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Distinct effects of voltage- and store-dependent calcium influx on stretch-induced differentiation and growth in
Jingli Ren1, Sebastian Albinsson, Per Hellstrand
1Department of Experimental Medical Science, Lund University, SE-221 84 Lund, Sweden.
Abstract:
Stretch of the vascular wall stimulates smooth muscle hypertrophy by activating the MAPK and Rho/Rho kinase (ROK) pathways. We investigated the role of calcium in this response. Stretch-stimulated expression of contractile and cytoskeletal proteins in mouse portal vein was inhibited at mRNA and protein levels by blockade of voltage-dependent Ca(2+) entry (VDCE). In contrast, blockade of store-operated Ca(2+) entry (SOCE) did not affect smooth muscle marker expression but decreased global protein synthesis. Activation of VDCE caused membrane translocation of RhoA followed by phosphorylation of its downstream effectors LIMK-2 and cofilin-2. Stretch-activated cofilin-2 phosphorylation depended on VDCE but not on SOCE. VDCE was associated with increased mRNA expression of myocardin, myocyte enhancer factor (MEF) -2A and -2D, and smooth muscle marker genes, all of which depended on ROK activity. SOCE increased ERK1/2 phosphorylation and c-Fos expression but had no effect on phosphorylation of LIMK-2 and cofilin-2 or on myocardin and MEF2 expression. Knockdown of MEF2A or -2D eliminated the VDCE-induced activation of myocardin expression and increased basal c-Jun and c-Fos mRNA levels. These results indicate that MEF2 mediates VDCE-dependent stimulation of myocardin expression via the Rho/ROK pathway. In addition, SOCE activates the expression of immediate-early genes, known to be regulated by MEF2 via Ca(2+)-dependent phosphorylation of histone deacetylases, but this mode of Ca(2+) entry does not affect the Rho/ROK pathway. Compartmentation of Ca(2+) entry pathways appears as one mechanism whereby extracellular and membrane signals influence smooth muscle phenotype regulation, with MEF2 as a focal point.
Insights
Vascular smooth muscle hypertrophy involves distinct calcium entry pathways. Voltage-dependent calcium entry (VDCE) activates Rho/ROK signaling and myocardin expression, while store-operated calcium entry (SOCE) influences immediate-early gene expression.
Area of Science:
- Cardiovascular Biology
- Cellular Physiology
- Molecular Biology
Background:
- Vascular smooth muscle hypertrophy is crucial for vascular wall remodeling.
- The MAPK and Rho/Rho kinase (ROK) pathways are implicated in stretch-induced smooth muscle hypertrophy.
- Calcium signaling plays a pivotal role in regulating cellular responses to mechanical stress.
Purpose of the Study:
- To elucidate the distinct roles of voltage-dependent calcium entry (VDCE) and store-operated calcium entry (SOCE) in mediating stretch-induced smooth muscle hypertrophy.
- To investigate the downstream signaling pathways activated by different calcium entry modes.
- To determine the involvement of transcription factors like MEF2 in calcium-mediated gene expression.
Main Methods:
- Utilized mouse portal vein model subjected to mechanical stretch.
- Employed pharmacological inhibitors to block VDCE and SOCE.
- Assessed protein and mRNA expression of contractile proteins, cytoskeletal elements, and transcription factors using Western blotting and RT-PCR.
- Performed gene knockdown experiments for MEF2A and MEF2D.
Main Results:
- Blockade of VDCE inhibited stretch-induced expression of contractile and cytoskeletal proteins, RhoA activation, and LIMK-2/cofilin-2 phosphorylation.
- VDCE activation led to increased expression of myocardin, MEF2A/D, and smooth muscle marker genes, dependent on ROK activity.
- SOCE blockade decreased global protein synthesis but did not affect smooth muscle marker expression.
- SOCE increased ERK1/2 phosphorylation and c-Fos expression, independent of Rho/ROK pathway.
- MEF2 knockdown abolished VDCE-induced myocardin expression and increased basal c-Jun/c-Fos mRNA.
Conclusions:
- VDCE is essential for the Rho/ROK-mediated stimulation of myocardin expression and smooth muscle-specific gene induction during stretch.
- SOCE regulates immediate-early gene expression via pathways distinct from Rho/ROK signaling.
- Compartmentalization of calcium entry pathways differentially controls smooth muscle phenotype, with MEF2 acting as a key mediator.
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