Introduction: Nexilin, encoded by the NEXN gene, is expressed in striated muscle where it localizes to Z-discs. Dense bodies and dense bands in smooth muscle cells (SMCs) are functional equivalents of Z-discs and provide attachment sites for actin. Myocardin Related Transcription Factors (MRTFs) act through the serum response factor (SRF) and so called CArG boxes, and play important roles for differentiation and function of smooth and striated muscles. The aims of this study were: 1) to study the expression and localization of Nexilin in SMCs, 2) to examine if Nexilin is regulated by MRTFs, and 3) to test if Nexilin promotes polymerization of actin and thus enhances contractile differentiation of SMCs.
Methods: The mRNA and protein expression of Nexilin were examined using expression databases and correlation analyses. The subcellular distribution of Nexilin was determined using immunofluorescence and immunoelectron microscopy in human detrusor specimens. Human bladder SMCs were transduced with adenoviruses for overexpression of MKL1, MKL2 and MYOCD, or transfected with siRNA against SRF. The expression of Nexilin (NEXN) was measured using RT-qPCR and western blotting. The involvement of the proximal promoter of NEXN was assayed using a promoter reporter. A short hairpin RNA was used for knockdown of NEXN in human bladder SMCs, and the expression of SMC markers was detected by RT-qPCR and western blotting. The ratio of filamentous to globular actin was measured using a sedimentation method followed by western blotting.
Results: Nexilin (NEXN) expression in human bladder smooth muscle was comparable to that in striated muscles. Imaging suggested that Nexilin localizes dense bodies and dense bands. Overexpression of MKL1, MKL2 and MYOCD increased the expression of Nexilin at both mRNA and protein levels, and SRF silencing reduced the expression of NEXN. Overexpression of MKL1 and MYOCD activated the proximal promoter of NEXN. NEXN silencing reduced expression of SMC markers, including Calponin (CNN1) and Transgelin (TAGLN). NEXN silencing also reduced actin polymerization.
Conclusions: Nexilin, targeted by MRTFs, is a dense body-/dense band-associated protein in human SMCs. Nexilin promotes actin polymerization and amplifies SMC differentiation, probably via dense bodies and dense bands. Source of
Funding: The Swedish Research Council (Vetenskapsrådet, http://www.vr.se/ (2017-01225_3 to KS, and 349-2007-8703 and 2017-01070 to UH)), the Heart Lung Foundation (20170323, KS), the Hillevi Fries Foundation (BU), the Gösta Jönsson Foundation (BU), the Crafoord Foundation (CR), and the Magnus Bergvalls Stiftelse (Magnus Bergvall Foundation) (CR).