Porphyrin homeostasis maintained by ABCG2 regulates self-renewal of embryonic stem cells.
BACKGROUND
Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined.


RESULTS
Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of gammaH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment.


CONCLUSIONS
The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and gammaH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells.
Susanto
Jimmy
J
Genomics Research Center, Academia Sinica, Taipei, Taiwan, Republic of China.
Lin
Yu-Hsing
YH
Chen
Yun-Nan
YN
Shen
Chia-Rui
CR
Yan
Yu-Ting
YT
Tsai
Sheng-Ta
ST
Chen
Chung-Hsuan
CH
Shen
Chia-Ning
CN
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology*; D055785:Gene Knockdown Techniques; D006657:Histones / Q000235:genetics / Q000502:physiology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006706:Homeostasis / Q000235:genetics / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D011166:Porphyrins / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D006657:Histones; D018398:Homeodomain Proteins; D007211:Indoles; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D011166:Porphyrins; D011524:Protoporphyrins; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; C501412:Abcg2 protein, mouse; C475540:Nanog protein, mouse; C028025:protoporphyrin IX; C010522:tryptoquivaline; C475961:gamma-H2AX protein, mouse