Inactivation of GSK-3β potentiates skeletal muscle PGC-1α signaling via TFEB

Cellular and molecular mechanisms (English)

Wessel Theeuwes
Maastricht University Medical Center+, NUTRIM School of Nutrition and Translational Research in Metabolism Department of Respiratory Medicine
BEKIJK PROGRAMMA
 
09 april 15:12 - 15:30 (Markgraaf 2)
Background: Aberrations in oxidative metabolism of the skeletal musculature is a debilitating feature often observed in chronic diseases, such as chronic obstructive pulmonary disease (COPD). Muscle mitochondrial biogenesis and oxidative metabolism are tightly controlled by the peroxisome proliferator-activated receptor-γ co-activator 1 (PGC-1) signaling network, which has been shown to be disturbed in the musculature of COPD patients. Our group previously showed potently increased PGC-1α abundance and oxidative metabolism following inactivation of glycogen synthase kinase (GSK)-3β in skeletal muscle cells. How inactivation of GSK-3β upregulates PGC-1α remains unclear. Several transcription factors have been reported to influence the transcriptional regulation of Pgc-1α, amongst others estrogen-related receptors (ERRs), myocyte enhancer factor (MEF)2 and transcription factor EB (TFEB).

Research Question: How does inactivation of GSK-3β mediate increased Pgc-1α mRNA expression in skeletal muscle cells?

Methods: GSK-3β was inactivated genetically or pharmacologically in fully differentiated C2C12 muscle cells, and associations between activity levels of key transcriptional regulators of PGC-1α, and PGC-1α gene expression were investigated. In addition, chromatin accessibility of the PGC-1α promoter was measured. Subsequently, candidate regulators were modulated using pharmacological or genetic approaches to address their requirement for increased Pgc-1α mRNA abundance and promoter activity in response to GSK-3β inhibition.

Results: Knock-down of GSK-3β did not alter alterations in chromatin accessibility of the PGC-1α promoter in C2C12 myotubes, suggesting a transcriptionally controlled mecahnism. Time-dependently, inactivation of GSK-3β increased Pgc-1α gene expression in fully differentiated C2C12 myotubes (10-fold), which was accompanied by increased gene expression of the ERR-branch of the PGC-1α-signaling cascade (Errα, ERRγ and the ERR target gene Perm1). However, pharmacological or genetic inhibition of ERRα and ERRγ did not prevent the induction of Pgc-1α mRNA levels mediated by inactivation of GSK-3β, indicating that ERRα and ERRγ are not required for induced Pgc-1α gene expression upon inactivation of GSK-3β. In addition, transcriptional activity of MEF2 remained unaltered upon inactivation of GSK-3β, suggesting that increased Pgc-1α mRNA levels upon inactivation of GSK-3β are not mediated by MEF2. Western blot analysis revealed decreased phosphorylation of TFEB upon pharmacological inactivation of GSK-3β, indicating TFEB activation. Interestingly, knock-down of TFEB completely prevented increases in Pgc-1α gene expression, PGC-1α promoter activity and PGC-1α protein abundance induced by inactivation of GSK-3β. Furthermore, mutation of a specific TFEB DNA binding site on the PGC-1α promoter blocked promoter activation upon pharmacological inhibition of GSK-3β.

Conclusion: We show that TFEB is required for increased Pgc-1α gene expression upon inactivation of GSK-3β. We show a novel interaction between inactivation of the GSK-3β protein, well-known to be involved in muscle mass regulation, and transcriptional regulation of Pgc-1α via TFEB in muscle cells. This highlights an intricate link between pathways involved in regulation of skeletal muscle energy production and those controlling muscle mass.

This work was financially supported by a grant from the Lung Foundation Netherlands (5.2.13.067).
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