[Frontiers in Bioscience 18, 685-695, January 1, 2013]

Homocysteine induces inflammatory transcriptional signaling in monocytes

Shu Meng1, Stephen Ciment1, Michael Jan1, Tran Tran1, Hung Pham1, Ramon Cueto1, Xiao-Feng Yang1, Hong Wang1

1Department of Pharmacology and Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, PA, 19140

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods
3.1. Identification of Hcy- and cytokine-induced genes in human MCs
3.2. TF selection and classification
3.3. TF occurrence in the promoter of Hcy- and pro-inflammatory cytokines-induced genes
4. Results
4.1. Hcy preferentially induced pro-inflammatory genes in human MC
4.2. Hcy shares features with pro-inflammatory cytokines in inducing inflammatory genes in human MC
4.3. Class 1 TFs exert high significant binding frequency on the promoter of Hcy-induced genes and are termed as putative Hcy-responsive TFs
4.4. Hcy-induced genes show similar TF binding profiles as IFNg -induced genes
4.5. Hcy transcriptional signaling resulted in inflammation and MC differentiation
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Here, we studied transcriptional regulation in homocysteine (Hcy)-induced gene expression in monocytes (MC). We identified 11 Hcy-induced genes, 17 anti-inflammatory cytokine interleukin 10-induced, 8 pro-inflammatory cytokine interferon gamma (IFN gamma)-induced and 8 pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha)-induced genes through literature search. Binding frequency of 36 transcription factors (TFs) implicated in inflammation and MC differentiation were analyzed within core promoter regions of identified genes, and classified into 3 classes based on the significant binding frequency to the promoter of Hcy-induced genes. Class 1 TFs exert high significant binding frequency in Hcy-induced genes. Class 2 and 3 TFs have low and no significant binding frequency, respectively. Class 1 TF binding occurrence in Hcy-induced genes is similar to that in IFN gamma -induced genes, but not that in TNF alpha -induced. We conclude that Hcy is a pro-inflammatory amino acid and induces inflammatory transcriptional signal pathways mediated by class 1 TF. We term class 1 TF as putative Hcy-responsive TFs.

2. INTRODUCTION

Hyperhomocysteinemia (HHcy) has been established as an independent risk factor for cardiovascular disease (CVD) (1, 2). Meta-analysis shows that an increase of 5 �mol/L in plasma homocysteine (Hcy) levels enhances the risk of CVD by 1.6- to 1.8-fold, which is similar to the risk seen with an increase of 20 mg/dL (0.52 mmol/L) in cholesterol concentration (3).

The causative role of HHcy in human CVD remains controversial. Several secondary prevention trials of Hcy-lowering therapy were reported to have no effect on combined endpoints of cardiovascular events (4). Hcy-lowering is found beneficial to reduce the risk of overall stroke in the HOPE 2 Trial. Indirect evidence for such a benefit was recently obtained a large population-based cohort study, which demonstrated that Hcy-lowering due to folic acid fortification significantly (5, 6).

Reports from our laboratory and others have established that HHcy accelerates vascular inflammation and atherosclerosis in mice (7-9). It has been suggested that HHcy accelerates atherosclerosis via inhibiting endothelial cell growth and impairing post-injury endothelialization (2, 10, 11), promoting vascular smooth muscle proliferation (12) and inflammatory monocyte (MC) differentiation (13).

MC activation and its recruitment into the artery wall are key cellular events during the development of atherosclerosis. MC can differentiate into macrophages and become lipid-laden foam cells in the arterial walls. Recent notions indicate that MCs can differentiate into inflammatory MC subsets and contribute to vessel wall inflammation (14). We recently reported that HHcy increases the differentiation of the inflammatory Ly6Chigh/middle MC subsets, promotes the accumulation of inflammatory MCs/macrophages in the vessel wall, and accelerates atherosclerosis in a HHcy-enhanced atherosclerotic mouse model of HHcy and hyperlipidemia (13). However, how HHcy promotes the differentiation of non-inflammatory MCs into inflammatory subsets remains unknown; and transcriptional mechanisms underlying HHcy-induced pro-inflammatory effects and MC differentiation has not been studied.

Several studies on the transcriptome (all the mRNAs being expressed) have showed that Hcy (≥100 μM), induces pro-inflammatory gene expression in endothelial cells (15, 16). The effect of Hcy on inflammatory gene expression has been investigated by several groups (17-21). These individual studies revealed that Hcy induces inflammatory gene expression in MC. However, a summarized analysis of Hcy's effects on inflammatory gene expression in MC is missing.

Transcription factors (TFs) are master genes that regulate gene expression and impact their associated pathways (22). Research to identify TF binding profile has become emerging frontier and fulfills an urgent need for the identification of therapeutic molecular targets. Transcriptional regulation of Hcy-modulated gene expression has not been studied in a systemic manner.

The complete human genome DNA sequence has been deposited in the National Institutes of Health (NIH) database, making the retrieval of experimentally-identified gene promoter sequences possible. In addition, nearly 2,000 TFs and their binding sequences have been experimentally characterized and placed in the searchable web-based transcription element search system (TESS) and other databases (23). These advances make the identification of TF binding patterns in the promoters of genes feasible.

In this study, we summarized Hcy- and cytokine-induced genes in human MC through extensive literature and data base search, analyzed their relevant TF binding profile, identified putative Hcy-responsive TFs and established a hypothetic model of Hcy transcriptional signaling (Figure 1). Our study is the first to use dynamic approaches to systemically summarize gene regulation findings in the literature, combining with database mining and bioinformatics analysis, leading to the identification of TF binding profiling and models of transcriptional signaling.

3. MATERIALS AND METHODS

3.1. Identification of Hcy- and cytokine-induced genes in human MCs

Extensive literature search was performed using the NIH/PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) to identify Hcy- and cytokines-induced genes. All Genes induced by Hcy, pro-inflammatory cytokine tumor necrosis factor α (TNFα), pro-inflammatory cytokine interferon g (IFNg ), and anti-inflammatory cytokine interleukin 10 (IL-10) in human MC were identified and listed in Figure 2. All selected genes were validated for their transcriptional regulation in their original publication. The Gene ID numbers of the identified genes were obtained from the NIH/National Center of Biotechnology Information (NCBI) gene database (http://www.ncbi.nlm.nih.gov/gene/). The fold change of gene expression was obtained from the original publication if specified, or derived from the published graphs if not specified. CD36 levels were determined by flow cytometry (24). SOD1 levels were examined by western blot (21). All other identified genes were examined by real-time PCR or northern blot (17-21). Further, in order to screen genes induced by both Hcy and pro-inflammatory cytokines, we searched for genes (25-33) induced by 20 cytokines included 5 MC differentiation cytokines (granulocyte macrophage colony stimulating factor (GM-CSF), CSF-1, TNFα, IL-4 and IFNg ), 11 pro-inflammatory cytokines (macrophage inflammatory protein-1α (MIP-1α), TNFβ, IL-1β, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-15 and IL-18) and 4 anti-inflammatory cytokines (transforming growth factor (TGFβ), IL-9, IL-10 and IL-11). The overall database mining strategy is illustrated in a flowchart shown in Figure 1.

3.2. TF selection and classification

36 TFs were selected based on their identified function implicated in inflammation and MC differentiation via literature search (Figure 3). The DNA sequences of 1,000 bp upstream of the transcription start site of the identified genes listed in Figure 2 were retrieved from the NIH/NCBI gene database and defined as putative core promoters. TF binding sites on the putative promoters were determined by using the publicly accessible TF database TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess) (34). Binding frequency denotes the number of the binding sites identified for the TF on the core promoter region. A confidence interval of binding frequency for each TF was established by evaluating the binding frequency on 4 housekeeping genes, including β-actin (ACTB), fructose-bisphosphate aldolase A (ALDOA), rho GDI 1 (ARHGDIA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (35), and 17 IL-10-induced genes (Figure 2B). These four genes are the mostly consistently expressed gene cross tissues in human and mouse as described in our recent database mining studies (35). An upper threshold of the confidence interval was set as mean+3X standard deviations (SD) of the TF binding frequencies in the promoters of 4 housekeeping genes and 17 IL-10-induced genes, and marked as the dashed lines. Binding frequency higher than the upper threshold of the confidence interval (p < 0.01) was defined as significant binding frequency. TFs with identified corresponding binding element were classified into three groups, class 1, 2 and 3, based on the pattern of significant binding frequency on Hcy-induced genes.

3.3. TF occurrence in the promoter of Hcy- and pro-inflammatory cytokines-induced genes

The occurrence of class 1 TFs in the promoter regions of Hcy- and pro-inflammatory cytokines TNFa and IFNg -induced genes were analyzed (Figure 4). TFs with at least one binding site on the promoter of indicated genes were recorded as occurrence positive (+), whereas, TFs that did not have binding sites on the promoter of indicated genes were recorded as occurrence negative (-). Ratio of occurrence positive on selected genes was calculated. Independent t test was used to determine the p value of paired groups. A probability value p < 0.05 was considered to be significant.

4. RESULTS

4.1. Hcy preferentially induced pro-inflammatory genes in human MC

Via extensive literature search, we identified 11 genes whose expressions are induced by pathogenic concentrations of Hcy (≥100μM) in cultured human PBMC or THP1 cells (a human monocytic leukemia cell line) (Figure 2A). Among these 11 genes, 8 are pro-inflammatory genes, including IL-8, MC chemoattractant protein-1 (MCP-1), IL-1β, IL-6, TNFα, IL-12β, chemokine (C-C motif) receptor 2 (CCR2) and cluster of differentiation 36 (CD36). All selected genes were validated for their transcriptional regulation in their original publication. The fold change of gene expression level is indicated in Figure 2A. Interestingly, Hcy also reduced the expression of 2 anti-inflammatory TFs peroxisome proliferator-activator receptor (PPAR) α and PPARγ (data not shown).

4.2. Hcy shares features with pro-inflammatory cytokines in inducing inflammatory genes in human MC

To compare the feature of Hcy, pro- and anti-inflammatory cytokines in inducing gene expression in human MC, we further examined genes induced by pro-inflammatory cytokines TNFα and IFNg , and anti-inflammatory cytokine IL-10 in human MC through literature search. We identified 8 genes induced by TNFα (32), 8 genes by IFNg (33, 36-41), and 17 genes induced by IL-10 (42) (Figure 2B). All selected genes were validated for their transcriptional regulation in their original publication. The fold change of gene expression level is indicated in Figure 2B. We found that TNFα and IFNg each induced specific inflammatory genes, respectively. Interestingly, we found that Hcy induced IFNg responsive gene CCR2 and TNFα responsive genes IL-1β and IL-8.

Since both HHcy (7) and pro-inflammatory cytokines have been shown to promote atherogenesis (43), we examined mRNA expression induced by 20 cytokines (three groups), including 5 MC differentiation cytokines, 11 pro-inflammatory cytokines and 4 immunosuppressive cytokines. We found that 5 Hcy-induced genes (CCR2, IL-1β, IL-6, IL-8 and MCP-1) are also induced by pro-inflammatory cytokines (Figure 5), but not by anti-inflammatory cytokines.

4.3. Class 1 TFs exert high significant binding frequency on the promoter of Hcy-induced genes and are termed as putative Hcy-responsive TFs

To identify a MC relevant transcriptional profile of Hcy-induced genes, we selected 36 TFs which are implicated in inflammation and MC differentiation (Figure 3). These include 6 TFs with identified functions in facilitating MC differentiation, such as E-twenty six (ETS), hematopoietic transcription factor PU.1 (PU-1), IFN consensus sequence binding protein/IFN regulatory factor 8 (ICSBP/IRF-8), krueppel-like factor-4 (KLF4), Maf musculoaponeurotic fibrosarcoma oncogene homolog B (MafB) and proto-oncogene c-maf (c-Maf) (44-46), and several TFs involved in modulating inflammation including NF-k B, nuclear factor of activated T-cells (NFAT), forkhead box O (FOXO) and heat shock transcription factor (HSF), as detailed in Figure 3. Since most of the important TFs bind to the promoter region 1,000 bp upstream of the transcription start site to fulfill their function (47), we defined putative promoter regions as 1,000 bp upstream of transcription start site. We examined the binding frequencies of these TFs on the putative promoter regions of 5 groups of genes; (1) housekeeping genes as the controls; (2) anti-inflammatory cytokine IL-10-induced genes as negative controls (Figure 2B); (3) Hcy-induced genes (Figure 2A); (4) pro-inflammatory cytokine IFNg -induced genes and (5) pro-inflammatory cytokine IFNg - and TNFα-induced genes (Figure 2B). A binding frequency higher than the upper threshold of the confidence interval (mean+3xSD of binding sites of 4 housekeeping genes and 17 IL-10-induced genes) was considered as the significant binding frequency. TFs were divided into 3 classes based on the pattern of binding frequency in Hcy-induced genes (Figure 6). Class 1 TFs are defined as TFs having significant binding frequency on more than 3 Hcy-induced genes, including HSF, myocyte enhancer factor-2 (MEF2), NFAT, NF-k B, and KLF4, which are termed as Hcy-responsive TF. Class 1 Hcy-responsive TFs have similar high significant binding frequency in the promoters of all IFNg -induced genes, but only on a few genes induced by TNFa (Figure 6A). Class 2 had 10 TFs which have significant binding frequency on less than three Hcy-induced genes, including androgen receptor, CCAAT-enhancer-binding proteins (C-EBP), ETS, early growth response protein 1 (EGR-1), glucocorticoid receptor, octamer-4 (Oct4), p53, signal transducer and activator of transcription 3(STAT3), MafB and PU.1 (Figure 6B). Class 3 contained 8 TFs, including activator protein 1(AP-1), cAMP response element binding (CREB), estrogen receptor, ETS related gene 1 (Erg-1), PPAR, retinoic acid receptor, specificity protein 1 (SP1) and TCF transcription factor/lymphoid enhancer-binding factor (TCF/LEF), which do not have significant binding frequency on Hcy-induced genes. The remaining 13 TFs, without identified binding sites in the promoters of Hcy-induced genes, were not included in the classification. Interestingly, class 1 TF binding sites are preferentially high in Hcy- and IFNg -induced genes (Figure 6B), whereas classes 2 and 3 TF binding sites are similarly distributed in the promoter of all 5 groups.

In addition, we analyze the binding site of these 36 TFs on 5,000 bp upstream of transcription start site promoter regions of the identified genes. There is no significant difference regarding binding frequency towards the interval identified between the 5,000 bp and 1000 bp promoter (data not shown). This finding supported the strategy to focus the TF analysis on the 1,000 bp upstream of transcription start site.

4.4. Hcy-induced genes show similar TF binding profiles as IFNg -induced genes

Because Hcy-induced genes were also induced by pro-inflammatory cytokines TNFα and IFNg (Figure 5), we further analyzed the biding profile of class 1 TFs in the promoters of genes induced by Hcy, TNFα and IFNg  As shown in Figure 4, class 1 TFs have significant binding frequency on Hcy-induced genes, except for TNFα. Among Hcy-induced genes, all 5 class 1 TFs have significant binding frequency on the promoter of CD36, IL-6 and IL-12β, 4 on IL-1β and IL-8, 3 on CCR2 and SOD1, 2 on MCP1, and 1 on other Hcy-induced genes. Similarly, class 1 TFs had significant binding frequencies in the promoters of all IFNg -induced genes. In contrast, class 1 TFs with significant binding frequencies was only identified in 5 of 8 genes induced by TNFa . The occurrence of class 1 TF's significant binding frequencies in the promoters of Hcy-induced genes was similar to that of IFNg -induced genes (p=0.242), but significantly different from that in the promoters of TNFα-induced genes (p=0.032) (Figure 4).

4.5. Hcy transcriptional signaling resulted in inflammation and MC differentiation

A working model of Hcy transcriptional signaling was established based on above results. As shown in Figure 7, Hcy induces pro-inflammatory gene expression (Figure 2) via class 1 TFs (Figure 6), the putative Hcy-responsive TFs. The Class 1 TF are known to mediate TNFa - (MEF2 and NF-k B), IFNg - (KLF4) and stress (HSF and NFAT)-induced inflammation and MC differentiation (Figure 3 & 7), and has high significant binding frequency in Hcy-induced genes (Figure 6A). Hcy can directly trans-activate genes via class 1 TFs and induce inflammation and MC differentiation. The classical signaling pathways of class 1 TFs related to inflammation and MC differentiation were also listed. In addition, we propose that Hcy promote inflammation via TNFa induction as indicated in Figure 2A & 4, which in turn leads to MEF2 and NF-k B transactivation and inflammatory response (Figure 7) in MC.

5. DISCUSSION

It has been reported that HHcy accelerates atherogenic process and that Hcy might cause vascular inflammation by inducing MC-derived inflammatory gene expression (7-9). MCs play critical roles in the development of atherosclerosis and can transmigrate across endothelial cells into the vessel wall contributing to vessel wall inflammation. Recent studies have shown that Hcy increased gene expression in cultured human and mouse MC (17-21, 24). However, previous gene expression studies were largely based on mRNA assessment, limited by individual model system, and lacked integrative analysis and mechanistic assessment. Hcy-relevant transcriptional signaling is unknown. Therefore, it is of important significance to profile Hcy-induced gene regulation and transcriptional signaling in MC.

In this study we developed a novel dynamic model system in combining intensive literature searching, database mining of experimental genomic data, pro-inflammatory and MC differentiation TF profiling and TF screening. We identified Hcy-relevant transcriptional signaling and reported four findings: 1) Hcy (≥100�M) preferentially induced 8 pro-inflammatory gene expression (Figure 2A), 2) Hcy induced 5 pro-inflammatory cytokines (CCR2, IL-1β, IL6, IL-8 and MCP1) which can also be induced by other pro-inflammatory cytokines (Figure 5), 3) class 1 TFs (HSF, MEF2, NF-AT, NF-k B, and KLF4) have high significant binding frequency in Hcy-induced genes and are putative Hcy-responsive TFs (Figure 6), and 4) HHcy may contribute to inflammation and MC differentiation via Class 1 TF transcriptional signaling (Figure 7).

Our data support the notion that Hcy functions as a pro-inflammatory molecule and induces MC-derived inflammation and MC differentiation. This conclusion is primarily based on the observation that Hcy preferentially induced pro-inflammatory genes in human MC (Figure 2A). In addition, we found that HHcy reduced the expression of anti-inflammatory genes, macrophage migration inhibitory factor (MIF) (48), PPARg and PPARα in human MC (49). Since PPARg and PPARα are TFs (50). these results suggest Hcy may promote inflammatory reaction, in part, by suppressing PPARg /α-associated anti-inflammatory signaling. Interestingly, Hcy induced antioxidant enzyme superoxide dismutase 1 (SOD1) and thioredoxin (Figure 2A). Hcy-induced SOD and thioredoxin responses might be compensatory and may be overpowered by the pro-inflammatory response. We propose that Hcy induces MC-derived inflammation via increasing pro-inflammation gene expression and suppressing anti-inflammatory gene expression.

Our study suggests that Hcy has a broader spectrum in cytokine induction than other pro-inflammatory cytokines, because five Hcy-induced genes, CCR2, IL-1β, IL-6, IL-8 and MCP-1 can also be induced by several other pro-inflammatory cytokines, but not by anti-inflammatory cytokines (Figure 5). Furthermore, we demonstrated that Hcy promotes inflammatory response not only via inducing pro-inflammatory gene expression as indicated in Figure 2A, but also through facilitating MC differentiation. As indicated in Figure 4 & 7, Hcy induces MC differentiation via trans-activating signaling similar to that of IFNg and stress (involving KLF4, HSF and NFAT). The Hcy-MC differentiation hypothesis is consistent with our recent findings showing that HHcy promotes the differentiation of inflammatory Ly6Chigh MC subset, increases the accumulation of inflammatory MCs/macrophages in atherosclerotic lesions, and accelerates atherosclerosis in mice (13).

TFs are master genes controlling gene expression. It is well-accepted that a greater number of a TF binding sites in a given promoter will result in a greater likelihood for the actual binding of this specific TF (51). Most important TFs bind to the putative core promoter region (1,000 bp upstream of the transcription start site) to fulfill their functions (47). We profiled 36 TFs which were involved in inflammation and MC differentiation. We identified 5 putative Hcy-responsive TFs (HSF, MEF2, NFAT, NF-k B, and KLF4, Figure 6) and established hypothetical Hcy transcriptional signaling (Figure 7). The characterization of TF binding profile and transcriptional signaling is novel and provided integrative view of Hcy-MC response and critical insights into the identification of key mechanisms determining MC-derived inflammation.

Our strategy to identify TF binding profile and transcriptional signaling is an important advance in merging bioinformatics information and experimental science. This study, together with our previous database mining works (35, 48, 52, 53), presented novel model systems of database mining in identifying disease related signaling pathways. Our research model is featured as; (1) hypothesis-driven, (2) intensively grounded in the literature, (3) summarized analysis and integrative for gene and TF regulation, (4) database mining on the NCBI databases, (5) well-characterized TFs in the searchable database TESS, (6) statistically rigorous analysis of available public databases (52).

In summary, we developed a working model of Hcy transcriptional signaling. As shown in Figure 3. Hcy can induce gene expression via 5 Hcy-responsive TFs, the class 1 TFs, which mediate TNFa -signaling (MEF2 and NF-k B), IFNg - and stress-signaling (KLF4, and HSF and NFAT). Hcy can directly trans-activate genes via class 1 TFs to induce the expression of genes involved in inflammation and MC differentiation.

In conclusion, our results demonstrate, for the first time, that Hcy induces pro-inflammatory gene expression via TF-dependent signaling pathways in MC, leading to MC differentiation and MC-mediated inflammation, thus contributing to vascular inflammation and atherosclerosis.

6. ACKNOWLEDGEMENTS

This work was supported in part by NIH Grants HL67033, HL77288, HL82774 and HL11076 (HW); HL94451 and HL108910 (XFY);

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Key Words: Homocysteine, Monocyte Differentiation, Inflammation, Transcription Factor, Gene Expression

Send correspondence to: Hong Wang, Department of Pharmacology, Temple University School of Medicine, 3500 North Broad Street, 10th floor, Philadelphia, PA 19140, USA, Tel: 215-707-5986, Fax: 215-707-5737, Email: hongw@temple.edu