A negative feedback of the HIF-1α pathway via interferon-stimulated gene 15 and ISGylation.
OBJECTIVE
The IFN-stimulated gene 15 (ISG15)- and ubiquitin-conjugation pathways play roles in mediating hypoxic and inflammatory responses. To identify interaction(s) between these two tumor microenvironments, we investigated the effect of ISG15 on the activity of the master hypoxic transcription factor HIF-1α.


METHODS
IFN and desferoxamine treatments were used to induce the expression of ISGs and HIF-1α, respectively. Interactions between HIF-1α and the ISG15 and ISGylation system were studied using knockdown of mRNA expression, immunoblotting, coimmunoprecipitation, and pull-down analyses. Effects of the ISG15 and ISGylation system on the HIF-1α-directed processes were examined using reporter, reverse transcription polymerase chain reaction (RT-PCR), and tumorigenic growth assays.


RESULTS
We found that the level of the free form of HIF-1α is differentially regulated by IFN treatment, and that the free ISG15 level is lower under hypoxia. Mechanism-directed studies have shown that HIF-1α not only interacts physically with ISG15, but is also ISGylated in multiple domains. ISG15 expression disrupts the functional dimerization of HIF-1α and -1β. Subsequently, expression of the ISG15 and/or ISGylation system attenuates HIF-1α-mediated gene expression and tumorigenic growth.


CONCLUSIONS
In summary, our results revealed cross-talk between inflammatory and hypoxic pathways through the ISGylation of HIF-1α. On the basis of these results, we propose a novel negative feedback loop for the HIF-1α-mediated pathway involving the regulation of HIF-1α via IFN-induced ISGylation.
Authors' Affiliations: Department and Graduate Institute of Microbiology, College of Medicine; Center for Biotechnology, National Taiwan University, Taipei; and Department of Internal Medicine, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan.
Yen-Hsiu
YH
Yeh
Yu-Chen
YC
Yang
Mei-Yi
MY
Hsieh
Yen-Cheng
YC
Yeh
Tsai-Kun
TK
Li
D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007372:Interferons / Q000494:pharmacology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020218:Response Elements; D015398:Signal Transduction; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014452:Ubiquitins; C074034:ISG15 protein, human; D007372:Interferons