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
It is well known that bone marrow-derived mesenchymal stem cells (MSCs) are involved in wound healing and regeneration responses. In this study, we globally profiled the proteome of MSCs to investigate critical factor(s) that may promote wound healing. Cysteine-rich protein 61 (Cyr61) was found to be abundantly present in MSCs. The presence of Cyr61 was confirmed by immunofluorescence staining and immunoblot analysis. Moreover, we showed that Cyr61 is present in the culture medium (secretome) of MSCs. The secretome of MSCs stimulates angiogenic response in vitro, and neovascularization in vivo. Depletion of Cyr61 completely abrogates the angiogenic-inducing capability of the MSC secretome. Importantly, addition of recombinant Cyr61 polypeptides restores the angiogenic activity of Cyr61-depleted secretome. Collectively, these data demonstrate that Cyr61 polypeptide in MSC secretome contributes to the angiogenesis-promoting activity, a key event needed for regeneration and repair of injured tissues.
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
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.
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.
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.
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
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 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
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.
The goal of this study was to examine the response of the cyclic corpus luteum of cows to the administration of a prostaglandin F2α analogue (PGF) during the transition of refractoriness to responsiveness by investigating ultrasonographic changes in the corpus luteum and changes in plasma progesterone concentration in cows following the administration of PGF 3 and 5 days after ovulation.
All cows that responded to PGF given on day 5 ovulated on day 9. In the cows that did not respond on day 5, the luteal cross-sectional area stagnated after treatment, whereas the plasma progesterone concentration continued to increase. In the cows that received PGF on day 3 of the oestrous cycle, the luteal cross-sectional area and the plasma progesterone concentration continued to increase.
The results of this study suggest that the transition of the bovine corpus luteum from refractoriness to responsiveness to PGF occurs during day 5 of the oestrous cycle because in 5 of 8 cows given PGF on day 5, early luteal regression was evident during the examination 24 hours after PGF administration.
In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.
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).
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
CCN-proteins are known to be involved in development, homeostasis and repair of mesenchymal tissues. Since these processes implicate recruitment of cells with the potential to be committed to various phenotypes, we studied the effect of CYR61/CCN1 and WISP3/CCN6 on migration of human bone marrow derived mesenchymal stroma cells (MSCs) in comparison to in vitro osteogenic differentiated MSCs using a modified Boyden chamber assay.
CYR61 and WISP3 were purified as fusion proteins with a C-terminal Fc-tag from baculovirus infected SF21 cells using protein G sepharose columns. CYR61 and WISP3 stimulated cell migration of undifferentiated MSCs in a dose-dependent manner. CYR61 and WISP3 had similar effects on committed osteogenic precursor cells. Checkerboard analysis revealed that CYR61 and WISP3 stimulated true directed cell migration (chemotaxis) of MSCs and committed osteogenic precursors. In MSCs the chemotactic activity of WISP3 but not CYR61 was mediated through integrin ανß5.
Our results indicate that CYR61 and WISP3 can function as soluble ligands transmitting chemotactic signals to human MSCs but differ in the involvement of integrin ανß5. This may be relevant for their possible role in connective tissue repair.
The corpus luteum (CL) is a transient endocrine organ that is essential for maintenance of pregnancy in both ruminants and primates. The cellular and endocrine mechanisms that regulate the CL in these species have commonalities and some distinct and intriguing differences. Both species have similar cellular content with large luteal cells derived from the granulosa cells of the follicle, small luteal cells from follicular thecal cells, and large numbers of capillary endothelial cells that form the vasculature that has an essential role in optimal CL function. Intriguingly, the large luteal cells in ruminants grow larger than in primates and acquire a capacity for high constitutive progesterone (P4) production that is independent of stimulation from LH. In contrast, the primate CL and the granulosa lutein cells from primates continue to require stimulation by LH/CG throughout the luteal phase. Although the preovulatory follicle of women and cows had similar size and steroidogenic output (10 to 20 mg/h), the bovine CL had about ten-fold greater P4 output compared to the human CL (17.4 vs. 1.4 mg/h), possibly due to the development of high constitutive P4 output by the bovine large luteal cells. The continued dependence of the primate CL on LH/CG/cAMP also seems to underlie luteolysis, as there seems to be a requirement for greater luteotropic support in the older primate CL than is provided by the endogenous LH pulses. Conversely, regression of the ruminant CL is initiated by PGF from the nonpregnant uterus. Consequently, the short luteal phase in ruminants is primarily due to premature secretion of PGF by the nonpregnant uterus and early CL regression, whereas CL insufficiency in primates is related to inadequate luteotropic support and premature CL regression. Thus, the key functions of the CL, pregnancy maintenance and CL regression in the absence of pregnancy, are produced by common cellular and enzymatic pathways regulated by very distinct luteotropic and luteolytic mechanisms in the CL of primates and ruminants.
corpus luteum; primate; progesterone; ruminant
Sclerostin, a secreted glycoprotein, regulates osteoblast function. Using yeast two-hybrid and direct protein interaction analyses, we demonstrate that sclerostin binds the Wnt-modulating and Wnt-modulated, extracellular matrix protein, cysteine-rich protein 61 (Cyr61, CCN1), which regulates mesenchymal stem cell proliferation and differentiation, osteoblast and osteoclast function, and angiogenesis. Sclerostin was shown to inhibit Cyr61-mediated fibroblast attachment, and Cyr61 together with sclerostin increases vascular endothelial cell migration and increases osteoblast cell division. The data show that sclerostin binds to and influences the activity of Cyr61.
Sclerostin; Cyr61; CCN1; osteoblasts; osteoclasts; high bone mass; LRP5; Wnt
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.
The corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.
Porcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage.
PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha.
We obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures.
Our data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.
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.
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
The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. As targeting of etiology-related pathogenic pathways may be more efficient than current standard heart failure treatment, we obtained the genomic expression profile of a DCM subtype characterized by cardiac inflammation to identify possible new therapeutic targets in humans. In this inflammatory cardiomyopathy (DCMi), a distinctive cardiac expression pattern not described in any previous study of cardiac disorders was observed. Two significantly altered gene networks of particular interest and possible interdependence centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. CYR61 overexpression, as in human DCMi hearts in situ, was similarly induced by inflammatory cytokines in vascular endothelial cells in vitro. APN was strongly downregulated in DCMi hearts and completely abolished cytokine-dependent CYR61 induction in vitro. Dysbalance between the CYR61 and APN networks may play a pathogenic role in DCMi and contain novel therapeutic targets. Multiple immune cell-associated genes were also deregulated (e.g., chemokine ligand 14, interleukin-17D, nuclear factors of activated T cells). In contrast to previous investigations in patients with advanced or end-stage DCM where etiology-related pathomechanisms are overwhelmed by unspecific processes, the deregulations detected in this study occurred at a far less severe and most probably fully reversible disease stage.
Electronic supplementary material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00109-006-0122-9 and is accessible for authorized users.
Human inflammatory cardiomyopathy; Genomic expression profiling; Molecular therapeutic targets; Molecular pathomechanisms; Dilated cardiomyopathy; Heart failure
The synthesis of progesterone by the corpus luteum is essential for the establishment and maintenance of early pregnancy. Regulation of luteal steroidogenesis can be broken down into three major events; luteinization (i.e., conversion of an ovulatory follicle), luteal regression, and pregnancy induced luteal maintenance/rescue. While the factors that control these events and dictate the final steroid end products are widely varied among different species, the composition of the corpus luteum (luteinized thecal and granulosa cells) and the enzymes and proteins involved in the steroidogenic pathway are relatively similar among all species. The key factors involved in luteal steroidogenesis and several new exciting observations regarding regulation of luteal steroidogenic function are discussed in this review.
Uterine fluid was obtained from eight clinical cases of pyometra with retained corpus luteum and nine additional samples of fluid were collected from animals slaughtered at an abattoir. These samples, along with uterine flushes from normal cows in their luteal phase were analyzed for prostaglandin of the F (PGF) and E (PGE) groups. Blood samples were also obtained from the clinical cases for analysis of 13,14-dihydro-15-keto PGF (PGFM) the major metabolite of PGF. Pyometrial exudate from clinical cases of abattoir samples had high concentrations of PGF (17.9 ng/mL) and PGE (33.2 ng/mL) and the total amount of PGF and PGE in the uterus was calculated to be several hundred times as great as in normal cows. Furthermore, clinical cases had elevated PGFM in their blood compared to that of controls, which suggests that at least some of the PGF was being absorbed from the uterus. These results are discussed in light of our current understanding of the maternal recognition of pregnancy in cattle.
A review of the current state of knowledge of oxytocin production by the preovulatory follicle and corpus luteum is presented. Corpora lutea of a number of mammalian species have been found to synthesize oxytocin. However, the synthesis and secretion of this nanopeptide by the corpus luteum of the ruminant has been most extensively studied because of the potential role of this peptide in facilitating luteal regression. While much information exists relative to various biochemical and endocrine factors that impact on oxytocin gene expression, this aspect about luteal synthesis of this peptide hormone remains enigmatic. Prostaglandin F-2α (PGF-2α) has been shown to be a primary endogenous hormone responsible for triggering luteal secretion of oxytocin. Details are provided regarding the PGF-2α-induced intracellular signal transduction pathway that ultimately results in exocytosis of luteal oxytocin. Evidence is also presented for potential autocrine/paracrine actions of oxytocin in regulating progesterone production by luteal and granulosa cells. Concluding remarks highlight aspects about luteal oxytocin production that require further research.