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Mechanism for Carbachol-Induced Secretion of Lacritin in Cultured Monkey Lacrimal Acinar Cells

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Mechanism for Carbachol-Induced Secretion of Lacritin in Cultured Monkey Lacrimal Acinar Cells Biochemistry and Molecular Biology Mechanism for Carbachol-Induced Secretion of Lacritin in Cultured Monkey Lacrimal Acinar Cells Ayumi Morimoto-Tochigi,1,2 Ryan D. Walkup,1,2 Emi Nakajima,1,2 Thomas R. Shearer,2 and Mitsuyoshi Azuma1,2 PURPOSE. Lacritin protein is highly expressed in the lacrimal acrimal acinar cells synthesize and secrete proteins into the gland, secreted into tear fluid, and detected only in primates. L tear fluid.1,2 Well-known tear proteins include lysozyme, The mechanism for lacritin secretion has not been fully inves- lactoferrin, lipocalin, and secretory IgA.3 Lysozyme was de- tigated, because a system for culturing primate lacrimal acinar creased in blepharitis patients,4 lactoferrin was lower in 5 cells had not been established. The purposes of the present Sjo¨gren and Stevens-Johnson syndromes, and reduced lipoca- study were (1) to develop a procedure to culture lacrimal lin has been found in patients with seborrheic blepharitis and 6 acinar cells from monkey and (2) to determine the mechanism meibomian gland dysfunction. Such observations have led to for the secretion of lacritin in the culture system. the concept that in addition to antibacterial function, certain tear proteins themselves can act as regulators of tear secretion, METHODS. Acinar cells from monkey lacrimal gland were cul- growth factors, or essential elements for renewal of ocular tured and characterized. Lacritin and other proteins were de- epithelia. tected by immunohistochemistry, immunocytochemistry, and Although less well studied, lacritin is the sixth most com- immunoblot analysis. Secreted proteins were also detected in mon mRNA in the National Eye Institute (NEI) human lacrimal the medium from stimulated acinar cells. mRNAs were deter- EST database and codes for a 12.3-kDa protein. Lacritin is mined by microarray and qPCR. Intracellular calcium levels highly expressed in human lacrimal and meibomian glands.7 were measured by calcium-4 assay. Monkey lacritin has also been cloned. It shows 89% amino acid RESULTS. Acinar cells cultured for 1 day contained adequate homology with human lacritin and is highly expressed in lac- rimal gland and moderately expressed in conjunctiva and mei- amounts of lacritin, lactoferrin, and lipocalin for use in lacritin 8 secretion studies. The cholinergic agonist carbachol (Cch) bomian gland. Lacritin orthologues have been reported in the Ensembl genome database (http://www.ensembl.org) in sev- stimulated the secretion of lacritin and increased intracellular 9 ϩ eral species. For example, the common shrew (Sorex ara- Ca2 . Cch-induced lacritin secretion was inhibited by the store- neus) and the cat (Felis catus) show 39% identity with human operated calcium (SOC) channel inhibitor YM58483 and the lacritin; however, to our knowledge, protein expression has PKC inhibitors GF109203 and Ro-32-0432. Cch-induced lacritin not been reported. Lacritin promotes protein secretion from secretion was not inhibited by MAPKK inhibitor U0126, al- cultured rat acinar cells10 and stimulates proliferation in cul- though p42/p44 MAPK was phosphorylated. Cch also en- tured human corneal cells.11,12 Lower levels of lacritin have hanced gene transcription, which was inhibited by U0126, been observed in blepharitis patients. These preliminary data GF109203, and calcium chelators. likewise suggest a role for lacritin in the maintenance of the CONCLUSIONS. Successful culture of monkey lacrimal acinar cells ocular surface and raise speculation that topical lacritin could showed that, among the prevalent tear proteins, the secretion be used in the treatment of dry eye.4 of lacritin involved the PKC/Ca2ϩ pathway, not the p42/p44 Monkey models are important for proof of concept, but the MAPK pathway. Induction of transcription by Cch involved the mechanism for secretion of lacritin is not clear, because no independent p42/p44 MAPK and PKC pathways. (Invest Oph- culture system for primate acinar cells has been established. thalmol Vis Sci. 2010;51:4395–4406) DOI:10.1167/iovs.09- Thus, the purposes of the present study were (1) to develop a 4573 culture procedure for acinar cells from monkey lacrimal gland and (2) to determine the mechanism for lacritin secretion in cultured monkey acinar cells. From the 1Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., Beaverton, Oregon; and the 2Department of Integrative MATERIALS AND METHODS Biosciences, Oregon Health and Science University, Portland, Oregon. TRS is a paid consultant for Senju Pharmaceutical Co., Ltd., a Experimental Animals company that may have a commercial interest in the results of this Lacrimal glands from rhesus monkeys (Macaca mulatta, 1–16 years of research and technology. MA and EN are employees of Senju Pharma- age) were obtained at necroscopy from the Oregon National Primate ceutical Co., Ltd. These potential conflicts of interest were reviewed, Research Center (Beaverton, OR) from protocols not related to the and management plans approved by the OHSU Conflict of Interest in Research Committee were implemented. present studies. Experimental animals were handled in accordance Submitted for publication September 1, 2009; revised December with the ARVO Statement for the use of Animals in Ophthalmic and 8, 2009, and February 10, 2010; accepted March 23, 2010. Vision Research and the Guiding Principles in the Care and Use of Disclosure: A. Morimoto-Tochigi, Senju Pharmaceutical Corp. Animals (DHEW Publication, NIH 80-23). (E); R.D. Walkup, Senju Pharmaceutical Corp. (E); E. Nakajima, Senju Pharmaceutical Corp. (E); T.R. Shearer, Senju Pharmaceutical Corp. Immunohistochemistry of Monkey Lacrimal Gland (C); M. Azuma, Senju Pharmaceutical Corp. (E) Corresponding author: Mitsuyoshi Azuma, Senju Laboratory of Lacrimal glands were fixed in formaldehyde and embedded in paraffin. Ocular Sciences, OHSU West Campus, 20000 NW Walker Road, Suite Three-micrometer sections were stained withH&E,orthey were JM508, Beaverton, OR 97006; [email protected]. blocked with 1% bovine serum albumin (BSA) in phosphate-buffered Investigative Ophthalmology & Visual Science, September 2010, Vol. 51, No. 9 Copyright © Association for Research in Vision and Ophthalmology 4395 Downloaded from iovs.arvojournals.org on 09/25/2021 4396 Morimoto-Tochigi et al. IOVS, September 2010, Vol. 51, No. 9 saline (PBS) for 1 hour at room temperature. The sections were then Immunoblot Analysis incubated for 2 hours at room temperature with primary antibodies for Denatured protein samples were separated on 4% to 12% Bis-Tris gels lactoferrin (dilutions: 1:200; Sigma-Aldrich), lacritin (1:200),8 lipocalin (NuPAGE; Invitrogen) with MES buffer (Invitrogen). The proteins were (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), or vesicle-associ- then electrotransferred from the gels to polyvinylidene fluoride (PVDF) ated membrane protein 2 (VAMP2, 1:100; Assay Designs, Ann Arbor, membranes (Millipore). The membranes were blocked with 5% skim MI). After the sections were rinsed with PBS, they were incubated with milk in Tris-buffered saline with 0.05% Tween 20 (TTBS) and incubated Alexa-Fluor 488-labeled anti-rabbit IgG (1:2000; Invitrogen Corp., overnight at 4°C with primary antibodies for lactoferrin (1:1000), Carlsbad, CA) or Alexa-Fluor 546-labeled anti-rabbit or anti-goat IgG lacritin (1:1000), lipocalin (1:100), GAPDH (1:1000; Sigma-Aldrich), (1:500; Invitrogen) for 1 hour at room temperature. Negative controls p42/p44 MAPK (1:1000; Cell Signaling Technology, Inc., Danvers, were treated in the same manner, but normal rabbit or goat IgG (1:200; MA), or phosphorylated p42/p44 MAPK (1:1000; Cell Signaling Tech- Santa Cruz Biotechnology) were used in place of the primary antibody. nology) in 1% BSA/TTBS. The membranes were then rinsed in TTBS Protocols for staining were the same as those for lacrimal gland tissue. and incubated for 1 hour at room temperature with HRP-conjugated Stained samples were photographed with an inverted microscope goat anti-rabbit secondary antibody (1:5000; Santa Cruz Biotechnology) (Axiovert 200, equipped with an AxioCam MRc5; Carl Zeiss Vision, or HRP-conjugated donkey anti-goat secondary antibody (1:5000; Santa GmbH, Hallbergmoos, Germany). Images were compiled in image Cruz Biotechnology). The protein bands were detected with chemilu- analysis software (Photoshop; Adobe, San Jose, CA). minescence (ECL Plus; GE Health Care Corp., Piscataway, NJ), and images were captured (FluorChem FC2 imager; Alpha Innotech Corp., Acinar Cell Culture and Characterization San Leandro, CA). Lacrimal acinar cells were isolated, as described previously,8 and cul- tured by using a modified protocol published by Hann et al.13 Collagen Translocation of PKC and Phosphorylated I has been shown to be a suitable matrix for culturing rat acinar cells.14 p42/p44 MAPK For this reason, monkey acinar cells in the present experiments were Subcellular fractionation was performed according to a published pro- plated on collagen I (0.01 mg/cm2; BD Biosciences, Franklin Lakes, tocol.15 The cells were sonicated in fractionation buffer containing 20 NJ)-coated plates with DMEM/Ham’s F12 (Invitrogen) containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 mM NaCl, 1 mM phenylmeth- ng/mL dexamethasone (Sigma-Aldrich.), 1 mM putrescine (Sigma-Al- ylsulfonyl fluoride, 1 mM dithiothreitol, phosphatase inhibitor cocktail drich), 50 ng/mL EGF (Invitrogen), 25 ␮g/mL L-ascorbic acid (Sigma- I and II, and protease inhibitor (Complete Mini-EDTA free; Roche, Aldrich), 1ϫ ITS (Invitrogen), and 25 ␮g/mL gentamicin (Invitrogen). Indianapolis, IN). The cytosol fraction was collected after centrifuga- For proliferation assays, the cells were plated at 1 ϫ 105 cells/well in tion at 16,000g for 40 minutes at 4°C. The pellets were rinsed twice 24-well plates, and the number of cells was counted daily with a with D-PBS, suspended in fractionation buffer containing 1% Triton hemocytometer, up to 5 days. To confirm proliferation, the acinar cells X-100, and sonicated. After 30 minutes’ incubation on ice, suspensions were stained with Ki-67 (1:1000; Abcam, Cambridge, UK). Acinar cells were centrifuged at 16,000g for 40 minutes at 4°C, to produce the were also stained with cytokeratin AE1/AE3 antibody (1:100; Millipore, supernatant membrane fraction.
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