Inositol Triphosphate Signaling Triggers Lysosome Biogenesis Via Calcium Release from Endoplasmic Reticulum Stores

Abstract

Lysosomes serve as cellular degradation and signaling centers that coordinate the turnover of macromolecules with cell metabolism. The adaptation of cellular lysosome content and activity via the induction of lysosome biogenesis is therefore key to cell physiology and to counteract disease. Previous work has established a pathway for the induction of lysosome biogenesis in signaling-inactive starved cells that is based on the repression of mTORC1-mediated nutrient signaling. How lysosomal biogenesis is facilitated in signaling-active fed cells is poorly understood. A recent study by Malek et al. (2022) partially fills this gap by unraveling a nutrient signaling-independent pathway for lysosome biogenesis that operates in signaling-active cells. This pathway involves the receptor-mediated activation of phospholipase C, inositol (1,4,5)-triphosphate (IP₃)-triggered release of calcium ions from endoplasmic reticulum stores, and the calcineurin-induced activation of transcription factor EB (TFEB) and its relative TFE3 to induce lysosomal gene expression independent of calcium in the lysosome lumen. These findings contribute to our understanding of how lysosome biogenesis and function are controlled in response to environmental changes and cell signaling and may conceivably be of relevance for our understanding and the treatment of lysosome-related diseases as well as for aging and neurodegeneration.

Lysosomes coordinate the degradative turnover of proteins, lipids, or defective organelles (e.g. via autophagy) with cell metabolism by responding to nutrient levels (e.g. amino acid levels) and extracellular signals such as insulin or growth factors. Lysosome dysfunction causes lysosomal diseases and neurodegeneration (Perera & Zoncu, 2016). To adapt to changing environmental conditions and to a variety of stresses, in particular starvation, cells and tissues can regulate the expression of genes encoding autophagy and lysosomal proteins via the transcription factor EB (TFEB) and related proteins of the MiT-TFE family. TFEB and its cousin TFE3 are kept inactive in the cytoplasm at steady-state via phosphorylation by multiple kinases including the mammalian target of rapamycin complex 1 (mTORC1) (Settembre et al., 2012), a major sensor of cellular nutrient status. Under conditions of starvation mTORC1 becomes inactive resulting in Mucolipin 1/TRPML1-mediated calcium efflux from the lysosome lumen, which then triggers TFEB dephosphorylation via the calcium-activated phosphatase calcineurin (Medina et al., 2015). However, as lysosomes are known to execute key functions, e.g. in signaling receptor degradation or lipid turnover, in signaling-active cells the question arises how lysosomal gene and protein expression are sustained under conditions of ample nutrient and growth factor supply.

We recently obtained insights into the mechanism that control lysosome biogenesis in fed signaling-active cells by studying the function of INPP5A, an IP₃-specific inositol 5-phosphatase implicated in cancer (Sekulic et al., 2010) and spinocerebellar ataxia (Liu et al., 2020). We found that cellular depletion of INPP5A increases the cellular content of degradative lysosomes and autophagosomes but not of other organelles. Biochemical and cell biological analysis showed that lysosome accumulation in INPP5A-depleted cells was not the result of altered (i.e. reduced) nutrient signaling via mTORC1, the induction of the ER stress response or lysosomal calcium release via Mucolipin 1/ TRPML1, pathways that had previously been implicated in lysosome biogenesis via TFEB. Instead, we found that INPP5A-depleted cells accumulate IP₃ (Malek et al., 2021), a signaling molecule that is generated by phospholipase C (PLC)-catalyzed cleavage of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] downstream of many G protein coupled receptors but also some receptor tyrosine kinases. IP₃ binds to and activates IP₃ receptors localized in the endoplasmic reticulum (ER) membrane, which act as ligand-gated calcium release channels. Consistently, we observed that genetic or pharmacological blockade of either PLC or IP₃ receptor function prevented the accumulation of lysosomes in the absence of INPP5A in cells. Conversely, pharmacological hyperactivation of PLC induced elevated cellular lysosome content in wild-type cells. Elevation of cytosolic calcium levels facilitate the activation of calcineurin, a calcium-controlled protein phosphatase, and thereby promotes the activation and nuclear translocation of TFEB. Active TFEB and TFE3 finally promote the expression of lysosomal genes including lysosomal acid ceramidase, and the lysosomal membrane proteins LAMP1, CD63, as well as the vacuolar ATPase. From these results a model emerges, whereby receptor-triggered generation of IP₃ from plasma membrane PI(4,5)P₂ initiates a cascade resulting in the calcineurin-dependent activation of lysosomal gene expression via nuclear TFEB (Figure 1) and TFE3.

Figure 1.

Model for the TFEB-dependent induction of lysosome biogenesis via IP₃-mediated calcium efflux from the endoplasmic reticulum in the absence of the IP₃ 5-phosphatase activity of INPP5A. In signaling-active wild-type (WT) control cells inactive phosphorylated TFEB is sequestered in the cytoplasm. In signaling-active cells depleted of INPP5A (INPP5AKD), IP₃-triggered activation of IP₃ receptors located in ER membranes causes a rise in cytosolic calcium levels and activation of the protein phosphatase calcineurin (Cn) to induce TFEB translocation to the nucleus. Active dephosphorylated nuclear TFEB promotes the expression of lysosomal genes and de novo lysosome formation.

How do these findings relate to other insights into the mechanisms that impinge on lysosome biogenesis and function? The INPP5A/IP₃-controlled pathway of lysosome biogenesis in signaling-active cells adds to previous works highlighting the role of calcium and calcineurin activity in TFEB/TFE3 nuclear translocation and activation. However, distinct from these previously described pathways IP₃ triggers TFEB-mediated lysosome biogenesis in fed cells independent of both mTORC1 activity and Mucolipin 1/ TRPML1-mediated calcium efflux from the lysosome lumen, a pathway that only occurs in signaling-inactive starved cells (Medina et al., 2015).

Hence, TFEB-mediated lysosome biogenesis relies on distinct calcium sources dependent on cellular nutrient status, i.e. the ER lumen in fed cells (Malek et al., 2022) versus the lysosome lumen in cells under starvation (Medina et al., 2015). Interesting possible implications arise from this distinction given that luminal ER calcium and lysosomal calcium are controlled by distinct mechanisms and are differentially regulated dependent on cell type and tissue. Moreover, as IP₃ is generated at the plasma membrane and IP₃ receptors may be confined to specialized sites of the ER, it seems tempting to speculate that TFEB-mediated lysosome biogenesis in fed cells is triggered by a local signaling circuit, e.g. at sites of ER-plasma membrane contact akin to excitation-contraction coupling in muscle. A further element of complexity is related to the fact that the ER forms membrane contacts with a variety of other organelles including lysosomes, endosomes, the plasma membrane, and mitochondria. While each of these organelles contribute to cytosolic calcium homeostasis on their own, it has also been reported that calcium can be funneled via membrane contacts between these organelles. For example, calcium derived from the ER lumen has been shown to refill lysosomal (Garrity et al., 2016) and mitochondrial calcium stores (Hirabayashi et al., 2017) in different cell types. Recent work has further revealed that under conditions of osmotic stress influx of extracellular calcium via functional coupling of the sodium-proton-exchanger NHE7 and the sodium-calcium exchanger NCX1 at the cell surface can trigger TFEB/TFE3 activation via calcineurin (Lopez-Hernandez et al., 2020). These collective findings suggest that the degradative capacity of cells via the autophagy/lysosome system is intimately linked to intracellular calcium homeostasis in signaling-active and in starved or stressed cells.

Ramping up the autophagy/lysosome system under conditions of hyperactive signaling, i.e. IP₃ accumulation downstream of sustained receptor-mediated PLC activation, might conceivably be part of a more extensive rewiring of membrane traffic and cell signaling. For example, it has been shown that IP₃-induced cytosolic calcium elevation in the absence of INPP5A causes the dissociation of of oxysterol binding protein (OSBP) from the Golgi complex and from VAP-containing membrane contact sites with the ER. As a result, lipid exchange, i.e. of cholesterol at these contact sites is disturbed, cholesterol and associated glycosphingolipids are depleted from the plasma membrane, and clathrin-independent endocytic trafficking is perturbed (Malek et al., 2021). These alterations are likely to impinge on receptor signaling, metabolism and cell growth. For example, the depletion of INPP5A in squamous cell carcinoma cells (Sekulic et al., 2010) may aid in sustaining cell growth and division when nutrients are scarce by promoting the lysosome-mediated degradation of proteins. These in turn serve to sustain mTORC1 activity. A more detailed understanding of the complex mechanisms that couple organelle homeostasis with cell signaling and growth will undoubtedly aid in the development of novel therapeutic options to combat metabolic diseases and cancer.