Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells

Abstract

Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA-binding protein, which is highly expressed in skeletal muscle. Abnormally expanded CUG-repeats in the DMPK gene cause myotonic dystrophy type 1 (DM1) by sequestration of MBNL1 to nuclear RNA foci and by upregulation of another RNAbinding protein, CUG-binding protein 1 (CUGBP1). We previously reported that a nonsteroidal antiinflammatory drug (NSAID), phenylbutazone, upregulates MBNL1 expression in DM1 mouse model by demethylation of MeR2, an enhancer element in Mbnl1 intron 1. NSAIDs inhibit cyclooxygenase (COX), which is comprised of COX-1 and COX-2 isoforms. In this study, we screened 29 NSAIDs in C2C12 myoblasts, and found that 13 NSAIDs enhanced Mbnl1 expression, where COX-1-selective NSAIDs upregulated Mbnl1 more than COX-2-selective NSAIDs. Consistently, knockdown of COX-1, but not of COX-2, upregulated MBNL1 expression in C2C12 myoblasts and myotubes, as well as in myotubes differentiated from DM1 patient-derived induced pluripotent stem cells (iPSCs). Luciferase assay showed that COX-1-knockdown augmented the MeR2 enhancer activity. Furthermore, bisulfite sequencing analysis demonstrated that COX-1-knockdown suppressed methylation of MeR2. These results suggest that COX-1 inhibition upregulates Mbnl1 transcription through demethylation of the MeR2 enhancer. Taken together, our study provides new insights into the transcriptional regulation of Mbnl1 by the COX-1-mediated pathway. Muscleblind-like (MBNL) is a multifunctional RNA binding protein that modulates diverse RNA metabolisms, including splicing, polyadenylation, stability, and localization of mRNA 1-3. There are three isoforms of MBNL in mammals (MBNL1, MBNL2, and MBNL3), in which MBNL1 is the most ubiquitously expressed isoform and is highly expressed in skeletal muscle 4. Expression of MBNL1 is elevated in skeletal muscle during myogenic differentiation 5. Knockout of Mbnl1 results in pathology reminiscent of myotonic dystrophy type 1 (DM1) with abnormal terminal muscle differentiation 6-8. DM1 is the most common form of muscular dystrophies in adults, and is caused by abnormal expansion of CTG repeats in the 3′ untranslated region of the myotonic dystrophy protein kinase (DMPK) gene on chromosome 19 9,10. The transcribed CUG repeats from the CTG repeats make abnormal RNA foci in the nucleus, leading to sequestration of MBNL1 and upregulation of another RNA binding protein, CUG-binding protein 1 (CUGBP1) 11. Downregulation of MBNL1 availability leads to aberrant regulation of alternative splicing of hundreds of genes, which causes various manifestations such as myotonia, progressive muscle wasting, cataracts, insulin resistance, cardiac arrhythmia, and intellectual deficits 8,12,13. Several therapeutic strategies for DM1 are currently under investigation 14-16. Induction of MBNL1 expression is one of the promising therapies for DM1. Overexpression of Mbnl1 in skeletal muscle with an adeno-associated

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