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Endocrinology Vol. 139, No. 3 1288-1299
Copyright © 1998 by The Endocrine Society

ARTICLES

Detection and Identification of Proteins Related to the Hereditary Dwarfism of the rdw Rat1

Masamichi Oh-Ishi, Akira Omori, Ji-Young Kwon, Takashi Agui, Tadakazu Maeda and Sen-Ichi Furudate

Department of Physics, School of Science (M.O., T.M.), and Department of Laboratory Animal Science, School of Medicine (S.F.), Kitasato University, 1–15-1 Kitasato, Sagamihara-shi, Kanagawa 228, Japan; Laboratory of Biopolymer Conformation Analysis, Mitsubishi Kasei Institute of Life Sciences (A.O.), 11 Minamiooya, Machida-shi, Tokyo 194, Japan; and Institute for Experimental Animal Science, Nagoya City University Medical School (J.-Y.K., T.A.), Mizuho-ku, Nagoya-shi, Aichi 467, Japan

Address all correspondence and requests for reprints to: Masamichi Oh-Ishi, Ph.D., Kitasato University School of Science, 1–15-1 Kitasato, Sagamihara-shi, 228 Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proteins having relations to hereditary dwarfism of the rdw rat (gene symbol: rdw) were searched for in various tissues of the rat with an improved two-dimensional gel electrophoresis technique followed by immunoblotting and microsequencing. Tissues inspected were cerebral cortex, cerebellum, brain trunk, hypothalamus, pituitary, thyroid gland, liver, testis, spleen, and thymus. Only pituitary and thyroid glands among those tissues showed abnormalities in protein contents. GH and PRL contents in the rdw pituitary were much less than in the normal one, which in the former were 1/15 and less than 1/30 times as much as in the latter, respectively, but the abnormalities in the rdw thyroid were far more serious than in the pituitary. At least 18 protein levels in the rdw thyroid were above, and 17 were below the normal. Those identified among the increased proteins were endoplasmin (GRP94), immunoglobulin heavy chain binding protein (BiP/GRP78), and heat shock protein 70 (hsp70), the contents of which respectively were 40 times, 10 times and more than 50 times as much in the rdw thyroid as in the normal tissue. Because BiP and endoplasmin are known to be ER resident proteins, and because all three belong to a chaperone protein family, accumulation of these proteins in the rdw thyroid suggests that protein folding and secreting disorders underlie the hypothyroidism of the rdw rat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE rdw rat has hereditary dwarfism and shows hypoplasia of a pituitary gland. The rat was isolated from a closed colony of Csk:Wistar-Imamichi rats by Koto et al. (1); the mode of inheritance of the rat is through an autosomal recessive trait. The rdw pituitary gland expresses 1/30 to 1/100 times as low messenger RNA (mRNA) levels for GH and PRL as a normal gland does but significantly higher mRNA levels for POMC and the ß subunit of TSH than those expressed in a normal tissue (2, 3).

Hereditary abnormalities similar to those of the rdw rat are found in the Snell mouse (dw) (4), Ames mouse (df) (5), little mouse (lit) (6), pygmy mouse (pg) (7), spontaneous dwarf rat (dr) (8), and dwarf rat (dw) (9). The lit mouse, dr and dw rats show isolated GH deficiency (6, 9, 10); the dw and df mice are deficient of PRL, TSH, and GH (11, 12). Causes of the disorder in the dw and df mice are respectively known to be malfunction and lack of activation of the Pit-1 gene encoding the POU-domain transcriptional factor, which controls the pituitary-specific expression of GH and PRL genes (13, 14, 15). The Pit-1 gene in the rdw rat, however, is not responsible for the hereditary dwarfism. The rdw Pit-1 mRNA level and Pit-1 nucleotide sequence are not different from those of normal Pit-1, and the Pit-1 protein content in the rdw pituitary is the same as in the normal tissue (2, 3).

The aim of this paper is to search for proteins that are related to the hereditary dwarfism of the rdw rat. The methods we adopted in this study are the following: 1) we used an improved method of two-dimensional gel electrophoresis (2-DE) with agarose gels in the first dimension to compare protein constituents of various tissues in the rdw rat, with those of the corresponding tissues in a normal rat; and 2) used immunoblotting and microsequencing to identify those protein spots that did not coincide with each other in the 2-DE patterns of the rdw and normal tissue extracts. Only thyroid and pituitary glands among the tissues inspected showed abnormalities in protein contents. The rdw pituitary gland contained GH 1/15 times and PRL less than 1/30 times as much as the normal pituitary did, but the abnormalities in the rdw thyroid were far more serious than in the pituitary. At least 18 protein levels in the rdw thyroid were above the normal, and 17 were below the normal. Those identified among the increased proteins were endoplasmin (GRP94), Ig heavy chain binding protein (BiP/GRP78), and heat shock protein 70 (hsp70). Contents of endoplasmin, BiP and hsp70, respectively, were 40 times, 10 times, and more than 50 times as much in the rdw thyroid as in the normal tissue. Because BiP and endoplasmin are known to be ER resident proteins (16), and because all three belong to a chaperone protein family, accumulation of these proteins in the rdw thyroid suggests that protein folding and secreting disorders underlie the hypothyroidism of the rdw rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
A colony of the rdw rat has been maintained at the laboratory animal facility of Kitasato University School of Medicine. The room where the colony is maintained is air conditioned at 24 ± 1 C maintained at 50–60% of relative humidity and artificially illuminated daily from 0600 h to 2000 h. The rdw rats are fed on CMF pellets (Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum.

We at first obtained the homozygous rdw rats (rdw/rdw) by mating male and female heterozygous rdw animals (+/rdw), which had previously been confirmed by a progeny test as bearing offspring with the rdw features (2). The genotypes of the offspring were a 1:2:1 mixture of +/+, +/rdw and rdw/rdw, and it was an easy task for us to phenotypically select the rdw/rdw rat from the rest. It being, however, quite difficult to distinguish by eye the +/rdw rat from the normal control, we routinely obtained the rdw/rdw and +/rdw rats in the following way. Because the rdw features can be healed over by transplantation of normal thyroid glands to the rdw/rdw infant rats, we raised the rdw/rdw infants to matured stage by transplanting thyroid glands from phenotypically normal animals, and mated the matured male rdw/rdw rats with female +/rdw rats, the offspring of which were a 1:1 mixture of genotype +/rdw and rdw/rdw. We phenotypically chose infants that have the rdw features as the rdw/rdw rats and the rest as the +/rdw ones. The normal control +/+ rats we used were offspring from a mating system of both normal +/+ female and male. The mating system have been confirmed by levels of T4 and TSH hormones (Furudate, S., unpublished data). Normal littermates were weaned at 3 weeks of age, but the rdw rats were weaned at 5 weeks of age according to their growth.

Sample preparation
Immediately following decapitation of the 13- to 25-week-old rdw rats and the 16- to 28-week-old normal control rats under light ether anesthesia, we excised various tissues from the killed animals from 0900 h to 1200 h in a day. Those tissues excised were cerebral cortex, cerebellum, brain trunk, hypothalamus, pituitary, thyroid gland, liver, testis, spleen, and thymus, which were cut into small pieces and kept frozen at -80 C until use. A frozen tissue piece about 10 mg in weight was homogenized with a Teflon glass homogenizer in an extraction medium of a 20-fold volume of the tissue pieces other than pituitary and of a 40-fold volume of a pituitary tissue piece. The extraction medium we used was a 5 M urea, 1 M thiourea solution containing 0.5% 2-mercaptoethanol and 0.1 mM N{alpha}-tosyl-L-lysylchloromethane hydrochloride (TLCK). The homogenate was centrifuged with a TOMY TMA-6 rotor at 15,000 rpm for 20 min, and the clear supernatant was subjected to the first-dimension isoelectric focusing of 2-DE.

Two-dimensional gel electrophoresis
We performed the 2-DE according to the procedure given by Oh-Ishi & Hirabayashi (17). The isoelectric focusing agarose gel for the first dimension electrophoresis was 260 mm in length and 3 mm in diameter in a glass tube. The slab gel for the second dimension electrophoresis was 12% polyacrylamide gel and was 195 mm in width, 120 mm in height, and 1.5 mm in thickness. The isoelectric focusing was conducted at 600 V for 18 h at 4 C, and the second dimension SDS electrophoresis was carried out according to the stacking system of Laemmli (18).

The sample volume for the first dimension isoelectric focusing was 100 µl for samples other than pituitary and 70 µl for pituitary. The slab gels after the second dimension electrophoresis were stained with PhastGel Blue R (Coomassie brilliant blue R 350: Pharmacia Biotech AB, Uppsala, Sweden).

To precisely compare protein constituents of the rdw and normal tissues, we repeated 2-DE three times per one tissue; the first is the normal tissue extract, the second the rdw one, and the third a 1: 1 mixture of the two.

The third 2-DE pattern is quite important, without which we are unable to discriminate as identical or dissimilar a pair of normal and rdw spots locating very close with each other in the first two 2-DE patterns. It also works for our identifying proteins in the rdw tissue, which are normal in 2-DE position but abnormal in protein content. When we look at either of the first two 2-DE patterns, we carefully see which protein is more and which is less in the pattern. When we find a protein spot in the normal 2-DE pattern that is denser, for example, than nearby protein spots but less dense in the rdw pattern, the spot would be a candidate of an abnormal protein, and we look for the corresponding spot in the third 2-DE pattern and compare the spot density with nearby spot densities. If the comparison comes up to our expectation that the 1:1 mixture 2-DE pattern is in between the first two, the candidate protein in the rdw tissue would likely be abnormal in protein content. The method of detecting protein content abnormalities, however, would fail if many spots in the rdw 2-DE pattern are abnormal in protein content in the same way at the same time, but we consider this highly unlikely.

Western blotting analyses
Anti-hsp70 (K-20) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-hsp70 is an affinity-purified goat polyclonal antibody raised against a synthetic peptide having a sequence from the 572nd to 591st amino acid residues of human hsp70. The antibody reacts with rat hsp70, but not with 70-kDa heat shock protein cognate type (hsc70). Antirat PRL rabbit antiserum (NIDDK-anti-rPRL-S-9) and antirat GH monkey antiserum (NIDDK-anti-rGH-S-5) were obtained from National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK). Antihuman thyroglobulin sheep antiserum was purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). The antithyroglobulin cross-reacts with rat thyroglobulin. For Western blotting analyses that followed 2-DE, proteins were transferred from a 2-DE gel onto a polyvinylidene difluoride (PVDF) membrane (Clear Blot Membrane-P: Atto Co., Ltd., Tokyo, Japan).

Antigen detection was carried out by alkaline phosphatase catalyzed color development with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer Mannheim GmbH, Mannheim, Germany).

In-gel digestion of proteins for internal amino acid sequencing
Our method of in-gel digestion of proteins was a variant of the Cleveland method (19) specially adapted for preparing peptide fragments of a target protein for internal amino-acid sequencing. The following is a brief description of the method we used in this study.

A protease pre-electrophoresed 1D slab gel was prepared beforehand: Staphylococcus aureus V8 protease of 1 µg in weight was dissolved in a sample buffer (125 mM Tris-HCl, pH 6.8, 1 mM EDTA, 1.5 mM DTT, 0.1% SDS) containing 10% glycerol, was put in a well of Laemmli’s 1D slab gel (9 cm x 9 cm x 0.2 cm) of SDS-15% polyacrylamide and was electrophoresed at 40 mA for 10 min into the stacking gel layer ahead of a target protein. A protein spot that contained a target protein was cut out from a 2-DE gel and was equilibrated with the sample buffer containing 10% sucrose for 30 min at room temperature before the gel piece was put in the well. The target protein in the gel piece was then electroeluted at 20 mA into the stacking gel, where the V8 protease had been preelectrophoresed. When the protease/protein mixture migrated to the boundary between the stacking and separation gels, electrophoresis was temporarily halted for 30 min at room temperature for the V8 protease to partially digest the target protein into peptide fragments. The digested fragments and the protease were then electrophoresed at 40 mA for 1 h to form a peptide map and the protease band in the separation gel. The peptide bands were blotted onto a siliconized glass-fiber membrane (Glassy-Bond: Biometra biomedizinische Analytik GmbH, Göttingen, Germany), and stained with PhastGel Blue R. Each peptide band on the membrane was then subjected to amino acid sequencing with Applied Biosystems 477A pulse-liquid phase sequencer as described by Omori et al. (20), and the amino acid sequences derived from the peptide bands let us identify the target protein in the protein identification resources (PIR) database.

Protein content determination from electrophoretic patterns
A Coomassie-stained 2-DE gel was interposed between two wet cellophane sheets and stretched with two styrene plastic frames (21) to be dried in a draft chamber at 60 C for 2 h. A 150 dpi gray-scale image of the dried gel, digitized with a Hewlett Packard ScanJet 3C scanner equipped with a transparency adapter, was processed by an image analysis software, NIH Image Version 1.57 (22), to give integrated densities of each protein spot and of molecular weight marker proteins of 1 µg each in weight in a same 2-DE gel. The ratio of the former density to the average of the latter ones was then compared with a calibration curve of integrated density ratios of actin vs. marker protein spots with known amount of actin from 1 µg to 50 µg. The comparison let us convert the integrated density ratio of a protein spot to the weight of the protein in the spot on the assumption that the protein and actin were similarly stained with PhastGel Blue R. The marker proteins we used were SDS-PAGE molecular standards, broad range, purchased from Bio-Rad Laboratories (Hercules, CA).

Northern blot analysis and sequencing of the TSH receptor (TSHr) mRNA
Size, expression level and sequence of a rdw TSHr gene were analyzed by Northern hybridization and complementary DNA (cDNA) sequencing. Tissues used were the +/? and rdw/rdw thyroid glands.

[{alpha}-32P]deoxycytidine triphosphate (dCTP), a pMosBlue vector, and a Hybond-N nylon hybridization membrane were purchased from Amersham International plc, Buckinghamshire, United Kingdom. A GeneClean II kit purchased from Bio 101, Inc. (Vista, CA) was used for isolating cDNA fragments from a 1% agarose gel. A DNA sequencing kit (Takara Shuzo Co., Tokyo, Japan) based on dideoxy chain-termination method (23) was used for cDNA sequencing.

Total RNA was prepared from thyroid tissues homogenized in a solution containing 4.4 M guanidine thiocyanate, 0.1 M ß-mercaptoethanol and 25 mM sodium citrate (pH 7.0). Two cDNA fragments, fragment 1 and 2, were synthesized by the RT-PCR method. The sense and antisense primers for cDNA fragment 1 were 5'-CTTCAATCCAAGGACATGCT-3' (569–588) and 5'-CTCTTGGCCGAAACCGATGA-3' (1088–1107), respectively, and those for cDNA fragment 2 were 5'-GGTTTCGGCCAAGAGCTCAA-3' (1093–1112) and 5'-TGAGCAGGAGAACAAGGGCG-3' (NOREF>1749–1768), respectively (24). A part of fragment 1 was labeled with [{alpha}-32 P]dCTP and was later used as a probe for Northern blot analysis. The two cDNA fragments were respectively electrophoresed in a 1% agarose gel, were isolated from the gel with a GeneClean II kit, and were cloned into pMosBlue vectors. Oligonucleotide primers that were complementary to the vector and labeled with fluorescein isothiocyanate were used in the cDNA sequencing of fragment 1 and 2.

For Northern blot analyses, 20 µg of the total RNA extracted from thyroid tissues was denatured in 50% formamide, 2.2 M formaldehyde at 65 C for 10 min, and electrophoresed in a 1% agarose gel containing 2.2 M formaldehyde. The RNA in the agarose gel was blotted onto a Hybond-N nylon hybridization membrane by capillary action in a 20x standard saline citrate (SSC) solution (1x SSC: 150 mM NaCl and 15 mM sodium citrate). The membrane was heated for 3 h at 80 C and prehybridized for 3 h in 50 mM sodium phosphate buffer (pH 6.5) containing 50% formamide, 5x SSC, 5x Denhardt’s solution (1x Denhardt’s solution: 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 1% SDS, and 100 µg/ml sonicated salmon sperm DNA. The prehybridization was followed by overnight incubation at 42 C with the [{alpha}-32 P]dCTP-labeled cDNA fragment 1. The membrane was washed two times at 37 C with 2x SSC containing 0.1% SDS, and was further washed two times at 65 C with 0.2x SSC containing 0.1% SDS. The membrane was then exposed to Kodak X-AR film for 36–48 h at -70 C. After the exposure was finished, the cDNA probe was washed off the membrane in boiling water for 5 min, and the amount of ß-actin mRNA on the membrane was assessed by the second Northern hybridization with a ß-actin DNA as a probe, the results of which were used for calibration of total mRNA amount on the membrane.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Protein content disparity in the rdw and normal pituitary glands
Figure 1Go shows 2-DE patterns of pituitary extracts of the +/+, rdw/rdw and +/rdw rats, and the upper right panel in Fig. 1Go is the 2-DE pattern of a 1:1 mixture of the +/+ and rdw/rdw pituitary extracts. Each protein spot in Fig. 1Go is named by the apparent molecular weight in the 2-DE pattern, i.e., the protein spots in Fig. 1Go denoted as p28, p25, and p23 respectively are 28K, 25K, and 23K in the apparent molecular weight. The +/+ and rdw/rdw patterns reveal general similarities between the +/+ and rdw/rdw pituitaries, but we still notice differences in several spots. Two dense spots named as p23 and p25 in the +/+ pituitary are faint (p23) or completely absent (p25) in the rdw/rdw tissue. The +/rdw panel in Fig. 1Go shows that spot p23 in the +/rdw pituitary looks as dense as spot p23 in the +/+ tissue, but spot p25 looks much thinner than p25 in the normal control. A spot named p28 in the +/+ pituitary is denser in the rdw/rdw one, which was observed in four cases out of five comparisons between the +/+ and rdw/rdw pituitaries; one exceptional case was that the p28 protein content in the +/+ tissue was about the same as in the rdw/rdw pituitary.



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  		Figure 1. Comparison of 2-DE patterns of the +/+, rdw/rdw and +/rdw pituitary extracts. The gels were stained with PhastGel Blue R. The upper left panel is the 2-DE pattern of a +/+ pituitary extract, the lower left that of a rdw/rdw tissue extract, the upper right that of a 1:1 mixture of the two and the lower right that of a +/rdw pituitary extract. Spots are labeled by the apparent molecular weights, i.e. spot p23, p25 and p28 are proteins of 23K, 25K, and 28K, respectively. The number and age of rats used are one 27-week-old +/+ rat, two 25-week-old rdw/rdw rats and one 25-week-old +/rdw rat, and the rdw/rdw and +/rdw rats are all littermates.

 
Spot p23 and p25 were respectively identified as GH and PRL by immunoblotting analyses with antirat PRL and antirat GH antibodies, the results of which are shown in Fig. 2Go. Table 1Go quantitatively shows dependence of GH, PRL, and p28 contents in the pituitary on the genotype +/+, +/rdw and rdw/rdw. It is difficult to make statistically meaningful remarks on the GH and PRL contents in the +/rdw pituitary using only two heterozygous rats having such large SDs of PRL data, but it appears that PRL production in the +/rdw pituitary decreases but GH production stays normal. Spot p28 has not yet been identified so far, but it is likely that the p28 production in the rdw pituitary increases above the normal level. Our conclusion drawn for pituitary is that PRL and GH contents in the rdw/rdw tissue are much less than those in the normal one.



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  		Figure 2. Identification of spot p25 and p23 in pituitary by Western blotting analyses. We repeated 2-DE three times using a pituitary of a same 27-week-old +/+ rat. A strip was cut out from each 2-DE gel around spot p25 and p23. One of the three gel strips was stained with PhastGel Blue R, and proteins in the other two gel strips were blotted to two pieces of PVDF membrane. Each of the membrane pieces was incubated with antirat PRL or antirat GH antibody, followed by alkaline phosphatase catalyzed color development with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indol phosphate. Top, PhastGel Blue R stained gel strip. Middle and bottom, PVDF membrane pieces incubated with antirat PRL and antirat GH antibodies, respectively. Filled arrows in the figures indicate positions of spot p23, and open arrows those of spot p25.

 

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  		Table 1. GH, PRL, and p28 contents in pituitary

 
Protein content disparity in the rdw and normal thyroid glands
Figure 3Go shows 2-DE patterns of thyroid extracts of the +/+, rdw/rdw rats and a 1:1 mixture of the two. A part of each panel in Fig. 3Go, where protein spots are too crowded to be clearly seen, is expanded and shown in Fig. 4Go, and an expanded view of the 2-DE pattern of the +/rdw thyroid extract is added as the lower right panel of Fig. 4Go.



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  		Figure 3. Comparison of 2-DE patterns of the normal and rdw thyroid extracts. The 2-DE gels were stained with PhastGel Blue R. Panels from top to bottom are the 2-DE patterns of the +/+, rdw/rdw thyroid extracts and a 1:1 mixture of the two, respectively. The +/+ rat was 27 weeks old, and the rdw/rdw rat 21 weeks old.

 


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  		Figure 4. Expanded views of the area below p100 and above p26 in Fig. 3Go, where spots are too crowded to be clearly seen. The upper left panel is the 2-DE pattern of a +/+ thyroid extract, the lower left that of a rdw/rdw tissue extract, the upper right that of a 1:1 mixture of the two and the lower right that of a +/rdw thyroid extract. The +/+ rat was 28 weeks old, the rdw/rdw 21 weeks old, and the +/rdw 25 weeks old.

 
If we compare Figs. 3Go and 4Go with Fig. 1Go, we quickly notice that the protein content disparity between normal and rdw tissues is more serious in the thyroid gland than in the pituitary. The most conspicuous in the rdw/rdw thyroid panel of Fig. 3Go is the presence of such big protein spots as p100, p74 and p69, which are either absent or faint in the +/+ thyroid. Detailed examination of Fig. 4Go further revealed minor but clear disparities between the normal and rdw/rdw thyroids. Fourteen peptides in the +/+ thyroid panel of Fig. 4Go named as p59a, p52a, p52b, p42b, p42c, p41a, p41b, p41c, p40a, p37, p33b, p33c, p32, and p26 are absent in the rdw/rdw thyroid, whereas nine peptides in the rdw/rdw thyroid named as p89, p70, p69, p59b, p49d, p42a, p40b, p39, and p33a are absent in the +/+ thyroid. Six proteins named as p58, p54, p51, p50, p49a, and p49b increased in the rdw/rdw thyroid above the normal level, but three proteins, p330, p49c, and p37, decreased. There are two pairs of normal and rdw spots locating very close in the +/+ and rdw/rdw 2-DE patterns; one is the pair of spot p42b and p42a, and the other that of spot p40a and p40b, but they are all distinguishable in the 1:1 mixture panel of Fig. 4Go. Spot p40a and p42b are unique to the normal thyroid gland, and spot p40b and p42a are found only in the rdw/rdw thyroid.

Though a heterozygous animal of autosomal recessive trait is in general considered to be phenotypically indistinguishable from a normal one, the +/rdw rat is clearly affected by the recessive rdw gene. Let us consider spot p100, p74 and p69, which dramatically increased in the rdw/rdw thyroid. It is conspicuous in Fig. 4Go that p100 in the +/rdw thyroid is about the same as in the +/+ thyroid, whereas p74 and p69 increased to the level in between those in the +/+ and rdw/rdw thyroids. Careful comparison of the +/rdw panel in Fig. 4Go with the +/+ and rdw/rdw panels further revealed minor but clear influences of the recessive rdw gene on the protein composition in the +/rdw thyroids. Among the fourteen peptides (p59a, p52a, p52b, p42b, p42c, p41a, p41b, p41c, p40a, p37, p33b, p33c, p32, and p26) that are absent in the rdw/rdw thyroid, nine peptides (p59a, p52b, p42c, p41c, p37, p33b, p33c, p32, and p26) are also absent in the +/rdw tissue, and the rest (p52a, p42b, p41a, p41b, and p40a) decreased in the +/rdw tissue below those in the +/+ thyroid. Among the nine peptides (p89, p70, p69, p59b, p49d, p42a, p40b, p39, and p33a) that are absent in the +/+ thyroid and present only in the rdw/rdw tissue, three peptides (p89, p69, and p49d) are also present, but others (p70, p59b, p42a, p40b, p39, and p33a) are absent in the +/rdw tissue. Among the eight peptides (p100, p74, p58, p54, p51, p50, p49a, and p49b) that increase in the rdw/rdw tissue, five peptides (p100, p51, p50, p49a, and p49b) in the +/rdw thyroid are the same as in the +/+ tissue, but other three (p74, p58, and p54) increased to the level in between those in the +/+ and rdw/rdw thyroids. The three peptides (p330, p49c, and p36) that decrease in the rdw/rdw tissue stayed normal in the +/rdw thyroid.

Spot p330 in Fig. 3Go was identified as thyroglobulin by immunoblotting analysis with antihuman thyroglobulin antiserum (data not shown). Thyroglobulin being the precursor protein of thyroid hormones, the decrease of thyroglobulin content in the rdw thyroid gland is a clear evidence of hypothyroidism in the rdw rat.

Spot p69 in the rdw thyroid was identified as hsp70 by Western blotting analysis with antihuman hsp70 antiserum, the result of which is shown in Fig. 5Go. The panels in the left column of Fig. 5Go are the Coomassie-stained 2-DE patterns of +/+, +/rdw and rdw/rdw thyroid extracts from top to bottom, and those in the right column are the corresponding patterns obtained by immunoblotting with antihuman hsp70 antiserum. Considering together the results shown in Fig. 4Go and 5Go, we conclude that hsp70, though absent in the normal tissue, is produced in the +/rdw and rdw/rdw thyroids. Note that the immunoblotting pattern of the +/rdw thyroid gave two spots (in the middle right panel of Fig. 5Go), but the lower spot of the two was not seen in the Coomassie-stained 2-DE pattern (in the middle left panel of Fig. 5Go). The +/rdw thyroid gland produces two types of hsp70, larger and smaller, but the production of the smaller hsp70 is too low to be detected by Coomassie brilliant blue staining.



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  		Figure 5. Identification of spot p69 in the +/+, +/rdw and rdw/rdw thyroids by Western blotting analyses with anti-hsp70. A strip of a 2-DE gel was cut out around spot p69, and proteins in the gel strip were blotted to a PVDF membrane. Left column, The gel strip was then stained with PhastGel Blue R to show positions of the proteins still remaining in the gel strip after blotting. Right column, The PVDF membrane was incubated with antihuman hsp70 polyclonal antibody, followed by alkaline phosphatase catalyzed color development with nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indol phosphate. Arrows in the panels indicate positions of hsp70. Though spot p100 can be seen faintly in the bottom right panel, we consider the poor Western blot to have been caused by nonspecific binding of the anti-hsp70 to such highly abundant protein as p100. The +/+ rat used was 27 weeks old, the +/rdw 25 weeks old, and the rdw/rdw 25 weeks old. The +/rdw and rdw/rdw rats were littermates.

 
Because spot p74 and p100 had not cross-reacted with either antihuman hsp70 or anti-hsp90a antibodies (data not shown), we carried out internal amino-acid sequencing of the two proteins. Figure 6Go shows the peptide maps of the two proteins digested by V8 protease, and peptide fragments indicated by arrows 1 to 4 in Fig. 6Go were submitted for amino-acid sequencing. Table 2Go shows the amino acid sequences of the four peptides together with the names of homologous proteins identified in the protein identification resources (PIR) database. Internal sequences of the p74 and p100 proteins coincided or were highly homologous with those of rat immunoglobulin heavy chain-binding protein (BiP/GRP78) and mouse endoplasmin (GRP94), respectively. Considering together the results shown in Fig. 4Go and Table 2Go, we conclude that production of both BiP and endoplasmin increases in the rdw/rdw thyroid, but only BiP production increases in the +/rdw thyroid above the normal level.



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  		Figure 6. Peptide maps obtained by in-gel digestion of p74 and p100 proteins in the rdw/rdw thyroid. Peptide bands numbered 1 to 4 were submitted for amino-acid sequencing.

 

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  		Table 2. N-terminal amino-acid sequences of fragment 1 to 4 in Fig. 6Go

 
Having respectively identified spot p330, p69, p74, and p100 as thyroglobulin, hsp70, BiP and endoplasmin, we determined contents of these proteins in the +/+, +/rdw and rdw/rdw thyroid glands. Table 3Go quantitatively shows the results. Because BiP and endoplasmin are known to be ER resident chaperone proteins, accumulation of the two proteins, being similar to the ER storage disease (ERSD) extensively studied in the cog mouse (25), suggests the rdw thyroid suffered from protein folding and secreting disorders in lumen of ER. Because hsp70 is also a chaperone protein in cell nucleus and/or cytoplasm, the influence of the rdw gene would not be limited in lumen of ER but be spread to the cytoplasm and/or cell nucleus.


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  		Table 3. Thyroglobulin, endoplasmin, BiP, and hsp70 contents in thyroid

 
Protein content disparities in tissues other than the thyroid and pituitary glands
We examined and compared the 2-DE patterns of various rdw tissue extracts other than thyroid and pituitary with those of the normal ones. Because the most conspicuous abnormalities we had found so far were the accumulation of endoplasmin and BiP in the rdw thyroid, we shall concentrate on these two proteins in showing our 2-DE results. Figure 7Go shows expanded 2-DE patterns of the normal and rdw brain tissue extracts from cerebral cortex, cerebellum, brain trunk, and hypothalamus, and Fig. 8Go those from liver, testis, spleen, and thymus. The upper left arrow and lower right arrowhead in Figs. 7Go and 8Go, respectively, point to endoplasmin and BiP positions. All tissues but hypothalamus and spleen produce neither endoplasmin nor BiP. Hypothalamus produces a small amount of endoplasmin but no BiP, and spleen produces both. In any case we could not see any difference between the 2-DE patterns of the normal and rdw tissues. Though the rdw thyroid suffered from protein folding and secreting disorders, we could not find any symptoms of such disorders in the rdw liver, testis, spleen, and thymus.



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  		Figure 7. Comparison of 2-DE patterns of the +/+ and rdw/rdw brain tissue extracts. Only part of the 2-DE patterns stained with PhastGel Blue R are shown where endoplasmin and BiP spots are to be seen. The basic end of the gel is to the left. Label A and T in the top left panel indicate positions of actin and tubulin, respectively. Upper left arrows and lower right arrowheads in the panels respectively indicate the positions of endoplasmin and BiP. The +/+ rat was 28 weeks old, and the rdw/rdw 21 weeks old.

 


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  		Figure 8. Comparison of 2-DE patterns of the +/+ and rdw/rdw tissues other than brain. Experimental conditions and the rats used are the same as those in Fig. 7Go.

 
Careful inspection of other areas of 2-DE patterns did not reveal any difference between the normal and rdw tissues, and we drew a conclusion that biochemical abnormalities related to the hereditary dwarfism of the rdw rat were limited to the thyroid and pituitary glands.

A question then arises as to which of the two glands is more responsible for the disorder in the rdw rat. To find a clue to the question, we have carried out Northern blot analysis and cDNA sequencing of the rdw TSHr gene.

Northern blot analysis and cDNA sequencing of the rdw TSH receptor
Figure 9Go shows the result of Northern blot analyses of the TSH receptor mRNA of the +/? and rdw/rdw thyroids. Rats used were 21 to 27 weeks old, and phenotypically normal animals were used as the normal standard because of their normal growth and maturation. Considering a slight difference in density of the ß-actin mRNA bands in Fig. 9Go, we conclude that the +/? and rdw/rdw TSHr mRNA are equal in size and expression level.



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  		Figure 9. Northern blot analyses of the +/? and rdw/rdw thyroid mRNA. Total RNA was extracted from thyroid glands of 21- to 27-week-old rats, and cDNA probes used were for TSHr and ß-actin mRNA. The expression level of ß-actin mRNA was used for calibration.

 
Nucleotide sequences of the rdw TSHr cDNA fragment 1 and 2 perfectly agreed with the rat TSHr sequence given in GenBank (data not shown). It should be noted that, at the present stage of study when we have finished sequencing only 50% of the rdw/rdw TSHr cDNA, it is too early to conclusively say the homozygous rdw TSHr is perfectly normal, but we can at least say that we have not yet find any abnormalities so far in the size, mRNA expression level, and nucleotide sequence of the rdw TSHr gene.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our aim in this paper was to search for proteins that were related to the hereditary dwarfism of the rdw rat. The following are our findings in this study: 1) Biochemical abnormalities in the rdw rat are limited to thyroid and pituitary glands, and no abnormalities have been seen in the 2-DE patterns of the rdw tissue other than the two glands. 2) PRL and GH contents in the rdw pituitary respectively are 1/15 times and less than 1/30 times as much as in the normal one, whereas the p28 protein is likely to be more in the rdw pituitary than in the normal tissue. 3) The 2-DE pattern of the rdw thyroid extract revealed that contents of more than thirty proteins changed in the tissue, but only a few of them have been identified in this study. 3a) Thyroglobulin content in the rdw thyroid decreases below that of the normal tissue. 3b) Production of endoplasmin, BiP and hsp70 in the rdw/rdw thyroid increases 40-fold, 10-fold, and more than 50-fold over the normal level, respectively. Because the three proteins are all chaperones, the rdw thyroid suffered from protein folding and secreting disorders. 3c) The +/rdw thyroid, on the other hand, produces BiP and hsp70 three times and more than twenty times as much as the normal tissue does, respectively, but the endoplasmin production stays normal. Topics to be discussed in this discussion are along the line of our findings summarized in the above.

The first point we would like to note is the improved 2-DE technique we used in the present study, with which we are able to search for rdw-related proteins in a very small piece of an animal tissue, as small as 10 mg, in a wide range of protein molecular weight (MW as large as ~500K) and isoelectric point (pI 3.0 ~9.5). Though O’Farrell’s 2-DE method (26) with its limited capability in analyzing high molecular weight proteins would be able to detect the presence of BiP (MW ~ 74K) and endoplasmin (MW ~ 100K), our method can do quantitative content analyses of the two proteins much better than O’Farrell’s, and thyroglobulin (MW ~ 330K) content analysis can only be done with our 2-DE technique. The range of the rdw-related proteins searched for in this study is therefore much wider than when we had used O’Farrell’s 2-DE technique.

Our first observation showed that constituent proteins of various tissues other than the pituitary and thyroid glands were normal in the rdw rat. Though the rdw thyroid accumulates chaperone proteins in large quantities, our study has shown that the rdw organs other than thyroid and pituitary glands are not influenced by the disorders. Accumulation of ER chaperones itself, however, is found in a variety of organs including liver (27), thyroid (28), pituitary (29), and bone (30), all of which more or less secrete proteins. Our observation 1) would therefore mean that the abnormal proteins we found in the rdw thyroid would not include proteins commonly required in the secretion pathways of these glands. It should also be noted that our first observation does not necessarily mean the rdw organs other than thyroid and pituitary are perfectly normal. They really are not normal in weight relative to the whole body. Koto et al. (1) weighed various organs of 10-week-old rdw rats, and reported that brain and testis of the rdw rat were almost the same in weight as normal organs, despite the fact that the rdw rats are approximately 40–60% of the normal controls in body weight. The rdw brain and testis are normal in the protein composition but abnormal in the growth rate relative to the whole body.

Knowing that the rdw related abnormalities are limited to the pituitary and thyroid, we would like to raise a question as to which gland is the cause of the biochemical disorder in the other; we conjecture that, contrary to Koto, et al.’s expectation (1), the reduction in GH and PRL production in the rdw pituitary would be the results of the reduction in thyroid hormone production and not vice versa. Reasons for our conjecture are the following:

1) As we have described in the Animals section, we are able to raise the rdw/rdw infant rats to matured stage by transplanting thyroid glands from phenotypically normal animals. If the normal thyroid glands had not been transplanted to the rdw/rdw infants, they were not able to grow to the matured stage in body weight and fertility. Though details of our transplantation experiment will be published elsewhere, it should be sufficient for us to say that, if hypoplasia in the pituitary were the primary cause of the disorder in the rdw thyroid, thyroid transplantation would be useless in healing the pituitary to raise the blood GH and PRL levels, and the rdw infant rats would not mature in body weight and fertility. We should have transplanted normal pituitaries instead.

2) The TSHß mRNA content in the rdw pituitary being about seven times as much as in the normal pituitary (3), the rdw pituitary would produce more TSH than the normal tissue does. As the thyroid transplantation result suggests, the rdw pituitary has a potency to respond to increased thyroid hormone levels and to produce active GH and PRL. The level of plasma thyroxine (NOREF>T4) in the rdw rat being about 1/7 of the normal level (3), hypoplasia in the rdw pituitary seems to be caused by the hypothyroidism, which retards the differentiation and growth of GH and PRL producing cells in the pituitary gland (31, 32, 33, 34).

3) Results of Northern blot analysis and cDNA sequencing about 50% in length do not yet prove the presence of abnormalities in the rdw TSH receptor. It should be noted that the rdw rat keeps holding a highly conserved proline residue at codon position 556 of the TSHr protein, but the hyt mouse, another animal model, has hypothyroidism and a defective TSHr gene, the point mutation in which leads to a replacement of Pro 556 with a leucine residue (35, 36).

4) The number of abnormal protein spots in a 2-DE gel being only three (GH, PRL, and p28) in the rdw pituitary and more than thirty in the thyroid, biochemical abnormalities found in the thyroid are more serious than those in the pituitary. The fact that the rdw pituitary does not accumulate chaperone proteins inside would also be a supporting data to our conjecture.

Our second finding on the decrease of GH and PRL contents in the rdw pituitary agrees with GH and PRL studies of the rdw rat previously done (2). The rdw rat, being deficient of GH and PRL, has hypoplasia in the pituitary, and mRNA levels of PRL and GH genes in the rdw pituitary are 1/30 to 1/100 of those of a normal tissue (2). The rdw rat resembles to the Snell and the Ames mice in having deficiencies of GH and PRL. The Pit-1 gene, being responsible for the abnormalities in the Snell and the Ames mice, was once considered to be so in the rdw rats, but there were no differences in the Pit-1 mRNA level, Pit-1 protein content or Pit-1 nucleotide sequences of the rdw and normal rats (3). Dwarfs of a stock of the Snell mice, called Snell-Bagg, have immunological deficiencies above the GH and PRL deficiencies (11), but our data showed that there were no abnormalities in the constituent proteins in the rdw thymus. We have not yet obtained any data that suggest immunological deficiencies in the rdw rat.

Our third finding on the protein composition of the rdw thyroid has shown that studying only a few major proteins of the rdw thyroid would be unwise, and we had better study more than thirty abnormal proteins as a whole in the rdw thyroid to clarify the real nature of the hypothyroidism of the rdw rat. Protein content abnormalities in the rdw thyroid is consistent with morphological studies of the rdw rat previously done (1, 37). The rdw rat suffered from hypothyroidism, which is similar to, but distinct from, that of the congenital goiter (cog) (38) and the hypothyroid (hyt) (39) mice. The cog thyroid, being congenital goiter, is more than ten times as large as a normal gland in absolute weight (38), but the rdw thyroid stays normal in relative weight to the whole body (1). While a normal thyroid has many large follicles containing eosinophilic colloidal substances, the rdw thyroid has numerous small follicles having no colloidal substances inside (1). The cog thyroid, on the other hand, is structurally disordered and composed of hypertrophied cells with no eosinophilic colloid inside (38, 40), and the hyt tissue has follicles filled some part with darkly stained colloid (35). Neither the cog nor hyt thyroids morphologically resemble to the rdw thyroid, and the hyt TSHr protein is different from the rdw counterpart in having a proline-to-leucine substitution at amino-acid position 556 (36). The rdw rat is a new animal model having hypothyroidism, which is similar to, but distinct from, that of the cog and hyt mice.

The observation (3a) on the decrease of the thyroglobulin content in the rdw thyroid suggests that thyroid hormone production would also be reduced in the rdw thyroid. However, SDS-PAGE showed that the molecular weight of the rdw thyroglobulin seemed quite similar to that of the normal protein (data not shown). Problems left to be clarified are 1) whether the thyroglobulin in the rdw thyroid has mutations in the amino-acid sequence or has abnormal posttranslational modifications, and 2) whether there are other disorders on the pathway from thyroglobulin production to thyroid hormone secretion. But we guess that the latter possibility is not high, because, if it were the case, the rdw secretory organs other than thyroid would also have troubles in secreting proteins, but the present study showed the rdw liver and spleen were normal.

Our finding (3b) of the increased levels of endoplasmin, BiP and hsp70 in the rdw thyroid suggests an accumulation of misfolded (41, 42, 43), aberrantly glycosylated (44, 45), or unassembled (43, 46, 47) proteins in the rdw thyroid. If these misfolded proteins, when bound to their chaperones, do not refold into their native conformation and do not let the chaperones be free, the free chaperone concentration in a cell would be lowered, and a mechanism to keep constant the free chaperone levels in the cell would continue producing chaperones to an abnormally high level of chaperone proteins as a whole (25). Because BiP and endoplasmin are ER resident chaperones, misfolded proteins would be accumulated in the lumen of ER of the thyroid, which resembles to the ERSD in the cog mouse (25). But, because hsp70 is also a chaperone located in cell nucleus and/or cytoplasm, those misfolded proteins would also be accumulated in the cytoplasm and/or cell nucleus of the rdw thyroid gland.

The last observation (3c) that BiP increases but endoplasmin stays normal in the +/rdw thyroid is not consistent with the report that BiP and endoplasmin were transcriptionally coregulated due to similar control elements (48). Though the two proteins have been considered to belong to a same group of ER-specific molecular chaperones (49), observation (3c) suggests that there are at least two classes in the group regulation mechanisms of chaperone syntheses and the syntheses of BiP and endoplasmin would be regulated independently in the +/rdw thyroid. The +/rdw thyroid would be a good model for studying the group regulation mechanism of chaperone syntheses.

Further work will be needed to identify many abnormal proteins in the rdw thyroid without which the entire picture of the hereditary inherited rdw hypothyroidism would stay in veil.


   Acknowledgments
 
We are grateful to the Rat Pituitary Hormone Distribution Program, NIDDK, NIH (Bethesda, MD) for providing the rat GH and PRL antibodies. We thank to Mr. O. Yoshida of Kitasato University School of Medicine for his technical assistance.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid 08680911 from the Ministry of Education, Science and Culture, Japan and a grant from the Foundation for Growth Science (to S. F.), and Kitasato Research Fund (to M.O.). Back

Received August 4, 1997.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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