Transcriptional and Posttranscriptional Inhibition of Lysyl Oxidase Expression by Cigarette Smoke Condensate in Cultured Rat Fetal Lung Fibroblasts

Gao, Song; Chen, Keyang; Zhao, Yinzhi; Rich, Celeste B.; Chen, Lijun; Li, Sandy J.; Toselli, Paul; Stone, Phillip J.; Li, Wande

doi:10.1093/toxsci/kfi212

Cited by 34 publications

(38 citation statements)

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“…6) and inhibiting some transporters like OATP1B1, OATP1B3, OCT1 and MATE1 (Fig. 1B) are similar to those used in previous studies with cultured cells, including human hepatic cells (Fields et al 2005;Nagaraj et al 2006;Xiao et al 2015); they also up-regulated the drug metabolizing enzymes CYP1A1 and CYP1B1, that, together with other AhR targets such as CYP1A2, constitute key targets induced by smoking in humans (Chang et al 2003;Dobrinas et al 2011;Thum et al 2006) and are in the range of CSC concentrations (20 to 120 µg/mL) previously hypothesized to be relevant for human smokers (Gao et al 2005). On the other hand, 10-40 µg/mL CSC corresponds to 0.768-3.072 µg/mL (4.73-18.92 µM) nicotine, when considering that CSC contains 7.68 % (weight/weight) nicotine (Eldridge et al 2015), and such nicotine concentrations are much higher than those (25 to 444 nM) commonly described in smoker blood (Russell et al 1980).…”

Section: Discussionsupporting

confidence: 79%

Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate

et al. 2016

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Smoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRG cells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B1, OATP2B1, OAT2, NTCP, OCT1 and BSEP, and enhanced that of MRP4. Such changes in transporter gene expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin, a reference activator of the aryl hydrocarbon receptor (AhR) pathway, and were counteracted, for some of them, by siRNA-mediated AhR silencing. This suggests that CSC alters hepatic drug transporter levels via activation of the AhR cascade. Importantly, drug transporter expression regulations as well as some transporter activity inhibitions occurred for a range of CSC concentrations similar to those required for inducing drug metabolizing enzymes and may therefore be hypothesized to be relevant for smokers. Taken together, these data established human hepatic transporters as targets of cigarette smoke, which could contribute to known alteration of pharmacokinetics and some liver adverse effects caused by smoking. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. SummarySmoking is well-known to impair pharmacokinetics, through inducing expression of drug metabolizing enzymes. In the present study, we demonstrated that cigarette smoke condensate (CSC) also alters activity and expression of hepatic drug transporters, which are now recognized as major actors of hepatobiliary elimination of drugs. CSC thus directly inhibited activities of sinusoidal transporters such as OATP1B1, OATP1B3, OCT1 and NTCP as well as those of canalicular transporters like P-glycoprotein, MRP2, BCRP and MATE1, in hepatic transporters-overexpressing cells. CSC similarly counteracted constitutive OATP, NTCP and OCT1 activities in human highly-differentiated hepatic HepaRG cells. In parallel, CSC induced expression of BCRP at both mRNA and protein level in HepaRG ce...

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Section: Discussionsupporting

confidence: 79%

Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate

et al. 2016

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“…A region of 1,865 bp of the rat LO promoter upstream of ATG was found to direct luciferase reporter gene expression. CSC suppressed luciferase activity in cells transiently transfected with the LO promoter-luciferase gene chimeric vector consistent with its effects on LO mRNA levels (16). Although several cis-elements have been characterized in the LO promoter region for human, mouse, and rat (17)(18)(19), the precise mechanisms for their activation remain to be understood.…”

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confidence: 74%

“…The resulting cDNA was then amplified by nested PCR using Platinum Taq High Fidelity DNA polymerase (Invitrogen) as well as the rat LO gene primers (reverse) and the adaptor primers (forward) provided by the manufacturer. The gene-specific antisense inner primer 5Ј-GACTCTCGAGGGTTGTCACGCAG-CAGCAGAATGG-3Ј and the nested PCR outer primer 5Ј-CAGATGGGCTTGGAGTCCTC-3Ј were designed for the RLM-RACE assay based on the sequence of rat LO cDNA (16,31). The 5Ј-RLM-RACE PCR products were analyzed on agarose gels and cloned into the pBluescript II SK (Ϫ) vector for sequencing as described (30).…”

Section: Methodsmentioning

confidence: 99%

“…SacI or XmaI (labeled with underline), and a specific primer sequence, followed by a number in parentheses indicating the 5Ј end position of each primer in the LO promoter region as follows: reverse, 5Ј-GTCACCCGGGATGATGCTCCCGGCTCGTC-CCTGCT-3Ј, corresponding to positions from Ϫ23 to ϩ2 in the rat LO gene sequence using the first nucleotide preceding the ATG codon as Ϫ1; forward 1, 5Ј-GTCAGAGCTCTGTT-TGCCCTGCACATCTTTAGTAA-3Ј (Ϫ3,979); forward 2, 5Ј-GTCAGAGCTCACTTTGGAGGAAATGAAAGAGG-GAC-3Ј (Ϫ3,237); forward 3, 5Ј-GTCAGAGCTCGAATGC-ACTAGGAAAGTCTGGAGGA-3Ј (Ϫ1,865); forward 4, 5Ј-GTCAGAGCTCAGTCACAACCTCCCCCATCCCAACG-3Ј (Ϫ709); forward 5, 5Ј-GTCAGAGCTCTATTTTGGCTTGGG-CCCATGGCCTG-3Ј (Ϫ598); forward 6, 5Ј-GTCAGAGCTCG-AGTGTGTCTCAGGATGTGTGTTCC-3Ј (Ϫ517); forward 7, 5Ј-GTCAGAGCTCGAGTGGTGGAGCTGTCCGCCTTGC-3Ј (Ϫ410); and forward 8, 5Ј-GTCAGAGCTCAGACACTG-TGCGCTCTCCCGGAC-3Ј (Ϫ277). LO gene-specific sequences for the primer design were based on the Rattus norvegicus chromosome 18 WGS supercontig NW_047513 (GenBank TM accession number) (16,31). LO promoter fragments obtained were restricted with SacI and XmaI and ligated into similarly restricted plasmid pGL3-Basic (Promega, Madison, WI) upstream of the firefly luciferase gene as described (16).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, environmental agents can also act upon the LO gene resulting in its transcriptional modification. For example, our recent studies indicated that cigarette smoke condensate (CSC), the particulate phase of smoke, inhibited synthesis of nascent LO transcripts leading to reduced levels of LO mRNAs in treated rat fetal lung fibroblasts (16). Transcription of the LO gene is driven by proximal promoter sequences, which interact with ubiquitous transcription factors.…”

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Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter

Gao

Zhao

Kong

et al. 2007

Journal of Biological Chemistry

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Lysyl oxidase (LO) stabilizes the extracellular matrix by crosslinking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from ؊78 to ؊51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5 Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region ؊709/؊598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment ؊612/؊580, which contains a nuclear factor I (NFI)-binding site at the region ؊594/؊580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at ؊147/؊133, were identified in the LO promoter region ؊804/ ؊1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation. Lysyl oxidase (LO)2 (EC 1.4.3.13) is a copper-dependent enzyme secreted by fibrogenic cells such as fibroblasts (1). This enzyme catalyzes the initiation of cross-linking of collagen and elastin, major structural components of the extracellular matrix (ECM), by oxidizing peptidyl lysine residues within these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde, leading to the formation of condensation products stabilizing polymeric collagen or elastin as insoluble fibers. Thus, LO plays a central role in ECM morphogenesis and tissue repair (1).In addition to the major function in stabilizing the ECM, LO also exhibits other biological activities. As reported, expression of transfected LO cDNA inhibited Ha-ras-induced cell transformation indicating an anti-tumorigenic effect of LO (2). LO can oxidize lysine residues in various globular proteins other than collagen and elastin (1). Oxidation of basic fibroblast growth factor (bFGF) by LO blocks the proliferation of bFGFstimulated cells and highly tumorigenic bFGF autocrine-transformed cells (3). Purified mature bovine LO displays chemotactic activity for monocytes and vascular smooth muscle cells (4, 5). LO and its oxidized substrates exist within the nuclei, potentially using histone H1 as a substrate and modulating the promoter activity of the collagen type III gene (6 -8). Increased LO activity is associated with fibrotic...

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Fusaric acid induces a notochord malformation in zebrafish via copper chelation

et al. 2015

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Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 10(5) M(-1). These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.

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Transcriptional and Posttranscriptional Inhibition of Lysyl Oxidase Expression by Cigarette Smoke Condensate in Cultured Rat Fetal Lung Fibroblasts

Cited by 34 publications

References 29 publications

Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate

Alteration of human hepatic drug transporter activity and expression by cigarette smoke condensate

Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter

Fusaric acid induces a notochord malformation in zebrafish via copper chelation

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