B cells from patients with Graves' disease aberrantly express the IGF-1 receptor: implications for disease pathogenesis

Graves' disease (GD) is an autoimmune process involving the thyroid and connective tissues in the orbit and pretibial skin. Activating anti-thyrotropin receptor Abs are responsible for hyperthyroidism in GD. However, neither these autoAbs nor the receptor they are directed against have been convincingly implicated in the connective tissue manifestations. Insulin-like growth factor-1 receptor (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD and when ligated with IgGs from these patients, express the T cell chemoattractants, IL-16, and RANTES. Disproportionately large fractions of peripheral blood T cells also express IGF-1R in patients with GD and may account, at least in part, for expansion of IGF-1R(+) memory T cells. We now report a similarly skewed B cell population exhibiting the IGF-1R(+) phenotype from the blood, orbit, and bone marrow of patients with GD. This expression profile exhibits durability in culture and is maintained or increased with CpG activation. Moreover, IGF-1R(+) B cells produce pathogenic Abs against the thyrotropin receptor. In lymphocytes from patients with GD, IGF-1 enhanced IgG production (p < 0.05) and increased B cell expansion (p < 0.02) in vitro while those from control donors failed to respond. These findings suggest a potentially important role for IGF-1R display by B lymphocytes in patients with GD in supporting their expansion and abnormal Ig production.     

Identification of ADAM10 as a major TNF sheddase in ADAM17-deficient fibroblasts

ADAM17 (a disintegrin and metalloprotease)-deficient murine fibroblasts stably transfected with proTNF cDNA release significant amounts of biologically active soluble TNF. The enzyme responsible for this activity is a membrane protein that hydrolyzes the peptide bond Ala⁷⁶:Val⁷⁷ within proTNF. Its activity is inhibited by 1,10-phenantroline and GM6001, insusceptible to TIMP-2 (tissue inhibitor of metalloproteinases-2), and stimulated by ionomycin. These characteristics match ADAM10. The moderate silencing of ADAM10 by shRNA resulted in a significant inhibition of TNF shedding. There was no correlation between the level of ADAM10 expression and the presence of active ADAM17. Our results indicate that ADAM10 may function as the TNF sheddase in cells which lack ADAM17 activity.     

Overexpression of sigma-1 receptor inhibits ADAM10 and ADAM17 mediated shedding in vitro

The sigma-1 receptor is a molecular chaperone protein highly enriched in the brain. Recent studies linked it to many diseases, such as drug addition, Alzheimer’s disease, stroke, depression, and even cancer. Sigma-1 receptor is enriched in lipid rafts, which are membrane microdomains essential in signaling processes. One of those signaling processes is ADAM17- and ADAM10-dependent ectodomain shedding. By using an alkaline phosphatase tagged substrate reporter system, we have shown that ADAM10-dependent BTC shedding was very sensitive to both membrane lipid component change and sigma-1 receptor agonist DHEAS treatment while ADAM17-dependent HB-EGF shedding was not; and overexpression of sigma-1 receptor diminished ADAM17- and ADAM10-dependent shedding. Our results indicate that sigma-1 receptor plays an important role in modifying the function of transmembrane proteases.     

Synovial fibroblasts from patients with rheumatoid arthritis, like fibroblasts from Graves' disease, express high levels of IL-16 when treated with Igs against insulin-like growth factor-1 receptor

We have reported recently that IgG from patients with Graves' disease (GD) can induce the expression of the CD4-specific T lymphocyte chemoattractant, IL-16, and RANTES, a C-C chemokine, in their fibroblasts. This induction is mediated through the insulin-like growth factor-1 receptor (IGF-1R) pathway. We now report that Abs from individuals with active rheumatoid arthritis (RA-IgG) stimulate in their synovial fibroblasts the expression of these same cytokines. IgG from individuals without known autoimmune disease fails to elicit this chemoattractant production. Furthermore, RA-IgG fails to induce IL-16 or RANTES expression in synovial fibroblasts from donors with osteoarthritis. RA-IgG-provoked IL-16 and RANTES production also appears to involve the IGF-1R because receptor-blocking Abs prevent the response. RA fibroblasts transfected with a dominant-negative mutant IGF-1R fail to respond to RA-IgG. IGF-1 and the IGF-1R-specific analog Des(1-3) also induce cytokine production in RA fibroblasts. RA-IgG-provoked IL-16 expression is inhibited by rapamycin, a specific macrolide inhibitor of the Akt/FRAP/mammalian target of rapamycin/p70(s6k) pathway, and by dexamethasone. GD-IgG can also induce IL-16 in RA fibroblasts, and RA-IgG shows similar activity in GD fibroblasts. Thus, IgGs from patients with RA, like those associated with GD, activate IGF-1R, and in so doing provoke T cell chemoattraction expression in fibroblasts, suggesting a potential common pathway in the two diseases. Immune-competent cell trafficking to synovial tissue is integral to the pathogenesis of RA. Recognition of this novel RA-IgG/fibroblast interaction and its functional consequences may help identify therapeutic targets.     

Nuclear translocation of insulin receptor substrate-1 by the insulin receptor in mouse embryo fibroblasts

Translocation of the insulin receptor substrate-1 (IRS-1) to the nuclei has been reported to occur in cells stimulated by insulin-like growth factor-1 (IGF-I) or expressing certain viral and cellular oncogenes. We show here that insulin can also induce nuclear translocation of IRS-1 in mouse embryo fibroblasts (MEF), that do not express the type 1 insulin-like growth factor receptor (IGF-IR). Only the A isoform of the insulin receptor (IR) can induce IRS-1 nuclear translocation, which is significant when the receptor is over-expressed. At physiological receptor levels, translocation occurs only in a fraction of cells, and only at high concentrations of ligand.     

Species specificity of ADAM10 and ADAM17 proteins in interleukin-6 (IL-6) trans-signaling and novel role of ADAM10 in inducible IL-6 receptor shedding

Hypomorphic ADAM17(ex/ex) mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17(ex/ex) mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.     

Divergent Sp1 protein levels may underlie differential expression of UDP-glucose dehydrogenase by fibroblasts: role in susceptibility to orbital Graves disease

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning -1436 nucleotides (nt) and -1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from -1387 to +71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease.     

Inhibitory role of TACE/ADAM17 cytotail in protein ectodomain shedding

AIM:To determine if the cytotail of the principal sheddase tumor necrosis factor-α converting enzyme (TACE;ADAM17) controls protein ectodomain shedding.METHODS:Site-directed mutagenesis was performed to derive TACE variants. The resulting TACE expression plasmids with amino acid substitutions in the extracel-lular,cysteine-rich disintegrin domain (CRD) and/or deleted cytotail,along with an expression vector for the enhanced green fluorescence protein were transfected into shedding-defective M1 mutants stably expressing transmembrane L-selectin or transforming growth factor (TGF)-α. The expression levels of the TACE substrates at the cell surface were determined by flow cytometry. RESULTS:Consistent with published data,a single point mutation (C600Y) in the CRD led to shedding defi-ciency. However,removal of the cytotail from the C600Y TACE variant partially restored ectodomain cleavage of TGF-α and L-selectin. Cytotail-deleted mutants with any other substituting amino acid residues in place of Cys600 displayed similar function compared with tail-less C600Y TACE.CONCLUSION:The cytotail plays an inhibitory role,which becomes evident when it is removed from an enzyme with another mutation that affects the enzyme function.     

Role of IGF-1 receptor in radiation response

Insulin-like growth factor 1 receptor (IGF-1R) is a transmembrane receptor tyrosine kinase involved in the development and progression of cancer whose activation strongly promotes cell growth and survival. IGF-1R exerts its main actions through the activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. In addition to their traditional roles, IGF-1R activation has been associated with increased radioresistance both in vitro and in vivo, although the molecular mechanisms behind this process are still unclear. Recently, IGF-1R has been associated to new partners as major vault proteins, BCL-2, BAX, or Ku70/80, related to radiochemotherapy resistance, regulation of apoptosis, and nonhomologous end-joining DNA repair. Here, we review these novel associations of IGF-1R trying to explain the resistance to radiotherapy mediated by IGF-1R. Finally, we revised the role of new therapies leading to block the receptor to enhance the efficacy of radiation.     

Multicomponent structure of the thyrotropin receptor: relationship to Graves' disease

The thyrotropin receptor is proposed to contain both a glycoprotein and a ganglioside component. Monoclonal antibodies have been developed against soluble thyroid TSH receptor preparations and using Graves' lymphocytes. These show that initial recognition of thyrotropin requires the glycoprotein component, but that monoclonal antibodies to this component block thyrotropin function (blocking antibodies) rather than mimic thyrotropin. Monoclonal antibodies which stimulate thyroid activity in cultured cell systems (cAMP increase) or mouse bioassays, all interact with gangliosides. Using monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, we show that two protein bands, molecular weights 18,000-23,000 and 50,000-55,000, are precipitated from detergent-solubilized preparations. Using a crosslinking procedure with 125I-labeled thyrotropin, we show that thyrotropin binding is related to the disappearance of the 18,000-23,000 molecular weight band on sodium dodecylsulfate gels and the appearance of a 30,000-33,000 molecular weight thyrotropin-membrane component complex. Higher molecular weight thyrotropin-membrane complexes of 75,000-80,000 and 250,000 are visualized when binding studies are performed at pH 7.4 in physiologic medium; larger amounts of the 30,000-33,000 complex are evident at pH 6.0 in a low salt medium. It is thus proposed that the glycoprotein component of the thyrotropin receptor is composed of two subunits with apparent molecular weights of 18,000-23,000 and 50,000-55,000; that the 18,000-23,000 subunit interacts with thyrotropin; and that different receptor subunits can exist depending on in vitro binding conditions. Using monoclonal-stimulating antibodies or natural autoimmune IgG preparations from patients' sera, we show that stimulating antibodies exhibit species-specific binding to human thyroid ganglioside preparations. Individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.     

Immunoglobulin activation of T cell chemoattractant expression in fibroblasts from patients with Graves' disease is mediated through the insulin-like growth factor I receptor pathway

Graves' disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1-3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific (125)I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.     

A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation

Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved. Using native bladder epithelium, in some cases transduced with adenoviruses encoding small interfering RNA, we observe that stimulation of apically localized A₁ adenosine receptors (A₁ARs) triggers a G_i-G_βγ-phospholipase C-protein kinase C (PKC) cascade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A₁AR activation. Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17^S811A mutant or expression of a tail-minus construct inhibits A₁AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17^S811A in bladder tissues impairs A₁AR-induced apical exocytosis. We conclude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphorylation state of Ser-811 in its cytoplasmic domain.     

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: interactions between alpha beta heterodimeric receptor halves

Examination of 125I-IGF-1 affinity cross-linking and beta-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated alpha beta heterodimeric IGF-1 receptors into an alpha 2 beta 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor [Morrison, B.D., Swanson, M.L., Sweet, L.J., & Pessin, J.E. (1988) J. Biol. Chem. 263, 7806-7813]. The formation of the alpha 2 beta 2 heterotetrameric IGF-1 receptor complex from the partially purified alpha beta heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified alpha beta heterodimeric insulin receptor complex. Incubation of the alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125I-IGF-1 binding of IGF-1 stimulation of protein kinase activity. In addition, IAN did not affect the Mn/MgATP-dependent noncovalent association of IGF-1 receptor alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state. However, IAN treatment of the alpha beta heterodimeric IGF-1 receptors inhibited the IGF-1-dependent covalent formation of the disulfide-linked alpha 2 beta 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated alpha beta heterodimeric IGF-1 receptor complexes into a disulfide-linked alpha 2 beta 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

Background

Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues.

Results

In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis.

Conclusions

Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.     

Igs from patients with Graves' disease induce the expression of T cell chemoattractants in their fibroblasts.

Thyroid-associated ophthalmopathy and dermopathy are connective tissue manifestations of Graves' disease (GD). Tissue remodeling is a prominent feature of both and is apparently driven by recruited T cells. In this study, we report that IgG isolated from patients with GD (GD-IgG) up-regulates T lymphocyte chemoattractant activity in GD-derived fibroblasts from orbit, thyroid, and several regions of skin. This chemoattractant activity, absent in fibroblasts from donors without known thyroid disease, is partially susceptible to neutralization by anti-IL-16 and anti-RANTES Abs. IL-16 is a CD4(+)-specific chemoattractant and RANTES is a C-C-type chemokine. IL-16 and RANTES protein levels, as determined by specific ELISAs, are substantially increased by GD-IgG in GD fibroblasts. Addition of the macrolide, rapamycin, to fibroblast culture medium blocked the up-regulation by GD-IgG of IL-16, implicating the FRAP/mTOR/p70(s6k) pathway in the induction of IL-16 expression. These findings suggest a specific mechanism for activation of fibroblasts in GD resulting in the recruitment of T cells. They may provide insight into a missing link between the glandular and extrathyroidal manifestations of GD.     

A quantitative analysis of orbital soft tissue in Graves' disease based on B-mode ultrasonography

Using B-mode ultrasonography, an attempt was made to measure the volume of extraocular muscles and retrobulbar fat in 31 patients (62 orbits) with Graves' disease. None of the patients had exophthalmometric measurements greater than 21 mm or had eye symptoms. The mean value of muscle volume of Graves' patients was significantly larger than that of normal controls (6.48 +/- 2.70 cm3 and 3.25 +/- 1.30 cm3, respectively, p less than 0.001). All of the patients had extraocular muscle swelling, although 2 of them had no extraocular muscle change for their unilateral eye. The extraocular muscle volume increased as the degree of the proptosis increased. The fat volume tended to increase in parallel with the degree of the proptosis. In the Graves' group with obvious proptosis (Hertel reading: 19--21 mm), the fat volume increased more significantly than in any other group. The ratio of extraocular muscle volume to retrobulbar fat volume was significantly higher in Graves' disease, but it did not increase as the degree of the proptosis increased. A significant correlation between proptosis and muscle volume plus fat volume was observed. No significant difference of the extraocular muscle volume was observed between the patients untreated and treated with antithyroid drugs. The data show a uniform enlargement of the extraocular muscles in Graves' disease and also suggest an involvement of increased retrobulbar fat volume in a group of obvious exophthalmos. The degree of the proptosis is in aclose proportion ot the quantitative change of the orbital soft tissue.