CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium Rong Li 1 , Rong Wen 2 , Tina Banzon 1 , Arvydas Maminishkis 1 , Sheldon S. Miller 1 * 1 National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida, United States of America Abstract Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTF’s 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 CNTFRa, LIFRb, gp130, and OsMRb, 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 TGFb2. 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/Bruch’s membrane complex and provide protection against neurodegenerative disease. Citation: Li R, Wen R, Banzon T, Maminishkis A, Miller SS (2011) CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium. PLoS ONE 6(9): e23148. doi:10.1371/journal.pone.0023148 Editor: Shukti Chakravarti, Johns Hopkins University, United States of America Received March 25, 2011; Accepted July 7, 2011; Published September 2, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This work was supported by the Intramural Research Program of the National Institutes of Health (SSM, RL, TB and AM) and National Institutes of Health grant R01-EY018586 (RW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: millers@nei.nih.gov Introduction Ciliary neurotrophic factor (CNTF) was discovered in chicken embryo extract where one third of its neurotrophic activity originated in the eye [1]. It has demonstrated a remarkable capacity for neuroprotection of rod photoreceptors in numerous retinal degeneration models across several species [2,3,4,5,6,7]. Recently, CNTF was found to promote cone outer segment regeneration and protect cone photoreceptors [8]. In addition, retinal injuries induce upregulation of CNTF, which is thought to protect photoreceptors [9,10,11]. These studies led to several CNTF clinical trials for retinal degenerative disorders. In a phase I clinical trial, CNTF-secreting implants (ECT) resulted in improved visual acuity in several patients with late stages of retinitis pigmentosa (RP) [12]. A recent clinical trial found that CNTF implant stabilizes vision loss in patients with geographic atrophy [13]. CNTF implants also prevent secondary cone degeneration in patients with RP [14]. CNTF is a member of the interleukin-6 (IL6) family of neuropoietic cytokines that includes IL11, leukemia inhibitory factor (LIF), oncostatin M (OsM), and cardiotrophin 1 (CT1) [15]. It binds to a receptor complex of three components: CNTF receptor a (CNTFRa) and two signal-transducing transmembrane subunits, LIF receptor b (LIFRb) and gp130. The schematic diagram in Figure 1 shows that LIFRb/gp130 heterodimers are shared by CNTF, CT1, and OsM, which can also signal through gp130/OsMRb receptor complex [16]. Previous studies suggested the absence of LIFRb in RPE resulting in a lack of responsiveness to CNTF (Song Y, et al. IOVS 2003;44:ARVO E-Abstract 390). However, the development of a human culture model of RPE that closely mimics native human adult RPE and the tight anatomical relationship between RPE and photoreceptors prompted us to reexamine the role of CNTF in RPE physiology. The RPE is a monolayer of polarized epithelial cells strategically located between the photoreceptors and the choroidal blood supply in the posterior part of the eye. It selectively transports ions, fluid, nutrients, and metabolic waste products between the neuronal retina and the choriocapillaris [17] and helps maintain the health and integrity of the distal retina by regulating the chemical composition and volume of the extracellular spaces that face the RPE apical and basolateral membranes [18,19,20]. Equally important are its roles in the visual cycle [21] and in photoreceptor outer segment renewal [22]. In the present study, we used primary cultures of hfRPE to understand how CNTF can regulate RPE function. These cultures molecularly and physiologically mimic native human tissue [17,18,19,23,24,25]. We report here that the apical membrane of hfRPE expresses all of the CNTF receptor subunits mediating binding and signal transduction. Activation of these receptors increases RPE survival. In addition, CNTF increases active, ion- linked fluid absorption across the RPE while modulating the polarized secretion of neurotrophic factors and cytokines to the apical bath. All of these responses, taken together, likely contribute to the protective effect of CNTF on photoreceptors [14,26,27]. PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e23148