Virology 413 (2011) 244–252

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Virology

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Identification of CD44 as a downstream target of noncanonical NF-κB pathwayactivated by Human T-cell leukemia virus type 1-encoded Tax protein

Jing Zhang a,⁎, Osamu Yamada a, Shinya Kida a, Yoshihisa Matsushita a, Shoji Yamaoka b,Haorile Chagan-Yasutan c, Toshio Hattori c

a Research and Development Center, FUSO Pharmaceutical Industries, LTD., 2-3-30 Morinomiya, Joto-ku, Osaka 536–8523, Japanb Department of Molecular Virology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japanc Division of Emerging Infectious Diseases, Department of Internal Medicine, Tohoku University, Sendai, Japan

⁎ Corresponding author. Fax: +81 6 6964 2706.E-mail address: j-zhang@fuso-pharm.co.jp (J. Zhang

0042-6822/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.virol.2011.02.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 November 2010Returned to author for revision21 January 2011Accepted 24 February 2011Available online 15 March 2011

Keywords:HTLV-1TaxCD44Noncanonical NF-κB signalingRelB

Our previous study showed Human T-cell leukemia virus type 1 Tax induces osteopontin (OPN) expression bytransactivating its promoter. As an extension, we investigated here the possible influence of Tax on CD44, animportant receptor for OPN. Co-expression of Tax, but not its NF-κB-defective mutant, significantly increasedthe reporter gene expression directed by CD44 promoter. Tax-mediated CD44 activation was largelydiminished by disrupting an element similar to the noncanonical κβ site found in other IKKα target genes, andfurther, co-transfection of RelB siRNA abolished CD44 induction by Tax, suggesting an involvement ofnoncanonical NF-κB pathway in Tax-mediated transactivation. Consistently, chromatin immunoprecipitationrevealed a specific interaction of CD44 promoter with RelB-containing complex. Together, these resultsindicate that D44 gene is one of the downstream target genes of aberrantly activated noncanonical NF-κBsignaling by Tax, providing an additional line of evidence explaining how Tax-induced NF-κB signaling isintegrated into a fate-determining cellular program.

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Introduction

Nuclear factor-κB (NF-κB) is a family of structurally relatedtranscription factors that form dimers composed of differentcombination of five subunits: NF-κB1 (p50/p105), NF-κB2 (p52/p100), RelA (p65), RelB and c-Rel (Ghosh and Karin, 2002; Karin andBen-Neriah, 2000). The NF-κB dimers are sequestered in thecytoplasm by interacting with a group of inhibitory IκB proteinsharboring ankyrin repeat domain (Baldwin, 1996). Upon stimulation,NF-κB can be activated by two independent pathways: the canonicaland noncanonical pathways. While activation of the canonicalpathway involves liberation of RelA:p50 from IκB following IκB kinase(IKK)-mediated phosphorylation and proteasomal degradation of IκB,activation of noncanonical pathway involves cleavage of NF-κB2/p100and dimerization of the mature p52 with RelB. In both cases, themature dimeric NF-κB complexes translocate to the nucleus andsubsequently transactivate the expression of a large array of targetgenes. Different forms of the NF-κB dimer exhibit distinct propertieswith respect to DNA binding preference and transcriptional capability.Whereas the RelA:p50 dimer preferentially binds to the sequence of5′-GGGRNNYYCC-3′ (R=purine, N=any nucleotide and Y=pyrim-idine), the RelB:p52 dimer recognizes the sequence of 5′-RGGA-

GAYTTR-3′ that is not recognized by the RelA:p50 dimer (Bonizziet al., 2004). Although activated concurrently in some circumstances,NF-κB pathways have distinct regulatory functions. In contrast to thecanonical one, thenoncanonicalNF-κBpathway is not activatedbysometypical inducer such as TNF-α, IL-1β, and dsRNA, but is rather activatedby signals involved in B cell maturation and lymphoid organogenesis,including lymphotoxin (LT) β receptor (LTβR) activation, engagementof BAFF-R (B cell-activating factor belonging to the TNF family receptor),CD40 ligand (Bonizzi and Karin, 2004). Deregulated activation of thenoncanonical pathway has been observed in autoimmune diseases andmultiple aggressive malignancies.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirusetiologically associated with adult T-cell leukemia (ATL) and HTLV-1-associatedmyelopathy/tropical spastic paraparesis (Gessain et al., 1985;Osame et al., 1986; Poiesz et al., 1980; Uchiyama et al., 1977). Althoughthe precise mechanisms of HTLV-1-associated leukemogenesis are notyet fully understood, it has been suggested that the transformingproperties of HTLV-1 are largely conferred by its regulatory protein Tax.Tax expression is sufficient to immortalize human T cells, and Taxexpression alone in transgenicmousewas shown to be adept enough inpromoting leukemogenesis in vivo (Hasegawa et al., 2006). Trans-formed cells isolated from late ATL patients, however, do not expressviral proteins, and whether continued expression of Tax is required fortransformation remains controversial. A currently accepted conceptionis that Tax protein is crucial for initiatingmalignant transformation, butmay be dispensable for maintenance of transformation. Tax initiates

Fig. 1. Transactivation of CD44 promoter by HTLV-1 Tax protein. HeLa (A) or Jurkat Tcells (C) were transfected with pCD44-luc1 along with the control vector or vectorexpressing each indicated Tax protein or its mutant. Luciferase activities in the lysateswere measured at 48 h post transfection. Renilla luciferase activities from cotransfectedpRL-TK were used to normalize the transfection efficiency. Normalized luciferaseactivity from an otherwise identical control transfection with pCn backbone was set as100%, and those in other transfectants are expressed as relative percentage. Results arepresented as the means and standard deviations of four independent triplicatetransfections. (B) Expression of Tax protein in each transfectants was confirmed byWestern blot analysis. Solid arrow indicates the signal of Tax protein.

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transformation largely through its abilities to transcriptionally deregu-late cellular gene expression and functionally inactive proteins involvedin cell-cycle progression and DNA repair. One of the major signalingpathways aberrantly activated by Tax involved in cell transformation isNF-κB (Higuchi and Fujii, 2009;Matsuoka and Jeang, 2011). ConstitutiveNF-κB activation is a hallmark of HTLV-1-transformed T-cell lines andfreshly isolated ATL cells (Arima et al., 1991; Ballard et al., 1988; LeungandNabel, 1988; Ruben et al., 1988). It is clear that Tax plays anessentialrole in HTLV-1-induced NF-κB activation, although a Tax-independentmechanism was suggested to be responsible for constitutive NF-κBactivation in ATL cells without detectable Tax expression. (Hironakaet al., 2004). Tax can activate NF-κB by both the canonical andnoncanonical pathways, with predominance for the former. In thecanonical pathway, Tax directly interacts with IKKγ and triggers thephosphorylation of IKKα and IKKβ, leading to degration of IκBα (Chuet al., 1999; Harhaj and Sun, 1999; Jin et al., 1999). On the contrary, Taxprovokes noncanonical pathway by activating and recruiting IKKα top100, stimulating phosphorylation, ubiquitination and processing top52. More recently, it was reported that regulation of NF-κB by Tax isinfluenced by awide range of post-translationalmodifications includingphosphorylation, ubiquitination, acetylation and sumoylation. Thesemodifications control Tax intracellular localization, assembly ofnuclear bodies and sequential steps in Tax-mediated NF-κB activation(Lodewick et al., 2009; Shembade et al., 2007a, 2007b). Additionally,multiple modulatory components in Tax-mediated NF-κB activationhave been newly identified, suggesting this signaling could be morecomplex than originally thought. For example, Tax1 binding protein,TAX1BP1, was demonstrated to be involved in the recruitment ofA20 deubiquitinase for termination of NF-κB signaling, and Tax maytarget TAX1BP1 to counteract the function of A20 and promotepersistent NF-κB activation (Iha et al., 2008; Shembade et al., 2007a,2007b). Also, Pin1, a cellular isomerase, was found to target phosphor-ylated Tax and be critical for Tax binding to IKKγ and its subsequentactivation of the NF-κB pathway (Peloponese et al., 2009). Target genesactivated by NF-κB include the genes for cell cycle regulators, cytokinesand their receptors, such as interleuin-2 and the alpha subunit of itshigh-affinity receptor, which promotes the proliferation and transfor-mation of infected cells via autocrine and/or paracrine stimulation. Inaddition, a number of anti-apoptotic genes, such as Bcl-xL (Nicot et al.,2000; Tsukahara et al., 1999), c-FLIP (Krueger et al., 2006) and HIAP(Wäldele et al., 2006), have been shown to be up-regulated by Tax viaNF-κB pathway. Tax-mediated anti-apoptotic functions favor a persis-tent viral infection and survival of infected cells, thereby contributing toHTLV-1-associated leukemogenesis.

CD44 is a broadly distributed cell surface glycoprotein that issynthesized in multiple isoforms due to alternative splicing of its pre-mRNA (Liu et al., 1997). CD44 binds to the extracellular matrixmacromolecule hyaluronan (HA) as well as other glycosaminoglycansincluding osteopontin (OPN). CD44 has been identified as a tumor-promoting molecule that is implicated in cancer cell growth, invasion,and metastasis. The pro-oncogenic property of CD44 is attributable toits association with and co-stimulation of signaling by a number ofgrowth factor receptors. CD44 can bind growth factors and presentthem to their authentic high-affinity receptors, thus promotingproliferation and invasiveness of cells. Indeed, there is accumulatingevidence suggesting that aberrant expression of CD44 and its variantforms is associated with aggressive stages of various human cancers(Paradis et al., 1999). Inhibition of CD44 by anti-CD44mAb or CD44-Fcfusion protein has been shown to block tumor growth, metastasis, andinvasion (Guo et al., 1994; Mummert et al., 2003).

We have recently shown that Tax induces OPN expression bytransactivating its promoter (Zhang et al., 2010). OPN is an autocrine/paracrine mediator, interacting with integrin αvβ3 or CD44 fortransduction of cell-matrix signaling directed to increase motility,invasion, and angiogenesis (Oates et al., 1997). To further clarify therelevance of OPN-CD44 loop in ATL pathogenesis, we investigated the

effect of Tax on CD44 expression. The results presented here show astrong transactivation of CD44 promoter by Tax, and the molecularbasis underlying the CD44 activation involves Tax-induced noncano-nical NF-κB signaling and a putative κβ-binding site selectivelyrecognized by RelB-containing complex.

Results

Transactivation of CD44 promoter by Tax

To explore the possible effect of Tax on the promoter of the CD44gene, we constructed a reporter vector (pCD44-luc1) inwhich a 2.0 kbDNA segment of CD44 promoter region was introduced into directlyupstream of the luciferase-coding gene. pCD44-luc1 was transfectedinto HeLa cells together with Tax-expressing plasmid and pRL-TK,which was used as an internal control to normalize the transfectionefficiency. To simultaneously gain some mechanistic insight, Taxmutants defective in their ability to activate either NF-κB (m148) orCREB pathway (m319) were also included in the reporter assay. Thecell lysates were collected and luciferase activities were measured48 h after transfection. As shown in Fig. 1A, both the wild type and theTax mutant m319 strongly enhanced luciferase expression from

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pCD44-luc1, while the Tax mutant m418 had no significant effect onCD44-driven gene expression. Western blot analysis showed acomparable expression of wild or mutant type Tax protein (Fig. 1B).Similar results were also observed with Jurkat T cells (Fig. 1C),suggesting that Tax-mediated activation of CD44 was not cell-typespecific. These results suggest that Tax transactivates CD44 promoterand its capacity to activate NF-κB pathway might be important forCD44 activation. To confirm this observation, we next assessed if Taxactivation of CD44 could be abrogated by blocking NF-κB activation inthese cells. For this purpose, we inhibited canonical NF-κB pathway byco-transfection of a construct (pRc-CMV-IκBαSR) expressing a “super-repressor” form of IκBαSS/AA (IκBα SR), which harbors serine-to-alanine substitutions at residues 32 and 36, thus not undergoingphosphorylation required for subsequent ubiquitination. Consistentwith a role for NF-κB, co-expression of IκBα SR potently blocked thestimulation of HIV-1 LTR activity by Tax, suggesting that IκBα SR wasexpressed and its dominant-negative function was intact in theemployed experimental condition. In contrast, Tax-mediated HTLV-1LTR activation was not significantly different irrespective of thepresence or absence of IκBα SR, indicating that IκBα SR specificallyblocks Tax-triggered NF-κB pathway without affecting CREB/ATFpathway (Fig. 2B). Unexpectedly, co-expression of IκBα SR had noeffect on Tax activation of CD44 (Fig. 2C), while over-expression ofNF-κB2 precursor protein p100,which has been reported to be able toefficiently interrupt Tax-mediated NF-κB activation (Béraud et al.,1994; Kanno et al., 1994), largely abolished the transactivation ofCD44 by Tax (Fig. 2D). Western blot showed expression level of Taxwas not affected by either IκBα SR or p100 (data not shown).

Determining Tax-responsive element in CD44 promoter

To dissect the transcription regulation of CD44 by Tax, we nextconstructed a series of related vectors pCD44-luc2, -luc3, and -luc4 inwhich the luciferase gene is directed under the control of a series of 5′end-truncated CD44 promoter. As shown in Fig. 3, Tax-mediated CD44up-regulation was observed in these reporter vectors, and the Tax-induced luciferase expression was even higher in pCD44-luc4, inwhich most of 5′-partial sequence of CD44 promoter was deleted (seeDiscussion). This result suggested that the proximal 375-bp regionmight contain regulatory element(s) responsible for the Tax activa-tion of CD44. A computer Database analysis indicated the presence ofbinding sites to multiple transcription factors including an elementwith sequence similarity to the classical κB binding site. In view of theabove-described results with Tax mutant m148, we hypothesized thispotential NF-κB-binding site might be responsible for the observedTax-mediated activation of CD44. To address this issue, we con-structed a mutant reporter vector pCD44-luc-mu1 by deleting theputative κB site and subsequently analyzed its activity in response toTax. Contrary to the expectation, disruption of the putative classicalκB site did not significantly affect the activity of CD44 promoter inresponse to Tax. Further inspection of the nucleotide sequence withinthis region revealed two sites that exhibit close sequence similarity(8/10 identity) to the noncanonical κβ binding site found in thestromal cell-derived factor-1 (SDF-1) gene, beginning at positions−279 (5′-GGGAGGGCTG-3′) and−360 (5′-CAGGTTCCCC-3′) nucleo-tides from the initiator ATG (Fig. 3) (Bonizzi et al., 2004). These twosites, designed here as κβ-like motifs 1 and 2, are arranged head tohead and separated by 73 nucleotides in distance. To examine therelevance of these two sites in CD44 promoter activation, we createdsingle-site mutants (pCD44-luc-mut2 and pCD44-luc-mut3), and

Fig. 2. Effects of ectopically expressed IκBα SR or p100 on Tax-induced CD44 activation.pHIVLTR-luc (A) or pHTLVLTR-luc (B) or pCD44-luc1 (C and D) was transfected into thecells along with pCn or pCnwtax, as well as the construct expressing IκBα SR or p100.Relative luciferase activities were determined and calculated as described for Fig. 1.Representative results are from three independent triplicate transfections.

Fig. 3. Identification of a 10-bp κB-like motif as the-Tax responsive element in CD44 promoter. Left: schematic illustration of the reporter vectors, the fragment of human CD44promoter is represented as heavy line, and the number at the 5′ end of each construct indicates the 5′-most end base position from the translational initiation codon of the CD44gene. The location of the putative canonical (κB-ca) and noncanonical κB sites as well as the p53-responsive element (p53 RE) are also indicated. Right: upper, alignment of the twoκB-like motifs in CD44 promoter with the noncanonical κB site identified in SDF promoter; lower, resulting constructs were transfected into cells along with pCn or pCnwtax and therelative luciferase activities were determined and calculated as described for Fig. 1. Representative data are from five independent triplicate transfections.

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double-site mutant (pCD44-luc-mut2+3), in which each of the twoκβ-like motifs was eliminated alone or together via site-directedmutagenesis. As shown in Fig. 3, Tax-mediated CD44 activation waslargely diminished by disruption of the κβ-likemotif 1,while ablation ofκβ-like motif 2 did not significantly affect the responsiveness of CD44promoter to ectopically expressed Tax protein. Combined mutation oftwo motifs in pCD44-luc-mut2+3 did not further attenuate CD44activation byTax. This observation suggests that the inducibility of CD44by Tax is likely mediated by a putative noncanonical κB site spanningnucleotides −279 to−270 in the promoter.

Fig. 4. Recruitment of RelB on the CD44 promoter. The chromatin complexes from Tax-transfected cells were co-immunoprecipitated with the antibody against p65, RelB ornonspecific rabbit IgG. The DNA from the resulting precipitated chromatin complexeswas amplified using the set of primers flanking the potential κB site in the CD44promoter (upper panel) or Bcl-xL promoter (lower panel).

CD44 promoter is recognized by RelB- but not RelA-containing complex

The data presented above suggested that a potential noncanonicalκB site in the proximal region of the promoter is responsible for theTax-mediated CD44 activation. We next asked whether CD44 gene isin fact a direct target of noncanonical NF-κB signaling pathwayinvolving specific recognition by RelB:p52 dimer. For this purpose, weperformed chromatin immunoprecipitation (ChIP) experiments withTax-transfected cells. Cross-linked chromatin was incubated withantibodies specific to the p65 or RelB, and the immunoprecipitatedDNAs were detected by PCR using primers flanking the potential κBsite in the proximal region of CD44 promoter. As shown in Fig. 4,amplified PCR product was detected in the immunoprecipitatesrecovered with anti-RelB antibody, while there was no amplifiedsignal detected from the chromatin recovered with either anti-p65 ornonspecific rabbit IgG. As a control, we analyzed the same immuno-precipitates for the presence of the promoter sequence for Bcl-xL, anantiapoptotic downstream target of Tax-activated canonical NF-κBsignaling (Bernal-Mizrachi et al., 2006; Tsukahara et al., 1999). Asexpected, we found efficient precipitation of the promoter fragmentby anti-p65, but a weak signal was obtained with anti-RelB antibody.This result indicates a selective recruitment of RelB-containing NF-κBdimers to the CD44 promoter.

Tax activation of CD44 was abrogated by silencing of RelB

We next took a different approach using siRNA-mediated silencingof RelB to further dissect the involvement of noncanonical NF-κBpathway in Tax-mediated CD44 activation. pCD44-luc1 plus Tax-expressing plasmid were transfected with or without the plasmidexpressing RelB siRNA. While the scramble siRNA had no significanteffect on RelB expression, RelB siRNA greatly reduced the expressionof RelB, resulting in a 62% decrease in protein level of RelB relative tothat co-transfected with the backbone control (Fig. 5A). Consistentwith the results obtained from ChIP experiment, Tax-induced CD44transactivation was largely diminished by siRNA-mediated knock-down of RelB, as assessed by the reporter assay which showed anaverage 51% decrease of CD44-directed luciferase expression in thepresence of RelB siRNA (Fig. 5B). This result further supports a key roleof the RelB subunit of the noncanonical NF-κB pathway in Taxactivation of CD44. Together with the observations shown above, it is

Fig. 5. The role of RelB in mediating the transactivating effect of Tax on CD44. Cells weretransfected with pCD44-luc1, Tax-expressing vector, and siRNA-expressing vector.(A) RelB protein level was efficiently down-regulated by siRNA. RelB expression wasassessed 72 h after transfection by Western blot. Actin was served as a control forprotein loading. (B) Relative luciferase activities were determined as described in Fig. 1.The results are from three independent triplicate experiments.

Fig. 6. Effect of HTLV-2 Tax2 on CD44 promoter activity. Cells were transfected withpCD44-luc1 (A) or pHIVLTR-luc (B), alongwith the control vector or vector expressing Taxor Tax2 protein. Relative luciferase activities were determined and calculated as describedfor Fig. 1. Representative results are from four independent triplicate transfections.

Fig. 7. Endogeneous CD44 expression in wild- and mutant-type Tax-expressing cells.Total RNAs were prepared from each cell lines expressing Tax or its mutant, andsubjected RT-PCR with a primer pair flanking the insert site of variant exons. Theamplified signals of 471 bp in length, corresponding to the predicted size of CD44standard form, is indicated. GAPDH served as an internal control.

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strongly suggested that the noncanonical, rather than canonical, NF-κB signaling is associated with Tax transactivation of CD44 gene.

HTLV-2 Tax2 does not transactivate CD44

Despite the similarity in multiple functions between HTLV-1 Taxand HTLV-2 Tax2, Tax 2 was found to be inactive in triggeringnoncanonical NF-κB signaling, and the reduced transforming activityof Tax2 relative to Tax may partially be attributable to its inability toactivate noncanonical NF-κB pathway (Higuchi et al., 2007). We nextinvestigated if Tax2 could also transactivate CD44. Different from thatobserved with Tax, CD44 promoter activity was not significantlyactivated by either Tax2A or Tax2B (Fig. 6A). The luciferase expressionunder the control of HIV-1 LTR, however, was similarly stimulated byTax and Tax2A or Tax2B (Fig. 6B), suggesting that Tax2A and Tax2Bwere expressed and their function were intact under the experimen-tal condition. This result indicates that Tax and Tax2 exhibit differentbehavior on CD44 induction, which is likely correlated with theirdistinct abilities to activate noncanonical NF-κB signaling.

Effect of Tax on endogenous CD44 RNA expression

CD44 has multiple isoforms resulting from alternative splicing of itsmRNA (Liu et al., 1997). We next investigated whether the expressionlevel or isoform pattern of CD44 is affected by Tax. Expression of CD44mRNA was determined by RT-PCR in HuhCn, Huhwtax, Huhm148 andHuhm319 cells, which were established by transfection of Huh-7 cellswithpCn,pCnwtax, pCnm148 andpCnm319 followedbyG418 selection(Zhang et al., 2007). Similar level of Tax expression in each cell lineswasconfirmed by Western blot analysis (data not shown). Compared withthat detected in HuhCn control, CD44 mRNA expression was enhancedin Huhwtax and Huhm319 cells, whereas the CD44 mRNA level wasnot elevated in Huhm148 cells (Fig. 7). This result is fully consistentwith that obtained from the reporter assay shown in Fig. 1, further

demonstrating that Tax induces CD44 gene expression and thisactivation is lined with its function to activate NF-κB pathway. Inaddition, the amplified 471-bp product corresponded to the predicted

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size of CD44 standard form (CD44s). In all cell lines irrespective of Taxexpression, there is no other band with different molecular weightdetected even after the amplification went to 35 cycles. The productswere also cloned and sequenced to verify the observation from gelelectrophoresis. The sequencing results confirmed that the amplifiedbands were CD44s (data not shown).

CD44 is activated by ligation of LTβR

We next investigated the effect of LTβR ligation on CD44expression using LTα1β2, a specific ligand for LTβR. Expression ofCD44mRNAwas determined by RT-PCR in each fraction of cells whichwere cultured in the absence or presence of LTα1β2 or TNF for 24 h,and it was found that LTα1β2 but not TNF up-regulated CD44expression (Fig. 8A). Similar results were also obtained in the reporterassay. As shown in Fig. 8B, incubation with 30 and 100 ng/ml of

Fig. 8. LTβR ligation induces CD44 expression. (A) 293 cells were either untreated orincubated with the indicated concentration of TNF or LTα1β2 for 24 h then RNA wasprepared and subjected to RT-PCR using primer pair specific for CD44. GAPDH wasserved as an internal control. Cells were transfected with pCD44-luc1 (B) or pE-selectin-luc (C), harvested and divided into several aliquots at 12 h post-transfection,which were further cultured for 16 h in the absence or presence of the indicatedconcentration of TNF or LTα1β2. Relative luciferase activities were determined asdescribed in Fig. 1. The results are from three independent triplicate experiments.

LTα1β2 for 16 h induced a 1.8- and 2.7-fold increase in CD44-directedluciferase expression, respectively; whereas TNF had no significanteffect. On the contrary, gene expression direct from E-selectin, one ofthe known target genes downstream of canonical NF-kappaBsignaling, was dramatically induced by TNF treatment, but onlymarginally elevated by LTα1β2 (Fig. 8C). This result indicates thatCD44 is a downstream target gene of LTβR-induced noncanonical,rather than TNF-induced canonical, NF-κB signaling, which is fullyconsistent with the fore-mentioned observations.

Correlation of increased CD44 and Tax expression in HTLV-1-transfromedcells

To clarify physiological relevance of CD44 activation by Tax, wenext examined the expression level of CD44 in different HTLV-1-transformed or ATL-derived cells. RNA was isolated and CD44 specifictranscripts were detected by RT-PCR. As shown in Fig. 9A, CD44mRNAis increased, albeit to different degree, in all Tax-positive cell lines(ATL-2, HUT-102, MT-2, MT-4 and TL-Su) when compared with JurkatT cell or Tax-negative cell lines (ATL-43 T, ED and TL-Om1). Westernblot revealed that elevated CD44 mRNA levels result in increasedamounts of CD44 protein in the Tax-expressing cells, whereas CD44proteinwas barely detectable in Tax-negative cells (Fig. 9B). Together,these results indicate that the augmented CD44 expression coincideswith the expression of the viral transactivator Tax, providing a furtherindication for Tax-mediated transactivation of the CD44 gene.

Discussion

Each NF-κB pathway has distinct biological functions that could beexecuted in part by selective gene activation. Noncanonical activationof NF-κB2 leads to transcription of a set of genes different from thoseregulated by canonical NF-κB activation, the target selectivity ofnoncanonical signaling depends on recognition of specific κB bindingsite by RelB:p52 dimer. Compared with IκBα degradation, theprocessing of the NF-κB2 p100 precursor occurs in a more regulatedmanner, which cannot be triggered in response to most canonicalstimuli. Cytokines such as LTβ, BAFF, CD40 ligand, as well as viral

Fig. 9. Correlation of increased CD44 and Tax expression in HTLV-1-transformed cells.(A) Total RNAs were extracted from the indicated cell lines and subjected to RT-PCRanalyses with the primer pairs specific for CD44 or Tax. GAPDH was served as aninternal control. (B) The protein level of CD44 was detected by Western blot usingCD44-specific antibody. Actin was served as a loading control.

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oncoproteins, are the few select stimuli able to activate thenoncanonical pathway. HTLV-1-encoded Tax protein is among thebest known of such viral oncoproteins, which acts as a pathologicalstimulator of p100 processing by physical recruiting IKKα to p100 andsubsequently triggering phosphorylation-dependent ubiquitylation.The aberrant activation of the noncanonical pathway by Tax has beenimplicated in T-cell transformation and HTLV-1-associated leukemo-genesis. Indeed, it was shown that Tax-activated noncanonicalsignaling plays an important role in the resistance to apoptosis,which is mediated by up-regulating a broader spectrum of down-stream antiapoptotic targets, such as survivin, FLIP, XIAP, and CIAP(Bernal-Mizrachi et al., 2006). We now extended this list byidentifying CD44, a transmembrane glycoprotein with multipletumor-promoting functions, as a downstream target of noncanonicalpathway activated by Tax. This conclusion was inferred based on thefollowing observations: 1) Tax transactivates CD44 promoter and itsability to activate NF-κB pathway is necessary for this activity; 2)CD44 activation by Tax is insensitive to a super repressor form of IκBαSS/AA, nevertheless being completely diminished in the presence ofover-expressed p100; 3) elimination of a element similar to thenoncanonical κβ site largely abolished the CD44 activation by Tax,while disruption of a putative classical κB site did not significantlyaffect the activity of CD44 promoter in response to Tax; 4) CD44promoter is recognized by RelB- but not by RelA-comtaining NF-κBcomplex; and 5) knockdown of RelB by siRNA attenuated Taxactivation on CD44 promoter. These results corroborate the conclu-sion that the transcriptional induction of CD44 by Tax is mediated viathe noncanonical, rather than the canonical, NF-κB pathway.

It iswell established that Tax inactivates the transcription function ofthe tumor suppressor p53 (Jeong et al., 2004; Pise-Masison et al., 2000,2001), and p53 was recently reported to repress CD44 transcription bydirect interaction with a element located at position −376 to −390(Godar et al., 2008). Thus, CD44 transactivation could be due to anindirect effect of Tax: that is, via antagonizing p53 and consequentlyderepressing the expression of CD44. In view of the fact that Tax-induced luciferase expression was also observed in pCD44-luc4, inwhich the upstream 1.6 kb region including the p53-responsiveelement was deleted, indicating that CD44 activation observed heredid not occur as a secondary consequence of p53 repression by Tax. Inaddition, the absence of the p53-responsice element in pCD44-luc4mayalso provide an explanation for the observation that both basal and Tax-induced luciferase activity is higher in pCD44-luc4 than those inreporter vectors pCD44-luc1, -luc2 and -luc3 (Fig. 3).

RelB-containing dimers are not bound and inhibited by IκBs, andIκBα SR was shown to sequester IκB-sensitive dimers withoutinterfering with the release of RelB:p52 (Saccani et al., 2003). Onthe other hand, given that p100 is one of the target genes up-regulated by canonical NF-κB signaling, the canonical inhibitor such asIκBα SRmay decrease the production of p100, as well as the processedform 52, thus cross-inhibiting the noncanonical signaling. Our datapresented here showed that although IκBα SR completely blocked thecanonical NF-κB activation (as evidenced by the vanished HIV1-LTRinduction), Tax-activated CD44 transcription nevertheless occurred inthe presence of IκBα SR (Fig. 2), indicating that noncanonical NF-κB-mediated CD44 activation could occur in the absence of the canonicalpathway. This observation is consistent to that previously reported bySaccani et al., who demonstrated that activation of the canonical andnoncanonical NF-κB pathways occurs in an independent fashion(Saccani et al., 2003). In that study, newly synthesized p100 was stillfound even when the canonical NF-κB-dependent up-regulation ofp100 was fully inhibited by IκBα SR.

Infectivity of free HTLV-1 virion is very poor, and the transmissionof HTLV-1 to target cells has been shown to require the formation of avirological synapse involving ligand-receptor interaction (Igakuraet al., 2003). In this regard, several studies have demonstrated apotential role of adhesionmolecules in HTLV-1 biology (Barnard et al.,

2005; Ceccaldi et al., 2006; Daenke et al., 1999). Likewise, since CD44is a cell surface adhesion molecule involved in cell–cell and cell–substrate interactions, it is thus possible that Tax-mediated CD44activation may facilitate cell-to-cell infection of HTLV, in addition toits contribution to leukemogenesis. Therefore, a positive feedbackloop involving CD44 and HTLV-1 Tax protein may be considered:CD44 is up-regulated by Tax on HTLV-1-infected cells, and, in turn,engagement of CD44 by its physiological ligand HA or OPN, whichitself is also up-regulated by Tax (Zhang et al., 2010), augments HTLV-1 infection by increasing cell-to-cell contact. Studies are under way toinvestigate this hypothesis. If confirmed, interrupting the CD44-ligandinteractionmay provide a novel strategy for the therapeutic control ofHTLV-1 infection.

In addition to HTLV-1-encoded Tax, noncanonical NF-κB pathwayis also activated by transforming proteins of other oncoviruses, suchas LMP protein from Epstein Barr virus (Luftig et al., 2004; Saito et al.,2003), vFLIP protein from Kaposi sarcoma herpes virus (Matta andChaudhary, 2004), and E6 protein from human papilloma vius (Jameset al., 2006), it is thus probable, but remains to be proven, that CD44 isalso aberrantly expressed in these viral infections, thus playing acrucial role in the oncogenesis by these viruses.

In summary, the data presented here indicate that CD44 is one ofthe downstream targets up-regulated by Tax-activated noncanonicalNF-κB signaling, which provides an additional line of evidenceexplaining how Tax-induced NF-κB signaling is integrated into afate-determining cellular program. Together with our prior findings(Zhang et al., 2010), these results suggest another important benefitconferred on leukemic cells by Tax: an increased resistance toapoptosis and responsiveness to mitogenic signals resulting fromelevated OPN and CD44 levels, further emphasizing the significance todevelop novel therapeutic modalities targeting Tax and/or NF-κBpathway for control of HTLV-1-associated malignancies.

Materials and methods

Plasmids

Control plasmid pCn, and plasmids encoding wild-type HTLV-1Tax (pCnwtax) or its mutants (pCnm148 and pCnm319) have all beendescribed previously (Yamaoka et al., 1996; Zhang et al., 2007). Forconstruction of pCD44-luc1, the promoter sequence (2021-bpfragment upstream of translation initiation site) was PCR amplifiedfrom human genomic DNA with primers 5′-ATAGGTACCTGAATC-CATGCTGTTCG-3′ and 5′-ATAAAGCTTGGTGTCCGGAGCGAACGG-3′.Various size deletions of the promoter were also generated by PCRwith various 5′ primers and the same fixed 3′ primer. The amplifiedproducts were digested with Kpn I and Hind III, and ligated withpGL3-Basic (Promega) that had been cut with the same enzymes.

For constructing the plasmid expressing siRNA against RelB, senseand antisense oligonucleotides of self-complementary hairpin sequencecontaining RelB target sequence (5′-GGATTTGCCGAATTAACAA-3′),which contains cohesive ends for BamH I amd EcoR I sites at the 5′-and 3′-ends, were synthesized and annealed. After a gel electrophoresispurification, the annealedoligonucleotideswere inserted into theBamHI and EcoR I sites of pShuttleU6 (Zhang et al., 2004). The sequence of theconstruct was confirmed by nucleotide sequencing.

Cells

Human hepatoma cell line Huh-7 and human cervical carcinomaHeLa cells were purchased from the American Type Culture Collection(ATCC) and maintained in Dulbecco's modified Eagle's medium(DMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS)and 50 u/ml penicillin and streptomycin. Jurkat T-lymphocytes weremaintained in RPMI1640 medium supplement with 10% FCS. The celllines HuhCn, Huhwtax, Huhm148 and Huhm319, were established by

251J. Zhang et al. / Virology 413 (2011) 244–252

transfecting Huh-7 cells with pCn, pCnwtax, pCnm148 and pCnm319,followed by selection in the presence of 600 μg/ml G418 (Geneticin,Invitrogen) (Zhang et al., 2007).

Chromatin immunoprecipitation (ChIP) assay

2×106 Huhwtax cells were fixed with formaldehyde, sonicated andchromatin complexes were immunoprecipitated with rabbit poyclonalantibody against p65 (sc-372), RelB (sc-226) or control rabbit IgG. TheDNA from immunoprecipitates was amplified using sets of primers forthe putative κB sites in the CD44 promoter (5′-GAAGAAAGC-CAGTGCGTCTC-3′ and 5′-GTGTCCGGAGCGAACGGAGG-3′), for the κBsite in the Bcl-XL promoter (5′-TCAGACAAACCACGCATTTG-3′ and 5′-GGTGGAGGCGTCCGAAACCC-3′).

Western blot analysis

Protein was electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel, transferred to Hybond-P PVDV Membrane (GEHealthcare). The blot was probed with the indicated specificantibodies, and signals were visualized with ECL Plus WesternBlotting Detection Reagents (GE Healthcare).

Transient transfection and luciferase assay

Cells were seeded at 1×105 in 1 ml medium per well of 12-wellplates 24 h before transfection. Indicated plasmid DNAs weretransfected into cells with FuGENE6 (Roche). For each transfection,pRL-TK (Promega) vector was co-transfected as an internal control tonormalize transfection efficiency. The cells were harvested at 48 hpost-transfection, and the cell lysates were prepared for luciferaseassay with Dual-Luciferase Reporter Assay System (Promega) accord-ing to the manufacturer's instruction. Luciferase activities weremeasured using a TD-20/20 Luminometer (Promega).

RT-PCR

Total RNAswaspreparedwith Trizol reagent (Invitrogen), and treatedwith RNase-free DNase (Promega). The DNA-free RNA was extractedwith phenol-chloroform and precipitated with ethanol. For reversetranscription reaction (RT), cDNAwas synthesized from1 μg of total RNAin 20 μl reaction volume with Superscript II reverse transcriptase(Invitrogen) at 42 °C for 1 h using oligo(dT) primer. The resulting cDNAwas amplified using primer pairs as follows: CD44, 5′-ACATCAGTCACA-GACCTGCC-3′ and 5′-GCAAACTGCAAGAATCAAAG-3′; Tax, 5′-ATGGCC-CACTTCCCAGGGTTTGGAC-3′ and 5′-TCAGACTTCTGTTTCTCGGAAATG-3′.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We thank Dr. Masahiro Fujii for kindly providing us Tax2A- andTax2B-expressing plasmids.

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