Growth hormone activates X-box binding protein 1 in a sexually dimorphic manner through the extracellular signal-regulated protein kinase and CCAAT/enhancer-binding protein β pathway in rat liver

Abstract

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein β (C/EBPβ) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPβ pathway.

GROWTH HORMONE (GH) is not only a critical hormone for growth but also plays numerous important physiological roles such as lipolysis, gluconeogenesis, and protein synthesis [1]. GH regulates expression of a significant number of genes in the liver, where GH receptors (GHRs) are abundantly expressed, and its effects on transcription have been extensively studied [2, 3]. GH secretion is highly pulsatile in adult rats and exhibits striking sex differences [4]. In adult male rats, GH is secreted episodically with a 3-h interval and with high amplitude, intervened by very low trough periods. In adult female rats, GH is secreted with a more frequent irregular pulsatility and with higher baseline levels. These sexually dimorphic patterns of GH secretion are responsible for the sex-specific expression of many genes in rat liver [5]. For example, GH has been shown to regulate the expression of insulin-like growth factor 1 (IGF-1) [6], the serine protease inhibitor Spi 2.1 [7], major urinary proteins [8], and the cytochrome P450 family, such as CYP2C11 and CYP2C12 [9, 10]. These genes are expressed in a gender-dependent manner.

GH signals mainly through activation of janus kinase 2 (JAK2)/signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase (MEK) pathways. STAT5B is a major mediator of GH in the sex-dependent transcriptional responses of the liver. The pulsatile plasma GH profile induces repeated cycles of STAT5B phosphorylation, and the phosphorylated (active) STAT5B level shows high variability in male rat liver. On the other hand, the level of active STAT5B is low and persistent in adult female rat liver [5, 11]. However, STAT5B is not sufficient to establish sexually dimorphic gene expressions, and additional transcription factors are required [11]. GH has been shown to promote phosphorylation of CCAAT/enhancer-binding protein β (C/EBPβ) through MEK/ERK signaling in mouse 3T3-F442A fibroblasts, and this phosphorylation is required in GH-stimulated activation of the c-fos promoter [12]. It is suggested that GH also activates c-JUN N-terminal kinase (JNK) [13], and GH-induced suppressor of cytokine signaling 3 (SOCS3) expression involves JNK activation [14].

We tried to identify GH-induced novel genes using the suppression subtractive hybridization (SSH) technique [15]. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The canonical unfolded protein response (UPR) was originally characterized as a pathway that responds to ER stress and upregulates ER chaperones or ER-associated degradation pathways [16]. Following ER stress, activating transcription factor 6 (ATF6) is activated through cleavage and induces expression of Xbp1 [17]. Inositol-requiring-1 (IRE1), a kinase/ribonuclease, undergoes autophosphorylation, leading to its activation, and removes a fragment from Xbp1 mRNA, switching the open reading frame. The resultant protein produced following this splicing event is a potent transcription factor, XBP1(S) [17-19].

The purpose of this study was to investigate the mode of action of GH on XBP1. We examined whether GH-induced activation of XBP1 is related to ER stress and whether naturally occurring Xbp1 expression depends on sexually dimorphic GH pulsatility. Then, the molecular mechanisms of Xbp1 expression induced by GH were investigated.

Materials and Methods

Materials

Recombinant rat GH (rGH) and prolactin (PRL) were supplied by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases (NHPP-NIDDK). Recombinant human GH (hGH) was a generous gift from Novo Nordisk Pharma (Tokyo, Japan). Radionucleotide [α-³²P] CTP (3000 Ci/mmol) was from PerkinElmer (Waltham, MA).

Animal preparation and in vivo experiments

Wistar rats weighing 150–200 g (7 weeks old) were used for in vivo experiments. They were housed in air-conditioned animal quarters with lights on from 08:00 to 20:00 and were given access to food and water ad libitum. All experimental protocols were reviewed and approved in advance by the Experimental Animal Ethics Committee of Nippon Medical School (Approval Number 18-140).

Male rats were hypophysectomized (HPX) approximately two weeks before use under ketamine (45 mg/kg)-xylazine (6 mg/kg) anesthesia. Hypophysectomy was performed via the parapharyngeal approach and HPX rats received a daily subcutaneous injection of dexamethasone phosphate (20 μg/kg) and L-thyroxine (20 μg/kg). Three days prior to GH administration, rats were fitted with an indwelling right atrial cannula via the external jugular vein under ketamine-xylazine anesthesia for systemic treatment with either recombinant GH or vehicle. Vehicle contained 30 mM NaHCO₃, 0.15 M NaCl, and 100 μg/mL rat albumin. On the day of the experiment, rats were sacrificed by decapitation at various times following intravenous injection of GH or vehicle. Tissue samples were removed from the liver and were immediately frozen in liquid nitrogen and stored at –80°C until analysis.

PCR select cDNA subtraction, cloning, and sequencing

Genes induced by GH in HPX rat livers were determined by the SSH technique [15] using the PCR-Select cDNA Subtraction Kit according to the manufacturer’s protocol (Clontech Laboratories, Inc., Palo Alto, CA). One microgram each of total RNA from GH or vehicle treated rat livers prepared using the SV Total RNA Isolation System (Promega Corporation, Madison, WI) was reverse transcribed using the SMART PCR cDNA Synthesis Kit (Clontech Laboratories, Inc.). The tester cDNAs were ligated to adaptors, whereas the driver cDNAs were unligated. Two subtractive libraries were prepared as follows. A forward subtractive library was prepared by subtracting vehicle treated adaptor-free cDNAs from GH treated adaptor-ligated cDNAs, and a reverse subtractive library was generated by subtracting GH treated adaptor-free cDNAs from vehicle treated adaptor-ligated cDNAs. The subtracted cDNAs were cloned into a pBluescriptSK+ vector (Toyobo, Osaka, Japan), and an aliquot of this library was plated onto nylon membranes. Forward or reverse subtracted cDNA probes were labeled with digoxigenin (DIG), and then the membranes were screened by differential hybridization following the instructions in the DIG High Prime DNA Labeling Kit (Roche Applied Science, Mannheim, Germany). Clones that hybridized specifically to the forward probe were sequenced by the BigDye Terminator cycle sequencing method using a 377 ABI Prism automated DNA sequencer (PerkinElmer) according to the manufacturers’ protocols. The sequences obtained were then compared to those in GenBank using BLAT in the UCSC Genome Browser.

RNA isolation and northern blotting

Total RNA was isolated from tissues using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Poly(A) RNA was isolated using the Micro-FastTrack Kit (Invitrogen, Carlsbad, CA). Then, 0.5 μg poly(A) RNA was electrophoresed on 1% agarose and 6% formamide gels, transferred to a Hybond-N+ membrane (GE Healthcare UK Ltd, Buckinghamshire, UK), and hybridized to radiolabeled complementary RNA (cRNA) probes derived from cDNA inserts encoding a portion of the 2.0-kb Xbp1 mRNA isoform as described previously [20].

Real time quantitative RT-PCR (qPCR)

Total RNA was treated with RNase-free DNase (Roche Applied Science) for 1 h at 37°C. Reverse transcription was performed on each RNA sample (1 μg) using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Shiga, Japan). The oligonucleotides utilized are listed in Table 1. qPCR was performed with SYBR-Premix Ex Taq (Takara) to a final volume of 10 μL. qPCR measurements were performed on an ABI Prism 7700 sequence detector system (Applied Biosystems, Foster City, CA). Final measurements were normalized by the target mRNA/Gapdh.

Table 1 Sequences of oligonucleotides

qPCR
2.5 kb Xbp1 forward	5'-CGATCTGTGAGACTCGGTTTG-3'
2.5 kb Xbp1 reverse	5'-TTAGGGTGGGTGGACGGTAG-3'
ERdj4 forward	5'-CGCACGGGTTATTAGAAATGG-3'
ERdj4 reverse	5'-TGAGGCTGGGACTTTCACAC-3'
Gapdh forward	5'-GATGCTGGTGCTGAGTATGTCG-3'
Gapdh reverse	5'-TGGTGCAGGATGCATTGCTG-3'
RT-PCR
Xbp1 forward	5'-GAACCAGAAACTCCAGCTAG-3'
Xbp1 reverse	5'-CATGACAGGGTCCAACTTGTCCAG-3'
EMSA
C/EBPβ (high affinity)	5'-GCTGCAGATTGCGCAATCTGCAGC-3'
wild	5'-CTCATTTCCTAAACACTCATTTTGTGAAATTCC-3'
1m	5'-CTCAAATCCTAAACACTCATTTTGTGAAATTCC-3'
2m	5'-CTCATTTCCTAAACACTCATTTTGTGATTTTCC-3'
12m	5'-CTCAAATCCTAAACACTCATTTTGTGATTTTCC-3'

RT-PCR analysis of Xbp1 mRNA splicing

Total RNA was treated with M-MLV reverse transcriptase (Takara) using the oligo (dT) primer and then amplified with Ex Taq polymerase (Takara) using the Xbp1 primers listed in Table 1. Cycle conditions were 94°C for 2 min, 26 cycles of 94°C for 10 s, 60°C for 10 s, and 72°C for 30 s, with a final extension for 10 min at 72°C. The 327- and 301-bp fragments representing unspliced and spliced Xbp1, respectively, were separated by electrophoresis on 3% agarose gels and visualized by ethidium bromide staining.

Construction of H4IIE GH expressing GHR and treatment of cells

A rat GHR expression plasmid was constructed by inserting RT-PCR products of rat GHR mRNA into pcDNA3.1. H4IIE hepatoma cells obtained from ATCC were transfected with the rat GHR expression plasmid using Fugene HD (Roche Applied Science), and stably transfected clones were selected by growing cells in G418, an antibiotic related to gentamicin. GHR mRNA levels of transfected clones were determined by qPCR, and the clone that expressed the highest GHR mRNA (H4IIE GHR) was used for further investigation.

H4IIE GHR cells were grown to confluence in alpha modified Eagle’s medium containing 10% fetal calf serum supplemented with 2 mM-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. Prior to treatment, cells were deprived of serum for 16–30 h in alpha modified Eagle’s medium. Cells were preincubated with a certain inhibitor or vehicle (DMSO, 0.1% final concentration) for 1 h prior to treatment with GH. The final concentration of the MEK inhibitor PD98059 (Cell Signaling Technology, Danvers, MA), JNK inhibitor SP600125 (Calbiochem, SanDiego, CA), and cycloheximide (CHX) (Calbiochem) was 50 μM, 10 μM, and 10 μg/mL, respectively, and GH was used at a concentration of 500 ng/mL.

Cell Culture and Transfections (Luciferase)

Reporter plasmids were constructed by inserting a 5' upstream fragment of Xbp1 into pGL3-basic (Promega). Mutations were introduced using the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene, Santa Clara, CA). The rat C/EBPβ expression plasmid was constructed by inserting the cDNA of rat C/EBPβ into pcDNA3.1. For transient transfections, cells were seeded in 24-well plates at a density of 8 × 10⁴ cells/well. Fugene HD transfection reagent (Roche Applied Science) was used at a ratio of 3:1 of Fugene HD/DNA (vol/wt), as described in the manufacturer’s protocol. Each well received a total of 500 ng DNA, including 250 ng luciferase reporter plasmid and 100 ng C/EBPβ expression plasmid. pRL-SV40-Luc plasmid (Renilla luciferase; 10 ng DNA) was included as an internal control for transfection efficiency. pcDNA3.1 DNA was used to adjust the total to 500 ng of DNA per well. Twenty-four hours after transfection, total cell extracts were prepared using 1× lysis buffer (Promega) for measuring luciferase activities. Firefly and Renilla luciferase activities were measured using a Dual Reporter Assay System (Promega) and Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany). Firefly luciferase activity values were divided by Renilla luciferase activity values to obtain normalized luciferase activities (mean ± standard error (SE) values for n = 3 separate transfections).

Electrophoretic mobility shift assay (EMSA)

The oligonucleotides were 3'-end labeled with digoxigenin-11-dideoxy-UTP using terminal transferase according to the manufacturer’s protocol (Roche Applied Science). Nuclear extracts of the pcDNA3.1 or C/EBPβ expression plasmid transfected HeLa cells were added to binding buffer (25 mM KCl, 16 mM HEPES (pH 7.9), 0.5 mg/mL BSA, 0.02 mM EDTA (pH 8.0), 0.67 mM dithiothreitol, 0.05 mg/mL poly [d(I-C)], 0.05 mg/mL poly L-lysine), and the mixture was incubated for 10 min at 25°C. Digoxigenin-labeled double-stranded oligonucleotide (Table 1) was added in the absence or presence of an unlabeled competitor, and the binding mixture was incubated for 30 min at 25°C. The mixture was kept on ice for 10 min and loaded onto the 4% polyacrylamide gels in 0.5× TBE. The gel was run in 0.5× TBE buffer at 100 V for 70 min. After electrophoresis, the gel was transferred onto a Hybond-N+ membrane (GE Healthcare UK Ltd.) and cross-linked by UV. Detection was performed using a chemiluminescent detection technique based upon an alkaline phosphatase-linked anti-digoxigenin Ab (DIG Gel Shift Kit, Roche). Imaging of the chemiluminescence was performed by LAS-3000mini (Fujifilm, Tokyo, Japan).

Protein extraction and western blotting

Protein extracts were prepared from rat livers. Rat tissues in the lysis buffer (10 mM HEPES, pH 7.9, 0.5% Triton X-100, 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, and 1 mM Na₃VO₄ containing Protease Inhibitor Cocktail (Roche Applied Science)) were homogenized using a Teflon-glass homogenizer. Cell lysates were centrifuged at 2,500 rpm for 10 min, and supernatants were used as cytoplasmic extracts. After washing the pellet in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, and 1 mM Na₃VO₄ containing Protease Inhibitor Cocktail), the pellet was suspended in buffer C (10 mM HEPES, pH 7.9, 500 mM NaCl, 0.1 mM EDTA, and 1 mM Na₃VO₄ containing Protease Inhibitor Cocktail), incubated for 15 min at 4°C, and centrifuged at 14,000 rpm for 10 min. Supernatants were used as nuclear extracts. Protein concentrations were determined using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA) with bovine gamma globulin as a standard.

Protein extracts were electrophoresed through SDS polyacrylamide gels. Gels were electrophoretically transferred to Immobilon PVDF membranes (Millipore, Bedford, MA) and reacted with the indicated antibodies. Immunoreactive proteins were visualized by the chemiluminescence detection system ImmunoStar LD (Wako, Osaka, Japan). The following antibodies were used: anti-C/EBPβ (sc-150, 1:1,000), anti-ERK1 (sc-93, 1:10,000), anti-ERK2 (sc-154, 1:10,000), and anti-STAT5B (sc-835, 1:10,000), and anti-XBP1 (sc-7160, 1:2,000) antibodies (Santa Cruz Biotechnology, Inc. Santa Cruz, CA), anti-active MAPK (V8031, 1:10,000) antibody (Promega), anti-XBP1(S) (619502, 1:1,000) antibody (BioLegend, San Diego, CA), anti-IRE1α (#3294, 1:2,000), anti-PERK (#3192, 1:1,000), anti-pT235C/EBPβ (#3084, 1:1,000) and anti-PARP (#9532, 1:1,000) antibodies (Cell Signaling Technology), and anti-pS724IRE1α (ab48187, 1:2,000) and anti-TATA binding protein (TBP) (ab818, 1:10,000) antibodies (Abcam, Cambridge, UK).

Statistical analysis

All results are expressed as the mean ± SE for each group. Main effects and interactions were analyzed by two-way ANOVA followed by Bonferroni’s test (SPSS version 25, IBM). Comparison between groups of XBP1 proteins was performed by Student’s t test. The level of significance was indicated as * for GH-treated vs. vehicle-treated rats or cells, # for GH-treated vs. untreated rats, and $ for inhibitor-treated vs. DMSO treated in vehicle treated cells. A double symbol (*, #, or $) indicates significance at p < 0.01, and a triple symbol indicates p < 0.001.

Results

GH increases Xbp1 mRNA levels in rat liver

We tried to identify genes induced by intravenous injection of GH in HPX male rat livers using the SSH technique as described in Materials and Methods. We found that one of the candidate clones induced by GH was a part of Xbp1 that has important roles in ER stress. To examine whether GH really induces Xbp1 mRNA, Xbp1 mRNA levels were determined by northern blotting. In rat liver, a 2.5-kb Xbp1 mRNA containing a longer 5'-untranslated region, which has the same coding region with 2.0-kb Xbp1 mRNA, is expressed in addition to the ubiquitously expressed 2.0-kb mRNA [21, 22]. The levels of both 2.5-kb and 2.0-kb Xbp1 mRNA were higher at 1 h after GH injection than those after vehicle injection (Fig. 1A). Next, we examined the time course of Xbp1 mRNA levels (Fig. 1B). In the absence of GH, northern blot analysis detected low levels of 2.0-kb Xbp1 mRNA, but not 2.5-kb Xbp1 mRNA. When GH was injected intravenously, 2.0-kb Xbp1 mRNA levels increased rapidly and transiently, declining to basal levels after 4 h. The 2.5-kb mRNA was detected between 0.5 and 2 h following GH injection and decreased to undetectable levels after 4 h. Injection of PRL did not significantly increase Xbp1 mRNA, indicating that the increase of Xbp1 mRNA level is GH specific (data not shown).

Fig. 1

Xbp1 mRNAs in response to GH were analyzed in HPX rat livers. A, Total RNA (7.5 μg/lane) isolated from HPX rat livers at 1 h after administration of vehicle or GH (1,000 μg/kg) was hybridized with cRNA probes for Xbp1 and Actb (β-actin). B, Poly(A) RNA (0.5 μg/lane) isolated from HPX rat livers prior to or following GH administration (200 μg/kg) at the indicated time points was hybridized with cRNA probes for Xbp1 and Actb. C, Total RNA samples were analyzed by RT-PCR using a primer set flanking the 26-nucleotide fragment in Xbp1 mRNA. An amplified fragment of 327 bp corresponding to the unspliced form of Xbp1(U) mRNA and a fragment of 301 bp corresponding to the spliced form of Xbp1(S) mRNA were identified. Gapdh served as a control. Results are representative of 4 independent experiments. D, Structures of Xbp1 mRNAs. Boxes indicate exons, and lines indicate introns. Large boxes indicate coding regions and small boxes indicate non-coding regions. Xbp1 mRNA undergoes site-specific cleavage by IRE1. It removes a 26-nucleotide fragment, resulting in a translational frame shift. Translation of the new reading frame converts XBP1(U) from an unspliced form, 267 amino acids (aa) in length, to an active spliced form, XBP1(S), that is 371 aa in length.

Xbp1 mRNA undergoes site-specific cleavage by IRE1, a process that is activated in the UPR pathway. This unconventional form of splicing removes a 26-nucleotide fragment, resulting in a translational frame shift (Fig. 1D). Translation of the new reading frame converts XBP1 from an unspliced form, XBP1(U), 267 amino acids (aa) in length, to an active spliced form, XBP1(S), that is 371 aa in length. RT-PCR analysis carried out with primers that amplify the region encompassing the splice junction detected both spliced and unspliced forms of Xbp1 mRNA in HPX rat livers. Both unspliced and spliced Xbp1 mRNAs increased immediately following injection of GH and reached maximum levels after 1–2 h (Fig. 1C). Injection of the vehicle compound did not increase expression levels of either mRNA.

ER stress response is not an event induced by GH

Because Xbp1 mRNA is induced and spliced by ER stress, we investigated whether injection of GH causes ER stress. In response to ER stress, IRE1α is phosphorylated at Ser724, PKR-like ER kinase (PERK) undergoes hyperphosphorylation [23], and ATF6 is activated through cleavage and induces Xbp1 expression [17]. When GH was injected, the level of phosphorylated IRE1α, which can be detected by the anti-phospho-IRE1α antibody, was unchanged (Fig. 2A), suggesting that IRE1α is active in HPX rat livers, but active IRE1α is not increased by injection of GH. Hyperphosphorylated PERK, which can be detected by an associated shift in mobility during SDS-polyacrylamide-gel electrophoresis, was not detected (Fig. 2A). This suggested that PERK was not activated by injection of GH. Cleaved ATF6 was not detected (data not shown). In addition, cleaved poly-(ADP ribose)-polymerase (PARP), which appears when ER stress is induced and is considered to be an early marker of apoptosis, did not appear following injection of GH (Fig. 2A). These results suggested that neither ER stress nor apoptosis was induced, even though the level of Xbp1 mRNA was increased by injection of GH.

Fig. 2

GH increased the XBP1(S) protein levels following STAT5B phosphorylation in rat livers without induction of the ER stress response. A, Cytoplasmic (Cyto) or nuclear (Nuc) extracts in the liver of HPX rats at indicated time points after GH injection were immunoblotted using anti-p-IRE1, anti-IRE1, anti-PERK, anti-STAT5B, anti-XBP1(S), anti-PARP, and anti-TBP antibodies. Anti-XBP1(S) antibody (BioLegend) which recognizes only XBP1(S) was used to detect 54 kD XBP1(S). H4IIE cells were treated with an ER stress inducer, thapsigargin (Tg), for positive control of phosphorylated PERK. Hyperphosphorylated PERK, which can be detected by an associated shift in mobility during SDS-polyacrylamide-gel electrophoresis was detected when Tg was incubated in H4IIE cells. p-STAT5B represents monophosphorylated form of STAT5B, and pp-STAT5B represents diphosphorylated form of STAT5B. B, Levels of ERdj4 mRNAs isolated from HPX rat livers prior to or following GH treatment (200 μg/kg) (red bar) or vehicle (blue bar) at the indicated time points were determined by qPCR analysis. Values were corrected using Gapdh as the reference gene and are presented relative to the averaged untreated HPX male liver level (white bar), which was set to 1. Data are shown as the mean ± SE of 4 rats. ** p < 0.01 for GH-treated vs. vehicle-treated rats. ^### p < 0.001 for GH-treated vs. untreated rats.

XBP1 has been shown to regulate a subset of ER resident chaperone genes. We examined changes in in vivo expression of one of those genes, ER-associated dnaJ protein 4 (ERdj4), following GH injection, because it is known that XBP1 highly induces ERdj4 mRNA and is essential for its induction by ER stress [24]. The qPCR revealed that ERdj4 mRNA level increased by 4.2-fold at 2 h after GH injection than that before injection and declined at 4 h, suggesting that XBP1(S) was increased by injection of GH to induce ERdj4 mRNA although we cannot exclude the possibility that GH induced expression of ERdj4 mRNA by other pathways (Fig. 2B).

We also investigated XBP1(S) protein as well as STAT5B protein levels. Immunoblotting revealed that STAT5B of cytosolic extracts was phosphorylated at 0.5–1 h after GH injection, and phosphorylated STAT5B appeared in nuclear extracts simultaneously (Fig. 2A). The XBP1(S) protein was increased at 0.5 h and reached its highest level at 2 h after injection of GH when phosphorylated STAT5B disappeared in the nucleus. Anti-XBP1(S) antibody (BioLegend) which recognizes only XBP1(S) was used to detect 54 kD XBP1(S). To ensure that 54 kD protein is XBP1(S), another anti-XBP1 antibody (Santa Cruz Biotechnology) which recognizes both XBP1(U) and XBP1(S) was used. It also detected 54 kD XBP1(S) (data not shown), which ascertained the 54 kD protein was XBP1(S).

XBP1(S) protein in the livers of normal adult male and female rats

Because sexually dimorphic patterns of GH secretion are responsible for the sex-specific expression of many genes in rat liver, we investigated whether XBP1(S) protein levels are sexually dimorphic. In normal male rats, XBP1(S) protein levels were highly variable individually, whereas in normal female rat livers, XBP1(S) protein levels were almost equal to the lowest level of normal male XBP1(S) (Fig. 3A).

Fig. 3

The levels of XBP1(S) proteins in rat livers were highly variable and sexually dimorphic. A, Nuclear extracts from normal female and male rat livers at ZT10 were immunoblotted using anti-XBP1(S) and anti-TBP antibodies. B, Nuclear extracts from normal male rat livers at indicated ZT were immunoblotted using anti-XBP1(S), anti-STAT5B, and anti-TBP antibodies. The numbers indicated are sampling times of ZT (h). Samples were loaded from highly phosphorylated STAT5B to low phosphorylated STAT5B at each ZT (left to right).

Because it was reported that a circadian clock regulated the activation of IRE1α and XBP1(S) levels in mouse livers [25], we investigated XBP1(S) protein levels in individual male rat livers at various zeitgeber times (ZT). The results showed that XBP1(S) protein levels of individual male rat liver nuclear extracts were highly variable even at the same ZT without a clear circadian rhythm. The pulsatilities of GH secretion are not synchronized in each rat, and the timing of GH pulses is variable in individual rat, as so the activation (phosphorylation) of STAT5B is [5, 11, 26]. The relationship between XBP1(S) levels and phosphorylated STAT5B was examined. The XBP1(S) levels were low when phosphorylated STAT5B protein levels were high, and phosphorylated STAT5B levels were low when XBP1(S) protein levels were high at any time of sampling (Fig. 3B).

GH-signaling pathways regulating Xbp1 expression

To investigate the molecular mechanism by which GH increases Xbp1 mRNA, we constructed H4IIE cells stably expressing GHR (H4IIE GHR) and examined whether GH increases 2.5-kb Xbp1 mRNA because expression of 2.5-kb Xbp1 mRNA depends on GH. When H4IIE GHR cells were treated with GH, 2.5-kb Xbp1 mRNA levels increased (Fig. 4A). XBP1(S) protein levels were also 2.4-fold higher after GH treatment than those after vehicle treatment (Fig. 4B). When CHX, an inhibitor of protein synthesis, was added to H4IIE GHR, Xbp1 mRNA levels increased and were further increased by GH treatment. These results suggest that Xbp1 is one of the immediate early genes and does not require de novo synthesis of transcription factors when GH increases Xbp1 mRNA levels (Fig. 4A). When H4IIE GHR cells were preincubated with the MEK inhibitor PD98059 for 1 h, GH did not increase Xbp1 mRNA levels. On the other hand, when cells were preincubated with the JNK inhibitor SP600125, the levels of Xbp1 mRNA with GH treatment were not different from the control. These results indicate that the MEK/ERK pathway contributes to the levels of Xbp1 mRNA stimulated by GH, whereas JNK does not.

Fig. 4

Analysis of signaling pathways regulating Xbp1 expression. A, H4IIE GHR cells were incubated for 1 h with DMSO, CHX, PD98059, or SP600125 and then GH (500 ng/mL) (red bar) or vehicle (blue bar) for an additional 1 h. Total RNA was assayed by qPCR for 2.5-kb Xbp1 mRNA. RNA levels were normalized to the Gapdh content of each cell and are presented relative to the averaged non-treated cell level, which was set to 1. Data are shown as the mean ± SE of 4 experiments. *** p < 0.001 for GH vs. vehicle. ^$$ p < 0.01 for treated vs. DMSO-treated. B, H4IIE GHR cells were treated with GH or vehicle for 2 h. Cells were lysed and used for immunoblotting with antibodies against XBP1(S) and TBP. Right panel: XBP1(S)/TBP levels were quantified. Data are shown as the mean ± SE of 4 experiments. *** p < 0.001 for GH vs. vehicle. C, H4IIE GHR cells were incubated with GH for 7.5–30 min as indicated. Cells were lysed and used for immunoblotting with antibodies that can detect phosphorylated ERK (p-ERK), ERK, phosphorylated C/EBPβ at the position of Thr189 of rat C/EBPβ (p-C/EBPβ), C/EBPβ, and TBP.

GH has been shown to promote phosphorylation of C/EBPβ at Thr188 through MEK/ERK signaling in mouse 3T3-F442A fibroblasts [12]. The C/EBPβ gene encodes three in-frame methionines, which can give rise to three translational products [27]. The molecular weight of each product is 38, 35, and 20 kDa (p38, p35, and p20, respectively) in rats. p38 and p35 C/EBPβ are transcriptional activators, and p20 C/EBPβ is a transcriptional inhibitor. We investigated the effect of GH on the phosphorylation of ERK and C/EBPβ in H4IIE GHR cells, using antibody that detects phosphorylated ERK and antibody that detects C/EBPβ phosphorylated at Thr189 of p38 C/EBPβ, corresponding to Thr188 of mouse C/EBPβ. As shown in Fig. 4C, GH induced the phosphorylation of both ERK1 and ERK2. p38 and p35 C/EBPβ were detected (Fig. 4C), whereas p20 C/EBPβ was not detected (data not shown). GH induced the phosphorylation of both p38 and p35 C/EBPβ (Fig. 4C). Thus, we hypothesized that Xbp1 expression was induced by GH through the ERK-C/EBPβ pathway.

GH induces Xbp1 expression through C/EBPβ

To investigate the transcriptional contribution to the increase of Xbp1 mRNA, –1,538 to –407 bp 5'-upstream of the 2.0-kb transcriptional initiation site was used to drive expression of the Firefly Luciferase reporter gene using H4IIE GHR cells. Many C/EBPβ consensus binding sites determined as T(T/G)NNGNAA(T/G) [28] were observed in this region. When the construct carrying this region was co-transfected with a C/EBPβ expression plasmid, relative luciferase activity was 3.9-fold higher than that of the pGL3-basic plasmid (Fig. 5A). This finding indicates that C/EBPβ activates the Xbp1 promoter. Then, a series of 5' deletions was constructed to map the sites contributing to C/EBPβ induction of Xbp1 promoter activity. As shown in Fig. 5A, stepwise deletion from –1,538 to –672 resulted in a minor decrease in Xbp1 promoter activity when C/EBPβ was overexpressed. This result suggests that five 3' terminal C/EBPβ binding sites were important for the induction of Xbp1 mRNA by C/EBPβ. To determine which of the five putative binding sites are important, we constructed plasmids containing point mutations on each putative C/EBPβ binding site. When either of the two proximal C/EBPβ binding sites was mutated (Fig. 5B), luciferase activity induced by C/EBPβ was reduced to 1.6-fold or 1.7-fold. When both were mutated, luciferase activity was the same as that of the pGL3-basic plasmid (Fig. 5A). These results indicate that these two proximal C/EBPβ binding sites are important for the induction of Xbp1 by C/EBPβ. These sites are highly conserved among humans, mice, and rats, and the rat sequence is a perfect match to the consensus sequence (Fig. 5B).

Fig. 5

Analysis of the Xbp1 gene promoter element required for activation by C/EBPβ. A, Scheme of the 5'-deletion and mutation constructs of the rat Xbp1 gene promoter fused to the luciferase gene (left). Putative C/EBPβ binding sites are indicated by rectangles, and mutated C/EBPβ binding sites are indicated by black rectangles. The numbers shown indicate the 5'-end position of the promoter sequence. H4IIE GHR cells were transfected with each plasmid and the C/EBPβ expression plasmid. Promoter activity was assayed as described in Materials and Methods. Luciferase activities are shown relative to the activity of the pGL3 basic vector transfected with the C/EBPβ expression plasmid/pcDNA3.1, which was set to 1. Data are shown as the mean ± SE of 3 separate transfections (right). B, Putative C/EBPβ binding elements on the human, mouse, and rat Xbp1 promoters are indicated by rectangles. The constructs 1m and 2m containing two base mutations are shown at the bottom. C, Nuclear extracts from HeLa cells transfected with pcDNA3.1 (lanes 1, 4, 7, and 10) and C/EBPβ expression plasmid (lanes 2, 3, 5, 6, 8, 9, 11, and 12) were assayed for their ability to bind wild type (lanes 1–3), 1m (lanes 4–6), 2m (lanes 7–9), and 12m (lanes 10–12) probes. A 125-fold excess of unlabeled competitor was added in some reactions (lanes 3, 6, 9, and 12). A red arrow indicates upper band and blue one indicates lower band, both of which are C/EBPβ specific complexes. A dashed arrow represents non-specific (ns) band that was detectable in lane 10.

Next, EMSA was used to examine whether C/EBPβ could bind directly to these binding sites (Fig. 5C). Nuclear extracts that were prepared from HeLa cells transfected with a C/EBPβ expression plasmid bound to the DNA probe, which had two proximal C/EBPβ binding sites, whereas those from HeLa cells transfected with pcDNA3.1 did not (shown as arrows in Fig. 5C, lane 2 compared to lane 1). When either of the two proximal C/EBPβ binding sites was mutated, the binding became weaker (Fig. 5C, lanes 5 and 8), and the binding was not detected when both were mutated (Fig. 5C, lane 11). The lower band in lane 8 was weaker than that in lane 5, suggesting that C/EBPβ preferentially binds to the downstream site, whereas luciferase activities were almost the same between the two mutants. It might be caused by the difference of cells used, i.e. H4IIE GHR cells were used in luciferase assay, and HeLa cells in EMSA. Binding was completely competed by an excess of unlabeled high affinity C/EBPβ binding probe (Fig. 5C, lanes 3, 6, and 9). Because we did not detect C/EBPβ in extracts of pcDNA3.1 transfected HeLa cells (data not shown), a band that was detectable in lane 10 was not supposed to be a complex with C/EBPβ, which meant non-specific. These results suggest that C/EBPβ binds to each of the two proximal C/EBPβ binding sites directly.

Discussion

In this study, we identified Xbp1 as a gene induced by GH in rat liver. A bolus injection of GH rapidly and transiently induced Xbp1 transcription to increase mRNA levels as well as XBP1(S) protein levels in the HPX male rat livers without activating major machineries of the ER stress response such as IRE1α, PERK, and ATF6. XBP1(S) protein predominated in male rat livers with remarkable variability among individuals and showed a reverse correlation to nuclear phosphorylated STAT5B. In addition, our in vitro study showed that the transcription of Xbp1 is induced by GH via the ERK-C/EBPβ pathway.

The existence of a sexually dimorphic pattern of GH secretion has been demonstrated in many species including rodents and humans. In rodents, the biological effects of a sexually dimorphic pattern of GH include sex differences in body weight gain, longitudinal bone growth [29], and hepatic steroid metabolism [30]. The GH secretory pattern is thought to be a major determinant of sexually dimorphic gene expression in rat liver. Some transcription factors are known to be GH-responsive and are suggested to be involved in the sex-specific gene expression. STAT5B is a major mediator of the sex-dependent transcriptional response to GH in the liver. The pulsatile plasma GH profile induces repeated cycles of STAT5B phosphorylation, and phosphorylated (active) STAT5B level shows high variability in male rat liver. Transcriptional repressor B cell lymphoma 6 (Bcl6) is a male specific rat liver gene product [31]. High variation in Bcl6 mRNA was observed, reflecting the suppressive effects of a recent GH pulse in male rat liver. The time course of GH injection into HPX rats revealed that Xbp1 mRNA began to increase when STAT5B was phosphorylated and declined when STAT5B was dephosphorylated, which was followed by the XBP1(S) protein increase (Figs. 1B and 2A). These results suggested that high variability of XBP1(S) levels and an inverse relationship between XBP1(S) protein and phosphorylated STAT5B in male rat livers are possibly caused by GH pulses. However, it is not necessarily supposed that XBP1(S) is induced by STAT5B.

We examined the effects of GH on Xbp1 expression in female rat liver. To eliminate endogenous GH secretion, female rats were treated with monosodium glutamate (MSG) at neonatal period, which destroys GH-releasing hormone (GH-RH) producing cells in the hypothalamic arcuate nucleus [32]. The levels of both Xbp1 mRNA and XBP1(S) protein increased 2 h after GH injection (Supplementary Fig. 1). Female rat liver cells responded enough to GH to induce Xbp1 mRNA and XBP1(S) protein. Therefore secretory pattern of GH is supposed to have a key role for the increase of XBP1(S) protein.

In pre-B acute lymphoblastic leukemia, Xbp1 is regulated positively by STAT5 and negatively by the transcriptional repressors BCL6, and BTB domain and CNC homolog 2 (BACH2) [33]. We carried out a luciferase assay to investigate whether constitutively active STAT5B induces expression of Xbp1. The result was that constitutively active STAT5B activated the Xbp1 promoter only 1.6 fold that was less than 3.9 fold of C/EBPβ (Supplementary Fig. 2), suggesting that STAT5B is involved in, but not very important for the activation of Xbp1 promoter. In addition, we were unable to locate putative binding sites (TTCN2-4GAA) for STAT upstream of the 2.5-kb Xbp1 transcription initiation site by using the TRANSFAC database [34]. It has been reported that β-Casein promoter that has STAT5 binding site is controlled by cooperative transactivation by STAT5A, C/EBPβ, and glucocorticoid receptor (GR) in mammary epithelial cells [35]. Because Xbp1 promoter does not have STAT5 binding site, it may not be controlled by cooperative transactivation of STAT5B and other factors.

Increased levels of mRNA do not necessarily indicate increased transcription. In order to determine whether transcription or post-transcriptional modifications contributed to the increase of Xbp1 mRNA levels, nascent unprocessed nuclear RNAs (heteronuclear (hn) RNAs) were analyzed. qPCR revealed that injection of GH rapidly increased the levels of unprocessed Xbp1 hnRNA compared with those prior to GH injection (Supplementary Fig. 3). The increase of Xbp1 mRNA levels detected in northern blotting experiments correlated with an increase in Xbp1 hnRNA, suggesting that the increase of Xbp1 mRNA levels following GH treatment is controlled primarily by RNA transcription, rather than by post-translational pathways including cytoplasmic mRNA stability modifications.

Tripartite motif-containing 24 (Trim24)/transcription initiation factor (TIF1α), Cut-like 2 (Cutl2)/cut homeobox 2 (Cux2), both of which display transcriptional repressor activity, and transcription factor hepatocyte nuclear factor-6 (HNF-6), are preferentially expressed in the adult female rat liver [36, 37]. However, a transcriptional activator expressed predominantly in male rat liver has not been reported yet. XBP1 is the first transcriptional activator whose transcription is increased by GH in rat liver.

Because changes in active IRE1α, PERK, or ATF6α levels were not detected after injection of GH into HPX rats, it is thought that Xbp1 mRNA is induced by ER stress-independent mechanisms by GH. It has been reported that transcription factors other than ATF6 induce Xbp1 expression. For example, HNF1α and HNF4α were found to induce Xbp1 expression in pancreatic β-cells [38, 39].

Studies using inhibitors have suggested that the MEK/ERK pathway, but not the JNK pathway, contributes to increased levels of Xbp1 mRNA by GH in H4IIE GHR cells. GH is known to promote phosphorylation of C/EBPβ by ERK in 3T3-F442A cells [12]. C/EBPβ contains multiple phosphorylation sites, and one of them is a highly conserved MAPK consensus site that corresponds to Thr189 in p38, Thr168 in p35, and Thr37 in p20. Phosphorylation of Thr188 of mouse C/EBPβ and Thr235 of human C/EBPβ (homologous to Thr188 in mouse C/EBPβ) by ERK is required for the induction of some genes such as c-fos by GH[12] and IL-6 by RAS [40]. In our study, GH did not increase C/EBPβ protein levels but promoted phosphorylation of C/EBPβ at Thr189 in p38 and Thr168 in p35. Overexpression of C/EBPβ increased C/EBPβ phosphorylated at these sites (Supplementay Fig. 4), and activated the Xbp1 promoter in H4IIE GHR cells. Therefore, it was considered that GH could induce Xbp1 mRNA by ERK-dependent phosphorylation of C/EBPβ. However, overexpression of mutant C/EBPβ where Thr189 was mutated to Ala (C/EBPβT189A) also increased luciferase activity as well as C/EBPβ (data not shown). This result coincides with the report that when Chinese hamster ovary cells expressing rat GHR (CHO-GHR) were transfected with human C/EBPβT235A (Thr235 is mutated to Ala), it increased luciferase activity mediated by the c-fos promoter as well as C/EBPβ. But, when CHO-GHR were transfected with C/EBPβT235A and treated with GH, luciferase expression was not increased significantly compared with those transfected with C/EBPβ [12]. In our study, when the C/EBPβ expressed H4IIE GHR cells were treated with GH, luciferase activity did not increase (data not shown). It may be caused by the difference of the cells used. Therefore, we could not examine whether phosphorylation of C/EBPβ at Thr189 is required in response to GH. And also we could not find the difference of phosphorylated CEBPβ levels between male and female rats, because GH induces phosphorylation of CEBPβ transiently. However, Verma et al. suggested the possible involvement of ERK in episodic growth hormone regulation of Cyp2c11 that is expressed male specific [41].

The luciferase assay revealed that the two proximal C/EBPβ binding sites of the Xbp1 promoter region are important for the induction of Xbp1 by C/EBPβ. It was reported that C/EBPβ protein levels increased when 3T3-L1 cells differentiated into adipocytes, and C/EBPβ induced the Xbp1 expression by directly binding to its proximal promoter region [42]. The binding site was the same as one of the putative binding sites important for the induction of Xbp1 in the H4IIE GHR cells of our study.

GH regulates the expression of numerous secretory products such as IGF-1 and ER resident proteins in the liver. Anabolic effects of GH suggest that GH regulates the expression of the Xbp1 gene in order to accommodate enhanced levels of protein traffic through the ER. XBP1(S) activates target genes, including chaperones and components of ER-associated degradation pathways [24, 43, 44]. We observed that the mRNA levels of ERdj4 increased following increases in the XBP1(S) protein by GH administration in the livers of HPX rats, suggesting that XBP1(S) induces expression of ERdj4. However, UPR is also involved in processes having little to do with protein folding. For example, XBP1 has been shown to be required for cardiac myogenesis, hepatogenesis, plasma cell differentiation, the development of secretory tissues, cytokines production, memory formation, glucose homeostasis, and thermogenesis [45-52]. Recently, XBP1 was found to suppress lipogenic gene expression and reduce hepatic triglyceride and diacylglycerol content in the livers of obese mice [53]. Because hepatic GH signaling is needed to regulate intrahepatic lipid metabolism [54-56], GH may control lipid metabolism by inducing XBP1.

Lack of Xbp1 is embryonically lethal in mice and causes hypoplastic fetal liver, suggesting that Xbp1 is essential for hepatic growth [47]. In rats, XBP1 was identified as a transcription factor enhanced in chemically induced hepatocellular carcinoma (HCC) and has been named hepatocarcinogenesis-related transcription factor (HTF). Both the 2.0-kb and 2.5-kb Xbp1 mRNAs were induced in HCC and during liver regeneration. Because anti-XBP1 antibody decreased the growth rate of cultured HCC cells, XBP1 was suggested to participate in hepatic cellular growth as well as hepatocarcinogenesis [22]. Because GH is important for normal liver growth [57], GH might regulate the growth of the liver by XBP1.

In conclusion, we demonstrated that GH increases the expression of Xbp1 and the levels of XBP1(S) protein in rat liver. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals, suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. We also identified the ERK-C/EBPβ signaling pathway for GH-induced Xbp1 transcription (Fig. 6). This would imply that XBP1 is a mediator of GH in the sex-dependent transcriptional response in rat liver. Further studies are needed to clarify the pathophysiological significance of the activation of XBP1 by GH in the liver.

Fig. 6

Signaling pathways of Xbp1 induction by GH. When GH binds its receptor, Janus kinase 2 (JAK2) is activated, and ERK and STAT5B are phosphorylated. Then, ERK phosphorylates C/EBPβ, that binds its consensus elements and induces Xbp1(U) mRNA. Phosphorylated STAT5B may participate the induction of Xbp1(U) mRNA. PERK, ATF6 and IRE1 are inactive when they bind to immunoglobulin heavy chain-binding protein (BiP) in the ER. Upon ER stress, these proteins are released and activated. GH dose not activate PERK, ATF6 or IRE1. In HPX male rat livers, IRE1 is constitutively phosphorylated (activated) and active IRE1 removes a fragment from Xbp1(U) mRNA, switching the open reading frame. The resultant protein produced following this splicing event is a potent transcription factor, XBP1(S). XBP1(S) induces expression of genes encoding chaperones such as ERdj4.

Acknowledgments

We thank Ms. Mitsuko Kajita for sequencing the cDNA of Xbp1 and Dr. Yoko Kato for the statistical analysis. We thank NHPD-NIDDK for providing rGH, and Novo Nordisk for hGH.

Disclosure

None of the authors have any potential conflicts of interest associated with this research.

Abbreviations

GH, growth hormone; ER, endoplasmic reticulum; IREα, inositol-requiring-1α; PERK, PKR-like ER kinase; ERdj4, ER-associated dnaJ protein 4; STAT5B, signal transducer and activator of transcription 5B; C/EBPβ, CCAAT/enhancer-binding protein β; ERK, extracellular signal-regulated protein kinase; PRL, prolactin; HPX, hypophysectomized; UPR, unfolded protein response; CHX, cycloheximide; cRNA, complementary RNA; qPCR, quantitative PCR; UTR, untranslated region; ZT, zeitgeber times.

Supplementary Fig. 1

Effects of GH on Xbp1 expression in MSG-treated female rat liver. A and B, To eliminate endogenous GH secretion, female rats were treated with MSG (4 g/kg, subcutaneous) every other day from the second day of birth for ten days. GH (2 mg/ kg) or vehicle was injected subcutaneously in MSG-treated female rats (23 weeks old). The levels of 2.5 kb-Xbp1 mRNA increased 62-fold (A), and the levels of XBP1(S) protein increased 13-fold (B) 2 h after GH injection. Each group contains 4 rats.

Supplementary Fig. 2

The effect of STAT5B on activation of Xbp1 promoter. A, –1,538 to –318 bp 5'-upstream of the 2.0-kb transcriptional initiation site was used to drive expression of the Firefly Luciferase reporter gene using HeLa cells. B, The constitutively active STAT5B expression plasmid or pcDNA3.1 was transfected. Luciferase activities are shown relative to the activity of the pGL3 basic vector transfected with the STAT5B expression plasmid/pcDNA3.1, which was set to 1. Data are shown as the mean ± SE of 3 separate transfections.

Supplementary Fig. 3

GH increased the Xbp1 hnRNAs expression in rat livers. A and B, Time course of accumulation of Xbp1 hnRNA at 0, 0.5, and 1 h after in vivo GH injection (20 μg/100 g), as assessed by qPCR using oligonucleotide primers that do not discriminate between 2.0-kb and 2.5-kb Xbp1 mRNA (A) and oligonucleotide primers specific for 2.5-kb mRNA (B). The values were corrected using Gapdh as the reference gene and were presented relative to the averaged untreated male liver level, which was set to 1. Transcripts were not observed in the absence of the reverse transcription step, indicating no contamination with chromosomal DNA. Data are shown as the mean ± SE of 3 rats. * p < 0.05 for GH-treated vs. vehicle-treated rats. C, The primer pairs are depicted as arrows on the gene maps. Boxes represent the exons, and the lines represent introns. The numerical designation of the exon is listed in each box.

Supplementary Fig. 4

C/EBPβ overexpressed in H4IIE GHR cells is phosphorylated. H4IIE GHR cells were transfected with a C/EBPβ expressing plasmid. Cells were lysed and used for immunoblotting with antibodies that can detect phosphorylated C/EBPβ at the position of Thr189 of rat C/EBPβ (p-C/EBPβ), C/EBPβ, and TBP. C/EBPβ was highly phosphorylated in C/EBPβ expressing H4IIE GHR cells compared with that of control.

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