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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

CCG-1423 mitigates the effect of actin depolymerization and MYOCD acts via the proximal CAV1 promoter.Panels A-D show effects of the MRTF (MKL) inhibitor CCG-1423 on caveolin and cavin mRNA expression in serum-depleted human coronary artery smooth muscle cells. 18S was used as a house-keeping gene for the qRT-PCR. Panels E (CAV1) and F (PTRF) shows an experiment where CCG-1423, latrunculin B and their combination were run in parallel with vehicle-treated controls. (G) Data in panels E and F was used to calculate fold repression by latrunculin B in the absence and presence of CCG-1423. Panel H shows a CAV1 promoter reporter assay. Three negative controls were included: cells transfected with empty vector (EV) followed by treatment with Ad-CMV-Null or Ad-CMV-MYOCD, as well as cells transfected with the CAV1 reporter plasmid (CAV1) followed by treatment with Ad-CMV-Null. Relative luciferase activity (RLU: relative luciferase units) was increased ≈7-fold by MYOCD in cells containing the reporter.
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pone.0133931.g005: CCG-1423 mitigates the effect of actin depolymerization and MYOCD acts via the proximal CAV1 promoter.Panels A-D show effects of the MRTF (MKL) inhibitor CCG-1423 on caveolin and cavin mRNA expression in serum-depleted human coronary artery smooth muscle cells. 18S was used as a house-keeping gene for the qRT-PCR. Panels E (CAV1) and F (PTRF) shows an experiment where CCG-1423, latrunculin B and their combination were run in parallel with vehicle-treated controls. (G) Data in panels E and F was used to calculate fold repression by latrunculin B in the absence and presence of CCG-1423. Panel H shows a CAV1 promoter reporter assay. Three negative controls were included: cells transfected with empty vector (EV) followed by treatment with Ad-CMV-Null or Ad-CMV-MYOCD, as well as cells transfected with the CAV1 reporter plasmid (CAV1) followed by treatment with Ad-CMV-Null. Relative luciferase activity (RLU: relative luciferase units) was increased ≈7-fold by MYOCD in cells containing the reporter.

Mentions: To further support involvement of myocardin-related coactivators in basal expression of caveolins and cavins, we used CCG-1423 (10 μM). This substance binds to RPEL motifs in MRTFs and inhibits their nuclear translocation thus providing for a pharmacological loss of function approach. In serum-depleted media, CCG-1423 inhibited expression of CAV1, CAV2, PTRF and SDPR 3-5-fold (Fig 5A–5D). We also examined if the effects of CCG-1423 and latrunculin B were additive. These experiments showed that latrunculin B and CCG-1423 were almost equipotent (Fig 5E and 5F) and that repression of CAV1 and PTRF by latrunculin B was almost absent in the presence of CCG-1423 (Fig 5G). This indicates that myocardin family coactivators mediate the effect of actin depolymerization on caveolin/cavin expression.


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)

CCG-1423 mitigates the effect of actin depolymerization and MYOCD acts via the proximal CAV1 promoter.Panels A-D show effects of the MRTF (MKL) inhibitor CCG-1423 on caveolin and cavin mRNA expression in serum-depleted human coronary artery smooth muscle cells. 18S was used as a house-keeping gene for the qRT-PCR. Panels E (CAV1) and F (PTRF) shows an experiment where CCG-1423, latrunculin B and their combination were run in parallel with vehicle-treated controls. (G) Data in panels E and F was used to calculate fold repression by latrunculin B in the absence and presence of CCG-1423. Panel H shows a CAV1 promoter reporter assay. Three negative controls were included: cells transfected with empty vector (EV) followed by treatment with Ad-CMV-Null or Ad-CMV-MYOCD, as well as cells transfected with the CAV1 reporter plasmid (CAV1) followed by treatment with Ad-CMV-Null. Relative luciferase activity (RLU: relative luciferase units) was increased ≈7-fold by MYOCD in cells containing the reporter.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133931.g005: CCG-1423 mitigates the effect of actin depolymerization and MYOCD acts via the proximal CAV1 promoter.Panels A-D show effects of the MRTF (MKL) inhibitor CCG-1423 on caveolin and cavin mRNA expression in serum-depleted human coronary artery smooth muscle cells. 18S was used as a house-keeping gene for the qRT-PCR. Panels E (CAV1) and F (PTRF) shows an experiment where CCG-1423, latrunculin B and their combination were run in parallel with vehicle-treated controls. (G) Data in panels E and F was used to calculate fold repression by latrunculin B in the absence and presence of CCG-1423. Panel H shows a CAV1 promoter reporter assay. Three negative controls were included: cells transfected with empty vector (EV) followed by treatment with Ad-CMV-Null or Ad-CMV-MYOCD, as well as cells transfected with the CAV1 reporter plasmid (CAV1) followed by treatment with Ad-CMV-Null. Relative luciferase activity (RLU: relative luciferase units) was increased ≈7-fold by MYOCD in cells containing the reporter.
Mentions: To further support involvement of myocardin-related coactivators in basal expression of caveolins and cavins, we used CCG-1423 (10 μM). This substance binds to RPEL motifs in MRTFs and inhibits their nuclear translocation thus providing for a pharmacological loss of function approach. In serum-depleted media, CCG-1423 inhibited expression of CAV1, CAV2, PTRF and SDPR 3-5-fold (Fig 5A–5D). We also examined if the effects of CCG-1423 and latrunculin B were additive. These experiments showed that latrunculin B and CCG-1423 were almost equipotent (Fig 5E and 5F) and that repression of CAV1 and PTRF by latrunculin B was almost absent in the presence of CCG-1423 (Fig 5G). This indicates that myocardin family coactivators mediate the effect of actin depolymerization on caveolin/cavin expression.

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