scholarly journals Polyamine-modulated c-Myc expression in normal intestinal epithelial cells regulates p21Cip1 transcription through a proximal promoter region

2006 ◽  
Vol 398 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Lan Liu ◽  
Xin Guo ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Bernard S. Marasa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Transcriptional Repression ◽  
Critical Role ◽  
Ectopic Expression ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Transcriptional Level ◽  
Intestinal Epithelial ◽  
Major Player ◽  
Stimulation Of

Maintenance of intestinal mucosal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. Our previous studies have shown that polyamines are essential for expression of the c-myc gene and that polyamine-induced c-Myc plays a critical role in stimulation of normal IEC (intestinal epithelial cell) proliferation, but the exact downstream targets of induced c-Myc are still unclear. The p21Cip1 protein is a major player in cell cycle control, which is primarily regulated at the transcriptional level. The current study was designed to determine whether induced c-Myc stimulates normal IEC proliferation by repressing p21Cip1 transcription following up-regulation of polyamines. Overexpression of the ODC (ornithine decarboxylase) gene increased levels of cellular polyamines, induced c-Myc expression and inhibited p21Cip1 transcription, as indicated by repression of p21Cip1 promoter activity and a decrease in p21Cip1 protein levels. In contrast, depletion of cellular polyamines by inhibiting ODC enzyme activity with α-difluoromethylornithine decreased c-Myc, but increased p21Cip1 transcription. Ectopic expression of wild-type c-myc not only inhibited basal levels of p21Cip1 transcription in control cells, but also prevented increased p21Cip1 in polyamine-deficient cells. Experiments using different p21Cip1 promoter mutants showed that transcriptional repression of p21Cip1 by c-Myc was mediated through Miz-1- and Sp1-binding sites within the proximal region of the p21Cip1 promoter in normal IECs. These findings confirm that p21Cip1 is one of the direct mediators of induced c-Myc following increased polyamines and that p21Cip1 repression by c-Myc is implicated in stimulation of normal IEC proliferation.

p53-Dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion

2007 ◽  
Vol 293 (1) ◽  
pp. C379-C389 ◽  
Author(s):  
Ai-Hong Zhang ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
Bernard S. Marasa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Intestinal Epithelial Cell ◽  
Critical Role ◽  
Ectopic Expression ◽  
Negative Control ◽  
Proximal Promoter ◽  
Intestinal Epithelial ◽  
Ndrg1 Gene ◽  
Factor Α

Normal intestinal mucosal growth requires polyamines that regulate expression of various genes involved in cell proliferation, growth arrest, and apoptosis. Our previous studies have shown that polyamine depletion stabilizes p53, resulting in inhibition of intestinal epithelial cell (IEC) proliferation, but the exact downstream targets of induced p53 are still unclear. The NDRG1 (N- myc downregulated gene-1) gene encodes a growth-related protein, and its transcription can be induced in response to stress. The current study tests the hypothesis that induced p53 inhibits IEC proliferation by upregulating NDRG1 expression following polyamine depletion. Depletion of cellular polyamines by inhibiting ornithine decarboxylase (ODC) with α-difluoromethylornithine not only induced p53 but also increased NDRG1 transcription as indicated by induction of the NDRG1 promoter activity and increased levels of NDRG1 mRNA and protein, all of which were prevented by using specific p53 siRNA and in cells with a targeted deletion of p53. In contrast, increased levels of cellular polyamines by ectopic expression of the ODC gene decreased p53 and repressed expression of NDRG1. Consistently, polyamine depletion-induced activation of the NDRG1-promoter was decreased when p53-binding sites within the NDRG1 proximal promoter region were deleted. Ectopic expression of the wild-type NDRG1 gene inhibited DNA synthesis and decreased final cell numbers regardless of the presence or absence of endogenous p53, whereas silencing NDRG1 promoted cell growth. However, overexpression of NDRG1 failed to directly induce cell death and to alter susceptibility to apoptosis induced by tumor necrosis factor-α/cycloheximide. These results indicate that NDRG1 is one of the direct mediators of induced p53 following polyamine depletion and that p53-dependent NDRG1 expression plays a critical role in the negative control of IEC proliferation.

scholarly journals Induced JunD in intestinal epithelial cells represses CDK4 transcription through its proximal promoter region following polyamine depletion

2007 ◽  
Vol 403 (3) ◽  
pp. 573-581 ◽  
Author(s):  
Lan Xiao ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
Bernard S. Marasa ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cells ◽  
Promoter Region ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Ectopic Expression ◽  
Proximal Promoter ◽  
Transcriptional Level ◽  
Intestinal Epithelial ◽  
Proximal Promoter Region

Maintenance of intestinal epithelial integrity requires cellular polyamines that regulate expression of various genes involved in cell proliferation, growth arrest and apoptosis. In prior studies, depletion of cellular polyamines has been shown to stabilize JunD, a member of the AP-1 (activator protein-1) family of transcription factors, leading to inhibition of intestinal epithelial cell proliferation, but the exact downstream targets of induced JunD remain elusive. CDK4 (cyclin-dependent kinase 4) is essential for the G1- to S-phase transition during the cell cycle and its expression is primarily controlled at the transcriptional level. In the present study, we show that induced JunD in IECs (intestinal epithelial cells) is a transcriptional repressor of the CDK4 gene following polyamine depletion. Increased JunD in polyamine-deficient cells was associated with a significant inhibition of CDK4 transcription, as indicated by repression of CDK4-promoter activity and decreased levels of CDK4 mRNA and protein, all of which were prevented by using specific antisense JunD oligomers. Ectopic expression of the wild-type junD also repressed CDK4-promoter activity and decreased levels of CDK4 mRNA and protein without any effect on CDK2 expression. Gel shift and chromatin immunoprecipitation assays revealed that JunD bound to the proximal region of the CDK4-promoter in vitro as well as in vivo, while experiments using different CDK4-promoter mutants showed that transcriptional repression of CDK4 by JunD was mediated through an AP-1 binding site within this proximal sequence of the CDK4-promoter. These results indicate that induced JunD in IECs represses CDK4 transcription through its proximal promoter region following polyamine depletion.

miR-449b-5p Inhibits Cell Proliferation and Migration of Breast Cancer via Targeting Flotillin-2

2020 ◽  
Vol 10 (8) ◽  
pp. 1094-1101
Author(s):  
Delin Wu ◽  
Xiaopeng Ma
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Breast Carcinoma ◽  
Critical Role ◽  
Ectopic Expression ◽  
Luciferase Reporter ◽  
Cell Growth And Migration ◽  
Cancer Pathogenesis ◽  
And Migration

Background: MicroRNAs (miRNAs) act as a critical role in cancer pathogenesis, while the potential of miR-449b-5p in breast carcinoma remains to be fully inquired. Therefore, we purposed to probe the mechanism governing miR-449b-5p in breast cancer. Methods: Reverse transcription-PCR (RTPCR) was adopted to examine miR-449b-5p expression level in breast carcinoma. The functional experiments were implemented to estimate the role of miR-449b-5p in cell growth and migration. The interplay of miR-449b-5p with FLOT2 was validated with luciferase reporter assay. Results: miR-449b-5p level was markedly lessened in the tissue samples and cell lines of breast carcinoma. Overexpression of miR-449b-5p contributed to suppression of cell growth and migration whereas induced apoptosis in SKBr-3 and MCF-7 cells. Moreover, luciferase reporter experiment suggested that FLOT2 had a negative correlation with miR-449b-5p expression. Functionally, ectopic expression of FLOT2 reversed repressive effects of miR-449b-5p mimic on malignant behaviors of breast carcinoma cells. Conclusion: miR-449b-5p hindered cell proliferation, migration and facilitated cell apoptosis of breast carcinoma through targeting FLOT2. Our findings may offer a potent target for the therapy of breast carcinoma.

scholarly journals lncRNA TUG1 modulates proliferation, apoptosis, invasion, and angiogenesis via targeting miR-29b in trophoblast cells

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Qian Li ◽  
Jing Zhang ◽  
Dong-Mei Su ◽  
Li-Na Guan ◽  
Wei-Hong Mu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Critical Role ◽  
Ectopic Expression ◽  
Neonatal Morbidity ◽  
Tube Formation ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Aberrant Expression ◽  
Lncrna Tug1

Abstract Background Pre-eclampsia (PE) is regarded as the leading cause of maternal and neonatal morbidity and mortality. Nevertheless, the potential mechanism for the regulation of trophoblast behaviors and the pathogenesis of PE remain largely elusive. Recently, accumulating evidence emphasized that aberrant expression of long non-coding RNAs (lncRNAs) functions as imperative regulators in human diseases, including PE. Thus, identifying PE-related specific lncRNAs to uncover the underlying molecular mechanism is of much significance. However, the functional roles and underlying mechanisms of lncRNAs in PE progression remain unclear. Method Placenta tissues obtained from patients with PE and healthy pregnant women were performed to measure TUG1 expression by qRT-PCR analysis. Transient transfections were conducted to alter TUG1 expression. Cell Counting Kit-8 (CCK-8) and flow cytometry assays were carried out to assess cell proliferation and apoptosis, respectively. Transwell and tube formation assays were performed to measure the capacity of cell invasion and angiogenesis. Moreover, the luciferase reporter assay was subjected to verify the binding relationship between TUG1 and miR-29b. Western blot analysis was performed to detect the expression of key proteins in the PI3K/AKT and ERK pathway. Results Here, we identified a lncRNA, TUG1, which was notably decreased in placental samples of PE patients. Functional experiments of loss- or gain-of-function assays also verified that ectopic expression of TUG1 promoted cell proliferation, invasion, and angiogenesis, but negatively regulated cell apoptosis, whereas TUG1 inhibition presented the opposite effects. Furthermore, mechanistic researches revealed that TUG1 could act as a molecular sponge for miR-29b, thus regulating MCL1, VEGFA, and MMP2 to modulate PE development. Conclusions Taken together, our findings demonstrated that TUG1 exerts as a critical role in PE progression, which might furnish a novel therapeutic marker for PE treatment.

Lysyl oxidase (LOX) in vascular remodelling

2014 ◽  
Vol 112 (10) ◽  
pp. 812-824 ◽  
Author(s):  
María Galán ◽  
Ingrid Martí-Pàmies ◽  
Saray Varona ◽  
Ricardo Rodríguez-Calvo ◽  
Ana María Briones ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Lysyl Oxidase ◽  
Critical Role ◽  
Specific Pattern ◽  
Proximal Promoter ◽  
Phenotypic Switching ◽  
Vascular Remodelling ◽  
Artery Ligation ◽  
Carotid Artery Ligation ◽  
Transgenic Lines

SummaryLysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that seems to play a critical role in vascular remodelling. However, the lack of viable LOX-deficient animal models has been an obstacle to deep in LOX biology. In this study we have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. The SM22 proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated by conventional methods (TgLOX). Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. Interestingly, cell proliferation was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of Myh10 (marker of SMC phenotypic switching), PCNA (marker of cell proliferation) and MCP-1, and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA and MCP-1 immunostaining. Our results give evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.

scholarly journals Induced TRPC1 expression sensitizes intestinal epithelial cells to apoptosis by inhibiting NF-κB activation through Ca2+ influx

2006 ◽  
Vol 397 (1) ◽  
pp. 77-87 ◽  
Author(s):  
Bernard S. Marasa ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
Kaspar M. Keledjian ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transient Receptor Potential ◽  
Intestinal Epithelial Cells ◽  
Critical Role ◽  
Ectopic Expression ◽  
Receptor Potential ◽  
Nuclear Factor Κb ◽  
Intestinal Epithelial ◽  
Store Depletion ◽  
Increased Susceptibility

Apoptosis occurs within crypts and at the intestinal luminal surface and plays a critical role in mucosal homoeostasis. NF-κB (nuclear factor-κB) is the central regulator of the transcription of genes involved in apoptosis, and its activity is highly regulated in the intestinal mucosa. We have recently demonstrated that TRPC1 (transient receptor potential canonical-1) is expressed in IECs (intestinal epithelial cells) and functions as a Ca2+ permeable channel activated by Ca2+ store depletion. The present study tests the hypothesis that TRPC1 channels are implicated in the regulation of apoptosis by inhibiting NF-κB through the induction of TRPC1-mediated Ca2+ influx in the IEC-6 line. The expression of TRPC1 induced by stable transfection of IEC-6 cells with the wild-type TRPC1 gene (IEC-TRPC1 cells) increased Ca2+ influx after Ca2+ store depletion and repressed NF-κB transactivation, which was associated with an increase in susceptibility to apoptosis induced by exposure to TNFα (tumour necrosis factor-α) plus CHX (cycloheximide) (TNF-α/CHX), or STS (staurosporine). By contrast, the induction of endogenous NF-κB activity, by the depletion of cellular polyamines, promoted resistance to apoptosis, which was prevented by the ectopic expression of the IκBα super-repressor. Furthermore, inhibition of TRPC1 expression by transfection with siRNA (small interfering RNA) targeting TRPC1 (siTRPC1) decreased Ca2+ influx, increased NF-κB transactivation, and prevented the increased susceptibility of IEC-TRPC1 cells to apoptosis. Decreasing Ca2+ influx by exposure to a Ca2+-free medium also induced NF-κB activity and blocked the increased susceptibility to apoptosis of stable IEC-TRPC1 cells. These results indicate that induced TRPC1 expression sensitizes IECs to apoptosis by inhibiting NF-κB activity as a result of the stimulation of Ca2+ influx.

scholarly journals Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Hao Zhang ◽  
Yang Zhang ◽  
Xinyue Zhou ◽  
Shaela Wright ◽  
Judith Hyle ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Critical Role ◽  
Ectopic Expression ◽  
Leukemia Cell ◽  
Therapeutic Strategies ◽  
Molecular Regulation ◽  
Leukemia Cells ◽  
Reporter Cell ◽  
Acute Leukemias ◽  
Regulation Network

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.

Polyamine-modulated expression of c-myc plays a critical role in stimulation of normal intestinal epithelial cell proliferation

2005 ◽  
Vol 288 (1) ◽  
pp. C89-C99 ◽  
Author(s):  
Lan Liu ◽  
Li Li ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Huifang M. Zhang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Growth ◽  
Epithelial Cell ◽  
Dna Synthesis ◽  
Intestinal Epithelial Cell ◽  
Small Intestinal Mucosa ◽  
Intestinal Epithelial ◽  
Epithelial Cell Proliferation ◽  
Myc Gene ◽  
Stimulation Of

The nuclear protein c-Myc is a transcription factor involved in the control of cell cycle. Our previous studies indicated that cellular polyamines are absolutely required for cell proliferation in crypts of small intestinal mucosa and that polyamines have the ability to stimulate expression of the c- myc gene. The current study went further to determine whether induced nuclear c-Myc plays a role in stimulation of cell proliferation by polyamines in intestinal crypt cells (IEC-6 line). Exposure of normal quiescent cells after 24-h serum deprivation to 5% dialyzed fetal bovine serum (dFBS) increased both cellular polyamines and expression of the c- myc gene. Increased c-Myc protein formed heterodimers with its binding partner, Max, and specifically bound to the Myc/Max binding site, which was associated with an increase in DNA synthesis. Depletion of cellular polyamines by pretreatment with α-difluoromethylornithine (DFMO) prevented increases in c- myc expression and DNA synthesis induced by 5% dFBS. c- Myc gene transcription and cell proliferation decreased in polyamine-deficient cells, whereas the natural polyamine spermidine given together with DFMO maintained c- myc gene expression and cell growth at normal levels. Disruption of c- myc expression using specific c- myc antisense oligomers not only inhibited normal cell growth (without DFMO) but also prevented the restoration of cell proliferation by spermidine in polyamine-deficient cells. Ectopic expression of wild-type c- myc by recombinant adenoviral vector containing c- myc cDNA increased cell growth. These results indicate that polyamine-induced nuclear c-Myc interacts with Max, binds to the specific DNA sequence, and plays an important role in stimulation of normal intestinal epithelial cell proliferation.

scholarly journals A Double-Bromodomain Protein, FSH-S, Activates the Homeotic Gene Ultrabithorax through a Critical Promoter-Proximal Region

2007 ◽  
Vol 27 (15) ◽  
pp. 5486-5498 ◽  
Author(s):  
Yuh-Long Chang ◽  
Balas King ◽  
Shu-Chun Lin ◽  
James A. Kennison ◽  
Der-Hwa Huang
Keyword(s):  
Hox Genes ◽  
Cultured Cells ◽  
Ectopic Expression ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Dependent Manner ◽  
Trithorax Group ◽  
Physiological Relevance ◽  
Bromodomain Proteins ◽  
Transgenic Embryos

ABSTRACT More than a dozen trithorax group (trxG) proteins are involved in activation of Drosophila HOX genes. How they act coordinately to integrate signals from distantly located enhancers is not fully understood. The female sterile (1) homeotic (fs(1)h) gene is one of the trxG genes that is most critical for Ultrabithorax (Ubx) activation. We show that one of the two double-bromodomain proteins encoded by fs(1)h acts as an essential factor in the Ubx proximal promoter. First, overexpression of the small isoform FSH-S, but not the larger one, can induce ectopic expression of HOX genes and cause body malformation. Second, FSH-S can stimulate Ubx promoter in cultured cells through a critical proximal region in a bromodomain-dependent manner. Third, purified FSH-S can bind specifically to a motif within this region that was previously known as the ZESTE site. The physiological relevance of FSH-S is ascertained using transgenic embryos containing a modified Ubx proximal promoter and chromatin immunoprecipitation. In addition, we show that FSH-S is involved in phosphorylation of itself and other regulatory factors. We suggest that FSH-S acts as a critical component of a regulatory circuitry mediating long-range effects of distant enhancers.

scholarly journals Hypercapnia Accelerates Wound Healing in Endothelial Cell Monolayers Exposed to Hypoxia

2013 ◽  
Vol 7 (1) ◽  
pp. 6-12 ◽  
Author(s):  
Takao Tsuji ◽  
Kazutetsu Aoshiba ◽  
Masayuki Itoh ◽  
Hiroyuki Nakamura ◽  
Kazuhiro Yamaguchi
Keyword(s):  
Cell Proliferation ◽  
Wound Healing ◽  
Endothelial Cell ◽  
Critical Role ◽  
Hypoxic Condition ◽  
Cell Monolayers ◽  
Hypoxic Conditions ◽  
Injury And Repair ◽  
Different Levels ◽  
Stimulation Of

Introduction: While tissue hypoxia is known to play a critical role in the process of vascular injury and repair, the effect of hypercapnia on this process remains uncertain. We investigated whether hypercapnia might influence endothelial cell wound healing under the influence of hypoxia. Materials and Methodology: Monolayers of human umbilical venous endothelial cells (HUVECs) were scratch-wounded and incubated under different levels of O2, CO2, and pH in the environment. Results: Inhibition of wound healing was observed in the HUVEC monolayers under the hypoxic condition as compared to the normoxic condition. Both hypercapnic acidosis and buffered hypercapnia, but not normocapnic acidosis improved the rate of wound healing under the influence of hypoxia. The beneficial effect of hypercapnia was associated with stimulation of cell proliferation, without effects on cell adhesion, migration or apoptosis. On the other hand, the stimulatory effect of hypercapnia on wound healing and cell proliferation was not noted under normoxic conditions. Conclusion: These results suggest that hypercapnia, rather than acidosis per se, accelerated the wound healing in HUVEC monolayers cultured under hypoxic conditions. The effect of hypercapnia on wound healing was due, at least in part, to the stimulation of cell proliferation by hypercapnia.

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