title: Autoregulatory Control of Smooth Muscle Myosin Light Chain Kinase Promoter by Notch Signaling.
abstract: Smooth muscle myosin light chain kinase (SM-MLCK) is the key enzyme responsible for phosphorylation of regulatory myosin light chain (MLC20), resulting in actin-myosin cross-bridging and force generation in vascular smooth muscle required for physiological vasoreactivity and blood pressure control. In this study, we investigated the combinatorial role of myocardin/serum response factor (SRF) and Notch signaling in the transcriptional regulation of MLCK gene expression. Promoter reporter analyses in rat A10 smooth muscle cells revealed a bimodal pattern of MLCK promoter activity and gene expression upon stimulation with constitutively active Notch1 in presence of myocardin or by Jagged1 ligand stimulation. An initial Notch1-induced increase in MLCK transcription was followed by loss in promoter sensitivity, which could be restored with further Notch1 dose escalation. Real-time PCR analyses revealed that endogenous levels of Hairy Related Transcription (HRT) factor 2 (HRT2) peaked concurrently with inhibitory concentrations of Notch1. Forced expression of HRT2 demonstrated simultaneous repression of both myocardin- and Notch1-induced MLCK promoter activity. HRT2-mediated repression was further confirmed by HRT2 truncations and siHRT2 treatments that rescued MLCK promoter activity and gene expression. Chromatin immunoprecipitation studies revealed both Jagged1 ligand- and Notch1-enhanced myocardin/SRF complex formation at the promoter CArG element. In contrast, heightened levels of HRT2 concomitantly disrupted myocardin/SRF and Notch transcription complex formation at respective CArG and CSL binding elements. Taken together, SM-MLCK promoter activity appears highly sensitive to the relative levels of Notch1 signaling, HRT2, and myocardin. These findings identify a novel Notch-dependent HRT2 autoregulatory circuit coordinating transcriptional regulation of SM-MLCK.
authors.affiliation: From the Department of Medicine, Case Cardiovascular Research Institute and University Hospitals Harrington Heart and Vascular Institute, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Sanchita
authors.initials: S
authors.lastname: Basu
authors.affiliation: From the Department of Medicine, Case Cardiovascular Research Institute and University Hospitals Harrington Heart and Vascular Institute, Case Western Reserve University, Cleveland, Ohio 44106 aaron.proweller@case.edu.
authors.forename: Aaron
authors.initials: A
authors.lastname: Proweller
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000096:biosynthesis* / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D000099251:Myocardin
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D009219:Myosin-Light-Chain Kinase; D009687:Nuclear Proteins; D051881:Receptor, Notch1; D015534:Trans-Activators; D000099251:Myocardin; C497763:Notch1 protein, rat
keywords: Jagged; Notch; Notch pathway; hairy-related transcription factors; myocardin; myosin light chain kinase; serum response factor (SRF); smooth muscle; transcription