Role of tissue-specific transcription factor LFB3 in a cyclic AMP-responsive enhancer of the urokinase-type plasminogen activator gene in LLC-PK1 cells.

A cyclic AMP (cAMP)-inducible enhancer in the pig urokinase-type plasminogen activator gene located 3.4 kb upstream of the transcription initiation site is composed of three protein-binding domains, A, B, and C. Domains A and B each contain a CRE (cAMP response element)-like sequence but require the adjoining C domain for full cAMP responsiveness. A tissue-specific transcription factor, LFB3/HNF1beta/vHNF1, binds to the C domain. Mutation analyses suggest that the imperfect CRE and LFB3-binding sequences are required for tight coupling of hormonal and tissue-specific regulation. CREB and ATF1 bind to domains A and B, and this binding is enhanced upon phosphorylation by cAMP-dependent protein kinase (protein kinase A [PKA]). Analysis in a mammalian two-hybrid system revealed that CREB/ATF1 and LFB3 interact and that transactivation potential is enhanced by PKA activation. Interestingly, however, phosphorylation of CREB at Ser-133 does not contribute to its interaction with LFB3. The region of LFB3 involved in its interaction with CREB/ATF1 lies, at least partly, between amino acids 400 and 450. Deletion of this region removed the ability of LFB3 to mediate cAMP induction of the ABC enhancer but did not impair its basal transactivation activity on the albumin promoter. Thus, the two activities are distinct functions of LFB3.

Cooperation of two PEA3/AP1 sites in uPA gene induction by TPA and FGF-2.

We have previously shown in NIH 3T3 fibroblasts that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) or fibroblast growth factor-2 (FGF-2) activates the Ras/Erk signaling pathway in NIH 3T3 fibroblasts, leading to the induction of the urokinase-type plasminogen activator (uPA) gene. In this study, we characterize cis-acting elements involved in this induction. DNase I hypersensitive (HS) site analysis of the uPA promoter showed that two regions were enhanced after TPA and FGF-2 treatment. One was located 2.4kb upstream of the transcription start site (-2.4kb), where a known PEA3/AP1 (AGGAAATGAGGTCAT) element is located. The other was located in a previously undefined far upstream region. Sequencing of this region revealed a similar AP1/PEA3 (GTGATTCACTTCCT) element at -6.9 kb corresponding to the HS site. Deletion analysis of the uPA promoter in transient transfection assays showed that both PEA3/AP1 elements are required for full inducibility, suggesting a synergism between the two elements. When the two sites were inserted together upstream of a minimal promoter derived from the thymidine kinase gene, expression of the reporter gene was more strongly induced by TPA and FGF-2 than with either of the two elements alone. Alone, the -6.9 element was more potent than the -2.4 element. The involvement of AP1 as well as Ets transcription factors was confirmed by examining different promoter constructs containing deletions in either the AP-1 or the PEA3 element, and by using an expression plasmid for dominant negative Ets-2. Electromobility shift analyses using specific antibodies showed that c-Jun and, JunD bind to both elements with or without induction. In addition, ATF-2 binds to the -2.4-kb element even without induction and c-Fos to the -6.9-kb element only after induction. Accordingly, overexpression of c-Fos caused induction from the -6.9-kb element, but reduced induction from the -2.4-kb element. The involvement of the Ets-2 transcription factor was shown by using expression plasmids for wild-type and dominant negative Ets-2.

Role of LFB3 in cell-specific cAMP induction of the urokinase-type plasminogen activator gene.

In previous work we suggested that a kidney-specific transcription factor LFB3 cooperates with cAMP-response element (CRE)-binding proteins within a cAMP regulatory unit comprised of three protein-binding domains and located 3.4 kilobase pairs upstream of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells (Menoud, P.-A., Matthies, R., Hofsteenge, J., and Nagamine, Y. (1993) Nucleic Acids Res. 21, 1845-1852). The two domains contain a CRE-like sequence, and the third domain is recognized by LFB3. The absolute requirement of LFB3 as well as the cooperation among the three domains for cAMP regulation were confirmed by transient transfection assays in F9 teratocarcinoma cells, in which the level of LFB3 was negligible. Suspecting a possible feedback regulation of LFB3 mRNA expression during cAMP-dependent uPA gene induction in LLC-PK1 cells, we measured LFB3 mRNA levels after cAMP treatment and found a strong reduction. This reduction was not due to a change in template activity of the LFB3 gene because run-on transcription showed no significant change in LFB3 gene transcription. RNA synthesis inhibitor-chase experiments indicated that the down-regulation was post-transcriptional. Interestingly, when the inhibitor was added at the same time as cAMP, the cAMP-induced decrease in LFB3 mRNA levels was abrogated, suggesting that ongoing RNA synthesis is required for the decrease. Similar effects on LFB3 mRNA metabolism were observed with all agents that induce uPA mRNA in LLC-PK1 cells, including 12-O-tetradecanoylphorbol-13-acetate, okadaic acid, colchicine, and cytochalasin. We discuss the significance of this regulation in uPA gene expression.

GABAA-receptors: drug binding profile and distribution of receptors containing the alpha 2-subunit in situ.

The highest structural diversity of GABAA-receptor subunits is observed among members of the alpha-subunit class. Using subunit-specific antisera, the receptors containing the alpha 2-subunit were characterized. Western blots revealed an apparent molecular size of 52 kDa for the alpha 2-subunit. Immunohistochemically, the alpha 2-subunit was most preponderant in areas which lack the alpha 1-subunit, e.g. striatum and olfactory bulb granule cell layer, suggesting that these two subunits represent largely distinct receptor subtypes. Pharmacologically, the receptor population which was immunoprecipitated by the alpha 2-subunit-specific antisera displayed a drug binding profile characterized by a low affinity for CL 218872, beta CCM and zolpidem. This is in striking contrast to the high affinities of these ligands displayed by receptors immunoprecipitated by the alpha 1-subunit-specific antiserum. Thus, the alpha 1- and the alpha 2-subunit characterize two GABAA-receptor populations which greatly differ in brain distribution and pharmacological profile.