Inherent Instability of Plasminogen Activator Inhibitor Type 2 mRNA Is Regulated by Tristetraprolin*

Hong YuDagger, Stan StasinopoulosDagger, Peter Leedman§, and Robert L. Medcalf

From the Department of Medicine, Monash University, Box Hill Hospital, Box Hill 3128, Victoria Australia and the § Laboratory for Cancer Medicine, Centre for Medical Research, School of Medicine and Pharmacology, Royal Perth Hospital Unit, Western Australian Institute for Medical Research, the University of Western Australia, Perth 6000 Western Australia, Australia

Received for publication, December 20, 2002, and in revised form, February 6, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasminogen activator inhibitor 2 (PAI-2)1 is a member of the plasminogen activator family of proteins and controls the activity of urokinase plasminogen activator (u-PA) in the extracellular compartment (1). PAI-2 is also a member of the OV-serpin family of serine protease inhibitors and has been considered as the most enigmatic member of this group of proteins (2). One of the most striking features of PAI-2 is that it exists in two molecular forms: a predominant non-glycosylated protein and a less abundant secreted glycosylated protein. The predominant intracellular location of PAI-2 has suggested additional functions for this u-PA inhibitor, and growing evidence has indicated an involvement for PAI-2 in the intracellular events associated with differentiation (3), proliferation (4, 5), signal transduction (6) and possibly apoptosis (7, 8). PAI-2 has also generated interest because of its impressive regulatory profile. PAI-2 gene transcription rates are markedly increased in response to the tumor promoter phorbol 12-myristate 13-acetate (PMA) (9), the phosphatase inhibitor, okadaic acid (10), tumor necrosis factor-alpha (TNF) (11, 12) and lipopolysaccharide (LPS) (13).

Notwithstanding the important contribution of transcriptional control (14, 15) to the regulation of PAI-2 expression, significant regulation also occurs at the post-transcriptional level, most notably at the level of PAI-2 mRNA stability (16, 17). Post-transcriptional regulation of gene expression has proven to be a particularly important component in the global control of gene regulation. It is now well established that the 3'-untranslated region (3'-UTR) of mRNA is critical in the decision-making process of transcript longevity (18). Most studies have focused on conserved AU-rich elements within the 3'-UTR of unstable cytokine or proto-oncogene transcripts. Typical elements are mostly of the AUUUA or UUAUUUAUU type or variants thereof (19, 20), and are mRNA destabilizing motifs that act, in part, by their ability to attract specialized RNA-binding proteins to elicit the decay process. These same elements have also been linked to other aspects of mRNA metabolism and processing including RNA trafficking (21) and translation (22), indicating that a given AU-rich element has pleiotropic effects.

A number of AU-rich element-binding proteins have been identified and shown to interact with these elements in many of these unstable transcripts including AUF-1 (23), AUH (24), the ELAV protein family members (25), and tristetraprolin (TTP) (26). AUF-1 for example, engages AU-rich elements in a variety of these genes, and the affinity of interaction correlates with the ability of these sequences to serve as mRNA instability elements (27). Members of the ELAV family including HuD, HuC, and HuR (HuA) can also interact with these same elements, but at least for HuR, this interaction is associated more with stabilization rather than destabilization of mRNA (28-30). Arguably the most powerful mRNA destabilizing protein identified is TTP. This protein is a critical regulator of TNF-alpha and GM-CSF mRNA instability (26, 31, 32). This has been decisively born out in TTP -/- mice, which develop a severe inflammatory phenotype because of the increase in cytokine expression resulting from the loss of negative regulation at the post-transcriptional level (26). TTP is therefore clearly a mRNA destabilizing protein that acts at least in part by promoting deadenylation and by attracting the exosome to the transcript for rapid decay (33).

Previous studies have addressed the mechanisms underlying the post-transcriptional regulation of the PAI-2 gene. Two functional mRNA instability elements have been identified within the PAI-2 transcript, one within the 3'-UTR of PAI-2 mRNA (16, 17), and the other within exon 4 of the coding region (34). Closer analysis of the 3'-UTR region of PAI-2 mRNA led to the identification of a nonameric AU-rich element (UUAUUUAUU) (ARE), 304-nt upstream from the poly(A) tail, as a mRNA destabilizing determinant (16). This element was subsequently shown to provide a binding site for a number of cytoplasmic and nuclear proteins (17), one of which was HuR, although the role of HuR in the post-transcriptional control of PAI-2 remains to be determined. To identify and characterize additional proteins that assemble on the PAI-2 ARE, a human leukocyte cDNA library was screened for PAI-2 AU-rich element-binding proteins using the yeast three-hybrid approach. This screen identified tristetraprolin (TTP) as a specific PAI-2 ARE-binding protein that associates with the PAI-2 AU-rich element and participates in the PAI-2 mRNA decay process. Since TTP is widely considered as a post-transcriptional regulator of cytokine gene expression in response to inflammatory signals our study suggests that the biological targets of TTP are not restricted to cytokines and implicates PAI-2 in a broader context of the inflammatory response associated with TTP.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Yeast Strains and Plasmid Constructs-- The reporter yeast strain L40-coat and the RNA bait plasmid vectors pIIIA/MS2-1 and pIIIA/MS2-2 (containing selection marker URA3, ADE2) (35, 36) were generous gifts from Dr. Marv Wickens of the University of Wisconsin. The TTP mammalian expression plasmid CMV.hTTPtag (31) (hereafter referred to as pTTP-HA) was provided by Dr. Paul Bohjanen (University of Minnesota). The wild-type PAI-2 expression plasmid pCI-PAI-2 contains the 1872-bp PAI-2 cDNA (9) inserted into the EcoR1 site of the expression plasmid pCl-neo (Promega, Madison, WI) (34).

The RNA bait plasmids were constructed as follows. The 5'-phosphorylated oligonucleotides of the ARE region (29 bp) of the human PAI-2 gene, were synthesized by Sigma Genosys: 5'-TTTTACTTTGTTATTTATTATTTTATATA-3' and 5'-TATATAAAATAATAAATAACAAAGTAAAA-3'. After annealing, the double-stranded ARE oligonucleotide was inserted into the SmaI site of the pIIIA/MS2-2 and pIIIA/MS2-1, creating ARE/MS2-2 and ARE/MS2-1, respectively (36). The insertion and orientation of the ARE in the plasmids was confirmed by PCR and sequence analysis (sequencing primer: 5'-CTGTCTCTATACTCCCCTATAG-3').

Construction of Globin/PAI-2 3'-UTR Constructs-- Plasmid pCMV-glo (hereafter referred to as pCI-glo), contains the rabbit beta -globin gene driven by the CMV promoter (37). Plasmid pCI-glo-PAI-2 is the same as pCI-glo, but contains the entire 3'-UTR of the PAI-2 mRNA inserted into the 3'-UTR of the globin reporter gene (16). Plasmid pCI-glo-ARE and pCI glo-2ARE are the same as pCI-glo, but contain either a single copy or two copies of the 29-nt PAI-2 ARE inserted into the 3'-UTR of the globin reporter gene, respectively. To prepare these plasmids, complementary oligonucleotides containing the single (29 nt) or tandem (58 nt) copy of the PAI-2 AU-rich element were synthesized. These oligonucleotides also included restriction sites for XhoI at each end to enable subcloning into the XhoI site of pCI-glo. Oligonucleotides were annealed, digested with XhoI, and inserted into the XhoI site of pCI-glo using standard techniques.

Screening cDNA Library Using the Yeast Three-hybrid System-- The yeast three-hybrid screening procedures were performed essentially as described elsewhere (35, 38, 39). The plasmids pIIIA/ARE-MS2-2 and pIIIA/ARE-MS2-1 were introduced into yeast L40-coat by transformation. A human leukocyte cell cDNA library (250 µg; Clontech) was transformed into L40/pIIIA/ARE-MS2-2 and L40/pIIIA/ARE-MS2-1. Double transformants (Trp+, Leu+, Ura+) were then plated onto Trp-, Leu-, His-, Ura- plates containing 5 mM 3-aminotriazole (3-AT).

As a negative selection procedure, candidate yeast clones were transferred onto plates containing 0.1% 5-fluoro-orotic acid (5-FOA) to eliminate the majority of RNA-independent false positive colonies (38). The same yeast clones were also transferred onto Trp-, Leu-, His- Ura- plates containing 5 mM 3-AT ("master plates"). The 5-FOA-sensitive colonies identified on the master plates were subsequently patched onto non-selective plates (Trp-, Leu-) to select for colonies in which the RNA plasmid had been removed ("RNA-dropout colonies"): After 3-4 days growth, single yeast colonies that had the RNA plasmid dropped out (pink color) were spotted back onto Leu- Trp- plates containing 0.1% 5-FOA to confirm that the RNA plasmid had been removed and were able to grow. In parallel, yeast were also transferred to Leu-, His- plates containing 5 mM 3-AT to retest for the RNA-dependent activation of HIS3.

In order to test candidate colonies for RNA sequence-specific activation of HIS3, the RNA dropout colonies expressing the putative RNA-binding protein cDNA, were retransformed with four different RNA plasmids: ARE/MS2-2, ARE/MS2-1, PAI-2 exon 4/MS2-2 as an unrelated RNA bait, and RNA bait vector pIIIA/MS2-2. After transformation, the single yeast colonies were picked and spotted onto Ura-, Trp-, Leu- plates to confirm the success of transformation and also onto Trp-, Ura-, Leu-, His- plates containing 5 mM 3-AT to test for RNA sequence-specific activation of HIS3. Positive colonies should grow only with RNA bait plasmids containing the PAI-2 ARE sequence, but not with baits containing irrelevant RNA sequence or empty RNA bait vector alone. cDNA plasmids from positive clones were isolated and subjected to DNA sequencing analysis (GAL4 AD sequencing primer: 5'-TACCACTACAATGGAT-3'). The cDNA sequences were then BLAST-searched for gene homology with the GenBankTM databases at NCBI.

Cell Culture and Gene Transfection-- HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, and 1× penicillin and streptomycin at 37 °C under 5% CO2. HEK293 cells were transiently co-transfected with the PAI-2 expression plasmid pCI-PAI-2 (17) and the TTP expression plasmid pTTP-HA, or globin/PAI-2 3'-UTR and TTP expression plasmids, by the calcium phosphate method (40), except that the transfection mixture was allowed to incubate with the cells for 16-24 h, and the glycerol shock was omitted (31). Fresh DMEM medium containing 10% heat-inactivated fetal calf serum was then added and cells maintained for a further 24 h. Total RNA was extracted for Northern blotting as described below.

Cross-linking of Protein to RNA and Immunoprecipitation-- Bluescript plasmids harboring the 29-nt PAI-2 AU-rich element (17) were used to transcribe the PAI-2 ARE RNA probes in vitro. Following linearization with XbaI, 1 µg of template was incubated for 2 h at 37 °C in the presence of 50 µCi of [alpha -32P]UTP (PerkinElmer Life Sciences), 10 µM UTP, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 20 units of RNase inhibitor (Promega) and 50 units of T7 RNA polymerase. RNA probes were purified on a 6% polyacrylamide-urea denaturing gel, eluted in a 500 mM NH4CH3COO, 1 mM EDTA solution for 6 h at room temperature, ethanol precipitated at -80 °C, and resuspended in water (500-1000 cps/µl) as previously described (16).

To prepare protein extracts for the UV cross-linking and immunoprecipitation, confluent cells were collected by trypsinization, washed three times with phosphate-buffered saline, then lysed for 5 min on ice in 100 µl/106 cells of cytoplasmic extraction buffer (CEB; 10 mM HEPES, pH 7.1, 3 mM MgCl2, 14 mM KCl, 0.2% Nonidet P-40, 5% glycerol, 1 mM dithiothreitol, 2 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 mM sodium orthovanadate, and 1 mM NaF). The nuclei were pelleted for 1 min at 1000 × g at 4 °C, and the supernatant containing the cytosolic fraction was aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. Nuclear protein extracts were prepared from isolated nuclei as previously described (41). Protein concentration of cell extracts was determined by the Bio-Rad protein dye reagent.

For the binding assays, 4 µg of protein extract were preincubated with 150 µg of heparin and 40 units of RNase inhibitor (RNasin; Promega) for 10 min at room temperature in CEB buffer before addition of the RNA probe (200 cps) in a total volume of 20 µl. After a further 30 min incubation at room temperature, samples were transferred to a 96-well microplate, which was placed on ice and irradiated for 15 min at a distance of 5 cm from a UV source (Ultra LUM model UVB-20). RNA not associated with protein was digested with 100 units of RNase T1 (Roche Molecular Biochemicals) for 20 min at room temperature. In some experiments the RNase T1-resistant RNA-protein complexes were further digested with 1 µg of RNase A (Roche Molecular Biochemicals) at 37 °C for 15 min.

The samples were diluted to 0.4 ml in CEB buffer, and protein A-Sepharose (Amersham Biosciences) added (total volume of 0.45 ml). These were incubated overnight at 4 °C in the presence of either preimmune rabbit serum (1:150 dilution) or crude polyclonal rabbit anti-HA (HA.11) antiserum (1:150 dilution) (BabCO, Richmond, CA). Immune complexes were recovered by centrifugation, washed three times with CEB buffer, resuspended in 50 µl of 2× SDS sample buffer, and subjected to SDS-PAGE on 10% acrylamide gels and autoradiography.

Northern Blotting-- Total RNA was purified from transfected cells as described by Chomczynski and Sacchi (42). 20-µg aliquots of RNA were electrophoresed through 1% agarose gels containing 20% formaldehyde and subsequently transferred to Hybond-N+ membranes (Amersham Biosciences). The filters were hybridized with 32P-labeled DNA probes as described (17). Membranes were processed by standard techniques and exposed to Kodak BioMax film (Eastman Kodak) at -80 °C with two intensifying screens.

The labeled cDNA probes used for hybridization were obtained as follows: 752 bp of the exon 1-6 PCR-amplified fragment of PAI-2 cDNA (9); the 1.1-kb HindIII TTP cDNA fragment of pTTP-HA (CMV.hTTPtag (31)); and the 380-bp HindIII/BamHI fragment of pCMV-glo containing the globin cDNA fragment (37).

Western Blotting-- Western blotting was performed as previously described (34). Cytoplasmic extracts (up to 50 µg) prepared from cells were separated by SDS-PAGE under reducing conditions and transferred to nylon membranes. Membranes were hybridized with antibodies directed against TTP (1:1000 dilution; kind gift from Dr. Andrew Clark) or PAI-2 (1:10,000 dilution; American Diagnostica). Secondary antibodies coupled to horseradish peroxidase were added (1:2,000 dilution) and the immunocomplexes assessed by enhanced chemiluminescence.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Three-hybrid Screening for PAI-2 AU-rich Element-binding Proteins-- The yeast three-hybrid approach was used to screen a human leukocyte cDNA library for proteins that interacted with the functional 29-nt AU-rich element within the 3'-UTR of PAI-2 mRNA. This genetic approach yielded seven positive clones that interacted with the PAI-2 AU-rich RNA bait in both an RNA-dependent and RNA sequence-dependent manner. Sequence analysis revealed that three of seven clones were identical, and all clones contained partial cDNA sequences (1399-1436 bp) that were homologous to human TTP (Fig. 1).


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Fig. 1.   Schematic description of human TTP cDNA clones identified by yeast three-hybrid screening. Schematic description of the TTP cDNA showing the relative positions of the two zinc finger domains and proline repeats. Five clones homologous to TTP were obtained following a yeast three-hybrid screen for PAI-2 AU-rich-binding proteins. The 5'-end points of the five TTP cDNA clones obtained following this screen are indicated.

The 5'-end point of the clones ended between -310 and -354 of the TTP cDNA. All clones had the identical 3' structure, with the exception of one clone (clone 5) that contained a 43-bp deletion at the terminus of the 3'-UTR (Fig. 1). TTP contains 326 amino acids with three tetraprolin repeats and two zinc fingers of the 2 Cys-Cys-Cys-His class. All clones contained the two zinc finger domains and two of the three tetraprolin domains suggesting the functional importance of these domains for PAI-2 AU-rich RNA binding activity.

TTP Interacts with the PAI-2 AU-rich Element in Vitro-- To confirm that TTP could directly interact with the PAI-2 ARE, RNA probes containing a single copy of the PAI-2 AU-rich element were incubated with cytoplasmic extracts prepared from either mock-transfected HEK293 cells, or cells transiently transfected with 5 µg of plasmid pTTP-HA. Samples were UV cross-linked then immunoprecipitated with anti-HA antibodies or preimmune serum, and subjected to SDS-PAGE (Fig. 2, lanes 1-4). Replicate samples were digested with RNase T1 either alone (lanes 5-8) or in combination with RNase A (lanes 9-12) prior to immunoprecipitation. As shown, extracts prepared from cells transfected with 5.0 µg of pTTP-HA did not produce an immunoprecipitable complex in the presence of preimmune serum (lane 2). However, protein-RNA complexes were detected when samples were immunoprecipitated with the anti-HA antibodies (lane 4). Following RNase T1 digestion, two complexes of ~120 and 55 kDa still remained (lane 8). When samples were digested with both RNase T1 and RNase A only a single complex of molecular mass 52 kDa was present (lane 12), although the 120-kDa complex was still present after longer exposure of gel to x-ray film. The 120-kDa complex most likely represents TTP associating with another protein and is more sensitive to RNase A possibly due to conformational changes. The slight reduction in molecular mass of the HA-containing RNA complex from 55 to 52 kDa is consistent with the further trimming of the RNA probe from the HA-tagged TTP protein by RNase A. Extracts derived from mock-transfected cells did not contain any HA-containing immune complexes. Taken together, these data indicate that TTP expressed in cells recognizes the AU-rich element in the PAI-2 3'-UTR.


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Fig. 2.   TTP binds to the PAI-2 ARE element. Cytoplasmic extracts were prepared from mock-transfected HEK293 cells or cells transiently transfected with 5 µg of plasmid pTTP-HA. Extracts (4 ug) were incubated with 32P-labeled RNA probes containing a single copy of the PAI-2 AU-rich element and subjected to UV cross-linking. Replicate samples were digested with RNase-T1 either alone (lanes 5-8) or in combination with RNase A (lanes 9-12). All samples were then immunoprecipitated with either rabbit preimmune serum (PI) or with rabbit anti-HA antibodies (HA) in the presence of protein-A Sepharose beads and subjected to SDS-PAGE. Gels were dried and autoradiographed. The open arrow to the right of the figure shows the HA-tagged TTP-RNA complex resistant to both RNase T1 and RNase A digestion.

Overexpression of TTP Destabilizes PAI-2 mRNA in HEK293 Cells-- It was important to determine whether TTP could influence the post-transcriptional regulation of the PAI-2 gene. TTP has previously been shown to influence cytokine gene expression by associating with AU-rich elements and accelerating the decay rate of the cytokine transcript. To determine whether TTP could act in a similar manner on PAI-2 mRNA, plasmids expressing PAI-2 (pCI-PAI-2) and TTP (pTTP-HA) were co-transfected into HEK293 cells. In this experiment, cells were transfected with a fixed amount of pCI-PAI-2 (5 µg) and increasing amounts (0, 0.005, 0.01, 0.05, 0.1, 1.0, 5.0 µg) of plasmid pTTP-HA.

As shown in Fig. 3 panel A, cells transfected with pCI-PAI-2 expressed a PAI-2 transcript (lane 2, middle blot). However, the intensity of this transcript decreased as the expression levels of TTP increased (lanes 3-9). It is also evident that levels of PAI-2 mRNA were significantly reduced at relatively low levels of TTP mRNA expression (e.g. lane 6). Furthermore, higher concentrations of TTP resulted in the formation of a stable but truncated PAI-2 transcript (lanes 7-9; indicated with arrow). The significance of the formation of this truncated PAI-2 transcript is unclear, but it is unlikely to be of physiological significance since this occurs only at levels of very high TTP levels. It is interesting to point out that high concentrations of TTP have also been shown to produce a stable truncated transcripts of TNF, GM-CSF, and IL-3 (43).


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Fig. 3.   Overexpression of TTP destabilizes PAI-2 mRNA and reduces levels of PAI-2 protein in HEK293 cells. HEK293 cells were transiently transfected with a constant amount of the PAI-2 expression plasmid pCI-PAI-2, together with increasing amounts (0-5.0 µg) of the TTP expression plasmid, pTTP-HA (pTTP). After a 24-h transfection, RNA was extracted and probed for PAI-2 and TTP mRNA by Northern blotting (panel A). Cytoplasmic proteins were extracted from a replicate series of transfected cells, and PAI-2 and TTP protein levels were assessed by Western blotting (panel B). As shown in panel A, expression of PAI-2 mRNA decreased with an increasing amount of TTP mRNA. This was also reflected at the protein level (panel B) with PAI-2 protein being reduced to undetectable levels with as little as 0.1 µg of plasmid pTTP-HA (lane 4). The lower panel in A represents the ethidium bromide-stained gel showing the relative levels of ribosomal RNA (rRNA). The lower panel in B is a Coomassie Blue-stained SDS-PAGE gel showing the equal loading of protein in all lanes.

TTP Expression Correlates with Reduced Levels of PAI-2 Protein-- To confirm that the reduced levels of PAI-2 mRNA following TTP expression correlated with a reduction in the synthesis of PAI-2 protein, a similar experiment was performed as described in Fig. 3 (panel A), and total cellular protein extracted and assessed for PAI-2 and TTP protein by SDS-PAGE and Western blotting. As shown in panel B, increasing levels of TTP protein correlated with decreasing levels of PAI-2 protein. It is also important to note that the levels of expressed TTP that conferred a marked reduction in PAI-2 protein occurred at levels of TTP that were below the detection limit of the Western blot assay (lane 4 of panel B). This suggests that PAI-2 mRNA is exquisitely sensitive to TTP; only a relative low concentration of TTP is sufficient to impart significant instability to PAI-2 mRNA and loss of PAI-2 protein biosynthesis.

The PAI-2 3'-UTR Confers TTP-mediated Instability to a Reporter Transcript-- To determine whether TTP regulates PAI-2 mRNA stability via the PAI-2 3'-UTR, we determined the ability of the full-length PAI-2 3'-UTR to destabilize a globin reporter transcript in a TTP-dependent manner. HEK293 cells were transfected with a constant amount (5 µg) of pCI-glo-PAI-2 and increasing amounts of pTTP-HA (0-5 µg). As shown in Fig. 4, panel A, the intensity of chimeric globin-PAI-2 mRNA levels decreased as the level of TTP increased in cells. In this experiment, TTP was more effective at destabilizing the chimeric globin-PAI-2 transcript than PAI-2 mRNA itself, since globin-PAI-2 mRNA levels were already substantially reduced in cells transfected with 0.01 µg of pTTP. This was observed in at least three independent experiments and suggests that stability elements outside the PAI-2 3'-UTR can modulate the destabilizing effect of TTP on PAI-2 mRNA. Also, akin to the results presented in Fig. 3, panel A, higher concentrations of TTP resulted in the accumulation of a truncated but stable globin-PAI-2 mRNA intermediate (indicated by arrow to the right of Fig. 3). The effect of TTP on the stability of the chimeric transcript was due to the presence of PAI-2 3'-UTR sequences, because TTP had no effect on globin transcripts devoid of the PAI-2 3'-UTR (panel B).


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Fig. 4.   The PAI-2 3'-UTR confers TTP-mediated mRNA instability to beta -globin mRNA. HEK293 cells were transiently co-transfected with a constant amount of expression plasmid pCI-Glo-PAI-2 containing the PAI-2 3'-UTR fused to the globin reporter transcript, together with increasing amounts (0-5.0 µg) of the TTP expression plasmid, pTTP-HA (pTTP). After a 24-h transfection, RNA was extracted and probed for globin and TTP mRNA by Northern blotting. As shown in panel A increasing levels of TTP mRNA correlated with a decrease in the levels of the globin reporter transcript (panel A). The arrow to the right of panel A indicates the position of the stable truncated globin-PAI-2 mRNA transcript formed in the presence of high levels of TTP. Similar transient co-transfection experiments performed using the wild-type globin expression plasmid with increasing amounts of pTTP-HA did not reduce globin mRNA levels nor did it result in the formation of a truncated transcript (panel B).

The PAI-2 AU-rich Element Independently Confers TTP-dependent mRNA Instability-- To determine whether the PAI-2 AU-rich element could independently confer TTP-mediated mRNA instability, HEK293 cells were transiently co-transfected with plasmids expressing the globin transcript alone or containing either a single or tandem copies of the 29-nt PAI-2 AU-rich element, together with increasing amounts of pTTP-HA. As seen in Fig. 5, globin transcripts devoid of additional 3' sequences were not destabilized by increasing levels of TTP (lanes 1-5). In contrast, globin transcripts containing the single copy of the PAI-2 AU-rich element were destabilized by TTP in a concentration-dependent manner (lanes 7-11). It is also apparent that the ability of the AU-rich element to confer TTP-dependent destabilization was less efficient than seen with the full-length PAI-2 3'-UTR (compare with Fig. 4, panel A). Interestingly, globin transcripts harboring two copies of the PAI-2 AU-rich element conferred TTP-dependent mRNA destabilization more effectively than the single copy AU-rich element (compare lanes 7-11 with lanes 13-17). These results indicate that the ARE within PAI-2 3'-UTR partially conveys the mRNA destabilizing function of TTP. However, since the single copy AU-rich element was not as effective as the full-length PAI-2 3'-UTR, it appears likely that complete TTP effect requires engagement with additional elements within the PAI-2 3'-UTR to implement the decay process.


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Fig. 5.   A single copy of the PAI-2 AU-rich element partially confers TTP-mediated destabilization to beta -globin. HEK293 cells were transiently co-transfected with a constant amount (5 µg) of expression plasmid pCI-Glo (lanes 1-5) or pCI-Glo-ARE (lanes 7-11) or pCI-Glo-2ARE (lanes 13-17) together with increasing amounts of plasmid, pTTP-HA (pTTP; 0-5.0 µg) as indicated in the figure. After 24 h, RNA was extracted and probed for globin and TTP mRNA by Northern blotting. As shown, a single copy of the PAI-2 AU-rich element was capable of conferring TTP-dependent instability to the globin reporter transcript (compare lanes 1-5 with 7-11). Two copies of the AU-rich element (2ARE) were even more effective than the single copy (lanes 13-17) at destabilizing the globin reporter transcript.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PAI-2 is subject to post-transcriptional regulation, and a significant component of this regulation relies on a functional AU-rich element within the 3'-UTR. In this study, we used the yeast three-hybrid approach to identify tristetraprolin as a PAI-2 AU-rich element-binding protein. TTP, also known as TIS11, G0S24, Nup475 (44-48), is the product of the immediate-early response genes Zfp-36 in mouse and ZFP36 in humans. TTP has previously been described as an mRNA destabilizing protein that associates with AU-rich elements within the 3'-UTR of cytokines, most notably TNF, GM-CSF, and IL-3 (26, 31, 43) and promotes deadenylation subsequent to mRNA degradation. TTP contains two zinc finger domains that have been shown to be necessary for TTP to interact with AU-rich elements in cytokine transcripts. Each of the five distinct TTP cDNA clones that were found to recognize the PAI-2 AU-rich element by the yeast three-hybrid approach were incomplete clones, but all contained both zinc finger domains. This is consistent with the possibility that the two zinc finger domains within the TTP molecule are also necessary for TTP to recognize the PAI-2 AU-rich element. One of the TTP clones was found to contain a 43-bp deletion at the 3'-end of the transcript. Whether this TTP variant defines a new closely related member of the TTP family of proteins requires further investigation.

UV cross-linking and immunoprecipitation assays demonstrated that TTP expressed in HEK293 cells formed an RNase T1- and RNase A-resistant 52-kDa complex with a single copy of the PAI-2 ARE. The size of the complex was consistent with that of the HA-tagged TTP protein coupled to the partly digested RNA probe. TTP was active in the turnover of PAI-2 mRNA decay since co-transfection of HEK293 cells with a PAI-2 expression plasmid with increasing amounts of a TTP expression plasmid resulted in destabilization of the PAI-2 transcript in a TTP-dependent manner. Furthermore, the effect of TTP was concentration-dependent, since the degree of PAI-2 instability correlated with an increase in the expression of TTP. In fact, PAI-2 mRNA levels were particularly sensitive to TTP since PAI-2 mRNA levels were substantially reduced in cells transfected with as little as 0.1 µg of pTTP-HA. Destabilization of PAI-2 mRNA by TTP was also accompanied with a concomitant reduction in the level of PAI-2 protein as assessed by Western blotting and in these experiments, PAI-2 protein was essentially abolished when cells were co-transfected with 0.1 µg of pTTP-HA. The 3'-UTR of PAI-2 was also shown to be necessary and sufficient to convey TTP-mediated instability while the PAI-2 AU-rich element in isolation was able to partially confer TTP-dependent destabilization to the globin reporter gene. The fact that the AU-rich element alone was less effective than the full-length PAI-2 3'-UTR at conferring TTP-dependent mRNA destabilization suggests that TTP requires additional elements within the PAI-2 3'-UTR to implement the decay process. Nonetheless, this study clearly identifies TTP as a potent post-transcriptional regulator of PAI-2 gene expression.

Curiously, overexpression of TTP resulted in the accumulation of a truncated PAI-2 transcript that appeared to be relatively stable. The significance of this is presently unclear, although overexpression of TTP as well as other members of this family of mRNA-binding proteins has been shown to produce stable truncated variants of TNF, GM-CSF, and IL-3 (43). This truncation has been previously mapped to occur within the TNF AU-rich element (43). As judged by the size of the truncated PAI-2 transcript, it also likely that the truncation is missing sequences 3' of the PAI-2 AU-rich region. This was further supported from Northern blot data using probes specific for regions immediately 5' or 3' of the AU-rich element, because only the 5'-probe hybridized with the truncated transcript (data not shown). We also suspect that this truncated transcript is unlikely to be of physiological significance since this effect was only seen in the presence of very high levels of TTP, levels unlikely to be achieved in vivo. Furthermore, truncations of these transcripts, including PAI-2, have not been reported in any physiological circumstance and as such are unlikely to be generated under natural conditions. It has been proposed that the formation of these stable truncated intermediates under high concentrations of TTP is a consequence of a sequestration of mRNA decay proteins by TTP, or due to excessive loading of TTP onto the transcript thereby preventing the exosome-mediated decay process (31).

Mice with a targeted deletion of the TTP gene display a complex inflammatory syndrome (26) consisting of inflammatory arthritis, dermatitis, cachexia, autoimmunity, and myeloid hyperplasia. TTP -/- animals were found to have high plasma levels of TNF protein while administration of anti-TNF antibodies largely reversed the phenotype, indicating that the inflammation was largely a consequence of the increase in the plasma levels of TNF. These findings therefore identified TTP as a component of a negative feedback loop that interferes with TNF production by destabilization of its mRNA. Since our study implicates TTP in the post-transcriptional regulation of PAI-2 gene expression, it would be predicted that endogenous PAI-2 levels would also be elevated in TTP -/- due to the loss of negative regulation, although any increase in PAI-2 expression that may be seen in these animals would need to be dissociated from the possible effects of TNF, since this cytokine itself is a powerful inducer of PAI-2 expression.

One of the long standing questions in the control of post-transcriptional gene expression is how transcripts that contain similar functional AU-rich elements and also provide binding sites for a common collection of RNA-binding proteins, still display a distinct pattern of regulation. In some cases, other regulatory domains have been shown exist within these transcripts that might act to temper the effect of the powerful AU-rich regulatory domains. On the other hand, although the same RNA-binding proteins can engage these transcripts, it is possible that the means by which these proteins modulate specific transcripts may differ for example, in the requirement for ancillary cofactors or specific phosphorylation events. The means by which TTP elicits its destabilizing effect on PAI-2 is unclear although it is likely that some of the requirements seen in the regulation of TNF by TTP would be similar for PAI-2. Nonetheless the fact remains that the time course of PAI-2 mRNA and protein accumulation is vastly different from that seen for TNF, and hence PAI-2 mRNA must contain additional regulatory domains (see below) or attracts other proteins to counteract the destabilizing effect of TTP in vivo.

We have identified TTP as an important regulator of PAI-2 gene expression, thereby extending the involvement of TTP beyond that of cytokine regulation. This therefore raises the possibility that PAI-2 may be an integral part of the inflammatory response since PAI-2 appears to be regulated by the same agents that regulate TNF. Our previous studies on the role of the AU-rich element within the PAI-2 3'-UTR led to the identification of the mRNA stabilizing protein HuR as a prominent PAI-2 AU-rich element-binding protein (17). Although there is a lack of direct evidence to implicate HuR in the stabilization of PAI-2 mRNA, it is tempting to speculate that HuR and TTP act in an opposing manner to maintain steady state levels of PAI-2 mRNA. Such a scenario has been postulated for the regulation of the GM-CSF transcript by HuR and TTP (49). Although clearly implicated in the regulation of PAI-2 gene expression, these proteins may not necessarily function in isolation, since a number of other unidentified proteins also associate with the PAI-2 AU-rich element and are likely to influence the destabilizing/stabilizing actions of TTP and HuR. This picture is further complicated by the fact that PAI-2 mRNA turnover is also influenced by instability elements residing within exon 4 of the PAI-2 coding region. A 28-nt sequence within exon 4 provides a binding site for an unidentified protein of 52 kDa. The role of this protein in the control of PAI-2 mRNA stability and its possible association with proteins associating with the PAI-2 AU-rich element is unknown, but it would not be surprising if some interaction occurs between PAI-2 mRNA-binding proteins to these instability elements.

The PAI-2 transcript is inherently unstable but the degree of instability can be modulated by agents that either increase (e.g. phorbol esters or Dioxin) or decrease (e.g. glucocorticoids) PAI-2 expression. It would be interesting to determine the role of TTP during modulation of PAI-2 mRNA stability by these agents and also to determine the effect of silencing of TTP expression (for example by siRNA) on the rate of PAI-2 mRNA accumulation under steady-state and regulated conditions.

Our previous REMSA studies using HT-1080 cells have shown that a number of cellular factors recognize the PAI-2 AU-rich element and during PMA+TNF treatment a transient increase in the binding activity of one particular mRNA- binding protein is observed (34). The time course of this transient increase in binding activity parallels the accumulation of TTP mRNA in the same cells (data not shown) raising the possibility that the transiently induced PAI-2 mRNA-binding protein is TTP itself. However, we have been unable to convincingly demonstrate that TTP is indeed the protein in question because of the limitation of the anti-TTP antibodies presently available.

In summary, we have identified TTP as a post-transcriptional regulator of PAI-2 gene expression that associates with the PAI-2 AU-rich element and implements the decay process. It would be interesting to determine whether disruption of TTP expression in cells changes the induction profile of the PAI-2 gene, thereby establishing a direct physiological role for TTP in PAI-2 gene expression in vivo. Further studies are required to determine the mechanisms by which TTP implements PAI-2 mRNA decay, and if there is any difference in the mechanism by which TTP orchestrates the decay of its target transcripts.

    ACKNOWLEDGEMENT

We thank Dr. Andrew Clark for supplying the anti-TTP antibodies.

    FOOTNOTES

* This study was supported by grants obtained from the National Health and Medical Research Council of Australia (to R. L. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

To whom correspondence should be addressed: Monash University Dept. of Medicine, Box Hill Hospital, Box Hill 3128 Victoria, Australia. Tel.: 61-3-9895-0318; Fax: 61-3-9895-0332; E-mail: Robert.Medcalf@med.monash.edu.au.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M213027200

    ABBREVIATIONS

The abbreviations used are: PAI-2, plasminogen activator inhibitor type 2; TNF, tumor necrosis factor; IL, interleukin; ARE, AU-rich motif; UTR, untranslated region; HA, hemagglutinin; TTP, tristetraprolin; HEK, human embryonic kidney cells; GM-CSF, granulocyte-macrophage colony-stimulating factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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