Published online before print August 3, 2004
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* Department of Molecular Biology, Pusan National University, Busan, Korea; and
Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan, Korea
1 Correspondence: Department of Molecular Biology, Pusan National University, Busan 607-935, Korea. E-mail: molecule85{at}pusan.ac.kr
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Key Words: monocyte • c-Jun • SHP
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Changes in gene expression during monocyte/macrophage differentiation have been investigated [5 6 7 ], and in monocyte-specific mRNAs, levels were reported to increase. During differentiation of HL-60 cells, which are usually used as a model to study monocyte/macrophage differentiation, Krichevsky et al. [8 ] described a cluster of mRNAs that are more associated to polysomes, which are more susceptible to translation into proteins.
Small heterodimer partner (SHP) is an orphan nuclear receptor, specifically expressed in liver and a limited number of other tissues, and its activities are in some ways opposite to those of retinoid X receptor (RXR) [9 ] and is also named with NROB2 according to the nuclear receptor nomenclature. Based on its ability to interact with a variety of nuclear receptors, distinct features distinguish SHP from RXR, the only known, common heterodimerization receptor [10 , 11 ]. First, SHP, unlike RXR, interacts with estrogen receptors (ERs), and agonistic ligands enhance, whereas antagonistic ligands inhibit, these interactions (for discussions, see refs. [10 , 12 ]). Second, the C terminus within SHP, including the putative dimerization helix, is dispensable for interactions, and a central ligand-binding domain region apparently forms the SHP-specific domain for interaction with receptors.
We investigated pathways leading to SHP production during HL-60 cell differentiation toward the monocyte lineage. Various studies have reported SHP to be a repressor of transcriptional activities of a number of nuclear receptors, including ligand responsive receptors, such as ER, thyroid hormone receptor, retinoic acid receptor, and RXR, and orphan receptors, such as constitutive androstane receptor, hepatocyte nuclear factor 4, and fetoprotein transcription factor [9 , 10 , 13 , 14 ]. Initial studies indicated that SHP is expressed predominantly in the liver, spleen, small intestine, and pancreas [11 ]. However, later studies with more sensitive approaches have demonstrated the presence of SHP mRNA in a wide variety of tissues, with highest expression in heart, brain, liver, lung, and adrenal gland [12 ]. Although very little is known about SHP gene regulation, some recent reports have suggested important roles for the bile acid receptor farnesoid X receptor and activated protein 1 (AP-1) transcription factors in regulating SHP gene expression and subsequent regulation of cholesterol homeostasis [15 , 16 ].
As the SHP gene promoter contains several 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (TRE), which are predicted to be c-Jun response elements, we investigated whether c-Jun subfamily members can regulate the SHP gene promoter. Mapping studies revealed that only one of the TRE on the SHP gene promoter is responsible for the c-Jun-mediated activation of the SHP promoter. Electrophoretic mobility shift assays (EMSAs) and mutational studies confirmed the binding of c-Jun to this element. In this study, we show that SHP plays a critical role in the monocytic differentiation, and c-Jun activation signaling induces its expression during differentiation. Cells expressing SHP stably led to transform the HL-60 leukemia cell differentiation into a monocyte-like character and to induce monocytic marker tumor necrosis factor 
(TNF-) protein expression.
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Plasmids
A full-length c-Jun cDNA was generated by reverse transcription followed by polymerase chain reaction (PCR) and verified by sequencing. c-Jun was subcloned into pCMX1 as BamHI and BamHI/KpnI fragments, respectively. pCMX1 was a gift from Jon Shuman (National Institutes of Health, Bethesda, MD) and was used in the transient transfection analysis. In vitro translation products were verified by [35S]Met incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The human p38 expression plasmid was provided by Jiahuai Han (The Scripps Research Institute, La Jolla, CA) [17
]. The SHP promoter, based on luciferase reporter plasmids, was provided by Heung Sik Choi (Chonnam University, Korea). The mammalian expression plasmids for mitogen-activated protein kinase kinase 1 (MKK1), MKK4, and MKK6 were provided by Han Do Kim (Pusan National University, Busan, Korea).
Western blot analysis
Untreated or TPA-treated HL-60 cells were harvested in Nonidet P-40 lysis buffer, and 30–50 µg nuclear protein was fractionated by SDS-PAGE. Proteins were electroblotted to Immobilon-P (Millipore, Bedford, MA), and membranes were blocked in Tris-buffered saline, 0.02% Tween 20, containing 5% nonfat milk. Specific antibody for SHP was prepared. The SHP cDNA (EcoRI) was subcloned into the vector pGEX (Pharmacia, Uppsala, Sweden) for protein expression in bacteria. Glutatione S-transferase (GST)-SHP was affinity-purified, and the antigen was excised from a 10% SDS-PAGE gel. Antisera were raised according to standard protocols, and appropriate reactivity was verified against recombinant proteins. The specificities of all antisera were verified against purified recombinant proteins. TNF- and TATA binding protein (TBP) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary detection used goat anti-rabbit-conjugated horseradish peroxidase (Amersham Pharmacia Biotech, Little Chalfont, UK) visualized with enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).
Gel mobility shift analysis
Nuclear extracts were prepared from HL-60 cells following stimulation with hormones or kinase inhibitors as indicated in the figure legends. Approximately 10 µg nuclear extract was immunoprecipitated with a SHP-specific antibody and incubated with a probe. A double-stranded oligonucleotide encoding the SHP TRE sequence (promoter positions –251 to –229) was used for gel shift analysis: 5'-TGTGCCCTGCAATGGCCACTTACA. Binding reactions were assembled without probe and held 5 min on ice followed by 5 min at room temperature. Probe was added with further room temperature incubation for 30 min. Samples were separated in 4% acrylamide, 0.5 x 0.045 M Tris, 0.045 M boric acid, 1.0 mM EDTA (pH 8.0), gels run at 200 V constant voltage.
Transient and stable transfection
HL-60 cells were transfected by the standard electroporation method. Cells were incubated with DNA precipitates for 16 h, washed, and maintained in complete medium 48 h prior to harvest. Relative luciferase and ß-galactosidase activities were determined. Basal promoter activity is reported as the activity observed after transfection of the reporter plus an appropriate amount of empty expression vector. In all cases, transfection data represent the mean of three independent experiments. For establishment of a SHP-expressing, stable cell line, HL-60 cells were transfected with 3 µg SHP expression plasmid [pcDNA3/hemagglutinin (HA)] using an electroporation method. At 48 h post-transfection, cells were cultured in the presence of 500 µg/ml G418 (Gibco-BRL). After 21 days in selective medium, individual G418-resistant colonies were isolated.
RNA interference (RNAi) expression
Individual DNA fragments containing sequences encoding the proteins of c-Jun and SHP to be targeted by RNAi were amplified by PCR from human genomic DNA and cloned in both orientations into the pCRII-Tri-n-octylphosphine oxide (TOPO) cloning vector by using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Sense and antisense clones were used as templates to generate single-stranded RNA (ssRNA) with a Ribomax kit (Promega, Madison, WI). ssRNA was resuspended in annealing buffer (5 mM KCl, 10 mM NaH2PO4), and equal quantities of sense and antisense ssRNAs were annealed by heating to 95°C for 5 min and slow cooling for 12–18 h to generate double-stranded RNA (dsRNA), which was stored at –80°C. Briefly, 2 x 106 cells were plated into a 60-mm diameter dish. After 1 h, FBS-containing medium was removed and replaced with 2 ml serum-free medium. Approximately 40 µg dsRNA (as little as 10 µg has been tested and found to be effective) was added per dish and mixed by swirling. After 30 min, 4 ml medium containing 10% FBS, 100 U penicillin per ml, and 100 µg streptomycin per ml was added. Cells were assayed at a specified day following addition of dsRNA. Western blotting was routinely carried out for RNAi-treated cells to evaluate the level of the targeted protein.
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Figure 1. The induction of monocytic differentiation increases mRNA and protein expression of SHP. (A) HL-60 cells were treated with TPA for 3 days. Approximately 50 µg cell lysate protein from each sample was separated on 10% SDS-PAGE and transferred to Immobilon-P membranes. Duplicate membranes were subjected to Western blot analysis by using antibodies specific for SHP or TBP as indicated. TBP expression is shown as a protein-loading control. (B) The mRNA expression of SHP is increased after monocytic differentiation of HL-60 cells. Total RNA (20 µg) extracted from HL-60 cells prior to and after TPA treatment was probed with SHP cDNAs. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression is shown directly below.
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Figure 2. c-Jun increases the gene expression of the SHP promoter. (A) SHP promoter reporter plasmids were transfected into HL-60 cells. Forty-eight hours after transfection in the absence or presence of several concentrations of TPA, as indicated, cells were harvested for luciferase (luc) activities. (B) HL-60 cells were transfected with 1 µg native SHP promoter-luciferase plasmid alone or along with 1 µg of the mammalian expression vector of c-Jun. Forty-eight hours after transfecting, luciferase assay was performed as described in Materials and Methods. All the transfection results were normalized to ß-galactosidase activity, and the presented results represented the average of three independent experiments, with fold induction over the level observed with the reporter alone. CREB, cyclic AMP response element-binding protein; C/EBPß, CCAAT/enhancer-binding protein ß. (C) HL-60 cells were transfected with a series of deleted promoter reporter plasmids and treated with TPA for 24 h. After harvesting the cells, luciferase assay was performed with normalization by ß-galactosidase activity.
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c-Jun binds on TRE sequence of the SHP promoter
To examine whether an increase of the transactivation of the SHP promoter by c-Jun expression results from the direct DNA binding activity of c-Jun on the SHP promoter, we applied to EMSA by using the TRE sequence of the SHP promoter. The increasing amounts of GST-c-Jun protein clearly bound to SHP-TRE, and the DNA protein complex was abolished by using the wild-type SHP-TRE oligonucleotide probe but not with the mutant one as shown in Figure 3
. To further confirm the induced c-Jun-SHP/TRE complex during monocytic differentiation by TPA treatment, we prepared the immunoprecipitated c-Jun protein by using antibody against c-Jun using TPA-treated HL-60 cells for each time interval. The result of Figure 3B
showed that the DNA protein complex of SHP-TRE and c-Jun was gradually increased in the differentiating cell extracts in a time-dependent manner. These results indicate that c-Jun increases the SHP expression at the transcriptional level through direct DNA binding following by TPA-induced monocytic differentiation.
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Figure 3. DNA binding of c-Jun on the SHP promoter. (A) Bacterially expressed and purified GST-c-Jun was incubated as indicated, followed by addition of a probe. For competition assays, the following nonradioactive competitor oligonucleotides were added to the reaction mixture at a 100-fold molar excess prior to the addition of the probe. (B) HL-60 cells were treated with TPA for 3 days. After preparation of nuclear extracts from pretreated cells and TPA-treated cells upon the time course, EMSA was examined with the SHP-TRE probe.
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Figure 4. ERK and JNK signaling increase the SHP expression. (A) Cultured cells were transfected with the indicated expression plasmids and assayed for reporter activity using a SHP promoter reporter. All results were normalized to ß-galactosidase activity and represent the mean of three independent experiments (error bars show SD of the mean). Cells were left untreated or treated with 20 nM TPA for 24 h in the absence or presence of 50 µM PD98059 (PD; ERK inhibitor), 5 µM SP600125 (SP; JNK inhibitor), and 10 µM SB203580 (SB; p38 kinase inhibitor). (B) A double-stranded oligonucleotide probe containing the TRE site of the SHP promoter was used in electrophoretic mobility shift analysis. Nuclear extracts (10 µg) of HL-60 cells treated with each kinase inhibitor were added in a binding reaction.
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Expression of the SHP transgene did not significantly affect the spontaneous differentiation rate of HL-60 cells in the absence of TPA. In the presence of TPA, however, SHP greatly accelerated monocytic differentiation; after 3 days in the presence of TPA, for example, only 
35% of control cells had differentiated into monocytes, whereas >95% of the SHP-overexpressing cells had done so (data not shown). In the other approach for the confirmation of monocytic differentiation in the SHP-stable transfectant cells, we investigated the monocyte-derived TNF- protein expression by using Western blot assay (Fig. 5A
). Thus, the level of SHP expression is apparently normally limiting for TPA-dependent monocytic differentiation.
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Figure 5. SHP and c-Jun expression are required for the induction of monocytic differentiation. (A) HL-60 cells were stably transfected with the mammalian expression plasmid encoding HA-SHP (SHP transfect) and expression plasmid without any insert as a control (Con. transfect), and were screened by Western blotting assay using anti-HA antibody. After isolation of the positive, HA-SHP-stable transfectant, Western blotting assay using TNF- and TBP antibodies was analyzed. TBP expression was counted as a protein-loading control. (B) Wild-type and RNAi-expressed cells were treated with TPA and determined the differentiation yield by scoring nonspecific esterase (NSE) activity, and quantified data are presented. RNAi can target specific genes for SHP and c-Jun. Silencing of endogenous SHP and c-Jun was monitored by Western blot (WB) analysis of whole-cell extracts from control or RNAi cells. All results are typical of three independent experiments.
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96% of the cells staining positively for nonspecific esterase (Fig. 5B)
, suggesting that the SHP or c-Jun overexpression is sufficient to mediate monopoiesis induced by TPA. To further determine the physiological requirements for SHP and c-Jun in monocytic differentiation, we have used RNAi methodology to reduce the level of endogenous SHP and c-Jun protein in HL-60 culture cells. RNAi causes degradation of a specific mRNA, which in turn, causes a reduction in the level of the encoded protein. In brief, RNAi was carried out by adding dsRNA, corresponding to a nucleotide region of the SHP and c-Jun mRNA to HL-60 cells and culturing the cells for 3 days prior to analysis. We refer to these cells as SHP and c-Jun RNAi cells. Western blot analysis of whole-cell extracts with an antibody directed against SHP and c-Jun showed that RNAi treatment resulted in near elimination of the SHP and c-Jun protein (Fig. 5B , lower). To monitor the physiological effect of the loss of SHP and c-Jun, we analyzed SHP and c-Jun RNAi cells for monocytic differentiation by using cytochemical determination of monocytic NSE. The addition of RNAi of SHP and c-Jun into cultured HL-60 cells was unable to induce monopoiesis (Fig. 5B) . In this case, less than one-third of the cells was positive upon NSE staining in a TPA-treated cell fraction. Loss of SHP and c-Jun caused a severe effect in monocytic differentiation of HL-60 cells by TPA treatment. Control cells were well induced to differentiate into monocytic cells by the same TPA treatment. Taken together, these results indicate that SHP and c-Jun are positive transcriptional regulators of monocytic differentiation.
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Kim et al. [27
] identified SHP as a novel transcription coactivator of nuclear factor (NF)-
B and have further presented the experimental results indicating that targeted expression of SHP appears to function as a distinct regulatory component of the transcriptional activities of NF-B in oxidized low-density lipoprotein-treated, resting macrophage cells.
Coexpression of Jun and SHP was also observed in heart, kidney, epididymus, prostate, and pancreas in addition to monocyte/macrophage. Based on this coexpression and its ability to transactivate the SHP promoter, we conclude that Jun is a potential regulator of SHP expression. The biological effects of TPA are mediated, at least in part, by the generation of active AP-1 complexes, which are predominantly composed of heterodimers of Jun/Fos. Activation of the Jun/Fos AP-1 complexes is often mediated by phosphorylation of the protein components, thus resulting in enhanced AP-1 function [9 , 10 , 13 , 14 ]. A variety of extracellular signals activates a combination activation of kinases and phosphatases, subsequently regulating positively and negatively a certain panel of transcription factors. This network cooperation of signaling components and transcription factors gives a clue to form a cascade action of gene expression machinery in a cellular differentiation procedure. Here, for the monocytic differentiation at the early differentiation stage, c-Jun may function as a master gene regulator for inducing the expression of some key transcription factors including SHP. c-Jun cooperates with PU.1 to regulate several monocytic genes via adjacent cis DNA elements or via direct interaction. Notably, c-Jun, JunB, and JunD levels increase during monocytic differentiation [28 ], and exogenous c-Fos or c-Jun induced partial monocytic differentiation in M1 and U937 cells and increased the responsiveness of U937 cells to TPA [28 , 29 ], an agent that stimulates the monocytic differentiation of several myeloid cell lines and directly induces the c-Jun promoter via binding sites for c-Jun:activating transcription factor 2 (ATF-2) dimers. TPA or other stimuli activate JNKs, which in turn, phosphorylate c-Jun and ATF-2 and increase their transactivating potency.
The issue of granulocyte versus monocytic lineage choice also must be resolved. The findings that Jun and Maf family members can each induce the monocyte lineage in cell lines and that these proteins interact with AP-1 consensus sites lead to the speculation that Maf:Maf, Maf:Jun, Maf:Fos, Jun:Jun, or Jun:Fos dimers initiate monopoiesis in conjunction with PU.1 [30 ]. The continued presence of C/EBPs to maintain elevated PU.1 levels may be required as well. In the absence of Maf or Jun proteins, C/EBPs and PU.1 may specify the granulocyte lineage. Many questions remain unanswered regarding the transcriptional regulation of monocyte development. There may be additional, important transcriptional regulators of myeloid genes remaining to be uncovered via detailed investigation of promoters and distal enhancers and be lineage-restricted coactivators or corepressors, which participate in lineage specification. With respect to key transcription factors, further clarification of the regulatory network is needed.
Received December 29, 2003; revised May 7, 2004; accepted May 14, 2004.
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and ERß J. Biol. Chem. 274,345-353-hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is mediated by the c-Jun N-terminal kinase pathway J. Biol. Chem. 276,15816-15822This article has been cited by other articles:
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