Autoregulatory Control of Smooth Muscle Myosin Light Chain Kinase Promoter by Notch Signaling

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.