0013.7227/92/1302-1077$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 2
Society
Printed
Expression of Thyroid CURTlSS
B. COOK,
lLDlK0
Hormone
KAKUCSKAt,
Receptor B2 in Rat Hypothalamus RONALD
M. LECHANr,
AND RONALD
J. KOENIG
Division of Endocrinology, University of Michigan Medical Center, Ann Arbor, MI 48109-0678; fDivision of Endocrinology, New England Medical Center Hospitals, Boston, MA 02111
ABSTRACT.
A polymerase chain reaction based assay was used to evaluate expression of thyroid hormone receptor 02 mRNA in rat hypothalamus. Expression was detected in the arcuate, ventromedial and paraventricular nuclei, as well as the median eminence. Trace expression was found in the dorsomedial nucleus, but no expression of thyroid hormone receptor L12 was detected in the lateral hypothalamus or the preoptic region. The results indicate that, contrary to previous belief, expression of thyroid hormone receptor I32 is not confiied to the anterior pituitary. Thyroid hormone receptors (TRs) are encoded by the two closely related prom-oncogenes erbAa and erbAJ3 (1,2). In the rat, alternative splicing of the erbAB primary transcript leads to the production of two distinct TRs, denoted TRDl and TRl32 (3). These proteins share identical DNA and ligand binding domains, but differ in their amino terminal regions (Fig. 1). While TRBl is expressed in essentially all T3 responsive tissues, previous evidence has suggested that expression of TRD2 is confined to the anterior pituitary (3). For example, in a study of erbA mRNA expression after gross dissection of rat brain, no evidence for expression of TR02 was found in brainstem, cerebellum, cerebral cortex, hippocampus, quadrigeminal plate, striatum or thalamus, even though expression was readily detected in anterior pituitary (4). However, using the same polymerase chain reaction (per) based assay as was used in the above study, we now report that TRt32 mRNA is expressed in rat hypothalamus, and that this expression is confined to specific nuclei within this region. 1
107
174
Special care was taken when dissecting the arcuate nucleus to assure that a rim of tissue at the basal margin of the arcuate was left behind to avoid inclusion of the pars tuberalis.
461
I31 FlG. 2. Coronal sections of rat brain showing location of tissue punches from the hypothalamus. AC, anterior commissure; AH, anterior hypothalamus; ARC, arcuate nucleus; DMN, dorsomedial nucleus; F, fomix; LH, lateral hypothalamus; ME, median eminence; MT, mammalothalamic tract; OC, optic chiasm; POA, preoptic nucleus; PVN, paraventricular nucleus; III, third ventricle; VMN, ventromedial nucleus.
FIG. 1. Schematic representation of rat TRDl and TRO2 proteins. TRf31 is 461 amino acids long; the DNA and ligand binding domains are noted. TRt32 is 514 amino acids long. These proteins contain unique amino terminal regions, but are identical in their DNA and hormone biding domains. The solid bar below TRl32 indicates the region amplified by per.
Materials
The anterior pituitary (devoid of neurointermediate lobe) and pooled tissue punches from each region in the brain were homogenized in 200 ~1 of 6 M urea, 3 M LiCl and 10 mM vanadyl ribonucleoside complex. The homogenates were incubated overnight at 4’C and then centrifuged at 14,000 g at 4’C for 30 min. The pellets were then redissolved in 10 mM Tris pH 7.5, 5 mM ethylenedinitrilotetraacetic acid, 0.1% sodium dodecyl sulfate. After extraction with phenol and chloroform, the RNA was precipitated from 0.1 M NaCl with 2.5 volumes of ethanol and redissolved in ribonuclease free water. An aliquot of each RNA sample was subjected to electrophoresis in a 1% agarose gel containing ethidium bromide to estimate total RNA content and quality (using the ratio of 28s to 18s RNA). The ratio of 28s to 18s RNA fluorescence was similar in all samples and consistently >l, indicating that RNA degradation did not significantly compromise the quality of RNA from any region. Reverse transcription of total RNA and subsequent per were performed as previously described (4). In brief, reverse transcription utilized -100 ng of total RNA, 500 ng of primer (corresponding to rat TRD2 noncoding strand nucleotides 673662). and 0.5 p.l of AMV reverse transcriptase in 25 l.tl reactions incubated at 40’C for 1 hour. Ten pl of the above reactions
and Methods
Adult male Sprague-Dawley rats (200-300 g) were anesthetized with pentobarbital(35 mg/kg ip) and decapitated by guillotine. The brains were rapidly removed, snap frozen in hexanes with dry ice, and the forebrains were sectioned coronally at 1.5 mm with stainless steel razor blades on a specially designed template. The brain sections were transferred to a glass slide and kept frozen on a slab of dry ice. Microdissection of discrete regions in the brain was performed by punching out the areas of interest using hollow needles under a Zeiss dissecting microscope. Anatomical landmarks including the third ventricle, median eminence, fornix, anterior commissure and optic chiasm were used to identify regions of interest, which were the arcuate, ventromedial, dorsomedial, medial preoptic and paraventricular nuclei of the hypothalamus, lateral hypothalamic nucleus, and median eminence (Fig. 2). Received
in
Iowa
City,
Iowa,
November
13,
1991.
1077
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 20:09 For personal use only. No other uses without permission. . All rights reserved.
in U.S.A.
RAPID
1078
COMMUNICATIONS
were then used in 50 ti per reactions. PCR was Derformed for 30 cycles; each cycle coisisted of denaturation at $4Y for 1 min 15 set and annealing/extension at 72-C for 3 min. The final extension was continued for 10 min. The per primers (200 ng of each per reaction) corresponded to nucleotides 210-239 and 577-548 (reverse) of TRB2. The former primer anneals to the unique portion of the TRB2 cDNA, while the latter primer anneals to sequences encoded by the first exon that is common to both TRl31 and 02. Ten ~1 of each per reaction were analyzed by electrophoresis through 2% agarose gels stained with ethidium bromide. The exnected reaction nroduct was 368 bu. Negative control reactions &lized water in place of RNA dur&g reverse transcription, and were carried through the subsequent analyses.
Results
Analysis of TRO2 mRNA expression in whole hypothalamus revealed a faint 368 bp per product (data not shown). This led to analvsis of individual hvnothalamic nuclei. to test the hypoth&is that expression mi& be confined to ce&n regions. Results of such a studv are shown in Fig. 3. The highest level of TRl32 mRNA expr&sion was found Tn the arcuate nucleus, followed by the ventromedial and paraventricular nuclei, and the Trace expression was found in the median eminence. dorsomedial nucleus, and no expression of TRB2 was detected in the lateral hypothalamus or the ureo~tic region. ExDression in the anterior pi&tary was conside&bly-greatey than in-any region of the hvpothalamus. To assess the reuroducibilitv of these findings; the arcuate nucleus and late&l hypothalimus were studied from twelve rats individuallv. In all cases the arcuate nucleus was positive and the lateral hypothalamus negative for the 368 bp TRB2 product (Fig. 4). Since per of lateral hypothalamic specimens with TRl31 primers was uniformly positive (data not shown), the negative results for TRB2 cannot be ascribed to technical difficulties.
PVP AD MRMLVMOAB ECNHN'NAPL 1353 872 603 %
FIG. 3. Expression of TR02 mRNA in regions of rat hypothalamus. RNA was prepared from microdissected regions of rat hypothalami, and was then subjected to reverse transcription and per using TR82 specific primers. The expected TR82 per product of 368 bp was detected in the median eminence (ME), as well as the arcuate (ARC). dorsomedial (DMN), p&av&ricular (PVN), and vdntro&dial (VMN) nuclei. TRB2 expression was not detected in the lateral
hypothalamus (LH) or preoptic nucleus (POA). Anterior
pm&ary (AP) was used as a positive control and water as a negative control (BL). @Xl74/HaeIII markers are shown at left.
Endo. Voll30.
Discussion Thyroid hormone alters gene expression by binding to nuclear receptor proteins (TRs), which in turn bind specific DNA sequences within target genes. The genes erbAa and erbAB encode closelv related functional TRs (1,2). Alternative splicing of the erbAd primary transcript results in production of two TRs. denoted Bl and 02. which differ only in their amino terminal’domains (Fig. 1). Although the purpose of multiple TRs is unknown, these proteins have been maintained over relatively large evolutionary distances. Thus, TRa and TRB are found in species as diverse as Xenopus (5) and humans (2,6). One simple hypothesis is that these TR isoforms are expressed in different tissues and that they therefore. may activate different target genes. Studies of TRal and TRBl mRNA expression in the rat have not yielded clearcut support for this hypothesis, in that both of these RNAs are expressed in all T3 responsive rat organs, although their ratio varies considerably (7). Thus, organ specific differences in expression of TRal and TRBl appear to be quantitative rather than qualitative.
FIG 4. Expression of TRB2 mRNA in arcuate nuclei (ARC) and lateral hypothalami (LH) from individual rats. Samples from individual rats were processed separately and analyzed as described in figure 3. The TR82 per product is consistently present in arcuate nucleus and absent from lateral hypothalamus. Quite different from this is the prevailing opinion regarding expression of TRl32. This cDNA was isolated from a GH3 (rat pituitary tumor) cell library, and Northern analysis showed expression in rat anterior pituitary but not in liver, heart, brown fat, kidney, adrenal, or skeletal muscle (3). Furthermore, a subsequent study using a reverse transcription/per based assay failed to demonstrate TRR2 mRNA expression in multiple regions of rat brain, even though TRBl is expressed at high levels in this orean (4). Thus. organ specific expression of TRB2 appeared To & regulated in a qualitative, rather than auantitative. manner. This suecested that TRB2 may play a inique role h T3 dependent pin&-y specific processes, such as down regulation of TSH expression. While this may indeed be the case, the current study clearly demonstrates TR132 mRNA expression is not confined to the anterior pituitary. This TR isoform also is expressed in multiple regions of the rat hypothalamus, albeit at levels below that in the anterior pituitary. It is of interest that one of the hypothalamic regions expressing TR112 mRNA is the paraventricular nucleus. This nucleus is the source of TRH that stimulates pituitary TSH production and release. In addition, although TRH is expressed in other regions of the central nervous system, including the lateral hypothalamus, it is only in the paraventricular nucleus that such expression is down regulated by T3 (8,9). One therefore may speculate that TRl32 may play a role in feedback regulation of thyroid hormone homeostasis at both the hypothalamic and pituitary levels. The presence of TRl32 in the arcuate is also of considerable interest in view of data suggesting that neurons herein may also be thyroid hormone responsive. Studies by F&kind, et al. have shown that the arcuate nucleus contains high concentrations of type II 5’-deiodinase, the enzyme which converts T4 to biologically active T3 (10).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 20:09 For personal use only. No other uses without permission. . All rights reserved.
1992 No 2
RAPID
COMMUNICATIONS
It should be noted that our studies do not formally prove that hypothalamic “TRl32” mRNA is absolutely identical to pituitary TRt32. Although these two per products are the same size and form with the same primers under high stringency conditions, the possibility remains that differences in alternative splicing result in subtle, undetected differences between pituitary and hypothalamic TRl32 mRNAs. In addition, our studies have been performed at the RNA level only; reagents currently do not exist to assess TRB2 expression at the protein level. Although it is reasonable to presume expression of TR82 mRNA correlates with that of the protein, quantitative differences may exist between these levels of gene expression.
This work DK37021.
Acknowledgment was supported by NIH grants DK44155
and
References Sap J, Munoz A, Darnm K, Goldberg Y, Ghysdael J, Leutz A, Beug H, Vennstrom B 1986 The c-erb-A protein is a high-affinity receptor for thyroid hormone. Nature 324:635640 Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A eene encodes a thvroid r hormone receptor. Nature 324:64l%46 Hodin RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen PR, Moore DD, Chin WW 1989 Identification
of a thyroid hormone receptor that is pituitary specific. Science 244:76-79 4. Cook CB, Koenig RJ 1990 Expression of erbAa and 8 mRNAs in regions of adult rat brain. Mol Cell Endocrinol 70: 13-20 5. Yaoita Y, Shi Y-B, Brown DD 1990 Xenopus laevis a and 8 thyroid hormone receptors. Proc Nat1 Acad Sci USA 87:7090-7094 6. Nakai A, Sakurai A, Bell GI, DeGroot LJ 1988 Characterization of a third human thvroid hormone receutor coexpressed with other thyroid hormone receptors in several tissues. Mol Endocrinol2: 1087- 1092 7. Hodin RA, Lazar MA, Chin WW 1990 Differential and tissue-specific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone. J Clin Invest 85:101-105 8. Dyess EM, Segerson TP, Liposits Z, Paul1 WK. Kaplan MM, Wu P, Jackson IMD, Lechan RM 1988 Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing hormone gene expression in the hvoothalamic uaraventricuar nucleus. Endocrinology -_ 12332291-2297 9. Sezerson TP. Kauer J. Wolfe HC. Mobtaker H. Wu P. Jar&on IMD; Lechan RM 1987 Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleur of the rat hypothalamus. Science 238:78-80 10. Riskind PN, Kolodny JM, Larsen PR 1987 The regional hypothalamic distribution of type II 5’-monodeiodinase in euthyroid and hypothyroid rats. Brain Res 420:194-198
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 20:09 For personal use only. No other uses without permission. . All rights reserved.
Vol. 130, No. 2
Society
Printed
Expression of Thyroid CURTlSS
B. COOK,
lLDlK0
Hormone
KAKUCSKAt,
Receptor B2 in Rat Hypothalamus RONALD
M. LECHANr,
AND RONALD
J. KOENIG
Division of Endocrinology, University of Michigan Medical Center, Ann Arbor, MI 48109-0678; fDivision of Endocrinology, New England Medical Center Hospitals, Boston, MA 02111
ABSTRACT.
A polymerase chain reaction based assay was used to evaluate expression of thyroid hormone receptor 02 mRNA in rat hypothalamus. Expression was detected in the arcuate, ventromedial and paraventricular nuclei, as well as the median eminence. Trace expression was found in the dorsomedial nucleus, but no expression of thyroid hormone receptor L12 was detected in the lateral hypothalamus or the preoptic region. The results indicate that, contrary to previous belief, expression of thyroid hormone receptor I32 is not confiied to the anterior pituitary. Thyroid hormone receptors (TRs) are encoded by the two closely related prom-oncogenes erbAa and erbAJ3 (1,2). In the rat, alternative splicing of the erbAB primary transcript leads to the production of two distinct TRs, denoted TRDl and TRl32 (3). These proteins share identical DNA and ligand binding domains, but differ in their amino terminal regions (Fig. 1). While TRBl is expressed in essentially all T3 responsive tissues, previous evidence has suggested that expression of TRD2 is confined to the anterior pituitary (3). For example, in a study of erbA mRNA expression after gross dissection of rat brain, no evidence for expression of TR02 was found in brainstem, cerebellum, cerebral cortex, hippocampus, quadrigeminal plate, striatum or thalamus, even though expression was readily detected in anterior pituitary (4). However, using the same polymerase chain reaction (per) based assay as was used in the above study, we now report that TRt32 mRNA is expressed in rat hypothalamus, and that this expression is confined to specific nuclei within this region. 1
107
174
Special care was taken when dissecting the arcuate nucleus to assure that a rim of tissue at the basal margin of the arcuate was left behind to avoid inclusion of the pars tuberalis.
461
I31 FlG. 2. Coronal sections of rat brain showing location of tissue punches from the hypothalamus. AC, anterior commissure; AH, anterior hypothalamus; ARC, arcuate nucleus; DMN, dorsomedial nucleus; F, fomix; LH, lateral hypothalamus; ME, median eminence; MT, mammalothalamic tract; OC, optic chiasm; POA, preoptic nucleus; PVN, paraventricular nucleus; III, third ventricle; VMN, ventromedial nucleus.
FIG. 1. Schematic representation of rat TRDl and TRO2 proteins. TRf31 is 461 amino acids long; the DNA and ligand binding domains are noted. TRt32 is 514 amino acids long. These proteins contain unique amino terminal regions, but are identical in their DNA and hormone biding domains. The solid bar below TRl32 indicates the region amplified by per.
Materials
The anterior pituitary (devoid of neurointermediate lobe) and pooled tissue punches from each region in the brain were homogenized in 200 ~1 of 6 M urea, 3 M LiCl and 10 mM vanadyl ribonucleoside complex. The homogenates were incubated overnight at 4’C and then centrifuged at 14,000 g at 4’C for 30 min. The pellets were then redissolved in 10 mM Tris pH 7.5, 5 mM ethylenedinitrilotetraacetic acid, 0.1% sodium dodecyl sulfate. After extraction with phenol and chloroform, the RNA was precipitated from 0.1 M NaCl with 2.5 volumes of ethanol and redissolved in ribonuclease free water. An aliquot of each RNA sample was subjected to electrophoresis in a 1% agarose gel containing ethidium bromide to estimate total RNA content and quality (using the ratio of 28s to 18s RNA). The ratio of 28s to 18s RNA fluorescence was similar in all samples and consistently >l, indicating that RNA degradation did not significantly compromise the quality of RNA from any region. Reverse transcription of total RNA and subsequent per were performed as previously described (4). In brief, reverse transcription utilized -100 ng of total RNA, 500 ng of primer (corresponding to rat TRD2 noncoding strand nucleotides 673662). and 0.5 p.l of AMV reverse transcriptase in 25 l.tl reactions incubated at 40’C for 1 hour. Ten pl of the above reactions
and Methods
Adult male Sprague-Dawley rats (200-300 g) were anesthetized with pentobarbital(35 mg/kg ip) and decapitated by guillotine. The brains were rapidly removed, snap frozen in hexanes with dry ice, and the forebrains were sectioned coronally at 1.5 mm with stainless steel razor blades on a specially designed template. The brain sections were transferred to a glass slide and kept frozen on a slab of dry ice. Microdissection of discrete regions in the brain was performed by punching out the areas of interest using hollow needles under a Zeiss dissecting microscope. Anatomical landmarks including the third ventricle, median eminence, fornix, anterior commissure and optic chiasm were used to identify regions of interest, which were the arcuate, ventromedial, dorsomedial, medial preoptic and paraventricular nuclei of the hypothalamus, lateral hypothalamic nucleus, and median eminence (Fig. 2). Received
in
Iowa
City,
Iowa,
November
13,
1991.
1077
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 20:09 For personal use only. No other uses without permission. . All rights reserved.
in U.S.A.
RAPID
1078
COMMUNICATIONS
were then used in 50 ti per reactions. PCR was Derformed for 30 cycles; each cycle coisisted of denaturation at $4Y for 1 min 15 set and annealing/extension at 72-C for 3 min. The final extension was continued for 10 min. The per primers (200 ng of each per reaction) corresponded to nucleotides 210-239 and 577-548 (reverse) of TRB2. The former primer anneals to the unique portion of the TRB2 cDNA, while the latter primer anneals to sequences encoded by the first exon that is common to both TRl31 and 02. Ten ~1 of each per reaction were analyzed by electrophoresis through 2% agarose gels stained with ethidium bromide. The exnected reaction nroduct was 368 bu. Negative control reactions &lized water in place of RNA dur&g reverse transcription, and were carried through the subsequent analyses.
Results
Analysis of TRO2 mRNA expression in whole hypothalamus revealed a faint 368 bp per product (data not shown). This led to analvsis of individual hvnothalamic nuclei. to test the hypoth&is that expression mi& be confined to ce&n regions. Results of such a studv are shown in Fig. 3. The highest level of TRl32 mRNA expr&sion was found Tn the arcuate nucleus, followed by the ventromedial and paraventricular nuclei, and the Trace expression was found in the median eminence. dorsomedial nucleus, and no expression of TRB2 was detected in the lateral hypothalamus or the ureo~tic region. ExDression in the anterior pi&tary was conside&bly-greatey than in-any region of the hvpothalamus. To assess the reuroducibilitv of these findings; the arcuate nucleus and late&l hypothalimus were studied from twelve rats individuallv. In all cases the arcuate nucleus was positive and the lateral hypothalamus negative for the 368 bp TRB2 product (Fig. 4). Since per of lateral hypothalamic specimens with TRl31 primers was uniformly positive (data not shown), the negative results for TRB2 cannot be ascribed to technical difficulties.
PVP AD MRMLVMOAB ECNHN'NAPL 1353 872 603 %
FIG. 3. Expression of TR02 mRNA in regions of rat hypothalamus. RNA was prepared from microdissected regions of rat hypothalami, and was then subjected to reverse transcription and per using TR82 specific primers. The expected TR82 per product of 368 bp was detected in the median eminence (ME), as well as the arcuate (ARC). dorsomedial (DMN), p&av&ricular (PVN), and vdntro&dial (VMN) nuclei. TRB2 expression was not detected in the lateral
hypothalamus (LH) or preoptic nucleus (POA). Anterior
pm&ary (AP) was used as a positive control and water as a negative control (BL). @Xl74/HaeIII markers are shown at left.
Endo. Voll30.
Discussion Thyroid hormone alters gene expression by binding to nuclear receptor proteins (TRs), which in turn bind specific DNA sequences within target genes. The genes erbAa and erbAB encode closelv related functional TRs (1,2). Alternative splicing of the erbAd primary transcript results in production of two TRs. denoted Bl and 02. which differ only in their amino terminal’domains (Fig. 1). Although the purpose of multiple TRs is unknown, these proteins have been maintained over relatively large evolutionary distances. Thus, TRa and TRB are found in species as diverse as Xenopus (5) and humans (2,6). One simple hypothesis is that these TR isoforms are expressed in different tissues and that they therefore. may activate different target genes. Studies of TRal and TRBl mRNA expression in the rat have not yielded clearcut support for this hypothesis, in that both of these RNAs are expressed in all T3 responsive rat organs, although their ratio varies considerably (7). Thus, organ specific differences in expression of TRal and TRBl appear to be quantitative rather than qualitative.
FIG 4. Expression of TRB2 mRNA in arcuate nuclei (ARC) and lateral hypothalami (LH) from individual rats. Samples from individual rats were processed separately and analyzed as described in figure 3. The TR82 per product is consistently present in arcuate nucleus and absent from lateral hypothalamus. Quite different from this is the prevailing opinion regarding expression of TRl32. This cDNA was isolated from a GH3 (rat pituitary tumor) cell library, and Northern analysis showed expression in rat anterior pituitary but not in liver, heart, brown fat, kidney, adrenal, or skeletal muscle (3). Furthermore, a subsequent study using a reverse transcription/per based assay failed to demonstrate TRR2 mRNA expression in multiple regions of rat brain, even though TRBl is expressed at high levels in this orean (4). Thus. organ specific expression of TRB2 appeared To & regulated in a qualitative, rather than auantitative. manner. This suecested that TRB2 may play a inique role h T3 dependent pin&-y specific processes, such as down regulation of TSH expression. While this may indeed be the case, the current study clearly demonstrates TR132 mRNA expression is not confined to the anterior pituitary. This TR isoform also is expressed in multiple regions of the rat hypothalamus, albeit at levels below that in the anterior pituitary. It is of interest that one of the hypothalamic regions expressing TR112 mRNA is the paraventricular nucleus. This nucleus is the source of TRH that stimulates pituitary TSH production and release. In addition, although TRH is expressed in other regions of the central nervous system, including the lateral hypothalamus, it is only in the paraventricular nucleus that such expression is down regulated by T3 (8,9). One therefore may speculate that TRl32 may play a role in feedback regulation of thyroid hormone homeostasis at both the hypothalamic and pituitary levels. The presence of TRl32 in the arcuate is also of considerable interest in view of data suggesting that neurons herein may also be thyroid hormone responsive. Studies by F&kind, et al. have shown that the arcuate nucleus contains high concentrations of type II 5’-deiodinase, the enzyme which converts T4 to biologically active T3 (10).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 20:09 For personal use only. No other uses without permission. . All rights reserved.
1992 No 2
RAPID
COMMUNICATIONS
It should be noted that our studies do not formally prove that hypothalamic “TRl32” mRNA is absolutely identical to pituitary TRt32. Although these two per products are the same size and form with the same primers under high stringency conditions, the possibility remains that differences in alternative splicing result in subtle, undetected differences between pituitary and hypothalamic TRl32 mRNAs. In addition, our studies have been performed at the RNA level only; reagents currently do not exist to assess TRB2 expression at the protein level. Although it is reasonable to presume expression of TR82 mRNA correlates with that of the protein, quantitative differences may exist between these levels of gene expression.
This work DK37021.
Acknowledgment was supported by NIH grants DK44155
and
References Sap J, Munoz A, Darnm K, Goldberg Y, Ghysdael J, Leutz A, Beug H, Vennstrom B 1986 The c-erb-A protein is a high-affinity receptor for thyroid hormone. Nature 324:635640 Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A eene encodes a thvroid r hormone receptor. Nature 324:64l%46 Hodin RA, Lazar MA, Wintman BI, Darling DS, Koenig RJ, Larsen PR, Moore DD, Chin WW 1989 Identification
of a thyroid hormone receptor that is pituitary specific. Science 244:76-79 4. Cook CB, Koenig RJ 1990 Expression of erbAa and 8 mRNAs in regions of adult rat brain. Mol Cell Endocrinol 70: 13-20 5. Yaoita Y, Shi Y-B, Brown DD 1990 Xenopus laevis a and 8 thyroid hormone receptors. Proc Nat1 Acad Sci USA 87:7090-7094 6. Nakai A, Sakurai A, Bell GI, DeGroot LJ 1988 Characterization of a third human thvroid hormone receutor coexpressed with other thyroid hormone receptors in several tissues. Mol Endocrinol2: 1087- 1092 7. Hodin RA, Lazar MA, Chin WW 1990 Differential and tissue-specific regulation of the multiple rat c-erbA messenger RNA species by thyroid hormone. J Clin Invest 85:101-105 8. Dyess EM, Segerson TP, Liposits Z, Paul1 WK. Kaplan MM, Wu P, Jackson IMD, Lechan RM 1988 Triiodothyronine exerts direct cell-specific regulation of thyrotropin-releasing hormone gene expression in the hvoothalamic uaraventricuar nucleus. Endocrinology -_ 12332291-2297 9. Sezerson TP. Kauer J. Wolfe HC. Mobtaker H. Wu P. Jar&on IMD; Lechan RM 1987 Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleur of the rat hypothalamus. Science 238:78-80 10. Riskind PN, Kolodny JM, Larsen PR 1987 The regional hypothalamic distribution of type II 5’-monodeiodinase in euthyroid and hypothyroid rats. Brain Res 420:194-198
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