Prokineticin 1 Research Articles

Prokineticin-1/endocrine gland-derived vascular endothelial growth factor is a survival factor for human multiple myeloma cells

Prokineticin-1 (PK1) has been identified as a mitogen-specific protein for the endothelium of steroidogenic glands. Here we report a novel function of PK1 in the regulation of multiple myeloma (MM) cells. PK1 activates multiple signals including mitogen-activated protein kinase (MAPK), PI3K-AKT, and Jak-STAT3, sphingosine kinase-1 (SPK1) in MM cells. Treatment of MM cells with PK1 causes a time- and dose-dependent phosphorylation of MAPK, AKT and STAT3 and upregulation of SPK1 expression and cellular activity. We also show that PK1 upregulates Mcl-1 expression in a time- and dose-dependent manner in human MM cell lines and in the cells of patients with MM. Pertussis toxin, a pan-PK1 receptor inhibitor, can block PK1-induced upregulation of Mcl-1, indicating it relates to a G-protein-coupled receptor. We also show that PK1 protects MM cells against apoptosis induced by starvation for fetal calf serum (FBS), or for FBS and IL-6. Taken together, PK1 activates multiple signaling pathways and, upregulates Mcl-1 expression, leading to proliferation and survival of MM cells.

Upregulation of Endocrine Gland-Derived Vascular Endothelial Growth Factor, But Not Vascular Endothelial Growth Factor in Human Ectopic Endometriotic Tissue

Previous studies have shown that the expression of vascular endothelial growth factor (VEGF) is critical for the development and growth of transplanted endometriosis-like lesions in mouse model systems. The angiogenic response induced by another growth factor, endocrine gland-derived VEGF (EG-VEGF), in the ovary is indistinguishable from that by VEGF. Also known as prokineticin 1 (PK1), EG-VEGF acts through G-protein coupled receptors, PKR1 and PKR2. It is upregulated in the periimplantation endometrium after gonadotropin stimulation and hormone replacement therapy. Some investigators believe that increased expression of EG-VEGF messenger RNA (mRNA) and protein in ectopic endometriotic tissues may be associated with the progression of the disease. The function of EG-VEGF in the uterus is largely unknown and its role in endometriosis is unclear. The aim of this case–control study was to investigate the expression of VEGF, EG-VEGF, and their receptors (PKR1 and PKR2) in eutopic and ectopic endometrial tissues. The investigators hypothesized that aberrant expression of EG-VEGF could be important to the pathogenesis of endometriosis. The subjects for study were infertile women with endometriosis; who were undergoing diagnostic laparoscopy for assessment of tubal patency. Samples of endometrial and endometriotic tissues were taken from women with and without endometriosis. The biopsied tissues were analyzed by quantitative real-time polymerase chain reaction analysis to compare mRNA expression in eutopic and ectopic endometrial tissues. Immunohistochemical staining (using H scoring) was used to determine the expression of EG-VEGF protein in 12 pairs of endometriotic samples. The expression of EG-VEGF mRNA in normal endometrium was 50-fold higher in the secretory than in the proliferative phase; mRNA expression in the PKR1 was 6-fold higher in the proliferative than in the secretory phase. The PKR2 transcript was detected in the normal proliferative but not in the normal secretory endometrium. The expression of eutopic PKR2 mRNA from patients with endometrioisis was 4-fold higher in the proliferative than in the secretory endometrium; no differences were noted in EG-VEGF and PKR1 expression between the proliferative and secretory eutopic endometria. Comparison of the normal and the endometriosis groups showed no significant difference in the expression of EG-VEGF in eutopic endometrium. Comparison of the eutopic and ectopic endometrial samples showed no significant difference in VEGF expression, whereas EG-VEGF expression was significantly higher for the ectopic samples. The expression of PKR1 and PKR2 was very low or undetectable. Higher H scores (more intense staining) were found in the stromal cells of ectopic endometriotic samples than in the paired eutopic endometrial samples; this was indicative of significantly higher EG-VEGF protein expression in the former. These findings suggest that EG-VEGF has an important role in ectopic endometrial angiogenesis and the pathogenesis of endometriosis.

Prokineticins in angiogenesis and cancer

This review will examine the roles of prokineticins in the different neoplasms that have been investigated and will discuss how and why the prokineticin family presents such interesting prospects in the research against cancer. Prokineticins belong to a family of highly conserved small peptides (8 kDa) discovered a decade ago in frog skin secretions and snake venom. The mammalian orthologs consist of two prokineticin peptides, prokineticin 1 (PROK1) and prokineticin 2 (PROK2) that signal through two G-protein coupled receptors: prokineticin receptor 1 (PROKR1) and prokineticin receptor 2 (PROKR2). Over the last decade of research, the PROK/PROKR system has been associated with a considerable number of physiological and pathological functions. Due to this wide spectrum of functions, notably potent angiogenic and immunoregulatory activities, numerous investigators have researched the PROK/PROKR system’s role in cancer development in a variety of tissues.

Natural Selection of Human Embryos: Impaired Decidualization of Endometrium Disables Embryo-Maternal Interactions and Causes Recurrent Pregnancy Loss

BackgroundRecurrent pregnancy loss (RPL), defined as 3 or more consecutive miscarriages, is widely attributed either to repeated chromosomal instability in the conceptus or to uterine factors that are poorly defined. We tested the hypothesis that abnormal cyclic differentiation of endometrial stromal cells (ESCs) into specialized decidual cells predisposes to RPL, based on the observation that this process may not only be indispensable for placenta formation in pregnancy but also for embryo recognition and selection at time of implantation.Methodology/Principal FindingsAnalysis of mid-secretory endometrial biopsies demonstrated that RPL is associated with decreased expression of the decidual marker prolactin (PRL) but increased levels of prokineticin-1 (PROK1), a cytokine that promotes implantation. These in vivo findings were entirely recapitulated when ESCs were purified from patients with and without a history of RPL and decidualized in culture. In addition to attenuated PRL production and prolonged and enhanced PROK1 expression, RPL was further associated with a complete dysregulation of both markers upon treatment of ESC cultures with human chorionic gonadotropin, a glycoprotein hormone abundantly expressed by the implanting embryo. We postulated that impaired embryo recognition and selection would clinically be associated with increased fecundity, defined by short time-to-pregnancy (TTP) intervals. Woman-based analysis of the mean and mode TTP in a cohort of 560 RPL patients showed that 40% can be considered “superfertile”, defined by a mean TTP of 3 months or less.ConclusionsImpaired cyclic decidualization of the endometrium facilitates implantation yet predisposes to subsequent pregnancy failure by disabling natural embryo selection and by disrupting the maternal responses to embryonic signals. These findings suggest a novel pathological pathway that unifies maternal and embryonic causes of RPL.

Prokineticin 1, homeobox A10, and progesterone receptor messenger ribonucleic acid expression in primary cultures of endometrial stromal cells isolated from endometrium of healthy women and from eutopic endometrium of women with endometriosis

To examine prokineticin 1 (PROK1), homeobox (HOX) A10, and P receptor (PR) messenger ribonucleic acid (mRNA) expression in primary cultures of endometrial stromal cells (ESC) obtained from eutopic endometrial samples of patients with endometriosis and to clarify whether in vitro steroid hormone dependence of PROK1 gene expression is altered in endometriosis. Prospective laboratory study. Tertiary university hospital. Twelve normal women (controls) and 12 patients affected by moderate to severe endometriosis in the midsecretory phase of the menstrual cycle. Endometrial specimens were obtained from control women and from women affected by endometriosis; ESC were isolated from endometrial biopsies, and primary cultures were established. Real-time polymerase chain reaction analysis of PROK1, HOXA10, and PR mRNA expression in ESC after 1-4 days of steroid hormone treatment and after decidual differentiation. Contrary to ESC from control women, in ESC obtained from women affected by endometriosis PROK1 and PR mRNA expression was not induced by 1-4 days of treatment with steroid hormones. Nevertheless, when ESC from both groups of women were differentiated to decidual phenotype, PROK1 mRNA was up-regulated and PR and HOXA10 mRNA were down-regulated to the same extent. Our results provide additional evidence for P resistance in endometriosis.

Prokineticin-1 (PROK1) modulates interleukin (IL)-11 expression via prokineticin receptor 1 (PROKR1) and the calcineurin/NFAT signalling pathway

Prokineticin-1 (PROK1) is a multifunctional secreted protein which signals via the G-protein coupled receptor, PROKR1. Previous data from our laboratory using a human genome survey microarray showed that PROK1–prokineticin receptor 1 (PROKR1) signalling regulates numerous genes important for establishment of early pregnancy, including the cytokine interleukin (IL)-11. Here, we have shown that PROK1–PROKR1 induces the expression of IL-11 in PROKR1 Ishikawa cells and first trimester decidua via the calcium–calcineurin signalling pathway in a guanine nucleotide-binding protein (Gq/11), extracellular signal-regulated kinases, Ca2+ and calcineurin–nuclear factor of activated T cells dependent manner. Conversely, treatment of human decidua with a lentiviral miRNA to abolish endogenous PROK1 expression results in a significant reduction in IL-11 expression and secretion. Importantly, we have also shown a regulatory role for the regulator of calcineurin 1 isoform 4 (RCAN1-4). Overexpression of RCAN1-4 in PROKR1 Ishikawa cells using an adenovirus leads to a reduction in PROK1 induced IL-11 indicating that RCAN1-4 is a negative regulator in the calcineurin-mediated signalling to IL-11. Finally, we have shown the potential for both autocrine and paracrine signalling in the human endometrium by co-localizing IL-11, IL-11Rα and PROKR1 within the stromal and glandular epithelial cells of non-pregnant endometrium and first trimester decidua. Overall we have identified and characterized the signalling components of a novel PROK1–PROKR1 signalling pathway regulating IL-11.

Prokineticin 1 modulates IL-8 expression via the calcineurin/NFAT signaling pathway

Prokineticins and their receptors are expressed in various cellular compartments in human endometrium, with prokineticin 1 (PROK1) showing a dynamic pattern of expression across the menstrual cycle and during pregnancy. Previous studies suggest that PROK1 can play an important role in implantation and early pregnancy by inducing vascular remodeling and increasing vascular permeability. Here we demonstrate that PROK1 induces the expression of IL-8, a chemokine with angiogenic properties, in endometrial epithelial Ishikawa cells stably expressing prokineticin receptor 1 and in human first trimester decidua. We also show that IL-8 promoter activity is induced by PROK1 and that this requires the presence of AP1 and NFAT motifs. The role of calcineurin/NFAT signaling pathway is confirmed by the use of specific chemical inhibitors. Additionally, PROK1 induces the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway. A modulatory role for RCAN1-4 is demonstrated by RCAN1-4 overexpression which results in the inhibition of PROK1-induced IL-8 expression whereas reduction in RCAN1-4 endogenous expression results in an increase in PROK1-induced IL-8 production. Our findings show that in endometrial cells PROK1 can activate the calcineurin/NFAT pathway to induce IL-8 expression and that this is negatively modulated by the induction of expression of RCAN1-4.

Prokineticin 1 mRNA expression in the endometrium of healthy women and in the eutopic endometrium of women with endometriosis

To examine prokineticin 1 (PROK1) mRNA expression in eutopic endometrial glands obtained from patients with or without endometriosis, to investigate the presence of additional endometrial abnormalities in women with endometriosis. Prospective laboratory study. University hospital. Twelve control women and 12 patients affected by endometriosis in the secretory phase of the menstrual cycle. Endometrial specimens were obtained from women affected (cases) or not (control group) by endometriosis. Endometrial glands were freshly isolated from endometrial biopsies. PROK1 mRNA expression levels by real-time polymerase chain reaction analysis. PROK1 mRNA was detectable in 4 of 12 (33%) samples obtained from women affected by endometriosis, whereas 10 of 12 (83%) samples obtained from normal women were positive for PROK1 detection by real-time polymerase chain reaction. Moreover, detectable PROK1 mRNA levels were 10 times lower in samples obtained from women with endometriosis than in samples obtained from control women. PROK1 is a newly discovered angiogenic factor implicated in the vascular function of peri-implantation endometrium and early pregnancy. An altered expression of PROK1 could be one of the several biochemical abnormalities characterizing eutopic endometrium in endometriosis.

Prokineticin 1 mediates fetal‐maternal dialogue regulating endometrial leukemia inhibitory factor

Implantation requires communication between a receptive endometrium and a healthy blastocyst. This maternal-embryonic crosstalk involves local mediators within the uterine microenvironment. We demonstrate that a secreted protein, prokineticin 1 (PROK1), is expressed in the receptive endometrium and during early pregnancy. PROK1 induces expression of leukemia inhibitory factor (LIF) in endometrial epithelial cells and first trimester decidua via a Gq-Ca2+-cSrc-mitogen-activated protein kinase kinase-mediated pathway. We show that human embryonic chorionic gonadotropin (hCG) induces sequential mRNA expression of PROK1 and LIF in an in vivo baboon model, in human endometrial epithelial cells, and in first-trimester decidua. We have used micro RNA constructs targeted to PROK1 to demonstrate that hCG-mediated LIF expression in the endometrium is dependent on prior induction of PROK1. Dual immunohistochemical analysis colocalized expression of the luteinizing hormone/chorionic gonadotropin receptor, PROK1, PROKR1, and LIF to the glandular epithelial cells of the first trimester decidual tissue. PROK1 enhances adhesion of trophoblast cells to fibronectin and laminin matrices, which are mediated predominantly via LIF induction. These data describe a novel signaling pathway mediating maternal-embryonic crosstalk, in which embryonic hCG via endometrial PROK1 may play a pivotal role in enhancing receptivity and maintaining early pregnancy.—Evans, J., Catalano, R. D., Brown, P., Sherwin, R., Critchley, H. O. D., Fazleabas, A. T., Jabbour, H. N. Prokineticin 1 mediates fetal-maternal dialogue regulating endometrial leukemia inhibitory factor.

Prokineticin-1: a novel mediator of the inflammatory response in third-trimester human placenta.

Prokineticin-1 (PK1) is a recently described protein with a wide range of functions, including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hemopoiesis. The aim of this study was to investigate the localization and expression of PK1 and PK receptor-1 (PKR1), their signaling pathways, and the effect of PK1 on expression of the inflammatory mediators cyclooxygenase (COX)-2 and IL-8 in third-trimester placenta. PK1 and PKR1 were highly expressed in term placenta and immunolocalized to syncytiotrophoblasts, cytotrophoblasts, fetal endothelium, and macrophages. PK1 induced a time-dependent increase in expression of IL-8 and COX-2, which was significantly reduced by inhibitors of Gq, cSrc, epidermal growth factor receptor (EGFR), and MAPK kinase. Treatment of third-trimester placenta with 40 nm PK1 induced a rapid phosphorylation of cSrc, EGFR, and ERK1/2. Phosphorylation of ERK1/2 in response to PK1 was dependent on sequential phosphorylation of cSrc and EGFR. Using double-immunofluorescent immunohistochemistry, PKR1 colocalized with IL-8 and COX-2 in placenta. These data suggest that PK1 may have a novel role as a mediator of the inflammatory response in placenta.

Prokineticin 1 Signaling and Gene Regulation in Early Human Pregnancy

Prokineticin 1 (PROK1) is a recently described protein with a wide range of functions including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hematopoiesis. The objective of this study was to investigate the role of PROK1 and prokineticin receptor 1 (PROKR1) in human endometrium during early pregnancy. PROK1 and PROKR1 expression is significantly elevated in first-trimester decidua, compared with nonpregnant endometrium. Expression of PROK1 and PROKR1 was localized in glandular epithelial and various cellular compartments within the stroma. To investigate the signaling pathways and target genes activated by PROK1, we generated an endometrial epithelial cell line stably expressing PROKR1 (Ishikawa PROKR1 cells). PROK1-PROKR1 interaction induced inositol phosphate mobilization and sequential phosphorylation of c-Src, epidermal growth factor receptor, and ERK 1/2. Gene microarray analysis on RNA extracted from Ishikawa PROKR1 cells treated with 40 nm PROK1 for 8 h revealed 49 genes to be differentially regulated. A number of these genes, including cyclooxygenase (COX)-2, leukemia inhibitory factor, IL-6, IL-8, and IL-11 are regulated in the endometrium during implantation and early pregnancy. We subsequently investigated the effect of PROK1 on expression of COX-2 in Ishikawa PROKR1 cells and first-trimester decidua. COX-2 mRNA and protein expression, and prostaglandin synthesis, were elevated in response to treatment with PROK1. Moreover, expression of COX-2 by PROK1 was dependent on activation of the Gq-phospholipase C-beta-cSrc-epidermal growth factor receptor-MAPK/ERK kinase pathway. These data demonstrate that PROK1 and PROKR1 expression is elevated in human decidua during early pregnancy and that PROK1-PROKR1 interaction regulates expression of a host of implantation-related genes.

Placental Expression of EG-VEGF and its Receptors PKR1 (Prokineticin Receptor-1) and PKR2 Throughout Mouse Gestation

Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be disregulated in pre-eclampsia (PE). Recently, we characterised the expression of EG-VEGF (endocrine gland-derived vascular endothelial growth factor), also known as prokineticin 1 (PK1) in human placenta during the first trimester of pregnancy and showed that this factor is likely to play an important role in human placentation. However, because it is impossible to prospectively study placentation in humans, it has been impossible to further characterise EG-VEGF expression throughout complete gestation and especially at critical gestational ages for PE development. In the present study, we used mouse placenta to further characterise EG-VEGF expression throughout gestation. We investigated the pattern of expression of EG-VEGF and its receptors, PKR1 and PKR2 at the mRNA and protein levels. Our results show that EG-VEGF and VEGF exhibit different patterns of expression and different localisations in the mouse placenta. EG-VEGF was mainly localised in the labyrinth whereas VEGF was mainly present in glycogen and giant cells. EG-VEGF mRNA and protein levels were highest before 10.5 days post coitus (dpc) whereas those of VEGF showed stable expression throughout gestation. PKR1 protein was localised to the labyrinth layer and showed the same pattern of expression as EG-VEGF whereas PKR2 expression was maintained over 10.5 dpc with both trophoblastic and endothelial cell localisations. Altogether these findings suggest that EG-VEGF may have a direct effect on both endothelial and trophoblastic cells and is likely to play an important role in mouse placentation.

Expression Pattern of Prokineticin 1 and Its Receptors in Bovine Ovaries During the Estrous Cycle: Involvement in Corpus Luteum Regression and Follicular Atresia

Prokineticin 1 (PROK1), also termed endocrine gland-derived vascular endothelial growth factor (endocrine gland-derived VEGF), is a newly identified protein assigned with diverse biologic functions. It binds two homologous G protein-coupled receptors, PROKR1 and PROKR2. To better understand the roles of PROK1 and its receptors in ovarian function, their expression was determined in follicles and corpora lutea (CLs) at different developmental stages. PROK1 mRNA levels were low at early luteal stage and midluteal stage, but increased sharply during natural or induced luteolysis. High PROK1 mRNA levels also were found in atretic follicles. This profile of PROK1 expression was opposite to that of the well-established angiogenic factor VEGF. Of the two receptor-type expressions, PROKR1 but not PROKR2 was correlated positively with its ligand. Immunohistochemical staining revealed that PROK1 was located mainly within the muscular layer of arterioles, and during regression it also was localized to macrophages and steroidogenic cells. The expression pattern of ITGB2 mRNA, a leukocyte cell marker, overlapped that of PROK1, thus suggesting that leukocyte infiltration may explain the elevated expression of PROK1 in atretic follicles and regressing CL. Indeed, flow cytometry analyses showed that nearly all beta-2 integrin chain (ITGB2)-positive cells also were stained with anti-PROK1 and that significantly more ITGB2/PROK1 double-stained cells were present in degenerating follicles and CL. Furthermore, when challenged in vitro with PROK1, adherent, mononuclear cell numbers and TNF levels were elevated, indicating that PROK1 triggers monocyte activation. Together, these data suggest that PROK1, acting via PROKR1, may be involved in the recruitment of monocytes to regressing CL and atretic follicles and their consequent activation therein.

Sensitization of Transient Receptor Potential Vanilloid 1 by the Prokineticin Receptor Agonist Bv8

Small mammalian proteins called the prokineticins [prokineticin 1 (PK1) and PK2] and two corresponding G-protein-coupled receptors [prokineticin receptor 1 (PKR1) and PKR2] have been identified recently, but the physiological role of the PK/PKR system remains mostly unexplored. Bv8, a protein extracted from frog skin, is a convenient and potent agonist for both PKR1 and PKR2, and injection of Bv8 in vivo causes a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]i in a population of isolated dorsal root ganglion (DRG) neurons, which we identified as nociceptors, or sensors for painful stimuli, from their responses to capsaicin, bradykinin, mustard oil, or proteases. Bv8 enhanced the inward current carried by the heat and capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) via a pathway involving activation of protein kinase Cepsilon (PKCepsilon), because Bv8 caused translocation of PKCepsilon to the neuronal membrane and because PKC antagonists reduced both the enhancement of current carried by TRPV1 and behavioral hyperalgesia in rodents. The neuronal population expressing PKRs consisted partly of small peptidergic neurons and partly of neurons expressing the N52 marker for myelinated fibers. Using single-cell reverse transcriptase-PCR, we found that mRNA for PKR1 was mainly expressed in small DRG neurons. Exposure to GDNF (glial cell line-derived neurotrophic factor) induced de novo expression of functional receptors for Bv8 in a nonpeptidergic population of neurons. These results show that prokineticin receptors are expressed in nociceptors and cause heat hyperalgesia by sensitizing TRPV1 through activation of PKCepsilon. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

Expression and Oxygen Regulation of Endocrine Gland-Derived Vascular Endothelial Growth Factor/Prokineticin-1 and Its Receptors in Human Placenta during Early Pregnancy

Angiogenesis is a key process of dynamic tissue remodeling occurring during placentation. Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be deregulated in preeclampsia. Recently a new angiogenic factor, endocrine gland-derived VEGF (EG-VEGF), also known as prokineticin 1 (PK1), has been identified, and its expression was shown to be restricted to endocrine glands, including the placenta. In this study we investigated the pattern of expression of EG-VEGF, its related factor Bv8/PK2, and their common receptors, PKR1 and PKR2, in human placenta during the first trimester of pregnancy. We also examined EG-VEGF and PKR1 regulation by oxygen tension in isolated trophoblast cells (TCs). Our results show that EG-VEGF, but not Bv8/PK2, is expressed in human placenta. EG-VEGF is mainly localized to the syncytiotrophoblast layer with the highest expression detected between the 8th and 10th wk of gestation. EG-VEGF expression within placental villi is different from that of VEGF, which is mainly localized in the cytotrophoblast and extravillous trophoblast cells. In TCs, PKR1 mRNA is about 80 times more abundant than PKR2 mRNA. Both EG-VEGF and PKR1 mRNAs appear to be regulated by hypoxia. These findings suggest that EG-VEGF has a direct effect on TCs via its receptor PKR1 and is likely to play an important role in human placentation. The expression pattern of EG-VEGF, its regulation by oxygen tension, and its complementary localization to that of VEGF suggest that this new factor might also be deregulated in preeclampsia.

Unique expression and regulatory mechanisms of EG-VEGF/prokineticin-1 and its receptors in the corpus luteum

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) or Prokineticin-1 (PK-1) is a novel cysteine-rich protein that belongs to the AVIT protein family. EG-VEGF/PK-1, described as selective angiogenic mitogen, is widely expressed in different tissues including steroidogenic endocrine glands. This review summarizes the expression and functions of EG-VEGF/PK-1 in corpus luteum (CL)-derived cells: endothelial and steroidogenic cell types. EG-VEGF/PK-1 mRNA is expressed by luteal steroidogenic cells of human, rat and bovine ovaries, but was absent from the luteal Endothelial cells CLEC. Luteal EC expressed high levels of both PK-receptors PK-R1 and PK-R2 - the two G protein-coupled PK-1 receptors. Interestingly, expression of EG-VEGF/PK-1 and VEGF were inversely regulated in human and bovine luteinized granulosa cells. EG-VEGF/PK-1 elevated [3H]-thymidine incorporation, MAPK activation and c-jun/fos mRNA expression and enhanced LEC proliferation. EG-VEGF/PK-1 also inhibited serum starvation-induced apoptosis in these cells. Stress conditions such as serum withdrawal, TNFalpha and chemical hypoxia markedly increase PK-R2 expression, whereas mRNA levels of PK-R1 remain unchanged, implying that the anti-apoptotic effect of PK-1 on LEC may be mediated via PK-R2. Besides its direct mitogenic and anti-apoptotic effects, EG-VEGF/PK-1 elevated VEGF mRNA expression in bovine luteal steroidogenic cells, which possesses only PK-R1. Together, these findings suggest an important role for PK-1 in luteal function by acting as a mitogen and survival factor in LEC. Nevertheless, the inverse regulation of EG-VEGF/PK1 and VEGF mRNA expression by ovarian cells and the distribution of its receptors may suggest that in addition to its angiogenic effects, EG-VEGF/PK-1 may also play other roles in ovary.

Discovery of an MIT-like atracotoxin family: Spider venom peptides that share sequence homology but not pharmacological properties with AVIT family proteins

This project identified a novel family of six 66–68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX–Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX–Hvf17, at concentrations up to 1 μM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca 2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX–Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed β-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.

PK1/EG-VEGF induces monocyte differentiation and activation

Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up-regulating antigen-presenting and costimulatory molecules. We report a novel function for prokineticin-1 (PK1)/endocrine gland-derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down-regulation of B7-1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)-12 and tumor necrosis factor alpha and down-regulated IL-10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte-derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.