The long non-coding RNA GAS5 regulates transforming growth factor β (TGF-β)–induced smooth muscle cell differentiation via RNA Smad–binding elements

Abstract

Smooth muscle cell (SMC) differentiation is essential for vascular development, and TGF-β signaling plays a critical role in this process. Although long non-coding RNAs (lncRNAs) regulate various cellular events, their functions in SMC differentiation remain largely unknown. Here, we demonstrate that the lncRNA growth arrest-specific 5 (GAS5) suppresses TGF-β/Smad3 signaling in smooth muscle cell differentiation of mesenchymal progenitor cells. We found that forced expression of GAS5 blocked, but knockdown of GAS5 increased, the expression of SMC contractile proteins. Mechanistically, GAS5 competitively bound Smad3 protein via multiple RNA Smad-binding elements (rSBEs), which prevented Smad3 from binding to SBE DNA in TGF-β-responsive SMC gene promoters, resulting in suppression of SMC marker gene transcription and, consequently, in inhibition of TGF-β/Smad3-mediated SMC differentiation. Importantly, other lncRNAs or artificially synthesized RNA molecules that contained rSBEs also effectively inhibited TGF-β/Smad3 signaling, suggesting that lncRNA-rSBE may be a general mechanism used by cells to fine-tune Smad3 activity in both basal and TGF-β-stimulated states. Taken together, our results have uncovered an lncRNA-based mechanism that modulates TGF-β/Smad3 signaling during SMC differentiation.

Glycoprotein M6B Interacts with TβRI to Activate TGF-β-Smad2/3 Signaling and Promote Smooth Muscle Cell Differentiation

“…Therefore, characterizing the regulation of vascular SMC differentiation is essential for both the prevention and treatment of these diseases. Although recent studies have improved the current understanding of SMC differentiation and cardiovascular system development [2][3][4][5][6][7][8][9][10][11][12], the molecular mechanisms governing SMC differentiation from pluripotent stem cells remain largely unknown.…”

LncRNA GAS5 attenuates fibroblast activation through inhibiting Smad3 signaling

“…LncRNA normally functions as a suppressive protein sponge for transcription factors [22,24]. lncRNA can also serve as a scaffold RNA which brings spatial proximity to different components thus facilitates protein complex formation and enhances their functions [48][49][50].…”

“…RNA immunoprecipitation (RIP) assay: RIP assay was performed as described [24]. Cells at 80-90% confluence in 15 cm 2 culture dishes were fixed with 1% Paraformaldehyde (PFA) and lysed in FA lysis buffer (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, pH7.5) containing 40U/ml RNAse inhibitor (Sigma-Aldrich, St. Louis, MO) and 1X Halt™ Protease Inhibitor Cocktail (Thermofisher Scientific, Grand Island, NY).…”

“…Growth Arrest Specific 5 (GAS5) is a well-known lncRNA that suppresses cell proliferation and induces apoptosis [22,23]. Our previous studies have identified GAS5 as a regulator in both Smad3-dependent cell differentiation and p53-dependent cell survival [24,25]. Since TGF-β signaling regulates both Smad-dependent fibroblast-myofibroblast transition and fibroblast proliferation during fibrosis, we hypothesized that GAS5 may be involved in TGF-β-induced fibroblast activation and fibrosis.…”

Long noncoding RNAs in key cellular processes involved in aortic aneurysms

“…LncRNA GAS5 (Growth arrest-specific 5), a critical regulator in hypertension-induced vascular remodeling [56], is another potential lncRNA that has been linked to AAA. LncRNA GAS5 can negatively regulate TGFβ/SMAD3 (SMAD family member 3) signaling, known to play a critical role in VSMC differentiation [57]. SMAD3 protein was Fig.…”