Rong Li

MicroRNA-204/211 alters epithelial physiology

MicroRNA (miRNA) expression in fetal human retinal pigment epithelium (hfRPE), retina, and choroid were pairwise compared to determine those miRNAs that are enriched by 10‐fold or more in each tissue compared with both of its neighbors. miRs‐184, 187, 200a/200b, 204/211, and 221/222 are enriched in hfRPE by 10‐ to 754‐fold compared with neuroretina or choroid (P<0.05). Five of these miRNAs are enriched in RPE compared with 20 tissues throughout the body and are 10‐ to 20,000‐fold more highly expressed (P<0.005). miR‐204 and 211 are the most highly expressed in the RPE. In addition, expression of miR‐204/211 is significantly lower in the NCI60 tumor cell line panel compared with that in 13 normal tissues, suggesting the progressive disruption of epithelial barriers and increased proliferation. We demonstrated that TGF‐β receptor 2 (TGF‐βR2) and SNAIL2 are direct targets of miR‐204 and that a reduction in miR‐204 expression leads to reduced expression of claudins 10, 16, and 19 (message/protein) consistent with our observation that anti‐miR‐204/211 decreased transepithelial resistance by 80% and reduced cell membrane voltage and conductance. The anti‐miR‐204‐induced decrease in Kir7.1 protein levels suggests a signaling pathway that connects TGF‐βR2 and maintenance of potassium homeostasis. Overall, these data indicate a critical role for miR‐204/211 in maintaining epithelial barrier function and cell physiology.—Wang, F. E., Zhang, C., Maminishkis, A., Dong, L., Zhi, C., Li, R., Zhao, J., Majerciak, V., Gaur, A. B., Chen, S., Miller, S. S. MicroRNA‐204/211 alters epithelial physiology. FASEB J. 24, 1552–1571 (2010). www.fasebj.org

Control of Chemokine Gradients by the Retinal Pigment Epithelium

PURPOSEnProinflammatory cytokines in degenerative diseases can lead to the loss of normal physiology and the destruction of surrounding tissues. In the present study, the physiological responses of human fetal retinal pigment epithelia (hfRPE) were examined in vitro after polarized activation of proinflammatory cytokine receptors.nnnMETHODSnPrimary cultures of hfRPE were stimulated with an inflammatory cytokine mixture (ICM): interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma. Western blot analysis and immunofluorescence were used to determine the expression/localization of the cytokine receptors on hfRPE. Polarized secretion of cytokines was measured. A capacitance probe technique was used to measure transepithelial fluid flow (J(V)) and resistance (R(T)).nnnRESULTSnIL-1R1 was mainly localized to the apical membrane and TNFR1 to the basal membrane, whereas IFN-gammaR1 was detected on both membranes. Activation by apical ICM induced a significant secretion of angiogenic and angiostatic chemokines, mainly across the hfRPE apical membrane. Addition of the ICM to the basal but not the apical bath significantly increased net fluid absorption (J(V)) across the hfRPE within 20 minutes. Similar increases in J(V) were produced by a 24-hour exposure to ICM, which significantly decreased total R(T).nnnCONCLUSIONSnChemokine gradients across the RPE can be altered (1) through an ICM-induced change in polarized chemokine secretion and (2) through an increase in ICM-induced net fluid absorption. In vivo, both of these factors could contribute to the development of chemokine gradients that help mediate the progression of inflammation/angiogenesis at the retina/RPE/choroid complex.

IFNγ regulates retinal pigment epithelial fluid transport

The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.

Integrin α5β1 mediates attachment, migration, and proliferation in human retinal pigment epithelium: relevance for proliferative retinal disease.

PURPOSEnThe aim of this study was to determine the expression and localization of integrin alpha5beta1 in human retinal pigment epithelium (RPE) and its ability to modulate RPE cell attachment, proliferation, migration, and F-actin cytoskeleton distribution.nnnMETHODSnExpression and localization of alpha5beta1 were analyzed on human RPE by immunoblot/immunofluorescence. Polarized secretion of fibronectin was measured. RPE attachments to different substrates were determined using cell attachment screening kits. BrdU incorporation and wound-healing assays were used to test hfRPE proliferation and migration. F-actin cytoskeleton was visualized with phalloidin.nnnRESULTSnIntegrin alpha5beta1 was detected in native adult and fetal human RPE. The alpha5-subunit is predominantly localized at the apical membrane of hfRPE, whereas the beta1-subunit is uniformly detected at the apical/basolateral membranes. The authors also found that hfRPE cultures secrete significant amounts of fibronectin to the apical bath. JSM6427, a specific integrin alpha5beta1 antagonist, significantly inhibited hfRPE cell attachment to fibronectin, but not laminin, or collagen I or IV. JSM6427 also showed a strong inhibitory effect on bFGF, PDGF-BB, and serum-induced cell migration and proliferation. Furthermore, JSM6427 induced significant disruption of the F-actin cytoskeleton of dividing RPE cells but had no effect on quiescent cells.nnnCONCLUSIONSnThe apical localization of alpha5beta1 and the secretion of fibronectin to the apical bath suggest the presence of an autocrine loop that can guide the migration of RPE. The strong inhibitory effects of JSM6427 on human RPE cell attachment, proliferation, and migration is probably mediated by F-actin cytoskeletal disruption in proliferating cells and suggests a potential clinical use of this compound in proliferative retinopathies.

CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium

Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTFs effects on retinal pigment epithelial (RPE) physiology. Therefore, we determined the expression and localization of CNTF receptors and the physiological consequence of their activation in primary cultures of human fetal RPE (hfRPE). Cultured hfRPE express CNTF, CT1, and OsM and their receptors, including CNTFRα, LIFRβ, gp130, and OsMRβ, all localized mainly at the apical membrane. Exogenous CNTF, CT1, or OsM induces STAT3 phosphorylation, and OsM also induces the phosphorylation of ERK1/2 (p44/42 MAP kinase). CNTF increases RPE survivability, but not rates of phagocytosis. CNTF increases secretion of NT3 to the apical bath and decreases that of VEGF, IL8, and TGFβ2. It also significantly increases fluid absorption (J V) across intact monolayers of hfRPE by activating CFTR chloride channels at the basolateral membrane. CNTF induces profound changes in RPE cell biology, biochemistry, and physiology, including the increase in cell survival, polarized secretion of cytokines/neurotrophic factors, and the increase in steady-state fluid absorption mediated by JAK/STAT3 signaling. In vivo, these changes, taken together, could serve to regulate the microenvironment around the distal retinal/RPE/Bruchs membrane complex and provide protection against neurodegenerative disease.

Retinal Pigment Epithelium: Cytokine Modulation of Epithelial Physiology

We have developed a robust and well-defined primary cell culture model of human fetal retinal pigment epithelium (hfRPE). This model was used to analyze how metabolic waste products produced in the retina can be disposed of by CO2/HCO3 and lactate transporters in the apical and basolateral cell membranes. It was also used to analyze RPE antioxidant mechanisms protective against disease processes, such as age-related macular degeneration or uveitis, and to define the impact of cytokines and, in particular, interferon gamma, on RPE function and physiology.