Inhibitor Of cAMP-dependent Protein Kinase Research Articles

Fustin suppressed melanoma cell growth via cAMP/PKA-dependent mechanism.

Melanoma, a cancer arising from melanocytes, requires a novel treatment strategy because of the ineffectiveness of conventional therapies in certain patients. Fustin is a flavanonol found in young fustic (Cotinus coggygria). However, little is known about its antimelanoma effects. Our study demonstrates that fustin suppresses the growth of B16 melanoma cells. Phalloidin staining of cytoskeletal actin revealed that fustin induced a conformational change in the actin structure of melanoma cells, accompanied by suppressed phosphorylation of myosin regulatory light chain 2 (MLC2), a regulator of actin structure. Furthermore, the protein kinase A (cAMP-dependent protein kinase) inhibitor H89 completely attenuated fustin-induced downregulation of phosphorylated myosin phosphatase targeting subunit 1 (MYPT1), which is involved in dephosphorylation of MLC2. In a mouse model, administration of fustin suppressed tumor growth in B16 melanoma cells without adverse effects. In conclusion, our findings suggest that fustin effectively suppresses melanoma cell growth both in vitro and in vivo.

PKIB, a Novel Target for Cancer Therapy.

The serine-threonine kinase protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent intracellular protein with multiple roles in cellular biology including metabolic and transcription regulation functions. The cAMP-dependent protein kinase inhibitor β (PKIB) is one of three known endogenous protein kinase inhibitors of PKA. The role of PKIB is not yet fully understood. Hormonal signaling is correlated with increased PKIB expression through genetic regulation, and increasing PKIB expression is associated with decreased cancer patient prognosis. Additionally, PKIB impacts cancer cell behavior through two mechanisms; the first is the nuclear modulation of transcriptional activation and the second is the regulation of oncogenic AKT signaling. The limited research into PKIB indicates the oncogenic potential of PKIB in various cancers. However, some studies suggest a role of PKIB in non-cancerous disease states. This review aims to summarize the current literature and background of PKIB regarding cancer and related issues. In particular, we will focus on cancer development and therapeutic possibilities, which are of paramount interest in PKIB oncology research.

DNA methylation patterns in CD4+ T-cells separate psoriasis patients from healthy controls, and skin psoriasis from psoriatic arthritis.

Psoriasis is an autoimmune/inflammatory disorder primarily affecting the skin. Chronic joint inflammation triggers the diagnosis of psoriatic arthritis (PsA) in approximately one-third of psoriasis patients. Although joint disease typically follows the onset of skin psoriasis, in around 15% of cases it is the initial presentation, which can result in diagnostic delays. The pathophysiological mechanisms underlying psoriasis and PsA are not yet fully understood, but there is evidence pointing towards epigenetic dysregulation involving CD4+ and CD8+ T-cells. The aim of this study was to investigate disease-associated DNA methylation patterns in CD4+ T-cells from psoriasis and PsA patients that may represent potential diagnostic and/or prognostic biomarkers. PBMCs were collected from 12 patients with chronic plaque psoriasis and 8 PsA patients, and 8 healthy controls. CD4+ T-cells were separated through FACS sorting, and DNA methylation profiling was performed (Illumina EPIC850K arrays). Bioinformatic analyses, including gene ontology (GO) and KEGG pathway analysis, were performed using R. To identify genes under the control of interferon (IFN), the Interferome database was consulted, and DNA Methylation Scores were calculated. Numbers and proportions of CD4+ T-cell subsets (naïve, central memory, effector memory, CD45RA re-expressing effector memory cells) did not vary between controls, skin psoriasis and PsA patients. 883 differentially methylated positions (DMPs) affecting 548 genes were identified between controls and "all" psoriasis patients. Principal component and partial least-squares discriminant analysis separated controls from skin psoriasis and PsA patients. GO analysis considering promoter DMPs delivered hypermethylation of genes involved in "regulation of wound healing, spreading of epidermal cells", "negative regulation of cell-substrate junction organization" and "negative regulation of focal adhesion assembly". Comparing controls and "all" psoriasis, a majority of DMPs mapped to IFN-related genes (69.2%). Notably, DNA methylation profiles also distinguished skin psoriasis from PsA patients (2,949 DMPs/1,084 genes) through genes affecting "cAMP-dependent protein kinase inhibitor activity" and "cAMP-dependent protein kinase regulator activity". Treatment with cytokine inhibitors (IL-17/TNF) corrected DNA methylation patterns of IL-17/TNF-associated genes, and methylation scores correlated with skin disease activity scores (PASI). DNA methylation profiles in CD4+ T-cells discriminate between skin psoriasis and PsA. DNA methylation signatures may be applied for quantification of disease activity and patient stratification towards individualized treatment.

Exploration of the Perturbation of PKIG in Lung Squamous Cell Carcinoma and the Role in Tumor Microenvironment Based on Bioinformatics Method

Lung cancer is the leading cause of cancer-related death worldwide, and patients have limited survival benefits from traditional treatments such as surgery, radiotherapy and chemotherapy. As a new treatment for lung cancer, immunotherapy has significantly prolonged the overall survival (OS) of patients. However, only some patients can benefit from it. We need to explore immunotherapy biomarkers more deeply to screen for advantages. The original data were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database, and the immunological and prognostic genes of lung squamous cell carcinoma (LUSC) were screened using R software and TIMER database. The expression of target genes was studied in TCGA and GEO databases, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and correlation analysis with tumor immune characteristics were performed by R software and TISIDB database. We screened out a gene related to immunity and prognosis, cAMP dependent protein kinase inhibitor γ (PKIG), which is significantly differentially expressed in LUSC and normal tissues, and has important reference value for the diagnosis and prognosis assessment of LUSC. PKIG differential genes are mainly concentrated in the regulation of humoral immune response and other processes. The expression of PKIG was positively correlated with the infiltration level of regulatory T cells (Tregs) (r=0.340, P<0.001). In addition, the expression level of PKIG was positively correlated with the expression of chemokines/chemokine receptors such as chemokine C-C motif ligand 2 (CCL2) (r=0.503, P<0.001), CXC chemokine ligand 12 (CXCL12) (r=0.386, P<0.001) and CXC-chemokine receptor 4 (CXCR4) (r=0.492, P<0.001), and immunoinhibitors such as programmed cell death protein 1 (PDCD1) (r=0.359, P<0.001), cytotoxic T-lymphocyte associated antigen 4 (CTLA4) (r=0.375, P<0.001) and T cell immunoglobulin and ITIM domains (TIGIT) (r=0.305, P<0.001) in LUSC. The immunological and prognostic gene PKIG in lung squamous cell carcinoma was screened through bioinformatics analysis. PKIG is highly correlated with LUSC prognosis and immune microenvironment, and is expected to be a potential biomolecular marker for LUSC immunotherapy.

Abstract 5999: Comparative and integrative analysis of transcriptomic and epigenome wide DNA methylation changes in African American prostate cancer

Abstract African American (AA) men have the highest incidence and mortality rate from Prostate cancer (PCa) than any other racial/ethnic group. To understand this racial disparity, molecular features of different types, including gene expression, DNA methylation and other genomic alterations, must be carried out in tumor samples from these 2 populations. To date, PCa genomic studies have largely under-represented tumor samples from AA men due to lack of inclusion of significant numbers in such studies. In this study we carried out genome-wide DNA methylation analysis in 63 clinically annotated fresh-frozen PCa and 50 normal prostate tissues from AA men using the Illumina Infunium 850K Human Methylation EPIC BeadChip array. mRNA expression database from a sub-set of the AA biospecimen were used to assess correlation of transcriptome and epigenomic datasets. Using bioinformatic analysis to integrate a comprehensive set of genomic datasets we identified probesets that were significant (p&amp;lt;0.01) and differentially methylated in AA PCa compared to normal prostate tissues and were significant (p&amp;lt;0.01) and inversely correlated with AA mRNA expression. GO analysis in this AA cohort showed significant differential methylation that were mostly hypermethylated genes with corresponding down-regulated mRNA expression for top genes associated with biological pathways in hemidesmosome assembly, mammary gland development, epidermis development, hormone biosynthetic process and cell communication. On the other hand, few significant differential top hypomethylated genes and corresponding up-regulated mRNA expression were identified in biological pathways of negative regulation of macrophage differentiation, cAMP-dependent protein kinase inhibitor activity, protein destabilization, transcription corepressor activity and fatty acid biosynthetic process. Furthermore, we identified significant and differential methylated genes in our AA PCa cohort that were associated with PCa progression when compared to the TCGA EA and AA dataset. These genes were AMIGO3, IER3, UPB1, GRM7, TFAP2C, TOX2, PLSCR2, ZNF292, ESR2, MIXL1, BOLL and FGF6. Overall, IPA of differential methylated promoter region are genes enriched for network involved in sex-steroid hormonal signals and tumor microenvironment signaling pathways. Our integrative analysis provides new candidate genes associated with prostate cancer progression in AA men as potential targets for improving prostate cancer treatment and addressing the racial disparity problem. Citation Format: Bernard Kwabi-Addo, Somiranjan Ghosh, Chad Creighton, Michael Ittmann. Comparative and integrative analysis of transcriptomic and epigenome wide DNA methylation changes in African American prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5999.

The TGR5 Agonist INT-777 Promotes Peripheral Nerve Regeneration by Activating cAMP-dependent Protein Kinase A in Schwann Cells.

Schwann cell (SC) myelination is a pivotal event in the normal physiological functioning of the peripheral nervous system (PNS), where myelination is finely controlled by a series of factors within SCs to ensure timely onset and correct myelin thickness for saltatory conduction. Among these, cyclic AMP (cAMP) is a promising factor for driving myelin gene expression in SCs. It has been shown that TGR5 activation is often associated with increased production of cAMP. Therefore, we speculated that the G-protein-coupled receptor (TGR5) might be involved in the PNS myelination. To test this hypothesis, sciatic nerve crush-injured mice were treated with INT-777, a specific agonist of TGR5, which significantly improved remyelination and functional recovery. Furthermore, rats that underwent sciatic nerve transection were treated with INT-777, which also promoted nerve regeneration and functional recovery. In primary SCs, the stimulatory effect of INT-777 on myelin gene expression was largely counteracted by H89, a potent inhibitor of cAMP-dependent protein kinase A (PKA). Additionally, INT-777 stimulated cell migration was blunted in the presence of H89. Overall, these data indicate that INT-777 is capable of promoting peripheral nerve regeneration and functional recovery after injury, and these benefits are likely due to the activation of the TGR5/cAMP/PKA axis. As such, INT-777, together with other TGR5 agonists, may hold great therapeutic potential for treating peripheral nerve injury.

A grooved porous hydroxyapatite scaffold induces osteogenic differentiation via regulation of PKA activity by upregulating miR-129-5p expression.

Hydroxyapatite scaffolds with different morphologies have been widely used in bone tissue engineering. Moreover, microRNAs (miRNAs) have been proven to be extensively involved in regulating bone regeneration. We developed grooved porous hydroxyapatite (HAG) scaffolds with good osteogenic efficiency. However, little is known about the role of miRNAs in HAG scaffold-mediated promotion of bone regeneration. The objective of this study was to reveal the mechanism from the perspective of differential miRNA expression. Scanning electron microscopy (SEM) was used to perform the coculture of cells and scaffolds. The miRNA profiles were generated by a microarray assay. A synthetic miR-129-5p mimic and inhibitor were used for overexpression or inhibition. The expression of osteogenic marker mRNAs and proteins was detected by quantitative real-time PCR (qRT-PCR), Western blotting, and immunofluorescence. An ALP activity kit and alizarin red staining (ARS) were used to measure ALP activity and mineral deposition formation. Cell migration ability was examined by wound healing and transwell assays. Protein kinase A (PKA) activity was measured by enzyme-linked immunosorbent assay (ELISA) after miR-129-5p transfection. Target genes were identified by a dual-luciferase reporter assay. H89 preculture evaluated the cross talk between miR-129-5p and PKA activity. Heterotopic implantation models, hematoxylin-eosin (HE), immunohistochemistry staining, and micro-CT were used to evaluate miR-129-5p osteogenesis in vivo. miRNAs were differentially expressed during osteogenic differentiation induced by HAG in vitro and in vivo. miR-129-5p was the only highly expressed miRNA both in vitro and in vivo. miR-129-5p overexpression promoted osteoblast differentiation and cell migration, while its inhibition weakened the effect of HAG. Moreover, miR-129-5p activated PKA to regulate the phosphorylation of β-catenin and cAMP-response element binding protein (CREB) by inhibiting cAMP-dependent protein kinase inhibitor alpha (Pkia). H89 prevented the effects of miR-129-5p on osteogenic differentiation and cell migration. HE, immunohistochemistry staining and micro-CT results showed that miR-129-5p promoted in vivo osteogenesis of the HAG scaffold. The HAG scaffold activates Pka by upregulating miR-129-5p and inhibiting Pkia, resulting in CREB-dependent transcriptional activation and accumulation of β-catenin and promoting osteogenic marker expression.

Arginine Promotes the Expression of Aquaporin-3 and Water Transport in Porcine Trophectoderm Cells Through NO- and cAMP-Dependent Mechanisms.

Dietary supplementation with L-arginine (Arg) has been shown to increase the volume of fetal fluids in gestating swine. Aquaporins (AQPs), known as water channel proteins, are essential for embryonic growth and development. It was not known if Arg mediates water transport through AQPs in porcine conceptus trophectoderm (pTr2) cells. pTr2 cells derived from pregnant gilts on day 12 of gestation were cultured in customized Arg-free Dulbecco's modified Eagle's Ham medium (DMEM) supplemented with either 0.00, 0.25, or 0.50 mM Arg. Arg treatment increased water transport and the expression of AQP3, which was abundantly expressed in pTr2 cells at both the mRNA and protein levels. Arg also increased the expression of iNOS and the synthesis of nitric oxide (NO) in pTr2 cells. The presence of Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME; an inhibitor of NO synthase) significantly attenuated the Arg-induced expression of AQP3. Furthermore, 0.50 mM Arg increased the concentrations of cAMP and the abundances of phosphorylated cAMP-dependent protein kinase A (PKA), phosphorylated PKA α/β/γ, and phosphorylated CREB. These effects of Arg were mimicked by Forskolin (a cell-permeable activator of adenylyl cyclase), but inhibited by H-89 (an inhibitor of cAMP-dependent protein kinase). The results of this study demonstrate that Arg regulates AQP3 expression and promotes water transport in pTr2 cells through NO- and cAMP-dependent signaling pathways.

An Epigenetic Signature is Associated with Serum 25-Hydroxyvitamin D in Colorectal Cancer Tumors.

Vitamin D has been widely associated with colorectal cancer (CRC) through different insights. This study aims to explore the association between serum 25-hydroxyvitamin D (25(OH)D) and the global DNA methylation in tumor from CRC patients. A genome-wide DNA methylation analysis is conducted in 20 CRC patients under categorical (10 patients have 25(OH)D <30ng mL-1 ; 10 patients with 25(OH)D ≥30 ng mL-1 )and continuous models of 25(OH)D. A total of 95 differentially methylated CpGs (DMCpGs) are detected under the categorical model (false discovery rate (FDR) <0.05), while 16 DMCpGs are found under the continuous model. Regional analysis showed eight vitamin D-associated differentially methylated regions (DMR). Between them, a DMR is the most significant at cAMP-Dependent Protein Kinase Inhibitor Alpha (PKIA) locus. Furthermore, seven genes, including PKIA gene, have more or equal than two significant DMCpGs. The protein networking analysis found pathways implicated in cell adhesion and extracellular matrix, as well as signaling transduction. This study identifies novel epigenetic loci associated with serum 25(OH)D status. Interestingly, also, a positive association between vitamin D and DNA methylation in the CRC context is found, suggesting a role in CRC. Further studies are warranted to clarify and replicate these results.

Black Carrot (Daucus carota ssp. sativus var. Atrorubens Alef.) Extract Protects against Ethanol-induced Liver Injury via the Suppression of Phosphodiesterase 4 mRNA Expression

We examined the protective effects of Black Carrot Extract (BCE) on Alcoholic Liver Disease (ALD) using in vivo and in vitro models. In an in vivo ethanol-Carbon Tetrachloride (CCl4)-treated rat model, BCE treatment suppressed serum alanine aminotransferase and aspartate aminotransferase activity. BCE also suppressed ethanol- and CCl4-induced alcoholic liver disease. Furthermore, we observed that the BCE or butanol-extracted fraction of BCE (BCE-BuOH) recovered the cell viability of in vitro ethanol-treated hepatocytes. BCE-BuOH also suppressed the production of reactive oxygen species induced by ethanol to the control level. Moreover, BCE-BuOH regulated the activities of three alcoholic metabolism-related enzymes: cytochrome P450 2E1 activity was suppressed at the posttranslational level, alcohol dehydrogenase activity was increased at the posttranslational level, and aldehyde dehydrogenase 2 activity was increased at the transcriptional level. Novel findings in this study include an increase in intracellular Cyclic Adenosine 3’,5’-Monophosphate (cAMP) levels in hepatocytes with the simultaneous addition of ethanol and BCE-BuOH and the suppression of changes in the activities of three enzymes upon treatment with an inhibitor of cAMP-dependent protein kinase. Our study also found that BCE-BuOH suppressed the expression of phosphodiesterase 4b mRNA, which increased intracellular cAMP levels. These results suggest that BCE is useful for the treatment of ALD.

Black Carrot (Daucus carota ssp. sativus var. Atrorubens Alef.) Extract Protects against Ethanol-induced Liver Injury via the Suppression of Phosphodiesterase 4 mRNA Expression

We examined the protective effects of Black Carrot Extract (BCE) on Alcoholic Liver Disease (ALD) using in vivo and in vitro models. In an in vivo ethanol-Carbon Tetrachloride (CCl4)-treated rat model, BCE treatment suppressed serum alanine aminotransferase and aspartate aminotransferase activity. BCE also suppressed ethanol- and CCl4-induced alcoholic liver disease. Furthermore, we observed that the BCE or butanol-extracted fraction of BCE (BCE-BuOH) recovered the cell viability of in vitro ethanol-treated hepatocytes. BCE-BuOH also suppressed the production of reactive oxygen species induced by ethanol to the control level. Moreover, BCE-BuOH regulated the activities of three alcoholic metabolism-related enzymes: cytochrome P450 2E1 activity was suppressed at the posttranslational level, alcohol dehydrogenase activity was increased at the posttranslational level, and aldehyde dehydrogenase 2 activity was increased at the transcriptional level. Novel findings in this study include an increase in intracellular Cyclic Adenosine 3’,5’-Monophosphate (cAMP) levels in hepatocytes with the simultaneous addition of ethanol and BCE-BuOH and the suppression of changes in the activities of three enzymes upon treatment with an inhibitor of cAMP-dependent protein kinase. Our study also found that BCE-BuOH suppressed the expression of phosphodiesterase 4b mRNA, which increased intracellular cAMP levels. These results suggest that BCE is useful for the treatment of ALD.

Resolvins D3, D4, and D5 activate selective signaling pathways to increase intracellular [Ca 2+ ] in rat conjunctival goblet cells

Purpose Resolvins (Rvs) D3, D4, and D5 are specialized proresolving mediators synthesized from the w3 fatty acid docosahexaenoic acid. The purpose of this study was to determine if RvD3, RvD4, or RvD5 interacts with rat conjunctival goblet cells and determine the signal transduction pathways utilized to increase intracellular [Ca2+] ([Ca2+]i). Methods Conjunctiva from male Sprague-Dawley rats was removed and placed in organ culture with RPMI media. Goblet cells were allowed to grow out of the tissue plug. First passage cells were used for all experiments. To measure [Ca2+]i, cells were trypsinized and transferred to 35-mm glass bottom dishes. The cells were incubated at 37°C for 1 hour with the calcium indicator fura2/AM with 250 mM sulfinpyrazone and 8 mM Pluronic acid F127. [Ca2+]i was measured with a ratio imaging system (InCytIm2; Intracellular Imaging, Cincinnati, OH) using wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. Cells were stimulated with RvD3, RvD4, and RvD5 from 10-12-10-8 M. Inhibitors were added 10-30 minutes prior to stimulation. Results RvD3, RvD4, and RvD5 each increased [Ca2+]i in a concentration dependent manner with peak concentrations of 10-9 M for each resolvin. Only RvD5-stimulated increase in [Ca2+]i was inhibited by BOC2, an inhibitor or the ALX/FPR2 receptor. [Ca2+]i stimulated by all three D-series resolvins was blocked by inhibition of phospholipase (PL) C with U73122___ and D with tert 1- butanol, but not A2 with aristolochic acid. Absence of extracellular Ca2+ also blocked the increase in [Ca2+]i stimulated by RvD3, RvD4, and RvD5. Only RvD3-stimulated increase in [Ca2+]i was blocked by H89, an inhibitor of cAMP dependent-protein kinase A. Conclusion RvD3, RvD4, and RvD5, in addition to RvD1 and RvD2, each play a role in the physiologic regulation of conjunctival goblet cells, but use selective intracellular signaling pathways.

Glucose deprivation affects the expression of genes encoding cAMP-activated protein kinase and related proteins in U87 glioma cells in ERN1 dependent manner.

Objective. The aim of this investigation was to study the expression of genes encoding cAMP-activated protein kinase catalytic and regulatory A subunits (PRKACA and PRKAR1A) and related proteins such as cAMP-dependent protein kinase inhibitors A and G (PKIA and PKIG), catalytic subunit A of protein phosphatase 3 (PPP3CA), A-kinase anchoring protein 12 (AKAP12), and praja ring finger ubiquitin ligase 2 (PJA2) in U87 glioma cells in response to glucose deprivation in both control U87 glioma cells and cells with ERN1 (endoplasmic reticulum to nucleus signaling 1) knockdown, the major pathway of the endoplasmic reticulum stress signaling, for evaluation of possible significance of glucose deprivation in ERN1 dependent regulation of glioma growth.Methods. The expression level of PRKA related genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation by real-time quantitative polymerase chain reaction.Results. It was shown that the expression level of PRKACA and PKIA genes was down-regulated in control glioma cells treated by glucose deprivation, but PJA2 gene was up-regulated. At the same time, the expression of four other genes (PRKAR1A, PKIG, AKAP12, and PPP3CA) was resistant to this experimental condition. Furthermore, ERN1 knockdown of glioma cells significantly modified the effect glucose deprivation on the expression almost all studied genes. Thus, treatment of glioma cells with inhibited ERN1 enzymatic activity by glucose deprivation lead to a more significant down-regulation of the expression level of PKIA and to suppression PRKAR1A gene expressions. Moreover, the ERN1 knockdown introduced up-regulation of PKIG and AKAP12 gene expressions in glioma cells treated by glucose deprivation and eliminated the sensitivity of PJA2 gene to this experimental condition.Conclusions. Results of this investigation demonstrated that ERN1 knockdown significantly modified the sensitivity of most studied PRKA related gene expressions to glucose deprivation and that these changes are a result of complex interactions of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the suppression of glioma cell proliferation and their possibly chemoresistance.

AKT Ser/Thr kinase increases V-ATPase–dependent lysosomal acidification in response to amino acid starvation in mammalian cells

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by the regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.

ENT1 inhibition attenuates apoptosis by activation of cAMP/pCREB/Bcl2 pathway after MCAO in rats

Background and purposeThe present study was designed to investigate the potential role and the mechanism of equilibrative nucleoside transporter 1 (ENT1) on neuronal apoptosis and neurological deficits after middle cerebral artery occlusion (MCAO) in rats. MethodsOne hundred and thirty-four male Sprague-Dawley rats were subjected to two hours of MCAO followed by reperfusion. The time course of the expression level of ENT1 and phosphorylation of CREB were detected by western blot and immunofluorescence staining. Another set of animals were administrated with NBTI, the ENT1 inhibitor, by daily intraperitoneal injection starting at 0.5 h post-MCAO, infarction volume and neurological deficits were measured both at 24 h and 72 h post MCAO. We further explored the neuroprotection machenism by using H89, cAMP dependent protein kinase inhibitor, the expression of Bcl-2, Bax, phosphorylated CREB and Cleaved caspase-3 were quantified by Western blot, neuronal apoptosis were analyed by TUNEL staining. ResultsThe endogenous expression of ENT1 were significantly increased and peaked at 12 h after MCAO. High-dose of NBTI (15 mg/kg) reduced brain infarction volume and improved neurologic deficits both at 24 h and 72 h post MCAO. Moreover, NBTI significantly increased the level of CREB phosphorylation and extracellular adenosine concentration, and decreased the neuronal apoptosis 24 h after MCAO. NBTI treatment reduced the expression of Bax and cleaved caspase-3, while up-regulated Bcl-2 compared with vehicle group. These effects were abolished by H89 pretreatment. ConclusionsENT1 inhibition prevented neuronal apoptosis and improves neurological deficits through cAMP/PKA/CREB/Bcl-2 signaling pathway after MCAO in rats. ENT1 might be an effective target in the treatment strategy for ischemic stroke.

Altered Steroid Milieu in AI-Resistant Breast Cancer Facilitates AR Mediated Gene-Expression Associated with Poor Response to Therapy.

Divergent roles for androgen receptor (AR) in breast cancer have been reported. Following aromatase inhibitor (AI) treatment, the conversion of circulating androgens into estrogens can be diminished by >99%. We wished to establish whether the steroid environment can dictate the role of AR and the implications of this for subsequent therapy. This study utilizes models of AI resistance to explore responsiveness to PI3K/mTOR and anti-AR therapy when cells are exposed to unconverted weak androgens. Transcriptomic alterations driven by androstenedione (4AD) were assessed by RNA-sequencing. AR and estrogen receptor (ER) recruitment to target gene promoters was evaluated using ChIP, and relevance to patient profiles was performed using publicly available data sets. Although BEZ235 showed decreased viability across AI-sensitive and -resistant cell lines, anti-AR treatment elicited a decrease in cell viability only in the AI-resistant model. Serum and glucocorticoid-regulated kinase 3 (SGK3) and cAMP-dependent protein kinase inhibitor β (PKIB) were confirmed to be regulated by 4AD and shown to be mediated by AR; crucially, reexposure to estradiol suppressed expression of these genes. Meta-analysis of transcript levels showed high expression of SGK3 and PKIB to be associated with poor response to endocrine therapy (HR = 2.551, P = 0.003). Furthermore, this study found levels of SGK3 to be sustained in patients who do not respond to AI therapy. This study highlights the importance of the tumor steroid environment. SGK3 and PKIB are associated with poor response to endocrine therapy and could have utility in tailoring therapeutic approaches.

Epidermal growth factor receptor controls glycogen phosphorylase in T cells through small GTPases of the RAS family

We recently uncovered a regulatory pathway of the muscle isoform of glycogen phosphorylase (PYGM) that plays an important role in regulating immune function in T cells. Here, using various enzymatic, pulldown, and immunoprecipitation assays, we describe signaling cross-talk between the small GTPases RAS and RAP1A, member of RAS oncogene family (RAP1) in human Kit 225 lymphoid cells, which, in turn, is regulated by the epidermal growth factor receptor (EGFR). We found that this communication bridge is essential for glycogen phosphorylase (PYG) activation through the canonical pathway because this enzyme is inactive in the absence of adenylyl cyclase type 6 (ADCY6). PYG activation required stimulation of both exchange protein directly activated by cAMP 2 (EPAC2) and RAP1 via RAS and ADCY6 phosphorylation, with the latter being mediated by Raf-1 proto-oncogene, Ser/Thr kinase (RAF1). Consistent with this model, PYG activation was EGFR-dependent and may be initiated by the constitutively active form of RAS. Consequently, PYG activation in Kit 225 T cells could be blocked with specific inhibitors of RAS, EPAC, RAP1, RAF1, ADCY6, and cAMP-dependent protein kinase. Our results establish a new paradigm for the mechanism of PYG activation, which depends on the type of receptor involved.

Efficient photocaging of a tight-binding bisubstrate inhibitor of cAMP-dependent protein kinase

Photocaging of a tight-binding bisubstrate inhibitor of cAMP-dependent protein kinase (PKA) with a nitrodibenzofuran-based group fully abolished its inhibitory potency. The affinity difference between the photocaged and the active inhibitor was over 5 orders of magnitude. The photocaged inhibitor disrupted the PKA holoenzyme in cell lysates upon photolysis under a 398 nm LED.

Possible Insulinotropic Action of Apolipoprotein A-I Through the ABCA1/Cdc42/cAMP/PKA Pathway in MIN6 Cells.

Aims/Introduction: We studied the mechanisms for the possible insulinotropic action of apolipoprotein (Apo) A–I in mouse insulinoma (MIN6) cells.Materials and Methods: The effects of ApoA-I on cAMP production and glucose-stimulated insulin secretion (GSIS), and the dose dependency (ApoA-I at 5, 10, 25, and 50 μg/ml) were determined using MIN6 cells. The effects of the small-interference ribonucleic acid (siRNA) of ATP-binding cassette transporter A1(ABCA1) and Cell division control protein 42 homolog (Cdc42) on the insulinotropic action of ApoA-I was studied, as well as mRNA and protein levels of ABCA1 and Cdc42. Then, the influence of cAMP inhibitor SQ22536, and the cAMP-dependent protein kinase inhibitor Rp-cAMPS on ApoA-I action were studied.Results: Addition of ApoA-I produced cAMP and increased insulin secretion, dose-dependently in high glucose concentration (25 mmmol/l). and ABCA1 protein and Cdc42 mRNA and protein were also enhanced. Specific ABCA1 and Cdc42 siRNA significantly decreased the effects of ApoA-I on insulin secretion compared with negative controls. Manifestations of ABCA1 and Cdc42 mRNA and protein were less than that of the negative control group. Both cAMP inhibiror (SQ22536) and protein kinases inhibitor (Rp-cAMPS) strongly inhibited the effects of ApoA-I on insulin secretion.Conclusions: We demonstrated that ApoA-I enhances glucose-stimulated insulin release in high glucose at least partially through the ABCA1/Cdc42/cAMP/ Protein kinase A (PKA) pathway.

MARCKS phosphorylation and amylase release in GLP-1-stimulated acini isolated from rat pancreas.

Little is known about the effects of glucagon-like peptide 1 (GLP-1) on the pancreatic exocrine gland. In the gland, secretagogues induce amylase release. That signal transduction is evoked mainly by an increase in intracellular Ca2+ levels and activation of protein kinase C (PKC). We previously demonstrated that myristoylated alanine-rich C kinase substrate (MARCKS), a PKC substrate, is involved in pancreatic amylase release. Here, we studied the effects of GLP-1 on MARCKS phosphorylation and amylase release in rat pancreatic acini. GLP-1 induced amylase release and MARCKS phosphorylation in isolated pancreatic acini. Inhibitors of cAMP-dependent protein kinase (PKA) suppressed those effects. Furthermore, a MARCKS-related peptide inhibited the GLP-1-induced amylase release. These findings suggest that GLP-1 induces amylase release through MARCKS phosphorylation via activation of PKA in isolated pancreatic acini.