MEF2 is regulated by CaMKIIδ2 and a HDAC4-HDAC5 heterodimer in vascular smooth muscle cells

Abstract

VSMCs (vascular smooth muscle cells) dedifferentiate from the contractile to the synthetic phenotype in response to acute vascular diseases such as restenosis and chronic vascular diseases such as atherosclerosis, and contribute to growth of the neointima. We demonstrated previously that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKII (Ca(2+)/calmodulin-dependent protein kinase) IIδ(2) in the medial wall and the expanding neointima [House and Singer (2008) Arterioscler. Thromb. Vasc. Biol. 28, 441-447]. These findings led us to hypothesize that increased expression of CaMKIIδ(2) is a positive mediator of synthetic VSMCs. HDAC (histone deacetylase) 4 and HDAC5 function as transcriptional co-repressors and are regulated in a CaMKII-dependent manner. In the present paper, we report that endogenous HDAC4 and HDAC5 in VSMCs are activated in a Ca(2+)- and CaMKIIδ(2)-dependent manner. We show further that AngII (angiotensin II)- and PDGF (platelet-derived growth factor)-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ(2) expression is suppressed or CaMKIIδ(2) activity is attenuated. The transcriptional activator MEF2 (myocyte-enhancer factor 2) is an important determinant of VSMC phenotype and is regulated in an HDAC-dependent manner. In the present paper, we report that stimulation of VSMCs with ionomycin or AngII potentiates MEF2's ability to bind DNA and increases the expression of established MEF2 target genes Nur77 (nuclear receptor 77) (NR4A1) and MCP1 (monocyte chemotactic protein 1) (CCL2). Suppression of CaMKIIδ(2) attenuates increased MEF2 DNA-binding activity and up-regulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSMCs. Suppression of HDAC4 expression and activity prevents AngII- and PDGF-dependent phosphorylation of HDAC5. Taken together, these results illustrate a mechanism by which CaMKIIδ(2) mediates MEF2-dependent gene transcription in VSMCs through regulation of HDAC4 and HDAC5.

Figure 1. Ca²⁺-dependent regulation of HDAC4 and HDAC5 in VSM

(A) Whole cell lysates from cultured VSM cells stimulated with 0.5 μM ionomycin for the indicated times were immunoblotted for phosphorylated HDAC4 (IB:P-HDAC4^ser632) or HDAC5 (IB:P-HDAC5^{ser 498}). The lysates were further immunoblotted for active CaMKII (IB: P-CaMKII). Total HDAC4, HDAC5, and CaMKII was immunoblotted to insure equal protein loading. The histogram represents quantification of four immunoblots of phosphorylated HDAC4 or HDAC5. (B) Nuclear and cytosolic fractions were isolated from cultured VSM cells treated with 0.5 μM ionomycin (Iono) as indicated. These lysates were resolved by SDS-PAGE and immunoblotted for total HDAC4 (IB:HDAC4). Histograms represent quantification of three separate experiments. Fractions were immunoblotted for Histone H3 and GAPDH respectively to distinguish the nuclear fraction from the cytosolic fraction. (C) VSM cells were stimulated with Iono as indicated and MEF2 DNA binding was determined as described in the “Methods”.* P< 0.05.

Figure 2. CaMKIIδ2 increases HDAC phosphorylation and MEF2 DNA binding

VSM cells were infected with adenovirus encoding constitutively active CaMKIIδ2 (CaCaMKIIδ2) for 16 hours. Levels of phosphorylated HDAC4 (IB:P-HDAC4^ser632) (A) or HDAC5 (IB:P-HDAC5^ser498) (B) were detected by immunoblotting as described previously. Immunoblotting for CaMKIIδ2 (IB:CaMKIIδ2) was performed to determine the extent of CaMKII overexpression. GAPDH immunoblotting (IB: GAPDH) was performed to insure equal protein loading. (C) MEF2 DNA binding was determined in VSM cells infected with increasing amounts of adenovirus encoding constitutively active CaMKIIδ2 (CaCaMKIIδ2) for 16 hours.

Figure 3. CaMKIIδ2 mediates Ca2+-dependent gene transcription

(A) MEF2 DNA binding was determined in VSM cells stimulated with 0.5 μM Iono for 10 min after adenoviral infection with either control shRNA (ShLuc) or shRNA targeting CaMKIIδ2 (Shδ2). (B) MEF2 DNA binding activity was determined in VSM cells stimulated with 0.5 μM Iono for 10 min after electroporation with either control (SiC) SiRNA or SiRNA targeting CaMKIIδ2 (Siδ). (C) Nur77 mRNA levels were determined by quantitative PCR in VSM cells stimulated with 0.25μM Iono for 1 hour after electroporation with either control (SiC) SiRNA or SiRNA targeting CaMKIIδ2 (Siδ). (D) MCP1 mRNA levels were determined by quantitative PCR in VSM cells stimulated with 0.25μM Iono for 1 hour after electroporation with either control (SiC) SiRNA or SiRNA targeting CaMKIIδ2 (Siδ). Each of these experiments was performed three separate times. * P<0.05.

Figure 4. Agonist-dependent regulation of HDAC4 and HDAC5 in VSM

(A) Whole cell lysates from cultured VSM cells stimulated with 100 nm Ang II (Left Panel) or 10 ng/ml PDGF (Right Panel) for the indicated times were immunoblotted for phosphorylated HDAC4 (IB:P-HDAC4^ser632) or HDAC5 (IB:P-HDAC5^{ser 498}). The lysates were also immunoblotted for active CaMKII (IB: P-CaMKII). Total HDAC4, HDAC5, and CaMKII were immunoblotted to insure equal protein loading. (B) Histograms represent quantification of four immunoblots of phosphorylated HDAC4 or HDAC5 as represented in panel A. (C) Nuclear and cytosolic fractions were isolated from cultured VSM cells treated with 100 nm Angiotensin II (Top Panel) or 10 ng/ml PDGF (Bottom Panel) as indicated. These lysates were resolved by SDS-PAGE and immunoblotted for total HDAC4 (IB:HDAC4). Fractions were immunoblotted for Histone H3 and GAPDH respectively to distinguish the nuclear fraction from the cytosolic fraction.

Figure 5. CaMKIIδ2 mediates HDAC4 and HDAC5 phosphorylation

(A) Levels of phosphorylated HDAC4 (IB:P-HDAC4^ser632)or HDAC5 (IB:P-HDAC5^ser498) were detected by immunoblotting in VSM cells stimulated with 100 nm Ang II for 10 min after adenoviral infection with either control shRNA (ShLuc) or shRNA targeting CaMKIIδ2 (Shδ2). Immunoblotting for CaMKIIδ2 (IB:CaMKIIδ2) was performed to determine the level of CaMKII knockdown. β-actin immunoblotting (IB:Pactin) was performed to insure equal protein loading. (B) Levels of phosphorylated HDAC4 (IB:P-HDAC4^ser632)or HDAC5 (IB:P-HDAC5^ser498) were detected by immunoblotting in VSM cells stimulated with 10 ng/ml PDGF for 10 min after adenoviral infection with either control shRNA (ShLuc) or shRNA targeting CaMKIIδ2 (Shδ2). (C) Histograms represent quantification of four experiments represented by panels A and B above. * indicates P<0.05. VSM cells were infected with 50 MOI kinase negative CaMKIIδ2 (KNδ2) and stimulated with 100 AngII for 10 min (D) or 10 ng/ml PDGF for 10 min (E) as indicated. The lysates were immunoblotted for phosphorylated HDAC4 or HDAC5. The lysates were further immunoblotted for CaMKIIδ2 and βactin to determine extent of CaMKIIδ2 overexpression and overall protein levels.

Figure 6. CaMKIIδ2 mediates agonist-dependent MEF2 activity

A) MEF2 DNA binding was determined in VSM cells stimulated with 100 nm Ang II or 10 ng/ml PDGF as indicated. This experiment was performed three separate times. B) MEF2 DNA binding was determined in VSM cells stimulated with 100 nm Ang II or 10 ng/ml PDGF for 10 min after adenoviral infection with either control shRNA (ShLuc) or shRNA targeting CaMKIIδ2 (Shδ2). This experiment was performed three separate times. (C) Nur77 mRNA levels were determined by quantitative PCR in VSM cells stimulated with 100 nm Ang II or 10 ng/ml PDGF for 1 hour after adenoviral infection with either control shRNA (ShLuc) or shRNA targeting CaMKIIδ2 (Shδ2). This experiment was performed three separate times. (D) MCP1 mRNA levels were determined by quantitative PCR in VSM cells stimulated with 100 nm Ang II or 10 ng/ml PDGF for 1 hour after electroporation with either control (SiC) SiRNA or SiRNA targeting CaMKIIδ2 (Siδ). This experiment was performed three separate times. * P< 0.05.

Figure 7. CaMKIIδ₂ physically interacts with HDAC4 and HDAC5

A) CaMKIIδ2 was immunoprecipitated (IP: CaMKIIδ₂) from untreated cultured VSM cells or cells stimulated with 100 nm AngII (Left Panel) or 0.5 μM Iono (Right Panel) for 10 min. The immunoprecipitates were resolved by SDS-PAGE and immunoblotted for HDAC4 (IB: HDAC4) or HDAC5 (IB: HDAC5). Because CaMKII runs at approximately 50 kd, the immunoprecipitates were immunoblotted with a CAMKII antibody conjugated with horseradish peroxidase (HRP) to directly detect CaMKII B) HDAC4 was immunoprecipitated (IP:HDAC4) from VSM cells treated as described above. The immunoprecipitates were immunoblotted for HDAC5 (IB:HDAC5) or HRP conjugated CaMKIIδ2 (IB:HRP-CaMKIIδ2). The immunoprecipitates were immunoblotted for HDAC4 to monitor the efficiency of the HDAC4 immunoprecipitation. C) HDAC4 was immunoprecipitated from cells stimulated with 0.5 μm Iono for 10 min and immunoblotted as indicated.

Figure 8. HDAC4 mediates agonist-dependent phosphorylation of HDAC5

A) Levels of phosphorylated HDAC5 were determined in VSM cells stimulated with 100 nm Ang II or 10 ng/ml PDGF for 10 min after electroporation with either control (SiC) SiRNA or SiRNA targeting HDAC4 (SiH4). The lysates were immunoblotted for total HDAC4 (IB:HDAC4) to determine the extent of HDAC4 protein suppression and CaMKIIδ2 (IB:CaMKIIδ2) as a loading control. The histogram (right panel) represents quantification of immunoblots from three separate experiments. B) Levels of phosphorylated HDAC5 were determined in VSM cells stimulated with 100 nm Ang II or 10 ng/ml PDGF for 10 min after transfection with either a GFP expressing plasmid as control (GFP) or a plasmid encoding mutant HDAC4 (3S>A) (mutH4). The lysates were also immunoblotted for total HDAC4 (IB:HDAC4) to determine the extent of HDAC4 overexpression and CaMKIIδ2 (IB:CaMKIIδ2) as a loading control. The histograms represent quantification of immunoblots from three separate experiments. * indicates P< 0.05.