Abstract

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-β/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-β-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-β-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo, BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development.

Highlights

  • The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process

  • transforming growth factor (TGF)-␤ is a central regulator for SMC fate determination during vascular development [14]

  • To determine whether brain cytoplasmic RNA 1 (BC1) is involved in SMC differentiation, we treated 10T1/2 cells with TGF-␤ to induce SMC differentiation in vitro [13, 14]

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Summary

Results

TGF-␤ is a central regulator for SMC fate determination during vascular development [14]. Smad activity via its selective inhibitor SIS3 impeded the expression of SMC markers that was enhanced because of knockdown of BC1 (Fig. 2, C–F, and Fig. S3, B and C) These data suggested that BC1 inhibited TGF-␤–induced SMC differentiation by suppressing Smad signaling. Smad was detected in the BC1–Smad complex pulled down with avidin beads but not in the control complex, indicating that BC1 directly and bound to Smad (Fig. 4, E and F) These data indicated that BC1 regulated Smad nuclear translocation/activity via physical binding to its protein. RSBE mutations restored the protein expression of SMC markers (␣SMA, CNN1, and SM22␣) that was attenuated by BC1 (Fig. 6, B and C) These data indicated that the rSBEs were essential for BC1 to inhibit TGF-␤–induced SMC differentiation of 10T1/2 cells. These data indicated that BC1 adversely impacted the normal development of the vascular system

Discussion
Reagents and cell culture
Construction of adenovirus
Western blot analysis
Cell morphology observation
Cell fractionation
ChIP assay
Luciferase reporter assay
In vitro transcription
RIP assay
Viral inoculation of mouse embryos
Histomorphometric analysis
Statistical analysis
Full Text
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