Abstract
A zinc-finger gene in autoimmune thyroid disease susceptibility region (ZFAT) was originally identified as a highly conserved immune-related transcriptional regulator containing one adenosine-thymidine (AT)-hook and 18 C2H2-type zinc-finger domains. Subsequently, roles of ZFAT in development, primitive haematopoiesis, angiogenesis, immune responses and several common diseases, such as multiple sclerosis, hypertension and cancer, have been demonstrated. Previously, we recorded a ZFAT protein expression in MOLT-4 human acute T-lymphoblastic leukaemia cells, while ZFAT knockdown activated caspases and induced apoptosis in these cells. Hence, the precise functions of ZFAT are of particular interest in cancer research. In this article, we have reviewed investigations on the roles of ZFAT in haematopoietic and angiogenesis, and discussed the possible involvement of ZFAT in haematopoietic malignancies.
Recent studies have indicated a central role for the aberrant expression of transcription factors in the pathobiology of haematopoietic malignancies. Deregulated expression of these transcription factors, which are often functionally normal, leads to abnormal proliferation and differentiation arrest of lymphoid progenitors (1).
Through linkage and association analyses in a cohort of Japanese patients with autoimmune thyroid disease (AITD) (2, 3), we identified a zinc-finger gene in the AITD susceptibility region (ZFAT) (3). ZFAT contains 18 zinc-fingers domains and one AT-hook and is an evolutionally conserved gene from fish to human. ZFAT protein is highly expressed in T- and B-cells in the lymphoid tissues in adult mice (4) and plays critical roles in peripheral T-cell homeostasis and its receptor-mediated responses (5). We previously reported that Zfat deficiency (Zfat−/−) in the mouse is embryonically lethal by embryonic day 8.5 and ZFAT is a critical transcriptional regulator for primitive haematopoiesis (6) and is functionally involved in regulation of apoptosis of mouse embryonic fibroblasts and MOLT-4 human acute T-lymphoblastic leukaemia cells (7, 8). We also found that ZFAT is expressed in human umbilical vein endothelial cells (HUVECs) and demonstrated that endothelial cell assembly and the branch point formation of capillary-like structures in HUVECs are impaired by the reduction of ZFAT expression through the use of ZFAT miRNA. These studies suggest that ZFAT is a key mediator of the development of specific cell lineages such as lymphocyte and endothelial cells, though the molecular and functional details in cancer development have not been determined. Here, we focus on the roles of ZFAT in the haematopoietic system and angiogenesis, and discuss the possible involvement of ZFAT in haematopoietic malignancies.
The Role of ZFAT in the Haematopoietic System
During embryonic development, mesodermal progenitors give rise to haemangioblasts, which have differentiation potential for both endothelial and haematopoietic lineages (9-11). Haemangioblasts arise in the primitive streak and then migrate into extra-embryonic yolk sacs to form blood islands (12, 13). Blood islands are the foci of haemangioblasts, which form a luminal layer of endothelial cells that produce haematopoietic progenitor cells, and are eventually assembled into a functional vascular network that transfers nutrients from the yolk sac to the embryo proper (14, 15). Recent studies have revealed that T-cell acute lymphocytic leukemia 1 (SCL/TAL1), a basic helix–loop–helix transcription factor, is essential for differentiation of haemangioblasts into haemogenic endothelium (9, 16-20). TAL1 also plays pivotal roles in vascular and haematopoietic development when in complex with LIM domain only 2 (LMO2) and GATA-binding protein-1 (GATA1) (17, 21-25). LMO2 functions as a bridging molecule between TAL1 and GATA1 in the DNA-binding complex (22). GATA1 also functions as a key molecule in the differentiation of erythroid lineages (26, 27). However, transcriptional regulation of upstream genes remains elusive. Recently, we found that Zfat−/− mice are embryonic-lethal, with impaired differentiation of haematopoietic progenitor cells in blood islands, precisely where ZFAT is highly expressed. Expression levels of Tal1, Lmo2 and Gata1 in Zfat−/− yolk sacs are greatly reduced compared with those of wild-type mice, and chromatin immunoprecipitation (ChIP)–polymerase chain reaction (PCR) analysis revealed Zfat binding to promoter regions of these genes in vivo. Furthermore, profound reductions in Tal1, Lmo2 and Gata1 protein expression were observed in Zfat−/− blood islands (6). We also found that vascular endothelial growth factor receptor-2 (Vegfr2), runt-related transcription factor 1 (Runx1) and integrin alpha 2b (platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41) are directly regulated by the Zfat targets Tal1, Lmo2 and Gata1 (6) (Figure 1). These results suggest that Zfat is indispensable for mouse embryonic development, and functions as a critical transcription factor in primitive haematopoiesis. Currently, we are generating knock-in reporter mice to elucidate ZFAT expression during embryogenesis.
Role of ZFAT in Angiogenesis
ZFAT mRNA was ubiquitously expressed in tissues, but had lower expression levels in the non-immune-related tissues than in the immune-related tissues (3). This suggests that ZFAT might also play physiological roles in non-immune-related cells. Recently, genetic variants of ZFAT are reportedly associated with adult height in Japanese and Korean populations (28, 29), equine body size (30, 31) and with several other diseases including hypertension and cancer (32, 33). It is of great interest that a genetic variant of ZFAT was found to be strongly associated with interferon-β responsiveness in multiple sclerosis (34). These findings suggest that ZFAT might have critical roles in non-immune-related cells involved in human diseases or altered physiological phenotypes.
Angiogenesis is the cellular mechanism by which the primitive vasculature is remodeled into a mature vascular bed comprising arteries, capillary networks and veins (35). Vascular remodeling is an active process of structural alteration that involves changes in cellular processes, including cell growth, cell death and cell penetration into the extracellular matrix (36). The mechanisms of vascular remodeling, in which endothelial cells play pivotal roles, include penetration via the sprouting and branching of vessels into avascular regions. This is observed under physiological and pathological conditions, such as wound healing, neovascularization in tumors, inflammation, autoimmune diseases and obesity (37-40). We previously found evident expression of ZFAT in HUVECs, and evaluated the physiological roles for ZFAT in the angiogenic responses of HUVECs (41). We established HUVEC transfectants expressing ZFAT miRNA through the use of lentiviruses. The relative growth rates for HUVECs and HUVEC transfectants with ZFAT miRNA were not significantly different, indicating that the ZFAT in HUVECs is not involved in proliferation or apoptosis, which are essential components of vascular remodeling. The HUVEC transfectants with the control miRNA manifested assembly into capillary-like structures and the branch point formation of interconnected capillary-like structures, whereas the capillary-like network formations of the HUVEC transfectants with ZFAT miRNA were dramatically impaired. We further quantified the number of the segments and the segment lengths between these transfectants. The number of the segments of the HUVEC transfectants with ZFAT miRNA decreased, indicating impaired branch point formation in the capillary-like structures caused by the reduction in ZFAT expression. Furthermore, the average segment length of the capillary-like structures in the HUVEC-transfectants with ZFAT miRNAs was significantly higher, indicating that impaired branch point formation due to the reduction of ZFAT expression culminated in the increase in the length of each segment in the capillary-like structure (Figure 2). Indeed, Lazrak et al. demonstrated that ectopic expression of wild-type TAL1 accelerated the formation of capillary-like structures in vitro and in vivo (42). These findings suggest that ZFAT may regulate TAL1 in HUVECs and play various physiological roles depending on the cell type and environmental conditions.
Role of ZFAT in Cancer Development
We previously showed that ZFAT regulates apoptosis in MOLT-4 human T-cell acute lymphocytic leukaemia (T-ALL) cells (8) and recently found ZFAT expression in other types of haematopoietic cancer cell lines (data not shown). Another recent study also demonstrated a correlation between ZFAT copy numbers and ovarian cancer (32). These studies indicate critical roles of ZFAT in cancer progression.
For example, the most frequent targets of genetic alterations in human lymphoid leukaemias are transcription factor genes with essential functions in blood cell development. During early haematopoietic development, TAL1 is required for the generation of all blood cell lineages (17) and haemogenic endothelium (9), but it is not required for the generation and function of haematopoietic stem cells during adult haematopoiesis (43). TAL1 is overexpressed in T-ALL as a result of the t(1;14) translocation or site-specific deletions in approximately one-fourth of childhood T-ALL cases (44). However, these two mechanisms cannot account for all instances of TAL1 overexpression in this disease, indicating that other mechanisms, such as ZFAT overexpression, are involved in these processes. The constitutive expression of ZFAT is suggested to induce subsequent tumour growth and angiogenesis through TAL1 activation (Figure 3). To confirm this hypothesis, we will investigate expression levels of ZFAT and TAL1 in different kinds of haematopoietic cancer cell lines.
Conclusion
In summary, ZFAT is an essential signalling molecule in haematopoietic development, angiogenesis and cancer. Deregulation of other ZFAT targets, such as LMO2 (45) and GATA1 (46) are also suggested to be critical in haematopoietic malignancies, and future studies will elucidate the detailed mechanism for the transcriptional regulation of ZFAT targets.
Footnotes
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Conflicts of Interest
The Authors declare no conflict of interests.
- Received April 4, 2013.
- Revision received June 3, 2013.
- Accepted June 4, 2013.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved