ZBP-89 and Sp1 contribute to Bak expression in hepatocellular carcinoma cells

The Specificity protein (Sp) family members are GC boxes and CACCC boxes binding transcription factors, which are reported to participate in development, differentiation and tumorigenesis [1, 2]. Sp1 is also a C2H2-type zinc fingers Krüppel-like factor, and it is the first identified transcription factor [3]. Numerous studies showed that Sp1 not only acted as a basal transcription factor, but also contributed to the regulation of many vital cellular genes [4, 5]. Overexpressed Sp1 differentially regulated a large number of genes which promoted cancer development, angiogenesis and metastasis [6]. Sp3 is very similar to Sp1 both in structure and regulatory function. Sp3 and Sp1 showed equal affinity to GC box, indicating the potential competition in genes expression regulation [7]. Reversely, Sp3 mediated transcriptional repression when it binded to GC-box, because Sp3 contained a suppressive domain [8]. Additionally, Sp3 lacked the multi-merization domain, and thus Sp3 did not have the ability to super-activate promoters by bridging multiple Sp binding sites [9].

ZBP-89, also known as ZNF148, is a Krüppel-type zinc-finger transcription factor that binds to GC-rich sequences to activate or suppress gene transcription [10]. ZBP-89 is also a member of the C2H2 zinc finger family subclass, as well as Sp family members. It is known that ZBP-89 participates in many genes transcription, such as Vimentin, Gastrin, p16, and Ornithine decarboxylase et al. These biological procedures are involved in cell growth, cell cycles, metabolism and T cell immunity. Study showed ZBP-89 competed with Sp1 for binding to induce Gastrin expression when ZBP-89 functions as a repressor [11]. Interestingly, because Sp1 itself did not bind to the promoter element, ZBP-89 interacted with Sp1 to suppress Vimentin expression in vitro [12]. In another case, ZBP-89 binded to the basal promoter of Pdcd4, also interacted with Sp family members to induce Pdcd4 protein expression [13].

Bak gene belongs to a large Bcl2 family, and acts as a pro-apoptosis protein to facilitate cellular apoptosis. It penetrates and makes pores in the mitochondrial membrane through forming heterodimer with Bax [14, 15]. The penetration releases cyto C into cytoplasm, and classic apoptosis signal pathway in the end [16, 17]. Currently, several cis-elements and associated factors are required to regulate expression of the bak gene. It was reported that Sp3 competed with Sp1 to transcriptional binding sites to increase Bak expression [18]. Up-regulation of Bak by butyrate in the colon was associated with increased Sp3 binding which did not contain a multimerization domain [18]. Instead, it contains an inhibitory domain located at N-terminal to its DNA-binding domain, which mediates transcriptional repression in some instances. Mithramycin A (MMA) is a GC-rich DNA binding chemical, which is also regarded as potent Sp family members inhibitor [19]. In the Bak promoter analysis, there are many GC-rich sequence domain, indicating the possibility regulation by MMA,Sp family and ZBP-89 transcription factor. Previously, we reported that ZBP-89 bound to Bak gene promoter, and epigenetically regulated its transcription companied with histone deacetylase 3 and DNA methyltransferase 1 [20]. Therefore, we speculate that ZBP-89 and Sp1/Sp3 are involved in hepatocellular carcinoma (HCC) and Bak induction. This study focuses on the elucidation of transcription factor Sp1/Sp3 and ZBP-89 involved Bak expression.

Bak is a major cell death initiator in the apoptotic signaling cascade [25]. It plays a significant role in the therapy and survival of HCC. It has been reported that different agents induce apoptosis in HCC cells by inducing Bak expression, but the mechanism responsible for its regulation in HCC is largely unknown.

Sp1 is a ubiquitous transcription factor and its regulated genes include those involved in proliferation, apoptosis, senescence and angiogenesis. Sp3, which competes with Sp1 for GC-box binding sites, is normally a weak transcriptional activator. However, it could function as a transcriptional activator with similar potency to Sp1 in the absence of acetyltransferases [26]. A similar switch of Sp1 for Sp3 has been observed at the Bak promoter following treatment with the HDAC inhibitors butyrate [18]. However, the relationship between Sp1, Sp3 and Bak is unknown. After the analysis of TCGA HCC database, we observed that Bak transcription in cancer tissue was higher than that in non-cancer tissues, and its transcription was correlated with Sp1 and Sp3 transcription level. No correlation was found between Bak and ZBP-89, neither Sp2 nor Sp4 (data not shown). Further analysis found Bak and Sp1 transcription level were significantly correlated with patients survival. These results illustrated that Bak played an important role in the hepatocarcinogenesis or HCC therapy. In Western blotting analyses, we showed that all selected cell lines (HepG2, Hep3B, PLC/PRF/5,Huh-7, HKCI3, HKCI-4, LO2, and MIHA) were able to constitutively express endogenous Bak. The base level of Bak can be greatly enhanced by MMA (30 nM) treatment and the increase in Bak is functional in terms of apoptotic induction in PLC/PRF/5 cells.

MMA functions as GC-rich DNA sequence binding chemical, acting as a Sp family members’ inhibitor. It was shown to induce apoptosis through increase of Bak expression in colon cancer cells [27]. We observed a regulation of Bak protein level after overexpression or knockdown of Sp1. Therefore we raised the question whether ZBP-89 could compete or cooperate with Sp1 to regulate the expression of the Bak gene. Indeed, Western blotting data showed MMA could not induce Bak expression if without ZBP-89 participation (Fig. 5). Additionally, when cells was treated with ZBP-89 siRNA, the consequence also turned down Bak expression even if Sp1 overexpression (Fig. 5, lane 1, 5th band). Results revealed that Bak expression required ZBP-89 and Sp1 to work simultaneously.

ZBP-89 and Sp family members are members of the zinc finger transcription factor and also subclasses of the C2H2 zinc finger family. ZBP-89 can efficiently inhibit cellular proliferation and induce apoptosis in human cancer cells [28]. In our previous study, we have identified that ZBP-89 might target at − 457 to − 407 region of Bak promoter to induce its expression and subsequently apoptosis [20]. Given the same bifunctional regulatory domains, ZBP-89 and Sp1 can interact to regulate a variety of genes. ZBP-89 appears to interact with Sp1, as binding to an overlapping site in DNA to regulate gastrin, two type I collagen genes [29], and ornithine decarboxylase [30]. In another aspect, many findings also suggested that Sp1 and ZBP-89 regulated target genes in a cooperative manner, like vimentin [12], Pdcd4 [13] and p21waf1/cip1 [31]. In agreement with peers’ finding, we further showed the cooperative function of ZBP-89 and Sp1 on Bak transcription. Besides, the study also revealed their association in patient survival, as well as the Bak expression level.

Our findings suggested that ZBP-89 and Sp1 overexpression induced Bak expression in a genetic manner. Increased Bak level was associated with poor patient survival, whereas high level of Sp1 is a beneficial factor for patient survival.