Mitogenic Effect of Orphan Receptor TR3 and Its Regulation by MEKK1 in Lung Cancer Cells

Abstract

TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.TR3 (also known as NGFI-B and nur77) (6,17,44), an immediate-early response gene, is an orphan member of the steroid/thyroid/retinoid receptor superfamily, whose members mainly act as transcriptional factors to positively or negatively regulate gene expression (22,41,67). The role of TR3 in cell proliferation was suggested by numerous observations showing that its expression is rapidly induced by several mitogenic inducers, including serum growth factor, epidermal growth factor (EGF), and fibroblast growth factor (6,9,13,17,35,44,58). However, whether TR3 expression has a causal role in promoting cell proliferation remains to be illustrated. Recent evidence also indicates that the expression of TR3 is required for apoptosis. TR3 was rapidly induced by T-cell receptor signaling in immature thymocytes and T-cell hybridomas (39, 60). Overexpression of a dominant-negative TR3 protein (60) or inhibition of TR3 expression by antisense TR3 mRNA (39) inhibited T-cell-receptor-induced apoptosis, whereas constitutive expression of TR3 resulted in massive cell death (57, 64).

Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt

“…The mitogenic effect of Nur77 likely occurs in the nucleus as it requires Nur77 DNA-binding and transactivation (Kolluri et al, 2003). A chimeric protein involving Nur77 subfamily member Nor-1 from a chromosomal translocation in chondrosarcoma is a potent transcriptional activator, suggesting that Nur77 transcriptional activation may be oncogenic (Labelle et al, 1995(Labelle et al, , 1999.…”

“…We have previously demonstrated a new paradigm in cancer cell apoptosis, in which Nur77 is induced, then translocates from the nucleus to the mitochondria, leading to release of cytochrome c from the mitochondria (Li et al, 2000). The apoptosis-associated nuclear egress of Nur77 has been observed in cancer cells from the lung, ovary, colon, stomach and breast (Dawson et al, 2001;Holmes et al, 2002Holmes et al, , 2003Liu et al, 2002;Wu et al, 2002;Jeong et al, 2003;Kolluri et al, 2003;Wilson et al, 2003;Cao et al, 2004;Jacobs et al, 2004;Lin et al, 2004;Lee et al, 2005). Nur77 mitochondrial localization is also involved in Sindbis virus-induced apoptosis (Lee et al, 2002).…”

“…The Nur77-dependent apoptotic process is triggered by a number of apoptosis inducers, including retinoidrelated molecule 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecar-boxylic acid (AHPN) (also called CD437) (Li et al, 2000) and its analogs (Li et al, 2000;Dawson et al, 2001;Holmes et al, 2002Holmes et al, , 2003Kolluri et al, 2003;Cao et al, 2004;Lin et al, 2004), etoposide (Li et al, 2000;Liu et al, 2002;Wilson et al, 2003), and insulin-like growth factor binding protein-3 (Lee et al, 2005). The importance of Nur77-dependent apoptotic pathway in mediating the anticancer activity of chemotherapeutic agents is supported by the positive correlation between Nur77 family member Nor-1 expression and survival in diffuse large B-cell lymphoma patients treated with therapeutic drugs (Shipp et al, 2002).…”

“…Nur77 mitochondrial targeting is induced by apoptotic retinoid-related compounds, TPA, a calcium ionophore, and chemotherapeutic agents, which are known to act through membrane-signaling pathways involving various kinases and phosphatases (Li et al, 2000;Kolluri et al, 2003). Thus, phosphorylation may play a role in regulating Nur77 subcellular trafficking.…”

“…Thus, phosphorylation may play a role in regulating Nur77 subcellular trafficking. In fact, Nur77 is heavily phosphorylated in various cell types and phosphorylation regulates Nur77 functions (Fahrner et al, 1990;Hazel et al, 1991;Davis et al, 1993;Hirata et al, 1993;Weih et al, 1996;Katagiri et al, 2000;Masuyama et al, 2001;Pekarsky et al, 2001;Kolluri et al, 2003). Nur77 is phosphorylated at its N-terminal A/B domain by Jun N-terminal kinase (JNK) (Kolluri et al, 2003) and at Ser350 (Ser351 in human Nur77) by Akt (Masuyama et al, 2001;Pekarsky et al, 2001).…”