Mapping of an inducible element in the T cell receptor V beta 2 promoter.

The murine V beta 2 promoter was analyzed for an element regulating phorbol ester inducibility of the TCR beta chain gene. In transient expression analysis of 5' nested deleted fragments of the V beta 2 promoter, the TPA-inducible element mapped between -85 and -42. The -85 to -62 oligo conferred 12-0-tetradecanoylphorbol-13-acetate (TPA) inducibility to the heterologous TPA-uninducible thymidine kinase promoter. The -85 to -62 region contained an AP-1 site (-85 to -72) and inverted repeat motif (-72 to -62). The AP-1 site required the 3' flanking inverted repeat region for conferring optimal inducibility. In vitro transcribed and translated jun/fos heterodimers bind to the V beta 2 AP-1 motif with a 16-fold lower affinity as compared to the collagenase AP-1 motif. This explains the inability of the V beta 2 AP-1 motif to confer optimal TPA inducibility by itself. The affinity of jun/fos heterodimers for the V beta 2 AP-1 motif was not increased by the presence in cis of the inverted repeat motif. The 3' flanking inverted repeat binds the ets transactivator but not jun/fos heterodimers. The demonstrated cooperativity between the AP-1 and the 3' flanking sequence to confer TPA inducibility can thus be explained by the individual contributions of jun/fos and ets transactivators.     

Interrupting activator protein-1 signaling in conscious rats can modify neuropeptide Y gene expression and feeding behavior of phenylpropanolamine

The mechanism for phenylpropanolamine (PPA)-induced anorexia has been attributed to its inhibitory action on hypothalamic neuropeptide Y (NPY), an orexigenic agent abundant in the brain. However, molecular mechanisms behind this effect are not well known. In this study, we investigated whether activator protein-1 (AP-1) signaling was involved. Rats were daily treated with PPA for 4 days. Changes in hypothalamic NPY, c- fos , c- jun , superoxide dismutase (SOD)-1, and SOD-2 mRNA contents were measured and compared. Results showed that c- fos and c- jun mRNA levels were increased following PPA treatment, which were relevant to a reduction in NPY mRNA level. To further determine if c- fos /c- jun genes were involved in PPA anorexia, infusions of antisense oligonucleotide into cerebroventricle were performed before daily PPA treatment in freely moving rats. Results showed that either c- fos or c- jun knock down could block PPA anorexia and restore NPY mRNA content to normal level. It is suggested that AP-1 signaling may participate in the central regulation of PPA-mediated appetite suppression via the modulation of NPY gene expression. Moreover, this modulation might be partly because of the neuroprotective effect of AP-1 since SOD gene was activated during PPA treatment.     

Characterization and purification of human fos protein generated in insect cells with a baculoviral expression vector.

We generated recombinant baculoviruses that contained the human fos gene and that, upon infection of insect cells, synthesized fos protein. The quantity of fos protein produced was at least 10 to 20 times higher than that observed in any mammalian cells reported so far. The fos protein made in insect cells manifested most of the characteristics of mammalian fos protein, which include (i) 55-kilodalton size, (ii) nuclear localization, (iii) phosphoesterification at serine residues, (iv) identical 35S tryptic peptide maps, (v) ability to make heterodimers with the nuclear jun oncoprotein, and (vi) cooperation with the jun protein to bind to a 12-O-tetradecanoyl-phorbol-13-acetate-responsive element. A 100- to 150-fold purification of the fos protein from infected insect cells was achieved in a single step by immunoaffinity chromatography. Availability of authentic fos protein made by baculoviral vectors in insect cells should allow a more rigorous analysis of its biochemical and biological properties.