Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL‐1 cardiac cell line

Abstract

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. K E Y W O R D Scardiomyocytes, hypertrophy, PDE5A isoforms, polyploidy

Novel landscape of HLA‐G isoforms expressed in clear cell renal cell carcinoma patients

“…However, although all HLA‐G isoforms are presumed to be generated by alternative splicing of a common pre‐mRNA, it remains possible that they could be derived from separate mRNA that are transcribed from distinct and internal promoters. These have been previously reported for several genes such as phosphodiesterase 5A (PDE5A) and TP53 (Campolo et al ., ; Lin et al ., ; Surget et al ., ).…”

Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

“…The α-signal DNA was synthesized as two short single strands complementary sequences containing at the 5′ and 3′ end the Xba I and Sal I sites (Fwd: ctag atg aat ata ttt tac ata ttt ttg ttt ttg ctg tca ttc gtt caa ggt agg ggt gtg ttt cgt cga g; Rev: tc gac tcg acg aaa cac acc cct acc ttg aac gaa tga cag caa aaa caa aaa tat gta aaa tat att cat), respectively, and then annealed. The 2.6 Kbp Sal I– Sma I PDE5A1 fragment was purified from p3XFLAG-CMV7 [ 36 ]. The two fragments were ligated into the Bluescript KS (Stratagene) plasmid digested with Xba I– Sma I.…”

“…pYG137/1 was also used to construct p3XFlagPDE5A1, p3XFlagPDE5A2 and p3XFlagPDE5A3 which contain the M. musculus Pde5a1, a2 and a3 spliced-variants of the Pde5 gene fused at their 5′ end to the short DNA sequence coding for the 3XFLAG peptide (Sigma), an immunogenic peptide for the affinity purification of PDEs from whole cellular extracts. These recombinant vectors were constructed in two stages from pα-PDE5A1 and the p3XFLAG-CMV7 vectors containing either Pde5a1, a2 or a3 in frame with the 3XFLAG coding sequence [ 36 ]. To start, pα-PDE5A1 was digested with Pml I, ligated and transformed in E. coli cells.…”

“…These expression vectors were used for the quantitative production of the Mus musculus PDE5 isoforms. We found that the expression of the M. musculus Pde5a1, a2 and a3 variants of the Pde5a gene, obtained from a pCDNA3.1-cloned cDNA embrio library [ 36 ], was well tolerated by K. lactis cells without major growth deficiencies. PDE5A proteins, produced in large amounts, displayed biochemical properties identical to those of the native murine isoforms [ 37 ].…”

Cardiac Cyclic Nucleotide Phosphodiesterases: Roles and Therapeutic Potential in Heart Failure

“…The one PDE5 subtype (PDE5A) is alternatively expressed as three isoforms (PDE5A1, PDE5A2, PDE5A3), although dispute persists concerning the physiological relevance of these to cardiac physiology. Whilst some studies have reported either no or minimal cardiac expression of PDE5 [56,[169][170][171], others have detected it within certain cell types [172][173][174]. In isolated cardiomyocytes, PDE5 appears restricted to the cytosol where it is anchored to the Z-lines and preferentially hydrolyses NO/cGMP (although this may change with disease) [92,175,176].…”