Knockdown of RhoQ, a member of Rho GTPase, accelerates TGF-ß-induced EMT in human lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related deaths worldwide, and the most common subtype of lung cancer is adenocarcinoma. RhoQ is a Rho family GTPase with primary sequence and structural similarities to Cdc42 and RhoJ. RhoQ is involved in neurite outgrowth via membrane trafficking and is essential for insulin-stimulated glucose uptake in mature adipocytes. However, the function of RhoQ in lung adenocarcinoma (LUAD) remains unclear. In this study, RhoQ siRNAs were introduced into A549 and PC-9 cells. Expression level of EMT-related genes and invasion ability were investigated using Western blot and transwell assay. To examine the relationship between RhoQ expression and prognosis of LUAD, Kaplan-Meier plotter was used. We discovered that suppressing RhoQ expression promoted TGF-ß-mediated EMT and invasion in LUAD cell lines. Furthermore, RhoQ knockdown increased Smad3 phosphorylation and Snail expression, indicating that RhoQ was involved in TGF/Smad signaling during the EMT process. Moreover, Kaplan-Meier plotter analysis revealed that low RhoQ levels were associated with poor overall survival in patients with LUAD. In conclusion, these findings shed light on RhoQ's role as a negative regulator of TGF-ß-mediated EMT in LUAD.

Repression of RhoJ expression promotes TGF-ß-mediated EMT in human non-small-cell lung cancer A549cells.

Non-small-cell lung cancer (NSCLC) accounts for most cancer-related deaths because of its strong metastatic ability. It is important to understand NSCLC's molecular mechanisms of metastasis. RhoJ, a protein that belongs to the Rho family of small GTPases, regulates endothelial motility, angiogenesis, and adipogenesis. Recently, bioinformatics analysis showed that NSCLC patients with lower RhoJ expression had a worse survival outcome than those with high RhoJ expression. However, little is known about RhoJ's role in NSCLC. In the present study, we demonstrated that RhoJ knockdown accelerated TGF-ßmediated epithelial-to-mesenchymal transition (EMT), an important cancer metastasis process, in A549 and PC-9 cells. Furthermore, using Matrigel-coated transwell chambers, we showed that RhoJ knockdown enhanced the invasion capacity of A549 cells that had undergone EMT. Also, reduced RhoJ expression increased Smad3 phosphorylation and Snail expression during the EMT process. Our results provide the first evidence of a potential novel role for RhoJ in the inhibition of EMT via modulation of the TGF-ß-Smad signaling pathway, and shed new light on the mechanisms underlying EMT in NSCLC.

Knockdown of RhoE Expression Enhances TGF-ß-Induced EMT (epithelial-to-mesenchymal transition) in Cervical Cancer HeLa Cells.

Cervical cancer with early metastasis of the primary tumor is associated with poor prognosis and poor therapeutic outcomes. Since epithelial-to-mesenchymal transition (EMT) plays a role in acquisition of the ability to invade the pelvic lymph nodes and surrounding tissue, it is important to clarify the molecular mechanism underlying EMT in cervical cancer. RhoE, also known as Rnd3, is a member of the Rnd subfamily of Rho GTPases. While previous reports have suggested that RhoE may act as either a positive or a negative regulator of cancer metastasis and EMT, the role of RhoE during EMT in cervical cancer cells remains unclear. The present study revealed that RhoE expression was upregulated during transforming growth factor-ß (TGF-ß)-mediated EMT in human cervical cancer HeLa cells. Furthermore, reduced RhoE expression enhanced TGF-ß-mediated EMT and migration of HeLa cells. In addition, we demonstrated that RhoE knockdown elevated RhoA activity and a ROCK inhibitor partially suppressed the acceleration of TGF-ß-mediated EMT by RhoE knockdown. These results indicate that RhoE suppresses TGF-ß-mediated EMT, partially via RhoA/ROCK signaling in cervical cancer HeLa cells.

FAD104, a regulator of adipogenesis, is a novel suppressor of TGF-ß-mediated EMT in cervical cancer cells.

Epithelial-to-mesenchymal transition (EMT) is a biological process in which epithelial cells translate into a mesenchymal phenotype with invasive capacities, contributing to tumour progression, metastasis, and the acquisition of chemotherapy resistance. To identify new therapeutic targets for cancers, it is important to clarify the molecular mechanism of induction of EMT. We have previously reported that fad104, a positive regulator of adipocyte differentiation, suppressed the invasion and metastasis of melanoma and breast cancer cells. In this study, we showed that FAD104 functions as a novel suppressor of transforming growth factor-ß (TGF-ß)-mediated EMT in cervical cancer cells. Expression of FAD104 is upregulated during TGF-ß-mediated EMT in human cervical cancer HeLa cells. Reduction of fad104 expression enhanced TGF-ß-mediated EMT and migration in HeLa cells. Conversely, overexpression of FAD104 suppressed TGF-ß-induced EMT. In addition, we showed that FAD104 negatively regulated phosphorylation of Smad2 and Smad3 but positively regulated phosphorylation of Smad1/5/8 via treatment with TGF-ß. These findings demonstrate that FAD104 is a novel suppressor of TGF-ß signalling and represses TGF-ß-mediated EMT in cervical cancer cells.

KCNMA1, a pore-forming α-subunit of BK channels, regulates insulin signalling in mature adipocytes.

KCNMA1 is a pore-forming α-subunit of the large conductance Ca2+ - and voltage-activated K+ channels, referred to as BK channels, which play key roles in various physiological functions. However, the role of KCNMA1 in mature adipocytes remains unclear. In this study, we reveal that kcnma1 expression is downregulated in white adipose tissue of mice fed a high-fat diet and in hypertrophied adipocytes. Furthermore, inhibition of kcnma1 expression or treatment with a BK channel blocker attenuated insulin-induced Akt phosphorylation in mature adipocytes. These results strongly indicate that KCNMA1 contributes to the regulation of insulin signalling in mature adipocytes.

FAD104, a Regulator of Adipogenesis and Osteogenesis, Interacts with the C-Terminal Region of STAT3 and Represses Malignant Transformation of Melanoma Cells.

Anchorage-independent growth is one of the defining characteristics of cancer cells. Many oncogenes and tumor suppressor genes are involved in regulating this type of growth. Factor for adipocyte differentiation 104 gene (fad104) is a regulator of adipogenesis and osteogenesis. Previously, we reported that fad104 suppressed metastasis as well as invasion of melanoma cells. However, it is unclear whether fad104 is involved in malignant transformation, which is associated with metastasis. In this study, we revealed that fad104 negatively regulated the colony forming activity of melanoma cells. The presence of the N-terminal region of FAD104 was required for the regulation of malignant transformation of melanoma cells. In addition, the deletion mutant of FAD104 that contained the N-terminal region and transmembrane domain interacted with signal transducer and activator of transcription 3 (STAT3) and suppressed STAT3 activity. However, the deletion mutant of FAD104 lacking the N-terminal region did not influence the interaction with STAT3 or suppress the STAT3 activity. Moreover, FAD104 interacted with the C-terminal region of STAT3. In summary, we demonstrated that fad104 suppressed anchorage-independent growth of melanoma cells, and that the N-terminal region of FAD104 is essential for inhibiting malignant transformation and STAT3 activity.

Fad24, a Positive Regulator of Adipogenesis, Is Required for S Phase Re-entry of C2C12 Myoblasts Arrested in G0 Phase and Involved in p27(Kip1) Expression at the Protein Level.

Factor for adipocyte differentiation 24 (fad24) is a positive regulator of adipogenesis. We previously found that human fad24 is abundantly expressed in skeletal muscle. However, the function of fad24 in skeletal muscle remains largely unknown. Because skeletal muscle is a highly regenerative tissue, we focused on the function of fad24 in skeletal muscle regeneration. In this paper, we investigated the role of fad24 in the cell cycle re-entry of quiescent C2C12 myoblasts-mimicked satellite cells. The expression levels of fad24 and histone acetyltransferase binding to ORC1 (hbo1), a FAD24-interacting factor, were elevated at the early phase of the regeneration process in response to cardiotoxin-induced muscle injury. The knockdown of fad24 inhibited the proliferation of quiescent myoblasts, whereas fad24 knockdown did not affect differentiation. S phase entry following serum activation is abrogated by fad24 knockdown in quiescent cells. Furthermore, fad24 knockdown cells show a marked accumulation of p27(Kip1) protein. These results suggest that fad24 may have an important role in the S phase re-entry of quiescent C2C12 cells through the regulation of p27(Kip1) at the protein level.

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.

KCNK10, a tandem pore domain potassium channel, is a regulator of mitotic clonal expansion during the early stage of adipocyte differentiation.

KCNK10, a member of tandem pore domain potassium channel family, gives rise to leak K+ currents. It plays important roles in stabilizing the negative resting membrane potential and in counterbalancing depolarization. We previously demonstrated that kcnk10 expression is quickly elevated during the early stage of adipogenesis of 3T3-L1 cells and that reduction of kcnk10 expression inhibits adipocyte differentiation. However, the molecular mechanism of KCNK10 in adipocyte differentiation remains unclear. Here we revealed that kcnk10 is induced by 3-isobutyl-1-methylxanthine, a cyclic nucleotide phosphodiesterase inhibitor and a potent inducer of adipogenesis, during the early stage of adipocyte differentiation. We also demonstrated that KCNK10 functions as a positive regulator of mitotic clonal expansion (MCE), a necessary process for terminal differentiation. The reduction of kcnk10 expression repressed the expression levels of CCAAT/enhancer-binding protein ß (C/EBPß) and C/EBPδ as well as the phosphorylation level of Akt during the early phase of adipogenesis. In addition, knockdown of kcnk10 expression suppressed insulin-induced Akt phosphorylation. These results indicate that KCNK10 contributes to the regulation of MCE through the control of C/EBPß and C/EBPδ expression and insulin signaling.

FAD104, a regulatory factor of adipogenesis, acts as a novel regulator of calvarial bone formation.

Osteogenesis is a complex process that is orchestrated by several growth factors, extracellular cues, signaling molecules, and transcriptional factors. Understanding the mechanisms of bone formation is pivotal for clarifying the pathogenesis of bone diseases. Previously, we reported that fad104 (factor for adipocyte differentiation 104), a novel positive regulator of adipocyte differentiation, negatively regulated the differentiation of mouse embryonic fibroblasts into osteocytes. However, the physiological role of fad104 in bone formation has not been elucidated. Here, we clarified the role of fad104 in bone formation in vivo and in vitro. fad104 disruption caused craniosynostosis-like premature ossification of the calvarial bone. Furthermore, analyses using primary calvarial cells revealed that fad104 negatively regulated differentiation and BMP/Smad signaling pathway. FAD104 interacted with Smad1/5/8. The N-terminal region of FAD104, which contains a proline-rich motif, was capable of binding to Smad1/5/8. We demonstrated that down-regulation of Smad1/5/8 phosphorylation by FAD104 is dependent on the N-terminal region of FAD104 and that fad104 functions as a novel negative regulator of BMP/Smad signaling and is required for proper development for calvarial bone. These findings will aid a comprehensive description of the mechanism that controls normal and premature calvarial ossification.

Targeted disruption of fad24, a regulator of adipogenesis, causes pre-implantation embryonic lethality due to the growth defect at the blastocyst stage.

In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown. In this study, we attempted to generate fad24-deficient mice by gene targeting. No fad24-null mutants were recovered after embryonic day 9.5 (E9.5). Although fad24-null embryos were detected in an expected Mendelian ratio of genotypes at E3.5, none of the homozygous mutants developed into blastocysts. In vitro culture experiments revealed that fad24-null embryos develop normally to the morula stage but acquire growth defects during subsequent stages. The number of nuclei decreased in fad24-deficient morulae compared with that in wild-type ones. These results strongly suggested that fad24 is essential for pre-implantation in embryonic development, particularly for the progression to the blastocyst stage.

Interactions of thyroid hormone receptor with Ku proteins and interleukin enhancer binding factor 3 modulate the promoter activity of thyroid-stimulating hormone alpha.

We previously identified Ku proteins and interleukin enhancer binding factor 3 (ILF3) as cofactors for the nuclear receptor farnesoid X receptor and liver receptor homolog-1, respectively. Here we provide further evidence that these cofactors modulate the promoter activity of the nuclear receptor thyroid hormone receptor (TR) target gene, thyroid-stimulating hormone alpha (TSHα), which is negatively regulated by the TR ligand triiodothyronine (T(3)). Ku proteins suppressed TSHα promoter activity independent of T(3), whereas ILF3 enhanced TSHα activity, especially in the presence of T(3). Taken together, our results suggest that Ku proteins and ILF3 function as co-regulators for TR-mediated TSHα expression.

Factor for adipocyte differentiation 158 gene disruption prevents the body weight gain and insulin resistance induced by a high-fat diet.

To clarify the molecular mechanism of adipocyte differentiation, we previously isolated a novel gene, factor for adipocyte differentiation (fad) 158, whose expression was induced during the earliest stages of adipogenesis, and its product was localized to the endoplasmic reticulum. We found that the knockdown of fad158 expression prevented the differentiation of 3T3-L1 cells into adipocytes. In addition, over-expression of fad158 promoted the differentiation of NIH-3T3 cells, which do not usually differentiate into adipocytes. Although these findings strongly suggest that fad158 has a crucial role in regulating adipocyte differentiation, the physiological role of the gene is still unclear. In this study, we generated mice in which fad158 expression was deleted. The fad158-deficient mice did not show remarkable changes in body weight or the weight of white adipose tissue on a chow diet, but had significantly lower body weights and fat mass than wild-type mice when fed a high-fat diet. Furthermore, although the disruption of fad158 did not influence insulin sensitivity on the chow diet, it improved insulin resistance induced by the high-fat diet. These results indicate that fad158 is a key factor in the development of obesity and insulin resistance caused by a high-fat diet.

Indispensable role of factor for adipocyte differentiation 104 (fad104) in lung maturation.

Factor for adipocyte differentiation 104 (fad104) is a regulator of adipogenesis and osteogenesis. Our previous study showed that fad104-deficient mice died immediately after birth, suggesting fad104 to be essential for neonatal survival. However, the cause of this rapid death is unclear. Here, we demonstrate the role of fad104 in neonatal survival. Phenotypic and morphological analyses showed that fad104-deficient mice died due to cyanosis-associated lung dysplasia including atelectasis. Furthermore, immunohistochemistry revealed that FAD104 was strongly expressed in ATII cells in the developing lung. Most importantly, the ATII cells in lungs were immature, and impaired the expression of surfactant-associated proteins. Collectively, these results indicate that fad104 has an indispensable role in lung maturation, especially the maturation and differentiation of ATII cells.

Interleukin enhancer-binding factor 3 functions as a liver receptor homologue-1 co-activator in synergy with the nuclear receptor co-activators PRMT1 and PGC-1α.

LRH-1 (liver receptor homologue-1), a transcription factor and member of the nuclear receptor superfamily, regulates the expression of its target genes, which are involved in bile acid and cholesterol homoeostasis. However, the molecular mechanisms of transcriptional control by LRH-1 are not completely understood. Previously, we identified Ku80 and Ku70 as LRH-1-binding proteins and reported that they function as co-repressors. In the present study, we identified an additional LRH-1-binding protein, ILF3 (interleukin enhancer-binding factor 3). ILF3 formed a complex with LRH-1 and the other two nuclear receptor co-activators PRMT1 (protein arginine methyltransferase 1) and PGC-1α (peroxisome proliferator-activated receptor γ co-activator-1α). We demonstrated that ILF3, PRMT1 and PGC-1α were recruited to the promoter region of the LRH-1-regulated SHP (small heterodimer partner) gene, encoding one of the nuclear receptors. ILF3 enhanced SHP gene expression in co-operation with PRMT1 and PGC-1α through the C-terminal region of ILF3. In addition, we found that the small interfering RNA-mediated down-regulation of ILF3 expression led to a reduction in the occupancy of PGC-1α at the SHP promoter and SHP expression. Taken together, our results suggest that ILF3 functions as a novel LRH-1 co-activator by acting synergistically with PRMT1 and PGC-1α, thereby promoting LRH-1-dependent gene expression.

RNA interference-mediated knockdown of the mouse gene encoding potassium channel subfamily K member 10 inhibits hormone-induced differentiation of 3T3-L1 preadipocytes.

Intramuscular fat (IMF) is an economically important trait of domestic meat animals; thus, it is important to identify the factors that influence the IMF content. In this study, we identified the gene associated with adipogenesis from all the positional candidate genes located in the quantitative trait loci (QTL) for IMF content on porcine chromosome 7. We analyzed the expression of the abovementioned genes during differentiation of mouse 3T3-L1 preadipocytes by using real-time polymerase chain reaction (PCR). Total cellular RNA was extracted before and 6, 12, 36, and 48 h and 4, 6, and 8d after treatment with standard hormonal inducers of differentiation-insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX). Six hours after induction, potassium channel subfamily K member 10 (KCNK10) gene expression in the preadipocytes was found to be 100-fold greater than that at the baseline; this expression declined until day 4 after the induction. Moreover, knockdown of the KCNK10 gene by transfection with short-hairpin RNA (shRNA) significantly decreased triacylglycerol accumulation on day 8 after the induction. An RNA interference study revealed that KCNK10 knockdown inhibited the differentiation of 3T3-L1 cells. These results indicate that KCNK10 plays an important role in the early stages of preadipocyte differentiation.

Fad104, a positive regulator of adipogenesis, negatively regulates osteoblast differentiation.

Fad104 (factor for adipocyte differentiation 104) is a novel gene expressed temporarily in the early stages of adipocyte differentiation. Previously, we showed that fad104 promotes adipocyte differentiation in mouse 3T3-L1 cells and mouse embryonic fibroblasts (MEFs). Furthermore, we reported that implanted wild-type MEFs could develop into adipocytes, whereas fad104-deficient MEFs could not. Interestingly, bone-like tissues were only observed in the implants derived from fad104-deficient MEFs. This result implies that fad104 is involved in osteoblast differentiation. However, the functions of fad104 during osteogenesis are unknown. In this paper, we show that fad104 negatively regulates osteoblast differentiation. During the differentiation process, the level of fad104 expression decreased. Deletion of fad104 facilitated osteoblast differentiation in MEFs, and elevated the level of runx2, a master regulator of osteoblast differentiation. Disruption of fad104 suppressed BMP-2-mediated adipocyte differentiation in MEFs. In conclusion, we demonstrate that fad104 reciprocally regulates differentiation of adipocytes and osteoblast; functions as a positive regulator in adipocyte differentiation and as a negative regulator in osteoblast differentiation.

Gelsolin, an actin regulatory protein, is required for differentiation of mouse 3T3-L1 cells into adipocytes.

To elucidate molecular mechanisms of adipocyte differentiation, we previously isolated TC10-like/ TC10betaLong (TCL/TC10betaL), which was transiently expressed in the early phase of adipogenesis of 3T3-L1 cells and seemed to be a positive regulator of adipogenesis. By using TCL/TC10betaL-overexpressing NIH-3T3 cells, we also isolated gelsolin as a gene whose expression was up-regulated by TCL/TC10betaL. However, the roles of gelsolin in adipocyte differentiation are unclear. In this paper we characterized the function of gelsolin in adipogenesis in 3T3-L1 cells. The level of gelsolin changed during adipocyte differentiation. Knockdown of the expression of gelsolin using RNAi inhibited adipocyte differentiation, and impaired the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein (C/EBP) alpha. Interestingly, the knockdown also impaired mitotic clonal expansion (MCE), and increased cell size, though it reduced levels of C/EBPbeta and C/EBPdelta, markers for the early stage of adipogenesis, only slightly. Gelsolin plays a crucial role in the differentiation of 3T3-L1 cells into adipocytes.

Repression of the promoter activity mediated by liver receptor homolog-1 through interaction with ku proteins.

Nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) plays a crucial role in the homeostasis of bile acids and cholesterol by controlling the expression of genes central to bile acid synthesis and efflux, reverse cholesterol transport, and high density lipoprotein-remodeling. However, the molecular mechanisms that modulate the transactivation activity of LRH-1 remain unclear. It is proposed that LRH-1's activity is regulated by post-modifications, the binding of small heterodimer partner (SHP), or the binding of coregulators. To search for cofactors that regulate the transactivation activity of LRH-1, we performed a pull-down assay using glutathione S-transferase (GST) fused to the N-terminal portion of LRH-1 and nuclear extracts from HeLa cells, and identified Ku proteins as interacting proteins with LRH-1. We also found that Ku proteins associate with LRH-1 through its DNA-binding domain and hinge region. Luciferase reporter assays revealed that Ku proteins repressed the SHP promoter activity mediated by LRH-1. Furthermore, Ku proteins suppressed the coactivating effect of peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1alpha (PGC-1alpha), an LRH-1 coactivator, on the LRH-1-mediated SHP promoter activity. Previously, we showed that Ku proteins interacted with nuclear receptor farnesoid X receptor (FXR; NR1H4) and decreased the expression of its target gene. In this study, we demonstrated that Ku proteins also interacted with not only LRH-1 but various nuclear receptors, such as the estrogen receptor, PPAR, and Rev-erb. Ku proteins may function as corepressors for various nuclear receptors including LRH-1.

TC10-like/TC10betaLong regulates adipogenesis by controlling mitotic clonal expansion.

To elucidate molecular mechanisms of adipocyte differentiation, we previously isolated TC10-like/TC10betaLong (TCL/TC10betaL), regulators of G protein signaling 2 (RGS2), factor for adipocyte differentiation (fad) 104 and fad158, which were transiently expressed in the early phase of adipogenesis. These four genes seem to be positive regulators of adipogenesis, since their knockdown resulted in the inhibition of adipocyte differentiation. When growth-arrested 3T3-L1 cells were induced to differentiate, they first reentered the cell cycle and underwent several rounds of cell division, a process known as mitotic clonal expansion (MCE). Although MCE is required for completion of the differentiation program, its molecular mechanisms are not fully understood. We examined the roles of these four genes during MCE. Knockdown of the expression of TCL/TC10betaL impaired MCE, while that of RGS2 or fad104 had a rather weak effect and that of fad158 had no effect. The suppression of TCL/TC10betaL inhibited the incorporation of bromodeoxyuridine (BrdU), indicating that DNA synthesis was prevented by the knockdown. Interestingly, the knockdown of TCL/TC10betaL inhibited the expression of the CCAAT/enhancer-binding protein (C/EBP) family, C/EBPbeta and C/EBPdelta, during MCE. The results strongly suggest that TCL/TC10betaL regulates adipocyte differentiation by controlling MCE and this regulatory effect is closely linked to C/EBPbeta and C/EBPdelta expression.