Biochemistry and Molecular Biology Mechanism for Carbachol-Induced of 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 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 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% 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 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 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

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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. After measurement of protein concen- Billerica, MA) to test for purity of the acinar cell cultures and absence trations with a protein assay (Bio-Rad), denatured proteins in LDS of fibroblast contamination. For immunocytochemistry of tear pro- sample buffer were applied to 4% and 12% Bis-Tris gels (NuPAGE; teins, the cells were plated at 2 ϫ 105 cells/well in 12-well plates, fixed Invitrogen) in MOPS buffer (Invitrogen). The protocol for immunoblot with Ϫ20°C 100% methanol, incubated for 3 minutes at Ϫ20°C, rinsed analysis was the same as just described, except anti-rabbit PKC␣,-␦, with PBS, and permeabilized for 15 minutes with 0.1% Triton X-100 in and -␨ antibodies (Santa Cruz Biotechnology) were used at 1:200 PBS. Protocols for staining were the same as described for lacrimal dilution. To test the nuclear translocation of phospho-p42/p44 MAPK, gland tissue. the cells were induced for 5 minutes with Cch and stained for immu- For measurement of tear proteins, acinar cells were plated at 2 ϫ nocytochemistry with a primary antibody against phospho-p42/p44 105 cells/well in 12-well plates. At the end of the culture period, the MAPK (1:1000). cells were harvested into RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25% sodium deoxycholate, 0.5% NP-40, 1 mM RNA Extraction and Reverse Transcription EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovana- Total RNA was extracted from cultured acinar cells (Trizol reagent; date, protease inhibitor (complete Mini-EDTA free; Roche Diagnostics Invitrogen), according to the manufacturer’s instructions. After phase Corp., Indianapolis, IN), and phosphatase inhibitor cocktail I and II separation, RNA was precipitated with 75% EtOH, bound to a fiberglass (EMD Biosciences Inc., San Diego, CA). The cells were then lysed by filter, washed, and collected with an RNA-extraction kit (RNAqueous; sonication, and protein concentrations were determined with a pro- Ambion, Austin, TX). RNA quality was determined on a bioanalyzer tein assay (Bio-Rad, Hercules, CA) with BSA standards. (model 2100; Agilent Technologies, Palo Alto, CA). Total RNA was treated with DNase (DNA-free; Ambion) according to the manufactur- Measurement of Secreted Proteins er’s protocol, with 1 U/␮L RNase inhibitor (SUPERase-In; Ambion). The concentration of total RNA was determined (RiboGreen reagent; In- Cells were plated at 4 ϫ 105 cells/well in 12-well plates or 1 ϫ 106 vitrogen), and RNA was reverse transcribed at a concentration of 1 cells/well in 6-well plates and cultured for 1 day. Protein secretion was ng/␮L with 10 U/␮L reverse transcriptase (SuperScript II; Invitrogen), induced by incubating the cells with carbachol (Cch; Sigma-Aldrich), according to the manufacturer’s instructions. For qPCR for NR4A1, PMA (EMD Biosciences) or ionomycin (EMD Biosciences) for 10 min- IL-6, and PTGS2, the DNase reaction was not performed, because utes at 37°C. The signaling pathways were tested by pretreating the probes crossed exon boundaries. The optimized RT reaction condi- cells for 30 minutes with the M1/M3 receptor antagonist atropine tions for the were 20 ␮M dNTP, 2 U/␮L reverse transcriptase (Sigma-Aldrich), the nicotinic receptor antagonist mecamylamine (SuperScript II; Invitrogen), and 0.5 U/␮L RNase inhibitor (SUPERase- (Sigma-Aldrich), the extracellular calcium chelator EGTA (Sigma-Al- In; Ambion) in 1ϫ first-strand buffer. drich), the intracellular calcium chelator BAPTA-AM (Invitrogen), the IP3 receptor antagonist 2-APB (Sigma-Aldrich), the SOC channel inhib- itor YM58483 (Sigma-Aldrich), the MAPKK inhibitor U0126 (Sigma- Quantitative Real-Time PCR Aldrich), and the PKC inhibitors GF109203 (EMD Biosciences) and Custom primers and probes for Rhesus macaque lacritin, lactoferrin, Ro-32-0432 (EMD Biosciences). Detached cells were removed by cen- and lipocalin were developed by Applied Biosystems (ABI; Foster City, trifugation, and the culture medium was concentrated and subjected to CA) by using gene expression assays (TaqMan; ABI; Table 1). Primers immunoblot analysis. and probes for IL-6 (Rh02621719_u1) and PTGS2 (Rh01573476_m1)

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TABLE 1. Primers and Probes for Quantitative PCR Quantification of gene transcripts in acinar culture samples was determined using the Pfaffl method16 for normalization to 18s rRNA or Lacritin GAPDH reference genes. PCR efficiencies for genes were determined ؅ ؅ Forward 5 -CCT CAA GCA GGC AGG AAC TA-3 to be similar and comparable, and the limit of detection for each Reverse 5؅-TCT TGC AAA GGC TTG TTC TGT TAG T-3؅ primer and probe pair was determined to be well under the reported Probe 5؅-CCC CCT GAA ATC CA-3؅ Lactoferrin expression amounts in each sample. For lacritin, lactoferrin, and li- Forward 5؅-GGT CCA CCT GAG GCC ATT G-3؅ pocalin, normalized expression levels were converted to the reported Reverse 5؅-ACA CAG CTG GCT GAG AAG AAC-3؅ absolute copy numbers relative to a standard curve of known serial Probe 5؅-CTG GCC ACA GCT GCC T-3؅ dilutions of a gel-purified and quantified PCR product (Quant-iT DNA Lipocalin HS kit; Invitrogen). Forward 5؅-CCA CCT CCT GGC CTC AGA-3؅ Reverse 5؅-CGT CAT GGC CTT CAG ATA CCA-3؅ Intracellular Calcium Measurements Probe 5؅-CCT GAC ACA TCC TGA ATC T-3؅ NR4A1 Acinar cells were seeded at a density of 3 ϫ 104 cells/well in 96-well Forward 5؅-GCA CTG CCA AAC TGG ACT AC-3؅ plates and cultured for 24 hours. The cells were then incubated at 37°C Reverse 5؅-CCC AGC ATC TTC CTT CCC AAA G-3؅ for 1 hour with the calcium-binding fluorophore calcium-4 (Molecular -Probe 5؅-TCC AAG TTT CAG GAG CTG GTG C-3؅ Devices; Sunnyvale, CA) in loading buffer (Hanks’ balanced salt solu GAPDH tion with 20 mM HEPES [pH 7.4]). After 20 seconds of initial back- ؅ ؅ Forward 5 -TGC ACC ACC AAC TGC TTA-3 ground fluorescent measurements, Cch was added. Fluorescence mea- Reverse 5؅-CAT GAG TCC TTC CAC GAT ACC AA-3؅ surements were performed on a microplate reader (Flexstation; Probe 5؅-CCC TGG CCA AGG TCA TCC ATG A-3؅ Molecular Devices) every 1.75 seconds for 3 minutes. Some samples were pretreated with EGTA for 10 minutes or BAPTA-AM, 2-APB, or YM58483 for 30 minutes. Results are expressed as the ratio of maxi- were purchased from ABI. NR4A1 and GAPDH FAM-BHQ-1 primers mum fluorescence to the average baseline fluorescence in each sample and probes were created (RealTimeDesign Software; Biosearch Tech- (F /F ). nologies, Novato, CA) (Table 1). Primer and probe for 18s rRNA (cat. max 0 no. 4333760F) were obtained from ABI. 18s rRNA was used as a reference gene and normalization factor in qPCR for determining Gene Chip Analysis relative amounts of the transcripts for lacritin, lactoferrin, and lipoca- Samples for microarray analysis were prepared from acinar cells incu- lin. GAPDH was used as the reference gene in the NR4A1, IL-6, and bated with or without 30 ␮M Cch for 1 hour. RNA extraction was PTGS2 qPCR experiments. All assays except NR4A1 and GAPDH con- performed as described earlier. Subjects consisted of replicate mon- tained 250 nM probe, 900 nM each primer, 1ϫ PCR master mix (ABI), keys (7 and 9 years) with a normal and Cch-incubated sample from and 1 ng cDNA. Reactions for NR4A1 were optimized at 300 nM each animal. RNA was checked for quality (2100 Bioanalyzer; Agilent) forward/400 nM reverse primers and 250 nM probe. GAPDH was and submitted for amplification, array hybridization, and detection optimized at 100 nM forward/200 nM reverse primers and 250 nM (Affymetrix Microarray Core at the OHSU Gene Microarray Shared probe. After an initial activation at 50°C for 2 minutes and 95°C for 10 Resource Facility). Samples were hybridized to Rhesus macaque ge- minutes, PCR reactions were run for 45 cycles at 95°C for 15 seconds nome arrays (Affymetrix; Santa Clara, CA). Array data were normalized and 60°C for 1 minute. Fluorescence measurements were taken at using the RMA algorithm and analyzed by ANOVA (Partek Genomics every cycle after extension (Chromo 4; Bio-Rad). Suite; Partek Inc., St. Louis, MO). Signal, P-value, and detection calls for

FIGURE 1. The presence of lacritin in acini from monkey lacrimal gland. (A) H & E staining of 1-year-old monkey lacrimal gland showing acini (black ar- row), ductal structures (arrowhead), and lumens (white arrows). (B) Immu- nohistochemistry showing the presence of proteins for lacritin, lactoferrin, and lipocalin with different localizations. Lac- ritin co-localized with the vesicle marker VAMP2. No visible staining with the pro- liferation marker Ki-67 was observed. Rabbit and goat IgG were negative con- trols. White arrows: lumens. Scale bar, 20 ␮m.

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specific probes were determined by using the MAS 5.0 algorithm Lactoferrin partially co-localized with lipocalin. Lack of Ki-67 (Affymetrix), and PKC isotype probes were selected by sequences that staining revealed that proliferation did not occur (Fig. 1B). align to the 3Ј end of target mRNA and are grade A annotations. Acinar cells cultured for 1 day attached to plates coated with collagen I (Fig. 2A). After spreading for 3 days, the cul- Statistical Analysis tured cells proliferated to a flattened morphology and subse- Data were analyzed by Student’s t-test or Dunnett’s test. P Ͻ 0.05 quently became confluent on day 5. was considered statistically significant (JMP software; JMP, Tokyo, The growth curves for acinar cells were similar among mon- Japan). keys of different ages (Fig. 2B). Approximately 50% of acinar cells were attached to the plate 1 day after inoculation. Cell prolifera- tion started on day 2 and continued gradually for 5 days. Cells RESULTS staining positive with the proliferation marker Ki-67 appeared at day 3 and disappeared at day 5, suggesting proliferation and then Establishment and Characterization of a Culture contact inhibition (Fig. 2A). All cultured acinar cells stained with Protocol for Acinar Cells from Monkey the epithelial cell marker cytokeratin during the culture period, Lacrimal Gland suggesting pure cultures of acinar cells (Fig. 2A). H&E–stained sections of monkey lacrimal gland showed acini Lacritin, lactoferrin, and lipocalin were detected by im- and ducts (Fig. 1A). These structures were similar to those munostaining in cultured acinar cells on day 1, but the observed in human lacrimal gland.17 Immunohistochemistry staining decreased with culture time (Fig 2A). Immunoblot visualized the major tear proteins lacritin, lactoferrin, and li- analysis performed on acinar cells at each day of cultivation pocalin in multilobed structures around the lumens of the acini confirmed an inverse relationship between the relative (Fig. 1B). The vesicle marker VAMP2 revealed that lacritin was amounts of these tear proteins and acinar cell proliferation located in the secretory vesicles (Fig. 1B).18 Lacritin partially (Fig. 2C). Lacritin slightly decreased in cells cultured for 1 co-localized with lactoferrin, but not with lipocalin (Fig. 1B). day. After 2 days, lacritin was significantly decreased and

FIGURE 2. Characterization of cultured acinar cells from monkey lacrimal gland. (A) Phase contrast microscopy showing growth of acinar cells in culture. Staining with Ki-67 (green) confirmed the proliferation of cultured cells at 3 days. Lacritin, lactoferrin, and lipocalin (red) were observed up to 3 days. Staining with cytokeratin (green) confirmed pure cultures of acinar cells. Lacritin co-localized with vesicle marker VAMP2 (red). Rabbit and goat IgG were negative controls. Scale bar, 20 ␮m. (B) Proliferation of cultured acinar cells from different monkeys at ages 7, 9, and 11 years. Data are the mean Ϯ SD (n ϭ 3). (C) Immunoblot analysis showed that lacritin, lactoferrin, and lipocalin in acinar cells decreased with culture time, whereas the control protein GAPDH was stable. (D) qPCR analysis showed decreased expression of mRNAs for three tear proteins. Data are the mean Ϯ SD (n ϭ 3).

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lipocalin showed punctuate staining in the cytoplasm, and lacto- ferrin showed grid-like staining with connections between patches. No co-localization between lacritin and lipocalin was observed in cultured cells. However, both proteins partially co- localized with lactoferrin (merged images in yellow). These re- sults in cultured cells were similar to that of lacrimal gland (Fig. 1B), suggesting that acinar cells at 1 day of culture retained their differentiated character as observed in vivo. Secretion of Lacritin from Cultured Acinar Cells The endogenous cholinergic agonist Ach induced secretion of lacritin from acinar cells in a dose-dependent manner, with a minimum effective concentration of 1 ␮M (Fig. 4A). Secretion of lactoferrin and lipocalin was also induced by Ach at similar concentrations, although different cellular distributions of lacritin, lactoferrin, and lipocalin were ob- served in the cultured cells (Fig. 3). Similar to Ach, the acetylcholine analogue Cch induced secretion of lacritin, lactoferrin, and lipocalin (Fig. 4A). To determine which subtypes of cholinergic receptors were responsible for Cch-induced secretion of lacritin, cells were pre- FIGURE 3. Phase-contrast microscopy and immunohistochemistry in treated before Cch induction with atropine, a muscarinic antag- acinar cells cultured for 1 day, showing the presence of lacritin, onist of M1 and M3 receptors, or with mecamylamine, an antag- ␮ lactoferrin, and lipocalin with differing localization. Scale bar, 20 m. onist of nicotinic receptors. Atropine, but not mecamylamine, showed inhibition of Cch-induced secretion of the three tear proteins (Fig. 4B). The nicotinic receptor agonist nicotine did not nearly depleted by 5 days. Lactoferrin and lipocalin were induce protein secretion. These results suggest that the musca- similarly decreased. The ubiquitous protein GAPDH was rinic receptors were involved in protein secretion induced by stable over the same culture period, suggesting that the cells Cch. Of the five muscarinic receptor subtypes, only mRNA for the become less differentiated as culture time increases. M3 receptor was significantly expressed in cultured monkey lac- Expression of mRNA for lacritin was higher than that of rimal acinar cells, as determined by microarray analysis (Table 2). lactoferrin and lipocalin (Fig. 2D). mRNAs for all three proteins The M3 receptor was therefore likely to be involved in Cch- decreased slightly on day 1 of culture and then showed major induced secretion of tear proteins, such as lacritin. decreases at day 2. These results followed their specific protein levels, suggesting that in addition to constitutive secretion in Involvement of Intracellular Ca2؉ in Cch-Induced cultured cells, control of tear protein secretion is partially regulated by transcriptional regulation. Secretion of Lacritin In view of these results, cells cultured for 1 day seemed most Free cytoplasmic Ca2ϩ was significantly increased in cultured appropriate for studies on acinar secretion. Acinar cells cultured acinar cells by Cch in a dose-dependent manner, starting at a for 1 day showed epithelial morphology and granular regions in minimum concentration of 3 ␮M (Fig. 5A). Since increased Ca2ϩ the cytoplasm (Fig. 3). Cellular distribution and co-localization of was associated with increased tear protein secretion (Fig. 4A), each tear protein were distinct in acinar cells cultured for 1 day. Ca2ϩ may be involved in the mechanism for protein secretion Lacritin showed patchy distribution throughout the cytoplasm, from acinar cells. Pretreatment with 100 ␮M of the IP3 receptor

FIGURE 4. Secretion of lacritin from cultured acinar cells. (A) Immuno- blot analysis showing a dose-depen- dent secretion of lacritin, lactoferrin, and lipocalin by cholinergic agonists Ach or Cch. (B) Pretreatment with the muscarinic receptor antagonist atropine, but not the nicotinic recep- tor antagonist mecamylamine, com- pletely inhibited secretion of tear proteins induced by Cch. The nico- tinic receptor agonist nicotine did not induce secretion of proteins. Data are representative of results in three experiments in cultured acinar cells from three different monkeys.

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TABLE 2. mRNAs for Muscarinic Receptor Isotypes in Monkey Lacrimal Acinar Cells

7y 9y Muscarinic Detection Probe Set ID Receptor Signal P Signal P Call

Mmu.3886.1.S1_at 1 5.8 0.466 2.5 0.696 Absent MmuSTS.1732.1.S1_at 2 5.1 0.837 4.7 0.697 Absent MmugDNA.35530.1.S1_at 3 77.1 0.002 78 0.0005 Present MmuSTS.804.1.S1_at 4 6.9 0.749 3.6 0.541 Absent MmugDNA.24600.1.S1_at 5 6.9 0.679 12.9 0.660 Absent

antagonist 2-APB completely inhibited the Cch-induced increase isotypes are involved in Cch-induced secretion of proteins. in intracellular Ca2ϩ (Fig. 5C) and protein secretion (Fig. 5B). This Monkey lacrimal acinar cells were therefore cultured with the ϩ result suggests that IP3 is an intracellular messenger for Ca2 direct PKC activator PMA. PMA induced protein secretion, mobilization in acinar cells treated with Cch. which was inhibited by 10 ␮M of the PKC inhibitors GF109203 2ϩ Depletion of Ca in the endoplasmic reticulum (ER) has and Ro-32-0432 (Fig. 8B). The extracellular chelator EGTA 2ϩ been shown to trigger influx of extracellular Ca via the SOC (5 mM) did not inhibit PMA-induced secretion of lacritin, be- 19 channel. In our cultured acinar cells, Cch-induced secretion cause PMA activates PKC directly. In contrast, the 125 ␮M of lacritin, lactoferrin, and lipocalin was completely inhibited intracellular chelator BAPTA-AM inhibited PMA-induced pro- by 5 mM EGTA (an extracellular Ca2ϩ chelator) or 125 ␮M 2ϩ tein secretion (Fig. 8B). Thus, physiological levels of intracel- BAPTA-AM (an intracellular Ca chelator; Fig. 6A). These ch- 2ϩ 2ϩ ϩ lular Ca or Ca mobilized from internal stores was neces- elators reduced intracellular Ca2 to normal concentrations ϩ sary for PMA-induced secretion of proteins. (Figs. 6B, 6C). We reasoned that the amount of Ca2 mobilized from the ER was not enough to trigger Cch-induced lacritin To further examine which specific isozymes of PKC were secretion and that influx of Ca2ϩ from the extracellular pool involved in secretion of tear proteins, we performed mi- was necessary. To confirm this hypothesis, calcium ionophore croarray analysis on acinar cells from monkeys of two ages ionomycin was tested and found to induce protein secretion in (Table 3). Relative mRNA levels from PKC isozymes from a a dose-dependent manner (Fig. 6D). The effect of ionophore 7-year-old monkey based on signal strength, P-value and was inhibited by BAPTA-AM (Fig. 6D). detection calls were ␨ Ͼ ␦ Ͼ ␯ Ͼ ␫ Ͼ ␩ Ͼ ␣ Ͼ ␧ Ͼ ␮ Ͼ ␪ In Cch-treated cells, pretreatment with the SOC channel (column 4). Cells from a 9-year-old monkey gave similar inhibitor YM58483 inhibited increased intracellular Ca2ϩ in a results with minor differences (column 6). PKC protein dose-dependent manner (Fig. 7B). YM58483 also inhibited lac- expression of the classic (␣), novel (␦), and atypical (␨) ritin secretion (Fig. 7A). These results suggested that the SOC subtypes was also demonstrated by immunoblot analysis channel was an entrance point for extracellular Ca2ϩ. (Fig. 8C). PKC␣ was primarily present in the cytoplasm, whereas PKC␦ and -␨ were located in both membrane and Involvement of PKC in Cch-Induced Secretion cytoplasm areas (Fig. 8C). PKC␣, but not PKC␦ and -␨, of Lacritin increased in the membrane fraction of cultured acinar cells Pretreatment with 10 ␮M of the PKC inhibitor GF109203 (an 1 minute after PMA treatment. Since previous studies have inhibitor of PKC␣,-␤,-␦,-␧, and -␨) or Ro-32-0432 (an inhibitor shown that translocation of PKC␣ to the membrane causes of PKC␣,-␤, and -␧) reduced Cch-induced lacritin secretion in activation,20 our data suggest that PKC␣, needing Ca2ϩ for monkey acinar cells (Fig. 8A), suggesting that various PKC activation, is involved in PMA-induced protein secretion.

2ϩ FIGURE 5. Increased intracellular Ca induced by Cch and inhibition by an IP3 receptor antagonist. (A) Free Ca2ϩ

(Fmax/F0 ratio) in the cytoplasm signifi- cantly increased in acinar cells after in- duction by Cch. Data are the mean Ϯ SD (n ϭ 3) at 3 minutes; *P Ͻ 0.05 relative to the control group (0 ␮M). (B) Pre- treatment with 100 ␮M of the IP3 recep- tor antagonist 2-APB inhibited Cch-in- duced protein secretion at 10 minutes and (C) inhibited increased intracellular Ca2ϩ at 3 minutes. Data are the mean Ϯ SD (n ϭ 3); #P Ͻ 0.05 relative to group treated without Cch. *P Ͻ 0.05 relative to group treated with Cch.

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2ϩ FIGURE 6. Involvement of Ca in the secretion of acinar proteins. (A) Five mM EGTA or 125 ␮M BAPTA-AM inhibited Cch-induced pro- tein secretion at 10 minutes. (B) EGTA (5 mM) or (C) BAPTA-AM (125 ␮M) significantly inhibited increased Ca2ϩ induced by Cch at 3 minutes. Data are the mean Ϯ SD (n ϭ 3). #P Ͻ 0.05 relative to group treated without Cch. *P Ͻ 0.05 relative to the group treated with Cch. (D) Ionomycin-induced pro- tein secretion at 10 minutes and inhi- bition by pretreatment with 125 ␮M BAPTA-AM.

Involvement of p42/p44 MAPK in Transcription, acinar cells treated with Cch. Phosphorylated p42/p44 MAPK but Not in Protein Secretion, in Acinar Cells translocated into the nucleus after 5 minutes (Fig. 10A). Microar- Cultured with Cch ray analysis further showed that some mRNAs were significantly upregulated by Cch in acinar cells cultured from two different Another kinase, p42/p44 MAPK has been shown to regulate Rhesus macaques. These were the nuclear receptor family genes protein secretion in rat lacrimal acini.21,22 When our monkey NR4A1, NR4A2, and NR4A3; the pleiotropic cytokines IL-6, LIF, acinar cells were treated with Cch, p42/p44 MAPK was phos- and CLCF1; the CXC chemokine family genes CXCL1 and CXCL2; phorylated in a dose-dependent manner (Fig. 9A). Pretreatment ␮ prostaglandin-endoperoxide synthase 2 (PTGS2), cysteine- with the 10 M MAPKK inhibitor U0126 completely inhibited ␤ p42/p44 MAPK phosphorylation (Fig. 9B). U0126 did not affect serine-rich nuclear protein 1 (CSRNP1); inhibin A(IN- protein secretion after induction by Cch (Fig. 9B) or PMA (Fig. HBA); salt-inducible kinase1 (SIK1), cancer protein 8B). Thus, unlike rat acinar cells, activation of p42/p44 MAPK 1(TC-1); and cyclin-L1 (CCNL1; Table 4). Increased expres- by Cch was not important for Cch-induced protein secretion in sion of NR4A1, IL-6, and PTGS2 was also confirmed by qPCR cultured monkey lacrimal acinar cells. analysis (Fig. 10B), since these three genes had been re- Previous studies showed that activation of p42/p44 MAPK- ported to be transcribed by activation of p42/p44 regulated gene expression in a fibroblast cell line from Chinese MAPK.24–26 Transcription of the three genes by Cch was hamster lung.23 The distribution of phosphorylated p42/p44 almost completely inhibited by BAPTA-AM, but phosphory- MAPK was therefore examined in our cultured monkey lacrimal lation of p42/p44 was not affected (Figs. 10B, 10C). This

FIGURE 7. Involvement of the SOC channel in influx of Ca2ϩ.(A) Dose- dependent inhibition of Cch-induced protein secretion by the SOC channel inhibitor YM58483 at 10 minutes and (B) inhibition of increased Ca2ϩ at 3 minutes. Data are the mean Ϯ SD (n ϭ 3). #P Ͻ 0.05 relative to the group treated without Cch. *P Ͻ 0.05 relative to the group treated with Cch.

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FIGURE 8. Involvement of PKC in protein secretion. (A) Application of 10 ␮M PKC inhibitor GF109203 or Ro-32-0432 inhibited protein secre- tion by Cch at 10 minutes. Data are representative of results in three ex- periments with acinar cells cultured from three different monkeys.(B) PMA (1 ␮M) induced secretion of lacritin, lactoferrin, and, lipocalin at 10 min- utes. BAPTA-AM at 125 ␮M, GF109203 at 10 ␮M (GF), and Ro-32-0432 at 10 ␮M (Ro) inhibited secretion of pro- teins induced by PMA, but 5 mM EGTA did not. (C) Immunoblots for PKC␣,-␦, and -␨ after induction by 1 ␮M PMA, showing increased PKC␣ only in the membrane fraction when measured at 0, 1, 5, and 10 minutes.

result suggests that Ca2ϩ may be necessary for transcription, nar cells responded similarly in some respects to these other but not for phosphorylation, of p42/p44. U0126 completely animal cells, but is more relevant to the human situation. For inhibited phosphorylation of p42/p44 and partially inhibited example, the morphology of lacrimal gland from monkey was transcription of the three genes. GF109203 or EGTA par- found to be similar to human (Fig. 1A), and the monkey has tially inhibited transcription, but not phosphorylation, sug- been used extensively as a model for human diseases and in gesting involvement of PKC in activation of transcription. drug trials because of the general similarities between monkeys Thus, transcription induced by Cch may be regulated by and humans. p42/p44 MAPK and PKC. Our cultured monkey acinar cells contained VAMP2-posi- tive granules that stained positive for lacritin, lactoferrin, and DISCUSSION lipocalin (Fig. 3). Such transport granules for tear proteins were also reported in rabbit.28 Our monkey acinar cells prolif- The most important conclusions of the present study were that erated with increased culture time, but lacritin-positive cells (1) monkey lacrimal acinar cells can be cultured and used to decreased after day 1, delineating the need for using cells at 24 study pathways controlling tear protein secretion; (2) acinar hours. Proliferation of acinar cells along with loss of differen- cells cultured with our protocol respond to cholinergic ago- tiated characteristics with cultured time was also observed in nists and secrete the tear proteins lacritin, lactoferrin, and rat and rabbit acinar cells.29,30 lipocalin; (3) the pathway for Cch-induced tear protein secre- ϩ Rabbit and rat acinar cells formed acinus-like structures tion utilizes Ca2 intracellular signaling and activation of PKC, when they were cultured on synthetic matrix (Matrigel; In- but not the p42/p44 MAPK pathway; (4) Cch-induced tran- vitrogen) for 2 to 3 days or 11 days, respectively, and they also scription of NR4A1, IL-6, and PTGS2 involves both the PKC secreted proteins such as ␤-hexosaminidase and peroxi- pathway and the p42/p44 MAPK pathway. dase.18,29 Acinus-like structures were not morphologically de- tected in our cultured monkey cells. Differences in culture Monkey Acinar Culture System conditions, such as different extracellular matrices and culture A culture protocol for lacrimal acinar cells from rat and rabbit periods, and/or different species may have a role in the forma- has been reported.13,27 Our culturing system for monkey aci- tion of acinus-like structures. Our cultured cells at 1 day,

TABLE 3. mRNAs for PKC Isozymes in Monkey Lacrimal Acinar Cells

7y 9y Gene Detection Symbol PKC Probe Set ID Signal P Signal P Call

PRKCZ ␨ MmugDNA.40989.1.S1_at 511.9 0.0002 519.9 0.0002 Present PRKCD ␦ MmuSTS.2318.1.S1_at 421.8 0.0008 358.1 0.0005 Present PRKD3 ␯ MmugDNA.40581.1.S1_at 343.4 0.0003 380.8 0.0002 Present PRKCI ␫ MmugDNA.23670.1.S1_at 315.9 0.0002 201.8 0.0003 Present PRKCH ␩ MmugDNA.19823.1.S1_at 236.3 0.0002 302.9 0.0002 Present PRKCA ␣ MmugDNA.7366.1.S1_at 163.2 0.0002 281.8 0.0002 Present PRKCE ␧ MmugDNA.10395.1.S1_at 83.7 0.0031 82 0.0026 Present PRKD1 ␮ MmuSTS.3032.1.S1_at 39.8 0.0193 46.4 0.0313 Present PRKCQ ␪ MmugDNA.10942.1.S1_at 7.3 0.8371 3.9 0.7493 Absent PRKCG ␥ MmugDNA.13748.1.S1_at 6.4 0.7805 4.7 0.8704 Absent PRKCB ␤1 MmugDNA.21588.1.S1_at 3.2 0.8286 2.3 0.8704 Absent

Data are listed in decreasing levels of expression in 7-year-old animal.

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growth. For example, lacritin enhanced tear secretion in rat lacrimal acinar cells10 and stimulated corneal and salivary duc- tal cell proliferation.11,12 Lacritin also induced expression of membrane mucin MUC16 in human corneal epithelial cells (Laurie GW, et al. IOVS 2006;47:ARVO E-Abstract 1606). Cho- linergic receptors have been observed in mouse lacrimal gland,31 and cholinergic agonists induced an increase in cyto- plasmic Ca2ϩ and activation of PKC, after secretion of tear proteins from lacrimal acinar cells from rats and rabbits.18,22 In the present experiments with monkey acinar cells, Cch activated the muscarinic receptor M3, causing production of IP3 (Fig. 11). IP3 interacted with the IP3 receptor on the ER, causing Ca2ϩ mobilization from the ER, and induced Ca2ϩ influx into the cytosol via the SOC channel. Increased cytoplas- mic Ca2ϩ activated the PKC necessary for protein secretion from the acinar cells. Preventing increased intracellular Ca2ϩ or activation of PKC inhibited protein secretion. Our experiments did not provide direct evidence to show which specific PKC isozymes are responsible for tear pro- tein secretion. Indirect evidence suggests that PKC␣ is likely to be one of the responsible isozymes. For example, of the classic isotypes, only PKC␣ was detected in our cultured acinar cells. Further, PMA caused translocation of PKC␣ to the membrane, a marker for activation of PKC.20 In previous FIGURE 9. Phosphorylation of p42/p44 MAPK was not involved in studies in the rat, PKC␣ and -␧ were both shown to be protein secretion. (A) Phosphorylation of p42/p44 MAPK was induced involved in Cch-induced secretion of proteins by lacrimal by Cch in a dose-dependent manner at 10 minutes. (B) The MAPKK 32–37 inhibitor U0126 (10 ␮M) completely inhibited Cch-induced phosphor- acinar cells. In the present monkey study, expression of ylation at 10 minutes but did not inhibit protein secretion. Data are PKC␧ was relatively low in microarray analysis, and protein representative of results three experiments in acinar cells cultured was not detected by immunoblot analysis (data not shown). from three different monkeys. Further experiments are needed to determine whether other PKC isozymes are involved in the induction of monkey however, did retain functional characteristics of acini ob- acinar cells. served in vivo, such as negligible proliferation and similar We also found that BAPTA-AM, but not EGTA, inhibited subcellular localization of tear proteins. Further, the monkey PMA-induced/PKC-activated protein secretion (Fig. 8). Our acinar cells cultured for 1 day responded to stimulators and results suggest that Ca2ϩ from intracellular stores, and not secreted lacritin, providing a useful culture system for study- influx from extracellular pools, is necessary for PMA-in- ing human-relevant, tear secretory mechanisms and their duced protein secretion. The source of increased cytosolic regulation. Ca2ϩ after PMA treatment has been reported to be cell-type dependent.38,39 All evidence taken together shows that in- Cell Signaling Pathway for Lacritin Secretion creased Ca2ϩ and PKC␣ activation probably play important Previous studies in other animal models indicated an associa- roles in the secretion of proteins from monkey lacrimal tion between lacritin and tear protein secretion and cell acinar cells.

FIGURE 10. Involvement of p42/p44 MAPK and PKC in transcription. (A) Immunohistochemistry showing translocation of phosphorylated p42/ p44 (red fluorescence, arrowheads) into the nucleus 5 minutes after treat- ment with Cch. (B) qPCR analysis after Cch-induction showing increased mR- NAs for NR4A1, IL-6, and PTGS2 and inhibition by treatment with 125 ␮M BAPTA-AM, 10 ␮M U0126, 10 ␮M GF109203, or 5 mM EGTA at 1 hour. Data are the mean Ϯ SD (n ϭ 3). #P Ͻ 0.05 relative to the group without Cch. *P Ͻ 0.05 relative to the Cch-treated group. (C) Phosphorylation of p42/p44 MAPK induced by Cch was completely inhibited by the MAPKK inhibitor U0126 at 10 ␮M, but not by 10 ␮M GF109203, 5 mM EGTA, or 125 ␮M BAPTA-AM at 10 minutes. Data are rep- resentative of results in three experi- ments in acinar cells cultured from three different monkeys.

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TABLE 4. Upregulated Genes Induced by Cch in Monkey Lacrimal Acinar Cells

Gene Change Ratio Symbol Gene Name Probe Set ID Cch vs. Nor P

NR4A1 Nuclear receptor subfamily 4, group MmugDNA.18685.1.S1_at 74.60 0.038 A, member 1 NR4A3 Nuclear receptor subfamily 4, group MmugDNA.15738.1.S1_at 21.19 0.021 A, member 3 IL-6 Interleukin 6 (interferon, beta 2) MmuSTS.4354.1.S1_at 19.23 0.019 IL-6 Interleukin 6 (interferon, beta 2) Mmu.12240.1.S1_at 17.95 0.001 PTGS2 Prostaglandin-endoperoxide MmugDNA.4494.1.S1_at 16.75 0.002 synthase 2 LIF Leukemia inhibitory factor MmugDNA.22098.1.S1_at 6.56 0.011 (cholinergic differentiation factor) CXCL2 Chemokine (C-X-C motif) ligand 2 MmuSTS.1895.1.S1_at 4.04 0.007 INHBA Inhibin, beta A MmugDNA.21962.1.S1_at 3.77 0.003 NR4A2 Nuclear receptor subfamily 4, group MmugDNA.28332.1.S1_at 3.62 0.002 A, member 2 CSRNP1 Cysteine-serine-rich nuclear protein 1 MmuSTS.2136.1.S1_at 3.45 0.038 SIK1 Salt-inducible kinase 1 MmugDNA.1154.1.S1_at 3.44 0.017 TC-1 Thyroid cancer protein 1 MmugDNA.14772.1.S1_at 3.35 0.0004 CLCF1 Cardiotrophin-like cytokine factor 1 MmuSTS.3418.1.S1_at 3.11 0.016 CXCL1 Chemokine (C-X-C motif) ligand 1 MmugDNA.37457.1.S1_at 2.96 0.017 CCNL1 Cyclin-L1 MmugDNA.4307.1.S1_at 2.90 0.011

Top 15 probes at P Ͻ 0.04 and change ratio Ͼ2. Nor, normal.

Tear proteins must be transported in secretory vesicles Lack of a Role for the p42/p44 MAPK Pathway in and fused with the plasma membrane for extracellular se- the Lacritin Secretion Pathway cretion.40 Indeed, the present experiments revealed that lacritin was located in the secretory vesicles (Fig. 1B). We In our cultured monkey acinar cells, p42/p44 MAPK was phos- speculate that cytoskeletal protein may be a key regulator phorylated after induction by Cch, and this phosphorylation was for these final secretory steps. Other studies have shown inhibited by U0126. Unexpectedly, this inhibition did not affect that a dominant negative for PKC␧ inhibits remodeling of protein secretion. This finding was different from those in another filamentous actin and decreases protein secretion in Cch- study in rat lacrimal acini induced by Cch, in which p42/p44 ϩ induced rabbit acinar cells.28 Ca2ϩ influx causes disassembly MAPK was phosphorylated by Pyk2 and c-Src through Ca2 and of filamentous actin under the plasma membrane, facilitating PKC.22 The MAPKK inhibitor U0126 reduced phosphorylation of access of vesicles to proper sites for exocytosis in chromaf- p42/p44 MAPK and enhanced protein secretion induced by Cch, fin cells.41–43 Thus, PKC and Ca2ϩ may induce protein suggesting negative regulation of protein secretion by activated secretion via modification of actin filaments in monkey aci- p42/p44 MAPK.21 Positive regulation was also observed by the nar cells. finding that U0126 inhibits secretion of goblet cell glycoconju-

FIGURE 11. Hypothesized signaling pathway for Cch-induced transcription and regulation of lacritin secretion.

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gates in rat conjunctival pieces induced by Cch.44 The role of 6. Yamada M, Mochizuki H, Kawai M, et al. Decreased tear lipocalin p42/p44 MAPK appears to be diverse and dependent on the concentration in patients with meibomian gland dysfunction. Br J animal species and tissue. Ophthalmol. 2005;89(7):803–805. In the present experiments on monkey acinar cells, phos- 7. Tsai PS, Evans JE, Green KM, et al. Proteomic analysis of human phorylated p42/p44 MAPK entered the nucleus after Cch in- meibomian gland . Br J Ophthalmol. 2006;90(3):372– duction (Fig. 10), suggesting the promotion of transcription. 377. Tear proteins were candidates for such transcription, since 8. Nakajima T, Walkup R, Tochigi A, et al. Establishment of an production of new proteins for replenishment is necessary appropriate animal model for lacritin studies: cloning and charac- terization of lacritin in monkey eyes. Exp Eye Res. 2007;85(5):651– after secretion. mRNA expression for lacritin, lactoferrin, and 658. lipocalin was not upregulated in response to Cch as deter- 9. Hubbard T, Barker D, Birney E, et al. The Ensembl genome data- mined by qPCR (data not shown), although the three genes base project. Nucleic Acids Res. 2002;30:38–41. already showed relatively high expression before induction. 10. Sanghi S, Kumar R, Lumsden A, et al. cDNA and genomic cloning Signal intensities in microarray analysis of cells cultured from of lacritin, a novel secretion enhancing factor from the human the seven-year-old monkey for lacritin, lactoferrin, and lipoca- lacrimal gland. J Mol Biol. 2001;310(1):127–139. lin were 18,391, 17,341, and 16,074, respectively (P Ͻ 0.01). 11. Ma P, Beck S, Raab R, et al. deglycanation of synde- These genes are most likely constitutively expressed at high can-1 is required for binding of the epithelial-restricted prosecre- levels, to maintain constant, continuously translated tear pro- tory mitogen lacritin. J Cell Biol. 2006;174(7):1097–1106. teins. 12. Wang J, Wang N, Xie J, et al. Restricted epithelial proliferation by In contrast, of the 14 genes showing highest change ratios lacritin via PKCalpha-dependent NFAT and mTOR pathways. J Cell in microarray analysis after our monkey cells acinar cells were Biol. 2006;174(5):689–700. cultured with Cch, 7 have been described as being regulated 13. Hann LE, Tatro JB, Sullivan DA. Morphology and function of lacri- through p42/p44 MAPK.24–26,45–47 In our experiments with mal gland acinar cells in primary culture. Invest Ophthalmol Vis monkey, transcription of three of these genes: NR4A1, IL-6, Sci. 1989;30(1):145–158. and PTGS2, was inhibited by U0126, GF109203, EGTA, or 14. 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