Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells |
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Authors: | Myoung Sup Shim April Nettesheim Angela Dixon Paloma B. Liton |
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Affiliation: | aDepartment of Ophthalmology, Duke Eye Center, Duke University, Durham, NC, 27705 |
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Abstract: | Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.The trabecular meshwork (TM) is a pressure-sensitive tissue located in the anterior segment of the eye and key regulator of intraocular pressure (IOP). Malfunction of this tissue results in improper drainage of aqueous humor outflow, leading to ocular hypertension, the major risk factor for developing glaucoma (1–3). The TM consists of an irregular lattice of collagen beams lined by TM endothelial-like cells, followed by a zone of loose connective tissue containing TM cells, through which aqueous humor must pass before leaving the eye. Changes in pressure gradients and fluid flow associated with eye movement, circadian rhythm, or the ocular pulse cause small and high variations in IOP, which are translated in continuous cycles of tissue deformation and relaxation. Cells in the TM are known to be able to sense these deformations as mechanical forces and respond to them by eliciting a variety of responses, including reorganization of actin cytoskeleton, changes in gene expression, secretion of cytokines, modulation of matrix metalloproteinases, and extracellular-matrix remodeling (reviewed in ref. 4). It is believed that these mechanoresponses are critical regulators of IOP homeostasis; however, the mechanosensors and the downstream mechanotransduction signaling in TM have still not been characterized.Our laboratory has identified the activation of macroautophagy (hereafter autophagy) as one of the responses elicited in TM cells following application of static or cyclic mechanical stretch (CMS) (5–7). Activation of autophagy was also observed quickly after pressure elevation in porcine perfused eyes (5), which prompted us to propose autophagy as a crucial physiological response to adapt to mechanical forces and maintain cellular homeostasis. The exact roles of autophagy in TM cells and tissue function are still under investigation. Most recently, we have shown the CMS-induced translocation of the autophagy marker LC3 (microtubule-associated protein 1 light chain 3) to the nuclear compartment, where it associates with the nucleolus and interacts with the ribophagy receptor NUFIP1 (nuclear FMR1 interacting protein 1), suggesting a potential role of CMS-induced autophagy in surveillance of the nucleolus activity (6). Furthermore, we have also recently provided direct evidence demonstrating autophagy as a regulator of TGF-β/SMAD-induced fibrogenesis in TM cells (8).Autophagy is a fundamental process for degradation or recycling of intracellular components, which is essential to maintain cellular homeostasis. Autophagy occurs constitutively at basal levels, but it is quickly activated upon several types of stress, such as nutrient depletion, pathogen infections, drug treatment, accumulation of aggregated proteins and damaged organelles, and mechanical stress (7, 9–11). The molecular mechanisms by which cells recognize stress and regulate autophagic activity are very complex and differ based on the stimuli. A variety of components, for example, receptor tyrosine kinases, second messengers (Ca2+ or cAMP [cyclic adenosine monophosphate]), protein kinases, and downstream autophagy-related (ATG) complexes, participate in the regulation of autophagy (10). Among them, the best characterized is MTOR (mechanistic target of rapamycin kinase), a negative regulator of autophagy (10, 12, 13). Although MTOR acts as a core sensor in autophagic regulation, numerous studies have shown the MTOR-independent autophagy activation upon different stresses (10). Indeed, our own study and that of King et al. showed that the induction of autophagy in response to mechanical stress occurs in an MTOR-independent manner (5, 14). The mechanosensor and the downstream signaling pathway responsible for the activation of autophagy in response to stretching have still not been identified.Primary cilium (PC) is a nonmotile cell-surface projection found almost ubiquitously in vertebrate cells, which acts as a cellular antenna that senses a wide variety of signals, including chemical and mechanical stimuli (15–19). PC plays a critical role in smell, sight, and mechanosensation. PC defects are associated with a number of human diseases termed ciliopathies. The most common feature in patients affected with ciliopathies include visual dysfunction (16). In particular, Lowe syndrome patients often develop ocular hypertension and glaucoma (20). Structurally, the PC consists of a microtubule-based core structure, called axoneme, and a basal body, which is a derivative of the centriole of centrosome from which axoneme is extended and surrounded by ciliary membrane (21). The ciliary membrane is a specialized domain extension of the plasma membrane enriched on signaling receptors and channels, including hedgehog (Hh) and Ca2+ pathways, which enables the PC to function as a signaling hub (16, 22, 23). Cargo trafficking into and out of the cilium is mediated by a specialized form of vesicle trafficking, named intraflagellar transport (IFT), that is composed of a multiprotein complex (16, 23).Recent studies have demonstrated the reciprocal relationship between PC and autophagy. Autophagy has been shown to both positively and negatively regulate ciliogenesis. Under nutrient-rich conditions, basal autophagy inhibits cilia growth by limiting trafficking of PC components to the basal body through direct degradation of IFT20 (24). In contrast, nutrient starvation triggers the autophagic degradation of oral-facial-digital syndrome 1 and promotes cilia growth (24, 25). Conversely, functional PC are required for activation of autophagy in response to starvation and fluid flow. In both cases, autophagy was initiated by the recruitment of ATG16L to the basal body, suggesting that this event is a hallmark for PC-induced autophagy. Intriguingly, the signaling pathway mediating PC-induced autophagy activation differed based on the stimuli. While Hh/smoothened (SMO) was reported to mediate activation of autophagy in response to starvation (24), the LKB1–AMPK–MTOR signaling pathway was found to regulate PC-induced autophagy and cell volume in kidney epithelial cells under shear stress (26, 27). Whether PC are also involved in the regulation of autophagy triggered by mechanical stretching has not yet been explored.The purpose of this study is to investigate a potential role of PC in stretch-induced autophagy in TM cells. Here, we report that PC acts as a mechanosensor for CMS-induced autophagy, and we identified AKT1 and SMAD2/3 as critical components of the signal mechanotransduction. |
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Keywords: | autophagy primary cilia glaucoma trabecular meshwork stretching |
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