The mechanism by which this family of miRNAs regulates VSMCs is dependent upon their mRNA targets. A bioinformatics approach incorporating sequence matching and mRNA secondary structure to predict mRNA targets (K. Ivey & D. Srivastava, unpublished; also see Methods) revealed multiple highly conserved binding sites for miR-143 in the 3′ UTR of Elk-1
and for miR-145 in the 3′ UTR of Myocd
(Supplementary Fig. 6a
). Growth signals repress smooth muscle gene expression by displacing Myocd from SRF with Elk-1, a ternary complex factor that acts as a myogenic repressor and an activator of VSMC proliferation22
. In this system, SRF serves as a platform for myogenic coactivators or corepressors that compete for a common docking site, thereby mediating VSMC phenotypic switching.
To determine whether Elk-1 and Myocd are direct targets of miR-143 or miR-145, respectively, we cloned the 3′ UTR of Elk-1
into the 3′ UTR of a CMV-driven luciferase reporter. In the presence of the Elk-1
3′ UTR, miR-143 repressed luciferase activity; this repression was diminished upon mutation of one of the two miR-143 binding sites (). The addition of an antagomiR to inhibit miR-143 in the A10 rat aortic VSMC line resulted in upregulation of Elk-1 protein, but not mRNA, consistent with translational repression of Elk-1 by miR-143 (, Supplementary Fig. 6b
). Furthermore, inhibition of miR-143 caused a doubling of the proliferative rate of VSMCs, demonstrating miR-143’s function in negatively regulating VSMC proliferation ().
miR-143 and miR-145 target a network of factors to promote VSMC differentiation and repress proliferation
The presence of putative miR-145 binding sites in the Myocd 3′ UTR seemed counter to the observed effects of miR-145 in potentiating Myocd’s reprogramming effects. When we cloned the Myocd
3′ UTR into a CMV-driven luciferase vector and introduced this into Cos cells, the constitutive luciferase activity decreased greater than 100-fold. Surprisingly, introduction of miR-145, but not miR-143, with the luciferase vector in Cos cells resulted in relief of the repression and an ~150-fold increase in luciferase activity compared to the CMV-luciferase-Myocd
3′ UTR-luciferase vector alone (). The increase in luciferase activity was largely lost upon mutation of the miR-145 binding site in the Myocd
3′ UTR (). In contrast, introduction of the same CMV-luciferase-Myocd
3′ UTR reporter did not cause a decrease in baseline luciferase activity in 293T cells. However, even in these cells, miR-145 consistently increased luciferase activity by ~1.5 fold (data not shown). Although antibodies to detect endogenous Myocd levels by western blot are not available, these findings are consistent with the recent observation that miRNAs can act as translational activators or repressors based upon the state of the cell cycle6
. While the mechanism for this remains unclear, it will be interesting to determine if miR-145 prevents binding of a repressive RNA-binding protein enriched in Cos cells.
While miR-145 may result in increased Myocd protein, its effects in potentiating Myocd-induced reprogramming of fibroblasts did not require presence of its binding site in Myocd’s 3′ UTR. miR-145’s potentiating effects could, however, be through effects on translation of endogenous Myocd mRNAs induced by the transfected Myocd protein; alternatively, miR-145 may promote differentiation through targets independent of Myocd. Indeed, our bioinformatics approach identified potential miR-145 binding sites in several other positive regulators of smooth muscle proliferation, including Kruppel-like factor 4 (Klf4) and Calmodulin kinase II-delta (CamkIIδ). Klf4 is a transcription factor involved in pluripotency30
that is also rapidly induced in post-injury proliferating VSMCs, where it interacts with enhancers in smooth muscle growth genes, inhibits smooth muscle differentiation genes, and represses Myocd expression31
. The miR-145 binding site in the 3′ UTR of Klf4
specifically mediated miR-145-dependent repression in luciferase assays ( and Supplementary Fig. 6c
). Furthermore, knockdown of miR-145 in rat A10 VSMCs resulted in an increase in Klf4 protein levels, but no change in Klf4
mRNA levels (, Supplementary Fig. 6d
). Similarly, a putative binding site in CamkII-δ, involved in multiple events including neointimal proliferation32,33
, was validated as a miR-145-repressed target by luciferase and western analysis in VSMCs ( and Supplementary Fig. 6e
). Numerous predicted targets for both miRNAs that were not validated in luciferase assays are shown (Supplementary Fig. 6f, g
Consistent with miR-145 repression of genes involved in VSMC proliferation, introduction of miR-145 was sufficient to suppress the proliferative response normally induced by platelet-derived growth factor (Pdgf-β) in cultured VSMCs (). In addition, lentiviral-mediated introduction of miR-145 into ligated mouse carotid arteries in vivo
increased expression of markers of VSMC differentiation (e.g., Calponin and Sm-α-actin), as well as Myocd, compared to control-infected injured carotid arteries (Supplementary Fig. 7
). These findings suggest that miR-145 may promote VSMC differentiation by directly repressing numerous transcription factors that promote the proliferative state while stabilizing factors that promote the differentiated state of VSMCs ().
Model of miR-143 and miR-145 regulation of smooth muscle cell proliferation and differentiation