| Krüppel-like factor4(KLF4), a zinc finger-containing transcriptionfactor, is expressed in a variety of tissues. KLF4both activates and repressesthe transcription of different genes depending on the cellular context, andthereby regulates numerous biological processes including proliferation,differentiation, development, inflammation, and apoptosis. Previous studieshave shown that the function of KLF4may be regulated not only at thetranscription level but also by post-translational modifications, such asphosphorylation, acetylation, sumoylation, and ubiquitylation. Furthermore,accumulating evidence suggests that KLF4can be induced by a variety ofstimuli, including serum starvation, oxidative stress, platelet-derived growthfactor (PDGF)-BB, butyrate, cyclosporine, selenium, transforming growthfactor (TGF)-β, interferon-γ, cyclic adenosine monophosphate (cAMP), andall-trans retinoic acid (ATRA). We have found in our laboratory that, besidesinduction of KLF4expression, ATRA promotes KLF4acetylation andphosphorylation, which subsequently transactivates SM22α and SM α-actinand promotes the differentiation process in vascular smooth muscle cells(VSMCs). Other studies also indicated that transcription factor stimulatingprotein-1(Sp1) participates in the transcription regulation of Klf4in VSMCs.Moreover, Yang et al showed that KLF4is subjected to autoregulation by itsown gene product.ATRA, a metabolite of dietary vitamin A (retinol), directly transactivatesdownstream target genes by binding to retinoic acid receptors (RARs) andretinoid X receptors (RXRs) which belong to the nuclear receptor superfamilyand which promote VSMC differentiation. RARs bind to retinoic acidresponse elements (RAREs) and, when bound by ligands, recruit a proteincomplex to activate transcription; in the absence of ligands, RARs associate with a co-repressor complex that silences transcription. With othertranscription factors, including Sp1/Sp3and STAT5(signal transducer andactivator of transcription5), RARs co-operatively transactivate the targetgenes. RARs are now considered to be an attractive research target fortreatment of VSMC proliferation disease. Clinical applications of ATRA havesuccessfully been applied in human diseases such as leukemia, cancer,restenosis and plaque formation. Despite these advances, the mechanisms bywhich ATRA functions to induce Klf4transcription are still largely unknown.In this study, we aimed to elucidate the molecular mechanisms of ATRAsignaling in the transactivation of Klf4expression in VSMCs.1RARα mediated ATRA-induced Klf4expressionATRA functions by binding to RARs and RXRs, which act astranscription factors upon ligand binding. There are three types of RARs:RARα, RARβ, and RARγ, each of which is encoded by their respective genes.In this study We show that RARα, but not RARβ or RARγ, mediatedATRA-induced Klf4expression in VSMCs. Results are as follows:1.1ATRA induced KLF4and RARα expressions in a concentration-andtime-dependent manner in VSMCsTo explore the actual relationship between KLF4and RARs inATRA-stimulated VSMCs, we first examined the expression of KLF4andRARs in response to ATRA signaling. ATRA increased KLF4and RARαexpressions in a concentration-and time-dependent manner. However, unlikeRARα, the expressions of RARβ and RARγ were little changed by the ATRAtreatment.1.2RARα mediated ATRA-induced Klf4expressionTo further define which RAR isoform mediated the induction of KLF4byATRA, we knocked down endogenous RARs by transfecting VSMCs withsiRNAs against RARα (si-RARα), RARβ (si-RARβ), RARγ (si-RARγ) or acontrol (si-NS), respectively. The induction of KLF4by ATRA wassignificantly reduced in mRNA and protein levels in the cells with RARαknockdown. When VSMCs were treated with a RARα antagonist (Ro41-5253) prior to the addition of ATRA, the blockade of RARα signaling partiallyreduced the response of KLF4to ATRA. Moreover, RARα overexpression,introduced by infection of the adenovirus vector pAd-GFP-RARα, furtherincreased the induction of KLF4by ATRA.To examine whether the RARα-mediated KLF4induction was dependenton its elevated promoter activity, CHO-K1cells were transientlyco-transfected with a Klf4promoter-reporter construct and RARα, RARβ orRARγ expression plasmid (GFP-RARα, GFP-RARβ or GFP-RARγ). Theluciferase activity assay showed that RARα overexpression significantlyincreased the activation of the Klf4promoter under ATRA treatment. Theinductive effect of ATRA was partially compromised by the presence ofincreasing amounts of RARα antagonist Ro41-5253, whereas pretreated withRARβ antagonist LE135or RARγ antagonist MM11253did not affectATRA-induced Klf4transcription. Collectively, these results suggest thatRARα, but not RARβ or RARγ, is essential for induction of Klf4transcriptionby ATRA.1.3ATRA induced KLF4expression via the proximal3GC boxes in the Klf4promoterTo identify the RARα-responsive elements in the Klf4promoter region,we constructed progressive5’-deletion constructs of the Klf4promoter fusedto the luciferase reporter gene. Using transfection of these constructs, weexamined the ability of ATRA to activate the expression of each, in CHO-K1cells. The results showed that the Klf4promoter proximal region-179to+20exhibited the greatest increase in luciferase activity, compared with control, inresponse to ATRA treatment. Sequence analysis revealed that there were3GCboxes in the Klf4promoter region within this region.To assess whether these GC boxes were required for ATRA-induced Klf4expression, we transfected the proximal promoter constructs (-179/+20)encoding various GC box mutations with ATRA stimulation. We observed thatthe mutation of a single GC box reduced the ATRA-stimulated activation ofKlf4promoter activity by64-80%. The simultaneous mutation of2GC boxes decreased ATRA-induced Klf4promoter activity by~85%. Furthermore, theelimination of all3GC boxes completely abrogated the response of the Klf4promoter to ATRA. This implies that the response to ATRA by the3GC boxesin the proximal region of the Klf4promoter was synergistic.1.4RARα recruited to the3GC boxes in the proximal Klf4promoter andpromoted Klf4transcriptionTo investigate whether RARα directly bound to the3GC boxes inATRA-stimulated VSMCs, we carried out the ChIP assay. The results showedthat ATRA stimulation significantly promoted the binding of RARα to theproximal region of the Klf4promoter, no binding of RARα was detected whendistal Klf4promoter region was amplified. Consistent with the results of theChIP assay, the oligonucleotide pull-down assay showed that the binding ofRARα to the GC1/2or GC3box was increased by ATRA stimulation, whilemutation in the GC boxes interrupted binding, indicating that the binding ofRARα to the GC boxes is specific.To further determine if the binding of RARα to GC boxes affected Klf4promoter activity, CHO-K1cells were co-transfected with GFP-RARα and theKlf4promoter-reporter construct (-179/+20wt), or with GFP-RARα and theKlf4promoter-reporter mutated construct (-179/+20mut). RARαoverexpression significantly elevated the activities of the Klf4proximalpromoter with integrative3GC boxes in response to ATRA stimulation.However, when all3GC boxes were mutated, the response of the Klf4promoter to RARα overexpression and ATRA was almost completelyabolished. Taken together, these results indicate that RARα mediatedATRA-induced Klf4expression in a GC box-dependent manner.2Association of KLF4, Sp1, or YB1with GC boxes facilitated Klf4promoter activitiesSeveral recent studies have reported that RARs can exert their effect viaRARE-independent regulatory mechanisms by interacting with othertranscription factors. We reasoned that, as a nuclear receptor mediatingATRA’s effect, RARα could interact with one or more GC box-binding factors to regulate Klf4expression. In this study we showed RARα wasrecruited to the Klf4promoter via its interaction with KLF4, Sp1, and Y boxbinding protein1(YB1), which are associated with GC boxes at the site, tocooperatively activate Klf4transcription. Results are as follows:2.1ATRA induced the interaction of RARα with Sp1, YB1or KLF4Several transcription factors, including Sp1and KLF4, have beendescribed as binding with the proximal Klf4promoter. To find new factorsmediating ATRA-stimulated KLF4expression, we affinity-purifiedDNA-binding transcription factors from VSMC extracts using as a probe abiotinylated double-stranded DNA fragment harboring nucleotides-179to+20of the Klf4promoter. The result showed that one protein of36kDa waspulled down by the Klf4(-179to+20) promoter fragment after ATRAstimulation. HCT mass spectrometry analysis with a database search, andfurther confirmation of the amino acid sequence by post-source decay peptidesequencing, showed that this protein was YB1.A Co-IP assay showed that ATRA stimulation significantly increased theinteraction of RARα with KLF4, Sp1, and YB1in VSMCs in vivo. To revealwhether RARα directly interacted with YB1, Sp1, and KLF4in vitro, GSTpull-down with overexpressed GFP-RARα was performed in vitro. Apparently,GFP-RARα physically bound to GST-KLF4, GST-Sp1, or GST-YB1in vitro,but not GST alone; ATRA treatment increased their bindings.2.2RARα cooperated with Sp1, YB1or KLF4and activated Klf4promoteractivityTo investigate the association of KLF4, Sp1, or YB1with the GC boxesin the proximal KLF4promoter, the oligonucleotide pull-down assaydemonstrated that the binding affinity of KLF4, Sp1, and YB1to GC boxes(GC1/2or GC3) in the proximal Klf4promoter were increased inATRA-stimulated VSMCs, while the mutation in GC boxes abrogated thebinding. The ChIP assay also showed that ATRA promoted the association ofKLF4, Sp1, and YB1with the proximal Klf4promoter in VSMCs.To evaluate the additive effects of the interaction of RARα with KLF4, Sp1or YB1on Klf4promoter activity, CHO-K1cells were co-transfectedwith a Klf4promoter reporter, along with various combinations of expressionplasmids for KLF4, Sp1, YB1, and RARα, and then treated with or without10μM of ATRA and followed by a luciferase assay. Transient expression ofKLF4, Sp1or YB1alone, as well as two or three combinations of theseexpression plasmids, increased Klf4promoter activity to a certain extent. Thestrongest activation was observed when all4expression plasmids for KLF4,Sp1, YB1, and RARα were co-transfected in the presence of ATRA.Altogether, these results demonstrated that the interaction of RARα withKLF4, Sp1, or YB1in the Klf4promoter region facilitated promoter activity.2.3KLF4, Sp1, or YB1directly bound to GC boxes and cooperativelytransactivated the Klf4promoterTo examine the physical binding of KLF4-Sp1-YB1to the GC boxes, weperformed an oligonucleotide pull-down assay using GST-KLF4, GST-Sp1,GST-YB1or GST-RARα with biotinylated double-stranded oligonucleotidescontaining the wild-type or mutant GC1/2or GC3box. GST-KLF4, GST-Sp1or GST-YB1directly bound to the GC1/2or GC3boxes, but not their mutants,in a concentration-dependent manner. However, we did not observe thephysical binding of GST-RARα to biotinylated probes containing GC boxes invitro.To further investigate the effect of KLF4, Sp1, and YB1on Klf4promoter activity, CHO-K1cells were co-transfected with a constant amountof pPac-Sp1plasmid, and increasing amounts of pEGFP-KLF4plasmid, alongwith the Klf4promoter-reporter construct pGL3-Klf4-luc. The stimulatoryeffect of Sp1on the Klf4promoter gradually increased with increasingamounts of pEGFP-KLF4. Likewise, when CHO-K1cells were co-transfectedwith a constant amount of pEGFP-KLF4and increasing amounts of pPac-Sp1,Sp1enhanced the stimulatory effect of KLF4on the Klf4promoter in aconcentration-dependent manner. These results suggested that both KLF4andSp1bound to the Klf4promoter to co-operatively activate its transcription. Inaddition, both Sp1and YB1, as well as both KLF4and YB1, also cooperated with each other to activate the Klf4promoter.2.4RARα is necessary for the binding of KLF4, Sp1and YB1to the proximalKlf4promoter induced by ATRANext, we knocked down endogenous RARα by transfecting VSMCs withsi-RARα. The ChIP assay showed that ATRA promoted the binding of KLF4,Sp1, and YB1to the GC box region of the Klf4promoter in si-NS-treatedVSMCs; downregulation of endogenous RARα by si-RARα reducedATRA-induced recruitments of these factors to the Klf4promoter. The resultssuggest that the presence of RARα or ATRA stimulation facilitated theassociation of KLF4, Sp1or YB1with the GC box region of the Klf4promoter.3ATRA promoted the binding of RARα to the Klf4promoter in aKLF4-Sp1-YB1-dependent mannerTo further determine the molecular mechanism of RARα-mediated KLF4transcription in the context of ATRA, we further investigated the associationof RARα and KLF4-Sp1-YB1complex with KLF4promoter inATRA-stimulated VSMCs.3.1ATRA stimulation promoted the binding of RARα to the KLF4-Sp1-YB1complexTo reveal the roles of ATRA in the interactions between RARα and KLF4,Sp1or YB1, we next performed a GST pull-down assay in vitro. We observedthat although KLF4, Sp1, and YB1formed a complex without ATRAtreatment, the addition of ATRA stimulation promoted the binding of RARα tothe KLF4-Sp1-YB1complex. Furthermore, To analyze the interactionsbetween KLF4, Sp1and YB1, GST pull-down assays with overexpressedGFP-KLF4, GFP-Sp1or GFP-YB1were performed in vitro. Apparently,GFP-KLF4, GFP-Sp1and GFP-YB1physically bound to any of the otherfactors. Simultaneously, we also analyzed the effect of ATRA on some othertarget genes of KLF4, Sp1, or YB1. In addition, the protein level of SM22α,p53and p21, the target genes of KLF4, YB1and Sp1, significantly increasedin VSMCs after ATRA stimulation for24h. 3.2ATRA promoted the binding of RARα to the Klf4promoter in aKLF4-Sp1-YB1-dependent mannerTo further examine whether RARα recruitment to the proximal Klf4promoter depended on the binding of the KLF4-Sp1-YB1complex to GCboxes on the Klf4promoter, we performed a ChIP assay in VSMCs whereKLF4, Sp1or YB1was knocked down. The result showed that higheroccupancy of RARα on the Klf4promoter was observed under ATRAstimulation. Knockdown of endogenous KLF4, Sp1or YB1by siRNAsignificantly attenuated the binding of RARα to the Klf4promoter, no bindingof RARα was detected when one pair of negative control primers were used.In addition, CHO-K1cells were co-transfected with expression constructs forKLF4, Sp1, YB1or RARα and then oligonucleotide pull-down assays wereperformed using GC1/2or GC3box sequence as probes. The results showedthat RARα bound to GC1/2or GC3box at the lower level, but theco-expression of KLF4, Sp1or YB1significantly increased the binding ofRARα to GC boxes when ATRA was present. These results suggested that thebinding of RARα to the Klf4promoter was KLF4-Sp1-YB1complex-dependent and that ATRA stimulation promoted the interaction ofRARα with KLF4, Sp1, and YB1.3.3KLF4, Sp1and YB1was necessary for RARα-mediated Klf4expression inATRA-stimulated VSMCsTo further validate the above results, we next transfected VSMCs withsiRNAs targeting Klf4, Sp1, YB1, or with an adenovirus expression vector forRARα, followed by ATRA treatment. Overexpression of RARα significantlyenhanced KLF4expression induced by ATRA. Knockdown of endogenousKLF4, Sp1or YB1, together with RARα overexpression, abrogated theinduction of KLF4expression by ATRA in VSMCs. Altogether, these resultsconfirmed that KLF4, Sp1and YB1was necessary for RARα-mediated Klf4expression in ATRA-stimulated VSMCs.4YB1interacted with GC box and promoted VSMC differetiationYB1, a member of the cold-shock domain (CSD) protein superfamily, is a multifunctional protein that participates in gene transcription, RNA splicing,and mRNA translation. YB-1preferentially binds to DNA containing the5’-CTGATTGG-3’ motif (so-called Y-box) or single-stranded DNA (ss-DNA,C/T-and GC/GA-rich sequences) and regulates the expression of a largenumber of gene products. Here, we first reported that YB1binds to doublestranded GGGCGG motif (known as GC box), which is considered a majorcontrolling sequence for a large number of gene transcription. In this study, wefurther investigated the molecular mechanisms of YB1interaction with GCbox in VSMCs. Results are as follows:4.1YB1bound to GC boxes directlyTo investigate the binding activity of YB1to GC box sequence, wedesigned various biotinylated double-stranded probes harboring GC boxsequences, the negative control probes containing CACCC motif sequences orthe probes containing mutated GC boxes. In addition, a Y-box probe and a4×GC probe were designed as positive controls. VSMC lysates, as well asGFP-YB1-transfected A293cell lysates or purified GST-YB1, were incubatedwith various probes and oligonucleotide pull-down assay was done to examinethe binding activity of YB1with GC boxes. The results showed thatendogenious YB1, as well as overexpressed GFP-YB1or purified GST-YB1,was associated with various probes containing GC boxes, including probesfrom promoters of differentiation related genes (SM22α, p21) andproliferation related gene (cyclin D1), and the mutation of GC box sequenceinterrupted its binding activity. The strongest binding was observed when4×GC-probe was used.4.2The interaction of YB1with GC box was independent on Cold ShockDomain (CSD) or ss-DNA binding regionTo identify the specific regions in YB1protein that are necessary for GCbox binding, we incubated biotinylated double-stranded probe a (SM22αpromoter) with various purified wt/truncated GST-tagged YB1in equalamounts under in vitro condition followed by oligonucleotide pull-down assay.Besides the full-length wt GST-YB1(1-324), probe a that harboring GC box retained GST-YB1(1-220), GST-YB1(125-324) and GST-YB1(125-220).Purified GST-YB1(125-220) directly bound to probe a and4×GC in aconcentration-dependent manner. Similarly, the full-length wt GFP-YB1(1-324) or various truncated GFP-tagged YB1were transfected into A293cellsand the cell lysates were used to perform oligonuleotide pull-down assay using4×GC probe. The result showed that the full-lengh GFP-YB1, GFP-YB1(125-220) and GFP-YB1(125-324) could bind the GC box. Taken together,we concluded that interaction of YB1with GC box is independent on CSD(65-125) or ss-DNA binding region (1-71), and the amino acids125-220sequence of YB1are sufficient for GC box binding.Next, to compare the binding affinity of YB1to ss(single-stranded)-DNA with that of to ds (double-stranded)-DNA, a ds-GC ora ss-GC probe were incubated with purified GST-YB1or GST-YB1(125-220),as well as overexpressed GFP-YB1or GFP-YB1(125-220), andoligonucleotide pull-down assay was performed. The result showed that wtGST-YB1or GFP-YB1could bind to both ds-GC and ss-GC probes;GST-YB1(125-220) and GFP-YB1(125-220), lacking of the ss-DNA bindingregion (15-71), only bound to ds-GC probe. These results strongly suggestedthat YB1(125-220) is necessary for binding GC box in vivo and in vitro.4.3C-terminal tail of YB1promotes VSMC differentiationTo investigate whether the C-terminal tail (125-324) of YB1is requiredfor the transactivation of GC-dependent target genes, VSMCs wereco-transfected with SM22α or p21promoter-reporter plasmids and GFP-YB1or GFP-YB1(125-324) expression plasmids. YB1C-terminal domainoverexpression significantly elevated the SM22α and p21promoter activities.Moreover, the adenovirus expression vector harboring GFP-tagged wt YB1orYB1C-terminal tail (125-324) coding region were designed and infected intoVSMCs. The full-length form of YB1was located in both the cytoplasm andnucleus with a speckled distribution in the majority of cells, whereas YB1C-terminal tail was located mainly in the nucleus. Phalloidin staining showedthat the actin filaments were recruited into thick and long actin bundles in YB1overexpressed VSMCs; VSMCs overexpressed with YB1C-terminaldomain markedly elongated, with increasing formation of thick stress fibersaligned along the long axis.To further identify the potential role of YB1C-terminal tail (125-324) inVSMC differentiation, VSMCs were infected for72h with Ad-GFP,Ad-GFP-YB1or Ad-GFP-YB1(125-324). Compared with Ad-GFP-infectedgroup, SM22α and p21expression levels significantly increased whileproliferating cell nuclear antigen (PCNA) level decrease, in Ad-GFP-YB1(125-324)-infected group. Next, BrdU incorporation assay was performed toexamine the effect of YB1and its C-terminal tail domain on VSMCproliferation. The relative BrdU incorporation was significantly increasedwhen endogenious YB1was knocked down using si-YB1, and markedlyinhibited by YB1or YB1C-terminal domain overexpression.Next, we sought to investigate the effect of YB1on VSMC migration.VSMCs were transfected with si-YB1or infected with Ad-GFP-YB1orAd-GFP-YB1(125-324) and boyden chamber or wounding cell migrationassays were performed to test the effects of YB1or its C-terminal domain onVSMC migration. The results showed that knockdown of endogenious YB1effectively enhanced the basal and transwell migration activity in VSMCs; incontrast, overexpression of C-terminal domain or wild-type YB1significantlysuppressed VSMC migration. These results indicated that C-terminal taildomain of YB1plays a key role in YB1-induced VSMC differentiation.CONCLUSIONS1ATRA induced KLF4and RARα expressions in VSMCs. RARα mediatedATRA-induced Klf4expression via the proximal3GC boxes in the Klf4promoter.2ATRA promoted the binding of RARα to the KLF4-Sp1-YB1complex.Association of KLF4, Sp1, or YB1with GC boxes facilitated Klf4promoter activities. 3ATRA promoted the binding of RARα to the Klf4promoter in aKLF4-Sp1-YB1-dependent manner. KLF4, Sp1and YB1was necessary forRARα-mediated Klf4expression in ATRA-stimulated VSMCs.4YB1physically interacted with GC boxes and promoted VSMCdifferetiation. |
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