• Mycobiology
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• 제38권1호
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• pp.26-32
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• 2010

The cyclic AMP (cAMP) pathway plays a major role in growth, sexual differentiation, and virulence factor synthesis of pathogenic fungi. In Cryptococcus neoformans, perturbation of the cAMP pathway, such as a deletion in the gene encoding adenylyl cyclase (CAC1), causes defects in the production of virulence factors, including capsule and melanin production, as well as mating. Previously, we performed a comparative transcriptome analysis of the Ras- and cAMP- pathway mutants, which revealed 163 potential cAMP-regulated genes (38 genes at a 2-fold cutoff). The present study characterized the role of one of the cAMP pathway-dependent genes (serotype A identification number CNAG_ 06576.2). The expression patterns were confirmed by Northern blot analysis and the gene was designated cAMP-regulated gene 1 (CAR1). Interestingly, deletion of CAR1 did not affect biosynthesis of any virulence factors and the mating process, unlike the cAMP-signaling deficient cac1$\Delta$ mutant. Furthermore, the car1$\Delta$ mutant exhibited wild-type levels of the stress-response phenotype against diverse environmental cues, indicating that Car1, albeit regulated by the cAMP-pathway, is not essential to confer a cAMP-dependent phenotype in C. neoformans.

• Animal cells and systems
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• 제9권3호
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• pp.173-179
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• 2005

Cyclic-AMP-dependent protein kinase (PKA) seems to function as a negative regulator of the c-Jun $NH_2-terminal$ kinase (JNK) signaling pathway. We demonstrate here that the activity of the PKA catalytic subunit (PKAc) is reduced in apoptotic PC12 pheochromocytoma cells. Apoptotic progress was inhibited by dibutyryl cyclic AMP (dbcAMP), an analog of cAMP. The rescue by dbcAMP was attributable to inhibition of the JNK but not of the p38 signaling pathway, due to the induction of PKA activity. JNK was present in immunocomplexes of PKAc, and PKAc phosphorylated JNK in vitro. Presence of p38 kinase, however, was not prominent in immunocomplexes of PKAc. Our data suggest that JNK is a target point of negative regulation by PKAc in the JNK signaling pathway.

• 약학회지
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• 제47권1호
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• pp.31-36
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• 2003

To investigate the effect of protein kinase on melanin production via cAMP-dependent pathway, we measured the melanin amount and tyrosinase activity in B16 melanoma cells stimulated by alpha-melanocyte stimulating hormone (MSH), forskolin and 8-Br-cAMP. MSH, forskolin and 8-Br-cAMP significantly increased both melanin production and tyrosinase activity in B16 cells. Melanin production and tyrosinase activity by MSH are significantly inhibited by cyclic AMP-dependent protein kinase inhibitor (KT5720) and protein kinase C down-regulation treated with PMA. Bisindolmaleimide (1$\mu$M), protein kinase C inhibitor, significantly inhibited melanin production and tyrosinase activity stimulated by MSH, forskolin and 8-Br-cAMP with the following order of potency: MSH＞forskolin＞8-Br-cAMP. Tyrosine kinase inhibitor, genistein and DHC, significantly inhibited both, but the inhibitory effect was more potent in 8-Br-cAMP-stimulated B16 cells than MSH-stimulated cells. NFkB inhibitor (parthenolide) significantly inhibited melanin production and tyrosinase activity. Neither melanin production nor tyrosinase activity induced by MSH, forskolin and 8-Br-cAMP were affected by KN-62 (calmodulin-dependent protein kinase II inhibitor), PD098059 (mitogen-activated protein kinase inhibitor, MAPKK) and worthmannin (phosphatidylinositol 3-kinase inhibitor). These results suggest that both protein kinase C and tyrosine kinase are involved in melanin production by cyclic AMP-dependent pathway and NFkB pathway may play an important role in cyclic AMP-dependent melanin production in B16 melanoma cells.

• Animal cells and systems
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• 제2권1호
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• pp.99-106
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• 1998

In this study, we have examined the role of cAMP in gap junctional communication (GJC) in preimplantation mouse embryos. GJC was monitored by Lucifer Yellow (LY) injected into one blastomere of compacted embryos. The speed of GJC was defined as the time taken for the last blastomere of the embryo to become visibly fluorescent. The median time for 8-cell embrvos (140 sec) was similar to that for 16-cell (135 sec). To determine whether cAMP and cAMP-dependent protein kinase (PKA) are involved in the regulation of GJC, the effects of PKA inhibitor (H8) and cAMP analogues (Rp-cAMP and 8-Br-cAMP) on dye transfer between blastomeres of compacted embryos were examined. Some of the embryos treated with either H8 or Rp-cAMP failed to transfer LY to all blastomeres within 10 min. In contrast, 8-Br-cAMP speeded up fluorescent dye transfer. The median time to fill all blastomeres with LY was 140 sec in untreated controls and 90 sec in siblings treated with 8-Br-cAMP. Inhibition of PKA by H8 or Rp-cAMP induced delay or arrest in embryo development after compaction, but the increase of intracellular cAMP showed no effect. These findings suggest that GJC in preimplantation mouse embryos is regulated by cAMP-PKA pathway and transient interference by PKA inhibitors induces the developmental delay beyond compaction.

• Journal of Microbiology
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• 제34권2호
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• pp.158-165
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• 1996

The signal transduction pathways through the RAS gene product and adenyl cyclease play a critical role in regulation of the cell cycle in yeast, Saccharomyces cerevisiae. We examined the genetic relationship between the spt3 gene and ras/cAMP pathway. A mutation in the SPT3 gene suppressed cell cycle arrest at the G1 phase caused by either an inactivation of the RAS or CYR1 gene which encodes a yeast homologue of human ras proto-oncogene or adenyl cyclase, respectively. The phenotypes such as sporulation and heat shock resistancy, that resulted from a partial inactivation of the RAS or CYR1 genes, were also suppressed by the spt3 mutation. Expression of the SSA1 gene encoding one of th heat shock proteins (Hsp70) can be induced by heat shock or nitrogen starvation. Expression of this gene is derepressed in cry1-2 and spt3 mutants. The bcy 1 mutation repressed by the bcy1 mutation, but not in spt3 mutants. These results suggest that the SPT gene is involved in expression of genes that are affected by the RAS/cAMP pathway.

• Oncology Letters
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• 제17권3호
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• pp.3589-3598
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• 2019

Colorectal cancer (CRC) is a complex disease involving numerous genetic abnormalities. One of the major characteristics of CRC is enhanced Wnt signaling caused by loss-of-function mutations in the adenomatous polyposis coli (APC) gene. Previously, it has been demonstrated that the majority of malignant phenotypes following APC deletion in adult murine small intestines could be rescued when Myc, a downstream target of the Wnt pathway, was deleted. This indicated that Myc is a critical regulator of CRC development following APC loss. Previous studies reported that cyclic adenosine 3',5'-monophosphate (cAMP) can influence the AKT/mammalian target of rapamycin (mTOR) survival pathway in cancer and Myc is a critical downstream molecule of AKT/mTOR signaling. Phosphodiesterase 4D (PDE4D), a member of the cAMP-specific PDE4 family, has been associated with drug resistance in CRC. However, the association between PDE4D and Myc remains unclear. To investigate the potential role of PDE4D in Myc regulation in CRC, the present study evaluated the expression levels of PDE4 subtypes in DLD-1 CRC cells. Additionally, the effects of PDE4 inhibitors on Myc expression and oncogenic properties were analyzed by western blot analysis, reverse transcription-quantitative polymerase chain reaction, colony formation and soft agar assays. It was demonstrated that cAMP/PDE4D signals serve a critical role in regulating Myc expression in DLD-1 CRC cells. Furthermore, PDE4D was identified to be a main hydrolyzer of cAMP and suppression of PDE4D using selective inhibitors of PDE4 increased intracellular cAMP levels, which resulted in a marked decrease in the oncogenic properties of DLD-1 cells, including colony formation, cell proliferation and anchorage-independent growth. Notably, the current data imply that cAMP represses Myc expression via the downregulation of AKT/mTOR signaling, which was abolished by high PDE4D activities in DLD-1 cells. Additionally, a natural polyphenol resveratrol in combination with forskolin elevated the concentration of cAMP and enhanced the expression of Myc and the malignant phenotype of DLD-1 cells, reproducing the effect of known chemical inhibitors of PDE4. In conclusion, the present study identified that cAMP/PDE4D signaling is a critical regulator of Myc expression in DLD-1 and possibly other CRC cells.

• Journal of Chest Surgery
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• 제25권4호
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• pp.364-382
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• 1992

In order to investigate the effect of intracellular cyclic GMP on the calcium channel, whole cell patch clamp technique with internal perfusion method was used in the single ventricular myocytes of the rabbit. Cyclic GMP, cGMP analogues, cAMP, isopernaline and forskolin were perfused into cells and their effects on the calcium current were analysed by applying depolarizing step pulse of 10 mV in amplitude for 200 msec from holding potential of -40 mV. Calcium currents usually activated from -30 mV and then reached a peak at +10 mV. Amplitude of the calcium current was standardized with membrane capacitance, 50 pF. Peak amplitude at +10 mV in control was -0.15 nA/50pF. When 100 mM cAMP was applied from the pipette, peak amplitude of calcium current increased to -0.32 nA and addition of 1 mM isoprenaline further increased its amplitude. In the presence of cGMP it alone also produced an increase of the calcium current to -0.52 nA/50pF and addition of isoprenaline or forskolin increased its magnitude to -[0.55~0.95] nA/50pF. Simultaneous application of cGMP and cAMP increased the calcium current to -0.67 nA/50pF. Among the cGMP analogues, 8-Br-cGMP was the most potent stimulant for the calcium current activation. From the above results it could be concluded tlat cGMP increases the calcium current not through cAMP dependent protein kinase nor cAMP dependent phosphodiesterase pathway, but through independent phosphorylation pathway, possibly cGMP dependent protein kinase pathway.

• 대한화장품학회지
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• 제37권3호
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• pp.275-282
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• 2011

MC1R의 길항제 역할을 하는 펩타이드와 tyrosinase를 저해하는 효능을 가지는 천연유래 물질을 유도체화하여 신규 미백소재를 개발하였다. 또한 이러한 새로운 기작에 의한 미백 효능을 평가할 수 있는 방법을 cAMP 신호전달 과정을 활용하여 제시하였다. 총 24종의 유도체를 합성하였고, cAMP assay의 결과를 통해 11종의 유도체를 선별하였으며, 선별된 유도체에 대하여 멜라닌 형성 억제 효능을 평가하여 본 평가 방법의 유효성을 확인하였다. 이 과정에서 RW를 서열에 포함하는 펩타이드 유도체가 특별히 멜라닌 형성 억제 효능이 높은 것을 알 수 있었다. 또한, cAMP assay는 MC1R의 antagonist라는 특정 타겟 물질을 디자인한 소재의 스크리닝 방법에는 활용할 수 있음을 알 수 있었다. 하지만, 미백소재로서의 가능성을 확인하기 위해서는 멜라닌 생성저해 시험과 병행하여야 할 것으로 판단된다.

• The Korean Journal of Physiology and Pharmacology
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• 제16권5호
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• pp.333-337
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• 2012

Gene expression of neuronal nitric oxide synthase (nNOS) changes in the hypothalamic paraventricular nucleus (PVN) depending on feeding conditions, which is decreased during food deprivation and restored by refeeding, and phosphorylated cAMP response element binding protein (pCREB) was suggested to play a role in its regulation. This study was conducted to examine if the fasting-induced down-regulation of the PVN-nNOS expression is restored by activation of cAMP-dependent protein kinase A (cAMP/PKA) pathway. Freely moving rats received intracerebroventricular (icv) injection of cAMP/PKA activator Sp-cAMP (40 nmol) or vehicle (sterilized saline) following 48 h of food deprivation. One hour after drug injections, rats were transcardially perfused with 4% paraformaldehyde, and the PVN tissues were processed for nNOS or pCREB immunohistochemistry. Sp-cAMP significantly increased not only nNOS but also pCREB immunoreactivities in the PVN of food deprived rats. Fastinginduced down-regulation of the PVN-nNOS was restored by 1 h after the icv Sp-cAMP. Results suggest that cAMP/PKA pathway may mediate the regulation of the PVN-nNOS expression depending on different feeding conditions.

• 대한한의학방제학회지
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• 제28권1호
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• pp.81-90
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• 2020

Objectives : This study was conducted to investigate the effects of major constituents of Epimedium koreanum Nakai (Icariin, epimedium A, epimedium B, and epimedium C) on melanin synthesis. Methods : We measured melanin contents, tyrosinase activity, and expression of Rab27a in B16F10 cells cultured with Epimedium koreanum Nakai ethanol extract (EKN) and their major constituents. After treatment with H89 and dibutyryl cAMP, which inhibit or promote the activation of PKA, we observed changes in melanin synthesis and tyrosinase activity stimulated by EKN. Results : Among them, EKN and icariin enhanced tyrosinase activity and melanin contents. We confirmed that EKN augmented melanin synthesis via cAMP/PKA pathway. Icariin-induced tyrosinase activity and melanin content were attenuated by PKA inhibitor H89, while melanogenic effect of icariin was further augmented by cAMP analog, dbc AMP. However, icariin did not affect the expression of small GTPase Rab27a involved in melanosome transport. Conclusions : These results suggest that icariin promotes melanogenesis through cAMP/PKA pathway but does not affect small GTPase Rab27a.