Transcription factor HMG box-containing protein 1 (HBP1) modulates mitotic clonal expansion (MCE) during adipocyte differentiation

2.1 Chemicals, reagents, and antibodies

Dulbecco's modified Eagle's medium (DMEM) and penicillin-streptomycin solution were purchased from Gibco Laboratory (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Antibodies against C/EBPα, C/EBPβ, and PPARγ were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against HBP1 and α-tubulin were purchased from Proteintech Group (Chicago, IL) and GeneTex Inc. (Irvine, CA), respectively. Antibodies against GFP, p16, p21, and p27, and the siRNA for mouse HBP1 were purchased from Santa Cruz Biotech (Santa Cruz, CA).

2.2 Protocol of adipocyte differentiation (Lii et al., 2012)

3T3-L1 preadipocytes and MEF (mouse embryonic fibroblast) cells were obtained from Bioresources Collection and Research Center (BCRC, Hsin-Chu, Taiwan) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/ml). Two days after reaching confluence (Day 0), the medium was changed to a differentiation medium (MDI) containing 10 µg/ml insulin, 0.25 mM 3-isobutyl-1-methylxanthin (IBMX), and 0.25 µM dexamethasone (DEX) (Ntambi & Young-Cheul, 2000). Two days later (Day 2), the medium was switched to DMEM containing 10 mg/L insulin and changed every other day till full differentiation.

2.3 Plasmids

Both pLKO-puro and pLKO-puro-HBP1 shRNA vectors were kindly provided by Dr. Amy S. Yee (Tufts University, MA). The pITA-GFP and pITA-HBP1-GFP expressing vectors were obtained from Dr. Xiaowei Zhang (Peking University, China).

2.4 Oil-red O staining

As described previously (Lii et al., 2012), the differentiated preadipocytes were washed twice with PBS and fixed with 10% formaldehyde for 1 hr at room temperature. After washing with distilled water three times, the cells were stained with 1.2 mg/ml Oil-red O/60% isopropanol solution for 10 min. Excess Oil-red O was washed off with distilled water. Stained oil droplets in 3T3-L1 cells were photographed under light microscopy or were extracted with isopropanol and had their absorbance measured at 490 nm.

2.5 Western blotting

Total cell lysates (50 µg) were separated by 10% SDS polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Nonspecific binding was blocked with 5% non-fat milk in TBST buffer (20 mM Tris base, 140 mM NaCl, pH 7.6, 0.1% Tween-20) for 1 hr, followed by incubation with primary antibodies at 4°C overnight. The next day, membranes were then incubated with appropriate secondary antibodies conjugated with horseradish peroxidase. The detection was achieved by measuring the chemiluminescence of the ECL blotting agent, and the images were visualized by densitometric scanning (Alliance 4.7, UVItec, Cambridge, UK).

2.6 Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR

Cellular RNA was extracted using a Quick-RNA™ MiniPrep kit (Zymo Research, Irvine, CA). The cDNA was synthesized by an iScript™ cDNA synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Real-time PCR was performed using an iQ™ SYBER® Green Supermix (Bio-Rad Laboratories). The primers and the RT-PCR protocol are described as before (Lee et al., 2013). The following primers were used: mouse HBP1, 5′-ACTCCTGTGATGAGCACATGGA-3′(F) and 5′-AGCAGTGCCAAATGGTGGAT-3′(R); C/EBPα, 5′-GAACAGCAACGAGTACCGGGTA-3′(F) and 5′-GCCATGGCCTTGACCAAGGAG-3′(R); C/EBPβ, 5′-GCAAGAGCCGCGACAAG-3′(F) and 5′-GGCTCGGGCAGCTGCTT-3′(R); PPARγ, 5′-TTTTCAAGGGTGCCAGTTTC-3′(F) and 5′-AATCCTTGGCCCTCTGAGAT-3′(R); aP2, 5′-AAGACAGCTCCTCCTCGAAGGTT-3′(F) and 5′-GCGTAAATGGGGATTTGGTCACCA-3′(R); 18S, 5′-GGGAGCCTGAGAAACGGC-3′(F) and 5′-CCGCTCCCAAGATCCAACTAC-3′(R).

2.6 Cell cycle analysis (Lee et al., 2013)

3T3-L1 cells were seeded onto six-well plates and grown until they reached confluence. On day 0, both control and HBP1 knockdown cells were treated with MDI for 6 hr and then the cells were trypsinized, washed with PBS, and fixed in cold 70% ethanol at −20°C. The fixed cells were collected and stained in a solution containing 0.5 ml of 4 µg/ml of PI, 0.5 mg/ml of RNase, and 1% Triton X-100 for 30 min at 4°C. The DNA content of these cells was analyzed using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ).

2.7 Statistical analysis

Our data are expressed as mean ± SD or mean ± SEM from at least three independent experiments. Statistical significance was analyzed using Student's t-test. Results were considered statistically significant at p < 0.05.

3.1 HBP1 expression level fluctuates during adipocyte differentiation process

To obtain clues regarding whether or not transcription factor HBP1 is involved in fat tissue formation, we first investigated the HBP1 expression pattern throughout the course of adipose formation in a cell culture model. Following standard protocol of hormonal stimulation for adipocyte differentiation (see section 2, above), 3T3-L1 preadipocyte did not display an obvious increase in HBP1 protein level until day 4, and this elevated HBP1 level was sustained till day 8 when fat drops were well-formed with a concomitant increase in PPARγ and C/EBPα expression (Figure 1a). A similar expression pattern was also observed in the mRNA level: HBP1 mRNA expression was down-regulated after differentiation medium (DMI) stimulation (within days 0 and 2) and then up-regulated gradually till terminal differentiation in 3T3-L1 cells (Figure 1b). This down-and-then-up fluctuation of the expression pattern of HBP1 during the course of adipocyte differentiation was also observed in MEF (mouse embryonic fibroblast) cells (Figure 1c), suggesting HBP1's role in the control of adipocyte differentiation and adipogenesis.

3.2 Reduced HBP1 expression impairs adipocyte differentiation

To understand if HBP1 expression level would affect the outcome of adipocyte differentiation, we used shRNA specific to HBP1 mRNA to knock down HBP1 expression in 3T3-L1 preadipocytes and score the fat drop formation following standard differentiation protocol. HBP1 knockdown (KD) (Figure 2a) significantly resulted in the reduced intensity of Oil red O staining (Figure 2b). A similar HBP1 effect was also noted happening in MEF cells (data not shown). The inhibitory effect of HBP1 KD on adipogenesis was further confirmed with reduced expression of the adipocyte markers, C/EBPα, PPARγ, and aP2, on day 8 of the differentiation program (Figure 2c). In contrast, the ectopic overexpression of HBP1 resulted in the increased accumulation of fat depots in MEF cells (with better transfection efficiency than 3T3-L1 cells) following standard differentiation protocol (Figure 2d–f). These data suggest that HBP1 indeed participates in the regulation of adipocyte differentiation process.

3.3 HBP1 regulates mitotic clonal expansion (MCE)

The growth-arrested 3T3-L1 preadipocytes were able to be induced to differentiate via synchronous reentry of the cell cycle with approximately two rounds of division (referred to as MCE) before terminal differentiation (Tang et al., 2003b). This MCE is shown to be a prerequisite for the expression of the adipocyte genes that give rise to the adipocyte phenotype in the culture model, such as 3T3-L1 preadipocyte (Tang et al., 2003b). Since HBP1 is known to be a negative regulator of G1-to-S transition during cell cycle progression (Shih et al., 2001; Tevosian et al., 1997), it is, therefore, rational to speculate as to the effect of HBP1 as a mediator of the MCE progression. Indeed, HBP1 KD hastened cell cycle progression as demonstrated by the earlier entry into the G2/M phase as compared with the none HBP1-KD control (Figure 3a), and it was accompanied by a concomitant increase in C/EBPβ expression (Figure 3b). These data suggest that HBP1 down-regulation and/or C/EBPβ up-regulation may be crucial for the reentry of growth-arrested preadipocytes into cell cycle mode to initiate MCE.

3.4 HBP1 modulates C/EBPβ expression during MCE stage

Since both HBP1 and C/EBPβ have been established to be involved in cell cycle regulation, we then examined whether or not C/EBPβ is a target gene of HBP1's modulation of the cell cycle program during the MCE stage. Observing the gene expression pattern of both transcription factors, we found that HBP1 and C/EBPβ expressed themselves reciprocally during the first 48 hr of the MCE stage: HBP1 expression was dramatically down-regulated upon stimulation of a differentiation medium (MDI: IBMX, DEX, and Insulin [Ntambi & Young-Cheul, 2000]) and then increased gradually at the late stage of MCE, whereas C/EBPβ expression was up-regulated following MDI stimulation and then decreased as the MCE stage progressed (Figure 4a). This observation means that C/EBPβ might be a target gene of HBP1 transcription repression. To test this hypothesis, we overexpressed HBP1 by transecting HEK-293T cells with the HBP1-GFP expressing plasmid followed by IBMX treatment, a direct inducer of C/EBPβ expression (Cao et al., 1991; Yeh et al., 1995). As shown in Figure 4b, ectopic overexpression of HBP1 resulted in a dose-dependent suppression of C/EBPβ protein levels regardless of the presence or absence of IBMX. In contrast, HBP1 siRNA led to increased expression of C/EBPβ protein in MEF cells (Figure 4c). A similar HBP1 effect pattern on C/EBPβ mRNA was also found in MEF cells (see Figure 4d), suggesting that HBP1 may function as a transcription repressor of C/EBPβ.

The treatment of MDI allows growth-arrested preadipocytes to enter proliferation mode. With that in mind, we further investigated the effect of MDI on HBP1 activation and found that HBP1 was transcriptionally regulated upon MDI administration; either MDI or IBMX alone suppressed HBP1 expression (Figure 5a) and a 2-kb HBP1 promoter activity (Figure 5b) in a dose-dependent manner. In contrast, DEX, a C/EBPδ activator (Cao et al., 1991), induced HBP1 expression at a high concentration (Figure 5a). These data indicate that HBP1 expression during an adipocyte differentiation program was tightly regulated by hormonal stimulation.

3.5 HBP1 regulates the transition from the cessation of the MCE to the onset of terminal differentiation

In trying to understand the role of HBP1 in adipocyte differentiation, so far, our data indicate that HBP1 KD accelerates the entry of growth-arrested preadipocyte into the cell cycle mode, leading to an impaired adipogenesis at the end. This observation prompted us to hypothesize that HBP1 expression might be required for the termination of the MCE and/or the onset of the terminal differentiation. Indeed, our experimentation showed that HBP1 KD led to delayed termination of the MCE as evidenced by an increased cell count at 48 and 72 hr (referred to as days 2 and 3) following MDI stimulation (Figure 6a). Similar to HBP1, C/EBPα is also antiproliferative (Freytag, Paielli, & Gilbert, 1994). Therefore, we examined the C/EBPα expression pattern during the time course of the MCE following MDI stimulation and found that C/EBPα and HBP1 displayed similar expression patterns. In particular, the expression of both genes increased at the late phase of the MCE (Figure 6b), which suggests both genes have an antiproliferative function. Finally, HBP1 KD resulted in the suppression of C/EBPα expression on day 2 (Figure 6c). These data indicate that HBP1 might regulate C/EBPα expression to terminate the cell cycle mode at the end of the MCE and then commit cells for terminal differentiation.

Several cell cycle inhibitors have been widely studied in the control of preadipocyte differentiation (Inoue et al., 2008; Phelps & Xiong, 1998; Reichert & Eick, 1999). HBP1 has been shown to be a positive regulator of cell cycle inhibitors p16 (Li et al., 2010; Wang, Pan, Chen, Huang, & Zhang, 2012) and p21 (Chen et al., 2016); therefore, we also examined the expression pattern of p16 and p21 along with p27 during preadipocyte differentiation. The result indicated that both p16 and p21 exhibited a similar fluctuating pattern with HBP1 (data not shown). In addition, HBP1 KD resulted in decreased expression of p16, p21, and p27 on day 2 of adipocyte differentiation, indicating that HBP1-mediated transcription of p16 and p21 may be also applicable in the context of HBP1 regulation of the MCE during adipocyte differentiation (Figure 6d).

Taken as a whole, our data support the consideration of a novel role of HBP1 in the regulation of adipocyte differentiation (Figure 7). HBP1 expression is down-regulated at the beginning of the MCE to ensure the up-regulation of C/EBPβ and re-entry into postconfluent mitosis. However, HBP1 expression is up-regulated at the end of the MCE to ensure the up-regulation of C/EBPα and the termination of the MCE as well as the onset of terminal differentiation.