Cyclic nucleotide regulation of PAI-1 mRNA stability. Identification of cytosolic proteins that interact with an a-rich sequence.

Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue 8-bromo-cAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator-inhibitor (PAI-1) mRNA. Previous studies utilizing HTC cells stably transfected with beta-globin:PAI-1 chimeric constructs demonstrated that at least two regions within the PAI-1 3'-untranslated region mediate the cyclic nucleotide-induced destabilization of PAI-1 mRNA; one of these regions is the 3'-most 134 nucleotides (nt) of the PAI-1 mRNA (Heaton, J. H., Tillmann-Bogush, M., Leff, N. S., and Gelehrter, T. D. (1998) J. Biol. Chem. 273, 14261-14268). In the present study, ultraviolet cross-linking analyses of this region demonstrate HTC cell cytosolic mRNA-binding proteins ranging from 38 to 76 kDa, with a major complex migrating at approximately 50 kDa. RNA electrophoretic mobility shift analyses demonstrate high molecular weight multiprotein complexes that specifically interact with the 134-nt cyclic nucleotide-responsive sequence. The 50, 61, and 76 kDa and multiprotein complexes form with an A-rich sequence at the 3' end of the cyclic nucleotide-responsive region; a 38-kDa complex forms with a U-rich region at the 5' end of the 134 nt sequence. Mutation of the A-rich region prevents both the binding of the 50-, 61-, and 76-kDa proteins and formation of the multiprotein complexes, as well as cyclic nucleotide-regulated degradation of chimeric globin:PAI-1 transcripts in HTC cells. These data suggest that the proteins identified in this report play an important role in the cyclic nucleotide regulation of PAI-1 mRNA stability.

Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences.

Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA. Although 8-bromo-cAMP causes a modest decrease in PAI-1 transcription, regulation is primarily the result of a 3-fold increase in the rate of PAI-1 mRNA degradation. To determine the cis-acting sequences required for cyclic nucleotide regulation, we have stably transfected HTC cells with chimeric genes containing sequences from the rat PAI-1 cDNA and the mouse beta-globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the beta-globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta-globin coding region and the PAI-1 3'-untranslated region (UTR) is destabilized in the presence of 8-bromo-cAMP, suggesting that this response is mediated by sequences in the PAI-1 3'-UTR. Analyses by deletion of sequences from this chimeric construct indicate that, whereas more than one region of the PAI-1 3'-UTR can confer cyclic nucleotide responsiveness, the 3'-most 134-nucleotide sequence alone is sufficient to do so. Insertion of PAI-1 sequences within the beta-globin 3'-UTR confirms that the 3'-most 134 nucleotides of PAI-1 mRNA can confer cyclic nucleotide regulation of stability on a heterologous transcript, suggesting that this sequence may play a major role in hormonal regulation of PAI-1 mRNA stability.

Characterization of a glial cell-specific DNA-protein complex formed with the human T cell lymphotropic virus type I (HTLV-I) enhancer.

Human T cell lymphotropic virus type I (HTLV-I) encodes the trans-activator, Tax, which facilitates viral transcription from three 21 bp repeated elements within the U3 region of the long terminal repeat (LTR). Electrophoretic mobility shift (EMS) analyses utilizing double-stranded (ds) oligonucleotides homologous to each of the 21 bp repeats and nuclear extracts derived from selected cell lines of lymphocytic, neuronal, and glial origin have demonstrated the differential binding of cellular factors to each of the three 21 bp repeats. Specifically, both a glial cell-specific DNA-protein complex (designated GCS) and 21 bp repeat-specific DNA-protein complexes (designated U1 and U2) were detected. The formation of the GCS DNA-protein complex may involve activating transcription factor (ATF)/cAMP-response element (CRE) binding protein (CREB) family member(s) while the formation of the U1 and U2 DNA-protein complexes may involve an Sp1-related factor. In addition, three ATF-CREB-related DNA-protein complexes common to each individual 21 bp repeat (designated C1-C3) were also detected. However, we demonstrated that the abundance of the C1 and C2 DNA-protein complexes detected with the individual 21 bp repeats and glial cell nuclear extract was relatively low compared to that obtained with lymphocyte, monocyte, or neuronal nuclear extracts. We also have demonstrated that the ATF-CREB factors participating formation of the GCS DNA-protein complex are distinct from those participating in formation of the C1-C3 DNA-protein complexes. Based on nucleotide sequence requirements and immunoreactivity, we suggest that the GCS DNA-protein complex may contain a novel glial cell type specific ATF-CREB-related factor(s). Furthermore, we demonstrate that the CRE modulator (CREM) protein in conjunction with the ATF/CREB factor, CREBP1, interact with each of the three 21 bp repeats to form the C3 DNA-protein complex. However, the abundance of the C3 DNA-protein complex formed utilizing the promoter proximal repeat is dramatically lower compared to either of the other two 21 bp repeat elements. Based on these observations, we suggest that the differential binding of cellular factors to each of the three 21 bp repeat elements may play a role in basal as well as Tax-mediated LTR-directed transcription within cell populations of either immune or nervous system origin.