CYP3A4 is the most important drug-metabolizing enzyme that is involved in biotransformation of more than 50% of drugs. Pregnane X receptor (PXR) dominantly controls CYP3A4 inducibility in the liver, whereas vitamin D receptor (VDR) transactivates CYP3A4 in the intestine by secondary bile acids. Four major functional PXR-binding response elements of CYP3A4 have been discovered and their cooperation was found to be crucial for maximal up-regulation of the gene in hepatocytes. VDR and PXR recognize similar response element motifs and share DR3(XREM) and proximal ER6 (prER6) response elements of the CYP3A4 gene. In this work, we tested whether the recently discovered PXR response elements DR4(eNR3A4) in the XREM module and the distal ER6 element in the CLEM4 module (CLEM4-ER6) bind VDR/RXRalpha heterodimer, whether the elements are involved in the intestinal transactivation, and whether their cooperation with other elements is essential for maximal intestinal expression of CYP3A4. Employing a series of gene reporter plasmids with various combinations of response element mutations transiently transfected into four intestinal cell lines, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP), we found that the CLEM4-ER6 motif interacts with VDR/RXRalpha heterodimer and partially cooperates with DR3(XREM) and prER6 in both basal and VDR-mediated inducible CYP3A4 regulation in intestinal cells. In contrast, eNR3A4 is involved only in the basal transactivation in intestinal cells and in the PXR-mediated rifampicin-induced transactivation of CYP3A4 in LS174T intestinal cells. We thus describe a specific ligand-induced VDR-mediated transactivation of the CYP3A4 gene in intestinal cells that differs from PXR-mediated CYP3A4 regulation in hepatocytes.

• MeSH
• chromatinová imunoprecipitace MeSH
• cytochrom P-450 CYP3A genetika   MeSH
• DNA primery MeSH
• genetická transkripce fyziologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• receptory kalcitriolu fyziologie   MeSH
• regulace genové exprese enzymů fyziologie   MeSH
• sekvence nukleotidů MeSH
• střeva enzymologie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Class C G-protein coupled receptors form obligatory dimers. Metabotropic glutamate receptors (mGluRs) are found commonly as homodimers. Alternative splicing of mGluR1 gene results in vivo in the expression of a long variant mGluR1a and at least two short variants mGluR1b and d. The amino acid sequences diverge within their carboxyl-termini six amino acid residues following RRKK motif. This four basic residue sequence was shown to have pronounced impact on function and trafficking of the short variants, while for mGluR1a the long C-terminus reduces the effects caused by presence of the RRKK motif. Here we investigated consequences of interactions between long mGluR1a and short mGluR1b variants. Our results show that mGluR1a interferes with mGluR1b trafficking to the cell surface in HEK293 transfected cells. Expression of a mGlu1a mutant incapable of activating G-proteins with mGluR1b mutated in the glutamate binding site led to the formation of a functional heterodimer. Moreover, we show that swapping long mGluR1a and/or short mGluR1b C-termini with corresponding regions in chimerical GB1 and GB2 gamma-amino butyric acid b (GABAb) receptor subunits do not exclude heterodimerization. These data reveal that the C-terminal ends of mGluR1 do not control subunit association, such that mGluR1 dimers with two distinct C-termini can form and function properly.

• MeSH
• alternativní sestřih genetika   MeSH
• exprese genu fyziologie   MeSH
• fosfatasy metabolismus   MeSH
• imunoprecipitace metody   MeSH
• lidé MeSH
• mutageneze fyziologie   MeSH
• receptory metabotropního glutamátu genetika   klasifikace   metabolismus   MeSH
• terciární struktura proteinů fyziologie   MeSH
• transfekce metody   MeSH
• transformované buněčné linie fyziologie   MeSH
• transport proteinů fyziologie   MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Congenital abnormalities, various diseases and injuries may result in the degeneration of articular cartilage. Recently, stem cell therapy has offered new treatment possibilities for this condition. The aim of our study was to verify the chondrogenic differentiation potential of human bone marrow mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (AMSCs) in vitro in the presence or absence of transforming growth factor beta (TGF-beta1). Human BMSCs and AMSCs from healthy donors were collected during orthopaedic surgeries and expanded in vitro to obtain a sufficient quantity of cells; their chondrogenic differentiation was studied in the pellet culture system. Spontaneous chondrogenesis occurred in both BMSC and AMSC pellet cultures and was similar in both TGF-beta1 treated and untreated pellet cultures. BMSC pellets contained more cells with a chondrogenic phenotype. The presence of TGF-beta1 led to a decrease in the levels of collagen type I mRNA and to increased levels of collagen type II mRNA only in the BMSC pellet culture. Our results demonstrate that although both mesenchymal cell types can be used in cartilage tissue engineering, the chondrogenic potential of human BMSCs is higher than that of AMSCs.

• MeSH
• finanční podpora výzkumu jako téma MeSH
• imunohistochemie metody   využití   MeSH
• kloubní chrupavka abnormality   patologie   růst a vývoj   MeSH
• kolagen typ II fyziologie   genetika   MeSH
• kostní dřeň fyziologie   chirurgie   MeSH
• kultivované buňky fyziologie   transplantace   MeSH
• lidé MeSH
• mezenchymální kmenové buňky fyziologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   využití   MeSH
• transformující růstový faktor beta1 terapeutické užití   MeSH
• tuková tkáň fyziologie   chirurgie   MeSH
• Check Tag
• lidé MeSH