Pro-Inflammatory Role of microRNA-200 in Vascular Smooth Muscle Cells from Diabetic Mice

Abstract

Objectives

Vascular smooth muscle cells (VSMC) from type 2 diabetic db/db mice (db/dbVSMC) exhibit enhanced pro-inflammatory responses implicated in accelerated vascular complications. We examined the role of microRNA-200 (miR-200) family members and their target Zeb1, an E-box binding transcriptional repressor, in these events.

Methods and Results

The expression levels of miR-200b, miR-200c and miR-429 were increased while protein levels of Zeb1 were decreased in VSMC and aortas from db/db mice relative to control db/+ mice. Transfection of miR-200 mimics into VSMC downregulated Zeb1 by targeting its 3′-UTR, upregulated the inflammatory genes cyclooxygenase-2 (COX-2) and monocyte chemoattractant protein-1, and promoted monocyte binding in db/+ VSMC. In contrast, miR-200 inhibitors reversed the enhanced monocyte binding of db/db VSMC. Zeb1 gene silencing with siRNAs also increased these pro-inflammatory responses in db/+ VSMC confirming negative regulatory role of Zeb1. Both miR-200 mimics and Zeb1 siRNAs increased COX-2 promoter transcriptional activity. Chromatin immunoprecipitation showed that Zeb1 occupancy at inflammatory gene promoters was reduced in db/dbVSMC. Furthermore, Zeb1 knockdown increased miR-200 levels demonstrating a feedback regulatory loop.

Conclusions

Disruption of the reciprocal negative regulatory loop between miR-200 and Zeb1 under diabetic conditions enhances pro-inflammatory responses of VSMC implicated in vascular complications.

INTRODUCTION

Pro-inflammatory and pro-atherogenic responses in vascular smooth muscle cells (VSMC) play key roles in the development of atherosclerosis^1-2. Diabetic conditions increase the expression of pro-inflammatory mediators such as receptor for advanced glycation end products (RAGE), Cyclooxygenase-2 (COX-2), cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and chemokines such as monocyte chemoattractant protein-1 (MCP-1) and Fractalkine (CX3CL1) in VSMC and other inflammatory cells ^3-8. Inflammatory mediators promote proliferation, oxidant stress, migration and monocyte-VSMC binding leading to VSMC dysfunction implicated in accelerated vascular complications of diabetes^9-10. Inflammatory gene expression is regulated by multiple signal transduction pathways leading to the activation of pro-inflammatory transcription factors such as Nuclear Factor-kappaB (NF-κB) and cAMP Responsive Element Binding Protein (CREB) under diabetic conditions in VSMC ^{3-5, 11}. Recent studies have also demonstrated the role of epigenetic mechanisms, including histone posttranslational modifications, in the upregulation of inflammatory genes in endothelial cells and VSMC under diabetic conditions ^{3, 12-13}. Enhanced expression of IL-6, MCP-1 and colony stimulating factor-1 (CSF-1) in VSMC derived from type 2 diabetic db/db mice (db/dbVSMC) was associated with reduced levels of the histone methyltransferase Suv39h1 and the corresponding repressive histone modification H3 lysine-9 tri-methylation (H3K9me3) at their promoters¹², suggesting that reversal of transcription repression could be a key mechanism for the enhanced expression of inflammatory genes in diabetes. In this study, we have uncovered a new cross talk mechanism between key miRNAs and their targets that further increases our understanding of molecular mechanisms involved in the de-repression of pathological genes in VSMC under diabetic conditions.

Zeb1 (also known as deltaEF1, TCF8 or Zfx1a) is a zinc finger transcription factor expressed in various cell types including VSMC ¹⁴. It contains multiple functional domains including two zinc fingers, a centrally located homeodomain, and others that interact with various transcription regulators. Zeb1 binds to canonical E-box elements [CACCT(G)] in target gene promoters and mediates gene repression via interaction with co-repressors including C-terminal-binding protein1 (CtBP1) ,CtBP2 and HDAC1. Zeb1 and its family member Zeb2 (also known as SIP1 or Zfx1b) regulate genes associated with epithelial-mesenchymal transition (EMT), fibrogenesis, chrondrogenesis, T-cell development and skeletal muscle differentiation, and the pathogenesis of cancer and renal complications such as diabetic nephropathy^14-17. However, the role of Zeb1 in inflammatory gene expression associated with diabetic vascular complications is not known.

Emerging evidence supports a role for microRNAs (miRNAs) in cardiovascular disease and diabetic complications^{13, 18-19}. miRNAs are 22-25 nucleotide non-coding RNAs that can repress target gene expression by post-transcriptional mechanisms. They act primarily through interaction with 3′-UTR of target mRNAs leading to downregulation or translation inhibition²⁰. We recently showed that inhibition of Suv39h1 protein levels by miR-125b was one of the mechanisms underlying enhanced pro-inflammatory responses in db/db VSMC²¹. However, the role of other miRNAs in these processes has not been examined. Lately, miR-200 family members have gained increased attention due to reports that they are downregulated in EMT implicated in cancer and metastasis ^22-23. The miR-200 family consists of five members sub-divided into Group I (miR-200a and miR-141) and Group II (miR-200b, miR200c and miR-429). They share common targets because the seed sequence between the two groups differs only by a single nucleotide. In mice, miR-200b, miR-200a and miR-429 are expressed as a single polycistronic transcript on chromosome 4 (chromosome 1 in humans), whereas miR-200c and miR-141 are expressed as a single transcript on chromosome 6 (chromosome 12 in humans). These miRNAs maintain epithelial cell phenotype by targeting transcription repressors Zeb1 and Zeb2 which repress E-cadherin. Zeb1/Zeb2 in turn negatively regulate miR-200 family members via E-box binding sites in their promoters. Dysregulation of this double negative feedback loop has been suggested to be a key mechanism in EMT and cancer^22-24. On the other hand, recent studies showed that downregulation of Zeb1 and Zeb2 by increased miR-200 plays a key role in enhanced fibrotic gene expression in mesangial cells related to diabetic nephropathy¹⁶. However, this pathway has not been examined in gene expression related to cardiovascular diseases.

Here we examined the role of miR-200 and its target Zeb1 in the enhanced inflammatory responses of db/dbVSMC relative to VSMC from db/+ mice (db/+VSMC). Our results showed that miR-200b, miR-200c and miR-429 levels are upregulated in db/dbVSMC leading to Zeb1 downregulation and enhanced inflammatory gene expression. miR-200 mimics directly targeted Zeb1 and promoted inflammatory responses in non-diabetic db/+VSMC. Furthermore, Zeb1 silencing also increased pro-inflammatory responses and expression of miR-200 members. Thus our studies identified novel pro-inflammatory effects associated with increased miR-200 and dysregulation of the reciprocal repression loop between Zeb1and miR-200 in VSMC under diabetic conditions.

MATERIAL AND METHODS

Expanded Materials and methods are available in the Online Supplement.

VSMC isolation

All animal protocols used were approved by the Institutional Animal Use and Care Committee. VSMC from thoracic aortas of Type 2 diabetic male db/db mice (10-12 weeks old) and age matched control littermates db/+ mice (Jackson Laboratories) were isolated by enzymatic digestion⁴ and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum. HeLa cells were cultured in DMEM supplemented with 10% serum.

Other methods

Western blotting, analyses of miRNA and mRNA by reverse transcriptase-real time quantitative PCR (RT-QPCR), transient transfections, luciferase assays, immunohistochemical staining, and monocyte-VSMC binding assays were performed as described in the Online Supplement.

Chromatin Immunoprecipitation (ChIP) Assays

ChIP assays were performed as described in the Online Supplement. ChIP-enriched DNA was amplified by SYBR green based QPCR using promoter specific primers (Table I, Online Supplement), analyzed by 2^−ΔΔCt method and results normalized to input were expressed as % of db/+VSMC¹².

Statistical Analysis

Statistical significance was calculated with Prism software (Graphpad, San Diego, CA) by t-tests when comparing two groups, or ANOVA for multiple groups. Data was presented as Means±SEM.

RESULTS

Levels of key miR-200 family members are increased in VSMC and aortas from db/db mice

To examine the functional role of miR-200 family members in db/dbVSMC, we first determined their expression levels by RT-QPCR analysis. Results showed that miR-200b, miR-429 and miR-200c levels were significantly increased in db/dbVSMC compared to db/+ VSMC (Fig 1A-C). On the other hand, miR-200a that is co-transcribed along with miR200b/429 was not detected in either db/+ or db/dbVSMC (not shown). Levels of miR-15b, unrelated to the miR-200 family, were not changed (Fig. 1D), indicating specificity. Expression of miR-200b and miR-429 was also significantly increased in aortas derived from db/db mice relative to db/+ mice (Fig 1E-F) establishing in vivo relevance.

Fig. 1. Altered levels of miR-200 family members and their target Zeb1 in VSMC and aortas from db/db mice.

A-F. Expression levels of indicated miRNAs in VSMC (A-D) and aortas (E-F) from db/+ and db/db mice analyzed by RT-QPCR using miScript assays. Results were normalized with internal controls U6 or actin and expressed as fold over db/+ (*, p<0.05, **, p<0.005, vs db/+, n=9-15 for A-D and n=5 for E-F). G-H. Immunohistochemical staining of aortas from db/+ and db/db mice with Zeb1 antibodies (brown color). Purple color indicates hematoxylin stain; scale bar 50 μm. Images were collected using an Olympus BX51 microscope and Zeb1 positive nuclei per field counted using Adobe Photoshop program (*, p<0.05 vs db/+, n=3). I-J. Zeb1 protein levels in cell lysates from db/+VSMC (db/+) and db/dbVSMC (db/db) were determined by immunoblotting with Zeb1 antibodies (I), and Zeb1 protein levels normalized with β-Actin protein were expressed as % of db/+ (**, p<0.005 vs db/+, n=9).

Zeb1 levels are decreased in VSMC and aortas from db/db mice

Zeb1 3′-UTR contains multiple miR-200 binding sites (Fig. I, Online Supplement). Studies in other cell types showed that miR-200 family members target E-box binding transcription repressor Zeb1 and modulate genes associated with cancer and diabetic nephropathy^{16, 22-23}. However, this inverse relationship has not been studied in VSMC and whether it can modulate processes associated with vascular complications has not been determined. We first examined whether the increased miR-200b and miR-429 noted in db/db mice was associated with downregulation of Zeb1 levels. Immunohistochemical staining with Zeb1 antibody revealed a significant decrease in Zeb1 positive nuclei in VSMC of aortas derived from db/db mice compared to db/+ (Fig. 1 G-H). Furthermore, immunoblotting of cell lysates showed that Zeb1 protein levels were significantly reduced in VSMC cultured from db/db mice compared to db/+ mice (Fig. 1 I-J). These results clearly demonstrated that Zeb1 expression is downregulated in VSMC of diabetic mice and established the reciprocal relation with increased miR-200 expression.

Zeb1 downregulation by miR-200b and miR-429

We next tested if Zeb1 is a direct target of these miRNAs in VSMC. We transfected non-diabetic db/+VSMC with miR-200b mimic (200b-M) and negative control mimic (NC-M) oligonucleotides and examined endogenous Zeb1 protein levels 48 hrs post transfection. Results showed that 200b-M significantly inhibited Zeb1 protein levels in db/+VSMC (Fig. 2A-B) compared to NC-M. Similarly, 429-M transfection also reduced Zeb1 protein levels relative to NC-M (Fig. II, Online Supplement). Next, we checked whether Zeb1 is targeted by miR-200b and miR-429 in VSMC by using luciferase reporters containing wild type Zeb1 3′-UTR with two miR-200b/429 binding sites and a Zeb1 mutant 3′-UTR (mUTR) harboring mutations in both miR-200b/429 binding sites¹⁶. These reporter plasmids were co-transfected with NC-M or 200b-M, and luciferase assays performed 48 hrs post-transfection. As shown in Fig. 2C, 200b-M significantly inhibited luciferase activity of Zeb1wild type 3′-UTR (UTR) construct, and this inhibition was abolished in mUTR. Similar results were obtained with 429-M (Fig. 2D). These results clearly demonstrate that Zeb1 is a direct target of miR-200b and miR-429 in VSMC, and that increased levels of these miRNAs could be a mechanism for downregulation of Zeb1 in db/dbVSMC.

Fig. 2. Regulation of Zeb1 and inflammatory genes by miR-200b and miR-429 in VSMC.

A. Immunoblotting of cell lysates from db/+VSMC transfected with negative control mimic (NC-M) and miR-200b mimic (200b-M) oligonucleotides, using indicated antibodies. B. Zeb1 protein levels were expressed as fold over NC-M (***, p<0.0002 vs NC-M, n=5). C-D. Luciferase activity in db/+VSMC cell lysates co-transfected with luciferase reporters containing wild type Zeb1 3′-UTR (UTR) with two miR-200 binding sites, or a mutant UTR (mUTR) harboring mutations in both the miR-200 binding sites, along with indicated oligonucleotides. Relative Renilla luciferase activities normalized with internal control (firefly luciferase) were expressed as % of NC-M (***, p<0.0002 vs NC-M, n=3-4). E-F. Expression of indicated genes in db/+VSMC transfected with NC-M or 200b-M (E) and NC-M or 429-M (F) was analyzed 48 hrs post transfection by RT-QPCR. Results were expressed as % of NC-M (*, p<0.05, **, p<0.005 and ***, p<0.0002 vs NC-M, n=9 for 200b-M and n=5 for 429-M). G. Location of key transcription factor binding sites in COX-2 promoter. Not drawn to scale. H. Luciferase activity of HeLa cells co-transfected with indicated COX-2 promoters expressing firefly luciferase reporter plasmids along with NC-M or 200b-M and Renilla luciferase vector. Luciferase activity was determined 48 hrs post transfection and expressed as fold over NC-M (***, p<0.0002 vs NC-M, n=3-6). WT-wild type; kBm-NF-κB mutant; CREm-CREB mutant; EBm-E-box mutant, COX-2 promoter.

Upregulation of inflammatory genes by miR-200b and miR-429

Since we noted the presence of E-boxes in the promoters of some inflammatory genes such as COX-2 which are upregulated in db/db VSMC, we next hypothesized that loss of Zeb1 might be a mechanism for de-repression of these genes. We therefore examined the potential functional roles of miR-200b and miR-429 in VSMC by transfecting db/+VSMC with NC-M and 200b-M or 429-M oligonucleotides. Inflammatory gene expression was analyzed 48 hrs post transfection. Results showed that Zeb1 mRNA expression was downregulated while levels of COX-2 and MCP-1 mRNAs were significantly upregulated in db/+VSMC transfected with 200b-M compared to NC-M (Fig. 2E). 200b-M, however, had no effect on other inflammatory genes such as CX3CL1 and RAGE that were also upregulated in db/dbVSMC (data not shown). Transfection of 429-M also inhibited Zeb1 and increased COX-2 and MCP-1 in db/+VSMC (Fig. 2F). Next, we examined whether miR-200b regulates the transcriptional activation of inflammatory gene promoters. COX-2 promoter contains cis-elements such as E-box, cyclic-AMP response element (CRE) and NF-κB upstream of transcription start site (Fig. 2G). We used plasmids containing luciferase reporter downstream of COX-2 wild type (WT) promoter or promoter with mutations in these binding sites²⁵ to determine if miR-200 affects their transcriptional activation. Co-transfection experiments showed that 200b-M significantly increased the luciferase activity of COX-2 WT promoter compared to NC-M, but failed to activate COX-2 promoter constructs with mutations in E-box (EBm), NF-κB (KBm) or CRE (CREm) binding sites (Fig. 2H) suggesting potential crosstalk between these three sites examined. These results demonstrate that miR-200 family has pro-inflammatory roles in VSMC and that downregulation of Zeb1 could be a key mechanism involved, at least in part.

Zeb1 gene silencing upregulates inflammatory genes in db/+VSMC

To examine the direct role of Zeb1 in inflammatory gene expression, we transfected db/+VSMC with two siRNAs targeting Zeb1 (siZeb1a and siZeb1b) or a non-targeting control (siNTC) oligonucleotides. Zeb1 siRNAs significantly inhibited Zeb1 mRNA levels (Fig. 3A) and also increased the expression of COX-2 (Fig. 3B) and MCP-1 (Fig. 3C) compared to siNTC. However, the expression of RAGE and another chemokine CX3CL1, which were shown to be upregulated in db/dbVSMC^{6, 26} was not affected (data not shown). Furthermore, co-transfection of COX-2 WT promoter luciferase reporter plasmid with siZeb1 (1:1 mixture of siZeb1a and siZeb1b) significantly increased COX2-WT (WT) promoter activity relative to siNTC (Fig. 3D). Furthermore, COX-2 E-box mutant promoter (EBm) activity was elevated relative to WT promoter in siNTC transfected cells, and this was further increased by siZeb1 (Fig. 3D). These results demonstrate that Zeb1 can repress these inflammatory genes in VSMC under normal conditions and that its downregulation in diabetes (via miR-200 or Zeb1 siRNAs) can relieve this repression to augment expression.

Fig. 3. Regulation of inflammatory gene expression by Zeb1 in VSMC.

A-C. Expression of Zeb1 (A), COX-2 (B) and MCP-1 (C) mRNAs in db/+VSMC transfected with siNTC (non targeting control) or two separate siRNA oligonucleotides targeting Zeb1 (siZeb1a or siZeb1b) was determined by RT-QPCR 72 hrs post transfection. Results normalized to internal control were expressed as % of siNTC (*, p<0.05, **, p<0.005, ***, p<0.0002 vs siNTC, n=7). D. COX-2 wild type promoter (WT) or an E-box mutant promoter (EBm) were co-transfected with siNTC, and siZeb1 oligonucleotides into db/+VSMC and luciferase assays performed 72 hrs post transfection. Relative luciferase activity normalized with Renilla luciferase was expressed as fold over siNTC (*, p<0.05, *** p<0.0002, n=8). E-G. Zeb1 occupancy at inflammatory gene promoters was analyzed by ChIP assays with db/+ and db/dbVSMC cell lysates using Zeb1 antibodies. ChIP enriched DNA was analyzed by QPCR using indicated promoter primers (Location of primers is shown in Fig. SIII, Online supplement). Results normalized to input DNA were expressed as % of db/+ cells (*, p<0.05, **, p<0.005, n=4). H. Reduced Zeb1 occupancy at inflammatory gene promoters in 200b-M transfected db/+VSMC, as determined by ChIP assays. ChIP assays were performed in triplicate using db/+VSMC transfected with NC-M or 200b-M and ChIP DNA was analyzed by the indicated promoter primers (**, p<0.005, n=3 vs NC-M).

Zeb1 binding to inflammatory gene promoters is reduced in db/dbVSMC

In order to further verify that a loss of Zeb1 at the MCP-1 and COX-2 promoters is associated with the increased expression of these genes in diabetic db/db VSMC, we next used ChIP assays to examine Zeb1 occupancy at these promoters. ChIP assays were performed with Zeb1 antibody, and ChIP DNA analyzed by QPCR with promoter specific primers (Fig. III, Online supplement). Results showed that Zeb1 occupancy was significantly reduced at both COX-2 (Fig. 3E) and MCP-1 promoters (Fig. 3F), with no significant change at the Cyclophilin A (CypA) promoter (Fig. 3G) in db/dbVSMC compared to db/+VSMC.

Since, transfection of 200b-M downregulates Zeb1 and increases inflammatory gene expression which mimicks the pro-inflammatory db/db phenotype, we examined if it also leads to reduced Zeb1 occupancy at inflammatory gene promoters. We performed ChIP assays with cell lysates from NC-M and 200b-M transfected db/+VSMC using Zeb1 antibody. Results showed that Zeb1 occupancy was greatly reduced at COX-2 and MCP-1 gene promoters in 200b-M transfected cells demonstrating that 200b-M effects can be mediated through Zeb1 downregulation (Fig. 3H). These results further support our hypothesis that enhanced expression of key VSMC inflammatory genes in diabetes could be due to reduced Zeb1 occupancy at their promoters.

COX-2 plays a role in enhanced monocyte binding in db/dbVSMC

Next we examined the functional relevance of miR-200b and Zeb1 mediated gene expression in pro-inflammatory responses such as monocyte-VSMC binding. Previous studies showed the role of MCP-1 in enhanced monocyte binding in db/dbVSMC relative to control db/+VSMC^{4, 6}. However, the involvement of increased COX-2 was not studied. We first confirmed that COX-2 mRNA (Fig. 4A) and protein (Fig. 4B-C) levels were significantly increased in db/dbVSMC relative to db/+VSMC. Then, both db/+VSMC and db/dbVSMC were pretreated with COX-2 specific inhibitor Celecoxib (1 and 10 μmol/L) or the vehicle DMSO (Ctrl) for one hour. Monocyte-VSMC binding assays were performed with fluorescently labeled mouse WEHI monocytes. Results showed that vehicle treated db/dbVSMC exhibited significantly enhanced monocyte binding compared to db/+VSMC, and this was blocked by pre-treatment with Celecoxib (Fig. 4D-E). These results demonstrate a key role for COX-2 in the enhanced monocyte binding displayed by the diabetic db/dbVSMC relative to control db/+ VSMC.

Fig. 4. Increased COX-2 levels regulate enhanced monocyte binding in db/db VSMC.

A. COX-2 mRNA expression was determined by RT-QPCR in db/+VSMC (db/+) and db/dbVSMC (db/db) (*, p<0.05 vs db/+, n=9). B. Representative immunoblots of cell lysates from db/+VSMC (db/+) and db/dbVSMC (db/db) performed with indicated antibodies. C. Intensity of COX-2 protein bands determined using a calibrated densitometer and expressed as fold over db/+VSMC (***, p<0.0002 vs db/+, n=6). D-E. COX-2 inhibitor Celecoxib blocks the enhanced monocyte binding in db/dbVSMC. Both db/+VSMC and db/dbVSMC were pretreated with vehicle (Ctrl) or indicated concentrations of Celecoxib for one hr, and monocyte-VSMC binding assays performed using fluorescently labeled WEHI mouse monocytes as described in the Online Methods section. Images of monocytes bound to VSMC monolayers were collected using a fluorescent microscope (D) and number of bound monocytes per field determined (E). Results were expressed as % of vehicle (Ctrl) treated db/+VSMC (**, p<0.005, n=4).

Regulation of monocyte-VSMC binding by miR-200b in diabetes

To evaluate the functional role of miR-200b, we transfected db/+VSMC with 200b-M or NC-M and performed monocyte binding 48 hrs post transfection. Results showed that 200b-M significantly increased the binding of WEHI mouse monocytes to db/+VSMC compared to NC-M transfected cells (Fig. 5 A-B). Next, we tested if the enhanced monocyte binding in diabetic db/dbVSMC can be blocked by miR-200b inhibitors. We transfected db/dbVSMC with miR-200b inhibitor (200b-I) that targets miR-200b hairpin, or negative control inhibitor (NC-I) oligonucleotides. In addition, db/+VSMC were also transfected with NC-I as a control for db/dbVSMC. Results showed that db/dbVSMC transfected with NC-I exhibited enhanced monocyte binding compared to db/+VSMC transfected with NC-I (Fig. 5C). Furthermore, transfection of db/dbVSMC with 200b-I significantly attenuated this enhanced monocyte binding, thus reversing the pro-inflammatory phenotype (Fig. 5C). These results demonstrate that increased miR-200b can promote, at least in part, the inflammatory responses in VSMC under diabetic conditions.

Fig. 5. Regulation of monocyte-db/+VSMC interactions by miR-200b.

A. miR-200b increases monocyte binding to db/+VSMC. Representative images of monocyte binding assays performed with db/+VSMC transfected with NC-M or 200b-M. B. Bound monocytes per field were expressed as % of NC-M transfected db/+VSMC (***, p<0.0002, n=8). C. MiR-200b Inhibitors reverse pro-inflammatory phenotype of db/dbVSMC. VSMC from db/db mice (db/db) were transfected with inhibitor targeting miR-200b hairpin (200b-I) or negative control inhibitor (NC-I). As controls, db/+VSMC were also transfected with NC-I. Monocyte binding assays were performed and results expressed as % of db/+VSMC transfected with NC-I (*, p<0.05, ***, p<0.0002 vs NC-I, n=8).

Zeb1 knockdown increases monocyte-VSMC binding in db/+VSMC

Since Zeb1 also negatively regulates COX-2 and MCP-1, we studied the direct effect of Zeb1 knockdown on monocyte-VSMC binding. As shown in Fig. 6A-B, transfection of siZeb1a or siZeb1b significantly increased monocyte-VSMC binding relative to siNTC confirming that Zeb1 is an endogenous repressor of key genes such as MCP-1 and COX-2 involved in VSMC-monocyte interactions.

Fig. 6. Zeb1 gene silencing increases monocyte binding and miR-200b/miR-429 in VSMC.

A. Increased monocyte binding by Zeb1 knockdown in db/+VSMC. Representative images of monocyte binding assays performed with db/+VSMC transfected with indicated siRNAs. B. Bound monocytes per field were expressed as % of siNTC transfected db/+VSMC (*, p<0.05, n=8). C. Western blot showing Zeb1 knockdown in db/+VSMC transfected with siZeb1 (1:1 mixture of siZeb1a+siZeb1b) relative to siNTC transfected cells. D-F. Expression of indicated miRNAs in db/+VSMC transfected with siNTC and siZeb1. Results were expressed as % of siNTC transfected db/+VSMC (*, p<0.05 vs siNTC, n=6). G. Schematic diagram showing the role of miR-200b-Zeb1 negative feedback loop in VSMC dysfunction under diabetic conditions.

Zeb1 knockdown upregulates miR-200b and miR-429

Studies in epithelial cells showed that Zeb1 can also negatively regulate miR-200 expression^23-24. Therefore, we examined if Zeb1 downregulation can upregulate miR-200b and miR-429 levels in a feedback mechanism in VSMC. Results showed that Zeb1 knockdown by siZeb1 (1:1 mixture of siZeb1a and siZeb1b) (Fig. 6C), significantly increased miR-200b (Fig. 6D) and miR-429 (Fig. 6E) levels in db/+VSMC but had no effect on miR-21 levels (Fig. 6F). These results demonstrate that Zeb1-and miR-200 cross talk and regulate each other through a double negative feedback loop in VSMC (Fig. 6G).

DISCUSSION

In this study, we demonstrated that miR-200 family members miR-200b, miR-200c and miR-429 were upregulated, while conversely protein levels of their target Zeb1 were downregulated in VSMC and aortas from type 2 diabetic db/db mice. Overexpression of miR-200 inhibited Zeb1 expression, while Zeb1 gene silencing increased miR-200b and miR-429, demonstrating the reciprocal negative regulation of each other in VSMC. Interestingly, miR-200 mimics and Zeb1 siRNAs increased the expression of inflammatory genes COX-2 and MCP-1 as well as monocyte binding in non-diabetic db/+VSMC, mimicking the enhanced pro-inflammatory phenotype of db/dbVSMC. Inhibition of miR-200 blocked enhanced pro-inflammatory responses in db/dbVSMC further supporting the key role of miR-200 in these events associated with diabetic vasculopathy.

Downregulation of miR-200 family members in EMT in cancer cells and renal epithelial cells is widely described^{17, 22-23}. However, only recently the role of miR-200 in vascular and renal cells has been investigated. Downregulation of miR-200b in endothelial cells by hypoxia²⁷ and streptozotocin induced diabetic retinopathy ²⁸ increased the expression of its targets Ets-1 and VEGF that promote angiogenesis. In contrast, increased levels of miR-200b and miR-200c were seen in diabetic mouse glomeruli^{16, 29} and in renal mesangial cells treated with TGF-β, resulting in Zeb1 and Zeb2 downregulation and increased fibrotic gene expression¹⁶, a key mechanism involved in diabetic nephropathy. Our results demonstrate that increased levels of miR-200b, miR-200c and miR-429 are present in VSMC of type 2 diabetic mice along with Zeb1 downregulation and enhanced inflammatory gene expression. These studies reveal the cell and disease-specific differences and complexities in the regulation of miR-200 family members, as well as the diverse roles played by them in various pathophysiological conditions.

Inflammatory genes are regulated by transcription factors such as NF-κB and CREB in VSMC, and activities of these are enhanced under diabetic conditions^{3-5, 13}. We found that COX-2 promoter activation by miR-200b required cis-elements NF-κB and CRE suggesting its interaction with pathways involved in the activation of NF-κB and CREB in VSMC. Furthermore, mutations in E-box increased the basal activity of the COX-2 promoter demonstrating that binding of repressors such as Zeb1 might inhibit its activity. However, miR-200b did not further enhance E-box mutant activity suggesting that E-box element in COX-2 promoter binds to both positive and negative regulatory transcription factors. Indeed, studies in other cell types showed that transcription factors USF-1 and USF-2 activate COX-2 promoter through binding to this E-box^30-31. This suggests that any alterations in the fine balance between activating and repressive transcription factors at promoters of pathologic genes might be one of mechanisms underlying enhanced inflammatory gene expression mediated by miR-200b in VSMC.

Zeb1 gene silencing (by siRNAs) also increased inflammatory genes and increased COX-2 WT promoter activation in VSMC. In addition, E-box mutant COX-2 promoter showed increased activity relative to WT in siNTC transfected cells, which was further enhanced by Zeb1 knockdown, demonstrating negative regulatory role of Zeb1 and E-box elements in VSMC. Furthermore, ChIP assays demonstrated reduced Zeb1 occupancy at both COX-2 and MCP-1 promoter in db/dbVSMC and in db/+VSMC transfected with 200b-M, further supporting the notion that Zeb1 inhibition can contribute to the enhanced inflammatory gene expression in db/dbVSMC. Interestingly, the MCP-1 proximal promoter region amplified in Zeb1 ChIP assays did not have a canonical E-box element, yet Zeb1 knockdown increased MCP-1 expression. This suggests that the MCP-1 promoter may have unidentified putative Zeb1 binding sites in its proximal promoter or away from the promoter. Previous studies have shown that Zeb1 binding as far as 16 kb away from transcription start site can influence Col1a2 gene expression ^15,32. Alternately, Zeb1 may interact with the MCP-1 promoter indirectly through protein-protein interactions with transcription repressors such as CtBP1 and CtBP2³³. Furthermore, Zeb1 can associate with repressor complexes formed by CtBP at gene promoters, which also includes other transcription repressors such as histone deacetylase 1 and lysine specific demethylase 1, a histone H3 lysine 4 demethylase^{14, 34-35}. In addition, increases in miR-200 levels in Zeb1 knockdown VSMC (Fig.6D-E) can further amplify inflammatory gene expression. Overall, our results suggest that Zeb1 can exert repressive effects at inflammatory gene promoters through multiple mechanisms in VSMC.

Evidence shows that Zeb1 negatively regulates the expression of miR-200 members through binding to E-box elements located upstream of their promoters in epithelial cells, while miR-200 in turn downregulates Zeb1 through 3′-UTR inhibition²³. This reciprocal negative regulatory loop has been implicated in pathological conditions including EMT ^22-24, renal fibrosis and diabetic nephropathy^{15-17, 19}. Our current studies demonstrate the operation of this reciprocal regulation and a novel pro-inflammatory function for miR-200 family members in VSMC. In contrast, Zeb1 knockdown by siRNAs or miR-200 transfection alone did not increase ECM genes such as Col1a2 and Col4a1 expression in VSMC (not shown) suggesting the requirement of additional transcription factors as shown previously in VSMC³² and MC¹⁶. Thus, a balance between Zeb1 and miR-200 members seems to regulate or fine tune the expression of inflammatory genes in VSMC while a dysregulation of this balance under diabetic conditions leads to inappropriate expression (Fig. 6G). However, from these studies it is not clear how Zeb1 and miR-200b expression is regulated in the diabetic state. Exposure of VSMC to high glucose (25 mmol/L) alone did not alter miR-200 and Zeb1 levels in VSMC (data not shown). This is in contrast to miR-200b downregulation by high glucose in human umbilical vein ECs and bovine retinal capillary ECs, mimicking miR-200b downregulation in retinas from streptozotocin induced diabetic rats²⁸, further suggesting cell and tissue specific differences in miRNA regulation. The observed changes in miRNAs in diabetic db/dbVSMC could be a cumulative effect of hyperglycemia, dyslipidemia, hyperinsulinemia and insulin resistance associated with this Type 2 diabetes model. Furthermore, diabetes activates multiple signaling pathways including oxidant stress, protein kinase C, tyrosine kinases, MAPKs and AGEs^{3, 5, 11} many of which are altered in this model ^{4, 6, 26} Involvement of these and other potential mechanisms including increased expression of growth factors such as TGF-β¹⁶ and PDGF-D³⁶ in disrupting the miR-200-Zeb1 loop in VSMC awaits further investigation.

Inflammatory genes in VSMC play key roles in the development of vascular complications by promoting VSMC migration, proliferation and monocyte binding. Previous results from in vitro and ex vivo studies showed that MCP-1 and CX3CL1, which are increased under diabetic conditions, mediate enhanced monocyte-VSMC binding and foam cell formation, key events in the pathogenesis of atherosclerosis^{4, 6}. Thus, miR-200-Zeb1 regulation of inflammatory genes such as MCP-1 and COX-2 could promote monocyte binding and subsequent differentiation in the sub-endothelial space and thereby contribute to accelerated vascular complications. Reports show that elevated COX-2 in db/dbVSMC was also associated with vascular smooth muscle hypercontractility related to hypertension^37-38. Therefore, miR-200 mediated COX-2 expression might also be involved in accelerated hypertension associated with diabetes.

In summary, we have identified a novel pro-inflammatory role for the negative feedback loop between miR-200 and Zeb1 in VSMC. Our previous studies showed the role of miR-125b in promoting inflammatory gene expression through downregulation of the epigenetic repressive histone methyltransferase Suv39h1 in db/db VSMC²¹. Together, these studies suggest that miRNA mediated targeting and downregulation of repressive mechanisms regulated by key epigenetic and transcription factors might augment pro-inflammatory responses of VSMC. These results could lead to the identification of potential new therapeutic targets for the accelerated vascular complications in diabetes.