The results of the present study shed new light on the posttranscriptional regulation of inflammatory mediator production at the levels of mRNA turnover and translation in activated bronchial epithelial cells. Using a comprehensive microarray analysis of changes in mRNA expression between nonactivated and IL-4/TNF-α-activated human bronchial epithelial BEAS-2B cells, we found (Table ) the following: (i) that activated epithelial cells express high levels of mRNAs for several cytokines and chemokines, such as IL-1-β, IL-6, granulocyte-macrophage colony-stimulating factor, MCP-1, IL-8, and exotaxin-3; (ii) expression of adhesion molecules (such as C3, ICAM-1, and VCAM-1) that are important for recruitment of innate immune cells is also greatly upregulated in activated BEAS-2B cells; and (iii) several genes related to airway remodeling and fibrosis are significantly upregulated in activated cells, including genes for collagens, serine protease inhibitors, and superoxide dismutase 2 (Table ). It is worth noting that mRNAs encoding five different collagens (COL1A1, COL5A1, COL6A1, COL7A1, and COL8A1) are rather abundant in BEAS-2B cells, suggesting a direct and active role of bronchial epithelium in airway remodeling. Collectively, these observations demonstrate that BEAS-2B cells stimulated with IL-4/TNF-α represent a useful in vitro cellular model for investigating molecular and cellular mechanisms of mRNA decay and translational control in airway inflammation. Moreover, these findings support the view that bronchial epithelium is a major source of many key inflammatory and remodeling molecules (3
) and thereby play a pivotal role in the pathogenesis of airway diseases.
Kinetic experiments conducted in this study measuring MCP-1 and IL-8 mRNA decay and in a previous study measuring exotaxin-1 mRNA decay (2
) support a critical role of mRNA turnover in the upregulation of chemokine gene expression during airway inflammation and remodeling. Intriguingly, while the stability of mRNAs encoding two key chemokines, MCP-1 and IL-8, increases in BEAS-2B cells activated by IL-4 and TNF-α (Fig. ), little change in the destabilization function of their AREs was observed (Fig. ). This finding is of particular interest because downregulation of ARE-mediated mRNA decay was often considered to account for the increased mRNA stability of an ARE-containing mediator mRNA in response to the proinflammatory stimulation (27
). Our findings that alteration of ARE-mediated decay is not always responsible for changes in stability of an ARE-containing message support the notion that an ARE plays different roles in different cell types or under different physiological conditions (5
). Given the fact that RNA-destabilizing elements can be anywhere in a message (e.g., 5′ UTR, protein coding region, or 3′ UTRs) (10
), it would be interesting to identify mRNA decay pathways that are involved in the observed mRNA stabilization in activated BEAS-2B cells.
Several lines of evidence from this study demonstrate an enhancement of global translation rate in activated bronchial epithelial cells. These include the following: (i) increased [35
S]methionine and cysteine incorporation into nascent polypeptides (Fig. ); (ii) increased ribosomes in the polysome fractions (Fig. ); and (iii) increased levels of several proteins critical for elevating translation efficiency (e.g., eIF4E, eRF1, and PABP) (Fig. ). It is worth noting that eIF4E, which is also a 5′ cap-binding protein, is the least abundant eIF and limits formation of the translation initiation complex (16
). Moreover, eIF4E has been found to be a major target for translational control by extracellular stimuli, such as stress, cytokines, growth factors, and mitogens (25
). Thus, availability of eIF4E greatly affects the translation profile of the cytoplasmic mRNA pool and is tightly regulated. As chronic airway inflammation is often associated with wound healing, tissue remodeling, and fibrosis, it is also worth noting that administration of eIF4E mRNA has been shown to augment wound healing in a rat model (62
). The observation that the level of hyperphosphorylated 4E-BP1 significantly increased upon IL-4/TNF-α stimulation (Fig. ) is fully consistent with the notion that activation of BEAS-2B cells triggers release of eIF4E proteins from the eIF4E/4E-BP1 complexes, making them available to elevate translation initiation efficiency. These data reveal a new example of translational control via altering eIF4E/4E-BP1 interaction and thus provide new insight into the mechanism by which global translation is enhanced in activated BEAS-2B cells.
eRF1 and PABP levels were also found to increase in activated BEAS-2B cells (Fig. ). eRF1 associates with eRF3, a PABP-interacting protein, to form a complex that is required for efficient translation termination (33
). Recently, it was proposed that interaction between the eRF1-eRF3 complex and PABP/poly(A) not only enhances efficient translation termination but also promotes translation reinitiation, thereby increasing the translation rate (33
). Thus, by upregulating the expression of factors necessary for translation, activated BEAS-2B cells can accommodate the elevated levels of mRNAs induced by IL-4/TNF-α treatment.
Recently, miRNAs, a group of evolutionarily conserved and abundant class of small silencing RNAs, have emerged as important posttranscriptional regulators of gene expression across species and in multiple biological processes (21
). In this study, we tested three different miRNAs exhibiting moderate to high abundance in BEAS-2B cells (data not shown) and found that miRNA-mediated mRNA silencing is compromised in human bronchial epithelial BEAS-2B cells following IL-4/TNF-α treatment, leading to an increase in protein levels of their targets (Fig. ). These findings open up a new avenue for studying mechanisms by which the modulation of miRNA function is accomplished to help cells reset their protein profile in response to external stimuli. One possibility is that the increased number of translation initiation complexes (see above) overcomes the translation initiation repression by miRNAs (46
). This interpretation is consistent with an in vitro study showing that the addition of purified initiation complex eIF4F, consisting of eIF4E, eIF4G, and eIF4A, to an ascites extract rescued mRNA from miRNA-mediated translation repression in vitro (46
). Another intriguing possibility is that under the physiological condition of activated BEAS-2B cells, Ago2-miRNA complex may somehow turn into a translation activator that promotes translation of mRNA targets. This notion is consistent with the findings of two recent reports showing that under specific serum-starvation conditions of some mammalian tissue culture cells, miRNAs including endogenous let-7 and miR-396-3 miRNAs and a synthetic miRNA positively promote translation of reporter mRNAs bearing the cognate miRNA target sites (69
Another important and novel finding in this study is that P bodies, which are closely linked to mRNA decay and translation repression, are abundant both in nonstimulated human bronchial epithelial BEAS-2B cells (Fig. ) and in the normal mouse bronchial epithelium (Fig. ). Prolonged treatment of IL-4 and TNF-α caused a significant decrease of P bodies in BEAS-2B cells (Fig. ). A similar reduction in the P-body population was observed in bronchial epithelium from mice exhibiting allergic airway inflammation (Fig. ). These findings suggest that decreased mRNA turnover, upregulation of translation, and the loss of P bodies are general features of activated bronchial epithelial cells during allergic airway inflammation. It is also worth noting that the bronchial epithelium from normal mice typically has 30 to 50 P bodies per epithelial cell (Fig. ), which is much more than many cultured mammalian cells (e.g., see references 15
, and 51
) (Fig. ). These observations suggest that bronchial epithelium is primed to elicit a response to airborne allergens or other insults by releasing factors and mRNPs from P bodies to the cytoplasm, e.g., for a boost of translation. Along this line, it is worth noting that eIF4E, the translation initiation factor that plays a rate-limiting role in formation of the translation initiation complex (see above), can be stored in P bodies (1
). The observation of a reduced number of P bodies in activated bronchial epithelial cells raises a possibility that translation initiation is boosted by releasing eIF4E from P bodies to the cytoplasm.
While the exact function of P bodies in mRNA turnover and translational control remains controversial and a subject of intensive experimentation (17
), our findings support a physiological role of P bodies as a cellular reservoir that allows storage and release of translation factors and dormant mRNPs in the cytoplasm of bronchial airway epithelium. General mRNA decay activity and translational silencing pathways may well be active in normal bronchial epithelium to keep certain mRNAs (e.g., those coding for inflammatory and remodeling mediators) from being effectively translated. Upon activation of the epithelium with inflammatory cytokines such as IL-4 and TNF-α, a concerted reduction of P bodies along with a downregulation of pathways that produce nontranslatable mRNPs promotes production of mediators. To our knowledge, this study represents the first report of a physiologically relevant modulation of mammalian P bodies in vivo. It will be important to determine the mechanism governing the loss of P bodies following the stimulation of bronchial epithelial cells.
In summary, our data suggest that a combination of decreasing mRNA decay, upregulating global translation, and decreasing P-body abundance results in persistently elevated production of inflammatory mediators in bronchial epithelium (Fig. ). This may represent an important amplification pathway in the regulation of inflammatory and remodeling genes in the airway epithelium. Thus, the present study reveals a potential new pathogenic mechanism operating at the levels of mRNA decay and translation in bronchial epithelium to sustain the chronic allergic inflammation and remodeling seen in allergic airway diseases. To the best of our knowledge, this study is among the first to demonstrate coordinate regulation of multiple posttranscriptional pathways following stimulation by factors largely thought to be activators of transcriptional pathways.
FIG. 8. Coordinated changes in mRNA turnover, translation, and RNA P bodies in bronchial epithelial cells following inflammatory stimulation. Coordinate regulation of multiple posttranscriptional pathways following stimulation by proinflammatory cytokines represents (more ...)