The International Journal of Biochemistry & Cell Biology 43 (2011) 1198–1207 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology jo ur nal homep ag e: www.elsevier.com/locate/biocel Mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells Ana Belén Fernández-Martínez a,∗ , María Isabel Arenas Jiménez b , Irene Sánchez Hernández a , María Laura García-Bermejo c , Victoria Moreno Manzano d , Elia Aguado Fraile c , Francisco Javier de Lucio-Caza ˜ na a a Departamento de Fisiología, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain b Departamento de Biología Celular, Universidad de Alcalá, Alcalá de Henares, Madrid, Spain c Departamento de Patología, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain d Cellular Reprogramming Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain a r t i c l e i n f o Article history: Received 12 January 2011 Received in revised form 4 April 2011 Accepted 18 April 2011 Available online 28 April 2011 Keywords: Hypoxia inducible factor-1 All-trans retinoic acid Retinoic acid receptor Human proximal tubular cells a b s t r a c t Hypoxia-inducible factor-1 (HIF-1) and all-trans retinoic acid (ATRA) afford protection in several experimental models of kidney disease. HIF-1 protein is degraded under normoxia but stabilized by hypoxia, which activates its transcription factor function. ATRA activates another set of transcription factors, the retinoic acid receptors (RAR) ,  and , which mediate its effects on target genes. ATRA also up-regulates the expression of RAR ,  and  at the transcriptional level. Here we demonstrate the presence of mutual regulation of hypoxic and retinoic acid related signalling in tubular proximal cells. In human proximal tubular HK-2 cells we have found that: (i) ATRA treatment induces HIF-1 under normoxic conditions and also synergizes with hypoxia leading to the over-expression of HIF-1 and vas- cular endothelial growth factor-A, a HIF-1-regulated renal protector. ATRA-induced HIF-1 expression involved stabilization of HIF-1 mRNA but not of HIF-1 protein. (ii) Expression of HIF-1 is an abso- lute requirement for the transcriptional up-regulation of RAR by ATRA. Transfection with HIF-1 siRNA abolished the induction by ATRA of the expression of both RAR mRNA and protein while treatment with HIF-1 inhibitor YC-1 results in the abolishment of ATRA-induced activity of a retinoic acid-response ele- ment (RARE) construct from the RAR promoter. (iii) Hypoxia up-regulates RAR through HIF-1 since this effect was inhibited by HIF-1 knockdown. In contrast to ATRA-induced RAR up-regulation, induc- tion of RAR expression by ATRA did not involve transcriptional up-regulation as hypoxia did not increase the expression of RAR mRNA or the activity of the RARE construct. These results suggest the presence of crosstalk between hypoxia/HIF-1 and ATRA/RAR that may be physiologically and pharmacologically relevant. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Transcriptional adaptive responses to hypoxia are controlled primarily through hypoxia-inducible factor (HIF). HIF is a het- erodimeric transcription factor consisting of a constitutively expressed  subunit and two  subunits, HIF-1 or HIF-2. Both  subunits are mainly regulated by oxygen at the protein level. HIF- is continuously synthesized but under aerobic con- ditions the HIF-1 subunit undergoes proline hydroxylation at Pro 402 and Pro 564 and is then recognized by von Hippel–Lindau (VHL)-dependent ubiquitin ligases and targeted for proteasomal ∗ Corresponding author at: Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain. Tel.: +34 9188 54515; fax: +34 9188 54590. E-mail address: anab.fernandez@uah.es (A.B. Fernández-Martínez). degradation (Bardos and Ashcroft, 2005; Brahimi-Horn et al., 2005). The proline hydroxylases (PHD) are inhibited under hypoxia, allowing accumulation of HIF-1, which translocates to the nucleus. Together with the  subunit and transcriptional coactivators, HIF-1 binds to hypoxia-responsive elements (HRE) in target genes (Lando et al., 2002). Several transcriptional targets are activated by HIF-1 that synergistically promotes survival under hypoxic conditions. These include enzymes involved in glucose uptake and metabolism, carbonic anhydrase IX, erythropoietin and vascular endothelial growth factor (VEGF) (Semenza, 2002). HIF-1 expression has also been shown to be increased in nor- moxic conditions by several cytokines and hormones by increasing HIF-1 transcription and/or translation (Zhou and Brüne, 2006) but not HIF-1 protein half-life. HIF-1 targets several genes encoding nuclear receptors. For instance, HIF-1 reduces the DNA binding activity of PPAR/RXR in cardiomyocytes (Belanger et al., 2007). The activation of Nur77 1357-2725/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2011.04.013