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Departments of Pharmacology (K.P., G.O., V.M.), Anatomy and Neurobiology (K.M.B., V.M.), and Gynecology and Obstetrics (G.O.), University of Vermont College of Medicine, Burlington, Vermont 05405; and Laboratoire de Cancerologie Experimentale, EA 2671, Faculté de Médecine Nord (L.O.), 13916 Marseille Cedex 20, France
Address all correspondence and requests for reprints to: Victor May, Ph.D., Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Given Health Science Building, Burlington, Vermont 05405. E-mail: vmay{at}zoo.uvm.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF, a pleiotropic cytokine first identified in tumor cells as a permeability factor in microvessels, is a potent endothelial cell mitogen that stimulates vasculogenesis and angiogenesis. Although several other members of the VEGF cysteine knot motif family have been identified, including placental growth factor (PlGF), VEGF-B, VEGF-C, and VEGF-D, the roles of VEGF in reproductive vascular function have been best studied. The human VEGF gene is organized into 8 exons and differential alternative splicing results in the synthesis of multiple VEGF isoforms of 121, 145, 165, 189, and 206 amino acids (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206, respectively); the corresponding murine forms are shorter by 1 amino acid (8). All of the VEGF isoforms except VEGF121 contain heparin-binding domains and can bind to extracellular matrix; VEGF165 is the more potent endothelial cell mitogen and the dominant variant in the uterus of many species (9).
The hormonal regulation of uterine VEGF messenger RNA (mRNA) by estrogen and progesterone appears consistent with the differential patterns of VEGF expression during the estrous cycle in rats and the menstrual cycle in humans. Estradiol administration to rats rapidly induces uterine VEGF mRNA expression (10); VEGF mRNA levels increase approximately 2-fold during proestrus and estrus (11). In the human menstrual cycle, VEGF mRNA expression is increased in late proliferative and luteal phases (12). Furthermore, during pregnancy, VEGF expression is altered in a temporally and spatially defined manner in areas of angiogenesis and vascular reactivity at implantation sites and in placenta (13). These studies support an essential role for VEGF in reproductive function.
VEGF glycoproteins associate as homo- or heterodimers before binding to
the various receptors identified to date. The best-characterized of
these high affinity cell surface receptors are VEGFR1 or Flt-1
(Fms-like tyrosine kinase-1) and VEGFR2 or Flk-1 (fetal liver
kinase)/kinase-insert domain receptor (KDR), two receptor
protein tyrosine kinases localized primarily on endothelial cells.
These receptors possess seven extracellular Ig-like domains, a single
transmembrane region, and an intracellular consensus tyrosine kinase
sequence that is interrupted by a kinase insert domain
(8). Receptor occupancy by VEGF results in receptor
autophosphorylation and the phosphorylation of downstream effectors,
including phospholipase C
and small GTP-binding proteins, and the
recruitment of adaptor proteins to the receptor to initiate the
angiogenic and other vascular responses (14). Yet, VEGFR1
appears to only facilitate cellular migration, whereas VEGFR2 induces
endothelial cell mitosis and proliferation, suggesting that the
signaling cascades initiated by each receptor may be different
(14).
More recently, however, the mitogenic VEGF165 was shown to bind selectively to neuropilins, a small family of type I transmembrane receptors first identified on neuronal growth cones as mediators of class III semaphorin molecules in axonal guidance (15, 16, 17, 18, 19, 20). The 2 neuropilins identified to date, neuropilin-1 (Npn-1) and neuropilin-2 (Npn-2), demonstrate 44% amino acid homology within structural domains that are unrelated to those found in VEGFR1 and VEGFR2 (21, 22). The extracellular domain contains 2 C1r/C19, Uegf, BMP1 (CUB) complement-binding motifs (domains a1 and a2), 2 coagulation factor V and VIII domains (domains b1 and b2), and a meprin, A5, Mu (MAM) domain (domain c) (19). The short intracellular cytoplasmic tail (40 amino acids) does not exhibit kinase activity or consensus phosphorylation sites (21), but is highly conserved among species, suggesting that it may have as yet undefined functions (18, 23). The ability of VEGF to bind Npn-1 and Npn-2 was first identified in expression cloning studies and has suggested that vascular growth and axonal guidance may share similar mechanistic principles (15).
The tissue expression of Npn-1 and Npn-2 is not always coincident. Although neuropilins have been identified on many nonneuronal cells, including endothelial cells, their roles in vascular growth, remodeling, and function have not been elucidated completely (15, 24, 25, 26). The neuropilins have been suggested to act as coreceptors; Npn-1, for example, has been shown to enhance VEGF binding to VEGFR2 and facilitate cellular chemotaxis (15). Neuropilin transgenic studies, however, have been central in implicating these receptors in vascular development and function. Homozygous Npn-1 knockouts are embryonically lethal due to cardiovascular failure (20); ectopic Npn-1 overexpression leads to vascular abnormalities, hemorrhaging, and embryonic death (23). Given the vascular adaptations that must accompany the periodicity of the female reproductive tract and pregnancy, we have examined rat uterine neuropilin expression and regulation to assess the potential roles of these proteins in the vascular and endometrial changes associated with reproductive functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) according to the following
parameters: initial denaturation, 94 C for 5 min (34 cycles);
denaturation, 94 C for 30 sec; annealing, primer-specific annealing
temperature for 30 sec; extension, 72 C for 45 sec; and final
extension, 72 C for 5 min. The amplified products were resolved on
1.6% agarose gels, stained with ethidium bromide, and visualized under
UV illumination. Complementary DNA synthesis in the absence of RNA
template or reverse transcriptase and amplification without template,
primers, or DNA polymerase failed to yield products.
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),
and the fragments were analyzed by ethidium bromide staining on a 1.6%
agarose gel. For sequencing and riboprobe synthesis, the amplified
products were gel-purified and polished with Pfu DNA
polymerase, and the blunt-ended products were ligated into the
EcoRV restriction site of pBluescript II
KS- cloning vector (Stratagene, La
Jolla, CA). The nucleotide sequence of the insert was verified by
automated fluorescent dideoxy dye terminator sequencing (Vermont Cancer
Center DNA Analysis Facility, Burlington, VT). The vectors were
linearized with NotI or HindIII for subsequent
synthesis of sense and antisense riboprobes with T7 or T3 DNA
polymerase.
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-32P]UTP-labeled antisense neuropilin
riboprobes at 65 C for 16 h. Membranes were washed under high
stringency and apposed to autoradiography film or analyzed by storage
phosphorimaging. To normalize the RNA loaded onto each lane, the
membranes were stripped in 1% SDS at 100 C and reprobed for 18S
ribosomal RNA (rRNA).
Immunocytochemistry
Uteri from 4% paraformaldehyde perfusion-fixed animals were
cryoprotected in 20% sucrose/PBS overnight, washed, and embedded in
Tissue-Tek OCT compound (Miles, Inc., Elkhart, IN). Cryosections (10
µm) were mounted onto subbed slides, and endogenous tissue peroxidase
activity was quenched with 0.3% hydrogen peroxide for 15 min. Tissue
nonspecific binding sites were blocked in 1:200 normal goat serum, and
the sections were incubated subsequently in affinity-purified rabbit
anti-Npn-1 (1:16,000) or anti-Npn-2 (1:4,000) for 16 h at 4 C
(antibodies provided by Dr. David D. Ginty, Department of Neuroscience,
The Johns Hopkins University School of Medicine, Baltimore, MD)
(17, 21). The sections were subsequently incubated with
biotinylated goat antirabbit IgG (1:400, 90 min) and the
avidin-biotinylated enzyme complex (1:200, 60 min; Vector Laboratories, Inc., Burlingame, CA) for the immunocytochemical
peroxidase reaction using diaminobenzidine and hydrogen peroxide as
substrates. Sections were viewed using a Leica Corp. DMRB
microscope (Rockleigh, NJ); no staining was observed upon omission of
primary or secondary antibodies.
Western blot analysis
Uteri were homogenized in 20 mM NaTes, pH 7.0,
containing 0.3 mg/ml phenylmethysulfonylfluoride, 2 µg/ml leupeptin,
and 1 µg/ml benzamidine. After low speed centrifugation to remove
debris, aliquots of the homogenates were removed for protein assay
using the bicinchoninic acid reagent (Pierce Chemical Co.,
Rockford, IL), and the samples were resuspended in Laemmli sample
buffer for fractionation on 8% SDS-PAGE gels. Proteins were
transferred to Nytran Plus membrane (Schleicher & Schuell, Inc., Keene, NH); the membranes were blocked in 5% nonfat dry
milk and incubated in Npn-1 antibody (0.5 µg/ml) or Npn-2 antibody (2
µg/ml) for 16 h at 4 C. The washed membranes were subsequently
incubated in 1:200 horseradish peroxidase-conjugated goat antirabbit
IgG for enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ).
Animals and treatments
Animal protocols were approved by the institutional animal care
and use committee at University of Vermont (Burlington, VT). Female
Sprague Dawley rats (200–250 g) were maintained on a 14-h light and
10-h dark schedule and provided with food and water ad
libitum. For estrous cycle studies, vaginal smears were obtained
daily between 0800–1000 h; only rats with four consecutive 4-day
estrus cycles were selected for study. For hormone replacement studies,
ovariectomized (OVX) animals were allowed to recover for 1 week before
the replacement regiment. OVX animals received daily for 7 days sc
injections of either 17ß-estradiol (4 µg/day; Merck & Co., Inc., Darmstadt, Germany), progesterone (1 mg/day;
Sigma, St. Louis, MO), or both steroids together. Control
OVX animals received sesame oil vehicle alone. Animals were killed by
decapitation, and trunk blood was collected for serum LH and estradiol
measurements by RIA. Reproductive tissues were collected rapidly and
prepared for total RNA extraction.
Statistics
ANOVA was used to determine differences among treatments, and
Newman-Keuls test was used in post-hoc analysis; analysis
was performed using SigmaStat 2.03 statistics software for Windows
(SPSS, Inc., Chicago, IL). Changes in mRNA levels were
evaluated using P < 0.05 as the level of
significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). For many
tissues, amplification using primers specific to the CUB region of
Npn-2 revealed expression similar to that of Npn-1 mRNA. Although the
relative levels of the 297-bp Npn-2 product appeared high in uterus,
ovary, and placenta, Npn-2 mRNA expression, by contrast, was barely
detectable in the uterine artery. Both VEGFR1 and VEGFR2 mRNAs were
also identified in these tissues, although VEGFR1 mRNA levels were
higher in ovary, and VEGFR2 levels were lower in uterus. Diagnostic
endonuclease restriction analyses were used to verify the
identities of the neuropilin- and VEGFR-amplified products. After
recovery and purification of the amplified products, specific
endonuclease restriction digestion of each product produced the
anticipated cleavage fragments, validating the accuracy of these
results (Fig. 2 and Table 2
). Further
subcloning of these isolated control amplified fragments into
pBluescript plasmids and fluorescence dideoxy dye terminator sequencing
verified the fidelity of the receptor products.
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).
Significantly, a smaller more than 90-kDa protein band for the soluble
form of Npn-1 was not apparent in rat uterus (data not shown). The
identification of these neuropilin proteins was consistent with the
neuropilin transcript studies and further supported a physiological
role for neuropilins in these tissues.
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); little or no staining was apparent
in either interstitial connective tissue or smooth muscle cells. The
endothelial cell Npn-1 staining was observed to extend into the
endometrial capillaries. The most prominent Npn-1 staining in the
endometrium, however, was in the glandular epithelial cells (Fig. 4B);
no staining was apparent in the stroma. By contrast, Npn-2
immunoreactivity was not apparent in myometrial vessels or endometrial
capillaries (Fig. 4C), and this apparent absence of staining in the
vascular structures was consistent with the very low levels of Npn-2
mRNA in uterine arteries (see Fig. 1). Similar to Npn-1, most of the
Npn-2 immunoreactivity in the uterus was confined to the glandular
epithelium (Fig. 4D).
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), and among
the different hormonal treatments, only progesterone significantly
induced Npn-1 mRNA levels. Normalized to tissue 18S rRNA levels,
progesterone increased Npn-1 mRNA levels approximately 2-fold
(P < 0.001). 17ß-Estradiol replacement for 7 days
after ovariectomy had no significant effect on uterine Npn-1 mRNA
expression, but may have induced a smaller 4-kb Npn-1 mRNA not
previously observed in OVX tissues; in combined estrogen and
progesterone treatments, estrogen appeared to have blunted the
progesterone-induced increase in Npn-1 mRNA levels to patterns observed
for estrogen alone (P < 0.001). When the blots were
stripped and reprobed for VEGFR2 mRNA, a comparable pattern of
expression was observed (Fig. 5).
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). Uterine Npn-2 mRNA levels
from progesterone-replaced animals did not appear different from those
in OVX animals; in the combined steroid treatment paradigm,
progesterone antagonized the inhibitory effects of estrogen and
restored Npn-2 mRNA expression to control OVX levels. These results
suggested that both Npn-1 and VEGFR2 receptor subtypes may be under
similar stimulatory progesterone control, whereas Npn-2 mRNA regulation
appeared divergent and under estrogen-mediated inhibition.
Altered Npn-1 and Npn-2 mRNA expression during the estrous
cycle
To assess whether the hormonal regulation of neuropilins is
reflected in the normal periodic changes in the uterus, uterine
neuropilin mRNA levels were also assessed during the rat estrous cycle.
Vaginal smears were prepared for four complete cycles to verify the
uterine stages and the tissues harvested for Northern analyses
(29). Npn-1 mRNA levels in the rat uterus appeared low
during proestrus and estrus despite increasing estrogen levels, as
shown by RIA levels of plasma estradiol and LH (data not shown).
Strikingly, the 6.5-kb form of Npn-1 mRNA was increased maximally more
than 7-fold when progesterone levels were highest at metestrus and was
elevated more than 4-fold (P < 0.004) at diestrus
after the progesterone peak (Fig. 6). The
levels of the other Npn-1 mRNA forms were also increased by
progesterone. These results were more dramatic than those of the
hormone replacement studies, suggesting that other physiological
regulators may have synergistic effects with progesterone in augmenting
neuropilin expression. The changes in Npn-2 mRNA levels were more
modest and were not significantly altered during the estrous cycle
(Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alternative exon usage in the VEGF gene has been shown to result in the synthesis of multiple tissue- and function-specific VEGF variants, and in the uterus, the mitogenic VEGF165 is the predominantly expressed isoform. The neuropilins, characterized originally as receptors for the semaphorin proteins associated with axonal formation and guidance, have been identified as selective high affinity binding sites of VEGF165 and represent an important new class of receptors essential for cardiovascular development, function, and growth (15). Although VEGF165 and semaphorin 3A (Sema3A) have distinct structures, biological activities, and neuropilin-binding domains, both molecules have similar high affinities for Npn-1 (15, 16). Both the CUB and coagulation domains of Npn-1 appear essential for semaphorin ligand binding; by contrast, only the coagulation domains are necessary and sufficient for binding of VEGF165 (21). The basic amino acid residues encoded by the VEGF gene alternative exon 7 confer the binding selectivity of the VEGF165 isoform to Npn-1 (31, 32); similar motifs are present in related protein family members, and recently, PlGF-2 and VEGF-B have been suggested to be endogenous ligands for Npn-1 (33, 34). Similar to Npn-1, Npn-2 binds VEGF165 and PlGF-2; surprisingly, VEGF145 discriminates between the two neuropilins and binds only Npn-2 with high affinity (35). Thus, a number of proteins implicated in vascular development and reproductive functions potentially mediate actions through selective binding of neuropilins.
The signal transduction mechanisms underlying the
VEGF/neuropilin-mediated angiogenic events remain unclear. Although the
amino acid sequence of the short cytoplasmic tail of Npn-1 is highly
conserved among species, suggesting that it may exhibit intracellular
signal transduction functions, the domain demonstrates neither kinase
activities nor significant homology with other known proteins. Although
studies have implicated Npn-1 cytoplasmic domain interactions with Rac1
or collapsin response mediator protein (CRMP), and
PSD-95/discs-large/ZO-1 (PDZ) domain proteins
(36, 37, 38), receptor chimeras have revealed that the
intracellular tail of Npn-1 is not required for biological activity
(21), leading to the suggestion that neuropilins function
as coreceptors for other signaling proteins. This role has been
strengthened by the demonstration of Npn-1-associated enhanced
VEGF165 binding to VEGFR2 and facilitated
VEGF165-mediated chemotaxis and mitosis
(15). The coexpression and coregulation of Npn-1 and
VEGFR2 in many tissues, including uterus, are consistent with that
possibility. More recently, Npn-1 has been demonstrated to form stable
complexes with plexins in mediating semaphorin-induced neuronal changes
(39). Whether plexin-related proteins are also potential
mediators in vascular functions is currently unclear. As a result, the
ligand-selective neuropilin signal will depend not only on the specific
protein variant, but also on the cell type and nature of the receptor
complexes (Fig. 7).
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Although Npn-2 transcripts and proteins were also identified in the uterus, similar to many tissues (22), Npn-1 and Npn-2 did not share identical tissue distribution patterns. Although morphological studies demonstrated Npn-1 protein expression in both endothelial and glandular cells of the uterus, Npn-2 was observed only in the endometrial glandular cells; the relatively low levels of Npn-2 mRNA observed in the uterine artery combined with the lack of Npn-2 immunoreactivity in the uterine vasculature of the present study are consistent with the absence of Npn-2 in uterine endothelial cells. Although structurally similar to Npn-1, the functions of VEGF165 and VEGF145 binding to Npn-2 have not been elucidated, but as for Npn-1, have been suggested to require interactions with VEGFR1 or VEGFR2.
Importantly, the current studies demonstrated gonadal steroid hormone regulation of Npn-1 and Npn-2 expression in the uterus. In OVX animals, Npn-1 mRNA expression was up-regulated by progesterone, a central modulator involved in the complex integration of female reproductive function. The regulation of vascular endothelial and glandular cell Npn-1 by progesterone may represent one of the many adaptations in the reproductive process in promoting VEGF-mediated uterine growth and proliferation to facilitate embryo implantation and maintain pregnancy. As the promoter regions of the neuropilin genes have not been identified, whether the actions of progesterone on Npn-1 expression represent direct or indirect regulatory effects remain to be established. Estrogen did not appear to regulate the expression of the predominant Npn-1 mRNA isoform, although other variants may be induced modestly. Estrogen and progesterone often exhibit opposing effects in uterine proliferation and function, and the functional implications of the estrogen attenuation of the progesterone-induced stimulated Npn-1 expression are the targets of current related studies. In this regard, Npn-1 is similar to uterine calcitonin/calcitonin gene-related peptide expression, which is up-regulated by progesterone, inhibited by estrogen, and implicated to be an important indicator of implantation (41, 42). The steroid hormone regulation of both Npn-1 and VEGFR2 mRNA suggest that the expression and interactions of these coreceptors are essential in coordinating the physiological responses.
The hormonal regulation of Npn-1 during the rat estrous cycle appears consistent with that for the hormone replacement studies; Npn-1 transcript levels were highest at metestrus and diestrus during periods of elevated progesterone levels. Significantly, progesterone has been reported to regulate VEGF mRNA in other tissues, including breast cells (43). The patterns of neuropilin transcript modulation differed from those of uterine VEGF mRNA, which was elevated maximally at estrus during peak estrogen levels; however, morphological analyses using in situ hybridization histochemistry demonstrated cell type-selective elevation of VEGF transcript levels in glandular cells during diestrus (11). Thus, the current results indicated that unlike VEGF, which was up-regulated by both gonadal steroid hormones, regulation of Npn-1 mRNA expression appeared to be driven predominantly by progesterone. By contrast, gonadal hormone Npn-2 transcript regulation differed considerably from that of Npn-1 mRNA; although estrogen did not appear to regulate significantly Npn-1 transcript expression, levels of uterine Npn-2 mRNA from OVX animals were decreased by estrogen. Furthermore, Npn-2 mRNA levels in uteri from progesterone-replaced animals did not differ from those in OVX control, and combined estrogen and progesterone replacement had no apparent effect.
The relative importance of neuropilins in modulating reproductive processes remains to be firmly established. Both the neuropilins and VEGF tyrosine kinase receptors are clearly important for vascular development and function. Npn-1- or VEGFR2-deficient mice, produced by targeted gene disruption, die in utero as a result of vascular developmental defects (20, 28, 44); furthermore, chimeras overexpressing Npn-1 were also embryonically lethal, exhibiting abnormalities, including excessive capillary and blood vessel growth and heart malformations (23). Whether inducible and tissue-specific targeted disruption of Npn-1 expression in reproductive tissues results in infertility remains to be studied.
Nevertheless, several key findings provide compelling evidence that both the neuropilin and VEGFR components are critical for normal reproductive functions, including the defined roles of specific isoforms of VEGF in reproduction; the distribution of neuropilins and VEGFR in the uterus; the established interactions between Npn-1 and VEGFR2 promoting VEGF binding, cellular mitosis, and chemotaxis; and the coregulation of uterine Npn-1 and VEGFR2 transcripts by gonadal steroid hormones. Our results are also in good agreement with recent studies demonstrating the coordinate expression of VEGFR2 and Npn-1 in uterus and implicating roles for these receptors in vascular permeability and angiogenesis during implantation in mice (9). Our preliminary work has also demonstrated Npn-1 expression in human endometrial tissues, and our continuing human studies under varying hormonal conditions may further clarify its roles in reproductive physiology. Unlike Npn-1, Npn-2 null mutants survive while demonstrating neuronal abnormalities (33, 45, 46); whether there are also subtle changes in vascular functions or fertility remains to be determined. In summary, we have shown the differential expression and hormonal regulation of neuropilins in rat uterus. A better understanding of the roles of VEGF receptors in normal reproduction may lead to new strategies to ameliorate reproductive abnormalities.
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Acknowledgments |
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Footnotes |
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Received July 31, 2000.
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