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Departments of Bacteriology and Immunology (M.L., A.R., M.H., O.R.) and Molecular/Cancer Biology Laboratory (K.P.), Haartman Institute, and the Departments of Clinical Chemistry and Obstetrics and Gynecology (A.R., K.N.), University of Helsinki, Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Mika Laitinen, Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21, University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: mplaitin{at}cc.helsinki.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VEGF/vascular permeability factor, is an endothelial cell mitogen that has been cloned and extensively characterized by several groups (8–14; reviewed in Ref.15). VEGF is a heparin-binding 40- to 46-kDa disulfide-linked dimeric glycoprotein that dissociates upon reduction into two 20- to 23-kDa subunits. (8, 10, 16). Analysis of the complementary DNA (cDNA) clones revealed that alternative messenger RNA (mRNA) splicing results in the generation of 121-, 165-, 189-, and 206-amino acid (aa)-encoding mRNA forms (17, 18). Moreover, placental cells and various carcinoma cells of female reproductive tract express a VEGF transcript giving rise to an additional 145-aa isoform (19, 20). VEGF121, VEGF145, and VEGF165 proteins appear to be secreted, whereas the VEGF189 form is not freely secreted but remains bound to heparan sulfate proteoglycans on the cell surface and in the extracellular matrix (20, 21). VEGF has significant homology with both platelet-derived growth factors A and B, with eight conserved cysteins located at distinct positions in the protein sequence (12, 13). Placenta growth factor was cloned from human placenta, and it shares a 53% identity with VEGF in the platelet-derived growth factor-like region (22). VEGF is also able to heterodimerize with placenta growth factor (23). Recently, two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), were shown to bind VEGF with high affinity and to transduce its cellular signal (24, 25, 26). Multiple growth factors, cytokines, hormones, as well as hypoxia regulate the expression of VEGF in various cell cultures (27, 28, 29, 30, 31, 32, 33).
VEGF-B and VEGF-C are recently identified endothelial cell growth factors (34, 35, 36). VEGF-B mRNA splicing results in transcripts encoding a soluble VEGF-B167 form and a cell surface/extracellular matrix-bound VEGF-B186 form (37). VEGF-B isoform monomers are able to form disulfide-linked homodimers, but they are also able to heterodimerize with VEGF (34, 37). VEGF-C is a secreted protein that stimulates the migration of endothelial cells and is also able to promote their mitogenesis (35, 36). Similar to VEGF, VEGF-C also increases vascular permeability (38). VEGF-C exerts its effects via VEGFR-2 and VEGFR-3 (35), whereas the cellular binding components for VEGF-B remain to be elucidated. VEGFR-2 is expressed by vascular endothelial cells, whereas VEGFR-3 is mainly found in the lymphatic endothelium (25, 39). Interestingly, VEGF-C induced specifically lymphatic, but not vascular, endothelial proliferation and vessel enlargement when overexpressed in the skin of transgenic mice (40).
As VEGF has been shown to be expressed in primate as well as human granulosa cells and CL (27, 41, 42), and VEGF-B and VEGF-C are expressed in human ovaries (34, 35, 36), we have studied the expression of VEGF, VEGF-B, and VEGF-C mRNAs in freshly isolated human granulosa-luteal (GL) cells and the regulation of the steady state levels of these transcripts in cultured GL cells. Our results provide evidence that ovarian GL cells express VEGF, VEGF-B, and VEGF-C and that the distinct VEGF isotypes are differentially regulated in cultured GL cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hormonal treatments of cultured human GL cells
Before hormone treatments, the cells were transferred to DMEM
supplemented with 2.5% FCS. To evaluate the effect of the culture age
of human GL cells, the cells were treated with 30 or 100 ng/ml purified
hCG (CR-127, obtained from the National Hormone and Pituitary
Distribution Program, NIDDK, NIH), 100 ng/ml recombinant human FSH
(rhFSH; Org 32489; Organon International BV, Oss, The Netherlands)
(46), 0.5–1 mM 8-bromo-cAMP (8-Br-cAMP; Sigma Chemical
Co., St. Louis, MO), 10 ng/ml 12-O-tetradecanoylphorbol
13-acetate (TPA; Sigma), or 1 µM PGE2 (Sigma)
for the indicated time periods on days 1–10 of culture. The time and
concentration dependence studies were performed on days 4–7 of
culture. Each experiment was performed at least three times, with
duplicate or triplicate cultures.
RNA extraction and Northern blotting
Total RNA from freshly isolated GL cells and cytoplasmic RNA
from cultured GL cells were extracted using the guanidine
isothiocyanate-cesium chloride method (47) and the modified Nonidet
P-40 lysis procedure (48), respectively. RNA was quantitated by
absorbance at 260 nm. For Northern blots 17–20 µg RNA from freshly
isolated human GL cells and 11–15 µg RNA from cultured GL cells were
size fractionated in 1.5% agarose gels and transferred to Hybond-N
nylon membranes (Amersham International, Aylesbury, UK). For dot blots,
1–2 µg cytoplasmic RNA were denatured in 7.5% formaldehyde and
6 x SSC (1 x SSC = 0.15 M NaCl and 0.015
M Na citrate, pH 7.0) at 50 C for 30 min and spotted onto
nylon membranes using a 96-well Minifold device (Schleicher and
Schuell, Keene, NH). The filters were baked for 1 h at 80 C and UV
cross-linked with a Reprostar II UV illuminator (Camag, Muttenz,
Switzerland) for 6 min before hybridizations.
Reverse transcription-PCR (RT-PCR), Southern blotting, cDNA
cloning, and analysis of cDNAs
VEGF cDNAs were synthesized by RT-PCR (49) from human GL and
JEG-3 cell RNA using oligonucleotides
5'-ATG-AAC-TTT-CTG-CTG-TCT-TGG-GT-3' (157–179) and
5'-TCA-CCG-CCT-CGG-CTT-GTC-AC-3' (785–>NOREF>804), designed according to
Sharkey et al. (44) with numbering according to Keck
et al. (13). RT reactions were performed as previously
described (43), and the cycling conditions for PCR were as follows:
denaturing at 95 C for 30 sec, annealing at 55 C for 30 sec, and
elongation at 72 C for 90 sec, with 45 cycles. The reagents for the PCR
were purchased from Perkin-Elmer/Cetus Corp. (Norwalk, CT) and used
according to the manufacturer’s instructions. Southern blots for
amplified VEGF cDNA fragments were prepared as previously described
(50). The cDNAs encoding VEGF121 and VEGF165
were ligated to the pGEM-T vector (Promega, Madison, WI) and verified
by sequencing the double stranded plasmid templates using cloned T7 DNA
polymerase (Sequenase 2.0, U.S. Biochemical Corp., Cleveland, OH).
Labeling of cDNA probes and hybridizations
As probes for filter hybridizations we used a human 581-bp VEGF
cDNA [nucleotides (nt) 57–638) (13), VEGF-B cDNA (nt 1–382) (34),
and VEGF-C cDNA (nt 495-1661) (35). Human cyclophilin (51) or rat
glyceraldehyde-6-phosphate dehydrogenase (GAPDH) (52) cDNAs were used
as controls for even loading in the filter hybridizations. All cDNA
inserts were labeled with [
-32P]deoxy-CTP (3000
Ci/mmol; Amersham) and a Prime-a-Gene kit (Promega). The probes were
purified with nick columns (Pharmacia, Uppsala, Sweden) and used at
1–3 x 106 dpm/ml in hybridization solution
containing 50% formamide, 6 x SSC, 0.1% Ficoll, 0.1%
polyvinylpyrrolidone, 0.1% BSA, 100 µg/ml salmon sperm DNA, 100
µg/ml yeast RNA, and 0.5% SDS. Dot, Northern, and Southern blots
were hybridized for 16 h at 42 C and washed three times for 20 min
each time with 1 x SSC-0.1% SDS at 50 C. Filters were exposed to
Fuji RX x-ray film (Fuji, Tokyo, Japan) with Trimax 16T intensifying
screens (3 M, Ferrania, Italy) at -70 C. The relative densities of dot
blot hybridization signals were determined using a transmission
densitometer (model 331, X-rite Co., Grand Rapids, MI). Alternatively,
Fujifilm IP-Reader Bio-Imaging Analyzer BAS 1500 (Fuji Photo Co.,
Tokyo, Japan) was used to analyze hybridization filters with the MacBas
software supplied by the manufacturer using a Macintosh (Apple
Computer, Cupertino, CA) personal computer.
Analysis of RNA data
For single comparisons between untreated and hormone stimulated
cultures, the data were analyzed using Student’s t test.
For multiple comparisons, the data were first analyzed by one-way
ANOVA, and statistical significance was determined by Scheffe’s
multiple comparison test using the Exstatics program (Select Micro
Systems, Yorktown Heights, NY) on a Macintosh (Apple Computer) personal
computer. The figures represent the mean ± range or the mean
± SEM of the values of duplicate or triplicate cultures,
respectively, as expressed in arbitrary densitometric units and
adjusted to a value of 1.0 for the mean of the first control
culture.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To
characterize the alternatively spliced VEGF transcripts in human GL
cells, we generated cDNA fragments by RT-PCR and analyzed the cDNAs by
Southern blotting using a 581-bp VEGF189 cDNA as a probe.
Figure 1B schematically shows the locations of the exonic regions in
the cDNA that encode for the five different VEGF forms. Southern
blotting analyses indicated that in addition to the 121-, 145-, and
165-aa forms found in GL cells, the JEG-3 cells also expressed
VEGF189.
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, A
and B, show that all these effectors increased the expression levels of
the VEGF transcripts. Simultaneous treatment with TPA and hCG did not
increase VEGF levels compared with the effect of TPA treatment alone
(Fig. 2B).
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shows that basal VEGF mRNA levels
declined steadily in all individual experiments during the 2- to 10-day
culture period. An 8-h treatment with hCG, 8-Br-cAMP, or TPA induced
VEGF transcript levels significantly [P < 0.05; by
hCG on days 5, 7, and 10 of culture (Fig. 3A) and by 8-Br-cAMP or TPA
on days 4, 7, and 9 of culture (Fig. 3B)]. Treatment with
PGE2 for 2 h raised VEGF transcripts significantly on
days 2, 4, and 7 of culture (Fig. 3C). Time-course studies indicated
that VEGF mRNA levels were induced only 2 or 3 h after treatment
(Figs. 4, A–C). Moreover, 2- and 6-h
treatments with 100 ng/ml rhFSH induced significantly VEGF mRNA levels
on day 7 of culture (data not shown). Figure 4, D–F, shows the
concentration dependence of the induction of VEGF mRNA levels by hCG,
8-Br-cAMP, and TPA. Maximal responses were observed with 10–100 ng/ml
hCG, 0.3–1 mM 8-Br-cAMP, and 10–100 ng/ml TPA. rhFSH and
PGE2 also induced VEGF mRNA levels in a dose-dependent
manner, with the maximal inductive effect for rhFSH at 30–300 ng/ml
and that for PGE2 at 0.3–1 mM (data not
shown).
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, A and
B).
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shows that a 24-h
treatment with 100 ng/ml hCG decreased VEGF-C transcript levels
moderately on day 5 and more clearly on day 7 of culture, whereas the
steady state mRNA levels of VEGF were induced by hCG in the same
experiment. Dot blot data from the same experiment (Figs. 5C and 6A
represent the Northern blot and dot blot data, respectively, from the
same experiment) and those from another experiment indicated that
VEGF-C mRNA levels were significantly inhibited (P <
0.05) by hCG between days 4–7 of culture (Fig. 6, A and B). The basal VEGF-C mRNA levels
tend to rise during the culture. In the experiment represented in Fig. 6A, there was a significant (P < 0.05) difference
between day 2 vs. day 5 as well as between day 5
vs. day 7 control values.
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, C and D, shows that hCG clearly decreased VEGF-C mRNA levels
in a concentration-dependent manner. Time dependence studies performed
on days 4–7 of culture showed that although VEGF-C mRNA levels were
decreased in some experiments as early as 3 h after hCG treatment,
the effect was statistically significant (P < 0.05) in
all individual experiments at the 24 to 48 h points (Fig. 7, A–C). As hCG increases intracellular
cAMP levels in human GL cells, we tested whether a cell-permeable cAMP
analog, 8-Br-cAMP, is also able to decrease VEGF-C mRNA levels in these
cells. Figure 7, D–F, shows three parallel experiments indicating that
stimulation with 0.5–1.0 mmol/liter 8-Br-cAMP decreased VEGF-C mRNA
levels in GL cells with essentially similar kinetics as hCG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The VEGF gene consists of eight exons, and as a result of alternative splicing, different transcripts encoding VEGF forms have been identified (17, 18, 19). We show that VEGF transcripts giving rise to VEGF121, VEGF145, and VEGF165 are produced in human GL cells. Furthermore, four alternatively spliced VEGF transcripts were identified in JEG-3 choriocarcinoma cells. These data are well in line with the earlier observations of others who detected splicing variants representing 121-, 145-, 165-, and 189-aa VEGF forms in human endometrium, myometrium, and endometrial carcinoma cell lines (19, 44). As no information has been available on the expression of VEGF145 other than in the endometrium and choriocarcinoma cells (19, 44) or in human epithelial ovarian carcinoma cells (20), the present study identifies the ovarian GL cell as a novel site for the expression of this isoform. The expression of alternatively spliced VEGF forms appear to be regulated in tissue-specific manner (18, 19). As VEGF121 and VEGF165 are known to have different receptor binding abilities (53), other alternatively spliced VEGF forms may have distinct, as yet unknown, biological effects.
The present study demonstrates that hCG and rhFSH induce VEGF mRNA levels in human GL cells. The VEGF mRNAs were clearly enhanced by hCG on day 5 of culture. At the beginning of the culture, human GL cells may be desensitized to gonadotropins because they are derived from hyperstimulated IVF patients (54). This is likely to explain why gonadotropin treatment was not able to increase VEGF mRNA levels during the early stages of culture. Using this cell culture model, we observed the same phenomenon for several gonadotropin-regulated hormonal parameters (51, 55, 56, 57). Our data on the stimulatory effect of hCG on VEGF mRNA levels are in line with the observations of Ravindranath et al. (27), suggesting that LH mediates the induction of VEGF expression in primate CL. As hCG stimulates human GL cells through the hCG/LH receptor, which binds both hCG and LH with high affinity (58), it is likely that LH is acting in a similar manner as hCG in GL cells. Similar to our results, Neulen et al. showed recently that hCG induces VEGF mRNA levels in cultured human GL cells (28). In contrast to their observations, we detected a clear increase in VEGF mRNAs when the cells were treated with rhFSH on days 5–7 of culture. This difference may be explained by the delayed responsiveness to rhFSH, compared with that to hCG, which is characteristic of this cell culture system (59). Our data clearly indicate that stimulation of both the FSH and LH/hCG receptor-activated pathways is able to induce VEGF mRNA levels in human GL cells.
The time-course studies indicated that hCG, 8-Br-cAMP, TPA, and PGE2 induced VEGF mRNA levels rapidly in cultured human GL cells, which is consistent with the results obtained with granulosa cells of human origin (28) and from other species (29, 60) as well as with other types of cells (30, 61) demonstrating that VEGF transcript levels are regulated as early as 1 h after treatment. Our results also show that the basal levels of VEGF mRNAs decreased during the culture period. Interestingly, in human ovarian sections, immunoreactivity for VEGF was found in highly vascularized CL, but not in degenerating CL (62).
A recent study has demonstrated that there are several potential activating protein-1- and -2-binding sites in the human VEGF gene promoter sequence, suggesting that both cAMP and TPA could induce transcription of the VEGF gene (17). The present data are consistent with these findings. We show that both 8-Br-cAMP and TPA are potent inducers of VEGF mRNA levels in cultured human GL cells. In bovine granulosa cell cultures, forskolin, an adenylate cyclase activator, and TPA have also been shown to induce VEGF mRNA levels (29). PGE2 has been found to be effective in enhancing VEGF mRNA levels in rat osteoblast and human synovial fibroblast cell cultures (30, 61). We show here that PGE2 increases VEGF mRNAs also in cultured human GL cells. Although the receptor isotypes binding PGE2 in these cells have not been characterized, it is possible that PGE2 may mediate this effect through a protein kinase A-dependent signaling pathway, as PGE2 increases cAMP levels in human granulosa cells (63).
VEGF-B is widely expressed in various tissues, most prominently in heart, skeletal muscle, and the pancreas (34). A recent study has further indicated that VEGF-B and VEGF-C might be important regulators of the reproductive system, as VEGF-B and VEGF-C mRNAs are highly expressed in human placenta (64). We show here that VEGF-B is expressed in human GL cells, and its expression level is rather stable in primary cultures of these cells. However, in contrast to VEGF and VEGF-C, VEGF-B mRNA levels were not found to be regulated by any hormonal treatment in vitro. As VEGF-B heterodimerized with VEGF when coexpressed in human embryo kidney, 293EBNA, cells (34), the overlapping expression of VEGF and VEGF-B in human GL cells suggests that these cells may produce natural VEGF-VEGF-B heterodimers. As VEGF165-VEGF-B heterodimers, in the absence of heparin, remain cell associated, VEGF-B may affect the bioavailability of VEGF.
VEGF-C transcripts are readily expressed in freshly isolated human GL cells, and hCG appears to decrease their steady state levels. In contrast to VEGF, the basal steady state expression of VEGF-C mRNAs is enhanced in GL cells during culture. During early stages of culture, the low levels of VEGF-C and the increased levels of VEGF may reflect the fact that these cells have been exposed in vivo to large doses of gonadotropins before oocyte harvest. As the cells regain their responsiveness to hCG by days 4–5 of culture, VEGF-C levels spontaneously increase, whereas VEGF mRNA levels continuously decrease. Interestingly, the expression pattern of basal VEGF-C mRNA resembles that of another granulosa cell-derived growth factor, bone morphogenetic protein-3, which is regulated in a similar manner by hCG (57). Our data indicate that after day 4 of culture, VEGF-C mRNA steady state levels are suppressed by hCG in a concentration- and time-dependent manner. We further show that 8-Br-cAMP decreases VEGF-C mRNAs in GL cells in a similar manner as hCG. Thus, we conclude that VEGF and VEGF-C transcript levels are differentially regulated and suggest that the protein kinase A signaling pathway may be responsible for these effects of hCG.
It is possible that VEGF, VEGF-B, and VEGF-C regulate angiogenesis in the CL. The present study shows that human GL cells express VEGF, VEGF-B, and VEGF-C mRNAs and that their expression is regulated differentially in vitro. Further studies are required to clarify the biological roles and cellular targets of these three growth factors in the ovary.
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Acknowledgments |
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Footnotes |
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Received May 16, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ßA-subunit messenger ribonucleic
acid levels by chorionic gonadotropin and recombinant
follicle-stimulating hormone in cultured human granulosa-luteal cells.
J Clin Endocrinol Metab 79:1670–1677[Abstract]
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J. Aaltonen, M. P. Laitinen, K. Vuojolainen, R. Jaatinen, N. Horelli-Kuitunen, L. Seppä, H. Louhio, T. Tuuri, J. Sjöberg, R. Bützow, et al. Human Growth Differentiation Factor 9 (GDF-9) and Its Novel Homolog GDF-9B Are Expressed in Oocytes during Early Folliculogenesis J. Clin. Endocrinol. Metab., August 1, 1999; 84(8): 2744 - 2750. [Abstract] [Full Text]
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F. Gaytán, C. Morales, L. García-Pardo, C. Reymundo, C. Bellido, and J.E. Sánchez-Criado A Quantitative Study of Changes in the Human Corpus Luteum Microvasculature during the Menstrual Cycle Biol Reprod, April 1, 1999; 60(4): 914 - 919. [Abstract] [Full Text]
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M. Stoner, F. Wang, M. Wormke, T. Nguyen, I. Samudio, C. Vyhlidal, D. Marme, G. Finkenzeller, and S. Safe Inhibition of Vascular Endothelial Growth Factor Expression in HEC1A Endometrial Cancer Cells through Interactions of Estrogen Receptor alpha and Sp3 Proteins J. Biol. Chem., July 21, 2000; 275(30): 22769 - 22779. [Abstract] [Full Text] [PDF]
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