Abstract
Type I myotonic dystrophy (DM1) is caused by a triplet repeat expansion in the 3'-untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Pathogenesis is closely linked with production of a toxic RNA from the mutant allele, which interferes with function of several RNA-binding proteins, including CUGBP1. Here we show that expression of a mutant DMPK 3'-UTR containing 960 CUG repeats is sufficient to increase expression and stability of an mRNA encoding the potent proinflammatory cytokine, tumor necrosis factor (TNF). CUGBP1 specifically recognizes sequences within the TNF 3'-UTR that are dissimilar from its canonical UG-rich binding site. Depletion of CUGBP1 from mouse myoblasts results in increased abundance of TNF mRNA through stabilization of the transcript. Moreover, activation of the protein kinase C pathway by treatment with phorbol ester, which has been shown previously to result in CUGBP1 phosphorylation, also causes TNF mRNA stabilization. Our results suggest that the elevated serum TNF seen in DM1 patients may be derived from muscle where it is induced by expression of toxic DMPK RNA. Importantly, overexpression of this potent cytokine could contribute to the muscle wasting and insulin resistance that are characteristic of this debilitating disease.
Highlights
Type I myotonic dystrophy (DM1) is caused by a triplet repeat expansion in the 3-untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene
Expression of Mutant DMPK 3Ј-UTR Stabilizes tumor necrosis factor (TNF) mRNA in Myoblasts—We wished to test whether the altered cellular conditions induced by an expanded CUG repeat in the DMPK gene can result in changes in TNF expression
We chose to examine TNF mRNA abundance in C2C12 mouse myoblasts because of the following: (i) muscle cells express DMPK, and muscle is the main tissue affected in DM1 patients; (ii) expression of a mutant DMPK 3Ј-UTR containing expanded CUG repeats has been shown to result in impaired differentiation and accumulation of nuclear foci in this cell line [26, 27]; and (iii) C2C12 cells constitutively express TNF at low levels [28]
Summary
Cell Culture, Transfection, and Selection of Cell Lines—Murine C2C12 myoblasts (ATCC CRL1772) were maintained at 37 °C, 5% CO2 at or below 60% confluency in Dulbecco’s modified Eagle’s media containing 10% fetal bovine serum, penicillin (10 units/ml), and streptomycin (10 g/ml). The pellet was incubated with 100 l of buffer A containing 1% Triton X-100 for 30 min to solubilize the membrane proteins. The samples were centrifuged for 5 min at 12,000 ϫ g, and the resulting supernatants containing solubilized membranes were used in Western blots to detect phosphorylated PKC. Western Blot Analysis—For CUGBP1 Westerns, 40 g of whole protein extract prepared by lysis of cells in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1.0% deoxycholate, 1% Triton X-100, 1 mM EDTA, and 0.1% SDS) was separated on a 10% SDS-polyacrylamide gel and blotted to polyvinylidene difluoride membrane. For detection of phosphorylated PKC by Western blotting, 10 g of membrane fraction proteins prepared as described above were separated on a 10% SDS-polyacrylamide gel and blotted to polyvinylidene difluoride membrane. Anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies were employed as appropriate, and detection was by the Supersignal Pico West kit (Pierce) or by ECL plus (GE Healthcare) using either x-ray film or a Bio-Rad VersaDoc imager
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