Liriope platyphylla has been though as an useful medical plant to improve the cough, sputum, neurodegenerative disorders, obesity, and diabetes in Korea and China from old times. In order to investigate the effects of Liriope platyphylla on expression and secretion of nerve growth factor (NGF), the mRNA expression and protein secretion were detected in the neuronal cell (B35) and neuroglial cell (C6) cultured with three differences concentration (5%, 10%, 15%) of Liriope platyphylla. In MTT assay and FACS anslysis, the some death of some B35 and C6 cells were observed in 15% extract-treated group, while other groups did not induce the death. Also, the mRNA expression of NGF were significantly increased in 5% and 10% extracts treated-group. Furthermore, the NGF protein concentration in supernatant collected from cultured cells showed the very similar pattern with mRNA expression. In order to verify the activity of secreted NGF, the culture supernatant collected from B35 and C6 cells cultured with Liriope platyphylla extracts for 24 hrs were treated into undifferentiated PC12 cells, and the differentiation level of PC12 cell were also observed with microscopes. The differentiation level of PC12 cell were significantly increased depend on the dose of extract. Therefore, these results suggested that the water extracts of Liriope platyphylla may contribute the regulation of NGF expression and secretion in the neuronal cell and be considered as an excellent candidate for a neurodegenerative disease-therapeutic drug.
TMP21은 AD의 원인으로 작용하는 A${\beta}$-42 펩타이드 생성에 중요한 ${\gamma}$-secretase 활성을 억제하는 p24 family에 속하는 type I 막 단백질이다. 본 연구에서는 TMP21이 세포의 성장과 분화에 중요한 NGF 수용체 신호전달과정에 미치는 영향을 분석하고자 인간의 TMP21 cDNA를 합성하고, CMV promoter 조절 하에 hTMP21를 클로닝하여, CMV/hTMP21 벡터를 제조하였다. 그리고 이들 벡터를 B35 neuroblastoma에서 과발현시킨 후 ${\gamma}$-secretase 구성단백질과 NGF 수용체 연관 단백질의 변화를 관찰하였다. 그 결과, 4종류의 ${\gamma}$-secretase 구성단백질의 발현은 vehicle transfectants보다 CMV/hTMP21 transfectants에서 유의적으로 감소하였다. 또한 NGF low affinity 수용체인 $p75^{NTR}$과 downstream 단백질인 RhoA의 양은 NGF를 처리하지 않은 TMP21 transfectants에서 유의적으로 증가하였으나 NGF 처리에 의해 감소되었다. High affinity NGF 수용체인 TrkA의 인산화도 NGF 처리가 없는 경우 유의적으로 감소하였으나 NGF 처리에 의해 증가되었다. 또한 downstream 신호전달 과정 중에서 ERK의 인산화는 TrkA와 유사한 발현변화를 나타내었으나 Akt 인산화는 NGF의 처리에 의해 더욱 증가하였다. 이러한 결과는 TMP21이 neuroblastoma에서 NGF 수용체 신호전달과정를 조절하는 중요한 단백질로서 작용함을 제시하며, AD의 작용기전 연구에 중요한 기초자료를 제공할 것으로 사료된다. Transmembrane protein 21 (TMP21) is a member of the p24 cargo protein family and has been shown to modulate ${\alpha}$-secretase-mediated A${\beta}$ production which was specifically observed in the brains of subjects with Alzheimer's disease (AD). In order to investigate whether TMP21 could affect nerve growth factor (NGF) receptor signaling pathway, the alteration of NGF receptors and their downstream proteins were detected in TMP21 over-expressed cells. CMV/hTMP21 vector used in this study was successfully expressed into TMP21 proteins in B35 cells after lipofectamin transfection. Expressed TMP21 proteins induced the down-regulation of ${\gamma}$-secretase complex components including Presenlin-1 (PS-1), PS-2, Nicastrin (NST), Pen-2 and APH-1. Also, the expression level of NGF receptor $p75^{NTR}$ and RhoA were significantly higher in CMV/hTMP21 transfectants than vehicle transfectants, while their levels returned to vehicle levels after NGF treatment. However, the phosphorylation of NGF receptor TrkA was dramtically decreased in NGF No-treated CMV/hTMP21 transfectants compared with vehicle transfectants, and increased in NGF treated CMV/hTMP21 transfectants. In TrkA downstream signaling pathway, the phosphorylation level of ERK was also decreased in CMV/hTMP21 transfectants, while the phosphorylation of Akt was increased in the same transfectants. Furthermore, NGF treatment induced the increase of phosphorylation level of Akt and ERK in CMV/hTMP21 transfectants. Therefore, these results suggested that over-expression of TMP21may simultaneously induce the up-regulation of $p75^{NTR}$/RhoA expression and the down-regulation of TrkA/ERK phosphorylation through the inhibition of ${\gamma}$-secretase activity.
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