Differential Response to L-Triiodothyronine of Anterior Pituitary Growth Hormone Messenger Ribonucleic Acid (mRNA) and β-Thyrotropin mRNA in a Hypothyroid Walker 256 Carcinoma-Bearing Rat Model of Nonthyroidal Disease*

We To address these issues, we asked whether an injected gene could be expressed in cardiac tissue and, if so, whether a gene coupled to a cellular promoter could be expressed at detectable levels and regulated appropriately. Such a finding would suggest an approach for targeting the expression of injected genes to specific cell types and for modulating their expression. The cellular promoter chosen for this study was derived from the rat a-cardiac myosin heavy chain (a-MHC) gene. In vivo, the expression of this gene is restricted to the heart (9) and its activity has been shown both in vivo (10) and in vitro (11, 12) to be positively regulated by thyroid hormone. By co-injecting rat cardiac and skeletal muscles with reporter genes linked to the a-MHC promoter and to a second viral promoter as an internal control, we demonstrated that the heart can be transfected in vivo with greater efficiency than skeletal muscle. Furthermore, we showed that the directly injected a-MHC promoter is active in heart, but not in skeletal muscle, and that its activity is regulated by the thyroid hormone status of the animal. MATERIALS AND METHODSPlasmids. pa-MHCluc contains the firefly luciferase coding region coupled to the rat a-MHC 5' flanking sequence, base pairs -613 to +32 (13). The latter, which includes a putative thyroid hormone response element (12), had been subcloned into ptz19R (United States Biochemical) as an Xba I-blunted EcoRI fragment. A HindIII-Kpn I fragment of this plasmid was subcloned into pXP2 (14), a promoterless luciferase vector, to construct pa-MHCluc. pRSVCAT, in which the coding sequence of the chloramphenicol acetyltransferase (CAT) gene is spliced to the long terminal repeat of the Rous sarcoma virus (RSV), has been described (15). pO-CAT (pCAT-Basic Vector; Promega) is a promoterless CAT construct.Animal Models. Adult female Wistar rats were anesthetized with inhaled methoxyfluorane and 0.1 ml/100g (body weight) of ketamine (50 mg/ml) and zylazine (10 mg/ml) intramuscularly. Animals receiving cardiac injections underwent exteriorization of the heart through a left thoracotomy (16) followed by injection of 50 /tl of a solution containing each plasmid at 2 ug/,l~as indicated in 20% (wt/vol) sucrose and 2% (vol/vol) Evans blue into the apex of the left ventricle through a 27-gauge needle. After expression of air from the chest, animals were ventilated briefly on a small-animal respirator and the incision was closed. In other rats, the belly of the adductor magnus muscle was injected under direct visualization.Two protocols were used in experiments involving thyroid hormone manipulations. (i) Rats were rendered hypothyroid by the administration of propylthiouracil (500 mg/liter in drinking water) (17) for 3 weeks, at which point their hearts were co-injected with 100 Ag of pRSVCAT and 100 pug of pa-MHCluc. The animals were then divided into two groups: one was continued on propylthiouracil and a second received 3,5,3'-triiodothyronine [T3; 200 ,ug/100 g (body weight), intraperitoneally] 2 hr aft...

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“…(i) Rats were rendered hypothyroid by the administration of propylthiouracil (500 mg/liter in drinking water) (17) …”

Section: Methodsmentioning

confidence: 99%

Hormonal modulation of a gene injected into rat heart in vivo.

Kitsis

Buttrick

McNally

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

163

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“…A related molecule, rat decrease in abundance of this receptor in anterior pituitary and liver of tumor-bearing rats when compared with controls. Differential regulation of thyroid hormone-induced cellular responses has also been demonstrated in these tissues in tumor rats (14,15). Although the differential response to thyroid hormone in tumor rats may be related to events that occur after the formation of hormone-receptor complexes, it is also possible that changes in expression of different receptor forms in thyroidresponsive tissues might explain altered responses to T3 in tumor rats.…”

mentioning

confidence: 99%

c-erb-A mRNA Correlates with T₃-Receptor Levels in Liver and Pituitary of Tumor Rats

Hupart¹,

Hodin²,

Lazar³

et al. 1993

Thyroid

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In the rat tumor model of the sick euthyroid syndrome, differential regulation of T3-induced cellular responses have been demonstrated in liver and anterior pituitary. These effects occur with a concomitant decrease in nuclear thyroid hormone receptor (TR) number as measured by the binding of 125I-labeled T3. To explore the possibility that these altered responses to T3 in tumor rats resulted from changes in the expression of different TR forms, we correlated the relative abundance of mRNAs encoding each receptor form with the concentration of TR measured by specific T3 binding. In anterior pituitary of tumor rats, TR beta-1 and beta-2 mRNA levels decreased to 51 and 45%, respectively, compared to controls; rat c-erb A alpha-2 mRNA, which encodes a TR-related DNA alpha-binding protein that does not bind T3, decreased to 46% of control. These findings correlate with a decrease in nuclear T3 binding capacity that has been shown to be 63% of control. The level of TR beta-1 mRNA, the only quantifiable TR form in liver, was decreased to 61% of control in the same hepatic tissue that revealed a 50% decrease in TR as measured by specific T3 binding. The coordinate down-regulation of all TR mRNA forms to a degree that parallels the decrease in TR number as measured by specific T3 binding suggests that the differential regulation of T3-mediated effects in illness is by a mechanism other than changing concentrations of specific receptor forms.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…model in cultured cells. GH-producing, thyroid hormone-responsive GC cells exhibit responses to heat stress which are similar to responses observed in anterior pituitary somatotrophs of a n in vivo tumor rat model of human disease (Hupart et al, 1990). These shared stress-induced responses include a reduction in T, nuclear receptor levels (Shapiro et al, 1989b) and a specific increase in T,-induced GH mRNA levels (Shapiro et al, 1989a).…”

Section: Effects On Gc Cell Growth Pyotein Content Andmentioning

confidence: 73%

The effects of chronic exposure to supraphysiological concentrations of 3,5,3′ triiodo‐L‐thyronine (T₃) on cultured GC cells

Reynolds

Surks

Shapiro

1991

Journal Cellular Physiology

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We have characterized the effects of supraphysiological concentrations of T3 on GC cells, a cultured cell line in which physiologic concentrations of T3 regulate cell growth, protein content, and growth hormone (GH) production. GC cells were exposed to 3 times (1.0 nM) and 80 times (25.0 nM) the physiologic concentration of T3 (0.3 nM) for either 4 d or for greater than 3 months. Both short and prolonged exposure to supranormal T3 concentrations supported maximal cell growth rate and induced significant increases in total protein (P less than 0.025) and GH production (P less than 0.01) per cell when compared to measurements in control GC cells. In addition, exposure to 1.0 nM and 25.0 nM T3 for greater than 3 months enhanced the toxicity of heat shock in a manner similar to previously described effects on GC cells due to T3 exposure of shorter duration. Thus, initial responses to raised T3 concentrations in cultured GC cells persisted without alteration when hormone exposure was prolonged for greater than 3 months.

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Differential Response to L-Triiodothyronine of Anterior Pituitary Growth Hormone Messenger Ribonucleic Acid (mRNA) and β-Thyrotropin mRNA in a Hypothyroid Walker 256 Carcinoma-Bearing Rat Model of Nonthyroidal Disease*

Cited by 7 publications

References 31 publications

Hormonal modulation of a gene injected into rat heart in vivo.

Hormonal modulation of a gene injected into rat heart in vivo.

c-erb-A mRNA Correlates with T₃-Receptor Levels in Liver and Pituitary of Tumor Rats

The effects of chronic exposure to supraphysiological concentrations of 3,5,3′ triiodo‐L‐thyronine (T₃) on cultured GC cells

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Differential Response to L-Triiodothyronine of Anterior Pituitary Growth Hormone Messenger Ribonucleic Acid (mRNA) and β-Thyrotropin mRNA in a Hypothyroid Walker 256 Carcinoma-Bearing Rat Model of Nonthyroidal Disease*

Cited by 7 publications

References 31 publications

Hormonal modulation of a gene injected into rat heart in vivo.

Hormonal modulation of a gene injected into rat heart in vivo.

c-erb-A mRNA Correlates with T3-Receptor Levels in Liver and Pituitary of Tumor Rats

The effects of chronic exposure to supraphysiological concentrations of 3,5,3′ triiodo‐L‐thyronine (T3) on cultured GC cells

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Product

Resources

About

c-erb-A mRNA Correlates with T₃-Receptor Levels in Liver and Pituitary of Tumor Rats

The effects of chronic exposure to supraphysiological concentrations of 3,5,3′ triiodo‐L‐thyronine (T₃) on cultured GC cells