miR-145 and miR-143 regulate smooth muscle cell fate and plasticity

Abstract

MicroRNAs (miRNAs) are regulators of myriad cellular events, but evidence for a single miRNA that can efficiently differentiate multipotent stem cells into a specific lineage or regulate direct reprogramming of cells into an alternative cell fate has been elusive. Here we show that miR-145 and miR-143 are co-transcribed in multipotent murine cardiac progenitors before becoming localized to smooth muscle cells, including neural crest stem-cell-derived vascular smooth muscle cells. miR-145 and miR-143 were direct transcriptional targets of serum response factor, myocardin and Nkx2-5 (NK2 transcription factor related, locus 5) and were downregulated in injured or atherosclerotic vessels containing proliferating, less differentiated smooth muscle cells. miR-145 was necessary for myocardin-induced reprogramming of adult fibroblasts into smooth muscle cells and sufficient to induce differentiation of multipotent neural crest stem cells into vascular smooth muscle. Furthermore, miR-145 and miR-143 cooperatively targeted a network of transcription factors, including Klf4 (Kruppel-like factor 4), myocardin and Elk-1 (ELK1, member of ETS oncogene family), to promote differentiation and repress proliferation of smooth muscle cells. These findings demonstrate that miR-145 can direct the smooth muscle fate and that miR-145 and miR-143 function to regulate the quiescent versus proliferative phenotype of smooth muscle cells.

Figure 1. miR-143 and miR-145 are cardiac and smooth muscle-specific miRNAs

(a–c) Whole mounts showing cardiac-specific β-gal activity in transgenic mouse embryos with 4.2 kb enhancer-lacZ construct (Supplementary Fig. 2a) at indicated time points. (d) Transverse section of (c) showing β-gal expression in pharyngeal mesoderm (pm), pharyngeal endoderm (pe), dorsal aorta (da), myocardium (mc), endocardium (ec). (e,f) β-gal expression in embryonic day (E) 15.5 (e) or post-natal (P21) (f) heart. Ao, aorta; pa, pulmonary artery. (g,h) Transverse sections of (f); co, coronary artery. (i,j) Section in-situ hybridization of miR-145 in P21 heart section. (h) and (j) represent higher magnification of boxed areas. (pcm, precardiac mesoderm; ht, heart; h, head; ot, outflow tract; rv, right ventricle; lv, left ventricle; cv, cardinal vein; ra, right atrium; la, left atrium).

Figure 2. SRF and Nkx2.5 directly regulate cardiac and smooth muscle expression of miR-143 and miR-145

(a–e) Lateral (a, b, c, e) or frontal (d) cardiac views of transgenic embryos containing indicated lacZ constructs and stained for β-Gal activity. (f) Fold-activation of luciferase activity directed by introduction of SRF, Myocd or Nkx2.5 expression vectors with the miR-143/145 enhancer in Cos cells. All changes were statistically significant (n=5). (g) miR-143 and miR-145 expression levels assessed by qPCR day 10 embyroid bodies (EBs) of indicated genotypes. (h) qPCR of miR-143 and miR-145 in Nkx2.5^+/− and Nkx2.5^−/− E9.5 hearts relative to WT. (i,j) qPCR of miRNAs in injured vessels (i) or atherosclerotic lesions (j) compared to normal arterial expression. Results shown in (f–j) are the average of three experiments. (ot, outflow tract; ra, right atrium; lv, left ventricle; rv, right ventricle; la, left atrium; dorsal aorta). *, p<0.05. Error bars indicate SD.

Figure 3. miR-145 directs vascular smooth muscle cell fate

(a) Immunocytochemistry of 10T1/2 fibroblasts using smooth muscle (Sm) α-actin antibodies (red) under conditions indicated; nuclear stain, Dapi (blue). (b) Quantification of Sm-α-actin positive cells (n=6). (c) qPCR of Sm gene expression in fibroblasts transfected with Myocd with or without anti-miR-145 or (d) fibroblasts transfected with 50 ng Myocd with or without miR-145 (n=5). (e) Western blot of calponin and Sm-α-actin. (f) Immunocytochemistry of neural crest stem cells (Joma1.3 NCCs) with or without miR-145 using antibodies indicated (green); tamoxifen (4OHT) was removed to allow differentiation. Quantification of percent Sm-α-actin + cells relative to total Dapi+ nuclei (blue) (n=6). (g) qPCR of Sm gene expression in NCCs with miR-145 expression (n=5); p75 is a marker of undifferentiated neural crest cells. (h) Western blot of Sm-α-actin and calponin. (i) Calcium flux [Ca²⁺]_i in SMCs derived from NCCs or rat aortic SMCs in response to endothelin-1 (Et-1) stimulation at 30 sec. Error bars indicating SD. *, p<0.05.

Figure 4. miR-143 and miR-145 target a network of factors to promote VSMC differentiation and repress proliferation

(a) Luciferase activity in Cos cells upon introduction of Elk-1 3′ UTR or mutant 3′ UTR (mut) downstream of a CMV-driven luciferase reporter with indicated miRNAs (n=5). (b) Elk-1 protein in cell lysates from A10 VSMCs transfected with a scrambled (scr) miRNA or anti-miR-143 or -miR-145 assessed by western blot. (c) Proliferation of VSMCs upon inhibition of miR-143 or miR-145 relative to control (5%FBS) (n=5). (d) Luciferase activity in Cos cells with Myocd 3′ UTR sequences with indicated miRNAs (n=5). The Myocd binding site (BS) was mutated in the context of a 4x concatemer. (e) Luciferase activity with wt or mutated Klf4-3′ UTR upon introduction of indicated miRNAs (n=5). (f) Analysis of Klf4 protein in cell lysates from A10 cells transfected with indicated anti-miRs by western blot. (g) Luciferase activity of wt or mutated CamkII-δ3′ UTR (n=5). (h) Western analysis for CamkII-δ protein in A10 cells transfected with scr miRNA or anti-miR-145. (i) Proliferation of VSMCs relative to control (n=5). Error bars indicate SD. Densitometry calculation performed by Image J. *, p<0.05.

Figure 5. Model of miR-143 and miR-145 regulation of smooth muscle cell proliferation and differentiation

miR-143 and miR-145 are positively regulated by SRF and function to repress multiple factors that normally promote the less differentiated, more proliferative smooth muscle phenotype (purple). These include Klf4, which also represses Myocd. miR-145 has a positive effect on Myocd activity to concurrently promote the more differentiated smooth muscle phenotype (pink), thereby also functioning to reinforce its own expression. Effects of miR-145 and miR-143 converge on SRF-dependent transcription by regulation of co-activators and co-repressors to dictate the proliferative or differentiated phenotype of VSMCs. Positive regulation of Myocd by miR-145 results in reinforcement of miR-145 and miR-143 expression and the differentiated phenotype. Dashed lines indicate indirect effects.