Differential regulation of interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-2 by microRNA-146a and NF-kappaB in stressed human astroglial cells and in Alzheimer disease

Abstract

Specific microRNAs (miRNAs), small non-coding RNAs that support homeostatic gene expression, are significantly altered in abundance in human neurological disorders. In monocytes, increased expression of an NF-κB-regulated miRNA-146a down-regulates expression of the interleukin-1 receptor-associated kinase-1 (IRAK-1), an essential component of Toll-like/IL-1 receptor signaling. Here we extend those observations to the hippocampus and neocortex of Alzheimer disease (AD) brain and to stressed human astroglial (HAG) cells in primary culture. In 66 control and AD samples we note a significant up-regulation of miRNA-146a coupled to down-regulation of IRAK-1 and a compensatory up-regulation of IRAK-2. Using miRNA-146a-, IRAK-1-, or IRAK-2 promoter-luciferase reporter constructs, we observe decreases in IRAK-1 and increases in miRNA-146a and IRAK-2 expression in interleukin-1β (IL-1β) and amyloid-β-42 (Aβ42) peptide-stressed HAG cells. NF-κB-mediated transcriptional control of human IRAK-2 was localized to between -119 and +12 bp of the immediate IRAK-2 promoter. The NF-κB inhibitors curcumin, pyrrolidine dithiocarbamate or CAY10512 abrogated both IRAK-2 and miRNA-146a expression, whereas IRAK-1 was up-regulated. Incubation of a protected antisense miRNA-146a was found to inhibit miRNA-146a and restore IRAK-1, whereas IRAK-2 remained unaffected. These data suggest a significantly independent regulation of IRAK-1 and IRAK-2 in AD and in IL-1β+Aβ42 peptide-stressed HAG cells and that an inducible, NF-κB-sensitive, miRNA-146a-mediated down-regulation of IRAK-1 coupled to an NF-κB-induced up-regulation of IRAK-2 expression drives an extensively sustained inflammatory response. The interactive signaling of NF-κB and miRNA-146a further illustrate interplay between inducible transcription factors and pro-inflammatory miRNAs that regulate brain IRAK expression. The combinatorial use of NF-κB inhibitors with miRNA-146a or antisense miRNA-146a may have potential as a bi-pronged therapeutic strategy directed against IRAK-2-driven pathogenic signaling.

FIGURE 1.

miRNA-146a up-regulation in AD superior temporal lobe neocortex compared with short PMI age-matched control brains. A, using fluorescent miRNA array and Northern dot-blot analysis, miRNA-146a showed consistent increases in short PMI AD superior temporal lobe neocortex (n = 36) when compared with age- and area-matched controls (n = 30). A related brain-enriched miRNA-132 showed no such increases. All values shown are relative to a control 5 S RNA within the same sample. All AD cases were approximately mid-stage AD (clinical dementia rating 1.5; see Fig. 1C). Clinical data of control and AD brains are shown in Table 1. For ease of comparison, a horizontal dashed line at 1.0 indicates the mean of miRNA-146a in control neocortex. B, miRNA-146a averaged a greater than 2.6-fold increase in the neocortical samples examined. Previous studies have shown comparable increases in AD affected hippocampal CA1 (29). For ease of comparison a horizontal dashed line at 1.0 indicates control miRNA-132 levels; *, p < 0.01 (ANOVA). C, miRNA-146a showed a graded increase in AD-affected superior temporal lobe neocortex as the clinical dementia rating (clinical dementia rating; an index of disease severity (37) advanced). D, these changes were confined to the hippocampus (HIPP) and neocortex (NCTX) of AD brains, areas targeted by AD neuropathology; control areas such as the brain stem (BST), cerebellum (CBM), and thalamus (THA) within the same brains showed no such elevations; a horizontal dashed line is included at 1.0 for ease of comparison. For D, n = 3–5 determinations from n = 16 brains; significance over control: *, p < 0.05; p < 0.01 (ANOVA).

FIGURE 2.

IRAK-2 is up-regulated in AD brain. A, shown is Western protein analysis of levels of β-actin, IRAK-1, IRAK-2, and IRAK-4 in 5 control (CON) and 5 select AD superior temporal lobe neocortex, age-matched with PMIs of <2.1 h selected from brains described in Table 1. B, quantified levels from A in the bar graph format are shown; note the inverse relationship between IRAK-1 and IRAK-2 in control and AD brains, which could be in part due to containment of an NF-κB binding site in the human IRAK-2 but not the human IRAK-1 immediate promoter (Fig. 3). No significant changes were observed in the levels of IRAK-4 or IRAK-M between control and AD brains (data not shown); a horizontal dashed line at 1.0 is included for ease of comparison. C, in comparison to stressed HNG primary cells, stressed HAG primary cells exhibit a greater up-regulation of miRNA-146a, perhaps due to their nature as an immune-responsive cell type (33, 34). Neuronal cells are stained with neuron-specific β-tubulin (red; λ = 690 nm), glial cells are stained with glial-specific glial fibrillary acidic protein (GFAP; green; λ = 525 nm), and nuclei are stained with Hoechst 33258 (blue; λ = 470 nm). Magnification, 20×. A horizontal dashed line at 1.0 corresponds to control levels of miRNA-132 in HNG cells; *, p < 0.05; **, p < 0.01. D, relative IRAK-1, IRAK-2, and miRNA-146a abundance in control and IL-1β+Aβ42-stressed HAG cells is shown. A horizontal dashed line at 1.0 corresponds to control levels of IRAK-1, IRAK-2, and miRNA-146a; n = 3–6; significance over control: *, p < 0.05; p < 0.01 (ANOVA).

FIGURE 3.

DNA sequence structure of the human IRAK-1, human IRAK-2 gene immediate promoters, and a highly stable miRNA-146a-IRAK-1 mRNA-3′-UTR interaction. A and B, the human IRAK-2 promoter contains a single NF-κB binding consensus sequence from −111 to −102 bp of the IRAK-2 promoter. This feature is missing from the immediate IRAK-1 promoter. Further studies showed that the IRAK-2, but not the IRAK-1 gene, is under NF-κB transcriptional control (Fig. 4). XhoI and BglII restriction sites (underlined) were used for ligation into pGL3 vectors (see text). The bent arrow at +1 indicates the start of transcription. AP2α, NF-κB, and Sp1 binding sites are bold and highlighted in yellow. C, the sequence of the 22 nucleotide miRNA-146a (highlighted in red) shows highly specific complementarity to the human IRAK-1 mRNA 3′-UTR (the target sequence is highlighted in yellow). 14 of 22 base pairs of the 5′ end of miRNA-146a align. The structural stability of the 22 nucleotide oligomer is −29.1 kcal/mol. This feature is absent from the IRAK-2 mRNA 3′-UTR (data not shown). Expression data suggest that the IRAK-1 mRNA 3′-UTR, but not the IRAK-2 mRNA, is regulated by miRNA-146a (28).

FIGURE 4.

Differential activation NF-κB and Sp1 and miRNA-146a, IRAK-1, or IRAK-2 gene promoter-luciferase reporter activities in IL-1β+Aβ42-stressed HAG primary cells. A, the NF-κB p50/p65 complex is up-regulated in IL-1β+Aβ42-stressed HAG primary cells: p65 (upper arrowhead), p50 (middle arrowhead). Sp1 activation is unchanged after any treatment condition (lower arrowhead). B, activation of miRNA-146a luciferase reporter expression by IL-1β+Aβ42 and inhibition by CAY10512, curcumin, or PDTC is shown. C, differential activation of IRAK-1 and IRAK-2 gene promoter-luciferase reporters (pGL3-promoter-IRAK-1, pGL3-promoter-IRAK-2 constructs) by IL-1β+Aβ42 and inhibition by CAY10512, curcumin, or PDTC is shown. n = 4–5; significance over control: *, p < 0.05 (ANOVA).

FIGURE 5.

Modulation of IRAK-1, IRAK-2, and miRNA-146a expression in IL-1β+ Aβ42 peptide-stressed HAG cells; attenuation by AM-146a and PDTC. A, we provide evidence that a complex interplay of miRNA-146a and NF-κB signaling regulates IRAK-1 and IRAK-2 signaling in IL-1β+Aβ42-stressed HAG primary cells. In HAG cells, IRAK-1 mRNA, IRAK-2 mRNA, and miRNA-146a were found to be induced about 1.1-, 3.1-, and 4.0-fold over controls. The effects of AM-146a on up-regulating IRAK-1 and PDTC on down-regulating IRAK-2 suggest that these are, respectively, miRNA-146a- and NF-κB-regulated genes. Further evidence for this is reflected in the structure of their immediate gene promoters (Fig. 3) and from the pattern of IRAK-1 and IRAK-2 protein abundance compared with the levels of an internal β-actin control in the same cell sample (B); n = 4–5; significance over control: *, p < 0.05, p < 0.01 (ANOVA).

FIGURE 6.

Schematic illustration of the TLR/IL-1R-IRAK-NFκB connectivity and signaling in AD brain and in IL-1β+Aβ42 peptide-stressed HAG cells. IRAK-1 and IRAK-2 occupy important early control points in TLR/IL-1R-NF-κB signaling pathway (6–8, 43, 44). A membrane-spanning TIR transduces signals triggered by extracellular toxins, peptides, and inflammatory cytokines via MyD88, IRAK-1, and IRAK-2. These ultimately trigger NF-κB-mediated inflammatory gene expression, neuropathology, and an altered innate immune response. The TLR/IL-1R shown here is generic and may represent multiple types of the IL-1 receptor sensor whose identities in the human brain are not well understood (6, 8, 31, 44). The normally high abundance of IRAK-1 in control aging human brain and HAG cells (Fig. 2) and the IRAK-1-NF-κB circuit (dotted lines) appears to be virtually shut down by an NF-κB-mediated up-regulation of miRNA-146a (a negative regulator of IRAK-1) and is associated with a shift to strong up-regulation of an NF-κB-activated IRAK-2. IRAK-2 transcription appears to be highly cell-specific as 10-fold differences in the same IRAK-2 immediate promoter construct between A549 and THP-1 cells have been noted (45). The current data support previous observations that the IRAK-2-NF-κB signaling loop is strongly self-reinforcing. This may contribute to the sustained activation of NF-κB and its pathogenic consequences in the neurodegenerative process (7, 8, 44). Although MyD88, NEMO, IKKα/β, IRAK-4, TRAF6, and other adaptor proteins are known to contribute to homeostatic TIR-IRAK-NF-κB signaling, their interactive role in this pathogenic scheme is currently not known and warrants further study. We have found no change in MyD88, IRAK-4, IRAK-M, or TRAF6 abundance in either AD or stressed HAG cells (Fig. 2, A and B; data not shown), suggesting that IRAK-2 up-regulation alone may be of primary importance in pathogenic signaling. The combinatorial use of miRNA-146a/AM146a (Fig. 5) along with selective NF-κB inhibitors (heavy horizontal black bars) may have significant potential as an effective multi-target therapeutic strategy against pathogenic IRAK-2-mediated inflammatory signaling that drives neurodegenerative processes.