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.
Highlights
The contractile activity of vascular smooth muscle cells (VSMCs)2 serves to regulate physiological vascular resistance and blood pressure and dysfunctional vasoconstriction or relaxation underlies peripheral hypertensive disease, the most common diagnosis and leading risk factor of cardiovascular mortality [1]
We investigated the combinatorial role of myocardin/serum response factor (SRF) and Notch signaling in the transcriptional regulation of myosin light chain kinase (MLCK) gene expression
These results suggest that the level of Notch1 signaling might coordinate both induction (10 ng ICN1 or 1:1 myocardin:ICN1) and repression (1:4 myocardin:ICN1) of Smooth muscle (SM-)MLCK promoter activity
Summary
Our previous identification of SM-MLCK as a novel Notch target gene included the demonstration that its promoter can be independently activated by either Notch or SRF/myocardin signals It remained undetermined whether these two activation pathways functionally interact to coordinate SM-MLCK transcription. Activated Notch or direct Jagged ligand stimulation enhances myocardin-induced SRF complex formation on the SM-MLCK promoter, but at relatively high levels can raise HRT2 content, which antagonizes both myocardin- and Notch-induced promoter activity through disruption of respective transcription complexes. Together, these findings suggest the operational presence of a Notch-dependent HRT2 autoregulatory loop coordinating transcriptional control of SM-MLCK gene expression
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