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Presence and Regulation of Endocrine Gland Vascular Endothelial Growth Factor/Prokineticin-1 and Its Receptors in Ovarian Cells

Department of Animal Sciences, The Hebrew University of Jerusalem (T.K., N.L., R.M.), Rehovot 76100, Israel; and Department of Obstetrics and Gynecology, Hadassah University Hospital (A.H.), Jerusalem 91905, Israel

Address correspondence and requests for reprints to: Rina Meidan, Department of Animal Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. E-mail: rina.meidan{at}huji.ac.il.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Endocrine gland vascular endothelial growth factor (EG-VEGF) is a novel angiogenic mitogen selective for endothelial cells (EC) in endocrine glands. EG-VEGF is identical to a protein previously cloned and termed prokineticin (PK)-1. The present study examined the expression of EG-VEGF/PK-1 and its receptors in ovarian steroidogenic cells and EC and compared the regulation of EG-VEGF/PK-1 and VEGF expression in SV40 transformed luteinized human granulosa cell line (SVOG). Normal granulosa or SVOG cells expressed EG-VEGF/PK-1 mRNA. Incubation of SVOG cells with forskolin augmented EG-VEGF/PK-1 expression in a dose-dependent manner. Chemical hypoxia induced by CoCl₂ and desferrioxamine mesylate (100 µM each) markedly reduced EG-VEGF/PK-1. In contrast, hypoxia significantly elevated VEGF mRNA (VEGF165, 189) and protein secretion. Thrombin, like hypoxia, also induced an opposite effect on VEGF and EG-VEGF/PK-1. Whereas EG-VEGF/PK-1 and VEGF were inversely regulated, steroidogenesis and EG-VEGF/PK-1 were positively correlated in SVOG cells. A distinct pattern of ovarian PK receptor (PK-R) expression was observed in which steroidogenic cells predominantly express PK-R1 receptors, whereas corpus luteum-derived EC express high levels of both PK-R1 and PK-R2. Therefore, acting via either PK-R2 or PK-R1, EG-VEGF/PK-1 may have angiogenic as well as nonangiogenic functions in the ovary.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE CORPUS LUTEUM (CL) is a transient endocrine gland that undergoes dynamic changes throughout its life span. CL formation, triggered by the LH surge, encompasses two major events: follicular granulosa and theca cells are converted into highly steroidogenic luteal cells synthesizing large amounts of progesterone (1). Concurrently, microvessels from follicular basal lamina invade the granulosa cell layer, and extensive angiogenesis ensues (2, 3, 4). At early to mid luteal stages, as much as 40% of the primate CL microvessels were found to contain proliferating endothelial cells (EC) (5). At mid stage, the vascular network of the CL is characterized by an intense degree of capillarization so that each luteal cell is in direct contact with two or three neighboring capillaries (4, 6). Therefore, factors affecting vascular growth are likely to play a major role in regulating luteal function. Vascular endothelial growth factor (VEGF), a potent mitogen for EC, is expressed in the CL in a time-dependent manner (7, 8, 9, 10). High mRNA expression for VEGF and VEGF receptor (VEGFR)-2 are found during the early to mid luteal phase and during rescue of the CL in pregnancy (8, 10). Suppression of luteal VEGF bioactivity in mice (11) and primates (3, 12) resulted in reduced EC proliferation and progesterone levels (13), demonstrating the role of VEGF as key regulator of luteal angiogenesis. Recently, LeCouter et al. (14) reported the identification of a new angiogenic mitogen selective for EC in endocrine glands. This 86-amino acid peptide, structurally nonrelated to VEGF, was termed endocrine gland-derived vascular endothelial growth factor (EG-VEGF) (14). EG-VEGF induces proliferation, migration, and fenestration of EC derived from adrenal capillaries but not of other EC types such as those derived from aorta, umbilical vein, and dermis (14). EG-VEGF is identical to prokineticin (PK)-1, which was previously cloned as a mammalian homolog of mamba intestinal toxin-1 (15). All members of the PK family, including frog skin peptide Bv8 and its mammalian homolog, PK-2, contain 10 cysteine residues in identical positions, suggesting that these peptides have a common evolutionary origin (16, 17, 18). The novel protein EG-VEGF/PK-1 regulates diverse biological functions such as contraction of gastrointestinal (GI) smooth-muscle cells and angiogenesis (14, 15). Two closely related G protein-coupled receptors for PK were cloned in human, rat, and bovine species [PK receptor (PK-R)-1 and PK-R2] (16, 17, 18). Expression of PK-Rs in heterologous systems shows that these receptors bind to and are activated by nanomolar concentrations of recombinant PK (16, 17, 18). Activation of PK-R leads to mobilization of calcium, stimulation of phosphoinositide turnover, and activation of p44/p42 MAPK signaling pathways that are consistent with the effects of PK on smooth-muscle contraction and angiogenesis (16, 17, 18, 19).

EG-VEGF/PK-1 mRNA was identified in ovarian samples and the intraovarian delivery of the peptide-promoted angiogenesis and cyst formation (14). A highly vascular endocrine gland, the CL can serve as an attractive model for the study of the EG-VEGF/PK-1 system. This work was undertaken to 1) study the expression of EG-VEGF/PK-1 and its receptors in luteal steroidogenic and EC types, and 2) compare the regulation of VEGF/PK-1 and VEGF expression in luteinized human granulosa cell lines and correlate it to the steroidogenic capacity of the cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

Medium 199 (M-199), MCDB 105 medium, hydrocortisone, forskolin, thrombin from bovine plasma, CoCl₂, deferoxamine mesylate (DFX), and progesterone antiserum were from Sigma (St. Louis, MO). Fetal calf serum (FCS), L-glutamine, gentamycin sulfate, and trypsin-EDTA solution A were from Biological Industries (Kibbutz Beit Hemeek, Israel). Bandeiraea simplicifolia lectin-1 (BS-1) was from Vector Laboratories (Burlingame, CA); collagenase type IV was from Worthington Biochemical Corp. (Freehold, NJ); and uncoated magnetic beads (Dynabeads M450) were from Dynal (Oslo, Norway). TRI reagent was from MRC (Cincinnati, OH). SuperScript II RNase H^- Reverse Transcriptase and Ultra Pure electrophoresis agarose were obtained from Invitrogen (Paisley, UK). Deoxynucleotide triphosphates and random hexamer oligodeoxynucleotides were from Fermentas (Vilnius, Lithuania). BioTaq DNA polymerase was obtained from Bioline GmbH (Luckenwalde, Germany). Oligonucleotide primers were synthesized by Sigma-Genosys (Cambridgeshire, UK). [1,2,6,7-³H]-Progesterone was purchased from NEN Life Science Products (Boston, MA). Human VEGF ELISA Development Kit was obtained from PeproTech (Rocky Hill, NJ).

Cell cultures

A line of human granulose-lutein cells immortalized with SV40 large T antigen (SVOG) was a generous gift from N. Auersperg (University of British Columbia, British Columbia, Canada). Cells were cultured in M-199/MCDB 105 (1:1), containing 10% FCS, 2 mM L-glutamine, 400 ng/ml hydrocortisone, and 50 µg/ml gentamycin sulfate (20). In all experiments, cells were grown to 80–90% confluence on 24-well plates. The cells were preincubated in M-199/MCDB 105 (1:1) with 1% FCS, 2 mM L-glutamine, 40 ng/ml hydrocortisone, and 50 µg/ml gentamycin sulfate for 3–6 h, and were then assayed in the same medium for the effects of forskolin (0.01–20 µM), thrombin (0.1–2.5 U), CoCl₂, and DFX (100 µM each) for 24 h. At the end of the incubation, media were collected for determination of progesterone and/or VEGF, and then RNA was extracted.

Human luteinized granulosa cells (hLGC) were obtained by follicle aspiration from six women who entered the in vitro fertilization program at Hadassah University Hospital, Jerusalem; cells were pooled before RNA extraction. The study protocol was reviewed by the appropriate institutional review committee of Hadassah University Hospital, and the investigation was performed in accordance with the guidelines expressed in the Declaration of Helsinki.

Enrichment of ovarian cell types

Bovine ovaries were collected at a local slaughterhouse. For the enrichment of luteal cell subpopulations, mid-cycle CL were dispersed using collagenase IV as described previously (21). Briefly, CL were sliced and dispersed in M-199 containing 0.5% BSA and collagenase (420 U/ml). Dispersed luteal cells were suspended in 1% BSA in PBS and mixed with epoxy magnetic beads precoated with BS-1 (the EC-specific lectin), at a bead-to-EC ratio of 1:3. The adherent cells were washed and concentrated using a magnet until the supernatant was free of cells. Both BS-1-positive cells (EC) and nonadherent cells (enriched luteal steroidogenic cells) were collected for RNA extraction. For the enrichment of follicular cells, healthy bovine follicles (estradiol in follicular fluids > 150 ng/ml) were collected as described previously (22, 23). Granulosa and theca cells were dispersed, as described previously, and collected separately. Enriched theca interna cells were obtained as follows: dissociated theca layer cells were incubated with BS-1-coated magnetic beads, and BS-1-adherent cells were than discarded (24). The remaining BS-1-negative cells (enriched theca interna cells) were pooled.

RNA isolation and RT-PCR

Total RNA was isolated from the cells and tissues using the TRI reagent according to the manufacturer’s instructions. Semiquantitative RT-PCR was performed as described previously (25), with glyceraldehyde 3-phosphate dehydrogenase (G3PDH) or ß-actin as a standard. Sequence analysis ascertained the identity of the PCR products. Computer searches and sequence alignments were performed using software from Genetics Computer Group (Madison, WI). For a list of primers, see Table 1.

The concentration of progesterone in SVOG culture media was determined by RIA as described previously (22). The sensitivity limit of the RIA was 3.9 pg/tube. VEGF concentrations in the same media were determined using a human VEGF ELISA Development Kit according to the manufacturer’s instructions. The lower limit of delectability was 12.5 pg/ml.

Statistical analyses

Data are presented as means ± SEM. Student’s t test was used to determine the statistical difference between treatments, as indicated in Results and Figs. 5–8. One-way ANOVA was used to estimate the statistical difference between PK-R expression in the various cell types as indicated in Fig. 2. A value of P < 0.05 was considered significant.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Effect of FCS on expression of EG-VEGF/PK-1 mRNA by SVOG cells. The cells were incubated with or without forskolin (FRS; 10 µM) in medium containing increasing concentrations of FCS (1, 5, and 10%) for 24 h. Total RNA was isolated, reverse transcribed, and amplified with G3PDH and with PK-1 primers. PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed. Data are the densitometric units of PK-1 relative to G3PDH from one representative experiment.

View larger version (31K): [in this window] [in a new window]   FIG. 6. Effect of forskolin (FRS; 10 µM, 24 h) on expression of EG-VEGF/PK-1, VEGF, StAR and P450scc mRNA, and progesterone production by SVOG cells. The mRNA levels were determined by semiquantitative RT-PCR using G3PDH as internal standard. PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed. A, The densitometric units of mRNA levels of EG-VEGF/PK-1 and VEGF (mean ± SEM; n = 3). B, mRNA levels of StAR. P450scc is presented as densitometric units and progesterone production is presented as percentage of control. The mean value of control was 0.84 ± 0.23 ng/ml. Data are mean ± SEM from three separate experiments. *, P < 0.05; **, P < 0.005 compared with control.

View larger version (25K): [in this window] [in a new window]   FIG. 7. Effect of chemical hypoxia on expression of EG-VEGF/PK-1, VEGF mRNA, and VEGF production by SVOG cells. The cells were incubated with 100 µM of CoCl2 or DFX for 24 h. The media were collected for determination of VEGF concentration. Total RNA was isolated, reverse transcribed, and amplified with EG-VEGF/PK-1 and with VEGF primers. ß-Actin was used as external standard. PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed. A, Densitometric analyses of EG-VEGF/PK-1 and VEGF relative to ß-Actin levels. B, VEGF production presented as pg/ml·24 h. Data (means ± SEM) are from three separate experiments. * P < 0.05; ** P < 0.01 compared with control.

View larger version (29K): [in this window] [in a new window]   FIG. 8. Effect of thrombin on the levels of EG-VEGF/PK-1 and VEGF mRNA in SVOG cells. The cells were incubated with different concentrations of thrombin (0.01–2.5 IU) for 24 h. Total RNA was isolated, reverse transcribed, and amplified with EG-VEGF/PK-1 and with VEGF primers. G3PDH was used as internal standard. PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed. Results of densitometric analysis of EG-VEGF/PK-1 and VEGF relative to G3PDH expression were normalized to respective control (without thrombin). Data are mean ± SEM from three separate experiments.

View larger version (37K): [in this window] [in a new window]   FIG. 2. Expression of PK-R1 () and PK-R2 () by bovine ovarian cells. The mRNA levels of PK-R were determined by semiquantitative RT-PCR using G3PDH as an internal standard. PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and photographed. Data are the mean ± SEM of the densitometric analysis of PK-R1 and PK-R2 relative to G3PDH expression. Top, Follicular cells, granulosa cells (GC) and theca interna cells (TC) (n = 5). Bottom, Luteal cells before enrichment on BS-1-coated beads (mix), enriched luteal steroidogenic cells (SC), and luteal EC (EC) (n = 12). *, Significant difference between PK-R1 and PK-R2; +, Significant difference in PK-R1 for either luteal or follicular cell types; #, Significant difference in PK-R2 for either luteal or follicular cell types.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

EG-VEGF/PK-1 mRNA was shown to be abundantly expressed in steroidogenic tissues, including ovaries (14, 15); however, whether the steroidogenic cells per se express this gene was unknown. We, therefore, examined the expression pattern of EG-VEGF/PK-1 in ovarian steroidogenic cells. Two preparations of human granulosa cells, those harvested from patients undergoing in vitro fertilization and transformed granulosa cells (SVOG), expressed the EG-VEGF/PK-1 mRNA (Fig. 1). Another steroidogenic cell line, human adrenocortical H295R cells, also expressed this gene (Fig. 1). PK-R1 mRNA was detected in all of these steroidogenic cell types, with SVOG cells expressing higher levels than H295R cells or hLGC (Fig. 1). PK-R2 mRNA was not detected in any of the human steroidogenic cell types examined here; PK-R2 mRNA was, however, readily detectable in human brain samples under the same experimental conditions (data not shown). EG-VEGF/PK-1 was shown to selectively affect EC derived from a steroidogenic gland and not from cells derived from a large blood vessel [e.g., human umbilical vein EC (HUVEC) or bovine aortic EC (BAEC)] (19). Therefore, it was of interest to examine the presence of PK-R in ovarian gland-derived EC vs. the other EC types. EC derived from bovine CL expressed high levels of both receptor types, PK-R1 and PK-R2 (Fig. 2). In contrast, neither PK-R1 nor PK-R2 mRNA could be detected in HUVEC. Steroidogenic bovine luteal cells and follicular cells predominantly expressed PK-R1 mRNA, similar to human cells (Fig. 2). Within preovulatory follicles, significantly higher levels were present in theca interna cells as compared with granulosa cells (Fig. 2). The residual expression of PK-R2 mRNA in steroidogenic cells is most probably due to contamination by EC because it corresponded to CD31 expression; in contrast, PK-R1 levels in these cells were not correlated with CD31 (data not shown). Like luteal EC, BAEC also expressed high levels of PK-R1 mRNA, however, PK-R2 was barely detected in these cells (Fig. 3).

View larger version (66K): [in this window] [in a new window]   FIG. 1. Presence of EG-VEGF/PK-1 and PK-R1 in human adrenocortical H295R cells, HUVEC, hLGC, and SV-transformed hLGC (SVOG) cells. Total RNA was reverse transcribed and amplified with G3PDH and EG-VEGF/PK-1, and PK-R1 primers, respectively. PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and photographed. Inverted images of RT-PCR products are presented.

View larger version (72K): [in this window] [in a new window]   FIG. 3. Expression of PK-R1 and PK-R2 by luteal EC (EC) and BAEC. The mRNA levels of PK-R1 and PK-R2 were determined by semiquantitative RT-PCR using G3PDH as an internal standard. Inverse picture of ethidium bromide-stained agarose gel shows a typical RT-PCR reaction with two replicates.

Effect of forskolin on EG-VEGF/PK-1 and VEGF in SVOG cells

cAMP is a potent stimulator of ovarian cell function. SVOG cells were, therefore, challenged with varying concentrations of forskolin for 24 h, as demonstrated in Fig. 4. Forskolin stimulated EG-VEGF/PK-1 mRNA, and maximal stimulation was attained at concentrations equal to or greater than 1 µM (Fig. 4). The presence of serum inhibited EG-VEGF/PK-1 mRNA (Fig. 5). Increasing serum concentrations from 1–10% decreased EG-VEGF/PK-1 mRNA in control as well as in forskolin-stimulated cells. Levels of EG-VEGF/PK-1 mRNA in forskolin-treated cells were 4.0, 2.1, and 0.2 in cells cultured in 1, 5, and 10% FCS, respectively (Fig. 5). All additional experiments were, therefore, carried out in 1% FCS.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Effect of forskolin on the levels of EG-VEGF/PK-1 mRNA in SVOG cells. The cells were incubated with basal media or in the presence of different concentrations of forskolin (0.01–20 µM) for 24 h. Total RNA was isolated, reverse transcribed, and amplified with G3PDH and EG-VEGF/PK-1 primers. PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and photographed. Data (mean ± range) are the densitometric units of EG-VEGF/PK-1 relative to G3PDH from two separate experiments.

Effect of hypoxia and thrombin on the expression of EG-VEGF/PK-1 and VEGF in SVOG cells

Hypoxia is a well-characterized inducer of VEGF and, consequently, of angiogenesis in both physiological and pathological conditions (27, 28); therefore, we next studied the effect of hypoxia on VEGF and EG-VEGF/PK-1 mRNA levels in SVOG cells. Chemical hypoxia was induced by the addition of CoCl₂ and DFX to the culture media. The transition metal CoCl₂ and the iron chelator DFX have been shown to mimic a hypoxic state in several hypoxia-responsive genes. CoCl₂ is thought to substitute for iron in the porphyrin ring of the O₂ sensor, binding O₂ with less affinity, thereby locking it in a deoxygenated conformation (29). Incubation of SVOG cells with CoCl₂ or DFX for 24 h significantly elevated expression of VEGF165 (Fig. 7) and VEGF 189 isoforms (data not shown). The amount of VEGF protein secreted into the culture media was also induced in cells treated with CoCl₂ or DFX (Fig. 7). The opposite occurred with EG-VEGF/PK-1 mRNA levels in SVOG cells, in which both agents almost completely eliminated mRNA levels (Fig. 7). Steroidogenesis was concurrently inhibited by chemical hypoxia: StAR, P450scc, and as a result, progesterone production, were inhibited by CoCl₂ and DFX by 50 and 40%, respectively (Table 2).

Regulation of PK-R1 mRNA in SVOG cells

Data presented in Table 3 show that PK-R1 mRNA was regulated differently than its ligand, EG-VEGF/PK-1, both in trend and in magnitude. Hypoxia induced by either CoCl₂ or DFX moderately but significantly elevated PK-R1. Thrombin and forskolin had no effect on PK-R1, at any of the doses examined.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The LH surge induces in the preovulatory follicles the expression of specific genes that have been shown to be critical for the ovulatory process (progesterone receptor, prostaglandin endoperoxide synthase-2, and several proteases, including the matrix metalloproteinases) (32, 33) and for the differentiation of granulosa and theca cells into progesterone-producing luteal cells. The latter involves up-regulation of the cholesterol synthetic pathways to increase substrate availability and the induction of StAR and steroidogenic enzymes (mainly P450scc) (34). SVOG cells do not express the LH/hGC receptor; however, by activating adenylyl cyclase, forskolin mimics the effects of gonadotropins in ovarian cells. The marked stimulatory effect of forskolin on EG-VEGF/PK-1 mRNA in SVOG cells we have observed here can, therefore, be of physiological significance. EG-VEGF/PK-1 is portrayed as a gonadotropin-induced gene and suggests a role for this protein in CL formation.

In many species including the human in vivo exposure to LH/hGC also augments the production of VEGF in granulosa cells and, hence, of angiogenesis in the ovary (7, 8, 35, 36). Under in vitro conditions as well, hCG was shown to elevate VEGF production, although this effect was evident after several days in culture (37, 38, 39). This may explain the lack of a forskolin-induced effect in the experiments shown here. The early rise of EG-VEGF/PK-1 by cAMP, before any increase in VEGF is observed, could nevertheless serve to augment VEGF signaling. This is supported by findings demonstrating that sphingosine1-phosphate transactivated VEGFR-2, thereby leading to phosphorylation of Akt and endothelial nitric oxide synthase (40). Like sphingosine 1-phosphate, EG-VEGF/PK-1 can stimulate the phosphorylation of these two enzymes (19).

Although LH/hCG may represent an ovary-specific activator of VEGF expression, a decline in local oxygen concentrations (hypoxia) is generally believed to be a primary initiator of new blood vessel formation in normal and pathological tissues. It is mainly mediated by hypoxia inducible factor 1 (HIF-1) (27, 28). The expression of HIF-1 correlates with hypoxia-induced angiogenesis as a result of the induction of the major HIF-1 target gene, VEGF (41). As expected VEGF, both its mRNA and protein secreted into the culture media, was elevated by hypoxic conditions inflicted on SVOG cells. In contrast to VEGF the expression of EG-VEGF/PK-1 was inhibited under these conditions. These findings contrast those reported by LeCouter et al. (14) using human adrenal carcinoma cell lines SW13 and H295R, in which both VEGF and EG-VEGF/PK-1 were increased by hypoxia; whether or not the different cell types may account for this discrepancy is unknown at present.

Data shown here indicated that thrombin could also affect the expression of VEGF as well as that of EG-VEGF/PK-1. As in hypoxia, there was an opposite effect on these two proteins. Thrombin elevated VEGF expression in SVOG cells, whereas EG-VEGF/PK-1 expression was down-regulated. Increasing serum concentrations up to 10% also inhibited EG-VEGF/PK-1 expression. It is unclear as yet which compound in serum is responsible for this inhibition, although it is notable that thrombin is abundant in serum. The classical role ascribed to thrombin has been as the key serine protease generated after the activation of the blood coagulation process. It is now recognized that thrombin, in addition to its role in fibrin clot formation, exhibits diverse bioregulatory functions through its interaction with specific cell-surface receptors (31, 42). During the female reproductive cycle, the ovary undergoes continuous growth, atresia, and repair (43, 44), thus providing multiple stages at which thrombin could exert a biological effect. The presence of thrombin receptors in granulosa cells and an active thrombin-generating system (31) indeed suggest a role for thrombin within the ovary. Thrombin is also a potent activator of angiogenesis, acting indirectly to up-regulate Vß3-integrin and VEGFRs in EC (45, 46, 47, 48) and directly by inducing VEGF expression in several cell types (30, 49), including in SVOG cells, as demonstrated in this study. Therefore, thrombin via VEGF or independently may serve as a promoter of the angiogenic process during CL development.

To date, very little is known about PK-R. The two genes located on different chromosomes share 87% homology. Several reports examined the binding affinities and activation of PK-R, with mammalian cells expressing either PK-R1 or PK-R2 bind both EG-VEGF/PK-1 and PK-2 with high affinities (in the nanomolar range) (16, 17, 18). Inconsistent results were published on the presence of PK-R in whole ovarian tissue, whereas we observed only the presence of PK-R1 (data not shown). Lin et al. (16) reported that PK-R2 was the abundant type, and Soga et al. (18) found only minimal expression of both receptors. A likely explanation for these inconsistencies is that the samples used were collected from different parts of the ovary (stroma or parenchyma) and at a different reproductive stage. By examining different cell types both in the follicle and CL, this study may provide a more accurate description of ovarian PK-R distribution.

A distinct pattern of ovarian PK-R expression was observed in this study: steroid-producing ovarian cells of human and bovine species predominantly express type I receptors. Ovarian EC types expressed the highest levels of both the PK-R1 and PK-R2. Therefore, among ovarian cells examined, only luteal EC expressed PK-R2. Likewise, adrenal cortex-derived EC also display both PK-R2 and PK-R1 (17). This pattern of PK-R expression raises some intriguing questions: EG-VEGF/PK-1 was described as a selective mitogen for EC, derived from endocrine glands; hence, could PK-R2 localized on luteal EC (and in adrenal cortex EC) mediate this angiogenic effect? Also, does PK-R1 present in many different cells types, such as BAEC, and do theca interna cells subserve different or similar functions? Examples of other pleiotropic ligands, for example, endothelin-1, come to mind. The widely expressed type A endothelin receptors are involved in diverse functions such as vasoconstriction, embryonic development, cardiovascular homeostasis (50, 51), and steroid production (52). PK has different functions: GI smooth muscle contraction (15), angiogenesis in selective EC types (14, 53), and PK-2 were also shown to affect circadian rhythm (54) and the sensitization of peripheral nociceptors (55). Whether both PK-Rs are involved in these effects, i.e. do the receptors have overlapping functions, is yet unknown. EG-VEGF/PK-1 induces the phosphorylation of p44/p42 MAPK in adrenal cortex capillary EC (17, 19), which express both receptor types. However, when expressed separately, these receptors exhibited different pattern of MAPK phosphorylation and inositol phosphate production (16).

HUVEC and BAEC are nonfenestrated cells derived from large blood vessels and are commonly used as models for EC. Yet unlike HUVEC, BAEC expressed PK-R (PK-R1); its role in the latter is presently unknown, but it may control vascular tone by increasing endothelial nitric oxide synthase activity (19).

In summary, data presented in this study show that EG-VEGF/PK-1 and VEGF are inversely regulated in ovarian granulosa cells. The inhibitory effects of classical proangiogenic cues, the effects of hypoxia and thrombin (as well as serum) on EG-VEGF/PK-1 expression in SVOG cells, could imply that this protein is not produced to solely support angiogenesis in the ovary and that it may affect other ovarian functions as well. The presence of PK-R1 in steroidogenic cells strongly suggests a role for EG-VEGF/PK-1 in these cells. The positive correlation between steroid production and EG-VEGF/PK-1 expression is also consistent with this notion. These putative ovarian-specific roles for EG-VEGF/PK-1 remain to be defined.

	Acknowledgments

 
We thank Drs. Gera Neufeld and Michal Lahav (Technion-Israel Institute of Technology, Haifa, Israel) for HUVEC and H295R cells and Dr. Israel Vlodavsky (Hadassah-Hebrew University Hospital, Jerusalem, Israel) for BAEC. We are also grateful to Dr. N. Auersperg (University of British Columbia, British Columbia, Canada) for providing SVOG cells.

	Footnotes

 
This study was supported by a grant from the The United States–Israel Binational Agricultural Research and Development Fund.

Abbreviations: BAEC, Bovine aortic EC; BS-1, Bandeiraea simplicifolia lectin-1; CL, corpus luteum; DFX, deferoxamine mesylate; EC, endothelial cells; EG-VEGF, endocrine gland-VEGF; FCS, fetal calf serum; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; GI, gastrointestinal; hGC, human granulosa cell; HIF-1, hypoxia inducible factor 1; hLGC, human luteinized granulosa cells; HUVEC, human umbilical vein EC; P450scc, P450 side-chain cleavage enzyme; PK, prokineticin; PK-R, PK receptor; StAR, steroidogenic acute regulatory protein; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

Received March 20, 2003.

Accepted May 12, 2003.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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