Airway fibrosis is a common complication of chronic asthma. This process is driven in part by the overproduction of TGF-β1 (5
) and other profibrotic mediators released by lung parenchyma as well as activated inflammatory cells (6
). Eos are a major contributor to the profibrotic milieu within the asthmatic lung (4
). Despite its importance, little is known regarding the mechanisms that underlie the production of TGF-β1 by activated Eos.
Here we document for the first time to our knowledge that Pin1 is a critical mediator of TGF-β1 mRNA stability and cytokine production in either in vivo or in vitro activated Eos. Pin1 regulates cytokine production at a posttranscriptional level by modulating the binding affinity, catabolism, and protein-protein interactions of multiple RNA-binding proteins, including AUF1, HuR, and TIA-1. Pin1 itself is controlled by PKC-α along with PP2A, which binds to and modulates PPIase activity in response to external stimulation. These observations have in vivo relevance, as Pin1 blockade or genetic ablation significantly reduced TGF-β1 production, collagen accumulation, and airway remodeling in animal models of asthma. Therefore, these findings are of considerable importance for understanding and possibly preventing fibrotic airway disease.
Using multiple pharmacological inhibitors and activators (Table ) and immunoprecipitation, we identified PKC-α and PP2A as probable upstream regulators of Pin1 in Eos. Inhibition of either PKC-α or PP2A mimicked the effect and kinetics of juglone on Pin1 isomerase activity (Figure E and Figure C) and TGF-β1 mRNA steady-state levels (Figure A, Figure A, and Figure B). Although we have only measured mRNA decay after juglone, the rapid and quantitatively similar reduction in TGF-β1 mRNA after PKC-α and PP2A inhibition suggests a similar mechanism (45
). In support of our findings, TGF-β1 mRNA levels were rapidly altered (15
) by PKC-α signaling in mesangial cells and T lymphocytes. Conventional PKCs (PKC-α, -β1, -βII, and -ζ) are highly expressed by Eos (46
), and PKC-α was heavily phosphorylated in BAL Eos after allergen challenge (Figure D). As juglone failed to alter PKC-α phosphorylation, our data suggest that Pin1 does not modulate PKC-α activity. However, as dephosphorylation of Ser16 within the WW domain activates Pin1 (21
), downstream phosphatases such as PP2A are likely participants in the regulation of Pin1. PP2A and PKC-α physically interact in mast cells (37
) and reciprocally modulate each other (47
). Direct PKC-α activation with 12(S)-HETE increased Pin1 PPIase activity and TGF-β1 mRNA expression, which was almost entirely suppressed by okadaic acid (Figure , F and G). In addition, the interaction between Pin1 and PP2A was increased by cell activation (Figure H). As PKC-α may control PP2A activity (47
), we propose that the sequential activation of coassociated PKC-α and PP2A modulates Pin1 isomerase activity in our system (50
). How PKC-α alters PP2A is unknown but presumably involves phosphorylation. Interestingly, phosphorylation of PP2A has been associated with decreased rather than increased phosphatase activity (51
TGF-β1 mRNA lacks multiple AREs, which are found in the 3′ UTRs of posttranscriptionally regulated cytokine mRNAs. The exosome is generally considered to be the site for ARE mRNA decay (52
). Our data demonstrate that cytokine mRNAs lacking multiple AREs decay at an intermediate rate and can be catabolized by the exosome as well. In addition, the observation that non–ARE-containing cytokine mRNAs can be regulated by AUF1 and HuR suggests considerable wobble in their target specificity. Previously we showed that Pin1 interacts with AUF1 and HuR in Eos and T cells and regulates the interaction of these AREBPs with GM-CSF mRNA (19
). Here, we extended those observations by demonstrating a similar interaction between Pin1, AUF1, and TGF-β1 mRNA (Figure ). AUF1 recognizes and binds multiple AU- and CU-rich regions as well as stem-loop structures (53
). Mutational analysis has shown that AUF1 bound with high affinity and specificity to UUAUUUUAU motifs in the 3′ UTR of phosphoenolpyruvate carboxykinase (PCK-6) mRNA (53
). Deletion of this sequence resulted in dramatically increased half-life (5-fold) of mutant transcripts. TGF-β1 mRNA contains a similar 3′ UTR sequence (CUAUUUUAU), which may be the binding site of AUF1. Similarly, computational analysis has identified 3 putative HuR motifs, 2 located in the coding region and 1 in the 3′ UTR of TGF-β1 mRNA (18
) (Figure ). Therefore, HuR and AUF1 may bind TGF-β1 mRNA on distinct and nonoverlapping sites. In support of this idea, we observed simultaneous RNA-binding protein interactions with TGF-β1 mRNA in cells treated with juglone (Figure E and Figure B). This is consistent with recent work showing the binding of both AUF1 and HuR to p21 and cyclin D1 mRNAs (42
). Many ARE-binding proteins contain potential isomerization sites. Of those, Pin1 directly interacts with AUF1 (19
) and indirectly via RNA with HuR (Figure C). Despite Ser-Pro sites, TIA-1 is not a ligand of Pin1 (Figure A). Therefore, we propose that only changes in AUF1 affinity are directly mediated by Pin1 isomerization. Activated Pin1, by isomerizing hyperphosphorylated AUF1 isoforms, likely decreases AUF1 binding affinity for TGF-β1 mRNA, which attenuates exosome-mediated decay (19
). As AUF1 controls the decay of many other mRNAs, this mechanism may apply to them as well. TIA-1 associates with TGF-β1 mRNA after exosome delivery, while HuR may act as a brake on this process, leading to the intermediate decay rate observed after Pin1 blockade. We have summarized these interactions in Figure .
Summary of the changes in TGF-β1 mRNA–AREBP interactions as a function of HA or HA plus juglone.
Within a few days of allergen challenge, Eos increase by 20- to 150-fold in both humans and animals (Table ) (54
). Depletion of Eos in humans with systemic steroids or anti–IL-5 or reduction of TGF-β1 expression in animal models markedly attenuated airway fibrosis (2
). These data strongly suggest that Eos are critically involved in airway remodeling and that targeted therapy could reduce this long-term sequela of asthma. Juglone-treated, challenged rats showed substantial reductions in airway Eos as well as their expression of TGF-β1. Downstream collagen gene expression and ECM deposition in airways was also largely suppressed in both acute and chronic asthma models. In the acute rat model, the infiltration of other immune cells was unaffected, suggesting a relatively selective effect on Eos. In vitro exposure to juglone or dominant-negative TAT-WW-Pin1 induced Eos apoptosis but had no effect on lymphocyte survival (43
). While the prodeath effects of low-dose juglone (0.1 μM) could be antagonized by exogenous GM-CSF, higher-dose juglone could not (19
). These data suggest that Pin1 is involved in both the production (19
) as well as downstream, prosurvival signaling of GM-CSF. Therefore, it is likely that attenuated Eos inflammation observed here after Pin1 blockade reflects enhanced apoptosis (29
). The combination of reduced airway Eos and attenuated TGF-β1 expression likely combine to reduce collagen production and airway remodeling in allergen-challenged animals. These data further support the concept that Eos are critical in airway remodeling and that the use of Pin1 inhibitors may be an important advance to delay or attenuate this process in chronic asthmatics.