Natural luteolysis involves multiple pulses of prostaglandin F2alpha (PGF) released by the nonpregnant uterus. This study investigated expression of 18 genes from five distinct pathways, following multiple low-dose pulses of PGF. Cows on Day 9 of the estrous cycle received four intrauterine infusions of 0.25 ml of phosphate-buffered saline (PBS) or PGF (0.5 mg of PGF in 0.25 ml of PBS) at 6-h intervals. A luteal biopsy sample was collected 30 min after each PBS or PGF infusion. There were four treatment groups: Control (n = 5; 4 PBS infusions), 4XPGF (4 PGF infusions; n = 5), 2XPGF-non-regressed (2 PGF infusions; n = 5; PGF-PBS-PGF-PBS; no regression after treatments), and 2XPGF-regressed (PGF-PBS-PGF-PBS; regression after treatments; n = 5). As expected, the first PGF pulse increased mRNA for the immediate early genes JUN, FOS, NR4A1, and EGR1 but unexpectedly also increased mRNA for steroidogenic (STAR) and angiogenic (VEGFA) pathways. The second PGF pulse induced immediate early genes and genes related to immune system activation (IL1B, FAS, FASLG, IL8). However, mRNA for VEGFA and STAR were decreased by the second PGF infusion. After the third and fourth PGF pulses, a distinctly luteolytic pattern of gene expression was evident, with inhibition of steroidogenic and angiogenic pathways, whereas, there was induction of pathways for immune system activation and production of PGF. The pattern of PGF-induced gene expression was similar in corpus luteum not destined for luteolysis (2X-non-regressed) after the first PGF pulse but was very distinct after the second PGF pulse. Thus, although the initial PGF pulse induced mRNA for many pathways, the second and later pulses of PGF appear to have set the distinct pattern of gene expression that result in luteolysis.
Repeated low doses of PGF produce distinct patterns of gene expression; the initial pulse increases expression of many genes, whereas second and later PGF pulses decrease expression of angiogenic and steroidogenic genes while further increasing genes involved in PGF production and the immune system.
corpus luteum; gene expression; hormone action; luteolysis; mRNA; prostaglandins
Prostaglandin F2alpha (PGF) causes luteolysis of the pig corpus luteum (CL) only after Day 12 of the estrous cycle. Recent evidence indicates that progesterone (P4) may protect the CL from cell death. The present study tested the hypothesis that acute inhibition of P4 by treatment with epostane (EPO; 3betaHSD inhibitor) in CL lacking luteolytic capacity (Day 9 CL) will allow PGF to induce responses associated with luteolysis. Multiple PGF-induced responses were evaluated, including genes involved in production of PGF and estradiol-17beta, apoptosis (caspase 3), and transcription (FOSB). These responses are associated with PGF-induced luteolysis and do not normally occur in CL lacking luteolytic capacity. Animals on Day 7 after estrus were divided into four groups: 1) control (C), 2) PGF, 3) EPO, and 4) PGF plus EPO (PGF+EPO). Treatment with EPO (10 mg/kg) or vehicle was given every 12 h for 36 h. Treatment with PGF (25 mg) or vehicle was given at 38 h, and CL were collected from all animals at 48 h. Some CL from each animal were frozen in liquid nitrogen for mRNA and protein analysis. Remaining CL were incubated in media for 2 h for determination of P4 and PGF production. EPO dramatically decreased production of P4 by luteal tissue (ng/mg tissue) by 90% and 95% in EPO and PGF+EPO groups, respectively, compared to C (P < 0.01). Low production of PGF by luteal tissue was found in C, PGF, and EPO groups; however, treatment with PGF+EPO dramatically increased (782%) luteal PGF production. Similar to intraluteal PGF production, increased mRNA for cyclooxygenase 2 (PTGS2) and phospholipase A2 (group IB; PLA2G1B) was found in the PGF+EPO, but not in the EPO or PGF, group. Aromatase (CYP19A1) mRNA was not induced by PGF or EPO; however, PGF+EPO caused a more than 40-fold increase in CYP19A1 mRNA (P < 0.01). CASP3 mRNA was increased (P < 0.01) by EPO (3.4-fold) and by PGF (2.7-fold) but was most dramatically increased by PGF+EPO (5.3-fold), whereas caspase activity was only increased by PGF (1.5-fold) or PGF+EPO (2.2-fold). Thus, these data support the hypothesis that elimination of the protective effect of intraluteal P4 does not directly cause luteolysis of the early CL but allows PGF to induce luteolytic responses in CL lacking luteolytic capacity.
Inhibition of intraluteal P4 using epostane sensitizes Day 9 corpora lutea of pigs to luteolytic effects of prostaglandin F2alpha (PGF) including induction of genes for intraluteal PGF and estradiol-17beta synthesis and apoptosis.
corpus luteum; corpus luteum function; luteolysis; ovary; porcine; progesterone; progesterone receptor; prostaglandin
Cysteine-rich protein 61 (Cyr61) is a secreted matrix-associated protein that regulates a broad spectrum of biological and cellular activities. This study aimed to investigate the role of Cyr61 in progressive kidney fibrosis induced by unilateral ureteral obstruction (UUO) surgery in mice. The expression of Cyr61 transcripts and proteins in the obstructed kidneys were increased from day 1 and remained high until day 10 after surgery. Immunohistochemistry indicated that Cyr61 was expressed mainly in renal tubular epithelial cells. The upregulated Cyr61 in UUO kidneys was reduced in mice treated with pan-transforming growth factor-β (TGF-β) antibody. The role of TGF-β in tubular Cyr61 upregulation after obstructive kidney injury was further supported by experiments showing that TGF-β1 stimulated Cyr61 expression in cultured tubular epithelial cells. Notably, the upregulation of Cyr61 in UUO kidneys was followed by a marked increase in monocyte chemoattractant protein 1 (MCP-1) transcripts and macrophage infiltration, which were attenuated in mice treated with anti-Cyr61 antibodies. This proinflammatory property of Cyr61 in inducing MCP-1 expression was further confirmed in tubular epithelial cells cultured with Cyr61 protein. The anti-Cyr61 antibody in UUO mice also reduced the levels of collagen type 1-α1 transcripts, collagen fibril accumulation evaluated by picrosirius red staining, and the levels of α-smooth muscle actin (α-SMA) transcripts and proteins on day 4 after surgery; however, the antifibrotic effect was not sustained. In conclusion, the TGF-β-mediated increase in tubular Cyr61 expression involved renal inflammatory cell infiltration through MCP-1 induction during obstructive kidney injury. The Cyr61 blockade attenuated kidney fibrosis in the early phase, but the antifibrotic effect could not be sustained.
The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of eosinophils and macrophages. The review highlights areas for future investigation of ovarian microvascular endothelial cells. The potential clinical applications of research directed on corpus luteum endothelial cells are intriguing considering reproductive processes in which vascular dysfunctions may play a role such as ovarian failure, polycystic ovary syndrome (PCOS), and ovarian hyperstimulation syndrome (OHSS).
Studies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation.
Several ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation.
Cells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells.
The findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion.
Prostaglandin F2alpha (PGF) induces luteolysis in cow by inducing a rapid reduction in progesterone production (functional luteolysis) followed by tissue degeneration (structural luteolysis). However the mechanisms of action of PGF remain unclear. Reactive oxygen species (ROS) play important roles in regulating the luteolytic action of PGF. The local concentration of ROS is controlled by superoxide dismutase (SOD), the main enzyme involved in the control of intraluteal ROS. Thus SOD seems to be involved in luteolysis process induced by PGF in cow.
To determine the dynamic relationship between PGF and ROS in bovine corpus luteum (CL) during luteolysis, we determined the time-dependent change of Copper/Zinc SOD (SOD1) in CL tissues after PGF treatment in vivo. We also investigated whether PGF and hydrogen peroxide (H2O2) modulates SOD1 expression and SOD activity in cultured bovine luteal endothelial cells (LECs) in vitro.
Following administration of a luteolytic dose of PGF analogue (0 h) to cows at the mid-luteal stage, the expression of SOD1 mRNA and protein, and total SOD activity in CL tissues increased between 0.5 and 2 h, but fell below the initial (0 h) level at 24 h post-treatment. In cultured LECs, the expression of SOD1 mRNA was stimulated by PGF (1–10 microM) and H2O2 (10–100 microM) at 2 h (P<0.05). PGF and H2O2 increased SOD1 protein expression and total SOD activity at 2 h (P<0.05), whereas PGF and H2O2 inhibited SOD1 protein expressions and total SOD activity at 24 h (P<0.05). In addition, H2O2 stimulated PGF biosynthesis at 2 and 24 h in bovine LECs. Overall results indicate that, SOD is regulated by PGF and ROS in bovine LECs. SOD may play a role in controlling intraluteal PGF and ROS action during functional and structural luteolysis in cows.
Bovine; Corpus luteum; Prostaglandin F2α; Hydrogen peroxide; Superoxide dismutase; Luteal endothelial cell
The hypotheses that PKCepsilon is necessary for: 1) PGF2alpha to inhibit LH-stimulated progesterone (P4) secretion, and 2) for the expression of key prostaglandin synthesizing/metabolizing enzymes were tested in bovine luteal cells in which PKCepsilon expression had been ablated using a validated siRNA protocol. Steroidogenic cells from Day -6 bovine corpus luteum (CL) were isolated and transfected to reduce PKCepsilon expression after 48, 72 and 96 h. A third tested hypothesis was that an increase in intracellular calcium concentration ([Ca(2+)]i) is the cellular mechanism through which PGF2alpha inhibits luteal progesterone. The hypothesis was tested with two pharmacological agents. In the first test, the dose-dependent effects on raising the [Ca(2+)]i with the ionophore, A23187, on basal and LH-stimulated P4 secretion in cells collected from early (Day -4) and mid-cycle (Day -10) bovine CL was examined. In the second test, the ability of PGF2alpha to inhibit LH-stimulated P4 secretion in Day-10 luteal cells was examined under conditions in which an elevation in [Ca(2+)]i had been buffered by means of the intracellular calcium chelator, Bapta-AM.
PKCepsilon expression was reduced 65 and 75% by 72 and 96 h after transfection, respectively. In cells in which PKCepsilon expression was ablated by 75%, the inhibitory effect of PGF2alpha on LH-stimulated P4 secretion was only 29% lower than in the LH-stimulated group. In contrast, it was reduced by 75% in the group where PKCepsilon expression had not been reduced (P < 0.05). Real time PCR analysis indicated that there were no differences in the expression of cyclooxygenase-2 (COX-2), aldoketoreductase 1B5 (AKR1B5), prostaglandin E synthase (PGES), hydroxyprostaglandin-15 dehydrogenase (PGDH) and PGE2 -9-reductase as a function of PKCepsilon down-regulation. Finally, LH stimulated secretion of P4 at each luteal stage (Day -4 and -10), and PGF2alpha inhibited this only in Day -10 cells (P < 0.05). When A23187 was used at concentrations greater than 0.1 μmol, the induced elevation in [Ca(2+)]i inhibited the effect of LH on secretion of P4 in Day -4 and -10 cells (P < 0.05, Fig. 5). The inhibitory effect of PGF2alpha on LH-stimulated P4 in Day -10 cells was reduced if an increase in [Ca(2+)]i was prevented with Bapta-AM. These results support the hypothesis that differential expression of PKCepsilon and an elevation of [Ca(2+)]i are important for acquisition of luteolytic response to PGF2alpha.
Cysteine-rich angiogenic inducer 61 (Cyr61) is an extracellular matrix protein involved in the transduction of growth factor and hormone signaling. Previously, we demonstrated that Cyr61 was highly expressed in prostate cancer (PCa) but that the expression levels were associated with a lower risk of PCa recurrence. In the present study, we demonstrate that serum Cyr61 is a potential biomarker that correlates with PCa aggressiveness. Furthermore, we also explore the potential mechanism underlying the changes in Cyr61 expression during PCa progression.
Cyr61 concentrations in the medium from PCa cell lines and in serum samples obtained from PCa patients were measured by sandwich ELISA. Serum Cyr61 levels were correlated with disease characteristics and the association between Cyr61 expression changes by several types of stimulation or stress and cAMP/cAMP-dependent protein kinase (PKA) pathway were examined.
There was a positive correlation between Cyr61 levels in cell supernatants and mRNA expression in these cell lines. Serum Cyr61 levels were significantly higher in non-organ-confined PCa patients (116.3 ± 140.2 ng/ml) than in organ-confined PCa patients (79.7 ± 56.1 ng/ml) (P = 0.031). Cyr61 expression was up-regulated in response to both lysophosphatidic acid and androgen treatments which promoted PCa cell invasion. Serum starvation and phosphoinositide-3-kinase inhibition also resulted in Cyr61 up-regulation; however, they suppressed cell proliferation. Cyr61 up-regulation was correlated with an increase in cAMP and suppressed by PKA inhibition.
These findings suggest that Cyr61 expression in PCa is regulated by the cAMP/PKA pathway and that circulating Cyr61 levels are a potential serum-based biomarker for characterizing PCa.
Cyr61; prostate cancer; serum marker; cAMP-dependent protein kinase
Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved.
The interactions between luteal, vascular endothelial, immune cells and its products: steroids, peptide hormones, prostaglandins (PGs), growth factors and cytokines play a pivotal role in the regulation of corpus luteum (CL) function. Luteal endothelial cells undergo many dynamic morphological changes and their action is regulated by cytokines. The aims are: (1) to establish in vitro model for bovine luteal endothelial cells examination; (2) to study the effect of cytokines: tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) on cell viability, leukotrienes (LTs) and PG synthases, and endothelin-1 (EDN-1) mRNA, protein expression and their secretion in bovine immortalized luteal endothelial (EnCL-1) cells.
The primary cultures of bovine luteal endothelial cells were immortalized by transfection with vector carrying the Simian virus 40 T-antigen (SV40 T-ag) sequence. Expression of SV40 T-ag gene in EnCL-1 cells was confirmed by RT-PCR and immunofluorescence staining showed the presence of endothelial cell markers: VE-cadherin and von Willebrand factor. EnCL-1 cells were stimulated by TNFalpha with IFNgamma (50 ng/ml each) for 24 h. Cell viability, mRNA expression (real time RT-PCR), protein expression (western blotting) for LTC4 synthase (LTC4S), LTA4 hydrolase (LTA4H), PGE2 and PGF2alpha synthases and endothelin-1 (EDN-1), and levels of LTs (B4 and C4) and PGs (E2 and F2alpha) and EDN-1 in the medium (EIA) were evaluated.
We received immortalized luteal endothelial cell line (EnCL-1). Cytokines did not change EnCL-1 cell viability but increased mRNA expression of LTC4S, LTA4H, PGE2 and PGF2alpha synthases and EDN-1. EDN-1/2/3, LTC4 and PGF2alpha synthases protein expression were elevated in the presence of TNFalpha/IFNgamma, and accompanied by increased EDN-1, LTC4 and PGF2alpha secretion. Cytokines had no effect on PGES and LTA4H protein expression, and PGE2 and LTB4 release.
TNFalpha and IFNgamma modulate EnCL-1 cell function. Moreover, established EnCL-1 cell line appears to be a good model for investigating the molecular mechanisms related to cytokines action and aa metabolites production in cattle.
Osteosarcoma is the most common primary malignant tumor in children and young adults, and its treatment requires effective therapeutic approaches because of a high mortality rate for lung metastasis. Epithelial to mesenchymal transition (EMT) has received considerable attention as a conceptual paradigm for explaining the invasive and metastatic behavior during cancer progression. The cysteine-rich angiogenic inducer 61 (Cyr61) gene, a member of the CCN gene family, is responsible for the secretion of Cyr61, a matrix-associated protein that is involved in several cellular functions. A previous study showed that Cyr61 expression is related to osteosarcoma progression. In addition, Cyr61 could promote cell migration and metastasis in osteosarcoma. However, discussions on the molecular mechanism involved in Cyr61-regulated metastasis in osteosarcoma is poorly discussed.
We determined that the expression level of Cyr61 induced cell migration ability in osteosarcoma cells. The Cyr61 protein promoted the mesenchymal transition of osteosarcoma cells by upregulating mesenchymal markers (TWIST-1 and N-cadherin) and inhibiting the epithelial marker (E-cadherin). Moreover, the Cyr61-induced cell migration was mediated by EMT. The Cyr61 protein elicited a signaling cascade that included αvβ5 integrin, Raf-1, mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK), and Elk-1. The reagent or gene knockdown of these signaling proteins could inhibit Cyr61-promoted EMT in osteosarcoma. Finally, the knockdown of Cyr61 expression obviously inhibited cell migration and repressed mesenchymal phenotypes, reducing lung metastasis.
Our results indicate that Cyr61 promotes the EMT of osteosarcoma cells by regulating EMT markers via a signal transduction pathway that involves αvβ5 integrin, Raf-1, MEK, ERK, and Elk-1.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-4598-13-236) contains supplementary material, which is available to authorized users.
Osteosarcoma; Cyr61; EMT; Migration
There is increasing evidence that the corpus luteum has an important role in regulating its own demise. A series of experiments was performed to study the effects of luteal concentrations of progesterone on the functions of steroidogenic luteal cells. In the first experiment, steroidogenic small luteal cells (SLCs) were separated from endothelial cells, and it was determined that it was the SLCs that contained receptors for oxytocin. Treatment with progesterone (95 μM) for as little as 1 h decreased (P < 0.05) the percentage of SLCs responding to oxytocin (10 μM) with an increase in intracellular concentrations of calcium, and this effect continued for the duration of the experiment. In a second experiment, the response to oxytocin was increased (P < 0.05) by 3 h (but not 1 h) following progesterone removal, with a further increase by 16 h. The ability of 1 μM prostaglandin F2alpha (PGF2alpha) to increase intracellular concentrations of calcium was also decreased (P < 0.05) by progesterone treatment. By 3 h following removal of progesterone, the percentage of steroidogenic large luteal cells (LLCs) responding to PGF2alpha was increased and not different from that observed in cells 16 h after progesterone removal. Finally, cyclodextrins (methyl-beta cyclodextrin [MbetaCD]) were used to remove cholesterol from the plasma membrane of luteal cells, and MbetaCD loaded with cholesterol was used to put cholesterol back into the plasma membrane of progesterone-treated cells. Treatment with MbetaCD reduced (P < 0.05) the responsiveness of SLCs to oxytocin and LLCs to PGF2alpha. Use of cholesterol-loaded MbetaCD returned the responsiveness of both SLCs and LLCs treated with progesterone to that observed in vehicle (no progesterone)-treated controls. In summary, intraluteal concentrations of progesterone inhibit the ability of oxytocin to increase intracellular concentrations of calcium in SLCs and the ability of PGF2alpha to increase intracellular concentrations of calcium in LLCs. The highest concentration of progesterone appears to act by influencing cholesterol content of the luteal cell membranes.
Intraluteal concentrations of progesterone inhibit increases in intracellular concentrations of calcium stimulated by oxytocin and prostaglandin F2α in ovine luteal cells.
corpus luteum; oxytocin; progesterone; prostaglandin F2alpha
The Hedgehog (Hh) pathway is well known for its involvement in angiogenesis and vasculogenesis during ontogeny. The ligand, Sonic hedgehog (SHH), plays an important role in vascular formation during development. However, SHH expression is upregulated on tumor cells and can impact the tumor microenvironment. We have investigated the effects of autocrine as well as paracrine Hh signaling on tumor cells as well as on endothelial cells, respectively.
Upon constitutive expression of SHH, breast cancer cells showed aggressive behavior and rapid xenograft growth characterized by highly angiogenic tumors that were spontaneously metastatic. In these cells, SHH caused activation of the Hh transcription factor, GLI1, leading to upregulated expression of the potent pro-angiogenic secreted molecule, CYR61 (cysteine rich angiogenic inducer 61). Silencing of CYR61 from these SHH-expressing Hh activated cells blunted the malignant behavior of the tumor cells and resulted in reduced tumor vasculature and limited hematogenous metastases. Thus, CYR61 is a critical downstream contributor to the Hh influenced pro-angiogenic tumor microenvironment. We also observed concomitant upregulation of SHH and CYR61 transcripts in tumors from patients with advanced breast cancer, further ratifying the clinical relevance of our findings. In summary, we have defined a novel, VEGF-independent, clinically relevant, pro-angiogenic factor, CYR61, that is a transcriptional target of Hh-GLI signaling.
SHH; CYR61; GLI; Hedgehog; angiogenesis; metastasis
Cysteine-rich angiogenic inducer 61 (Cyr61) is a novel molecule that has been shown to be increased in the synovial tissues of patients with rheumatoid arthritis (RA). The present study was conducted in order to investigate the role of Cyr61 in the pathogenesis of RA. A human genome-wide gene assay was used to screen gene expression in synovial tissues obtained from four patients with RA and three patients with osteoarthritis (OA). To examine the role of Cyr61 in the phenotype of RA-fibroblast-like synovial (FLS) cells, Cyr61 expression in RA-FLS cells was knocked down using small interfering RNA (siRNA). Normal FLS cells transduced with lentiviral vectors encoding Cyr61 cDNA were used to further explore the effects of this molecule on FLS cell apoptosis, proliferation and invasion. The study found that the Cyr61 gene was highly expressed in the synovial cells from patients with RA compared with those from patients with OA. Downregulation of Cyr61 by siRNA led to impaired cell proliferation and invasion. Furthermore, it decreased the levels of matrix metalloproteinase (MMP)-3 and MMP-13, and induced apoptosis in RA-FLS cells. Conversely, overexpression of Cyr61 in normal FLS cells led to opposite effects. In conclusion, these results indicate that Cyr61 is capable of promoting RA-FLS cell proliferation and invasion via the suppression of apoptosis and the regulation of MMP expression. Therefore, Cyr61 may be a good target molecule for the treatment and prevention of RA.
rheumatoid arthritis; fibroblast-like synovial cell; cysteine-rich angiogenic inducer 61; cell proliferation; cell invasion
Prostaglandin F2α (PGF2α) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF2α administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF2α injection. The number of PMNs was increased at 5 min after PGF2α administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF2α directly stimulated P-selectin protein expression at 5–30 min in luteal endothelial cells (LECs). Moreover, PGF2α enhanced PMN adhesion to LECs, and this enhancement by PGF2α was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF2α is crucial in PMN migration. In conclusion, PGF2α rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF2α may involve an acute inflammatory-like response due to rapidly infiltrated PMNs.
Prostaglandin F2α (PGF2α) induces luteolysis in cows and causes
infiltration of immune cells, which resembles inflammatory immune response. Since the
general immune response is mediated by the lymphatic system, we hypothesized that
luteolysis is associated with generation of an immune response that involves lymphatic
vessels in the bovine corpus luteum (CL). The CL was obtained from Holstein cows at the
mid-luteal phase (days 10–12, ovulation = day 0) by ovariectomy at various time points
after PGF2α injection. Lymphatic endothelial cell (LyEC) marker, endothelial
hyaluronan receptor 1 (LYVE1), levels decreased significantly 12 h after PGF2α
injection. Podoplanin, another LyEC marker, decreased from 15 min after PGF2α
injection. PGF2α also diminished mRNA expression of lymphangiogenic factors,
such as vascular endothelial growth factor (VEGF) C, VEGFD and VEGF receptor 3 (VEGFR3).
During PGF2α-induced luteolysis, the levels of mRNA expression of tumor
necrosis factor α (TNFα; the major pro-inflammatory cytokine) and chemokine (C-X-C motif)
ligand 1 (neutrophil chemokine) were increased. On the other hand, chemokine (C-C motif)
ligand 21, which regulates outflow of immune cells from tissues via the lymphatic vessels
during an immune response, was decreased. This study demonstrated that the lymphatic
network in the CL is disrupted during luteolysis and suggests that during luteolysis,
immune cells can induce a local immune response in the CL without using the lymphatic
Corpus luteum; Cow; Luteolysis; Lymphatic vessel; Tumor necrosis factor α
Luteinizing hormone (LH) regulates several ovarian functions. However, the
luteoprotective mechanisms of LH involved in the maintenance of bovine corpus luteum (CL)
function are not well understood. Since prostaglandin F2α (PGF), PGE2 and progesterone
(P4) are well documented as antiapoptotic factors in the bovine CL, we hypothesized that
LH protects the CL by stimulating the local production and action of PGF, PGE2 and P4.
Cultured bovine luteal cells obtained at the mid-luteal stage (days 8–12 of the estrous
cycle) were treated with LH (10 ng/ml), onapristone (OP: a specific P4 receptor
antagonist, 100 μM) and indomethacin [INDO; a cyclooxygenase (COX) inhibitor, 100 μM] for
24 h. LH with and without OP significantly increased the mRNA and protein expressions of
COX-2, PGF synthase and carbonyl reductase (P<0.05) but not the mRNA and protein
expressions of COX-1 and PGE synthase in bovine luteal cells. In addition, these
treatments significantly increased PGF and P4 production (P<0.05) but not PGE2
production. Luteal cell viability was significantly increased by LH alone (P<0.05), but
LH-increased cell viability was reduced by LH in combination with INDO as well as OP
(P<0.05). The overall results suggest that LH prevents luteal cell death by stimulating
luteal PGF and P4 production and supports CL function during the luteal phase in
Corpus luteum; Luteinizing hormone; Progesterone; Prostaglandin E2; Prostaglandin F2α
Background and purpose: A prostamide analogue, bimatoprost, has been shown to be effective in reducing intraocular pressure, but its precise mechanism of action remains unclear. Hence, to elucidate the molecular mechanisms of this effect of bimatoprost, we focused on pharmacologically characterizing prostaglandin FP receptor (FP) and FP receptor variant (altFP) complexes.
Experimental approach: FP receptor mRNA variants were identified by reverse transcription-polymerase chain reaction. The FP-altFP4 heterodimers were established in HEK293/EBNA cells co-expressing FP and altFP4 receptor variants. A fluorometric imaging plate reader was used to study Ca2+ mobilization. Upregulation of cysteine-rich angiogenic protein 61 (Cyr61) mRNA was measured by Northern blot analysis, and phosphorylation of myosin light chain (MLC) by western analysis.
Key results: Six splicing variants of FP receptor mRNA were identified in human ocular tissues. Immunoprecipitation confirmed that the FP receptor is dimerized with altFP4 receptors in HEK293/EBNA cells co-expressing FP and altFP4 receptors. In the studies of the kinetic profile for Ca2+ mobilization, prostaglandin F2α (PGF2α) elicited a rapid increase in intracellular Ca2+ followed by a steady state phase. In contrast, bimatoprost elicited an immediate increase in intracellular Ca2+ followed by a second phase. The prostamide antagonist, AGN211335, selectively and dose-dependently inhibited the bimatoprost-initiated second phase of Ca2+ mobilization, Cyr61 mRNA upregulation and MLC phosphorylation, but did not block the action of PGF2α.
Conclusion and implications: Bimatoprost lacks effects on the FP receptor but may interact with the FP-altFP receptor heterodimer to induce alterations in second messenger signalling. Hence, FP-altFP complexes may represent the underlying basis of bimatoprost pharmacology.
prostaglandin; prostamide; intraocular pressure; receptor dimerization; calcium signalling
Bovine luteal parenchymal cells express class II major histocompatibility complex (MHC) molecules and stimulate class II MHC-dependent activation of T cells in vitro. The ability of a class II MHC-expressing cell type to elicit a response from T cells in vivo is also dependent on expression of costimulatory molecules by the antigen presenting cell and delivery of a costimulatory signal to the T cell. Whether bovine luteal parenchymal cells express costimulatory molecules and can deliver the costimulatory signal is currently unknown.
Bovine luteal tissue was collected during the early (day 5; day of estrus = day 0), mid (day 11–12), or late (day 18) luteal phase of the estrous cycle, and at 0, 0.5, 1, 4, 12 or 24 hours following administration of PGF2alpha to cows on day 10 of the estrous cycle. Northern analysis was used to measure CD80 or CD86 mRNA concentrations in luteal tissue samples. Mixed luteal parenchymal cell cultures and purified luteal endothelial cell cultures were prepared, and real-time RT-PCR was used to examine the presence of CD80 and CD86 mRNA in each culture type. Monoclonal antibodies to CD80 and CD86 were added to a mixed luteal parenchymal cell-T cell co-culture in vitro T cell proliferation assay to assess the functional significance of costimulatory molecules on activation of T lymphocytes by luteal parenchymal cells.
Northern analysis revealed CD80 and CD86 mRNAs in luteal tissue, with greatest steady-state concentrations at midcycle. CD80 and CD86 mRNAs were detected in mixed luteal parenchymal cell cultures, but only slight amounts of CD80 (and not CD86) mRNA were detected in cultures of luteal endothelial cells. Luteinizing hormone, PGF2alpha and TNF-alpha were without effect on concentrations of CD80 or CD86 mRNA in mixed luteal parenchymal cells cultures. Anti-CD80 or anti-CD86 monoclonal antibodies inhibited T cell proliferation in the in vitro T cell proliferation assay.
It can be concluded from this study that parenchymal cells within the bovine CL express functional costimulatory molecules that facilitate interactions between with T cells, and these components of the antigen presentation pathway are expressed maximally in the midcycle CL.
Disturbed uterine involution impairs ovarian function in the first weeks after calving. This study analyzed the long-term effect of metritis on luteal function of 47 lactating Holstein-Friesian cows during the first four postpartum estrous cycles. Cows with abnormal uterine enlargement and malodorous lochia were classified as having metritis (group M, n = 18), and all others were considered healthy (group H, n = 29). Luteal size was measured once between days 9 and 13 of the first (group H, n = 11; group M, n = 12), second (group H, n = 23; group M, n = 18) and fourth (group H, n = 11; group M, n = 7) postpartum luteal phases. Serum progesterone concentration was measured at the same time. Sixteen cows (group H, n = 9; group M, n = 7) underwent transvaginal luteal biopsy for gene expression analysis of steroidogenic regulatory proteins during the second and fourth cycles. Cows with persistence of the corpus luteum (CL) underwent determination of luteal size, luteal biopsy and serum progesterone measurement once between days 29 and 33, followed by prostaglandin treatment to induce luteolysis. The same procedures were repeated once between days 9 and 13 of the induced cycle.
The cows in group M had smaller first-cycle CLs than the cows in group H (p = 0.04), but progesterone concentrations did not differ between groups. Luteal size, progesterone concentration and gene expression did not differ between the two groups during the second and fourth cycles. Compared with healthy cows (10%), there was a trend (p = 0.07) toward a higher prevalence of persistent CLs in cows with metritis (33%). Persistent CLs were limited to the first cycle. Persistent CLs and the induced cyclic CLs did not differ with regard to the variables investigated.
An effect of metritis on luteal activity was apparent in the first postpartum estrous cycle. However, after the first postpartum cycle, no differences occurred in analyzed parameters between metritis and control cows. Therefore, a metritis is able to impair luteal activity transiently, but does not seem to have a long-term effect on luteal function.
Metritis; Luteal size; Progesterone; Persistent corpus luteum; Gene expression; Bovine
Prostaglandin-F2α (PGF2α) is a product of the cyclooxygenase pathway and is a local signaling molecule that activates a G-protein-coupled prostanoid receptor named FP. FP receptors can stimulate T-cell factor (Tcf) transcriptional activation by stabilization of β-catenin and can upregulate the expression of mRNA encoding cysteine-rich protein 61 (Cyr61), a secreted extracellular matrix protein that stimulates angiogenesis. We now show in both HEK cells and human microglial cells that the induction of Cyr61 protein expression by the human FP receptor utilizes a novel mechanism involving the activation of Ras and Raf followed by a MEK/ERK independent activation of Tcf signaling. The upregulation of Cyr61 in microglial cells may contribute to glioma tumorigenesis and could be a potential therapeutic target.
G-protein coupled receptors; prostaglandin; cyclooxygenase; glioma; T-cell factor; cysteine-rich protein 61; mitogen activated protein kinase; MAPK kinase; β-catenin; microglial cells
Cysteine-rich 61 (Cyr61), a member of the CCN protein family, possesses diverse functionality in cellular processes such as adhesion, migration, proliferation, and survival. Cyr61 can also function as an oncogene or a tumour suppressor, depending on the origin of the cancer. Only a few studies have reported Cyr61 expression in colorectal cancer. In this study, we assessed the Cyr61 expression in 251 colorectal cancers with clinical follow up.
We examined Cyr61 expression in 6 colorectal cancer cell lines (HT29, Colo205, Lovo, HCT116, SW480, SW620) and 20 sets of paired normal and colorectal cancer tissues by western blot. To validate the association of Cyr61 expression with clinicopathological parameters, we assessed Cyr61 expression using tissue microarray analysis of primary colorectal cancer by immunohistochemical analysis.
We verified that all of the cancer cell lines expressed Cyr61; 2 cell lines (HT29 and Colo205) demonstrated Cyr61 expression to a slight extent, while 4 cell lines (Lovo, HCT116, SW480, SW620) demonstrated greater Cyr61 expression than HT29 and Colo205 cell lines. Among the 20 cases of paired normal and tumour tissues, greater Cyr61 expression was observed in 16 (80%) tumour tissues than in normal tissues. Furthermore, 157 out of 251 cases (62.5%) of colorectal cancer examined in this study displayed strong Cyr61 expression. Cyr61 expression was found to be associated with pN (p = 0.018). Moreover, Cyr61 expression was associated with statistically significant cancer-specific mortality (p = 0.029). The duration of survival was significantly lesser in patients with Cyr61 high expression than in patients with Cyr61 low expression (p = 0.001). These results suggest that Cyr61 expression plays several important roles in carcinogenesis and may also be a good prognostic marker for colorectal cancer.
Our data confirmed that Cyr61 was expressed in colorectal cancers and the expression was correlated with worse prognosis of colorectal cancers.
Colorectal cancer; Cyr61; Immunohistochemistry; Prognosis
There is clear evidence for intraluteal production of prostaglandins (PGs) in numerous species and under a variety of experimental conditions. In general, secretion of PGs appears to be elevated in the early corpus luteum (CL) and during the period of luteolysis. Regulation of intraluteal PG production is regulated by a variety of factors. An autoamplification pathway in which PGF-2alpha stimulates intraluteal production of PGF-2alpha has been identified in a number of species. The mechanisms underlying this autoamplification pathway appear to differ by species with expression of Cyclooxygenase-2 (Cox-2) and activity of phospholipase A2 acting as important physiological control points. In addition, a number of other responses that are induced by PGF-2alpha (decreased luteal progesterone, increased endothelin-1, increased cytokines) also have been found to increase intraluteal PGF-2alpha production. Thus, regulation of intraluteal PG production may serve to initiate or amplify physiological signals to the CL and may be important in specific aspects of luteal physiology particularly during luteal regression.
AIMS: To characterise the human cyr61 gene (cyr61H) and determine its chromosomal locality. To compare expression of cyr61H in human tumour cell lines with that of two other structurally related genes, novH (nephroblastoma overexpressed gene) and CTGF (connective tissue growth factor), that are likely to play a role in the control of cell proliferation and differentiation. METHODS: To isolate the human cyr61 gene, placental genomic and HeLa cDNA libraries were screened with murine cyr61 cDNA. The nucleotide sequence of the complete cyr61H cDNA was established. Both Southern blotting of a panel of somatic cell hybrids and in situ hybridisation on chromosomes were performed to map the cyr61H gene. Expression of cyr61H, novH, CTGF, and novH was analysed by northern blotting in both human neuroblastomas and glioblastoma cell lines. RESULTS: Genomic and cDNA clones encompassing the cyr61H gene were isolated and characterised. Comparison of mouse and human cyr61 sequences indicated that their genomic organisation is highly conserved. Alignment of coding sequences highlighted the conservation of cyr61 regions that might be critical for its biological function. The data showed that the cyr61H gene is assigned to chromosome 1p22.3 and that different levels of cyr61H, CTGF, and novH mRNA have been detected in several human tumour cell lines derived from the nervous system. CONCLUSIONS: The human cyr61 gene belongs to an emerging family of genes including CTGF/fisp12 and nov. The murine cyr61 encodes an extracellular cysteine rich protein that exhibits chemotactic activity, promotes attachment and spreading of cells, and potentiates the mitogenic effect of growth factors. Assignment of the cyr61H gene to chromosome 1p22.3 will allow studies to determine whether human pathologies derived from the nervous system or from other tissues are associated with chromosomal abnormalities involving this region. Although the coding regions of cyr61H, CTGF, and novH are highly homologous, a growing body of evidence suggests that expression of these genes is regulated differentially, and that a balance between expression of these genes might represent a key element in determining the stage of differentiation and/or the malignant potential of tumour cells.
The development and regression of corpus luteum (CL) is characterized by an intense angiogenesis and angioregression accompanied by luteal tissue and extracellular matrix (ECM) remodelling. Vascular Endothelial Growth Factor (VEGF) is the main regulator of angiogenesis, promoting endothelial cell mitosis and differentiation. After the formation of neovascular tubes, the remodelling of ECM is essential for the correct development of CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). During luteal regression, characterized by an apoptotic process and successively by an intense ECM and luteal degradation, the activation of Ca++/Mg++-dependent endonucleases and MMPs activity are required. The levels of expression and activity of VEGF, MMP-2 and -9, and Ca++/Mg++-dependent endonucleases throughout the oestrous cycle and at pregnancy were analyzed.
Different patterns of VEGF, MMPs and Ca++/Mg++-dependent endonuclease were observed in swine CL during different luteal phases and at pregnancy. Immediately after ovulation, the highest levels of VEGF mRNA/protein and MMP-9 activity were detected. On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed.
Our findings, obtained from a precisely controlled in vivo model of CL development and regression, allow us to determine relationships among VEGF, MMPs and endonucleases during angiogenesis and angioregression. Thus, CL provides a very interesting model for studying factors involved in vascular remodelling.