Expression, Structure/function, Regulation, and Roles of PDE3 Isoforms

PDE3 同工型的表达、结构/功能、调节和作用

• 批准号: 8158022
• 负责人: VINCENT MANGANIELLO
• 金额: $ 168.91万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158022
• 关键词: Protein Isoforms; Regulation; Role; Structure

Many cardiac functions, from contractility to gene expression and structural remodeling, are regulated by multiple spatially and functionally distinct pools of cAMP. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP, regulate the amplitude, duration, and compartmentation of cAMP-mediated signaling. Our data suggest that PDE3A is a component of a molecular scaffold that may integrate cyclic AMP and SERCA2 transduction pathways in cardiac muscle. Immunohistochemical staining of human myocardium indicates that PDE3A is co-localized with desmin, AKAP18 and SERCA2 to sarcomere Z-bands, while PDE3B is co-localized with mitochondrial proteins (COX4, ATP synthase, Cyt-C). During sucrose gradient centrifugation of mouse cardiac membranes, PDE3A co-fractionates with sarcoplasmic reticulum (SR) Ca+2 ATPase 2 (SERCA2) and phospholamban, and immunochemical staining indicates that PDE3A co-localized with SERCA2 and desmin in mouse heart. In addition, Western blots and LC-MS/MS analysis of PDE3A immunoprecipitates indicates that murine PDE3A coimmunoprecipitated with SERCA2. Similarly, in solubilized human and murine cardiac microsomes, PDE3A co-immunoprecipitates with SERCA2 and other signaling molecules thought to be components of an AKAP/SERCA2 macromolecular regulatory complex, including phospholamban, PKARII, PP2A, and AKAP18, but not AKAP LBC. In human myocardial microsomes, the PKA catalytic subunit (PKAc) (plus ATP) phosphorylates the isoforms PDE3A1 and PDE3A2 but not PDE3A3, and significantly increases PDE3 catalytic activity. cAMP or PKAc significantly increases Ca2+ uptake into SR vesicles, and cilostamide, a PDE3-selective inhibitor, potentiates the effect of cAMP. In murine cardiac microsomes, cilostamide increased the effect of cAMP on Ca2+ uptake into SR vesicles. SERCA2 Ca2+ -ATPase activity and Ca2+ uptake and Ca2+ content were increased in SR vesicles prepared from PDE3A-knockout (KO) mice, compared to wild type. In lysates from KO hearts, SERCA2 expression was increased and that of PLB decreased, and phosphorylation of PLB at Ser-16 (pPLB/PLB ratio) was increased. In KO lysates, due to the loss of PDE3A activity, PKA was activated, as evidenced by increased phosphorylation of PKA substrates and PLB. In collaborative studies, these PDE activity changes in PDE3A-/- hearts were associated with elevations in contractility and relaxation properties of isolated hearts, as well as increased Ca2+ transient amplitudes in isolated cardiac myocytes, without differences in L-type Ca2+ currents (ICa,L). Ca2+ transients and SR Ca2+ content were normalized to the WT levels by dialysis of myocytes with the PKA inhibitor RpcAMP. PDE3 inhibition had no effect on cardiac contractility, Ca2+ transients, or SR Ca2+ content in PDE3A-/- preparations but increased these same parameters in WT hearts to levels indistinguishable from PDE3A-/-, establishing that PDE3A is the PDE3 isoform regulating basal heart function in heart, as a component of a macromolecular complex which regulates cAMP levels in microdomains containing SERCA2-PLN-PDE3A complexes. Taken together, these data suggest that, as a component of SERCA2-containing macromolecular complexes in murine and human myocardium, PDE3A regulates a discrete cAMP pool important in regulating contractility by modulating Ca2+ uptake into the SR. In addition to effects on contractility, PDE3 is thought to affect vascular smooth muscle relaxation and proliferation. The function and role of individual PDE3A and PDE3B isoforms in regulation of these processes is, however, is largely unclear, primarily due to the lack of isoform-selective PDE3 inhibitors. Using VSMCs (cultured vascular smooth muscle myocytes) expanded from the aortas of PDE3A KO and PDE3B KO mice, we examined the role of PDE3A and B isoforms in regulation of VSMC growth, and the mechanisms by which PDE3 isoforms may affect signaling pathways that mediate PDGF-induced VSMC proliferation. Cultured VSMCs expanded from the aortas of PDE3A KO mice exhibited marked inhibition of serum- and PDGF-induced DNA synthesis when compared to VSMCs expanded from PDE3A WT type and PDE3B KO mice. Growth inhibition was accompanied by selective inhibition of ERK phosphorylation in PDE3A KO VSMCs, most likely due to a combination of increased site-specific Raf-1ser259 inhibitory phosphorylation as well as excessive dephosphorylation of ERKs by elevated MKP-1 (MAP kinase phosphatase 1). Furthermore, PDE3A KO VSMCs exhibited elevated basal PKA activity, upregulation of CREB and p53 and its phosphorylation, and elevated p21 expression, together with reduction of cyclin D1 and Rb levels and Rb phosphorylation. Adenoviral infection with inactive CREB (mCREB) partially restored growth effects of serum in PDE3A KO VSMCs. In contrast, exposure of PDE3A WT VSMCs to Vp16 CREB (active CREB) was associated with inhibition of cell growth, effects similar to those observed in untreated PDE3A KO VSMCs. Transfection of PDE3A KO VSMCs with p53 siRNA reduced p21 and MKP-1 elevations and completely restored growth, without affecting cyclin D1 levels and Rb phosphorylation. We conclude that PDE3A. not PDE3B, regulates VSMC growth via two complimentary signaling pathways, i.e., PKA-mediated inhibition of MAPK signaling via Raf-1 site-specific inhibitory phosphorylation and PKA-CREB-mediated induction of p21, leading to GO/G1 cell cycle arrest, as well as by induction of p53 which induces MKP-1, p21and Wip1, leading to inhibition of G1 to S cell cycle progression. Although other studies using cAMP agonists and pharmacologic inhibitors of PDE3 and PDE4 isoforms have reported inhibition of VSMC growth, the exact role of PDE3A and PDE3B isoforms, however, was difficult to discern, in large part because of the lack of availability of isoform-specific PDE3A and PDE3B inhibitors. Taken together, our data and those of others, suggest that PDE3A isoforms may play a major role in cardiovascular function by regulating cardiac contractility and peripheral vasodilation as well as VSMC growth. Future in vivo studies with balloon injury models are needed to test the protective effect of PDE3A deletion on vessel wall thickening, smooth muscle cell migration and growth, since, under basal conditions, PDE3A KO mice do not appear to be grossly compromised in terms of growth, development and cardiovascular status. PDE3A may be an important regulator of cell cycle progression in other cells. Female PDE3A KO mice are sterile, presumably because increased cAMP/PKA signaling in oocytes maintains meiotic arrest at prophase I of the first meiotic division, and thereby inhibits oocyte maturation and prevents fertilization.Our results suggest that Polo-like kinase 1 (Plk1) may be a target of PKA, and involved in meiotic arrest of PDE3A KO oocytes. In cultured, G2/M-arrested PDE3A KO murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A KO oocytes; Cdc2 and Plk1 were activated, and phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A KO oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A KO oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Taken together, our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility.

从收缩到基因表达和结构重塑的许多心脏功能受到多个cAMP的多个库池的调节。通过水解训练营（PDES）的环状核苷酸磷酸二酯酶（PDE）调节营地介导的信号传导的幅度，持续时间和分隔。我们的数据表明，PDE3A是分子支架的组成部分，该分子支架可能会在心肌中整合环状AMP和SERCA2转导途径。人心肌的免疫组织化学染色表明PDE3A与Desmin，Akap18和Serca2共定位于肌节Z-循环，而PDE3B与线粒体蛋白（Cox4，ATP Synthase，ATP Synthase，cyt-c）共定位。在小鼠心脏膜的蔗糖梯度离心过程中，PDE3A与肌浆网（SR）CA+2 ATPase 2（SERCA2）和磷脂式染色共同分级，以及免疫化学染色表明PDE3A与Serca2和Desmin in hears Heart in Mouse Heart in Mouse Heart in hears Heart in heart heart Heart in hears in house Heart in heart heart Heart in heart Heart in heart heart Heart in鼠标心脏。此外，PDE3A免疫沉淀物的Western印迹和LC-MS/MS分析表明，鼠PDE3A与SERCA2共免疫沉淀。同样，在溶解的人和鼠心脏微粒体中，PDE3A与SERCA2和其他信号分子共免疫沉淀，被认为是AKAP/SERCA2大分子调节复合物的组成部分，包括磷脂，PKARIBAN，PKARIBAN，PKARIII，PP2A和AKAP18，但NOT BC。在人体心肌微粒体中，PKA催化亚基（PKAC）（加上ATP）磷酸化同工型PDE3A1和PDE3A2，但没有PDE3A3，并显着增加了PDE3催化活性。 CAMP或PKAC显着增加Ca2+摄取SR囊泡，而Cilostamide（一种PDE3选择性抑制剂）增强了CAMP的作用。在鼠心脏微粒体中，西洛雄增加了CAMP对Ca2+摄取SR囊泡的影响。与野生型相比，用PDE3A -KNOCKOUT（KO）小鼠制备的SR囊泡中SERCA2 Ca2+ ATPase活性和Ca2+摄取和Ca2+含量增加。在KO心脏的裂解物中，SERCA2表达增加并降低PLB的表达，并且在Ser-16（PPLB/PLB比）的PLB磷酸化增加。在KO裂解物中，由于PDE3A活性的损失，PKA被激活，这可以通过PKA底物和PLB的磷酸化增加证明。在协作研究中，PDE3A - / - 心脏中的这些PDE活性变化与隔离心脏的收缩力和放松特性的升高以及孤立心肌细胞中Ca2+瞬时幅度的增加相关，而L型Ca2+电流没有差异（ICA，ICA，ICA，ICA，ICA，，ICA，ICA，， L）。用PKA抑制剂RPCAMP通过肌细胞透析将Ca2+瞬变和SR Ca2+含量标准化为WT水平。 PDE3抑制对PDE3A - / - 制剂中的心脏收缩性，Ca2+瞬变或SR Ca2+含量没有影响在心脏中，作为大分子复合物的组成部分，它调节含有SERCA2-PLN-PDE3A复合物的微型域中的cAMP水平。综上所述，这些数据表明，作为鼠和人心肌中含有SERCA2的大分子复合物的组成部分，PDE3A调节一个离散的营地池，通过调节Ca2+对SR的摄取来调节收缩力很重要。 除了对收缩性的影响外，PDE3还被认为会影响血管平滑肌松弛和增殖。但是，单个PDE3A和PDE3B同工型在这些过程中的调节中的功能和作用在很大程度上不清楚，这主要是由于缺乏同工型选择性PDE3抑制剂。使用VSMC（培养的血管平滑肌肌细胞）从PDE3A KO和PDE3B KO小鼠的主动脉扩展，我们检查了PDE3A和B同型在VSMC生长调节中的作用，以及PDE3 Isoforms可能会影响PDE3 Isoforms PDGF的机制的作用。 - 诱导的VSMC增殖。与从PDE3A WT型和PDE3B KO小鼠相比，培养的VSMC从PDE3A KO小鼠的主动脉扩展，表现出明显的抑制血清和PDGF诱导的DNA合成的DNA合成。生长抑制作用伴随着PDE3A KO VSMC中ERK磷酸化的选择性抑制作用，这很可能是由于位点特异性RAF-1SER259抑制性磷酸化的组合以及MAP KINAPE-1的MAP KINase Chastasase 1）的结合。此外，PDE3A KO VSMC表现出升高的基础PKA活性，CREB和p53及其磷酸化的上调，p21表达升高，以及降低细胞周期蛋白D1和RB水平以及RB磷酸化的降低。无效CREB（MCREB）的腺病毒感染部分恢复了PDE3A KO VSMC中血清的生长作用。相比之下，PDE3A WT VSMC暴露于VP16 CREB（活动CREB）与细胞生长的抑制有关，其作用与未经处理的PDE3A KO VSMC中观察到的作用相似。 p53 siRNA的PDE3A KO VSMC转染可降低p21和MKP-1升高，并完全恢复生长，而不会影响细胞周期蛋白D1水平和RB磷酸化。我们得出的结论是PDE3A。不是PDE3B，而是通过两种免费信号通路来调节VSMC的生长，即PKA介导的MAPK信号通过RAF-1位点特异性抑制性磷酸化和PKA-CREB介导的P21诱导，导致GO/G1细胞周期停滞，例如以及通过诱导MKP-1，p21和WIP1的p53诱导，从而抑制G1对S细胞周期进程。尽管使用cAMP激动剂和PDE3和PDE4同工型的其他研究的研究据报道抑制了VSMC生长，但PDE3A和PDE3B同工型的确切作用很难辨别，因为在很大程度上缺乏相同的同工型特性特异性特异性特异性特异性的作用PDE3A和PDE3B抑制剂。综上所述，我们的数据和其他数据表明，PDE3A同工型通过调节心血管收缩性和外周血管舒张以及VSMC增长来在心血管功能中起主要作用。需要使用气球损伤模型的未来体内研究来测试PDE3A缺失对容器壁增厚，平滑肌细胞迁移和生长的保护作用，因为在基础条件下，PDE3A KO小鼠似乎并未以生长的严重损害，发展和心血管状况。 PDE3A可能是其他细胞中细胞周期进程的重要调节剂。雌性PDE3A KO小鼠是无菌的，大概是因为卵母细胞中的CAMP/PKA信号增加在第一种减数分裂分裂的先知I中保持减数分裂停滞，从而抑制卵母细胞的成熟并预防受精。成为PKA的靶标，并参与PDE3A KO卵母细胞的减数分裂骤停。在培养的，G2/M rarted的PDE3A KO鼠卵母细胞中，PKA活性升高与CDC2和PLK1的失活以及抑制组蛋白H3（S10）（S10）的磷酸化以及Cdc25b（S323）和CDC2（S323）和CDC2（Thr14/Tyr14/Tyr14/Tyr14/Tyr15）的磷酸化抑制作用有关。在WT卵母细胞中，PKA活性瞬时降低，然后增加到PDE3A KO卵母细胞中观察到的活性。观察到观察到CDC2和PLK1，并观察到组蛋白H3（S10）的磷酸化以及Cdc25b（S323）和Cdc2（Thr14/Tyr15）的去磷酸化。 在WT卵母细胞中，PKAC迅速转移到核中，然后转移到纺锤体中，但是在PDE3A KO卵母细胞中，PKAC仍留在细胞质中。 PLK1通过PDE3A KO卵母细胞与PKA抑制剂RP-cAMP孵育。 PDE3A与PLK1在WT卵母细胞中共定位，并在WT卵巢和HeLa细胞中与PLK1共免疫沉淀。 PKAC磷酸化的RPLK1和HeLa细胞PLK1并在体外抑制PLK1活性。综上所述，我们的结果表明，PKA诱导的PLK1抑制作用对于卵母细胞减数分裂和女性不育可能至关重要。