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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 and cavin protein expression is regulated by actin polymerization/ depolymerization.Human coronary artery smooth muscle cells (hCASMC) were treated with the actin stabilizing substance Jasplakinolide in the presence of 5% growth supplement “serum” or the actin de-polymerizing agent Latrunculin B in “starved” cells (2% growth supplement). Panel A shows representative western blots for caveolins (-1 and -2) and for cavins (-1, -2 and -3, official gene symbols: PTRF, SDPR and PRKCDBP). HSP90 was used as a loading control. Panels B-E show summarized western blot data for caveolin-1 and (B, C) and for cavin-1 (PTRF, D, E) in the presence of either Jasplakinolide or Latrunculin B, respectively. Panel F shows TMM-normalized mRNA read counts from 44 human coronary artery samples showing a comparatively low expression of MURC (cavin-4), CAV3 and SDPR. Data are presented as means±S.E.M. **P<0.01, ***P<0.001. In panel F all differences are significant except CAV1 vs. EHD2, CAV2 vs. PRKCDBP and CAV2 vs. PACSIN2.
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pone.0133931.g001: Caveolin and cavin protein expression is regulated by actin polymerization/ depolymerization.Human coronary artery smooth muscle cells (hCASMC) were treated with the actin stabilizing substance Jasplakinolide in the presence of 5% growth supplement “serum” or the actin de-polymerizing agent Latrunculin B in “starved” cells (2% growth supplement). Panel A shows representative western blots for caveolins (-1 and -2) and for cavins (-1, -2 and -3, official gene symbols: PTRF, SDPR and PRKCDBP). HSP90 was used as a loading control. Panels B-E show summarized western blot data for caveolin-1 and (B, C) and for cavin-1 (PTRF, D, E) in the presence of either Jasplakinolide or Latrunculin B, respectively. Panel F shows TMM-normalized mRNA read counts from 44 human coronary artery samples showing a comparatively low expression of MURC (cavin-4), CAV3 and SDPR. Data are presented as means±S.E.M. **P<0.01, ***P<0.001. In panel F all differences are significant except CAV1 vs. EHD2, CAV2 vs. PRKCDBP and CAV2 vs. PACSIN2.

Mentions: The activity of two myocardin family members, MRTF-A (MKL1) and-B (MKL2), is controlled by changes in actin polymerization. This depends on their binding to globular (G)-actin in the cytosol which inhibits their nuclear translocation. We therefore first examined whether caveolae proteins are regulated by changes in actin polymerization. Primary human coronary artery smooth muscle cells (hCASMCs) were either treated with jasplakinolide, a drug that increases filamentous (F-) actin, or with latrunculin B which induces actin depolymerization. Due to poor stability of latrunculin B in serum, the latter treatment was done in serum-depleted media (2%), which in and of itself increased expression of caveolae proteins. CAV1 protein expression was increased by jasplakinolide and, in serum-depleted media, reduced by latrunculin B (Fig 1A, summarized data in B and C). Similar effects were seen for PTRF (cavin-1, Fig 1A and summarized data in D and E). PRKCDBP (cavin-3) was readily detectable (Fig 1A), but did not change significantly. We also blotted for CAV2 (Fig 1A), CAV3 (not shown), SDPR (cavin-2, Fig 1A) and MURC (cavin-4, not shown). With the exception of CAV2, which was regulated in the same manner as CAV1 (Fig 1A), these other caveolae proteins were difficult to detect (exemplified by blot for SDPR in Fig 1A). To address if this was due to low expression of CAV3, SDPR and MURC, TMM-normalized read counts from RNA sequencing of 44 coronary artery samples were plotted. As shown in Fig 1F, CAV3, SDPR and MURC were expressed at lower levels than other caveolae genes. They were therefore omitted from subsequent western blot analyses.


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 and cavin protein expression is regulated by actin polymerization/ depolymerization.Human coronary artery smooth muscle cells (hCASMC) were treated with the actin stabilizing substance Jasplakinolide in the presence of 5% growth supplement “serum” or the actin de-polymerizing agent Latrunculin B in “starved” cells (2% growth supplement). Panel A shows representative western blots for caveolins (-1 and -2) and for cavins (-1, -2 and -3, official gene symbols: PTRF, SDPR and PRKCDBP). HSP90 was used as a loading control. Panels B-E show summarized western blot data for caveolin-1 and (B, C) and for cavin-1 (PTRF, D, E) in the presence of either Jasplakinolide or Latrunculin B, respectively. Panel F shows TMM-normalized mRNA read counts from 44 human coronary artery samples showing a comparatively low expression of MURC (cavin-4), CAV3 and SDPR. Data are presented as means±S.E.M. **P<0.01, ***P<0.001. In panel F all differences are significant except CAV1 vs. EHD2, CAV2 vs. PRKCDBP and CAV2 vs. PACSIN2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526231&req=5

pone.0133931.g001: Caveolin and cavin protein expression is regulated by actin polymerization/ depolymerization.Human coronary artery smooth muscle cells (hCASMC) were treated with the actin stabilizing substance Jasplakinolide in the presence of 5% growth supplement “serum” or the actin de-polymerizing agent Latrunculin B in “starved” cells (2% growth supplement). Panel A shows representative western blots for caveolins (-1 and -2) and for cavins (-1, -2 and -3, official gene symbols: PTRF, SDPR and PRKCDBP). HSP90 was used as a loading control. Panels B-E show summarized western blot data for caveolin-1 and (B, C) and for cavin-1 (PTRF, D, E) in the presence of either Jasplakinolide or Latrunculin B, respectively. Panel F shows TMM-normalized mRNA read counts from 44 human coronary artery samples showing a comparatively low expression of MURC (cavin-4), CAV3 and SDPR. Data are presented as means±S.E.M. **P<0.01, ***P<0.001. In panel F all differences are significant except CAV1 vs. EHD2, CAV2 vs. PRKCDBP and CAV2 vs. PACSIN2.
Mentions: The activity of two myocardin family members, MRTF-A (MKL1) and-B (MKL2), is controlled by changes in actin polymerization. This depends on their binding to globular (G)-actin in the cytosol which inhibits their nuclear translocation. We therefore first examined whether caveolae proteins are regulated by changes in actin polymerization. Primary human coronary artery smooth muscle cells (hCASMCs) were either treated with jasplakinolide, a drug that increases filamentous (F-) actin, or with latrunculin B which induces actin depolymerization. Due to poor stability of latrunculin B in serum, the latter treatment was done in serum-depleted media (2%), which in and of itself increased expression of caveolae proteins. CAV1 protein expression was increased by jasplakinolide and, in serum-depleted media, reduced by latrunculin B (Fig 1A, summarized data in B and C). Similar effects were seen for PTRF (cavin-1, Fig 1A and summarized data in D and E). PRKCDBP (cavin-3) was readily detectable (Fig 1A), but did not change significantly. We also blotted for CAV2 (Fig 1A), CAV3 (not shown), SDPR (cavin-2, Fig 1A) and MURC (cavin-4, not shown). With the exception of CAV2, which was regulated in the same manner as CAV1 (Fig 1A), these other caveolae proteins were difficult to detect (exemplified by blot for SDPR in Fig 1A). To address if this was due to low expression of CAV3, SDPR and MURC, TMM-normalized read counts from RNA sequencing of 44 coronary artery samples were plotted. As shown in Fig 1F, CAV3, SDPR and MURC were expressed at lower levels than other caveolae genes. They were therefore omitted from subsequent western blot analyses.

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