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Shear-stress-induced PI3P synthesis at the PC depends on PI3KC2α a Representative confocal images upon shear-stress conditions of PI3KC2α knocked down HK2 cells (siPI3KC2α), compared to control cells (siCTRL), immunostained for ARL13B, PI3P (using FYVE-GST indirect recombinant peptide) and DAPI (N = 80 cells, from five independent experiments). Scale bar, 10 μm. b Schematic drawing showing the 150 µm² circular area centred at the cell nucleus used as identifier of the PC basal body area. c Quantification of the PI3P-positive structures at the PC area, as defined in (b) and shown in (a), in siCTRL or siPI3KC2α HK2 cells, upon static (ctrl) and shear-stress (96 h) conditions (mean ± SEM, N = 80 cells, from five independent experiments). NS: not significant, ***p < 0.001 in two-tailed Student’s t test. d Western blot analysis and quantification of WIPI2 protein levels in lysates of polarized siCTRL or siPI3KC2α HK2 cells, upon static (ctrl) and shear-stress (96 h) conditions. Bar graph denotes average protein levels normalized to actin (mean ± SEM, from three independent experiments). NS: not significant, ***p < 0.001 in two-tailed Student’s t test. e WIPI2 mRNA levels quantifications from total lysates of polarized siCTRL or siPI3KC2α HK2 cells, upon static (ctrl) and shear-stress (96 h) conditions. Bar graphs denote fold change of average mRNA levels relative to ctrl and normalized to actin mRNA (mean ± SEM, from three independent experiments). ***p < 0.001 in two-tailed Student’s t test. f Representative confocal acquisition of WIPI2GFP transfected HK2 cells upon shear-stress conditions immunostained for ARL13B, PI3P (using FYVE-GST indirect recombinant peptide) and DAPI showing PI3P and WIPI2GFP colocalization at the base of PC (arrowhead). Scale bar, 10 μm.
Source publication
Cells subjected to stress situations mobilize specific membranes and proteins to initiate autophagy. Phosphatidylinositol-3-phosphate (PI3P), a crucial lipid in membrane dynamics, is known to be essential in this context. In addition to nutriments deprivation, autophagy is also triggered by fluid-flow induced shear stress in epithelial cells, and t...
Citations
... In kidney proximal tubules, the primary cilium, a microtubulebased organelle present at the apical surface of epithelial cells, acts as a flow sensor to integrate variations in flow rates of the glomerular ultrafiltrate. We and others have demonstrated an interplay between macroautophagy (hereafter referred to as autophagy) and primary cilia [8][9][10] . Recently, we showed that primary cilium-dependent autophagy is activated through an LKB1-AMPK signaling pathway to regulate metabolism and cell volume of kidney epithelial cells 11,12 . ...
Shear stress generated by urinary fluid flow is an important regulator of renal function. Its dysregulation is observed in various chronic and acute kidney diseases. Previously, we demonstrated that primary cilium-dependent autophagy allows kidney epithelial cells to adapt their metabolism in response to fluid flow. Here, we show that nuclear YAP/TAZ negatively regulates autophagy flux in kidney epithelial cells subjected to fluid flow. This crosstalk is supported by a primary cilium-dependent activation of AMPK and SIRT1, independently of the Hippo pathway. We confirm the relevance of the YAP/TAZ-autophagy molecular dialog in vivo using a zebrafish model of kidney development and a unilateral ureteral obstruction mouse model. In addition, an in vitro assay simulating pathological accelerated flow observed at early stages of chronic kidney disease (CKD) activates YAP, leading to a primary cilium-dependent inhibition of autophagic flux. We confirm this YAP/autophagy relationship in renal biopsies from patients suffering from diabetic kidney disease (DKD), the leading cause of CKD. Our findings demonstrate the importance of YAP/TAZ and autophagy in the translation of fluid flow into cellular and physiological responses. Dysregulation of this pathway is associated with the early onset of CKD.
... Phosphoinositide 3-kinases (PI3Ks) are a type of enzyme that catalyzes the formation of second messengers for a variety of signaling pathways (8). This family can be classified into three subtypes based on structure and substrate specificity (9). ...
Background
Phosphoinositide 3-kinases (PI3Ks) are lipid enzymes that regulate a wide range of intracellular functions. In contrast to Class I and Class III PI3K, which have more detailed descriptions, Class II PI3K has only recently become the focus of functional research. PIK3C2A is a classical member of the PI3Ks class II. However, the role of PIK3C2A in cancer prognosis and progression remains unknown.
Methods
The expression pattern and prognostic significance of PIK3C2A in human malignancies were investigated using multiple datasets and scRNA-seq data. The PIK3C2A expression in renal clear cell carcinoma (KIRC) was then validated utilizing Western blot. The functional role of PIK3C2A in KIRC was assessed using combined function loss experiments with in vitro experiments. Furthermore, the correlation of PIK3C2A expression with tumor immunity was investigated in KIRC. The TCGA database was employed to investigate PIK3C2A functional networks.
Results
Significant decrease in PIK3C2A expression in KIRC, demonstrated that it potentially influences the prognosis of diverse tumors, particularly KIRC. In addition, PIK3C2A was significantly correlated with the T stage, M stage, pathologic stage, and histologic grade of KIRC. Nomogram models were constructed and used to predict patient survival based on the results of multivariate Cox regression analysis. PIK3C2A knockdown resulted in significantly increased KIRC cell proliferation. Of note, PIK3C2A expression demonstrated a significant correlation with the infiltrating levels of primary immune cells in KIRC.
Conclusion
These findings support the hypothesis that PIK3C2A is a novel biomarker for tumor progression and indicates dynamic shifts in immune infiltration in KIRC. Furthermore, aberrant PIK3C2A expression can influence the biological activity of cancer cells.
... Later on, it controls their maturation by recruiting effector proteins that promote their fusion with lysosomes [99,100]. Of note, in some physiological contexts such mechanical shear stress, autophagic pools of PI (3) P may also be synthesized by class II PI3Ks, including PI3KC2α [101]. Added to its functions in vesicle traffic, Vps34 has scaffolding properties. ...
Simple Summary
The PI3K/AKT pathway is one of the most important signaling nodes in cancer. While class I PI3K roles in cancer are well known, class II and III functions in physiology and physiopathology are poorly studied. Moreover, the interactions between pathways controlled by all three classes are not fully understood. The understanding of the mechanisms behind their cooperative functions could be key for efficient PI3K combination targeting in cancer therapy. This review will focus on the recent advances on the roles of the different classes of PI3K in cancer biology, their cross-regulations and their targeting in preclinical models.
Abstract
Phosphatidylinositol-3-kinase (PI3K) enzymes, producing signaling phosphoinositides at plasma and intracellular membranes, are key in intracellular signaling and vesicular trafficking pathways. PI3K is a family of eight enzymes divided into three classes with various functions in physiology and largely deregulated in cancer. Here, we will review the recent evidence obtained during the last 5 years on the roles of PI3K class I, II and III isoforms in tumor biology and on the anti-tumoral action of PI3K inhibitors in preclinical cancer models. The dependency of tumors to PI3K isoforms is dictated by both genetics and context (e.g., the microenvironment). The understanding of class II/III isoforms in cancer development and progression remains scarce. Nonetheless, the limited available data are consistent and reveal that there is an interdependency between the pathways controlled by all PI3K class members in their role to promote cancer cell proliferation, survival, growth, migration and metabolism. It is unknown whether this feature contributes to partial treatment failure with isoform-selective PI3K inhibitors. Hence, a better understanding of class II/III functions to efficiently inhibit their positive and negative interactions with class I PI3Ks is needed. This research will provide the proof-of-concept to develop combination treatment strategies targeting several PI3K isoforms simultaneously.
... PI3K-C2α is a phosphoinositide 3-kinase and phosphorylates PtdIns and PtdIns4P to produce PtdIns3P and PtdIns(3,4)P 2 , respectively ( Figure 1) [1]. PI3K-C2α is involved in a wide range of biological events such as endocytosis [111][112][113], exocytosis [114], mitosis [115], and autophagy [116,117]. Recently, a specific accumulation of PI3K-C2α is observed in the pericentriolar recycling endocytic compartment (PC-REC) at the ciliary base (Figure 1), where it produces a local pool of PtdIns3P [58]. In PI3K-C2α-depleted cells, the reduction in pericentriolar PtdIns3P altered the organization of PC-REC around the ciliary base, reduced RAB11 activation and impaired the ciliary targeting of RAB8 [58]. ...
Primary cilia are microtube-based organelles that extend from the cell surface and function as biochemical and mechanical extracellular signal sensors. Primary cilia coordinate a series of signaling pathways during development. Cilia dysfunction leads to a pleiotropic group of developmental disorders, termed ciliopathy. Phosphoinositides (PIs), a group of signaling phospholipids, play a crucial role in development and tissue homeostasis by regulating membrane trafficking, cytoskeleton reorganization, and organelle identity. Accumulating evidence implicates the involvement of PI species in ciliary defects and ciliopathies. The abundance and localization of PIs in the cell are tightly regulated by the opposing actions of kinases and phosphatases, some of which are recently discovered in the context of primary cilia. Here, we review several cilium-associated PI kinases and phosphatases, including their localization along cilia, function in regulating the ciliary biology under normal conditions, as well as the connection of their disease-associated mutations with ciliopathies.
... The class III PI3K complex consists of several proteins including vacuolar protein sorting (VPS) 34, VPS15, Beclin-1, and Atg14 that drive the early stages of phagophore growth. The VPS34 subunit of the PI3K complex produces phosphatidylinositol-3-phosphate (PI3P) simultaneously with the formation of omegasomes [115]. It has been shown that experimentally induced overexpression of Beclin 1 can decrease the accumulation of α-Syn aggregates and reduce the related cell damage. ...
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. It is characterized by the accumulation of α-Synuclein aggregates and the degeneration of dopaminergic neurons in substantia nigra in the midbrain. Although the exact mechanisms of neuronal degeneration in PD remain largely elusive, various pathogenic factors, such as α-Synuclein cytotoxicity, mitochondrial dysfunction, oxidative stress, and pro-inflammatory factors, may significantly impair normal neuronal function and promote apoptosis. In this context, neuroinflammation and autophagy have emerged as crucial processes in PD that contribute to neuronal loss and disease development. They are regulated in a complex interconnected manner involving most of the known PD-associated genes. This review summarizes evidence of the implication of neuroinflammation and autophagy in PD and delineates the role of inflammatory factors and autophagy-related proteins in this complex condition. It also illustrates the particular significance of plasma and serum immune markers in PD and their potential to provide a personalized approach to diagnosis and treatment.
... Regarding the molecular events required for autophagosome formation following primary cilium sensing, it has been reported that many ATGs (except ULK1 and Beclin1) can be recruited at the primary cilium upon serum starvation or shear stress (Pampliega et al., 2013;Boukhalfa et al., 2020). In contrast to bulk autophagy initiation, where the ULK and PI3KIII complexes are essential for PI3P formation, autophagy induction in kidney epithelial cells submitted to fluid flow has been shown to be dependent on PI3P synthesis by the class II PI3Kα (PI3K-C2α) (Boukhalfa et al., 2020). ...
... Regarding the molecular events required for autophagosome formation following primary cilium sensing, it has been reported that many ATGs (except ULK1 and Beclin1) can be recruited at the primary cilium upon serum starvation or shear stress (Pampliega et al., 2013;Boukhalfa et al., 2020). In contrast to bulk autophagy initiation, where the ULK and PI3KIII complexes are essential for PI3P formation, autophagy induction in kidney epithelial cells submitted to fluid flow has been shown to be dependent on PI3P synthesis by the class II PI3Kα (PI3K-C2α) (Boukhalfa et al., 2020). Importantly, the PI3K-C2α kinase controls ciliogenesis and is relocated at the primary cilium upon shear stress (Franco et al., 2014;Boukhalfa et al., 2020). ...
... In contrast to bulk autophagy initiation, where the ULK and PI3KIII complexes are essential for PI3P formation, autophagy induction in kidney epithelial cells submitted to fluid flow has been shown to be dependent on PI3P synthesis by the class II PI3Kα (PI3K-C2α) (Boukhalfa et al., 2020). Importantly, the PI3K-C2α kinase controls ciliogenesis and is relocated at the primary cilium upon shear stress (Franco et al., 2014;Boukhalfa et al., 2020). Moreover, PI3KC2α ± mice exhibit defects in autophagic stimulation and cell-size regulation in kidney tubular cells (Boukhalfa et al., 2020). ...
The maintenance of cellular homeostasis in response to extracellular stimuli, i.e., nutrient and hormone signaling, hypoxia, or mechanical forces by autophagy, is vital for the health of various tissues. The primary cilium (PC) is a microtubule-based sensory organelle that regulates the integration of several extracellular stimuli. Over the past decade, an interconnection between autophagy and PC has begun to be revealed. Indeed, the PC regulates autophagy and in turn, a selective form of autophagy called ciliophagy contributes to the regulation of ciliogenesis. Moreover, the PC regulates both mitochondrial biogenesis and lipophagy to produce free fatty acids. These two pathways converge to activate oxidative phosphorylation and produce ATP, which is mandatory for cell metabolism and membrane transport. The autophagy-dependent production of energy is fully efficient when the PC senses shear stress induced by fluid flow. In this review, we discuss the cross-talk between autophagy, the PC and physical forces in the regulation of cell biology and physiology.
... Many studies have identified a critical requirement for Vps34 expression in multiple mammalian tissues for the constitutive or induced generation of autophagy (Jaber et al., 2012;Parekh et al., 2017;Parekh et al., 2013;Zhou et al., 2010). Nevertheless, situations where autophagy can be induced in a Vps34-independent manner have also been described (Boukhalfa et al., 2020;Martinez-Martin et al., 2017). To measure the contribution of Vps34 to autophagy in TECs, we used an autophagy reporter strain that expresses transgenic LC3 (microtubule-associated protein 1A/1B-light chain 3) that is dually labeled with EGFP and RFP . ...
The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.
... Class II PI3Ks comprise PI3KC2α, PI3KC2β and PI3KC2γ isoforms and are unique in their ability to directly synthesize PI 3,4-bisphosphate (PI(3,4)P 2 ) from PI 4-monophosphate (PI(4)P) in vivo. In addition, they have been suggested to contribute to cellular PI(3)P production on endosomes and in the autophagy/lysosomal pathway 7,[12][13][14] . PI3KC2α and PI3KC2β are ubiquitously expressed, whereas PI3KC2γ is mostly expressed in exocrine glands and liver. ...
... Apart from the synthesis of PI(3,4)P 2 at plasma membrane endocytic sites and at the cytokinetic bridge 20 , PI3KC2α has been implicated in contributing to the formation of endosomal pools of PI(3)P in a cell-typeand/or context-specific manner 7,12,14,17,21,27,38 . A major alternative route of PI(3)P formation on endosomes involves Vps34 (refs. ...
... PITCOINs thus are first-in-class potent and selective inhibitors of PI3KC2α catalytic function and represent unique pharmacological tools to further decipher the multiple roles of PI3KC2α in biology. The observation that acute inhibition of PI3KC2α causes antithrombotic effects that occur concomitantly with a reduction in PI(3)P levels and changes in platelet membrane morphology (that is, the OCS) opens the possibility that PITCOIN3 and related compounds may serve as a new class of shear stress-dependent 12,27 antithrombotic agents that, unlike current therapies, may not increase bleeding risk. Future studies will be directed to explore this exciting perspective and to further improve PITCOINs, for example, with respect to cell membrane permeability. ...
Phosphatidylinositol 3-kinase type 2α (PI3KC2α) and related class II PI3K isoforms are of increasing biomedical interest because of their crucial roles in endocytic membrane dynamics, cell division and signaling, angiogenesis, and platelet morphology and function. Herein we report the development and characterization of PhosphatidylInositol Three-kinase Class twO INhibitors (PITCOINs), potent and highly selective small-molecule inhibitors of PI3KC2α catalytic activity. PITCOIN compounds exhibit strong selectivity toward PI3KC2α due to their unique mode of interaction with the ATP-binding site of the enzyme. We demonstrate that acute inhibition of PI3KC2α-mediated synthesis of phosphatidylinositol 3-phosphates by PITCOINs impairs endocytic membrane dynamics and membrane remodeling during platelet-dependent thrombus formation. PITCOINs are potent and selective cell-permeable inhibitors of PI3KC2α function with potential biomedical applications ranging from thrombosis to diabetes and cancer.
... Vesicles enriched with phosphoInositol-3-kinase type C II α (PI3KC2α), found at the base of the cilia just beyond the TZ, are suggested to generate PI(3)P (Franco et al., 2014). Shear stress increases PI(3)P level at the cilia base, which is essential for maintaining autophagy in the kidney epithelial cells (Boukhalfa et al., 2020). PI3KC2α is also implicated in the transport of PC2 ...
Cilium, a tiny microtubule‐based cellular appendage critical for cell signalling and physiology, displays a large variety of receptors. The composition and turnover of ciliary lipids and receptors determine cell behaviour. Due to the exclusion of ribosomal machinery and limited membrane area, a cilium needs adaptive logistics to actively reconstitute the lipid and receptor compositions during development and differentiation. How is this dynamicity generated? Here, we examine whether, along with the Intraflagellar‐Transport, targeted changes in sector‐wise lipid composition could control the receptor localisation and functions in the cilia. We discuss how an interplay between ciliary lipid composition, localised lipid modification, and receptor function could contribute to cilia growth and signalling. We argue that lipid modification at the cell‐cilium interface could generate an added thrust for a selective exchange of membrane lipids and the transmembrane and membrane‐associated proteins. The review discusses how an interplay between ciliary lipid composition, localized lipid modification, and receptor function could contribute to cilia growth and signaling. It extends a hypothesis that lipid modification at the cell‐cilium interface could generate an added thrust for a selective exchange of membrane lipids, receptors, and associated proteins.
... Intriguingly, in KECs submitted to shear stress, primary cilium-dependent autophagy has been demonstrated to act independently of ULK1 and BECN1. PtdIns3P, the key lipid involved in autophagosome biogenesis, is produced by the basal body localized class II PIK3C2A [73], which regulates ciliogenesis [74] and promotes the synthesis of a local pool of PtdIns3P upon shear stress [73]. Renal tubular epithelial cells from the Pik3c2a ± mouse model show decreased autophagic activation and loss of cell-size control [73]. ...
... Intriguingly, in KECs submitted to shear stress, primary cilium-dependent autophagy has been demonstrated to act independently of ULK1 and BECN1. PtdIns3P, the key lipid involved in autophagosome biogenesis, is produced by the basal body localized class II PIK3C2A [73], which regulates ciliogenesis [74] and promotes the synthesis of a local pool of PtdIns3P upon shear stress [73]. Renal tubular epithelial cells from the Pik3c2a ± mouse model show decreased autophagic activation and loss of cell-size control [73]. ...
... PtdIns3P, the key lipid involved in autophagosome biogenesis, is produced by the basal body localized class II PIK3C2A [73], which regulates ciliogenesis [74] and promotes the synthesis of a local pool of PtdIns3P upon shear stress [73]. Renal tubular epithelial cells from the Pik3c2a ± mouse model show decreased autophagic activation and loss of cell-size control [73]. However, how primary cilia control AMPK-mediated autophagy induction in the absence of phosphorylation of its targets -ULK1 and BECN1 -remains an open question. ...
Macroautophagy/autophagy is a self-degradative process necessary for cells to maintain their energy balance during development and in response to nutrient deprivation. Autophagic processes are tightly regulated and have been found to be dysfunctional in several pathologies. Increasing experimental evidence points to the existence of an interplay between autophagy and cilia. Cilia are microtubule-based organelles protruding from the cell surface of mammalian cells that perform a variety of motile and sensory functions and, when dysfunctional, result in disorders known as ciliopathies. Indeed, selective autophagic degradation of ciliary proteins has been shown to control ciliogenesis and, conversely, cilia have been reported to control autophagy. Moreover, a growing number of players such as lysosomal and mitochondrial proteins are emerging as actors of the cilia-autophagy interplay. However, some of the published data on the cilia-autophagy axis are contradictory and indicate that we are just starting to understand the underlying molecular mechanisms. In this review, the current knowledge about this axis and challenges are discussed, as well as the implication for ciliopathies and autophagy-associated disorders.
