Positive correlation between type 1 and 2 iodothyronine deiodinases activities in human goiters

Type 1 (D1) and 2 (D2) iodothyronine deiodinases are selenocysteine-containing enzymes that catalyze the deiodination of T4 to T3 in the thyroid and in peripheral tissues. Despite their importance to the plasma T3 pool in human beings, there are few studies about their behavior in human thyroids. In order to better understand iodothyronine deiodinase regulation in the thyroid gland, we studied thyroid tissue samples from follicular adenoma (AD, n = 5), toxic diffuse goiter (TDG, n = 6), nontoxic multinodular goiter (NMG, n = 40), papillary thyroid carcinoma (PTC, n = 8), and surrounding normal tissues (NT, n = 7) from 36 patients submitted to elective thyroidectomy. D1 and D2 activities were determined by quantification of the radioiodine released by 12?I-rT3 or 12?I-T4 under standardized conditions, and expressed as pmol rT3 deiodinated per minute and mg protein (pmol rT3 min?1 mg?1 ptn) and fmol T4 deiodinated per minute and mg protein (fmol T4 min?1 mg?1 ptn), respectively. D1 activity detected in TDG and AD tissues were significantly higher than in NT, PTC or NMG samples. D2 activity was also significantly higher in TDG and AD samples than in PTC, NMG, or NT. There was great variability in D1 and D2 enzymatic activities from distinct patients as well as from different areas from the same goiter. There was a positive correlation (P < 0,0001, r = 0.4942) between D1 and D2 activities when all samples were taken into account, suggesting that-in the thyroid-these two iodothyronine deiodinases may have related regulatory mechanisms, even if conditioned by other as yet unknown factors.     

Intense expression of the b7-2 antigen presentation coactivator is an unfavorable prognostic indicator for differentiated thyroid carcinoma of children and adolescents

Previous observations suggest that an immune response against thyroid carcinoma could be important for long-term survival. We recently found that infiltration of thyroid carcinoma by proliferating lymphocytes is associated with improved disease-free survival, but the factors that control lymphocytic infiltration and proliferation are largely unknown. We hypothesized that the antigen presentation coactivators (B7-1 and B7-2), which are important in other immune-mediated thyroid diseases, might be important in lymphocytic infiltration of thyroid carcinoma. To test this, we determined B7-1 and B7-2 expression by immunohistochemistry [absent (grade 0) to intense (grade 3)] in 27 papillary (PTC) and 8 follicular (FTC) thyroid carcinomas and 9 benign thyroid lesions. B7-1 and B7-2 were expressed by the majority of PTC and FTC (78% of PTC and 100% of FTC expressed B7-1; 88% of PTC and 88% of FTC expressed B7-2). B7-1 expression was more intense in PTC (1.4 +/- 0.2; P = 0.01) and FTC (2.6 +/- 0.2; P < 0.001) than in benign tumors (0.57 +/- 0.30) or presumably normal adjacent thyroid (0.07 +/- 0.07) and was more intense in carcinoma that contained lymphocytes (1.95 +/- 0.21) than in carcinoma that did not (1.08 +/- 0.26; P = 0.016). B7-2 expression was of similar intensity in benign and malignant tumors (PTC, 1.6 +/- 0.2; FTC, 2.1 +/- 0.4; benign, 1.86 +/- 0.4), but was more intense than in presumably normal adjacent thyroid (0.64 +/- 0.25; P 相似文献   

Over-expression of hepatocyte growth factor/scatter factor (HGF/SF) and the HGF/SF receptor (cMET) are associated with a high risk of metastasis and recurrence for children and young adults with papillary thyroid carcinoma

OBJECTIVE: The study determined if hepatocyte growth factor/scatter factor (HGF/SF) or the HGF/SF receptor (cMET) might be important for metastasis in thyroid cancer. DESIGN: We examined HGF/SF and cMET expression by immunohistochemistry in a retrospective group of benign and malignant thyroid lesions from children and young adults, and correlated the intensity of expression with clinical outcome. PATIENTS: Patients included 42 children and young adults with papillary thyroid carcinomas (PTC), seven with follicular thyroid carcinomas (FTC), two with medullary thyroid carcinomas (MTC), 14 with benign thyroid disorders, and two with normal thyroids. MEASUREMENTS: Expression of cMET was graded from 0 (absent) to 4 (intense); and HGF/SF expression was graded from 0 (absent-minimal) to 3 (diffuse and intense). RESULTS: cMET staining was greater in PTC (mean intensity 2.3 +/- 0.4 vs. 0.8 +/- 0.2, P < 0.005) and FTC (2.4 +/- 0.6 vs. 0.8 +/- 0.2, P = 0.04) than benign lesions (0.8 +/- 0.2) or normal thyroids (0.4 +/- 0.5). PTC with intense cMET staining had shorter disease free survival (P = 0.05) and increased HGF/SF staining (r = 0.39, P = 0.017). HGF/SF correlated with the extent of disease at diagnosis (r = 0.33, P = 0.049). Patients with PTC were stratified into quartiles based on combined cMET and HGF/SF staining. Those with intense cMET and HGF/SF staining were younger (P = 0.05), and had reduced disease free survival (P = 0.03). CONCLUSIONS: We conclude that increased cMET and HGF/SF expression is associated with a high risk for metastasis and recurrence in children and young adults with papillary thyroid carcinoma.     

Effects of substitution and high-dose thyroid hormone therapy on deiodination, sulfoconjugation, and tissue thyroid hormone levels in prolonged critically ill rabbits

To delineate the metabolic fate of thyroid hormone in prolonged critically ill rabbits, we investigated the impact of two dose regimes of thyroid hormone on plasma 3,3'-diiodothyronine (T(2)) and T(4)S, deiodinase type 1 (D1) and D3 activity, and tissue iodothyronine levels in liver and kidney, as compared with saline and TRH. D2-expressing tissues were ignored. The regimens comprised either substitution dose or a 3- to 5- fold higher dose of T(4) and T(3), either alone or combined, targeted to achieve plasma thyroid hormone levels obtained by TRH. Compared with healthy animals, saline-treated ill rabbits revealed lower plasma T(3) (P=0.006), hepatic T(3) (P=0.02), and hepatic D1 activity (P=0.01). Substitution-dosed thyroid hormone therapy did not affect these changes except a further decline in plasma (P=0.0006) and tissue T(4) (P=0.04). High-dosed thyroid hormone therapy elevated plasma and tissue iodothyronine levels and hepatic D1 activity, as did TRH. Changes in iodothyronine tissue levels mimicked changes in plasma. Tissue T(3) and tissue T(3)/reverse T(3) ratio correlated with deiodinase activities. Neither substitution- nor high-dose treatment altered plasma T(2). Plasma T(4)S was increased only by T(4) in high dose. We conclude that in prolonged critically ill rabbits, low plasma T(3) levels were associated with low liver and kidney T(3) levels. Restoration of plasma and liver and kidney tissue iodothyronine levels was not achieved by thyroid hormone in substitution dose but instead required severalfold this dose. This indicates thyroid hormone hypermetabolism, which in this model of critical illness is not entirely explained by deiodination or by sulfoconjugation.     

Characterization of thyrotropin-induced increase in iodothyronine monodeiodinating activity in mice

To further characterize the effect of TSH administration on thyroid iodothyronine monodeiodinating activity, we have evaluated the in vitro conversion of T4 to T3 (outer ring deiodination) and T3 to 3,3'-diiodothyronine (T2; inner ring deiodination) by mouse thyroid, liver, and kidney homogenates, comparing tissues from TSH-treated mice (0.1-200 mU bovine TSH, ip, for 1-3 days) with tissues from saline-treated controls. The in vitro conversion activity was studied in the presence of 1-20 mM dithiothreitol; most of the studies were carried out at 4 mM. Studies were carried out at optimal pH 6.5 for outer ring and 7.8 for inner ring deiodination. The iodothyronine monodeiodinase in mouse thyroid is similar to the ones in liver and kidney. It is heat labile (inactivated at 56 C for 5 min), inhibited by propylthiouracil (0.2 mM) and ipodate (0.2 mM), and unaffected by methimazole (up to 20 mM), ascorbate (up to 0.1 M) or KI (up to 20 mM). The mean +/- SE baseline rates of T4 to T3 and T3 to T2 conversion were 100 +/- 6.3 and 56.5 +/- 2.9 pmol/mg thyroid protein X 30 min at 37 C, respectively. A significant increase in each conversion activity was found after TSH treatment (0.2 U, ip, daily for 3 days); T4 to T3 conversion rose to 282 +/- 15.4, and T3 to T2 increased to 153 +/- 7.4 pmol/mg thyroid protein (P less than 0.001). A 12.8% increase in thyroid weight was found in the TSH-treated group (P less than 0.03 compared with saline control group). Similar but less marked increased in monodeiodinating activities were seen in the liver. A minimal but significant increase in inner ring monodeiodination with no significant increase in T4 to T3 converting activity was found in kidney, which, in the mouse, has markedly less outer ring deiodinase than liver or thyroid. The iodothyronine monodeiodinating activities did not increase until 12 h in thyroid and 48 h in liver after the first dose of TSH. Significant increases in T4 to T3 and T3 to T2 conversion were seen with doses of TSH as low as 0.1 mU (ip, daily for 3 days), and there was a linear dose-response thereafter. The decay of the increased iodothyronine monodeiodinating activities after a single dose of TSH (0.2 U) appeared to be linear, with a decay t 1/2 of 1.3 days for T4 to T3 conversion and about 1.0 day for T3 to T2 conversion.(ABSTRACT TRUNCATED AT 400 WORDS)     

The expression of vascular endothelial growth factor and the type 1 vascular endothelial growth factor receptor correlate with the size of papillary thyroid carcinoma in children and young adults.

Vascular endothelial growth factor (VEGF) is essential for the growth of many solid tumors, but there are little data regarding VEGH in childhood thyroid cancers. We examined the relationships between VEGF, the type 1 VEGF receptor (FLT-1) and clinical outcome for a group of thyroid cancers in children and young adults. The expression of VEGF and FLT-1 were determined by immunohistochemistry using archival, paraffin-embedded thyroid tissue blocks and compared with the retrospective clinical outcome for each patient. The study included 67 children and young adults with papillary thyroid carcinoma (PTC, n = 42), follicular thyroid carcinoma (FTC, n = 8), benign lesions (n = 15), or controls (n = 2). VEGF expression was greater in PTC (mean intensity 2.23 +/- 0.25, p = 0.002) and FTC (2.8 +/- 0.73, p = 0.01) than benign lesions (1.0 +/- 0.27), and correlated with PTC size (r = 0.42, p = 0.008). FLT-1 expression was greater in PTC (mean intensity 2.8 +/- 0.17) than FTC (1.9 +/- 0.25, p = 0.015) and benign lesions (1.7 +/- 0.32, p = 0.002); and correlated with PTC size (r = 0.41, p = 0.01) as well as VEGF expression (r = 0.52, p = 0.002). Recurrent disease developed exclusively in patients with PTC which expressed VEGF (7/28, 95% CI 10.6%-44.2%). PTC that did not express VEGF (0/8, 95% CI = 0%-31.2%) did not recur; however, the difference was not statistically significant (p = 0.15). We conclude that the expression of VEGF and FLT-1 are directly correlated with the size of PTC in children and young adults.     

Effect of administration of growth hormone on plasma and intracellular levels of thyroxine and tri-iodothyronine in thyroidectomized thyroxine-treated rats.

We have studied the effects of the administration of GH on plasma levels and peripheral production of tri-iodothyronine (T3) from thyroxine (T4) in thyroidectomized male Wistar rats given a continuous i.v. infusion of T4 (1 microgram/100 g body weight per day) and GH (120 micrograms per day) for 3 weeks. Tracer doses of 131I-labelled T3 and 125I-labelled T4 were added to the infusion. At isotopic equilibrium (10 days after the addition of 125I-labelled T4) the rats were bled and perfused. The plasma appearance rate for T3 was higher (10.6 +/- 1.3 vs 8.4 +/- 2.8 pmol/h per 100 g body weight, P = 0.05) and plasma TSH was lower (246 +/- 24 vs 470 +/- 135 pmol/l, P less than 0.01) in GH-treated rats. The amount of T3 in liver (12.3 +/- 2.8 vs 5.5 +/- 1.7 pmol/g wet weight, P less than 0.01), kidney (11.5 +/- 1.4 vs 6.5 +/- 1.4 pmol/g wet weight, P less than 0.01) and pituitary (8.8 +/- 2.7 vs 4.8 +/- 0.5 pmol/g wet weight, P less than 0.01) was higher than in controls, mainly as a result of an increased local production of T3 from T4, but plasma-derived T3 was also higher in most organs. We found an increased intracellular T3 concentration in the pituitary which may be responsible for the lower plasma TSH concentration in the GH-treated rats. Since the increase in locally produced T3 is found particularly in liver, kidney and pituitary, typical organs that express 5'-deiodinase activity, we suggest that GH acts on thyroid hormone metabolism by stimulating type-I deiodinase activity.     

Localization and expression of thyroid hormone receptors normal and neoplastic human thyroid

The aim of this study was to investigate the regional expression of thyroid hormone nuclear receptor forms (TR(alpha) and TR(beta)) and isoform (TR(alpha1) and TR(beta2)) mRNAs in normal and neoplastic (benignant and malignant) human thyroid tissue. Tumor specimens from patients with thyroid carcinomas (papillary: 5 cases; follicular: 5 cases; anaplastic: 2 cases), thyroid follicular adenomas (7 cases) and tissue from normal thyroid glands (12 cases) were analyzed by in situ hybridization and semiquantitative RT-PCR for the expression of TR(alpha1) and beta, as well as for the isoform alpha2 that does not bind the hormone. In normal tissues, TR(alpha2) was expressed at lower levels compared to TR(alpha1) (alpha1/alpha2 = 4.3). In papillary and follicular carcinomas, the expression of TR(alpha1) and TR(beta) did not change as compared with normal thyroid tissue and adenomas (0.87 +/- 0.15 SD vs 0.89 +/- 0.17 densitometric units, DU, and 0.15 +/- 0.02 vs 0.14 +/- 0.03 DU, respectively). However, the expression of TR(alpha2) was significantly higher in differentiated carcinomas compared to normal thyroid tissue and adenomas (0.47 +/- 0.05 vs 0.20 +/- 0.05 DU, p < 0.05) with alpha1/alpha2 = 1.4. In anaplastic carcinoma all TRs were absent. We concluded that both normal and pathological thyroid tissues, with the exception of anaplastic carcinoma, express all TRs in thyreocites and that differentiated thyroid carcinomas are associated in enhancing the expression of TR(alpha2) mRNA.     

3,5,3'-Triiodothyronine thyrotoxicosis due to increased conversion of administered levothyroxine in patients with massive metastatic follicular thyroid carcinoma

OBJECTIVE: Some patients with massive metastatic thyroid carcinoma exhibit T(3) thyrotoxicosis. We investigated the prevalence and cause of T(3) thyrotoxicosis and the clues to the diagnosis. DESIGN: Serum free T(3) (FT(3)), free T(4) (FT(4)), and TSH were measured in patients with massive metastases from papillary, follicular, or medullary thyroid carcinomas (31, 20, and seven patients, respectively). Patients without recurrence served as controls. Thyrotoxic patients were reexamined 1 wk after withdrawal of levothyroxine. Type 1 and type 2 iodothyronine deiodinase (D1 and D2) activities were measured in three tumor tissues from thyrotoxic patients. MAIN OUTCOME: The serum FT(3) level and FT(3)/FT(4) ratio in the follicular carcinoma (FC) group were significantly higher than those in the papillary carcinoma group or patients without recurrence. Four patients (20%) in the FC group but none in the other groups demonstrated T(3) thyrotoxicosis or a FT(3)/FT(4) ratio greater than 3.5. One week after withdrawal of levothyroxine, both FT(3) and FT(4) levels decreased. Retrospective measurements of FT(3) in frozen stored sera demonstrated that FT(3) exceeded the upper normal limit when FT(4) began to decrease but remained within the normal range. Tumor tissues showed high D1 and D2 activities. CONCLUSIONS: Twenty percent of patients with massive metastatic FC exhibited T(3) thyrotoxicosis, most likely due to increased conversion of T(4) to T(3) by tumor expressing high D1 and D2 activities. Occasional measurement of serum FT(3) in addition to FT(4) and TSH is recommended in patients with massive metastatic FC, especially when serum FT(4) decreases on fixed doses of levothyroxine.     

Thyroxine to triiodothyronine hyperconversion thyrotoxicosis in patients with large metastases of follicular thyroid carcinoma.

OBJECTIVE: We experienced two cases of follicular thyroid carcinoma with distant metastases, which showed high levels of free triiodothyronine (T(3)) while free thyroxine (T(4)) levels remained in the low or normal range. In this report, we described the detail of these cases and examined the cause of T(3) thyrotoxicosis. DESIGN: In one of the cases, quantitative measurement of types I and II iodothyronine deiodinase mRNAs was performed using a surgically dissected tissue from the primary tumor and a distant metastasis. MAIN OUTCOME: In the both tissues, types I and II iodothyronine deiodinase mRNAs were expressed in the same level as in the normal thyroid tissues. CONCLUSION: These results suggest that T(3) thyrotoxicosis in our patients was caused by hyperconversion of administered levothyroxine to T(3). In the follow-up of patients with distant metastases of follicular carcinoma, not only free T(4), but also free T(3) should be tested to avoid the excessive administration of levothyroxine.     

Matrix metalloproteinase (MMP) expression by differentiated thyroid carcinoma of children and adolescents

The factor(s) that control metastasis of thyroid carcinoma are unknown, but the matrix metalloproteinases (MMPs) are excellent candidates. MMP-1, membrane-type-1 MMP (MT1-MMP), and tissue inhibitor of MMP-1 (TIMP-1) have all been implicated, but the site of production and importance are disputed. In vitro, normal thyroid cells secrete TIMP-1, while thyroid cancer cells secrete TIMP-1 and MMP-1. However, previous pathological studies identified MMP-1 and TIMP-1 only in the stroma surrounding thyroid carcinoma. These data suggest that thyroid carcinoma or tumor-associated inflammatory cells might secrete a factor(s) which stimulates MMP-1 or TIMP-1 expression by surrounding tissues. We hypothesized that MMP-1, MT1-MMP, and TIMP-1 would be directly expressed by thyroid carcinoma and might promote invasion or metastasis. We used immunohistochemistry to determine the expression of MMP-1, MT1-MMP, and TIMP-1 in 32 papillary thyroid carcinoma (PTC), 10 follicular thyroid carcinoma (FTC) and 13 benign thyroid lesions from children and adolescents. The intensity of staining was graded from absent (grade 0) to intense (grade 3). Average MMP-1 expression (mean relative intensity units+/-SE) was significantly greater among PTC (1.97+/-0.15; p=0.004) and FTC (2.2+/-0.25; p=0.006) compared to benign lesions (1.30+/-0.15); but there was no relationship between MMP-1 expression and invasion, metastasis, or recurrence. Expression of MT1-MMP and TIMP-1 was similar for benign and malignant lesions; but recurrent PTC expressed lower levels of TIMP-1 when compared to non-recurrent PTC (p=0.049). Only the expression of TIMP-1 correlated with the presence of tumor-associated lymphocytes (r=0.35, p=0.032). We conclude that MMP-1, MT1-MMP and TIMP-1 are all expressed by thyroid carcinoma and could be important in promoting recurrence.     

Nitrotyrosine,inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) are increased in thyroid tumors from children and adolescents

Nitric oxide (NO) is a reactive cell signal that controls vascular tone and is generated by inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NO synthase (NOS). We hypothesized that NO could be important for growth of thyroid tumors and tested this hypothesis, by staining 41 papillary thyroid carcinoma (PTC), 9 follicular thyroid carcinoma (FTC), and 15 benign thyroid lesions for iNOS, eNOS and nitrotyrosine (N-TYR). Staining intensity was determined by 2 blinded, independent examiners, and quantified from grade 1 (absent) to grade 4 (intense). Average N-TYR staining of benign adenomas (2.5+/-0.42, p=0.009), PTC (3.10+/-0.12, p=0.001), FTC (2.44+/-0.30, p=0.001), and autoimmune lesions (3.25+/-0.48, p=0.019) were greater than that of multinodular goiter (1.0 for all 3) and surrounding normal thyroid (1.1+/-0.1). Average iNOS staining of benign adenomas (2.6+/-0.37), PTC (2.7+/-0.16), FTC (2.4+/-0.26) and autoimmune lesions (3.5+/-0.29) were all greater than that of surrounding normal thyroid (1.1+/-0.1, p<0.008), but there were too few multinodular goiters to achieve a significant difference (no.=2, 3.0+/-1.0). Average eNOS staining of benign adenomas (2.9+/-0.40), multinodular goiters (3.5+/-0.5), PTC (3.24+/-0.18), FTC (3.5+/-0.50), and autoimmune lesions (2.8+/-0.6) were also greater than that of surrounding normal thyroid (mean=1.4+/-0.2, p<0.001). N-TYR staining correlated with that of vascular endothelial growth factor (VEGF, r=0.36, p=0.007) and the number of lymphocytes/high power field (r=0.39, p=0.004). Recurrent disease developed only from carcinoma with moderate-intense N-TYR staining, but there were too few recurrent tumors to achieve statistical significance (p=0.08). We conclude that NO is produced by benign adenomas, PTC and FTC suggesting that NO could be important in vascularization of thyroid tumors and autoimmune thyroid diseases.     

Treatment of type II amiodarone-induced thyrotoxicosis by either iopanoic acid or glucocorticoids: a prospective,randomized study

Amiodarone-induced thyrotoxicosis (AIT) may occur either in the presence of underlying thyroid disease (type I AIT) or in apparently normal thyroid glands (type II AIT). Type II AIT, a destructive thyroiditis, often favorably responds to glucocorticoids. Iopanoic acid (IopAc) is an iodinated cholecystographic agent that inhibits deiodinase activity and reduces the conversion of T(4) toT(3). It has recently been reported that cholecystographic agents restore euthyroidism in patients with type II AIT. We describe the results of a prospective randomized study conducted in 12 patients with type II AIT treated with either iopanoic acid (group A, n = 6) or glucocorticoids (group B, n = 6). Serum free T(3) levels normalized rapidly in both groups after 7 d, from 0.75 +/- 0.20 ng/dl (11.5 +/- 3.1 pmol/liter) to 0.46 +/- 0.10 ng/d (7.1 +/- 1.7 pmol/liter), P < 0.01, and from 0.58 +/- 0.10 ng/dl (9.0 +/- 1.2 pmol/liter) to 0.34 +/- 0.03 ng/dl (5.2 +/- 0.5 pmol/liter), P < 0.003, in groups A and B, respectively (P = NS). Serum free T(4) levels reduced at 6 months in group B [from 2.70 +/- 0.32 ng/dl (35.1 +/- 4.1 pmol/liter) to 1.0 +/- 0.04 ng/dl (13.4 +/- 0.6 pmol/liter), P < 0.0001] but not in group A (from 2.90 +/- 0.6 ng/dl (38.0 +/- 7.5 pmol/liter) to 2.30 +/- 0.4 ng/dl (35.6 +/- 6.1 pmol/liter, P = 0.39; P = 0.005 group B vs. group A). All patients in both groups became euthyroid and had their amiodarone-induced destructive thyroiditis cured as defined by normalization of both serum free T(4) and free T(3) levels, during both drugs therapy. However, patients in group B were cured more rapidly than patients in group A (43 +/- 34 d vs. 221 +/- 111 d, respectively, P < 0.002). This study shows that, albeit both drugs are effective, glucocorticoids are probably the drug of choice for more rapidly curing type II AIT.     

Iodothyronine deiodinase activities in FRTL5 cells: predominance of type I 5'-deiodinase

FRTL5 cells, a thyroid follicular cell line derived from normal rat thyroid, has been extensively used as a model system to study various aspects of the physiology of the thyroid epithelium. The capacity of these cells to metabolize iodothyronines and to generate T3 from T4 has not been previously examined. Here we studied the deiodination of T4, T3, and rT3 in homogenates of FRTL5 cells. By far, these homogenates were more potent catalyzing the 5'-deiodination (outer ring) of T4 and rT3 than the inner ring deiodination of T4 or T3. Both the production of rT3 and the degradation of newly formed T3 from T4 were very limited. Thus, when T4 was used as a substrate, T3 and iodide accumulated in a linear fashion with time, and initially the amounts of iodide and T3 were approximately equal. rT3 and 3,3'-diiodothyronine were rapidly deiodinated by these homogenates, with the 3'-deiodination of 3,3'-diiodothyronine occurring at a slower rate than the 5'-deiodination of rT3. The iodothyronine 5'-deiodinase activity corresponded to type I, as indicated by the following: the Km for T4 and T3 was in the micromolar range; rT3 was a better substrate than T4 (maximum velocity = 101 vs. 19 pmol/min.mg protein; Km = 0.83 vs. 3.1 microM, respectively); and the kinetics of inhibition by 6n-propyl-2-thiouracil were uncompetitive and substrate dependent, suggesting ping-pong kinetics. The type I 5'-deiodinase activity of FRTL5 cells was distinctly stimulated by TSH. This stimulation seems to be mediated by cAMP and requires serum as a permissive factor. In conclusion, 1) FRTL5 cells exhibit both inner and outer ring iodothyronine-deiodinating activities; 2) iodothyronine 5'-deiodination is by far more active; 3) the 5'-deiodination has been characterized as type I deiodinase based on substrate preference, enzyme kinetics, and inhibitors; 4) in all respect iodothyronine deiodination by FRTL5 cell homogenates proceeded with marked similarity to that in homogenates or microsomes of thyroid glands from several species; and 5) the FRTL5 type I deiodinase is more active than that reported in thyroid tissue and as active as that reported in liver and kidney, the prototype of type I deiodinase-containing tissues. The present studies indicate that FRTL5 cells are an excellent model system to study cellular and biochemical aspects of the regulation of this enzyme as well as its regulation by TSH and putative serum factors.     

A common variation in deiodinase 1 gene DIO1 is associated with the relative levels of free thyroxine and triiodothyronine

INTRODUCTION: Genetic factors influence circulating thyroid hormone levels, but the common gene variants involved have not been conclusively identified. The genes encoding the iodothyronine deiodinases are good candidates because they alter the balance of thyroid hormones. We aimed to thoroughly examine the role of common variation across the three deiodinase genes in relation to thyroid hormones. METHODS: We used HapMap data to select single-nucleotide polymorphisms (SNPs) that captured a large proportion of the common genetic variation across the three deiodinase genes. We analyzed these initially in a cohort of 552 people on T(4) replacement. Suggestive findings were taken forward into three additional studies in people not on T(4) (total n = 2513) and metaanalyzed for confirmation. RESULTS: A SNP in the DIO1 gene, rs2235544, was associated with the free T(3) to free T(4) ratio with genome-wide levels of significance (P = 3.6 x 10(-13)). The C-allele of this SNP was associated with increased deiodinase 1 (D1) function with resulting increase in free T(3)/T(4) ratio and free T(3) and decrease in free T(4) and rT(3). There was no effect on serum TSH levels. None of the SNPs in the genes coding for D2 or D3 had any influence on hormone levels. CONCLUSIONS: This study provides convincing evidence that common genetic variation in DIO1 alters deiodinase function, resulting in an alteration in the balance of circulating free T(3) to free T(4). This should prove a valuable tool to assess the relative effects of circulating free T(3) vs. free T(4) on a wide range of biological parameters.     

Thyroid T4 5'-deiodinase activity in normal and abnormal human thyroid glands

To investigate the activity of the thyroid gland to convert T4 to T3, we measured the activity of thyroid T4 5'-deiodinase in the following human thyroid glands: 9 normal glands, 5 Hashimoto's thyroiditis, 13 follicular adenomas, 11 methimazole (MMI)-treated Graves' disease (GD), 11 propranolol iodide-treated GD, and 8 propylthiouracil (PTU)-treated GD. The enzyme activity was determined by the ability of 100,000 X g pellet of the thyroid homogenate to convert T4 to T3 in vitro. Normal thyroids showed the enzyme activity of 1.59 +/- 0.18 (mean +/- SEM) pmol T3/mg protein/min. Euthyroid Hashimoto's thyroiditis displayed the enzyme activity of 1.01 +/- 0.15 pmol T3/mg protein/min, which was similar to the normal thyroid enzyme activity. The hypothyroid gland of Hashimoto's thyroiditis showed the enzyme activity of 1.8 pmol T3/mg protein/min. Follicular adenomas showed a wide range of enzyme activity with the mean level of 3.24 +/- 0.82 pmol T3/mg protein/min that did not differ significantly from that of the normal thyroids. Interestingly, one adenoma, despite TSH suppression that ordinarily decreases enzyme activity, showed the greatest activity of 11.0 pmol T3/mg protein/min. Graves' thyroids following treatment with MMI, PTU, and propranolol-iodide showed enzyme activities of 4.61 +/- 0.53, 3.95 +/- 0.43, and 3.51 +/- 0.46 pmol T3/mg protein/min, respectively; all these values were greater than that of the normal thyroids (P less than 0.01), but did not differ significantly when compared with each other. In summary, thyroid glands with Hashimoto's thyroiditis had activities of T4 to T3 conversion similar to the normal thyroid glands.(ABSTRACT TRUNCATED AT 250 WORDS)     

A polymorphism in type I deiodinase is associated with circulating free insulin-like growth factor I levels and body composition in humans

The interaction between the GH-IGF-I axis and thyroid hormone metabolism is complex and not fully understood. T(4) stimulates IGF-I activity in animals in the absence of GH. On the other hand, GH replacement therapy results in an increase in serum T(3) and a decrease in T(4) and rT(3) levels, suggesting a stimulation of type I deiodinase (D1) activity. Recently, we demonstrated the association of two polymorphisms in D1 (D1a-C/T; T = 34%, and D1b-A/G; G = 10%) with serum iodothyronine levels. Haplotype alleles were constructed, suggesting a lower activity of the D1 haplotype 2 allele (aT-bA) and a higher activity of the haplotype allele 3 (aC-bG). In this study, we investigated whether genetic variations in D1 are associated with the IGF-I system.In 156 blood donors and 350 elderly men, the association of the D1 haplotype alleles with circulating IGF-I and free IGF-I levels was studied. In addition, potential associations with muscle strength and body composition were investigated in the elderly population. Finally, the relation between serum iodothyronine levels and IGF-I levels was studied.In blood donors, haplotype allele 2 was associated with higher levels of free IGF-I (302.9 +/- 22.9 vs. 376.3 +/- 19.1 pg/ml, P = 0.02). In elderly men, haplotype allele 2 also showed an allele dose increase in free IGF-I levels (P(trend) = 0.01) and an allele dose decrease in serum T(3) levels (P(trend) = 0.01), independent of age. Carriers of the D1a-T variant also had a higher isometric grip strength (P = 0.047) and maximum leg extensor strength (P = 0.07) as well as a higher lean body mass (P = 0.03).In blood donors, T(4) and free T(4) were negatively correlated with total IGF-I levels (R = -0.18, P = 0.03 and R = -0.24, P = 0.003), whereas T(3) to T(4) and T(3) to reverse T(3) ratios were positively correlated with total IGF-I (R = 0.31, P < 0.001 and R = 0.18, P = 0.03). Free IGF-I showed a negative correlation with T(4) (R = -0.26, P = 0.001) and T(4)-binding globulin (R = -0.31, P < 0.001) and a positive correlation with T(3) to T(4) ratio (R = 0.21, P = 0.01).In conclusion, a polymorphism that results in a decreased D1 activity is associated with an increase in free IGF-I levels. The pathophysiological significance of this association with IGF-I is supported by an increased muscle strength and muscle mass in carriers of the D1 haplotype 2 allele in a population of elderly men. The association of D1 haplotype allele 2 with serum T(3) levels in the elderly population suggests a relative increase in its contribution to circulating T(3) in old age.     

Silencing of the maternally imprinted tumor suppressor ARHI contributes to follicular thyroid carcinogenesis

The two most common subtypes of thyroid cancer, follicular thyroid carcinoma (FTC) and papillary thyroid carcinoma, have been extensively studied, but our fundamental understanding of the molecular events in thyroid epithelial oncogenesis is still limited. Unreported data from our previous published global gene expression analysis revealed that the tumor suppressor gene aplysia ras homolog I (ARHI) is frequently underexpressed in FTCs. In this study, we elucidated the frequency and mechanism of ARHI silencing in benign and malignant thyroid neoplasia. We demonstrated that underexpression of ARHI occurs principally in FTCs (P = 0.0018), including its oncocytic variant (11 of 13), even at minimally invasive stage but not classic papillary thyroid carcinoma (two of seven) or follicular adenoma (FA) (three of 14). FTCs show strong allelic imbalance with reduction in copy number/loss of heterozygosity (LOH) in 69%, compared with less than 10% for FAs. In combination with our LOH data, bisulfite sequencing in a subset of samples revealed that FA displays a symmetric methylation pattern, likely representing one unmethylated allele and one presumptively imprinted allele, whereas FTC shows a virtually complete methylation pattern, representing LOH of the nonimprinted allele with only the hypermethylated allele remaining. Furthermore, we showed that pharmacologic inhibition of histone deacetylation but not demethylation could reactivate ARHI expression in the FTC133 FTC cell line. Therefore, our data suggest that silencing of the putative maternally imprinted tumor suppressor gene ARHI, primarily by large genomic deletion in conjunction with hypermethylation of the genomically imprinted allele, serves as a key early event in follicular thyroid carcinogenesis.     

Thyroid cancers express CD-40 and CD-40 ligand: cancers that express CD-40 ligand may have a greater risk of recurrence in young patients.

The immune response might suppress thyroid cancer recurrence. Although the factors that control this are unknown, CD-40 and CD-40 ligand might be important. To test this, we stained 36 papillary (PTC) and four follicular (FTC) thyroid carcinomas for CD-40 (n = 37) and CD-40 ligand (n = 36) and graded staining from absent (grade 0) to intense (grade 3). Follicular cells of the majority of thyroid tumors expressed CD-40 (30/37, 81%) and CD-40 ligand (15/24, 69%). Cancers from young patients (< or =21 years of age) that expressed CD-40 contained more numerous lymphocytes/high-power field (36 +/- 11) than cancers that failed to express CD-40 (4 +/- 3, p = 0.01), but there was no correlation with clinical outcome. Among young patients, CD-40 ligand expression was more intense in multifocal (1.1 +/- 0.2 vs. 0.45 +/- 0.2, p = 0.037), aggressive (1.14 +/- 0.14 vs. 0.65 +/- 0.2, p = 0.05) and recurrent tumors (1.2 +/- 0.2 vs. 0.65 +/- 0.2, p = 0.05) and associated with reduced disease-free survival (p = 0.03). We conclude that the majority of thyroid cancers express CD-40 and CD-40 ligand. In patients < or =21 years of age, tumors with intense expression of CD-40 ligand are more often multifocal, aggressive, and recurrent.