Abstract
Endothelin, encoded by ET1, is a vasoactive substance primarily synthesized in vascular endothelial cells (VECs). Elevation of endothelin levels, due to transcriptional hyperactivation, has been observed in a host of cardiovascular diseases. We have previously shown that serum response factor (SRF) is a regulator of ET1 transcription in VECs. Here we report that angiotensin II (Ang II) induced ET1 transcription paralleled activation of glycogen synthase kinase 3 (GSK3) in cultured VECs. GSK3 knockdown or pharmaceutical inhibition attenuated Ang II induced endothelin expression. Of interest, the effect of GSK3 on endothelin transcription relied on the conserved SRF motif within the ET1 promoter. Further analysis revealed that GSK3 interacted with and phosphorylated SRF at serine 224. Phosphorylation of SRF by GSK3 did not influence its recruitment to the ET1 promoter. Instead, GSK3-mediated SRF phosphorylation potentiated its interaction with MRTF-A, a key co-factor for SRF, which helped recruit the chromatin remodeling protein BRG1 to the ET1 promoter resulting in augmented histone H3 acetylation/H3K4 trimethylation. Consistently, over-expression of a constitutively active GSK enhanced Ang II-induced ET1 transcription and knockdown of either MRTF-A or BRG1 abrogated the enhancement of ET1 transcription. In conclusion, our data highlight a previously unrecognized mechanism that contributes to the transcriptional regulation of endothelin. Targeting this GSK3-SRF axis may yield novel approaches in the intervention of cardiovascular diseases.
Highlights
The vascular endothelium functions as a physical barrier separating the circulation from the basal lamina but synthesizes and secretes a host of substances contributing to the maintenance and regulation of internal microenvironment either locally or systemically (Rafii et al, 2016)
Active GSK3β, in which serine 9 was substituted by alanine (S9A), dominant negative GSK3β, in which arginine 96 was substituted by alanine (R96A), phosphorylation-defective serum response factor (SRF), in which serine 224 was substituted by alanine (S224A), and phosphorylation mimetic SRF in which serine 224 was substituted by aspartic acid (S224D), were generated by a QuikChange kit (Thermo Fisher Scientific, Waltham, MA, United States) and verified by direct sequencing as previously described (Bechard and Dalton, 2009)
When cultured endothelial cells were exposed to angiotensin II (Ang II) treatment, ET1 expression levels were significantly up-regulated as measured by qPCR (Figure 1A) and Enzyme-Linked Immunosorbent Assay (ELISA) (Figure 1B) in keeping with previous findings (Weng et al, 2015a,b; Yu et al, 2015)
Summary
The vascular endothelium functions as a physical barrier separating the circulation from the basal lamina but synthesizes and secretes a host of substances contributing to the maintenance and regulation of internal microenvironment either locally (via paracrine) or systemically (via endocrine) (Rafii et al, 2016). Endothelin, encoded by ET1, is a polypeptide derived from. Regulation of ET1 Transcription by GSK3 endothelial cells (Yanagisawa et al, 1988). Endothelin primarily signals through one of the two cognate receptors, ETRA and ETRB. Whereas binding of endothelin to ETRA leads to contraction of vascular smooth muscle cells, the endothelinETRB interaction is thought to trigger endothelin clearance and vasorelaxation (Abman, 2009). ETR antagonists have been demonstrated to be effective in the intervention of a host of cardiovascular and metabolic diseases (Enevoldsen et al, 2020)
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