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1.
Life Sci ; 283: 119857, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34339715

RESUMO

AIM: Diabetic cardiomyopathy (DCM) accomodates a spectrum of cardiac abnormalities. This study aims to investigate whether DCM is associated with changes in cyclic adenosine 3'-5' monophosphate (cAMP) signaling, particularly cyclic nucleotide phosphodiesterases (PDEs). MAIN METHODS: Type 1 diabetes (T1D) was induced in rats by streptozotocin (STZ, 65 mg/kg) injection. Myocardial remodeling, structure and function were evaluated by histology and echocardiography, respectively. We delineated the sequential changes affecting cAMP signaling and characterized the expression pattern of the predominant cardiac PDE isoforms (PDE 1-5) and ß-adrenergic (ß-AR) receptors at 4, 8 and 12 weeks following diabetes induction, by real-time quantitative PCR and Western blot. cAMP levels were measured by immunoassays. KEY FINDINGS: T1D-induced DCM was associated with cardiac remodeling, steatosis and fibrosis. Upregulation of ß1-AR receptor transcripts was noted in diabetic hearts at 4 weeks along with an increase in cAMP levels and an upregulation in the ejection fraction and fraction shortening. However, ß2-AR receptors expression remained unchanged regardless of the disease stage. Moreover, we noted an early and specific upregulation of cardiac PDE1A, PDE2A, PDE4B, PDE4D and PDE5A expression at week 4, followed by increases in PDE3A levels in diabetic hearts at week 8. However, DCM was not associated with changes in PDE4A gene expression irrespective of the disease stage. SIGNIFICANCE: We show for the first time differential and time-specific regulations in cardiac PDEs, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Diester Fosfórico Hidrolases/metabolismo , Animais , AMP Cíclico/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Cardiomiopatias Diabéticas/metabolismo , Masculino , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Ratos , Ratos Wistar , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Estreptozocina/farmacologia
2.
Int J Mol Sci ; 22(15)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34361022

RESUMO

Gamma-aminobutyric acid (GABA) is considered the primary inhibitory neurotransmitter in the human cortex. However, whether GABA regulates melanogenesis has not been comprehensively elucidated. In this study, we reveal that GABA (20 mM) significantly inhibited α-melanocyte-stimulating hormone (α-MSH)-induced extracellular (from 354.9% ± 28.4% to 126.5% ± 16.0%) and intracellular melanin contents (from 236.7% ± 11.1% to 102.7% ± 23.1%) in B16F10 melanoma cells, without inducing cytotoxicity. In addition, α-MSH-induced hyperpigmentation in zebrafish larvae was inhibited from 246.3% ± 5.4% to 116.3% ± 3.1% at 40 mM GABA, displaying no apparent cardiotoxicity. We also clarify that the GABA-mediated antimelanogenic properties were related to the direct inhibition of microphthalmia-associated transcription factor (MITF) and tyrosinase expression by inhibiting cyclic adenosine monophosphate (cAMP) and cAMP response element-binding protein (CREB). Furthermore, under α-MSH stimulation, GABA-related antimelanogenic effects were mediated through the GABAA and GABAB receptors, with subsequent inhibition of Ca2+ accumulation. In B16F10 melanoma cells and zebrafish larvae, pretreatment with bicuculline, a GABAA receptor antagonist, and CGP 46381, a GABAB receptor antagonist, reversed the antimelanogenic effect of GABA following α-MSH treatment by upregulating Ca2+ accumulation. In conclusion, our results indicate that GABA inhibits α-MSH-induced melanogenesis. Hence, in addition to the health benefits of GABA in the central nervous system, it could ameliorate hyperpigmentation disorders.


Assuntos
Melaninas/biossíntese , Receptores de GABA-B/metabolismo , alfa-MSH/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Sinalização do Cálcio , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Fator de Transcrição Associado à Microftalmia/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Receptores de GABA-A/metabolismo , Peixe-Zebra
3.
Gene ; 802: 145870, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34363886

RESUMO

Leydig cells (LCs) are testosterone-generating endocrine cells that are located outside the seminiferous tubules in the testis, and testosterone is fundamental for retaining spermatogenesis and male fertility. In buffalo, adult Leydig cells (ALCs) are developed by immature Leydig cells (ILCs) in the postnatal testes. However, the genes/pathways associated to the regulation of testosterone secretion function during the development of postnatal LCs remains comprehensively unidentified. The present study comparatively analyzed the transcriptome profiles of ILC and ALC in buffalo with significant differences in testosterone secretion. Differentially expressed genes (DEGs) analysis identified 972 and 1,091 annotated genes that were significantly up- and down-regulated in buffalo ALC. Functional enrichment analysis showed that cAMP signaling being the most significantly enriched pathway, and testosterone synthesis and lipid transport-related genes/pathways were upregulated in ALC. Furthermore, gene set enrichment analysis (GSEA) shows that cAMP signaling and steroid hormone biosynthesis were activated in ALC, demonstrating that cAMP signaling may serve as a positive regulatory pathway in the maintenance of testosterone function during postnatal development of LCs. Protein-protein interaction (PPI) networks analysis highlighted that ADCY8, ADCY2, POMC, CHRM2, SST, PTGER3, SSTR2, SSTR1, NPY1R, and HTR1D as hub genes in the cAMP signaling pathway. In conclusion, this study identified key genes and pathways associated in the regulation of testosterone secretion function during the ILC-ALC transition in buffalo based on bioinformatics analysis, and these key genes might be deeply involved in cAMP generation to influencing testosterone levels in LCs. The results suggest that ALCs might increase testosterone levels by enhancing cAMP production than ILCs. Our data will enhance the understanding of developmental mechanism studies related to testosterone function and provide preliminary evidence for molecular mechanisms of LCs regulating spermatogenesis.


Assuntos
Búfalos/genética , Células Intersticiais do Testículo/fisiologia , Testículo/citologia , Testosterona/fisiologia , Animais , Búfalos/fisiologia , Separação Celular/veterinária , AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Redes e Vias Metabólicas , RNA-Seq/veterinária , Transdução de Sinais , Espermatogênese/genética , Esteroides/biossíntese , Testosterona/metabolismo , Transcriptoma
4.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360641

RESUMO

The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Guanosina Tetrafosfato/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/química , Proteínas de Ligação a DNA/química , Escherichia coli , Espectrometria de Fluorescência
5.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445119

RESUMO

The sinoatrial (SA) node is the physiological pacemaker of the heart, and resting heart rate in humans is a well-known risk factor for cardiovascular disease and mortality. Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the basal cardiac pacemaker function and provides control over normal spontaneous beating of SANCs through (PDE3 + PDE4)-dependent modulation of local subsarcolemmal Ca2+ releases (LCRs).


Assuntos
Relógios Biológicos/fisiologia , Miócitos Cardíacos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Nó Sinoatrial/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , AMP Cíclico/metabolismo , Humanos
6.
Life Sci ; 282: 119843, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34298037

RESUMO

AIMS: Ischemia/reperfusion (I/R) occurs in renal artery stenosis, partial nephrectomy and most commonly during kidney transplantation. It brings serious consequences such as DGF (Delayed Graft Function) or organ dysfunction leading to renal failure and ultimate death. There is no effective therapy to handle the consequences of Renal Ischemia/Reperfusion (I/R) injury. Cyclic nucleotides, cAMP and cGMP are the important second messengers that stimulate intracellular signal transduction for cell survival in response to growth factors and peptide hormones in normal tissues and in kidneys plays significant role that involves vascular tone regulation, inflammation and proliferation of parenchymal cells. Renal ischemia and subsequent reperfusion injury stimulate signal transduction pathways involved in oxidative stress, inflammation, alteration in renal blood flow leading to necrosis and apoptosis of renal cell. MATERIALS AND METHODS: An extensive literature review of various search engines like PubMed, Medline, Bentham, Scopus, and EMBASE (Elsevier) databases was carried out. To understand the functioning of Phosphodiesterases (PDEs) and its pharmacological modulation in Renal Ischemia-Reperfusion Injury. KEY FINDINGS: Current therapeutic options may not be enough to treat renal I/R injury in group of patients and therefore, the current review has discussed the general characteristics and physiology of PDEs and preclinical-studies defining the relationship between PDEs expression in renal injury due to I/R and its outcome on renal function. SIGNIFICANCE: The role of PDE inhibitors in renal I/R injury and the clinical status of drugs for various renal diseases have been summarized in this review.


Assuntos
Nefropatias , Rim/enzimologia , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/metabolismo , Traumatismo por Reperfusão , Transdução de Sinais/efeitos dos fármacos , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Humanos , Nefropatias/tratamento farmacológico , Nefropatias/enzimologia , Nefropatias/patologia , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/enzimologia
7.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299326

RESUMO

Ziziphus jujuba extracts possess a broad spectrum of biological activities, such as antioxidant and anticancer activities in melanoma cancers. Nevertheless, the compounds contain high antioxidant capacities and anticancer activities in melanoma cells, shown to be effective in hyperpigmentation disorders, but whether flavonoid glycosides from Z. jujuba regulate anti-melanogenesis remains unclear. In this study, we evaluated the anti-melanogenic activity of five flavonoid glycosides from Z. jujuba var. inermis (Bunge) Rehder seeds, including jujuboside A (JUA), jujuboside B (JUB), epiceanothic acid (EPA), betulin (BTL), and 6'''-feruloylspinosin (FRS), in B16F10 melanoma cells and zebrafish larvae. According to our results, JUB, EPA, and FRS potently inhibited α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis and prevented hyperpigmentation in zebrafish larvae. In particular, under α-MSH-stimulated conditions, FRS most significantly inhibited α-MSH-induced intracellular and extracellular melanin content in B16F10 melanoma cells. Additionally, JUB, EPS, and FRS remarkably downregulated melanogenesis in α-MSH-treated zebrafish larvae, with no significant change in heart rate. Neither JUA nor BTA were effective in downregulating melanogenesis in B16F10 melanoma cells and zebrafish larvae. Furthermore, JUB, EPA, and FRS directly inhibited in vitro mushroom tyrosinase enzyme activity. JUB, EPA, and FRS also downregulated cyclic adenosine monophosphate (cAMP) levels and the phosphorylation of cAMP-response element-binding protein (CREB), and subsequent microphthalmia transcription factor (MITF) and tyrosinase expression. In conclusion, this study demonstrated that JUB, EPA, and FRS isolated from Z. jujuba var. inermis (Bunge) Rehder seeds exhibit potent anti-melanogenic properties by inhibition of the cAMP-CERB-MITF axis and consequent tyrosinase activity.


Assuntos
Flavonoides/farmacologia , Glicosídeos/farmacologia , Ziziphus/metabolismo , alfa-MSH/metabolismo , Animais , Antioxidantes/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Flavonoides/isolamento & purificação , Glicosídeos/isolamento & purificação , Larva , Melaninas/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Melanoma Experimental , Fosforilação/efeitos dos fármacos , Extratos Vegetais/farmacologia , Sementes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra , alfa-MSH/antagonistas & inibidores
8.
Mol Med Rep ; 24(4)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34328196

RESUMO

Inflammation and oxidative stress have indispensable roles in the development of acute lung injury (ALI). MicroRNA (miRNA/miR)­351­5p was initially identified as a myogenesis­associated miRNA; however, its role in lipopolysaccharide (LPS)­induced ALI remains unclear. The aim of the present study was to investigate the role and potential mechanisms of miR­351­5p in ALI. ALI was induced through a single intratracheal injection of LPS for 12 h, and miR­351­5p agomir, antagomir or their corresponding negative controls were injected into the tail vein before LPS stimulation. Compound C, 2',5'­dideoxyadenosine and H89 were used to inhibit AMP­activated protein kinase (AMPK), adenylate cyclase and protein kinase A (PKA), respectively. miR­351­5p levels in the lungs were significantly increased in response to LPS injection. miR­351­5p antagomir alleviated, while miR­351­5p agomir aggravated LPS­induced oxidative stress and inflammation in the lungs. The present results also demonstrated that miR­351­5p antagomir attenuated LPS­induced ALI via activating AMPK, and that the cAMP/PKA axis was required for the activation of AMPK by the miR­351­5p antagomir. In conclusion, the present study indicated that miR­351­5p aggravated LPS­induced ALI via inhibiting AMPK, suggesting that targeting miR­351­5p may help to develop efficient therapeutic approaches for treating ALI.


Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Inflamação/genética , Lipopolissacarídeos/toxicidade , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Oxidativo/genética
9.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207309

RESUMO

Secondary hyperparathyroidism and abnormalities in tryptophan (TRP) metabolism are commonly observed in chronic kidney disease (CKD). The present study aimed to establish potential interactions between endogenous parathyroid hormone (PTH) and activation of the bone kynurenine (KYN) pathway in relation to bone turnover and strength in young rats after one month (CKD-1) and three months (CKD-3) of experimental CKD. TRP, KYN, KYN/TRP ratio and bone turnover markers (BTMs) were measured in trabecular and cortical bone tissue. Expression of aryl hydrocarbon receptor (AhR) and the genes involved in osteogenesis was determined in femoral bone. Biomechanical testing of femoral diaphysis and femoral neck was also performed. Activation of the KYN pathway in trabecular bone during CKD development intensified the expression of genes related to osteogenesis, which led to a decrease in cyclic adenosine monophosphate (cAMP) and BTMs levels, resulting in a stiffer and mechanically weaker femoral neck. In contrast, reduction of the KYN pathway in cortical bone allowed to unblock the PTH-dependent anabolic activating transcription factor 4/parathyroid hormone 1 receptor (PTH1R/ATF4) axis, led to cAMP accumulation, better bone turnover and strength in the course of CKD development. In summary, the paracrine KYN pathway in bone can interfere with the anabolic effects of PTH on bone through disrupting PTH-dependent molecular signaling.


Assuntos
Fêmur/metabolismo , Cinurenina/metabolismo , Comunicação Parácrina , Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Uremia/metabolismo , Fator 4 Ativador da Transcrição/metabolismo , Animais , Osso Esponjoso/metabolismo , Osso Cortical/metabolismo , AMP Cíclico/metabolismo , Masculino , Ratos , Ratos Wistar , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo
10.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198681

RESUMO

Lack of adult cells' ability to produce sufficient amounts of elastin and assemble functional elastic fibers is an issue for creating skin substitutes that closely match native skin properties. The effects of female sex hormones, primarily estrogen, have been studied due to the known effects on elastin post-menopause, thus have primarily included older mostly female populations. In this study, we examined the effects of female sex hormones on the synthesis of elastin by female and male human dermal fibroblasts in engineered dermal substitutes. Differences between the sexes were observed with 17ß-estradiol treatment alone stimulating elastin synthesis in female substitutes but not male. TGF-ß levels were significantly higher in male dermal substitutes than female dermal substitutes and the levels did not change with 17ß-estradiol treatment. The male dermal substitutes had a 1.5-fold increase in cAMP concentration in the presence of 17ß-estradiol compared to no hormone controls, while cAMP concentrations remained constant in the female substitutes. When cAMP was added in addition to 17ß-estradiol and progesterone in the culture medium, the sex differences were eliminated, and elastin synthesis was upregulated by 2-fold in both male and female dermal substitutes. These conditions alone did not result in functionally significant amounts of elastin or complete elastic fibers. The findings presented provide insights into differences between male and female cells in response to female sex steroid hormones and the involvement of the cAMP pathway in elastin synthesis. Further explorations into the signaling pathways may identify better targets to promote elastic fiber synthesis in skin substitutes.


Assuntos
Monofosfato de Adenosina/farmacologia , Derme/fisiologia , Elastina/biossíntese , Estradiol/farmacologia , Caracteres Sexuais , Pele Artificial , Engenharia Tecidual , Adulto , Meios de Cultura , AMP Cíclico/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Masculino , Receptores de Superfície Celular/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto Jovem
11.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199197

RESUMO

In the cochlea, non-sensory supporting cells are directly connected to adjacent supporting cells via gap junctions that allow the exchange of small molecules. We have previously shown that the pharmacological regulation of gap junctions alleviates cisplatin (CDDP)-induced ototoxicity in animal models. In this study, we aimed to identify specific small molecules that pass through gap junctions in the process of CDDP-induced auditory cell death and suggest new mechanisms to prevent hearing loss. We found that the cyclic adenosine monophosphate (cAMP) inducer forskolin (FSK) significantly attenuated CDDP-induced auditory cell death in vitro and ex vivo. The activation of cAMP/PKA/CREB signaling was observed in organ of Corti primary cells treated with FSK, especially in supporting cells. Co-treatment with gap junction enhancers such as all-trans retinoic acid (ATRA) and quinoline showed potentiating effects with FSK on cell survival via activation of cAMP/PKA/CREB. In vivo, the combination of FSK and ATRA was more effective for preventing ototoxicity compared to either single treatment. Our study provides the new insight that gap junction-mediated intercellular communication of cAMP may prevent CDDP-induced ototoxicity.


Assuntos
Comunicação Celular , Cisplatino/efeitos adversos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Junções Comunicantes/metabolismo , Ototoxicidade/metabolismo , Transdução de Sinais , Células A549 , Animais , Comunicação Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Colforsina/farmacologia , Colforsina/uso terapêutico , Conexina 26/metabolismo , Junções Comunicantes/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Células HeLa , Perda Auditiva/induzido quimicamente , Perda Auditiva/tratamento farmacológico , Perda Auditiva/prevenção & controle , Humanos , Camundongos , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Gânglio Espiral da Cóclea/efeitos dos fármacos , Gânglio Espiral da Cóclea/patologia , Tretinoína/farmacologia , Tretinoína/uso terapêutico
12.
Int J Mol Sci ; 22(14)2021 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-34299073

RESUMO

Theasinensin A (TSA) is a major group of catechin dimers mainly found in oolong tea and black tea. This compound is also manufactured with epigallocatechin gallate (EGCG) as a substrate and is refined after the enzyme reaction. In previous studies, TSA has been reported to be effective against inflammation. However, the effect of these substances on skin melanin formation remains unknown. In this study, we unraveled the role of TSA in melanogenesis using mouse melanoma B16F10 cells and normal human epidermal melanocytes (NHEMs) through reverse transcription polymerase chain reaction (RT-PCR), Western blotting analysis, luciferase reporter assay, and enzyme-linked immunosorbent assay analysis. TSA inhibited melanin formation and secretion in α-melanocyte stimulating hormone (α-MSH)-induced B16F10 cells and NHEMs. TSA down-regulated the mRNA expression of tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and Tyrp2, which are all related to melanin formation in these cells. TSA was able to suppress the activities of certain proteins in the melanocortin 1 receptor (MC1R) signaling pathway associated with melanin synthesis in B16F10 cells: cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB), protein kinase A (PKA), tyrosinase, and microphthalmia-associated transcription factor (MITF). We also confirmed α-MSH-mediated CREB activities through a luciferase reporter assay, and that the quantities of cAMP were reduced by TSA in the enzyme linked immunosorbent assay (ELISA) results. Based on these findings, TSA should be considered an effective inhibitor of hyperpigmentation.


Assuntos
Benzopiranos/farmacologia , AMP Cíclico/metabolismo , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Fenóis/farmacologia , Animais , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Fosforilação , Transdução de Sinais
13.
FASEB J ; 35(8): e21723, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34224609

RESUMO

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.


Assuntos
Canais de Cálcio/metabolismo , Espermatozoides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Sinalização do Cálcio , AMP Cíclico/metabolismo , Feminino , Fertilização In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Transdução de Sinais , Capacitação Espermática/fisiologia , Motilidade Espermática/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores
14.
Anticancer Res ; 41(8): 3769-3778, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281836

RESUMO

BACKGROUND/AIM: Cholangiocarcinoma is a lethal disease with increasing incidence worldwide. New therapeutic compounds are urgently needed for this disease. Here, the inhibitory effect of adenosine on cholangiocarcinoma cells was studied. MATERIALS AND METHODS: Western blot analysis was used to study autophagy and flow cytometry to analyze cell death and the cell cycle. RESULTS: Cholangiocarcinoma and immortalized cholangiocytes responded to adenosine differently, and adenosine inhibited cholangiocarcinoma cell growth by inducing autophagy. Adenosine failed to activate adenylyl cyclase in cholangiocarcinoma cell lines, but activated this enzyme in immortalized cholangiocytes. Adenosine treatment activated AMPK and led to phosphorylation of its downstream proteins including ULK and Raptor. In addition, autophagy induced by adenosine appeared to be a survival mechanism. The combination of adenosine with autophagy inhibitors greatly increased cell death, as compared to the use of either agent alone. Interestingly, immortalized cholangiocytes were more resistant to adenosine. CONCLUSION: Adenosine may have potential for application in cholangiocarcinoma treatment.


Assuntos
Adenosina/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/fisiologia , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , AMP Cíclico/metabolismo , Humanos , Hidroxicloroquina/farmacologia , Macrolídeos/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Regulatória Associada a mTOR/metabolismo
15.
Mol Cell ; 81(14): 2929-2943.e6, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34166608

RESUMO

The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ativação do Canal Iônico/fisiologia , Íons/metabolismo , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Linhagem Celular , Microscopia Crioeletrônica/métodos , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Marca-Passo Artificial
16.
J Biol Chem ; 297(1): 100881, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34144038

RESUMO

GPR17 is a G-protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Such evidence is primarily drawn from mouse knockout studies and suggests GPR17 as a potential novel therapeutic target for the treatment of metabolic diseases. However, links between human GPR17 genetic variants, downstream cellular signaling, and metabolic diseases have yet to be reported. Here, we analyzed GPR17 coding sequences from control and disease cohorts consisting of individuals with adverse clinical metabolic deficits including severe insulin resistance, hypercholesterolemia, and obesity. We identified 18 nonsynonymous GPR17 variants, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, membrane localization, and downstream signaling profiles of nine GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). These nine GPR17 variants had similar protein expression and subcellular localization as wild-type GPR17; however, they showed diverse downstream signaling profiles. GPR17-G136S lost the capacity for agonist-mediated cAMP, Ca2+, and ß-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT, but showed impaired Ca2+ and ß-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling, but unaffected agonist-stimulated ß-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with diverse signaling profiles, including differential signaling perturbations that resulted in GPCR signaling bias. Our findings provide a framework for structure-function relationship studies of GPR17 signaling and metabolic disease.


Assuntos
Síndrome Metabólica/genética , Mutação de Sentido Incorreto , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Cálcio/metabolismo , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Transporte Proteico , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestinas/metabolismo
17.
J Biol Chem ; 297(1): 100907, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34166681

RESUMO

Endosomal signaling downstream of G-protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. However, our knowledge of the functional consequences of intracellular signaling is incomplete. To begin to address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger generated by active GPCRs, cyclic AMP (cAMP), with unbiased mass-spectrometry-based analysis of the phosphoproteome. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP elevation. We next determined that the same amount of cAMP produced from the endosomal membrane led to more robust changes in phosphorylation than the plasma membrane. Remarkably, this was true for the entire repertoire of 218 identified targets and irrespective of their annotated subcellular localizations (endosome, cell surface, nucleus, cytosol). Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A by cAMP-responsive kinases as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness to cAMP by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.


Assuntos
AMP Cíclico/metabolismo , Endossomos/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Animais , Células HEK293 , Humanos , Fosfoproteínas/química , Fosforilação , Domínios Proteicos , Proteína Fosfatase 2/metabolismo , Proteoma/química
18.
J Biol Chem ; 297(1): 100908, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34171357

RESUMO

The cAMP response element-binding protein (CREB) is an important regulator of cell growth, metabolism, and synaptic plasticity. CREB is activated through phosphorylation of an evolutionarily conserved Ser residue (S133) within its intrinsically disordered kinase-inducible domain (KID). Phosphorylation of S133 in response to cAMP, Ca2+, and other stimuli triggers an association of the KID with the KID-interacting (KIX) domain of the CREB-binding protein (CBP), a histone acetyl transferase (HAT) that promotes transcriptional activation. Here we addressed the mechanisms of CREB attenuation following bursts in CREB phosphorylation. We show that phosphorylation of S133 is reversed by protein phosphatase 2A (PP2A), which is recruited to CREB through its B56 regulatory subunits. We found that a B56-binding site located at the carboxyl-terminal boundary of the KID (BS2) mediates high-affinity B56 binding, while a second binding site (BS1) located near the amino terminus of the KID mediates low affinity binding enhanced by phosphorylation of adjacent casein kinase (CK) phosphosites. Mutations that diminished B56 binding to BS2 elevated both basal and stimulus-induced phosphorylation of S133, increased CBP interaction with CREB, and potentiated the expression of CREB-dependent reporter genes. Cells from mice harboring a homozygous CrebE153D mutation that disrupts BS2 exhibited increased S133 phosphorylation stoichiometry and elevated transcriptional bursts to cAMP. These findings provide insights into substrate targeting by PP2A holoenzymes and establish a new mechanism of CREB attenuation that has implications for understanding CREB signaling in cell growth, metabolism, synaptic plasticity, and other physiologic contexts.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteína Fosfatase 2/química , Animais , Sítios de Ligação , Células Cultivadas , Células HeLa , Humanos , Camundongos , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/metabolismo , Transdução de Sinais , Ativação Transcricional
19.
Commun Biol ; 4(1): 776, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34163006

RESUMO

Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can "re-route" the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling.


Assuntos
Adrenomedulina/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Hormônios Peptídicos/fisiologia , Receptores Acoplados a Proteínas G/agonistas , Proteína Semelhante a Receptor de Calcitonina/fisiologia , Células Cultivadas , AMP Cíclico/metabolismo , Células Endoteliais/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Receptores de Adrenomedulina/agonistas , Receptores de Adrenomedulina/análise , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia
20.
Arch Insect Biochem Physiol ; 107(4): e21825, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34164848

RESUMO

Mating triggers physiological and behavioral changes in female insects. In many species, females experience postmating behavioral and physiological changes that define a post-mated state. These changes are comprised of several conditions, including long-term refractoriness to re-mating and increased production and laying of eggs. Here, we report that mating led to several changes in brown planthopper (BPH) females, including increased octopamine (OA), cAMP concentrations, and activities of several enzymes. Mating also led to changes in the expression of several genes acting in female physiology, including those in the cAMP/PKA signal transduction pathway. OA injections into virgin females led to similar changes. RNAi silencing of the gene encoding tyramine ß-hydroxylase, involved in the final step in OA synthesis, led to decreased expression of these genes, and reduced the cAMP/PKA signaling. At the whole-organism level, the RNAi treatments led to reduced fecundity, body weights, and longevity. RNAi silencing of genes acting in OA signaling led to truncated ovarian development, egg maturation, and ovarian vitellogenin (Vg) uptake. The impact of these decreases is also registered at the population level, seen as decreased population growth. We infer that OA signaling modulates the postmating state in female BPH and possibly other hemipterans.


Assuntos
Hemípteros/fisiologia , Oxigenases de Função Mista/metabolismo , Octopamina/metabolismo , Comportamento Sexual Animal/fisiologia , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Longevidade , Ovário/crescimento & desenvolvimento , Oviposição
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