The biological function of Id proteins is achieved through antagonizing the activity of their associated bHLH transcription factors (1
). Although the molecular mechanism involved in this process has been well dissected, additional regulators should work in concert with Ids in view of the spatio-temporal variation of Id-controlled gene expression. In this study, the LIM-only protein FHL2, a well-known cofactor or adaptor of several transcription factors (19
), was identified as a novel Id2 interactor. In addition, the interaction of FHL2 with three other members of the Id protein family (Id1, Id3 and Id4) was demonstrated in GST-pulldown in vitro
assays. We further show that FHL2 blocks Id–E47 coupling by a competitive binding mechanism, which subsequently represses the inhibitory effect of Ids on E47-mediated transcription. Finally, the FHL2 regulation of the E47–Id2 transcriptome was extended to the proliferation of the neuroblastma cells. These findings underscore an important function of FHL2 in regulating the Id2 signaling pathway.
The LIM domain-containing proteins can function as adaptors or scaffolds to support the assembly of multimeric protein complexes and can operate as competitors, autoinhibitors and localizers (38
). As a member of the LIM protein family, FHL2 has been shown to associate with several transcriptional factors or signaling transducers such as AR, CBP/p300, β-catenin, FOXO1, AP1 and TRAF6, to regulate signal transduction and gene expression (19
). In most cases, FHL2 protein appears to participate in functional complexes to modulate the tissue-specific activity of activators or repressors. One possible explanation for the dual role of FHL2 in a transcription or regulating signaling pathway might be that FHL2 acts to stabilize the functional complexes as a type of bridging factor. This explanation was supported by the fact, like that the formation of a ternary complex by FHL2, CBP/p300, and β-catenin could synergistically activate AR-mediated transcription (28
), and that FHL2 interacts with titin, a protein that plays a crucial role as organizer of the sarcomere, and functions as an adaptor molecule that links the metabolic enzymes MM-creatine kinase, adenylate cyclase and phosphofructokinase to titin, thereby helping to recruit metabolic enzymes needed for energy provision during muscle contraction (40
). In dramatic contrast, we observed that FHL2 acts as a competitor of E47 for Id2 binding and subsequently allows E47 to recruit to its target DNA and execute its transcriptional activity ( and ). Although different epitopes of Id2 are required for FHL2 and E47 binding, the binding site for FHL2 or E47 of Id2 may be masked by a conformational change of Id2 when it is occupied by FHL2 or E47. In addition, Id2 may not provide enough surface space to simultaneously interact with multiple proteins; after all, Id2 is only 18 kDa. Indeed, such competitive binding is also observed for other FHL2-interacting molecules. For example, the cytoplasmic domain of TRAF6, which is formed by few amino acid numbers, can provide a binding surface for only FHL2 or RANK. As a consequence, FHL2 antagonizes the RANK–TRAF6 interaction and blunts the RANK–TRAF6 signaling (41
Emerging evidence has linked FHL2 to cell-cycle progression. However, the function of FHL2 in cell proliferation is particularly intriguing, because it may exert a positive or negative effect on cell-cycle-regulated processes in a tissue-dependent fashion. This dual nature of FHL2 can be explained by the finding that FHL2 can function as a repressor or activator of transcriptional activity depending on the cell type. For example, FHL2 promoted SKI-induced proliferation of UCD-Mel-N melanoma cells and enhanced proliferation of MDA-MB-231 human breast cancer cells by regulating cell-cycle-dependent p21 expression (42
). Loss of FHL2 downregulated the expression of cyclin D1 and greatly reduced the proliferative capacity of mouse embryo fibroblasts (44
). In contrast, FHL2 antagonized the proliferation of differentiated myoblasts by downregulating cyclin D1 (45
) and suppressed liver cancer cell growth through a TGF β-like pathway (24
). In this study, we demonstrated an antagonistic effect of FHL2 on Id2-driven proliferation of neuroblastoma cell types. The p57Kip2, IGF2
genes, belonging to the human chromosome 11p15.5-imprinted cluster, which have crucial functions in differentiation, the cell cycle and oncogenesis (46–48
), are within the small group of E47–Id2 targets (27
). In accordance with a previous report (27
), we also demonstrated that the expression of these genes was strongly induced by E47 and inhibited by Id2. Among them, p57Kip2
is one of the three members of the Cip/Kip family of cyclin-dependent kinase inhibitors and a well-known cell-cycle regulator (49
), the induction of which is essential for E47-mediated inhibition of the cell cycle in neuroblastoma cells (27
). Ectopic expression of FHL2 in neuroblastoma cells strongly induced the expression of p57Kip2, IGF2
genes by regulating the Id2–E47 transcriptome (). Given the oncogenic activity of Id2 in tumors from the nervous system, our data suggest that FHL2 possesses a tumor suppressor function in these cell types.
Id family proteins were implicated in the control of differentiation in organisms from fly to human (1
). There is general agreement with the notion that differentiation of a variety of cell types requires elimination of the Id function. Thus, the functional inhibitors of Id proteins might possess the potential to facilitate cell differentiation. Here, we demonstrated a reverse expression relation between Id2 and its functional repressor FHL2 in differentiated neuroblastoma cells induced by RA (). In addition, independent evidences suggested a diverse role of FHL2 and Id proteins during the differentiation process of myoblasts and osteoblasts (45
). These limited data implied that a refined regulation mechanism of Id function by FHL2 is required during the processes of development and cellular differentiation.
In the past decade, numerous reports demonstrated the aberrant accumulation of Id proteins in solid tumors and Ids facilitating tumorigenesis by repressing cell differentiation, stimulating cell proliferation and promoting tumor neoangiogenesis (5–9
). As a functional suppressor of Id2, FHL2 is frequently deregulated, which includes being overexpressed and down-regulated in various types of tumors (21
). The most common mechanism selected by tumor cells to activate Id2 function is to elevate the expression of Id2 gene. In this study, we demonstrated that the expression of FHL2 is very weak and even negligible in human neuroblastoma cells. Based on our findings, we suggest that tumor cells may target another level in Id biology regulation. The fact that RA induced the reverse expression of Id2 and its functional repressor FHL2 () indicated that FHL2 may be an important stalker against Id2 protein activity in neuroblastoma cells. Loss of FHL2 will release the oncogenic activity of Id2 and may contribute to tumor progression. Testing the ratio of FHL2 to Id2 expression in human neuroblastoma cells might be beneficial to differentiate different clinopathological stages.
In conclusion, these findings provide definite evidence for FHL2–Id2 interaction and its corresponding functional effect. The functional activity of FHL2 acting as an repressor of the oncogenic activity of Id2 in neuroblastoma cells suggests that loss of FHL2 may be involved in tumorigenesis and progression of the nervous system.