Pros and Cons of Pharmacological Manipulation of cGMP-PDEs in the Prevention and Treatment of Breast Cancer

The cyclic nucleotides, cAMP and cGMP, are ubiquitous second messengers responsible for translating extracellular signals to intracellular biological responses in both normal and tumor cells. When these signals are aberrant or missing, cells may undergo neoplastic transformation or become resistant to chemotherapy. cGMP-hydrolyzing phosphodiesterases (PDEs) are attracting tremendous interest as drug targets for many diseases, including cancer, where they regulate cell growth, apoptosis and sensitization to radio- and chemotherapy. In breast cancer, PDE5 inhibition is associated with increased intracellular cGMP levels, which is responsible for the phosphorylation of PKG and other downstream molecules involved in cell proliferation or apoptosis. In this review, we provide an overview of the most relevant studies regarding the controversial role of PDE inhibitors as off-label adjuvants in cancer therapy.

The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

<p>Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study. The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest. Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro. The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.</p>

The role of follicular-secreted factors in the maintenance of meiotic arrest in rats

<p>Meiosis is the process by which diploid germ cells develop into competent haploid gametes. In female mammals, meiosis is characterised by two periods of arrest, the duration of which is species-specific. This study investigated the first period of meiotic arrest which occurs at the diplotene stage of prophase I. This period of arrest has important implications for artificial reproductive technologies as the maintenance of meiotic arrest in the in vitro situation has been correlated with improved embryological outcomes. Despite there being extensive evidence that the somatic cells of the follicle (granulosa and cumulus cells) produce meiosis-inhibiting factors, the factors themselves and the mechanisms through which they act are unclear. Recent evidence implicates C-type natriuretic peptide (CNP) and oestradiol in the regulation of meiotic arrest in mouse oocytes. In this proposed hypothesis, CNP is produced by the granulosa cells and activates its cognate receptor, NPR2, on cumulus cells. This results in the production of cyclic guanosine monophosphate (cGMP) in cumulus cells which is transferred to the oocyte via gap junctions. In the oocyte, cGMP slows the rate of hydrolysis of cyclic adenosine monophosphate (cAMP) by phosphodiesterase 3A resulting in elevated intra-oocyte cAMP levels. By maintaining high levels of cAMP in the oocyte, maturation-promoting factor (MPF) activity is inhibited, preventing re-entry into the cell cycle, thus maintaining meiotic arrest. The overall objective of this study was to investigate the validity of this aforementioned hypothesised regulatory pathway in another mammalian species, the rat. Four fundamental components of this pathway were chosen to be investigated and these framed the four aims of this study. The aims of this study were to investigate in cultured rat cumulus cell-oocyte complexes (COCs) the short and long-term effects of CNP and oestradiol, both alone and in combination on (1) gap junction permeability using a validated gap junction assay, (2) intracellular cGMP levels using a direct competitive immunoassay, (3) mRNA expression levels of key cumulus cell-derived genes (Npr2, the receptor for CNP; and Pde4b and Pde4d, phosphodiesterases) using an optimised multiplex TaqMan qPCR reaction, and (4) duration of meiotic arrest. Overall, the results of this study indicated that the assessed treatments did not alter gap junction permeability in rat COCs in vitro. Whilst treatment with CNP and oestradiol appeared to increase the intracellular levels of cGMP in COCs, this requires further investigation. Notably, this study confirmed the role of steroid hormones in upregulating Npr2 expression. Indirect evidence suggests that PDE4D in particular, is a major regulator of cyclic nucleotide levels in the cumulus cells. Finally, treatment of rat COCs with CNP and oestradiol increased the duration of meiotic arrest in oocytes incubated in vitro. The results of this study provide the first evidence that the hypothesised regulatory pathway proposed above is also relevant in the rat. Nonetheless, further investigation of the effects of CNP and oestradiol on the modulation of intracellular cGMP levels are required to fully validate the model.</p>

Abstract P239: Identification Of Binding Sites For Phosphodiesterase 5 (PDE5) On Rho-related BTB Domain Containing 1 (RhoBTB1) As A Guide To Identifying Novel RhoBTB1-Binding Partners

In-depth studies of molecular pathways have provided insights to explore therapeutic targets for treating hypertension as a complex disease. We previously identified PDE5 as an interacting partner with RhoBTB1 protein in aortic smooth muscle cells (SMCs). RhoBTB1 regulates PDE5 activity and therefore intracellular cGMP levels, and restoration of RhoBTB1 expression in RhoBTB1-deficient states results in improvement of cardiovascular status in a mouse model of hypertension. Here we identified the specific regions of RhoBTB1 that are responsible for the recruitment of PDE5. Our goal is to use this as a guide to identify additional RhoBTB1 interacting proteins which play important roles in cardiovascular function in the hope their identification may open novel therapeutic avenues against hypertension and associated pathologies. We hypothesize that RhoBTB1 acts as an adaptor for Cullin-3 E3 ring ubiquitin ligase complex, where Cullin-3 functions as “scaffold” which delivers PDE5 (and other targets) to Cullin-3 complex followed by ubiquitination and proteasomal degradation. We generated several truncations in full length RhoBTB1, splitting it into its GTPase, BTB1, BTB2, Carboxyl-terminal (CT), BTB1-BTB2, and BTB1-BTB2-CT domains. To check the preferred binding interface for PDE5, we co-transfected HEK293 cells with epitope tagged PDE5 and separately tagged truncations of RhoBTB1, and analyzed the interaction between PDE5 and RhoBTB1 domains using co-immunoprecipitation assays. Our result shows that BTB1-BTB2-C domain of RhoBTB1 is the preferred binding region for PDE5. The BTB1-BTB2 domain lacking the CT was unable to bind to PDE5. Next, we will utilize stable isotope labeling by amino acids in cell culture (SILAC) followed by proteomic profiling to identify additional targets and/or adaptors involved in the binding of PDE5 and RhoBTB1. Similarly, we will tag specific domains of RhoBTB1 using the ascorbate peroxidase (APEX2) fusion system followed by proteomic profiling.

C-type natriuretic peptide stimulates function of the murine Sertoli cells via activation of the NPR-B/cGMP/PKG signaling pathway

C-type natriuretic peptide (CNP) is an important regulator of the male reproductive process. Our previous investigations showed that CNP can significantly stimulate the mRNA expression of androgen-binding protein (Abp) and transferrin (Trf) in the rat Sertoli cells, but the pathways responsible for this process remain to be elucidated. We predict that CNP binds the natriuretic peptide receptor B (NPR-B) to regulate expression of ABP and TRF through the intracellular cyclic guanosine monophosphate (cGMP) pathway. To address this question, in this study, we first confirmed the expression and localization of CNP and NPR-B in rat testes by immunohistochemistry and western blotting. Then, ELISA and real-time PCR were performed to investigate the signaling pathway of CNP in Sertoli cells in rat testes. Our results showed that CNP was mainly localized in the germ cells and Leydig cells, and its receptor, NPR-B, was mostly expressed in the Sertoli cells and vascular endothelial cells. CNP supplementation in the Sertoli cell medium was accompanied by an increase in the amount of intracellular cGMP and in the production of Abp and Trf mRNA, whereas inhibition of PKG with KT5823 led to a decrease in the expression of Abp and Trf mRNA. Moreover, Abp and Trf mRNA were no longer elevated when we used liposome-mediated RNA interference technology to silence the NPR-B gene in a mouse Sertoli cell line (TM4). These results suggest that CNP contributes to the regulation of ABP and TRF in the Sertoli cells through the NPR-B/cGMP/PKG signaling pathways.

ANP and BNP Exert Anti-Inflammatory Action via NPR-1/cGMP Axis by Interfering with Canonical, Non-Canonical, and Alternative Routes of Inflammasome Activation in Human THP1 Cells

Dysregulated inflammasome activation and interleukin (IL)-1β production are associated with several inflammatory disorders. Three different routes can lead to inflammasome activation: a canonical two-step, a non-canonical Caspase-4/5- and Gasdermin D-dependent, and an alternative Caspase-8-mediated pathway. Natriuretic Peptides (NPs), Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP), binding to Natriuretic Peptide Receptor-1 (NPR-1), signal by increasing cGMP (cyclic guanosine monophosphate) levels that, in turn, stimulate cGMP-dependent protein kinase-I (PKG-I). We previously demonstrated that, by counteracting inflammasome activation, NPs inhibit IL-1β secretion. Here we aimed to decipher the molecular mechanism underlying NPs effects on THP-1 cells stimulated with lipopolysaccharide (LPS) + ATP. Involvement of cGMP and PKG-I were assessed pre-treating THP-1 cells with the membrane-permeable analogue, 8-Br-cGMP, and the specific inhibitor KT-5823, respectively. We found that NPs, by activating NPR-1/cGMP/PKG-I axis, lead to phosphorylation of NLRP3 at Ser295 and to inflammasome platform disassembly. Moreover, by increasing intracellular cGMP levels and activating phosphodiesterases, NPs interfere with both Gasdermin D and Caspase-8 cleavage, indicating that they disturb non-canonical and alternative routes of inflammasome activation. These results showed that ANP and BNP anti-inflammatory and immunomodulatory actions may involve the inhibition of all the known routes of inflammasome activation. Thus, NPs might be proposed for the treatment of the plethora of diseases caused by a dysregulated inflammasome activation.

NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis

Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1 −/− /NOX2 −/− /NOX4 −/− ), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP—a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride–driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

NHERF3 is necessary for Escherichia coli heat-stable enterotoxin-induced inhibition of NHE3: differences in signaling in mouse small intestine and Caco-2 cells

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood death from diarrhea and the leading cause of Traveler’s diarrhea. E. coli heat-stable enterotoxin (ST) is a major virulence factor of ETEC and inhibits the brush border Na/H exchanger NHE3 in producing diarrhea. NHE3 regulation involves multiprotein signaling complexes that form on its COOH terminus. In this study, the hypothesis was tested that ST signals via members of the Na/H exchanger regulatory factor (NHERF) family of scaffolding proteins, NHERF2, which had been previously shown to have a role, and now with concentration on a role for NHERF3. Two models were used: mouse small intestine and Caco-2/BBe cells. In both models, ST rapidly increased intracellular cGMP, inhibited NHE3 activity, and caused a quantitatively similar decrease in apical expression of NHE3. The transport effects were NHERF3 and NHERF2 dependent. Also, mutation of the COOH-terminal amino acids of NHERF3 supported that NHERF3-NHERF2 heterodimerization was likely to account for this dual dependence. The ST increase in cGMP in both models was partially dependent on NHERF3. The intracellular signaling pathways by which ST-cGMP inhibits NHE3 were different in mouse jejunum (activation of cGMP kinase II, cGKII) and Caco-2 cells, which do not express cGKII (elevation of intracellular Ca2+ concentration [Ca2+]i). The ST elevation of [Ca2+]i was from intracellular stores and was dependent on NHERF3-NHERF2. This study shows that intracellular signaling in the same diarrheal model in multiple cell types may be different; this has implications for therapeutic strategies, which often assume that models have similar signaling mechanisms.