The Orphan Nuclear Receptor SHP Inhibits Apoptosis During the Monocytic Differentiation by Inducing P21waf1

EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 41, No. 6, 429-439, June 2009 The orphan nuclear receptor SHP inhibits apoptosis during the monocytic differentiation by inducing p21WAF1 KyeongJin Kim1, Yoon Ha Choi2, pendent kinase inhibitor p21; monocytes; nuclear re- Hyeong Hoe Kim3,4 and JaeHun Cheong1,4 ceptor subfamily 0, group B, member 2 1Department of Molecular Biology Pusan National University Introduction Busan 609-735, Korea 2Department of Life Science The molecular mechanisms regulating cell differen- Division of Molecular and Life Science tiation involve a fine and complex balance of proteins and signal transduction pathways that Pohang University of Science and Technology modulate the progression through control of cell Pohang 790-784, Korea cycle and cell survival (Scott et al., 1994; Cory, 3Department of Experimental Medicine 1995). Human macrophages are differentiated into College of Medicine, Pusan National University noncycling cells arrested at the G1 checkpoint that Busan 601-721, Korea 4 derive from peripheral blood cycling human mono- Corresponding authors: Tel, 82-51-510-2277; cytes. Although little is known regarding the mole- Fax, 82-51-513-9258; E-mail, [email protected] (J.H. Cheong), cular mechanisms governing the monocyte differen- Tel, 82-51-240-7414; E-mail, [email protected] (H.H. Kim) tiation to macrophage, promonocytic human cell DOI 10.3858/emm.2009.41.6.048 lines, can be triggered to differentiate to human macrophages by defined stimuli (Oberg et al., Accepted 22 January 2009 1993; Henkel et al., 1999). Agents such as phorbol esters (PMA) induce their exit from the cell cycle at Abbreviation: SHP, small heterodimer partner G1, leading to their differentiation, which is characterized by the appearance of macrophage-like features such as cell surface integrins, adherence to plastic, and the production of reactive oxygen Abstract intermediates (Freytag, 1988; Baeuerle and Henkel, 1994; Casini and Pelicci, 1999). U937, human Small heterodimer partner (SHP) is an atypical member leukemic lymphoma cell line with monoblastic of nuclear receptor superfamily that lacks a DNA-bind- characteristics, can be induced to differentiate ing domain. In previous study, we showed that SHP, towards morphologically mature macrophage-like c-jun, p65 of NF-κB subunits, and p21WAF1 ex- cells after treatment with PMA (Larsson et al., 1988; pression was increased during monocytic differ- Lubbert et al., 1991). entiaton with the exposure of human leukemia cells to Treatment of myeloid leukemia cells with PMA is a differentiation agent, PMA. In this study, c-Jun and associated with changes in the expression of p65 were shown to mediate the transcriptional activa- certain early- and late-response genes and level of tion of the SHP promoter. In addition, SHP induced the modification of several transcription factors by cell cycle regulatory protein levels and cooperatively signal pathway. PMA down-regulates c-myc trans- cripts in HL-60 cells and induces expression of the increased an induction of p21WAF1 expression with c-jun gene (Sherman et al., 1990; Szabo et al., p65. Furthermore, SHP protected differentiated cells 1991). Activation of Jun/AP-1 contributes to induc- from etoposide-induced cellular apoptosis through tion of c-jun transcription and monocytic differen- the induction and cytoplasmic sequestration of tiation (Angel et al., 1988; Szabo et al., 1991). The p21WAF1. Complex formation between SHP and differentiated macrophages, in contrast to cycling p21WAF1 was demonstrated by means of coimmuno- monocyte, contain significant levels of NF-κB in the precipitation. These results suggest that SHP pro- nuclei. The differentiation to macrophages triggered longs a cellular survival of differentiating monocytes by plastic adherence or phorbol esters leads to a through the transcriptional regulation of target genes progressive degree of NF-κB nuclear translocation, of cell survival and differentiation. a hallmark of differentiated macrophages. NF-κB plays a key role in cell differentiation by conferring Keywords: apoptosis; cell differentiation; cyclin-de- cell survival that in the case of macrophages is 430 Exp. Mol. Med. Vol. 41(6), 429-439, 2009 mediated through p21WAF1/Cip1 (Pennington et al., for the early stages of the differentiation program 2001). (Jiang et al., 1994; Steinman et al., 1994; Halevy et Small heterodimer partner (SHP) is an atypical al., 1995; Andres and Walsh, 1996). orphan nuclear receptor that lacks a conventional In this study, SHP-overexpressed cell represents DNA binding domain and consists only of putative resistance to apoptosis; moreover, p21 protein is ligand binding domain (Seol et al., 1996). SHP is significantly accumulated in the cytoplamic com- expressed in a wide variety of tissues, with highest partment as a basal level and induced rapidly by expression in heart, brain, liver, lung, and adrenal the apoptosis signal in the SHP-overexpressed cell gland (Johansson et al., 2000). It has been reported line. Additionally, it was confirmed that SHP was that SHP represses the transcriptional activity of a induced from an early phase of monocytic diffe- number of nuclear receptors such as constitutive rentiation prior to a dramatic increase of p21. androstane receptor (CAR), esterogen receptor Together, this study shows that SHP increased by (ER), thyroid hormone receptor (TR), Retinoid X signal of differentiation or apoptosis plays a role to receptor (RXR), hepatocyte nuclear factor 4α (HNF protect monocytic cells from cell death through 4α), androgen receptor (AR), liver receptor homolog rapid and significant accummulation of p21 in the 1 (LRH-1), ER-related receptor-γ (ERRγ), glucocor- cytoplasmic compartment. ticoid receptor (GR), and liver X receptor (LXR) (Seol et al., 1996; Lee et al., 2000; Johansson et al., 2000; Gobinet et al., 2001; Borgius et al., 2002; Results Brendel et al., 2002; Lee and Moore, 2002; Sanyal et al., 2002). Although SHP is well known as the c-Jun and NF-κB p65 increase the transcriptional repressor of various NRs, recently it is identified activation of the SHP promoter that SHP roles as a novel transcription coactivator To investigate the influence on the basal level of of nuclear factor, NF-κB and appears to functions cellular proteins related to monocytic differen- as a distinct regulatory component of the trans- tiation, U937 cells were stimulated with PMA and criptional activities of NF-κB in oxidized LDL-treated, assayed with immunoblotting. As shown as Figure resting macrophage cells (Kim et al., 2001b). It 1A, c-Jun was induced and phosphorylated during was reported that during the procedure of promo- the progression of differentiation. Furthermore, the nocyte cell differentiation of HL-60 leukemia cells level of p65 of NF-κB subunits, SHP, and p21WAF1 toward the monocyte lineage by phorbol ester, were increased by the treatment of PMA. To gain mRNA and protein levels of SHP were increased in insight that the differentiation signaling effects on a time-dependent manner. It seems that SHP plays the transcriptional activation of the SHP promoter, a fundamental role in the processes of monocytic transient transfection assay was analyzed using differentiation (Choi et al., 2004). However, precise the SHP promoter as a reporter construct with functions of SHP in monocytic differentiation have PMA. The result of Figure 1B shows that promoter not been clearly elucidated. In the previous report, activity of SHP is positively regulated by the the role for SHP in the protection of cells against treatment of PMA as a dose-dependent manner. Nur77-mediated apoptosis has been proposed Cycling primary human monocytes have no (Yeo et al., 2005). Anti-apoptosis function of SHP NF-κB in their nuclei. However, their differentiation would be related with the resistance of phorbol into macrophages triggered by PMA significantly ester-differentiated monocytes against apoptosis. leads to nuclear translocation of NF-κB. Its trans- A number of transcription factors have been location into the nucleus by the PMA stimuli implicated in monocyte differentiation together with regulates various genes including monocytic diffe- the transcriptional regulation of the downstream rentiation and cell survival programs (Pennington target gene expression, including p21WAF1 (hereafter et al., 2001). To investigate whether NF-κB regu- referred to as p21) (Freytag, 1988; Nguyen et al., lates transcriptional level of SHP, p65 expression 1993; Scott et al., 1994; Nerlov and Graf, 1998; vector and SHP luciferase-based reporter were Wang et al., 1998; Kelly et al., 2000). p21 inhibits cotransfected. As a result, p65 increased the cell cycle progression by binding to G1 cyclin-CDK transactivation of the SHP promoter and p65 and complexes through its N-terminal domain. In addi- c-Jun increased the expression level of SHP tion, recent studies have shown that p21 promotes synergistically (Figure 1C). It is supported by the the association of Cdk4 with D-type cyclins, and report that SHP plays a role as a coactivator of p65 targets Cdk4 and cyclin D1 to the nucleus (LaBaer in macrophage cells (Kim et al., 2001b). Taken et al., 1997). Thus, the cell cycle inhibitory activity together, these results may indicate that the of p21 is intimately correlated with its nuclear loca- expression level of SHP is associated with trans- lization and this property appears to be responsible criptional induction. SHP inhibits apoptosis via p21 induction 431 p21 induced by SHP in the late monocytic SHP synergistically

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