ABSTRACT
Transcriptional activation by thyroid hormone (T3) requires interactions between the T3 receptor (TR) and T3 response elements (TREs) composed of two copies of sequences related to AGGTCA. Direct repeats of this sequence are a functional TRE when spaced by 4 but not by 5 bp (DR4 versus DR5). TR bound as monomers, homodimers and heterodimers with retinoid X receptor (RXR) to both DR4 and DR5, with an approximately 10-fold greater affinity for DR4 due to reduced dissociation of the protein-DNA complex. We explored DNA bending as an additional variable which could influence the transcriptional outcome of the TR-TRE interaction. Circular permutation indicated a large distortion of the DNA following TR binding, but phasing analysis strongly suggested that this was due only in small part to DNA bending. Phasing analysis indicated that both TR/RXR and TR homodimer induced bends of approximately 10 degrees in DR4, but caused little bending of DR5. Moreover, the TR homo- and heterodimers bent DR4 in opposite directions. These results indicate that in addition to regulating the affinity and spacing requirement for DNA binding by TR, the TR dimer partner may also modulate transcription by influencing the direction of the bending induced by TR binding to DNA, although this effect may be subtle, due to the modest degree of bending.
Subject(s)
DNA/metabolism , Nucleic Acid Conformation , Receptors, Retinoic Acid/metabolism , Receptors, Thyroid Hormone/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , DNA/chemistry , Molecular Sequence Data , Protein Binding , Rats , Repetitive Sequences, Nucleic Acid , Retinoid X ReceptorsABSTRACT
Adipocyte differentiation of 3T3-L1 cells is a complex process which is inhibited by retinoic acid (RA). Since RA acts by nuclear receptors which directly regulate gene expression, we postulate that the primary targets of RA action in this system are genes which are regulated early in adipose conversion. In this study, we demonstrate the use of the differential display technique to search for early events in adipose commitment which are sensitive to RA. A mRNA was identified on the basis of its RA-dependent gene expression 24 h after initiation of a standard differentiation protocol. Molecular cloning of the cDNA revealed it to be identical to the ras recision gene (rrg), for lysyl oxidase. Indeed, two mRNAs identical to those recognized by lysyl oxidase probes were expressed in preadipocytes and tandemly repressed with 24 h of exposure to differentiation conditions. Lysyl oxidase activity was similarly reduced in the media of differentiated cells. RA completely blocked the differ-entiation-related reduction in rrg/lysyl oxidase gene expression, although RA had no independent stimulatory effect on rrg/lysyl oxidase expression in cells not exposed to differentiating conditions. Thus, differential display has been successfully used to identify rrg/lysyl oxidase as an early marker for adipose conversion that is responsive to RA.
Subject(s)
Adipocytes/cytology , Gene Expression Regulation/drug effects , Protein-Lysine 6-Oxidase/biosynthesis , Tretinoin/pharmacology , 3T3 Cells , Adipocytes/metabolism , Animals , Base Sequence , Cell Differentiation , Cloning, Molecular , DNA Primers , Kinetics , Mice , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Sequence Homology, Nucleic Acid , Time Factors , Transcription, GeneticABSTRACT
Activators of peroxisome proliferator activated receptor (PPAR) regulate fatty acid metabolism and can induce adipocyte differentiation. We show here that the gamma subtype of PPAR is expressed at high levels in adipose tissue in contrast to a variety of other tissues, where little gene expression was noted. In addition, PPAR gamma is present at low levels in 3T3-L1 preadipocytes and is induced dramatically during adipocyte conversion using either normal differentiating conditions (fetal calf serum, dexamethasone, isobutyl-methylxanthine, and insulin) or the PPAR activator, WY-14,643. Thus PPAR gamma may be important for adipose cell development and function.
Subject(s)
Adipose Tissue/metabolism , Gene Expression , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , 3T3 Cells , Animals , Cell Differentiation , Cell Line , Mice , RNA, Messenger/metabolismABSTRACT
In proximal tubule cells of the mouse kidney, transcription of a number of genes is induced by androgens. Although a great deal of molecular and genetic information on the induction process has accumulated, the lack of an appropriate cell culture system for DNA transfection studies has hampered efforts to identify and characterize in detail the molecular factors that mediate the response. In the present paper, we have examined a primary renal epithelial cell culture system. We show that in response to androgens, these cells undergo induction of five messenger RNAs that are induced in the intact kidney; thus, the effects of androgens on renal gene expression derive from a direct action of the hormone on proximal tubule cells. The response does not occur in cells from Tfm animals, indicating that androgen receptor is required. Differences in patterns of androgen-inducible messenger RNA expression between mouse species are reflected by similar differences in the cultured cells. Interestingly, the kinetics of induction in culture seem to be distinct from those in the intact kidney, suggesting that a factor or factors differing between the whole animal and tissue culture environments influence the response. Transient transfection experiments with the primary cells showed that the 5'-flanking region of the androgen-regulated RP2 gene, which includes nucleotides -1500 to +42 and contains a glucocorticoid response element that binds the androgen receptor, does not mediate androgen-responsive transcription; thus, this region, and the glucocorticoid response element within it, are either insufficient for, or are not involved in, the gene's response to hormone.
