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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

Caveolin-1 and cavin-1 conform to patterns of regulation established for classical smooth muscle differentiation markers.Aortae from three different control mice were isolated. One piece was immediately frozen and the other was used for isolating smooth muscle cells which were subsequently cultured in vitro. (A) Western blots show higher expression of cavin-1 (Ptrf) and -3 (Prkcdbp) and caveolin-1 (Cav1) in whole aortae than in cultured cells from the same vessels. Panels B-D show summarized western blot data. Data are presented as means±SEM.**P<0.01, ***P<0.001. Panel E shows staining (in red) for calponin (left), caveolin-1 (middle) and cavin-3 (Prkcdbp, right) in consecutive sections from a saccular aortic aneurysm induced by AngII. This aneurysm had a diameter exceeding 2 mm and the lumen is indicated by an L. The aneurysm bulges to the left whereas the right side is normal. The partitions between normal and diseased areas are indicated by dotted yellow lines. A progressive reduction, from the normal to the dilated side, of medial staining for calponin, caveolin-1 and cavin-3 is evident, leaving only a thin red rim just below the endothelium at the aneurysmal site. This is shown at higher magnification in panel F, giving views of caveolin-1 staining in distal control aorta (left) and in the aneurysm (right). Caveolin-1-positive capillary structures are present in the adventitia of the aneurysm whereas medial staining is drastically reduced. Nuclei were stained with DAPI throughout. In panel F, autofluorescence (green) was used to visualize elastic lamellae showing loss of elastic fibers in the aneurysm to the right. Images are representative of three aneurysms and three control aortae. Scale bars (white) in E and F represent 200 and 20 μm, respectively.
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pone.0133931.g007: Caveolin-1 and cavin-1 conform to patterns of regulation established for classical smooth muscle differentiation markers.Aortae from three different control mice were isolated. One piece was immediately frozen and the other was used for isolating smooth muscle cells which were subsequently cultured in vitro. (A) Western blots show higher expression of cavin-1 (Ptrf) and -3 (Prkcdbp) and caveolin-1 (Cav1) in whole aortae than in cultured cells from the same vessels. Panels B-D show summarized western blot data. Data are presented as means±SEM.**P<0.01, ***P<0.001. Panel E shows staining (in red) for calponin (left), caveolin-1 (middle) and cavin-3 (Prkcdbp, right) in consecutive sections from a saccular aortic aneurysm induced by AngII. This aneurysm had a diameter exceeding 2 mm and the lumen is indicated by an L. The aneurysm bulges to the left whereas the right side is normal. The partitions between normal and diseased areas are indicated by dotted yellow lines. A progressive reduction, from the normal to the dilated side, of medial staining for calponin, caveolin-1 and cavin-3 is evident, leaving only a thin red rim just below the endothelium at the aneurysmal site. This is shown at higher magnification in panel F, giving views of caveolin-1 staining in distal control aorta (left) and in the aneurysm (right). Caveolin-1-positive capillary structures are present in the adventitia of the aneurysm whereas medial staining is drastically reduced. Nuclei were stained with DAPI throughout. In panel F, autofluorescence (green) was used to visualize elastic lamellae showing loss of elastic fibers in the aneurysm to the right. Images are representative of three aneurysms and three control aortae. Scale bars (white) in E and F represent 200 and 20 μm, respectively.

Mentions: Our findings argued that several caveolae genes share transcriptional regulatory mechanisms with classical smooth muscle differentiation marker genes. Smooth muscle markers, including the MYOCD coactivator, are known to decrease when cells are isolated and cultured in vitro as well as in various injury models in vivo [53]. We therefore next addressed if selected caveolae proteins follow this pattern of regulation. First we examined expression of caveolin-1, cavin-1 and cavin-3 in intact mouse aortae and in cultured cells from the same specimens. This demonstrated reductions of these proteins compared to total protein remaining on the gels after transfer (Fig 7A–7D). We also examined caveolin-1 and cavin-3 staining in angiotensin II-induced aortic aneurysms in mice (the cavin-1 antibody was not ideal for staining of mouse tissues). Blood pressure was significantly elevated in angiotensin II treated mice (104±9.6 (day 0) vs. 127±14mmHg (day 21)) and weight gain was similar in both groups (3.7±0.5 (saline) vs 2.6 ±0.7g (Ang II)). Similar to the myocardin target calponin (red in left panel of Fig 7E), reduced staining for caveolin-1 and cavin-3 was seen in all three aneurysms compared to adjacent normal media (red in the two panels to the right in Fig 7E). This was also evident when aneurysms were compared with distal normal aorta at higher magnification (Fig 7F, right vs. left). Caveolins and cavins therefore adhere to patterns of expression seen for classical smooth muscle differentiation markers.


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)

Caveolin-1 and cavin-1 conform to patterns of regulation established for classical smooth muscle differentiation markers.Aortae from three different control mice were isolated. One piece was immediately frozen and the other was used for isolating smooth muscle cells which were subsequently cultured in vitro. (A) Western blots show higher expression of cavin-1 (Ptrf) and -3 (Prkcdbp) and caveolin-1 (Cav1) in whole aortae than in cultured cells from the same vessels. Panels B-D show summarized western blot data. Data are presented as means±SEM.**P<0.01, ***P<0.001. Panel E shows staining (in red) for calponin (left), caveolin-1 (middle) and cavin-3 (Prkcdbp, right) in consecutive sections from a saccular aortic aneurysm induced by AngII. This aneurysm had a diameter exceeding 2 mm and the lumen is indicated by an L. The aneurysm bulges to the left whereas the right side is normal. The partitions between normal and diseased areas are indicated by dotted yellow lines. A progressive reduction, from the normal to the dilated side, of medial staining for calponin, caveolin-1 and cavin-3 is evident, leaving only a thin red rim just below the endothelium at the aneurysmal site. This is shown at higher magnification in panel F, giving views of caveolin-1 staining in distal control aorta (left) and in the aneurysm (right). Caveolin-1-positive capillary structures are present in the adventitia of the aneurysm whereas medial staining is drastically reduced. Nuclei were stained with DAPI throughout. In panel F, autofluorescence (green) was used to visualize elastic lamellae showing loss of elastic fibers in the aneurysm to the right. Images are representative of three aneurysms and three control aortae. Scale bars (white) in E and F represent 200 and 20 μm, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133931.g007: Caveolin-1 and cavin-1 conform to patterns of regulation established for classical smooth muscle differentiation markers.Aortae from three different control mice were isolated. One piece was immediately frozen and the other was used for isolating smooth muscle cells which were subsequently cultured in vitro. (A) Western blots show higher expression of cavin-1 (Ptrf) and -3 (Prkcdbp) and caveolin-1 (Cav1) in whole aortae than in cultured cells from the same vessels. Panels B-D show summarized western blot data. Data are presented as means±SEM.**P<0.01, ***P<0.001. Panel E shows staining (in red) for calponin (left), caveolin-1 (middle) and cavin-3 (Prkcdbp, right) in consecutive sections from a saccular aortic aneurysm induced by AngII. This aneurysm had a diameter exceeding 2 mm and the lumen is indicated by an L. The aneurysm bulges to the left whereas the right side is normal. The partitions between normal and diseased areas are indicated by dotted yellow lines. A progressive reduction, from the normal to the dilated side, of medial staining for calponin, caveolin-1 and cavin-3 is evident, leaving only a thin red rim just below the endothelium at the aneurysmal site. This is shown at higher magnification in panel F, giving views of caveolin-1 staining in distal control aorta (left) and in the aneurysm (right). Caveolin-1-positive capillary structures are present in the adventitia of the aneurysm whereas medial staining is drastically reduced. Nuclei were stained with DAPI throughout. In panel F, autofluorescence (green) was used to visualize elastic lamellae showing loss of elastic fibers in the aneurysm to the right. Images are representative of three aneurysms and three control aortae. Scale bars (white) in E and F represent 200 and 20 μm, respectively.
Mentions: Our findings argued that several caveolae genes share transcriptional regulatory mechanisms with classical smooth muscle differentiation marker genes. Smooth muscle markers, including the MYOCD coactivator, are known to decrease when cells are isolated and cultured in vitro as well as in various injury models in vivo [53]. We therefore next addressed if selected caveolae proteins follow this pattern of regulation. First we examined expression of caveolin-1, cavin-1 and cavin-3 in intact mouse aortae and in cultured cells from the same specimens. This demonstrated reductions of these proteins compared to total protein remaining on the gels after transfer (Fig 7A–7D). We also examined caveolin-1 and cavin-3 staining in angiotensin II-induced aortic aneurysms in mice (the cavin-1 antibody was not ideal for staining of mouse tissues). Blood pressure was significantly elevated in angiotensin II treated mice (104±9.6 (day 0) vs. 127±14mmHg (day 21)) and weight gain was similar in both groups (3.7±0.5 (saline) vs 2.6 ±0.7g (Ang II)). Similar to the myocardin target calponin (red in left panel of Fig 7E), reduced staining for caveolin-1 and cavin-3 was seen in all three aneurysms compared to adjacent normal media (red in the two panels to the right in Fig 7E). This was also evident when aneurysms were compared with distal normal aorta at higher magnification (Fig 7F, right vs. left). Caveolins and cavins therefore adhere to patterns of expression seen for classical smooth muscle differentiation markers.

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