Real-time visualization of processive myosin 5a-mediated vesicle movement in living astrocytes.

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.

Myosin V plays an essential role in the thyroid hormone-dependent endocytosis of type II iodothyronine 5'-deiodinase.

In astrocytes, thyroxine modulates type II iodothyronine 5'-deiodinase levels by initiating the binding of the endosomes containing the enzyme to microfilaments, followed by actin-based endocytosis. Myosin V is a molecular motor thought to participate in vesicle trafficking in the brain. In this report, we developed an in vitro actin-binding assay to characterize the thyroid hormone-dependent binding of endocytotic vesicles to microfilaments. Thyroxine and reverse triiodothyronine (EC(50) levels approximately 1 nm) were >100-fold more potent than 3,5,3'-triiodothyronine in initiating vesicle binding to actin fibers in vitro. Thyroxine-dependent vesicle binding was calcium-, magnesium-, and ATP-dependent, suggesting the participation of one or more myosin motors, presumably myosin V. Addition of the myosin V globular tail, lacking the actin-binding head, specifically blocked thyroid hormone-dependent vesicle binding, and direct binding of the myosin V tail to enzyme-containing endosomes was thyroxine-dependent. Progressive NH(2)-terminal deletion of the myosin V tail and domain-specific antibody inhibition studies revealed that the thyroxine-dependent vesicle-tethering domain was localized to the last 21 amino acids of the COOH terminus. These data show that myosin V is responsible for thyroid hormone-dependent binding of primary endosomes to the microfilaments and suggest that this motor mediates the actin-based endocytosis of the type II iodothyronine deiodinase.

Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase.

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.

Thyroid hormone regulates the extracellular organization of laminin on astrocytes.

Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmembrane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astrocyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of the microfilaments. In this study, the effect of thyroid hormone on the secretion and topology of laminin in astrocytes was examined. Linear arrays of secreted laminin were observed on the surface of the T4-treated astrocytes within 10 h after seeding the cells onto poly-D-lysine-coated coverslips and became an organized meshwork by 24 h. In contrast, little if any laminin was identified on the surface of either hormone-deficient or T3-treated cells until 36 h after seeding and then was restricted to punctate deposits. Secretion of laminin into the medium by hormone-deficient and T3-treated cells was significantly greater than that by T4-treated cells. Conversely, deposition of laminin into the extracellular matrix was significantly greater in T4-treated cells than in hormone-deficient and T3-treated cells. Thyroid hormone had no effect on the production of laminin by astrocytes. These data show that T4 regulates the extracellular deposition and organization of laminin on the surface of astrocytes and provide a mechanism by which this morphogenic hormone can influence neuronal migration and axonal projection in the developing brain.

Thyroid hormone regulates the expression of laminin in the developing rat cerebellum.

In the rat cerebellum, migration of neurons from the external granular layer to the internal granular layer occurs postnatally and is dependent upon the presence of thyroid hormone. In hypothyroidism, many neurons fail to complete their migration and die. Key guidance signals to these migrating neurons are provided by laminin, an extracellular matrix protein that is fixed to the surface of astrocytes. Expression of laminin in the brain is developmentally timed to coincide with neuronal growth spurts. In this study, we examined the role of thyroid hormone on the expression and distribution of laminin in the rat cerebellum. We show that laminin content steadily increased 2- to 3-fold from birth to maximal levels on postnatal day 8-10 then steadily decreased to a plateau by postnatal day 12 in the euthyroid cerebellum. Immunoreactive laminin appeared in the molecular layer of the euthyroid cerebellum by postnatal day 4, reached maximal intensity by postnatal day 8-10, and was gone by postnatal day 14. In contrast, laminin content in the hypothyroid cerebellum remained unchanged from birth until postnatal day 10 and then increased to maximal levels over the next two days; maximal levels were approximately 35% less than those levels in the euthyroid cerebellum. Laminin staining did not appear in the molecular layer of the hypothyroid rat cerebellum until postnatal day 10, reached maximal intensity by postnatal day 15 and disappeared by postnatal day 18, despite the continued presence granular neurons in the external granular layer. These data indicate that the disruption of the timing of the appearance and regional distribution of laminin in the absence of thyroid hormone may play a major role in the profound derangement of neuronal migration observed in the cretinous brain.

The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat.

Type II iodothyronine deiodinase is a short-lived, membrane-bound enzyme found in rat brain, brown adipose tissue, and cAMP-stimulated astrocytes. Recently, a full-length complementary DNA (cDNA) encoding a 30-kDa, type II-like selenodeiodinase was cloned from frog, and a homologous partial cDNA (rBAT 1.1), containing two in-frame selenocysteine codons (UGA), was isolated from rat brown adipose tissue. Importantly, the rBAT 1.1 cDNA was derived from a 7.5-kb messenger RNA (mRNA) and did not encode a functional selenoenzyne unless an enabling selenocysteine insertion sequence was appended to the presumed coding region and this cDNA. In this study we determined whether the native 7.5-kb SeD2 mRNA in rat tissues programmed the synthesis of the native type II deiodinase using specific antibodies that were raised against the C-terminus of full-length, 30-kDa SeD2 protein and against the catalytic core of SeD2. Direct analysis of the translation products programmed by the native SeD2 mRNA in cAMP-stimulated astrocytes was performed using antisense deoxynucleotides and hybrid selection strategies. (Bu)2cAMP-stimulated rat astrocytes expressed both type II deiodinase activity (approximately 2500 U/mg protein) and contained abundant levels of the 7.5-kb SeD2 mRNA. However, no immunoreactive 30-kDa SeD2 protein was identified by Western analysis, immunoprecipitation, or immunocytochemistry, and the specific C-terminus antiserum failed to immunoprecipitate deiodinase activity from (Bu)2cAMP-stimulated astrocytes, brown adipose tissue or brain. Instead, the native 7.5-kb SeD2 mRNA encoded a 15-kDa protein that terminated at the first UGA codon and contained the catalytically inactive, N-terminal 129 amino acids of SeD2. These data show that the native 7.5-kb SeD2 mRNA in stimulated astrocytes does not encode D2.

Thyroid hormone-regulated actin polymerization in brain.

Thyroid hormones play an important role in the growth and development of the brain. Central to the proper integration of neuronal circuitry is the ability of the growing neurite to interpret guidance cues during its migration. The action cytoskeleton is especially rich in the growth cone, and is a likely target for thyroid hormone regulation. This brief review summarizes work showing that thyroxine, but not T3, dynamically regulates the polymerization of the actin cytoskeleton in astrocytes. The ability of T4 to enhance actin polymerization, without directly affecting gene expression, has a profound effect on the ability of the cell to interact with laminin, the major extracellular matrix protein in the developing brain. T4 also regulates the formation of key cell contacts with extracellular matrix guidance cues. These processes are likely to participate in thyroid hormone's regulation of brain development.

Inflammatory thyroid disorders.

Inflammatory disorders of the thyroid, including autoimmune thyroiditis, are among the most common endocrine abnormalities encountered in clinical practice. The association of pain with these disorders, however, is relatively uncommon. Despite this observation, painful thyroid disorders comprise a significant component of the spectrum of thyroid disease. A rational approach to such patients, including history, physical examination, laboratory evaluation, radionuclide or ultrasonographic imaging, and fine needle aspiration biopsy, will allow the appropriate diagnosis to be made in the vast majority of cases.

Catalytic activity of type II iodothyronine 5'-deiodinase polypeptide is dependent upon a cyclic AMP activation factor.

Type II iodothyronine 5'-deiodinase is an approximately 200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3'-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5'-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5'-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of approximately 60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5'-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane.

Degradation and recycling of the substrate-binding subunit of type II iodothyronine 5'-deiodinase in astrocytes.

Thyroxine dynamically regulates levels of type II iodothyronine 5'-deiodinase (5'D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5'D-II is an approximately 200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T4), p29 is slowly endocytosed and transported to the lysosomes. T4 treatment rapidly activates an actin-mediated endocytotic pathway and targets the enzyme to the endosomes. In this study, we have characterized the influence of T4 on the intracellular trafficking of 5'D-II. We show that T4 accelerates the rate of 5'D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5'D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane approximately 2 h after enzyme internalization in the presence of T4, indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T4-treated cell, 5'D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T4, enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways.

Selenium-regulated translation control of heterologous gene expression: normal function of selenocysteine-substituted gene products.

In eukaryotes, the synthesis of selenoproteins depends on an exogenous supply of selenium, required for synthesis of the novel amino acid, selenocysteine, and on the presence of a "selenium translation element" in the 3' untranslated region of mRNA. The selenium translation element is required to re-interpret the stop codon, UGA, as coding for selenocysteine incorporation and chain elongation. Messenger RNA lacking the selenium translation element and/or an inadequate selenium supply lead to chain termination at the UGA codon. We exploited these properties to provide direct translational control of protein(s) encoded by transfected cDNAs. Selenium-dependent translation of mRNA transcribed from target cDNA was conferred by mutation of an in-frame UGU, coding for cysteine, to UGA, coding for either selenocysteine or termination, then fusing the mutated coding region to a 3' untranslated region containing the selenium translation element of the human cellular glutathione peroxidase gene. In this study, the biological consequences of placing this novel amino acid in the polypeptide chain was examined with two proteins of known function: the rat growth hormone receptor and human thyroid hormone receptor beta 1. UGA (opal) mutant-STE fusion constructs of the cDNAs encoding these two polypeptides showed selenium-dependent expression and their selenoprotein products maintained normal ligand binding and signal transduction. Thus, integration of selenocysteine had little or no consequence on the functional activity of the opal mutants; however, opal mutants were expressed at lower levels than their wild-type counterparts in transient expression assays. The ability to integrate this novel amino acid at predetermined positions in a polypeptide chain provides selenium-dependent translational control to the expression of a wide variety of target genes, allows facile 75Se radioisotopic labeling of the heterologous proteins, and permits site-specific heavy atom substitution.

Thyroxine-dependent regulation of integrin-laminin interactions in astrocytes.

Adhesive interactions among the extracellular matrix protein laminin, cell surface receptors known as integrins, and the microfilament network play a fundamental role in the regulation of neural cell migration during brain development. The disturbed neuronal migration that occurs when thyroid hormone is lacking during early neonatal life contributes to the profound morphological alterations characteristic of the cretinous brain. We have previously shown that thyroid hormone determines the organization of the microfilament network in astrocytes by regulating the polymerization of F-actin fibers. In this paper, we examined whether T4-dependent alterations in microfilament organization affected astrocyte-laminin interactions. We show that T4-treated astrocytes readily attached to laminin, whereas attachment of thyroid hormone-deficient cells to laminin was delayed. T4-dependent cell attachment to laminin was completely abolished by blocking integrin recognition sites with site-specific peptides or by depolymerizing the microfilaments with dihydrocytochalasin B. We also show that T4 was required for integrin clustering and focal contact formation in astrocytes attached to laminin. Thus, T4 dynamically regulates interactions between integrins and laminin via modulation of microfilament organization in astrocytes. The T4-dependent regulation of laminin-integrin interactions provides a mechanism by which this morphogenic hormone can influence neuronal migration and development.

Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells.

The brain has abundant nuclear T3-binding sites and contains messenger RNAs (mRNAs) encoding multiple thyroid hormone receptor (TR) isoforms; the cellular distribution of these different TR isoforms is unknown. To determine whether the TR isoforms are differentially expressed in neuronal and astroglial cells, we examined the relative abundance of the mRNAs encoding TR alpha 1, c-erbA alpha 2, and TR beta 1 in primary cultures of fetal rat brain and in several cell lines of neural and glial origin. Additionally, the TR isoform polypeptides were identified by immunocytochemistry using isoform-specific antibodies. Northern blot analysis showed that fetal brain cell cultures contain mRNAs encoding the T3-binding isoforms TR alpha 1 and TR beta 1 as well as the mRNA encoding the non-T3-binding c-erbA alpha 2. c-erbA alpha 2 mRNA was most abundant, comprising more than 85% of the TR mRNAs in the primary cultures. Neuronal enrichment by antimitotic selection increased TR beta 1 mRNA approximately 3-fold, decreased c-erbA alpha 2 mRNA 70%, and had little or no effect on TR alpha 1 mRNA. Neuronal depletion resulted in the complete loss of TR beta 1 mRNA without changing c-erb alpha 2 or TR alpha 1 mRNA levels. Primary cultures of rat astrocytes, the astrocytoma cell line C6, and the pheochromocytoma cell line PC12 contained only the c-erbA alpha 2 mRNA. Immunocytochemistry using isoform-specific anti-sera revealed that TR beta 1 was exclusively localized to neuronal nuclei, and c-erbA alpha 2 was only found in the nuclei of astrocytes. These results show that TR beta 1 is localized to the nuclei of neuronal cells, and that c-erbA alpha 2 is restricted to the nuclei of astrocytes.

Structural requirements of iodothyronines for the rapid inactivation and internalization of type II iodothyronine 5'-deiodinase in glial cells.

3,3'5,5'-Tetraiodothyronine (T4), but not 3,3'5-triiodothyronine (T3), acutely regulates the activity of the plasma membrane-bound enzyme, type II iodothyronine 5'-deiodinase (5'D-II), by inducing internalization of the enzyme through an extranuclear, energy-dependent mechanism that requires an intact actin cytoskeleton. The affinity label, N-bromoacetyl-L-T4, binds to 5'D-II and irreversibly inhibits the enzyme but does not initiate internalization. To determine the structural elements of T4 which are required for enzyme internalization, T4 analogs were modified in the alanine side chain and were then evaluated for their ability to induce enzyme internalization, to inhibit enzyme activity, and to promote actin polymerization in hypothyroid cells. The analogs studied showed marked variability in their ability to inactivate 5'D-II. The rank order of potency for enzyme inactivation was T4 > COOH-blocked analogs > NH3 and COOH blocked analogs >> NH3 blocked analogs (EC50 values range from 1 to > 1000 nM). In contrast, all T4 analogs tested and T4 were excellent competitive inhibitors of 5'D-II with respect to substrate (Ki values ranged from 4 to 27 nM). The differential capability of iodothyronines to inactivate the enzyme was not related to their ability to enter the cell, since Ki values measured in intact glial cells were equivalent to those measured in cell sonicates. The power of the T4 analogs to inactivate 5'D-II was paralleled by their ability to polymerize actin in hypothyroid cells and to induce 5'D-II binding to F-actin. The data show that modification of the alanine side chain markedly alters the ability of T4 analogs to induce 5'D-II inactivation and actin polymerization. A net negative charge on the alanine side chain of T4 is detrimental for the hormone-dependent inactivation of 5'D-II and polymerization of actin, whereas uncharged or positively charged molecules retain significant activity.

The thyroid gland is a major source of circulating T3 in the rat.

In rats, the respective contribution of the thyroid and peripheral tissues to the pool of T3 remains unclear. Most, if not all, of the circulating T3 produced by extrathyroidal sources is generated by 5'-deiodination of T4, catalyzed by the selenoenzyme, type I iodothyronine 5'-deiodinase (5'D-I). 5'D-I in the liver and kidney is almost completely lost in selenium deficiency, resulting in a marked decrease in T4 deiodination and an increase in circulating T4 levels. Surprisingly, circulating T3 levels are only marginally decreased by selenium deficiency. In this study, we used selenium deficiency and thyroidectomy to determine the relative contribution of thyroidal and extrathyroidal sources to the total body pool of T3. Despite maintaining normal serum T4 concentrations in thyroidectomized rats by T4 replacement, serum T3 concentrations remained 55% lower than those seen in intact rats. In intact rats, restricting selenium intake had no effect on circulating T3 concentrations. Decreasing 5'D-I activity in the liver and kidney by > 90% by restricting selenium intake resulted in a further 20% decrease in serum T3 concentrations in the thyroidectomized, T4 replaced rats, suggesting that peripheral T4 to T3 conversion in these tissues generates approximately 20% of the circulating T3 concentrations. While dietary selenium restriction markedly decreased intrahepatic selenium content (> 95%), intrathyroidal selenium content decreased by only 27%. Further, thyroid 5'D-I activity actually increased 25% in the selenium deficient rats, suggesting the continued synthesis of this selenoenzyme over selenoproteins in other tissues in selenium deficiency. These data demonstrate that the thyroid is the major source of T3 in the rat and suggest that intrathyroidal T4 to T3 conversion may account for most of the T3 released by the thyroid.

Thyroxine targets different pathways of internalization of type II iodothyronine 5'-deiodinase in astrocytes.

In the brain, thyroid hormone dynamically regulates levels of the short-lived plasma membrane protein, type II iodothyronine 5'-deiodinase. In cultured astrocytes, thyroxine modulates deiodinase levels by activating cytoskeletal-plasma membrane interactions that increase the rate of inactivation of the enzyme. Here we characterized the effects of these thyroxine-dependent cytoskeletal interactions upon the route of internalization of the deiodinase by following the intracellular transit of the affinity-labeled substrate-binding subunit of the deiodinase (p29). Thyroxine rapidly induced the inactivation of the deiodinase and initiated the binding of p29 to F-actin. By 40 min, > 75% of the p29 had been transported to an endosomal pool, which was followed by dissociation of the F-actin-p29 complex. There was no significant accumulation of p29 in the dense lysosomes seen in the presence of thyroxine. In the absence of thyroxine, p29 was internalized and transported to the dense lysosomes at a rate parallel to the inactivation rate of the deiodinase (t1/2 0.75 and 0.64 h, respectively) without involvement with the microfilaments. These data demonstrate that thyroxine targets type II iodothyronine 5'-deiodinase to an endosomal pool by activating specific protein-F-actin interactions involved in microfilament-mediated intracellular protein trafficking.

Thyroid hormone-dependent redistribution of the 55-kilodalton monomer of protein disulfide isomerase in cultured glial cells.

In addition to the effects of thyroid hormone that are mediated through interaction with chromatin-associated receptors, T4 modulates the activity of the cellular content of the membrane-associated protein type II iodothyronine 5'-deiodinase (5'D-II) by regulating its degradation through an actin-dependent extranuclear mechanism. Under the influence of thyroid hormone, the substrate-binding subunit of 5'D-II is translocated from the plasma membrane to an intracellular microfilament-associated pool. In glial cells, a 55-kilodalton (kDa) protein (glial-p55), which was shown to be identical to the 55-kDa monomer of protein disulfide isomerase (PDI) also demonstrates a similar T4-dependent association to the F-actin microfilaments. To explore the role of glial-p55 in the extranuclear effect of thyroid hormone in glial cells, the effects of thyroid hormone on the subcellular localization of glial-p55 were further examined. The current study demonstrates the presence of two pools of glial-p55. While the majority of glial-p55 is associated with endoplasmic reticulum and represents PDI, approximately 25% of glial-p55 is cytosolic in the absence of thyroid hormone. Cytosolic glial-p55 is lost from the cells after mild permeabilization with saponin, and treatment of cells with T4 causes the shift of glial-p55 from the cytosolic pool to the subcellular fractions that contain the actin cytoskeleton. Crude microsomal preparations were prepared which contain membranes, microfilaments, and other particulate cell structures. In the absence of thyroid hormone, glial cells lack an intact actin cytoskeleton, and glial-p55 is easily removed from these preparations by conditions that remove extrinsic membrane proteins like PDI, such as alkaline pH and detergent extraction. In contrast, glial-p55 is not removed from the crude microsomes prepared from thyroid hormone-replete glial cells that contain an intact actin cytoskeleton. Since previous work in our laboratory indicated that glial-p55 becomes actin associated in a thyroid-dependent manner along with the substrate-binding subunit of 5'D-II, this study suggests that the 55-kDa monomer of PDI may play a role in the thyroid hormone-dependent regulation of actin polymerization and the degradation of 5'D-II.

Effects of selenium deficiency on thyroid hormone economy in rats.

In selenium-deficient rats, peripheral T4 to T3 conversion is markedly decreased due to the loss of the selenoprotein, type I iodothyronine 5'-deiodinase (5'D-I). Despite the marked increase in circulating T4 that results from this loss of 5'D-I, serum T3 concentrations in selenium-deficient rats remain in the normal range. To determine the physiological mechanism(s) that maintains circulating T3 when peripheral T4 to T3 conversion is impaired, we examined the interrelationships between selenium intake and the metabolism of T3 and T4 in the rat. In euthyroid rats, selenium deficiency caused the expected loss of 5'D-I, with a 52% increase in serum T4, which paralleled an increase in the T4 biological half-life. Consistent with the prolonged t1/2 of T4, short term thyroidectomy (48 h) in selenium-deficient rats failed to decrease serum T4 concentrations to the levels observed in short term thyroidectomized, selenium-supplemented rats. Short term thyroidectomy also caused an expected 33% decrease in liver 5'D-I and a 44% increase in brain type II iodothyronine 5'-deiodinase (5'D-II) activities in selenium-supplemented rats. However, in selenium-deficient rats, short term thyroidectomy did not affect 5'D-I or 5'D-II activities. In contrast to the selenium-dependent changes in circulating T4 levels, little or no change in circulating T3 concentrations occurred. There was a 20% increase in the T3 half-life in selenium-deficient rats. The serum T3 sulfate concentration was increased, and T3 deiodination was reciprocally decreased in the selenium-deficient rats. These data suggest that increased T3 sulfate generation in selenium-deficient rats may lead to greater T3 availability through enterohepatic recycling of the iodothyronine and may explain why there are only minor changes in serum T3 concentrations in selenium-deficient rats.

Dissociation of actin polymerization and enzyme inactivation in the hormonal regulation of type II iodothyronine 5'-deiodinase activity in astrocytes.

T4 dynamically regulates the levels of type II iodothyronine 5'-deiodinase in the brain. Using an astrocyte cell culture model, we have shown that thyroxine increases inactivation of this enzyme through a mechanism using the actin cytoskeleton. In the absence of T4, the filamentous actin (F-actin) stress fibers are absent, and deiodinase inactivation is relatively slow. T4 increases inactivation of type II 5'-deiodinase by 1) restoring the F-actin stress fibers, 2) promoting the binding of the enzyme to F-actin, and 3) stimulating enzyme internalization. To determine whether inactivation of the deiodinase is due solely to the restoration of stress fibers by T4 or also involves direct thyroxine-mediated enzyme-F-actin interactions, we examined the effects of retinoids on both actin polymerization and type II 5'-deiodinase activity in cultured astrocytes, as these hormones have been shown to alter cytoskeletal organization in other tissues. In thyroid hormone-deficient astrocytes, retinoic acid increased F-actin levels, with no change in total cell actin. The F-actin content increased approximately 40% within 30 min after the addition of retinoic acid. After a plateau of 6-8 h, the F-actin content increased further to approximately 90% of the total cell actin and was associated with the reappearance of stress fibers. Only this latter retinoid-stimulated increase in F-actin content was blocked by actinomycin-D. Restoration of the F-actin stress fibers by retinoids did not increase the turnover of the type II 5'-deiodinase (t1/2, 1.99 h-1) or promote binding of the enzyme to F-actin in the absence of T4. Similarly, retinoids did not affect the rapid T4-mediated turnover (t1/2, 0.18 h-1) of type II 5'-deiodinase. These data show that an intact F-actin cytoskeleton in the absence of T4 is inadequate to alter the inactivation of type II 5'-deiodinase and that specific T4-enzyme-F-actin interactions are necessary to initiate the rapid inactivation/internalization of this enzyme.