Cystic Fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung arising from profound expression of inflammatory genes, including interleukin-8 (IL-8). We have previously reported that IL-8 mRNA is stabilized in CF lung epithelial cells, resulting in concomitant hyperexpression of IL-8 protein. However, the mechanistic link between mutations in CFTR and acquisition of the proinflammatory phenotype in the CF airway has remained elusive. We hypothesized that specific microRNAs (miRNAs) might mediate this linkage. To identify the potential link, we screened an miRNA library for differential expression in ⌬F508-CFTR and wild type CFTR lung epithelial cell lines. Of 22 differentially and significantly expressed miRNAs, we found that expression of miR-155 was more than 5-fold elevated in CF IB3-1 lung epithelial cells in culture, compared with control IB3-1/S9 cells. Clinically, miR-155 was also highly expressed in CF lung epithelial cells and circulating CF neutrophils biopsied from CF patients. We report here that high levels of miR-155 specifically reduced levels of SHIP1, thereby promoting PI3K/Akt activation. However, overexpressing SHIP1 or inhibition of PI3K in CF cells suppressed IL-8 expression. Finally, we found that phospho-Akt levels were elevated in CF lung epithelial cells and were specifically lowered by either antagomir-155 or elevated expression of SHIP1. We therefore suggest that elevated miR-155 contributes to the proinflammatory expression of IL-8 in CF lung epithelial cells by lowering SHIP1 expression and thereby activating the PI3K/Akt signaling pathway. These data suggest that miR-155 may play an important role in the activation of IL-8-dependent inflammation in CF.

MicroRNAs (miRNAs) are naturally occurring, highly conserved families of transcripts (*22 nucleotides in length) that are processed from larger hairpin precursors. miRNAs primarily regulate gene expression by promoting messenger RNA (mRNA) degradation or repressing mRNA translation. miRNAs have been shown to be important regulators of a variety of cellular processes involving development, differentiation, and signaling. Moreover, various human diseases, including cancer and immune dysfunction, are associated with aberrant expression of miRNAs. This review will focus on how the multifunctional miRNA, miR-155, regulates inflammatory diseases, including cancer and pulmonary disorders, and also how miR-155 expression and biogenesis are regulated. We will also provide examples of miR-155-regulated networks in coordination with other noncoding RNAs, including long noncoding RNAs as well as coding mRNAs acting as competing endogenous RNAs.

mRNAs encoding proinflammatory chemokines are regulated posttranscriptionally via adenine-uridine-rich sequences (AREs) located in the 3′ untranslated region of the message, which are recognized by sequence-specific RNA-binding proteins. One ARE binding protein, tristetraprolin (TTP), has been implicated in regulating the stability of several ARE-containing mRNAs, including those encoding TNF-α and GM-CSF. In the present report we examined the role of TTP in regulating the decay of the mouse chemokine KC (CXCL1) mRNA. Using tetR-regulated control of transcription in TTP-deficient HEK293 cells, KC mRNA half-life was markedly decreased in the presence of TTP. Deletion and site-specific mutagenesis were used to identify multiple AUUUA sequence determinants responsible for TTP sensitivity. Although a number of studies suggest that the destabilizing activity of TTP is subject to modulation in response to ligands of Toll/IL-1 family receptors, decay mediated by TTP in 293 cells was not sensitive to stimulation with IL-1α. Using primary macrophages from wild-type and TTP-deficient mice, KC mRNA instability was found to be highly dependent on TTP. Furthermore, LPS-mediated stabilization of KC mRNA is blocked by inhibition of the p38 MAPK in macrophages from wild-type but not TTP-deficient mice. These findings demonstrate that TTP is the predominant regulator of KC mRNA decay in mononuclear phagocytes acting via multiple 3′-untranslated region-localized AREs. Nevertheless, KC mRNA remains highly unstable in cells that do not express TTP, suggesting that additional determinants of instability and stimulus sensitivity may operate in cell populations where TTP is not expressed.

IL-10 has been reported to inhibit the expression of LPS-induced proinflammatory cytokines and chemokines by altering the rate of specific mRNA decay although the molecular target(s) for its action remain unknown. In the present study, using primary peritoneal exudate macrophages and a cell culture model in which a tetracycline-responsive promoter controls transcription of CXC ligand 1 (KC) mRNA, we demonstrate that LPS promotes a time-dependent increase in KC mRNA stability. Although IL-10 had no direct effect on mRNA decay, this treatment antagonized the stabilizing action of LPS. The mechanisms involved were further explored using a cell-free mRNA degradation system. A 5′-capped, polyadenylated in vitro transcript derived from the 3′-untranslated region of KC mRNA exhibited time-dependent decay in the presence of protein extracts prepared from untreated RAW264.7 macrophages. Extracts prepared from LPS-treated RAW264.7 cells had reduced decay activity and this change was antagonized if the cells were costimulated with IL-10. A substrate in which the AU-rich element motifs were mutated exhibited minimal decay that did not vary using extracts prepared from cells treated with LPS or LPS and IL-10. A nonadenylated RNA substrate was also degraded and that activity was diminished by LPS. In concert, these findings demonstrate that KC mRNA stability is regulated by LPS-induced alterations in activities that govern both deadenylation and degradation of the mRNA body. The effects of IL-10 on KC mRNA stability reflect antagonism of the response to LPS.

Cystic fibrosis (CF) is characterized by a massive proinflammatory phenotype in the lung, caused by mutations in the CFTR gene. IL-8 and other proinflammatory mediators are elevated in the CF airway, and the immediate mechanism may depend on disease-specific stabilization of IL-8 mRNA in CF lung epithelial cells. MAPK signaling pathways impact directly on IL-8 protein expression in CF cells, and we have hypothesized that the mechanism may also involve stabilization of the IL-8 mRNA. To test this hypothesis, we have examined the effects of pharmacological and molecular inhibitors of p38, and downstream MK2, ERK1/2, and JNK, on stability of IL-8 mRNA in CF lung epithelial cells. We previously showed that tristetraprolin (TTP) was constitutively low in CF and that raising TTP destabilized the IL-8 mRNA. We therefore also tested these effects on CF lung epithelial cells stably expressing TTP. TTP binds to AU-rich elements in the 3'-UTR of the IL-8 mRNA. We find that inhibition of p38 and ERK1/2 reduces the stability of IL-8 mRNA in parental CF cells. However, neither intervention further lowers TTP-dependent destabilization of IL-8 mRNA. By contrast, inhibition of the JNK-2 pathway has no effect on IL-8 mRNA stability in parental CF cell, but rather increases the stability of the message in cells expressing high levels of TTP. However, we find that inhibition of ERK1/2 or p38 leads to suppression of the effect of JNK-2 inhibition on IL-8 mRNA stability. These data thus lend support to our hypothesis that constitutive MAPK signaling and proteasomal activity might also contribute, along with aberrantly lower TTP, to the proinflammatory phenotype in CF lung epithelial cells by increasing IL-8 mRNA stability and IL-8 protein expression.

We have observed over expression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or post transcriptional regulation. UCSC genome browser analysis of PACS-1 mRNA revealed two 8 base target sequences at the 3’ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and northern blot studies showed reduced or loss of expression of the two miRNAs in cervical cancer cell lines and primary tumors indicating dysregulation of these two miRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response (DDR), S-phase cell cycle arrest and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, K-382-p53 acetylation and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or 449a or PACS-1 cDNA transfection led to the reversal of DDR and restoration of cell growth. Release of cells post 24_h serum starvation showed PACS-1 nuclear localization at G1-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response resulting in the development of chemo-resistant tumors.

Cystic fibrosis (CF) is due to mutations in the CFTR gene and is characterized by hypersecretion of the proinflammatory chemokine IL-8 into the airway lumen. Consequently, this induces the highly inflammatory cellular phenotype typical of CF. Our initial studies revealed that IL-8 mRNA is relatively stable in CF cells compared with those that had been repaired with [WT]CFTR (wild-type CFTR). Relevantly, the 3′-UTR of IL-8 mRNA contains AU-rich sequences (AREs) that have been shown to mediate posttranscriptional regulation of proinflammatory genes upon binding to ARE-binding proteins including Tristetraprolin (TTP). We therefore hypothesized that very low endogenous levels of TTP in CF cells might be responsible for the relative stability of IL-8 mRNA. As predicted, increased expression of TTP in CF cells resulted in reduced stability of IL-8 mRNA. An in vitro analysis of IL-8 mRNA stability in CF cells also revealed a TTP-induced enhancement of deadenylation causing reduction of IL-8 mRNA stability. We conclude that enhanced stability of IL-8 mRNA in TTP-deficient CF lung epithelial cells serve to drive the proinflammatory cellular phenotype in the CF lung.

Certain pro-inflammatory chemokine mRNAs containing adenine/uridine-rich sequence elements (AREs) in their 3 untranslated regions (3-UTRs) are known to exhibit constitutive instability and sensitivity to proinflammatory stimuli resulting in the stabilization of the message. Using tetR-regulated transcription we now show that the 3-UTR of the mouse CXCL1 (KC) mRNA contains at least two ARE motifs that are structurally and functionally distinct. A fragment of 77 nucleotides containing 4 clustered AUUUA pentamers located at the 5-end of the KC 3-UTR is only modestly unstable yet promotes markedly enhanced, post-transcriptional protein production in response to either interleukin-1␣ (IL-1␣) or lipopolysaccharide (LPS), suggesting translational regulation. In contrast, a fragment containing 3 isolated AUUUA pentamers corresponding to the residual 3 400 nucleotides of the KC 3-UTR confers both instability and is stabilized in response to IL-1␣. Although the clustered AUUUA pentamers in the upstream region are required for stimulus sensitivity, mutation of all three pentamers in the downstream region has little or no effect on either instability or stimulus sensitivity. The upstream region is comparably stabilized in response to either IL-1␣ or LPS, whereas the AUUUA-independent downstream determinant is differentially more sensitive to IL-1␣. Finally, using UV-induced RNA cross-linking, these functionally independent sequences exhibit different patterns of interaction with RNA-binding proteins. Collectively, these findings document the presence of multiple independent determinants of KC mRNA function and demonstrate that these operate via distinct mechanisms.