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Section of Pediatric Endocrinology/Diabetology, Wells Center for Pediatric Research, Department of Pediatrics (S.F., R.S., D.W.K., P.Z., O.H.P.); Laboratory Animal Resource Center, Indiana University School of Medicine (C.V.); and Department of Microbiology and Immunology and The Walther Cancer Institute (S.C., G.H., H.E.B.), Indiana University School of Medicine, Indianapolis, Indiana 46202
Address all correspondence and requests for reprints to: Dr. Ora H. Pescovitz, Riley Hospital, Room A5984, 702 Barnhill Drive, Indianapolis, Indiana 46202. E-mail: opescovi{at}iupui.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse pro-GHRH molecule contains the GHRH peptide, (amino acids
30–73 of the preprohormone) and a C-terminal region that we have
designated GHRH-related peptide (GHRH-RP; amino acids 74–104 of the
preprohormone; Fig. 1
). GHRH, which
primarily functions to stimulate GH secretion, is widely believed to be
the only biologically active peptide produced after posttranslational
processing of pro-GHRH. However, due to the marked homologies of the
peptide sequences and structural organization of the genes for VIP and
PACAP compared with that for GHRH (1), we hypothesized that pro-GHRH is
also processed to produce more than one biologically active
peptide.
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The goals of the present study were to produce a transgenic (Tg) mouse that constitutively expresses GHRH-RP to characterize the effect(s) of chronic exposure to this peptide in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) were
designed. Primer A (5'-AGACGAATTCATGCTGCT CTGGGT GCTC-3') contains an
EcoRI site, at the 5' position for cloning, and 10
nucleotides (nt) complementary to the first 10 found in the prepro-GHRH
cDNA signal sequence. Primers B
(5'-CCACATGCTGTCTTCCTGTCGCTGCATCCTGAAGGG-3') and
C (5'-CCCTTCAGGATGCAGCGACAGGAAGACAGCATGTGG-3')
both contain 18 nt that are complementary to the 3'-end of the signal
sequence (in bold above) and 18 nt that are complementary to
the 5'-end of the GHRH-RP coding sequence (underlined
above and in Fig 1.). Primer D (5'-TTCGGAAGTCGCCTGCGAAC TCAGCTGCAGA) is
complementary to the last 18 nt of the GHRH-RP-coding region, which
includes the endogenous stop codon. A SalI restriction site
was added to this primer for cloning. All four primers were used for
the first round of PCR. Products from the first round of PCR were
isolated by agarose gel electrophoresis and purified using QIAEX gel
purification kit (QIAGEN, Valencia, CA). The expected
sizes of amplified products are 115 bp using primers A and B and 118 bp
for primers C and D. These products were heated to 95 C before the
second PCR was initiated. The second round of PCR, using primers A and D, resulted in a single 215-bp product (SS-RP) that was purified and verified by automated sequence analysis. SS-RP cDNA was then restriction digested with EcoRI and SalI enzymes and subcloned into the multiple cloning site of the pCI vector (Promega Corp., Madison, WI). The cytomegalovirus immediate-early enhancer/promoter region contained in the original pCI vector was replaced with the mouse phosphoglycerate kinase (PGK-1) promoter. The new plasmid (pCI-SS-RP) was digested with BglII and BamHI restriction enzymes. The resulting 1.1-kb DNA fragment was sequenced and used for microinjection.
TM4 cell transfection
TM4 cells, a mouse Sertoli cell line, were plated in
75-cm2 tissue culture flasks and maintained in
growth medium [DMEM containing 15% FBS and 1%
penicillin/streptomycin antibiotic solution (Life Technologies, Inc., Gaithersburg, MD)] at 37C in 5%
CO2. Cells (2 x 107
cells) were transfected using Lipofectamine Plus reagents (Life Technologies, Inc.; Lipofectamine, 30 µg/flask; Plus reagent,
30 µl/flask) and 30 µg/flask of the pCI-SS-RP vector DNA for
5 h. After the transfection, cells were incubated in growth medium
for an additional 48 h, at which time cell lysates were
prepared.
Western blot analysis
Transfected TM4 cell lysates (20 µg) were separated on a 12%
SDS-polyacrylamide gel and electrotransferred to nitrocellulose
(NitroPure, MSI, Westboro, MA), and the resulting membrane was probed
with a polyclonal primary antibody (Genemed Synthesis, Inc., South San
Francisco, CA) directed against rat GHRH-RP. After washing, peptides
were visualized using a horseradish peroxidase-conjugated second
antibody coupled with enhanced chemiluminescence detection
(Amersham Pharmacia Biotech, Aylesbury, UK). A lysate
prepared from TM4 cells transfected with the empty vector served as a
negative control. Western gels were repeated three times with similar
results.
Transgenic mice
All procedures involving animals were approved by the
institutional animal care and use committee at Indiana University
School of Medicine and followed the USPHS Guide for the Care and Use of
Laboratory Animals. For generation of Tg mice, the 1.1-kb SS-RP
transgene construct was injected into the male pronucleus of fertilized
C57BL/6xC3H F1 mice eggs, which were then transferred to the oviducts
of pseudopregnant recipient C3H mice.
Southern blot and PCR analysis of genomic DNA
Genomic DNA was extracted from tail tissue, digested with
EcoRI and BamHI or EcoRI and
SalI restriction enzymes, and analyzed by Southern blotting
(6). Products were separated on a 1% agarose gel, transferred to a
nylon membrane (MagnaGraph, Micron Separations, Inc., Westboro, MA) and
hybridized at 42 C with the full-length
32P-labeled 215-bp SS-RP probe. Genomic DNA was
extracted from tail tissue and analyzed by PCR for the presence of the
SS-RP transgene using primers A and D.
RT-PCR and Northern blot analysis of Tg mouse tissues
Heart, spleen, kidney, adrenal, gut, lung, brain, and testis or
ovary were harvested from Tg and non-Tg animals. A portion of each
tissue was fixed in 10% neutral buffered formalin or Bouin’s fixative
(testes only) and processed for histological examination. Total RNA was
extracted from the remaining tissue using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH), reverse transcribed, and
analyzed by PCR using primers A and D for detection of the transgene or
with primers that specifically recognize the endogenous pro-GHRH
message.
For detection of endogenous pro-GHRH transcripts, two primers were used: 5'-CACGTAGATGC CATCTTCACCA-3', which corresponds to the first 22 nt of the GHRH-coding region, and 5'-TCAAGCGTCCGCTGAAGGCTT-3', which hybridizes to the last 21 nt of the GHRH-RP-coding region. The expected size of the resulting PCR product is 221 bp and identifies sequences specific to pro-GHRH but excluded from the transgene. Primer pairs were designed to span exon/intron borders. PCR reactions containing no reverse transcriptase or cDNA served as negative controls. The pCI-SS-RP vector was used as a positive control for detection of the transgene. Separate PCR reactions were run to detect the transgene and endogenous GHRH transcripts in mouse tissues. Testis was used as a positive control for endogenous GHRH messenger RNA (mRNA).
Sequence analysis of SS-RP mRNA from Tg mice
Total RNA was extracted from Tg mice and subjected to RT-PCR
analysis using primers A and D above. PCR products were cloned into the
pCR2.1 vector DNA using the reagents supplied in the TA Cloning Kit
(Invitrogen, Carlsbad, CA). Plasmid DNA was checked by
restriction enzyme digestion and sequenced (Davis Sequencing, Davis,
CA).
For Northern blot analysis, total RNA (10 µg) was electrophoresed on a denaturing agarose gel and transferred to a nylon membrane. Blots were then hybridized with a 32P-labeled SS-RP probe, either alone or in combination with a 600-bp 32P-labeled cDNA probe for mouse SCF (provided by Dr. David Williams, Indiana University School of Medicine). After hybridization, blots were washed and exposed to x-ray film for 2–3 days at -70 C. Northern blots in each experiment contained tissues from Tg and non-Tg littermate mice of the same sex. Experiments were repeated five times using animals that were derived from both founders and with similar or identical results.
Hematopoietic testing
Spleen and bone marrow cells were harvested from 5-week-old Tg
and non-Tg (littermates) mice (n = 4 males and 3–4
females/group). In vitro testing for nucleated cellularity,
myeloid progenitor cell colony formation, and percentage of myeloid
progenitor cells in S phase, as assessed by high specific activity
tritiated thymidine incorporation, was performed as described
previously (7). Briefly, single cell suspensions were prepared, and the
cells were plated (5 x 104/ml for bone
marrow and 5 x 105/ml for spleen) in 1%
methylcellulose containing 30% FBS, 1 U/ml recombinant human
erythropoietin, 5% pokeweed mitogen mouse spleen cell-conditioned
medium, 0.1 mM hemin, and 50 ng/ml recombinant
murine steel factor. Colonies were scored after 7 days incubation at 37
C in 5% O2 and 5%
CO2.
Behavioral testing
Four groups of animals, 6 C3H males, 10 C3H females, 6 Tg males,
and 9 Tg females, were examined between 1000–1200 h at 38, 50, and 65
days of age. Control and Tg animals were weighed and evaluated for
righting ability, postural response, balance, and open field activity,
using standard behavioral and strength testing procedures (8).
Statistical analysis
Results of the hematopoietic testing were analyzed using
Student’s t test, and the results shown in Tables 1 and 2
represent the mean ± SEM of three or four
animals per group, each tested individually.
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Results |
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). On
completion of the second round of PCR, a single product was identified,
containing the GHRH signal peptide-coding region positioned directly 5'
to the coding region for the GHRH-RP portion of the prohormone, and
cloned into the pCI expression vector.
To verify that the transgene expressed the GHRH-RP peptide, Western
analysis using protein extracts prepared from TM4 cells transfected
with the SS-RP expression vector were performed. Western blots of cell
lysates confirmed that the transgene encodes for a peptide of the
correct size (3 kDa) which cross-reacts with GHRH-RP antiserum (Fig. 2). Additionally, GHRH-RP was detected in
the culture medium from these cells, indicating that the peptide was
secreted (data not shown). To detect the peptide in the transfected
cells, the blots were allowed to develop for 30 min, which resulted in
overexposure of both the marker and GHRH-RP-positive control lanes. No
peptide of this size was detected in cells transfected with the empty
vector.
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) and PCR analysis (Fig. 3B). Founder
animals were then crossed with wild-type C3H mice to produce hemizygous
Tg offspring. In successive generations, Tg animals were identified by
PCR analysis only.
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Using RT-PCR, the SS-RP mRNA was detected in all tissues examined,
heart, liver, spleen, kidney, lung, adrenal, testis or ovary, prostate,
and seminal vesicles. No transcript was detected in controls. Figure 4 shows a representative RT-PCR analysis
of RNA extracted from a Tg and a non-Tg animal. RNA extracted from the
hypothalamus of Tg mice also expressed the SS-RP transgene. Similar
transgene expression was observed in animals that were derived from
both of the founder animals. Progeny from both parental lines were used
for subsequent RT-PCR, Northern blotting, and phenotypic analyses. TA
cloning and sequence analysis confirmed that the mRNA transcribed from
the SS-RP transgene in vivo contained the correct SS-RP
sequence (data not shown).
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). In addition to the pro-GHRH band of
the predicted size, a larger PCR product was detected in these
tissues.
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) compared with those from control mice,
in which no SCF signal was detected. Northern analysis failed to detect
either SS-RP or SCF mRNA in adrenal tissue. SCF transcripts of
approximately 6.5 kb were detected in all of the tissues examined,
except for the liver and lung, in which transcripts of 5.7 and 10 kb,
respectively, were detected. Northern blots were probed for SCF, either
alone or in combination with SS-RP. Experiments were repeated three
times with identical results.
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). However, the proliferation of these cells was greatly
increased. The most impressive increase in proliferation was observed
in the spleen, where the percentage of cells in S phase increased by
more than 50-fold for all cell types examined (Table 2). In the bone
marrow, the percentage of CFU-GM, BFU-E, and CFU-GEMM cells in S phase
increased 94%, 46%, and 47%, respectively, compared with controls,
in which no increase in cycling was detected. Interestingly, in the Tg
animals there was also a significant reduction in the nucleated
cellularity in the spleen compared with that in control mice, resulting
in a reduced spleen to body weight ratio (Table 1). |
Discussion |
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A transgenic mouse model was generated that constituitively expresses GHRH-RP mRNA. There is concomitant overexpression of SCF and increased blood progenitor cell cycling rates. GHRH-RP Tg animals do not produce excess GHRH or GH, because the animals exhibited normal body and organ weights.
We were unable to consistently detect GHRH-RP peptide in tissues from the Tg animals. This may be due to several factors. First, we are currently using an antibody that is directed against rat GHRH-RP, which may not be as sensitive for detection of the peptide in the mouse. Second, the stability of the peptide in vivo has not been determined. Indeed, if it is degraded quickly, detection would be difficult. However, results of Western blot analysis of transfected cells confirm that the correct peptide product is expressed. Furthermore, this GHRH-RP is also detected in the medium from these cultured cells, suggesting that it is a secreted peptide (data not shown).
There are two mouse PGK genes under the regulatory control of two distinct promoters. Both genes produce phosphoglycerate kinase, a key enzyme in the glycolytic pathway. The first promoter, PGK-1 is X-linked, ubiquitously expressed, and constituitively active in all cells, including cell types in the hematopoietic system. This is in contrast to the PGK-2 promoter that is autosomally linked and is expressed only in germ cells during spermatogenesis when the PGK-1 promoter is quiescent due to X-chromosome inactivation (14, 15, 16). For the generation of SS-RP Tg mice, the PGK-1 promoter was used, rather than the testis-specific PGK-2, so as to evaluate the effect of GHRH-RP in a variety of tissues.
SCF is produced by stromal cells of the hematopoietic system, by the gonads, and by neurons, skin keratinocytes, and epithelial cells in the gut (11). In the GHRH-RP Tg animals SCF mRNA was detected at much greater abundance in all tissues examined, except for adrenal, compared with tissues from control mice. The fact that we were not able to detect either the transgene or SCF mRNA in adrenal tissue from the Tg animals does not preclude its presence. Although we did not pool adrenals from several animals, it is likely that if we did so, both mRNAs would have been seen, as we readily detected the transgene using RT-PCR in the adrenals of the Tg mice. The 6.5-kb SCF transcript expressed in these Tg animals is identical in size to that previously reported (17). A smaller, 5.7-kb SCF transcript was also detected in the liver, whereas in the lung, the SCF message was greater than 10 kb in size. These results possibly indicate that in these tissues differential processing of SCF mRNA occurs. Further investigation would be needed to answer this question. However, endogenous SCF transcripts of different sizes have previously been described (18, 19), and our results may not represent novel findings.
GHRH-RP Tg mice are characterized by pronounced increases in the mitotic activity of granulocyte-macrophage, erythroid, and multilineage progenitor cells in both the bone marrow and spleens. It has previously been shown that SCF, when combined with other growth factors, enhances colony formation of multipotential stem/progenitor cells and more committed progenitor cells (10, 11, 17, 20, 21). Although SCF normally promotes the survival of hematopoietic stem and progenitor cells (10, 11, 22, 23), the increases in mitotic activity observed in the Tg animals did not result in hypercellularity in either the spleen or bone marrow, nor was there an increase in organ weights. Recently, an activating mutation of the human SCF receptor has been identified and cloned. Hematopoietic testing of the individual with this mutation determined that similar to GHRH-RP mice reported here, there was a marked increase in colony formation for erythroid and myeloid progenitor cells. Further analysis of this patient revealed that the rate of programmed cell death was approximately twice that measured in controls (Dr. Yigal Dror, personal communication). An analogous mechanism may be active in the GHRH-RP mice. Apoptotic rates may be increased in the spleen and bone marrow of the Tg mice, resulting in a decrease in organ to body weights and no increase in the absolute number of cells.
GHRH transgenic mice exhibit a hematopoietic phenotype similar to that reported here for GHRH-RP animals (24). GHRH Tg mice were generated by insertion of a transgene containing the cDNA for the entire pro-GHRH molecule, including the coding sequence for GHRH-RP. The GHRH mice had increased cycling rates for CFU-GM, BFU-E, and CFU-GEMM progenitor cells and no significant change in the absolute numbers of progenitor cells per organ. In contrast to GHRH-RP animals, GHRH mice did not have reduced cellularity in their hematopoietic organs, had excessive linear growth, and developed pituitary tumors. We speculate that in the GHRH Tg animals, increased GHRH leads to their overgrowth and tumor formation via stimulation of the GH axis, whereas increased GHRH-RP in these animals may be responsible at least in part for the increase in cell cycling rates. It should be noted that SCF expression was not evaluated in the GHRH animals.
A role for GHRH in hematopoiesis has previously been reported. The GHRH gene is expressed in blood cells (25, 26, 27), and the mature GHRH peptide has been shown to alter chemotaxis (28), natural killer cell activity (28, 29), and the proliferation of circulating lymphocytes (30, 31). Thus, based on our findings, we propose that in addition to GHRH, the GHRH-RP peptide may be involved in the regulation of hematopoiesis, having either a complimentary or overlapping role.
Few details are known concerning the proteolytic processing of the prepro-GHRH molecule. In cultures of hypothalamic neurons, we have recently determined that pro-GHRH is processed into several peptide products. In addition to GHRH, at least two other smaller peptides were observed. One of these peptides was 3.6 kDa in size, comigrated with 125I-labeled synthetic GHRH-RP when analyzed by HPLC, and was immunoprecipitated using GHRH-RP antisera (32). The second smaller peptide was approximately 2.2 kDa in size and has yet to be characterized. These results suggest that GHRH-RP is produced in vivo and may undergo further enzymatic processing to produce smaller, possibly more active, peptides. Sequence analysis and amino acid synthesis of these peptides will allow us to confirm their identity and characterize their physiological relevance.
Our laboratory has previously reported that GHRH mRNA is expressed in rat testicular germ cells (33, 34) and that its expression is regulated by a tissue-specific promoter (35). Subsequent in vitro studies demonstrated that both GHRH (36) and GHRH-RP-like immunoreactivity are detectable in testicular germ cells and that GHRH-RP is capable of stimulating SCF expression in cultured Sertoli cells (2). The results that are presented here demonstrate that GHRH-RP is also a possible regulator of SCF expression in the hematopoietic system. However, to confirm the physiological role of GHRH-RP, an animal model in which the peptide has been ablated would be of great value. To this end, we are currently developing a GHRH-RP knockout mouse that will be used to better define the actions of GHRH-RP and allow us to verify our hypothesis.
In conclusion, this report is the first in vivo evidence that more than one biologically active peptide is contained within the pro-GHRH molecule. These findings also support the likelihood that this second peptide, GHRH-RP, may participate in the regulation of hematopoiesis via its stimulatory effect on SCF expression. The GHRH-RP Tg mice provide a valuable animal model that can be used to better determine the individual roles of the peptide products of the GHRH gene.
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Acknowledgments |
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Footnotes |
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2 S.F. and R.S. should both be considered as first authors on this
manuscript.
Received September 1, 1999.
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References |
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