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Hypoxia- inducible factors (HIFs) are transcription factors consisting of an oxygen-sensitive α-subunit binding to a stable β-subunit. HIFs regulate multiple signaling pathways that could contribute to fibrogenesis, supporting their potential role in hypoxia-mediated renal fibrosis. We previously reported that HIF-1 is upregulated and required for transforming growth factor (TGF)-β induction of collagen in renal tubular cells. Here, we performed in vitro and in vivo studies of potential glomerular crosstalk between TGF-β and normoxic HIF signaling. HIF-α has two major isoforms, HIF-1α and HIF-2α with different target gene sets. In cultured human mesangial cells, TGF-β1 treatment increased both HIF-1α and HIF-2α expression in normoxia. TGF-β1 did not increase HIF-1α/2α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances HIF-1α/2α expression through translation. TGF-β receptor (ALK5) kinase activity was required for increased, TGF-β-stimulated HIF-α expression in response to TGF-β, and inhibiting PI3-kinase markedly decreased HIF-α expression. Blocking HIF-1α/2α expression using siRNA decreased basal and TGF-β1-stimulated type I collagen expression, while overexpressing nondegradable HIF-α increased the collagen response, with HIF-2α being significantly more effective than HIF-1α. In adriamycin-induced mouse glomerulosclerosis, HIF-2α target genes were upregulated in sclerosing glomeruli. Taken together, our data demonstrate potential signaling interaction between TGF-β and HIFs to promote renal fibrogenesis in normoxia and suggest that the HIF-2α isoform is more important during glomerulosclerosis. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β is a major mediator of kidney fibrosis. In the pastdecade it was recognized that, besides canonical Smad signaling, many other signaling pathways participate in the process of TGF-β-induced fibrogenesis. One such pathway involves mammalian targetof rapamycin complex (mTORC)1. We recently reported that thehypoxia-inducible factor (HIF)-1 is essential for TGF-β-induced collagenexpression regardless of ambient oxygen tension. A modulatorof HIF expression other than oxygen tension is mTORC1. We thereforesought to evaluate a possible role for mTORC1 activity inTGF-β-induced fibrogenesis, mTORC1 activity was increased inhuman mesangial cells treated with TGF-β in a TGF-β receptordependentmanner. Short hairpin (sh)RNA to Smad3 decreased, whileoverexpression of Smad3 increased, the mTORC1 activity, suggestingthat TGF-β stimulation of mTORC1 also requires Smad3. Pretreatmentwith rapamycin or shRNA for a regulatory molecule of mTORC1, Raptor, reduced TGF-β-induced COL1A2-luc activity andcollagen I protein expression, mTORC1 inhibition also prevented theTGF-β-stimulated increase in both hypoxia-responsive element(HRE) activity and HIF-1α protein expression, while activation of mTORC1 by active Rheb increased basal but not TGF-β-inducedHRE activity, shRNA to Smad3 reduced HRE activity, while overexpressionof Smad3 increased HIF-1α protein expression and activityin an mTORC1-dependent manner. Lastly, overexpression ofHIF-1α bypassed the inhibitory effect of mTORC1 blockade oncollagen expression. These results suggest that Smad3/mTORC1interaction to promote HIF-1 expression is a key step in normoxickidney fibrogenesis. [ABSTRACT FROM AUTHOR]