Masayoshi Imagawa

Wnt4 and Wnt5a promote adipocyte differentiation.

The roles of the non‐canonical Wnt pathway during adipogenesis are not well known, though Wnt10b is known to function as a negative regulator for adipogenesis by activating the canonical Wnt pathway. We focused on the roles of Wnt4, Wnt5a and Wnt6, which are thought to be part of the non‐canonical Wnt pathway. The expression of these genes changed dramatically at the initial stage of adipogenesis. Furthermore, the inhibition of Wnt4 or Wnt5a expression prevented the accumulation of triacylglycerol and decreased the expression of adipogenesis‐related genes. Wnt4 and Wnt5a have crucial roles in adipogenesis as positive regulators.

Silencer binding proteins function on multiple cis-elements in the glutathione transferase P gene

The glutathione transferase P (GST-P) gene is specifically expressed during chemical hepatocarcinogenesis of the rat, whereas mRNA of this gene is virtually undetectable in normal liver. We have previously identified a stretch of DNA, that acted negatively in transcription, at 400 bp upstream from the cap site of the rat GST-P gene. Further characterization has revealed that this negative fragment functions in an orientation and position independent manner, suggesting that it is acting as a silencer. This silencer consists of multiple negative elements to which nuclear factors bind. This silencer is active not only in rat non-hepatoma and hepatoma cells but also in human and mouse cell lines, suggesting that these elements function as general regulators of basal gene expression. At least two proteins bind to this silencer fragment, one of which, designated SF-A (Silencer Factor A), has been partially purified. SF-A binds to several regions in this silencer, and likely plays an important role on negative regulation of this gene.

FAD24 Acts in Concert with Histone Acetyltransferase HBO1 to Promote Adipogenesis by Controlling DNA Replication

Preadipocytes differentiate into adipocytes through approximately two rounds of mitosis, referred to as mitotic clonal expansion (MCE), but the events early in the differentiation process are not fully understood. Previously, we identified and characterized a novel gene, fad24 (factor for adipocyte differentiation 24), induced to express at the early stages of adipocyte differentiation. Although fad24 clearly has crucial roles in adipogenesis, its precise functions remain unknown. Here we show that the knockdown of fad24 by RNAi in 3T3-L1 preadipocytes repressed MCE. Moreover, FAD24 interacts with HBO1, a histone acetyltransferase and positive regulator of DNA replication initiation. The knockdown of hbo1 repressed MCE and adipogenesis, indicating that FAD24 acts in concert with HBO1 to promote adipogenesis by controlling DNA replication. Regarding the molecular mechanisms behind the regulation of DNA replication by fad24, we revealed that FAD24 co-localizes with HBO1 to chromatin during late mitosis, which is when the prereplication initiation complex is assembled. Furthermore, chromatin immunoprecipitation experiments indicated that FAD24 localizes to origins of DNA replication with HBO1. When fad24 expression was inhibited during adipocyte differentiation, the recruitment of HBO1 to origins of DNA replication was reduced. Thus, FAD24 controls DNA replication by recruiting HBO1 to origins of DNA replication and is required for MCE during adipocyte differentiation.

Histone acetyltransferase MOZ acts as a co-activator of Nrf2-MafK and induces tumour marker gene expression during hepatocarcinogenesis.

HATs (histone acetyltransferases) contribute to the regulation of gene expression, and loss or dysregulation of these activities may link to tumorigenesis. Here, we demonstrate that expression levels of HATs, p300 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] were decreased during chemical hepatocarcinogenesis, whereas expression of MOZ (monocytic leukaemia zinc-finger protein; MYST3)--a member of the MYST [MOZ, Ybf2/Sas3, Sas2 and TIP60 (Tat-interacting protein, 60 kDa)] acetyltransferase family--was induced. Although the MOZ gene frequently is rearranged in leukaemia, we were unable to detect MOZ rearrangement in livers with hyperplastic nodules. We examined the effect of MOZ on hepatocarcinogenic-specific gene expression. GSTP (glutathione S-transferase placental form) is a Phase II detoxification enzyme and a well-known tumour marker that is specifically elevated during hepatocarcinogenesis. GSTP gene activation is regulated mainly by the GPE1 (GSTP enhancer 1) enhancer element, which is recognized by the Nrf2 (nuclear factor-erythroid 2 p45 subunit-related factor 2)-MafK heterodimer. We found that MOZ enhances GSTP promoter activity through GPE1 and acts as a co-activator of the Nrf2-MafK heterodimer. Further, exogenous MOZ induced GSTP expression in rat hepatoma H4IIE cells. These results suggest that during early hepatocarcinogenesis, aberrantly expressed MOZ may induce GSTP expression through the Nrf2-mediated pathway.

peg10, an imprinted gene, plays a crucial role in adipocyte differentiation

An imprinted gene, paternally expressed gene (peg) 10, was isolated as one of the genes expressed early in adipogenesis. The expression of peg10 was elevated after the addition of inducers, and was detected in adipocyte differentiable 3T3‐L1 cells, but not observed in the non‐adipogenic cell line NIH‐3T3. Moreover, the knockdown of peg10 by RNA interference (RNAi) inhibited the differentiation of 3T3‐L1 cells into lipid‐laden adipocytes. Interestingly, peg10 RNAi‐treatment reduced the expressions of C/EBPβ and C/EBPδ, and inhibited mitotic clonal expansion. These findings strongly indicate that peg10 plays a crucial role at the immediate early stage of adipocyte differentiation.

A novel gene, fad49, plays a crucial role in the immediate early stage of adipocyte differentiation via involvement in mitotic clonal expansion

Adipogenesis is accomplished via a complex series of steps, and the events at the earliest stage remain to be elucidated. To clarify the molecular mechanisms of adipocyte differentiation, we previously isolated 102 genes expressed early in mouse 3T3‐L1 preadipocyte cells using a PCR subtraction system. About half of the genes isolated appeared to be unknown. After isolating full‐length cDNAs of the unknown genes, one of them, named factor for adipocyte differentiation 49 (fad49), appeared to be a novel gene, as the sequence of this clone showed no identity to known genes. FAD49 contains a phox homology (PX) domain and four Src homologyu20033 (SH3) domains, suggesting that it may be a novel scaffold protein. We found that the PX domain of FAD49 not only has affinity for phosphoinositides, but also binds to its third SH3 domain. Expression of fad49 was transiently elevated 3u2003h after differentiation was induced, and diminished 24u2003h after induction. Induction of the fad49 gene was observed in adipocyte differentiable 3T3‐L1 cells, but not in non‐adipogenic NIH‐3T3 cells. RNAi‐mediated knockdown of fad49 significantly impaired adipocyte differentiation. Moreover, the knockdown of fad49 by RNAi inhibited mitotic clonal expansion, and reduced the expression of CCAAT/enhancer‐binding proteinu2003β (C/EBPβ) and C/EBPδ at the immediate early phase. Taken together, these results show that fad49, a novel gene, plays a crucial role in the immediate early stage of adipogenesis.

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression.

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

Crucial roles of D-type cyclins in the early stage of adipocyte differentiation

Cyclin D2 was isolated as one of the genes expressed early in adipogenesis. The expression of cyclin D2 increased temporarily early on and then again late in the differentiation process. The expression of cyclin D1 and cyclin D3, the other D-type cyclins, was also transiently induced early during adipocyte differentiation. RNAi (RNA interference)-mediated knockdown of cyclin D1, D2, or D3 inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Moreover, the knockdown of cyclin D1 or D3 significantly inhibited mitotic clonal expansion (MCE), while silencing of the cyclin D2 gene had a milder effect on MCE. Each of the D-type cyclins seems to play a crucial role in adipocyte differentiation by regulating MCE.

Fad104, a Positive Regulator of Adipocyte Differentiation, Suppresses Invasion and Metastasis of Melanoma Cells by Inhibition of STAT3 Activity

Metastasis is the main cause of death in patients with cancer, and understanding the mechanisms of metastatic processes is essential for the development of cancer therapy. Although the role of several cell adhesion, migration or proliferation molecules in metastasis is established, a novel target for cancer therapy remains to be discovered. Previously, we reported that fad104 (factor for adipocyte differentiation 104), a regulatory factor of adipogenesis, regulates cell adhesion and migration. In this report, we clarify the role of fad104 in the invasion and metastasis of cancer cells. The expression level of fad104 in highly metastatic melanoma A375SM cells was lower than that in poorly metastatic melanoma A375C6 cells. Reduction of fad104 expression enhanced the migration and invasion of melanoma cells, while over-expression of FAD104 inhibited migration and invasion. In addition, melanoma cells stably expressing FAD104 showed a reduction in formation of lung colonization compared with control cells. FAD104 interacted with STAT3 and down-regulated the phosphorylation level of STAT3 in melanoma cells. These findings together demonstrate that fad104 suppressed the invasion and metastasis of melanoma cells by inhibiting activation of the STAT3 signaling pathway. These findings will aid a comprehensive description of the mechanism that controls the invasion and metastasis of cancer cells.

Nuclear Factor 1 Family Members Interact with Hepatocyte Nuclear Factor 1α to Synergistically Activate L-type Pyruvate Kinase Gene Transcription

Transcription of hepatic L-type pyruvate kinase (L-PK) gene is cell type-specific and is under the control of various nutritional conditions. The L-PK gene contains multiple cis-regulatory elements located within a 170-bp upstream region necessary for these regulations. These elements can synergistically stimulate L-PK gene transcription, although their mechanisms are largely unknown. Because nuclear factor (NF) 1 family members bind to specific cis-regulatory elements known as L-IIA and L-IIB and hepatocyte nuclear factor (HNF) 1α binds to the adjacent element L-I, we examined the functional and physical interactions between these two transcription factors. Reporter gene assay showed that these two factors synergistically activated the L-PK promoter containing the 5′-flanking region up to –189. Although two NF1-binding sites are required for the maximum synergistic effect of NF1 family members with HNF1α, significant functional interaction between the two factors was observed in the L-PK promoter containing two mutated NF1-binding sites and also in the promoter containing only the HNF1α-binding site, raising the possibility that NF1 proteins function as HNF1α co-activators. Chromatin immunoprecipitation assay revealed that both NF1 proteins and HNF1α bound to the promoter region of the L-PK gene in vivo. In vitro binding assay confirmed that NF1 proteins directly interacted mainly with the homeodomain of HNF1α via their DNA-binding domains. This interaction enhanced HNF1α binding to the L-I element and was also observed in rat liver by co-immunoprecipitation assay. Thus, we conclude that cooperative interaction between NF1 family members and HNF1α plays an important role in hepatic L-PK transcription.