Tuomas Ryhänen

Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy‐mediated proteolysis in human retinal pigment epithelial cells

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.

Efflux Protein Expression in Human Retinal Pigment Epithelium Cell Lines

PurposeThe objective of this study was to characterize efflux proteins (P-glycoprotein (P-gp), multidrug resistance proteins (MRP1–6) and breast cancer resistance protein (BCRP)) of retinal pigment epithelium (RPE) cell lines.MethodsExpression of efflux proteins in two secondary (ARPE-19, D407) and two primary (HRPEpiC and bovine) RPE cell lines was measured by quantitative RT-PCR and western blotting. Furthermore, activity of MRP1 and MRP5 of ARPE-19 cell line was assessed with calcein-AM and carboxydichlorofluorescein (CDCF) probes.ResultsSimilar efflux protein profile was shared between ARPE-19 and primary RPE cells, whereas D407 cell line was notably different. D407 cells expressed MRP2 and BCRP, which were absent in other cell lines and furthermore higher MRP3 transcript expression was found. MRP1, MRP4 and MRP5 were identified from all human RPE cell lines and MRP6 was not expressed in any cell lines. The pattern of efflux protein expression did not change when ARPE-19 cells were differentiated on filters. The calcein-AM and CDCF efflux tests provided evidence supporting MRP1 and MRP5 activity in ARPE-19 cells.ConclusionsMRP1, MRP4 and MRP5 are the main efflux transporters in RPE cell lines. There are differences in efflux protein expression between RPE cell lines.

Geldanamycin increases 4-hydroxynonenal (HNE)-induced cell death in human retinal pigment epithelial cells

Development of age-related macular degeneration (AMD) is associated with functional abnormalities and cell death in retinal pigment epithelial (RPE) cells attributable to oxidative stress. To minimize the adverse effects of oxidative stress, cells activate their defence systems, e.g., via increased expression of heat shock protein (Hsp), activation of stress sensitive AP-1 and NF-kappaB transcription factors. In this study, we examined the accumulation of Hsp70 protein, activation of AP-1 and NF-kappaB transcription factors in human ARPE-19 cells subjected to a 4-hydroxynonenal (HNE)-induced oxidative stress. In addition, the influence of Hsp90 inhibitor geldanamycin (GA) was studied in HNE-treated cells. Mitochondrial metabolic activity and apoptosis were determined to evaluate cell death in the ARPE-19 cells. The ARPE-19 cells showed increased accumulation of Hsp70 protein before of the cytotoxic hallmarks appearing in response to HNE. In contrast, increased DNA-binding activities of AP-1 or NF-kappaB transcription factors were not seen under HNE insults. Interestingly, GA significantly increased cell death in the HNE-treated cells, which was involved in caspase-3 independent apoptosis. This study reveals that the Hsps have an important role in the cytoprotection of RPE cells subjected to HNE-derived oxidative stress.

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE -derived diseases, drug testing and targeted drug therapy.

Radicicol but not geldanamycin evokes oxidative stress response and efflux protein inhibition in ARPE-19 human retinal pigment epithelial cells

Drug delivery to retinal cells has represented a major challenge for ophthalmologists for many decades. However, drug targeting to the retina is essential in therapies against retinal diseases such as age-related macular degeneration, the most common reason of blindness in the developed countries. Retinal cells are chronically exposed to oxidative stress that contributes to cellular senescence and may cause neovascularization in the most severe age-related macular degeneration cases. Various pre- and clinical studies have revealed that heat shock protein 90 (HSP90) inhibitors, such as geldanamycin and radicicol, are promising drugs in the treatment of different malignant processes. In this study, our goal was to compare the effects of 0.1 microM, 1 microM or 5 microM geldanamycin or radicicol on the oxidative stress response, cytotoxicity, and efflux protein activity (a protein pump which removes drugs from cells) in ARPE-19 (human retinal pigment epithelial, RPE) cells. Our findings indicate that geldanamycin and radicicol increased HSP70 and HSP27 expression analyzed by western blotting. Cellular levels of protein carbonyls were increased in response to 0.1 microM (P=0.048 for 24 h, P=0.018 for 48 h) or 5 microM (P=0.030 for 24 h, P=0.046 for 48 h) radicicol but not to geldanamycin analyzed by ELISA assay. In addition, HNE-protein adducts were accumulated in the RPE cells exposed to 0.1 microM or 5 microM radicicol but not to geldanamycin analyzed by western blotting. However, MTT assay revealed that 5 microM geldanamycin reduced cellular viability 20-30% (P<0.05 for 24 h, P<0.01 for 48 h), but this was not observed at any radicicol concentration in RPE cells. Interestingly, the increased oxidative stress response was associated with efflux protein inhibition (20-30%) when the cells were exposed to 1 microM or 5 microM (P<0.05) radicicol, but not in geldanamycin-treated RPE cells. These novel findings help in understanding the influence of HSP90 inhibition and regulatory mechanisms of drug delivery to retinal cells.

Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells

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.

Influence of Hsp90 and HDAC inhibition and tubulin acetylation on perinuclear protein aggregation in human retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein-dependent retrograde trafficking to a juxtanuclear location. We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation. These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration.

Increased IL-6 levels are not related to NF-κB or HIF-1α transcription factors activity in the vitreous of proliferative diabetic retinopathy

PURPOSE The purpose was to assess the activity of nuclear factor (NF)-κB and hypoxia inducible factor (HIF)-1α transcription factors and the expression levels of inflammation markers [interleukin (IL)-6 and IL-8] in the vitreous of patients suffering from proliferative diabetic retinopathy (PDR) scheduled for elective vitreous surgery in a single academic-based retina practice in a prospective clinical study. METHODS Twenty-seven patients with PDR were enrolled in the study. The severity of retinopathy was classified (0, 1, 2, 3, 4) and the activity of neovascularization was graded (0, 1, 2, 3, 4) by the surgeon intraoperatively. Samples of the vitreous were collected during surgery, and the activity of NF-κB and HIF-1α transcription factors and the expression levels of IL-6 and IL-8 were measured. RESULTS The majority of samples fell into the retinopathy class 3 (n = 12) or 4 (n = 13). The level of IL-6 increased from 68.9 ± 46.8 pg/ml to 102.7 ± 94.1 pg/ml, and IL-8 increased from 165.1 ± 136.0 pg/ml to 521.0 ± 870.9 pg/ml (mean ± S.D., nonsignificant change: normality test followed with Mann-Whitney Rank Sum Test). According to the neovascularization activity, the samples fell into grade 1 (n = 7), 2 (n = 12) or 3 (n = 7). In IL-6, there was a statistically significant increase (P < .05) from grade 2 to 3: 58.6 ± 40.3 pg/ml and 158.4 ± 102.5 pg/ml, respectively (Kruskal-Wallis One-Way Analysis of Variance on Ranks followed with Dunns Method). The level of IL-8 was as follows: in grade 1: 118.0 ± 62.4 pg/ml, in grade 2: 192.3 ± 127.1 pg/ml and in grade 3: 884.3 ± 1161.0 pg/ml (statistically nonsignificant change). There was a statistically significant linear regression between IL-6 and IL-8 (P < .001): IL-6 = 51.88 pg/ml + (0.092*IL-8), r = 0.772. Increased activity of the NF-κB and HIF-1α transcription factors was not observed. CONCLUSION Interleukin-6 is a candidate to indicate activity of neovascularization process in PDR. It might be a new molecular therapeutic target to regulate innate immunity response in vitreous.

Influence of selective estrogen receptor modulators on interleukin-6 expression in human retinal pigment epithelial cells (ARPE-19)

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.

Cis‐urocanic acid, a novel anti‐inflammatory and cytoprotective drug, decreases effectively UVB‐induced IL‐6 secretion and cytotoxicity in human corneal and conjunctival epithelial cells in vitro

Purpose Urocanic acid (UCA) is a major UV-absorbing chromophore in the epidermis and has been suggested to act as one of the initiators of UV-induced immunosuppression. The anti-inflammatory and cytoprotective effects of cis-UCA were studied in human corneal and conjunctival epithelial cells in response to UVB-irradiation in vitro. Methods Human corneal epithelial cells (HCE-2) and human conjunctival epithelial cells (HCEC) were exposed to 10, 100, 1000, and 5000 µg/ml concentrations of cis-UCA (BioCis Pharma, Turku, Finland) with and without UVB-radiation (4 x Philips TL 20W/12 lamps; total irradiation dose 153 mJ/cm2). Secreted interleukin-6 (IL-6) levels were analyzed with ELISA assay. Cell viability was measured by a colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results The 100 µg/ml and 1000 µg/ml concentrations of cis-UCA significantly suppressed IL-6 secretion induced by UVB-irradiation in both cell types. In addition, the same concentrations improved the viability of the UVB-irradiated cells when analyzed by MTT assay. No significant alterations in IL-6 expression levels or viability were observed in response to 10, 100, and 1000 µg/ml cis-UCA only, while 5000 µg/ml cis-UCA evoked cytotoxicity in both cell types. Conclusion Our findings suggest that cis-UCA is a promising novel drug to suppress UVB-induced inflammation and cellular damage in human corneal and conjunctival epithelial cells.