Sustained EC activation may adversely contribute to the pathogenesis of both acute and chronic inflammatory disease states. Herein, we provide evidence that miR-181b is dynamically regulated in response to proinflammatory stimuli and functions to suppress the expression of an enriched set of NF-κB target genes associated with inflammatory disease states, such as adhesion molecules (e.g., VCAM-1, E-selectin), chemokines and chemokine receptors (e.g., CCL1, CCL7, CX3CL1, CXCL1, CCR2), and other key inflammatory mediators (e.g., COX-2, PAI-1). In support, using complementary gain- and loss-of-function approaches, we demonstrated that miR-181b inhibits downstream NF-κB signaling by directly targeting importin-α3 in vitro and in vivo, an effect that inhibits nuclear accumulation of p50 and p65, and reduced expression of adhesion molecules, leukocyte accumulation, and vascular inflammation in vivo. Importantly, systemic delivery of miR-181b inhibited activation of the NF-κB pathway and reduced mortality of endotoxemic mice by reducing NF-κB nuclear translocation. Moreover, systemic overexpression of a dominant-negative IκBα completely blocked the inhibitory effect of miR-181b on LPS-induced NF-κB activation in the lungs. In light of these findings, these studies identify miR-181b as a novel regulator of EC activation and downstream NF-κB signaling in vitro and in vivo.
ECs perform multiple functions that are critical to vascular homeostasis, including controlling leukocyte trafficking, regulating vessel wall permeability, and maintaining blood fluidity. The recruitment of leukocytes and extravasation into the blood vessel wall are essential early events in normal inflammatory responses and related disease states (9
). Our findings that miR-181b can potently inhibit adhesion molecules, chemokines, and other NF-κB–responsive mediators suggest that it may serve to dampen the early stages of vascular inflammation. Furthermore, miR-181b–mediated inhibition of NF-κB targets was observed for several major physiologic proinflammatory mediators, such as TNF-α and LPS (Figures , , , and ). Remarkably, miR-181b expression was also rapidly reduced in response to these stimuli in vitro and in vivo, an effect that may allow for enhanced NF-κB signaling. Consistent with this hypothesis, use of miR-181b inhibitors increased (a) leukocyte adhesion to stimulated EC monolayers; (b) leukocyte accumulation and lung injury scores; (c) NF-κB activity in lung lysates; and (d) expression of NF-κB target genes (VCAM-1
) in vivo (Figure F and Figure ). The effect of miR-181b was determined to be specific for the NF-κB signaling pathway, as the majority of the TNF-α–inducible genes examined were inhibited by miR-181b and were “phenocopied” by overexpression of a dominant-negative IκBα (Figure ). Moreover, interrogation of the entire set of over 800 miR-181b–reduced genes identified by microarray analysis revealed 6 biological signaling pathways associated with NF-κB activation. In addition, miR-181b had no inhibitory effect on phosphorylation of the MAPK downstream mediators, ERK, p38, and JNK (Supplemental Figure 3B). Interestingly, in the presence of LPS, systemic delivery of miR-181b mimics failed to inhibit NF-κB–regulated targets COX-2 and IL-1β and NF-κB activity in PBMCs (Supplemental Figure 9). These findings indicate that the maintenance of miR-181b expression in the vascular endothelium is a key feature in controlling NF-κB signaling events and vascular inflammation.
miR-181b belongs to the miR-181 family, which consists of 4 members, miR-181a, miR-181b, miR-181c, and miR-181d. The biological functions of this miRNA family were first identified when miR-181a was recognized as a contributor to hematopoietic lineage commitment and differentiation (72
). Later studies revealed that increased miR-181a activity in primary embryonic lymphatic ECs resulted in substantially reduced levels of Prox1
mRNA and protein and, consequently, regulated vascular development and neo-lymphangiogenesis (74
). miR-181b was defined as a regulator of the B cell primary antibody repertoire based upon its ability to restrict the activity of activation-induced cytidine deaminase (75
). Altered expression levels of both miR-181a and miR-181b have been detected in multiple tumors and leukemia/lymphoma (76
), raising the intriguing possibility that dysregulation of NF-κB signaling in these cancers may be associated with defective miR-181 expression and/or function. Indeed, reduced expression of miR-181b has been observed in several primary human cancers, including astrocytic tumors, gastric, lung, and prostate cancer, glioma cells, acute myeloid leukemia, and chronic lymphocytic leukemia (76
), an effect associated with a poor prognosis in several cancer subtypes (77
). In contrast, miR-181b has been found overexpressed in some cancers (87
). For example, a recent study identified CYLD as a target of miR-181b-1 in an Src-transformed mammary epithelial cell line, an effect that potentiated NF-κB expression (88
). A number of important differences exist between this study and ours, including the following: (a) cell type–specific differences — transformed (MCF-10A) cells versus primary ECs; (b) use of different stimuli — Src-oncoprotein increased versus cytokine/LPS decreased miR-181b expression; and (c) cell type–specific effects on CYLD
gene expression — we observed no significant effect of miR-181b on CYLD expression in HUVECs (Supplemental Figure 10). The use of miR-181b, as well as other microRNAs, for therapeutic purposes will require careful scrutiny as this nascent field progresses. Finally, members of the miR-181 family may have nonredundant functions, as was suggested by evidence of one study in which miR-181a, but not miR-181c, promoted CD4 and CD8 double-positive T cell development when ectopically expressed in thymic progenitor cells (90
). In the present study, we found that miR-181b is the dominant miR-181 family member expressed in ECs and is capable of potently suppressing EC activation by targeting importin-α3 in vitro and in vivo.
The role of miRNAs in ECs related to inflammatory and immune responses has only recently begun to emerge. miR-126, an EC-enriched miRNA, was found to regulate endothelial expression of VCAM-1 by directly targeting its 3′ UTR (91
). A more recent microarray-based study showed that miR-31 and miR-17-3p are inducible by TNF-α and target the 3′ UTRs of E-selectin and ICAM-1, respectively, to reduce their expression (35
). The above 2 studies suggest miRNAs can reduce proinflammatory gene expression in ECs by targeting the 3′ UTR of these genes. Recently, an antiinflammatory role was demonstrated for miR-10a in ECs (34
). The expression of endothelial miR-10a was lower in the athero-susceptible regions of the aortic arch than in the nearby regions of the descending thoracic aorta (34
). miR-10a was reported to inhibit NF-κB–mediated EC activation in vitro by reducing the expression of mitogen-activated kinase kinase kinase 7 and β-transducin repeat-containing gene, 2 key regulators of TNF-α degradation; furthermore, an inverse association was observed between the expression of miR-10a and mitogen-activated kinase kinase kinase 7, β-transducin repeat-containing gene, and nuclear p65 (34
). Thus, in response to inflammatory stimuli, the expression of several miRNAs would be expected to be differentially regulated in a spatial and temporal manner to fine-tune the output of the NF-κB signaling pathway via multiple upstream, downstream, and feedback regulatory mechanisms. While some miRNAs have been reported to directly alter the expression of other miRNAs in pathophysiological states, we found no effect of systemically delivered miR-181b in vivo on the expression of any of the miRNAs implicated in affecting NF-κB activation, including the following: miR-10a (targets mitogen-activated kinase kinase kinase 7 [MAP3K7; TAK1] and β-transducin repeat-containing gene [β-TRC]), miR-146a (targets TNF receptor-associated factor 6 [TRAF6] and IL-1 receptor-associated kinase 1 [IRAK1]), miR-155 (targets IKKβ and IKKε), miR-31 (targets E-selectin), and miR-17-3p (targets ICAM-1) (Supplemental Figure 8). Considering these findings, we have identified an unexpected role for miR-181b in its unique ability to inhibit downstream NF-κB nuclear translocation via targeting importin-α3, an effect that is distinct from that of other miRs that have been implicated in regulating upstream NF-κB signals.
Importins are a family of proteins involved in nuclear translocation. We demonstrate by a combination of experimental approaches, including bioinformatics, 3′ UTR reporter assays, miRNP-IP, and in vivo expression and functional analyses, that importin-α3 is necessary and sufficient to mediate miR-181b’s inhibitory effect on NF-κB and NF-κB–responsive gene expression in response to proinflammatory stimuli. Indeed, overexpression of importin-α3 (lacking its 3′ UTR) effectively rescued miR-181b–mediated inhibition of NF-κB–induced activity in vitro and in vivo (Figure F and Supplemental Figure 5I). Conversely, siRNA knockdown of importin-α3 “phenocopied” miR-181b’s inhibitory effects on NF-κB activity and targets (VCAM-1 and E-selectin) in vitro and in vivo (Supplemental Figure 5, A–G). Importin-α3 has been previously implicated in regulating nuclear translocation of p50 and p65 in other cell systems; for example, alteration of importin-α3 expression is the mechanism by which prohibitin reduces NF-κB activation in intestinal epithelial cells (92
). Because we did not observe any effect of miR-181b on IKK complex proteins or IκBα phosphorylation (Figure , C–E, and Supplemental Figure 3A), miR-181b–mediated inhibitory effects are not likely to be associated with these upstream events. Indeed, multiple prediction databases did not reveal miR-181b–binding sites in the 3′ UTRs of IκBα, IKKα, IKKβ, or IKKγ. In addition, while miR-181b inhibits the expression of upstream receptors implicated in NF-κB signaling, such as TRAF1, TNFRSF11B, and IL1R1 (Figure ), we found that miR-181b could not inhibit the 3′ UTRs for these genes (Supplemental Figure 4D). Indeed, these genes are known to be NF-κB responsive (93
Accumulating studies have implicated circulating miRNAs in acute inflammatory diseases such as sepsis, a life-threatening condition associated with poor outcome. We identified patients with sepsis or sepsis plus sepsis/ARDS as having reduced circulating plasma levels for miR-181b compared with control patients admitted to the ICU without sepsis (Figure ). Furthermore, after adjusting for the APACHE II score (a measure of severity of disease at presentation to the ICU), miR-181b was independently associated with sepsis or sepsis plus sepsis/ARDS. These findings coupled with the improved survival observed in miR-181b “rescue” studies in septic mice (Figure I) suggest that therapies directed at restoring miR-181b expression may ameliorate this acute inflammatory process. To date, 3 other miRNAs have been identified in plasma as potential biomarkers for sepsis patients. miR-150, initially identified from leukocyte profiling, was also reduced in plasma of sepsis patients and correlated with increasing severity of Sequential Organ Failure Assessment (SOFA) score (96
). Circulating levels of miR-146a and miR-223 were also found reduced in sepsis patients compared with patients with SIRS or healthy controls (97
). Collectively, these findings indicate that miRNAs correlate with varying levels of severity of sepsis for patients admitted to the ICU.
In summary, we have identified miR-181b as a cytokine-responsive miRNA that regulates the expression of key NF-κB–regulated genes involved in the endothelial response to inflammation in vitro and in vivo by regulating the NF-κB signaling pathway. These findings also revealed an unexpected mechanistic role for this miRNA in targeting downstream NF-κB signaling by directly targeting importin-α3. These studies support the possibility that miR-181b may serve as an important “inflamiR” by controlling critical aspects of EC homeostasis under physiologic or pathologic conditions. Strategies aimed at restoring miR-181b expression may provide a novel therapeutic approach to limiting acute inflammatory disease states.