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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

Actin depolymerization regulates caveolins and cavins at the mRNA level in human coronary artery smooth muscle cells.Cells were treated with either Latrunculin B (Lat; depolymerizes actin) or Jaspakinolide (Jasp; polymerizes actin) for 24h. qRT-PCR was performed to detect caveolin-1 (CAV1) and caveolin-2 (CAV2) mRNA levels, which were reduced by Lat (A) and only marginally affected by Jasp (B). Latrunculin B similarly reduced mRNA expression of cavin-1 and -2 (PTRF and SDPR) but slightly increased mRNA expression of cavin-3 (PRKCDBP, panel C). Treatment with Jasp did not affect cavin-1 (PTRF, not shown), modestly reduced the mRNA level of cavin-2 and slightly increased the mRNA expression of cavin-3 (D). 18S was used as housekeeping gene throughout. Panel E shows concentration-response curves for LatB and vehicle (DMSO). CAV1 and PTRF were measured at each Lat concentration and compared to vehicle-treated (DMSO) control cells. Controls for CAV1 and PTRF did not change with increasing DMSO (except at the highest concentration of DMSO) and were pooled for simplicity. Data are presented as means±S.E.M throughout. *P<0.05, **P<0.01, ***P<0.001.
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pone.0133931.g002: Actin depolymerization regulates caveolins and cavins at the mRNA level in human coronary artery smooth muscle cells.Cells were treated with either Latrunculin B (Lat; depolymerizes actin) or Jaspakinolide (Jasp; polymerizes actin) for 24h. qRT-PCR was performed to detect caveolin-1 (CAV1) and caveolin-2 (CAV2) mRNA levels, which were reduced by Lat (A) and only marginally affected by Jasp (B). Latrunculin B similarly reduced mRNA expression of cavin-1 and -2 (PTRF and SDPR) but slightly increased mRNA expression of cavin-3 (PRKCDBP, panel C). Treatment with Jasp did not affect cavin-1 (PTRF, not shown), modestly reduced the mRNA level of cavin-2 and slightly increased the mRNA expression of cavin-3 (D). 18S was used as housekeeping gene throughout. Panel E shows concentration-response curves for LatB and vehicle (DMSO). CAV1 and PTRF were measured at each Lat concentration and compared to vehicle-treated (DMSO) control cells. Controls for CAV1 and PTRF did not change with increasing DMSO (except at the highest concentration of DMSO) and were pooled for simplicity. Data are presented as means±S.E.M throughout. *P<0.05, **P<0.01, ***P<0.001.

Mentions: The observed effects of drugs that affect actin polymerization could in principle depend on altered protein synthesis, altered protein degradation or both. There is ample evidence in the literature that caveolae proteins are regulated at the level of protein degradation. We found however that depolymerization of actin with latrunculin B reduced the mRNA levels for CAV1 and CAV2 (5-fold, Fig 2A) as determined by qRT-PCR. Polymerization of actin with jasplakinolide modestly increased CAV1 whereas the effect on CAV2 failed to reach significance (Fig 2B). Latrunculin B also reduced expression of PTRF and of SDPR (5-fold, Fig 2C). PRKCDBP (cavin-3) expression was instead slightly increased. Actin polymerization with jasplakinolide failed to induce PTRF (not shown) and slightly reduced expression of SDPR (Fig 2D). Modest induction of PRKCDBP was however seen (Fig 2D). The discrepant effect of jasplakonolide on PTRF protein and mRNA was addressed in an experiment where protein synthesis was inhibited by cycloheximide (not shown). While PTRF tended to be stabilized by jasplakinolide at an early time (P = 0.07 at 42h), the drug combination was cytotoxic upon longer incubation, precluding in depth characterization of any protein stabilizing effect. Concentration-response curves indicated slightly higher sensitivity of PTRF than of CAV1 to latrunculin B (Fig 2E). Time-course studies disclosed maximal PTRF repression at 12h whereas maximal CAV1 repression was seen at 24h (not shown); repression then subsided with time but was maintained for at least 48h in both cases. We therefore conclude that the mRNA expression of caveolins and cavins is regulated by latrunculin B-induced actin depolymerization.


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)

Actin depolymerization regulates caveolins and cavins at the mRNA level in human coronary artery smooth muscle cells.Cells were treated with either Latrunculin B (Lat; depolymerizes actin) or Jaspakinolide (Jasp; polymerizes actin) for 24h. qRT-PCR was performed to detect caveolin-1 (CAV1) and caveolin-2 (CAV2) mRNA levels, which were reduced by Lat (A) and only marginally affected by Jasp (B). Latrunculin B similarly reduced mRNA expression of cavin-1 and -2 (PTRF and SDPR) but slightly increased mRNA expression of cavin-3 (PRKCDBP, panel C). Treatment with Jasp did not affect cavin-1 (PTRF, not shown), modestly reduced the mRNA level of cavin-2 and slightly increased the mRNA expression of cavin-3 (D). 18S was used as housekeeping gene throughout. Panel E shows concentration-response curves for LatB and vehicle (DMSO). CAV1 and PTRF were measured at each Lat concentration and compared to vehicle-treated (DMSO) control cells. Controls for CAV1 and PTRF did not change with increasing DMSO (except at the highest concentration of DMSO) and were pooled for simplicity. Data are presented as means±S.E.M throughout. *P<0.05, **P<0.01, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133931.g002: Actin depolymerization regulates caveolins and cavins at the mRNA level in human coronary artery smooth muscle cells.Cells were treated with either Latrunculin B (Lat; depolymerizes actin) or Jaspakinolide (Jasp; polymerizes actin) for 24h. qRT-PCR was performed to detect caveolin-1 (CAV1) and caveolin-2 (CAV2) mRNA levels, which were reduced by Lat (A) and only marginally affected by Jasp (B). Latrunculin B similarly reduced mRNA expression of cavin-1 and -2 (PTRF and SDPR) but slightly increased mRNA expression of cavin-3 (PRKCDBP, panel C). Treatment with Jasp did not affect cavin-1 (PTRF, not shown), modestly reduced the mRNA level of cavin-2 and slightly increased the mRNA expression of cavin-3 (D). 18S was used as housekeeping gene throughout. Panel E shows concentration-response curves for LatB and vehicle (DMSO). CAV1 and PTRF were measured at each Lat concentration and compared to vehicle-treated (DMSO) control cells. Controls for CAV1 and PTRF did not change with increasing DMSO (except at the highest concentration of DMSO) and were pooled for simplicity. Data are presented as means±S.E.M throughout. *P<0.05, **P<0.01, ***P<0.001.
Mentions: The observed effects of drugs that affect actin polymerization could in principle depend on altered protein synthesis, altered protein degradation or both. There is ample evidence in the literature that caveolae proteins are regulated at the level of protein degradation. We found however that depolymerization of actin with latrunculin B reduced the mRNA levels for CAV1 and CAV2 (5-fold, Fig 2A) as determined by qRT-PCR. Polymerization of actin with jasplakinolide modestly increased CAV1 whereas the effect on CAV2 failed to reach significance (Fig 2B). Latrunculin B also reduced expression of PTRF and of SDPR (5-fold, Fig 2C). PRKCDBP (cavin-3) expression was instead slightly increased. Actin polymerization with jasplakinolide failed to induce PTRF (not shown) and slightly reduced expression of SDPR (Fig 2D). Modest induction of PRKCDBP was however seen (Fig 2D). The discrepant effect of jasplakonolide on PTRF protein and mRNA was addressed in an experiment where protein synthesis was inhibited by cycloheximide (not shown). While PTRF tended to be stabilized by jasplakinolide at an early time (P = 0.07 at 42h), the drug combination was cytotoxic upon longer incubation, precluding in depth characterization of any protein stabilizing effect. Concentration-response curves indicated slightly higher sensitivity of PTRF than of CAV1 to latrunculin B (Fig 2E). Time-course studies disclosed maximal PTRF repression at 12h whereas maximal CAV1 repression was seen at 24h (not shown); repression then subsided with time but was maintained for at least 48h in both cases. We therefore conclude that the mRNA expression of caveolins and cavins is regulated by latrunculin B-induced actin depolymerization.

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