Cyclic Nucleotide Regulation of PAI-1 mRNA Stability

Tillmann-Bogush, Maribeth; Heaton, Joanne H.; Gelehrter, Thomas D.

doi:10.1074/jbc.274.2.1172

Cited by 42 publications

(18 citation statements)

References 44 publications

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“…As shown in Fig. 3B, the RNA-protein complex migrates with an apparent molecular mass of ϳ50 kDa, similar in size to the major band seen with HTC cytoplasmic proteins (23). Binding is dependent on protein concentration (Fig.…”

Section: Affinity Purification and Identification Of Pai-1 Mrna-bindingmentioning

confidence: 86%

“…Preparation of Radiolabeled and Unlabeled RNA-All DNA constructs used as templates to prepare radiolabeled or unlabeled RNA have been described previously (23). 32 P-Labeled sense strand RNA was prepared by published methods (24) using as template either plasmid DNA linearized with the appropriate restriction enzyme or PCR products, including a T3 RNA polymerase site.…”

Section: Methodsmentioning

confidence: 99%

“…R-EMSA and UV Cross-linking Analysis-R-EMSA and UV crosslinking analyses were carried out essentially as described previously (23). For R-EMSA, HTC cell polysomes or purified recombinant protein were incubated with 32 P-labeled RNA (ϳ200,000 dpm/reaction) for 20 min at room temperature in buffer containing 10 mM Hepes (pH 7.6), 5 mM MgCl 2 , 40 mM KCl, 5% gylcerol, 1 mM dithiotreitol, 8 units of RNase inhibitor, and 10 -25 g of tRNA.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were grown to confluence in T-150 flasks and harvested by trypsinization. HTC cell polysomes were isolated as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

“…Most of these proteins interact with the A-rich portion of the PAI-1 CRS. Mutations in the A-rich regions abolish both RNA-protein complex formation and cyclic nucleotide regulation of message decay in transfected HTC cells, suggesting that these RNA-binding proteins may be important regulators of mRNA stability (23).…”

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confidence: 99%

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Identification and cDNA Cloning of a Novel RNA-binding Protein That Interacts with the Cyclic Nucleotide-responsive Sequence in the Type-1 Plasminogen Activator Inhibitor mRNA

Heaton

Dlakic

Dlakić

et al. 2001

Journal of Biological Chemistry

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Incubation of HTC rat hepatoma cells with 8-bromocAMP results in a 3-fold increase in the rate of degradation of type-1 plasminogen activator inhibitor (PAI-1) mRNA. We have reported previously that the 3-most 134 nt of the PAI-1 mRNA is able to confer cyclic nucleotide regulation of message stability onto a heterologous transcript. R-EMSA and UV cross-linking experiments have shown that this 134 nt cyclic nucleotide-responsive sequence (CRS) binds HTC cell cytoplasmic proteins ranging in size from 38 to 76 kDa. Mutations in the A-rich region of the CRS both eliminate cyclic nucleotide regulation of mRNA decay and abolish RN-protein complex formation, suggesting that these RNA-binding proteins may be important regulators of mRNA stability. By sequential R-EMSA and SDS-PAGE we have purified a protein from HTC cell polysomes that binds to the PAI-1 CRS. N-terminal sequence analysis and a search of protein data bases revealed identity with two human sequences of unknown function. We have expressed one of these sequences in E. coli and confirmed that the recombinant protein interacts specifically with the PAI-1 CRS. Mutation of the A-rich portion of the PAI-1 CRS reduces binding by the recombinant PAI-1 RNA-binding protein.The amino acid sequence of this protein includes an RGG box and two arginine-rich regions, but does not include other recognizable RNA binding motifs. Detailed analyses of nucleic acid and protein data bases demonstrate that blocks of this sequence are highly conserved in a number of metazoans, including Arabidopsis, Drosophila, birds, and mammals. Thus, we have described a novel RNA-binding protein that identifies a family of proteins with a previously undefined sequence motif. Our results suggest that this protein, PAI-RBP1, may play a role in regulation of mRNA stability.Regulation of mRNA stability is an important component of the regulation of gene expression and is known to have a significant role in normal physiology and development (1-5). Our understanding of the regulation of message degradation has been enhanced by the identification of consensus cis-acting sequences that are involved in determining message stability and of some proteins that interact with them (4, 6). Although it is known that many stimuli alter mRNA stability and some cis-acting sequences responsible have been identified, in few cases have trans-acting factors been isolated (2, 7-10). In contrast, a broad spectrum of RNA-binding proteins that are involved in RNA processing, cellular localization, and translation have been identified, and structural domains involved in RNA recognition have been described (11,12). In many cases RNAbinding proteins contain short signature domains that bind RNA and anchor the protein such that functional domains align (13,14). Much less is known about the mechanisms by which RNA-binding proteins regulate mRNA stability.Plasminogen activators (PAs) 1 are serine proteases that catalyze the conversion of plasminogen to the broad spectrum protease, plasmin. Plasmin is the major fibrinolytic en...

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Section: Affinity Purification and Identification Of Pai-1 Mrna-bindingmentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Cells were grown to confluence in T-150 flasks and harvested by trypsinization. HTC cell polysomes were isolated as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Identification and cDNA Cloning of a Novel RNA-binding Protein That Interacts with the Cyclic Nucleotide-responsive Sequence in the Type-1 Plasminogen Activator Inhibitor mRNA

Heaton

Dlakic

Dlakić

et al. 2001

Journal of Biological Chemistry

Self Cite

125

120

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Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-?1

et al. 1999

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Transcription of the plasminogen activator inhibitor type-1 (PAI-1) gene appears to be growth state regulated in several cell types (e.g. , Ryan and Higgins, 1993, J Cell Physiol 155:376-384; Mu et al., 1998, J Cell Physiol 174:90-98). Transit of serum-stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G(0)) provided a model system to assess the kinetics and mechanisms underlying PAI-1 expression in a growth "activated" phenotype. PAI-1 mRNA transcripts increased by more than 20-fold during the G(0)-->G(1) transition; induced expression had immediate-early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI-1 gene paralleled the steady-state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI-1 mRNA levels were up-regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor-beta1 (TGF-beta1) effectively attenuated the more than ninefold serum-associated PAI-1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum-mediated PAI-1 transcription, PAI-1 induction upon addition of TGF-beta1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)- or TGF-beta1-stimulated PAI-1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI-1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non-FBS- or TGF-beta1-treated cultures), moreover, effectively stimulated PAI-1 induction whereas puromycin was ineffective. Although TGF-beta1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF-beta1, prior to serum stimulation, resulted in a 10- to 12-fold increase in PAI-1 expression coincident with exit out of G(0). These data support a model in which PAI-1 gene expression is closely associated with creation of the growth-activated state and that cell cycle controls appear to be superimposed on the time course of the serum-induced expression of the PAI-1 gene.

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Urokinase‐mediated posttranscriptional regulation of urokinase‐receptor expression in non small cell lung carcinoma

Montuori

Mattiello

Mancini

et al. 2003

Intl Journal of Cancer

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The urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in the proteolytic cascade required for tumor cell dissemination and metastasis, and are highly expressed in many human tumors. We have recently reported that uPA, independently of its enzymatic activity, is able to increase the expression of its own receptor in uPAR-transfected kidney cells at a posttranscriptional level. In fact, uPA, upon binding uPAR, modulates the activity and/or the level of a mRNA-stabilizing factor that binds the coding region of uPAR-mRNA. We now investigate the relevance of uPA-mediated posttranscriptional regulation of uPAR expression in non small cell lung carcinoma (NSCLC), in which the up-regulation of uPAR expression is a prognostic marker. We show that uPA is able to increase uPAR expression, both at protein and mRNA levels, in primary cell cultures obtained from tumor and adjacent normal lung tissues of patients affected by NSCLC, thus suggesting that the enzyme can exert its effect in lung cells. We investigated the relationship among the levels of uPA, uPAR and uPARmRNA binding protein(s) in NSCLC. Lung tissue analysis of 35 NSCLC patients shows an increase of both uPA and uPAR in tumor tissues, as compared to adjacent normal tissues, in 27 patients (77%); 19 of these 27 patients also show a parallel increase of the level and/or binding activity of a cellular protein capable of binding the coding region of uPAR-mRNA. Therefore, in tumor tissues, a strong correlation is observed among these 3 parameters, uPA, uPAR and the level and/or the activity of a uPAR-mRNA binding protein. We then suggest that uPA regulates uPAR expression in NSCLC at a posttranscriptional level by increasing uPAR-stability through a cellular factor that binds the coding region of uPAR-mRNA.

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Cyclic Nucleotide Regulation of PAI-1 mRNA Stability

Cited by 42 publications

References 44 publications

Identification and cDNA Cloning of a Novel RNA-binding Protein That Interacts with the Cyclic Nucleotide-responsive Sequence in the Type-1 Plasminogen Activator Inhibitor mRNA

Identification and cDNA Cloning of a Novel RNA-binding Protein That Interacts with the Cyclic Nucleotide-responsive Sequence in the Type-1 Plasminogen Activator Inhibitor mRNA

Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-?1

Urokinase‐mediated posttranscriptional regulation of urokinase‐receptor expression in non small cell lung carcinoma

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