Hypoxia-Inducible Factor-1α Activates the Transforming Growth Factor-β/SMAD3 Pathway in Kidney Tubular Epithelial Cells.
Kidney injury, including chronic kidney disease and acute kidney injury, is a worldwide health problem. Hypoxia and transforming growth factor-β (TGF-β) are well-known factors that promote kidney injury. Hypoxia-inducible factor (HIF) and SMAD3 are their main downstream transcriptional factors. Hypoxia-HIF pathway and TGF-β/SMAD3 pathway play a crucial role in the progression of kidney injury. However, reports on their interactions are limited, and the global transcriptional regulation under their control is almost unknown.



Kidney tubular epithelial cells were cultured and stimulated by hypoxia and TGF-β. We detected global binding sites of HIF-1α and SMAD3 in cells using chromatin immunoprecipitation-sequencing (ChIP-Seq), and measured the gene expression using RNA-sequencing (RNA-Seq). ChIP-quantitative PCR (qPCR) was used to quantitatively evaluate bindings of SMAD3.



ChIP-Seq revealed that 2,065 and 5,003 sites were bound by HIF-1α and SMAD3, respectively, with 614 sites co-occupied by both factors. RNA-Seq showed that hypoxia and TGF-β stimulation causes synergistic upregulation of 249 genes, including collagen type I alpha 1 (COL1A1) and serpin peptidase inhibitor, clade E, member 1, which are well-known to be involved in fibrosis. Ontology of the 249 genes implied that the interaction of HIF-1α and SMAD3 is related to biological processes such as fibrosis. ChIP-qPCR of SMAD3 at HIF-1α binding sites near COL1A1 and SERPINE1 indicated that HIF-1α promotes the bindings of SMAD3, which is induced by TGF-β.



These findings suggest that HIF-1α induced by hypoxia activates the TGF-β/SMAD3 pathway. This mechanism may promote kidney injury, especially by upregulating genes related to fibrosis.
Division of Genome Science, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan.
Natsuki
N
Kushida
Seitaro
S
Nomura
Imari
I
Mimura
Takanori
T
Fujita
Shogo
S
Yamamoto
Masaomi
M
Nangaku
Hiroyuki
H
Aburatani
D015687:Cell Hypoxia; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics; D000090602:Collagen Type I, alpha 1 Chain; D004847:Epithelial Cells / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D017423:Sequence Analysis, RNA; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D059467:Transcriptome; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
C000716127:COL1A1 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C051321:SERPINE1 protein, human; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta