assessing-urinary-tract-junction-obstruction-defects-methylene-blue

Assessing Urinary Tract Junction Obstruction Defects by Methylene Blue Dye Injection

Urinary tract junction obstruction defects are congenital anomalies inducing hydronephrosis and hydroureter. Murine urinary tract junction obstruction ...

National Institutes of Health

The relation of Wnt/beta-catenin signaling to osteoarthritis progression has been revealed with little information on the underlying molecular mechanism. In this study we found overexpression of Lef1 in cartilage tissue of osteoarthritic patients and elucidated molecular mechanism of NF-kappaB-mediated Lef1 gene regulation in chondrocytes. Treatment of IL-1beta augmented Lef1 upregulation and nuclear translocation of NF-kappaB in chondrocytes. Under IL-1beta signaling, treatment of NF-kappaB nuclear translocation inhibitor SN-50 reduced Lef1 expression. A conserved NF-kappaB-binding site between mouse and human was selected through bioinformatic analysis and mapped at the 14 kb upstream of Lef1 transcription initiation site. NF-kappaB binding to the site was confirmed by chromatin immunoprecipitation assay. Lef1 expression was synergistically upregulated by interactions of NF-kappaB with Lef1/beta-catenin in chondrocytes. Our results suggest a pivotal role of NF-kappaB in Lef1 expression in arthritic chondrocytes or cartilage degeneration.

NF-kappaB regulates Lef1 gene expression in chondrocytes.

Although the involvement of Wnt/beta-catenin signaling pathway in the regulation of COX-2 expression has been suggested, the underlying molecular mechanisms are still unclear. Here, we demonstrate that lymphoid enhancer-binding factor 1 (Lef1) in concert with beta-catenin is associated with the transcriptional regulation of the gene encoding COX-2 in mouse chondrocytes. Our results show that co-overexpression of Lef1 and beta-catenin synergistically upregulates COX-2 expression. Furthermore, decrease of Lef1 expression using Lef1 siRNA results in the downregulation of COX-2 expression. Using bioinformatic analysis, we identified a conserved Lef1-binding site that is mapped at the 3' region of the genomic COX-2 locus. In vivo and in vitro binding of Lef1 at the predicted binding site was proved using chromatin immunoprecipitation assays and electrophoretic mobility shift assays, respectively. Moreover, binding of Lef1 and beta-catenin to the Lef1-binding site transactivates COX-2 promoter activity. Our results indicate a pivotal role of Lef1 in the regulation of COX-2 transcription in arthritic chondrocytes.

Lef1 regulates COX-2 transcription in chondrocytes.

An association between Wnt/beta-catenin signaling and MMP13 expression has been reported but there has been little information about the underlying mechanism. Here, we investigated the role of Lef1 in IL-1beta-mediated MMP13 regulation in mouse chondrocytes. Lef1 and beta-catenin synergistically upregulated MMP13 transcription while knock-down of Lef1 using Lef1 siRNA downregulated IL-1beta-mediated MMP13 expression. Lef1 binding site was mapped to the 3' region of the MMP13 genomic locus and binding of Lef1/beta-catenin to the site was confirmed by chromatin immunoprecipitation (ChIP) assays and electrophoretic mobility shift assays (EMSAs). Furthermore, Lef1/beta-catenin binding to the Lef1 binding site transactivated MMP13 promoter activity. Our results suggest a pivotal role of Lef1/beta-catenin in MMP13 regulation in chondrocytes, which might be associated with matrix loss of degenerated arthritic cartilage.

Transcriptional regulation of MMP13 by Lef1 in chondrocytes.

Efficient transcription depends upon efficient physical and functional interactions between transcriptosome complexes and DNA. We have previously shown that IL-1beta-induced lymphoid enhancer binding factor 1 (Lef1) regulates the transcription of its target genes COX2 and MMP13 in mouse chondrocytes by binding to the Lef1 binding sites located in the 3' region. In this study, we investigated how the 3' region-bound Lef1 regulates expression of target genes. IL-1beta stimulation induced gene looping in COX2 and MMP13 genomic loci, which is mediated by the physical interaction of Lef1 with its binding partners, including beta-catenin, AP-1, and NF-kappaB. As shown by chromosome conformation capture (3C) assay, the 5' and 3' genomic regions of these genes were juxtaposed in an IL-1beta-stimulation dependent manner. Lef1 played a pivotal role in this gene looping; Lef1 knockdown decreased the incidence of gene looping, while Lef1 overexpression induced it. Physical interactions between the 3' region-bound Lef1 and promoter-bound transcription factors AP-1 or NF-kappaB in COX2 and MMP13, respectively, were increased upon stimulation, leading to synergistic up-regulation of gene expression. Knockdown of RelA or c-Jun decreased the formation of gene loop and down-regulated cyclooxygenase 2 (COX2) or matrix metalloproteinase 13 (MMP13) transcription levels. However, overexpression of RelA or c-Jun along with Lef1 increased the looping and their expression levels. Our results indicate a novel function of Lef1, as a mediator of gene looping between 5' and 3' regions. Gene looping may serve to delineate the transcription unit in the inducible gene transcription of mammalian cells.

Lymphoid enhancer binding factor 1 regulates transcription through gene looping.

Control of organ size by cell proliferation and survival is a fundamental developmental process, and its deregulation leads to cancer. However, the molecular mechanism underlying organ size control remains elusive in vertebrates. In Drosophila, the Hippo (Hpo) signaling pathway controls organ size by both restricting cell growth and proliferation and promoting cell death. Here we investigated whether mammals also require the Hpo pathway to control organ size and adult tissue homeostasis. We found that Mst1 and Mst2, the two mouse homologs of the Drosophila Hpo, control the sizes of some, but not all organs, in mice, and Mst1 and Mst2 act as tumor suppressors by restricting cell proliferation and survival. We show that Mst1 and Mst2 play redundant roles, and removal of both resulted in early lethality in mouse embryos. Importantly, tumors developed in the liver with a substantial increase of the stem/progenitor cells by 6 months after removing Mst1 and Mst2 postnatally. We show that Mst1 and Mst2 were required in vivo to control Yap phosphorylation and activity. Interestingly, apoptosis induced by TNFalpha was blocked in the Mst1 and Mst2 double-mutant cells both in vivo and in vitro. As TNFalpha is a pleiotropic inflammatory cytokine affecting most organs by regulating cell proliferation and cell death, resistance to TNFalpha-induced cell death may also contribute significantly to tumor formation in the absence of Mst1 and Mst2.

Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression.

Type II collagen is the major component of articular cartilage and is mainly synthesized by chondrocytes. Repeated sub-culturing of primary chondrocytes leads to reduction of type II collagen gene (Col2a1) expression, which mimics the process of chondrocyte dedifferentiation. Although the functional importance of Col2a1 expression has been extensively investigated, mechanism of transcriptional regulation during chondrocyte dedifferentiation is still unclear. In this study, we have investigated the crosstalk between cis-acting DNA element and transcription factor on Col2a1 expression in primary chondrocytes. Bioinformatic analysis revealed the potential regulatory regions in the Col2a1 genomic locus. Among them, promoter and 3' untranslated region (UTR) showed highly accessible chromatin architecture with enriched recruitment of active chromatin markers in primary chondrocytes. 3' UTR has a potent enhancer function which recruits Lef1 (Lymphoid enhancer binding factor 1) transcription factor, leading to juxtaposition of the 3' UTR with the promoter through gene looping resulting in up-regulation of Col2a1 gene transcription. Knock-down of endogenous Lef1 level significantly reduced the gene looping and subsequently down-regulated Col2a1 expression. However, these regulatory loci become inaccessible due to condensed chromatin architecture as chondrocytes dedifferentiate which was accompanied by a reduction of gene looping and down-regulation of Col2a1 expression. Our results indicate that Lef1 mediated looping between promoter and 3' UTR under the permissive chromatin architecture upregulates Col2a1 expression in primary chondrocytes.

Looping mediated interaction between the promoter and 3' UTR regulates type II collagen expression in chondrocytes.

Congenital anomalies of the kidney and urinary tract (CAKUT) affect about 1 in 500 births and are a major cause of morbidity in infants. Duplex collecting systems rank among the most common abnormalities of CAKUT, but the molecular basis for this defect is poorly understood. In mice, conditional deletion of Wnt5a in mesoderm results in bilateral duplex kidney and ureter formation. The ureteric buds (UBs) in mutants emerge as doublets from the intermediate mesoderm (IM)-derived nephric duct (ND) without anterior expansion of the glial cell line-derived neurotrophic factor (Gdnf) expression domain in the surrounding mesenchyme. Wnt5a is normally expressed in a graded manner at the posterior end of the IM, but its expression is down-regulated prior to UB outgrowth at E10.5. Furthermore, ablation of Wnt5a in the mesoderm with an inducible Cre at E7.5 results in duplex UBs, whereas ablation at E8.5 yields normal UB outgrowth, demonstrating that Wnt5a functions in IM development well before the formation of the metanephros. In mutants, the posterior ND is duplicated and surrounding Pax2-positive mesenchymal cells persist in the nephric cord, suggesting that disruption of normal ND patterning prompts the formation of duplex ureters and kidneys. Ror2 homozygous mutants, which infrequently yield duplex collecting systems, show a dramatic increase in incidence with the additional deletion of one copy of Wnt5a, implicating this receptor in non-canonical Wnt5a signaling during IM development. This work provides the first evidence of a role of Wnt5a/Ror2 signaling in IM extension and offers new insights into the etiology of CAKUT and possible involvement of Wnt5a/Ror2 mutations.

Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension.

The Wnt5a null mouse is a complex developmental model which, among its several posterior-localized axis defects, exhibits multiple kidney phenotypes, including duplex kidney and loss of the medullary zone. We previously reported that ablation of Wnt5a in nascent mesoderm causes duplex kidney formation as a result of aberrant development of the nephric duct and abnormal extension of intermediate mesoderm. However, these mice also display a loss of the medullary region late in gestation. We have now genetically isolated duplex kidney formation from the medullary defect by specifically targeting the progenitors for both the ureteric bud and metanephric mesenchyme. The conditional mutants fail to form a normal renal medulla but no longer exhibit duplex kidney formation. Approximately 1/3 of the mutants develop hydronephrosis in the kidneys either uni- or bilaterally when using Dll1Cre. The abnormal kidney phenotype becomes prominent at E16.5, which approximates the time when urine production begins in the mouse embryonic kidney, and is associated with a dramatic increase in apoptosis only in mutant kidneys with hydronephrosis. Methylene blue dye injection and histologic examination reveal that aberrant cell death likely results from urine toxicity due to an abnormal ureter-bladder connection. This study shows that Wnt5a is not required for development of the renal medulla and that loss of the renal medullary region in the Wnt5a-deleted kidney is caused by an abnormal ureter-bladder connection.

Article	Year
NF-kappaB regulates Lef1 gene expression in chondrocytes. Biochemical and biophysical research communications\| PubMed ID: 17445771	2007
Lef1 regulates COX-2 transcription in chondrocytes. Biochemical and biophysical research communications\| PubMed ID: 17945188	2007
Transcriptional regulation of MMP13 by Lef1 in chondrocytes. Biochemical and biophysical research communications\| PubMed ID: 17971297	2007
Lymphoid enhancer binding factor 1 regulates transcription through gene looping. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 19783677	2009
Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20080598	2010
Looping mediated interaction between the promoter and 3' UTR regulates type II collagen expression in chondrocytes. PloS one\| PubMed ID: 22815835	2012
Non-canonical Wnt5a/Ror2 signaling regulates kidney morphogenesis by controlling intermediate mesoderm extension. Human molecular genetics\| PubMed ID: 25082826	2014
Hydronephrosis in the Wnt5a-ablated kidney is caused by an abnormal ureter-bladder connection. Differentiation; research in biological diversity\| PubMed ID: 27923152	2017

Kangsun Yun

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