Endocrine Abstracts

Glucocorticoid excess-induced muscle wasting is characterised by activation of the unfolded protein response (UPR) via its three canonical arms (PERK, IRE1, and ATF6) yet underlying causal mechanism remains unclear. Here we examine how glucocorticoids regulate the endoplasmic reticulum (ER) UPR, aiming to mitigate adverse effects of glucocorticoids. We previously identified links between glucocorticoids and ADP-ribosylation, an NAD⁺-dependent modification involving mono-(MAR) or poly-(PAR) ADP-ribose. In terminally differentiated murine muscle cells(C2C12s), we show dexamethasone(1μM) inhibits PAR (0.61-fold change ± 0.13 sem, P < 0.01; n = 8) with a corresponding increase in NAD⁺(20% increase, P < 0.02; n = 8). Conversely, Mono-ADP-ribose increases (1.34FC ± 0.20, P < 0.001; n = 6) under the same conditions. To understand whether increment of MAR is from breakdown of PAR, we used a PAR gluco-hydrolyser inhibitor and found MAR upregulation is unaffected by inhibition of PAR degradation. This demonstrates MAR is not a breakdown product of PAR, but a distinct signaling modification. Analysis of subcellular fractionations of muscle cells reveals PAR is decreased in ER, mitochondria, and nuclei with dexamethasone (0.75FC ± 0.08), (0.85FC ± 0.07), (0.68FC ± 0.1), respectively. MAR is significantly elevated in all organelles in response to dexamethasone (nucleus: 1.61FC ± 0.30 ; mitochondria: 2.37FC ± 0.68; microsomes: 3.36FC ± 1.18; cytoplasm: 3.28FC ± 1.14, P < 0.05; n = 3). These data highlight the compartmental-specific signaling of this post-translational modification. Based on dexamethasone-induced elevation of MAR and UPR-stress markers in the ER, we examined transcriptional responses of PARPs1-16 to glucocorticoids finding PARP16 to be most upregulated (1.82FC ± 0.18, P < 0.05; n = 3). Crucially, inhibiting PARP16 reverses dexamethasone-induced activation of all three arms of UPR (p-eif2a/eif2a: 0.3FC ± 0.31; sXBP1: 0.15FC ± 0.13; ATF6: 0.76FC ± 0.23, P < 0.05, n = 3), and MAR. Our findings uncover compartment-specific regulation of ADP-ribosylation by glucocorticoids. Importantly, we define PARP16–ADP-ribosylation–Unfolded Protein Response axis as a key pathway driving muscle atrophy.