• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.2
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• pp.238-246
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• 2001

Bone morphogenetic proteins(BMPs), members of the transforming growth factor $\beta$(TGF-$\beta$) superfamily were first identified as the factors that induce ectopic bone formation in vivo, when implanted into muscular tissue. Especially BMP-2 inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. In the molecular mechanism of the signal transduction of TGF-$\beta$ and related factors, intracellular signaling proteins were identified as Smad. In previous study, it has been reported that Smad 1 and Smad 5, which belong to the R-Smad family mediate BMP signaling, were involved in the induction of osteoblast differentiation in C2C12 cells. To understnad the role of Smads involved in osteogenic transdifferentiation in C2C12 cell, in present study, after we stably transfected C2C12 cells with each. Smad(Smad 1,Smad 5) expression vector, cultured for 3 days and stained for alkaline phophatase activity. ALP activity positive cells appeared in the Smad 1, Smad 5 stably transfected cell even in the abscence of BMP. After transiently co-transfected C2C12 cells with each Smad expression vector and ALP promoter, it was examined that Smad 1 and Smad 5 expression vector had increased about 2 fold ALP promoter activity in the abscence of BMP. These result suggested that both Smad 1 and Smad 5 were involved in the intracellular BMP signals which induce osteoblast differentiation in C2C12 cells. The effect of BMP on C2C12 cells with Smad 1, Smad 5 transfected were studied by using northern blot analysis. the treatment of BMP upregulated ALP mRNA level in three groups, especially upregulation of ALP was larger in Smad 1, Smad 5 transfected cell than control group. Pretreatment with cycloheximide($10{\mu}g/ml$), a protein synthesis inhibitor resulted in blocking the ALP gene expression even in BMP(100ng/ml) treated cell. These results suggested that Smad increased the level of ALP mRNA via the synthesis of a certain transcriptional regulatory protein.

• Journal of Life Science
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• v.32 no.11
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• pp.833-840
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• 2022

Chronic inflammation has been shown to be closely associated with tumor development and progression. Nuclear factor kappa B (NF-κB) is composed of a family of five transcription factors. NF-κB signaling plays a crucial role in the inflammatory response and is often found to be dysregulated in various types of cancer, making it an attractive target in cancer therapeutics. In this study, CDC-like kinase 3 (CLK3) was identified as a novel kinase that regulates the NF-κB signaling pathway. Our data demonstrate that CLK3 inhibits the canonical and non-canonical NF-κB pathways. Luciferase assays following the transient or stable expression of CLK3 indicated that this kinase inhibited NF-κB activation mediated by Tumor necrosis factor-alpha (TNFα) and Phorbol 12-myristate 13-acetate (PMA), which are known to activate NF-κB signaling via the canonical pathway. Consistent with data on the ectopic expression of CLK3, CLK3 knockdown using shRNA constructs increased NF-κB activity 1.5-fold upon stimulation with TNFα in HEK293 cells compared with the control cells. Additionally, overexpression of CLK3 suppressed the activation of this signaling pathway induced by NF-κB-inducing kinase (NIK) or CD40, which are well-established activators of the non-canonical pathway. To further examine the negative impact of CLK3 on NF-κB signaling, we performed Western blotting following the TNFα treatment to directly identify the molecular components of the NF-κB pathway that are affected by this kinase. Our results revealed that CLK3 mitigated the phosphorylation/activation of transforming growth factor-α-activated kinase 1 (TAK1), inhibitor of NF-κB kinase alpha/beta (IKKα/α), NF-κB p65 (RelA), NF-κB inhibitor alpha (IκBα), and Extracellular signal-regulated kinase 1/2-Mitogen-activated protein kinase (ERK1/2-MAPK), suggesting that CLK3 inhibits both the NF-κB and MAPK signaling activated by TNFα exposure. Further studies are required to elucidate the mechanism by which CLK3 inhibits the canonical and non-canonical NF-κB pathways. Collectively, these findings reveal CLK3 as a novel negative regulator of NF-κB signaling.