (100 ng) and FLAG-Smad6 (100 ng and 200 ng) expressexpressing Renilla luciferase (Promega) was co-transfected in eaadding pcDNA3 empty vector. The transfected cells were harvesnormalized to Renilla luciferase levels as internal control. (B) OPGng) and FLAG-Smad6 (200 ng) expression plasmids as indicated.expression levels of HA-tagged Hoxb7, c8, and FLAG-tagged Sma

Besides the target genes regulated by Hoxc8 [34], wenoted that Hoxc8 can activate OPG gene transcription whenoverexpressed in prostate cancer cells (unpublished data),opposite to its roles in osteoblast cells [33]. This observationpromoted us to examine the effect of Smad6 on Hoxc8-mediated gene activation in this biological context. Inter-estingly, our results demonstrate that Smad6 blockedHoxc8-induced OPG transcription dose dependently inLNCaP cells (Fig. 3A). Since Smad6 forms a complex withHoxc8 on DNA target [31], it is likely that Smad6 switchesHoxc8 from activator to repressor in LNCaP cells. We alsoexamined Hoxb7 (Fig. 3B), which also activated OPGpromoter, similar to Hoxc8, likely due to the functionalredundancy between these two neighboring Hox proteins.Again, Smad6 exerted inhibitory effect on Hoxb7 activity ata similar dose (Fig. 3B).

To further explore the effects of Smads on Hox-mediatedgene activation, another reporter system was used. Hoxd10had been shown to be an activator for Hoxd9 gene transcrip-tion; the core element that mediates the transactivation byHoxd10 had been mapped to a 90 bp Hox cross-talk region(HCR) within the Hoxd9 promoter [37]. We therefore used theluciferase reporter (pTHCR) [37] containing the core element toexamine whether the transactivation by Hoxd10 is regulatedby Smads. Consistent with the previous findings [37], over-expression of Hoxd10 resulted in 5- to 8-fold increase of thereporter activity (Fig. 4). Importantly, Smad1 blocked the

. (A) OPG-luc reporter plasmid (50 ng) was co-transfected withion plasmids into LNCaP cells. 5 ng pRL-SV40 plasmidch well as internal control. Total DNA was kept constant byted 48 h later. Reporter activities were measured and-luc reporter (50 ng) was co-transfected with HA-Hoxb7 (100Experiments were repeated three times in triplicate. Thed6 are shown in the lower panels by Western blot.

Fig. 4 – Smad1 and Smad6 inhibit Hoxd10 transcriptional activities. (A) Schematic representation of pTHCR reporter. Thepromoter region contains a 90 bp HCR (Hox-cross talk region) element that is activated by Hoxd10 (Ref. 37). (B) Smad1 inhibitsHoxd10-induced transactivation dose dependently. pTHCR reporter plasmid (25 ng) was co-transfected with Hoxd10 (50 ng),and Smad1 (50 ng, 100 ng, and 150 ng) expression plasmids into NIH3T3 cells. 5 ng pRL-SV40 plasmid expressing Renillaluciferase (Promega) was co-transfected in eachwell as internal control. Total DNAwas kept constant by adding pcDNA3 emptyvector. The transfected cells were harvested 48 h later. Reporter activities were measured and normalized to Renilla luciferaselevels as internal control. (C) Smad6 inhibits Hoxd10-induced transactivation. NIH3T3 cells were transfected with indicatedplasmids. (D) Smad2 does not regulate Hoxd10 transcriptional activity. Experiments were repeated three times in triplicate.

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transactivation by Hoxd10 in a dose-dependent manner whencoexpressed (Fig. 4B). We also examined the effects of Smad2.However, unlike Smad1, Smad2 did not have effects onHoxd10-induced transactivation at similar doses (Fig. 4D),suggesting that Smad2 was not involved in regulating Hoxd10transcriptional activity. Smad6 also inhibited activation by

Hoxd10, similar to Smad1 (Fig. 4C). As our gel-shift resultsshowed that Smad1 reduced Hoxd10 binding to DNA whileSmad6 formed a ternary complex with Hoxd10 and DNA, ourfindings indicate that Smad1 and Smad6 may regulate theactivator function of Hoxd10 through different mechanisms.Smad1 inhibited Hoxd10 activity by preventing it from binding

Fig. 5 – Smad1 inhibits Hoxb4 target gene expression during early Xenopus development. (A) Diagram showing animal capassay carried out in frog embryos. (B) Smad1 inhibits Hoxb4-induced Irx5 gene expression. Embryoswere injectedwithmRNAsencoding Smad1 or Hoxb4 at the two-cell stage. The animal capswere removed at the blastula stage and incubated until controlembryos reached neurula stage 19. Total RNA was then extracted and analyzed for Irx5 expression by RT-PCR. EF1α PCR wasused as a loading control. Lane 1, uninjected control caps; lanes 2–4, caps injected with Smad1 mRNA (lane 2), Hoxb4 mRNA(lane 3) and combinedmRNAs (lane 4); lanes 5 and 6, whole-embryo controls, in the absence (lane 5, −RT) or presence (lane 6) ofreverse transcriptase in the RT-PCR reaction. (C) Smad2 is not involved in Hoxb4 target gene expression. Embryoswere injectedwith indicated mRNAs. Then the same procedures were carried out as in (B). The doses of mRNAs used for embryo injectionwere as follows: Hoxb4 2 ng, Smad1 1 ng, and Smad2 1 ng.

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to the DNA target. Smad6, by forming a complex with Hoxd10,could switch it from activator to repressor.

Smad1 modulates Hoxb4 transcriptional activities in vivo

Both BMPs and Hox proteins are key factors that mediatemany similar morphological events during vertebrate devel-opment. The similar expression patterns of BMPs and Hoxproteins inmany embryonic tissues imply that theymay exertfunctions together to mediate developmental processes. Weattempted to investigate whether BMP/Smad signals canregulate Hox target gene transcription in vivo, particularlyfor Hox proteins as activators. It was noted that Hoxb4upregulates Irx5 expression during early Xenopus development[49]. Irx family had been shown to be essential for neural plateformation [50,51]. Our aimwas to examinewhether Smads canmodulate Hoxb4-induced Irx5 gene transcription. In theanimal cap assay, we injected Hoxb4 mRNA together with orwithout Smad1mRNA into the animal poles of frog embryos atthe two-cell stage; the animal caps were removed at theblastula stage when endogenous Hoxb4 expression is notinitiated. The Irx5 gene expression was examined at theneurula stage by RT-PCR. As shown in Fig. 5B, Irx5 expressionwas not detected at significant levels in uninjected animalcaps (lane 1), which have no endogenous Hoxb4 expression.Overexpressing Hoxb4 clearly induced Irx5 expression. Smad1

significantly blocked the Irx5 induction by Hoxb4 (lane 4). Wethen compared the effects of Smad1 and Smad2. UnlikeSmad1, Smad2 had no obvious effect on Hoxb4-mediatedIrx5 upregulation (Fig. 5C, lane 6). These in vivo results suggestthat Smad1 inhibits Hoxb4-induced gene transactivationduring early development. In addition, these results areconsistent with those of the in vitro reporter assays (Fig. 4)and suggest that Smad1 antagonizes the activator function ofHox proteins by dislodging them from DNA targets.

Discussion

BMPs are involved in multiple morphological events duringembryonic development, including mesoderm patterning,skeletogenesis, limb patterning, and apoptosis [2]. While theBMP/Smad signaling cascade is well characterized, the path-way itself does not explain how BMPs exert such diversefunctions. The answer lies in the cross-talk between BMP andother signaling pathways, as well as distinct transcriptionfactors recruited by the BMP pathway. Initially, we identifiedHoxc8 as a transcription repressor in BMP-mediated osteo-pontin and osteoprotegerin gene transcription [30,33]. In thisreport, we demonstrated that Smad1, Smad4, and Smad6interact with the representative members of the other 12 Hoxparalogs, similar to Hoxc8. More importantly, we determined

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that Smad1 prevented Hox binding to DNA and inhibitedtransactivation by Hox proteins, and that Smad6 formed DNA-binding complexes with Hox proteins and inhibited Hox-induced gene transcription. Our results suggest that Hoxproteins act as general downstream DNA-binding proteinsrecruited by BMP/Smad pathway and their transcriptionalactivities are regulated by Smad proteins.

In the gel-shift assay, all of the Hox proteins tested, exceptHoxd13, bound to osteopontin promoter element (OPN5)containing TAAT sequence. Smad1, Smad4, and Smad6interacted with most of the Hox proteins, in ways similar totheir interactions with Hoxc8 (Fig. 1A). For Hoxd13, we usedalternative DNA target containing a TTAT core element thathad been shown to preferentially bind with Abd-B-like Hoxproteins [43]. Again, Smad1 and Smad4 inhibited Hoxd13binding to DNA (Fig. 1B). In our experiments, Smad1/4 alwaysprevented Hox–DNA complex formation. On the other hand,studies on the BMP target geneMsx2 indicate that the presenceof both homeoproteins and Smad1/4 on the promoter elementis required for the gene activation [52]. It is likely that theSmad binding sites flanking the Hox binding site are critical todetermine whether Smad1/4 form complexes with Hoxproteins on the DNA elements or prevents them from bindingto DNA. To confirm the interactions, we performed PCA assayto test three Hox proteins (Hoxa1, Hoxb4, and Hoxd10) for theirinteractions with Smad1 in mammalian cells. AlthoughSmad1 inhibits their DNA binding at different levels (Fig. 1A),it interacts with those Hox proteins in mammalian cellssimilarly (Fig. 2). We propose that Smad1 may interact withmost of the Hox proteins directly, though it is also possiblethat cofactorsmay exist for a subset of Hox proteins to interactwith Smad1. Hox proteins and Smad1 are predominantlycolocalized in the nucleus (Fig. 2B). The cytoplasm localizationof Hox proteins (Fig. 2B) suggests that they may undergoposttranslational modifications. Smad6 formed complexeswith most of the Hox proteins on the OPN5 element (Fig. 1A),but failed to form a complex with Hoxd13 on the TTAT target(Fig. 1B). It is possible that the complex formation betweenSmad6 and Hox proteins is dependent on the identity of DNA.This situation could be similar to that of Hoxa10–Pbx1acomplex, which is only formed on selective DNA sequence[43,53,54]. Our results suggest that Smad1 and Smad6 interactwith and modulate the DNA-binding activities of most Hoxproteins, if not all. We did not examine other R-Smads.Different R-Smads share highly conserved MH1 and MH2domains, therefore interactions may also exist between Hoxproteins and TGF-β-specific R-Smads as well as Smad5 andSmad8 in BMP pathway.

Although Hox proteins are well known as transcriptionfactors, their transcription mechanisms are not well charac-terized. Cofactors are often required for Hox-mediated tran-scription. So far the only well-characterized cofactors are non-Hox homeoproteins [55]. Pbx proteins interact with Hoxproteins (paralog 1–10) and act as partners to offer the DNA-binding specificities for different Hox proteins [53,54]. Meisproteins interact with AbdB-like Hox proteins (paralog 9–13)[56]. Another class of cofactors identified are histone acetyl-transferases (HATs) and histone deacetylases (HDACs), whichinteract with either Hox–Pbx complex or Hox proteins alone[57,58] and mediate transcription by chromatin structure

remodeling [59]. Our results indicate that Smad6 formsheterodimers with Hox proteins and always as Hox co-repressor, and that Smad1 inhibits Hox binding to DNA andopposes their transcriptional activities. Specifically, bothSmad1 and Smad6 inhibited transactivation by Hoxd10 (Fig.4). The mechanisms for their actions are likely to be different.Smad1 inhibited Hoxd10 activities mainly by preventing itfrom binding to the DNA target, whereas Smad6 could switchHoxd10 from transcription activator to repressor by forming acomplex on the DNA element. The inhibitory role of Smad6 onHox protein-mediated transactivation was further supportedby another reporter assay, in which Smad6 blocked bothHoxc8- and Hoxb7-induced OPG promoter activations inLNCaP cells (Fig. 3). Therefore, our results indicate thatSmad1 and Smad6 oppose the transactivation induced byHox proteins.

Finally, we investigated the effects of Smads on Hoxtranscriptional activity during Xenopus development. Again,Hoxb4 examined in the experiments is an activator. We foundthat Smad1 inhibited Hoxb4-mediated Irx5 gene transcriptionduring early Xenopus development (Fig. 5). The results areconsistent with those of the reporter assays (Fig. 4). AlthoughSmad4 showed stronger interaction with Hoxb4 (Fig. 1A, lane20), we did not find obvious effects of Smad4 on Hoxb4activities (data not shown). It is likely that Smad4 only servesas cofactor for Smad1–Hox interactions. Without Smad1,Smad4 alone is not sufficient to regulate Hox activities.

For both Hoxd10- and Hoxb4-induced transactivation,Smad2 was also tested but showed no obvious effects. Itseems that Smad2 does not regulate Hox-mediated genetranscription in these two specific cases (Figs. 4D and 5C).However, in view of so many Hox proteins possessing distinctfunctions with their specific targets, it is still possible thatTGF-β-specific Smads are involved in regulating Hox functionsin other biological contexts. Both BMPs and Hox proteins arekey regulators during vertebrate development. The facts thatmany developmental processes require both BMPs and Hoxproteins imply the intrinsic functional connections betweenthe two regulators. However, little is known about theirrelations beyond their mutual regulation of gene expression.In our studies, we characterized the interactions between Hoxproteins and BMP-specific Smads and the regulations of Hoxtranscriptional activities by Smads. We propose the modelthat Hox proteins act as downstream DNA-binding proteins inthe BMP signaling pathway. Their transcriptional activities areregulated by Smads through their physical interactions.

Acknowledgments

We thank Dr. S. W. Michnick and Odyssey Thera, Inc., forkindly providing YFP1 and YFP2 expression plasmids forprotein-fragment complementation assay. We thank Dr. V.Zappavigna for kindly providing Hoxa1 cDNA and pTHCRreporter, Dr. M. Mallo for Hoxa2 cDNA, Dr. K. R. Humphriesfor Hoxb3 and b4 cDNAs, Dr. M. Featherstone for Hoxd10cDNA, Dr. S. S. Potter for Hoxa11 cDNAs, Dr. C. Largman forHoxb6, b7, a9, d12, and d13 cDNAs, Dr. H. Le Mouellic forHoxc8 cDNA, Dr. R. Derynck for Smad1, 2, and 4 expressionplasmids, and Dr. A. Hemmati-Brivanlou for Smad6 cDNA.

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This work was supported by National Institutes of HealthGrant DK57501 (to X.C.).

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