The cDNA of a human lymphocyte cyclic-AMP phosphodiesterase (PDE IV) reveals a multigene family (original) (raw)
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1 The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T-lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)-2 and interferon (IFN)-y evaluated. 2 In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (-65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3 Reverse transcription-polymerase chain reaction (RT-PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4 Functionally, rolipram inhibited phytohaemagglutinin-(PHA) and anti-CD3-induced proliferation of CD4+ and CD8+ T-lymphocytes, and the elaboration of IL-2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL-2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T-cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHAand anti-CD3-induced T-cell proliferation, and PHA-induced IL-2 release. 5 When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co-mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL-2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis. 6 Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL-2 biosynthesis and proliferation in human CD4+ and CD8+ T-lymphocytes. However, the ability of rolipram to suppress markedly mitogen-induced IL-2 generation without affecting T-cell proliferation suggests that growth and division of T-lymphocytes may be governed by mediators in addition to IL-2. Finally, T-cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors.
Cyclic nucleotide phosphodiesterases from purified human CD4+ and CD8+ T lymphocytes
Clinical <html_ent glyph="@amp;" ascii="&"/> Experimental Allergy, 1995
Background CD4"^ and CDB"^ T-lymphocytes are suggested to differentially affect airway inflammation in asihma. Agents whieh inerease intracelluiar cAMP levels, sueh as PDE inhibitors, have been shown to diminish lymphocyte growth and differentiation, and to affeet eytokine expression. DiflTerenees in the PDE isoenzyme profile between CD4"^ and CD8^ cells might form a basis to differentially modify their functions by PDE inhibitors. Objective The study investigates and compares the PDE isoenzyme activity profiles of human peripheral blood CDA^ and CDS"^ T-lymphocytes. Methods CD4"^ and CD8"^ T-lymphoeytes were puritied (>98%) from peripheral blood mononuciear cells by negative selection. PDE isoenzyme activity profiles were investigated using PDE isoenzyme selective inhibitors and activators. Results In CD4^ and CD8^ T-lymphoeyte homogenates. PDE IV and PDE III activities were the predominant PDE isoenzyme activities at 0-5 ;iM eyelie nueleotide substrate concentrations. PDE IV was localized in the soluble fraction whereas PDE III was membrane bound. Low PDE I. II and V aetivities were detected. About 20% of total eAMP hydrolysing capacity at 0 5/JM CAMP was insensitive to PDE isoenzyme selective inhibitors and activators and therefore could not be assigned to PDE 1-IV. The PDE isoenzyme pattern was not different between CD4"^ and CD8"^ T-lymphocytes. Moreover, representative inhibitors of PDE III and IV aetivity inhibited cAMP hydrolysis in soluble fractions of both T-lymphocyte subsets with similar potency. Enzyme kinetic analysis similarly did not reveal differences between CD4"^ and CD8"T -lymphoeytes. Conclusion Normal CD4"^ and CD8^ T-lymphocytes are likely to be equally sensitive targets for the effects of PDE inhibitors.
Multiple high-affinity cAMP-phosphodiesterases in human T-lymphocytes
Biochemical Pharmacology, 1991
Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that inactivate intracellular cyclic AMP (CAMP). Because the functions of T-lymphocytes are modulated by CAMP levels, the isozymes of PDE in these cells are potential targets for new drugs designed to modify the body's immunity through selective alteration of T-lymphocyte PDE activity. Cyclic GMP and 3(2H)-pyridazinone-4,5-dihydro-6-[4-(lfi-imidazol-l-yl)phenyl]-5-methyl-monohydrochloride (CI-930) selectively inhibit the catalytic activity of one of the two high affinity CAMP-PDE isozyme families known to occur in mammals, whereas d,f-1,4-[3-butoxy-4-methoxybenzyl]-Z-imidazolidinone (Ro 20-1724) selectively inhibits the other. The objectives of this investigation were: (1) to determine whether human T-lymphocytes contain one or both of these pharmacologically distinguishable high-affinity CAMP-PDEs, and (2) to determine the effects of selective inhibitors of these PDEs on lymphocyte blastogenesis. High-affinity CAMP-PDE was found in both the soluble and particulate fractions of T-lymphocyte sonicates. Cyclic GMP and CI-930 inhibited PDE in the particulate fraction better than in the soluble fraction, but the converse was found for Ro 20-1724. CI-930 or Ro 20-1724, used alone, attenuated T-lymphocyte blastogenesis, but neither suppressed it completely. In combination, the same PDE inhibitors caused greater suppression of blastogenesis than either produced alone. The results indicate that human T-lymphocytes contain both CI-930-and Ro 20-1724-inhibitable isozymes. Either of the isozymes can modulate human Tlymphocyte blastogenesis, but inhibition of both isozymes produces synergistic antiblastogenic effects, Intracellular cyclic AMP (CAMP) is an essential regulator of lymphocyte blastogenesis [l]. The only enzymes known to catalyze the inactivation of this second messenger are the phosphodiesterases 5 To whom correspondence should be sent at the following address:
Biochemical and Biophysical Research Communications, 2000
We have identified and characterised a novel member of the PDE7 family of cyclic nucleotide phosphodiesterases (PDE), which we have designated PDE7B. Mouse and human full-length cDNAs were isolated encoding a protein of 446 and 450 amino acids, respectively. The predicted protein sequence of PDE7B showed highest homology (70% identity) to that of PDE7A. Northern blot analysis identified a single 5.5-kb transcript with highest levels detected in brain, heart, and liver. Kinetic analysis of the mouse and human purified recombinant enzymes show them to specifically hydrolyse cAMP with a K m of 0.1 and 0.2 M respectively. Inhibitor studies show sensitivity to dipyridamole, IC 50 of 0.51 and 1.94 M, and IBMX, IC 50 of 3.81 and 7.37 M, for the mouse and human enzymes, respectively. This shows that dipyridamole is not selective for cGMP over cAMP PDEs as previously believed. Other standard PDE inhibitors including zaprinast, rolipram, and milrinone do not significantly inhibit PDE7B.
Intracellular Compartmentalization of PDE4 Cyclic AMP-Specific Phosphodiesterases
Methods, 1998
on chromosome 19p13.2. The catalytic unit of the The PDE4 cyclic AMP-specific phosphodiesterase family various PDE4 family members is encoded by Ç7 excomprises a large number of different isoforms encoded by ons, the sequences of which show high homology for four distinct genes, with additional complexity arising the four different PDE4 families (4A, 4B, 4C, 4D). through alternate mRNA splicing. This generates a number However, the extreme 3 exons, which encode the Cof distinct PDE4 isoforms with unique N-terminal regions. terminal tail of the various PDE4 enzymes, are very The range of such splice variants emanating from the four different and encode amino acid sequences that show PDE4 genes appears to be highly conserved across species. no similarity between the C-terminal regions of One key role for such regions appears to be their potential members of any of the four PDE4 gene families. Nevto target isoforms to specific intracellular sites. Evidence ertheless, within a particular PDE4 gene family, all for such a targeting role for these N-terminal regions can active isoforms appear to have identical C-terminal be gleaned by a variety of techniques. These include subcelregions. This observation has been usefully exploited lular fractionation, confocal microscopy, binding assays to (4-9) to generate antibodies/antisera that are able show association with proteins having src homology 3 (SH3) to detect all active PDE4 isoforms of a particular domains, and generation of chimeric constructs of these N-PDE4 gene family. Similarly, generic reverse tranterminal regions with proteins that are normally expressed scription polymerase chain reaction (RT-PCR) primin the cytosol. ᭧ 1998 Academic Press ers can also be designed to identify the presence of transcripts for all active members of a particular PDE4 gene family without a priori knowledge of the range of splice variants present (10, 11). This is because alternative mRNA splicing appears, for active The PDE4 cyclic AMP specific phosphodiesterase species, to be uniquely associated with 5 domain family comprises a large number of different isoswaps. It should, however, be noted, that regions of forms encoded by four distinct genes with additional sequence within the putative catalytic domain are complexity arising through alternate mRNA splicing PDE4 subfamily specific and have also been usefully (1-3). The genes are complex structures with many exploited to generate generic RT-PCR primers exons. For example, the human PDE4A gene spans aimed, again, at amplifying/detecting transcripts for 50 kb, comprises at least 17 exons, and is located all active members arising from a particular PDE4 gene subfamily. Thus, both molecular and immunological methods can be employed to detect active 1 To whom correspondence and reprint requests should be addressed.
Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A
Proceedings of the National Academy of Sciences, 2000
We report here the cloning, expression, and characterization of human PDE11A1, a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits <50% amino acid identity with the catalytic domains of all other PDEs, being most similar to PDE5, and has distinct biochemical properties. The human PDE11A1 cDNA isolated contains a complete open reading frame encoding a 490-amino acid enzyme with a predicted molecular mass of 55,786 Da. At the N terminus PDE11A1 has a single GAF domain homologous to that found in other signaling molecules, including PDE2, PDE5, PDE6, and PDE10, which constitutes a potential allosteric binding site for cGMP or another small ligand. Tissue distribution studies indicate that PDE11A mRNA occurs at highest levels in skeletal muscle, prostate, kidney, liver, pituitary, and salivary glands and testis. PDE11A is expressed as at least three major transcripts of Ϸ10.5, Ϸ8.5, and Ϸ6.0 kb, thus suggesting the existence of multiple subtypes. This possibility is further supported by the detection of three distinct proteins of Ϸ78, Ϸ65, and Ϸ56 kDa by Western blotting of human tissues for PDE11A isoforms. Recombinant human PDE11A1 hydrolyzes both cGMP and cAMP with K m values of 0.52 M and 1.04 M, respectively, and similar V max values. Therefore, PDE11A represents a dual-substrate PDE that may regulate both cGMP and cAMP under physiological conditions. PDE11A is sensitive to the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) as well as zaprinast and dipyridamole, inhibitors that are generally considered relatively specific for the cGMP-selective PDEs, with IC50 values of 49.8 M, 12.0 M, and 0.37 M, respectively.
British Journal of Pharmacology, 1996
1 The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T-lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)-2 and interferon (IFN)-y evaluated. 2 In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (-65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3 Reverse transcription-polymerase chain reaction (RT-PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4 Functionally, rolipram inhibited phytohaemagglutinin-(PHA) and anti-CD3-induced proliferation of CD4+ and CD8+ T-lymphocytes, and the elaboration of IL-2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL-2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T-cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHAand anti-CD3-induced T-cell proliferation, and PHA-induced IL-2 release. 5 When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co-mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL-2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis. 6 Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL-2 biosynthesis and proliferation in human CD4+ and CD8+ T-lymphocytes. However, the ability of rolipram to suppress markedly mitogen-induced IL-2 generation without affecting T-cell proliferation suggests that growth and division of T-lymphocytes may be governed by mediators in addition to IL-2. Finally, T-cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors.
Multiple splice variants of phosphodiesterase PDE4C cloned from human lung and testis
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1997
Ž. Four closely related cyclic-nucleotide specific phosphodiesterase PDE4 genes have been identified in both humans and rats: PDE4A, 4B, 4C and 4D. We have now cloned cDNAs for multiple splice variants of human PDE4C. Two splice Ž. variants, PDE4C-791 and PDE4C-426, were isolated from a fetal lung library. The longest open reading frame ORF of Ž. w 791 amino acids aa is encoded by PDE4C-791, which is similar to a recently described cDNA Engels, P.
Biochemical Journal, 2008
We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related ‘long’ PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC50 of 11±1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC50 of 101±7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissu...