Kidney organoid derived from renal tissue stem cells is a useful tool for histopathological assessment of nephrotoxicity in a cisplatin-induced acute renal tubular injury model.

Organoids derived from renal tissue stem cells (KS cells) isolated from the S3 segment of adult rat nephrons have previously been developed and evaluated. However, data regarding the histopathological evaluation of these organoids are limited. Therefore, in this study, we performed histopathological examinations of the properties of these organoids and evaluated the nephrotoxicity changes induced by cisplatin treatment. We observe that the tubular structure of the organoids was generally lined by a single layer of cells, in concordance with previous findings. Microvilli were exclusively observed under electron microscopy on the luminal side of this tubular structure. Moreover, the luminal side of the tubular structure was positive for aquaporin-1 (Aqp1), a marker of the proximal renal tubule. Cisplatin treatment induced cell death and degeneration, including cytoplasmic vacuolation, in cells within the tubular structure of the organoids. Cisplatin toxicity is associated with the induction of γ-H2AX (a marker of DNA damage) and the drop of phospho-histone H3 (a marker of cell division) levels. During the nephrotoxicity assessment, the kidney organoids displayed various features similar to those of the natural kidney, suggesting that it is possible to use these organoids in predicting nephrotoxicity. The histological evaluation of the organoids in this study provides insights into the mechanisms underlying nephrotoxicity.

Stabilization of P/CAF, as a ubiquitin ligase toward MDM2, suppresses mitotic cell death through p53-p21 activation in HCT116 cells with SIRT2 suppression.

We previously reported that the suppression of SIRT2, an NAD + -dependent protein deacetylases, induces p53 accumulation via degradation of p300 and the subsequent MDM2 degradation, eventually leading to apoptosis in HeLa cells. The present study identified a novel pathway of p53 accumulation by SIRT2 suppression in HCT116(p53+/+) cells in which SIRT2 suppression led to escape from mitotic cell death caused by spindle assembly checkpoint activation induced by microtubule inhibitors such as nocodazole but not apoptosis or G1 or G2 arrest. We found that SIRT2 interacts with P/CAF, a histone acetyltransferase, which also acts as a ubiquitin ligase against MDM2. SIRT2 suppression led to an increase of P/CAF acetylation and its stabilization followed by a decrease in MDM2 and activation of the p53-p21 pathway. Depression of mitotic cell death in HCT116(p53+/+) cells with SIRT2 suppression was released by suppression of P/CAF or p21. Thus, the P/CAF-MDM2-p53-p21 axis enables the escape from mitotic cell death and confers resistance to nocodazole in HCT116(p53+/+) cells with SIRT2 suppression. As SIRT2 has attracted attention as a potential target for cancer therapeutics for p53 regulation, the present study provides a molecular basis for the efficacy of SIRT2 for future cancer therapy based on p53 regulation. These findings also suggest an undesirable function of the SIRT2 suppression associated with activation of the p53-p21 pathway in the suppression of mitotic cell death caused by spindle assembly checkpoint activation.

MPP8 and SIRT1 crosstalk in E-cadherin gene silencing and epithelial-mesenchymal transition.

As a critical developmental process, epithelial-mesenchymal transition (EMT) involves complex transcriptional reprogramming and has been closely linked to malignant progression. Although various epigenetic modifications, such as histone deacetylation and H3K9 methylation, have been implicated in this process, how they are coordinated remains elusive. We recently revealed that MPP8 couples H3K9 methylation and DNA methylation for E-cadherin gene silencing and promotes tumor cell migration, invasion, and EMT. Here, we show that MPP8 cooperates with the class III HDAC SIRT1 in this process through their physical interaction. SIRT1 antagonizes PCAF-catalyzed MPP8-K439 acetylation to protect MPP8 from ubiquitin-proteasome-mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl-H3K9 on target promoters. Consequently, disabling either MPP8 methyl-H3K9 binding or SIRT1 interaction de-represses E-cadherin and reduces EMT phenotypes, as does knockdown of MPP8 or SIRT1 in prostate cancer cells. These results illustrate how SIRT1 and MPP8 reciprocally promote each other's function and coordinate epithelial gene silencing and EMT.

A kidney injury molecule-1 (Kim-1) gene reporter in a mouse artificial chromosome: the responsiveness to cisplatin toxicity in immortalized mouse kidney S3 cells.

BACKGROUND: Kidney injury molecule-1 (Kim-1) has been validated as a urinary biomarker for acute and chronic renal damage. The expression of Kim-1 mRNA is also activated by acute kidney injury induced by cisplatin in rodents and humans. To date, the measurement of Kim-1 expression has not fully allowed the detection of in vitro cisplatin nephrotoxicity in immortalized culture cells, such as human kidney-2 cells and immortalized proximal tubular epithelial cells. METHODS: We measured the augmentation of Kim-1 mRNA expression after the addition of cisplatin using immortalized S3 cells established from the kidneys of transgenic mice harboring temperature-sensitive large T antigen from Simian virus 40. RESULTS: A mouse Kim-1 gene luciferase reporter in conjunction with an Hprt gene reporter detected cisplatin-induced nephrotoxicity in S3 cells. These two reporter genes were contained in a mouse artificial chromosome, and two luciferases that emitted different wavelengths were used to monitor the respective gene expression. However, the Kim-1 reporter gene failed to respond to cisplatin in A9 fibroblast cells that contained the same reporter mouse artificial chromosome, suggesting cell type-specificity for activation of the reporter. CONCLUSIONS: We report the feasibility of measuring in vitro cisplatin nephrotoxicity using a Kim-1 reporter gene in S3 cells.

Methyl-H3K9-binding protein MPP8 mediates E-cadherin gene silencing and promotes tumour cell motility and invasion.

H3K9 methylation has been linked to a variety of biological processes including position-effect variegation, heterochromatin formation and transcriptional regulation. To further understand the function of H3K9 methylation, we have identified and characterized MPP8 as a methyl-H3K9-binding protein. MPP8 displays an elevated expression pattern in various human carcinoma cells, whereas knocking-down MPP8 results in the loss of cellular mesenchymal marker as well as the reduction of tumour cell migration and invasiveness, suggesting that MPP8 contributes to tumour progression. Following characterization demonstrates that MPP8 targets the E-cadherin gene promoter and modulates the expression of this key regulator of cell behaviour and tumour progression through its methyl-H3K9 binding. Furthermore, MPP8 interacts with H3K9 methyltransferases GLP and ESET, as well as DNA methyltransferase 3A. MPP8 knockdown decreases DNA methylation on E-cadherin CpG island attended by the loss of DNMT3A localization, indicating MPP8 also directs DNA methylation. Together, our results suggest a model by which MPP8 recognizes methyl-H3K9 marks and directs DNA methylation to repress tumour suppressor gene expression and, in turn, has an important function in epithelial-to-mesenchymal transition and metastasis.

The same enhancer regulates the earliest Emx2 expression in caudal forebrain primordium, subsequent expression in dorsal telencephalon and later expression in the cortical ventricular zone.

We have analyzed Emx2 enhancers to determine how Emx2 functions during forebrain development are regulated. The FB (forebrain) enhancer we identified immediately 3' downstream of the last coding exon is well conserved among tetrapods and unexpectedly directed all the Emx2 expression in forebrain: caudal forebrain primordium at E8.5, dorsal telencephalon at E9.5-E10.5 and the cortical ventricular zone after E12.5. Otx, Tcf, Smad and two unknown transcription factor binding sites were essential to all these activities. The mutant that lacked this enhancer demonstrated that Emx2 expression under the enhancer is solely responsible for diencephalon development. However, in telencephalon, the FB enhancer did not have activities in cortical hem or Cajal-Retzius cells, nor was its activity in the cortex graded. Emx2 expression was greatly reduced, but persisted in the telencephalon of the enhancer mutant, indicating that there exists another enhancer for Emx2 expression unique to mammalian telencephalon.

Cisplatin Induces Cell Death in Rat Adult Kidney Stem/ Progenitor Cell-Derived Kidney Organoids.

Background: The use of stem/ progenitor cell-derived organoids to evaluate the toxicity of chemical substances has widely increased. Organoids with nephron-like structures (NLS) can be derived from rat adult kidney stem/ progenitor cells (rKS cells) using three-dimensional culture. In this study, we examined the effects of cisplatin, an anticancer drug that induces nephrotoxicity in vivo, on rKS cell-derived NLS. Methods: Twelve organoids were simultaneously derived from three-dimensionally cultured rKS cells in Matrigel matrices. The surface area of each organoid was measured using microscopy-based imaging, and the morphological changes of NLS were monitored using an image analysis method. NLS were exposed to cisplatin, and their associated effects were assessed. Results: NLS elongated over time. The surface areas of the 12 organoids were almost constant. Cisplatin exposure induced cell death in NLS and inhibited the increase in the surface area of the organoids. Conclusion: Cisplatin exposure induces damage to NLS derived from rKS cells. Thus, the organoids may be valuable as an in vitro model to assess nephrotoxicity.

Ski co-repressor complexes maintain the basal repressed state of the TGF-beta target gene, SMAD7, via HDAC3 and PRMT5.

The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co-repressors and interact with other co-repressors such as N-CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c-Myb. In particular, the inhibition of TGF-beta-induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF-beta target gene, in TGF-beta-untreated HepG2 cells. Knockdown of these components using siRNA led to up-regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF-beta-responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.

Establishment of human immortalized mesenchymal stem cells lines for the monitoring and analysis of osteogenic differentiation in living cells.

Mesenchymal stem cells (MSCs) are expected to be useful in bone regeneration treatment for various diseases and conditions, including cleft lip and palate, fracture, and bone absorption. However, to date, MSCs have failed to produce satisfactory results in clinical settings. This is primarily due to the low rate of induced osteogenic differentiation. To realize MSC potential, it is necessary to establish methods for the isolation of MSC-derived living osteoblasts. However, no osteoblast markers have been reported to date. In an attempt to develop a method for the assessment of osteoblast differentiation, we established reporter human immortalized MSC (hiMSC) lines for in vitro monitoring of bone gamma-carboxyglutamate protein (BGLAP, osteocalcin) expression. To this end, we successfully knocked-in an enhanced green fluorescent protein (EGFP) gene cassette immediately downstream of the first ATG of BGLAP via CRISPR-Cas9, and established hiMSC lines expressing EGFP to monitor osteogenic differentiation. On differentiation day 7, EGFP-positive cells were collected by flow cytometric cell sorting, and the expression of EGFP and endogenous BGLAP was analyzed. During osteogenic differentiation, EGFP upregulation was found to correlate with expression of endogenous BGLAP. Moreover, mineralization was confirmed using Alizarin red-S staining after two weeks of osteogenic differentiation of the modified hiMSC lines. The modified hiMSC lines, as well as the derived differentiated osteoblasts obtained herein, are valuable tools for the monitoring osteoblast gene and protein expression, and can be used to develop novel methods for isolating living osteoblasts.

In vitro histone demethylase assays.

Histone methylation plays important roles in chromatin structure, transcription, and epigenetic state of the cell. Tremendous discoveries recently demonstrated that methylation mark is not static but is dynamically regulated by both histone methyltransferases and the histone demethylases. Two families of histone demethylases have been identified to remove methyl groups from lysine side chain through different reaction mechanisms in presence of distinct cofactors. Amine oxidase LSD1 family requires flavin adenine dinucleotide (FAD) whereas dioxygenase Jmjc domain-containing proteins family relies on Fe(II) and alpha-ketoglutarate. Identification of these enzymes opened a new era in understanding how chromatin dynamic is regulated and further understanding the regulation of these enzymes will provide significant insights into fundamental mechanisms of many biological processes and human diseases. This chapter describes different assay conditions and detection methods for different family of histone demethylases. We also summarize step-by-step protocols for purification and preparation of various histone substrates for histone demethylase assays.

Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome.

The site-specific excision of a target DNA sequence for genetic knockout or lineage tracing is a powerful tool for investigating biological systems. Currently, site-specific recombinases (SSRs), such as Cre or Flp recombination target cassettes, have been successfully excised or inverted by a single SSR to regulate transgene expression. However, the use of a single SSR might restrict the complex control of gene expression. This study investigated the potential for expanding the multiple regulation of transgenes using three different integrase systems (TP901-1, R4, and Bxb1). We designed three excision cassettes that expressed luciferase, where the luciferase expression could be exchanged to a fluorescent protein by site-specific recombination. Individual cassettes that could be regulated independently by a different integrase were connected in tandem and inserted into a mouse artificial chromosome (MAC) vector in Chinese hamster ovary cells. The transient expression of an integrase caused the targeted luciferase activity to be lost and fluorescence was activated. Additionally, the integrase system enabled the specific excision of targeted DNA sequences without cross-reaction with the other recombination targets. These results suggest that the combined use of these integrase systems in a defined locus on a MAC vector permits the multiple regulation of transgene expression and might contribute to genomic or cell engineering.

Evaluation of an Hprt-Luciferase Reporter Gene on a Mammalian Artificial Chromosome in Response to Cytotoxicity.

BACKGROUND: Hypoxanthine guanine phosphoribosyltransferase (Hprt) is known as a house-keeping gene, and has been used as an internal control for real-time quantitative RT-PCR and various other methods of gene expression analysis. To evaluate the Hprt mRNA levels as a reference standard, we engineered a luciferase reporter driven by a long Hprt promoter and measured its response to cytotoxicity. METHODS: We constructed a reporter vector that harbored a phiC31 integrase recognition site and a mouse Hprt promoter fused with green-emitting luciferase (SLG) coding sequence. The Hprt-SLG vector was loaded onto a mouse artificial chromosome containing a multi-integrase platform using phiC31 integrase in mouse A9 cells. We established three independent clones. RESULTS: The established cell lines had similar levels of expression of the Hprt-SLG reporter gene. Hprt-SLG activity increased proportionately under growth conditions and decreased under cytotoxic conditions after blasticidin or cisplatin administration. Similar increases and decreases in the SLG luminescent were observed under growth and cytotoxic conditions, respectively, to those in the fluorescent obtained using the commercially available reagent, alamarBlue. CONCLUSION: By employing a reliable and stable expression system in a mammalian artificial chromosome, the activity of an Hprt-SLG reporter can reflect cell numbers under cell growth condition and cell viability in the evaluation of cytotoxic conditions.

In vitro histone demethylase assays.

Histone methylation plays pivotal roles in modulating chromatin structure and dynamics and in turn regulates genomic processes that require access to the DNA template. The methylation status at different sites is dynamically regulated by histone methyltransferases and demethylases. During the past decade, two classes of proteins have been characterized to actively remove methyl groups from lysine residues through different mechanisms. The LSD1/KDM1 family of amine oxidases require flavin adenine dinucleotide (FAD) for reaction, while the larger Jumonji C (JmjC) family of hydroxylases utilize Fe(II) and α-ketoglutarate as cofactors to demethylate histones. Since their discoveries, histone demethylases have been implicated in the precise control of gene expression program during development, cell identity, and fate decision. Several demethylases have also been linked to various human diseases such as neurological disorders and cancer. This chapter describes several in vitro assay conditions and detection methods for two classes of histone demethylases. We also discuss the protocols to prepare various substrates for different histone demethylase assays.

Gene expression in hepatomas.

Gene expression changes in accordance with cell growth, differentiation and carcinogenesis. To elucidate the molecular mechanisms for hepatocarcinogenesis as well as maintenance of normal hepatocytes, it is important to identify the genes that have altered expression with carcinogenesis. We established a new and efficient cDNA subtraction method via two cDNA populations. By using this method along with rat hepatomas made by the Soh-Farber protocol, we identified a number of genes, some of which are activated in hepatocellular carcinoma (HCC). These genes include ones which code for a transcription factor and a metabolic enzyme. One particular gene can be used as a tumour marker. Our method is beneficial for the isolation of a wide range of HCC-related genes in rats which, in turn, enables easy identification of their human counterparts. In this review, we describe details of our method and the isolated genes. We also briefly describe transcription factors in the liver.

MPP8 mediates the interactions between DNA methyltransferase Dnmt3a and H3K9 methyltransferase GLP/G9a.

DNA CpG methylation and histone H3 lysine 9 (H3K9) methylation are two major repressive epigenetic modifications, and these methylations are positively correlated with one another in chromatin. Here we show that G9a or G9a-like protein (GLP) dimethylate the amino-terminal lysine 44 (K44) of mouse Dnmt3a (equivalent to K47 of human DNMT3A) in vitro and in cells overexpressing G9a or GLP. The chromodomain of MPP8 recognizes the dimethylated Dnmt3aK44me2. MPP8 also interacts with self-methylated GLP in a methylation-dependent manner. The MPP8 chromodomain forms a dimer in solution and in crystals, suggesting that a dimeric MPP8 molecule could bridge the methylated Dnmt3a and GLP, resulting in a silencing complex of Dnmt3a-MPP8-GLP/G9a on chromatin templates. Together, these findings provide a molecular explanation, at least in part, for the co-occurrence of DNA methylation and H3K9 methylation in chromatin.

The Ski-binding protein C184M negatively regulates tumor growth factor-beta signaling by sequestering the Smad proteins in the cytoplasm.

Ski is a transcriptional co-repressor and is involved in the negative regulation of tumor growth factor-beta (TGF-beta) signaling. To understand more fully the role of Ski in TGF-beta signaling, we searched for novel Ski-interacting proteins. The identified C184M protein consists of 189 amino acids and contains the leucine-rich region. An association between Ski and C184M involving the leucine-rich region of C184M and the C-terminal coiled-coil motif of Ski was confirmed by glutathione S-transferase pull-down and immunoprecipitation assays. The C184M protein is located in the cytosol, and the C184M and Ski signals are co-localized in the cytoplasm when C184M was co-expressed with Ski in CV-1 cells. The cytoplasmic C184M-Ski complex inhibited the nuclear translocation of Smad2. Consistent with this, the activity of promoter containing the Smad-binding sites was repressed by C184M, and the TGF-beta-induced growth inhibition of mink lung Mv1Lu cells was attenuated by the ectopic expression of C184M. Thus, C184M inhibits TGF-beta signaling in concert with Ski. In hepatocytes, which express significant levels of C184M, the Ski signals were found only in the cytoplasm, supporting the notion that C184M forms a complex with Ski in the cytosol.

Requirement of the co-repressor homeodomain-interacting protein kinase 2 for ski-mediated inhibition of bone morphogenetic protein-induced transcriptional activation.

Multiple co-repressors such as N-CoR/SMRT, mSin3, and the c-ski proto-oncogene product (c-Ski) mediate the transcriptional repression induced by Mad and the thyroid hormone receptor by recruiting the histone deacetylase complex. c-Ski also binds directly to Smad proteins, which are transcriptional activators in the transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP) signaling pathways, and inhibits TGF-beta/BMP-induced transcriptional activation. However, it remains unknown whether other co-repressor(s) are also involved with Ski in the negative regulation of the TGF-beta/BMP signaling pathways. Here, we report that the co-repressor homeodomain-interacting protein kinase 2 (HIPK2) directly binds to both c-Ski and Smad1. HIPK2 efficiently inhibited Smad1/4-induced transcription from the Smad site-containing promoter. A dominant negative form of HIPK2, in which the ATP binding motif in the kinase domain and the putative phosphorylation sites were mutated, enhanced Smad1/4-dependent transcription and the BMP-induced expression of alkaline phosphatase. Furthermore, the c-Ski-induced inhibition of the Smad1/4-dependent transcription was suppressed by a dominant negative form of HIPK2. The HIPK2 co-repressor activity may be regulated by an uncharacterized HIPK2 kinase. These results indicate that HIPK2, together with c-Ski, plays an important role in the negative regulation of BMP-induced transcriptional activation.

p53 suppresses c-Myb-induced trans-activation and transformation by recruiting the corepressor mSin3A.

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2, and NLK.

The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. Here we report that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps.