• Laboraroty Animal Research
• /
• 제34권4호
• /
• pp.264-269
• /
• 2018

Cell cycle dysfunction can cause severe diseases, including neurodegenerative disease and cancer. Mutations in cyclin-dependent kinase inhibitors controlling the G1 phase of the cell cycle are prevalent in various cancers. Mice lacking the tumor suppressors $p16^{Ink4a}$ (Cdkn2a, cyclin-dependent kinase inhibitor 2a), $p19^{Arf}$ (an alternative reading frame product of Cdkn2a,), and $p27^{Kip1}$ (Cdkn1b, cyclin-dependent kinase inhibitor 1b) result in malignant progression of epithelial cancers, sarcomas, and melanomas, respectively. Here, we generated knockout mouse models for each of these three cyclin-dependent kinase inhibitors using engineered nucleases. The $p16^{Ink4a}$ and $p19^{Arf}$ knockout mice were generated via transcription activator-like effector nucleases (TALENs), and $p27^{Kip1}$ knockout mice via clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9). These gene editing technologies were targeted to the first exon of each gene, to induce frameshifts producing premature termination codons. Unlike preexisting embryonic stem cell-based knockout mice, our mouse models are free from selectable markers or other external gene insertions, permitting more precise study of cell cycle-related diseases without confounding influences of foreign DNA.

• Bulletin of the Korean Chemical Society
• /
• 제32권spc8호
• /
• pp.2893-2894
• /
• 2011

• Molecules and Cells
• /
• 제21권2호
• /
• pp.237-243
• /
• 2006

Brain-derived neurotrophic factor (BDNF) is a neuromodulator of nociceptive responses in the dorsal root ganglia (DRG) and spinal cord. BDNF synthesis increases in response to nerve growth factor (NGF) in trkA-expressing small and medium-sized DRG neurons after inflammation. Previously we demonstrated differential activation of multiple BDNF promoters in the DRG following peripheral nerve injury and inflammation. Using reporter constructs containing individual promoter regions, we investigated the effect of NGF on the multiple BDNF promoters, and the signaling pathway by which NGF activates these promoters in PC12 cells. Although all the promoters were activated 2.4-7.1-fold by NGF treatment, promoter IV gave the greatest induction. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294003, protein kinase A (PKA) inhibitor, H89, and protein kinase C (PKC) inhibitor, chelerythrine, had no effect on activation of promoter IV by NGF. However, activation was completely abolished by the MAPK kinase (MEK) inhibitors, U0126 and PD98059. In addition, these inhibitors blocked NGF-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2. Taken together, these results suggest that the ERK1/2 pathway activates BDNF promoter IV in response to NGF independently of NGF-activated signaling pathways involving PKA and PKC.

• 대한의생명과학회지
• /
• 제15권2호
• /
• pp.141-146
• /
• 2009

Paclitaxel, an antimicrotubule agent, binds to beta-tubulin in the microtubule and stabilizes the polymer, thereby repressing dynamic instability. Here, we have demonstrated that microtubule cytoskeletal architecture involved in regulation of the COX-2 expression in chondrocyte treated with paclitaxel. Paclitaxel enhanced COX-2 expression and prostaglandin E2 production, as indicated by the Western blot analysis, reverse transcriptase PCR(RT-PCR) and immunofluorescence staining, and $PGE_2$ assay, respectively. In our previous data have shown that paclitaxel treatment stimulated activation of ERK-1/2 and p38 kinase(Im et al., 2009). SB203580, an inhibitor of p38 kinase, blocked the induction of COX-2 expression by paclitaxel. Also PD98059, an inhibitor of ERK-1/2 kinase was blocked the induced COX-2 expression. These results indicate that activation of ERK-1/2 and p38 kinase is required for COX-2 expression induced by paclitaxel in rabbit articular chondrocytes.

• Archives of Pharmacal Research
• /
• 제21권3호
• /
• pp.315-319
• /
• 1998

Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM $LACI_3$ and significantly inhibited by $10\mu$M verapamil and $1\mu$M nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular $Ca^{2+}$ concentration, and the influx of extracellular $Ca^{2+}$ was mediated through voltage-dependent $Ca^{2+}$ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin ($1\mu$M) and sodium nitroprusside ($1\mu$M) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosino kinase are involved in the vanadate-induced contraction which required the influx of extracellular $Ca^{2+}$ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.

• Archives of Pharmacal Research
• /
• 제29권12호
• /
• pp.1114-1118
• /
• 2006

(+)-Eudesmin [4,8-bis(3,4-dimethoxyphenyl)-3,7 -dioxabicyclo[3.3.0]octane] was isolated from the stem bark of Magnolia kobus DC. var. borealis Sarg. and found to have neuritogenic activity. $50\;{\mu}M$ (+)-eudesmin induced neurite outgrowth and enhanced nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. At this concentration, (+)-eudesmin also enhanced NGF-induced neurite-bearing activity and this activity was partially blocked by various protein kinase inhibitors. These included PD98059, a mitogen-activated protein kinase (MAPK) kinase inhibitor. GF109203X, a protein kinase C (PKC) inhibitor and H89, a protein kinase A (PKA) inhibitor. These results suggest that (+)-eudesmin can induce neurite outgrowth from PC12 cells by stimulating up-stream MAPK, PKC and PKA pathways.

• The Korean Journal of Physiology and Pharmacology
• /
• 제5권2호
• /
• pp.139-146
• /
• 2001

L-type $Ca^{2+}$ channels play an important role in regulating cytosolic $Ca^{2+}$ and thereby regulating hormone secretions in neuroendocrine cells. Since hormone secretions are also regulated by various kinds of protein kinases, we investigated the role of some kinase activators and inhibitors in the regulation of the L-type $Ca^{2+}$ channel currents in rat pituitary $GH_3$ cells using the patch-clamp technique. Phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator, and vanadate, a protein tyrosine phosphatase (PTP) inhibitor, increased the $Ba^{2+}$ current through the L-type $Ca^{2+}$ channels. In contrast, bisindolylmaleimide I (BIM I), a PKC inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, suppressed the $Ba^{2+}$ currents. Forskolin, an adenylate cyclase activator, and isobutyl methylxanthine (IBMX), a non-specific phosphodiesterase inhibitor, reduced $Ba^{2+}$ currents. The above results show that the L-type $Ca^{2+}$ channels are activated by PKC and PTK, and inhibited by elevation of cyclic nucleotides such as cAMP. From these results, it is suggested that the regulation of hormone secretion by various kinase activity in $GH_3$ cells may be attributable, at least in part, to their effect on L-type $Ca^{2+}$ channels.

• Biomolecules & Therapeutics
• /
• 제29권1호
• /
• pp.41-51
• /
• 2021

Src family kinases (SFKs), an important group of non-receptor tyrosine kinases, are suggested to be excessively activated during various types of tissue fibrosis. The present study investigated the effect of KF-1607, an orally active and a newly synthesized Src kinase inhibitor (SKI) with proposed low toxicity, in preventing the progression of renal interstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed in 6-week-old male C57BL/6 mice to induce renal interstitial fibrosis. Either KF-1607 (30 mg/kg, oral gavage) or PP2 (2 mg/kg, intraperitoneal injection), a common experimental SKI, was administered to mice for seven days, started one day prior to surgery. UUO injury-induced SFK expression, including Src, Fyn, and Lyn kinase. SFK inhibition by KF-1607 prevented the progression of tubular injury in UUO mice, as indicated by decreases in albuminuria, urinary KIM-1 excretion, and kidney NGAL protein expression. Renal tubulointerstitial fibrosis was attenuated in response to KF-1607, as shown by decreases in α-SMA, collagen I and IV protein expression, along with reduced Masson's trichrome and collagen-I staining in kidneys. KF-1607 also inhibited inflammation in the UUO kidney, as exhibited by reductions in F4/80 positive-staining and protein expression of p-NFκB and ICAM. Importantly, the observed effects of KF-1607 were similar to those of PP2. A new pan Src kinase inhibitor, KF-1607, is a potential pharmaceutical agent to prevent the progression of renal interstitial fibrosis.

• Archives of Pharmacal Research
• /
• 제26권5호
• /
• pp.411-415
• /
• 2003

To investigate the mechanisms involved in hemin-induced febrile response, the rectal temperature of rats were measured after intracerebroventricular (i.c.v.) injections of hemin, with or without antagonists. Hemin ($10\mu\textrm{g}$) elicited a significant febrile response, which lasted from 30 min, to more than 6 h, after its administration, but this was not the case with biliverdin (i.c.v.) and bilirubin (i.c.v.). The hemin-induced febrile response was significantly inhibited by pretreatment with an inhibitor of tyrosine kinase (genistein), but not by pretreatment with an inhibitor of protein kinase C (chelerythrine) and a scavenger of iron (deferoxamine). These results suggest that tyrosine kinase is involved in the hemin-induced febrile response.

• Animal cells and systems
• /
• 제14권3호
• /
• pp.147-154
• /
• 2010

It is well-established that phosphoinositide 3-kinase (PI3-kinase) regulates myogenesis by inducing transcription of myogenin, a key muscle regulatory factor, at the initiation of myoblast differentiation. In this study, we investigated the role of PI3-kinase in cells that have committed to differentiation. PI3-kinase activity increases during myogenesis, and this increase is sustained during the myogenic process; however, its function after the induction of differentiation has not been investigated. We show that LY294002, a PI3-kinase inhibitor, blocked myoblast fusion even after myogenin expression initially increased. In contrast to the inhibitory effects of LY294002 on myogenin mRNA levels during the initiation of differentiation, LY294002 blocked the accumulation of myogenin protein without affecting its mRNA level after differentiation was induced. Treatment with cycloheximide, a translation inhibitor, or actinomycin D, a transcription inhibitor, indicated that the stability of myogenin protein is lower than that of its mRNA. LY294002 inhibited the activities of several important translation factors, including eukaryotic elongation factor-2(eEF2), by altering their phosphorylation status. In addition, LY294002 blocked the incorporation of [$^{35}S$]methionine into newly synthesized proteins. Since myogenin has a relatively short half-life, LY294002-mediated inhibition of post-transcriptional processes resulted in a rapid depletion of myogenin protein. In summary, these results suggest that PI3-kinase plays an important role in regulating the expression of myogenin through post-transcriptional mechanisms after differentiation has been induced.