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Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1.

Villeneuve LM, Kato M, Reddy MA, Wang M, Lanting L, Natarajan R - Diabetes (2010)

Bottom Line: Conversely, miR-125b inhibitors showed opposite effects.Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells.In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT

Objective: Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to "metabolic memory." Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice.

Research design and methods: We used luciferase assays with Suv39h1 3'untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays.

Results: We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3'UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

Conclusions: These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.

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Model for the mechanisms related to miR-125b mediated decrease in SUV39H1, leading to loss of repression of inflammation in diabetic VSMC. Diabetic conditions in the db/db VSMC can promote the decrease of Suv39h1 (via increased miR-125b) and concomitant loss of repressive chromatin marks such as H3K9 trimethylation (K9me3) relative to nondiabetic db+ cells. This can lead to a more open chromatin state (right) characterized by active chromatin marks such as K4methylation (K4me) and related HMTs, K9acetylation (K9ac) and related coactivators and histone acetyl transferases (HATs) generally associated with active gene expression. Together such events can result in increased expression of inflammatory genes. Various combinations of histone modifications are likely to be involved.
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Figure 7: Model for the mechanisms related to miR-125b mediated decrease in SUV39H1, leading to loss of repression of inflammation in diabetic VSMC. Diabetic conditions in the db/db VSMC can promote the decrease of Suv39h1 (via increased miR-125b) and concomitant loss of repressive chromatin marks such as H3K9 trimethylation (K9me3) relative to nondiabetic db+ cells. This can lead to a more open chromatin state (right) characterized by active chromatin marks such as K4methylation (K4me) and related HMTs, K9acetylation (K9ac) and related coactivators and histone acetyl transferases (HATs) generally associated with active gene expression. Together such events can result in increased expression of inflammatory genes. Various combinations of histone modifications are likely to be involved.

Mentions: In this study, we uncovered a novel miR-dependent mechanism for the epigenetic regulation of pathologic genes in MVSMC of type 2 diabetic mice. There was increased expression of miR-125b as well as key inflammatory genes in MVSMC derived from db/db mice relative to control db/+ mice, and both these increases persisted even after culturing the MVSMC for several passages in vitro, possibly due to “metabolic memory” in this type 2 diabetic mouse model. In addition, immunoblotting of cell lysates and immunohistochemistry of aortic sections showed a decrease in Suv39h1 K9 HMT in diabetic db/db mice relative to db/+. We recently showed that decreased Suv39h1 in db/db MVSMC can play a role, at least in part, in the increased expression of inflammatory genes in these cells (22). Interestingly, we now found that Suv39h1 is a target of miR-125b, and that increased miR-125b can subsequently decrease H3K9me3 repressive marks at inflammatory gene promoters, leading to a corresponding increase in expression of inflammatory genes such as IL-6 and MCP-1. The miR-125b mimic specifically downregulated H3K9me3, but not H3K9me2 and H3K9me1. Furthermore, increasing miR-125b levels (with miR-125b mimic) in control db/+ MVSMC functionally led to increased monocyte-MVSMC binding, mimicking the diabetic phenotype. Since miR-125b could mimic key diabetic phenotypes, aberrant regulation of miR-125b may be an underlying mechanism for sustained vascular dysfunction, at least in this type 2 diabetic model, by targeting and downregulating a key chromatin HMT that normally keeps key inflammatory genes in the repressed state (Fig. 7). Additional histone modifications, such as H3K4-acetylation and H3K4me activating marks, as suggested in previous studies (24–25,43), and/or key histone demethylases, may also cooperate to orchestrate dynamic events at these gene promoters along with this miR-mediated loss of repression to promote chronic vascular inflammation and accelerated atherosclerosis seen in diabetes (Fig. 7).


Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1.

Villeneuve LM, Kato M, Reddy MA, Wang M, Lanting L, Natarajan R - Diabetes (2010)

Model for the mechanisms related to miR-125b mediated decrease in SUV39H1, leading to loss of repression of inflammation in diabetic VSMC. Diabetic conditions in the db/db VSMC can promote the decrease of Suv39h1 (via increased miR-125b) and concomitant loss of repressive chromatin marks such as H3K9 trimethylation (K9me3) relative to nondiabetic db+ cells. This can lead to a more open chromatin state (right) characterized by active chromatin marks such as K4methylation (K4me) and related HMTs, K9acetylation (K9ac) and related coactivators and histone acetyl transferases (HATs) generally associated with active gene expression. Together such events can result in increased expression of inflammatory genes. Various combinations of histone modifications are likely to be involved.
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Related In: Results  -  Collection

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Figure 7: Model for the mechanisms related to miR-125b mediated decrease in SUV39H1, leading to loss of repression of inflammation in diabetic VSMC. Diabetic conditions in the db/db VSMC can promote the decrease of Suv39h1 (via increased miR-125b) and concomitant loss of repressive chromatin marks such as H3K9 trimethylation (K9me3) relative to nondiabetic db+ cells. This can lead to a more open chromatin state (right) characterized by active chromatin marks such as K4methylation (K4me) and related HMTs, K9acetylation (K9ac) and related coactivators and histone acetyl transferases (HATs) generally associated with active gene expression. Together such events can result in increased expression of inflammatory genes. Various combinations of histone modifications are likely to be involved.
Mentions: In this study, we uncovered a novel miR-dependent mechanism for the epigenetic regulation of pathologic genes in MVSMC of type 2 diabetic mice. There was increased expression of miR-125b as well as key inflammatory genes in MVSMC derived from db/db mice relative to control db/+ mice, and both these increases persisted even after culturing the MVSMC for several passages in vitro, possibly due to “metabolic memory” in this type 2 diabetic mouse model. In addition, immunoblotting of cell lysates and immunohistochemistry of aortic sections showed a decrease in Suv39h1 K9 HMT in diabetic db/db mice relative to db/+. We recently showed that decreased Suv39h1 in db/db MVSMC can play a role, at least in part, in the increased expression of inflammatory genes in these cells (22). Interestingly, we now found that Suv39h1 is a target of miR-125b, and that increased miR-125b can subsequently decrease H3K9me3 repressive marks at inflammatory gene promoters, leading to a corresponding increase in expression of inflammatory genes such as IL-6 and MCP-1. The miR-125b mimic specifically downregulated H3K9me3, but not H3K9me2 and H3K9me1. Furthermore, increasing miR-125b levels (with miR-125b mimic) in control db/+ MVSMC functionally led to increased monocyte-MVSMC binding, mimicking the diabetic phenotype. Since miR-125b could mimic key diabetic phenotypes, aberrant regulation of miR-125b may be an underlying mechanism for sustained vascular dysfunction, at least in this type 2 diabetic model, by targeting and downregulating a key chromatin HMT that normally keeps key inflammatory genes in the repressed state (Fig. 7). Additional histone modifications, such as H3K4-acetylation and H3K4me activating marks, as suggested in previous studies (24–25,43), and/or key histone demethylases, may also cooperate to orchestrate dynamic events at these gene promoters along with this miR-mediated loss of repression to promote chronic vascular inflammation and accelerated atherosclerosis seen in diabetes (Fig. 7).

Bottom Line: Conversely, miR-125b inhibitors showed opposite effects.Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells.In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT

Objective: Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to "metabolic memory." Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice.

Research design and methods: We used luciferase assays with Suv39h1 3'untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays.

Results: We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3'UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.

Conclusions: These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.

Show MeSH
Related in: MedlinePlus