ATM and SIRT6/SNF2H Mediate Transient H2AX Stabilization When DSBs Form by Blocking HUWE1 to Allow Efficient γH2AX Foci Formation.

In response to DNA double-strand breaks (DSBs), H2AX is rapidly phosphorylated at Ser139 to promote DSB repair. Here we show that H2AX is rapidly stabilized in response to DSBs to efficiently generate γH2AX foci. This mechanism operated even in quiescent cells that barely expressed H2AX. H2AX stabilization resulted from the inhibition of proteasome-mediated degradation. Synthesized H2AX ordinarily underwent degradation through poly-ubiquitination mediated by the E3 ligase HUWE1; however, H2AX ubiquitination was transiently halted upon DSB formation. Such rapid H2AX stabilization by DSBs was associated with chromatin incorporation of H2AX and halting of its poly-ubiquitination mediated by the ATM kinase, the sirtuin protein SIRT6, and the chromatin remodeler SNF2H. H2AX Ser139, the ATM phosphorylation site, was essential for H2AX stabilization upon DSB formation. Our results reveal a pathway controlled by ATM, SIRT6, and SNF2H to block HUWE1, which stabilizes H2AX and induces its incorporation into chromatin only when cells are damaged.

Development of cancer-initiating cells and immortalized cells with genomic instability.

Cancers that develop after middle age usually exhibit genomic instability and multiple mutations. This is in direct contrast to pediatric tumors that usually develop as a result of specific chromosomal translocations and epigenetic aberrations. The development of genomic instability is associated with mutations that contribute to cellular immortalization and transformation. Cancer occurs when cancer-initiating cells (CICs), also called cancer stem cells, develop as a result of these mutations. In this paper, we explore how CICs develop as a result of genomic instability, including looking at which cancer suppression mechanisms are abrogated. A recent in vitro study revealed the existence of a CIC induction pathway in differentiating stem cells. Under aberrant differentiation conditions, cells become senescent and develop genomic instabilities that lead to the development of CICs. The resulting CICs contain a mutation in the alternative reading frame of CDKN2A (ARF)/p53 module, i.e., in either ARF or p53. We summarize recently established knowledge of CIC development and cellular immortality, explore the role of the ARF/p53 module in protecting cells from transformation, and describe a risk factor for genomic destabilization that increases during the process of normal cell growth and differentiation and is associated with the downregulation of histone H2AX to levels representative of growth arrest in normal cells.

Artemis-dependent DNA double-strand break formation at stalled replication forks.

Stalled replication forks undergo DNA double-strand breaks (DSBs) under certain conditions. However, the precise mechanism underlying DSB induction and the cellular response to persistent replication fork stalling are not fully understood. Here we show that, in response to hydroxyurea exposure, DSBs are generated in an Artemis nuclease-dependent manner following prolonged stalling with subsequent activation of the ataxia-telangiectasia mutated (ATM) signaling pathway. The kinase activity of the catalytic subunit of the DNA-dependent protein kinase, a prerequisite for stimulation of the endonuclease activity of Artemis, is also required for DSB generation and subsequent ATM activation. Our findings indicate a novel function of Artemis as a molecular switch that converts stalled replication forks harboring single-stranded gap DNA lesions into DSBs, thereby activating the ATM signaling pathway following prolonged replication fork stalling.

The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs.

BACKGROUND: It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. RESULTS: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. CONCLUSION: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. SIGNIFICANCE: Cellular transformation renders cells more susceptible to some DNA-damaging agents. Anti-cancer drugs generally target cancer cells rather than normal somatic cells. However, the factors that determine this differential sensitivity are poorly understood. Here we show that Arf/p53-dependent down-regulation of H2AX induced the selective survival of normal cells after drug treatment, resulting in the preferential targeting of cancer cells. Treatment with camptothecin, a topoisomerase I inhibitor, caused normal cells to down-regulate H2AX and become quiescent, a process mediated by both Arf and p53. In contrast, transformed cells that harbor mutations in either Arf or p53 do not down-regulate H2AX and are more sensitive to drugs unless they have developed drug resistance. Such transformation-associated changes in H2AX expression rendered cancer cells more susceptible to drug-induced damage (by two orders of magnitude). Thus, the expression of H2AX and γH2AX (phosphorylated form of H2AX at Ser-139) is a critical factor that determines drug sensitivity and should be considered when administering chemotherapy.

Induction of cancerous stem cells during embryonic stem cell differentiation.

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.

The quiescent cellular state is Arf/p53-dependent and associated with H2AX downregulation and genome stability.

Cancer is a disease associated with genomic instability and mutations. Excluding some tumors with specific chromosomal translocations, most cancers that develop at an advanced age are characterized by either chromosomal or microsatellite instability. However, it is still unclear how genomic instability and mutations are generated during the process of cellular transformation and how the development of genomic instability contributes to cellular transformation. Recent studies of cellular regulation and tetraploidy development have provided insights into the factors triggering cellular transformation and the regulatory mechanisms that protect chromosomes from genomic instability.

Hepatic stellate cells mediate differentiation of dendritic cells from monocytes.

BACKGROUND: We have previously reported that human umbilical cord blood (UCB)-nucleated cells differentiate into hepatocyte-like cells when cultured in a 5-cytokine cocktail medium. We further found that UCB cells rather differentiated into dendritic-shaped cells by coculture with a human stellate cell (HSC) line, LI90. METHODS: Monocytes from UCB and adult peripheral blood were cocultured with LI90 or rat primary HSCs in a cell-culture insert. Monocytes were also cultured with LI90-conditioned medium containing secreted factors, which were analyzed by a cytokine array. RESULTS: In the coculture with LI90, resulting dendritic-shaped cells from monocytes expressed dendritic cell (DC) markers and activated allogeneic T cells, indicating that the dendritic-shaped cells were DCs. LI90 in the cytokine cocktail medium secreted various inflammatory factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Fibroblast growth factor-2 in the cytokine cocktail was responsible for GM-CSF production from LI90 cells and for differentiation of monocytes into DCs in the LI90 coculture. Moreover, the coculture of monocytes with activated HSCs derived from damaged rat liver induced the differentiation of DCs, whereas quiescent HSCs derived from normal liver scarcely induced such a change. CONCLUSION: These results suggest that activated HSCs are involved in differentiation of monocytes into DCs in the liver.

Onset of quiescence following p53 mediated down-regulation of H2AX in normal cells.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.

Transcription-dependent activation of ataxia telangiectasia mutated prevents DNA-dependent protein kinase-mediated cell death in response to topoisomerase I poison.

Camptothecin (CPT) is a topoisomerase I inhibitor, derivatives of which are being used for cancer chemotherapy. CPT-induced DNA double-strand breaks (DSBs) are considered a major cause of its tumoricidal activity, and it has been shown that CPT induces DNA damage signaling through the phosphatidylinositol 3-kinase-related kinases, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase). In addition, CPT causes DNA strand breaks mediated by transcription, although the downstream signaling events are less well characterized. In this study, we show that CPT-induced activation of ATM requires transcription. Mechanistically, transcription inhibition suppressed CPT-dependent activation of ATM and blocked recruitment of the DNA damage mediator p53-binding protein 1 (53BP1) to DNA damage sites, whereas ATM inhibition abrogated CPT-induced G(1)/S and S phase checkpoints. Functional inactivation of ATM resulted in DNA replication-dependent hyperactivation of DNA-PK in CPT-treated cells and dramatic CPT hypersensitivity. On the other hand, simultaneous inhibition of ATM and DNA-PK partially restored CPT resistance, suggesting that activation of DNA-PK is proapoptotic in the absence of ATM. Correspondingly, comet assay and cell cycle synchronization experiments suggested that transcription collapse occurring as the result of CPT treatment are converted to frank double-strand breaks when ATM-deficient cells bypass the G(1)/S checkpoint. Thus, ATM suppresses DNA-PK-dependent cell death in response to topoisomerase poisons, a finding with potential clinical implications.

DPPA4 modulates chromatin structure via association with DNA and core histone H3 in mouse embryonic stem cells.

Developmental pluripotency associated 4 (DPPA4) is one of the uncharacterized genes that is highly expressed in embryonic stem (ES) cells. DPPA4 is associated with active chromatin and involved in the pluripotency of mouse ES cells. However, the biological function of DPPA4 remains poorly understood. In this study, we performed fluorescence recovery after photobleaching (FRAP) analysis to examine the dynamics of DPPA4 in ES cells. FRAP analysis showed that the mobility of DPPA4 is similar to that of histone H1. In addition, biochemical analysis with purified proteins and immunoprecipitation analysis showed that DPPA4 directly binds to both DNA and core histone H3. The analysis using truncated proteins indicated that DPPA4 is associated with DNA via the N-terminal region and histone H3 via the C-terminal region. In vitro assembled chromatin showed resistance to micrococcal nuclease (MNase) digestion in the presence of DPPA4. Moreover, MNase assay and FRAP analysis with the truncated proteins implies that DPPA4 binding to both DNA and histone H3 is necessary for the chromatin structure resistant to MNase and for the proper localization of DPPA4 in ES cell nuclei. These results suggest that DPPA4 modulates the chromatin structure in association with DNA and histone H3 in ES cells.

DNA lesions induced by replication stress trigger mitotic aberration and tetraploidy development.

During tumorigenesis, cells acquire immortality in association with the development of genomic instability. However, it is still elusive how genomic instability spontaneously generates during the process of tumorigenesis. Here, we show that precancerous DNA lesions induced by oncogene acceleration, which induce situations identical to the initial stages of cancer development, trigger tetraploidy/aneuploidy generation in association with mitotic aberration. Although oncogene acceleration primarily induces DNA replication stress and the resulting lesions in the S phase, these lesions are carried over into the M phase and cause cytokinesis failure and genomic instability. Unlike directly induced DNA double-strand breaks, DNA replication stress-associated lesions are cryptogenic and pass through cell-cycle checkpoints due to limited and ineffective activation of checkpoint factors. Furthermore, since damaged M-phase cells still progress in mitotic steps, these cells result in chromosomal mis-segregation, cytokinesis failure and the resulting tetraploidy generation. Thus, our results reveal a process of genomic instability generation triggered by precancerous DNA replication stress.

Proteasome inhibition suppresses DNA-dependent protein kinase activation caused by camptothecin.

The ubiquitin-proteasome pathway plays an important role in DNA damage signaling and repair by facilitating the recruitment and activation of DNA repair factors and signaling proteins at sites of damaged chromatin. Proteasome activity is generally not thought to be required for activation of apical signaling kinases including the PI3K-related kinases (PIKKs) ATM, ATR, and DNA-PK that orchestrate downstream signaling cascades in response to diverse genotoxic stimuli. In a previous work, we showed that inhibition of the proteasome by MG-132 suppressed 53BP1 (p53 binding protein1) phosphorylation as well as RPA2 (replication protein A2) phosphorylation in response to the topoisomerase I (TopI) poison camptothecin (CPT). To address the mechanism of proteasome-dependent RPA2 phosphorylation, we investigated the effects of proteasome inhibitors on the upstream PIKKs. MG-132 sharply suppressed CPT-induced DNA-PKcs autophosphorylation, a marker of the activation, whereas the phosphorylation of ATM and ATR substrates was only slightly suppressed by MG-132, suggesting that DNA-PK among the PIKKs is specifically regulated by the proteasome in response to CPT. On the other hand, MG-132 did not suppress DNA-PK activation in response to UV or IR. MG-132 blocked the interaction between DNA-PKcs and Ku heterodimer enhanced by CPT, and hydroxyurea pre-treatment completely abolished CPT-induced DNA-PKcs autophosphorylation, indicating a requirement for ongoing DNA replication. CPT-induced TopI degradation occurred independent of DNA-PK activation, suggesting that DNA-PK activation does not require degradation of trapped TopI complexes. The combined results suggest that CPT-dependent replication fork collapse activates DNA-PK signaling through a proteasome dependent, TopI degradation-independent pathway. The implications of DNA-PK activation in the context of TopI poison-based therapies are discussed.

Role of Parp-1 in suppressing spontaneous deletion mutation in the liver and brain of mice at adolescence and advanced age.

Poly(ADP-ribose) polymerase-1 knockout (Parp-1(-/-)) mice show increased frequency of spontaneous liver tumors compared to wild-type mice after aging. To understand the impact of Parp-1 deficiency on mutations during aging, in this study, we analyzed spontaneous mutations in Parp-1(-/-) aged mice. Parp-1(-/-) mice showed tendencies of higher mutation frequencies of the red/gam genes at 18 months of age, compared to Parp-1(+/+) mice, in the liver and brain. Complex-type deletions, accompanying small insertion were observed only in Parp-1(-/-) mice in the liver and brain. Further analysis in the liver showed that the frequency of single base deletion mutations at non-repeat or short repeat sequences was 5.8-fold higher in Parp-1(-/-) than in Parp-1(+/+) mice (p<0.05). A 3.2-fold higher tendency of the deletion frequency of two bases or more was observed in Parp-1(-/-) mice compared to Parp-1(+/+) mice (p=0.084). These results support the model that Parp-1 is involved in suppressing imprecise repair of endogenous DNA damage leading to deletion mutation during aging. The mutation frequencies of the gpt gene in the brain were found to be 3-fold lower in Parp-1(-/-) than in Parp-1(+/+) mice at 4 months of age (p<0.01), implying that Parp-1 may be positively involved in imprecise DNA repair in the brain. On the other hand, the frequencies of gpt mutation showed an increase at 18 months of age in the Parp-1(-/-) (p<0.05) but not in Parp-1(+/+) brains, suggesting that Parp-1 deficiency causes an increase of point mutations in the brain by aging.

A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver.

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or alpha-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

Vascular endothelial growth factor promotes proliferation and function of hepatocyte-like cells in embryoid bodies formed from mouse embryonic stem cells.

BACKGROUND/AIMS: Embryoid bodies (EBs) formed from embryonic stem cells (ESCs) differentiate into hepatocyte-like cells (HLCs), and are thus thought to be a useful cell source for drug testing and bioartificial liver. The aim of this study was to induce proliferation and function of ESC-derived HLCs in EBs using HLC-endothelial cell interaction. METHODS: EBs were cultured in the presence of vascular endothelial growth factor (VEGF) and/or VEGF receptor (VEGFR) inhibitors. To reproduce HLC-endothelial cell interaction, we overexpressed VEGF in ESC-derived HLCs under the control of Cyp7a1 gene in EBs. RESULTS: VEGF added to the cultured EBs increased the proliferation of ESC-derived endothelial cells, resulting in the promotion of proliferation and function of ESC-derived HLCs. In EBs, the VEGFR2 inhibitor suppressed expression of albumin and endothelial cell marker genes, whereas the inhibitor for both VEGFR1 and VEGFR2 suppressed expression of Cyp7a1 and hepatocyte growth factor (Hgf) genes. Upon exposure to VEGF, the endothelial cells in EBs increased Hgf mRNA expression. Forced VEGF expression in ESC-derived HLCs in EBs induced angiogenesis around the HLCs and resulted in an increase in the amount of HLCs. CONCLUSIONS: VEGF indirectly induces the proliferation and function of ESC-derived HLCs through VEGFR1 and VEGFR2 signaling in endothelial cells.

Involvement of CCAAT/enhancer binding protein-beta (C/EBPbeta) in epigenetic regulation of mouse methionine adenosyltransferase 1A gene expression.

Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the main methyl donor in cellular transmethylation reactions and the aminopropyl moiety in polyamine biosynthesis. In mammals, two different genes, MAT1A and MAT2A, encode catalytic polypeptides of liver-specific MAT I/III and ubiquitous MAT II, respectively. Reverse transcription-polymerase chain reaction showed that MAT1A gene expression was at a detectable level in embryonic day 14 mouse fetal liver and subsequently increased. Bisulfite genomic sequencing indicated that the methylation status of 10CpG sites in the MAT1A promoter proximal region was appreciably correlated with the gene expression in mouse developing liver and in adult hepatic cells; hepatic stellate cells and hepatocytes. When mouse hepatoma-derived Hepa-1 cells showing extremely low expression of MAT1A gene were treated with 5-aza-2'-deoxycytidine and trichostatin A, MAT1A gene expression was enhanced. In addition, in vitro methylation of the MAT1A promoter region suppressed the MAT1A promoter activity in reporter assay. Next, we performed electrophoretic mobility shift assay and found that the transcriptional factor CCAAT/enhancer binding protein-beta (C/EBPbeta) specifically binds to a putative binding site of C/EBPbeta in the MAT1A promoter. Suppression of C/EBPbeta expression by short hairpin RNA decreased the MAT1A promoter activity and MAT1A gene expression, and inhibition of C/EBPbeta binding to MAT1A by site-directed mutagenesis also showed similar results. Western blot analysis and chromatin immunoprecipitation assay indicated that C/EBPbeta binding is dependent on DNA methylation status. Based on these findings, we conclude that C/EBPbeta plays an important role in epigenetic regulation of the mature hepatic gene MAT1A.

Hepatic progenitor cells in the mouse extrahepatic bile duct after a bile duct ligation.

The intrahepatic bile duct has been suggested to be a source of hepatic progenitor cells in the severely damaged liver. In contrast, little attention has been paid to the question of whether hepatic progenitor cells exist in the extrahepatic bile duct (EHBD). In the present study, we examined the phenotypic changes of the mouse EHBD following bile duct ligation. After bile duct ligation, the number of c-Kit-positive epithelial cells increased in the EHBD. The ligated EHBD expressed mRNA for hepatic progenitor cell markers, including c-Kit and Thy-1. Hepatocyte markers such as albumin and cytochrome P450 7a1 were also transiently detected in the EHBD after a bile duct ligation. In a culture of EHBD cells, we detected hepatic progenitor cells that were positive for both staining with anti-albumin antibodies and Dolichos biflorus agglutinin, a biliary epithelial cell-specific lectin. Furthermore, hepatic progenitor cells positive for both c-Kit and albumin were found in the cultured EHBD population. Additionally EHBD-derived hepatocyte-like cells were also observed in the culture. A transplantation study revealed that EHBD cells integrate into the parenchyma and are albumin positive. These data suggest that hepatic progenitor cells emerge in the EHBD following bile duct ligation, that subsequently give rise to hepatocyte-like cells. We also observed that the gall bladder transiently expressed hepatocyte markers after bile duct ligation. Our results suggest a potential of the EHBD and gall bladder as useful transplantable sources for liver injury.

Developmental pluripotency-associated 4 (DPPA4) localized in active chromatin inhibits mouse embryonic stem cell differentiation into a primitive ectoderm lineage.

Because embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state and differentiate into various cell types, ES cells are expected to be useful for cell replacement therapy and basic research on early embryogenesis. Although molecular mechanisms of ES cell self-renewal have been studied, many uncharacterized genes expressed in ES cells remain to be clarified. Developmental pluripotency associated 4 (Dppa4) is one such gene highly expressed in both ES cells and early embryos. Here, we investigated the role of Dppa4 in mouse ES cell self-renewal and differentiation. We generated Dppa4-overexpressing ES cells under the control of tetracycline. Dppa4 overexpression suppressed cell proliferation and formation of embryoid bodies and caused massive cell death in differentiating ES cells. Quantitative reverse transcription-PCR analysis showed that Dppa4 overexpression does not support ES cell self-renewal but partially inhibits ES cell differentiation. Suppression of Dppa4 expression by short hairpin RNA induced ES cell differentiation into a primitive ectoderm lineage. DPPA4 protein was localized in the ES cell nucleus associated with chromatin. Micrococcal nuclease digestion analysis and immunocytochemistry revealed that DPPA4 is associated with transcriptionally active chromatin. These findings indicate that DPPA4 is a nuclear factor associated with active chromatin and that it regulates differentiation of ES cells into a primitive ectoderm lineage.

The intrinsic structure of glucose transporter isoforms Glut1 and Glut3 regulates their differential distribution to detergent-resistant membrane domains in nonpolarized mammalian cells.

The hexose transporter family, which mediates facilitated uptake in mammalian cells, consists of more than 10 members containing 12 membrane-spanning segments with a single N-glycosylation site. We previously demonstrated that glucose transporter 1 is organized into a raft-like detergent-resistant membrane domain but that glucose transporter 3 distributes to fluid membrane domains in nonpolarized mammalian cells. In this study, we further examined the structural basis responsible for the distribution by using a series of chimeric constructs. Glucose transporter 1 and glucose transporter 3 with a FLAG-tagged N-terminus were expressed in detergent-resistant membranes and non-detergent-resistant membranes of CHO-K1 cells, respectively. Replacement of either the C-terminal or N-terminal cytosolic portion of FLAG-tagged glucose transporter 1 and glucose transporter 3 did not affect the membrane distribution. However, a critical sorting signal may exist within the N-terminal half of the isoforms without affecting transport activity and its inhibition by cytochalasin B. Further shortening of these regions altered the critical distribution, suggesting that a large proportion or several parts of the intrinsic structure, including the N-terminus of each isoform, are involved in the regulation.

Twist relates to tubular epithelial-mesenchymal transition and interstitial fibrogenesis in the obstructed kidney.

Epithelial-mesenchymal transition (EMT) is a critical step in renal fibrosis. It has been recently reported that a transcription factor, Twist, plays a pivotal role in metastasis of breast tumors by inducing EMT. In this study, we examined whether Twist relates to renal fibrogenesis including EMT of tubular epithelia, evaluating Twist expression level in the unilateral ureteral obstruction (UUO) model. Kidneys of mice subjected to UUO were harvested 1, 3, 7, and 10 days after obstruction. Compared with control kidneys, Twist mRNA-level significantly increased 3 days after UUO (UUO day 3 kidney) and further augmented until 10 days after UUO. Twist expression increased in tubular epithelia of the dilated tubules and the expanded interstitial areas of UUO kidneys, where cell-proliferating appearances were frequently found in a time-dependent manner. Although a part of tubular cells in whole nephron segment were immunopositive for Twist in UUO day 7 kidneys, tubular epithelia downstream of nephron more frequently expressed Twist than upstream of nephron. In UUO day 7 kidneys, some tubular epithelia were confirmed to coexpress Twist and fibroblast-specific protein-1, a marker for EMT, indicating that Twist is involved in tubular EMT under pathological state. Twist was expressed also in a number of alpha-smooth muscle actin-positive myofibroblasts located in the expanded interstitial area of UUO kidneys. From these findings, the present investigation suggests that Twist is associated with tubular EMT, proliferation of myofibroblasts, and subsequent renal fibrosis in obstructed kidneys.