Intracellular Cyclic Adenosine Monophosphate Research Articles

Phosphodiesterase 8 (PDE8): Distribution and Cellular Expression and Association with Alzheimer's Disease.

Phosphodiesterase 8 (PDE8), as a member of PDE superfamily, specifically promotes the hydrolysis and degradation of intracellular cyclic adenosine monophosphate (cAMP), which may be associated with pathogenesis of Alzheimer's disease (AD). However, little is currently known about potential role in the central nervous system (CNS). Here we investigated the distribution and expression of PDE8 in brain of mouse, which we believe can provide evidence for studying the role of PDE8 in CNS and the relationship between PDE8 and AD. Here, C57BL/6J mice were used to observe the distribution patterns of two subtypes of PDE8, PDE8A and PDE8B, in different sexes in vivo by western blot (WB). Meanwhile, C57BL/6J mice were also used to demonstrate the distribution pattern of PDE8 in selected brain regions and localization in neural cells by WB and multiplex immunofluorescence staining. Furthermore, the triple transgenic (3×Tg-AD) mice and wild type (WT) mice of different ages were used to investigate the changes of PDE8 expression in the hippocampus and cerebral cortex during the progression of AD. PDE8 was found to be widely expressed in multiple tissues and organs including heart, kidney, stomach, brain, and liver, spleen, intestines, and uterus, with differences in expression levels between the two subtypes of PDE8A and PDE8B, as well as two sexes. Meanwhile, PDE8 was widely distributed in the brain, especially in areas closely related to cognitive function such as cerebellum, striatum, amygdala, cerebral cortex, and hippocampus, without differences between sexes. Furthermore, PDE8A was found to be expressed in neuronal cells, microglia and astrocytes, while PDE8B is only expressed in neuronal cells and microglia. PDE8A expression in the hippocampus of both female and male 3×Tg-AD mice was gradually increased with ages and PDE8B expression was upregulated only in cerebral cortex of female 3×Tg-AD mice with ages. However, the expression of PDE8A and PDE8B was apparently increased in both cerebral cortex and hippocampus in both female and male 10-month-old 3×Tg-AD mice compared WT mice. These results suggest that PDE8 may be associated with the progression of AD and is a potential target for its prevention and treatment in the future.

Pacemaker Channels and the Chronotropic Response in Health and Disease

Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease—including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating β-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate–sensitive hyperpolarization–activated current (I f ) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate–dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond.

A cAMP-biosensor-based assay for measuring plasma arginine–vasopressin levels

Arginine–vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.

Phosphodiesterase Inhibitors of Natural Origin

Abstract: Phosphodiesterases (PDEs) function to hydrolyze intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), regulating a variety of intracellular signal transduction and physiological activities. PDEs can be divided into 11 families (PDE1~11) and the diversity and complex expression of PDE family genes suggest that different subtypes may have different mechanisms. PDEs are involved in various disease pathologies such as inflammation, asthma, depression, and erectile dysfunction and are thus targets of interest for several drug discovery campaigns. Natural products have always been an important source of bioactive compounds for drug discovery, over the years several natural compounds have shown potential as inhibitors of PDEs. In this article, phosphodiesterase inhibitors of natural origin have been reviewed with emphasis on their chemistry and biological activities.

A phosphodiesterase 4 (PDE4) inhibitor, amlexanox, reduces neuroinflammation and neuronal death after pilocarpine-induced seizure

Epilepsy, a complex neurological disorder, is characterized by recurrent seizures caused by aberrant electrical activity in the brain. Central to this study is the role of lysosomal dysfunction in epilepsy, which can lead to the accumulation of toxic substrates and impaired autophagy in neurons. Our focus is on phosphodiesterase-4 (PDE4), an enzyme that plays a crucial role in regulating intracellular cyclic adenosine monophosphate (cAMP) levels by converting it into adenosine monophosphate (AMP). In pathological states, including epilepsy, increased PDE4 activity contributes to a decrease in cAMP levels, which may exacerbate neuroinflammatory responses. We hypothesized that amlexanox, an anti-inflammatory drug and non-selective PDE4 inhibitor, could offer neuroprotection by addressing lysosomal dysfunction and mitigating neuroinflammation, ultimately preventing neuronal death in epileptic conditions. Our research utilized a pilocarpine-induced epilepsy animal model to investigate amlexanox's potential benefits. Administered intraperitoneally at a dose of 100 ​mg/kg daily following the onset of a seizure, we monitored its effects on lysosomal function, inflammation, neuronal death, and cognitive performance in the brain. Tissue samples from various brain regions were collected at predetermined intervals for a comprehensive analysis. The study's results were significant. Amlexanox effectively improved lysosomal function, which we attribute to the modulation of zinc's influx into the lysosomes, subsequently enhancing autophagic processes and decreasing the release of inflammatory factors. Notably, this led to the attenuation of neuronal death in the hippocampal region. Additionally, cognitive function, assessed through the modified neurological severity score (mNSS) and the Barnes maze test, showed substantial improvements after treatment with amlexanox. These promising outcomes indicate that amlexanox has potential as a therapeutic agent in the treatment of epilepsy and related brain disorders. Its ability to combat lysosomal dysfunction and neuroinflammation positions it as a potential neuroprotective intervention. While these findings are encouraging, further research and clinical trials are essential to fully explore and validate the therapeutic efficacy of amlexanox in epilepsy management.

TIMP2 protects against sepsis-associated acute kidney injury by cAMP/NLRP3 axis-mediated pyroptosis.

The tissue inhibitor of metalloproteinases 2 (TIMP2) has emerged as a promising biomarker for predicting the risk of sepsis-associated acute kidney injury (SA-AKI). However, its exact role in SA-AKI and the underlying mechanism remains unclear. In this study, we investigated the impact of kidney tubule-specific Timp2 knockout mice on kidney injury and inflammation. Our findings demonstrated that Timp2-knockout mice exhibited more severe kidney injury than wild-type mice, along with elevated levels of pyroptosis markers NOD-like receptor protein 3 (NLRP3), Caspase1, and gasdermin D (GSDMD) in the early stage of SA-AKI. Conversely, the expression of exogenous TIMP2 in TIMP2-knockout mice still protected against kidney damage and inflammation. In in vitro experiments, using recombinant TIMP2 protein, TIMP2 knockdown demonstrated that exogenous TIMP2 inhibited pyroptosis of renal tubular cells stimulated by lipopolysaccharide (LPS). Mechanistically, TIMP2 promoted the ubiquitination and autophagy-dependent degradation of NLRP3 by increasing intracellular cyclic adenosine monophosphate (cAMP), which mediated NLRP3 degradation through recruiting the E3 ligase MARCH7, attenuating downstream pyroptosis, and thus alleviating primary tubular cell damage. These results revealed the renoprotective role of extracellular TIMP2 in SA-AKI by attenuating tubular pyroptosis, and suggested that exogenous administration of TIMP2 could be a promising therapeutic intervention for SA-AKI treatment.NEW & NOTEWORTHY Tissue inhibitor of metalloproteinase 2 (TIMP-2) has been found to be the best biomarker for predicting the risk of sepsis-associated acute kidney injury (SA-AKI). However, its role and the underlying mechanism in SA-AKI remain elusive. The authors demonstrated in this study using kidney tubule-specific knockout mice model of SA-AKI and primary renal tubule cells stimulated with lipopolysaccharide (LPS) that extracellular TIMP-2 promoted NOD-like receptor protein 3 (NLRP3) ubiquitination and autophagy-dependent degradation by increasing intracellular cyclic adenosine monophosphate (cAMP), thus attenuated pyroptosis and alleviated renal damage.

Novel Scaffold Agonists of the α2A Adrenergic Receptor Identified via Ensemble-Based Strategy.

The α2A adrenergic receptor (α2A-AR) serves as a critical molecular target for sedatives and analgesics. However, α2A-AR ligands with an imidazole ring also interact with an imidazoline receptor as well as other proteins and lead to undesirable effects, motivating us to develop more novel scaffold α2A-AR ligands. For this purpose, we employed an ensemble-based ligand discovery strategy, integrating long-term molecular dynamics (MD) simulations and virtual screening, to identify new potential α2A-AR agonists with novel scaffold. Our results showed that compounds SY-15 and SY-17 exhibited significant biological effects in the preliminary evaluation of protein kinase A (PKA) redistribution assays. They also reduced levels of intracellular cyclic adenosine monophosphate (cAMP) in a dose-dependent manner. Upon treatment of the cells with 100 μM concentrations of SY-15 and SY-17, there was a respective decrease in the intracellular cAMP levels by 63.43% and 53.83%. Subsequent computational analysis was conducted to elucidate the binding interactions of SY-15 and SY-17 with the α2A-AR. The binding free energies of SY-15 and SY-17 calculated by MD simulations were -45.93 and -71.97 kcal/mol. MD simulations also revealed that both compounds act as bitopic agonists, occupying the orthosteric site and a novel exosite of the receptor simultaneously. Our findings of integrative computational and experimental approaches could offer the potential to enhance ligand affinity and selectivity through dual-site occupancy and provide a novel direction for the rational design of sedatives and analgesics.

CAMP regulates the progesterone receptor gene expression through the protein kinase A pathway during decidualization in human immortalized endometrial stromal cells

Decidualization, a crucial process for successful pregnancy establishment and maintenance, involves endometrial stromal cell differentiation. This process is orchestrated by estradiol (E2), progesterone, and other stimuli that increase intracellular cyclic adenosine monophosphate (cAMP) levels. The intracellular progesterone receptor (PR), encoded by the PGR gene, has a key role in decidualization.This study aimed to understand the role of sex steroids and cAMP in regulating PGR expression during the in vitro decidualization of the human immortalized endometrial stromal cell line, T-HESC. We subjected the cells to individual and combined treatments of E2, medroxyprogesterone (MPA), and cAMP. Additionally, we treated cells with PR and estrogen receptor antagonists and a protein kinase A (PKA) inhibitor. We evaluated the expression of PGR isoforms and decidualization-associated genes by RT-qPCR.Our findings revealed that cAMP induced PGR-B and PGR-AB expression by activating the PKA signaling pathway, while MPA downregulated their expression through the PR. Furthermore, downstream genes involved in decidualization, such as those coding for prolactin (PRL), insulin-like growth factor-binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1), exhibited positive regulation via the cAMP-PKA pathway. Remarkably, MPA-activated PR signaling induced the expression of IGFBP1 and DKK1 but inhibited that of PRL.In conclusion, we have demonstrated that the PKA signaling pathway induces PGR gene expression during in vitro decidualization of the T-HESC human endometrial stromal cell line. This study has unraveled some of the intricate regulatory mechanisms governing PGR expression during this fundamental process for implantation and pregnancy maintenance.

Expression of the umami taste receptor T1R1/T1R3 in porcine testis of: Function in regulating testosterone synthesis and autophagy in Leydig cells

Testosterone is a vital male hormone responsible for male sexual characteristics. The taste receptor family 1 subunit 3 (T1R3) regulates testosterone synthesis and autophagy in non-taste cells, and the links with the taste receptor family 1 subunit 1 (T1R1) for umami perception. However, little is known about these mechanisms. Thus, we aimed to determine the relationship between the umami taste receptor (T1R1/T1R3) and testosterone synthesis or autophagy in testicular Leydig cells of the Xiang pig. There was a certain proportion of spermatogenic tubular dysplasia in the Xiang pig at puberty, in which autophagy was enhanced, and the testosterone level was increased with a weak expression of T1R3. Silenced T1R3 decreased testosterone level and intracellular cyclic adenosine monophosphate (cAMP) content and inhibited the messenger RNA (mRNA) expression levels of testosterone synthesis enzyme genes [steroidogenic acute regulatory protein (StAR), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (3β-HSD1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and hydroxysteroid 17-beta dehydrogenase 3 (17β-HSD3)]. In addition, T1R3 increased the number of acidic autophagy bubbles and upregulated the expression levels of autophagy markers [Microtubule-associated protein 1 A/1B-light chain 3 (LC3) and Beclin-1] in testicular Leydig cells of the Xiang pig. Using an umami tasting agonist (10 mM L-glutamate for 6 h), the activation of T1R1/T1R3 enhanced the testosterone synthesis ability by increasing the intracellular cAMP level and upregulated the expression levels of StAR, 3β-HSD1, CYP17A1 and 17β-HSD3 in Leydig cells. Furthermore, the number of acidic autophagy bubbles decreased in the T1R1/T1R3-activated group with the downregulation of the expression levels of the autophagy markers, including LC3 and Beclin-1. These data suggest that the function of T1R1/T1R3 expressed in testicular Leydig cells of the Xiang pig is related to testosterone synthesis and autophagy.

Beraprost sodium attenuates the development of myocardial fibrosis after myocardial infarction by regulating GSK-3β expression in rats.

The aim of this study was to elucidate the mechanism of beraprost sodium (BPS) in the intervention of myocardial fibrosis after myocardial infarction (MI) through glycogen synthase kinase-3β (GSK-3β) and to provide new ideas for intervention in myocardial fibrosis. MI model rats given BPS and cardiac fibroblasts (CFs) treated with BPS and TGF-β. HE staining and Masson staining were used to detect the pathological changes of myocardial tissue. Fibrotic markers were detected by immunohistochemical staining. The expressions of GSK-3β, cAMP response element binding protein (CREB), and p-CREB were analyzed by qPCR and western blot analysis. EDUstaining was used to detect the proliferation of CFs. The promoter activity of GSK-3β was detected by luciferase assay. Chromatin immunoprecipitation assay was used to detect the binding levels of GSK-3β promoter and Y-box binding protein 1 (YBX1). The levels of intracellular cyclic adenosine monophosphate (cAMP) were analyzed by enzyme-linked immunosorbent assay (ELISA). After operation, BPS improved myocardial fibrosis and upregulated GSK-3β protein expression in male SD rats. BPS can down-regulate α-smooth muscle actin (α-SMA) level and up-regulate GSK-3β protein expression in CFs after TGF-β stimulation. Furthermore, GSK-3β knockdown can reverse the effect of BPS on TGF-β-activated CFs, enhance α-SMA expression, and promote the proliferation of CFs. BPS could regulate GSK-3β expression by promoting the binding of GSK-3β promoter to YBX1. BPS induced upregulation of p-CREB and cAMP, resulting in reduced fibrosis, which was reversed by the knockdown of GSK-3β or prostaglandin receptor (IPR) antagonists. BPS treatment increased the binding of YBX1 to the GSK-3β promoter, and GSK-3β protein expression was upregulated, which further caused the upregulation of p-CREB and cAMP, and finally inhibited myocardial fibrosis.

Shear stress in the intervillous space promotes syncytial formation of iPS cells-derived trophoblasts†.

The intervillous space of human placenta is filled with maternal blood, and villous trophoblasts are constantly exposed to the shear stress generated by maternal blood pressure and flow throughout the entire gestation period. However, the effects of shear stress on villous trophoblasts and their biological significance remain unknown. Here, using our recently established naïve human pluripotent stem cells-derived cytotrophoblast stem cells (nCTs) and a device that can apply arbitrary shear stress to cells, we investigated the impact of shear stress on early-stage trophoblasts. After 72h of exposure to 10dyn/cm2 shear stress, nCTs became fused and multinuclear, and mRNA expression of the syncytiotrophoblast (ST) markers, such as glial cell missing 1, endogenous retrovirus group W member 1 envelope, chorionic gonadotropin subunit beta 3, syndecan 1, pregnancy specific beta-1-glycoprotein 3, placental growth factor, and solute carrier family 2 member 1 were significantly upregulated compared to static conditions. Immunohistochemistry showed that shear stress increased fusion index, human chorionic gonadotropin secretion, and human placental lactogen secretion. Increased microvilli formation on the surface of nCTs under flow conditions was detected using scanning electron microscopy. Intracellular cyclic adenosine monophosphate significantly increased under flow conditions. Moreover, transcriptome analysis of nCTs subjected to shear stress revealed that shear stress upregulated ST-specific genes and downregulated CT-specific genes. Collectively, these findings indicate that shear stress promotes the differentiation of nCTs into ST.

Novel Melanocortin-3 and -4 Receptor Functional Variants in Asian Children With Severe Obesity.

Genetic variants in melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) genes are strongly associated with childhood obesity. This study aims to identify and functionally characterize MC3R and MC4R variants in an Asian cohort of children with severe early-onset obesity. Whole-exome sequencing was performed to screen for MC3R and MC4R coding variants in 488 Asian children with severe early-onset obesity (body mass index for age ≥97th percentile). Functionality of the identified variants were determined via measurement of intracellular cyclic adenosine monophosphate (cAMP) concentrations and luciferase activity. Four MC3R and 2 MC4R heterozygous nonsynonymous rare variants were detected. There were 3 novel variants: MC3R c.151G > C (p.Val51Leu), MC4R c.127C > A (p.Gln43Lys), and MC4R c.272T > G (p.Met91Arg), and 3 previously reported variants: MC3R c.127G > A (p.Glu43Lys), MC3R c.97G > A (p.Ala33Thr), and MC3R c.437T > A (p.Ile146Asn). Both MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants demonstrated defective downstream cAMP signaling activity. The MC4R c.127C > A (p.Gln43Lys) variant showed reduced cAMP signaling activity at low substrate concentration but the signaling activity was restored at high substrate concentration. The MC3R c.151G > C (p.Val51Leu) variant did not show a significant reduction in cAMP signaling activity compared to wild-type (WT) MC3R. Coexpression studies of the WT and variant MC3R/MC4R showed that the heterozygous variants did not exhibit dominant negative effect. Our functional assays demonstrated that MC3R c.127G > A (p.Glu43Lys) and MC4R c.272T > G (p.Met91Arg) variants might predispose individuals to early-onset obesity, and further studies are needed to establish the causative effect of these variants in the pathogenesis of obesity.

Unkeito promotes follicle development by restoring reduced follicle-stimulating hormone responsiveness in rats with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common disorder resulting in irregular menstruation and infertility due to improper follicular development and ovulation. PCOS pathogenesis is mediated by downregulated follicle-stimulating hormone receptor (FSHR) expression in granulosa cells (GCs); however, the underlying mechanism remains elusive. Unkeito (UKT) is a traditional Japanese medicine used to treat irregular menstruation in patients with PCOS. In this study, we aimed to confirm the effectiveness of UKT in PCOS by focusing on follicle-stimulating hormone (FSH) responsiveness. A rat model of PCOS was generated by prenatal treatment with 5α-dihydrotestosterone. Female offspring (3-week-old) rats were fed a UKT mixed diet or a normal diet daily. To compare the PCOS phenotype in rats, the estrous cycle, hormone profiles, and ovarian morphology were evaluated. To further examine the role of FSH, molecular, genetic, and immunohistological analyses were performed using ovarian tissues and primary cultured GCs from normal and PCOS model rats. UKT increased the number of antral and preovulatory follicles and restored the irregular estrous cycle in PCOS rats. The gene expression levels of FSHR and bone morphogenetic protein (BMP)-2 and BMP-6 were significantly decreased in the ovarian GCs of PCOS rats compared to those in normal rats. UKT treatment increased FSHR staining in the small antral follicles and upregulated Fshr and Bmps expression in the ovary and GCs of PCOS rats. There was no change in serum gonadotropin levels. In primary cultured GCs stimulated by FSH, UKT enhanced estradiol production, accompanied by increased intracellular cyclic adenosine monophosphate levels, and upregulated the expression of genes encoding the enzymes involved in local estradiol synthesis, namely Cyp19a1 and Hsd17b. Furthermore, UKT elevated the expression of Star and Cyp11a1, involved in progesterone production in cultured GCs in the presence of FSH. UKT stimulates ovarian follicle development by potentiating FSH responsiveness by upregulating BMP-2 and BMP-6 expression, resulting in the recovery of estrous cycle abnormalities in PCOS rats. Restoring the FSHR dysfunction in the small antral follicles may alleviate the PCOS phenotype.

AaRgs1 and AaRgs2 differential regulate fungal development, stress response and appressorium-like formation in Alternaria alternata

Alternaria alternata can recognize and respond to physichemical cues from the host plant epidermis through a complex signaling network to initiate infectious structures formation. The primarily functions of RGS as a negative regulator of G protein signaling has been characterized in several model fungi, but its biological function in A. alternata remains unclear. In present study three RGS proteins were identified in A. alternata by Blast alignment and designed as AaRgs1, AaRgs2 and AaRgs3. Targeted genes deletion demonstrated that AaRgs1 and AaRgs2 play the opposite function in vegetative growth, melanin production and secretion, stress response and appressorium-like formation rate in A. alternata, while AaRgs3 had no certain regulatory effect. Differently, AaRgs1 negatively regulated tolerance to high-osmosis stress response and cell wall synthesis inhibitors by down regulating the expression level of AaHog1 and AaSlt2. AaRgs2 acts as a negative role in regulating appressorium-like formation rates through decreasing intracellular cyclic adenosine monophosphate (cAMP) accumulation. Deletion of AaRgs1 and AaRgs2 also resulted in significantly reducing penetrating ability and pathogenicity. The findings indicated that RGS proteins play multiple roles in fungal development, stress response and appressorium-like formation of A. alternata.

67-kDa laminin receptor mediates oolonghomobisflavan B-induced cell growth inhibition in melanoma

BackgroundOolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (–)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PurposeOHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. MethodsThe effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. ResultsCell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. ConclusionsTaken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.

Co-treatment of Naringenin and Ketoprofen-RGD Suppresses Cell Proliferation via Calmodulin/PDE/cAMP/PKA Axis Pathway in Leukemia and Ovarian Cancer Cells.

Naringenin (Nar) has anti-inflammatory and anticarcinogenic properties. Arginine-glycine- aspartate (RGD) is a tripeptidic sequence used as an integrin ligand and targeting system for delivering chemotherapeutic agents to cancer cells. In this study, the inhibitory effects of Nar and ketoprofen-RGD on leukemia and ovarian cancer cells (K562 and SKOV3) were explored for the first time, focusing on their proliferation activity and their anti-inflammatory capacity. Analyses were conducted on the calmodulin (CaM)-dependent phosphodiesterase 1 (PDE1) activation by ketoprofen-RGD, Nar, and their combination. These drugs' effects on protein kinase A (PKA) activation, intracellular cyclic adenosine monophosphate (cAMP) level, and PDE1 inhibition were identified. Later, it was also evaluated if ketoprofen-RGD alone or in combination with Nar had anti-inflammatory effects. Nar improved the antagonizing consequences of ketoprofen-RGD on the CaM protein, which hinders PDE1, improving PKA activity and cAMP levels. A mixture of ketoprofen-RGD and Nar and ketoprofen-RGD alone diminished K562 and SKOV3 cell viability through the cAMP/PKA pathway by inhibiting PDE1 and CaM. These two compounds showed anti-inflammatory effects on both cell lines. This study indicated for the first time that combining ketoprofen-RGD and Nar can be a promising anti-inflammatory therapeutic regimen for treating leukemia and ovarian cancer.

Δ9-Tetrahydrocannabinol Treatment Modifies Insulin Secretion in Pancreatic Islets from Prediabetic Mice Under Hypercaloric Diet.

Background: The endocannabinoid system over-activation is associated with type-2 diabetes mellitus onset, involving physiological, metabolic, and genetic alterations in pancreatic islets. The use of Δ9-Tetrahydrocannabinol (THC) as treatment is still controversial since its effects and mechanisms on insulin secretion are unclear. The aim of this study was to evaluate the effects of THC treatment in pancreatic islets from prediabetic mice. Methods: Prediabetes was induced in mice by hypercaloric diet, and then treated with THC for 3 weeks. Blood glucose and body weight were determined, after behavior tests. Histological changes were evaluated in whole pancreas; in isolated islets we analyzed the effect of THC exposure in glucose-stimulated insulin secretion (GSIS), gene expression, intracellular cyclic adenosine monophosphate (cAMP), and cytosolic calcium changes. Results: THC treatment in prediabetic mice enhanced anxiety and antidepressive behavior without changes in food ingestion, decreased oral-glucose tolerance test, plasma insulin and weight, with small alterations on pancreatic histology. In isolated islets from healthy mice THC increased GSIS, cAMP, and CB1 receptor (CB1r) expression, meanwhile calcium release was diminished. Small changes were observed in islets from prediabetic mice. Conclusions: THC treatment improves some clinical parameters in prediabetic mice, however, in isolated islets, modifies GSIS, intracellular calcium and gene expression, suggesting specific effects related to diabetes evolution.

Stress granule activation attenuates lipopolysaccharide-induced cardiomyocyte dysfunction

BackgroundSepsis is the leading cause of death in intensive care units. Sepsis-induced myocardial dysfunction, one of the most serious complications of sepsis, is associated with higher mortality rates. As the pathogenesis of sepsis-induced cardiomyopathy has not been fully elucidated, there is no specific therapeutic approach. Stress granules (SG) are cytoplasmic membrane-less compartments that form in response to cellular stress and play important roles in various cell signaling pathways. The role of SG in sepsis-induced myocardial dysfunction has not been determined. Therefore, this study aimed to determine the effects of SG activation in septic cardiomyocytes (CMs).MethodsNeonatal CMs were treated with lipopolysaccharide (LPS). SG activation was visualized by immunofluorescence staining to detect the co-localization of GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and T cell-restricted intracellular antigen 1 (TIA-1). Eukaryotic translation initiation factor alpha (eIF2α) phosphorylation, an indicator of SG formation, was assessed by western blotting. Tumor necrosis factor alpha (TNF-α) production was assessed by PCR and enzyme-linked immunosorbent assays. CMs function was evaluated by intracellular cyclic adenosine monophosphate (cAMP) levels in response to dobutamine. Pharmacological inhibition (ISRIB), a G3BP1 CRISPR activation plasmid, and a G3BP1 KO plasmid were employed to modulate SG activation. The fluorescence intensity of JC-1 was used to evaluate mitochondrial membrane potential.ResultsLPS challenge in CMs induced SG activation and resulted in eIF2α phosphorylation, increased TNF-α production, and decreased intracellular cAMP in response to dobutamine. The pharmacological inhibition of SG (ISRIB) increased TNF-α expression and decreased intracellular cAMP levels in CMs treated with LPS. The overexpression of G3BP1 increased SG activation, attenuated the LPS-induced increase in TNF-α expression, and improved CMs contractility (as evidenced by increased intracellular cAMP). Furthermore, SG prevented LPS-induced mitochondrial membrane potential dissipation in CMs.ConclusionSG formation plays a protective role in CMs function in sepsis and is a candidate therapeutic target.

The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

BackgroundLignocellulose is the most abundant renewable resource in the world and has attracted widespread attention. It can be hydrolyzed into sugars with the help of cellulases and hemicellulases that are secreted by filamentous fungi. Several studies have revealed that the Ras small GTPase superfamily regulates important cellular physiological processes, including synthesis of metabolites, sporulation, and cell growth and differentiation. However, it remains unknown how and to what extent Ras small GTPases participate in cellulase production.ResultsIn this study, we found that the putative Ras small GTPase RSR1 negatively regulated the expression of cellulases and xylanases. Deletion of rsr1 (∆rsr1) significantly increased cellulase production and decreased the expression levels of ACY1-cAMP-protein kinase A (PKA) signaling pathway genes and the concentration of intracellular cyclic adenosine monophosphate (cAMP). Loss of acy1 based on ∆rsr1 (∆rsr1∆acy1) could further increase cellulase production and the expression levels of cellulase genes, while overexpression of acy1 based on ∆rsr1 (∆rsr1-OEacy1) significantly reduced cellulase production and transcriptional levels of cellulase genes. In addition, our results revealed that RSR1 negatively controlled cellulase production via the ACY1-cAMP-PKA pathway. Transcriptome analysis revealed significantly increased expression of three G-protein coupled receptors (GPCRs; tre62462, tre58767, and tre53238) and approximately two-fold higher expression of ACE3 and XYR1, which transcriptionally activated cellulases with the loss of rsr1. ∆rsr1∆ tre62462 exhibited a decrease in cellulase activity compared to ∆rsr1, while that of ∆rsr1∆tre58767 and ∆rsr1∆tre53238 showed a remarkable improvement compared to ∆rsr1. These findings revealed that GPCRs on the membrane may sense extracellular signals and transmit them to rsr1 and then to ACY1-cAMP-PKA, thereby negatively controlling the expression of the cellulase activators ACE3 and XYR1. These data indicate the crucial role of Ras small GTPases in regulating cellulase gene expression.ConclusionsHere, we demonstrate that some GPCRs and Ras small GTPases play key roles in the regulation of cellulase genes in Trichoderma reesei. Understanding the roles of these components in the regulation of cellulase gene transcription and the signaling processes in T. reesei can lay the groundwork for understanding and transforming other filamentous fungi.

Brain and eye involvement in McCune-Albright Syndrome: clinical and translational insights.

McCune-Albright Syndrome (MAS) is a rare mosaic (post-zygotic) genetic disorder presenting with a broad continuum clinical spectrum. MAS arises from somatic, activating mutations in the GNAS gene, which induces a dysregulated Gsα-protein signaling in several tissues and an increased production of intracellular cyclic adenosine monophosphate (cAMP). Overall, MAS is a rare disorder affecting less than 1/100,000 children and, for this reason, data establishing genotype-phenotype correlations remain limited. Affected individuals clinically present with a variable combination of fibrous dysplasia of bone (FD), extra-skeletal manifestations (including cafeí-au-lait spots) and precocious puberty which might also be associated to broad hyperfunctioning endocrinopathies, and also gastrointestinal and cardiological involvement. Central nervous system (CNS) and eye involvement in MAS are among the less frequently described complications and remain largely uncharacterized. These rare complications mainly include neurodevelopmental abnormalities (e.g., delayed motor development, cognitive and language impairment), CNS anomalies (e.g., Chiari malformation type I) and a wide array of ophthalmological abnormalities often associated with vision loss. The pathophysiological mechanisms underlying abnormal neurological development have not been yet fully elucidated. The proposed mechanisms include a deleterious impact of chronically dysregulated Gsα-protein signaling on neurological function, or a secondary (damaging) effect of (antenatal and/or early postnatal) hypercortisolism on early pre- and post-natal CNS development. In this Review, we summarize the main neurological and ophthalmological features eventually associated with the MAS spectrum, also providing a detailed overview of the potential pathophysiological mechanisms underlying these clinical complications.