Reciprocal regulation of cilia and autophagy via the MTOR and proteasome pathways

Abstract

Abbreviations: 3-MA, 3-methyladenine; ANKS6, ankyrin repeat and sterile a motif domain containing 6; ATG/atg, autophagy-related; Baf, bafilomycin A 1 ; DAPI, 4 0 -6-diamidino-2-phenylindole; Ac-TUBA, acetylated-tubulin a; CQ, chloroquine; CF, confluence; FBS, fetal bovine serum; HK2, human kidney proximal tubular cells; IFT, intraflagellar transport; K D , knockdown; KAP3, kinesin family-associated protein 3; KIF3A/3B, kinesin family member 3A/3B; KO, knockout; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 b; LTA, lotus tetragonolobus agglutinin; MEF, mouse embryonic fibroblast; MTOR, mechanistic target of rapamycin; OFD1, oral-ficial-digital syndrome 1; PBS, phosphate-buffered saline; PKD, polycystic kidney disease; Rapa, rapamycin; RKRB, Krebs-Henseleit saline containing 25 mM NaHCO 3 ; RPS6KB1, ribosomal protein S6 kinase; 70kDa, polypeptide 1; SD, standard deviation.Primary cilium is an organelle that plays significant roles in a number of cellular functions ranging from cell mechanosensation, proliferation, and differentiation to apoptosis. Autophagy is an evolutionarily conserved cellular function in biology and indispensable for cellular homeostasis. Both cilia and autophagy have been linked to different types of genetic and acquired human diseases. Their interaction has been suggested very recently, but the underlying mechanisms are still not fully understood. We examined autophagy in cells with suppressed cilia and measured cilium length in autophagy-activated or -suppressed cells. It was found that autophagy was repressed in cells with short cilia. Further investigation showed that MTOR activation was enhanced in cilia-suppressed cells and the MTOR inhibitor rapamycin could largely reverse autophagy suppression. In human kidney proximal tubular cells (HK2), autophagy induction was associated with cilium elongation. Conversely, autophagy inhibition by 3-methyladenine (3-MA) and chloroquine (CQ) as well as bafilomycin A 1 (Baf) led to short cilia. Cilia were also shorter in cultured atg5-knockout (KO) cells and in atg7-KO kidney proximal tubular cells in mice. MG132, an inhibitor of the proteasome, could significantly restore cilium length in atg5-KO cells, being concomitant with the proteasome activity. Together, the results suggest that cilia and autophagy regulate reciprocally through the MTOR signaling pathway and ubiquitin-proteasome system.

Autophagy and Tubular Cell Death in the Kidney

“…For example, the transcription factor, signal transducer and activator of transcription 1 (STAT1), and MAPK8 (JUNK1) are present in cysts and can potentially mediate cell death by apoptosis or necrosis, and autophagy 127,128 . A recent study has further demonstrated a reciprocal regulation between cilia and autophagy 129 . Essentially, the cells with shorter cilia had lower levels of autophagy and, conversely, inhibition of autophagy reduced cilia growth.…”

Autophagy and primary cilia: dual interplay

“…Thus, recent studies in a paradigm of kidney epithelial cell differentiation have demonstrated a positive regulatory effect of ciliogenesis on basal autophagy [25]. In this context, the decreased basal autophagy resulting from a block in ciliogenesis can be restored by suppressing mTOR signaling, a well-characterized endogenous repressor of autophagy [25], whereas a similar intervention cannot overcome the defect in starvation-induced autophagy in fibroblasts lacking PC [15]. These divergences suggest that the mechanisms behind cilia-mediated autophagy are stimulus- and/or cell type dependent and could originate from a diversification in the receptor composition of the ciliary membrane according to the cell type or cell conditions.…”

“…Although the increase in autophagy observed with these drugs could be secondary to the longer PC or to their effect in other cellular process, the idea that autophagy could regulate PC length both at the level of growth or at the level of disassembly is attractive. Adding complexity to the autophagy/PC interplay, the consequences of autophagy inhibition on PC growth have been shown to be dependent in part on the ability of different cell types to engage proteasome degradation upon autophagy blockage, although the specific proteins targeted by the proteasome under these conditions are yet to be identified [25]. …”

Aldosterone controls primary cilium length and cell size in renal collecting duct principal cells

“…The mammalian target of rapamycin complex‐1 (mTORC1) was recently identified as a regulator of renal Na + and K + handling, and has been reported to be involved in the control of cilium growth . We therefore assessed the effects of aldosterone of mTORC1 activation.…”