Spatial expression of aquaporin 5 in mammalian cornea and lens, and regulation of its localization by phosphokinase A

Abstract

Purpose: Aquaporins (AQPs) play a significant role in the movement of water across the plasma membrane. In the eye, the cornea and lens are avascular with unique microcirculatory mechanisms to meet the metabolic demands. We have previously shown that AQP0 and AQP1 water channels participate in maintaining lens transparency and homeostasis. In the present investigation, we explored the expression and spatial distribution of AQP5 in the cornea and lens, and its regulation during membrane localization.

Methods: AQP5 expression and cellular localization were investigated by reverse transcription polymerase chain reaction (RT-PCR) using gene-specific primers, and by western blot and immunocytochemistry analyses using specific antibodies. AQP5 phosphorylation was studied using calf intestinal alkaline phosphatase for dephosphorylation. Effects of phosphokinase A (PKA) agonist cyclic AMP (cAMP), and antagonist H-89 on AQP5 expression and localization were studied in vitro using MDCK (Madin-Darby Canine Kidney) cells, and ex vivo using isolated corneas from wild type mice.

Results: RT-PCR revealed the presence of AQP5 transcripts in the cornea, lens epithelial cells and fiber cells. Western blotting identified the presence of both non-phosphorylated and phosphorylated forms of AQP5 protein. Immunostaining showed the distribution of AQP5 in the epithelial layer and stromal keratocytes of the cornea, and epithelial and fiber cells of the lens. In vitro and ex-vivo experiments revealed PKA-induced AQP5 internalization; PKA inhibition prevented such internalization.

Conclusions: This is the first report on the spatial expression of AQP5 in the corneal keratocytes and lens epithelial cells, as well as on the regulation of AQP5 localization by PKA in the corneal epithelial cells. PKA-mediated regulation of AQP5 holds promise for therapeutic intervention to control corneal and lens diseases.

Figure 1

Schematic diagram of mouse AQP5 transmembrane topology. NPA (blue circles) represents the highly conserved aquaporin signature sequence. H1–H6, membrane-spanning helices; A–E, loops; loops B and E form pore helices. NH₂- and COOH- amino and carboxyl terminal domains, respectively. Two consensus phosphorylation motifs are present, one at amino acid residues RRTSP at 153–157 in loop D and another, RKKT at 256–259 at the COOH-terminal domain.

Figure 2

Reverse-transcription polymerase chain reaction (RT–PCR) analysis of AQP5. A: In mouse lens. Lanes: 1 Wild type (WT) lacrimal gland (positive control), 2. AQP5 knockout (AQP5-KO) lacrimal gland, 3. WT cornea, 4. WT cornea + RNase, 5. AQP5-KO cornea, 6. WT lens epithelium, 7. AQP5-KO lens epithelium, 8. WT lens epithelium + RNase, 9. WT lens cortex, 10. WT lens cortex + RNase, 11. AQP5-KO lens cortex, MW-Molecular weight marker. B: RT–PCR analysis of AQP1 in mouse lens epithelial cells. Lanes: 1. WT lens epithelium, 2.WT lens epithelium + RNase, 3. AQP5-KO lens epithelium, 4. AQP5-KO lens epithelium + RNase, MW-Molecular weight marker. C: RT–PCR analysis of AQP0 in mouse lens fiber cells. Lanes: 1. WT lens cortex, 2. WT lens cortex + RNase, 3. AQP5-KO lens cortex, 4. AQP5-KO lens cortex + RNase, MW-molecular weight marker.

Figure 3

Immunoblot analyses of corneal and lens membrane proteins to identify the expression of AQP5. A: From rabbit: Lanes; 1. total cornea, 2. total lens. B: From mouse: Lanes: 1. lacrimal gland (+ve control), 2. total cornea, 3. total lens, 4. lens epithelial cell membrane, 5. lens cortex fiber cell membrane, 6. lens nuclear fiber cell membrane. C: AQP5-KO mouse lens fiber cell membrane proteins. Lanes: 1. AQP0, 2. AQP5, expressions studied using anti-AQP0 and anti-AQP5 antibodies, respectively. D: Immunoblot of dephosphorylation studies in WT corneal membrane proteins using anti-AQP5 antibody. Lanes: 1. WT untreated proteins (arrow - ~28 kDa; arrowhead - ~34 kDa), 2. WT proteins treated with calf intestinal alkaline phosphatase; the 34 kDa band disappeared, presumably due to dephosphorylation.

Figure 4

Immunolocalization of AQP5 in mouse cornea. A: Schematic diagram of a mammalian cornea showing the five layers. B: AQP5 localization (green) in central corneal epithelial cells and stromal keratocytes. C: AQP5 knockout mouse central corneal epithelial cells and stromal keratocytes showing lack of immunoreactivity. D: AQP5 localization (red) in the limbal area of the cornea; the window in D is enlarged and shown as E. E: AQP5 (red) in limbal stromal keratocytes. F: AQP5 knockout mouse corneal stromal keratocytes in the limbal area with no immunoreactivity. B, C: FITC conjugated secondary antibody. D, E, F: Texas Red conjugated secondary antibody; blue, nuclear stain DAPI. Epi: epithelium; Str: stroma; En: endothelium; arrows- antibody binding.

Figure 5

Immunolocalization of AQP5 protein in comparison with AQP1 or AQP0 in mouse lens. A: AQP1 expression in the WT lens anterior epithelial cells; a window of anterior epithelial cells is enlarged and shown below, in the same figure. B: AQP5 expression in the WT lens epithelial cells. C: (negative control), AQP5 knockout mouse lens section showing lack of immunoreactivity in the epithelial cells. D: WT lens outer cortex fiber cells with AQP5 expression. E: WT lens inner cortex fiber cells with AQP5 expression. F: AQP0 expression in the lens inner cortex fiber cells (very intense immunoreactivity compared to AQP5 expression shown in E). G: WT lens equatorial region showing AQP5 in the epithelial and fiber cells. H: The window shown in G is enlarged to provide a clear view of anti-AQP5 antibody binding to the narrow end of the fiber cells. I, J: AQP5 knockout mouse lens sections showing lack of immunoreactivity in the cells at the equatorial and anterior regions, respectively. A-J: FITC conjugated secondary antibody; green, antibody binding indicating AQP expression; blue, nuclear stain DAPI. White arrows – antibody binding. Yellow arrows – narrow side of the fiber cell; Red arrows – broader side of the fiber cell; A-C and G-J Sagittal sections; D-F: Cross sections.

Figure 6

Effects of PKA activator mp-cAMP or PKA inhibitor H-89 on AQP5 protein localization. MDCK cells expressing AQP5 were exposed to: mp-cAMP (A); H-89 (B); H-89 first and then stimulated with mp-cAMP (C). D-G: Mouse corneal epithelial AQP5 regulation studies in organ culture. Mouse eyes were dissected out and cultured in MEM containing 2% FBS with mp-cAMP (D, E) or H-89 (F, G) for 30 min (D, F) or 6 h (E, G). MDCK cells expressing AQP5 (A-C) and cryosections of cultured eyes were immunostained for corneal AQP5 expression (D-G). Epi: epithelium; Str: stroma.