Targeting of thyroglobulin to transcytosis following megalin-mediated endocytosis: evidence for a preferential pH-independent pathway.

TG internalized from the colloid by megalin, bypasses the lysosomal pathway and is transported across thyrocytes by transcytosis. Although most of the intracellular mechanisms responsible for targeting of ligands to transcytosis are unknown, for certain ligands a role of lysosomal pH has been established. Thus, ligands that undergo lysosomal degradation dissociate from their receptors due to the low pH of endosomes, whereas certain ligands that undergo transcytosis fail to dissociate because they bind to their receptors at acidic pH. Here we studied the role of pH in TG transcytosis. We first investigated the effect of pH on megalin binding to TG in solid phase assays and found that, although megalin bound to TG at various pH values (ranging from 4-8), optimal binding was seen at acidic pH (ranging from 4.5-6). We then studied the effect of chloroquine (CQ) and ammonium chloride (AC), which increase endosomal pH, on transcytosis of TG across Fisher rat thyroid (FRTL-5 cells). Transcytosis assays were performed using FRTL-5 cells cultured on filters in dual chambered devices, with megalin expression only on the upper surface of the layers. TG was added to the upper chamber and transcytosed TG was measured in fluids collected from the lower chamber after incubation at 37 C. Treatment of FRTL-5 cells with CQ or AC did not affect binding and uptake of TG, but it did reduce T3 release from exogenously added TG, used as a measure of TG degradation in the lysosomal pathway. Treatment with CQ or AC resulted in an increase of transcytosis of TG across FRTL-5 cells, but only to a minimal extent (15-20%). The effects of CQ or AC and those of a megalin competitor (the monoclonal antibody 1H2, which reduced transcytosis) were not additive, suggesting that CQ and AC act on the megalin-mediated pathway. In conclusion, because TG binding to megalin is greatest at acidic pH, it is possible that TG does not dissociate from megalin in the lysosomal pathway. However, the pH-dependence of TG binding to megalin does not account for much of transcytosis, which probably occurs largely because of other mechanisms of targeting.

Impaired thyroglobulin (Tg) secretion by FRTL-5 cells transfected with soluble receptor associated protein (RAP): evidence for a role of RAP in the Tg biosynthetic pathway.

Secretion of thyroglobulin (Tg) by thyrocytes requires several endoplasmic reticulum (ER)-resident molecular chaperones. The receptor-associated protein (RAP), a known molecular chaperone, binds to Tg in thyroid cells shortly after biosynthesis. Here we investigated whether RAP is involved in Tg secretion by FRTL-5 cells. For this purpose, we studied Tg secretion by FRTL-5 cells transfected with a soluble RAP chimera, as a mean for interfering with endogenous RAP. We used a RAP-human IgG Fc (RAP-Ig) chimeric cDNA, which was designed in order to exclude the ER retention sequence of RAP and to allow generation of a secreted form of RAP. FRTL-5 cells were transiently transfected with the RAP-Ig cDNA or, as control, with a CD8-Ig cDNA. Media were collected at 24, 48 and 72 h after transfection. Secretion of fusion proteins and of Tg in the media was measured by ELISA. As expected, under standard culture conditions, RAP was not secreted into the media by FRTL-5 cells, even though it could be detected by Western blotting in cell extracts. In transfection experiments, fusion proteins were present in the media of FRTL-5 cells transfected with either RAP-Ig or CD8-Ig, indicating that transfection was successful. Although Tg was found in the media of FRTL-5 cells transfected with either CD8-Ig or RAP-Ig, a lower amount was found in cells transfected with RAP-Ig. Therefore, we concluded that RAP is involved in Tg secretion by FRTL-5 cells suggesting that RAP may function as a Tg molecular chaperone.

Binding of the low density lipoprotein receptor-associated protein (RAP) to thyroglobulin (Tg): putative role of RAP in the Tg secretory pathway.

The 39-44 kDa protein known as the receptor-associated protein binds to members of the low density lipoprotein receptor family and is found within cells that express these receptors. The receptor-associated protein has been shown to prevent premature binding of ligands to the receptors in the endoplasmic reticulum and to promote proper folding and transport of the receptors in the secretory pathway. In the thyroid, megalin (a low-density lipoprotein receptor family member) serves as an endocytic receptor for thyroglobulin. Here we present evidence that the receptor-associated protein can bind to thyroglobulin, which suggests a novel function of the receptor-associated protein, namely binding of certain megalin ligands possibly during the biosynthetic pathway. In solid-phase assays thyroglobulin was shown to bind to the receptor-associated protein with moderately high affinity (mean between K(d) and K(i) = 39.8 nM), in a calcium-dependent and saturable manner. The receptor-associated protein also bound to a native carboxyl-terminal 230-kDa thyroglobulin polypeptide, which markedly reduced binding of intact thyroglobulin to the receptor associated protein, indicating that the receptor-associated protein binding sites of thyroglobulin are located in the carboxyl-terminal portion of the molecule. In addition to thyroglobulin, the receptor-associated protein specifically bound to another megalin ligand, namely lipoprotein lipase. Because lipoprotein lipase markedly reduced receptor-associated protein binding to thyroglobulin, we concluded that the receptor-associated protein uses the same binding site/s to bind to thyroglobulin and lipoprotein lipase. Evidence of thyroglobulin binding to the receptor-associated protein was also obtained in vivo and in cultured thyroid cells. Thus, anti-receptor-associated protein antibodies precipitated intact thyroglobulin from extracts prepared from rat thyroids and cultured thyroid cells (FRTL-5 cells). Chase experiments after inhibition of protein synthesis in FRTL-5 cells showed that thyroglobulin interacts with the receptor-associated protein shortly after the beginning of thyroglobulin biosynthesis.

Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level.

BACKGROUND: Phosphoinositide 3-kinase (PI3-K) is implicated in various cellular processes involving signaling, including intracellular trafficking. PI3-K has been shown to play a part in both receptor- and non-receptor-mediated transcytosis across cultured kidney cells and undifferentiated thyroid cells. OBJECTIVE: To investigate the role of PI3-K in transcytosis of thyroglobulin (Tg) across differentiated cultured Fisher rat thyroid cells (FRTL-5 cells) - a process known to be mediated by megalin, a member of the low-density lipoprotein receptor family. DESIGN: We studied the effect of the microbial product wortmannin, a specific inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells. METHODS: Transcytosis experiments were performed using FRTL-5 cells cultured as tight layers on filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper cell surface. Tg was added to the upper chamber and cells were incubated at 37 degrees C. Transcytosed Tg was measured in fluids collected from the lower chamber. To study the role of PI3-K, cells were pre-incubated with wortmannin. RESULTS: Pre-incubation of FRTL-5 cells with wortmannin did not affect Tg binding and uptake, but resulted in a considerable increase in Tg transcytosis (by 40-75%, depending on the concentration of wortmannin), suggesting that PI3-K exerts an inhibitory effect on Tg transcytosis. In experiments in which a monoclonal antibody against megalin was used to reduce Tg transcytosis, pre-incubation with wortmannin did not increase Tg transcytosis from its reduced levels, indicating that PI3-K is involved in the megalin-mediated pathway. Wortmannin did not affect the extent of release of tri-iodothyronine from exogenously added Tg by FRTL-5 cells, which was used as a measure of Tg degradation in the lysosomal pathway, indicating that the effect of PI3-K on transcytosis occurs after diversion of Tg from the lysosomal pathway. CONCLUSIONS: PI3-K exerts an inhibitory role on megalin-mediated Tg transcytosis across cultured thyroid cells. PI3-K action takes place at a post-sorting level, after Tg bypassing of the lysosomal pathway.

Identification of thyroglobulin in orbital tissues of patients with thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is thought to have an autoimmune pathogenesis because of its association with autoimmune thyroid disease, in particular with Graves' disease. Nevertheless, the nature of the autoimmune reaction is unclear, and a target orbital autoantigen has not been conclusively identified. A widely discussed hypothesis is that antigens constitutively shared by the thyroid and orbital tissues are targets of an autoimmune reaction. It has been also postulated that a thyroid-soluble antigen, namely thyroglobulin (Tg), is transported to orbital tissues through the lymphatics, where it accumulates and elicits autoimmune damages in susceptible individuals. Here we have investigated whether Tg is present in orbital tissues from patients with TAO. Retrobulbar tissue specimens were obtained from three patients with Graves' disease and TAO who underwent decompressive orbitotomy, and at autopsy from two patients with no thyroid or eye disease. All patients with TAO had been previously treated with radioiodine to control Graves' hyperthyroidism. Western blot analysis with a monoclonal anti-Tg antibody showed the presence of intact Tg, both in soluble and membrane-associated fractions of orbital tissue extracts from the patients with TAO, in amounts estimated to range from approximately 320 to approximately 900 pg/microg of tissue protein. In contrast, Tg was not detected in orbital tissue extracts from patients with no thyroid or eye disease. Tg was also demonstrated in orbital tissue extracts from two of three patients with TAO by enzyme-linked immunosorbent assay (ELISA), in amounts estimated to range from approximately 450 to approximately 1000 pg/microg of protein. In addition, Tg in orbital tissue extracts from patients with TAO was immunoprecipitated by a rabbit anti-Tg antibody, suggesting that it retained its native conformation. An anti-thyroxine (T4) antibody captured in solid-phase Tg from orbital tissue extracts, showing that it contained thyroid hormone residues and had therefore originated in the thyroid. Tg-anti-Tg immune complexes were not found in orbital tissues, suggesting that if an autoimmune reaction to Tg occurs in TAO, it is likely to be cell mediated.

Megalin in thyroid physiology and pathology.

Megalin, a member of the low density lipoprotein endocytic receptor family, is expressed on the apical surface of thyroid epithelial cells, directly facing the follicle lumen, where colloid is stored in high concentrations. Studies in vivo and with cultured thyroid cells have provided evidence that megalin expression on thyroid cells is TSH-dependent. Thyroglobulin (Tg), the major protein component of the colloid and the precursor of thyroid hormones, binds to megalin with high affinity and megalin mediates in part its uptake by thyrocytes. Tg internalized by megalin avoids the lysosomal pathway and is delivered by transepithelial transport (transcytosis) to the basolateral membrane of thyrocytes, from which it is released into the bloodstream. This process competes with pathways leading to thyroid hormone release from Tg molecules, which occurs following internalization of Tg molecules from the colloid by other means of uptake (fluid phase endocytosis or endocytosis mediated by low affinity receptors) that result in proteolytic cleavage in the lyosomes. During transcytosis of Tg, a portion of megalin (secretory component) remains complexed with Tg and enters the circulation, where its detection may serve as a tool to identify the origin of serum Tg in patients with thyroid diseases. Tg endocytosis via megalin is facilitated by the interaction of Tg with cell surface heparan sulfate proteoglycans, which occurs via a carboxyl terminal heparin binding site of Tg functionally related with a major megalin binding site. Although autoantibodies against megalin can be found in the serum of approximately 50% of patients with autoimmune thyroiditis, a role of megalin in this and other thyroid diseases remains to be established.

Role of thyroglobulin endocytic pathways in the control of thyroid hormone release.

Thyroglobulin (Tg), the thyroid hormone precursor, is synthesized by thyrocytes and secreted into the colloid. Hormone release requires uptake of Tg by thyrocytes and degradation in lysosomes. This process must be precisely regulated. Tg uptake occurs mainly by micropinocytosis, which can result from both fluid-phase pinocytosis and receptor-mediated endocytosis. Because Tg is highly concentrated in the colloid, fluid-phase pinocytosis or low-affinity receptors should provide sufficient Tg uptake for hormone release; high-affinity receptors may serve to target Tg away from lysosomes, through recycling into the colloid or by transcytosis into the bloodstream. Several apical receptors have been suggested to play roles in Tg uptake and intracellular trafficking. A thyroid asialoglycoprotein receptor may internalize and recycle immature forms of Tg back to the colloid, a function also attributed to an as yet unidentified N-acetylglucosamine receptor. Megalin mediates Tg uptake by thyrocytes, especially under intense thyroid-stimulating hormone stimulation, resulting in transcytosis of Tg from the colloid to the bloodstream, a function that prevents excessive hormone release.

Circulating thyroglobulin transcytosed by thyroid cells in complexed with secretory components of its endocytic receptor megalin.

After its endocytosis from the colloid, some thyroglobulin (Tg) is transcytosed intact across thyrocytes, accounting in part for its presence in the circulation. We previously showed that megalin (gp330), an endocytic Tg receptor, mediates apical to basolateral Tg transcytosis. Here we investigated whether a portion of megalin remains combined with Tg after its transcytosis, using studies with cultured thyroid cells and in vivo observations. FRTL-5 cells, a rat thyroid cell line, cultured on filters in dual chambers form tight junctions and exhibit features of polarity, with expression of megalin exclusively on the upper (apical) surface. After the addition of unlabeled Tg to the upper chamber and incubation at 37 C, some Tg was transcytosed intact across FRTL-5 cells into the lower chamber. Two antimegalin ectodomain antibodies precipitated transcytosed Tg in fluids collected from the lower chamber. After the addition of Tg to surface-biotinylated FRTL-5 cells, an anti-Tg antibody and the two antimegalin ectodomain antibodies precipitated high molecular mass biotinylated material in fluids collected from the lower chamber, corresponding to much of the megalin ectodomain, as well as smaller amounts of lower molecular mass material. The results indicate that Tg transcytosed across FRTL-5 cells remains complexed with megalin ectodomain components, which we refer to as megalin secretory components. In aminotriazole-treated rats, which develop increased megalin-mediated Tg transcytosis, antimegalin antibodies precipitated some of the Tg in the serum. Tg was also precipitated by antimegalin antibodies in sera from patients with Graves' disease, in which we found increased megalin expression on the apical surface of thyrocytes. In contrast, in thyroidectomized patients with metastatic papillary thyroid carcinoma, in whom Tg is directly secreted by neoplastic thyroid cells into the circulation rather than transcytosed, serum Tg was not precipitated by antimegalin antibodies. The detection of Tg-megalin complexes may help identify the source of serum Tg in patients with thyroid diseases.

Binding of rat thyroglobulin to heparan sulfate proteoglycans.

We previously showed that rat thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits Tg binding to megalin (gp330), an endocytic Tg receptor found on the apical surface of thyrocytes. Cooperation between cell surface receptors and heparin-like molecules, namely heparan sulfate proteoglycans (HSPGs), can facilitate cell surface binding of some heparin-binding proteins. Based on our previous findings indicating that heparin and megalin-binding sites of rat Tg are functionally related, here we investigated whether rat Tg binds to HSPGs, which are expressed by thyroid cells. We showed in solid phase assays that unlabeled rat Tg binds to a heparan sulfate (HS) preparation in a dose-dependent, saturable manner, with moderately high affinity (Kd approximately 19 nM, Ki approximately 25 nM). Binding was inhibited by heparin and by HS itself. We then studied the role of HSPGs in Tg binding to FRTL-5 cells, a differentiated Fisher rat thyroid cell line. As previously reported, after incubation of FRTL-5 cells with unlabeled rat Tg at 4 degrees C, heparin released virtually all the cell-bound Tg. Co-incubation of Tg with HS or with a preparation of HSPGs resulted in a reduction of binding by 35%-40%. When FRTL-5 cells were preincubated with heparitinase or heparinase I, which released 20%-30% of cell surface HSPGs, Tg binding was reduced to a similar extent. An antibody against a Tg heparin-binding site functionally related to a major megalin-binding site virtually abolished Tg binding to HS and to FRTL-5 cells, supporting the hypothesis that combined interactions of Tg with HSPGs and with megalin are involved in Tg binding to rat thyroid cells.

Megalin-mediated transcytosis of thyroglobulin by thyroid cells is a calmodulin-dependent process.

Megalin, a multiligand receptor expressed on the apical surface of thyroid cells, mediates transepithelial transport (transcytosis) of thyroglobulin (Tg) across thyrocytes, resulting in diversion of Tg from the lysosomal pathway and reduction of the extent of thyroid hormone release from internalized Tg molecules. The calcium regulatory protein calmodulin facilitates some forms of transcytosis. Here we investigated the role of calmodulin in megalin-mediated transcytosis of Tg by thyroid cells. For this purpose, we studied the effect of calmodulin antagonists on Tg transcytosis by Fisher rat thyroid cells (FRTL-5), an established, differentiated thyroid cell line. FRTL-5 cells were cultured on permeable filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper surface. Unlabeled Tg was added to the upper chamber at 37 degrees C, and transcytosed Tg was detected by enzyme-linked immunosorbent assay (ELISA) in fluids collected 1 hour later from the lower chamber. To study the role of calmodulin in Tg transcytosis, cells were preincubated with one of two calmodulin antagonists, either trifluoperazine or W7. Both antagonists markedly reduced transcytosis of Tg by FRTL-5 cells. These inhibitory effects and those of a monoclonal antimegalin antibody were not additive, indicating that calmodulin acts on the megalin-mediated pathway. Furthermore, trifluoperazine increased the extent of triiodothyronine (T3) release from exogenously added Tg by FRTL-5 cells, indicating that Tg transported in the calmodulin-dependent, megalin-mediated pathway, bypasses the lysosomal pathway.

Role of megalin (gp330) in transcytosis of thyroglobulin by thyroid cells. A novel function in the control of thyroid hormone release.

When thyroglobulin (Tg) is endocytosed by thyrocytes and transported to lysosomes, thyroid hormones (T4 and T3) are released. However, some internalized Tg is transcytosed intact into the bloodstream, thereby avoiding proteolytic cleavage. Here we show that megalin (gp330), a Tg receptor on thyroid cells, plays a role in Tg transcytosis. Following incubation with exogenous rat Tg at 37 degrees C, Fisher rat thyroid (FRTL-5) cells, a differentiated thyroid cell line, released T3 into the medium. However, when cells were incubated with Tg plus either of two megalin competitors, T3 release was increased, suggesting that Tg internalized by megalin bypassed the lysosomal pathway, possibly with release of undegraded Tg from cells. To assess this possibility, we performed experiments in which FRTL-5 cells were incubated with either unlabeled or (125)I-labeled Tg at 37 degrees C to allow internalization, treated with heparin to remove cell surface-bound Tg, and further incubated at 37 degrees C to allow Tg release. Intact 330-kDa Tg was released into the medium, and the amount released was markedly reduced by megalin competitors. To investigate whether Tg release resulted from transcytosis, we studied FRTL-5 cells cultured as polarized layers with tight junctions on permeable filters in the upper chamber of dual chambered devices. Following the addition of Tg to the upper chamber and incubation at 37 degrees C, intact 330-kDa Tg was found in fluids collected from the lower chamber. The amount recovered was markedly reduced by megalin competitors, indicating that megalin mediates Tg transcytosis. We also studied Tg transcytosis in vivo, using a rat model of goiter induced by aminotriazole, in which increased release of thyrotropin induces massive colloid endocytosis. This was associated with increased megalin expression on thyrocytes and increased serum Tg levels, with reduced serum T3 levels, supporting the conclusion that megalin mediates Tg transcytosis. Tg transcytosis is a novel function of megalin, which usually transports ligands to lysosomes. Megalin-mediated transcytosis may regulate the extent of thyroid hormone release.

Identification of a heparin-binding region of rat thyroglobulin involved in megalin binding.

We recently showed that thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits binding of Tg to its endocytic receptor megalin (gp330). Here we have identified a heparin-binding region in the carboxyl-terminal portion of rat Tg and have studied its involvement in megalin binding. Rat thyroid extracts, obtained by ammonium sulfate precipitation, were separated by column fractionation into four Tg polypeptides, with apparent masses of 660, 330, 210, and 50 kDa. As assessed by enzyme-linked immunoadsorbent assays and ligand blot binding assays, megalin bound to intact Tg (660 and 330 kDa) and, to a even greater extent, to the 210-kDa Tg polypeptide. Furthermore, the 210-kDa Tg polypeptide inhibited megalin binding to intact Tg by approximately 70%. Solid phase assays showed binding of biotin-labeled heparin to intact Tg and to the 210-kDa Tg polypeptide. We characterized the 210-kDa Tg polypeptide by matrix-assisted laser desorption/ionization mass spectrometry analysis and found that it corresponds to the carboxyl-terminal portion of rat Tg. We developed a synthetic peptide corresponding to a 15-amino acid sequence in the carboxyl-terminal portion of rat Tg (Arg(689)-Lys(703)), containing a heparin-binding consensus sequence (SRRLKRP) and demonstrated heparin binding to this peptide. A rabbit antibody raised against the peptide recognized intact Tg in its native conformation and under denaturing conditions. This antibody markedly reduced heparin-binding to intact Tg, indicating that the region of native Tg corresponding to the peptide is involved in heparin binding. Furthermore, the anti-Tg peptide antibody almost completely inhibited binding of megalin to Tg, suggesting that the Tg region containing the peptide sequence is required for megalin binding. Physiologically, Tg binding to megalin on thyroid cells may be facilitated by Tg interaction with heparin-like molecules (heparan sulfate proteoglycans) via adjacent binding sites.

Serum antibodies against megalin (GP330) in patients with autoimmune thyroiditis.

Megalin (gp330) is a multiligand receptor found on the apical surface of selected epithelial cells, including thyroid cells. We recently showed that megalin is a high-affinity receptor for thyroglobulin. Megalin is capable of inducing autoantibodies, as shown in the rat model, Heymann nephritis. Based on this consideration and on the knowledge that autoantibodies against several thyroid antigens develop in patients with autoimmune thyroid diseases, we searched for antimegalin antibodies in 78 patients with autoimmune and nonautoimmune thyroid diseases. We developed an assay, based on flow cytometry, to measure binding of serum IgGs to L2 cells, a rat carcinoma cell line that expresses abundant megalin. After incubation of L2 cells with serum samples and then with fluorescein isothiocynate-conjugated antihuman IgG Fc-specific antibody, the mean fluorescence intensity (MFI) was determined. Using results obtained in sera from 32 normal subjects, we established a cutoff value for MFI (50.62), above which, tests were considered positive. Significantly elevated values were found in 18 patients, including 13 of 26 patients with autoimmune thyroiditis (50.0%) and in 2 of 19 patients with Graves' disease (10.5%). Furthermore, 2 of 19 patients with nontoxic goiter (10.5%) and 1 of 14 patients with differentiated thyroid cancer (7.14%) had MFI values greater than 50.62, associated with the presence of circulating antithyroid autoantibodies. As a control cell line, we used Chinese hamster ovary cells, which do not express megalin. We found that, among the 18 patients with positive tests for binding to L2 cells, only 1 patient with nontoxic goiter had significant binding of serum IgGs to Chinese hamster ovary cells. Binding of serum IgGs to L2 cells was significantly reduced by coincubation with purified megalin in 15 of 18 positive patients (83.33%) and by a rabbit antimegalin antibody in 11 patients (61.11%). Further and more conclusive evidence that positive tests (MFI >50.62) for binding to L2 cells were attributable to serum antimegalin antibodies was demonstrated by immunoprecipitation experiments. After incubation of serum samples with L2 cell extracts, incubation with antihuman IgG Fc-specific agarose beads resulted in immunoprecipitation of megalin in all the 18 positive patients, but not in normal subjects, as assessed by Western blotting using a monoclonal antibody against megalin. Furthermore, the intensity of the band corresponding to megalin precipitated by serum IgGs in the above 18 patients was significantly correlated with the L2 binding MFI. This is the first clear-cut demonstration of antibodies against megalin in humans. Further studies are needed to determine whether antimegalin antibodies have pathogenic significance or diagnostic value in autoimmune thyroid diseases.

Megalin (gp330) is an endocytic receptor for thyroglobulin on cultured fisher rat thyroid cells.

We recently reported that megalin (gp330), an endocytic receptor found on the apical surface of thyroid cells, binds thyroglobulin (Tg) with high affinity in solid phase assays. Megalin-bound Tg was releasable by heparin. Here we show that Fisher rat thyroid (FRTL-5) cells, a differentiated rat thyroid cell line, can bind and endocytose Tg via megalin. We first demonstrated that FRTL-5 cells express megalin in a thyroid-stimulating hormone-dependent manner. Evidence of Tg binding to megalin on FRTL-5 cells and on an immortalized rat renal proximal tubule cell line (IRPT cells), was obtained by incubating the cells with 125I-Tg, followed by chemical cross-linking and immunoprecipitation of 125I-Tg with antibodies against megalin. To investigate cell binding further, we developed an assay in which cells were incubated with unlabeled Tg at 4 degrees C, followed by incubation with heparin, which released almost all of the cell-bound Tg into the medium. In solid phase experiments designed to illuminate the mechanism of heparin release, we demonstrated that Tg is a heparin-binding protein, as are several megalin ligands. The amount of Tg released by heparin from FRTL-5 and IRPT cells, measured by enzyme-linked immunosorbent assay (ELISA), was markedly reduced by two megalin competitors, receptor-associated protein (RAP) and 1H2 (monoclonal antibody against megalin), indicating that much of the Tg released by heparin had been bound to megalin ( approximately 60-80%). The amount inhibited by RAP was considered to represent specific binding to megalin, which was saturable and of high affinity (Kd approximately 11.2 nM). Tg endocytosis by FRTL-5 and IRPT cells was demonstrated in experiments in which cells were incubated with unlabeled Tg at 37 degrees C, followed by heparin to remove cell-bound Tg. The amount of Tg internalized (measured by ELISA in the cell lysates) was reduced by RAP and 1H2, indicating that Tg endocytosis is partially mediated by megalin.

Alternating antineutrophil cytoplasmic antibody specificity: drug-induced vasculitis in a patient with Wegener's granulomatosis.

We describe a patient who presented with Wegener's granulomatosis associated with antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) with a cytoplasmic immunofluorescence pattern (cANCA), whose ANCA type changed to antimyeloperoxidase antibodies with a perinuclear immunofluorescence pattern (pANCA) when treated with propylthiouracil, and changed back to anti-PR3 antibodies with cANCA after the medication was discontinued. The patient developed flares of vasculitis symptoms associated with rises in either type of ANCA. Tests for antimyeloperoxidase ANCA were repeatedly negative before the drug was started, strongly implicating the drug as the cause of the episode. This case demonstrates that patients with idiopathic ANCA-positive vasculitis may quickly develop a superimposed drug-associated ANCA-positive vasculitis. Iatrogenic vasculitis should be suspected when a patient with idiopathic vasculitis with one type of ANCA develops the other type of ANCA.

Experimental Goodpasture's syndrome in Wistar-Kyoto rats immunized with alpha3 chain of type IV collagen.

BACKGROUND: Glomerulonephritis and lung hemorrhage of autoimmune Goodpasture syndrome develop due to immune reactions against epitope(s) of the non-collagenous (NC1) domain of alpha3-chain of type IV collagen [alpha3(IV) NC1]. Whether thymic mechanisms have a role in the loss of tolerance to the Goodpasture epitope has not been established. We studied the renal and pulmonary effects of immunization with different forms (monomer, dimer, or hexamer) of alpha3(IV) NC1 collagen in Wistar-Kyoto (WKY) rats, and assessed whether the intrathymic inoculation of the antigen may protect against anti-GBM disease. METHODS: WKY rats were immunized with bovine alpha3(IV) monomer, dimer, or hexamer, or with alpha3(IV) NC1 synthetic peptide. Renal function, kidney and lung immunohistology, and circulating and tissue bound antibodies to type IV collagen chains were analyzed. Effects of intrathymic inoculation of antigen on subsequent disease induction were analyzed in WKY rats given alpha3(IV) NC1 dimer or GBM preparation intrathymically 48 hours before immunization. RESULTS: Proteinuria, linear IgG deposition in GBM, and crescentic glomerulonephritis developed in WKY rats immunized with alpha3(IV) NC1 dimer or hexamer. Lesions were dose-dependent upon injections of 10 to 100 microgram dimer. The alpha3(IV) NC1 monomer induced less severe proteinuria and no crescents. Pulmonary hemorrhage was detectable in 35% of rats immunized with 25 to 100 microgram alpha3(IV) NC1 dimer; alpha3(IV) synthetic peptide (36 carboxyl terminal) did not induce disease. Rats injected intrathymically with up to 100 microgram alpha3(IV) NC1 dimer or with GBM 48 hours before immunization were not protected against subsequent development of proteinuria and glomerulonephritis. CONCLUSIONS: These findings document that glomerulonephritis and lung hemorrhage can be elicited in WKY rats by immunization with alpha3(IV) NC1. Failure of the intrathymic inoculation of antigen to prevent disease suggests that immunological tolerance cannot be achieved by this intervention, in contrast to other autoimmune conditions, and may imply independent roles for cellular and humoral nephritogenic pathways in anti-GBM nephritis.

Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70.

The Wilms tumor suppressor WT1 encodes a zinc finger transcription factor that is expressed in glomerular podocytes during a narrow window in kidney development. By immunoprecipitation and protein microsequencing analysis, we have identified a major cellular protein associated with endogenous WT1 to be the inducible chaperone Hsp70. WT1 and Hsp70 are physically associated in embryonic rat kidney cells, in primary Wilms tumor specimens and in cultured cells with inducible expression of WT1. Colocalization of WT1 and Hsp70 is evident within podocytes of the developing kidney, and Hsp70 is recruited to the characteristic subnuclear clusters that contain WT1. The amino-terminal transactivation domain of WT1 is required for binding to Hsp70, and expression of that domain itself is sufficient to induce expression of Hsp70 through the heat shock element (HSE). Substitution of a heterologous Hsp70-binding domain derived from human DNAJ is sufficient to restore the functional properties of a WT1 protein with an amino-terminal deletion, an effect that is abrogated by a point mutation in DNAJ that reduces binding to Hsp70. These observations indicate that Hsp70 is an important cofactor for the function of WT1, and suggest a potential role for this chaperone during kidney differentiation.

Megalin (gp330): a putative endocytic receptor for thyroglobulin (Tg).

Megalin (gp330) is a large glycoprotein receptor found mainly on a group of absorptive epithelial cells, including renal proximal tubule, epididymal and thyroid cells. Megalin has been shown to bind multiple, unrelated ligands, mainly in vitro, and to mediate endocytosis of ligandsin cultured cells. However, physiologic ligands of megalin are largely unknown. In the present study we have demonstrated that purified rat megalin binds rat thyroglobulin (Tg) in solid phase assays, with anestimated Kd of 9.2+/-0.6 nM. Binding was calcium dependent and was almost completely inhibited by excess Tg, by three megalin ligands - lactoferrin, lipoprotein lipase and apolipoprotein J- and by the receptor associated protein (RAP), which inhibits binding of all megalin ligands. Three anti-megalin antibodies partially inhibited Tg binding to megalin. 125I labeled Tg bound to megalin was released by EDTA and heparin; the released product was shown by SDS-PAGE and autoradiography to be 660 kD (dimeric) Tg. However, an immunoblotting experiment showed binding of megalin both to monomeric (330 kD) and dimeric Tg. We propose that megalin, which is known to mediate ligand endocytosis and is found on the apical surface of thyrocytes, may participate in the endocytosis of Tg from the colloid, a process that is required for hormone release from Tg.

Immortalized rat proximal tubule cells produce membrane bound and soluble megalin.

Megalin (gp330), a glycoprotein receptor found on renal proximal tubule cells and several other epithelial cells, is deduced to be a type I integral membrane protein, but may also exist as a cell surface form lacking a cytoplasmic domain. Furthermore, soluble megalin products have been detected in urine, and in culture medium of a rat yolk sac carcinoma cell line, combined with receptor associated protein (RAP). Permanent renal cell lines expressing megalin were unavailable until the recent description of two immortalized rat proximal tubule cell lines (IRPTC). The present study demonstrated megalin on IRPTC surface by immunofluorescence, without surface staining for RAP, which was, however, readily detected within cells. Antibodies to ectodomain megalin epitopes immunoprecipitated megalin products both from cell lysates and culture medium, whereas antibodies to cytoplasmic domain epitopes precipitated megalin only from lysates. Western blots showed two major megalin products in medium, a prominent band at approximately 200 kDa, and a fainter band above 400 kDa, slightly below intact megalin in cell lysates. Anti-receptor associated protein antibodies immunoprecipitated megalin from IRPTC lysates, but not from media. We propose that portions of megalin are spontaneously produced by IRPTC, probably either by cleavage in the ectodomain or release of forms lacking a cytoplasmic domain.