Transient Transfection Analysis Research Articles

Breast cancer cell resistance to hormonal and targeted therapeutics is correlated with the inactivation of the NR6A1 axis.

Aim: Resistance to hormonal and targeted therapies in breast cancer limits treatment efficacy. Epigenetic alterations, including changes mediated by DNA methyltransferases, play a key role in this process. Previously, we identified that resistance to tamoxifen and rapamycin is associated with the suppression of DNMT3A. This study aims to further explore the mechanisms underlying this suppression, with a focus on identifying NR6A1 as a novel regulatory factor. Methods: Acquisition of resistant breast cancer cell sublines, MTT-test, immunoblotting, transient transfection and reporter analysis, lentiviral infection, qRT-PCR, and analysis of methylation using bisulfite pyrosequencing. Results: Our findings indicate that the development of cross-resistance in breast cancer cells to hormonal and targeted therapies involves a shift in cell signaling to alternative AKT pathways, marked by a localized suppression of the NR6A1/DNMT3A axis and associated DNA methylation changes. We demonstrated the critical role of NR6A1 downregulation in resistance development. Additionally, we observed activation of Snail - a key regulator in the epithelial-mesenchymal transition - as a mediator of the effects of NR6A1 depletion, establishing a direct link between Snail expression and resistance formation. Conclusion: The coordinated suppression of NR6A1 and DNMT3A may contribute to sustaining the resistant phenotype in breast cancer cells. This pathway could serve as a predictive marker, helping guide the selection of optimal therapeutic strategies for breast cancer treatment in the future.

Negative regulation of activation-induced cytidine deaminase gene transcription in developing B cells by a PU.1-interacting intronic region

Activation-induced cytidine deaminase (AID, encoded by Aicda) plays a key role in somatic hypermutation and class switch recombination in germinal center B cells. However, off-target effects of AID are implicated in human leukemia and lymphoma. A mouse model of precursor B cell acute lymphoblastic leukemia driven by deletion of the related transcription factors PU.1 and Spi-B revealed C->T transition mutations compatible with being induced by AID. Therefore, we hypothesized that PU.1 negatively regulates Aicda during B cell development. Aicda mRNA transcript levels were increased in leukemia cells and bone marrow pre-B cells lacking PU.1 and/or Spi-B, relative to wild type cells. Using chromatin immunoprecipitation, PU.1 was found to interact with a negative regulatory region (R2–1) within the first intron of Aicda. CRISPR-Cas9-induced mutagenesis of R2–1 in cultured pre-B cells resulted in upregulation of Aicda in response to lipopolysaccharide stimulation. Mutation of the PU.1 interaction site and neighboring sequences resulted in reduced repressive ability of R2–1 in transient transfection analysis followed by luciferase assays. These results show that a PU.1-interacting intronic region negatively regulates Aicda transcription in developing B cells.

PuNAC100-like/PubHLH61 co-regulate PuLOX3 mediating ester biosynthesis in cold-stored ‘Nanguo’ pears

Refrigeration plays a vital role in prolonging the marketing supply period of ‘Nanguo’ pear; however, it induces a loss of esters, which seriously affects the aromatic quality of the fruit. This study characterized the key enzyme genes and transcription factors closely correlated with ester loss of cold-stored ‘Nanguo’ pears. However, their transcriptional regulation is largely unknown. We found that the expression of PuLOX3, PuNAC100-like, and PubHLH61 was notably repressed and was accompanied by aroma fading in cold-stored ‘Nanguo’ pears. The overexpression or silencing of PuNAC100-like or PubHLH61 led to the overexpression or suppression of PuLOX3. Yeast-hybridization, electrophoretic mobility shift assay, and transient transfection analyses showed that the nuclear-localized PuNAC100-like directly bound to the PuLOX3 promoter successfully, activating its expression. Yeast two-hybrid and bimolecular fluorescence complementation indicated that PuNAC100-like interacted with PubHLH61. In addition, the transcriptional regulation of PubHLH61 on the PuNAC100-like was also observed. Collectively, these findings revealed that the suppressed expression of PuNAC100-like and PubHLH61 down-regulated the transcription of PuLOX3 under low temperature stress and thus induced a decrease in aroma quality in cold-stored ‘Nanguo’ pears.

Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata

Cunninghamia lanceolata (C. lanceolata) is an important timber tree species in southern China that requires gene function studies to understand its traits. In this study, we investigated the callus induction rates of immature zygotic embryos from reciprocal hybrids between genotypes B46 and B49. With zygotic embryo development, the callus induction rates showed an increasing trend, followed by a decreasing trend. Moreover, the rate of callus induction in genotype B46 × B49 immature zygotic embryos was greater than in genotype B49 × B46. Callus from C. lanceolata with genotype B46 × B49 was selected as the donor material for protoplast isolation. By using an enzymatic digestion solution containing cellulase, macerozyme, and pectinase, combined with an osmotic stabilizer, we obtained 9.76 × 106 protoplasts/mL with 92.7% viability. We subsequently transformed plasmids into C. lanceolata callus protoplasts and observed the location of the H2B-eYFP fusion protein in the nucleus. To achieve transient transfection of C. lanceolata callus protoplasts, we compared transfection efficiencies at different concentrations of PEG4000, PEG6000, or PEG8000 in a modified MMg solution. We found that 20% (w/v) PEG6000 mediated the transient transfection of C. lanceolata callus protoplasts with a 6.70% efficiency. This study provides a technical foundation for future research on transient transfection and functional analysis of C. lanceolata genes.

MiR-16-5p regulates aerobic glycolysis and tumorigenesis of NSCLC cells via LDH-A/lactate/NF-κB signaling

Background and aimCancer cells exhibit Warburg effect, characterized by increased glycolysis followed by fermentative conversion of pyruvate to lactate. Upregulation of Lactate Dehydrogenase–A (LDH-A) is elucidated to be a dominant molecular mediator of the phenomenon. Also, microRNA (miRNA) dysregulation participates in malignant progression and dissemination in several cancers. miR-16-5p is considerably reduced in lung cancers (LC), suggesting its tumor-suppressive role. However, its role in the regulation of aerobic glycolysis remains unknown. Our study aims to identify the regulatory roles of miR-16-5p/LDH-A in Non-small cell lung cancer (NSCLC). Main methodsWe evaluated the differential expression of LDH-A and its prognostic potential in NSCLC tissues using online databases. We performed Tissue analysis using Immunohistochemistry (IHC); In-vitro cellular analysis including transient transfection, cellular proliferation, migration, and colony forming analysis. We also performed cell survival, metabolic, cell cycle, apoptotic, ROS generation and Immunocytochemistry (ICC) analyses to identify the role of miR-16-5p/LDH-A in aerobic glycolysis and tumorigenesis of NSCLC. Key findingsWe have identified that miR-16-5p directly targets LDH-A by binding to the complementary binding regions present in its 3′-UTR region, leading to degradation, sequentially leading to reduced lactate accumulation, glucose uptake and ATP levels. Our study also demonstrated the role of lactate accumulation in promoting NSCLC tumorigenesis via activation of NF-κB signaling pathway. However, miR-16-5p mediated targeting of LDH-A downregulates the expression of NF-κB associated genes, along with increased ROS generation, apoptosis, and cell cycle arrest. SignificanceIn conclusion, our findings identify miR-16-5p/LDH-A/lactate/NF-κB as an important link between metabolism and NSCLC cells tumorigenesis.

Salicylic acid treatment alleviates diminished ester production in cold-stored ‘Nanguo’ pear by promoting the transcription of PuAAT

Ester loss due to chilling injury adversely affects the quality of ‘Nanguo’ pears. Salicylic acid (SA) is a well-known regulator of many physiological processes. Nevertheless, the regulatory mechanism underlying SA-mediated biosynthesis of esters remains largely unknown. Herein, we reported that SA treatment increased the kinds of esters and content of main and total esters in cold-stored ‘Nanguo’ pears. Yeast-hybridization, subcellular localization assay, transient transfection and β-glucuronidase analysis showed that the nuclear-localized PuMYB308-like and PuMYB6-like directly bound and activated PuAAT, moreover, overexpression of PuMYB308-like and PuMYB6-like promoted the expression of PuAAT as well as the main volatile content, and these functions were further enhanced by SA treatment. Furthermore, PuNAC68-like was found to be a negative regulator that inhibited the transcription of PuAAT. However, this effect was suppressed by SA treatment. Collectively, this study uncovered the transcriptional mechanism underlying SA promoted the transcription of PuAAT thus mediating ester formation.

Cardioprotective Effect of Stem-Leaf Saponins From Panax notoginseng on Mice With Sleep Derivation by Inhibiting Abnormal Autophagy Through PI3K/Akt/mTOR Pathway.

Sleep deprivation (SD) may lead to serious myocardial injury in cardiovascular diseases. Saponins extracted from the roots of Panax notoginseng, a traditional Chinese medicine beneficial to blood circulation and hemostasis, are the main bioactive components exerting cardiovascular protection in the treatment of heart disorders, such as arrhythmia, ischemia and reperfusion injury, and cardiac hypertrophy. This study aimed to explore the protective effect of stem-leaf saponins from Panax notoginseng (SLSP) on myocardial injury in SD mice. SD was induced by a modified multi-platform method. Cardiac morphological changes were assessed by hematoxylin and eosin (H&E) staining. Heart rate and ejection fraction were detected by specific instruments. Serum levels of atrial natriuretic peptide (ANP) and lactate dehydrogenase (LDH) were measured with biochemical kits. Transmission electron microscopy (TEM), immunofluorescent, and Western blotting analysis were used to observe the process and pathway of autophagy and apoptosis in heart tissue of SD mice. In vitro, rat H9c2 cells pretreated with rapamycin and the effect of SLSP were explored by acridine orange staining, transient transfection, flow cytometry, and Western blotting analysis. SLSP prevented myocardial injury, such as morphological damage, accumulation of autophagosomes in heart tissue, abnormal high heart rate, serum ANP, and serum LDH induced by SD. In addition, it reversed the expressions of proteins involved in the autophagy and apoptosis and activated PI3K/Akt/mTOR signaling pathway that is disturbed by SD. On H9c2 cells induced by rapamycin, SLSP could markedly resume the abnormal autophagy and apoptosis. Collectively, SLSP attenuated excessive autophagy and apoptosis in myocardial cells in heart tissue induced by SD, which might be acted through activating PI3K/Akt/mTOR signaling pathway.

Evidence Against the Causal Relationship Between a Putative Cis-Regulatory Variant of MYH3 and Intramuscular Fat Content in Pigs.

Improving meat quality has become the main goal of modern pig breeding. Intramuscular fat content (IMF) is an important trait influencing meat quality of livestock, but the molecular mechanism behind this trait is still unclear. Recently, Cho et al. reported the discovery of the first causal mutation affecting IMF and red flesh color (a*) in pigs, namely XM_013981330.2:g.−1805_−1810del, a 6-bp deletion variant in the porcine MYH3 promoter region. The objective of this study was to reassess the causality of this mutation for its potential commercial application. By Sanger sequencing, we firstly identified several new variants (including a 4-bp deletion) at or near the 6-bp deletion site, which formed four haplotypes in multiple breeds. Unexpectedly, the 6-bp deletion allele, previously determined as the MYH3 Q allele because of its significantly positive effect on IMF and a*, was found not only in Chinese indigenous breeds, but also in four western commercial breeds with relatively lower IMF levels, including Duroc, Large White, Landrace and Pietrain. More surprisingly, we found that the MYH3 Q allele and the haplotypes harboring it had no significant effects on IMF, marbling and color score in three large-scale divergent pig populations: the heterogeneous F6 and F7 pigs and commercial crossbred Duroc × (Landrace × Yorkshire) pigs. Transient transfection analysis in porcine satellite cells showed that the 6-bp deletion variants had a negligible effect on transcription of reporter gene, but could attenuate the MRF (myogenesis regulatory factors)-induced increase in luciferase activity of the MYH3 promoter vector. The MYH3 protein level in muscle did not differ significantly among the haplotype groups. Therefore, our results cannot support the causal relationship between the 6-bp deletion in MYH3 and IMF trait, suggesting that the causal mutation for the IMF QTL on SSC12 needs to be further identified.

Inhibitory effects of Polygonum cuspidatum on the Epstein-Barr virus lytic cycle

Polygonum cuspidatum is widely used as a medicinal herb in Asia. In this study, we examined the ethanolic extract of Polygonum cuspidatum roots (PcE) for their capacity to inhibit the EBV lytic cycle. The cell viability was determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method. The expression of Epstein-Barr virus (EBV) lytic proteins was analyzed by flow cytometric assay. Real-time quantitative PCR was used to measure the EBV virion production. Transient transfection analysis was subsequently performed to assess the transcription of lytic genes, including BRLF1 and BZLF1. Results showed that the PcE inhibited the transcription of EBV immediate early genes, the expression of EBV lytic proteins, including Rta, Zta, and EA-D and reduced the production of virus particles, showing that PcE is useful for preventing the proliferation of the virus.

Nuclear Pregnane X Receptor Transduces Glucose Signal to Suppress Estrogen Sulfotransferase Gene in Human Liver Cells

Estrogen sulfotransferase (SULT1E1) metabolically inactivates estrogen and SULT1E1 expression is tightly regulated by multiple nuclear receptors. Previously, it was found that nuclear receptors HNF4a and RORa form as complexes binding to the SULT1E1 enhancer (−943/−922 bp) to activate gene expression in high glucose (450 mg/dL) treated human liver cells. However, the mechanism by which low glucose signal (40 mg/dL) mediated SULT1E1 suppression in human liver cells remain mysterious. Here, we have demonstrated that low glucose signal stimulates PXR phosphorylation at Ser350 on its ligand binding domain, preventing its heterodimerization with RXR and enabling it to interact with the HNF4a/RORa binding complexes on the enhancer, thereby suppressing SULT1E1 gene transcription. SULT1E1 was significantly repressed at both mRNA and protein levels in low glucose cultured human liver cells and this suppression was found to be PXR dependent. Vaccinia‐related kinase 1 (VRK1) was found to phosphorylate PXR at Ser350 in low glucose treated cells. Transient transfection analysis of SULT1E1 promoter‐luciferase reporter genes revealed that phosphorylated PXR significantly suppressed SULT1E1 promoter activity via the enhancer. Chromatin immunoprecipitation and gel shift assays elucidated that phosphorylated PXR bound to the SULT1E1 enhancer and disrupted the formation of the HNF4a/RORa binding complexes on it at low glucose condition. The physical interactions among these nuclear receptors were subsequently demonstrated by a series of co‐immunoprecipitation assays. Our study has shown that xenobiotic sensing nuclear receptor PXR transduces low glucose signal by its conserved phosphorylation to suppress SULT1E1 expression in human liver cells.Support or Funding InformationThis work was supported by National Institutes of Health intramural research program Z01ES1005‐01.

Follicular dendritic cell-secreted protein gene expression is upregulated and spread in nifedipine-induced gingival overgrowth.

Follicular dendritic cell-secreted protein (FDC-SP) is secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium (JE). Its expression could be controlled during inflammatory process of gingiva; however, responsible mechanism for gingival overgrowth and involvement of FDC-SP in clinical condition is still unclear. We hypothesized that JE-specific genes are associated with the initiation of drug-induced gingival enlargement (DIGE) called gingival overgrowth, and investigated the changes of JE-specific gene's expression and their localization in overgrown gingiva from the patients. Immunohistochemical analysis revealed that the FDC-SP localization was spread in overgrown gingival tissues. FDC-SP mRNA levels in GE1 and Ca9-22 cells were increased by time-dependent nifedipine treatments, similar to other JE-specific genes, such as Amelotin (Amtn) and Lamininβ3 subunit (Lamβ3), whereas type 4 collagen (Col4) mRNA levels were decreased. Immunocytochemical analysis showed that FDC-SP, AMTN, and Lamβ3 protein levels were increased in GE1 and Ca9-22 cells. Transient transfection analyses were performed using luciferase constructs including various lengths of human FDC-SP gene promoter, nifedipine increased luciferase activities of -345 and -948FDC-SP constructs. These results raise the possibility that the nifedipine-induced FDC-SP may be related to the mechanism responsible for gingival overgrowth does not occur at edentulous jaw ridges.

Efficient isolation of protoplasts from freesia callus and its application in transient expression assays

Freesia hybrida is one of the most famous cut flower variety domesticated from wild species in Freesia genus, distinguished for its colorful flower colors and sweet scents. It has potential to be a model plant to unravel the molecular basis of plant specialized metabolites biosynthesis in family Iridaceae, which are crucial to genetically modify ornamental traits such as flower color and flower scents. However, the lengthy life cycle propagated from tissue culture and extremely low transformation rate severely decelerate functional characterization of genes through transgenic method in this iridaceous plant. Using calluses induced from young flower buds from one popular cultivar of Freesia hybrida, ‘Red River®’, an efficient protoplast isolation method was established and optimized, which was further employed in the gene transient expression system. Results demonstrated that the newly explored transient system was versatile and could be applied in numerous aspects, such as protein immunoblotting, enzyme activity, protein subcellular localization, protein–protein interaction, protein–DNA interaction and regulatory gene transregulation property assays. Moreover, calluses induced from another cultivar of Freesia hybrida, ‘Ambiance’ could also be used to isolate protoplasts for gene transient transfection analysis, indicating the wide applicability of this system. Results in this study provided new methods for the elucidation of gene function in Freesia in vivo, which might also be inspirational in other species with long life cycles or hardly to be transformed, such as plants in family Iridaceae. Freesia protoplast isolation and transfection methods were established and illustrated the utilization in protein immunoblotting, enzyme activity, subcellular localization, protein-protein or protein-DNA interaction, and investigation of transactivation capacities of regulators.

Tumor necrosis factor-α regulates human follicular dendritic cell-secreted protein gene transcription in gingival epithelial cells.

Follicular dendritic cell-secreted protein (FDC-SP) is a secreted protein expressed in follicular dendritic cells, periodontal ligament and junctional epithelium. To elucidate the transcriptional regulation of the human FDC-SP gene by tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with chimeric constructs of the FDC-SP gene promoter linked to a luciferase reporter gene, gel mobility shift and chromatin immunoprecipitation assays using Ca9-22 gingival epithelial cells. TNF-α (10ng/ml) induced FDC-SP mRNA and protein levels at 3hr and reached maximum at 12hr. In transient transfection assays, TNF-α (12hr) increased the LUC activities of constructs between -116FDCSP and -948FDCSP including the human FDC-SP gene promoter. Transcriptional stimulations by TNF-α were partially inhibited in the -345FDCSP constructs that included 3-bp mutations in the YY1, GATA, CCAAT enhancer-binding protein 2 (C/EBP2) and C/EBP3. Transcriptional activities induced by TNF-α were inhibited by tyrosine kinase, MEK1/2 and phosphoinositide 3-kinase inhibitors. The results of ChIP assays showed that YY1, GATA and C/EBPβ transcription factors interacted with the YY1, GATA, C/EBP2 and C/EBP3 elements that were increased by TNF-α. These studies show that TNF-α stimulates human FDC-SP gene transcription by targeting YY1, GATA, C/EBP2 and C/EBP3 in the FDC-SP gene promoter.

IL-1β and TNF-α regulate mouse amelotin gene transcription in gingival epithelial cells

Amelotin (AMTN) is an enamel protein expressed in maturation-stage ameloblasts and junctional epithelium. To clarify the transcriptional regulation of the AMTN gene by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), we conducted real-time PCR, Western blotting, transient transfection analyses with luciferase constructs including various lengths of the mouse AMTN gene promoter, and gel shift and chromatin immunoprecipitation assays using mouse gingival epithelial GE1 cells. The levels of AMTN mRNA and protein in GE1 cells were increased after 6 h of stimulation with IL-1β (1 ng/mL) and TNF-α (10 ng/mL). IL-1β and TNF-α induced luciferase activities of the constructs between -116AMTN and -705AMTN including the mouse AMTN gene promoter. Transcriptional activation by IL-1β and TNF-α was partially inhibited in -460AMTN including 3-bp mutations in the CCAAT-enhancer-binding protein 1 (C/EBP1), C/EBP2 and Yin Yang 1 (YY1) elements. Transcriptional activities induced by IL-1β and TNF-α were inhibited by tyrosine kinase, MEK1/2 and PI3-kinase inhibitors. Results of ChIP assays showed that IL-1β and TNF-α increased C/EBPβ and YY1 binding to the C/EBP1, C/EBP2 and YY1 elements. These results demonstrate that IL-1β and TNF-α increase AMTN gene transcription via the C/EBP1, C/EBP2 and YY1 elements in the mouse AMTN gene promoter.

TGFβ1-induced Amelotin gene expression is downregulated by Bax expression in mouse gingival epithelial cells.

Amelotin (AMTN) is induced upon initiation of apoptosis by transforming growth factor beta1 (TGFβ1) and is mediated by Smad3 in gingival epithelial cells (GE1 cells). This upregulation of AMTN gene expression is temporary, and the mechanism responsible is still unclear. The present study investigated the transcriptional downregulation of TGFβ1-induced AMTN gene expression in GE1 cells during the progression of apoptosis. To examine time-dependent changes in the levels of AMTN, Smad3 and Bax mRNA induced by TGFβ1, real-time PCR analyses were performed. Immunocytochemistry was carried out to detect the expression of Smad3 and Bax. Transient transfection analyses were performed using mouse AMTN gene promoter constructs of various lengths including Smad response elements (SBEs), in the presence or absence of TGFβ1. Changes in Smad3 binding to SBEs resulting from overexpression of Bax were examined using ChIP assays. Overexpression of Bax dramatically downregulated the levels of TGFβ1-induced AMTN mRNA and transcription of the AMTN gene. Smad3 binding to SBEs in the mouse AMTN gene promoter was induced by overexpression of Smad3 or TGFβ1, and this was inhibited by Bax overexpression. These results show that the levels of AMTN mRNA induced by TGFβ1 and Smad3 are decreased by robust expression of Bax in gingival epithelial cells.

Tumor necrosis factor-α stimulates human amelotin gene transcription in gingival epithelial cells.

Amelotin (AMTN) is an enamel protein that is localized in the basal lamina of ameloblasts in their maturation stage and the internal basal lamina of junctional epithelium (JE) and it is suggested that AMTN could be involved in the dentogingival attachment. To elucidate the transcriptional regulation of human AMTN gene in inflamed gingiva, we have analyzed the effect of tumor necrosis factor-α (TNF-α) on the expression of AMTN gene in Ca9-22 and Sa3 human gingival epithelial cells. Total RNAs were extracted from Ca9-22 and Sa3 cells after stimulation by TNF-α (10ng/ml). AMTN mRNA and protein levels were measured by real-time PCR and Western blotting. Transient transfection analyses were completed using the various lengths of human AMTN gene promoter constructs with or without TNF-α. Gel mobility shift and chromatin immunoprecipitation assays were performed to investigate the transcription factors bindings to the human AMTN gene promoter by TNF-α. TNF-α (10ng/ml) increased AMTN mRNA and protein levels after 12h. TNF-α induced luciferase activities of human AMTN gene promoter constructs (- 211AMTN, - 353AMTN, and - 501AMTN). TNF-α-induced luciferase activities were partially inhibited in the mutation - 353AMTN constructs that included 3-bp mutations in CCAAT enhancer-binding protein 1 (C/EBP1), C/EBP2 and Ying Yang 1 (YY1) elements. Transcriptional activities induced by TNF-α were inhibited by protein kinase A, Src-tyrosine kinase, MEK1/2, p38 kinase, NF-κB, and PI3-kinase inhibitors. Gel shift assays showed that TNF-α increased nuclear proteins binding to two types of C/EBP elements (C/EBP1 and C/EBP2) and YY1 element. The results of the chromatin immunoprecipitation assays showed that C/EBPβ binding to C/EBP1 and C/EBP2, and YY1 binding to YY1 were increased by TNF-α. These findings demonstrated that TNF-α stimulates AMTN gene transcription in human gingival epithelial cells via C/EBP1, C/EBP2, and YY1 elements in the human AMTN gene promoter.

Calcium hydroxide regulates transcription of the bone sialoprotein gene via a calcium-sensing receptor in osteoblast-like ROS 17/2.8 cells.

Bone sialoprotein (BSP) is a glycoprotein associated with mineralized tissues. In this study, we investigated the regulation of Bsp transcription by calcium hydroxide [Ca(OH)2 ] in rat osteosarcoma-derived osteoblast-like ROS 17/2.8 cells and stromal bone marrow cells. Application of Ca(OH)2 (0.4mM) increased the levels of runt-related transcription factor 2 (Runx2) and BspmRNAs at 3 and 6h and the level of BSP protein at 12h. Transient transfection analyses were performed using chimeric constructs encompassing different regions of the rat Bsp gene promoter ligated to a luciferase reporter gene. It was found that Ca(OH)2 increased the luciferase activities of the pLUC3 and pLUC4 constructs. Introduction of 2-bp mutations to the luciferase construct showed that the effects of Ca(OH)2 were mediated by cAMP response-element (CRE) and fibroblast growth factor 2 (FGF2) response element (FRE). Luciferase activities induced by Ca(OH)2 were blocked by protein kinase C (PKC), protein kinase A (PKA), phosphoinositide-3-kinase (PI3-K), and extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitors and by calcium-sensing receptor (CASR) antagonists. Gel-shift analyses showed that Ca(OH)2 increased binding of nuclear protein to CRE and FRE. Dexamethasone-induced mineralization in stromal bone marrow cells was abrogated by CASR antagonists. These studies demonstrate that Ca(OH)2 regulates Bsp transcription via the CASR by targeting CRE and FRE in the rat Bsp gene promoter.

Ligand-dependent and -independent regulation of human hepatic sphingomyelin phosphodiesterase acid-like 3A expression by pregnane X receptor and crosstalk with liver X receptor

Pregnane X receptor (PXR) mainly regulates xenobiotic metabolism and detoxification. Additionally, it exerts pleiotropic effects on liver physiology, which in large parts depend on transrepression of other liver-enriched transcription factors. Based on the hypothesis that lower expression levels of PXR may reduce the extent of this inhibition, an exploratory genome-wide transcriptomic profiling was performed using HepG2 cell clones with different expression levels of PXR. This screen and confirmatory real-time RT-PCR identified sphingomyelin phosphodiesterase acid-like (SMPDL) 3A, a novel nucleotide phosphodiesterase and phosphoramidase, as being up-regulated by PXR-deficiency. Transient siRNA-mediated knock-down of PXR in HepG2 cells and primary human hepatocytes similarly induced mRNA up-regulation, which translated into increased intracellular and secreted extracellular protein levels. Interestingly, ligand-dependent PXR activation also induced SMPDL3A in HepG2 cells and primary human hepatocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of PXR to the previously identified liver X receptor (LXR)-binding DR4 motif as well as to an adjacent ER8 motif in intron 1 of SMPDL3A. Constitutive binding of the unliganded receptor to the intron 1 chromatin indicated ligand-independent repression of SMPDL3A by PXR. Transient transfection and reporter gene analysis confirmed the specific role of these motifs in PXR- and LXR-dependent activation of the SMPDL3A intronic enhancer. PXR inhibited LXR mainly by competition for binding sites. In conclusion, this study describes that a decrease in PXR expression levels and ligand-dependent activation of PXR and LXR increase hepatic SMPDL3A levels, which possibly connects these receptors to hepatic purinergic signaling and phospholipid metabolism and may result in drug-drug interactions with phosphoramidate pro-drugs.

Amelotin gene expression is temporarily being upregulated at the initiation of apoptosis induced by TGFβ1 in mouse gingival epithelial cells.

Amelotin (AMTN) is expressed and secreted by ameloblasts in the maturation stage of amelogenesis and persist with low levels in the junctional epithelium (JE) of erupted teeth. The purpose of this study is to investigate the transcriptional regulation of the AMTN gene by transforming growth factor beta1 (TGFβ1) in gingival epithelial (GE1) cells in the apoptosis phase. Apoptosis was evaluated by the fragmentation of chromosomal DNA and TUNEL staining. A real-time PCR was carried out to examine the AMTN mRNA levels induced by TGFβ1 and Smad3 overexpression. Transient transfection analyses were completed using the various lengths of mouse AMTN gene promoter constructs with or without TGFβ1. Chromatin immunoprecipitation (ChIP) assays were performed to investigate the Smad3 bindings to the AMTN gene promoter by TGFβ1. TGFβ1-induced apoptosis in GE1 cells were detected at 24 and 48h by DNA fragmentation and TUNEL staining. AMTN mRNA levels increased at 6h and reached maximum at 24h in GE1 cells. Luciferase activities of the mouse AMTN gene promoter constructs were induced by TGFβ1. The results of the ChIP assays showed that there was an increase in Smad3 binding to Smad-binding element (SBE)#1 and SBE#2 after stimulation by TGFβ1. Immunohistochemical localization of AMTN was detected in the JE, and the AMTN protein levels in Smad3-deficient mice were decreased compared with wild-type mice. AMTN mRNA levels were induced at the initiation of apoptosis by TGFβ1, which mediated through the Smad3 bindings to SBEs in the mouse AMTN gene promoter.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells.