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Myocardin Family Members Drive Formation of Caveolae.

Krawczyk KK, Yao Mattisson I, Ekman M, Oskolkov N, Grantinge R, Kotowska D, Olde B, Hansson O, Albinsson S, Miano JM, Rippe C, Swärd K - PLoS ONE (2015)

Bottom Line: The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB.Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs.The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medical Science, BMC D12, Lund University, Lund, Sweden.

ABSTRACT
Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

No MeSH data available.


Related in: MedlinePlus

Induction of caveolins by adenoviral transduction of MRTF-A and myocardin.(A) Adenoviral transduction of human coronary artery smooth muscle cells with MRTF-A (MKL1, 20 MOI) increased expression of caveolin-1 and caveolin-2, but not caveolin-3 mRNA compared to empty virus. (B) Transduction of myocardin (MYOCD, 100 MOI) increased mRNA expression of all three caveolins (-1 to -3). (C) A dose-response curve with increasing concentrations of myocardin (1 to 300 MOI) demonstrated a similar threshold of sensitivity of caveolin-1 mRNA to that of the prototypical myocardin target calponin (CNN1). (D) Western blotting of MYOCD-transduced cells demonstrated increases of caveolins and cavins at the protein level. The magnitude of induction was similar to that of the well-established myocardin targets tropomyosin (TPM1) and SM22α (TAGLN). Summarized data from the western blots is shown in panels E through J (n = 8). 18S was used as a house-keeping gene for the qRT-PCR and HSP90 as loading control for western blotting. Data are presented as means±S.E.M. **P<0.01, ***P<0.001.
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pone.0133931.g003: Induction of caveolins by adenoviral transduction of MRTF-A and myocardin.(A) Adenoviral transduction of human coronary artery smooth muscle cells with MRTF-A (MKL1, 20 MOI) increased expression of caveolin-1 and caveolin-2, but not caveolin-3 mRNA compared to empty virus. (B) Transduction of myocardin (MYOCD, 100 MOI) increased mRNA expression of all three caveolins (-1 to -3). (C) A dose-response curve with increasing concentrations of myocardin (1 to 300 MOI) demonstrated a similar threshold of sensitivity of caveolin-1 mRNA to that of the prototypical myocardin target calponin (CNN1). (D) Western blotting of MYOCD-transduced cells demonstrated increases of caveolins and cavins at the protein level. The magnitude of induction was similar to that of the well-established myocardin targets tropomyosin (TPM1) and SM22α (TAGLN). Summarized data from the western blots is shown in panels E through J (n = 8). 18S was used as a house-keeping gene for the qRT-PCR and HSP90 as loading control for western blotting. Data are presented as means±S.E.M. **P<0.01, ***P<0.001.

Mentions: The high sensitivity of caveolin and cavin synthesis to latrunculin B could reflect involvement of myocardin family coactivators [29, 47]. We therefore next tested if caveolins and cavins are transcriptional targets of these coactivators. MRTF-A (MKL1) and myocardin (MYOCD) were overexpressed using adenoviruses. Compared to empty vector, transduction of MRTF-A (20 MOI) in the presence of serum caused 4-fold induction of CAV1 and CAV2 while CAV3 was unchanged (Fig 3A). Transduction of myocardin (100 MOI) increased the mRNA levels for all caveolins with close to 10-fold induction of CAV3 (Fig 3B).


Myocardin Family Members Drive Formation of Caveolae.

Krawczyk KK, Yao Mattisson I, Ekman M, Oskolkov N, Grantinge R, Kotowska D, Olde B, Hansson O, Albinsson S, Miano JM, Rippe C, Swärd K - PLoS ONE (2015)

Induction of caveolins by adenoviral transduction of MRTF-A and myocardin.(A) Adenoviral transduction of human coronary artery smooth muscle cells with MRTF-A (MKL1, 20 MOI) increased expression of caveolin-1 and caveolin-2, but not caveolin-3 mRNA compared to empty virus. (B) Transduction of myocardin (MYOCD, 100 MOI) increased mRNA expression of all three caveolins (-1 to -3). (C) A dose-response curve with increasing concentrations of myocardin (1 to 300 MOI) demonstrated a similar threshold of sensitivity of caveolin-1 mRNA to that of the prototypical myocardin target calponin (CNN1). (D) Western blotting of MYOCD-transduced cells demonstrated increases of caveolins and cavins at the protein level. The magnitude of induction was similar to that of the well-established myocardin targets tropomyosin (TPM1) and SM22α (TAGLN). Summarized data from the western blots is shown in panels E through J (n = 8). 18S was used as a house-keeping gene for the qRT-PCR and HSP90 as loading control for western blotting. Data are presented as means±S.E.M. **P<0.01, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526231&req=5

pone.0133931.g003: Induction of caveolins by adenoviral transduction of MRTF-A and myocardin.(A) Adenoviral transduction of human coronary artery smooth muscle cells with MRTF-A (MKL1, 20 MOI) increased expression of caveolin-1 and caveolin-2, but not caveolin-3 mRNA compared to empty virus. (B) Transduction of myocardin (MYOCD, 100 MOI) increased mRNA expression of all three caveolins (-1 to -3). (C) A dose-response curve with increasing concentrations of myocardin (1 to 300 MOI) demonstrated a similar threshold of sensitivity of caveolin-1 mRNA to that of the prototypical myocardin target calponin (CNN1). (D) Western blotting of MYOCD-transduced cells demonstrated increases of caveolins and cavins at the protein level. The magnitude of induction was similar to that of the well-established myocardin targets tropomyosin (TPM1) and SM22α (TAGLN). Summarized data from the western blots is shown in panels E through J (n = 8). 18S was used as a house-keeping gene for the qRT-PCR and HSP90 as loading control for western blotting. Data are presented as means±S.E.M. **P<0.01, ***P<0.001.
Mentions: The high sensitivity of caveolin and cavin synthesis to latrunculin B could reflect involvement of myocardin family coactivators [29, 47]. We therefore next tested if caveolins and cavins are transcriptional targets of these coactivators. MRTF-A (MKL1) and myocardin (MYOCD) were overexpressed using adenoviruses. Compared to empty vector, transduction of MRTF-A (20 MOI) in the presence of serum caused 4-fold induction of CAV1 and CAV2 while CAV3 was unchanged (Fig 3A). Transduction of myocardin (100 MOI) increased the mRNA levels for all caveolins with close to 10-fold induction of CAV3 (Fig 3B).

Bottom Line: The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB.Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs.The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Medical Science, BMC D12, Lund University, Lund, Sweden.

ABSTRACT
Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.

No MeSH data available.


Related in: MedlinePlus