Differential regulation of HOXA9 expression by nuclear factor kappa B (NF-κB) and HOXA9

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Gene 408 (2008) 187 – 195 www.elsevier.com/locate/gene

Differential regulation of HOXA9 expression by nuclear factor kappa B (NF-κB) and HOXA9 Chinmay M. Trivedi a,b,1 , Rekha C. Patel b , Chandrashekhar V. Patel a,⁎ a

Department of Cell and Developmental Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC-29209, United States b Department of Biological Sciences, University of South Carolina, Columbia, SC-29208, United States Received 30 July 2007; received in revised form 30 October 2007; accepted 2 November 2007 Available online 13 November 2007 Received by A.J. van Wijnen

Abstract HOXA9 is a homeobox transcription factor expressed in endothelial cells (EC) and its expression is rapidly downregulated during EC activation by inflammatory signals like tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS). Recently, we have shown that HOXA9 overexpression prevents EC activation by inhibiting NF-κB activity, which suggests that HOXA9 downregulation is an essential event for EC activation. The present study is directed towards understanding the mechanism of HOXA9 regulation during EC activation. Here we show that nuclear factor-κB (NF-κB) activation is an essential step for HOXA9 downregulation. Deletion analyses of HOXA9 promoter in EC and NF-κB knockout cells have shown that NF-κB is a major transcription factor that is absolutely required for HOXA9 downregulation. Our 5′ deletion analysis of HOXA9 promoter shows that NF-κB response element is localized within first 400 nucleotides, while minimal basal promoter is within 100 nucleotides upstream of its transcriptional start site. We demonstrate that HOXA9 regulates its own expression by positive feedback mechanism. To define mechanism by which HOXA9 autoregulates its expression, we show that HOXA9 DNA binding and transactivation domains are essential. © 2007 Elsevier B.V. All rights reserved. Keywords: Atherosclerosis; Endothelial cells; Homeodomain; Inflammation; Transcriptional control of gene expression; Gene regulation; Cell signaling

1. Introduction Atherosclerosis, the principle cause of heart attack, is responsible for 20% of the mortality in the United States Abbreviations: (EC), Endothelial Cells; (ICAM-1), Intracellular adhesion molecule-1; (VCAM-1), Vascular cell adhesion molecule-1; (HUVECs), Human umbilical vein endothelial cells; (HOX), Homeobox; (TNF-α), Tumor necrosis factor-α; (LPS), Lipopolysaccharide; (PMA), Phorbol myristate acetate; (IL-1β. ), Interleukin-1β; (NF-κB), Nuclear Factor κB. ⁎ Corresponding author. Department of Cell and Developmental Biology and Anatomy, School of Medicine, University of South Carolina, Bldg 4, Rm. C16, 6439, Garners Ferry Road, Columbia, SC-29209, United States. Tel.: +1 803 733 3274; fax: +1 803 733 1533. E-mail address: [email protected] (C.V. Patel). 1 Present address: Cardiovascular Institute, University of Pennsylvania School of Medicine, 943, BRB II, 421, Curie Blvd, Philadelphia, PA-19104, United States, Tel.: +1 215 746 6324; fax: +1 215 573 2094. The authors have no conflicting financial interests. 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.11.001

(Rosamond et al., 2007). EC activation is a key event for the onset of inflammatory disease processes including atherosclerosis (Ross, 1993). Activated ECs are characterized by the expression of leukocyte adhesion molecules like, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, which are induced by inflammatory signals such as TNF-α, interleukin-1β (IL-1β), and LPS (Iademarco et al., 1993; Caldenhoven et al., 1994; Read et al., 1997). Majority of transcriptionally regulated genes expressed in the endothelium in response to inflammatory mediators such as TNF-α, LPS, and IL-1β contained an NF-κB binding site in their promoters, including ICAM-1, VCAM-1, and E-selectin (Degitz et al., 1991; Iademarco et al., 1993; Schindler and Baichwal, 1994; Read et al., 1997). NF-κB activation is regarded as one of the most important and early events of EC activation (Collins et al., 1995). Activated NF-κB has been shown to be present in human atherosclerotic plaques

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but absent in normal vessels devoid of atherosclerosis (Iiyama et al., 1999). Inhibition of NF-κB activation results in highly efficient inhibition of EC activation (Collins and Cybulsky, 2001). Although the mechanisms of TNF-α dependent NF-κB activation are well established, TNF-α mediated repression is poorly understood during EC activation. TNF-α is known to downregulate expression of several atheroprotective genes in EC and thereby it facilitates the progression of atherosclerosis. For example, endothelial nitric oxide synthase (eNOS), an important vasorelaxation factor, expression is downregulated by TNF-α during atherogenesis (Yoshizumi et al., 1993). TNF-α is also known to downregulate eNOS promoter activity (Neumann et al., 2004). Similarly, TNF-α inhibits expression of argininosuccinate synthase (AS), which is essential for atheroprotective eNOS production (Goodwin et al., 2007). TNF-α regulates expression of genes through several different signaling pathways, including NF-κB signaling pathway (Aggarwal, 2003). Intriguingly, various studies have shown that several atheroprotective genes like PPARα and PPARγ are downregulated during EC activation in response to TNF-α and this downregulation is NF-κB dependent (Delerive et al., 1999; Ruan et al., 2002). We have shown that a novel homeobox transcription factor, HOXA9, is rapidly downregulated during EC activation in response to TNF-α (Patel et al., 1999). Recently, we have shown that HOXA9 downregulation is an essential event for EC activation. HOXA9 overexpression inhibits inflammatory cytokine dependent inducible expression of leukocyte adhesion molecules in EC (Trivedi et al., 2007b). Further, we demonstrate that continued expression of HOXA9 inhibits the EC activation pathway by interfering with the transcriptional activity of NF-κB (Trivedi et al., 2007b). Thus, HOXA9 maintains EC in a quiescent state by negatively regulating NF-κB activity. To further understand the regulation of HOXA9 expression during EC activation, we show that TNF-α and LPS downregulates HOXA9 expression in NF-κB dependent manner. Further we show that HOXA9 overexpression positively regulates its own expression through its DNA binding and transactivation domains. Together, our results indicate that HOXA9 expression in EC is rapidly downregulated in response to inflammatory signals and this is a prerequisite step for EC activation. 2. Materials and methods 2.1. Plasmids HOXA9 promoter (3.7 kb) was PCR amplified from human endothelial cell genomic DNA by using primers 5′-CGG GGT ACC CCG GGG GCG TGG TAG GAA TTC T-3′, and 5′-CGG GGT ACC CCG ACA ACT TGG TGG CAC-3′ and cloned into the pGEM-T Easy vector (Promega). The PCR product was sub-cloned into the pGL3 basic by using KpnI restriction enzyme sites. HOXA9-pcDNA3.1+ vector with N-terminus FLAG tag sequence, MEKK expression constructs, and RelA-

GFP expression construct were described earlier (Trivedi et al., 2007b) HOXA9 DNA binding mutant (N51S) was a kind gift from Mark Kamps, MD, PhD (University of California, San Diego). 2.2. Deletion mutants Deletion mutants from the 5′ end of the HOXA9 promoter were created using PCR amplification corresponding to the following locations relative to the transcriptional start site: − 2400, − 1300, − 1000, − 700, − 400, − 100. The six deletion mutants were then sub-cloned into pGL3 basic to allow for their promoter activity to be tested in a cell culture system using Human umbilical vein endothelial cells (HUVECs), bovine aortic endothelial cells (BAECs), mouse endothelial cells (MECs,), and NIH 3T3 cells. 2.3. Reagents Human TNF-α was purchased from Promega and LPS, PMA and endothelial cell growth supplement was purchased from Sigma. 2.4. Cell culture HUVECs were grown on gelatin-coated tissue culture plates in Ham's F-12K medium (Irvine Scientific) containing Kaighn's nutrient mixture, 10% heat-inactivated fetal bovine serum (Life-Technologies), 50 μg/ml porcine intestinal heparin (Sigma), 50 μg/ml endothelial cell growth supplement (Sigma) and 10 mM L-glutamine (Life-Technologies). NIH 3T3 wt, p65–p50 double knockout NIH 3T3 cells, and p50–p52 double knock out NIH 3T3 cells were a generous gift from Dr. David Baltimore PhD, Caltech Institute, Pasadena, CA. BAECs, MECs, and all types of NIH 3T3 cells were maintained in Dulbecco's Modified Minimal Essential Medium (DMEM) with 10% fetal bovine serum. HUVECs passages 2 and 4, and BAEC passages 4 and 6 were used in all experiments. All cell types were grown under 5% CO2 at 37 °C. 2.5. Transient transfections HUVECs were transfected by SuperFect transfection reagent (Qiagen) as per manufacturer's protocol. In brief, each well of a six well plate containing a sub-confluent culture was transfected with total of 3 μg of DNA, including 0.2 μg of phRL-SV40 (Promega), and 12 μl of SuperFect transfection reagent in 1 ml of DMEM without serum. Total amount of DNA was maintained by adding empty pcDNA3.1+ vector when appropriate. After 4 h of transfection, cells were washed twice with PBS and Ham's F-12K medium was added as described in cell culture. 4 h after transfection, TNF-α (10 ng/ml) or LPS (5 ng/ml) or PMA (20 ng/ml) or TGF-β (1 nM) treatment was given for 16 h. BAECs and NIH 3T3 were transiently transfected by Effectene transfection reagent (Qiagen) as per manufacturer's protocol. MECs were transiently transfected with Fugene-6 (Roche) as per manufacturer's protocol.

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2.6. Measurement of dual luciferase activity HUVECs, BAECs, MECs, or NIH 3T3 cells were lysed with passive lysis buffer (Promega) and lysates were analyzed for both firefly and Renilla luciferase activity using a dual luciferase reporter assay kit (Promega) according to the manufacturer's protocol. Zylux (Fisher Scientific) luminometer was used according to the manufacturer's instructions. 2.7. Real time PCR analysis mRNA was collected from transiently transfected HUVECs with or without HOXA9 by TRIZOL (Invitrogen). cDNA was synthesized using SuperScript III RTS First-Strand cDNA Synthesis Kit as per manufacturer's protocol (Invitrogen). Quantitative PCR was performed by using SYBR green, as described previously (Trivedi et al., 2007a). 2.8. Statistical analysis Quantitative data is expressed as mean ± S.E. Statistical analysis was performed with the use of the Student's t test. Differences were considered significant when probability values were less than 0.05. 3. Results 3.1. HOXA9 promoter activity is downregulated during EC activation by TNF-α and LPS There is a rapid and nearly complete (N 8 fold) downregulation in the steady state level of HOXA9 mRNA in response to TNF-α treatment for 4 h (Patel et al., 1999). Similar downregulation of HOXA9 mRNA was observed in EC treated with either IL-1β or LPS but not PMA (Patel et al., 1999). Based on the primer extension assay, it has been shown that the transcription initiation site of HOXA9 was localized to the exon-1 (Patel et al., 1999). This finding together with the HOXA9 transcript size of 2.1 kb suggested that the exon-1 and the HOXA9 homeodomain-coding exon were sufficient to generate the HOXA9 transcript and that HOXA9 may be transcribed from a novel promoter element immediately upstream of the exon-1 (Patel et al., 1999). To determine the TNF-α dependent regulatory region in HOXA9 promoter, we have cloned the 3.7 kb region upstream of HOXA9 exon-1 transcription initiation site in to pGL3 basic luciferase vector. We determined the 3.7 kb HOXA9 promoter activity in resting and TNF-α or LPS activated HUVECs. As seen in Fig. 1A, TNF-α or LPS treatment of the transfected cells resulted in a 60–70% reduction in HOXA9 promoter activity, while treatment with transforming growth factor (TGF)-β had no effect. TNF-α or LPS treatment upregulated control NF-κB luciferase reporter vector activity (Fig. 1B) but had no effect on pGL3-SV40 or pGL3 basic luciferase control vectors (Fig. 1C, D). This data suggests that TNF-α and LPS downregulate HOXA9 at transcriptional level during EC activation. We observed similar results in other endothelial cells like BAECs

Fig. 1. TNF-α and LPS suppresses HOXA9 promoter activity. HUVECs were transfected with 3.7 kb HOXA9 promoter-pGL3 basic luciferase (A) or NF-κB luciferase (B) or pGL3-SV40 luciferase (C) or pGL3-basic luciferase (D) reporter vector. 4 h post-transfection, the cells were treated with TNF-α (10 ng/ml) or LPS (5 ng/ml) for 16 h and luciferase activity was measured. Measuring Renilla luciferase activity of co-transfected phRL-SV40 normalized the transfection efficiencies.

and MECs (Supplementary Fig. 1) and non-endothelial cells like NIH 3T3, HT1080, HEK 293 (Supplementary Fig. 1). 3.2. Constitutive NF-κB activation downregulates HOXA9 promoter activity EC activation in response to inflammatory signals has been shown to be mediated by activation of the transcription factor NF-κB (Shu et al., 1993; Lewis et al., 1994; Rahman et al., 2002). Although other transcription factors are involved in activation of EC, NF-κB activity has been shown to be essential (Collins et al., 1995). MEKK (Mitogen Activated protein/ERK Kinase Kinase) and TIRAP (Toll-Interleukin-1 Receptor (TIR) domain containing Adaptor Protein), known to be an upstream activator of the IKK complex in the NF-κB activation pathway but downstream of TNF-α and LPS respectively (Read et al., 1997; Hagemann and Blank, 2001).

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3.3. RelA (p65) overexpression inhibits HOXA9 promoter activity Overexpression of RelA (p65) results in activation of NF-κB due to formation of active RelA homodimers in the absence of any inflammatory signals (Chen et al., 2001; Hoffmann et al., 2002). In order to determine if RelA overexpression can inhibit HOXA9 promoter activity in the absence of any proinflammatory signals, we transiently co-transfected HOXA9 promoter luciferase construct with or without RelA expression plasmid. Cotransfection of the RelA expression plasmids resulted in a significant suppression (N 80%) of HOXA9 promoter activity (Fig. 3A), while it upregulated NF-κB reporter activity by 11 fold (Fig. 3B). p65 overexpression had no effect on control pGL3 luciferase vectors (Fig. 3C, D). MEKK or TIRAP overexpression activates NF-κB activation cascade at a

Fig. 2. MEKK and TIRAP overexpression inhibits HOXA9 promoter activity. HUVECs were co-transfected with 3.7 kb HOXA9 promoter-pGL3 basic luciferase (A) or NF-κB luciferase (B) or pGL3-SV40 luciferase (C) or pGL3basic luciferase (D) reporter vector and either pcDNA3.1+ control vector, flagMEKK-pcDNA3.1+, or flag-TIRAP-pcDNA3.1+. Luciferase activity was measured 24 h after transfection. Measuring Renilla luciferase activity of cotransfected phRL-SV40 normalized the transfection efficiencies.

MEKK or TIRAP overexpression causes activation of IKK complex leading to constitutive IκBα phosphorylation and degradation, which result in constitutive activation of NF-κB. In order to determine effect of constitutive NF-κB activity on HOXA9 promoter, we transiently co-transfected HOXA9 promoter luciferase construct with or without MEKK or TIRAP. As seen in Fig. 2A, cotransfection of the MEKK or TIRAP expression plasmids resulted in a significant suppression (N 70–80%) of HOXA9 promoter activity, while it upregulated NF-κB reporter activity by 32–37 fold (Fig. 2B). Control pGL3 luciferase vector activity was unaffected by MEKK or TIRAP overexpression (Fig. 2C, D). These data suggest that HOXA9 promoter activity is regulated through activation of NF-κB pathway.

Fig. 3. RelA/p65 overexpression suppresses HOXA9 promoter activity. HUVECs were transiently co-transfected with 3.7 kb HOXA9 promoter-pGL3 basic luciferase (A) or NF-κB luciferase (B) or pGL3-SV40 luciferase (C) or pGL3-basic luciferase (D) reporter vector with or without RelA/p65-pEGFP. Luciferase activity was measured 24 h after transfection. Measuring Renilla luciferase activity of co-transfected phRL-SV40 normalized the transfection efficiencies.

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relatively upstream point, whereas RelA overexpression bypasses all the earlier signaling steps and results in nuclear localization of RelA homodimers, thereby leading to NF-κB activation (Hoffmann et al., 2003). Since HOXA9 promoter activity is inhibited by NF-κB activity induced by both these means, it strongly suggests that NF-κB may act at a downstream of its nuclear localization step. 3.4. NF-κB responsive region is present in first 400 bp of HOXA9 promoter We found one consensus NF-κB binding site (− 1505 to − 1496 bp) in HOXA9 promoter region (Fig. 4A). To determine the functional importance of NF-κB binding and to locate the cis-regulatory element(s) present within the 5′-flanking sequence of the HOXA9 promoter that confers NF-κB responsiveness, we generated a series of 5′-deletion constructs of the HOXA9 promoter (Fig. 4A). Based on the deletion constructs analysis, our data suggests that minimal basal

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promoter of HOXA9 is within first 100 bp upstream of transcriptional start site (Fig. 4B). Forward oriented 0.1 kb HOXA9 promoter is approximately 150 fold more active than control pGL3 basic and reverse oriented HOXA9 promoter (data not shown). HUVECs were transiently transfected with all the 5′deletion constructs with or without MEKK or RelA expression plasmid and treated with or without TNF-α. All the 5′-deletion constructs of HOXA9 promoter were downregulated by TNF-α, MEKK, and RelA, except 100 bp HOXA9 promoter (Fig. 4C). This data suggests that NF-κB responsive region should be between − 400 to − 100 bp of HOXA9 promoter. 3.5. NF-κB is absolutely required for HOXA9 downregulation We wanted to determine whether NF-κB is absolutely required for downregulation of HOXA9 promoter in response to TNF-α. We have observed downregulation of HOXA9 promoter activity in various cell types, including NIT 3T3, in response to TNF-α (Supplementary Fig. 1). We transiently

Fig. 4. TNF-α responsive region is present in first 400 bp of HOXA9 promoter: (A) Deletion constructs of HOXA9 promoter (B) Minimal basal promoter of HOXA9 is within 100 nucleotides upstream of its transcriptional start site: 5′ deletion constructs of HOXA9 promoter luciferase were transiently transfected in HUVECs. 24 h post-transfection luciferase activities were measured. Measuring Renilla luciferase activity of co-transfected phRL-SV40 normalized the transfection efficiencies. (C) HUVECs were transiently co-transfected with various 5′ deletion constructs of HOXA9 promoter luciferase reporter with or without MEKK-pcDNA3.1+ or RELA/p65-pEGFP. TNF-α (10 ng/ml) treatment was initiated 4 h post-transfection for 16 h. Luciferase activity was measured 24 h after transfection. Measuring Renilla luciferase activity of co-transfected phRL-SV40 normalized the transfection efficiencies.

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Fig. 5. TNF-α mediated downregulation of HOXA9 promoter is NF-κB dependent: 3.7 kb HOXA9 promoter-pGL3 basic luciferase (A) or pGL3-SV40 luciferase (B) or pGL3-basic luciferase (C) reporter vector was transiently transfected in NIH 3T3 wt, p65–p50 double knockout (DKO) NIH 3T3, or p50–p52 DKO NIH 3T3 cells. 4 h post-transfection, the cells were treated with TNF-α (10 ng/ml) for 16 h and luciferase activity was measured. Measuring Renilla luciferase activity of cotransfected phRL-SV40 normalized the transfection efficiencies.

transfected 3.7 kb promoter luciferase construct of HOXA9 in wild type NIH 3T3, p65–p50 double knockout (DKO) NIH 3T3, and p50–p52 DKO NIH 3T3 cells. As shown in Fig. 5A, HOXA9 promoter activity is not downregulated by TNF-α in p65–p50 DKO cells, while TNF-α downregulates HOXA9 promoter activity in NIH 3T3 WT and p50–p52 DKO cells. Activity of control pGL3 luciferase plasmids is unaffected in DKO cells (Fig. 5B, C). This data clearly suggests that HOXA9 downregulation absolutely requires NF-κB (p65). 3.6. HOXA9 overexpression upregulates its own promoter in a dose dependent manner Recently we showed that overexpression of HOXA9 inhibits EC activation by inhibiting NF-κB activity (Trivedi et al., 2007b). Therefore, HOXA9 acts as a negative regulator of NFκB during EC activation. Figs. 1–3 show that activation of NFκB inhibits its negative regulator HOXA9 at transcriptional level. Why NF-κB is downregulating HOXA9? To address this question, we wanted to determine what maintains basal expression level of HOXA9 in quiescent state of EC. Multiple examples exist of HOX gene autoregulation in systems where these genes must be continuously expressed to maintain the differentiated state of a cell (Care et al., 1996; Chariot et al., 1999; Yaron et al., 2001). We tested whether HOXA9 exhibited autoregulation in EC. 3.7 kb HOXA9 promoter luciferase construct was co-transfected either with HOXA9-pcDNA3.1+ or HOXA2-pcDNA3.1+. In cotransfection experiments, a 4-fold increase in 3.7 kb HOXA9 promoter activity was observed (Fig. 6A). Real time PCR analysis showed that overexpression of HOXA9 upregulated endogenous HOXA9 mRNA by two fold in EC (Fig. 6B). This provides an insight about a novel autoregulation of HOXA9 in EC. Based on this data, we hypothesize that NF-κB activation is inhibiting its negative regulator (HOXA9) and therefore positive autoregulation of HOXA9. This dual regulation of HOXA9 causes its near

complete and rapid downregulation during EC activation by NF-κB. 3.7. HOXA9 DNA binding and transactivation domains are essential for upregulation of its own promoter Homeobox genes are known to regulate transcription of several genes through their homeodomain, which confers sequence specific DNA binding (Levine et al., 1984). We transfected HUVECs with HOXA9 promoter luciferase construct with or without HOXA9 DNA binding mutant (N51S)

Fig. 6. HOXA9 overexpression upregulates its transcription in a dose dependent manner. (A) HUVECs were co-transfected with HOXA9 promoter luciferase reporter vector and either pcDNA3.1+ control vector, flag-HOXA9-pcDNA3.1+, or flag-HOXA2-pcDNA3.1+. Luciferase activity was measured 24 h posttransfection. Measuring Renilla luciferase activity of co-transfected phRL-SV40 normalized the transfection efficiencies. The fold induction represents the ratio of normalized luciferase values of HOXA9 transfected and untransfected HUVECs. (B) HUVECs were transfected with pcDNA3.1+ control vector or flag-HOXA9-pcDNA3.1+ and mRNA levels were measured by real time PCR 24 h post-transfection.

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Fig. 7. HOXA9 DNA binding and transactivation domains are required for autoregulation: (A) HUVECs were co-transfected with 3.7 kb HOXA9 promoter luciferase reporter vector and either pcDNA3.1+ control vector, flag-HOXA9-pcDNA3.1+, or flag-HOXA9 DBM-pcDNA3.1+ or flag-HOXA9 Δ11-pcDNA3.1+. Luciferase activity was measured 24 h post-transfection. Measuring Renilla luciferase activity of co-transfected phRL-SV40 normalized the transfection efficiencies. The fold induction represents the ratio of normalized luciferase values of HOXA9 transfected and untransfected HUVECs. (B) Regulation of HOXA9 in EC: Under quiescent state of EC, basal HOXA9 protein level is maintain due to positive feedback mechanism and autoregulation by HOXA9. Sustained expression of HOXA9 acts as a negative regulator of NF-κB activity and thereby prevents EC activation. Under activated state of EC, TNF-α and LPS dependent inflammatory signaling pathway leads to activation of NF-κB, which causes the inhibition of its negative regulator HOXA9 transcription and therefore positive autoregulation of HOXA9. This dual regulation of HOXA9 causes its near complete and rapid downregulation during EC activation.

construct. As shown in Fig. 7A, DNA binding mutant of HOXA9 is unable to upregulate its promoter activity. This clearly suggests that HOXA9 DNA binding is essential for upregulation of HOXA9 promoter activity. Further, we tested whether HOXA9 transactivation domain is required for upregulation of HOXA9 promoter activity. We transfected HUVECs with HOXA9 promoter luciferase construct with or without HOXA9 Δ11 (first 11 amino acids are deleted) construct. Our data suggests that HOXA9 activation domain is essential for upregulation of HOXA9 promoter activity (Fig. 7A). 4. Discussion Our results suggest a novel role and regulation of HOXA9 expression during EC activation. A novel HOXA9 promoter is downregulated by inflammatory cytokines like TNF-α and LPS (Fig. 1A). Based on our previous and present findings, we hypothesized that HOXA9 is constitutively expressed and helps to maintain a quiescent, non-activated EC state by negatively regulating TNF-α dependent NF-κB activity (Trivedi et al., 2007b). HOXA9 autoregulation (Figs. 6–7) may help to preserve a sufficient, steady state level of the short-lived HOXA9 transcript and rapid turnover of HOXA9 protein (Zhang et al., 2003). In response to inflammatory cytokines like TNF-α, transcription of HOXA9 is suppressed leading to a rapid and dramatic reduction of HOXA9 mRNA, and ultimately protein, allowing EC to progress to an activated state. HOXA9 is known to regulate various functions of ECs. HOXA9 overexpression increases EC migration and tube

forming activity by upregulating EphB4 receptor expression (Bruhl et al., 2004). siRNA mediated inhibition of HOXA9 expression causes downregulation of eNOS, VEGF-R2, and VE-cadherin, which results in inhibition of angiogenesis (Rossig et al., 2005). Previous studies reported that TNF-α downregulates eNOS and VEGF-R expression in ECs thereby inhibits angiongenesis (Yoshizumi et al., 1993; Patterson et al., 1996). Interestingly, our results show that TNF-α downregulates HOXA9 at transcriptional level in ECs (Fig. 1A). This finding might suggest that TNF-α inhibits eNOS and VEGF-R expression by downregulating HOXA9. Histone deacetylase (HDAC) inhibitors blocks endothelial differentiation of adult progenitor cells by downregulating HOXA9 expression at both transcriptional and protein level (Rossig et al., 2005). Previously we have shown that HOXA9 overexpression increases HDAC activity in ECs (Trivedi et al., 2007b). It is possible that HOXA9 maintains its steady state expression in adult progenitor cells by recruiting HDAC activity. Based on our present data, we can hypothesize that inflammatory cytokines like TNF-α and LPS might block endothelial differentiation of adult progenitor cells by downregulating HOXA9 and thereby decreasing overall HDAC activity. Interestingly, granulocyte-macrophage colony-stimulating factor (GM-CSF) dependent downregulation of HOXA9 is essential for differentiation of myelomonocytic progenitor cells to granulocytes or monocytes (Calvo et al., 2000). Previous studies documented that TNF-α and LPS activates ECs by inducing expression of leukocyte adhesion molecules like ICAM-1, VCAM-1, and E-selectin, which are not

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expressed by quiescent ECs (Ross, 1993). Our data demonstrates that TNF-α and LPS causes downregulation of HOXA9 and it is an essential event for transition of ECs from quiescent to active state. Sustained activation of TNF-α signaling has been implicated in the pathogenesis of a wide-spectrum of human diseases, including atherosclerosis (Gimbrone, 1999). TNF-α either mediate apoptosis, survival, differentiation or proliferation through the activation of pathways involving NF-κB, JUN N-terminal kinase (JNK), p42/p44 mitogen-activated protein kinase (MAPK) and p38 MAPK. NF-κB is a pivotal transcription factor for activation and progression of atherogenesis (Aggarwal, 2003). Levels of NF-κB components and their extent of NF-κB activation are low in regions of the mouse aorta with a low probability of atherosclerotic lesion formation (Iiyama et al., 1999). Our study identified that TNF-α and LPS downregulates HOXA9 in NF-κB dependent manner. Activation of NF-κB by overexpression of MEKK or TIRAP or p65 (NF-κB) downregulates HOXA9 promoter activity (Figs. 2–3). Importantly, TNF-α or LPS did not downregulate HOXA9 promoter in NF-κB (p50–p65) knockout cells, which suggest that NF-κB is absolutely required for HOXA9 downregulation (Fig. 5). Further we determined whether NF-κB regulates HOXA9 expression by directly binding to its promoter. We found one consensus NF-κB binding site (5′-GGGRNYYYCC-3′, where R is a purine, Y is a pyrimidine and N is any nucleic acid) in HOXA9 promoter at − 1505 to − 1496 bp from transcriptional start site. To understand the functional importance of this NFκB binding site, we generated several 5′ deletion constructs. Analysis of deletion constructs revealed that NF-κB response element is present in first 400 bp region of HOXA9 promoter. As shown in Fig. 4A, there are no consensus NF-κB binding sites in first 400 bp region. This suggests that NF-κB DNA binding is not required for HOXA9 downregulation (Fig. 4). Previous work suggests that NF-κB DNA binding is not required for regulation of gene expression. For example, in sertoli cells TNF-α dependent activated NF-κB interacts with SF-1 that is bound to the SF-1 binding site (SF-1RE) within the MIS promoter, and recruits HDACs to repress the gene expression (Hong et al., 2003). NF-κB also downregulates Sox9 and MyoD expression by indirect mechanism involving mRNA destabilization (Sitcheran et al., 2003). Exact mechanism of NF-κB dependent downregulation of HOXA9 transcription remains to be elucidated. Multiple examples exist of HOX gene autoregulation in systems where these genes must be continuously expressed to maintain the differentiated state of a cell (Care et al., 1996; Chariot et al., 1999; Yaron et al., 2001). We wanted to determine how HOXA9 maintains its basal expression in quiescent ECs. Our results demonstrated that HOXA9 positively autoregulates its promoter activity as well as endogenous transcription in quiescent EC and thereby maintains its basal expression (Fig. 6). This is an interesting result as it suggests that HOXA9 might be maintaining its steady state expression to keep ECs quiescent. Our previous results support this hypothesis as HOXA9 overexpression is known to inhibit EC activation by

negatively regulating NF-κB pathway (Trivedi et al., 2007b). HOXA9 is shown to regulate expression of various genes through its 60 amino acid DNA binding homeodomain (Calvo et al., 2000; Bruhl et al., 2004). Our data suggests that HOXA9 DNA binding is essential for its autoregulation (Fig. 7A). Previously it has been shown that HOXA9 regulates EphB4, eNOS, VEGF-R2, and VE-Cadherin expression by directly binding to their promoter regions (Bruhl et al., 2004; Rossig et al., 2005). Two sequence motifs (TAAT and TTAT/C) were described as HOXA9 binding sites. There are numerous TAAT and TTAT/C binding motifs are present in HOXA9 promoter, which makes it difficult to analyze their functional importance. However it has been shown that HOXA9 forms triple complexes with members of the EXD/PBX or MEIS family, which enhances DNA binding affinity and specificity. 5′-A/G-T-G-AT-T-T/A-A-T/C-G-3′ is the suggested consensus sequence, surrounding the HOXA9 binding motif (shown in bold letters), for HOXA9 triple complex DNA binding (Shen et al., 1999). HOXA9 promoter sequence analysis shows that there is one consensus HOXA9 triple complex binding site (− 1 to − 10 bp from transcriptional start site). Currently we are pursuing functional importance of this binding site by mutagenesis and ChIP approach. Further experiments are required to understand how HOXA9 interaction with other transcriptional co-factors affects its ability to autoregulate its expression in quiescent EC. Our results suggest a very novel and important function of HOXA9 during EC activation. We propose that HOXA9 maintains its basal expression through autoregulation and thereby keeps EC in a quiescent state by negatively regulating NF-κB dependent inflammatory pathway. TNF-α and LPS dependent inflammatory signaling activates NF-κB pathway, which results in the activation of EC. Interestingly, NF-κB activation leads to inhibition of its negative regulator HOXA9 and thereby abolishes HOXA9 autoregulation. This dual form of regulation leads to almost complete and rapid downregulation of HOXA9 during EC activation. Therefore, HOXA9 downregulation is an essential event for EC activation in response to inflammatory pathways. Acknowledgments C.M.T.a,b,c has been awarded American Heart Association Predoctoral Fellowship, AHA #01315366U. This work was supported by grants from the AHA to C.V.P.a,⁎ AHA # 01604124. We thank David Baltimore, PhD, Caltech Institute, Pasadena, CA for providing p50-p65 DKO and p50-p52 DKO cells. We also thank Warner C. Greene, MD, PhD, (University of California, San Francisco, CA) for providing RelA expression construct and Mark Kamps, MD, PhD, (University of California, San Diego, CA) for providing HOXA9 DNA binding mutant (N51S). Appendix A. Supplementary data Supplementary Fig. 1. TNF-α downregulates HOXA9 promoter activity in various endothelial and non-endothelial cell lines: BAEC, MEC, HT1080, NIH3T3, HEK293 cells were

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