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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

Myocardin drives formation of caveolae.Coronary smooth muscle cells, cultured on polycarbonate membranes, were transduced with Ad-CMV-MYOCD or Ad-CMV- for 96h and prepared for electron microscopy. Left micrographs in panel A show control cells (Ad-CMV-) where caveolae were rare. Rough endoplasmic reticulum was very prominent in these cultured cells. Right micrographs show caveolae in cells transduced with MYOCD (black arrowheads). Panel B shows quantification of the number of caveolae per μm membrane in cells from three independent transductions. The total membrane lengths examined for Ad-CMV- and Ad-CMV-MYOCD were 501 and 340 μm, respectively. Panel C shows the diameters of caveolae in control and MYOCD transduced cells. Panel D shows area of rough endoplasmic reticulum compared to total cell area. Scale bars in the upper micrographs of panel A represent 500 nm whereas those in the lower micrographs represent 200 nm.
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pone.0133931.g006: Myocardin drives formation of caveolae.Coronary smooth muscle cells, cultured on polycarbonate membranes, were transduced with Ad-CMV-MYOCD or Ad-CMV- for 96h and prepared for electron microscopy. Left micrographs in panel A show control cells (Ad-CMV-) where caveolae were rare. Rough endoplasmic reticulum was very prominent in these cultured cells. Right micrographs show caveolae in cells transduced with MYOCD (black arrowheads). Panel B shows quantification of the number of caveolae per μm membrane in cells from three independent transductions. The total membrane lengths examined for Ad-CMV- and Ad-CMV-MYOCD were 501 and 340 μm, respectively. Panel C shows the diameters of caveolae in control and MYOCD transduced cells. Panel D shows area of rough endoplasmic reticulum compared to total cell area. Scale bars in the upper micrographs of panel A represent 500 nm whereas those in the lower micrographs represent 200 nm.

Mentions: Our findings so far established that myocardin-related coactivators control the expression of genes from at least three families of importance for the biogenesis of caveolae. We therefore directly examined if myocardin drives formation of caveolae. hCASMCs were transduced with adenovirus encoding myocardin or empty vector. They were then processed for electron microscopy and the density of caveolae was determined. Overexpression of myocardin led to a higher density of membrane caveolae compared with control (Fig 6A). Summarized data established a 5-fold increase of the density of caveolae after transduction of myocardin (bar graph in Fig 6B). The diameters of caveolae were similar (Fig 6C) and the relative area of the endoplasmic reticulum was unchanged (Fig 6D).


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)

Myocardin drives formation of caveolae.Coronary smooth muscle cells, cultured on polycarbonate membranes, were transduced with Ad-CMV-MYOCD or Ad-CMV- for 96h and prepared for electron microscopy. Left micrographs in panel A show control cells (Ad-CMV-) where caveolae were rare. Rough endoplasmic reticulum was very prominent in these cultured cells. Right micrographs show caveolae in cells transduced with MYOCD (black arrowheads). Panel B shows quantification of the number of caveolae per μm membrane in cells from three independent transductions. The total membrane lengths examined for Ad-CMV- and Ad-CMV-MYOCD were 501 and 340 μm, respectively. Panel C shows the diameters of caveolae in control and MYOCD transduced cells. Panel D shows area of rough endoplasmic reticulum compared to total cell area. Scale bars in the upper micrographs of panel A represent 500 nm whereas those in the lower micrographs represent 200 nm.
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Related In: Results  -  Collection

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pone.0133931.g006: Myocardin drives formation of caveolae.Coronary smooth muscle cells, cultured on polycarbonate membranes, were transduced with Ad-CMV-MYOCD or Ad-CMV- for 96h and prepared for electron microscopy. Left micrographs in panel A show control cells (Ad-CMV-) where caveolae were rare. Rough endoplasmic reticulum was very prominent in these cultured cells. Right micrographs show caveolae in cells transduced with MYOCD (black arrowheads). Panel B shows quantification of the number of caveolae per μm membrane in cells from three independent transductions. The total membrane lengths examined for Ad-CMV- and Ad-CMV-MYOCD were 501 and 340 μm, respectively. Panel C shows the diameters of caveolae in control and MYOCD transduced cells. Panel D shows area of rough endoplasmic reticulum compared to total cell area. Scale bars in the upper micrographs of panel A represent 500 nm whereas those in the lower micrographs represent 200 nm.
Mentions: Our findings so far established that myocardin-related coactivators control the expression of genes from at least three families of importance for the biogenesis of caveolae. We therefore directly examined if myocardin drives formation of caveolae. hCASMCs were transduced with adenovirus encoding myocardin or empty vector. They were then processed for electron microscopy and the density of caveolae was determined. Overexpression of myocardin led to a higher density of membrane caveolae compared with control (Fig 6A). Summarized data established a 5-fold increase of the density of caveolae after transduction of myocardin (bar graph in Fig 6B). The diameters of caveolae were similar (Fig 6C) and the relative area of the endoplasmic reticulum was unchanged (Fig 6D).

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