Reducing Akt2 in retinal pigment epithelial cells causes a compensatory increase in Akt1 and attenuates diabetic retinopathy.

The retinal pigment epithelium (RPE) plays an important role in the development of diabetic retinopathy (DR), a leading cause of blindness worldwide. Here we set out to explore the role of Akt2 signaling-integral to both RPE homeostasis and glucose metabolism-to DR. Using human tissue and genetically manipulated mice (including RPE-specific conditional knockout (cKO) and knock-in (KI) mice), we investigate whether Akts in the RPE influences DR in models of diabetic eye disease. We found that Akt1 and Akt2 activities were reciprocally regulated in the RPE of DR donor tissue and diabetic mice. Akt2 cKO attenuated diabetes-induced retinal abnormalities through a compensatory upregulation of phospho-Akt1 leading to an inhibition of vascular injury, inflammatory cytokine release, and infiltration of immune cells mediated by the GSK3ß/NF-κB signaling pathway; overexpression of Akt2 has no effect. We propose that targeting Akt1 activity in the RPE may be a novel therapy for treating DR.

Regulation of Autophagy by the Glycogen Synthase Kinase-3 (GSK-3) Signaling Pathway.

Autophagy is a vital cellular mechanism that benefits cellular maintenance and survival during cell stress. It can eliminate damaged or long-lived organelles and improperly folded proteins to maintain cellular homeostasis, development, and differentiation. Impaired autophagy is associated with several diseases such as cancer, neurodegenerative diseases, and age-related macular degeneration (AMD). Several signaling pathways are associated with the regulation of the autophagy pathway. The glycogen synthase kinase-3 signaling pathway was reported to regulate the autophagy pathway. In this review, we will discuss the mechanisms by which the GSK-3 signaling pathway regulates autophagy. Autophagy and lysosomal function are regulated by transcription factor EB (TFEB). GSK-3 was shown to be involved in the regulation of TFEB nuclear expression in an mTORC1-dependent manner. In addition to mTORC1, GSK-3ß also regulates TFEB via the protein kinase C (PKC) and the eukaryotic translation initiation factor 4A-3 (eIF4A3) signaling pathways. In addition to TFEB, we will also discuss the mechanisms by which the GSK-3 signaling pathway regulates autophagy by modulating other signaling molecules and autophagy inducers including, mTORC1, AKT and ULK1. In summary, this review provides a comprehensive understanding of the role of the GSK-3 signaling pathway in the regulation of autophagy.

Role of the Transcriptional Repressor Zinc Finger with KRAB and SCAN Domains 3 (ZKSCAN3) in Retinal Pigment Epithelial Cells.

Lysosomes are important for proper functioning of the retinal pigment epithelial (RPE) cells. RPE cells have a daily burden of phagocytosis of photoreceptor outer segments (POS) and also degrade cellular waste by autophagy. Here, we identified the role of Zinc-finger protein with KRAB and SCAN domains 3 (ZKSCAN3) in co-ordinate regulation of lysosomal function and autophagy in the RPE. Our studies show that in the RPE, ZKSCAN3 is predominantly nuclear in healthy cells and its nuclear expression is reduced upon nutrient deprivation. siRNA-mediated knockdown of ZKSCAN3 results in de-repression of some of the ZKSCAN3 target genes. Knockdown of ZKSCAN3 also resulted in an induction in autophagy flux, increase in the number of functional lysosomes and accompanied activation of lysosomal cathepsin B activity in ARPE-19 cells. We also demonstrated that inhibition of P38 mitogen-activated protein kinase (MAPK) retains ZKSCAN3 in the nucleus in nutrient-deprived cells. In summary, our studies elucidated the role of ZKSCAN3 as a transcriptional repressor of autophagy and lysosomal function in the RPE.

Role of Nuclear Factor of Activated T Cells (NFAT) Pathway in Regulating Autophagy and Inflammation in Retinal Pigment Epithelial Cells.

Nuclear factor of activated T cells (NFAT) family of transcription factors are substrates of calcineurin and play an important role in integrating Ca2+ signaling with a variety of cellular functions. Of the five NFAT proteins (NFAT1-5), NFAT1-4 are subject to dephosphorylation and activation by calcineurin, a Ca2+-calmodulin-dependent phosphatase. Increased levels of intracellular Ca2+ activates calcineurin, which in turn dephosphorylates and promotes nuclear translocation of NFAT. We investigated the functions of NFAT proteins in the retinal pigment epithelial cells (RPE). Our results show that NFAT-mediated luciferase activity was induced upon treatment with the bacterial endotoxin, lipopolysaccharide (LPS) and treatment with the NFAT peptide inhibitor, MAGPHPVIVITGPHEE (VIVIT) decreased LPS-induced NFAT luciferase activity. LPS-induced activation of NFAT-regulated cytokines (IL-6 and IL-8) is inhibited by treatment of cells with VIVIT. We also investigated the effects of NFAT signaling on the autophagy pathway. Our results show that inhibition of NFAT with VIVIT in cells deprived of nutrients resulted in cytosolic retention of transcription Factor EB (TFEB), decreased expression of TFEB-regulated coordinated Lysosomal Expression and Regulation CLEAR network genes and decreased starvation-induced autophagy flux in the RPE cells. In summary, these studies suggest that the NFAT pathway plays an important role in the regulation of autophagy and inflammation in the RPE.

The role of lipocalin-2 in age-related macular degeneration (AMD).

Lipocalins are a family of secreted adipokines which play important roles in various biological processes. Lipocalin-2 (LCN-2) has been shown to be involved in acute and chronic inflammation. This particular protein is critical in the pathogenesis of several diseases including cancer, diabetes, obesity, and multiple sclerosis. Herein, we discuss the general molecular basis for the involvement of LCN-2 in acute infections and chronic disease progression and also ascertain the probable role of LCN-2 in ocular diseases, particularly in age-related macular degeneration (AMD). We elaborate on the signaling cascades which trigger LCN-2 upregulation in AMD and suggest therapeutic strategies for targeting such pathways.

Nutrient deprivation and lysosomal stress induce activation of TFEB in retinal pigment epithelial cells.

BACKGROUND: Induction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB. METHODS: Expression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes. RESULTS: Our results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes. CONCLUSIONS: Collectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.

The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H+ -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding ßA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.

Modulation of V-ATPase by ßA3/A1-Crystallin in Retinal Pigment Epithelial Cells.

We have previously demonstrated that ßA3/A1-crystallin, a member of the ß/γ-crystallin superfamily, is expressed in the astrocytes and retinal pigment epithelial (RPE) cells of the eye. In order to understand the physiological functions of ßA3/A1-crystallin in RPE cells, we generated conditional knockout (cKO) mice where Cryba1, the gene encoding ßA3/A1-crystallin, is deleted specifically from the RPE using the Cre-loxP system. By utilizing the cKO model, we have shown that this protein is required by RPE cells for proper lysosomal degradation of photoreceptor outer segments (OS) that have been internalized in phagosomes and also for the proper functioning of the autophagy process. We also reported that ßA3/A1-crystallin is trafficked to lysosomes, where it regulates endolysosomal acidification by modulating the activity of the lysosomal V-ATPase complex. Our results show that the V-ATPase activity in cKO RPE is significantly lower than WT RPE. Since, V-ATPase is important for regulating lysosomal pH, we noticed that endolysosomal pH was higher in the cKO cells compared to the WT cells. Increased lysosomal pH in cKO RPE is also associated with reduced Cathepsin D activity. Cathepsin D is a major lysosomal aspartic protease involved in the degradation of the OS and hence we believe that reduced proteolytic activity contributes to impaired degradation of OS in the cKO RPE. Reduced lysosomal activity in the cKO RPE also contributes to the incomplete degradation of the autophagosomes. Our results also suggest that ßA3/A1-crystallin regulates V-ATPase activity by binding to the V0 subunit of the V-ATPase complex. Taken together, these results suggest a novel mechanism by which ßA3/A1-crystallin regulates lysosomal function by modulating the activity of V-ATPase.

Increased Lipocalin-2 in the retinal pigment epithelium of Cryba1 cKO mice is associated with a chronic inflammatory response.

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration (AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice. We have recently shown that these mice, which lack ßA3/A1-crystallin specifically in RPE, have defective lysosomal clearance. The age-related increase in LCN2 in the cKO mice is accompanied by increases in chemokine (C-C motif) ligand 2 (CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD-like pathology.

Lipid raft endocytosis and exosomal transport facilitate extracellular trafficking of annexin A2.

Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the plasma membrane and the endosomal system. Within the plasma membrane, AnxA2 associates in a Ca(2+) dependent manner with cholesterol-rich lipid raft microdomains. Here, we show that the association of AnxA2 with the lipid rafts is influenced not only by intracellular levels of Ca(2+) but also by N-terminal phosphorylation at tyrosine 23. Binding of AnxA2 to the lipid rafts is followed by the transport along the endocytic pathway to be associated with the intralumenal vesicles of the multivesicular endosomes. AnxA2-containing multivesicular endosomes fuse directly with the plasma membrane resulting in the release of the intralumenal vesicles into the extracellular environment, which facilitates the exogenous transfer of AnxA2 from one cell to another. Treatment with Ca(2+) ionophore triggers the association of AnxA2 with the specialized microdomains in the exosomal membrane that possess raft-like characteristics. Phosphorylation at Tyr-23 is also important for the localization of AnxA2 to the exosomal membranes. These results suggest that AnxA2 is trafficked from the plasma membrane rafts and is selectively incorporated into the lumenal membranes of the endosomes to escape the endosomal degradation pathway. The Ca(2+)-dependent exosomal transport constitutes a novel pathway of extracellular transport of AnxA2.

Signal transducer and activator of transcription 6 (STAT6) is a novel interactor of annexin A2 in prostate cancer cells.

Annexin A2 (AnxA2) is a multifunctional Ca(2+)-dependent phospholipid-binding protein, and its overexpression is implicated in malignant transformation of several cancers. In prostate cancer, however, the expression of AnxA2 is lost in prostate intraepithelial neoplasia and reappears in the high-grade tumors, suggesting a complex regulation of AnxA2 in the prostate microenvironment. Since a majority of the biological functions of AnxA2 are mediated by its interaction with other proteins, we performed a yeast two-hybrid assay to search for novel interactors of AnxA2. Our studies revealed that signal transducer and activator of transcription 6 (STAT6), a member of the STAT family of transcription factors, is a binding partner of AnxA2. We confirmed AnxA2-STAT6 interaction by in vitro co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies and demonstrated that AnxA2 interacts with phosphorylated STAT6. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that AnxA2 is associated with the STAT6 DNA-binding complex, and luciferase reporter assays demonstrated that AnxA2 upregulates the activity of STAT6. Upon interleukin-4 treatment, AnxA2 stabilizes the cytosolic levels of phosphorylated STAT6 and promotes its nuclear entry. These findings suggest that AnxA2-STAT6 interactions could have potential implications in prostate cancer progression. This report is the first to demonstrate the interaction of AnxA2 with STAT6 and suggests a possible mechanism by which AnxA2 contributes to the metastatic processes of prostate cancer.