Expression of Reduced Nicotinamide Adenine Dinucleotide ...

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Departments of Pathology (B.C., M.N., M.T.), Clinical Biology (L.L., J.M.B.), and Nuclear Medicine. (M.S.), Institut Gustave-Roussy, 94805 Villejuif; and INSERM ...
0021-972X/01/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2001 by The Endocrine Society

Vol. 86, No. 7 Printed in U.S.A.

Expression of Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase (ThoX, LNOX, Duox) Genes and Proteins in Human Thyroid Tissues* BERNARD CAILLOU, CORINNE DUPUY, LUDOVIC LACROIX, MARIA NOCERA, ´ E OHAYON, DANIELLE DE ` ME, JEAN-MICHEL BIDART, MONIQUE TALBOT, RENE MARTIN SCHLUMBERGER, AND ALAIN VIRION. Departments of Pathology (B.C., M.N., M.T.), Clinical Biology (L.L., J.M.B.), and Nuclear Medicine (M.S.), Institut Gustave-Roussy, 94805 Villejuif; and INSERM Unite´ 486 (C.D., R.O., D.D., A.V.), Faculte´ de Pharmacie, 92296 Chaˆtenay-Malabry Cedex, France ABSTRACT The large homolog of NADPH oxidase flavoprotein LNOX2, and probably LNOX1, are flavoproteins involved in the thyroid H2O2 generator. Western blot analysis of membrane proteins from normal human thyroid, using antipeptide antibodies, indicated that LNOX1,2 are 164-kDa glycoproteins and that N-glycosylated motifs account for at least 10 –20 kDa of their total apparent molecular mass. Northern blot analysis of 23 different human tissues demonstrated that LNOX2 messenger RNA (mRNA) is strongly expressed only in the thyroid gland, although blast analysis of expressed sequence tags databases indicated that LNOX genes are also expressed in some nonthyroid cells. We investigated LNOX1,2 gene and protein expressions in normal and pathological human thyroid tissues using real-time kinetic quantitative PCR and antipeptide antibodies, respectively. In normal tissue, LNOX1,2 are localized at the apical pole of thyrocytes. Immu-

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HE IODINE PATHWAY comprises several steps in thyroid cells that involve 1/an iodide transport from blood to follicular lumen via the basolateral sodium/iodide (Na⫹/I-) symporter (NIS) (1, 2), and the apical chlorideiodide porter (pendrin) (3, 4), 2/the incorporation of iodide in tyrosyl residues of thyroglobulin (Tg) molecules at the apical pole, followed by the coupling of hormonogenic iodotyrosyl residues into thyroid hormone residues; these latter two reactions are catalyzed by the thyroid peroxidase (TPO) in the presence of H2O2 (5), and 3/Tg hydrolysis and hormone secretion (6). TPO has no biological activity in the absence of H2O2, which is likely to be the limiting factor for Tg iodination when iodide supply is normal (7). The H2O2 generator has been studied in porcine and human thyroid tissues and is a calcium-dependent and transmembrane system that functions by transferring electrons from NADPH to molecular oxygen (8, 9). It comprises a flavoprotein with FAD as cofactor (10), which possibly requires other components for its Received June 9, 2000. Revision received December 6, 2000. Rerevision received March 8, 2001. Accepted March 13, 2001. Address all correspondence and requests for reprints to: A. Virion, M.D., INSERM Unite´ 486, Faculte´ de Pharmacie, 92296 ChaˆtenayMalabry Cedex, France. E-mail: [email protected]. * Supported by grants from the Projets Institut Fe´de´ratif de Recherche, 1999, Institut Gustave-Roussy, Electricite´ de France, and from INSERM, “Aide aux Projets Exceptionnels” no. 4X014E.

nostaining for LNOX1,2 was heterogeneous, inside a given follicle, with 40 – 60% of positive follicular cells. Among normal and pathological tissues, variations of LNOX1 and LNOX2 mRNA levels were parallel, suggesting a similar regulation of both gene expressions. Whereas LNOX mRNAs seemed slightly affected in benign disease, the expression of protein was highly variable. In multinodular goiters, 40 – 60% of cells were stained. In hypofunctioning adenomas, LNOX immunostaining was highly variable among follicles, whereas sodium/iodide (Na⫹/I-) symporter immunostaining was decreased. In hyperfunctioning thyroid tissues, only few cells (0 –10%) were weakly stained, whereas sodium/iodide symporter staining was found in the majority of follicular cells. In conclusion, LNOX proteins are new apical glycoproteins with a regulation of expression that differs from other thyroid markers. (J Clin Endocrinol Metab 86: 3351–3358, 2001)

activity (11). We recently purified a novel flavoprotein from pig thyroid tissue, which constitutes the main component of the thyroid NADPH oxidase (11, 12). Availability of peptide microsequences permitted the cloning of porcine and of human complementary DNAs (cDNAs) encoding 1207- and 1210-amino acid proteins, respectively (12). The encoded protein displayed a theoretical molecular mass of 138 kDa and was designated p138Tox for p138 thyroid oxidase (12). Its gene was mapped on human chromosome 15q15 (12). Two cDNAs (GenBank database accession numbers AF230495 and AF 230496) encoding two flavoproteins of 1,551 (thyroid oxidase ThOX1) and 1,548 amino acids (ThOX2) have recently been cloned from human thyroid tissue (13). Expression of both genes was up-regulated by cAMP in cultured human thyrocytes. Human p138Tox corresponds to the carboxyl fragment of human ThOX2, lacking the first 338 residues. ThOX1 is identical to the previously cloned dualoxidase Duox1 (GenBank accession number AF213465), and its sequence is 83% similar to ThOX2. Whether ThOX1/ Duox1 participates to the Ca2⫹/NADPH-dependent H2O2 generator in thyroid tissue is still unknown. The C-terminal region of these novel proteins is very similar to the flavoproteins of the NOX family (14 –16). Although strongly expressed in thyrocytes, these membrane flavoproteins are not absolutely thyroid-specific, and the designations “thyroid oxidase”, “p138Tox”, and “ThOX” are therefore inadequate

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(17). Because ThOX/Duox flavoproteins constitute a new family of long homologs of the NOX flavoproteins and probably form a part of NADPH:O2-oxidoreductase systems, we will call them LNOX (large NOX) rather than Duox and/or ThOX (17). The present study was designed to analyze LNOX gene and protein expression in normal human thyroid tissue and in a series of thyroid tissues from patients with benign diseases, in which we already studied Tg, TPO, NIS, and PDS gene expression (18, 19). To this aim, we developed a realtime kinetic quantitative PCR to analyze gene expression (19), and we used an immunohistochemical method based on antipeptide antibodies to detect proteins (18). Materials and Methods Northern blot analysis The full coding sequence of the human p138Tox cDNA, corresponding to nucleotides 1015–3633 in the coding sequence of human LNOX2, was radiolabeled with [32P]␣-deoxycycidine triphosphate by random priming extension and separated from unincorporated nucleotides by filtration on G-50 Sephadex column. Hybridization of the LNOX2 and actin probes to three human Multiple Tissue Northern Blots, and membrane washing, were made according to the manufacturer’s instructions (CLONTECH Laboratories, Inc.). Detection of signals was performed by Electronic Autoradiography using a Packard Instant Imager (Packard Instrument Company, Meriden, CT).

Peptide synthesis, production, and characterization of antipeptide antisera to LNOX2 An antipeptide rabbit polyclonal antibody was produced, tested by enzyme-linked immunosorbent assay (ELISA), and purified by Eurogentec (Seraing, Belgium). A 14-amino acid peptide encompassing the L410-T423 portion of LNOX2 (LRDYWPGPGKFSRT) was synthesized by a conventional solid-phase method. For control experiments, the V404-T417 homologous peptide of LNOX1 (VRDFWPGPLKFSRT) was also synthesized by the same method. The purity and identity of peptides were verified by reverse phase-high-performance liquid chromatography and mass spectrometry. The L410-T423 LNOX2 peptide was conjugated to keyhole limpet hemocyanine on a cysteine residue added to the C-terminal threonine. Two rabbits were immunized by intradermal injections of the synthetic peptide-carrier conjugate. After two boosts at 4-week intervals, animals were bled, and their sera specimens were tested in an ELISA. Antisera, at various dilutions, were verified for their capacity to react with the L410-T423LNOX2 and with the V404-T417 homologous peptide of LNOX1 (100 ng in PBS) coated (4 C, 16 h) on microtiter plates. Antibody binding was then revealed by peroxidase-labeled antirabbit antibody (1:1000 dilution). Both animals produced antisera that were reactive in the ELISA assay. Antiserum with the highest affinity for the peptide, which was approximately 2 ⫻ 10⫺7 mol/L as determined in the competitive inhibition experiment, was purified by affinity chromatography (EAH-Sepharose coupled to the peptide). The purity estimated by SDS-PAGE was higher than 80%. ELISA assay showed that purified antiserum, obtained after immunization with the L410-T423 LNOX2 synthetic peptide-carrier conjugate, also reacted with the homologous V404-T417 LNOX1 synthetic peptide. The concentration of the nondiluted purified antibody was 0.7 mg/mL.

TABLE 1. Primer pair and TaqMan probe sequences Genes

Genbank accession number

LNOX1

AF230495

LNOX2

AF230496

FIG. 1. Expression profile of LNOX2 mRNA in human tissues. Northern blots were done with polyA⫹ RNA from: heart (lane 1), brain (lane 2), placenta (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), pancreas (lane 8), spleen (lane 9), thymus (lane 10), prostate (lane 11), testis (lane 12), ovary (lane 13), small intestine (lane 14), colon without mucosa (lane 15), peripheral blood leukocyte (lane 16), stomach (lane 17), thyroid (lane 18), spinal cord (lane 19), lymph node (lane 20), trachea (lane 21), adrenal gland (lane 22), and bone marrow (lane 23). Upper panel, Hybridization with human LNOX2 probe; lower panel, hybridization with human ␤-Actin cDNA control probe.

Primers and probes

Sens 4112 Antisens 4218 Probe Sens 2742 Antisens 2848 Probe

CCA GCA TGG GGC FAM-AGC CCG GCA CCT TGG FAM-AGC

ATC CGC AGC ATC GGC AGC

ATC TGG GTG ATC CTC GTG

TAT AAC GTG TAT TGG GTG

GGG C AAG GGA AAT AAG

GGC GCG GAG C-TAMRA GGT T GCG GAG C-TAMRA

EXPRESSION OF LNOX GENES IN THYROID Western blot Cos-7 cells were transiently transfected using the FuGENE 6 (Roche Molecular Biochemicals, Meylan, France) according to the manufacturer’s instructions. Transfected cells were incubated for 48 h, harvested, washed, and directly solubilized in SDS-PAGE sample buffer. Partially purified particulate fraction from thyroid tissue was prepared by differential centrifugation as previously described (9). Deglycosylation of membrane protein. Particulate fraction (200 ␮g) was treated for 10 min at 37 C in 50 mmol/L phosphate buffer, pH 7.4 (60 ␮L final vol) containing the antiprotease cocktail, 2.5 mmol/L EDTA, 0.1% SDS, before the addition of 10 U/mL N-glycosidase F (1 365 169; Roche Molecular Biochemicals). After a first incubation for 1 h, 10 or 25 U/mL N-glycosidase F was added again, and the incubation was prolonged for 1 h. Controls were performed without N-glycosidase F. The reaction was stopped by adding 1 vol of gel loading buffer (60 ␮L), and the samples were incubated overnight, at room temperature, before electrophoresis. SDS-PAGE and immunoblot analysis. Protein samples (25 ␮g) were boiled for 2 min in sample buffer (2% SDS, 5% ␤-mercaptoethanol and 10% TABLE 2. Blastn analysis of EST databases LNOX1 3⬘-UTR

Cerebellum Lung Testis Tongue epithelium Colon Gall bladder Pancreatic islet Prostate Uterus

LNOX2 3⬘-UTR

gb兩AA323168.1兩 gb兩AW292740.1兩 emb兩AL045997.2兩 gb兩AW275608.1兩

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glycerol) and were subjected to SDS-PAGE (19) using a 4 –12% linear gradient acrylamide slab minigel (Invitrogen Corporation/Novex, Carlsbad, CA) and electrotransferred (20) to 0.2 ␮m Protan BA 83 nitrocellulose sheets (Schleicher & Schuell, Inc., Ecquevilly, France) for immunodetection. Immunodetection of LNOX1,2 was achieved by preincubating membrane in TBS containing 5% fat free dry milk, and 0.1% Tween for 2 h at room temperature, and then overnight with the purified antipeptide antibody (1:1000 dilution, i.e. 0.7 ␮g/mL) in TBS with 1.5% milk, 0.1% Tween buffer, and 0.5 mmol/L dithiothreitol, with or without 10 ␮g/mL peptide. Antigen antibody complexes were detected using goat antirabbit IgG conjugated to alkaline phosphatase (Promega Corp., Madison, WI) and bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma, St. Louis, MO).

Tissue samples Thyroid tissue samples were selected after histological examination and classified according to World Health Organization recommendations (21). Tissue samples selected for real-time quantitative PCR were as follows: normal tissues (n ⫽ 5), multinodular goiters (n ⫽ 5), hyperfunctioning tissues, including Graves’ thyroid tissues (n ⫽ 5) and toxic adenomas (n ⫽ 8), and benign hypofunctioning follicular adenomas (n ⫽ 24). Specimens were frozen at ⫺80 C in isopentane and stored in liquid nitrogen before use. Except for the patients with Graves’ disease or toxic adenoma, in whom serum TSH was undetectable, thyroid samples were obtained from euthyroid subjects whose serum TSH concentrations were in the normal range at the time of surgery.

Immunohistochemistry gb兩AW849575.1兩 gb兩AA344113.1兩 gb兩W52149.1兩 gb兩AA687426.1兩 gb兩AW804496.1兩

Database and accession number of EST identical or highly similar to the 3⬘ untranslated region of LNOX1 and LNOX2 mRNA.

Immunohistochemistry was performed on Duboscq-Brasil-fixed, paraffin-embedded tissues blocks of 30 thyroid samples, selected from the above series, and including normal thyroid samples (n ⫽ 5), multinodular goiters (n ⫽ 5), Graves’ thyroid tissues (n ⫽ 4), toxic adenomas (n ⫽ 4), and hypofunctioning benign adenomas (n ⫽ 12, including 2 Hu¨rthle cell adenomas). Briefly, 5-␮m sections were initially deparaffinized by serial passages in xylene and in alcohol series. Endogenous peroxidase

FIG. 2. Comparison of LNOX1 and LNOX2 mRNA level in normal and pathological thyroid tissues. Individual LNOX1 (white bars) and LNOX2 (black bars) mRNA concentrations in normal thyroid (A), Graves’ thyroid tissue (B), toxic adenoma (C), multinodular goiter (D), hypofunctioning adenoma (E), are presented in arbitrary units.

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FIG. 3. LNOX1 (A) and LNOX2 (B) gene expression according to the histological types. Each plot represents the expression level as an x-fold difference, relative to the calibrator (normal thyroid). Lines, Median values. mRNA values according to the histological group are presented under the figure: mean (SD); median [minimum-maximum]; n, number of samples.

activity was quenched by incubation in 0.03% of hydrogen peroxide, in 0.1 mol/L Tris-HCl buffer 1X (pH 7.6) for 5 min. Subsequently, microwave/pressure cooker pretreatment (three cycles of 5 min each) was performed in 1 mmol/L EDTA buffer (pH 8). Sections were subsequently incubated for 30 min at room temperature with the anti-LNOX antiserum (1:25 dilution) or the anti-NIS antiserum (1:50 dilution). Sections were then washed three times in Tris-HCl 1X buffer for 5 min each time and incubated with a peroxidaseconjugated goat antirabbit antibody for 15 min (DAKO Corp., Carpinteria, CA). After three further washes, peroxidase staining was revealed in diaminobenzidine tetrahydrochloride (Polysciences Inc, Warrington, PA) with 0.1% of hydrogen peroxide, in Tris buffer, 0.01 mol/L (pH 7.2). Sections were counterstained with hematoxylin, dehydrated, and mounted. Negative controls were obtained by studying nonthyroid tissues and by studying thyroid tissues incubated with preimmune antisera and immune sera preabsorbed with an excess of the corresponding peptide. The percentage of positively stained cells was assessed semiquantitatively by light microscopy observation.

Determination of messenger RNA (mRNA) level using realtime PCR Total RNA was isolated from tissue samples using the DNA/RNA extraction Midi kit according to the manufacturer’s instructions (QIAGEN, Hilden, Germany). The quality of RNA was assessed by

conventional gel electrophoresis. One microgram of total RNA from each sample was reverse transcribed in a 20-␮L vol reaction using 50 U Murine Moloney Virus reverse transcriptase, 20 U ribonuclease inhibitor (Perkin-Elmer Corp. PE Applied Biosystems, Foster City, CA), 1 mmol/L dA/T/C/G (Amersham Pharmacia Biotech, Uppsala, Sweden), 5 mmol/L MgCl2, 10 mmol/L Tris HCl pH 8.3, 10 mmol/L KCl, and 50 pmol/L random hexamers (Perkin-Elmer Corp. PE Applied Biosystems). The cDNAs were then diluted 1:20 in nuclease-free H2O (Promega Corp.). Two pairs of oligonucleotide primers were designed to be, respectively, LNOX1- and LNOX2-specific and intron-spanning, using the computer program Primer Express (Perkin-Elmer Corp. PE Applied Biosystems). TaqMan probe sequence was designed to hybridize to a 100% identical region of LNOX1 and LNOX2 cDNA. They were purchased from Perkin-Elmer Corp. PE Applied Biosystems, and their sequences are presented in Table 1. PCR reaction was carried out to produce amplicons that were subsequently analyzed by gel electrophoresis and sequencing. Real-time quantitative PCR was achieved in 96 sample tubes/assay using a cDNA equivalent of 20 ng/total RNA䡠50 ␮L per tube with the TaqMan PCR core reagent kit, according to the manufacturer’s instructions: 1⫻ buffer A, 5 mmol/L MgCl2, 200 ␮mol/L dA/C/G, 400 ␮mol/L dU, 1.25 U AmpliTaq Gold DNA polymerase, 2.5 U uracyl N-glycosylase, 200 nmol/L TaqMan probe, and 400 nmol/L of each primer. PCR was developed on the ABI Prism 7700 Sequence Detector (Perkin-Elmer Corp. PE Applied Biosystems). To normalize for

EXPRESSION OF LNOX GENES IN THYROID differences in the amount of total RNA added to the reaction, amplification of 18S ribosomal RNA was performed as an endogenous control. Primers and probes for 18S RNA were purchased from Perkin-Elmer Corp. PE Applied Biosystems. The mRNA content of each target gene was simultaneously determined in a one-run assay. Standard curves for the LNOX1 and LNOX2 genes and 18S ribosomal RNA were constructed from PCR of calibrator cDNA strand serially diluted in nuclease-free H2O. The efficiency of the standard curves, as determined by their slope, allowed us to quantify the LNOX1 and LNOX2 gene expression profiles in each thyroid specimen by using the comparative threshold cycle method, according to the manufacturer’s instructions. The calibrator was constituted from one sample of normal tissue: it was used as the 1⫻ sample, and all other levels were expressed as an n-fold difference relative to the calibrator. The intraassay coefficient of variation was less than 1%. PCR control experiments, performed with each LNOX cDNA as template and with each pair of primers designed to be LNOX1- or LNOX2-specific, showed no interference of LNOX1 cDNA in quantification of LNOX2 cDNA, and vice versa.

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Statistical methods The expression of LNOX genes detected in each histological type was compared with that found in normal samples. mRNA expression patterns were compared using the t test, and correlation between these expression profiles was studied using Spearman’s rank-order correlation coefficient for each histological type. The level of significance chosen was fixed at 5%.

Results Northern blot analysis of LNOX2 gene expression

Among 23 different human tissues (Fig. 1), a high expression of an approximately 7-kb mRNA was evidenced in the thyroid gland, and a markedly lower expression in the trachea. No expression was observed in the other tissues. Colon polyA⫹ RNA, provided by CLONTECH Laboratories, Inc., were purified from tissues without mucosa. Blast analysis of expressed sequence tags (EST) databases

GenBank “expressed sequence tags” database (dbest) was searched using the sequences of the 3⬘-untranslated region (3⬘-UTR) of LNOX1 and of LNOX2 cDNA as queries. Table 2 reports EST from normal human tissues, other than the thyroid gland, with strong or complete similarity with the 3⬘-UTR sequences of LNOX1 and LNOX2 mRNA. Blast analysis indicates that LNOX1 gene is also expressed in the cerebellum, lung, testis, and squamous epithelium of the tongue. LNOX2 gene expression is found in the uterus, colon, prostate, gall-bladder, and pancreatic islets. Expression of LNOX1 and LNOX2 mRNAs in thyroid tissues FIG. 4. Correlation between LNOX1 and LNOX2 mRNA levels in the 47 thyroid tissues. Open circles correspond to hyperfunctioning tissues.

FIG. 5. Immunoblot analysis of LNOXs. A, A partially purified particulate fraction of normal thyroid tissue (fraction 2) was treated with (⫹) or without N-glycosidase F (⫺). C, Partially purified particulate fraction (25 ␮g) from normal thyroid tissue (1), total proteins (25 ␮g) from Cos-7 cells transfected with pREP4 expression vector alone (2), or Cos-7 cells transfected with pREP4 expression vector containing LNOX1 coding sequence cDNA (3). Immunoblot analyses were done with purified antipeptide antibodies incubated in the absence (A and C) or in the presence (B and D) of 10 ␮g/mL peptide. The position of proteins of known molecular mass (expressed in kDa) is indicated on the sides.

Figure 2 shows the LNOX1 and LNOX2 mRNA levels in 47 thyroid tissues. Figure 2A shows that, among the 5 specimens of normal tissue, LNOXI gene expression displayed

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FIG. 6. Immunostaining of LNOX1,2 and of NIS. A, Immunostaining for LNOX1,2 in normal thyroid tissue. The staining is localized in the apical membrane of follicular cells (magnification, ⫻400). B, Immunostaining for LNOX1,2 in normal thyroid tissue. The staining is heterogeneous inside a given follicle and from one follicle to another. Thick arrow, Positive cells; thin arrows, negative cells (magnification, ⫻100). C, Immunostaining for LNOX1,2 in hyperfunctioning tissue from Graves’ disease. No, or very weak, staining is observed (magnification, ⫻250). D, Immunostaining for NIS in hyperfunctioning tissue from Graves’ disease. The basolateral membrane of the majority of follicular cells is

EXPRESSION OF LNOX GENES IN THYROID

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variations (1- to 5-fold) and that even greater variations (1to 11-fold) affected LNOX2 gene expression. Considerable variations of both LNOX mRNA levels were also observed among the 5 Graves’ tissues (Fig. 2B), 8 toxic adenomas (Fig. 2C), 5 multinodular goiters (Fig. 2D), and 24 hypofunctioning adenomas (Fig. 2E). The mean mRNA levels of LNOX1 (Fig. 3A) and of LNOX2 (Fig. 3B) in pathological tissues were not statistically different from normal. Figure 4 shows that the expression of the two LNOX genes was significantly correlated (r ⫽ 0.6; P ⬍ 0.0001) in the 47 samples. However, values corresponding to hyperfunctioning tissues were mainly distributed under the curve, reflecting a lower proportion of LNOX2 mRNA in these samples.

lower in larger follicles. As in normal tissues, immunostaining was observed in groups of contiguous high cells that seem to define a hyperfunctioning area in the follicle. Morphologically flattened cells were negative (Fig. 6E). In hypofunctioning adenomas, LNOX immunostaining showed a higher number of positive cells than in hyperfunctioning thyroid tissues. Immunostaining was highly variable among adenomas and seemed to be related to the size of the follicles, ranging from a weak staining of a minority of cells in macrofollicular adenomas (not shown) to an intense staining in microfollicular adenomas (Fig. 6G). In Hu¨rthle cell adenomas, immunostaining was similar to that in normal tissue (Fig. 6F).

Western blot analysis

Comparison of LNOXs and NIS immunostaining

Immunoblot analysis of membrane proteins from normal tissue was performed using purified antiserum to LNOX2 peptide. A protein with a molecular mass of 164 ⫾ 3 kDa (mean ⫾ sd, n ⫽ 14) was detected in untreated membranes (Fig. 5A). N-glycosidase F treatment of membrane proteins induced a shift of the molecular mass to 148 ⫾ 9 kDa (n ⫽ 9). The 10 –20 kDa shift of the molecular mass was not increased by an additional treatment with N- glycosidase F (25 U/mL), suggesting that N-deglycosylation was maximal (not shown). Preincubation of the antibody with an excess of synthetic peptide prevented the labeling of these proteins (Fig. 5B). Although obtained after immunization with the L410-T423 LNOX2 synthetic peptide, the antiserum also detected, as a 162 ⫾ 3 kDa (n ⫽ 3) polypeptide, the recombinant human LNOX1 expressed in Cos-7 cells after transfection with pREP4 expression vector containing its full coding cDNA (Fig. 5C). An excess of synthetic peptide again prevented labeling the of these proteins (Fig. 5D).

The comparison of immunostaining for LNOXs and NIS on serial tissue sections from different pathological conditions demonstrated an inverse relationship between the number of LNOX-positive cells and of NIS-positive cells. In Graves’ thyroid tissues and in toxic adenomas, the number of LNOX-positive cells was low (0 –10%) and that of NISpositive cells was high (⬎80%) (Fig. 6, C and D). In contrast, in hypofunctioning microfollicular adenomas, the number of LNOX-positive cells was high, whereas the number of NISpositive cells was very low or null (Fig. 6, G and H).

Expression of LNOX proteins in thyroid tissues

In normal thyroid tissue, immunohistochemistry showed that LNOX proteins were localized at the apical membrane of thyrocytes (Fig. 6A). The staining was heterogeneous (Fig. 6B). Indeed, inside a given follicle, 40 – 60% of follicular cells, i.e. cells that are morphologically high and likely correspond to functionally active cells, were immunostained. Positive cells were contiguous and seemed to define a particular functioning area of the follicle. In contrast, flattened cells, which are usually considered as inactive, were negative. Stromal cells, lymphocytes, intrafollicular macrophages, and vascular endothelial cells did not react with anti-LNOX antibodies. In hyperfunctioning tissues, immunohistochemistry showed that only few follicular cells (0 –10%) were positive (Fig. 6C). Furthermore, these positive cells were weakly immunostained for LNOX1,2. In multinodular goiters, immunostaining was highly variable from one follicle to the other, but the average number of positive follicular cells was similar to that of normal thyroid tissues (40 – 60%). LNOX immunostaining was related to the size of the follicles: the great majority of cells were positive in small follicles, and the proportion of positive cells was

Discussion

Previous studies have shown that the p138Tox/LNOX2 mRNA expression was restricted to the thyroid gland in pig (12) and dog tissues, with a weak expression in dog stomach (13), whereas LNOX1 was found only in dog thyroid (13). In this study, Northern blot analysis was extended to 23 different human tissues. LNOX2 mRNA was present only in the thyroid gland and trachea. Expression in trachea was weak and may be attributable to a contamination by thyroid tissue. These data confirm that LNOX2 constitutes a new differentiation marker of thyrocytes. However, Northern blot studies were not sensitive enough to detect the probably low levels of LNOX2 mRNA evidenced in prostate and pancreas by a blast analysis of EST database. It is likely that only few cells from these tissues express LNOX genes. LNOX2 gene is expressed in rat colon (17), and probably also in human colon, as attested by numerous EST isolated from this tissue. Blast analysis revealed also the presence of LNOX1 mRNA in nonthyroid tissues such as the cerebellum, lung, testis, and tongue epithelium. The availability of a polyclonal antibody permitted us to analyze LNOX expression in various thyroid tissues. As shown by ELISA analysis, the antibody raised against LNOX2 peptide also reacted with the homologous LNOX1 peptide. It also reacted with recombinant human LNOX1 in Western blot analyses. LNOX1,2 were detected as immunoreactive proteins with an apparent molecular mass of 164 kDa; but in the absence of specific antibody, it was not possible to determine the relative proportion of each LNOX

strongly stained (magnification, ⫻100). E, Immunostaining for LNOX1,2 in multinodular goiter. The staining is restricted to high active columnar follicular cells. The flattened inactive follicular cells (arrow) are not stained (magnification, ⫻250). F, Immunostaining for LNOX1,2 in a Hu¨rthle cells adenoma that is similar to normal tissues (magnification, ⫻250). G, Immunostaining for LNOX1,2 in a hypofunctioning microfollicular adenoma. The majority of follicular cells are strongly stained (magnification, ⫻250). H, Immunostaining for the NIS in the same hypofunctioning microfollicular adenoma as in Fig. 5G. No staining is observed (magnification, ⫻250).

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protein in thyroid tissue. Western blot analyses demonstrated that LNOX proteins are actually glycoproteins, and that N-glycosylation motifs account for 10 –20 kDa of their molecular mass. The predicted molecular mass of the unglycosylated LNOX proteins being 177 kDa (13), the glycosylated molecules should exhibit a molecular mass of at least 187–197 kDa. It is known that binding of higher amount of SDS to hydrophobic proteins than to marker proteins accelerates their relative rate of migration during electrophoresis, leading to an underestimation of their molecular mass. Because LNOX1,2 have seven hydrophobic stretches, such a mechanism may be responsible for their low apparent molecular mass in our experiments. Alternatively, a maturation or a degradation of the protein involving the release of a 20 –30 kDa peptide cannot be excluded. The immunohistochemical study confirmed that LNOX1,2 are located at the apical pole of thyrocytes (13). This localization is in agreement with the well-known localization of iodide organification (22), and of the H2O2 generator system (23–25) to which at least LNOX2 is likely to participate. In normal tissue, 40 – 60% of follicular cells were stained and corresponded to high, columnar cells, which are usually considered as functionally active cells. In contrast, flattened cells, which are usually considered as functionally inactive, were not stained. In both multinodular goiters and in hypofunctioning adenomas, the proportion of positive cells and the intensity of staining were much higher in microfollicles usually considered as active than in macrofollicles usually considered as inactive. These data suggest that LNOX expression may be related to the functional activity of the follicle, and inside a given follicle to the functional activity of each cell. Quantitative PCR measurements of each mRNA revealed that the levels of the two LNOX mRNAs varied in parallel, suggesting that the two genes, closely linked on chromosome 15, are regulated by similar mechanisms (Fig. 4). However, the expression of each LNOX gene may also be more specifically modulated, as suggested by a lower proportion of LNOX2 mRNA in hyperfunctioning tissues. Although a positive effect of cAMP has been previously observed on the expression of the H2O2 generator (26), of NADPH oxidase activity (27), and recently of LNOX1 and LNOX2 mRNAs and proteins in cultured thyrocytes (12, 13, 17), a decrease in LNOX2 mRNA level in the hyperstimulated thyroid gland of rat treated by an antithyroid drug has been reported (17). Despite a dependent expression of all known thyroid functional genes upon cAMP stimulation, other mechanisms occur in vivo and may account for the absence of increased expression of LNOX2 mRNAs in hyperstimulated glands. Average LNOX1 and LNOX2 mRNA levels seemed to be unchanged in hyperfunctioning human tissues (Fig. 3), in contrast to that of NIS and TPO, which are increased (28). However, the considerable individual variations of LNOX1 and of LNOX2 mRNA levels within each group of tissues made this comparison poorly relevant. Further studies are therefore necessary to assess the consequences of hyperstimulation of human thyroid on each LNOX at the mRNA level. By contrast, the substantial decrease of LNOX immunostaining in all Graves’ thyroid tissues and toxic adenoma demonstrates the existence of mechanisms that may protect hyperstimulated thyrocytes from oxidative stress resulting from an overexpression of NADPH oxidase. This is in agree-

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