Brain Research 887 (2000) 440–443
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Short communication
Expression of stanniocalcin in the epithelium of human choroid plexus
a a a b
´
Annika M. Franzen , Ke-zhou Zhang , Johan A. Westberg , Wan-Ming Zhang ,
a c a ,
*
Johanna Arola , Henrik S. Olsen , Leif C. Andersson
a
Department of Pathology, Haartman Institute, Helsinki University Central Hospital, P.O. Box 21, FIN-00014 Helsinki, Finland
b
Department of Clinical Chemistry, Helsinki University Central Hospital, P.O. Box 21, FIN-00014 Helsinki, Finland
c
Human Genome Science, Inc. Rockville, MD, USA
Accepted 19 September 2000
Abstract
Stanniocalcin (STC) is a 28 kD glycoprotein hormone originally found in bony fish in which it regulates calcium / phosphate homeostasis and protects against hypercalcemia. The recently characterized mammalian STC shows about 70% homology with fish STC. The epithelial cells of proximal tubuli in human and rat kidney and brain neurons have been found to express STC. Here we show that the epithelium of the choroid plexus, already at 16 weeks of fetal age, and of plexus papillomas, synthesize and express STC. Our findings suggest that STC may be of importance for the distribution of calcium and phosphate between the cerebrospinal fluid and blood. 2000 Elsevier Science B.V. All rights reserved.
Theme: Cellular and molecular biology
Topic: Staining, tracing, and imaging techniques
Keywords: Stanniocalcin; Blood–brain barrier; Phosphate homeostasis; Calcium transport
The choroid plexus (CP) is a specialized secretory protects against hypercalcemia by lowering the uptake of organ, which produces cerebrospinal fluid (CSF). CP calcium via the gills [7,10] and the intestinal tract [18] and consists of invaginated fronds of leptominges around a increases reabsorption of phosphate in the kidney [11]. highly vascularized, fibrous stroma. The plexus is covered Elevated environmental concentration of calcium is the by an ependyma-derived epithelium forming a single layer main trigger of STC production in fish [20,21]. STC was of polarized, cuboidal cells. considered exclusive to fish until the cDNAs for human [3]
Primary neoplasms originating in the epithelium of the and mouse [4] STC were cloned.
CP are rare. They mostly consist of benign papillomas, STC has remained highly conserved during evolution. which occur both in childhood, and at adult age. Usually Human STC cDNA shares a 72% nucleotide sequence they come to attention due to their propensity of obstruct- homology with fish STC and the protein shows 80% ing CSF circulation or producing excess CSF. Malignant similarity with conserved cysteins [3]. The ultimate func-tumors derived from the CP, i.e. carcinomas, are exceed- tion(s) of STC in mammalians remains to be clarified but it ingly rare and occur mostly in infancy or childhood. appears to regulate the phosphate / calcium balance. In-Stanniocalcin (STC) is a 28 KD glycoprotein originally travenous infusion of recombinant human STC to rats was discovered in fish [23]. Fish STC is synthesized in a found to increase the reabsorption of phosphate in the specialized organ, the Corpuscle of Stannius which is kidney [22]. Histochemical investigations have revealed a located adjacent to the kidney [16]. STC plays a major role constitutive expression of STC in the epithelial cells of the in regulating the calcium / phosphate homeostasis in fish. It proximal tubuli in human [6], rat [8,24] and mouse kidney [25]. Expression of STC mRNA has been found in various tissues, including the heart muscle, prostate and gastroin-testinal tract [3].
*Corresponding author. Fax:1358-919-126-675.
E-mail address: leif.andersson@helsinki.fi (L.C. Andersson). We originally reported a high expression of STC in
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A.M. Franzen et al. / Brain Research 887 (2000) 440 –443 441
terminally differentiated neurons in mouse and human To study whether the STC in the epithelium of CP is brain [26]. Glial cells did not express detectable levels of synthesized locally or taken up from the circulation we STC but we observed a strong positive immunohistochemi- performed in situ hybridization. The anti-sense single-cal staining of the epithelial cells of the CP. strand RNA probe for STC gave a strong reactivity In this study we show that the epithelium of CP confined to the epithelial cells (Fig. 1c) while no specific constitutively synthesizes and expresses STC. The neo- reactivity was obtained with the control (sense) probe (Fig. plastic epithelium of CP papillomas was also found to 1d).
express STC. When five cases of CP papillomas were
immunohistoch-Biopsies of CP from ten patients, five with papilloma emically stained, a similar expression of STC in the and five with miscellaneous diseases, were studied. In neoplastic epithelium was seen in all cases although the addition, CP tissue was obtained from autopsies of two staining intensity showed more variation than in the fetuses at 16 and 21 weeks of gestation age (Table 1). The normal CP epithelium (Fig. 1f).
tissue was fixed in 4% buffered formaldehyde for 12–48 h, The epithelial cells of CP localize in between the blood routinely processed and embedded in paraffin. Four mm and CSF. Circulating STC (4.5 to 11.6 ng / ml) has been thick sections were mounted on 3-aminopropyl-triethoxy- found in human serum [19]. We used an STC immuno-silane (APES) (Sigma, St. Louis, MO, USA) coated slides assay to measure the amount of STC in the CSF collected and dried for 12 h at 378C. Immunohistochemical stainings at the Department of Occupational Health for diagnostic of deparaffinized and rehydrated sections with rabbit purposes from five adult persons suffering from headache antibodies to human STC [26] were performed by using a with no known organic cause. All samples contained less commercial Elite ABC Kit (Vectastain, Vector Laborator- than 2 ng / ml STC (the detection limit of our assay, data ies, Burlingame, CA, USA) as described in detail [26]. not shown).
Antibodies to STC pre-absorbed with recombinant STC The CP not only produces the CSF, but it also consti-protein and normal rabbit serum served as controls. tutes an essential part of the blood-CSF barrier. In this For in situ hybridization, single-stranded anti-sense localization, the epithelial cells of the CP are actively RNA probes were generated in vitro by T7 RNA poly- regulating the distribution of molecules between the blood merase from the pcDNA3 vector (Invitrogen) in which a and CSF. In this respect the epithelium shares many human stc cDNA was cloned in sense and anti-sense properties with renal tubular cells. The CP has been called direction and labeled with digoxigenin (Boehringer Mann- a ‘kidney’ in the brain with the difference, however, that heim). A sense strand was used as a control. The hybridi- the kidney clears waste products from the blood while the zation and detection were carried out as described [27]. CP transports waste products from CSF into the blood
Staining with rabbit antibodies to human STC revealed a [15].
strong reactivity in the epithelial cells of normal CP. A Co-expression of a variety of proteins involved in cytoplasmic, slightly granular pattern with most intense epithelial barrier functions has been found in CP and staining close to the apical surface was seen (Fig. 1a). kidney tubular cells. These include isoforms of the plasma
21
Expression of STC was observed in the epithelium of CP membrane Ca pump [17], the anionic transport protein already at 16 (Fig. 1e) and 21 (not shown) weeks of fetal [1], the angiotensin-converting enzyme [5], the
1 1
development, i.e. stages at which most of the proliferating Na K ATPase [14], and the recently characterized in-1
and migrating brain neurons do not yet contain amounts of wardly rectifying K channel [13]. This study adds STC to STC detectable by immunohistochemistry [26]. the list.
Given the analogous transport functions, it is interesting that the epithelial cells of both the proximal tubuli of Table 1
mammalian kidney and the epithelium of CP shown here Choroid plexus tissue used in the study
express STC. The function of STC, regulating the calcium / Case Age / Sex Reason for CP resection
phosphate homeostasis in fish, appears conserved through
1 2 / M Papilloma evolution. Addition of recombinant human STC to isolated
2 35 / M Papilloma
pig duodenal epithelium increased the cross-epithelial
3 59 / F Papilloma
transport of phosphate [12]. Recent studies indicate that
4 62 / F Papilloma
the sodium dependent phosphate co-transporter (NaPi-2) is
5 65 / F Papilloma
6 1 / F Meningeal angiomatosis a target of action of STC in mammalian kidney [22]. 7 31 / M Astrocytoma, G2 Whether the NaPi is expressed also in the choroid plexus
8 49 / M Meningeoma
remains to be established. It is, however, tempting to
9 55 / F Metastatic melanoma
speculate that STC in the choroid plexus may be involved
10 60 / F Metastatic breast cancer
in the regulation of the phosphate / calcium homeostasis in
Autopsy material: the CSF [2,9]. The local activity of STC may also have
11 Fetus 16 weeks of GA bearings on the pathogenesis of the calcified foci frequent-12 Fetus 21 weeks of GA
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442 A.M. Franzen et al. / Brain Research 887 (2000) 440 –443
Fig. 1. Immunohistochemical staining of normal CP with antibodies to human STC (a), and with STC antibodies preabsorbed with recombinant STC protein as a control (b). In situ hybridization with an stc anti-sense probe on normal CP (c), and with the sense probe as a control (d). Immunohistochemical staining of fetal CP (16 weeks) with antibodies to STC (e). Immunohistochemical staining of CP papilloma with STC antibodies (f). The scale bar represents 200mm in (a), (b), (c), (d) and 100mm in (e) and (f).
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