Rationale Skeletal-muscle wasting with accompanying cachexia is a life threatening complication in congestive SIB 1893 heart failure (CHF). export. Mice lacking PKD1 in skeletal myocytes were resistant to Ang II-induced muscle wasting. Conclusion We propose that elevated Ang II serum concentrations as occur in CHF patients could activate the PKD1/HDAC5/TFEB/MuRF1 pathway to induce skeletal muscle wasting. expression in muscle is not well understood. To search for novel transcription factors involved in Ang II-induced expression we performed a cDNA expression screen. The basic helix-loop-helix (bHLH) transcription factor EB (TFEB) was identified as potent inducer. TFEB activity was regulated via the Ang II/protein kinase D1 (PKD1)/histone deacetylase-5 (HDAC5) signal transduction pathway. Inhibiting TFEB abolished Ang II-induced atrophy in vitro. We suggest that Ang II-induced skeletal muscle SIB 1893 wasting could be mediated at least in part by the PKD1/HDAC5/TFEB/MuRF1 pathway. METHODS An expanded Materials and Methods section is included in the Online Supplement. RESULTS To discover novel regulators of expression we performed a cDNA expression screen using a luciferase reporter controlled by the human expression by TFEB has not been reported. To confirm the results from the cDNA expression screen SIB 1893 we generated cDNA expression constructs of TFEB and tested if overexpression of TFEB activates Hs_expression (Figure 1A). Because MuRF1 is primarily contained in skeletal muscle and heart14 whereas TFEB is ubiquitously expressed 21 22 quantitative real-time PCR (qRT-PCR) was used to test if was also expressed in striated muscle. To investigate whether is expressed in a fiber-type specific manner we quantitated its expression in muscle primarily containing fast twitch/type II fibers (expression with expression in liver and spleen both organs were included into the analysis. Our data showed that expression in skeletal muscle and the heart is similar with its expression in liver where the function is well described.18 No evidence was found for fibre type related differences in expression. However because TFEB was contained in all skeletal muscle and all parts of the heart analyzed TFEB could contribute to transcriptional regulation of in muscles (Figure 1B). To test if TFEB increases endogenous MuRF1 mRNA expression and protein content in myocytes we used qRT-PCR and Western blot analysis of lysates from C2C12 myoblasts transfected with cDNA expression plasmids encoding TFEB. Overexpressed TFEB increased endogenous MuRF1 mRNA expression (Figure 1C) and protein content (Figure 1D) in these cells. In addition to MuRF1 we analyzed the effect of TFEB on and expression homologous MuRF family members that are also restricted to striated muscles. In contrast to and expression (Figure 1C). Loss-of-function experiments were performed to investigate if TFEB is required for basal expression in C2C12 myoblasts. The siRNA mediated TFEB knockdown led to reduced MuRF1 mRNA expression and protein content in C2C12 myoblasts in vitro (Figure 1E and F). Figure 1 A cDNA expression screen identified the transcription factor EB (TFEB) as activator of the human MuRF1 promoter To uncover cis-regulatory elements in the expression site-directed mutagenesis was used to mutate these E-box SIB 1893 motifs from CANNTG to ATNNTG known to inhibit E-box functionality 23 in the ?543 bp Hs_expression whereas mutation of E-box 2 had only minor effects (Figure 2C). These data indicate that E-box 1 and 3 in the human expression. We next used chromatin-immunoprecipitation followed by qRT-PCR (ChIP-PCR) to elucidate if TFEB binds to the conserved E-box motifs E-box 1 2 and 3 in the endogenous expression via conserved E-box elements Although the function and Goat polyclonal to IgG (H+L). regulation of TFEB in non-muscle cells is well described 18 25 its function in myocytes is not well understood. We next performed immunocytochemistry and immunofluorescence microscopy to investigate subcellular localization of TFEB in C2C12 myoblasts. We generated cDNA expression plasmids encoding wild-type or mutant TFEB (Figure 3A) and transfected them into C2C12 cells. Overexpressed wild-type TFEB was localized in the nucleus cytosol and vesicular structures (Figure 3B Figure.