p53 alters intracellular Ca2+ signaling through regulation of TRPM4.

Altered expression of transient receptor potential channel melastatin 4 (TRPM4) contributes to several diseases, including cardiac conduction disorders, immune diseases, and cancer. Yet the underlying mechanisms of TRPM4 expression changes remain elusive. In this study, we report that loss of tumor suppressor protein p53 or p63γ function or mutation of a putative p53 response element in the TRPM4 promoter region increase TRPM4 promoter activity in the colorectal cancer cell line HCT 116. In cells that lack p53 expression, we observed increased TRPM4 mRNA and protein levels and TRPM4-mediated Na+ currents. This phenotype can be reversed by transient overexpression of p53. In the prostate cancer cell line LNCaP, which expresses p53 endogenously, p53 overexpression decreases TRPM4-mediated currents. As in other cancer cells, CRISPR-Cas9 mediated knockout of TRPM4 in p53 deficient HCT 116 cells results in increased store-operated Ca2+entry. The effect of the TRPM4 knockout is mimicked by p53 mediated suppression of TRPM4 in the parental cell line expressing TRPM4. In addition, a TRPM4 knockout-mediated shift in cell cycle is abolished upon loss of p53. Taken together, these findings indicate that p53 represses TRPM4 expression, thereby altering cellular Ca2+ signaling and that TRPM4 adds to cell cycle shift dependent on p53 signaling. One sentence summary: TRPM4 is repressed in the p53 pathway leading to reduced currents and increased calcium signaling.

How to Obtain a Mega-Intestine with Normal Morphology: In Silico Modelling of Postnatal Intestinal Growth in a Cd97-Transgenic Mouse.

Intestinal cylindrical growth peaks in mice a few weeks after birth, simultaneously with crypt fission activity. It nearly stops after weaning and cannot be reactivated later. Transgenic mice expressing Cd97/Adgre5 in the intestinal epithelium develop a mega-intestine with normal microscopic morphology in adult mice. Here, we demonstrate premature intestinal differentiation in Cd97/Adgre5 transgenic mice at both the cellular and molecular levels until postnatal day 14. Subsequently, the growth of the intestinal epithelium becomes activated and its maturation suppressed. These changes are paralleled by postnatal regulation of growth factors and by an increased expression of secretory cell markers, suggesting growth activation of non-epithelial tissue layers as the origin of enforced tissue growth. To understand postnatal intestinal growth mechanistically, we study epithelial fate decisions during this period with the use of a 3D individual cell-based computer model. In the model, the expansion of the intestinal stem cell (SC) population, a prerequisite for crypt fission, is largely independent of the tissue growth rate and is therefore not spontaneously adaptive. Accordingly, the model suggests that, besides the growth activation of non-epithelial tissue layers, the formation of a mega-intestine requires a released growth control in the epithelium, enabling accelerated SC expansion. The similar intestinal morphology in Cd97/Adgre5 transgenic and wild type mice indicates a synchronization of tissue growth and SC expansion, likely by a crypt density-controlled contact inhibition of growth of intestinal SC proliferation. The formation of a mega-intestine with normal microscopic morphology turns out to originate in changes of autonomous and conditional specification of the intestinal cell fate induced by the activation of Cd97/Adgre5.

Repertoire and Neutralizing Activity of Antibodies Against Hepatitis C Virus E2 Peptide in Patients With Spontaneous Resolution of Hepatitis C.

Neutralizing antibodies can prevent hepatitis C virus (HCV) infection, one of the leading causes of cirrhosis and liver cancer. Here, we characterized the immunoglobulin repertoire of memory B-cell antibodies against a linear epitope in the central front layer of the HCV envelope (E2; amino acids 483-499) in patients who were infected in a single-source outbreak. A reverse transcription polymerase chain reaction-based immunoglobulin gene cloning and recombinant expression approach was used to express monoclonal antibodies from HCV E2 peptide-binding immunoglobulin G-positive memory B cells. We identified highly mutated antibodies with a neutralizing effect in vitro against different genotype isolates sharing similar gene features. Our data confirm the importance of VH1-69 use for neutralizing activity. The data offer a promising basis for vaccine research and the use of anti-E2 antibodies as a means of passive immunization.

Loss of Msh2 and a single-radiation hit induce common, genome-wide, and persistent epigenetic changes in the intestine.

BACKGROUND: Mismatch repair (MMR)-deficiency increases the risk of colorectal tumorigenesis. To determine whether the tumors develop on a normal or disturbed epigenetic background and how radiation affects this, we quantified genome-wide histone H3 methylation profiles in macroscopic normal intestinal tissue of young radiated and untreated MMR-deficient VCMsh2LoxP/LoxP (Msh2-/-) mice months before tumor onset. RESULTS: Histone H3 methylation increases in Msh2-/- compared to control Msh2+/+ mice. Activating H3K4me3 and H3K36me3 histone marks frequently accumulate at genes that are H3K27me3 or H3K4me3 modified in Msh2+/+ mice, respectively. The genes recruiting H3K36me3 enrich in gene sets associated with DNA repair, RNA processing, and ribosome biogenesis that become transcriptionally upregulated in the developing tumors. A similar epigenetic effect is present in Msh2+/+ mice 4 weeks after a single-radiation hit, whereas radiation of Msh2-/- mice left their histone methylation profiles almost unchanged. CONCLUSIONS: MMR deficiency results in genome-wide changes in histone H3 methylation profiles preceding tumor development. Similar changes constitute a persistent epigenetic signature of radiation-induced DNA damage.

Bistable Epigenetic States Explain Age-Dependent Decline in Mesenchymal Stem Cell Heterogeneity.

The molecular mechanisms by which heterogeneity, a major characteristic of stem cells, is achieved are yet unclear. We here study the expression of the membrane stem cell antigen-1 (Sca-1) in mouse bone marrow mesenchymal stem cell (MSC) clones. We show that subpopulations with varying Sca-1 expression profiles regenerate the Sca-1 profile of the mother population within a few days. However, after extensive replication in vitro, the expression profiles shift to lower values and the regeneration time increases. Study of the promoter of Ly6a unravels that the expression level of Sca-1 is related to the promoter occupancy by the activating histone mark H3K4me3. We demonstrate that these findings can be consistently explained by a computational model that considers positive feedback between promoter H3K4me3 modification and gene transcription. This feedback implicates bistable epigenetic states which the cells occupy with an age-dependent frequency due to persistent histone (de-)modification. Our results provide evidence that MSC heterogeneity, and presumably that of other stem cells, is associated with bistable epigenetic states and suggest that MSCs are subject to permanent state fluctuations. Stem Cells 2017;35:694-704.

Stem cell competition in the gut: insights from multi-scale computational modelling.

Three-dimensional (3D) computational tissue models can provide a comprehensive description of tissue dynamics at the molecular, cellular and tissue level. Moreover, they can support the development of hypotheses about cellular interactions and about synergies between major signalling pathways. We exemplify these capabilities by simulation of a 3D single-cell-based model of mouse small intestinal crypts. We analyse the impact of lineage specification, distribution and cellular lifespan on clonal competition and study effects of Notch- and Wnt activation on fixation of mutations within the tissue. Based on these results, we predict that experimentally observed synergistic effects between autonomous Notch- and Wnt signalling in triggering intestinal tumourigenesis originate in the suppression of Wnt-dependent secretory lineage specification by Notch, giving rise to an increased fixation probability of Wnt-activating mutations. Our study demonstrates that 3D computational tissue models can support a mechanistic understanding of long-term tissue dynamics under homeostasis and during transformation.

Transient receptor potential melastatin 4 channel contributes to migration of androgen-insensitive prostate cancer cells.

Impaired Ca2+ signaling in prostate cancer contributes to several cancer hallmarks, such as enhanced proliferation and migration and a decreased ability to induce apoptosis. Na+ influx via transient receptor potential melastatin 4 channel (TRPM4) can reduce store-operated Ca2+ entry (SOCE) by decreasing the driving force for Ca2+. In patients with prostate cancer, gene expression of TRPM4 is elevated. Recently, TRPM4 was identified as a cancer driver gene in androgen-insensitive prostate cancer.We investigated TRPM4 protein expression in cancer tissue samples from 20 patients with prostate cancer. We found elevated TRPM4 protein levels in prostatic intraepithelial neoplasia (PIN) and prostate cancer tissue compared to healthy tissue. In primary human prostate epithelial cells (hPEC) from healthy tissue and in the androgen-insensitive prostate cancer cell lines DU145 and PC3, TRPM4 mediated large Na+ currents. We demonstrated significantly increased SOCE after siRNA targeting of TRPM4 in hPEC and DU145 cells. In addition, knockdown of TRPM4 reduced migration but not proliferation of DU145 and PC3 cells. Taken together, our data identify TRPM4 as a regulator of SOCE in hPEC and DU145 cells, demonstrate a role for TRPM4 in cancer cell migration and suggest that TRPM4 is a promising potential therapeutic target.

CK2 kinase activity but not its binding to CK2 promoter regions is implicated in the regulation of CK2α and CK2ß gene expressions.

Protein kinase CK2, a ubiquitous serine/threonine kinase in control of a variety of crucial cellular functions, is composed of catalytic α- and α'-subunits and non-catalytic ß-subunits which form holoenzymes such as CK2(αß)2, CK2αα'ß2, or CK2(α'ß)2. In addition, there is ample evidence for the occurrence of the individual subunits beside the holoenzyme. While the CK2 subunits are well analyzed on the protein level, only little is known about the regulation of their transcription. The existence of multiple forms of CK2 subunits raised the question about a mutual regulation of their expression. Here we defined two 5'-upstream regions of the CK2α and the CK2ß genes, respectively, as sequences with promoter activities. We found that CK2α and CK2α' stimulated the expression of the reporter constructs whereas, CK2ß was inactive. Using chromatin immunoprecipitation assays, we were unable to detect binding of endogenous CK2 subunits to these promoter sequences in vivo. However, it turned out that inhibition of the kinase activity of CK2 attenuated the promoter activity indicating that CK2α and CK2α' might regulate their gene expression indirectly by phosphorylation reactions. Thus, we have shown here (i) that under normal physiological conditions CK2 does not bind to CK2 promoter regions and (ii) that the CK2 kinase activity is implicated in the regulation of its own expression.

p53 and cell cycle dependent transcription of kinesin family member 23 (KIF23) is controlled via a CHR promoter element bound by DREAM and MMB complexes.

The microtubule-dependent molecular motor KIF23 (Kinesin family member 23) is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21(WAF1/CIP1) was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR) in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth.

One function--multiple mechanisms: the manifold activities of p53 as a transcriptional repressor.

Maintenance of genome integrity is a dynamic process involving complex regulation systems. Defects in one or more of these pathways could result in cancer. The most important tumor-suppressor is the transcription factor p53, and its functional inactivation is frequently observed in many tumor types. The tumor suppressive function of p53 is mainly attributed to its ability to regulate numerous target genes at the transcriptional level. While the mechanism of transcriptional induction by p53 is well characterized, p53-dependent repression is not understood in detail. Here, we review the manifold mechanisms of p53 as a transcriptional repressor. We classify two different categories of repressed genes based on the underlying mechanism, and novel mechanisms which involve regulation through noncoding RNAs are discussed. The complete elucidation of p53 functions is important for our understanding of its tumor-suppressor activity and, therefore, represents the key for the development of novel therapeutic approaches.

The retinal dehydrogenase/reductase retSDR1/DHRS3 gene is activated by p53 and p63 but not by mutants derived from tumors or EEC/ADULT malformation syndromes.

Retinol and its metabolites have important roles in many processes including embryonic development, cellular differentiation, apoptosis and maintenance of epithelia. Retinal short-chain dehydrogenase/reductase retSDR1, also known as dehydrogenase/reductase member 3 (DHRS3), is involved in maintaining the cellular supply of retinol metabolites. We observe that retSDR1 expression is activated by members of the p53 family. Particularly p53 and TAp63γ regulate transcription through two separate response elements in the retSDR1 promoter. Both proteins bind the promoter in vitro and in vivo. Induction of DNA damage leads to recruitment of p53 and p63 to the retSDR1 promoter. A tumor-derived p53 mutant is unable to activate retSDR1 transcription. As mutants of p63 in humans exhibit phenotypes that cause several autosomal dominantly inherited syndromes leading to developmental malformations, we tested the transcriptional response of TAp63γ mutants derived from the EEC, SHFM and ADULT syndromes. EEC syndrome-specific mutations of TAp63γ fail to transactivate retSDR1 and an ADULT syndrome-derived mutant stimulates retSDR1 transcription significantly less than the wild-type variant of p63. Taken together, the results suggest a potential role of the p53/p63-mediated retSDR1 activation in tumor suppression as well as in developmental processes.

The CCN3 gene coding for an extracellular adhesion-related protein is transcriptionally activated by the p53 tumor suppressor.

The CCN3 protein (Nov, Nephroblastoma overexpressed) is a member of the CCN family (Cyr61, CTGF, Nov) of growth regulators and exerts antiproliferative properties. We show here that the tumor suppressor p53 transcriptionally upregulates the CCN3 gene. p53 is an important transcription factor contributing to cell cycle arrest and apoptosis after cell damage through the regulation of numerous target genes. We show that CCN3 mRNA and protein are upregulated following p53 expression. A DNA binding-deficient p53 mutant fails to regulate CCN3. CCN3 protein is located in the perinuclear space after induction and is also exported to the extracellular matrix. Furthermore, the CCN3 promoter is inducible by p53 and the response element is located in the first exon of the CCN3 gene. Chromatin immunoprecipitations show that p53 binds to the CCN3 promoter in vivo. As CCN3 was shown to inhibit cell growth, transcriptional induction by p53 may serve as an antiproliferative signal in the extracellular matrix. Furthermore, CCN3 depletion was also reported to reduce collagen type IV-dependent adhesion of melanocytes. Thus, elevated levels of CCN3 protein regulated by p53 might influence cell adhesion.

Gene expression of cyclin-dependent kinase subunit Cks2 is repressed by the tumor suppressor p53 but not by the related proteins p63 or p73.

Cks2 proteins are essential components of cyclin/cyclin-dependent kinase complexes and contribute to cell cycle control. We identify Cks2 as a transcriptional target downregulated by the tumor suppressor p53. Cks2 expression was found to be repressed by p53 both at the mRNA and the protein levels. p53 downregulates transcription from the Cks2 promoter in a dose-dependent manner and in all cell types tested. This repression appears to be independent of p53 binding to the Cks2 promoter. In contrast to p53, neither p63 nor p73 proteins can repress Cks2 transcription. Thus p53, rather than its homologues p63 and p73, may contribute to control of the first metaphase/anaphase transition of mammalian meiosis by downregulation of Cks2 expression.

Expression of cyclin-dependent kinase subunit 1 (Cks1) is regulated during the cell cycle by a CDE/CHR tandem element and is downregulated by p53 but not by p63 or p73.

Cks1 is a member of the cyclin-dependent kinase subunit family. These proteins are essential components of cyclin/cyclin-dependent kinase (cdk) complexes contributing to cell cycle control in all eukaryotes. Cks1 protein is found overexpressed in a number of tumors. Expression of Cks1 mRNA starts in late G1 reaching a peak in S/G2-phases of the cell cycle. We find that this expression pattern depends on transcriptional regulation and is controlled by a combination of a cell cycle-dependent element (CDE) together with a cell cycle genes homology region (CHR) in the Cks1 promoter. Furthermore, we observe Cks1 mRNA and protein to be downregulated after induced expression of the tumor suppressor p53. This repression is due to p53 downregulating transcription from the Cks1 promoter. p53-dependent repression is seen in a dose-dependent manner and in several cell types of different origin. In contrast to p53, its homologues p63 and p73 do not significantly repress transcription from the Cks1 promoter. The Cks1 promoter does not contain a p53 binding site. For some promoters the CCAAT box-binding transcription factor NF-Y had been implicated in p53-dependent repression. NF-Y is the main activator for Cks1 transcription but does not influence p53-dependent repression from the Cks1 promoter. Generally, the observation that the potential oncogene Cks1 is downregulated by the tumor suppressor p53 corresponds well with the idea that p53 employs multiple ways in order to halt the cell cycle.

Identification of Tcf-4 as a transcriptional target of p53 signalling.

T-cell factor (Tcf)-4 is a main transcription factor to pass on Wnt/beta-catenin signalling. The tumour suppressor protein p53 contributes as a transcription factor to cell-cycle arrest and apoptosis induction. Mutations of components in p53 and Wnt/beta-catenin signalling networks play a part in tumour formation. Here, we identify the Tcf-4 gene as a downstream effector of p53. Induction of wild-type p53 in a tet-off regulated human colon cell system leads to the reduction of Tcf-4 mRNA and protein levels. Also, mRNA of the Tcf-4 target gene uPAR is downregulated after p53 induction. Expression of a luciferase reporter controlled by the Tcf-4 promoter is repressed by wild-type p53, but not by a p53 mutant deficient in DNA binding. Such a regulation is seen in cell lines of different origin. These findings directly link Wnt/beta-catenin signalling and p53 tumour suppressor function and may provide a mechanism by which loss of p53 function contributes to progression in the adenoma/carcinoma sequence in colon tumours. Furthermore, since Tcf-4 is expressed in many tissues and downregulation of Tcf-4 by p53 is seen in several different cell types, this regulation likely plays a role in proliferation control of all tissues that can express p53 and Tcf-4.

Differential regulation of transcription and induction of programmed cell death by human p53-family members p63 and p73.

The p53 tumor suppressor acts as a transcription factor and has a central function in controlling apoptosis. With p63 and p73 two genes coding for proteins homologous to p53 have been identified. We describe the properties of seven human p63 and p73 proteins as transcriptional activators of p21WAF1/CIP1 expression and apoptotic inducers in direct comparison to p53 in the same assay systems employing DLD-1-tet-off colon cells. Programmed cell death is detected in cells expressing high levels of p53 and p73alpha. Cells overexpressing TAp63alpha, TAp63gamma, TA*p63alpha, TA*p63gamma, DeltaNp63alpha, and DeltaNp63gamma display low or no detectable apoptosis.

A single cell cycle genes homology region (CHR) controls cell cycle-dependent transcription of the cdc25C phosphatase gene and is able to cooperate with E2F or Sp1/3 sites.

The cdc25C phosphatase participates in regulating transition from the G2 phase of the cell cycle to mitosis by dephosphorylating cyclin-dependent kinase 1. The tumor suppressor p53 down-regulates expression of cdc25C as part of G2/M checkpoint control. Transcription of cdc25C oscillates during the cell cycle with no expression in resting cells and maximum transcription in G2. We had identified earlier a new mechanism of cell cycle-dependent transcription that is regulated by a cell cycle-dependent element (CDE) in conjunction with a cell cycle genes homology region (CHR). The human cdc25C gene was the first example. CDE/CHR tandem elements have since been found in promoters of many cell cycle genes. Here we show that the mouse cdc25C gene is regulated by a CHR but does not hold a CDE. Therefore, it is the first identified gene with CHR-dependent transcriptional regulation during the cell cycle not relying on a CDE located upstream of it. The CHR leads to repression of cdc25C transcription early in the cell cycle and directs a release of this repression in G2. Furthermore, we find that this CHR can cooperate in cell cycle-dependent transcription with elements placed directly upstream of it binding E2F, Sp1 or Sp3 transcription factors.