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Biomarkers and Prevention Research Branch, National Cancer Institute (L.M.M., A.M., M.J.M., F.C.), Rockville, Maryland 20850; the Department of Histology and Pathology, University of Navarra (L.M.M.), Pamplona, Spain; and the Center for Biologics Evaluation and Research, United States Food and Drug Administration (E.J.U.), Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Luis M. Montuenga, Biomarkers and Prevention Research Branch, National Cancer Institute, 9610 Medical Center Drive, Rockville, Maryland 20850. E-mail montuengal{at}bprb.nci.nih.gov
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apart from the vasorelaxant effect, several other functions have been reported for this peptide hormone. AM has been implicated in the regulation of renal function by means of its potent natriuretic and diuretic properties (14), has bronchodilatory effects (15), and inhibits the release of aldosterone, ACTH, and insulin (24, 28). Recent data suggest the involvement of AM in the regulation of growth. AM stimulates DNA synthesis and cell proliferation of Swiss 3T3 fibroblasts acting via the elevation of intracellular cAMP (32). Low concentrations (10–100 nM) of AM induced the cell cycle progression from G0 to G1 phase and the expression of c-fos messenger RNA (mRNA) in cultured rat aorta muscle cells (20). Moreover, our recent data show that numerous malignant cell lines express not only AM mRNA but also the mRNA for its receptor (25), suggesting the existence of an autocrine or paracrine type of growth regulation in human tumors. We also demonstrated that an inactivating anti-AM monoclonal antibody inhibits tumor cell growth in a dose-dependent manner, an effect that could be reversed by exogenous AM (25). Consistent with this proposed involvement in tumor growth control, AM has been found to be expressed in a variety of human malignancies of both neural and pulmonary lineage (23, 29).
An increasing number of peptides have been shown to be implicated in the long term modulation of growth and differentiation, not only in normal tissues but also in cancer, wound repair, and embryogenesis (3, 22, 26). These observations together with the recent data linking AM to the control of normal and tumor cells prompted us to investigate the possible role of AM in embryogenesis. Although the localization of AM in adult mammalian tissues and human neoplasms has been the object of some studies, the expression pattern during embryogenesis has not been reported. To gain further insight into the role of AM in embryonic development, we analyzed its expression in the mouse [embryonic days 8–16 (E8-16)] and the rat (E10-18) embryo. In the present study we show the expression of AM, AM-R, and PAMP during rodent embryogenesis by means of molecular, biochemical, and histochemical techniques.
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Materials and Methods |
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Antibodies
A previously reported, well characterized rabbit antiserum (no.
2343) was used to localize AM immunoreactivity in embryonic tissues
(23). For the localization of PAMP, an antibody (no. 2463) was raised
in rabbits against a synthetic peptide (P070) that represents the
C-terminal eight amino acids of PAMP linked to a YY residue
(YYWNKWALSR-NH2). The P070 peptide was C-terminally
amidated to conform with the C-terminus of native PAMP. The peptide was
linked to key limpet hemocyanin via glutaraldehyde cross-linkage before
immunization of the rabbits. The antibody was characterized for binding
specificity using a solid phase RIA with [125I]protein A
as the detector. In brief, test peptides were passively absorbed onto
individual wells (100 ng/well; overnight at 4 C) of a 96-well
polyvinylchloride microtiter plate (Dynatech Laboratories, Chantilly,
VA), after which the plate was coated with 1% BSA in PBS to minimize
nonspecific binding. Test peptides included PAMP, P070, AM, CGRP,
gastrin-releasing peptide, glucagon-like peptide-1, vasoactive
intestinal peptide, IBE1, IBE2, substance P, arginine vasopressin, GRF,
cholecystokinin, amylin, gastrin, oxytocin, calcitonin, 
MSH,
pancreatic polypeptide, peptide tyrosine-tyrosine, and Tabanus
atratus hypotrehalosemic hormone (Peninsula Laboratories, Belmont,
CA); BSA (Sigma Chemical Co., St. Louis, MO) was used as a negative
control. Most of these test peptides were chosen because they are also
C-terminally amidated. The antibody was tested at serial 2-fold
dilutions ranging from 1:100 to 1:204,800. Very slight cross-reactivity
was observed with glucagon-like peptide-1 and IBE2, and this was
completely abolished when the antibody was preabsorbed with the peptide
at 1 nmol/ml. The rest of the peptides tested gave negative results,
except for PAMP and P070, which in our assay were readily recognized by
the polyclonal antibody diluted up to 1:25,600.
Immunocytochemical technique
For the immunocytochemical localization of AM and PAMP in
paraffin sections, the avidin-biotin complex technique was employed.
Optimal dilution for anti-P072 was 1:1000, whereas the anti-P070 was
used at a 1:600 dilution. After overnight treatment with the primary
antiserum, sections were incubated with biotinylated goat antirabbit
antiserum (Vector Laboratories) diluted 1:200. The sections were then
treated with the avidin-biotin peroxidase complex (Vector Laboratories)
diluted 1:100 in PBS. Peroxidase activity was demonstrated using
diaminobenzidine-H2O2 as substrate. Finally,
the sections were lightly counterstained with Gills’ hematoxylin.
Solid phase absorption controls were carried out by preincubation (overnight at 4 C) of the primary antiserum with the equivalent peptide previously adsorbed to the wall of a polystyrene tube at a concentration of 10 µg/ml. Another negative control included the use of nonimmune swine serum as first layer and gave no immunocytochemical reaction.
Reverse transcription-PCR (RT-PCR)
The oligonucleotide primers were synthesized using a
MilliGen/Biosearch 8700 DNA synthesizer (Millipore, Marlborough, MA).
Primer sets for AM detection were as follows: sense (AM 250–270),
5'-AAG-AAG-TGG-AAT-AAG-TGG-GCT-3'; antisense (AM 523–542),
5'-TGT-GAA-CTG-GTA-GAT-CTG-GT-3'; and nested probe antisense (AM
430–450), 5'-TCT-GGC-GGT-AGC-GCT-TGA-CTC-3', with a predicted product
of 293 bp. For human AM-R amplification, the following rat primers were
selected from the published sequence (16): sense, AM-R-(476–497),
5'-AGC-GCC-ACC-AGC-ACC-GAA-TAC-G-3'; antisense, AM-R-(923–946),
5'-AGA-GGA-TGG-GGT-TGG-CGA-CAC-AGT-3'; and antisense probe,
AM-R788-811, 5'-GGT-AGG-GCA-GCC-AGC-AGA-TGA-CAA-3', yielding a 471-bp
product. Procedures for RT-PCR using these primers have been described
previously (24). A Perkin-Elmer 9600 Thermocycler (Norwalk, CT) was
used to amplify the samples for 35 cycles, with annealing temperatures
of 55 and 60 C, respectively, for the ligand and its receptor. AM
full-length probe was also generated by the PCR technique from normal
human lung complementary DNA (cDNA; Clontech, Palo Alto, CA). PCR was
performed using sense primer AM-(25–45)
(5'-CCA-CTT-CGG-GCT-TCT-CAC-TGC-3') and antisense primer
AM-(1304–1327) (5'-CAC-GCG-AAC-AAC-TTT-ACA-CCT-3') to obtain a 1303-bp
product. Unique conditions were used during the PCR, with a
(NH4)2SO4-based buffer [10
mM (NH4)2SO4, 10
mM KCl, 10 mM Tris-HCl (pH 8.4), 0.01%
gelatin, and 0.1% Triton X-100] containing 3.75 mM
MgCl2. The annealing temperature was 55 C, and 35 cycles
were performed. The PCR product was then cloned into the pCRII vector
(Invitrogen, San Diego, CA) following the manufacturer’s
procedures.
In situ hybridization
Detection of the mRNA of both AM and its membrane receptor
protein was performed using in situ hybridization. The
full-length cDNAs for AM and AM-R (16) were ligated into the expression
vectors pCRII and pcDNA1, respectively, and used to generate
riboprobes. Both plasmids were linearized with EcoRV and
BamHI and used as a template to synthesize
digoxigenin-labeled sense and antisense RNA transcripts. Hybridization
was performed in a moist chamber at 46 C for 20 h in a 20-µl
volume containing the probe. After stringency washes, visualization of
digoxigenin was performed using digoxigenin detection kit (Boehringer
Mannheim, Indianapolis, IN). Sense probes were used as controls.
Western blotting
To extract the proteins, the tissue samples were immersed in
2 x tricine sample buffer (with 8% SDS; Novex, San Diego, CA)
containing a 1-µM final concentration of each of the
following protease inhibitors: Pefabloc (Centerchem, Stamford, CT) and
bestatin and phosphoramidon (Sigma Chemical Co.). The tissue was then
homogenized, sonicated, and clarified by ultracentrifugation, and the
final protein concentration was determined (BCA kit, Bio-Rad
Laboratories, Richmond, CA). Tissue extracts were diluted to an
approximate protein concentration of 35 µg/50 µl, heated to 95 C
for 3 min, and loaded into the sample well.
Tissue extracts were electrophoretically separated on a gradient 10–20% tricine, SDS-PAGE gel (Novex) and run at 100 V for 2 h under reducing (5% ß-mercaptoethanol) conditions. Five nanograms of synthetic AM were added to a separate well as a positive control. Transfer blotting was accomplished in the same apparatus equipped with a titanium plate electrode and transferred to a polyvinylidene difluoride membrane (Immobilon PVDF, Millipore) at 30 V for 3 h. The membrane was blocked overnight in 1% BSA-PBS, incubated for 1 h in a 1:1000 dilution of rabbit anti-AM (bleed 2343), washed three times in PBS, exposed to 1 x 106 cpm [125I]protein A for 30 min at 4 C, washed six times in PBS, dried, and autoradiographed overnight at -80 C on Kodak XAR5 film (Eastman Kodak, Rochester, NY). The specificity control consisted of a duplicate membrane incubated in antigen-preabsorbed (10 nmol/ml) antiserum.
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, A–D).
Table 1 summarizes the immunocytochemical detection of
AM in the organogenetic period of the mouse. The distribution of AM-
and PAMP-like immunoreactivities in the different tissues throughout
the multiple stages of development of the two species studied were
parallel; AM-like immunoreactivity was generally stronger than
PAMP-like immunoreactivity (Fig. 1, D and E). The distribution for AM
mRNA was coincident with the results obtained by immunocytochemical
techniques. We also detected the mRNA of the AM-R protein in several
tissues of the murine embryo at various stages.
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, A and B).
The resulting 293- and 471-bp RT-PCR products for AM and AM-R mRNA were
confirmed by Southern blotting with antisense nested probes. The
majority of fetal tissues tested were shown to express AM and AM-R mRNA
by RT-PCR, including the whole embryo, placenta, yolk sac, heart, lung,
liver, brain, and gut. Tissue extracts from the same organs were
examined for AM immunoreactivity by Western blot analysis using a
previously characterized rabbit antiserum (23). Figure 2C illustrates
the electrophoretic profile of the AM-like peptides identified.
Molecular mass species of 18, 14, and 6 kDa were identified in tissue
lysates and presumably represent AM precursor, processed intermediates,
and the authentic peptide, respectively. The specificity of our immune
detection assay was confirmed by an antibody absorption control that
eliminated the specific bands.
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and 3, A–C). Maternal
decidual cells and the embryonic cells of the ectoplacental cone were
intensely stained; the trophoblastic giant cells were particularly
distinct (Fig. 3B) The cells of the chorionic plate were also
immunoreactive for AM, but the intensity was lower than that for the
other cell types examined. At the same stage, the visceral yolk sac,
especially the vacuolated and microvillous border of the endodermal
cells, was immunostained for AM. By nonradioactive in situ
hybridization showing a blue/black precipitate, AM and AM-R mRNA were
also localized to several cell types of the developing placenta,
especially the giant trophoblastic cells (Fig. 3, D–H).
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) and also expressed the receptor mRNA. The
developing heart was the organ that showed higher levels of
immunoreactivity throughout all stages of the mouse development. The
myocytes of both atrial and ventricular walls, including the elaborate
trabeculae formed around E10 in the ventricular chamber, were strongly
stained for both AM and AM mRNA (Fig. 4, B–D). The differentiating
smooth muscle fibers found in the large arteries starting on E11 were
strongly immunoreactive for AM (data not shown).
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), showed a detectable level
of AM-like material by day 10. In this stage, the maturing cells of the
ventral horn of the spinal cord showed AM-like immunoreactivity (Fig. 4E). As development continued, both the maturing postmitotic neural
cells of the mantle layer and the neuropil of the marginal layer
stained for AM in E11 embryos. On E12 and the following days, the
difference between differentiated stained and less differentiated
unstained areas of the central nervous system was more clear (Fig. 4F).
From E12 on, specific groups of neurons positive for AM could be seen
in the central nervous system, for example in the mantle layer of the
spinal cord (Fig. 4F). mRNA for AM and for its receptor was shown in
these neurons by in situ hybridization techniques (Fig. 4, G
and H). Neural crest-derived dorsal root ganglia and their segmental
nerve trunks became increasingly immunostained from E11-12 on (Fig. 4, I–K). In the earlier stages (Fig. 4I) only the trunks, but not neural
cell bodies, were detectable with the anti-AM antibodies, whereas in
E14 embryos, both neurons and nerve trunks were strongly stained (Fig. 4K). Throughout the rest of the development, the pattern of staining
that was first seen in the nervous system on E12 remained,
i.e. AM was localized to the neuropil, individual or grouped
neurons within the central nervous system, and dorsal root ganglia. A
certain segmental progression in the degree of staining of dorsal root
ganglia could also be detected within a given stage. Slight
immunoreactivity was observed in the Rathke’s pouch on E11 and in the
forming adenohypophysis in later stages of embryogenesis. Choroid plexi
were also strongly immunostained for AM throughout development. In the
eye, a discrete immunocytochemical signal was detected in the outer
neuroblastic layer of the retina on E14-15. In situ
hybridization showed that the mRNA for AM was present in the same
cells. The lens epithelium and the lens fibers were stained with the
antibodies against AM and, more strongly, against PAMP (data not
shown).
Skeletal and integumentary tissues. The presence of
AM-immunoreactive material was observed at low levels in a diffuse
pattern on groups of mesenchymal cells from E9 on. The mRNA for AM was
present in the same cells at this stage. The expression of AM by the
mesenchymal cells seems to follow precise patterns of spacio-temporal
distribution, as the degree of staining of this tissue varied from one
region to the other and during development. In the E12-13 stage, when
the differentiation of the cartilaginous skeleton proceeds from
condensed mesenchyma/precartilage toward more mature chondrocytic
cells, AM-like material started to be expressed. Precondensing
mesenchymal cells were immunocytochemically negative for AM (Fig. 4I).
Maturing cartilage cells were increasingly positive from E13 on (Fig. 4J), and hypertrophic cartilage cells found from E15 were also highly
stained (Fig. 4K). In situ hybridization showed that AM and
AM-R mRNAs were clearly present in maturing chondrocytes (Fig. 4, G and
H). Osteoblasts of the developing bone in the later stages of
development were strongly immunoreactive for AM. During tooth
development, AM-like expression could be observed on E15-16 in the
condensing mesenchyme and the columnar cells of the inner layer of the
enamel epithelium as well as in the underlying forming bone (Fig. 4L).
After differentiation, the striated muscle cells were also
immunoreactive for AM. This was particularly evident between E12 and
E14, when the tongue elongated and increased in volume, and the
striated muscle fibers differentiated from the myotubes (Fig. 1B).
AM-like expression could be observed in the outer layer of the early
skin, the periderm, from the E10 stage and was remarkably strong in the
epidermal cells of the more differentiated skin on day 14 and
throughout the developmental stages that followed (Fig. 4F). AM mRNA
and the mRNA for the receptor could also be found by in situ
hybridization (Fig. 4, G and H). The cells that form the cortex of the
hair follicles were immunostained, whereas the mesenchyma that will
form the papilla was negative (data not shown).
Embryonic internal organs. Apart from the strong
immunostaining found in the heart throughout development, other
internal organs expressed AM in the mouse embryo. In the gut,
development progressed in a proximal to distal direction, so that at
one time point different segments of the gut were at various stages of
maturation. Initially, when the lumen of the gut was small and was
lined by a pseudostratified columnar epithelium, no AM staining could
be observed (Fig. 5A). In E13 embryos, a slight apical
immunostaining was present in the epithelial cells that formed the
inner lining of the midgut loops. As the gut matured, the peripheral
mesenchyme differentiated toward smooth muscle, whereas the
subepithelial mesenchymal tissue originated the lamina propria,
muscularis mucosa, and submucosa. Prominent AM expression was found on
E13 in the mesenchymal cells that will form the gut muscle layer. From
E14 on, the presence of AM in the epithelium increased. On E16, the
more differentiated simple columnar intestinal epithelium showed a
marked expression of AM located mainly in the apical region of the
enterocytes (Fig. 5B). On days 11–12, bilobed megakaryocytes
expressing AM were particularly detectable in the developing liver
(data not shown). Although the pancreatic primordia were initially
negative for AM, on E14 a number of cells in the developing pancreatic
ducts became immunoreactive for AM (Fig. 4D). The primordia of the
endocrine islets that could be seen on E14 budding off the ducts were
also intensely positive for AM (Fig. 4D). On E10.5, when the early lung
bud was already formed, no expression was seen in the airway
epithelium, and very little expression was found in the mesenchyme that
surrounds it (Fig. 4E). On E11-12, the epithelium of the primordia of
the bronchial tree showed increasing staining. Mesenchymal expression
of AM in the lung was also increasingly present during these stages and
was particularly notable in E13-14 embryos (Fig. 4F). In the lung of
these embryos, the cells surrounding the developing bronchi that will
differentiate into smooth muscle cells stained particularly strongly.
Smooth muscle fibers of the forming arteries also expressed high levels
of AM-immunoreactive material at this stage. By day 14, differentiation
of the tubular epithelial system into the prospective bronchial system
(central) and the prospective respiratory system (peripheral) had
started. In E14-15 embryos. AM immunostaining was present in the latter
epithelia and absent in the former. The pattern of staining reversed in
later stages. In E15-16 embryos, a very clear staining was found in the
epithelial lining of developing bronchi; the smooth muscle and the
cartilage plates and rings of the bronchi and trachea were also stained
(Fig. 5H). During the formation of the kidney, the first AM-expressing
cells were found on E13-14 in the epithelium of the metanephric duct
and later in the metanephric collecting tubules, but not in the
mesenchyma, from which primitive glomeruli forms (Fig. 5, I and J).
Slight AM immunoreactivity was found in the epithelium of the
developing nephrons at the late S-shape body stage and in the final
stages of development in the capillary and mesangial cell tufts of the
developing glomeruli. The primordia of the adrenal glands expressed
AM-immunoreactive material from E12-13 (Fig. 5K). Later in development,
immunoreactivity in both the medulla and cortex of the adrenals could
be found. The mRNA for AM followed the same pattern of distribution at
this stage (Fig. 5L).
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, C and D, and
5C). Nevertheless, the levels of immunocytochemical signal for PAMP
were lower than those for AM.
AM and PAMP expression in the developing rat
The distribution of AM and PAMP immunoreactivity in the rat embryo
was parallel to what we have described in the mouse when equivalent
stages were compared (some representative examples can be found in
Figs. 1, E and F, and 6). The pattern of appearance and
distribution of AM immunoreactivity in the rat was very similar to that
in the mouse, but occurred 1.5–2 days latter in development.
Nevertheless, some differences were found between the two species. In
the embryonic rat, endothelial cells expressed AM earlier and in a
broader pattern than those in the mouse. AM-immunoreactive endothelial
cells could be found as early as the E10 stage in the rat embryo and
were also found in some very small vessels, such as those in developing
glomeruli, on E16-17. The smaller blood vessels were less frequently
stained for AM in the developing mouse embryo. A clear AM staining for
differentiated hepatocytes was found in the E17 rat embryo, which was
less apparent in the equivalent stage of mouse development. In general,
the later stages of development of rat embryos showed higher levels of
AM immunoreactivity than the equivalent stages in the mouse. In
situ hybridization for mRNA as well as for AM-R showed a parallel
anatomical distribution. Our Western blot results (Fig. 2C) showed the
presence of immunoreactive adrenomedullin in several organs of the E16
and E18 rat embryo. RT-PCR of isolated total mRNA also confirmed the
presence of AM and AM-R message in the same stages of the rat embryo
(Fig. 2, A and B).
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Discussion |
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To strengthen the conclusions drawn from the analysis of our immunocytochemical data concerning localization of AM, we have studied the distribution of PAMP encoded in the same precursor. The difference in intensity of immunostaining between AM and PAMP may reflect variations in intracellular levels of both peptides, possibly as the result of regulation of posttranslational processes. Alternatively, these differences may be due to the characteristics of the two antisera; in fact, solid phase RIA characterization studies (see Materials and Methods) showed that the antiserum used to detect PAMP (no. 2463) has lower titer than the one against AM (no. 2343). Moreover, we performed in situ hybridization studies using a specific probe to detect adrenomedullin mRNA. The three different approaches (localization of AM, PAMP, and their mRNA) show similar, if not identical, patterns of distribution. A further verification of our data comes from the simultaneous study of embryos of two species that also show a very analogous spatio-temporal pattern of AM expression. Finally, Western blot and RT-PCR analysis of total embryos and dissected late embryonic tissues were performed. Our biochemical results also show that both adrenomedullin and its receptor are present in different organs during development.
In general, the organs that express AM during murine development correspond to those described for adults, determined by RIA, Northern blot, and immunocytochemistry (10, 11, 23), with heart and adrenals showing higher levels of expression in both cases. Nevertheless, comparison of the distribution of immunocytochemical staining suggests that the relative AM expression in embryonic tissues in late organogenesis is even broader than that in adults.
The gene that codes for a membrane receptor protein specific for AM has recently been cloned from rat lung (16). Our results show a widespread distribution of the receptor in many tissues and organs of the developing mouse and rat. To date, the cellular distribution of this receptor has not been reported, with the exception of rat pancreas and lung (23, 24). The fact that the distribution of the AM receptor in most organs colocalizes with the ligand supports the hypothesis that AM may act locally in a short range autocrine or paracrine mode. Several other growth factors, such as platelet-derived growth factor (PDGF) or the several transforming growth factors-ß (TGFßs), have been proposed to work in an autocrine/paracrine manner on the basis of a close juxtaposition, or even identity, of the cells expressing the receptors and the cells that secrete these factors (1, 4).
The formation of organs during embryogenesis is highly dependent on the sequential and time-regulated expression of a number of growth factors (26). The pattern of distribution of AM-like immunoreactivity and its mRNA during murine development is very similar to that of other well established peptide growth factors. The distribution of AM immunoreactivity shown in our study is very similar to that reported for the TGFß isoforms 1, 2, and 3 in the E12-18.5 mouse embryos (2, 27), suggesting that AM and the TGFßs could be involved in analogous functions. AM distribution also shows some coincident locations with other active peptides such as PDGF, the fibroblast growth factors, insulin-like growth factors I and II, and the recently discovered cardiotropin, a member of the leukemia inhibitory factor/interleukin-6 family. On a comparative basis with the data available on the above-mentioned growth factors, AM seems to be the peptide that is expressed in a larger variety of cells and tissues throughout development, which suggests that this peptide is an important requirement for development to proceed in most organs.
An interesting finding is that AM is intensely expressed in tissues and organs where strong mesenchymal-epithelial interactions take place, such as kidney, lung, tooth primordia, and hair follicles. The expression of AM in these structures is tightly regulated and restricted to particular structures in a given developmental stage. In the kidney, for example, AM is initially expressed only in the ureteric bud-derived metanephric ducts. Later in development, the differentiating nephron tubules and the glomeruli tufts begin to express AM. Other growth factors, such as insulin-like growth factors I and II, TGFß1 and -2, and PDGF are also expressed in the developing kidney in a very specific spatial and temporal pattern (8).
In adult mammals, AM has been shown to have multiple and diverse functions. A broad spectrum of functions, such as in vivo and in vitro regulation of embryonic cell proliferation, differentiation, and migration, is also a common feature for other thoroughly studied growth factors (for a review, see Ref.26). Our results on the localization of AM and its receptor together with the available data on the diverse functions of AM have lead us to formulate the hypothesis that AM could be involved in the regulation of mammalian morphogenesis, possibly through the control of cell proliferation, differentiation, and migration.
Several reports have shown that AM acts as a true growth factor in several normal and malignant tissues. The potent mitogenic effect of AM on embryonic mouse fibroblasts has been reported recently (32). AM also stimulates growth in tumor cell lines via an autocrine loop (25) and promotes cell cycle progression from the G0 to the G1 phase and the expression of c-fos mRNA in vascular smooth muscle cells (20). These reports coincide by showing a cAMP-dependent signal of transduction pathway effecting growth for AM. Interestingly, AM seems to act also as a suppressor of mitogenesis in cultured rat mesangial cells and vascular smooth muscle (5). This duality of regulatory functions could be due to the participation of different protein kinase isoforms in the cellular response to AM (12).
A functional relationship between the expression of AM and differentiation can be deduced by the anatomical distribution of AM immunoreactivity and mRNA. In many embryonic organs, AM expression seems to correlate with the onset and progression of the differentiation process. For example, in the formation of the cartilaginous templates that precede bone, AM and AM-R seem to be expressed in the more mature cell types in the chondrocyte lineage, including the hypertrophic cells, whereas the condensing mesenchymal cells that are present in the previous stages express very low, if any, levels of the peptide and its receptor. The expression of growth factors in the formation of cartilage has been the object of a number of studies (7). Interestingly, a relationship between cAMP and terminal differentiation and calcification of cultured chondrocytes has been clearly established (13), which could support the involvement of AM in the regulation of this process through its cAMP-dependent signal transduction system. Various other active factors that affect chondrocyte differentiation also increase the cAMP concentration in chondrocytes (17). The localization of AM, its mRNA, and the mRNA of its receptor to the growing skeletal structures and the fact that AM elicits its biological actions via the elevation of cAMP levels suggest an involvement of AM in embryonic chondrocyte maturation. Further investigation is required into the proper in vitro and in vivo models to clarify the exact role of AM in chondro- and osteogenesis. Another example in which the expression of AM seems to be associated with the degree of differentiation is neural tissue. Although in the early neural tube, neither immunocytochemical signal of AM nor mRNA of the ligand or the receptor is present, as development proceeds, AM starts to be expressed in many of the more mature and differentiated regions of the central nervous system. The expression of AM in the dorsal root ganglia neurons is also increased in the later stages of development, especially from the moment when most of the dorsal root ganglia neurons are probably formed (21).
Our immunocytochemical results as well as the data on localization of the mRNA of AM and its receptor consistently show that trophoblastic cells, in particular the giant trophoblastic cells, express AM and AM-R at high levels. A possible autocrine or paracrine loop that could regulate the physiology of this cell type is proposed. Murine giant trophoblast cells as well as their human counterparts share in vivo and in vitro invasive properties with malignant cells (33). The AM regulatory role on these cells could be similar to that of TGFß, which is known to be produced by trophoblast cells and seems to be involved in the regulation of trophoblast invasiveness via the induction of tissue inhibitor of metalloproteases and by decreasing collagenase IV activity (6). Invasiveness by trophoblastic cells is also regulated by leukemia inhibitory factor and epidermal growth factor via the stimulation of matrix metalloproteases and other proteinases (9).
In summary, our study has shown a widespread expression of the recently discovered multifunctional peptide AM, its receptor, and its gene-related peptide PAMP throughout the organogenetic stages of mouse and rat embryos. Based on the sites of expression and by analogy with other multifunctional growth factors for which functional studies have been performed, we propose that the AM regulatory system may play an important role in a variety of key processes in embryonic development, namely in the control of growth, differentiation, and invasion. Further functional evaluation, on the basis of our distributional study, will clarify the validity of our hypothesis.
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Acknowledgments |
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Received July 31, 1996.
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A. Martinez, E. Zudaire, S. Portal-Nunez, L. Guedez, S. K. Libutti, W. G. Stetler-Stevenson, and F. Cuttitta Proadrenomedullin NH2-Terminal 20 Peptide Is a Potent Angiogenic Factor, and Its Inhibition Results in Reduction of Tumor Growth Cancer Res., September 15, 2004; 64(18): 6489 - 6494. [Abstract] [Full Text] [PDF]
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 S. Iimuro, T. Shindo, N. Moriyama, T. Amaki, P. Niu, N. Takeda, H. Iwata, Y. Zhang, A. Ebihara, and R. Nagai Angiogenic Effects of Adrenomedullin in Ischemia and Tumor Growth Circ. Res., August 20, 2004; 95(4): 415 - 423. [Abstract] [Full Text] [PDF]
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 S. D. Brain and A. D. Grant Vascular Actions of Calcitonin Gene-Related Peptide and Adrenomedullin Physiol Rev, July 1, 2004; 84(3): 903 - 934. [Abstract] [Full Text] [PDF]
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 M. Zhou, Z. F. Ba, I. H. Chaudry, and P. Wang Adrenomedullin binding protein-1 modulates vascular responsiveness to adrenomedullin in late sepsis Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2002; 283(3): R553 - R560. [Abstract] [Full Text] [PDF]
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 M. J. S. de Miguel and M. A. Burrell Immunocytochemical Detection of Orexin A in Endocrine Cells of the Developing Mouse Gut J. Histochem. Cytochem., January 1, 2002; 50(1): 63 - 70. [Abstract] [Full Text] [PDF]
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 T. Shindo, Y. Kurihara, H. Nishimatsu, N. Moriyama, M. Kakoki, Y. Wang, Y. Imai, A. Ebihara, T. Kuwaki, K.-H. Ju, et al. Vascular Abnormalities and Elevated Blood Pressure in Mice Lacking Adrenomedullin Gene Circulation, October 16, 2001; 104(16): 1964 - 1971. [Abstract] [Full Text] [PDF]
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D. Naot, K. E. Callon, A. Grey, G. J. S. Cooper, I. R. Reid, and J. Cornish A Potential Role for Adrenomedullin as a Local Regulator of Bone Growth Endocrinology, May 1, 2001; 142(5): 1849 - 1857. [Abstract] [Full Text]
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H. Iwasaki, M. Shichiri, F. Marumo, and Y. Hirata Adrenomedullin Stimulates Proline-Rich Tyrosine Kinase 2 in Vascular Smooth Muscle Cells Endocrinology, February 1, 2001; 142(2): 564 - 572. [Abstract] [Full Text] [PDF]
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 M. Garayoa, A. Martínez, S. Lee, R. Pío, W. G. An, L. Neckers, J. Trepel, L. M. Montuenga, H. Ryan, R. Johnson, et al. Hypoxia-Inducible Factor-1 (HIF-1) Up-Regulates Adrenomedullin Expression in Human Tumor Cell Lines during Oxygen Deprivation: A Possible Promotion Mechanism of Carcinogenesis Mol. Endocrinol., June 1, 2000; 14(6): 848 - 862. [Abstract] [Full Text]
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 T. Udono, K. Takahashi, M. Nakayama, O. Murakami, Y. K. Durlu, M. Tamai, and S. Shibahara Adrenomedullin in Cultured Human Retinal Pigment Epithelial Cells Invest. Ophthalmol. Vis. Sci., June 1, 2000; 41(7): 1962 - 1970. [Abstract] [Full Text]
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P. G. Andreis, A. Markowska, H. C. Champion, G. Mazzocchi, L. K. Malendowicz, and G. G. Nussdorfer Adrenomedullin Enhances Cell Proliferation and Deoxyribonucleic Acid Synthesis in Rat Adrenal Zona Glomerulosa: Receptor Subtype Involved and Signaling Mechanism Endocrinology, June 1, 2000; 141(6): 2098 - 2104. [Abstract] [Full Text] [PDF]
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 J. P. Hinson, S. Kapas, and D. M. Smith Adrenomedullin, a Multifunctional Regulatory Peptide Endocr. Rev., April 1, 2000; 21(2): 138 - 167. [Abstract] [Full Text]
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 J. H. Youson The Agnathan Enteropancreatic Endocrine System: Phylogenetic and Ontogenetic Histories, Structure, and Function Integr. Comp. Biol., April 1, 2000; 40(2): 179 - 199. [Abstract] [Full Text] [PDF]
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 F. Yoshihara, T. Nishikimi, T. Horio, C. Yutani, N. Nagaya, H. Matsuo, T. Ohe, and K. Kangawa Ventricular adrenomedullin concentration is a sensitive biochemical marker for volume and pressure overload in rats Am J Physiol Heart Circ Physiol, February 1, 2000; 278(2): H633 - H642. [Abstract] [Full Text] [PDF]
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T. H. Elsasser, J. L. Sartin, A. Martínez, S. Kahl, L. Montuenga, R. Pío, R. Fayer, M. J. Miller, and F. Cuttitta Underlying Disease Stress Augments Plasma and Tissue Adrenomedullin (AM) Responses to Endotoxin: Colocalized Increases in AM and Inducible Nitric Oxide Synthase within Pancreatic Islets Endocrinology, November 1, 1999; 140(11): 5402 - 5411. [Abstract] [Full Text]
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N. Jiménez, A. Calvo, A. Martínez, D. Rosell, F. Cuttitta, and L. M. Montuenga Expression of Adrenomedullin and Proadrenomedullin N-terminal 20 Peptide in Human and Rat Prostate J. Histochem. Cytochem., September 1, 1999; 47(9): 1167 - 1178. [Abstract] [Full Text]
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E. B. Pewitt, R. Haleem, and Z. Wang Adrenomedullin Gene Is Abundantly Expressed and Directly Regulated by Androgen in the Rat Ventral Prostate Endocrinology, May 1, 1999; 140(5): 2382 - 2386. [Abstract] [Full Text]
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L. M. Montuenga, J. M. Mariano, M. A. Prentice, F. Cuttitta, and S. B. Jakowlew Coordinate Expression of Transforming Growth Factor-{beta}1 and Adrenomedullin in Rodent Embryogenesis Endocrinology, September 1, 1998; 139(9): 3946 - 3957. [Abstract] [Full Text] [PDF]
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 B. L. Jensen, S. Gambaryan, E. Schmaus, and A. Kurtz Effects of dietary salt on adrenomedullin and its receptor mRNAs in rat kidney Am J Physiol Renal Physiol, July 1, 1998; 275(1): F55 - F61. [Abstract] [Full Text] [PDF]
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V. A. Cameron and A. M. Fleming Novel Sites of Adrenomedullin Gene Expression in Mouse and Rat Tissues Endocrinology, May 1, 1998; 139(5): 2253 - 2264. [Abstract] [Full Text] [PDF]
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Y. Isumi, N. Minamino, T. Katafuchi, M. Yoshioka, T. Tsuji, K. Kangawa, and H. Matsuo Adrenomedullin Production in Fibroblasts: Its Possible Function as a Growth Regulator of Swiss 3T3 Cells Endocrinology, May 1, 1998; 139(5): 2552 - 2563. [Abstract] [Full Text] [PDF]
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 J. Cornish, K. E. Callon, C. Q.-X. Lin, C. L. Xiao, T. B. Mulvey, D. H. Coy, G. J. S. Cooper, and I. R. Reid Dissociation of the effects of amylin on osteoblast proliferation and bone resorption Am J Physiol Endocrinol Metab, May 1, 1998; 274(5): E827 - E833. [Abstract] [Full Text] [PDF]
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A. MartInez, T. H. Elsasser, C. Muro-Cacho, T. W. Moody, M. J. Miller, C. J. Macri, and F. Cuttitta Expression of Adrenomedullin and Its Receptor in Normal and Malignant Human Skin: A Potential Pluripotent Role in the Integument Endocrinology, December 1, 1997; 138(12): 5597 - 5604. [Abstract] [Full Text] [PDF]
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J. Cornish, K. E. Callon, D. H. Coy, N.-Y. Jiang, L. Xiao, G. J. S. Cooper, and I. R. Reid Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo Am J Physiol Endocrinol Metab, December 1, 1997; 273(6): E1113 - E1120. [Abstract] [Full Text] [PDF]
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