Expression Of Cyclic Nucleotide Phosphodiesterase Research Articles

Methods to Assess Phosphodiesterase and/or Adenylyl Cyclase Activity Via Heterologous Expression in Fission Yeast.

Heterologous expression of cyclic nucleotide phosphodiesterases (PDEs) and adenylyl cyclases (ACs) in the fission yeast Schizosaccharomyces pombe can be used in combination with PKA-repressed reporters to either carry out high throughput screens for small molecule inhibitors of these target enzymes or to assess hit compounds and their analogs from such screens. Here, we describe two methods for testing panels of such compounds. The first uses a growth assay for which growth in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA) occurs in response to inhibiting PDE activity to activate PKA. The second uses mass spectrometry to directly measure the impact of compound treatment to study compounds that modulate either PDE or AC activity.

Abstract 5344: Subtyping of glioblastoma stem-like cancer cells with neural lineage markers predicts invasiveness and correlates with survival

Abstract Glioblastoma multiforme (GBM) is a poorly treated human brain cancer with few established clinically useful molecular prognostic markers. GBM gene expression and microRNA profiling recently revealed developmental subtypes that potentially correlates with prognosis and treatment response. However, the clinically significant subset of glioblastoma stem-like cells (GSC) still lacks detailed molecular characterization and correlation to patient survival. We report that molecular classification of patient-derived GSCs using neural lineage markers show association with differential xenograft invasiveness, and also demonstrate significant correlation to survival in both the mouse model and human patients. Immunoblot array of neural lineage markers classified five independently isolated human GSC lines into three classes exhibiting differential expression of oligodendrocyte progenitor cells (OPC), astrocyte progenitor cells (APC), and neural progenitor cells (NPC) markers. After orthotopic injection into immunodeficient mice, Ki-67 proliferative index independent xenograft infiltration was observed: class I GSCs (OPC and NPC positive) created focal lesions, class II GSCs (NPC positive) formed minimally invasive lesions, and class III GSCs (APC positive) established highly infiltrative lesions. The OPC marker, cyclic nucleotide phosphodiesterase (CNP), exhibited high expression in focal xenografts versus low expression in invasive xenografts. Differential CNP expression significantly correlated with mouse model survival, and CNP assay of a large clinically annotated human GBM tissue microarray also showed differential patient survival. Therefore, GSC molecular characterization with neural lineage markers revealed a novel and potentially useful clinical prognosis marker, and suggests clinical importance for patient-specific GSC subtyping. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5344. doi:1538-7445.AM2012-5344

Alterations in cyclic nucleotide phosphodiesterase activities in omental and subcutaneous adipose tissues in human obesity

Objective: To elucidate the activity and expression of cyclic nucleotide phosphodiesterase (PDE) families in omental (OM) and subcutaneous (SC) adipose tissue and adipocytes, and to study alterations in their activity in human obesity.Design: Cross-sectional, translational research study.Patients: In total, 25 obese and 9 non-obese subjects undergoing gastrointestinal surgery participated in the study.Results: Inverse correlations between PDE activities and body mass index (BMI) were seen in both SC and OM adipose tissue. Inverse correlations between total PDE and PDE3 activity and BMI were seen in OM adipocytes but not in SC adipocytes. In both SC and OM adipose tissue of obese patients, total PDE and PDE3 activities were decreased compared with the controls. In SC adipose tissue of Type 2 diabetes (T2D) patients, the PDE activity not inhibitable by PDE3 or PDE4 inhibitors (PDEn) was increased compared with obese non-diabetic patients. In addition to PDE3 and 4 isoforms, PDE7B, PDE9A and PDE10A proteins were also detected in adipose tissue or adipocytes.Conclusions: Multiple PDE families are present in human adipose tissue and their activities are differentially affected by obesity and T2D.

Differential expression of cyclic nucleotide phosphodiesterases 4 in developing rat lung

During the perinatal period, lungs undergo changes to adapt to air breathing. The genes involved in these changes are developmentally regulated by various signaling pathways, including the cyclic nucleotide cAMP. As PDE4s are critical enzymes for regulation of cAMP levels, the objective of this study was to investigate PDE4's ontogeny in developing rat lung during the perinatal period. Pulmonary PDE4 activity, PDE4A-D, PDE4B, and PDE4D variant expression levels, PDE4B and PDE4D protein levels, and PDE4D localization in distal lung were determined. PDE4 activity increased towards term, dropped at birth, and increased thereafter to reach a plateau at the end of the second week of life. PDE4B2 and PDE4D long forms demonstrated a pattern of expression that increased markedly at birth. After birth, PDE4D was expressed in alveolar epithelial and mesenchymal cells. The study, therefore, evidenced striking variations in expression patterns among the PDE4 family that differed from changes in global PDE4 activity.

Cyclic nucleotide signaling in polycystic kidney disease

Increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP) stimulate cell proliferation and fluid secretion in polycystic kidney disease. Levels of this molecule are more sensitive to inhibition of phosphodiesterases (PDEs), whose activity far exceeds the rate of cAMP synthesis by adenylyl cyclase. Several PDEs exist, and here we measured the activity and expression of PDE families, their isoforms, and the expression of downstream effectors of cAMP signaling in the kidneys of rodents with polycystic kidney disease. We found a higher overall PDE activity in kidneys from mice as compared with rats, as well as a higher contribution of PDE1, relative to PDE4 and PDE3, to total PDE activity of kidney lysates and lower PDE1, PDE3, and PDE4 activities in the kidneys of cystic as compared with wild-type mice. There were reduced amounts of several PDE1, PDE3, and PDE4 proteins, possibly due to increased protein degradation despite an upregulation of their mRNA. Increased levels of cGMP were found in the kidneys of cystic animals, suggesting in vivo downregulation of PDE1 activity. We found an additive stimulatory effect of cAMP and cGMP on cystogenesis in vitro. Cyclic AMP-dependent protein kinase subunits Ialpha and IIbeta, PKare, the transcription factor CREB-1 mRNA, and CREM, ATF-1, and ICER proteins were upregulated in the kidneys of cystic as compared with wild-type animals. Our study suggests that alterations in cyclic nucleotide catabolism may render cystic epithelium particularly susceptible to factors acting on Gs-coupled receptors. This may account, in part, for increased cyclic nucleotide signaling in polycystic kidney disease and contribute substantially to disease progression.

Expression of MBP, PLP, MAG, CNP, and GFAP in the Human Alcoholic Brain

Chronic and excessive alcohol misuse results in neuropathological damage in the cerebral cortex. The damage includes white matter loss, brain atrophy, and selective loss of neurons in the superior frontal gyrus. Chronic alcohol misuse also results in alterations in the expression of a number of genes, including a selective reprogramming of myelin gene expression in the frontal cortex. The expression of cyclic nucleotide phosphodiesterase, glial fibrillary acidic protein, myelin-associated glycoprotein, myelin basic protein, and myelin proteolipid protein were assessed in the superior frontal gyrus and the primary motor cortex of control, uncomplicated alcoholic, and cirrhotic alcoholic cases. Overall, the expression of cyclic nucleotide phosphodiesterase, glial fibrillary acidic protein, myelin-associated glycoprotein, and myelin basic protein were significantly lower in the cirrhotic alcoholic cases compared with controls, with a similar tendency for myelin proteolipid protein. There was a strong correlation between the expression of the proteins studied and the brain weight of the individual case, but this interaction did not confound the overall analysis. There was no significant difference between controls and uncomplicated alcoholics. The loss of myelin proteins occurred without gross changes in brain pathology or brain weight and was not restricted to pathologically susceptible brain regions. It is not possible to determine whether the loss of myelin proteins in cirrhotic alcoholics is the result of cirrhosis per se or the combination of alcohol misuse and liver cirrhosis. Future studies comparing cases with alcoholic and nonalcoholic cirrhosis of the liver disease are required to elucidate this further.

Localization and regulation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase by cyclic AMP-dependent protein kinase

Calmodulin-dependent cyclic nucleotide phosphodiesterase is one of the key enzymes involved in the complex interactions, which occur between the cyclic nucleotide and Ca2+ second-messenger systems. In eye, cAMP regulation is important in a variety of physiological processes such as aqueous humor regulation, photoreceptor signal transduction and retinal blood flow. Bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was purified to apparent homogeneity and the isolated enzyme had a significantly higher affinity for calmodulin and Ca2+. Immunohistology revealed calmodulin-dependent cyclic nucleotide phospho-diesterase expression in corneal epithelium, retina and optic nerve of the eye. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase and the following observations were made. Firstly, the phosphorylation resulted in the incorporation of 1 mol of phosphate per mol of subunit, resulting in higher calmodulin and Ca2+ concentration requirement for calmodulin-dependent cyclic nucleotide phosphodiesterase activation. Secondly, Ca2+ and calmodulin prevented the phosphorylation. Thirdly, the phosphorylation of calmodulin-dependent cyclic nucleotide phosphodiesterase could be reversed by the calmodulin-dependent phosphatase, calcineurin. Analysis of the complex regulatory properties of the calmodulin-dependent cyclic nucleotide phosphodiesterase in the eye has led to the suggestion that fluxes of cAMP and Ca2+ during cell activation are closely coupled and that calmodulin-dependent cyclic nucleotide phosphodiesterase plays a key role in this signal coupling phenomenon.

Role of cyclic nucleotide signaling in oocyte maturation

The development of the ovarian follicle, oocyte maturation, and ovulation require a complex set of endocrine, paracrine, and autocrine inputs that are translated into the regulation of cyclic nucleotide levels. Changes in intracellular cAMP mediate the gonadotropin regulation of granulosa and theca cell functions. Likewise, a decrease in cAMP concentration in the oocyte has been associated with the resumption of meiosis. Using pharmacological and molecular approaches, we determined that the expression of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, is compartmentalized in the ovarian follicle of all species studied, with PDE3 present in the oocytes and PDE4s in granulosa cells. The PDE3 expressed in the mouse oocyte was cloned, and the protein expressed in a heterologous system had properties similar to those of a PDE3A derived from somatic cells. Inhibition of the oocyte PDE3 completely blocked oocyte maturation in vitro and in vivo, demonstrating that the activity of this enzyme is essential for oocyte maturation. Heterologous expression of PDE3A in Xenopus oocyte causes morphological changes distinctive of resumption of meiosis (GVBD), as well as activation of mos translation and MAPK phosphorylation. Using mRNA and antibody microinjection in the Xenopus eggs, we have shown that PDE3 is downstream from the kinase PKB/Akt in the pathway that mediates IGF-1 but not progesterone-induced meiotic resumption. The presence of a similar regulatory module in mammalian oocytes is inferred by pharmacological studies with PDE3 inhibitors and measurement of PDE activity. Thus, PDE3 plays an essential role in the signaling pathway that controls resumption of meiosis in amphibians and mammals. Understanding the regulation of this enzyme may shed some light on the signals that trigger oocyte maturation.

In situ expression of PLP/DM‐20, MBP, and CNP during embryonic and postnatal development of the jimpy mutant and of transgenic mice overexpressing PLP

We analyzed by in situ hybridization the spatiotemporal expression of dm-20, myelin basic protein (MBP) and 2′-3′ cyclic nucleotide phosphodiesterase (CNP) during embryonic and postnatal development of the normal mouse and two plp/dm-20 mutants: the jimpy mouse and a transgenic mouse overexpressing the plp gene. In the central nervous system (CNS) of the normal mouse, dm-20 mRNA was detected at embryonic day (E)9.5 in the laterobasal plate of the diencephalon. The pattern of expression of CNP transcript was superimposable on that of dm-20, but appeared slightly later, at E12.5. MBP mRNA was detected even later (E14.5), and, in addition, only in the caudal (rhombencephalon and spinal cord) territories of expression of dm-20 and CNP. These observations support our previous proposals: (1) dm-20-expressing cells in the germinative neuroepithelium are precursors of oligodendrocytes, and (2) oligodendrocytes emerge from distinct pools of precursors along the neural tube (Timsit et al., 1995). In the jimpy mutant, despite the mutation in the plp gene, cells of the oligodendrocyte lineage developed normally. It is only at the time of myelin deposition that oligodendrocytes die. During embryonic development of the transgenic mutant overexpressing plp, there were no alterations in the spatiotemporal pattern or the level of expression of dm-20 in the CNS, in contrast to the higher levels of dm-20 observed in the peripheral nervous system (PNS). J. Neurosci. Res. 50:190–201, 1997. © 1997 Wiley-Liss, Inc.

In situ expression of PLP/DM-20, MBP, and CNP during embryonic and postnatal development of the jimpy mutant and of transgenic mice overexpressing PLP.

We analyzed by in situ hybridization the spatiotemporal expression of dm-20, myelin basic protein (MBP) and 2'-3' cyclic nucleotide phosphodiesterase (CNP) during embryonic and postnatal development of the normal mouse and two plp/dm-20 mutants: the jimpy mouse and a transgenic mouse overexpressing the plp gene. In the central nervous system (CNS) of the normal mouse, dm-20 mRNA was detected at embryonic day (E)9.5 in the laterobasal plate of the diencephalon. The pattern of expression of CNP transcript was superimposable on that of dm-20, but appeared slightly later, at E12.5. MBP mRNA was detected even later (E14.5), and, in addition, only in the caudal (rhombencephalon and spinal cord) territories of expression of dm-20 and CNP. These observations support our previous proposals: (1) dm-20-expressing cells in the germinative neuroepithelium are precursors of oligodendrocytes, and (2) oligodendrocytes emerge from distinct pools of precursors along the neural tube (Timsit et al., 1995). In the jimpy mutant, despite the mutation in the plp gene, cells of the oligodendrocyte lineage developed normally. It is only at the time of myelin deposition that oligodendrocytes die. During embryonic development of the transgenic mutant overexpressing plp, there were no alterations in the spatiotemporal pattern or the level of expression of dm-20 in the CNS, in contrast to the higher levels of dm-20 observed in the peripheral nervous system (PNS).

Differential expression of cyclic nucleotide phosphodiesterase 3 and 4 activities in human T cell clones specific for myelin basic protein.

Abstract Little is known concerning the relative distribution and function of the different cyclic nucleotide phosphodiesterases (PDEs) in lymphocytes. Recent reports, however, have indicated that specific PDE4 inhibitors were effective in treatment of experimental allergic encephalomyelitis, an animal model of multiple sclerosis. The therapeutic effect of PDE4 inhibitors is thought to be related to inhibition of autoreactive CD4+ T cells specific for myelin basic protein (MBP) or other myelin proteins. Human autoreactive CD4+ T lymphocyte clones (TCC), specific for the immunodominant MBP epitope (amino acids 83-99), contain PDE3 and PDE4, two PDEs that exhibit a high affinity for cAMP. Amplification of TCC mRNA by reverse transcription-PCR indicated that TCC PDE3 mRNA was of the PDE3B, not PDE3A, subtype. Different TCC contained different proportions of PDE3 and PDE4, and their activities increased during Ag (MBP) stimulation. Specific PDE3 (cilostamide) and PDE4 (rolipram) inhibitors suppressed [3H]thymidine incorporation in TCC. Since it is believed that many autoimmune diseases are at least partially mediated by autoreactive CD4+ T cells, these observations may have important implications not only for the treatment of multiple sclerosis but also for other autoimmune diseases.

Cell layer-specific expression of cyclic nucleotide phosphodiesterases in rat arteries: Molecular cloning using isolated cell layers from paraformaldehyde-fixed tissues

We describe a method for the isolation of small quantities of large poly (A) + mRNA from blood vessels of the rat as well as from distinct cell layers of the rat aorta. The poly (A) + mRNA isolated by this method is suitable for use in reverse transcription polymerase chain reaction (RT-PCR) amplification of low abundance messages. In this method, anesthetized rats are perfused with ice-cold phosphate-buffered paraformaldehyde to allow for the in situ fixation of many of the main arteries of the rat. Following the in situ fixation of the rat vasculature, selected blood vessels can be removed, cleaned, and poly (A) + mRNA purified. In addition, the distinct cell layers of the paraformaldehyde-fixed aorta can be mechanically separated and poly (A) + mRNA purified selectively from each. The application of this method to the study of enzymes involved in cyclic nucleotide-mediated cell-signaling is illustrated by the cloning of two cyclic AMP phosphodiesterases from rat arteries, and from the selective amplification of message for these enzymes from different cell layers isolated from the rat aorta. This method should be applicable to determine if selected mRNAs are present in selected blood vessels of the rat, or within distinct cell layers of particular large blood vessels.

Differential regulation of the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene during oligodendrocyte development

The two major isoforms of 2′,3′-cyclic nucleotide phosphodiesterase (CNP), 48 and 46 kDa, have recently been shown to be produced from a single gene by alternative splicing. In addition, messenger RNA encoding the larger isoform is transcribed from a separate promoter, approximately 1 kb upstream from that encoding the smaller isoform. We have investigated the expression of these two CNP isoforms and have found that they are differentially expressed during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only the mRNA encoding the larger protein is found. At the time of oligodendrocyte differentiation, however, both CNP mRNAs are induced. These patterns of CNP expression are likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation.

Differential localization of calmodulin-dependent enzymes in rat brain: evidence for selective expression of cyclic nucleotide phosphodiesterase in specific neurons.

High-affinity antibodies against calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase and protein phosphatase (calcineurin) were purified and characterized. Rabbit anti-phosphodiesterase antibody did not react with other phosphodiesterases or with the regulatory subunits of cAMP-dependent protein kinase. Affinity-purified goat anti-calcineurin antibody recognized both the 61-kDa catalytic subunit and the 18-kDa Ca2+-binding subunit of the phosphatase. Neither antibody reacted with CaM, several CaM-binding proteins (calmodulin-dependent protein kinase, myosin light chain kinase, fodrin), or other cytosolic proteins from brain. The antibodies were used to compare the cellular localization of these two CaM-dependent enzymes in rat brain. Both calcineurin and phosphodiesterase were found predominantly in nerve cells; however, phosphodiesterase was restricted to very specific neuronal populations. Phosphodiesterase was prominent in the somatic cytoplasm and dendrites of regional output neurons--e.g., cerebellar Purkinje cells and hippocampal and cortical pyramidal cells. The extensive and uniform staining in the dendrites was consistent with postsynaptic localization and suggested an important function for this enzyme in neurons that integrate multiple convergent inputs. Calcineurin was present in virtually all classes of neurons, with immunoreactivity confined primarily to cell bodies. Both diffuse cytoplasmic staining and characteristic punctate staining of cell bodies were observed; the latter suggested compartmentalization of calcineurin at or near the plasma membrane. The results of this study demonstrate that calcineurin and phosphodiesterase are differentially localized in the central nervous system. Thus, the expression and compartmentalization of CaM-binding proteins may be highly regulated and specific for particular differentiated nerve cell types.