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Age‐related macular degeneration (AMD) is an emerging cause of blindness in aged people worldwide. One of the key signs of AMD is the degeneration of the retinal pigment epithelium (RPE), which is indispensable for the maintenance of the adjacent photoreceptors. Because of impaired energy metabolism resulting from constant light exposure, hypoxia, and oxidative stress, accumulation of drusen in AMD‐affected eyes is observed. Drusen contain damaged cellular proteins, lipoprotein particles, lipids and carbohydrates and they are related to impaired protein clearance, inflammation, and extracellular matrix modification. When autophagy, a major cellular proteostasis pathway, is impaired, the accumulations of intracellular lipofuscin and extracellular drusen are detected. As these aggregates grow over time, they finally cause the disorganisation and destruction of the RPE and photoreceptors leading to visual loss. In this review, the role of autophagy in drusen biogenesis is discussed since impairment in removing cellular waste in RPE cells plays a key role in AMD progression. In the future, means which improve intracellular clearance might be of use in AMD therapy to slow the progression of drusen formation. [ABSTRACT FROM AUTHOR]

The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes. [ABSTRACT FROM AUTHOR]

Background: Epiphora is a common complaint of nasolacrimal duct obstruction (NLDO) in adults. The precise pathogenesis of NLDO is still unknown, but inflammatory processes are believed to be predisposing factors. Endoscopic dacryocystorhinostomy (EN-DCR) is an effective surgical technique for treating symptomatic NLDO. The purpose of the procedure is to relieve the patient's symptoms by creating an opening, ie, a rhinostoma, between the lacrimal sac and the nasal cavity. Although the success rates after EN-DCR are high, the procedure sometimes fails due to onset of a fibrotic process at the rhinostomy site. The aim of this prospective comparative study was to investigate inflammation-related gene expression in the nasal mucosa at the rhinostomy site. Methods: Ten participants were consecutively recruited from eligible adult patients who underwent primary powered EN-DCR (five patients) or septoplasty (five controls). Nasal mucosa specimens were taken from the rhinostomy site at the beginning of surgery for analysis of gene expression. Specimens were taken from the same site on the lateral nasal wall for controls. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed for the inflammatory genes interleukin (IL)-6, IL-1β, and CCL2, and because of a clear trend of increased inflammation in the EN-DCR samples, a wider PCR array was performed to compare inflammation-related gene expression in EN-DCR subjects and corresponding controls. Results: Our qRT-PCR results revealed a clear trend of increased transcription of IL-6, IL-1β, and CCL2 (P=0.03). The same trend was also evident in the PCR array, which additionally revealed notable differences between EN-DCR subjects and controls with regard to expression of several other inflammation-related mediators. At 6-month follow-up, the success rate after primary EN-DCR was 60%, ie, in three of five patients. Conclusion: The present study demonstrates that there is an intense inflammation gene expression response in the nasal mucosa of patients undergoing EN-DCR. [ABSTRACT FROM AUTHOR]

The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a mounting cause of loss of sight in the elderly in the developed countries, a trend enhanced by the continual ageing of the population. AMD is a multifactorial and only partly understood, malady. Unfortunately, there is no effective treatment for most AMD patients. It is known that oxidative stress (OS) damages the retinal pigment epithelium (RPE) and contributes to the progression of AMD. We review here the potential importance of two OS-related cellular systems in relation to AMD. First, the nuclear factor erythroid 2-related factor 2 (NFE2L2; NRF2)-mediated OS response signalling pathway is important in the prevention of oxidative damage and a failure of this system could be critical in the development of AMD. Second, epithelial-to-mesenchymal transition (EMT) represents a change in the cellular phenotype, which ultimately leads to the fibrosis encountered in RPE, a characteristic of AMD. Many of the pathways triggering EMT are promoted by OS. The possible interconnections between these two signalling routes are discussed here. From a broader perspective, the control of NFE2L2 and EMT as ways of preventing OS-derived cellular damage could be potentially valuable in the therapy of AMD. [ABSTRACT FROM AUTHOR]

Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1-3), all producing an identical polymer from UDP-N-acetylglucosamine and UDPglucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1-3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ~10-fold with 1 mM glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mM had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDPsugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50- fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars. [ABSTRACT FROM AUTHOR]

All-trans retinoic acid (RA) compromises epidermal differentiation and causes keratinocyte hyperproliferation through mechanisms not completely understood, but may involve the regulatory matrix molecule hyaluronan. In this work, the influences of all-trans RA on epidermal morphology and hyaluronan metabolism were examined in organotypic and monolayer cultures of rat epidermal keratinocytes (REKs). All-trans RA treatment of organotypic REK cultures (10 days) increased the synthesis of hyaluronan, the expression of hyaluronan synthases Has2 and Has3, and the CD44 receptor, with hyperplasia of the epidermis. The hyperplasia and hyaluronan production induced by all-trans RA were blocked with (1) AG1478, an inhibitor of the EGFR; (2) UO126, an inhibitor of the MAPK/ERK kinase, and (3) GM6001, an inhibitor of the matrix metalloproteinases. These effects were consistent with the findings that all-trans RA upregulated heparin-binding epidermal growth factor-like growth factor mRNA expression and increased the phosphorylation of EGFR and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, the activation of EGFR and ERK1/2 was seen already 30 minutes after all-trans RA treatment, suggesting that the activation of this signaling pathway is a primary response to all-trans RA. These results indicate that the effects of all-trans RA on keratinocyte proliferation and hyaluronan synthesis are partly mediated through EGFR signaling.Journal of Investigative Dermatology (2008) 128, 797–807. doi:10.1038/sj.jid.5701098; published online 18 October 2007 [ABSTRACT FROM AUTHOR]

Hyaluronan is a multifunctional glycosaminoglycan up to 107 Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4–5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a progressive sight-impairing disease of the elderly. The pathogenic mechanisms of AMD are not well understood although both genetic and many environmental factors have been associated with the development of AMD. One clinical hallmark of AMD is the detrimental aggregation of damaged proteins. Recently, it has been suggested that the weakening of autophagy clearance is an important mechanism in the pathogenesis of AMD. Autophagy is important in the removal of damaged or no longer needed cellular material and its recycling. A considerable number of autophagy-targeting microRNAs (miRNAs), small RNA molecules and epigenetic regulators have been found to be either up- or down-regulated in AMD patients and experimental models. The important role of autophagy-targeting miRNAs is supported by several studies and can open the prospect of the use of these miRNAs in the therapy for AMD., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Age-related macular degeneration is an eye disease that is the main cause of legal blindness in the elderly in developed countries. Despite this, its pathogenesis is not completely known, and many genetic, epigenetic, environmental and lifestyle factors may be involved. Vision loss in age-related macular degeneration (AMD) is usually consequence of the occurrence of its wet (neovascular) form that is targeted in the clinic by anti-VEGF (vascular endothelial growth factor) treatment. The wet form of AMD is associated with the accumulation of cellular waste in the retinal pigment epithelium, which is removed by autophagy and the proteosomal degradation system. In the present work, we searched for the association between genotypes and alleles of single nucleotide polymorphisms (SNPs) of autophagy-related genes and wet AMD occurrence in a cohort of Finnish patients undergoing anti-VEGF therapy and controls. Additionally, the correlation between treatment efficacy and genotypes was investigated. Overall, 225 wet AMD patients and 161 controls were enrolled in this study. Ten SNPs (rs2295080, rs11121704, rs1057079, rs1064261, rs573775, rs11246867, rs3088051, rs10902469, rs73105013, rs10277) in the mTOR (Mechanistic Target of Rapamycin), ATG5 (Autophagy Related 5), ULK1 (Unc-51-Like Autophagy Activating Kinase 1), MAP1LC3A (Microtubule Associated Protein 1 Light Chain 3 α), SQSTM1 (Sequestosome 1) were analyzed with RT-PCR-based genotyping. The genotype/alleles rs2295080-G, rs11121704-C, rs1057079-C and rs73105013-T associated with an increased, whereas rs2295080-TT, rs2295080-T, rs11121704-TT, rs1057079-TT, rs1057079-T, rs573775-AA and rs73105013-C with a decreased occurrence of wet AMD. In addition, the rs2295080-GG, rs2295080-GT, rs1057079-TT, rs11246867-AG, rs3088051-CC and rs10277-CC genotypes were a positively correlated cumulative number of anti-VEGF injections in 2 years. Therefore, variability in autophagy genes may have an impact on the risk of wet AMD occurrence and the efficacy of anti-VEGF treatment.

Increased oxidative stress and mitochondrial damage are observed in protein aggregation diseases, such as age-related macular degeneration (AMD). We have recently reported elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in the retinal pigment epithelial cells (RPE) of the dry AMD-resembling NFE2L2 / PGC1α double knockout (dKO) mouse model. Here, we provide evidence of a disturbance in the autolysosomal machinery handling mitochondrial clearance in the RPE cells of one-year-old NFE2L2 / PGC1α -deficient mice. Confocal immunohistochemical analysis revealed an upregulation of autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as numerous mitophagy markers, such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN) together with damaged mitochondria. However, we detected no evidence of increased autolysosome formation in transmission electron micrographs or of colocalization of lysosomal marker LAMP2 (lysosome-associated membrane protein 2) and the mitochondrial marker ATP synthase β in confocal micrographs. Interestingly, we observed an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells together with autofluorescence aggregates. Our results reveal that there is at least a relative decrease of mitophagy in the RPE cells of NFE2L2 / PGC1α dKO mice. This further supports the hypothesis that mitophagy is a putative therapy target in AMD-like pathology.

Age-related macular degeneration (AMD) is a complex eye disease in which decline in autophagy leads to the accumulation of sequestosome 1/p62 (SQSTM1/p62)-labeled waste material inside the retinal pigment epithelial (RPE) cells, and the condition results in activation of the inflammasome signaling and IL-1β secretion. Here, we have studied the role of SQSTM1/p62 in the production of IL-6, IL-8, and MCP-1 in the presence or absence of IL-1β. SQSTM1/p62 was either overexpressed or silenced in ARPE-19 cells, which were then exposed to IL-1β. Alternatively, bafilomycin A was used to demonstrate the functional decline of autophagy with increased SQSTM1/p62 levels. The protein concentration of SQSTM1/p62 was measured using the western blot technique, and interleukin levels were determined by ELISA. In IL-1β-loaded RPE cells, SQSTM1/p62 depletion and overexpression increased the production of MCP-1 and IL-8, respectively. Neither knock-down nor overexpression of SQSTM1/p62 induced the release of IL-6. Our data suggest that SQSTM1/p62 is a significant factor in inflammatory responses, especially following the inflammasome activation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In this review we will discuss the links between autophagy, a mechanism involved in the maintenance of cellular homeostasis and controlling cellular waste management, and the DNA damage response (DDR), comprising various mechanisms preserving the integrity and stability of the genome. A reduced autophagy capacity in retinal pigment epithelium has been shown to be connected in the pathogenesis of age-related macular degeneration (AMD), an eye disease. This degenerative disease is a major and increasing cause of vision loss in the elderly in developed countries, primarily due to the profound accumulation of intra- and extracellular waste: lipofuscin and drusen. An abundance of reactive oxygen species is produced in the retina since this tissue has a high oxygen demand and contains mitochondria-rich cells. The retina is exposed to light and it also houses many photoactive molecules. These factors are clearly reflected in both the autophagy and DNA damage rates, and in both nuclear and mitochondrial genomes. It remains to be revealed whether DNA damage and DDR capacity have a more direct role in the development of AMD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Processing of misfolded proteins is important in order for the cell to maintain its normal functioning and homeostasis. Three systems control the quality of proteins: chaperone-mediated refolding, proteasomal degradation of ubiquitinated proteins, and finally, when the two others fail, aggrephagy, as selective form of autophagy, degrades ubiquitin-labelled aggregated cargos. In this route misfolded proteins gradually form larger aggregates, aggresomes and they eventually become double membrane-wrapped organelles called autophagosomes, which become degraded when they fuse to lysosomes, for reuse by the cell. The stages, the main molecules participating in the process, and the regulation of aggrephagy are discussed here, as is the role of protein aggregation in protein accumulation diseases. In particular, we emphasize that both Alzheimer's disease and age-related macular degeneration, two of the most common pathologies in the aged, are characterized by altered protein clearance and deposits. Based on the hypothesis that manipulations of autophagy may be potentially useful in these and other aggregation-related diseases, we will discuss some promising therapeutic strategies to counteract protein aggregates-induced cellular toxicity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Purpose: In this work, we investigated the ability of pinosylvin (PS), 3,5-dihydroxy-trans-stilbene, to modulate oxidative stress in human RPE cells. PS, a stilbenoid polyphenol, occurs in high concentrations in bark byproducts and therefore represents an attractive bioactive compound for health-promoting applications., Methods: First, we evaluated the toxicity range of PS by exposing ARPE-19 cells to 0.1-200 µM concentrations of PS for 24 h followed by the cell viability test. In the next stage, the ARPE-19 cells were preincubated in PS for 24 h followed by hydroquinone (HQ) exposure without PS for another 24 h. The cell viability test was conducted after HQ exposure. To elucidate the potential mechanisms behind PS-mediated protection against oxidative stress, the ARPE-19 cells were treated with 5 µM PS for 6 h, and mRNA was extracted at four time points (2 h, 6 h, 12 h, 24 h) to determine changes in the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), sequestosome 1 (p62/SQSTM1), heme oxygenase-1 (HO-1), and glutathione S-transferase pi 1 (GSTP1) genes. To clarify the molecular mechanism behind PS-mediated protection further, the ARPE-19 cells were transfected with p62 and Nrf2 siRNAs for 24 h, and the roles of p62, Nrf2, and its target gene HO-1 in conferring protection against oxidative stress were studied with quantitative real-time PCR (qRT-PCR) and the cell viability test., Results: PS treatment at concentrations of 5 and 10 µM significantly enhanced cell survival from oxidative stress. The expression levels of an enzyme with antioxidative, anti-inflammatory, and immunomodulatory properties, HO-1, were increased by PS treatment and correlated strongly with cell survival. PS treatment did not elevate the expression levels of Nrf2 or its target genes, p62 or GSTP1, even though it had a clear effect on the expression of HO-1, another gene controlled by Nrf2. RNA interference analysis further confirmed the important role of Nrf2 and HO-1 in PS-mediated protection against oxidative stress whereas the role of p62 seemed to be insignificant at the gene expression and cell viability levels., Conclusions: Our results suggest that PS treatment conferred protection against oxidative stress through the induction of HO-1 in human RPE cells. Consequently, PS-stilbene compounds, which can be isolated in significant amounts from bark waste, may possess health-promoting properties against aging-related diseases associated with oxidative stress such as age-related macular degeneration (AMD) and Alzheimer's disease. These natural compounds may offer opportunities for high-value use of bark waste in diverse health-related applications.

Macroautophagy is an important route in cellular maintenance, in the breakdown and reuse of intracellular materials. It is closely related to endocytosis, the means by which the cell can absorb extracellular material, as both macroautophagy and endocytosis have converging steps and common participating molecules. The point where autophagosomes and endosomes fuse with lysosomes to permit for the final degradation of their contents is important. One of the most substantial molecules in the maturation of autophagosomes/endosomes is Rab7, a member of small GTPases. Rab7 designates the maturation of endosomes and also autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes. Rab7 is an effective multifunctional regulator of autophagy and endocytosis. Since many aggregation-based diseases, e.g. age-related macular degeneration of the eye (AMD) and Alzheimer's disease are due of malfunctioning in the autophagic process, the management of Rab7 activity might hold potential as a therapeutic target against these diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Age-related macular degeneration (AMD) is the most common reason for blindness in developed countries. AMD essentially involves chronic oxidative stress, increased accumulation of lipofuscin in retinal pigment epithelial (RPE) cells, and extracellular drusen formation, as well as presence of chronic inflammation in the retina. The capacity to prevent the accumulation of cellular cytotoxic protein aggregates is decreased in senescent cells, which may evoke lipofuscin accumulation into lysosomes in postmitotic RPE cells. The formation of lipofuscin, in turn, decreases the lysosomal enzyme activity and impairs the autophagic clearance of damaged proteins destined for cellular removal. 5'-Adenosine monophosphate-activated protein kinase (AMPK) is a well-known inhibitor of mammalian target of rapamycin (mTOR) that subsequently evokes induction of autophagy. This review examines the novel potential therapeutic targets on the AMPK-mTOR axis and the ways in which autophagy clearance can suppress or prevent RPE degeneration and development of AMD.

Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Since estrogen and selective estrogen receptor modulators can inhibit inflammatory responses, we studied the regulatory role of several selective estrogen receptor modulators on interleukin-6 (IL-6) expression in human retinal pigment epithelial cells (ARPE-19). ARPE-19 cells were exposed to lipopolysaccharide with simultaneous exposure to different selective estrogen receptor modulators with the secretion of IL-6 cytokine being analyzed by enzyme-linked immunosorbent assay (ELISA). We demonstrate that 17beta-estradiol and HM-D, a novel selective estrogen receptor modulator compound, clearly reduced the IL-6 expression levels after lipopolysaccharide exposure in ARPE-19 cells. Molecular effects of selective estrogen receptor modulators and estrogen on the estrogen response element-mediated transcription were studied using MCF-7 and ARPE-19 cell lines carrying the estrogen response element-luciferase reporter gene. Estrogen and HM-D stimulated the activity of estrogen response element-reporter gene in MCF-7 cells but did not affect the activity in ARPE-19 cells. In addition, HM-D did not activate estrogen receptor alpha when studied by nuclear receptor peptide estrogen receptor alpha ELISA in ARPE-19 cells. These results indicate that estrogen and HM-D can suppress the lipopolysaccharide-induced inflammatory response but signalling is not mediated through estrogen response element transcription in human retinal pigment epithelial cells., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Purpose: The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress-induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the functional roles of p62 and HSP70 were evaluated in conjunction with proteasome inhibitor-induced autophagy in human RPE cells (ARPE-19)., Methods: The p62, HSP70, and ubiquitin protein levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay., Results: Proteasome inhibition evoked the accumulation of perinuclear aggregates that strongly colocalized with p62 and HSP70. The p62 perinuclear accumulation was time- and concentration-dependent after MG-132 proteasome inhibitor loading. The silencing of p62, rather than Hsp70, evoked suppression of autophagy, when related to decreased LC3-II levels after bafilomycin treatment. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently, we showed that hsp70 mRNA depletion increased cell death in ARPE-19 cells. Here, we demonstrate that p62 mRNA silencing has similar effects on cellular viability., Conclusions: Our findings open new avenues for understanding the mechanisms of proteolytic processes in retinal cells, and could be useful in the development of novel therapies targeting p62 and HSP70.

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.

Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor beta, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2'-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor beta reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor beta suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor beta through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation.