Identification and characterization of the hypoxia-responsive element in human stanniocalcin-1 gene
Introduction
Stanniocalcin-1 (STC1) is a hypocalcemic glycoprotein hormone originally discovered in teleost fishes (Gerritsen and Wagner, 2005). The hormone is known to act in an endocrine fashion for the regulation of plasma Ca2+ homeostasis. The mammalian form of STC1 has been identified in the last decade and the hormone may have evolved to act as an autocrine/paracrine factor (De Niu et al., 2000, Ishibashi and Imai, 2002). The expression of the mammalian STC1 is found to be regulated in numerous developmental and pathophysiological processes, including pregnancy, lactation, organogenesis, and cerebral ischemia (Chang et al., 1995, Chang et al., 2003, Deol et al., 2000, Ishibashi and Imai, 2002, Olsen et al., 1996, Stasko and Wagner, 2001a, Stasko and Wagner, 2001b, Stasko et al., 2001, Varghese et al., 1998, Varghese et al., 2002, Wagner and DiMattia, 2006, Zhang et al., 1998, Zhang et al., 2000). Recently there is growing evidence to suggest that the mammalian STC1 is involved in carcinogenesis (Chang et al., 2003). Considerable numbers of studies have demonstrated that hypoxia and/or VEGF can activate STC1 gene expression in several cancerous tissues and cell-line models (Fujiwara et al., 2000, Ismail et al., 2000, Liang and Richardson, 2003, Okabe et al., 2001, Watanabe et al., 2002, Welcsh et al., 2002, Yeung et al., 2005) and angiogenic endothelial cells (Bell et al., 2001, Gerritsen et al., 2002, Kahn et al., 2000, Liu et al., 2003, Wary et al., 2003). The use of STC1 expression as a prognostic marker for human breast, hepatocellular colorectal cancers and melanoma has also been suggested (Findeisen et al., 2008, Fujiwara et al., 2000, McCudden et al., 2004, Paulitschke et al., 2009, Wascher et al., 2003).
Currently STC1 receptor has not yet been cloned, however, using both electron microscopy and receptor binding studies, McCudden et al. (2002) revealed that mitochondrion is the cellular target of the hormone. Since STC1 is identified as one of the target genes in hypoxia and was demonstrated to be a stimulator of mitochondrial respiration, these observations have prompted a question regarding the possible role of STC1 to HIF-1-mediated Warburg effect in solid tumors (Chang et al., 2003, Maxwell et al., 2001). Using murine model, a possible biological function of STC1 was reported (Westberg et al., 2007a, Westberg et al., 2007b). In their studies, STC1 expression was able to provoke tolerance development in mouse brains and hearts against ischemia. The data provide an insight into the possible role of STC1 in cell survival at hypoxic conditions. Recently Block et al. (2008) demonstrated that STC-1 reduced the number of apoptotic lung cancer epithelial cells, following hypoxia. In concurring with their findings, we have reported that STC1 expression can be regulated by p53 and NFκB in apoptotic nasopharyngeal and colon cancer cells (Lai et al., 2007, Law et al., 2008b). In considering the important roles of the transcriptional factors (i.e. p53 and NFκB) and mitochondria in tumor hypoxia (Gogvadze et al., 2008, Royds et al., 1998, Hammond and Giaccia, 2006), the induction of STC1 in hypoxic tumor microenvironments has suggested its possible role in tumor progression.
With the benefit of hindsight, we are interested in the understanding of the transcriptional regulation of STC1 in hypoxic cancer cells. By using small interference RNA approach, we previously demonstrated the involvement of HIF-1 in the regulation of STC1 expression, using nasopharyngeal cancer cell-lines (Yeung et al., 2005). In another previous report by Manalo et al. (2005), it was shown that the overexpression of HIF-1α upregulated STC1 expression in arterial endothelial cells. However, these data have inferred but did not prove the direct transactivation role of HIF-1α in the regulation of STC1 expression. Additionally no information of HIF-1 binding motif on STC1 gene promoter region has been reported. In the present study, we aimed to test whether HIF-1 is involved in the transactivation of human STC1 gene promoter. With the use of a luciferase reporter system and promoter mutagenesis, we demonstrated that the upstream region between −2322/−2335, contains an authentic HIF-1 binding motif that mediates increased STC1 promoter activity. ChIP assays were conducted to illustrate the binding of HIF-1α to STC1 gene promoter. In addition our data demonstrated that p300 was required for a productive interaction with HIF-1 and the upregulation of STC1 mRNA and protein expression.
Section snippets
Effects of HIF-1α overexpression to STC1 gene promoter-driven luciferase activity and STC1 expression in normoxic CNE2 cells
The human nasopharyngeal carcinoma cell-line, CNE-2 were grown in RPMI 1640 supplemented with 10% FBS (HyClone®, Perbio) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) (Invitrogen). The day before transfection, CNE2 cells were plated into 6-well tissue culture dishes at a density reaching 70–80% confluence by the time of transfection. Transfection was performed using LipofectAMINE™ 2000 reagent (Invitrogen). For STC1 promoter luciferase assay, the pGL3-basic vector (Promega) or
An overexpression of HIF-1α in normoxic CNE2 cells stimulated STC1 promoter-driven luciferase activity
Fig. 1 shows the cloned ∼3 kb human STC1 promoter sequence (−2724/+225), displaying the putative HIF-1α, p53 and NKκB binding sites. According to the information of the core HRE sequence –CGTG– (Wenger et al., 2005), one putative HIF binding site (–ggacaTCGTGttct–) was found to locate at the upstream region of −2322/−2335, relative to the transcriptional start site of the human STC1 promoter. Comparatively the other hypoxia-responsive transcriptional factor binding sites (i.e. p53 and NKκB),
Discussion
Our previous study revealed that HIF-1α is involved in the activation of STC1 gene expression in various human cancer cell lines (Yeung et al., 2005). This finding has been supported by studies from other laboratories (Eisenhofer et al., 2004, Lal et al., 2001, Westberg et al., 2007a, Westberg et al., 2007b). Current evidence, however, only inferred but did not prove the direct role of HIF-1 in STC1 gene transactivation. To facilitate a better understanding of HIF-1-induced STC1 expression, it
Acknowledgement
This work was supported by the Faculty Research Grant, Hong Kong Baptist University (C.K.C. Wong).
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