Sp1, Instead of AhR, Regulates the Basal Transcription of Porcine CYP1A1 at the Proximal Promoter.
ABSTRACT: Pigs are commonly used as an animal model to evaluate the toxic effects of exogenous compounds. Cytochrome P450 1A1 (CYP1A1) metabolizes numerous exogenous compounds and is abundantly expressed in the liver, kidneys, and intestines. The high amino acid similarity between human and porcine CYP1A1 indicates that they probably have the same metabolic characteristics. Therefore, understanding the regulatory mechanism of CYP1A1 expression in pigs is particularly important for predicting the toxicology and metabolic kinetics of exogenous chemicals. Currently, the transcriptional regulation of porcine CYP1A1 has rarely been studied, especially regarding basal transcription. In this study, we first confirmed that the key regulatory elements of porcine CYP1A1 basal transactivation are in the proximal promoter region using promoter truncation analysis via a dual luciferase assay in a porcine kidney cell line LLC-PK1. Two overlapping cis-elements, the xenobiotic response element (XRE) and GC box, in this proximal region potentially play key roles in the basal transactivation of porcine CYP1A1. Furthermore, using electrophoretic mobility shift assay and chromatin immunoprecipitation, the GC box binding protein Sp1 was confirmed to bind to the proximal promoter of porcine CYP1A1, instead of AhR, the XRE binding protein. In LLC-PK1 cells, by knocking down either Sp1 or AhR, the expression of porcine CYP1A1 at the mRNA level and protein level was significantly downregulated, suggesting both proteins are important for porcine CYP1A1 expression. However, promoter activity analysis in LLC-PK1 cells treated with an AhR agonist and antagonist confirmed that AhR does not participate in the basal regulation of porcine CYP1A1 at the proximal promoter. In conclusion, our study revealed that the proximal promoter is the key regulatory region for porcine CYP1A1 basal expression. Although AhR plays an important role in the transactivation of porcine CYP1A1 expression, the key determinant transcription factor for its basal transactivation is Sp1 at the proximal promoter of porcine CYP1A1.
Project description:We have cloned a new mammalian unconventional myosin, porcine myosin-VI from the proximal tubule cell line, LLC-PK1 (CL4). Porcine myosin-VI is highly homologous to Drosophila 95F myosin heavy chain, and together these two myosins comprise a sixth class of myosin motors. Myosin-VI exhibits ATP-sensitive actin-binding activities characteristic of myosins, and it is associated with a calmodulin light chain. Within LLC-PK1 cells, myosin-VI is soluble and does not associate with the major actin-containing domains. Within the kidney, however, myosin-VI is associated with sedimentable structures and specifically locates to the actin- and membrane-rich apical brush border domain of the proximal tubule cells. This motor was not enriched within the glomerulus, capillaries, or distal tubules. Myosin-VI associates with the proximal tubule cytoskeleton in an ATP-sensitive fashion, suggesting that this motor is associated with the actin cytoskeleton within the proximal tubule cells. Given the difference in association of myosin-VI with the apical cytoskeleton between LLC-PK1 cells and adult kidney, it is likely that this cell line does not fully differentiate to form functional proximal tubule cells. Myosin-VI may require the presence of additional elements, only found in vivo in proximal tubule cells, to properly locate to the apical domain.
Project description:Enteric coronaviruses (CoVs) are major pathogens that cause diarrhea in piglets. To date, four porcine enteric CoVs have been identified: transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and HKU2-like porcine enteric alphacoronavirus (PEAV). In this study, we investigated the replicative capacity of these four enteric CoVs in LLC-PK1 cells, a porcine kidney cell line. The results showed that LLC-PK1 cells are susceptible to all four enteric CoVs, particularly to TGEV and PDCoV infections, indicating that LLC-PK1 cells can be applied to porcine enteric CoV research in vitro, particularly for coinfection studies.
Project description:Polyacetylenic compounds isolated from Panax species are comprised of non-polar C17 compounds, exhibiting anti-inflammatory, antitumor, and antifungal activities. Panaxynol represents the major component of the essential oils of ginseng. We investigated whether panaxynol isolated from Panax vietnamensis (Vietnamese ginseng, VG) could prevent cisplatin-induced renal damage induced in vitro and in vivo. Cisplatin-induced apoptotic cell death was observed by staining with annexin V conjugated with Alexa Fluor 488, and western blotting evaluated the molecular mechanism. Panaxynol at concentrations above 0.25 ?M prevented cisplatin-induced LLC-PK1 porcine renal proximal tubular cell death. LLC-PK1 cells treated with cisplatin demonstrated an increase in apoptotic cell death, whereas pretreatment with 2 and 4 ?M panaxynol decreased this effect. Cisplatin demonstrated a marked increase in the phosphorylation of c-Jun N-terminal kinase (JNK), P38, and cleaved caspase-3. However, pretreatment with 2 and 4 ?M panaxynol reversed the upregulated phosphorylation of JNK, P38, and the expression of cleaved caspase-3. We confirmed that the protective effect of panaxynol isolated from P. vietnamensis in LLC-PK1 cells was at least partially mediated by reducing the cisplatin-induced apoptotic damage. In the animal study, panaxynol treatment ameliorated body weight loss and blood renal function markers and downregulated the mRNA expression of inflammatory mediators.
Project description:Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The upstream E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues, including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide -300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.
Project description:In the present study we examined the ability of 3,3',4,4',5-pentachlorinated biphenyl [PCB126 (polychlorinated biphenyl 126)], a prototypical AHR (aryl hydrocarbon receptor) agonist, and 2,2',4,6,6'-PCB (PCB104), which does not activate AHR, to induce the recruitment of ERalpha (oestrogen receptor alpha) to CYP1A1 (cytochrome P4501A1 gene) and CYP1B1 promoters in T-47D human breast cancer cells and other cell lines. PCB126 treatment strongly induced CYP1A1 and CYP1B1 mRNA expression that was unaffected by co-treatment with E2 (17beta-oestradiol). PCB104 failed to induce changes in either CYP1A1 or CYP1B1 expression levels. ChIP (chromatin immunoprecipitation) assays show that PCB126, but not PCB104, increased the promoter occupancy by ERalpha to CYP1A1 and CYP1B1 promoters. Co-treatment with PCB126+E2 significantly enhanced the promoter occupancy of ERalpha at CYP1A1, whereas co-treatment with PCB126+4-hydroxytamoxifen or ICI182,780 did not. Competitive binding studies revealed that neither PCB126 nor PCB104 bound to ERalpha. HEK-293 cells (human embryonic kidney-293 cells) stably transfected with ERalpha showed significantly higher PCB126-induced CYP1A1 expression compared with empty vector controls, whereas no increase was observed in cells stably transfected with ERalpha lacking its N-terminal AF1 (activation function-1) domain (ERalphaDeltaAF1). Despite no increase in AHR-mediated gene expression, ChIP assays revealed that ERalphaDeltaAF1 was present at CYP1A1 and CYP1B1 promoters. HC11 mouse mammary cells stably expressing shRNA (small-hairpin RNA) against ERalpha showed an 8-fold reduction in PCB126-dependent Cyp1a1 expression. Our results provide further evidence that AHR agonists induce ERalpha promoter occupancy at AHR target genes through indirect activation of ERalpha, and support a role for ERalpha in AHR transactivation.
Project description:Shiga toxin (Stx) producing Escherichia coli (STEC) cause the edema disease in pigs by releasing the swine-pathogenic Stx2e subtype as the key virulence factor. Stx2e targets endothelial cells of animal organs including the kidney harboring the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Gal?1-4Gal?1-4Glc?1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAc?1-3Gal?1-4Gal?1-4Glc?1-1Cer). Since the involvement of renal epithelial cells in the edema disease is unknown, in this study, we analyzed the porcine kidney epithelial cell lines, LLC-PK1 and PK-15, regarding the presence of Stx-binding GSLs, their sensitivity towards Stx2e, and the inhibitory potential of Gb3- and Gb4-neoglycolipids, carrying phosphatidylethanolamine (PE) as the lipid anchor, towards Stx2e. Immunochemical and mass spectrometric analysis revealed various Gb3Cer and Gb4Cer lipoforms as the dominant Stx-binding GSLs in both LLC-PK1 and PK-15 cells. A dihexosylceramide with proposed Gal?1-4Gal-sequence (Gal2Cer) was detected in PK-15 cells, whereas LLC-PK1 cells lacked this compound. Both cell lines were susceptible towards Stx2e with LLC-PK1 representing an extremely Stx2e-sensitive cell line. Gb3-PE and Gb4-PE applied as glycovesicles significantly reduced the cytotoxic activity of Stx2e towards LLC-PK1 cells, whereas only Gb4-PE exhibited some protection against Stx2e for PK-15 cells. This is the first report identifying Stx2e receptors of porcine kidney epithelial cells and providing first data on their Stx2e-mediated damage suggesting possible involvement in the edema disease.
Project description:Recovery from acute kidney injury involving tubular epithelial cells requires proliferation and migration of healthy cells to the area of injury. In this study, we show that palladin, a previously characterized cytoskeletal protein, is upregulated in injured tubules and suggest that one of its functions during repair is to facilitate migration of remaining cells to the affected site. In a mouse model of anti-neutrophilic cytoplasmic antibody involving both tubular and glomerular disease, palladin is upregulated in injured tubular cells, crescents and capillary cells with angiitis. In human biopsies of kidneys from patients with other kidney diseases, palladin is also upregulated in crescents and injured tubules. In LLC-PK1 cells, a porcine proximal tubule cell line, stress induced by transforming growth factor-?1 (TGF-?1) leads to palladin upregulation. Knockdown of palladin in LLC-PK1 does not disrupt cell morphology but does lead to a defect in cell migration. Furthermore, TGF-?1 induced increase in the 75 kDa palladin isoform occurs in both the nucleus and the cytoplasm. These data suggest that palladin expression is induced in injured cells and contributes to proper migration of cells in proximal tubules, possibly by regulation of gene expression as part of the healing process after acute injury.
Project description:Two low-molecular-mass Ni-binding fractions first isolated from human kidneys [Templeton & Sarkar (1985) Biochem. J. 230, 35-42] are further characterized. Both components are acidic and are readily separated from each other by gel chromatography on Bio-Gel P-2. After equilibration with 63Ni the largest complex constitutes about 30% of the radioactive 63Ni and is an approx. 3.5 kDa peptide and the smallest species comprise short oligosaccharides containing 70% of the radioactivity. Both of these components are found in human, bovine and porcine kidneys as well as in a porcine proximal tubule-like cell line LLC-PK1. There is a small variation in amino acid composition between species. The oligosaccharides are reducing sugars and contain sulphate, glucosamine, glucuronic acid and iduronic acid with two to four overall negative charges. The monosaccharide composition was determined by h.p.l.c. with pulsed amperometric detection of the acid hydrolysates and by gas chromatography. In the LLC-PK1 cell line the acidic peptide is both intracellular and extracellular, whereas the oligosaccharides are only intracellular. The concentration of extracellular peptide, as measured by 63Ni binding, is found to increase after exposure of the cells to low micromolar concentrations of Ni, whereas the oligosaccharide concentrations, also measured by 63Ni binding, remain constant. The oligosaccharide component is decreased by 40% in the presence of NH4Cl, suggesting that is derived from degradation of internalized heparan sulphate.
Project description:Cis-dichlorodiammineplatinum II (CDDP) is a chemotherapeutic agent that induces nephrotoxicity by different mechanisms, including oxidative stress, mitochondrial dysfunction, autophagy, and endoplasmic reticulum stress. This study aimed to evaluate if the protective effects of the antioxidant alpha-mangostin (αM) in CDDP-induced damage in proximal tubule Lilly laboratory culture porcine kidney (LLC-PK1) cells, are related to mitochondrial function preservation. It was found that αM co-incubation prevented CDDP-induced cell death. Furthermore, αM prevented the CDDP-induced decrease in cell respiratory states, in the maximum capacity of the electron transfer system (E) and in the respiration associated to oxidative phosphorylation (OXPHOS). CDDP also decreased the protein levels of voltage dependence anion channel (VDAC) and mitochondrial complex subunits, which together with the reduction in E, the mitofusin 2 decrease and the mitochondrial network fragmentation observed by MitoTracker Green, suggest the mitochondrial morphology alteration and the decrease in mitochondrial mass induced by CDDP. CDDP also induced the reduction in mitochondrial biogenesis observed by transcription factor A, mitochondria (TFAM) decreased protein-level and the increase in mitophagy. All these changes were prevented by αM. Taken together, our results imply that αM's protective effects in CDDP-induced toxicity in LLC-PK1 cells are associated to mitochondrial function preservation.
Project description:Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants that are metabolized to carcinogenic dihydrodiol epoxides (PAHDE) by cytochrome P450 1B1 (CYP1B1). This metabolism occurs in bone marrow (BM) mesenchymal stem cells (MSC), which sustain hematopoietic stem and progenitor cells (HSPC). In BM, CYP1B1-mediated metabolism of 7, 12-dimethylbenz[a]anthracene (DMBA) suppresses HSPC colony formation within 6?h, whereas benzo(a)pyrene (BP) generates protective cytokines. MSC, enriched from adherent BM cells, yielded the bone marrow stromal, BMS2, cell line. These cells express elevated basal CYP1B1 that scarcely responds to Ah receptor (AhR) inducers. BMS2 cells exhibit extensive transcriptome overlap with leptin receptor positive mesenchymal stem cells (Lepr+ MSC) that control the hematopoietic niche. The overlap includes CYP1B1 and the expression of HSPC regulatory factors (Ebf3, Cxcl12, Kitl, Csf1 and Gas6). MSC are large, adherent fibroblasts that sequester small HSPC and macrophage in the BM niche (Graphic abstract). High basal CYP1B1 expression in BMS2 cells derives from interactions between the Ah-receptor enhancer and proximal promoter SP1 complexes, boosted by autocrine signaling. PAH effects on BMS2 cells model Lepr+MSC niche activity. CYP1B1 metabolizes DMBA to PAHDE, producing p53-mediated mRNA increases, long after the in vivo HSPC suppression. Faster, direct p53 effects, favored by stem cells, remain possible PAHDE targets. However, HSPC regulatory factors remained unresponsive. BP is less toxic in BMS2 cells, but, in BM, CYP1A1 metabolism stimulates macrophage cytokines (Il1b?>?Tnfa> Ifng) within 6?h. Although absent from BMS2 and Lepr+MSC, their receptors are highly expressed. The impact of this cytokine signaling in MSC remains to be determined.