• Animal cells and systems
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• v.9 no.3
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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.

• Animal cells and systems
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• v.13 no.3
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• pp.289-295
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• 2009

Cyclic AMP-mediated signaling pathways regulate a number of cellular functions. In this study, we examined the regulatory role of cAMP signaling pathways in chondrogenesis of chick limb bud mesenchymal cells in vitro. Forskolin, which increases cellular cAMP levels by the activation of adenylate cyclase, enhanced chondrogenic differentiation. Inhibition of PKA with specific inhibitors (H89 or KT5720) blocked pre-cartilage condensation stage, indicating that chondrogenesis is regulated by the increase in cellular cAMP level and subsequent activation of PKA. Downstream signaling pathway of PKA leading to gene expression was investigated by examination of several nuclear transcription factors. Forskolin treatment increased transcription level for a cartilage-specific marker gene Sox9. However, inhibition of PKA with H89 led to restore expression of Sox9, indicating PKA activity was required to regulate the expression of Sox9 in chondrogenesis. In addition, CREB was highly phosphorylated at early stage of mesenchyme culture, and followed by progressive dephosphorylation. CBP and ATF, another CRE related proteins were transiently expressed at the early stage of chondrogenesis with a pattern similar to CREB phosphorylation. Electrophoretic mobility shift assays confirmed that the binding activity of CREB to the CRE is closely correlated to the phosphorylation pattern of CREB. Therefore, cAMP-mediated signal transduction to nuclear events for the induction of genes appeared to be required at the early stage of chick limb bud chondrogenesis.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2021.04a
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• pp.54-54
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• 2021

The Melanoma Research Coalition reported melanoma affects humans of various races. This study was conducted to confirm the inhibitory effect of melanogenesis in B16 F10 cells of Nypa fruticans Wurmb of ethyl acetate fraction (NEF). Nypa fruticans Wurmb is an important component of the East Asian mangrove vegetation. It belongs to Araceae family. Traditionally, N. fruticans was used to treat various diseases such as asthma, sore throat, liver disease, a pain reliever, and can also be used as sedative and carminative. The present study, the inhibitory effect on melanogenesis was determined by Western blotting and RT-qPCR. The level of expression of tyrosinase, TRP-1, and TRP-2 is regulated by microphthalmia-associated transcription factor (MITF) and cAMP, and cAMP affects the activity of protein kinase A (PKA). Activated PKA stimulates the phosphorylation of cAMP-reactive element-binding protein (CREB) in the nucleus, thereby increasing the amount of MITF expression and enhancing melanogenesis. Western blotting and RT-qPCR analysis showed that NEF treatment decreased the expression of tyrosinase. Similarly, TRP-1 and TRP-2 levels were decreased, which were decreased significantly at compared with the untreated control. Also, NEF attenuated the IBMX mediated increase in the intracellular cAMP level and the phosphorylation of PKA. In conclusion, NEF significantly inhibited the expressions of melanogenesis through cAMP/PKA/CREB signaling pathways.

• Journal of Microbiology and Biotechnology
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• v.26 no.5
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• pp.967-974
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• 2016

Zearalenone (ZEA) is an estrogenic mycotoxin that is produced by several Fusarium species, including Fusarium graminearum. One of the ZEA biosynthetic genes, ZEB2, encodes two isoforms of Zeb2 by alternative transcription, forming an activator (Zeb2L-Zeb2L homooligomer) and an inhibitor (Zeb2L-Zeb2S heterodimer) that directly regulate the ZEA biosynthetic genes in F. graminearum. Cyclic AMP-dependent protein kinase A (PKA) signaling regulates secondary metabolic processes in several filamentous fungi. In this study, we investigated the effects of the PKA signaling pathway on ZEA biosynthesis. Through functional analyses of PKA catalytic and regulatory subunits (CPKs and PKR), we found that the PKA pathway negatively regulates ZEA production. Genetic and biochemical evidence further demonstrated that the PKA pathway specifically represses ZEB2L transcription and also takes part in posttranscriptional regulation of ZEB2L during ZEA production. Our findings reveal the intriguing mechanism that the PKA pathway regulates secondary metabolite production by reprograming alternative transcription.

• International Journal of Stem Cells
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• v.16 no.3
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• pp.315-325
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• 2023

Background and Objectives: Glioblastoma (GBM) is an aggressive primary brain tumor characterized by its heterogeneity and high recurrence and lethality rates. Glioblastoma stem cells (GSCs) play a crucial role in therapy resistance and tumor recurrence. Therefore, targeting GSCs is a key objective in developing effective treatments for GBM. The role of Parathyroid hormone-related peptide (PTHrP) in GBM and its impact on GSCs remains unclear. This study aimed to investigate the effect of PTHrP on GSCs and its potential as a therapeutic target for GBM. Methods and Results: Using the Cancer Genome Atlas (TCGA) database, we found higher expression of PTHrP in GBM, which correlated inversely with survival. GSCs were established from three human GBM samples obtained after surgical resection. Exposure to recombinant human PTHrP protein (rPTHrP) at different concentrations significantly enhanced GSCs viability. Knockdown of PTHrP using target-specific siRNA (siPTHrP) inhibited tumorsphere formation and reduced the number of BrdU-positive cells. In an orthotopic xenograft mouse model, suppression of PTHrP expression led to significant inhibition of tumor growth. The addition of rPTHrP in the growth medium counteracted the antiproliferative effect of siPTHrP. Further investigation revealed that PTHrP increased cAMP concentration and activated the PKA signaling pathway. Treatment with forskolin, an adenylyl cyclase activator, nullified the antiproliferative effect of siPTHrP. Conclusions: Our findings demonstrate that PTHrP promotes the proliferation of patient-derived GSCs by activating the cAMP/PKA signaling pathway. These results uncover a novel role for PTHrP and suggest its potential as a therapeutic target for GBM treatment.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.2
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• pp.113-123
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• 2022

Diarylpropionitrile (DPN), a selective agonist for estrogen receptor β (ERβ), has been reported to regulate various hormonal responses through activation of ERβ in tissues including the mammary gland and brain. However, the effect of DPN on melanogenesis independent of ERβ has not been studied. The aim of this study is to examine the possibility of anti-melanogenic effect of DPN and its underlying mechanism. Melanin contents and cellular tyrosinase activity assay indicated that DPN inhibited melanin biosynthesis in alpha-melanocyte stimulating hormone-stimulated B16F10 melanoma cell line. However, DPN had no direct influence on in vitro tyrosinase catalytic activity. On the other hand, 17β-estradiol had no effect on inhibition of melanogenesis, suggesting that the DPN-mediated suppression of melanin production was not related with estrogen signaling pathway. Immunoblotting analysis showed that DPN down-regulated the expression of microphthalmia-associated transcription factor (MITF), a central transcription factor of melanogenesis and its down-stream genes including tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Also, DPN attenuated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). Additionally, DPN suppressed the melanin synthesis in UVB-irradiated HaCaT conditioned media culture system suggesting that DPN has potential as an anti-melanogenic activity in physiological conditions. Collectively, our data show that DPN inhibits melanogenesis via downregulation of PKA/CREB/MITF signaling pathway.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.29-29
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• 2003

Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of $\beta$$_2$-adrenoceptor ($\beta$$_2$-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of $\beta$$_2$-AR-coupled $G_{i}$ proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the $\beta$$_2$-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the $\beta$$_2$-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables $\beta$$_2$-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in $\beta$$_2$-AR-induced cAMP formation. Blocking $G_{i}$ or $G_{$\square$$\square$}$ signaling with pertussis toxin or $\beta$ARK-ct, a peptide inhibitor of $G_{$\square$$\square$}$, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of $\beta$$_2$-AR-PKA siglaling sequentially involves $G_{i}$, $G_{$\square$$\square$}$, and PI3K. Thus, PI3K constitutes a key downstream event of $\beta$$_2$-AR- $G_{i}$ signaling, which confines and negates the concurrent $\beta$$_2$-AR/Gs-mediated PKA signaling.gnaling.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2023.04a
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• pp.56-56
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• 2023

The cone of Pinus rigida × Pinus taeda (PRT), a plant in the Pinaceae family, has long been used in traditional medicine to treat hemostasis, bruises, and burns. Previous research has shown that regulating oxidation-reduction reactions in reactive oxygen species can help inhibit melanogenesis, the process of melanin synthesis, which is a common target for addressing hyperpigmentation. Inhibiting tyrosinase is also known to be effective in this regard. Based on these findings, we conducted an investigation into the inhibitory effect of the ethyl acetate fraction of PRT (ERT) on melanogenesis in B16 F10 cells. We know that the expression levels of melanin biosynthesis-related proteins, including tyrosinase, TRP-1, and TRP-2, are regulated by MITF (microphthalmia-associated transcription factor) and cAMP, with cAMP affecting the activity of protein kinase A (PKA). PKA can reduce melanogenesis, and CREB reduces the phosphorylation of melanin-producing enzymes. In addition, the MAPK signaling pathway, composed of ERK, JNK, p38, and other factors, is also known to play a role in the inhibition of melanogenesis in melanocytes. Our immunoblotting results showed that ERT inhibited the expression of melanin production-related proteins (tyrosinase, TRP-1, TRP-2, and MITF) that were significantly increased by a-MSH treatment to promote melanin production. Furthermore, the phosphorylation levels of factors related to cAMP/PKA/CREB and MAPK signaling pathways were significantly reduced without affecting the total form. In conclusion, we believe that treatment with ERT can inhibit melanin synthesis by modulating the phosphorylation of cAMP/PKA/CREB and MAPK signaling pathways at the cellular level. These findings suggest the potential of ERT as a raw material for functional cosmetics and pharmaceuticals, thanks to its antioxidant activity and ability to inhibit melanogenesis. We thought that these findings of ERT as a natural plant resource will inspire further research and development in this area.

• Journal of Acupuncture Research
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• v.25 no.2
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• pp.119-127
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• 2008

목적 : 골관절염치료에 빈용되는 유근피 약침액의 처치가 생쥐의 파골세포에 있어서 골재흡수의 저해에 미치는 영향을 알아보고자 하였다 방법 : 생쥐에게 유근피 약침액을 두개골 세포에 전, 후 처치하여 골재흡수에 대한 유근피 약침액의 억제 활성능을 검토하였다. 결론 : 염증성 cytokine 중 $IL-1{\beta}$ 유도인자인 PGE와 LPS처리로 $IL-1{\beta}$생성이 증가되었으나, 유근피 약침액 처치군은 이를 억제하였다. 유근피 약침액 전처리군에서도 $IL-1{\beta}$ 생성이 억제되었다. 유근피 약침액 처치군은 PGE2유발 $IL-1{\beta}$ 전사를 억제하였으며, PGE2 유발 $IL-1{\beta}$ 유도는 cAMP antagonist인 Rp-cAMP와 protein kinase A(PKA)저해제에 의해서도 억제되어 $IL-1{\beta}$ 발현에 cAMP, PKA 신호전달경로가 관여함을 시사하였다. 본 연구에서 유근피 약침액은 강한 항 관절염효과와 골재흡수 저해 활성이 있으며, 관절염 치료, 예방에 유의함을 밝힌 것으로 사료된다.

• Molecules and Cells
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• v.41 no.3
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• pp.234-243
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• 2018

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of $GSK3{\beta}$, increased ${\beta}-catenin$ phosphorylation at Ser675 site and upregulated nuclear ${\beta}-catenin$ content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, ${\beta}-catenin$ abrogated the liraglutide-induced activation of cAMP, AKT, ${\beta}-catenin$, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through $cAMP/PKA/{\beta}-catenin$ and $PI3K/Akt/GSK3{\beta}$ signaling pathways.