고혈당에 노출된 세포에서 고장성 반응에 대한 TonEBP의 표현과 핵 분포의 변화 = The Abundance and Nuclear Distribution of TonEBP in Response to Changes of Tonicity in Hyperglycemic Cells

다국어 초록 (Multilingual Abstract)

Background: TonEBP(Tonicity-responsive enhancer binding protein) regulates the transcription of tonicity responsive genes, such as sodium-myo-inositol and the sodium- chloride-betaine co- transporters(SMIT & BGT1), heat shock protein 70(HSP70) and aldose reductase(AR). To characterize the signals that activate TonEBP in hyperglycemic human retinal pigment epithelial(hRPE) cells, the abundance and nuclear distribution of TonEBP were studied in response to changes of tonicity in culture media. Methods: After the cultures reached confluence, the hRPE cells were exposed for 3 days to 25mM glucose and 100mM NaCl, both with and without 20μM tolrestat. The expressions of AR, SMIT and HSP 70 were determined by northern blot, and the abundances of TonEBP by western blot. The nuclear distributions of TonEBP were observed by fluorescence microscopy, after immunostaining. Results: The AR and SMIT mRNA levels in hyperglycemic and hypertonic media were decreased compared to those in hypertonic media alone. These decreased AR and SMIT mRNA expressions were also observed to be significantly prevented in those cells incubated with tolrestat. Stimulation of TonEBP in hypertonic medium occurs due to a combination of an increased abundance of TonEBP and an increased distribution into the nucleus from the cytoplasm. However, the expressions
and nuclear distributions of TonEBP in hyperglycemic and hypertonic media were not different from those in hypertonic media alone.
Conclusion: The expressions of AR and SMIT genes that may influence the development of diabetic complication were down-regulated by the intracellular accumulation of sorbitol in sustained hyperglycemia. TonEBP does not play a key role in the hypertonicity-induced transcriptional regulation of AR and SMIT in hyperglycemic cells, due to the intracellular accumulation of sorbitol and depletion of myo-inositol

국문 초록 (Abstract)

연구배경: TonEBP는 AR, SMIT, BGT1와 HSP70 등의 유전자들의 전사를 조절하는 것으로 알려져 있으며 세포 전체의 표현 양 증가와 세포 핵으로의 재분포 (nuclear redistribution)에 의해 활성화된다고 한다. 그러나 지속적인 고혈당 환경에서는 폴리올 경로의 활성화로 인해 세포내 소르비톨의 축적과 마이오이노시톨의 감소가 일어나고 이로 인해 AR과 SMIT 유전자 표현 양이 이차적으로 하향 조절된다고 한다. 이에 고혈당 환경에서 AR과 SMIT 표현 양의 변화에 TonEBP의 영향을 알고자 고혈당에 노출된 사람 망막 색소 상피 세포에서 고장성 반응에 의한 AR, SMIT, HSP70의 변화와 TonEBP의 표현 양과 핵 분포의 변화 관계를 비교 관찰하였다.방법: 사람 망막 색소 상피 세포를 25mM 포도당, 100mM NaCl 및 두 성분 혼합 환경에 노출시키면서 tolrestat 20μM를 투여한 경우와 투여하지 않은 경우를 3일 동안 배양하여 AR, SMIT과 HSP70는 northern 블롯으로, TonEBP는 western 블롯으로 비교하였으며 TonEBP의 핵 분포 변화는 면역염색법으로 관찰하였다.결과: 고혈당과 고장성 혼합 환경에서 고장성 단독 환경에 비해 AR과 SMIT 유전자의 표현 정도가 의미 있게 감소되고 tolrestat를 투여한 경우에 이를 억제할 수 있다는 것을 확인하였다. TonEBP는 고혈당 환경과 tolrestat투여와 관계없이 고장성 환경에 노출된 경우에 증가되고 세포 핵 부위에 주로 분포되는 것을 확인하였으며 고혈당과 고장성 혼합 환경에서 AR과 SMIT의 감소에도 불구하고 변화가 나타나지 않는 것을 발견할 수 있었다.
결론: 당뇨병의 만성 합병증 발생에 영향을 미친다고 알려진 AR과 SMIT 유전자는 지속적인 고혈당 상태에서 세포내 소르비톨 축적에 의해 하향 조절된다. 또한 고혈당 환경에서 AR과 SMIT 유전자 표현 양은 이들 유전자의 전사 조절 인자인 TonEBP의 표현 양이나 핵 분포 변화에 영향을 직접적으로 받지 않고 폴리올 경로의 활성화로 인해 세포내 소르비톨과 마이오이노시톨 농도에 따라 이차적으로 조절될 수 있다는 것을 알 수 있었다.
이로서 고혈당 상태에서의 이와 같은 TonEBP의 기능 변화가 당뇨병 합병증 발생의 위험 요소 중 하나가 될 수 있다고 생각된다.

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