Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Abstract

These authors contributed equally to this publication.Keywords: autophagy, bafilomycin, bone, chondrocyte, lysosome, MTOR, MTORC1Abbreviations: 3-MA, 3-methyladenine; ACAN, aggrecan; ACP5/TRAP, acid phosphatase 5, tartrate-resistant; ACTB, actin, b; ALPL, alkaline phosphatase, liver/bone/kindey; ATG, autophagy related; ATG5cKO, ATG5 conditional knockout in chondrocytes; BC, avidin-biotin complex; Baf, bafilomycin A 1 ; bGP, b-glycerophosphate; BrdU, bromodeoxyuridine; C5.18 cells, RCJ 3.1C5.18 rat mesenchymal cell line; COL2A1, collagen, type II, a 1; COL10A1, collagen, type X, a 1; CQ, chloroquine; DAPI, 4 0 , 6-diamidino-2-phenylindole, dihydrochloride; Dox, doxycycline; EIF4EBP1, eukaryotic translation initiation factor 4E binding protein 1; FBS, fetal bovine serum; GAG, glycosaminoglycan; IGF1, insulin-like growth factor 1 (somatomedin C); LAMP2, lysosomalassociated membrane protein 2; MEFs, mouse embryonic fibroblasts; MAP1LC3A, microtubule-associated protein 1 light chain 3 a;MTOR, mechanistic target of rapamycin (serine/threonine kinase); MTORC, MTOR complex; p-, phosphorylated-; PFA, paraformaldehyde; RPS6, ribosomal protein S6; RPS6KB1/p70(S6K)-a/PS6K/S6K1, ribosomal protein S6 kinase, 70kDa, polypeptide 1; RPTOR, regulatory associated protein of MTOR, complex 1; SGK1, serum/glucocorticoid regulated kinase 1; TSC, tuberous sclerosis complex; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; v-ATPase, vacuolar-type H C -ATPase.Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H C -ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A 1 or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy

“…Unexpectedly, in this work we observed that lysosomal storage enhances, rather than inhibits, mTORC1 signaling in LSD chondrocytes. Similar observations were recapitulated pharmacologically by others (36), suggesting a chondrocyte-specific response of mTORC1 to lysosomal dysfunction.…”

The vacuolar H+ ATPase V0 subunit D2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation

“…A recent study of the role of V-ATPase in long bone growth yielded results that appear to contradict our observations of a role for this enzyme in expression of hypertrophy-associated genes (Newton et al, 2015). In this study, organ cultures of metatarsal bones from 3.5-dayold mice provided the source of chondrocytes; bafilomycin A1 was found to cause an increase in the length of the bones, the size of hypertrophic chondrocytes and the length of the zone positive for collagen type X.…”

Beclin-1- mediated autophagy may be involved in the elderly cognitive and affective disorders in streptozotocin-induced diabetic mice

“…The protein of p62, a protein associated with autophagosomes and degraded in the autolysosome, is commonly regarded as autophagy substrate protein [45, 46], as a mainly negative regulator of autophagy, activated mTORC1 inhibits autophagy induction through blocking of the initial inducer of autophagy [47, 48]. The p62 is the scaffold protein implicated in selective autophagy which is induced by the stress of cells.…”