• Development and Reproduction
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• v.26 no.1
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• pp.1-12
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• 2022

This study aimed to investigate the signal transduction of phosphorylation sites at the carboxyl (C)-terminal region of equine luteinizing hormone/chorionic gonadotropin receptor (eLH/CGR). The eLH/CGR has a large extracellular domain of glycoprotein hormone receptors within the G protein-coupled receptors. We constructed a mutant (eLH/CGR-t656) of eLH/CGR, in which the C-terminal cytoplasmic tail was truncated at the Phe656 residue, through polymerase chain reaction. The eLH/CGR-t656 removed 14 potential phosphorylation sites in the intracellular C-terminal region. The plasmids were transfected into Chinese hamster ovary (CHO)-K1 and PathHunter Parental cells expressing β-arrestin, and agonist-induced cAMP responsiveness was analyzed. In CHO-K1 cells, those expressing eLH/CGR-t656 were lower than those expressing eLH/CGR wild-type (eLH/CGR-wt). The EC₅₀ of the eLH/CGR-t656 mutant was approximately 72.2% of the expression observed in eLH/CGR-wt. The maximal response in eLH/CGR-t656 also decreased to approximately 43% of that observed in eLH/CGR-wt. However, in PathHunter Parental cells, cAMP activity and maximal response of the eLH/CGR-t656 mutant were approximately 173.5% and 100.8%, respectively, of that of eLH/CGR-wt. These results provide evidence that the signal transduction of C-terminal phosphorylation in eLH/CGR plays a pivotal role in CHO-K1 cells. The cAMP level was recovered in PathHunter Parental cells expressing β-arrestin. We suggest that the signal transduction of the C-terminal region phosphorylation sites is remarkably different depending on the cells expressing β-arrestin in CHO-K1 cells.

• BMB Reports
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• v.55 no.2
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• pp.98-103
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• 2022

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of β-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator.

• Animal Bioscience
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• v.35 no.3
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• pp.399-409
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• 2022

Objective: Follicle-stimulating hormone (FSH) is the central hormone involved in mammalian reproduction, maturation at puberty, and gamete production that mediates its function by control of follicle growth and function. The present study investigated the mutations involved in the regulation of FSH receptor (FSHR) activation. Methods: We analyzed seven naturally-occurring mutations that were previously reported in human FSHR (hFSHR), in the context of equine FSHR (eFSHR); these include one constitutively activation variant, one allelic variant, and five inactivating variants. These mutations were introduced into wild-type eFSHR (eFSHR-wt) sequence to generate mutants that were designated as eFSHR-D566G, -A306T, -A189V, -N191I, -R572C, -A574V, and -R633H. Mutants were transfected into PathHunter EA-parental CHO-K1 cells expressing β-arrestin. The biological function of mutants was analyzed by quantitating cAMP accumulation in cells incubated with increasing concentrations of FSH. Results: Cells expressing eFSHR-D566G exhibited an 8.6-fold increase in basal cAMP response, as compared to that in eFSHR-wt. The allelic variation mutant eFSHR-A306T was not found to affect the basal cAMP response or half maximal effective concentration (EC₅₀) levels. On the other hand, eFSHR-D566G and eFSHR-A306T displayed a 1.5- and 1.4-fold increase in the maximal response, respectively. Signal transduction was found to be completely impaired in case of the inactivating mutants eFSHR-A189V, -R572C, and -A574V. When compared with eFSHR-wt, eFSHR-N191I displayed a 5.4-fold decrease in the EC₅₀ levels (3,910 ng/mL) and a 2.3-fold decrease in the maximal response. In contrast, cells expressing eFSHR-R633H displayed in a similar manner to that of the cells expressing the eFSHR-wt on signal transduction and maximal response. Conclusion: The activating mutant eFSHR-D566G greatly enhanced the signal transduction in response to FSH, in the absence of agonist treatment. We suggest that the state of activation of the eFSHR can modulate its basal cAMP accumulation.

• BMB Reports
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• v.56 no.3
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• pp.196-201
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• 2023

Renal fibrosis is the final manifestation of chronic kidney disease (CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha (HIF-2α) is an important regulator of chronic hypoxia, and the late-stage renal tubular HIF-2α activation exerts protective effects against renal fibrosis. However, its specific role in progressive renal fibrosis remains unclear. Here, we investigated the effects of the long-term tubular activation of HIF-2α on renal function and fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelial cells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg) mice and wild-type (WT) controls through a 6-week adenine diet. Tg mice were maintained on doxycycline (DOX) for the diet period to induce Tg HIF-2α expression. Primary TECs isolated from Tg mice were treated with DOX (5 ㎍/ml), transforming growth factor-β1 (TGF-β1) (10 ng/ml), and a combination of both for 24, 48, and 72 hr. Blood was collected to analyze creatinine (Cr) and blood urea nitrogen (BUN) levels. Pathological changes in the kidney tissues were observed using hematoxylin and eosin, Masson's trichrome, and Sirius Red staining. Meanwhile, the expression of fibronectin, E-cadherin and α-smooth muscle actin (α-SMA) and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was observed using western blotting. Our data showed that serum Cr and BUN levels were significantly lower in Tg mice than in WT mice following the adenine diet. Moreover, the protein levels of fibronectin and E-cadherin and the phosphorylation of p38 MAPK were markedly reduced in the kidneys of adenine-fed Tg mice. These results were accompanied by attenuated fibrosis in Tg mice following adenine administration. Consistent with these findings, HIF-2α overexpression significantly decreased the expression of fibronectin in TECs, whereas an increase in α-SMA protein levels was observed after TGF-β1 stimulation for 72 hr. Taken together, these results indicate that long-term HIF-2α activation in CKD may inhibit the progression of renal fibrosis and improve renal function, suggesting that long-term renal HIF-2α activation may be used as a novel therapeutic strategy for the treatment of CKD.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.2
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• pp.247-253
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• 1997

Development of soybean cultivars with great nodulation and high nitrogen fixation activity, derived mostly from mutagenesis, may decrease inputs of chemical fertilizer nitrogen into the soil-plant system. Soybean seeds (cv. Jangyupkong, Hwanggeumkong, and Geomjungkong 1) were treated with three different levels of EMS (ethyl methanesulfonate) concentration(30, 50, and 70mM). Increasing the doses of EMS resulted in decreased field emergence rate of seeds, whereas it did not increase M$_2$ mutation frequencies. This indicated that the most efficient concentration of EMS was 30mM for generating mutants. Extensive mutagenesis of Sinpaldalkong 2 with 30mM EMS was undertaken to isolate soybean mutants with greater nodulation. Approximately 8, 200 M$_2$ families were screened for greater nodulation on 5 mM nitrate after inoculation with Bradyrhizobium japonicum strain YCK213-KFCC-10728. Mutant SS-2 nodulated more than the wild type. Comparison of supernodulation between SS-2 and two nts mutants(nts 1007 and nts 1116) revealed that SS-2 showed the supernodulation character at an earlier growth stage than the two nts mutants. Further studies should be needed to characterize the difference in timing of nodulation between SS-2 and nts mutants.

• Biomolecules & Therapeutics
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• v.31 no.1
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• pp.73-81
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• 2023

Sirtuins (SIRTs) belong to the nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase family. They are key regulators of cellular and physiological processes, such as cell survival, senescence, differentiation, DNA damage and stress response, cellular metabolism, and aging. SIRTs also influence carcinogenesis, making them potential targets for anticancer therapeutic strategies. In this study, we investigated the anticancer properties and underlying molecular mechanisms of a novel SIRT1 inhibitor, MHY2251, in human colorectal cancer (CRC) cells. MHY2251 reduced the viability of various human CRC cell lines, especially those with wild-type TP53. MHY2251 inhibited SIRT1 activity and SIRT1/2 protein expression, while promoting p53 acetylation, which is a target of SIRT1 in HCT116 cells. MHY2251 treatment triggered apoptosis in HCT116 cells. It increased the percentage of late apoptotic cells and the sub-G1 fraction (as detected by flow cytometric analysis) and induced DNA fragmentation. In addition, MHY2251 upregulated the expression of FasL and Fas, altered the ratio of Bax/Bcl-2, downregulated the levels of pro-caspase-8, -9, and -3 proteins, and induced subsequent poly(ADP-ribose) polymerase cleavage. The induction of apoptosis by MHY2251 was related to the activation of the caspase cascade, which was significantly attenuated by pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. Furthermore, MHY2251 stimulated the phosphorylation of c-Jun N-terminal kinase (JNK), and MHY2251-triggered apoptosis was blocked by pre-treatment with SP600125, a JNK inhibitor. This finding indicated the specific involvement of JNK in MHY2251-induced apoptosis. MHY2251 shows considerable potential as a therapeutic agent for targeting human CRC via the inhibition of SIRT1 and activation of JNK/p53 pathway.

• Journal of Life Science
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• v.33 no.1
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• pp.8-14
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• 2023

Peroxidasin (PXDN), a multidomain heme peroxidase containing extracellular matrix (ECM) motifs, as well as a catalytic domain, catalyzes the sulfilimine crosslink of collagen IV (Col IV) to reinforce Col IV scaffolds. We previously reported that PXDN is required for endothelial cell (EC) survival and growth signaling through sulfilimine crosslink-dependent matrix assembly. In this study, we examined whether peroxidase activity is required for PXDN function in ECs. First, we constructed a mutant PXDN by point mutation of two highly conserved amino acids, Q823 and D826, which are present in the active site of the peroxidase domain. After isolation of HEK293 clones highly expressing the mutant protein, conditioned medium (CM) was obtained after incubating the cells in serum-free medium for 24 hours and then analyzed by Western blot analysis under nonreducing conditions. The results revealed that the mutant PXDN formed a trimer and that it was cleaved by proprotein convertase-like wild-type (WT) PXDN. However, peroxidase activity was not detected in the CM containing the mutant PXDN, in contrast to that of WT PXDN. In addition, the sulfilimine crosslink ability of the mutant PXDN was lost. Moreover, the CM containing the mutant PXDN failed to promote the growth of PXDN-depleted ECs, unlike the CM containing WT PXDN. These results suggest that the peroxidase activity of PXDN affects EC growth by forming a sulfilimine crosslink.

• Journal of Ginseng Research
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• v.47 no.3
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• pp.376-384
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• 2023

Background: Hepatic lipid disorder impaired mitochondrial homeostasis and intracellular redox balance, triggering development of non-alcohol fatty liver disease (NAFLD), while effective therapeutic approach remains inadequate. Ginsenosides Rc has been reported to maintain glucose balance in adipose tissue, while its role in regulating lipid metabolism remain vacant. Thus, we investigated the function and mechanism of ginsenosides Rc in defending high fat diet (HFD)-induced NAFLD. Methods: Mice primary hepatocytes (MPHs) challenged with oleic acid & palmitic acid were used to test the effects of ginsenosides Rc on intracellular lipid metabolism. RNAseq and molecular docking study were performed to explore potential targets of ginsenosides Rc in defending lipid deposition. Wild type and liver specific sirtuin 6 (SIRT6, 50721) deficient mice on HFD for 12 weeks were subjected to different dose of ginsenosides Rc to determine the function and detailed mechanism in vivo. Results: We identified ginsenosides Rc as a novel SIRT6 activator via increasing its expression and deacetylase activity. Ginsenosides Rc defends OA&PA-induced lipid deposition in MPHs and protects mice against HFD-induced metabolic disorder in dosage dependent manner. Ginsenosides Rc (20mg/kg) injection improved glucose intolerance, insulin resistance, oxidative stress and inflammation response in HFD mice. Ginsenosides Rc treatment accelerates peroxisome proliferator activated receptor alpha (PPAR-α, 19013)-mediated fatty acid oxidation in vivo and in vitro. Hepatic specific SIRT6 deletion abolished ginsenoside Rc-derived protective effects against HFD-induced NAFLD. Conclusion: Ginsenosides Rc protects mice against HFD-induced hepatosteatosis by improving PPAR-α-mediated fatty acid oxidation and antioxidant capacity in a SIRT6 dependent manner, and providing a promising strategy for NAFLD.

• Journal of Life Science
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• v.32 no.3
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• pp.241-248
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• 2022

NF-κB acts as a critical transcription factor in inflammation and innate immunity, and it is also closely involved in cell survival and tumorigenesis via induction of anti-apoptotic genes. In these processes, NF-κB cooperates with multiple other signaling molecules and pathways, and although many studies have demonstrated that Hsp90 regulates NF-κB activity, the exact mechanism is unclear. In this study, we investigated the relationship between Hsp90 and IKKγ in the regulation of NF-κB using expression plasmids of IKK complex components. Wild-type and deletion mutants of IKKγ were expressed together with Hsp90, and the combined regulatory effect of Hsp90 and IKKγ on NF-κB activation was assayed. The results show that Hsp90 activates NF-κB by promoting the phosphorylation and degradation of IκBα and that activation of NF-κB by NIK and LPS was increased by Hsp90. IKKγ elevated the effect of Hsp90 on NF-κB activation by increasing phosphorylation and degradation of IκBα. The positive regulation on NF-κB by Hsp90 and IKKγ was also proved in analysis with IKKβ-EE, the constitutively active form of IKKβ. In experiments with the deletion mutants of IKKγ, the N-terminal IKKβ binding domain, C-terminal leucine zipper, and zinc finger domains of IKKγ were found not necessary for the positive regulation of NF-κB activity. Additionally, the expression of pro-inflammatory cytokines was synergistically elevated by Hsp90 and IKKγ. These results indicate that inhibiting the interaction between Hsp90 and IKKγ is a possible strategic method for controlling NF-κB and related diseases.

• Korean Journal of Breeding Science
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• v.51 no.4
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• pp.318-325
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• 2019

Salt stress is a significant factor limiting growth and productivity in crops. However, little is known about the response and resistance mechanism to salt stress in maize. The objective of this research was to develop an enhanced salt-tolerant silage maize by mutagenesis with gamma radiation. To generate gamma radiation-induced salt-tolerant silage maize, we irradiated a KS140 inbred line with 100 Gy gamma rays. Salt tolerance was determined by evaluating plant growth, morphological changes, and gene expression under NaCl stress. We screened 10 salt-tolerant maize inbred lines from 2,248 M₂ mutant populations and selected a line showing better growth under salt stress conditions. The selected 140RS516 mutant exhibited improved seed germination and plant growth when compared with the wild-type under salt stress conditions. Enhanced salt tolerance of the 140RS516 mutant was attributed to higher stomatal conductance and proline content. Using whole-genome re-sequencing analysis, a total of 328 single nucleotide polymorphisms and insertions or deletions were identified in the 140RS516 mutant. We found that the expression of the genes involved in salt stress tolerance, ABP9, CIPK21, and CIPK31, was increased by salt stress in the 140RS516 mutant. Our results suggest that the 140RS516 mutant induced by gamma rays could be a good material for developing cultivars with salt tolerance in maize.