ARTICLE |
Correspondence to: Per Hellman, Dept. of Surgery, University Hospital, S-751 85 Uppsala, Sweden.
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Summary |
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We used riboprobes and monoclonal antibodies to characterize tissue distribution of the human 550-kD homologue to gp330/megalin, primarily identified in the rat kidney. Human gp330/megalin mRNA and protein are readily identified in human parathyroid cells, placental cytotrophoblasts, kidney proximal tubule cells, and epididymal epithelial cells. The immunoreactivity is found on the surface of the cells and is heterogeneously downregulated in parathyroid hyperplasia and adenomas. Cells of the proximal kidney tubule and epididymis express the protein on their luminal aspect. Moreover, the protein is expressed in Type II pneumocytes, mammary epithelial and thyroid follicular cells, and the ciliary body of the eye. Sequence analysis of cDNA fragments, obtained by RT-PCR, revealed identical nucleotide sequences in parathyroid, kidney, placenta, epididymis, and lung. Immunohistochemistry for parathyroid hormone-related protein (PTHrP) revealed partial co-expression with human gp330/megalin in parathyroid, placenta, and mammary gland. The findings substantiate human gp330/megalin expression in a variety of human tissues expected to possess calcium-sensing functions. It may constitute a protein of utmost importance to adult and fetal calcium homeostasis, although other important functions may also be coupled to this exceptionally large protein with highly restricted tissue distribution. (J Histochem Cytochem 45:383-392, 1997)
Key Words: human, Ca2+ sensing, tissue distribution, parathyroid hormone-related, protein, immunohistochemistry, in situ hybridization
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Introduction |
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The parathyroid glands exert an overall regulatory role in systemic calcium homeostasis by their unique ability to respond to changes in the extracellular Ca2+ concentration (
The antibody-reactive protein was isolated from human placenta and revealed as a 550-kD single-chain glycoprotein (
The present investigation utilized immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR), and in situ hybridization to establish more closely the human tissue distribution of the 550-kD, putative Ca2+-sensing protein, and compared this expression with that of PTHrP.
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Materials and Methods |
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Tissues
Pathological human parathyroid tissue was obtained during surgery for primary hyperparathyroidism (HPT). Biopsy specimens of normal parathyroid glands were acquired during thyroid surgery. Normal tissue specimens from kidney, epididymis, testis, mammary gland, adrenal gland, smooth and striated muscle, skin, liver, ileum, jejunum, colon, spleen, thyroid, pancreas, lymph node, eye, and lung were removed at surgery. Human placenta was acquired from uncomplicated pregnancies at full term. All tissue pieces intended for immunohistochemistry or RNA preparation were snap-frozen in liquid nitrogen and stored at -70C until processing. Cells intended for immunoprecipitation were prepared from parathyroid adenomatous and chief cell hyperplastic glands of patients with primary HPT, from human placenta, and from normal kidney as previously described (
Monoclonal Antibodies
The production and characterization of the mouse monoclonal anti-parathyroid antibodies E11 and G11 have previously been described (
Immunostaining
Cryosections (6 µm) were fixed in acetone and exposed for 15 min to 1% H2O2 in PBS and 10% normal rabbit serum in PBS. For examination of human gp330/megalin expression, the sections were then incubated for 30 min with MAb E11 (5 µg/ml) in PBS containing 2% bovine serum albumin (BSA, fraction V; Sigma Chemical, St Louis, MO). A general finding with the MAb was that only acetone-fixed cryosectioned tissue could be used for immunohistochemistry. For example, the paraformaldehyde/glutaraldehyde-fixed specimens used for in situ hybridizations were uninformative with Mab E11. After exposure to a secondary rabbit anti-mouse antibody (Dakopatts; Glostrup, Denmark) for 30 min, the sections were treated with anti-mouse peroxidase-anti-peroxidase (PAP) complexes (Dakopatts). For investigation of PTHrP expression, sections were exposed to 10 µg/ml of a murine MAb recognizing human PTHrP(38-64) (Oncogene Science; New York, NY) followed by a biotinylated secondary rabbit anti-mouse antibody (Jackson Laboratories; West Grove, PA) and peroxidase-conjugated biotin (Vector; Los Angeles, CA). Staining was visualized in all sections with 3-amino-9-ethylcarbazol and counterstaining was performed with Mayer's hematoxylin. Negative controls included omission of the primary antibodies and substitution with mouse IgG1 fractions or an irrelevant murine monoclonal IgG MAb specific for collagen Type II.
In Situ Hybridization
Tissues intended for in situ hybridization were fixed for 16-20 hr on ice in 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium phosphate (pH 7.0). Dehydration by increasing concentrations of ethanol and xylene was followed by embedding in Histovax (Histolab Products; Gothenburg, Sweden) as described earlier (
A 377-BP fragment of a 2.8-KB cDNA clone denoted pCAS-2, encoding a portion of human gp330/megalin (-[35S]-UTP (>37 TBq/mmol) or
-[33P]-UTP (15.2 TBq/mmol; Amersham International, Amersham, UK) utilizing a commercially available kit (Maxiscript; Ambion, Austin, TX). Transcripts were subjected to limited alkaline hydrolysis to yield about 100 nucleotide fragments. The sections were hybridized with the probes (2 x 106 cpm/20 µl) overnight at 45C in the presence of 50% formamide (
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Reverse Transcriptase-Polymerase Chain Reaction
Total RNA was prepared from parathyroid, placenta, kidney, epididymis, liver, jejunum, ileum, colon, and lung utilizing the acid phenol/chloroform method (
Two pairs of primers were utilized (Figure 1). The 1192/1193 pair yielded a PCR product of 660 BP, and the 5/6 pair resulted in a 770-BP product. These parts of the pCAS-2 clone encode portions immediately outside and inside of the transmembrane segment of human gp330/megalin, respectively (
Immunoprecipitation and SDS-PAGE
A total of 2-5 x 106 parathyroid, placental, or renal cells were incubated for 30 min at 37C in 2 ml methionine-depleted DMEM containing 10% dialyzed fetal calf serum. [35S]-Methionine (0.5 mCi, <1000Ci/mmol; Amersham) was added, and the cells were labeled for 4 hr. The labeling was terminated by centrifugation, after which the cells were lysed on ice in 1 ml of 0.01 M Tris-HCl (pH 8.0), 4% Triton X-100, 2% aprotinin (Bayer; Leverkusen, Germany), 0.5 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine hydrochloride. The lysate was centrifuged at 35,000 x g for 1 hr at 4C and then subjected to antibody precipitation. Cell lysates were incubated with 10 µl normal rabbit serum overnight at 4C. Immunoglobulins were removed by the addition of protein A-Sepharose (50 µl; Pharmacia-UpJohn) and centrifugation. Aliquots of the supernatants were incubated for 4-12 hr with 1 µg MAb E11 or MAb G11. An MAb against a plant protein was used as a negative control. The immunoglobulins were recovered by exposure to protein A-Sepharose, washed with 0.01 M Tris-HCl (pH 8.0) containing 0.1% Triton X-100 and once with 0.01 M Tris-HCl (pH 8.0) containing 0.5 M NaCl and 0.1% Triton X-100. The immunoprecipitated proteins were eluted by heating at 95C in SDS loading buffer under reducing conditions. SDS-PAGE was carried out as described (
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Results |
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Immunohistochemistry
The chief cells of normal human parathyroid glands demonstrated intense cell surface staining with MAb E11 (Figure 2A). Pathological parathyroid tissue invariably showed reduced and often irregularly distributed immunoreactivity (Figure 2d) (
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Human placental tissue obtained at term demonstrated intense E11 staining, which was confined only to the cytotrophoblasts (Figure 2G). Although some intracellular staining was present, the most intense staining was seen on the surface, as previously described (
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PTHrP expression was demonstrated in several of the same cells as human gp330/megalin. Thus, human gp330/megalin and PTHrP were co-expressed in parathyroid chief cells, placental cytotrophoblasts, and mammary duct epithelial cells. Although parathyroid chief cells from normal parathyroid glands were intensely positive for both proteins, there was no clear correlation between PTHrP and the downregulated human gp330/megalin expression of parathyroid adenoma (Figure 2D and Figure 2F) or nodular hyperplasia. Apart from the human gp330/megalin-expressing placental cytotrophoblasts, there was also intense PTHrP reactivity within stromal cells of the placental villi (Figure 2I). The ducts of the mammary gland showed intense PTHrP immunoreactivity, also seen with MAb E11 (Figure 3G).
Intense PTHrP staining was also seen in epididymis, but in other areas than the human gp330/megalin immunostaining. The PTHrP staining was confined to the corpus and cauda, and generally failed to appear on the outermost layer of the epithelial lining (Figure 3F). Kidney glomeruli were negative for PTHrP and weak staining was seen in proximal tubule cells, whereas intense reactivity was obvious in the collecting ducts (not shown). The only cells in the eye staining positive with the anti-PTHrP antibody were cells in the conjunctiva. Therefore, cells in the pigment epithelium were negative, as was the normal thyroid. The lung generally showed weak reactivity except for scattered cells in the alveolar lining.
In Situ Hybridization
The parathyroid tissues revealed variably intense hybridization with the anti-sense pCAS-2 probe (Figure 2B and Figure 2E). Although some background activity was present with the sense probe, this usually caused no problems in interpreting the data. Parathyroid adenomas and nodules of hyperplastic glands displayed substantially lower anti-sense probe signal, whereas the normal parathyroid glands and the rim of normal tissue of parathyroid adenoma elicited considerably more intense and homogeneous staining. This was also true for some areas of diffuse hyperplasia, which surrounded the larger nodules of the primary hyperplastic glands.
In placenta, the anti-sense pCAS-2 probe signaled expression of human gp330/megalin mRNA in the cytotrophoblasts only, but the interpretation in the kidney was more difficult. However, after comparison with unspecific sense pCAS-2 signals as well as parallel sections stained with hematoxylin/eosin, it was evident that the human gp330/megalin mRNA was confined solely to proximal tubule cells in the kidney and was thus correlated with the immunohistochemical E11 staining. Epididymal epithelial cells in the caput (Figure 3E) also demonstrated intense hybridization signals with the anti-sense riboprobe, and only low background with the sense RNA (not shown). No specific signal was detected in human lung. Moreover, in parallel to the findings at immunohistochemistry, in situ hybridization of small intestine and colon was difficult to interpret because of high background signals with the sense probe.
Polymerase Chain Reaction
Polymerase chain reaction of first strand cDNA from human parathyroid, epididymis, kidney, lung, and full term placenta revealed the expected bands of 660 BP with the 1192/1193 oligonucleotide primer pair and 770 BP with the 5/6 pair. Sequencing of these products revealed identical nucleotide sequences in all tissues, corresponding to the pCAS-2 clone (cf Figure 1). However, PCR analysis of first strand cDNA from jejunum, ileum, and colon displayed no bands specific for human gp330/megalin.
Immunoprecipitation
The E11 and G11 antibodies precipitated a single protein with a similar size of approximately 550 kD from placenta, kidney, and parathyroid (Figure 4).
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Discussion |
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Extracellular calcium is the predominant regulator of cytoplasmic calcium concentration ([Ca2+]i) in parathyroid cells (
In situ hybridization with the pCAS-2 anti-sense riboprobe closely corresponded to the immunohistochemical staining with the E11 and G11 antibodies. A concomitant decrease in human gp330/megalin mRNA and protein expression was demonstrated in the pathological parathyroid parenchyma of adenoma and hyperplasia, suggesting that the reduced gp330/megalin protein expression does not occur at the translational level. This decrease is compatible with the characteristic right shift in the set-point for PTH release and [Ca2+]i, due to decreased sensitivity to changes in ambient Ca2+, previously recognized as a characteristic feature of the pathological parathyroid tissue in all entities of HPT (
In situ hybridization of epididymis, kidney, and placenta also displayed concordance with the immunohistochemical investigations. In epididymis the signals were confined to the luminal side of the ductus epididymis as well as the rete testis, but no clear immunostaining was seen in visible sperm cells. The function of human gp330/megalin in epididymis is unclear, but it is intriguing that the protein is directed towards a fluid-containing space in this organ, as in the kidney, placenta, thyroid, eye, and the mammary gland. In the proximal tubule cells, gp330/megalin has been proposed to facilitate uptake of other proteins from the primary urine (
In the thyroid gland, weaker expression of human gp330/megalin was detected within follicular cells, where PTHrP appeared absent. In the human eye, clear expression of gp330/megalin was seen in the choroid layer in the ciliary body but also stretched into the corresponding layer of retina. These cells produce the vitreous humor and are proposed to be equipped with proteins regulating the Ca2+ concentration in this liquid (
Human gp330/megalin immunostaining was also detected in human lung tissue and the nucleotide sequence was at least partially identified. Although we were unable to achieve specific in situ hybridizations with human lung tissue, the PCR analysis and immunohistochemistry substantiate previous findings of gp330/megalin expression in Type II pneumocytes. In these cells, calcium has been shown to stimulate PTHrP and phospholipid secretion (
In virtually all tissues expressing human gp330/megalin, the protein is located on the cell surface, ideal for a receptor function. In contrast, hithertho published and unpublished data have shown that malignant tumors such as parathyroid carcinoma, choriocarcinoma (e.g., JEG-3 cells), renal cancer, and pulmonary adenocarcinoma display a markedly different and mainly intracellular localization of the protein (
The results in this study indicate that only one form of gp330/megalin is expressed throughout the body, since the E11 antibody precipitated only one protein version from the investigated tissues and the PCR analyses demonstrated identical sequences. Furthermore, Northern blots of total RNA from human parathyroid, kidney, epididymis, and placenta have earlier shown only one species of hybridizing mRNA (
Evidence obtained thus far strongly suggests the involvement of CaR, and possibly also human gp330/megalin, in sensing of extracellular Ca2+ concentrations and thereby in the regulation of [Ca2+]i and PTH release in the parathyroid gland. Although available evidence can be considered circumstantial, functional studies appear to strongly suggest that human gp330/megalin is involved in sensing of extracellular Ca2+ concentrations and regulation of [Ca2+]i also in placental cytotrophoblasts (
In conclusion, the distribution of human gp330/megalin is demonstrated in a number of tissues claimed to possess calcium sensing mechanisms (
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