Characterization and Semiquantitative Analyses of Pendrin Expressed in Normal and Tumoral Human Thyroid Tissues

Abstract

The gene mutated in Pendred syndrome (PDS), the PDS gene, is expressed in the inner ear, kidney, and thyroid. It encodes a membrane protein named pendrin that is endowed with the function of anion transporter or exchanger. It has been postulated that in the thyroid pendrin could participate in the transport of iodide from the cell to the lumen of follicles. We generated antipeptide antibodies directed against the C- terminal sequence of human pendrin 1) to characterize the protein expressed in the human thyroid, and 2) to analyze its expression level in relation to the functional activity of thyroid tissue. In denaturing conditions, a single molecular species of 110-115 kDa was identified in human thyroid membrane fractions. After treatment of thyroid membranes with N-glycosidase F, pendrin had an apparent molecular mass of 85 kDa. Analyzed by ultracentrifugation on sucrose gradient in nondenaturing conditions, pendrin sedimented as a main 120- to 140-kDa component. Pendrin was assayed by semiquantitative Western blot in thyroid membrane fractions from 25 hyper- or hypofunctioning tumors and paired normal tissue samples. Pendrin was increased 2-fold in toxic adenomas, was not significantly altered in follicular adenoma, and was decreased, on the average, by 35% in papillary carcinomas compared with levels in paired normal tissue. The variations in the pendrin tissue content and PDS transcript levels, assayed by RT-PCR on duplicate samples of the same tumors, were similar. In conclusion, we show that pendrin expressed by the human thyroid gland is a mainly monomeric glycoprotein and that the level of expression of pendrin, although somewhat related, only moderately varied with the functional status of the thyroid tissue.

Evidence for Transcriptional and Posttranscriptional Alterations of the Sodium/Iodide Symporter Expression in Hypofunctioning Benign and Malignant Thyroid Tumors

“…Protein was assayed by the Lowry method after solubilization in 0.1% sodium deoxycholate. The total amount of protein per mass of tissue and the proportion of membrane protein (100,000 ϫ g pellet) obtained from tumors (FA, PTC, or TA) and paired NT samples were very similar; 16 protein of the crude membrane fractions represented 25 to 35% of total protein. Membrane proteins (1 to 180 g of protein) were subjected to electrophoresis on 7.5% acrylamide gel in the presence of sodium dodecyl sulfate (SDS) and electrotransferred onto Immobilon P membranes (Millipore Corp., Bedford, MA).…”

“…To determine whether the dissociation between transcript and protein levels was a general feature of hypofunctioning tumors, transcripts of the PDS gene and the corresponding protein, pendrin (another membrane protein with an iodide transport activity), were assayed in the same tumors and NT samples, as previously described. 16 Transcripts from the PDS gene and pendrin were detected in all samples. Data from four paired samples are reported in Figure 5B.…”

“…16 Tissue samples weighing 50 to 200 mg, quickly frozen in liquid nitrogen and stored at Ϫ80°C, consisted of paired samples, benign or malignant thyroid tumors, and surrounding normal tissue (NT). Pathological and clinical annotations were anonymously associated with thyroid tissue samples classified according to World Health Organization recommendations.…”

“…Immune complexes were revealed by the enhanced chemiluminescence method using the enhanced chemiluminescent substrate from Amersham Biosciences (Orsay, France) and exposed to Kodak X-Omat AR films (Eastman Kodak Co., Rochester, NY). In a second step, the same membranes were incubated with a monoclonal antibody (mAb) directed against the ␣-subunit of Na ϩ ,K ϩ -adenosine triphosphatase (Na ϩ ,K ϩ -ATPase) at a final dilution of 1:25,000 16 and then with a biotinylated goat anti-mouse Ig from Amersham Biosciences. Immune complexes were visualized using streptavidin conjugated to alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate as substrate.…”

“…Immune complexes were visualized using streptavidin conjugated to alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate as substrate. Western blot signals from the enhanced chemiluminescence (hNIS) and alkaline phosphatase (Na ϩ ,K ϩ -ATPase ␣-subunit) reactions were quantified as previously reported 16 and expressed in luminance values. The ␣-subunit of the Na ϩ ,K ϩ -ATPase was used to control the amount of protein subjected to the hNIS Western blot assay.…”