Calcitriol Treatment Decreases Cell Migration, Viability and ß-Catenin Signaling in Oral Dysplasia.

Nearly 90% of oral cancers are characterized as oral squamous cell carcinoma (OSCC), representing the sixth most common type of cancer. OSCC usually evolves from oral potentially malignant disorders that, in some cases, are histologically consistent with a oral dysplasia. The levels of 1α,25 dihydroxyvitamin D3 (1,25-(OH)2D3; calcitriol), the active form of vitamin D3, have been shown to be decreased in patients with oral dysplasia and OSCC. Moreover, treatment with 1,25-(OH)2D3 has been proven beneficial in OSCC by inhibiting the Wnt/ß-catenin pathway, a signaling route that promotes cell migration, proliferation, and viability. However, whether this inhibition mechanism occurs in oral dysplasia is unknown. To approach this question, we used dysplastic oral keratinocyte cultures and oral explants (ex vivo model of oral dysplasia) treated with 1,25-(OH)2D3 for 48 h. Following treatment with 1,25-(OH)2D3, both in vitro and ex vivo models of oral dysplasia showed decreased levels of nuclear ß-catenin by immunofluorescence (IF) and immunohistochemistry (IHC). Consistently, reduced protein and mRNA levels of the Wnt/ß-catenin target gene survivin were observed after treatment with 1,25-(OH)2D3. Moreover, 1,25-(OH)2D3 promoted membranous localization of E-cadherin and nuclear localization of vitamin D receptor (VDR). Functionally, DOK cells treated with 1,25-(OH)2D3 displayed diminished cell migration and viability in vitro.

Nuclear accumulation of ß-catenin is associated with endosomal sequestration of the destruction complex and increased activation of Rab5 in oral dysplasia.

Potentially malignant lesions, commonly referred to as dysplasia, are associated with malignant transformation by mechanisms that remain unclear. We recently reported that increased Wnt secretion promotes the nuclear accumulation of ß-catenin and expression of target genes in oral dysplasia. However, the mechanisms accounting for nuclear re-localization of ß-catenin in oral dysplasia remain unclear. In this study, we show that endosomal sequestration of the ß-catenin destruction complex allows nuclear accumulation of ß-catenin in oral dysplasia, and that these events depended on the endocytic protein Rab5. Tissue immunofluorescence analysis showed aberrant accumulation of enlarged early endosomes in oral dysplasia biopsies, when compared with healthy oral mucosa. These observations were confirmed in cell culture models, by comparing dysplastic oral keratinocytes (DOK) and non-dysplastic oral keratinocytes (OKF6). Intriguingly, DOK depicted higher levels of active Rab5, a critical regulator of early endosomes, when compared with OKF6. Increased Rab5 activity in DOK was necessary for nuclear localization of ß-catenin and Tcf/Lef-dependent transcription, as shown by expression of dominant negative and constitutively active mutants of Rab5, along with immunofluorescence, subcellular fractionation, transcription, and protease protection assays. Mechanistically, elevated Rab5 activity in DOK accounted for endosomal sequestration of components of the destruction complex, including GSK3ß, Axin, and adenomatous polyposis coli (APC), as observed in Rab5 dominant negative experiments. In agreement with these in vitro observations, tissue immunofluorescence analysis showed increased co-localization of GSK3ß, APC, and Axin, with early endosome antigen 1- and Rab5-positive early endosomes in clinical samples of oral dysplasia. Collectively, these data indicate that increased Rab5 activity and endosomal sequestration of the ß-catenin destruction complex leads to stabilization and nuclear accumulation of ß-catenin in oral dysplasia.

Wnt/ß-Catenin Signaling in Oral Carcinogenesis.

Oral carcinogenesis is a complex and multifactorial process that involves cumulative genetic and molecular alterations, leading to uncontrolled cell proliferation, impaired DNA repair and defective cell death. At the early stages, the onset of potentially malignant lesions in the oral mucosa, or oral dysplasia, is associated with higher rates of malignant progression towards carcinoma in situ and invasive carcinoma. Efforts have been made to get insights about signaling pathways that are deregulated in oral dysplasia, as these could be translated into novel markers and might represent promising therapeutic targets. In this context, recent evidence underscored the relevance of the Wnt/ß-catenin signaling pathway in oral dysplasia, as this pathway is progressively "switched on" through the different grades of dysplasia (mild, moderate and severe dysplasia), with the consequent nuclear translocation of ß-catenin and expression of target genes associated with the maintenance of representative traits of oral dysplasia, namely cell proliferation and viability. Intriguingly, recent studies provide an unanticipated connection between active ß-catenin signaling and deregulated endosome trafficking in oral dysplasia, highlighting the relevance of endocytic components in oral carcinogenesis. This review summarizes evidence about the role of the Wnt/ß-catenin signaling pathway and the underlying mechanisms that account for its aberrant activation in oral carcinogenesis.

Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gßγ signaling, ßARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, ßARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and ß-myosin heavy chain (ß-MHC), was prevented by AG538, PTX, ßARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/ßγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin.

GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum-mitochondria coupling.

Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)-mitochondria communication, as it allows for a more efficient transfer of Ca(2+) into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER-mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3h of GLP-1 treatment, paralleled by increased Ca(2+) transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca(2+) increases in GLP-1 treated cells. Inhibiting both Ca(2+) release from the ER and Ca(2+) entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER-mitochondria communication in VSMC, resulting in higher mitochondrial activity.

Inhibition of PORCN Blocks Wnt Signaling to Attenuate Progression of Oral Carcinogenesis.

PURPOSE: Oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, referred to as oral dysplasia. We recently reported that oral dysplasia is associated with aberrant activation of the Wnt/ß-catenin pathway, due to overexpression of Wnt ligands in a Porcupine (PORCN)-dependent manner. Pharmacologic inhibition of PORCN precludes Wnt secretion and has been proposed as a potential therapeutic approach to treat established cancers. Nevertheless, there are no studies that explore the effects of PORCN inhibition at the different stages of oral carcinogenesis. EXPERIMENTAL DESIGN: We performed a model of tobacco-induced oral cancer in vitro, where dysplastic oral keratinocytes (DOK) were transformed into oral carcinoma cells (DOK-TC), and assessed the effects of inhibiting PORCN with the C59 inhibitor. Similarly, an in vivo model of oral carcinogenesis and ex vivo samples derived from patients diagnosed with oral dysplasia and OSCC were treated with C59. RESULTS: Both in vitro and ex vivo oral carcinogenesis approaches revealed decreased levels of nuclear ß-catenin and Wnt3a, as observed by immunofluorescence and IHC analyses. Consistently, reduced protein and mRNA levels of survivin were observed after treatment with C59. Functionally, treatment with C59 in vitro resulted in diminished cell migration, viability, and invasion. Finally, by using an in vivo model of oral carcinogenesis, we found that treatment with C59 prevented the development of OSCC by reducing the size and number of oral tumor lesions. CONCLUSIONS: The inhibition of Wnt ligand secretion with C59 represents a feasible treatment to prevent the progression of early oral lesions toward OSCC.

PKD2 regulates autophagy and forms a protein complex with BECN1 at the primary cilium of hypothalamic neuronal cells.

The primary cilium, hereafter cilium, is an antenna-like organelle that modulates intracellular responses, including autophagy, a lysosomal degradation process essential for cell homeostasis. Dysfunction of the cilium is associated with impairment of autophagy and diseases known as "ciliopathies". The discovery of autophagy-related proteins at the base of the cilium suggests its potential role in coordinating autophagy initiation in response to physiopathological stimuli. One of these proteins, beclin-1 (BECN1), it which is necessary for autophagosome biogenesis. Additionally, polycystin-2 (PKD2), a calcium channel enriched at the cilium, is required and sufficient to induce autophagy in renal and cancer cells. We previously demonstrated that PKD2 and BECN1 form a protein complex at the endoplasmic reticulum in non-ciliated cells, where it initiates autophagy, but whether this protein complex is present at the cilium remains unknown. Anorexigenic pro-opiomelanocortin (POMC) neurons are ciliated cells that require autophagy to maintain intracellular homeostasis. POMC neurons are sensitive to metabolic changes, modulating signaling pathways crucial for controlling food intake. Exposure to the saturated fatty acid palmitic acid (PA) reduces ciliogenesis and inhibits autophagy in these cells. Here, we show that PKD2 and BECN1 form a protein complex in N43/5 cells, an in vitro model of POMC neurons, and that both PKD2 and BECN1 locate at the cilium. In addition, our data show that the cilium is required for PKD2-BECN1 protein complex formation and that PA disrupts the PKD2-BECN1 complex, suppressing autophagy. Our findings provide new insights into the mechanisms by which the cilium controls autophagy in hypothalamic neuronal cells.

Methods for studying primary cilia in heart tissue after ischemia-reperfusion injury.

Cardiovascular diseases are the leading cause of death and disability worldwide. After heart injury triggered by myocardial ischemia or myocardial infarction, extensive zones of tissue are damaged and some of the tissue dies by necrosis and/or apoptosis. The loss of contractile mass activates a series of biochemical mechanisms that allow, through cardiac remodeling, the replacement of the dysfunctional heart tissue by fibrotic material. Our previous studies have shown that primary cilia, non-motile antenna-like structures at the cell surface required for the activation of specific signaling pathways, are present in cardiac fibroblasts and required for cardiac fibrosis induced by ischemia/reperfusion (I/R) in mice. I/R-induced myocardial fibrosis promotes the enrichment of ciliated cardiac fibroblasts where the myocardial injury occurs. Given discussions about the existence of cilia in specific cardiac cell types, as well as the functional relevance of studying cilia-dependent signaling in cardiac fibrosis after I/R, here we describe our methods to evaluate the presence and roles of primary cilia in cardiac fibrosis after I/R in mice.

Integrating the effects of sucrose intake on the brain and white adipose tissue: Could autophagy be a possible link?

Excess dietary sucrose is associated with obesity and metabolic diseases. This relationship is driven by the malfunction of several cell types and tissues critical for the regulation of energy balance, including hypothalamic neurons and white adipose tissue (WAT). However, the mechanisms behind these effects of dietary sucrose are still unclear and might be independent of increased adiposity. Accumulating evidence has indicated that dysregulation of autophagy, a fundamental process for maintenance of cellular homeostasis, alters energy metabolism in hypothalamic neurons and WAT, but whether autophagy could mediate the detrimental effects of dietary sucrose on hypothalamic neurons and WAT that contribute to weight gain is a matter of debate. In this review, we examine the hypothesis that dysregulated autophagy in hypothalamic neurons and WAT is an adiposity-independent effect of sucrose that contributes to increased body weight gain. We propose that excess dietary sucrose leads to autophagy unbalance in hypothalamic neurons and WAT, which increases caloric intake and body weight, favoring the emergence of obesity and metabolic diseases.

Autophagy in aging-related oral diseases.

Autophagy is an intracellular degradation mechanism that allows recycling of organelles and macromolecules. Autophagic function increases metabolite availability modulating metabolic pathways, differentiation and cell survival. The oral environment is composed of several structures, including mineralized and soft tissues, which are formed by complex interactions between epithelial and mesenchymal cells. With aging, increased prevalence of oral diseases such as periodontitis, oral cancer and periapical lesions are observed in humans. These aging-related oral diseases are chronic conditions that alter the epithelial-mesenchymal homeostasis, disrupting the oral tissue architecture affecting the quality of life of the patients. Given that autophagy levels are reduced with age, the purpose of this review is to discuss the link between autophagy and age-related oral diseases.

PKD2/polycystin-2 induces autophagy by forming a complex with BECN1.

Macroautophagy/autophagy is an intracellular process involved in the breakdown of macromolecules and organelles. Recent studies have shown that PKD2/PC2/TRPP2 (polycystin 2, transient receptor potential cation channel), a nonselective cation channel permeable to Ca2+ that belongs to the family of transient receptor potential channels, is required for autophagy in multiple cell types by a mechanism that remains unclear. Here, we report that PKD2 forms a protein complex with BECN1 (beclin 1), a key protein required for the formation of autophagic vacuoles, by acting as a scaffold that interacts with several co-modulators via its coiled-coil domain (CCD). Our data identified a physical and functional interaction between PKD2 and BECN1, which depends on one out of two CCD domains (CC1), located in the carboxy-terminal tail of PKD2. In addition, depletion of intracellular Ca2+ with BAPTA-AM not only blunted starvation-induced autophagy but also disrupted the PKD2-BECN1 complex. Consistently, PKD2 overexpression triggered autophagy by increasing its interaction with BECN1, while overexpression of PKD2D509V, a Ca2+ channel activity-deficient mutant, did not induce autophagy and manifested diminished interaction with BECN1. Our findings show that the PKD2-BECN1 complex is required for the induction of autophagy, and its formation depends on the presence of the CC1 domain of PKD2 and on intracellular Ca2+ mobilization by PKD2. These results provide new insights regarding the molecular mechanisms by which PKD2 controls autophagy.Abbreviations: ADPKD: autosomal dominant polycystic kidney disease; ATG: autophagy-related; ATG14/ATG14L: autophagy related 14; Baf A1: bafilomycin A1; BCL2/Bcl-2: BCL2 apoptosis regulator; BCL2L1/BCL-XL: BCL2 like 1; BECN1: beclin 1; CCD: coiled-coil domain; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GOLGA2/GM130: golgin A2; GST: glutathione s-transferase; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PKD2/PC2: polycystin 2, transient receptor potential cation channel; RTN4/NOGO: reticulon 4; RUBCN/RUBICON: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; UVRAG: UV radiation resistance associated; WIPI2: WD repeat domain, phosphoinositide interacting 2.

New emerging roles of Polycystin-2 in the regulation of autophagy.

Polycystin-2 (PC2) is a calcium channel that can be found in the endoplasmic reticulum, the plasmatic membrane, and the primary cilium. The structure of PC2 is characterized by a highly ordered C-terminal tail with an EF-motif (calcium-binding domain) and a canonical coiled-coil domain (CCD; interaction domain), and its activity is regulated by interacting partners and post-translational modifications. Calcium mobilization into the cytosol by PC2 has been mainly associated with cell growth and differentiation, and therefore mutations or dysfunction of PC2 lead to renal and cardiac consequences. Interestingly, PC2-related pathologies are usually treated with rapamycin, an autophagy stimulator. Autophagy is an intracellular degradation process where recycling material is sequestered into autophagosomes and then hydrolyzed by fusion with a lysosome. Interestingly, several studies have provided evidence that PC2 may be required for autophagy, suggesting that PC2 maintains a physiologic catabolic state.

Role of Autophagy in the Microenvironment of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma, the most common type of oral cancer, affects more than 275,000 people per year worldwide. Oral squamous cell carcinoma is very aggressive, as most patients die after 3 to 5 years post-diagnosis. The initiation and progression of oral squamous cell carcinoma are multifactorial: smoking, alcohol consumption, and human papilloma virus infection are among the causes that promote its development. Although oral squamous cell carcinoma involves abnormal growth and migration of oral epithelial cells, other cell types such as fibroblasts and immune cells form the carcinoma niche. An underlying inflammatory state within the oral tissue promotes differential stress-related responses that favor oral squamous cell carcinoma. Autophagy is an intracellular degradation process that allows cancer cells to survive under stress conditions. Autophagy degrades cellular components by sequestering them in vesicles called autophagosomes, which ultimately fuse with lysosomes. Although several autophagy markers have been associated with oral squamous cell carcinoma, it remains unclear whether up- or down-regulation of autophagy favors its progression. Autophagy levels during oral squamous cell carcinoma are both timing- and cell-specific. Here we discuss how autophagy is required to establish a new cellular microenvironment in oral squamous cell carcinoma and how autophagy drives the phenotypic change of oral squamous cell carcinoma cells by promoting crosstalk between carcinoma cells, fibroblasts, and immune cells.

Nuclear localization of ß-catenin and expression of target genes are associated with increased Wnt secretion in oral dysplasia.

OBJECTIVES: To evaluate the localization of ß-catenin in oral dysplastic cells, the expression of target genes upregulated in oral dysplasia, and the role of Wnt ligands in these events. MATERIALS AND METHODS: Subcellular localization of total and non-phosphorylated (transcriptionally active) ß-catenin was evaluated by immunofluorescence and biochemical fractionation in dysplastic oral keratinocytes (DOK), non-dysplastic oral keratinocytes (OKF6), oral squamous carcinoma cells (CAL27) and primary oral keratinocytes. Tcf/Lef-dependent transcription was measured by luciferase reporter assays. Expression of target genes, survivin and cyclin D1, was evaluated by RT-qPCR and Western blotting. Wnt secretion was inhibited with the inhibitor of porcupine, C59. Wnt3a and ß-catenin were evaluated in biopsies by tissue immunofluorescence. RESULTS: Immunofluorescence and fractionation experiments showed augmented nuclear ß-catenin (total and transcriptionally active) in DOK, when compared with OKF6 and CAL27 cells. Intriguingly, conditioned medium from DOK promoted nuclear accumulation of ß-catenin and Tcf/Lef-dependent transcription in OKF6 and primary oral keratinocytes, suggesting the participation of secreted factors. Treatment of DOK with C59 decreased Wnt3a secretion, nuclear ß-catenin and the expression of survivin and cyclin D1 at both mRNA and protein levels. Accordingly, DOK secreted higher Wnt3a levels than OKF6, and inhibition of Wnt3a secretion prevented DOK-induced Tcf/Lef-dependent transcription in OKF6. These observations were confirmed in clinical samples, since tissue immunofluorescence analysis showed simultaneous expression of Wnt3a and nuclear ß-catenin in oral dysplasia, but not in healthy mucosa biopsies. CONCLUSION: These data indicate that secretion of Wnt ligands is critical for ß-catenin nuclear localization and expression of target genes in oral dysplasia.

Polycystin-2 Is Required for Starvation- and Rapamycin-Induced Atrophy in Myotubes.

Muscle atrophy involves a massive catabolism of intracellular components leading to a significant reduction in cellular and tissue volume. In this regard, autophagy, an intracellular mechanism that degrades proteins and organelles, has been implicated with muscle breakdown. Recently, it has shown that polycystin-2 (PC2), a membrane protein that belongs to the transient receptor potential (TRP) family, is required for the maintenance of cellular proteostasis, by regulating autophagy in several cell types. The role of PC2 in the control of atrophy and autophagy in skeletal muscle remains unknown. Here, we show that PC2 is required for the induction of atrophy in C2C12 myotubes caused by nutrient deprivation or rapamycin exposure. Consistently, overexpression of PC2 induces atrophy in C2C12 myotubes as indicated by decreasing of the myogenic proteins myogenin and caveolin-3. In addition, we show that inhibition of mTORC1, by starvation or rapamycin is inhibited in cells when PC2 is silenced. Importantly, even if PC2 regulates mTORC1, our results show that the regulation of atrophy by PC2 is independent of autophagy. This study provides novel evidence regarding the role of PC2 in skeletal muscle cell atrophy.

Palmitic Acid Reduces the Autophagic Flux and Insulin Sensitivity Through the Activation of the Free Fatty Acid Receptor 1 (FFAR1) in the Hypothalamic Neuronal Cell Line N43/5.

Chronic consumption of high fat diets (HFDs), rich in saturated fatty acids (SatFAs) like palmitic acid (PA), is associated with the development of obesity and obesity-related metabolic diseases such as type II diabetes mellitus (T2DM). Previous studies indicate that PA accumulates in the hypothalamus following consumption of HFDs; in addition, HFDs consumption inhibits autophagy and reduces insulin sensitivity. Whether malfunction of autophagy specifically in hypothalamic neurons decreases insulin sensitivity remains unknown. PA does activate the Free Fatty Acid Receptor 1 (FFAR1), also known as G protein-coupled receptor 40 (GPR40); however, whether FFAR1 mediates the effects of PA on hypothalamic autophagy and insulin sensitivity has not been shown. Here, we demonstrate that exposure to PA inhibits the autophagic flux and reduces insulin sensitivity in a cellular model of hypothalamic neurons (N43/5 cells). Furthermore, we show that inhibition of autophagy and the autophagic flux reduces insulin sensitivity in hypothalamic neuronal cells. Interestingly, the inhibition of the autophagic flux, and the reduction in insulin sensitivity are prevented by pharmacological inhibition of FFAR1. Our findings show that dysregulation of autophagy reduces insulin sensitivity in hypothalamic neuronal cells. In addition, our data suggest FFAR1 mediates the ability of PA to inhibit autophagic flux and reduce insulin sensitivity in hypothalamic neuronal cells. These results reveal a novel cellular mechanism linking PA-rich diets to decreased insulin sensitivity in the hypothalamus and suggest that hypothalamic autophagy might represent a target for future T2DM therapies.

Autophagy and oxidative stress in non-communicable diseases: A matter of the inflammatory state?

Non-communicable diseases (NCDs), also known as chronic diseases, are long-lasting conditions that affect millions of people around the world. Different factors contribute to their genesis and progression; however they share common features, which are critical for the development of novel therapeutic strategies. A persistently altered inflammatory response is typically observed in many NCDs together with redox imbalance. Additionally, dysregulated proteostasis, mainly derived as a consequence of compromised autophagy, is a common feature of several chronic diseases. In this review, we discuss the crosstalk among inflammation, autophagy and oxidative stress, and how they participate in the progression of chronic diseases such as cancer, cardiovascular diseases, obesity and type II diabetes mellitus.

New Roles of the Primary Cilium in Autophagy.

The primary cilium is a nonmotile organelle that emanates from the surface of multiple cell types and receives signals from the environment to regulate intracellular signaling pathways. The presence of cilia, as well as their length, is important for proper cell function; shortened, elongated, or absent cilia are associated with pathological conditions. Interestingly, it has recently been shown that the molecular machinery involved in autophagy, the process of recycling of intracellular material to maintain cellular and tissue homeostasis, participates in ciliogenesis. Cilium-dependent signaling is necessary for autophagosome formation and, conversely, autophagy regulates both ciliogenesis and cilium length by degrading specific ciliary proteins. Here, we will discuss the relationship that exists between the two processes at the cellular and molecular level, highlighting what is known about the effects of ciliary dysfunction in the control of energy homeostasis in some ciliopathies.

Hyperosmotic stress stimulates autophagy via polycystin-2.

Various intracellular mechanisms are activated in response to stress, leading to adaptation or death. Autophagy, an intracellular process that promotes lysosomal degradation of proteins, is an adaptive response to several types of stress. Osmotic stress occurs under both physiological and pathological conditions, provoking mechanical stress and activating various osmoadaptive mechanisms. Polycystin-2 (PC2), a membrane protein of the polycystin family, is a mechanical sensor capable of activating the cell signaling pathways required for cell adaptation and survival. Here we show that hyperosmotic stress provoked by treatment with hyperosmolar concentrations of sorbitol or mannitol induces autophagy in HeLa and HCT116 cell lines. In addition, we show that mTOR and AMPK, two stress sensor proteins involved modulating autophagy, are downregulated and upregulated, respectively, when cells are subjected to hyperosmotic stress. Finally, our findings show that PC2 is required to promote hyperosmotic stress-induced autophagy. Downregulation of PC2 prevents inhibition of hyperosmotic stress-induced mTOR pathway activation. In conclusion, our data provide new insight into the role of PC2 as a mechanosensor that modulates autophagy under hyperosmotic stress conditions.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.