1 The Division of Endocrinology, Metabolism and Nutrition, Endocrine Research
Unit, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, MN
55905, USA
2 The University of Aarhus, Department of Molecular Biology, Science Park,
Gustav Wieds Vej 10C, DK-8000, Aarhus C, Denmark
3 The Department of Pediatric and Adolescent Medicine, and Department of
Biochemistry and Molecular Biology, Mayo Clinic and Mayo Foundation, 200 First
Street SW, Rochester, MN 55905, USA
* Author for correspondence (e-mail: conover.cheryl{at}mayo.edu)
Accepted 20 November 2003
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SUMMARY |
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Key words: Pregnancy associated plasma protein A, Insulin-like growth factor, Gene targeting, Metalloproteinase
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Introduction |
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Human PAPPA has an elongated zinc-binding motif, with residues coordinating
the catalytic zinc ion of the active site, and a structurally important
methionine residue located downstream in the so-called Met-turn, both of which
are strictly conserved within the metzincin superfamily of metalloproteinases
(Stocker et al., 1995;
Boldt et al., 2001
;
Overgaard et al., 2003
).
Metzincins are remarkably similar in their tertiary structure, although they
have only limited sequence identity. PAPPA is distinct from the other four
metzincin groups (astacins, serralysins, adamalycins or reprolysins, and
matrix metalloproteinases) because of a characteristic residue directly
following the zinc-binding motif, and the unusual distance between the
zinc-binding motif and the Met-turn (Boldt
et al., 2001
). The overall sequence identity between murine and
human PAPPA is 91% (Soe et al.,
2002
), with the coding of all residues of the zinc binding and
Met-turn consensus conserved in exon 4
(Overgaard et al., 2003
).
In this study we generated PAPPA-null mice by gene targeting and demonstrate a crucial role for PAPPA during fetal development.
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Materials and methods |
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Male chimeras from these three clones (designated E3, E7, D10) were then cross-bred with C57Bl/6 females and germ-line transmission was obtained for all three. Heterozygous mutants were identified by Southern analysis of tail tip DNA. After transmission of the mutations, intercrosses between heterozygous progeny yielded homozygous mutants for E3, E7 and D10. Littermates obtained by breeding heterozygous males and females were used for all phenotypic analyses.
Genotyping
Southern analysis
ES cells and mouse tail tip DNA were digested with BamHI, run on a
0.8% agarose gel and transferred to Hybond (Amersham Pharmacia, Arlington
Heights, IL). Membranes were prehybridized for 1 hour at 65°C in RapidHyb
and then hybridized overnight at 65°C in the same solution containing
32P-labeled 3' probe (see
Fig. 1A). Membranes were washed
at 65°C in 1xSSC/0.1% SDS, 0.3xSSC/0.1% SDS and
0.1xSSC/0.1% SDS, and then exposed to film. With this probe, homologous
recombination in ES cells would be expected to show both the wild-type 15 kb
fragment and a mutant 2.6 kb fragment of BamHI-digested DNA. For
mouse tail DNA, we would expect wild-type mice to have a single 15 kb
fragment, heterozygous mice to have both 15 kb and 2.6 kb fragments, and
homozygous mutants to have single 2.6 kb fragments (see insert
Fig. 1A).
PCR
PCR on mouse tail DNA was performed using primers: 5'-ATG ATT CAT GAG
ATT GGG CAT AG-3' and 5'-TGT TGT AAG GAG TGT TGA AGA AGC-3',
to detect exon 4 in the mouse PAPPA gene; and 5'-AGG ATC TCC
TGT CAT CTC ACC TTG CTC CTG-3' and 5'-AAG AAC TCG TCA AGA AGG CGA
TAG AAG GCG-3', to detect neo. PCR reactions containing these
primers generated fragments in ethidium bromide-stained agarose gels of 223 bp
for the endogenous exon 4-containing PAPPA gene and 492 kb for the
recombinant neo-containing gene.
PCR-based sexing of mouse embryos was performed according to the method of
McClive and Sinclair (McClive and
Sinclair, 2001), using yolk sac DNA and primer pairs for
Sry, the master sex determining gene on the Y chromosome, and
myogenin, a control gene. It is known that there is a great deal of
variability in embryo sizes even among littermates, and that male embryos may
develop faster than females. Therefore, yolk sacs from embryos were sexed by
PCR to rule out possible gender bias.
RT-PCR
Total RNA was extracted from whole embryos and tissues using RNeasy Mini
kit (Qiagen, Valencia, CA) and treated with DNase (DNA-free, Ambion, Austin,
TX). RNA (400 ng) was reverse-transcribed using TaqMan Reverse Transcription
reagents (PE Biosystems, Foster City, CA), according to manufacturer's
instructions. Primer sequences for assessment of PAPPA mRNA
expression were as above for a predicted PCR product of 223 bp. Having
established the linear range, amplifications were performed for 32 cycles. The
initial denaturation was performed at 94°C for 5 minutes, cycles were at
94°C for 30 seconds, 62°C for 30 seconds and 72°C for 1 minute,
and full-length products were obtained by a final elongation period of 10
minutes at 72°C. PCR reaction products were analyzed by agarose gel
electrophoresis and visualized by ethidium bromide staining.
Primary cell cultures
Primary cultures of mouse embryo fibroblasts (MEF) were derived from E13.5
day embryos from heterozygous matings. Tissue from each embryo was used for
genotyping. Embryos were washed, minced, trypsinized and single cell
suspensions plated in high glucose DMEM, containing glutamine, penicillin,
streptomycin, ß-mercaptoethanol and 10% ES cell-tested FCS. Cells at
passage 2-4 were used for experiments.
IGFBP4 protease activity assay
Primary cultures of MEF were washed and changed to serum-free medium. After
24 hours, conditioned medium was collected for cell-free assay. IGFBP4
proteolysis was assayed as described previously
(Conover et al., 1995;
Conover et al., 2001
;
Lawrence et al., 1999
;
Overgaard et al., 2000
), by
incubating MEF-conditioned medium samples at 37°C for 6 hours with
125I-IGFBP4 in the absence and presence of 5 nM IGF2. Proteins were
separated by SDS-PAGE and visualized by autoradiography.
Cell proliferation
[3H]Thymidine incorporation was performed as described
previously (Conover et al.,
1995; Ortiz et al.,
2003
). MEF cultures were grown to 80% confluence, washed twice and
changed to 0.1% FCS for 48 hours prior to experimental additions.
[methyl-3H]Thymidine (0.5 µCi/ml; DuPont-NEN, Boston, MA) was
added for 22-26 hours after the experimental additions. For the experiments in
Table 2, cultures were washed
three times immediately before addition of IGFs. For the experiments in
Fig. 5B, 25 nM IGFBP4 or
IGFBP3±5 nM IGF were directly added to the 48-hour-conditioned medium.
Results are calculated as the percentage of total counts in the incubation
medium that are incorporated into acid-precipitable material.
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Skeletal staining
Embryos were eviscerated, skinned, fixed in ethanol and then stained with
Alcian Blue 8GS (cartilaginous elements) and Alizarin Red S (mineralized
elements) at 37°C for 3-5 days, as adapted from McLeod
(McLeod, 1980). The tissues
were cleared with 1% KOH and the skeletons stored in glycerol.
In situ hybridization
Whole-mount embryos were hybridized with digoxigenin-labeled RNA probes as
described previously (Fuchtbauer et al., 1995). cDNA clones, all contained in
vectors with the T7 promoter, were used as templates for in vitro
transcription. Murine PAPPA clone E11 (Soe
et al., 2002) was used for PAPPA. Rat IGF2 and mouse
IGFBP4 cDNAs were kindly provided by Dr C. Bondy (NIH-NICHD,
Bethesda, MD) and Dr S. Drop (Rotterdam, The Netherlands), respectively.
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Results |
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Skeletal development
Investigation of the effect of PAPPA gene disruption on skeletal
development during embryogenesis indicated developmental delay in the
appearance of ossification centers in the clavicle, facial and cranial bones,
vertebrae, and the digits of the forelimbs and hindlimbs
(Fig. 2). The initiation of
mineralization occurs between E12.5 and E13.5 in the mouse. As expected
(Huang et al., 1997), the
first bone to mineralize in both wild-type and PAPPA-/- mice was
the clavicle. At E13.5, the middle portion of the clavicle was clearly
mineralized in the wild type, whereas mineralization was just initiated in
PAPPA-/- littermates (Fig.
2A). The delay in mineralization occurred in bones that form via
intramembranous ossification (cranial vault) and endochondral ossification
(vertebrae, metatarsals) (Fig.
2B,C,D). At E16.5, no caudal vertebrae were mineralized in the
PAPPA-/- mouse, whereas three to four caudal vertebral elements
were undergoing mineralization in wild-type littermates. Likewise an
additional metatarsal had initiated mineralization in the wild type compared
with the PAPPA-/- mice. The frontal and interparietal bones of the
cranial vault were both undergoing mineralization at E16.5, but these
processes were far less complete in PAPPA-/- mice. Examination of
several skeletons from E13.5 to E18.5 littermates indicated ossification
delays of approximately 1 day.
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Discussion |
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In many instances, ablation of one member of a gene family has resulted in compensation by one or more other members. We can argue against complete functional redundancy in this case because specific IGFBP4 protease activity is abolished and PAPPA-null mice have a distinct phenotype, i.e. proportional dwarfism. In addition, there was no apparent compensation in other components of the IGF system in the PAPPA-/- mice, when assessed as follows.
(1) In situ hybridization demonstrated the widespread pattern of
IGF2 and IGFBP4 mRNA expression during normal embryogenesis,
as has been previously reported
(Stylianopoulou et al., 1988;
Lee et al., 1990
;
Schuller et al., 1993
;
Pintar et al., 1998
), with no
apparent change in spatial pattern or level of mRNA in the PAPPA-deficient
embryos. Similarly, there was no significant difference in steady-state levels
of IGF2 and IGFBP4 mRNA in wild-type and PAPPA-/-
embryos as determined by northern analysis.
(2) Serum levels of IGF1 were not different between wild-type and PAPPA-deficient mice.
(3) Fibroblasts from PAPPA-null E13.5 embryos were as responsive as those from wild-type embryos to growth stimulation by IGF1 and IGF2, indicating that capacity for type I IGF receptor signaling was not altered in the absence of PAPPA gene expression.
Thus, IGF ligands are expressed, type I IGF receptors are present and operational, and the potential reservoir function of IGFBP4 is preserved in both wild-type and PAPPA-/- embryos. The major difference is the presence or absence of PAPPA and its proteolytic activity.
Although PAPPA-deficient mice were significantly smaller than wild-type littermates, the organ-to-body weight ratios were normal. Thus, the impact of the PAPPA mutation could be on control of the timing, rate and/or duration of the growth process early in embryonic development that is important for size control. Skeletal development experiments indicated striking differences in embryonic size and ossification between wild-type and PAPPA-null embryo littermates as early as E13.5. These findings, together with the findings of developmental delay as early as E12.5 as assessed by in situ hybridization and proportional reduction in organ weights, indicate that the impact of PAPPA gene disruption on embryonic growth occurred prior to organogenesis (E10-E14 in the mouse).
The phenotype of the PAPPA-null mouse is strikingly similar to that of the
IGF2-null mouse. Thus, mice lacking the ability to express the IGF2
gene are small at birth and remain 60% of the size of wild-type littermates
during post-natal life (DeChiara et al.,
1990). They also show a similar delay in skeletal ossification
(Liu et al., 1993
). The
principal period of IGF2 impact on body size has been suggested to
occur during E9-E10 (Burns and Hassan,
2001
). In the absence of the IGF2 gene there are
significant decreases in cell proliferation during this period of time that
could account for the smaller size of the embryo, detected at
E12.5.
Double mutation studies indicated that IGF2 signals through type I insulin and
IGF receptors in early embryogenesis (Baker
et al., 1993
; Louvi et al.,
1997
), and a similar growth-deficient phenotype occurs with
disruption of insulin receptor substrate 1, a key intracellular
transducer of type I insulin and IGF receptor signaling
(Araki et al., 1994
;
Tamemoto et al., 1994
). The
most straightforward explanation for the observed alterations in fetal growth
in PAPPA-deficient mice is that PAPPA is necessary for amplification of
receptor-mediated IGF2 signaling through specific IGFBP4 proteolysis during a
crucial period in embryogenesis. This model is supported by the similar
phenotype of PAPPA-, IGF2- and insulin receptor substrate 1-null mice.
Interestingly, studies by Pintar et al. indicate that IGFBP4-null mice are
10-15% smaller than wild-type mice (Pintar
et al., 1998
). This would support the view of IGFBP4 as an
important local reservoir of IGFs that can then be released at definitive
times and discrete regions through proteolysis. Furthermore, this model is
supported by our in vitro studies with fibroblasts from wild-type and
PAPPA-/- embryos, demonstrating the importance of PAPPA-induced
IGFBP4 proteolysis in regulating endogenous and exogenous IGF bioactivity.
Currently, the only identified function of PAPPA is as an IGFBP protease.
However, contributions of other IGF system components, as well as possible
IGF-independent effects of PAPPA, remain to be determined.
A major conclusion from our genetic data is that PAPPA is clearly involved
in optimal fetal growth and development. This growth regulatory mechanism may
also underlie the association between PAPPA levels and fetal development
recently reported in humans (Smith et al.,
2002). Furthermore, PAPPA-deficient mice provide a unique model
for testing hypotheses concerning the role of PAPPA in regulating IGF action
postnatally, e.g. during bone remodeling and vascular repair (Z. T. Resch, R.
D. Simari and C. A. Conover, unpublished).
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ACKNOWLEDGMENTS |
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