Binding loci of RelA-containing nuclear factor-kappaB dimers in promoter regions of PHM1-31 myometrial smooth muscle cells (original) (raw)
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The Journal of Clinical Endocrinology & Metabolism, 2004
In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory factors, many of which are regulated by the nuclear factor κB (NF-κB) family of transcription factors. The expression and DNA-binding activity of NF-κB in the myometrium during gestation and parturition were examined. Levels of c-Rel, p50, and p105 NF-κB species were dramatically reduced in pregnant myometrium compared with nonpregnant (NP) controls, whereas expression of the RelA subunit remained uniform. Importantly, during labor, expression of all subunits was observed to be significantly reduced in all myometrial samples studied relative to NP levels. Moreover, for RelA, c-Rel, and p50 subunits, there was a gradient of expression between laboring upper (corpus) and lower uterine segment myometrium. No RelB or p52 subunits could be detected. ...
NF-κB function in the human myometrium during pregnancy and parturition
Histology and Histopathology, 2010
Interactions between the nuclear factor kappaB (NF-κB) family of proteins (RelA, RelB, c-Rel, p50 and p52) and DNA are vital for cells to function normally; for example, in the human myometrium, NF-κB-regulated pro-inflammatory mediators, including TNFα, IL-1ß, IL-8 and COX-2 are associated with the onset of labour. NF-κB, however, regulates the expression of over 400 genes, although it is unlikely these would all be activated in concert by a single inducer. At present, defining the role of the NF-κB RelA:p50 dimer, which governs a number of inflammatory promoters, is at the forefront of the parturition research field. However, to overlook the function of other family members and how they may regulate alternative signalling networks within reproductive tissues, only serves to ensure we will never fully understand the molecular circuitry influenced by this family of transcription factors. Consequently this review highlights other mechanisms by which the NF-κB family of regulators have been shown to function in other systems and how they may readily translate to understanding the regulation underpinning human parturition.
NFκB and AP-1 Drive Human Myometrial IL8 Expression
Mediators of Inflammation, 2012
The uterine expression of the chemokine IL8 increases dramatically with the onset of labour both at term and preterm. The IL8 promoter contains binding sites for the transcription factors nuclear factor-kappa B (NFκB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein (CEBP). In this study we investigated the roles of these transcription factors in IL1B regulation of the IL8 gene in human myometrium. Using chromatin immune precipitation (ChIP) assay, we showed that each of NFκB, CEBP, and AP-1 binds to the IL8 promoter upon IL1B stimulation. To examine the relative importance of each site in IL8 gene expression, site-directed mutagenesis of each of these sites was performed. We found that the NFκB site was essential for basal and IL1B-stimulated gene expression. Mutation of the AP-1 site reduced both basal and IL1B-stimulated expression but to a lesser extent. Mutation of the CEBP site had no effect upon basal expression but eliminated the IL1B response. Small interfering RNA (siRNA) silencing of NFκB abolished the IL8 response to IL1B significantly; siRNA against AP-1 reduced it to a lesser extent whilst knockdown of CEBP enhanced the response. Our data confirms a central and essential role for NFκB in regulation of IL8 in human myometrium.
Endocrinology, 2005
In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory mediators, many of which are regulated by the nuclear factor B (NF-B) family of transcription factors. Recently, we have provided evidence that the RelA NF-B subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the G␣s/cAMP/protein kinase A pathway. TNF␣ is a potent activator of NF-B, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNF␣ was observed to repress expression of G␣s while, at the same time, stimulating NF-B activity. Furthermore, this effect could be
Distribution of NF-kappaB-binding sites across human chromosome 22
Proceedings of the National Academy of Sciences USA, 2003
We have mapped the chromosomal binding site distribution of a transcription factor in human cells. The NF-kappaB family of transcription factors plays an essential role in regulating the induction of genes involved in several physiological processes, including apoptosis, immunity , and inflammation. The binding sites of the NF-kappaB family member p65 were determined by using chromatin immunoprecipi-tation and a genomic microarray of human chromosome 22 DNA. Sites of binding were observed along the entire chromosome in both coding and noncoding regions, with an enrichment at the 5 end of genes. Strikingly, a significant proportion of binding was seen in intronic regions, demonstrating that transcription factor binding is not restricted to promoter regions. NF-kappaB binding was also found at genes whose expression was regulated by tumor necrosis factor , a known inducer of NF-kappaB-dependent gene expression, as well as adjacent to genes whose expression is not affected by tumor necrosis factor. Many of these latter genes are either known to be activated by NF-kappaB under other conditions or are consistent with NF-kappaB's role in the immune and apoptotic responses. Our results suggest that binding is not restricted to promoter regions and that NF-kappaB binding occurs at a significant number of genes whose expression is not altered, thereby suggesting that binding alone is not sufficient for gene activation.
NF-κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term
Journal of Cellular and Molecular Medicine, 2011
. cDNA microarray analysis showed that NF-B increased expression of 38 genes principally related to immunity and inflammation. IL-1 stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL-1 proving a central role for NF-B. We conclude that NF-B is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.
Activation of the transcription factor nuclear factor-κB in human inflamed synovial tissue
Arthritis & Rheumatism, 1996
Establishment and maintenance of pregnancy in the pig involves intricate communication between the developing conceptuses and the maternal endometrium. This process occurs during trophoblast elongation which is spaciotemporally associated with conceptus synthesis and release of IL1B concomitant with pregnancy-specific endometrial up-regulation of IL-1 receptors, providing the potential for activation of the transcription factor, NFKB. The objective of the current investigation was to determine changes in expression and cellular localization of NFKB and associated factors during the estrous cycle and early pregnancy in the pig. In situ hybridization was used to localize changes in PGR, ESR1, and TNFRSF11A during the peri-implantation period. Quantitative RT-PCR was utilized to demonstrate gene expression changes for NFKB1, RELA, TNFRSF11A, TLR4, NFKBIA and NFKBIB. Transcription factor ELISA demonstrated an overall increase in RELA during the peri-implantation period in both cyclic and pregnant gilts. While the presence of TNFSF11A and TLR4 were both detected, TLR4 expression changes were temporally associated with NFKB expression and activation. Collectively, these data demonstrate that NFKB activation may occur during the period of uterine receptivity in both the cyclic and pregnant endometrium.
Endocrinology, 2004
The specific binding of transcription factors to DNA has been shown to be inhibited by chromatin structure and increased by cooperative interactions with other proteins. Consequently, in situ analysis using chromatin immunoprecipitation offers the most accurate view of transcriptional control. Transient transfection studies and in vitro analyses of IL-1induced cox-2 transcription in a number of cell types have indicated regulation by either nuclear factor B (NF-B) or CCAAT/enhancer binding protein (C/EBP), or both acting cooperatively. To determine the mechanisms of COX-2 (cyclooxygenase or prostaglandin endoperoxide synthase) induction in cultured human myometrial cells in situ, we examined the cross-linking of the RelA subunit of NF-B and C/EBP to the cox-2 promoter and flanking sequences. As a control, we inspected the interaction of these transcription factors with the IL-8 gene, which has been shown in other cell types to be activated by the cooperative interaction of NF-B and C/EBP. Indeed, both transcription factors were cross-linked to the il-8 promoter after IL-1 treatment, but only RelA was cross-linked to cox-2 DNA. The il-8 promoter was also found to physically interact with proteins cross-linked to sites further upstream. IL-1 treatment also increased polymerase II crosslinking to both promoters and increased histone H4 acetylation at specific sites. These results indicate that modification of chromatin structure is part of the response to IL-1 stimulation. Chromatin immunoprecipitation thus provides critical insight into the mechanisms of COX-2 and IL-8 expression in human myometrial cells. (Endocrinology 145: 1248-1254, 2004)
NFκB and AP-1 Drive Human Myometrial IL8 Expression
Mediators of Inflammation, 2012
The uterine expression of the chemokine IL8 increases dramatically with the onset of labour both at term and preterm. The IL8 promoter contains binding sites for the transcription factors nuclear factor-kappa B (NFκB), activator protein-1 (AP-1), and CCAAT/enhancer-binding protein (CEBP). In this study we investigated the roles of these transcription factors in IL1B regulation of the IL8 gene in human myometrium. Using chromatin immune precipitation (ChIP) assay, we showed that each of NFκB, CEBP, and AP-1 binds to the IL8 promoter upon IL1B stimulation. To examine the relative importance of each site in IL8 gene expression, site-directed mutagenesis of each of these sites was performed. We found that the NFκB site was essential for basal and IL1B-stimulated gene expression. Mutation of the AP-1 site reduced both basal and IL1B-stimulated expression but to a lesser extent. Mutation of the CEBP site had no effect upon basal expression but eliminated the IL1B response. Small interfering RNA (siRNA) silencing of NFκB abolished the IL8 response to IL1B significantly; siRNA against AP-1 reduced it to a lesser extent whilst knockdown of CEBP enhanced the response. Our data confirms a central and essential role for NFκB in regulation of IL8 in human myometrium.