The miR-143/145 Cluster Is a Novel Transcriptional Target of Jagged-1/Notch Signaling in Vascular Smooth Muscle Cells

Chunxiang Zhang,Lucy Liaw,Sarah M. Peterson,Sumithra Urs,Joshua M. Boucher

doi:10.1074/jbc.m111.221945

Abstract

Activation of Notch signaling by Jagged-1 (Jag-1) in vascular smooth muscle cells (VSMC) promotes a differentiated phenotype characterized by increased expression of contractile proteins. Recent studies show that microRNAs (miR)-143/145 regulates VSMC phenotype. The serum response factor (SRF)/myocardin complex binds to CArG sequences to activate miR-143/145 transcription, but no other regulators are known in VSMC. Using miR arrays, we found miR-143/145 induced following expression of a constitutively active Notch1 intracellular domain (N1ICD). We hypothesized that miR-143/145 is required for Jag-1/Notch-induced VSMC differentiation. Activation of Notch receptors by Jag-1 caused CBF1-dependent up-regulation of miR-143/145, increased differentiation, and decreased proliferation. Conversely, inhibiting basal Notch signaling decreased steady state levels of miR-143/145. Using SRF knockdown, we found that Jag-1/Notch induction of miR-143/145 is SRF independent, although full acquisition of contractile markers requires SRF. Using miR-143/145 promoter reporter constructs we show Jag-1/Notch increases promoter activity, and this is dependent on intact CBF1 consensus sites within the promoter. Chromatin immunoprecipitation (ChIP) assays revealed that N1ICD-containing complexes bind to CBF1 sites in the miR-143/145 promoter. We also identified N1ICD complex binding to CBF1 sites within the endogenous human miR-143/145 promoter. Using miR-143/145-interfering oligonucleotides, we demonstrate that Jag-1/Notch signaling requires induction of both miR-143 and miR-145 to promote the VSMC contractile phenotype. Thus, miR-143/145 is a novel transcriptional target of Jag-1/Notch signaling in VSMC. We propose miR-143/145 as activated independently by Jag-1/Notch and SRF in parallel pathways. Multiple pathways converging on miR-143/145 provides potential for fine-tuning or amplification of VSMC differentiation signals.

Highlights

Aberrant Notch signaling in the vasculature is associated with vascular defects including abnormal structure, leakiness, Alagille syndrome, and CADASIL [10, 11]
Expression of Jag-1 by the endothelium promotes vascular smooth muscle cells (VSMC) differentiation by activating Notch3 [15], while the physiological functions of Delta like-1 (Dll-1)/Notch signaling in VSMC are not known
The miR-143/145 Cluster Is Differentially Regulated by Notch Signaling in Human VSMC—We previously reported that activation of Notch signaling by expression of the Notch1 intracellular domain (N1ICD) enhanced the contractile phenotype in human primary VSMC [29]

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—All experiments were conducted using primary human aortic VSMC (Lonza, Walkersville, MD) cultured in smooth muscle growth media (SmGM) (Lonza) between passages 5–7. VSMC at passage 5 were co-transfected with 2 ␮g of pGL3 (luciferase vector) and 3 ␮g of either wild type or CArG box mutant reporter A or reporter B before being immediately plated on Jag-1 or control Fc for 24 h. MiR-143/145 Is a Novel Jagged-1/Notch Target transduced cells were incubated with mouse monoclonal anti-V5 antibody at 1:1000 for a biological control, while N1ICD transduced cell lysates were incubated with a human IgG antibody (Sigma) at 1:1000 for an antibody control or anti-V5 for an experimental group. Cells were fixed in 4% paraformaldehyde, washed in 4N HCl for 30 min and stained using Alexa-Fluor 488 conjugated mouse anti-BrdU monoclonal antibody (Millipore) diluted to 1:200 for 1 h at room temperature.

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS