Vps34 PI 3-kinase controls thyroid hormone production by regulating thyroglobulin iodination, lysosomal proteolysis and tissue homeostasis

BACKGROUND: The production of thyroid hormones (T3, T4) depends on the organization of the thyroid in follicles, which are lined by a monolayer of thyrocytes with strict apico-basal polarity. This polarization supports vectorial transport of thyroglobulin for storage into, and recapture from, the colloid. It also allows selective addressing of channels, transporters, ion pumps and enzymes to their appropriate basolateral (NIS, SLC26A7 and Na+/K+-ATPase) or apical membrane domain (Anoctamin, SLC26A4, DUOX2, DUOXA2 and TPO). How these actors of T3/T4 synthesis reach their final destination remains poorly understood. The PI 3-kinase (PI3K) isoform Vps34/PIK3C3 is now recognized as a main component in the general control of vesicular trafficking and of cell homeostasis via the regulation of endosomal trafficking and autophagy. We recently reported that conditional Vps34 inactivation in proximal tubular cells in the kidney prevents normal addressing of apical membrane proteins and causes abortive macroautophagy. // METHODS: Vps34 was inactivated using a Pax8-driven Cre recombinase system. The impact of Vps34 inactivation in thyrocytes was analyzed by histological, immunolocalization and mRNA expression profiling. Thyroid hormone synthesis was assayed by 125I injection and serum plasma analysis. // RESULTS: Vps34cKO mice were born at the expected Mendelian ratio and showed normal growth until postnatal day 14, then stopped growing and died at around 1 month of age. We therefore analyzed thyroid Vps34cKO at postnatal day 14. We found that loss of Vps34 in thyrocytes causes: (i) disorganization of thyroid parenchyma, with abnormal thyrocyte and follicular shape and reduced PAS+ colloidal spaces; (ii) severe non-compensated hypothyroidism with extremely low T4 levels (0.75 ± 0.62 g/dL) and huge TSH plasma levels (19,300 ± 10,500 mU/L); (iii) impaired 125I organification at comparable uptake and frequent occurrence of follicles with luminal thyroglobulin but non-detectable T4-bearing thyroglobulin; (iv) intense signal in thyrocytes for the lysosomal membrane marker, LAMP-1, as well as thyroglobulin and the autophagy marker, p62, indicating defective lysosomal proteolysis, and (v) presence of macrophages in the colloidal space. // CONCLUSIONS: We conclude that Vps34 is crucial for thyroid hormonogenesis, at least by controlling epithelial organization, Tg iodination as well as proteolytic T3/T4 excision in lysosomes

TRH: Pathophysiologic and clinical implications

Thyrotropin releasing hormone is thought to be a tonic stimulator of the pituitary TSH secretion regulating the setpoint of the thyrotrophs to the suppressive effect of thyroid hormones. The peptide stimulates the release of normal and elevated prolactin. ACTH and GH may increase in response to exogenous TRH in pituitary ACTH and GH hypersecretion syndromes and in some extrapituitary diseases. The pathophysiological implications of extrahypothalamic TRH in humans are essentially unknown. The TSH response to TRH is nowadays widely used as a diganostic amplifier in thyroid diseases being suppressed in borderline and overt hyperthyroid states and increased in primary thyroid failure. In hypothyroid states of hypothalamic origin, TSH increases in response to exogenous TRH often with a delayed and/or exaggerated time course. But in patients with pituitary tumors and suprasellar extension TSH may also respond to TRH despite secondary hypothyroidism. This TSH increase may indicate a suprasellar cause for the secondary hypothyroidism, probably due to portal vessel occlusion. The TSH released in these cases is shown to be biologically inactive

Metabolism of thyroxine-binding globulin in man. Abnormal rate of synthesis in inherited thyroxine-binding globulin deficiency and excess.

It has been previously suggested that inherited thyroxine-binding globulin (TBG) abnormalities in man may be due to mutations at a single X-chromosome-linked locus controlling TBG synthesis. However, abnormalities in TBG degradation have not been excluded. The availability of purified human TBG and its successful labeling with radioiodide allowed us to examine such possibility. Human TBG was purified by affinity chromatography, labeled under sterile conditions with 131I or 125I,, and mixed with [125I]thyroxine (T4) or [131I]T4, respectively, before their intravenous injection. Blood and urine samples were collected over a 10-day period, and the turnover parameters were calculated. In eight normal volunteers mean values +/-SD for TBG and T4 respectively, were as follows: Half time (t1/2) 5.3 +/- 0.4 and 7.0 +/- 0.6 days; distribution space (DS) 7.2 +/- 1.0 and 10.8 +/- 1.2 liters; and total daily degradation (D) 0.211 +/- 0.053 and 0.088 +/- 0.011 mumol/day. In all subjects, t1/2 of TBG was shorter than that of T4; and the DS was smaller. 2.4 mol of TBG was degraded for each mole of T4. In five of six subjects from four families, comprising hemizygous and heterozygous carriers of TBG absence, decrease, and excess, the t1/2 and DS for TBG were within the normal range. The D of TBG was proportional to the serum concentration of the protein. Changes in the T4 kinetics in these patients were compatible with euthyroidism and with the known alterations in the extrathyroidal T4 pool associated with the changes in serum TBG concentration. A striking decrease in the t1/2 of TBG was found only in a patient with acquired diminution in TBG concentration and in patients with thyrotoxicosis or other conditions apparently unrelated to thyroid dysfunction. TBG t1/2 was 2.5 days in a patient with multiple myeloma and 3.6 days in two patients with thyrotoxicosis. Decreased TBG t1/2 was also observed in three of six patients with nonthyroidal pathology and was associated with an increase in TBG D disproportionate to their level of serum TBG. These studies indicate that changes in TBG concentration in patients with X-chromosome-linked TBG abnormalities are due to alterations in its rate of synthesis. In other conditions, abnormalities of TBG degradation and/or rate of synthesis may be found

Radioimmunoassays specific for the tertiary and primary structures of thyroxine-binding globulin (TBG): measurement of denatured TBG in serum.

Antisera prepared by immunization of rabbits with human T4-binding globulin (TBG) contained two populations of antibodies: one directed against determinants of the native molecule, and the other directed against antigenic sites present only in denatured TBG. These two populations of antibodies were present in all nine antisera prepared in this or other laboratories that were tested. Exploiting this property of anti-TBG sera and using radioiodinated denatured TBG as a tracer, a RIA was developed which measures specifically denatured TBG in the presence of native TBG. The RIA for measuring denatured TBG used purified native TBG, which was denatured by reduction and pyridylethylation (RP-TBG) before labeling with 125I. Native TBG was measured using the same antiserum, but the 125I-labeled tracer was unmodified TBG. The sensitivity of the native TBG RIA was 0.25 ng purified native TBG. Equivalent amounts of native TBG in serum, desialylated TBG, and deglycosylated TBG produced superimposeable standard curves. The cross-reactivity with RP-TBG was less than 0.02%. The denatured TBG RIA had a sensitivity of 1 ng, and superimposeable curves were produced with equivalent concentrations of RP-TBG and heat-denatured native TBG. The cross-reactivity of 0.8% with native and deglycosylated TBG was, at least in part, due to denatured TBG in the purified preparations. The specificity of the two RIAs is due to the existence of distinct and exclusive antigenic determinants in native TBG and denatured TBG which are probably located on the surface of the tertiary structure and internally at the primary structure of the molecule, respectively. Heat and acid pH treatments of serum produced a progressive loss in immunoreactive native TBG, proportional to the loss of T4-binding capacity. A reciprocal and quantitative increase in denatured TBG, as measured in the denatured TBG RIA, was found. T4 partially protected the native TBG from denaturation. Denatured TBG was detected in sera from normal adults. The mean value was 6.05 +/- 2.25 (+/- SD) micrograms/dl (n = 11). Similar values were found in 8 pregnant women, 5 men with familial partial TBG deficiency, and 15 hypothyroid 7 hepatic, and 8 renal failure patients.(ABSTRACT TRUNCATED AT 400 WORDS)Journal ArticleResearch Support, U.S. Gov't, P.H.S.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Cross-talk between PI3K and estrogen in the mouse thyroid predisposes to the development of follicular carcinomas with a higher incidence in females.

It is well known that thyroid disease is more frequent in women than in men; however, the molecular basis for this gender-based difference is still poorly understood. The activation of phosphoinositide 3-kinase (PI3K), through different mechanisms including loss of the PTEN tumor suppressor, is being increasingly recognized as a major player in the development of thyroid neoplastic lesions. Loss of Pten in the mouse thyroid results in a significant increase in the thyrocyte proliferative index, which is more prominent in the female mice. In this study, we show that 52% of the Pten(-/-) female mice, but only 12% of the males, develop follicular adenomas by 1 year of age. In addition, 50% of female mutants, but only 35% of males older than 1 year of age develop invasive, and often metastatic, follicular carcinomas. Mutant females have a significantly shorter overall survival compared with male mutants. Hormonal manipulation experiments established a direct role of estrogens in controlling the increased thyrocyte proliferation index in mutant females. Furthermore, while genetic ablation of one Cdkn1b allele accelerated the development of neoplastic lesions, it also abolished the gender differences in survival and reduced the difference in neoplastic lesion development rate, underlining a key role of p27 in mediating estrogen action in the thyroid follicular cells. These data, based on a clinically relevant model of thyroid follicular carcinoma, provide, to the best of our knowledge, for the first time in vivo evidence that circulating estrogens are directly responsible for the increased female susceptibility to thyroid disease, at least on activation of the PI3K pathway, and provide new insights into the gender-based differences characterizing thyroid neoplastic disorders

TBG deficiency: description of two novel mutations associated with complete TBG deficiency and review of the literature

Thyroxine-binding globulin (TBG) is the main thyroid hormone transport protein in serum. Inherited TBG defects lead to a complete (TBG-CD) or a partial (TBG-PD) deficiency and have a diagenic transmission, being clinically fully expressed only in hemizygous males and in homozygous females. In the present study, seven patients from two unrelated families with TBG-CD were studied and two novel TBG mutations were documented. In particular, a T insertion at the 5\u2032 donor splice site of exon 0, between nucleotides 2 and 3 at the beginning of intron 1 (g.IVS1+2_3insT) was found in one family and was named TBG-Milano. The other novel mutation is a T deletion at nucleotide 214 of exon 1, which leads to a frameshift at codon 50 with a premature stop codon at position 51 (c.214delT, P50fsX51) and was named TBG-Nikita. According to the X-linked transmission of the defect, females harboring the mutation showed a reduction in TBG levels with normal TSH and total thyroid hormone values at the lower limit of normal. Males harboring either TBG-Milano or TBG-Nikita, showed normal TSH values and low levels of total thyroid hormones and lacked TBG. In conclusion, we report two novel mutations of the TBG gene associated with a complete TBG defect. The first mutation lies at the 5\u2032 donor splice site of exon 0 and probably alters the start of translation, while the second is a single nucleotide deletion and leads to a premature stop codon