Activation of p38 signaling increases utrophin A expression in skeletal muscle via the RNA-binding protein KSRP and inhibition of AU-rich element-mediated mRNA decay: implications for novel DMD therapeutics

Abstract

Several therapeutic approaches are currently being developed for Duchenne muscular dystrophy (DMD) including upregulating the levels of endogenous utrophin A in dystrophic fibers. Here, we examined the role of post-transcriptional mechanisms in controlling utrophin A expression in skeletal muscle. We show that activation of p38 leads to an increase in utrophin A independently of a transcriptional induction. Rather, p38 controls the levels of utrophin A mRNA by extending the half-life of transcripts via AU-rich elements (AREs). This mechanism critically depends on a decrease in the functional availability of KSRP, an RNA-binding protein known to promote decay of ARE-containing transcripts. In vitro and in vivo binding studies revealed that KSRP interacts with specific AREs located within the utrophin A 3' UTR. Electroporation experiments to knockdown KSRP led to an increase in utrophin A in wild-type and mdx mouse muscles. In pre-clinical studies, treatment of mdx mice with heparin, an activator of p38, causes a pronounced increase in utrophin A in diaphragm muscle fibers. Together, these studies identify a pathway that culminates in the post-transcriptional regulation of utrophin A through increases in mRNA stability. Furthermore, our results constitute proof-of-principle showing that pharmacological activation of p38 may prove beneficial as a novel therapeutic approach for DMD.

Anisomycin Activates Utrophin Upregulation Through a p38 Signaling Pathway

“…Anisomycin is known to activate the p38 MAPK pathway, which has been implicated in the regulation of the utrophin gene 19, 21. Two of the strongest utrophin mRNA‐inducing agents identified in the Cmap data, emetine and anisomycin, were p38 MAPK pathway activators ( Table 1).…”

“…Amirouche et al . 21 showed that p38‐mediated phosphorylation of the KH‐type splicing regulatory protein leads to increased stabilization of utrophin A mRNA that in turn caused an increase in utrophin protein (C2C12 cells). Our lab and others have established anisomycin as a potent p38 activator even at low doses 32, 33…”

Converging pathways involving microRNA-206 and the RNA-binding protein KSRP control post-transcriptionally utrophin A expression in skeletal muscle

“…The full-length 3′-UTR of the mouse utrophin A mRNA (∼2.1 kb) (39) and the full-length 3′-UTR of the mouse KSRP mRNA (∼1642 kb) were isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) and subcloned downstream of the Firefly luciferase gene driven by the cytomegalovirus (CMV) promoter (pGL4). Cells were cotransfected with 250 ng Firefly luciferase and with 50 ng of Renilla luciferase reporter (phRGtk-luc) to control for transfection efficiency.…”

“…The experimental protocols were all approved by the University of Ottawa Institutional Animal Care and User Committee. Electrotransfer of pEGP-mmu-miR-206 or pEGP-mmu-miR-Null (Cell Biolabs Inc., San Diego, CA, USA) into tibialis anterior (TA) muscles was performed as described elsewhere (39,40) while the animals were under anesthesia. Ten days after electroporation, the mice were euthanized and muscles were frozen in liquid nitrogen.…”

“…Several studies have clearly shown via transgenic technology (32–35) or pharmacological agents (36–38), the ability of increased utrophin A to functionally compensate for the lack of dystrophin in dystrophic fibers. In recent work, we identified another pathway also causing downregulation of utrophin A via post-transcriptional events (39). In this case, we showed using a variety of complementary approaches that K-homology splicing regulator protein (KSRP), a member of the AU-rich element binding protein (ARE-BP) family, binds directly to conserved AREs in the 3′-UTR of utrophin A mRNAs thereby increasing their destabilization and decay.…”