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Conflict of interest
Introduction Heme is an essential molecule for cellular metabolism involving oxygen and electron transfer . However, free heme is a potent pro-oxidant that catalyzes the generation of reactive oxygen species (ROS) leading to cellular damage . Heme oxygenase-1 (HO-1) is an inducible rate-limiting enzyme in the cellular catabolism of heme to biliverdin, carbon monoxide, and ferrous iron. Biliverdin is rapidly converted to bilirubin by biliverdin reductase . Since the transcription of HO-1 gene is induced by substrate heme, HO-1 is thought to be a cytoprotective enzyme against oxidative stress . Although the beneficial effects of HO-1 induction have been reported in a number of Aloperine australia and tissues, a line of evidence indicate that the increased HO-1 expression may lead to the progression of some diseases such as carcinogenesis [4,5]. Heme-dependent induction of HO-1 has been extensively studied [2,6]. The expression of HO-1 is regulated by E1 and E2 enhancers, each of which contains the Maf recognition element (MARE). Under non-stimulated conditions, BACH1 (BTB and CNC homolog 1)/MafK heterodimer binds to MAREs, and thereby maintains a quiescent state of the HO-1 gene . Heme promotes displacement of BACH1/MafK from MARE  and dissociation of NRF2 from kelch-like ECH-associated protein (KEAP1) in the cytosol, leading to nuclear localization of NRF2. Nuclear NRF2 with small Maf binds to MARE . HSF1 is a master regulator of heat shock response in vertebrates and activates heat shock genes by binding to heat shock element (HSE) [9,10]. In the 5′ flanking region of HO-1 gene, multiple HSEs are present in E1 and the proximal promoter region . In rat cells, the proximal promoter region of the HO-1 gene contains the functional HSE, and HO-1 expression was increased by heat shock treatment . Thus, rat HO-1 is defined as heat shock protein (HSP) 32. Similarly, the proximal regions of the human and mouse HO-1 genes contain HSEs, but human and mouse HO-1s are apparently not induced by heat shock , except for human Hep3B hepatoma cells . These discrepancies in heat-induced HO-1 expression among different species and cell lines have not been clarified yet. Although HSF1 and NRF2 are regulated by distinct mechanisms, it is becoming apparent that they engage in crosstalk by sharing overlapping transcriptional targets, including HSP70 and HO-1 . In order to investigate the participation of HSF1 and NRF2 in the regulation of HO-1 expression, we examined the heat shock- and heme-induced HO-1 expression by using Hsf1-knockout and Nrf2-knockout mouse embryonic fibroblast cells.
Materials and methods
Discussion The present study demonstrated that heme-induced HO-1 mRNA and protein expressions were about 2-fold higher in Hsf1-knockout cells than that of the wild-type cells (Fig. 1). The heme-induced HO-1 expression in the wild-type and Hsf1-knockout cells depended on the concentration of heme and the incubation time. Under the conditions used in this experiment, more than 2-fold higher expression of heme-induced HO-1 expression was observed in HSF1-knockout cells compared to that in the wild-type cells. These results indicated negative regulation of heme-induced HO-1 expression by HSF1. A possible role for HSF1 in the negative regulation of HO-1 gene expression has been reported for human Hep3B hepatoma cells stimulated with 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and arsenate . However, the molecular mechanism underlying the 15d-PGJ2-induced HO-1 expression has not been elucidated. Another finding we showed here was that heat-induced HO-1 mRNA and protein expressions were observed only in Nrf2-knockout cells and not in the wild-type cells (Fig. 2). Experiments of temperature dependency and time course of HO-1 expression after heat shock demonstrated that the highest expression of HO-1 was observed in Nrf2-knockout cells treated at 41 °C for 1 h followed by recovery for 3 h. In addition, the heat-induced HO-1 expression in Nrf2-knockout cells was significantly reduced when HSF1 expression was knocked down by siRNA (Fig. 3A). These results indicated that NRF2 repressed the heat-induced and HSF1-dependent HO-1 expression.