Rho and smooth muscle cell phenotype

Smooth muscle cell (SMC) phenotypic modulation from the mature ’contractile’ to a less differentiated ’synthetic’ phenotype involves not only altered expression but also a reorganisation of contractile and cytoskeletal proteins. Objective: To investigate the role of RhoA, a known regulator of the actin cytoskeleton, in SMC phenotypic regulation. Methods: Rho transcription (RT-PCR), expression (Western analysis) and activation (membrane translocation or Rho ’pull-down’ assay) was investigated in cultured rabbit aortic SMC during phenotypic modulation, and under the influence of known SM-regulatory proteins (thrombin, heparin and TGF- β). Rho’s effect on cell morphology was examined by transient transfection of ’synthetic’ state SMC with either constitutively active Rho (Val14RhoA) or its inhibitor, C3 transferase. Results: RhoA transcription was elevated in the first 3 days of primary culture, and protein expression peaked at 2 days post-confluence when SMC return to a more ’contractile’ state. However, RhoA showed augmented activation at three time-points in primary culture: the transition point when SMCs enter logarithmic growth and are highly motile, upon reaching quiescence, and when they return to a more ’contractile’ state. Thrombin, heparin and TGF-β activated RhoA in ’synthetic’ state SMCs. Transfection with Val14RhoA caused a dramatic decrease in SMC size and a reorganization of cytoskeletal proteins, reminiscent of the ’contractile’ phenotype. Specific inhibition of endogenous Rho by C3 transferase resulted in an almost complete loss of contractile proteins. Conclusion: These data indicate that Rho is an important determining factor of SMC functional state.

A role for Rho in smooth muscle phenotypic regulation

The role of the small GTP-binding protein Rho in the process of smooth muscle cell (SMC) phenotypic modulation was investigated using cultured rabbit aortic SMCs. Both Rho transcription and Rho protein expression were high for the first 3 days of culture (contractile state cells), with expression decreasing after change to the synthetic state and peaking upon return to the contractile phenotype. Activation of Rho (indicated by translocation to the membrane) also peaked upon return to the contractile state and was low in synthetic state SMCs. Transient transfection of synthetic state rabbit SMCs with constitutively active Rho (val14rho) caused a dramatic decrease in cell size and reorganization of cytoskeletal proteins to resemble those of the contractile phenotype; alpha-actin and myosin adopted a tightly packed, highly organized arrangement, whereas vimentin localized to the immediate perinuclear region and focal adhesions were enlarged. Conversely, specific inhibition of endogenous Rho, by expression of C3 transferase, resulted in the complete loss of actin and myosin filaments without affecting the distribution of vimentin. Focal adhesions were reduced in number. Thus, Rho plays a key role in regulating SMC phenotypic expression.

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

The chicken ovalbumin upstream promoter-transcription factors modulate genes and pathways involved in skeletal muscle cell metabolism

The chicken ovalbumin upstream promoter-transcription factors ( COUP-TFs) are orphan members of the nuclear hormone receptor ( NR) superfamily. COUP-TFs are involved in organogenesis and neurogenesis. However, their role in skeletal muscle ( and other major mass tissues) and metabolism remains obscure. Skeletal muscle accounts for similar to 40% of total body mass and energy expenditure. Moreover, this peripheral tissue is a primary site of glucose and fatty acid utilization. We utilize small interfering RNA ( siRNA)-mediated attenuation of Coup-TfI and II ( mRNA and protein) in a skeletal muscle cell culture model to understand the regulatory role of Coup-Tfs in this energy demanding tissue. This targeted NR repression resulted in the significant attenuation of genes that regulate lipid mobilization and utilization ( including Ppar alpha, Fabp3, and Cpt-1). This was coupled to reduced fatty acid beta-oxidation. Additionally we observed significant attenuation of Ucp1, a gene involved in energy expenditure. Concordantly, we observed a 5-fold increase in ATP levels in cells with siRNA-mediated repression of Coup-TfI and II. Furthermore, the expression of classical liver X receptor ( LXR) target genes involved in reverse cholesterol transport ( Abca1 and Abcg1) were both significantly repressed. Moreover, we observed that repression of the Coup-Tfs ablated the activation of Abca1, and Abcg1 mRNA expression by the selective LXR agonist, T0901317. In concordance, Coup-Tf-siRNA-transfected cells were refractory to Lxr-mediated reduction of total intracellular cholesterol levels in contrast to the negative control cells. In agreement Lxr-mediated activation of the Abca1 promoter in Coup-Tf-siRNA cells was attenuated. Collectively, these data suggest a pivotal role for Coup-Tfs in the regulation of lipid utilization/cholesterol homeostasis in skeletal muscle cells and the modulation of Lxr-dependent gene regulation.