단핵구에서 TNF-a Gene의 전사 활성을 증가시키는 HIF-1과 NF-kB의 기능적 상호작용 = Functional Interaction of HIF-1 and NF-kB Increasing the Transcriptional Activation of TNF-a Gene in Monocytes

국문 초록 (Abstract)

배경: 종양 괴사 인자 알파(TNF-α)는 광범위하고 다양한 면역 조절 기능을 수행하는 다면발현(pleiotropic) 사이토카인이다. 단핵구와 대식세포는 염증 및 면역 조절에 중추적인 역할을 한다.
NF-κB와 HIF-1은 다른 방식으로 TNF-α 유전자발현을 증가하는 것으로 알려져 있다.
방법: NF-κB와 HIF-1의 세포 내 발현을 위해인간 단핵구 세포주인 U937 세포를 표준전기천공방법으로 형질 전환시켰고, 두 단백질의 상호작용을 확인하기 위해 mammalian two-hybrid assay와Co-immunoprecipitation assay로 분석하였다.
또한, NF-κB와 HIF-1이 binding 하는 TNF-α 유전자의 프로모터를 확인하기 위해 chromatin immunoprecipitation 분석을 시행하였다.
결과: 두 전사인자 NF-κB와 HIF-1의 직접 단백질 상호 작용에 의해 프로모터 활성도에 따른TNF-α 유전자 발현이 협동적으로 증가하는 것으로 나타났다. 저산소 상태에서 HIF-1과 NF-κB에의한 TNF-α 프로모터의 활성도가 상당히 증가되었고, tandem NF-κB/HIF-1 binding site가 강력한인핸서로 작용하는 것이 TNF-α 프로모터 내에서확인되었다. NF-κB의 Rel도메인과 HIF-1의 N-TD 도메인은 물리적으로 서로 연결되어야 했다. Hypoxia 치료에서도 생체 내 NF-κB와 HIF-1 단백질상호 작용이 상당히 증가되었고, 두 전사 인자는hypoxia 자극에 의해 염색질 TNF-α 프로모토에결합되었다.
결론: 이러한 결과는 TNF-α gene활성화를 위한 다양한 세포외 신호 전달이 NF-κB/HIF-1 신호경로를 통해 전사 조절이 될 수 있음을 나타낸다.

다국어 초록 (Multilingual Abstract)

Background:Tumor necrosis factor alpha (TNF-a) is a pleiotropic cytokine fulfilling a broad variety of immunoregulatory functions. Monocytes and macrophages play a pivotal role in inflammation and immune regulation. NF-kB and HIF-1 are known to increase expression of the TNF-a gene in a separate way.
Methods:Human monocytic leukemia, U937 cells, were transfected using the standard electroporation method for intracellular expression of NF-kB and HIF-1. We performed analysis using the mammalian two-hybrid assay and co-immunoprecipitation assay for detection of protein interaction of both proteins. In addition, chromatin immunoprecipitation analysis was performed for examination of NF-kB and HIF-1 binding on the TNF-a gene promoter.
Results:Here we show that NF-kB and HIF-1 cooperatively induced an increase in expression of the TNF-a gene dependent on promoter activity by the direct protein interaction of these two transcription factors. Hypoxia signaling induced marked enhancement of the transactivation of TNF-a promoter by HIF-1 and NF-kB. A tandem NF-kB/HIF-1 binding site was identified within the TNF-a promoter, which acted as a strong enhancer element. Physical association of the Rel domain of NF-kB and the N-TD domain of HIF-1 was required. Hypoxia treatment also resulted in a significant increase in the protein interaction of NF-kB and HIF-1 in vivo. Both transcription factors were recruited on the chromatin TNF-a promoter dependent on hypoxia stimuli.
Conclusion:The results of this study indicate that a variety of extracellular signals for activation of TNF-a gene expression might converge on the transcriptional regulation through the NF-kB/HIF-1 signaling pathway.

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