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PI3K/MAPK 관련 신호전달인자의 발현 특성 분석을 통한 인간배아줄기세포의 자가재생산 이해
이규원 ( Kyu Won Lee ),육진영 ( Jin Yong Yook ),조이숙 ( Yee Sook Cho ) 한국조직공학·재생의학회 2007 조직공학과 재생의학 Vol.4 No.3
Understanding the molecular mechanism that regulates pluripotency and self-renewal in human embryonic stem cells (hESCs) is important for developing treatments for degenerative diseases. However, so far it remains largely unknown. To characterize a unique signature of transcriptional profiles regulating hESC self-renewal, we selected the genes involved in PI3K and MAPK signaling and tested for semi-quantitative RT-PCR analysis of mRNA expression levels. We set out to compare the transcriptional profile of undifferentiated CHA3-hESCs with those of retinoic acid (RA)-differentiated hESCs or embryoid bodies (EBs). 5 μM RA as general differentiation inducer of hESCs was treated on CHA3 hESCs for 4 days. Down-regulation of hESC-specific markers such as AP, Oct4, Nanog, Sox2, SSEA-3, Tra1-60, and Tra-1-81 were confirmed on RA-differentiated hESCs. Down-regulation of Nanog and Oct4 mRNAs was confirmed in 14- and 21-day-old EBs, respectively, whereas Sox-2 had no change up to 28 days. Our results showed that PI3K and MAPK signaling related genes are highly expressed in undifferentiated hESCs. We confirmed their differential expression pattern between undifferentiated and differentiated hESCs. Specifically, the expression of AKT1 mRNA was significantly down-regulated in both RA-differentiated hESCs and EBs. Downstream target of mTOR p70S6K1 and 2 was significantly down-regulated in EBs, comparing with undifferentiated hESCs. While expression of PDK1 did not changed, the expression of its downstream target, p90Rsk1 was significantly decreased upon differentiation. We also found that the expression level of ERK1, 2 and MAPK4 genes is significantly decreased upon differentiation. Interestingly, Down-regulation of ERK activity induced by treatment of ERK specific inhibitor, PD98059, did not affect the undifferentiated state of hESCs in our experimental conditions. Disruption of MAPK pathways using specific inhibitors such as U0126, JNK inhibitor II, SB202190 affected to commitment and early differentiation stages of EBs. These results imply that the differential transcription of PI3K and MAPK signaling related genes may reflect the state of hESC self-renewal and govern differentiation process.
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