Human cervical carcinoma (HeLa) cells and hepatocellular carcinoma (HepG2) cells were obtained from the American Tissue Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ and 95% air incubator.

Site-directed mutagenesis to inactivate the CREB binding site was carried out within the p-210 construct containing 262 bp of the 5′-flanking sequence of the USP22 gene using the MutanBEST kit (Takara, Shiga, Japan) according to the manufacturer’s protocols. The following primers: 5′-GTagCGTAATCTCCGTCCGC-3′ (sense) and 5′-CCCCCCCCCCCCTCCCGGCC-3′ (antisense) were utilized and the mutation was confirmed by DNA sequencing.

CREB-1 full-length cDNA was obtained by reverse transcription PCR (RT-PCR). Briefly, total RNA was obtained from HeLa cells and reverse-transcribed with SuperScript™ II primed by oligo(dT)₁₅ (Takara). cDNA products were amplified using the following PCR primers: 5′-AGGATCCATGACCATGGAATCTGGAGCC-3′ (sense) and 5′-GGAATTCTTAATCTGATTTGTGGCAGTA-3′ (antisense), digested with EcoRI and BamHI, and inserted into pCDNA 3.1(+).

Cells were plated in 24-well plates 24 h before transfection, with each well treated with 0.5 μg of promoter construct, 0.1 μg of pRL-TK (Promega, Madison, W, USA), and Lipofectamine 2000 (Invitrogen). All transfection experiments were repeated five times, with cells lysed 24 h after transfection and lysates used for the dual luciferase assays (Promega) according to the manufacturer’s protocol. The normalized luciferase activity was expressed as a ratio of firefly luciferase activity to Renilla luciferase for each sample.

ChIP assays were performed using the EZ-ChIP kit according to the manufacturer’s instructions (Pierce, Rockford, IL, USA). Approximately 10⁸ cells were fixed by adding formaldehyde to a final concentration of 1% and incubated for 30 min at room temperature with modest shaking. Thereafter, the cells were washed twice with cold phosphate-buffered saline, the pellet was resuspended and lysed, and the nuclei were isolated and sonicated until the chromatin had an average length of 400–500 bp. Following centrifugation, the supernatant was incubated with 1 μg of anti-CREB1 antibody or rabbit IgG overnight at 4°C for immunoprecipitation. The following day, magnetic protein-G beads were added and further incubated at 4°C for 1 h. After appropriate washing, the antibody-transcription factor-DNA complex was eluted from the beads, formaldehyde cross-links were reversed, and proteins were digested with proteinase K at 67°C overnight. DNA was purified and used for PCR with the primers, 5′-GGGCGGGCCCTCGTTAGCTT-3′ and 5′-GGAGGCTGCAAGGCAGGCAC-3′.

One day before transfection, HeLa cells were plated in 24-well plates at a density of 5×10⁴ cells/well, and then transfected with 20 nM of siControl Non-Targeting siRNA duplex (siControl) or human CREB1-specific siRNA duplex (sc-29281; Santa Cruz Biotechnology, Santa Cruz, CA, USA), using an siRNA reagent system (Santa Cruz Biotechnology) according to the manufacturer’s instructions. After 24 h, the siRNA-transfected cells were harvested for real-time PCR or transfected with p-210 for the luciferase assay.

Cells were grown to confluency and RNA was extracted using TRIzol^® reagent (Invitrogen). cDNA was synthesized from 1 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen) and random hexamers in a total volume of 20 μl according to the manufacturer’s instructions. The GAPDH gene was used as an endogenous control to normalize for RNA loading. qRT-PCR was performed using the SYBR-Green PCR Master Mix (Toyobo, Osaka, Japan) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), with USP22 primers as previously described and validated (11).

Cells were lysed with 1X SDS sample buffer, and the protein was separated by 10% SDS-PAGE, and electroblotted onto a nitrocellulose membrane. The membrane was then incubated with the anti-USP22 antibody (Santa Cruz Biotechnology), the anti-CREB1 antibody (Santa Cruz Biotechnology), or the anti-GAPDH antibody (Santa Cruz Biotechnology) and visualized using the Pierce ECL system according to the manufacturer’s instructions.

Data are presented as mean ± SEM. Statistical differences between sample means were determined using an unpaired, two-tailed Student’s t-test, with P<0.05 deemed significant.

Analysis of the basic USP22 promoter region by the TFSEARCH program revealed a putative CREB binding site (GTGAGCTAA) at position −113 to −105 (Fig. 1A) with a 100 point score, which is highly conserved among several mammalian species, including the mouse and rat. To evaluate the impact of this CREB motif on USP22 promoter activity, site-directed mutagenesis was introduced within the basic promoter p-210, and the resulting plasmids were transiently transfected into the HeLa and HepG2 cells. The p-210/CREBmut construct showed an ~45% decrease in luciferase activity in both the HeLa and HepG2 cells compared to the wild-type p-210 (Fig. 1B), suggesting that the CREB site is critical for constitutive USP22 promoter activity in the cells tested.

To determine whether CREB directly binds to the USP22 promoter in vivo, chromatin immunoprecipitation (ChIP) was performed using cultured HeLa cells. Chromatin was sonicated into ~400–500-bp fragments and precipitated using the anti-CREB-1 antibody or rabbit IgG (negative control). The precipitated DNA was subjected to PCR using primers designed to amplify a 272-bp fragment of the USP22 promoter encompassing the CREB site. Following immunoprecipitation, the samples treated with the anti-CREB-1 antibody yielded a 272-bp PCR product, while no PCR amplification product was detected following IgG incubation (Fig. 2). These results indicate that CREB-1 constitutively occupies this region of the USP22 promoter, and thus, is likely to contribute to basal transcriptional activation.

To determine whether CREB is necessary for basal USP22 transcription, siRNA-mediated knockdown was employed. CREB-specific siRNA resulted in an ~80% reduction in CREB protein expression relative to the control (Fig. 3A), which resulted in a significant decrease in p-210 (WT) luciferase activity, with no change in luciferase activity observed in the control group (Fig. 3B). To confirm CREB regulation of the USP22 promoter via the CREB site, HeLa cells were co-transfected with CREB siRNA and the p-210/CREBmut plasmid. This showed that siRNA-mediated knockdown had no significant impact on p-210/CREBmut promoter activity (Fig. 3C). Furthermore, the effect of CREB deletion on endogenous USP22 expression was determined by monitoring USP22 expression following CREB siRNA transfection via qRT-PCR. The results showed that siRNA-mediated knockdown of CREB led to a significant reduction in USP22 mRNA expression levels (Fig. 3D).

Since it was now established that CREB is required for USP22 transcription, the effect of exogenous CREB on USP22 transcriptional activity was further examined through the transfection of HeLa cells with CMV-driven CREB expression plasmids pCDNA/CREB (Fig. 3E). The results showed that forced expression of exogenous CREB did not affect USP22 promoter activity (Fig. 3F). Accordingly, exogenous CREB did not promote endogenous USP22 mRNA expression (Fig. 3G). These results suggested that CREB behaves as a constitutive rather than an inducible element for USP22 transcription.

It has been reported that CREB transcriptional activity is controlled by several upstream signaling pathways. Therefore, HeLa cell USP22 promoter activity was pharmacologically targeted using H-89, staurosporine and wortmannin to selectively inhibit PKA, PKC and PI3K, respectively. Based on published reagent concentrations (12–14), HeLa cells were treated with 30 nM of H-89, 10 nM of staurosporine, or 10 nM of wortmannin for 6 or 12 h following a 24-h transfection with p-210. The dual luciferase assay showed that H-89, but not staurosporine or wortmannin, decreased promoter activity in a time-dependent manner (Fig. 4A), thus, indicating that the PKA signaling pathway could play a positive role in the regulation of USP22 transcription.

To determine whether H-89 suppresses endogenous USP22 expression, HeLa cells were treated with 15, 30 or 60 nM of H-89 for 12 h, with both USP22 mRNA and protein expression levels analyzed. As shown in Fig. 4B and C, 30 nM of H-89 induced a very significant decrease in USP22 mRNA and protein expression in the HeLa cells. Furthermore, HeLa cells were treated with forskolin, which raises intracellular levels of cAMP. Unexpectedly, incubation with 10 μM of forskolin did not increase USP22 promoter activity or endogenous mRNA expression (Fig. 4D and E). These results indicate that the PKA pathway perhaps plays a constitutive rather than an inducible role in USP22 promoter activity.

To delineate the region(s) that respond to H-89, a series of 5′ truncated promoter fragments derived from p-210 were developed and the HeLa cells were transfected. A dual luciferase assay showed that USP22 promoter activity was serially decreased when the 5′ terminal of P-120 was continuously cut. Furthermore, when the 5′ terminal of P-120 was cut by 92 bp (P-118), a loss of H-89 responsiveness was noted (Fig. 4F), suggesting that the H-89 response region is located between −129 bp and −118 bp which contains the functional CREB binding site. To determine whether H-89 decreases USP22 expression via the CREB binding site, HeLa cells were transfected with wild-type p-210 or p-210/CREBmut and incubated with H-89 for 12 h. A dual luciferase assay showed that a mutation of the CREB binding site abolished H-89 responsiveness (Fig. 4G), collectively suggesting that the PKA pathway regulates USP22 expression and its effects may be partly mediated by CREB.