Histone deacetylase inhibition mitigates fibrosis-driven disease progression in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a blistering disease caused by mutations in the gene encoding type VII collagen (C7). RDEB is associated with fibrosis, which is responsible for severe complications. The phenotypic variability observed in RDEB siblings suggests that epigenetic modifications contribute to disease severity. Identifying epigenetic changes may help to uncover molecular mechanisms underlying RDEB pathogenesis and new therapeutic targets. OBJECTIVES: To investigate histone acetylation in RDEB skin and to explore histone deacetylase inhibitors (HDACis) as therapeutic molecules capable of counteracting fibrosis and disease progression in RDEB mice. METHODS: Acetylated histone levels were detected in human skin by immunofluorescence and in RDEB fibroblasts by ELISA. The effects of Givinostat and valproic acid (VPA) on RDEB fibroblast fibrotic behaviour were assessed by collagen-gel contraction assay, Western blot and immunocytofluorescence for α-smooth muscle actin, ELISA for released transforming growth factor-ß1 (TGF-ß1). RNA-seq was performed in HDACi- and vehicle-treated RDEB fibroblasts. VPA was systemically administered to RDEB mice, and effects on overt phenotype were monitored. Fibrosis was investigated in the skin using histological and immunofluorescence analyses. Eye and tongue defects were examined microscopically. Mass spectrometry proteomics was performed on skin protein extracts from VPA-treated RDEB and control mice. RESULTS: Histone acetylation decreases in RDEB skin and primary fibroblasts. RDEB fibroblasts treated with HDACis lowered fibrotic traits including contractility, TGF-ß1 release, and proliferation. VPA administration to RDEB mice mitigated severe manifestations affecting eyes and paws. These effects were associated with fibrosis inhibition. Proteomic analysis of mouse skin revealed that VPA almost normalised protein sets involved in protein synthesis and immune response, processes linked to the increased susceptibility to cancer and bacterial infections observed in RDEB patients. CONCLUSIONS: Dysregulated histone acetylation contributes to RDEB pathogenesis by facilitating the progression of fibrosis. Repurposing of HDACi could be considered for disease-modifying treatments of RDEB.

Evaluation of the Therapeutic Effects of Harmine on Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid carcinoma (ATC) is an extremely difficult disease to tackle, with an overall patient survival of only a few months. The currently used therapeutic drugs, such as kinase inhibitors or immune checkpoint inhibitors, can prolong patient survival but fail to eradicate the tumor. In addition, the onset of drug resistance and adverse side-effects over time drastically reduce the chances of treatment. We recently showed that Twist1, a transcription factor involved in the epithelial mesenchymal transition (EMT), was strongly upregulated in ATC, and we wondered whether it might represent a therapeutic target in ATC patients. To investigate this hypothesis, the effects of harmine, a ß-carboline alkaloid shown to induce degradation of the Twist1 protein and to possess antitumoral activity in different cancer types, were evaluated on two ATC-derived cell lines, BHT-101 and CAL-62. The results obtained demonstrated that, in both cell lines, harmine reduced the level of Twist1 protein and reverted the EMT, as suggested by the augmentation of E-cadherin and decrease in fibronectin expression. The drug also inhibited cell proliferation and migration in a dose-dependent manner and significantly reduced the anchorage-independent growth of both ATC cell lines. Harmine was also capable of inducing apoptosis in BHT-101 cells, but not in CAL-62 ones. Finally, the activation of PI3K/Akt signaling, but not that of the MAPK, was drastically reduced in treated cells. Overall, these in vitro data suggest that harmine could represent a new therapeutic option for ATC treatment.

Anterior Cruciate Ligament Revision Plus Lateral Extra-Articular Procedure Results in Superior Stability and Lower Failure Rates Than Does Isolated Anterior Cruciate Ligament Revision but Shows No Difference in Patient-Reported Outcomes or Return to Sports.

PURPOSE: To determine whether comparative clinical studies demonstrate significant advantages of revision anterior cruciate ligament reconstruction (RACLR) combined with a lateral extra-articular procedure (LEAP), with respect to graft rupture rates, knee stability, return to sport rates, and patient-reported outcome measures, compared with isolated RACLR. METHODS: A systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews & Meta-Analyses Guidelines. A PubMed search was conducted using the key words "revision anterior cruciate ligament reconstruction" combined with any of the following additional terms, "lateral extra-articular tenodesis" OR "anterolateral ligament reconstruction" OR "Lemaire." All relevant comparative clinical studies were included. Key clinical data were extracted and evaluated. RESULTS: Eight comparative studies (seven Level III studies and a one Level IV study) were identified and included. Most studies reported more favorable outcomes with combined procedures with respect to failure rates (0%-13% following RACLR+LEAP, and 4.4%-21.4% following isolated RACLR), postoperative side-to-side anteroposterior laxity difference (1.3-3.9 mm following RACLR+LEAP and 1.8-5.9 mm following isolated RACLR), and high-grade pivot shift (0%-11.1% following RACLR+LEAP and 10.2%-23.8% in patients following isolated RACLR). There were no consistent differences between isolated and combined procedures with respect to return to sport or patient-reported outcome measures. CONCLUSIONS: This systematic review demonstrates that the addition of a LEAP to RACLR was associated with an advantage with respect to ACL graft failure rates and avoidance of high-grade postoperative knee laxity across almost all included studies. LEVEL OF EVIDENCE: IV, Systematic review of level III to IV studies.

Structural Characterization of Murine Phosphodiesterase 5 Isoforms and Involvement of Cysteine Residues in Supramolecular Assembly.

Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms' oligomeric assembly and the loss of enzymatic activity when modified.

Cellular Redox Metabolism Is Modulated by the Distinct Localization of Cyclic Nucleotide Phosphodiesterase 5A Isoforms.

3'-5' cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionarily conserved cAMP and/or cGMP hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5-being a regulator of vascular smooth muscle contraction-is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of the MmPDE5A2 is responsible for its mitochondrial localization. Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)+/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors.

Phosphodiesterases Expression during Murine Cardiac Development.

3'-5' cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.

A2A Receptor Dysregulation in Dystonia DYT1 Knock-Out Mice.

We aimed to investigate A2A receptors in the basal ganglia of a DYT1 mouse model of dystonia. A2A was studied in control Tor1a+/+ and Tor1a+/- knock-out mice. A2A expression was assessed by anti-A2A antibody immunofluorescence and Western blotting. The co-localization of A2A was studied in striatal cholinergic interneurons identified by anti-choline-acetyltransferase (ChAT) antibody. A2A mRNA and cyclic adenosine monophosphate (cAMP) contents were also assessed. In Tor1a+/+, Western blotting detected an A2A 45 kDa band, which was stronger in the striatum and the globus pallidus than in the entopeduncular nucleus. Moreover, in Tor1a+/+, immunofluorescence showed A2A roundish aggregates, 0.3-0.4 µm in diameter, denser in the neuropil of the striatum and the globus pallidus than in the entopeduncular nucleus. In Tor1a+/-, A2A Western blotting expression and immunofluorescence aggregates appeared either increased in the striatum and the globus pallidus, or reduced in the entopeduncular nucleus. Moreover, in Tor1a+/-, A2A aggregates appeared increased in number on ChAT positive interneurons compared to Tor1a+/+. Finally, in Tor1a+/-, an increased content of cAMP signal was detected in the striatum, while significant levels of A2A mRNA were neo-expressed in the globus pallidus. In Tor1a+/-, opposite changes of A2A receptors' expression in the striatal-pallidal complex and the entopeduncular nucleus suggest that the pathophysiology of dystonia is critically dependent on a composite functional imbalance of the indirect over the direct pathway in basal ganglia.

Phosphodiesterase Inhibitors: Could They Be Beneficial for the Treatment of COVID-19?

In March 2020, the World Health Organization declared the severe acute respiratory syndrome corona virus 2 (SARS-CoV2) infection to be a pandemic disease. SARS-CoV2 was first identified in China and, despite the restrictive measures adopted, the epidemic has spread globally, becoming a pandemic in a very short time. Though there is growing knowledge of the SARS-CoV2 infection and its clinical manifestations, an effective cure to limit its acute symptoms and its severe complications has not yet been found. Given the worldwide health and economic emergency issues accompanying this pandemic, there is an absolute urgency to identify effective treatments and reduce the post infection outcomes. In this context, phosphodiesterases (PDEs), evolutionarily conserved cyclic nucleotide (cAMP/cGMP) hydrolyzing enzymes, could emerge as new potential targets. Given their extended distribution and modulating role in nearly all organs and cellular environments, a large number of drugs (PDE inhibitors) have been developed to control the specific functions of each PDE family. These PDE inhibitors have already been used in the treatment of pathologies that show clinical signs and symptoms completely or partially overlapping with post-COVID-19 conditions (e.g., thrombosis, inflammation, fibrosis), while new PDE-selective or pan-selective inhibitors are currently under study. This review discusses the state of the art of the different pathologies currently treated with phosphodiesterase inhibitors, highlighting the numerous similarities with the disorders linked to SARS-CoV2 infection, to support the hypothesis that PDE inhibitors, alone or in combination with other drugs, could be beneficial for the treatment of COVID-19.

Dystonia: Sparse Synapses for D2 Receptors in Striatum of a DYT1 Knock-out Mouse Model.

Dystonia pathophysiology has been partly linked to downregulation and dysfunction ofdopamine D2 receptors in striatum. We aimed to investigate the possible morpho-structuralcorrelates of D2 receptor downregulation in the striatum of a DYT1 Tor1a mouse model. Adultcontrol Tor1a+/+ and mutant Tor1a+/- mice were used. The brains were perfused and free-floatingsections of basal ganglia were incubated with polyclonal anti-D2 antibody, followed by secondaryimmune-fluorescent antibody. Confocal microscopy was used to detect immune-fluorescent signals.The same primary antibody was used to evaluate D2 receptor expression by western blot. The D2receptor immune-fluorescence appeared circumscribed in small disks (0.3-0.5 µm diameter), likelyrepresenting D2 synapse aggregates, densely distributed in the striatum of Tor1a+/+ mice. In theTor1a+/- mice the D2 aggregates were significantly smaller (µm2 2.4 ± SE 0.16, compared to µm2 6.73± SE 3.41 in Tor1a+/+) and sparse, with ~30% less number per microscopic field, value correspondentto the amount of reduced D2 expression in western blotting analysis. In DYT1 mutant mice thesparse and small D2 synapses in the striatum may be insufficient to "gate" the amount ofpresynaptic dopamine release diffusing in peri-synaptic space, and this consequently may result ina timing and spatially larger nonselective sphere of influence of dopamine action.

Metabolic role of cGMP in S. cerevisiae: the murine phosphodiesterase-5 activity affects yeast cell proliferation by altering the cAMP/cGMP equilibrium.

In higher eukaryotes, cAMP and cGMP are signal molecules of major transduction pathways while phosphodiesterases (PDE) are a superfamily of cAMP/cGMP hydrolysing enzymes, modulatory components of these routes. Saccharomyces cerevisiae harbours two genes for PDE: Pde2 is a high affinity cAMP-hydrolysing enzyme, while Pde1 can hydrolyse both cAMP and cGMP. To gain insight into the metabolic role of cGMP in the physiology of yeast, the murine Pde5a1 gene encoding a specific cGMP-hydrolysing enzyme, was expressed in S. cerevisiae pdeΔ strains. pde1Δ and pde2Δ PDE5A1-transformed strain displayed opposite growth-curve profiles; while PDE5A1 recovered the growth delay of pde1Δ, PDE5A1 reversed the growth profile of pde2Δ to that of the untransformed pde1Δ. Growth test analysis and the use of Adh2 and Adh1 as respiro-fermentative glycolytic flux markers confirmed that PDE5A1 altered the metabolism by acting on Pde1-Pde2/cyclic nucleotides content and also on the TORC1 nutrient-sensing cascade. cGMP is required during the log-phase of cell proliferation to adjust/modulate cAMP levels inside well-defined ranges. A model is presented proposing the role of cGMP in the cAMP/PKA pathway. The expression of the PDE5A1 cassette in other mutant strains might constitute the starting tool to define cGMP metabolic role in yeast nutrient signaling.

Extracellular Vesicle-Induced Differentiation of Neural Stem Progenitor Cells.

Neural stem progenitor cells (NSPCs) from E13.5 mouse embryos can be maintained in culture under proliferating conditions. Upon growth-factor removal, they may differentiate toward either neuronal or glial phenotypes or both. Exosomes are small extracellular vesicles that are part of the cell secretome; they may contain and deliver both proteins and genetic material and thus play a role in cell-cell communication, guide axonal growth, modulate synaptic activity and regulate peripheral nerve regeneration. In this work, we were interested in determining whether NSPCs and their progeny can produce and secrete extracellular vesicles (EVs) and if their content can affect cell differentiation. Our results indicate that cultured NSPCs produce and secrete EVs both under proliferating conditions and after differentiation. Treatment of proliferating NSPCs with EVs derived from differentiated NSPCs triggers cell differentiation in a dose-dependent manner, as demonstrated by glial- and neuronal-marker expression.

Phosphodiesterase-10A Inverse Changes in Striatopallidal and Striatoentopeduncular Pathways of a Transgenic Mouse Model of DYT1 Dystonia.

We report that changes of phosphodiesterase-10A (PDE10A) can map widespread functional imbalance of basal ganglia circuits in a mouse model of DYT1 dystonia overexpressing mutant torsinA. PDE10A is a key enzyme in the catabolism of second messenger cAMP and cGMP, whose synthesis is stimulated by D1 receptors and inhibited by D2 receptors preferentially expressed in striatoentopeducuncular/substantia nigra or striatopallidal pathways, respectively. PDE10A was studied in control mice (NT) and in mice carrying human wild-type torsinA (hWT) or mutant torsinA (hMT). Quantitative analysis of PDE10A expression was assessed in different brain areas by rabbit anti-PDE10A antibody immunohistochemistry and Western blotting. PDE10A-dependent cAMP hydrolyzing activity and PDE10A mRNA were also assessed. Striatopallidal neurons were identified by rabbit anti-enkephalin antibody.In NT mice, PDE10A is equally expressed in medium spiny striatal neurons and in their projections to entopeduncular nucleus/substantia nigra and to external globus pallidus. In hMT mice, PDE10A content selectively increases in enkephalin-positive striatal neuronal bodies; moreover, PDE10A expression and activity in hMT mice, compared with NT mice, significantly increase in globus pallidus but decrease in entopeduncular nucleus/substantia nigra. Similar changes of PDE10A occur in hWT mice, but such changes are not always significant. However, PDE10A mRNA expression appears comparable among NT, hWT, and hMT mice.In DYT1 transgenic mice, the inverse changes of PDE10A in striatoentopeduncular and striatopallidal projections might result over time in an imbalance between direct and indirect pathways for properly focusing movement. The decrease of PDE10A in the striatoentopeduncular/nigral projections might lead to increased intensity and duration of D1-stimulated cAMP/cGMP signaling; conversely, the increase of PDE10A in the striatopallidal projections might lead to increased intensity and duration of D2-inhibited cAMP/cGMP signaling.SIGNIFICANCE STATEMENT In DYT1 transgenic mouse model of dystonia, PDE10A, a key enzyme in cAMP and cGMP catabolism, is downregulated in striatal projections to entopeduncular nucleus/substantia nigra, preferentially expressing D1 receptors that stimulate cAMP/cGMP synthesis. Conversely, in DYT1 mice, PDE10A is upregulated in striatal projections to globus pallidus, preferentially expressing D2 receptors that inhibit cAMP/cGMP synthesis. The inverse changes to PDE10A in striatoentopeduncular/substantia nigra and striatopallidal pathways might tightly interact downstream to dopamine receptors, likely resulting over time to increased intensity and duration respectively of D1-stimulated and D2-inhibited cAMP/cGMP signals. Therefore, PDE10A changes in the DYT1 model of dystonia can upset the functional balance of basal ganglia circuits, affecting direct and indirect pathways simultaneously.

Chronic phosphodiesterase type 5 inhibition has beneficial effects on subcutaneous adipose tissue plasticity in type 2 diabetic mice.

Different adipose tissue (AT) depots are associated with multiple metabolic risks. Phosphodiesterase type 5 (PDE5) is involved in adipocyte physiology and PDE5 inhibition may affect adipogenesis and ameliorate white AT quality. The aim of this study is to investigate the distribution of AT and the composition of the stroma-vascular fraction (SVF) of subcutaneous AT (SAT) in type 2 diabetic mice after prolonged treatment with a PDE5 inhibitor, Sildenafil. 18 db/db mice were treated with Sildenafil or vehicle for 12 weeks. AT distribution was monitored and SAT was processed for isolation of SVF by flow cytometry. Sildenafil induced an overall reduction in AT, mainly in visceral AT (VAT), compared with SAT. In Sildenafil-treated mice, the mean change in body weight from baseline positively correlated with VAT, but not with SAT. Characterization of SVF of SAT showed an increase in the frequency of M2 macrophages and endothelial cells in treated mice. Sildenafil improved the maintenance of SAT homeostasis and distribution.

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

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.

Egr-1 Maintains NSC Proliferation and Its Overexpression Counteracts Cell Cycle Exit Triggered by the Withdrawal of Epidermal Growth Factor.

In adult mammals, neural stem cells (NSCs) reside in specialized niches at the level of selected CNS regions, such as the subventricular zone (SVZ). The signaling pathways that reg-ulate NSC proliferation and differentiation remain poorly understood. Early growth response protein 1 (Egr-1) is an important transcription factor, widely studied in the adult mammalian brain, mediating the activation of target genes by a variety of extracellular stimuli. In our study, we aimed at testing how Egr-1 regulates adult NSCs derived from mouse SVZ and, in particular, the interplay between Egr-1 and the proliferative factor EGF. We demonstrate that Egr-1 expression in NSCs is induced by growth factor stimulation, and its level decreases after EGF deprivation or by using AG1478, an inhibitor of the EGF/EGFR signaling pathway. We also show that Egr-1 overexpression rescues the cell proliferation decrease observed either after EGF removal or upon treatment with AG1478, suggesting that Egr-1 works downstream of the EGF pathway. To better understand this mechanism, we investigated targets downstream of both the EGF pathway and Egr-1, and found that they regulate genes involved in NSC proliferation, such as cell cycle regulators, cyclins, and cyclin-dependent kinase inhibitors.

The oligomeric assembly of the phosphodiesterase-5 is a mixture of dimers and tetramers: A putative role in the regulation of function.

BACKGROUND: Phosphodiesterases (PDEs) are a superfamily of evolutionary conserved cyclic nucleotides (cAMP/cGMP) hydrolysing enzymes, components of transduction pathways regulating crucial aspects of cell life. PDE5, one of these families, is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Despite its medical relevance, PDE5 macromolecular structure has only been solved for the isolated regulatory and catalytic domains. The definition of the quaternary structure of the full length PDE5 (MmPDE5A1), produced in large amounts in the yeast Kluyveromyces lactis, could greatly enhance the knowledge on its assembly/allosteric regulation and the development of new inhibitors for clinical-therapeutic applications. METHODS: Small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (PAGE) and western blot (WB) were used to assess the assembly of PDE5A1. RESULTS: The full length MmPDE5A1 isoform is a mixture of dimers and tetramers in solution. We also report data showing that dimers and tetramers also coexist in vivo in platelets, blood components naturally containing high levels of PDE5. CONCLUSIONS: This is the first time that structural studies on the full length protein evidenced the assembly of PDE5 in tetramers in addition to the expected dimers. GENERAL SIGNIFICANCE: The assembly of PDE5 in tetramers in platelets, beside the dimers, opens the possibility to alternative assembly/allosteric regulation of this enzyme, as component of large signaling complexes, in all cellular districts in which PDE5 is present.

Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 µM) and DC-TA-46 (0.5 µM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc.

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

BACKGROUND: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. RESULTS: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. CONCLUSIONS: To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

Characterization of the transcription factor encoding gene, KlADR1: metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like KlADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc-finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Scadr1Δ the transcription of ADH2 in a ScAdr1-dependent fashion.

Modulation of cAMP/cGMP signaling as prevention of congenital heart defects in Pde2A deficient embryos: a matter of oxidative stress.

Phosphodiesterase 2A (Pde2A) is a dual-specific PDE that breaks down both cAMP and cGMP cyclic nucleotides. We recently highlighted a direct relationship between Pde2A impairment, a consequent increase of cAMP, and the appearance of mouse congenital heart defects (CHDs). Here we aimed to characterize the pathways involved in the development of CHDs and in their prevention by pharmacological approaches targeting cAMP and cGMP signaling. Transcriptome analysis revealed a modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, transcription, and oxidative stress in hearts from Pde2A-/- embryos. Metoprolol and H89 pharmacological administration prevented heart dilatation and hypertabeculation in Pde2A-/- embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. Amelioration of cardiac defects was also observed by using the antioxidant NAC, indicating oxidative stress as one of the molecular mechanisms underpinning the CHDs. In addition, Sildenafil treatment recovered cardiac defects suggesting the requirement of cAMP/cGMP nucleotides balance for the correct heart development.