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Animal Model |
From the Department of Laboratory Medicine and Pathology, MayoClinic, Rochester, Minnesota
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-subunit (SU)-null mice that develop
thyroid-stimulating hormone (TSH) cell hyperplasia with
p18-null mice. The resulting offsprings developed accelerated
enlargement of the anterior lobe with predominantly TSH cell
hyperplasia. Immunohistochemical and histological analyses of these
mice along with p27/p18 double-null mice, p18-null
mice, and p27-null mice showed evidence of TSH,
adrenocorticotropic hormone, prolactin, and luteinizing
hormone hyperplasia. To determine whether there were alterations of p27
and the target proteins implicated in the ubiquitin degradation of p27
and other cyclin-dependent kinase inhibitors, we examined
expression of SKP 2, Grb 2, and Jab 1 in the
pituitaries of null mice. In the SU-null mice there were decreased
levels of SKP 2 and elevated levels of Grb 2 expression by Western blot
analysis. Immunohistochemical analysis of the pituitary showed elevated
Grb 2 in SU-null and p18/SU double-null mice. Jab 1 levels were
not different from controls in the pituitary. These results show that
1) the p18/SU double-null mice represent a good model to study the
rapid development of anterior pituitary hyperplasia, and 2)
various proteins important in p27 and other cyclin-dependent kinase
inhibitor protein degradation are altered in the pituitary of
SU-null and p18/SU double-null mouse models.
-subunit gene9
and
transgenic mice expressing the growth hormone-releasing hormone with a
metalloproteinase-driven promotor crossed with p27-null
mice10
have also been used to study anterior pituitary
hyperplasia.9,10 The levels of p27 proteins are decreased in many human cancers compared to normal tissues and have prognostic significance, suggesting that p27 may be a tumor suppressor gene. However, there are few mutations in the p27 gene and the mRNA levels are relatively unchanged compared to the decreased levels of p27 protein in tumors.11,12 These observations suggest that the proteins regulating posttranslational degradation of p27 may be potential targets to explain the mechanism of down-regulation of p27 and other cyclin-dependent kinase inhibitory (CDKI) cell-cycle genes during tumor development.
Although it has been shown that the ubiquitin-proteasome system13,14 regulated short-lived CKDI proteins such as p27, the role of various proteins in the degradation are currently being investigated.15 In studies with p27, the jun-activated protein Jab 1,16 various F-box proteins including SKP 217-20 and the signal-transducing adaptor protein Grb 221,22 have been shown to have a regulatory roles in p27 degradation.
In this study, we targeted pituitary hyperplasia to the anterior
pituitary of p18-deficient mice by creating double-null animals with
loss of the p18 and -subunit genes. These mice as well as p27-null
and p18/p27 double-null mice were used to examine expression of some of
the major proteins that play a role in p27 and other CDKI
ubiquitin-mediated degradation of CDKIs.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The p27 mice with a C57BL/6 background (a gift from Dr. M. L.
Fero and J. L. Roberts, Fred Hutchinson Cancer Center, Seattle,
WA), the p18 mice with a C57BL/6 background (a gift from Drs.
D. S. Franklin and Y. Xiang, University of North Carolina, Chapel
Hill, NC), and the -subunit of glycoprotein hormones (SU)
mice had a background of C57BL/6J (a gift from Dr. S. A. Camper,
University of Michigan, Ann Arbor, MI) were all maintained in a
specialized mouse barrier facility at the Mayo Clinic.
F2 mice heterozygous for p27, p18, or SU were generated from F1 mice
in each group. Mice were genotyped and the resulting F1 mice were
intercrossed to created double-null animals. The p18/SU double-null
mice were derived from crossing p18-null mice with SU heterozygous
mice. The p27/p18 double-null mice were derived from crossing p27 and
p18 heterozygous mice. All animals were mainly B6 in their genetic
background. Genotyping was done by polymerase chain reaction (PCR).
All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
PCR Genotyping
Tail snips were taken from all mice at 4 weeks of age and genomic DNA extracted for genotyping. The PCR reactions contained 1.25 U of Taq polymerase, 1x PCR buffer, 1.5 of mmol/L magnesium chloride (Promega, Madison, WI), 100 ng of each primer, and 1 [µ]l of genomic DNA in a total volume of 25 [µ]l unless otherwise specified. All PCR products were resolved on a 2% agarose gel stained with ethidium bromide.
Two sets of primers were used to amplify the intact -subunit
wild-type allele and the disrupted nonfunctional allele. The wild-type
sense primer (5'-GCA TAT CCC ACT CCC GCC AGG) located within exon 3 was
used in conjunction with an antisense primer (5'-CAA TTA AAG AGG ATA
AAT GCA GGT GTC GCC) within intron 3 resulting in a 220-bp product.
Detection of the disrupted or null allele used a sense primer (5'-TGC
CTT TTG TAT TAT CAG GGT ACC TAG ACT) and an antisense primer located
within the neo gene (5'-CGC CTT CTA TCG CCT TCT TGA CGA GTT
CTT) resulting in a 1.1-kb product.23
Twice the amount of
Taq polymerase was used for the null allele PCR (2.5 U). The
-subunit-null mice and the p18/SU double-null mice required
genetic sexing because of hypogonadism and hypothyroidism. Males were
identified through PCR detection of the sry gene on the Y
chromosome.24
The sense primer (5'-GTC TAG AGA GCA TGG
ACGG GCC) and antisense primer (5'-ACA GGT GTG CAG CTC TAC TCC)
amplified a 381-bp product in males only.25
PCR for the
wild-type and null alleles of the -subunit gene as well as the
sex-determining PCR shared the same PCR profile: 95°C for 5 minutes;
30 cycles at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2
minutes followed by 10 minutes of final extension at 72°C.
Detection of p27 wild-type allele and p27-null allele used one common sense primer (5'-TGG AAC CCT GTG CCA TCT CTA T). The antisense primers used were specific for the p27 wild-type allele (5'-GAG CAG ACG CCC AAG AAG C) and the neo-disrupted allele (5'-CCT TCT ATG GCC TTC TTG ACG). PCR was performed for 40 cycles with an annealing temperature of 57°C. The wild-type allele resulted in a 1300-bp product and the null allele in a 600-bp product.6
A sense primer for the p18 gene (5'-AGC CAT CAA ATT TAT TCA TGT TGC AGG) was used in combination with the wild-type antisense primer (5'-CCT CCA TCA GGC TAA TGA CC) and null antisense primer (5'-CCA GCC TCT GAG CCC AGA AAG CGA AGG). PCR amplification was performed at an annealing temperature of 60°C for 35 cycles and produced a 600-bp wild-type amplicon or a 400-bp null amplicon.7
Anatomical and Histological Analysis
Animals were monitored daily and sacrificed at preplanned intervals or when they showed signs of morbidity. Body weight and specific organ weights were obtained at the time of sacrifice and genotyping was rechecked in some cases. Portions of specific tissues, including pituitaries and liver, were frozen in liquid nitrogen, stored at -70°C, and used for protein analysis and other studies.
For histological analysis tissues were fixed in 10% buffered formalin, dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in paraffin blocks. Hematoxylin and eosin (H&E) stains were performed on all blocks and other sections were used for immunohistochemical analysis. Mitotic figures in the pituitary were counted in 50 fields at a final magnification of x400.
Antibodies and Immunohistochemistry
Antibodies for prolactin (PRL) (used at 1/1000), growth hormone (GH) (used at 1/1000), thyroid stimulating hormone (TSH) (used at 1/500), and luteinizing hormone (LH) (used at 1/500) were obtained from the National Pituitary Distribution Agency, Bethesda, MD. Adrenocorticotropic hormone (ACTH) (used at 1/500) was obtained from DAKO (Santa Barbara, CA).
Antibodies to p27 (1/1000; Transduction Laboratories, Lexington, KY), SKP 2 (1/100; Santa Cruz Inc., Santa Cruz, CA), Jab 1 (1/500, Santa Cruz, Inc.), and Grb 2 (1/500; Santa Cruz, Inc.) were used for immunohistochemistry. Immunohistochemical staining was done as previously reported23,24 with antigen retrieval used for p27, SKP 2, Jab 1, and Grb 2. Antigen retrieval was performed in an 800-W microwave oven using 0.1 mol/L of citrate buffer, pH 6.0, for 5 minutes followed by 20 minutes of cooling at room temperature. Immunohistochemical staining was done by incubation with the primary antibody overnight and the reaction product was developed with diaminobenzidine.26,27 The avidin-biotin complex peroxidase kits were obtained from Vector Laboratories (Burlingame, CA). Semiquantitation of immunohistochemical staining was done by grading the nuclear immunoreactivity as 0, negative; +, rare or <5% cells positive; ++, focal or <25% cells positive; and +++, diffuse or >25% of cells positive. Four to five pituitaries were analyzed for each group.
Western Blotting
Western blots were done as previously reported.27,28 Briefly, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done and the electrophoresed proteins subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA) and subjected to immunoblot analysis for p27 (1/1000 dilution), SKP 2 (1/400), Jab 1 (1/500), Grb 2 (1/250), and ß-actin (1/1000; Sigma Chemical Co., St. Louis, MO). The reaction product was detected with enhanced chemiluminescence (Amersham, Arlington Heights, IL). ß-Actin was used to normalize for equal loading of the gels. Densitometric analysis of the bands was done with a Fluor-S multimager (Bio-Rad, Hercules, CA). Results were expressed as relative densitometry units, and all data were normalized with ß-actin. A linear response was obtained using different amounts of proteins on the gel. Each experiment was repeated two to three times.
Statistical Analysis
Results were expressed as the mean ± SEM. Statistical analysis was done with the Wilcoxon rank sum test.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Genotype analyses by PCR were used to characterize the mice
resulting from the various crosses (Figure 1)
. Null mice that were deficient in the
SU gene developed anterior pituitary hyperplasia in a time-dependent
manner as previously reported.25,29
The p18- and p27-null
mice developed enlarged intermediate lobes in a time-dependent manner.
Animals sacrificed at 1 year of age from the SU-null group had
pituitaries weighing 73 ± 20 mg. The average weights of p18- and
p27-null mice pituitaries were 6.1 ± 1.5 mg and 12.8 ± 5.4
mg, respectively.
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SU double-null mice had a more rapid growth of their
anterior pituitaries compared to either the p18-null or SU-null
animals (Figure 2)
. When the mice were
sacrificed by 5 months of age, the pituitaries were sevenfold larger
than the SU-null mice sacrificed at the same time
(P < 0.05) and fivefold larger than the
p18-null mice sacrificed at 11 months of age on average (Figure 2)
.
Mice that were p18 heterozygous and SU-null sacrificed at 5 months
of age had pituitaries that were slightly larger than those of the
SU-null mice (Figure 2)
.
|
. The pituitaries of p27
heterozygous/p18-null mice were slightly larger than the control mice
at the same age (Figure 3)
.
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Histological analysis revealed diffuse and nodular or adenomatous
hyperplasia of the pituitary (Figure 4
; A
to F). For p18- and p27-null mice, this was mainly intermediate lobe
ACTH cell hyperplasia (Table 1
and Figure 4, A and B
), although one PRL-positive hyperplastic gland was present
in 1 of 10 p18-null mice examined by immunohistochemistry (Figure 4E)
.
The SU-null mice had TSH cell hyperplasia in the anterior lobe of
the pituitary. The p18/SU double-null mice had mainly anterior lobe
TSH cell hyperplasia when sacrificed at 5 months of age with less
developed intermediate lobe ACTH cell hyperplasia at this age (Figure 4, C and D)
.
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, Figure 4B
) at the
time of sacrificing at 2 to 3 months of age. Nodular LH cell
hyperplasia was present in two of six pituitaries from p27
heterozygous/p18-null mice (Table 1
, Figure 4F
) along with intermediate
lobe ACTH cell hyperplasia.
Analysis of mitotic activity showed between one to five mitoses per 50
high-power fields in the p27-null, p18-null, SU-null, as well as the
double-null mice whereas none to one mitoses per 50 high-power fields
were present in the anterior pituitary of control mice.
Expression of p27 Regulatory Proteins
When pituitary tissues for p27-null, p18-null, and SU-null mice
were analyzed by Western blots for p27, SKP 2, the highest levels of
Grb 2 and Jab 1, the SU-null mice had the lower levels of SKP 2 and
Grb 2 protein that were 3.5-fold higher than the control wild-type
liver. The p27-null mice pituitary tissue had Grb 2 levels that were
2.3-fold higher than the control mice (Figure 5, A and B)
. The levels of Jab 1 were not
significantly different in any of the pituitary tissues from p27-,
p18-, or SU-null mice by Western blot analysis (Figure 5)
.
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.
Only nuclear staining was evaluated. Staining of the pituitaries showed
the highest levels of Grb 2 in the SU- and p18/SU double-null
mice (Table 2)
. p27 immunoreactivity was
not present in the pituitaries of the p27-null and p27/p18 double-null
mice. Jab 1 levels were similar to wild type in the pituitary by
Western blotting and by immunohistochemistry.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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SU genes. This new model
more closely simulates pituitary lesions developing spontaneously in
rodents and humans and serves as a model to analyze the pathogenesis of
anterior pituitary neoplasia.
The TSH cell hyperplasia in the SU-null mice and the p18/SU
double-null mice was a secondary hyperplasia resulting from the
knockout of the -subunit gene that led to thyroid atrophy and
pituitary TSH cell hyperplasia.23,25
Although ACTH intermediate lobe hyperplasia was most common in our studies, one hyperplastic PRL cell lesion was found in a p18-null male mouse. In the p27 heterozygous/p18-null mice two animals developed nodular hyperplasia of LH-producing cells. These findings indicate that hyperplastic lesions with these models involve various pituitary cell lineage, including ACTH, TSH, PRL, and LH. The presence of increased numbers of mitoses in the pituitary tissues of null mice compared to the controls supports the observation that there was pituitary cell hyperplasia and not just hypertrophy in these tissues.
The development of hyperplastic pituitaries in mice expressing one allele of a CDKI gene such as p27 or p18 in our studies is consistent with the concept of haplo-insufficiency for tumor suppressor genes observed by Fero and colleagues.34 In their studies of mice heterozygous for p27, animals treated with radiation or carcinogens subsequently developed enlarged pituitaries more rapidly than the untreated control littermates. These observations indicate that the absence of one copy of a tumor suppressor gene may be sufficient to lead to hyperplasia/neoplasia in specific target tissues.34
Although enlarged pituitaries in rodents are often referred to as
tumors or adenomas,4-8
the distinction between
pituitaries with diffuse and adenomatous hyperplasia from true
neoplasms is often difficult. Although the use of X chromosomal-linked
genes such as the androgen receptor and other genes have been used
successfully to support clonality of pituitary tumors in
humans,35,36
these techniques have not been used
extensively in rodents. We are currently developing methods to
determine clonality in hyperplastic and neoplastic rodent pituitaries.
However, for the purpose of the current studies, the morphological
features of the enlarged pituitaries in mice with SU deletions and
in the p27- and p18-null mice were considered to represent hyperplastic
glands. These observations are supported by our observations that
pituitaries weighing 100 mg in SU-null mice were reduced to 10 to 15
mg in 2 weeks of thyroxine treatment (unpublished
observations).25,29
We analyzed the expression of various proteins that influence p27
ubiquitin-mediated degradation in the pituitary of p27-, p18-, and
SU-null mice. In the pituitary of p27-null mice, Grb 2 levels were
2.3 times greater than control. Interestingly, the SU-null mice had
markedly elevated levels of Grb 2, but decreased levels of SKP 2 by
Western blotting.
Immunohistochemical analysis done with the pituitary tissues also
showed high levels of Grb 2 in the pituitary of SU-null and
p18/SU double-null mice. Although several studies have indicated an
inverse relationship between p27 and SKP 2 expression in cultured cells
and tissues,17,18,20
we did not detect a marked increase
in SKP 2 in p27-null mice in the pituitary. The Jab 1/CSN5 protein is a
regulator of p27 transport from the nucleus to the cytoplasm and Jab 1
has an important role in G1 progression and cell
proliferation.16
More recent studies have shown that
overexpression of Grb 2 accelerates Jab 1-mediated degradation of
p27,21
indicating that Grb 2 participates in a negative
regulation of p27 that may link the signal transduction pathway with
the cell-cycle regulatory pathway.22
The markedly elevated
levels of Grb 2 in the pituitary of SU-null mice in the presence of
relatively normal levels of Jab 1 suggests an important role of Grb 1
in p27 degradation.
In the pituitary of SU-null and p18/SU double-null mice Grb 2
levels were higher than in the control wild-type mice, suggesting that
Grb 2 expression with possible increased degradation of p27. The
immunoreactivity for SKP 2 was relatively high in the pituitaries of
most groups compared to wild-type mice except for the SU-null mice
by Western blotting. A growing body of evidence has implicated SKP 2 as
an oncogenic factor in human cancers.17
Other studies have
shown that SKP 2 was required for the ubiquitin-mediated degradation of
p27.18
Our analysis in the pituitary suggests that SKP 2
may have a role in pituitary p27 regulation, but it probably has other
functions in these cells, because it was highly expressed in p27-null
mice. Previous studies from our laboratory showed lower levels of p27
in anterior pituitary ACTH cells compared to other cell
types.37,38
Because of these differences, which were also
observed by other investigators subsequently,39
it is
possible that the ubiquitin-mediated degradation of p27 by proteins
such as Grb 2, Jab 1, and SKP 2 may vary in different types of anterior
pituitary cell types and this may influence pituitary hyperplasia and
tumor. In situ studies with double localization of proteins
or laser capture microdissection combined with
immunophenotyping26
will be needed to address this
question.
Other recent studies with a mouse knock-in model have observed a second proteolytic pathway for controlling p27, which is activated by mitogens and degrades p27 exclusively during the G1 phase of the cell cycle indicating the complexity of p27 regulation.40
In summary, we have described a model of p18- and SU double-null
mice that develop accelerated anterior pituitary TSH cell hyperplasia
and shows alterations in some proteins related to p27 and other CDKI
ubiquitin-mediated degradation. These null mice can be used as a model
to study the molecular pathogenesis of anterior pituitary hyperplasia
and tumor development.
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Acknowledgements |
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SU-null mice; and the
National Pituitary Agency for antibodies to pituitary hormones. |
Footnotes |
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Supported in part by National Institutes of Health grant CA90249 and a grant from the Jarislowsky Foundation.
Accepted for publication November 27, 2001.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-subunit produces hypogonadal and hypothyroid mice. Genes Dev 1995, 9:2007-2019-subunit knockout mice after thyroxine and 17 ß-estradiol treatment: role of apoptosis. Endocr Pathol 1998, 9:261-274[Medline]
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