Journal of Histochemistry and Cytochemistry, Vol. 50, 433-435, March 2002, Copyright © 2002, The Histochemical Society, Inc.


BRIEF REPORT

Human Lipocalin-1, a Physiological Scavenger of Lipophilic Compounds, Is Produced by Corticotrophs of the Pituitary Gland

Petra Wojnara, Stephan Dirnhoferb,c, Peter Ladurnerd, Peter Bergere, and Bernhard Redla
a Department of Molecular Biology, University Innsbruck, Innsbruck, Austria
b Institute of Pathology, University Innsbruck, Innsbruck, Austria
c Institute of Pathology, University Basel, Basel, Switzerland
d Department of Zoology, Faculty of Sciences, University Innsbruck, Innsbruck, Austria
e Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria

Correspondence to: Bernhard Redl, Institut für Molekularbiologie, Universität Innsbruck, Fritz Pregl Str. 3, A-6020 Innsbruck, Austria. E-mail: bernhard.redl@uibk.ac.at


  Summary
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Lipocalin-1 (Lcn-1), a member of the lipocalin superfamily that binds a broad array of different chemical classes of lipophilic ligands, is believed to act as a physiological scavenger of potentially harmful lipophilic molecules. Thus far, it was thought to be produced exclusively by a number of exocrine glands and tissues, including lachrymal and lingual glands, prostate, secretory glands of the tracheobronchial tract, and sweat glands. Using Northern blotting analysis, we were able to demonstrate Lcn-1 expression by the human pituitary gland. Moreover, double immunolabeling with antibodies against Lcn-1 and pituitary gland hormones and detection with fluorophore-conjugated secondary antibodies revealed that Lcn-1 is specifically produced by corticotrophs, clearly indicating that its distribution is not restricted to exocrine tissues. (J Histochem Cytochem 50:433–435, 2002)

Key Words: lipocalin, pituitary gland, immunohistochemistry, adenohypophysis, corticotrophs


  Introduction
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Summary
Introduction
Literature Cited

LIPOCALINS are members of an expanding superfamily of typically small secretory proteins characterized by their ability to bind hydrophobic ligands and their common structure, consisting of a single eight-stranded antiparallel ß-sheet, which forms a hydrogen-bonded ß-barrel enclosing an internal ligand binding site (Flower et al. 2000 ). Lipocalins exhibit great functional diversity, with roles in retinol transport, olfaction, coloration, pheromone transport, prostaglandin synthesis, regulation of cell homeostasis, modulation of the immune response, and as general clearance factors of endogenous and exogenous compounds (Flower 1996 ).

Human Lcn-1 (also called tear lipocalin or von Ebner's gland protein) is known to bind an unusual large variety of lipophilic molecules of different chemical classes, including fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol, retinol, and lipid peroxidation products such as isoprostanes, arachidonic acid and metabolites of arachidonic acid (Redl et al. 1992 ; Glasgow et al. 1995 ; Lechner et al. 2001 ). Functionally, it is suggested to act as a physiological scavenger of putative harmful lipophilic ligands (Redl 2000 ; Lechner et al. 2001 ). Thus far, human Lcn-1 was found to be expressed by a number of exocrine glands, including lachrymal glands, linqual glands, prostate, secretory glands of the tracheobronchial tract, and sweat glands (Redl 2000 ).

This study presents the first evidence that Lcn-1 is also produced by endocrine organs, because we were able to localize Lcn-1-producing cells in human pituitary gland, which were further identified as corticotrophs.

In a first step we examined expression of Lcn-1 by Northern blotting analysis using an Lcn-1-specific cDNA probe (Redl et al. 1992 ) and (poly)A+ RNA isolated from human pituitary glands. As shown in Fig 1, the probe hybridized with a single RNA species of about 0.7 kb in poly(A)+ RNA from pituitary gland, but no hybridizing band was detected in RNA isolated from thyroid gland (Fig 1, Lane 2). Because the human genome contains at least two different genes, LCN1 and LCN1b, which potentially encode Lcn-1 mRNA (Lacazette et al. 2000 ), we analyzed whether the mRNA expressed in pituitary gland is encoded by LCN1 or LCN1b. Therefore, we performed RT-PCR from pituitary gland cDNA using primers 5'-GTGGACTCAGACTCCGGAG-3' and 5'-GAGGAGCCAAGGTGTCCCC-3' corresponding to nucleotides 42–60 and 591–610 of the lachrymal gland-specific Lcn-1 cDNA sequence (Redl et al. 1992 ). After cloning of the PCR product into vector pGEM, a sequence analysis of clones derived from five independent PCR reactions revealed that the sequence of the amplified cDNA corresponded exactly to the published sequence of tear lipocalin (GenBank M90424), which is encoded by LCN1 (Redl 2000 ).



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Figure 1. Expression of Lcn-1 in the human pituitary gland detected by Northern blotting analysis. mRNA was isolated, separated on a 1% agarose gel, and blotted onto a nylon membrane. Lane 1, 2 µg of pituitary mRNA; Lane 2, 2 µg of thyroid gland mRNA. The blot was hybridized with an Lcn-1 encoding cDNA from human lachrymal gland.

Samples for immunohistochemical analysis were obtained from autopsies of two male donors and one female donor aged 65, 71, and 74 years, respectively, all of whom died of cardiac failure. Preliminary immunohistochemical investigations on sections (4 µm) of formalin-fixed and paraffin-embedded human pituitary glands, using an Lcn-1-specific antiserum, indicated production of Lcn-1 in the anterior lobe (adenohypophysis) of the glands (data not shown). In a next step, double labeling immunohistochemical experiments were performed to identify the exact localization of Lcn-1 within the specific cell populations of the anterior pituitary gland. For this purpose, cryostat sections (20 µm) of paraformaldehyde (4% in PBS)-fixed tissues were rinsed in 50 mM Tris-HCl-buffered saline, pH 7.2 (TBS), 0,4% Triton X-100. After incubation with 10% normal horse serum (Dakopatts; Copenhagen, Denmark), the free-floating sections were incubated with a polyclonal primary rabbit anti-Lcn-1 (Redl et al. 1992 ) at a dilution of 1:2000 and mouse monoclonal antibodies against growth hormone (GH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), adrenocorticotropic hormone (ACTH), and follicle-stimulating hormone (FSH) (Berger et al. 1988 , Berger et al. 1990 ) at dilutions of 1:500 to 1:20,000, for 24 hr at 4C. Sections were then washed in TBS–Triton three times for 5 min. Rabbit anti-Lcn-1 was visualized using Cy3 anti-rabbit (1:400) secondary antibodies (Chemicon; Temecula, CA). Mouse monoclonal antibodies against the pituitary hormones were detected with Cy2 rabbit anti-mouse IgG antibodies (1:800; Chemicon). Incubations with secondary antibodies were performed at ambient temperature for 90 min. Sections were then washed in TBS–Triton three times for 10 min, mounted using fluoromount-6, and investigated with a Zeiss LSM 510 confocal laser scanning microscope equipped with argon (488 nm) and HeNe (543 nm) lasers to detect Cy2 and Cy3, respectively. The double-immunolabeled sections were studied by sequential operation of the two lasers to avoid simultaneous excitation of the fluorescent dyes. A bandpass filter (505–530) was used for Cy2 to further prevent crosstalk between the channels. The Cy2 signal was displayed on the monitor in green color and the Cy3 signal in red. As depicted in Fig 2, a clear co-localization of Lcn-1 with ACTH (in yellow) was found when both images were superimposed, whereas no co-localization signal was observed with the other hormones studied (data not shown). As expected for secretory compounds, for both antigens, Lcn-1 and ACTH, a typical cytoplasmic staining was found, whereas the nucleus became apparent as a dark round area on the upper side of the cell. As indicated in Fig 2, not all of the corticotrophs showed positive staining for Lcn-1, suggesting that production of this protein is restricted to a subset of these cells.



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Figure 2. Confocal double immunofluorescence detection of Lcn-1 (red) and ACTH (green). (A) A single corticotroph cell of the human pituitary gland. The images represent a 3.2-µm confocal projection of a Z-stack. Co-localized areas in the cytoplasm appear yellow. Bar = 5 µm. (B) A cell population of the human pituitary gland. The images represent a 7.2-µm confocal projection of a Z-stack. Bar = 10 µm.

Expression of a lipid scavenger, such as Lcn-1, by cells of the pituitary gland is novel but not fully unexpected, because potential ligands of Lcn-1 are known to induce various effects in the pituitary gland, such as the modulation of hormone secretion and the activation or inhibition of ion channels (Won and Orth 1994 ; Perez et al. 1998 ; Denson et al. 2000 ). Therefore, production of a lipid scavenger might be useful to overcome the harmful action of these molecules in general. However, the specific expression of Lcn-1 in corticotrophs suggests a more specific physiological activity of this protein in the pituitary gland, which should be investigated in further studies.


  Acknowledgments

Supported by the Austrian Science Fund (FWF) grant P12628 (to BR) and P13652 (to PB).

We would like to thank F. Marx for helpful discussions on preparing the manuscript, and G. Sperk and A. Wieselthaler for technical advice.

Received for publication July 23, 2001; accepted October 31, 2001.


  Literature Cited
Top
Summary
Introduction
Literature Cited

Berger P, Klieber R, Panmoung W, Madersbacher S, Wolf H, Wick G (1990) Monoclonal antibodies against the free subunits of human chorionic gonadotrophin. J Endocrinol 125:301-309[Abstract]

Berger P, Panmoung W, Khaschabi D, Mayregger B, Wick G (1988) Antigenic features of human follicle stimulating hormone delineated by monoclonal antibodies and construction of an immunoradiometric assay. Endocrinology 123:2351-2359[Abstract]

Denson DD, Wang X, Worrell RT, Eaton DC (2000) Effects of fatty acids on BK channels in GH3 cells. Am J Physiol 279:C1211-C1219[Abstract/Free Full Text]

Flower DR (1996) The lipocalin protein family: structure and function. Biochem J 318:1-14[Medline]

Flower DR, North ACT, Sansom CE (2000) The lipocalin protein family: structural and sequence overview. Biochim Biophys Acta 1482:9-24[Medline]

Glasgow BJ, Abduragimov AR, Farahbakhsh ZT, Faull KF, Hubbell WL (1995) Tear lipocalins bind a broad array of lipid ligands. Curr Eye Res 14:363-372[Medline]

Lacazette E, Gachon AM, Pitiot G (2000) A novel human odorant-binding protein gene family resulting from genomic duplicons at 9q34: differential expression in the oral and genital spheres. Hum Mol Genet 9:289-301[Abstract/Free Full Text]

Lechner M, Wojnar P, Redl B (2001) Human tear lipocalin acts as an oxidative-stress induced scavenger of potentially harmful lipid peroxidation products in a cell culture system. Biochem J 356:129-135[Medline]

Perez FR, Camina JP, Menendez C, Beuras A, Casabiell X, Casanueva FF (1998) Cis-unsaturated free acids block VIP-mediated GH and PRL secretion by perturbing the cAMP/protein kinase A pathway. Pituitary 1:25-32[Medline]

Redl B (2000) Human tear lipocalin. Biochim Biophys Acta 1482:241-248[Medline]

Redl B, Holzfeind P, Lottspeich F (1992) cDNA cloning and sequencing reveals human tear prealbumin to be a member of the lipophilic-ligand carrier protein superfamily. J Biol Chem 267:20282-20287[Abstract/Free Full Text]

Won JG, Orth DN (1994) Role of lipoxygenase metabolites of arachidonic acid in the regulation of adrenocorticotropin secretion by perfused rat anterior pituitary cells. Endocrinology 135:1496-1503[Abstract]