IκB-ζ mRNA Research Articles

Tacrolimus Inhibits TNF-α/IL-17A-Produced pro-Inflammatory Effect on Human Keratinocytes by Regulating IκBζ.

Psoriasis is a chronic autoimmune disease that is predominantly mediated by T-lymphocytes and keratinocytes. Tacrolimus is T cell-targeted immunosuppression drug that has been widely used in topical therapy of psoriasis; however, the pharmacologic effect of tacrolimus on human keratinocytes has not been fully clarified. This study aimed to investigate the potential regulatory effect of tacrolimus on TNF-α/ IL-17A-costimulated human keratinocytes in the mimic psoriatic microenvironment. The cultured normal human keratinocytes (NHKs) were divided into the following groups: control, TNF-α/IL-17A, tacrolimus, and TNF-α/IL-17A + tacrolimus. Cultured cells and supernatant were collected after 24h, and then real-time quantitative PCR, western blot, and ELISA analysis were performed. Foreskin tissues were treated by using TNF-α, IL-17A, and tacrolimus 0.03% ointment and then cultured for 24h, and immunohistochemistry was performed. NHKs expressed significant IL-36γ, CCL-20, IL-1β, S100-A9, and CXCL-1 mRNA after TNF-α/IL-17A treatment. Tacrolimus significantly inhibited TNF-α/IL-17A-induced IL-36γ, CCL-20, IL-1β, and S100-A9 expression at gene level and IL-36γ and CCL-20 expression at protein level. We further discovered TNF-α/IL-17A induced significant IκBζ mRNA and protein expression in NHKs, which could be inhibited by tacrolimus. Tacrolimus can inhibit pro-inflammatory synergistic action of TNF-α/IL-17A on human keratinocytes by regulating IκBζ expression.

Dimethyl fumarate dampens IL-17-ACT1-TBK1 axis-mediated phosphorylation of Regnase-1 and suppresses IL-17–induced IκB-ζ expression

The signaling elicited by the cytokine interleukin-17A (IL-17) is important for antimicrobial defense responses, whereas excessive IL-17 production leads to autoimmune diseases such as psoriasis and multiple sclerosis. IL-17–induced stabilization of mRNAs has been recognized as a unique and important feature of IL-17 signaling. Previously, we demonstrated that IL-17 signaling protein ACT1 is required to counteract constitutive inhibitor of nuclear factor kappa B zeta (IκB-ζ) mRNA degradation by the ribonuclease Regnase-1. However, information about the mechanism of mRNA stabilization in IL-17–stimulated cells remains insufficient. In the present study, we aimed to clarify the mechanism in more detail and identify an agent that can inhibit IL-17–induced mRNA stabilization. Experiments using small interfering RNA and an inhibitor of TANK-binding kinase 1 (TBK1) revealed that TBK1 was required for IκB-ζ mRNA stabilization through Regnase-1 phosphorylation. Intriguingly, this TBK1-mediated phosphorylation of Regnase-1 was suppressed by the addition of dimethyl fumarate (DMF), an electrophilic small molecule that has been used to treat IL-17–related autoimmune diseases. Confocal microscopic observation of the cellular localization of ACT1 revealed that DMF treatment resulted in the disappearance of ACT1 nuclear dots and perinuclear accumulation of ACT1. These results suggested that DMF is a small molecule that compromises IL-17–induced activation of the ACT1-TBK1 pathway, thereby inhibiting IL-17–induced mRNA stabilization.

IκB-ζ Expression Requires Both TYK2/STAT3 Activity and IL-17-Regulated mRNA Stabilization.

Cytokine IL-17A (IL-17) acts on various cell types, including epidermal keratinocytes, and induces antimicrobial peptide and chemokine production to elicit antibacterial and antifungal defense responses. Excess IL-17 leads to inflammatory skin diseases such as psoriasis. The IκB family protein IκB-ζ mediates IL-17-induced responses. However, the mechanism controlling IκB-ζ expression in IL-17-stimulated cells remains elusive. In this study, we showed that JAK kinase TYK2 positively regulates IL-17-induced IκB-ζ expression. TYK2-deficient mice showed reduced inflammation and concomitant reduction of IκB-ζ mRNA compared with wild-type mice in imiquimod-induced skin inflammation. The analysis of the IκB-ζ promoter activity using human cell lines (HaCaT and HeLa) revealed that catalytic activity of TYK2 and its substrate transcription factor STAT3, but not IL-17, is required for IκB-ζ promoter activity. In contrast, IL-17-induced signaling, which did not activate STAT3, posttranscriptionally stabilized IκB-ζ mRNA via its 3'-untranslated region. IL-17 signaling protein ACT1 was required to counteract constitutive IκB-ζ mRNA degradation by RNase Regnase-1. These results suggested that transcriptional activation by TYK2-STAT3 pathway and mRNA stabilization by IL-17-mediated signals act separately from each other but complementarily to achieve IκB-ζ induction. Therefore, JAK/TYK2 inhibition might be of significance in regulation of IL-17-induced inflammatory reactions.

IκBζ is a key transcriptional regulator of IL-36–driven psoriasis-related gene expression in keratinocytes

Proinflammatory cytokine signaling in keratinocytes plays a crucial role in the pathogenesis of psoriasis, a skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes and infiltration of inflammatory cells. Although IL-17A and TNFα are effective therapeutic targets in psoriasis, IL-36 has recently emerged as a proinflammatory cytokine. However, little is known about IL-36 signaling and its downstream transcriptional responses. Here, we found that exposure of keratinocytes to IL-36 induced the expression of IκBζ, an atypical IκB member and a specific transcriptional regulator of selective NF-κB target genes. Induction of IκBζ by IL-36 was mediated by NF-κB and STAT3. In agreement, IL-36-mediated induction of IκBζ was found to be required for the expression of various psoriasis-related genes involved in inflammatory signaling, neutrophil chemotaxis, and leukocyte activation. Importantly, IκBζ-knockout mice were protected against IL-36-mediated dermatitis, accompanied by reduced proinflammatory gene expression, decreased immune cell infiltration, and a lack of keratinocyte hyperproliferation. Moreover, expression of IκBζ mRNA was highly up-regulated in biopsies of psoriasis patients where it coincided with IL36G levels. Thus our results uncover an important role for IκBζ in IL-36 signaling and validate IκBζ as an attractive target for psoriasis therapy.

IL-1-induced post-transcriptional mechanisms target overlapping translational silencing and destabilizing elements in IκBζ mRNA.

During further molecular analysis concerning the function of the translational silencing element (TSE) identified in our publication, we noticed that a plasmid different from the one intended had been used erroneously in one part of the study. The mistake led to the use of a plasmid that contained the TSE sequence in antisense instead of sense orientation, downstream of the luciferase coding sequence in the bicistronic vector employed for experiments in Fig. 8A. Therefore, the conclusions drawn from this experiment that the TSE does not silence cap-independent translation and may function in a spatially restricted fashion (see under “Abstract,” line 10; see under “Results,” p. 29173, right column) have no basis. The discussion of this point (see under “Discussion,” p. 29176, left column) is obsolete. We are very sorry for this error and apologize for any problems it may have caused. Other conclusions and results of the publication are not affected by it. Concerning the question addressed by the experiment in Fig. 8, our most recent experiments demonstrate that the TSE does indeed silence even IRES-dependent translation.

Screening of posttranscriptional regulatory molecules of IκB-ζ

Posttranscriptional regulatory elements in the 3′-untranslated region (UTR) of mRNAs play an important role in mRNA stabilization. Induction of IκB-ζ, a critical transcriptional regulator in the innate immune response, is mediated via specific mRNA stabilization by lipopolysaccharide (LPS) and interleukin (IL)-1β. It is known that the 3′-UTR of IκB-ζ, especially 165 nucleotides after the stop codon, plays a crucial role in mRNA stability. Herein, we show that AU-rich elements and miRNA targets in these 165 3′-UTR nucleotides are dispensable for stability of IκB-ζ mRNA. Additionally, NF-κB activation is important for IκB-ζ transcription, but dispensable for IκB-ζ mRNA stability. Interestingly, high-throughput screening results show that MyD88, a signal molecule responsive to LPS/IL-1β stimulation, is key for stabilizing IκB-ζ mRNA expression. Moreover, MyD88-deficient macrophages exhibited a decreased half-life of IκB-ζ mRNA expression. These results indicate that the LPS/IL-1β-MyD88 axis plays a crucial role for stabilization of IκB-ζ mRNA.

Effects of flavonoids on senescence-associated secretory phenotype formation from bleomycin-induced senescence in BJ fibroblasts

During senescence, cells express molecules called senescence-associated secretory phenotype (SASP), including growth factors, proinflammatory cytokines, chemokines, and proteases. The SASP induces a chronic low-grade inflammation adjacent to cells and tissues, leading to degenerative diseases. The anti-inflammatory activity of flavonoids was investigated on SASP expression in senescent fibroblasts. Effects of flavonoids on SASP expression such as IL-1α, IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2 and MMP-3 and signaling molecules were examined in bleomycin-induced senescent BJ cells. In vivo activity of apigenin on SASP suppression was identified in the kidney of aged rats. Among the five naturally-occurring flavonoids initially tested, apigenin and kaempferol strongly inhibited the expression of SASP. These flavonoids inhibited NF-κB p65 activity via the IRAK1/IκBα signaling pathway and expression of IκBζ. Blocking IκBζ expression especially reduced the expression of SASP. A structure-activity relationship study using some synthetic flavones demonstrated that hydroxyl substitutions at C-2′,3′,4′,5 and 7 were important in inhibiting SASP production. Finally, these results were verified by results showing that the oral administration of apigenin significantly reduced elevated levels of SASP and IκBζ mRNA in the kidneys of aged rats. This study is the first to show that certain flavonoids are inhibitors of SASP production, partially related to NF-κB p65 and IκBζ signaling pathway, and may effectively protect or alleviate chronic low-grade inflammation in degenerative diseases such as cardiovascular diseases and late-stage cancer.

Transcriptional and post-transcriptional regulation of IκB-ζ upon engagement of the BCR, TLRs and FcγR

IκB-ζ is a nuclear IκB protein robustly induced in macrophages and fibroblasts upon TLR or IL-1R stimulation. IκB-ζ associates with NF-κB in the cell nucleus and is essential for the induction of a subset of secondary response genes represented by IL-6. Here, we analyzed induction of IκB-ζ in mouse B cells and found that IκB-ζ is induced by BCR or TLR stimulation. Similar to TLR stimulation, BCR stimulation elicited NF-κB-mediated transcriptional activation and mRNA stabilization of IκB-ζ via a cis-element in IκB-ζ mRNA. Proteasome inhibitors inhibited transcriptional activation but not post-transcriptional activation, indicating independency of the two signals. Co-stimulation of the BCR and TLR9 or TLR7, but not TLR2/1, synergistically induced IκB-ζ. Co-engagement of inhibitory Fcγ receptor suppressed BCR-mediated IκB-ζ expression but not that induced by TLR stimulation alone or co-stimulation of TLR and the BCR. The PI3K inhibitor LY294002 inhibited BCR-mediated, but not TLR-mediated, induction of IκB-ζ, consistent with the role of PI3K in BCR signaling and its suppression by FcγR. Analysis of IκB-ζ-deficient B cells demonstrated that IκB-ζ was essential upon stimulation of BCR or TLR for the expression of several genes including IL-10 and CTLA4. IκB-ζ-deficient B cells exhibited impaired proliferation and enhanced up-regulation of CD86 following stimulation of TLR9, but not the BCR, indicating critical roles for IκB-ζ in TLR signaling in B cells. Strict regulatory mechanisms for the induction of IκB-ζ via multiple pathways and its essential function upon stimulation indicate that IκB-ζ plays an important role in B cells.

Interleukin-17 (IL-17) and IL-1 Activate Translation of Overlapping Sets of mRNAs, Including That of the Negative Regulator of Inflammation, MCPIP1

Changes in gene expression during inflammation are in part caused by post-transcriptional mechanisms. A transcriptome-wide screen for changes in ribosome occupancy indicated that the inflammatory cytokine IL-17 activates translation of a group of mRNAs that overlaps partially with those affected similarly by IL-1. Included are mRNAs of IκBζ and of MCPIP1, important regulators of the quality and course of immune and inflammatory responses. Evidence for increased ribosome association of these mRNAs was also obtained in LPS-activated RAW264.7 macrophages and human peripheral blood mononuclear cells. Like IL-1, IL-17 activated translation of IκBζ mRNA by counteracting the function of a translational silencing element in its 3'-UTR defined previously. Translational silencing of MCPIP1 mRNA in unstimulated cells resulted from the combined suppressive activities of its 5'-UTR, which contains upstream open reading frames, and of its 3'-UTR, which silences independently of the 5'-UTR. Only the silencing function of the 3'-UTR was counteracted by IL-17 as well as by IL-1. Translational silencing by the 3'-UTR was dependent on a putative stem-loop-forming region previously associated with rapid degradation of the mRNA. The results suggest that translational control exerted by IL-1 and IL-17 plays an important role in the coordination of an inflammatory reaction.

Identification of interleukin‐1 receptor‐associated kinase 1 as a critical component that induces post‐transcriptional activation of IκB‐ζ

IκB-ζ, an essential inflammatory regulator, is specifically induced by Toll-like receptor ligands or interleukin (IL)-1β by post-transcriptional activation mediated via a 165-nucleotide element in IκB-ζ mRNA. Here, we analyzed the Toll-like receptor-IL-1 receptor signaling components involved in the post-transcriptional regulation of IκB-ζ with mutated estrogen receptor [ER(T2)] fusion proteins. Upon 4-hydroxytamoxifen treatment, the ER(T2) fusion proteins with IL-1 receptor-associated kinase (IRAK)1 and IRAK4 elicited specific activation of a reporter gene for the post-transcriptional regulation of IκB-ζ. The tumor necrosis factor receptor-associated factor (TRAF)6-ER(T2) protein activated nuclear factor-κB, but not post-transcriptional regulation, indicating that activation of IRAK1/4, but not of TRAF6, is sufficient to activate the 165-nucleotide element-mediated post-transcriptional mechanism. Interestingly, the post-transcriptional mechanism was not activated in TRAF6-deficient cells, indicating an essential role for TRAF6. Thus, the signaling pathway leading to nuclear factor-κB activation and the post-transcriptional activation bifurcates at IRAK1, suggesting a new pathway activated by IRAK1.

IL-1-induced Post-transcriptional Mechanisms Target Overlapping Translational Silencing and Destabilizing Elements in IκBζ mRNA*[S

The inflammatory cytokine IL-1 induces profound changes in gene expression. This is contributed in part by activating translation of a distinct set of mRNAs, including IκBζ, as indicated by genome-wide analysis of changes in ribosomal occupancy in IL-1α-treated HeLa cells. Polysome profiling of IκBζ mRNA and reporter mRNAs carrying its 3' UTR indicated poor translation in unstimulated cells. 3' UTR-mediated translational silencing was confirmed by suppression of luciferase activity. Translational silencing was unaffected by replacing the poly(A) tail with a histone stem-loop, but lost under conditions of cap-independent internal initiation. IL-1 treatment of the cells caused profound shifts of endogenous and reporter mRNAs to polysome fractions and relieved suppression of luciferase activity. IL-1 also inhibited rapid mRNA degradation. Both translational activation and mRNA stabilization involved IRAK1 and -2 but occurred independently of the p38 MAPK pathway, which is known to target certain other post-transcriptional mechanisms. The translational silencing RNA element contains the destabilizing element but requires additional 5' sequences and is impaired by mutations that leave destabilization unaffected. These differences in function are associated with differential changes in protein binding in vitro. Thus, rapid degradation occurs independently of the translational silencing effect. The results provide evidence for a novel mode of post-transcriptional control by IL-1, which impinges on the time course and pattern of IL-1-induced gene expression.

Induction of Neutrophil Gelatinase-associated Lipocalin Expression by Co-stimulation with Interleukin-17 and Tumor Necrosis Factor-α Is Controlled by IκB-ζ but neither by C/EBP-β nor C/EBP-δ

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding antimicrobial protein that is up-regulated in epithelial tissues during inflammation. We demonstrated previously that the gene encoding NGAL (LCN2) is strongly up-regulated by interleukin (IL)-1β in an NF-κB-dependent manner but not by tumor necrosis factor (TNF)-α, another potent activator of NF-κB. This is due to an IL-1β-specific synthesis of the NF-κB-binding co-factor IκB-ζ, which is essential for NGAL induction. We demonstrate here that NGAL is strongly induced by stimulation with TNF-α in the presence of IL-17, a pro-inflammatory cytokine produced by the newly discovered subset of CD4+ T helper cells, TH-17. In contrast to the murine NGAL orthologue, 24p3/lipocalin 2, we found no requirement for C/EBP-β or C/EBP-δ for NGAL induction by IL-17 and TNF-α as neither small interfering RNAs against the two C/EBP mRNAs nor mutation of the C/EBP sites in the LCN2 promoter abolished IL-17- and TNF-α-induced up-regulation of NGAL. NGAL induction is governed solely by NF-κB and its co-factor IκB-ζ. This was demonstrated by a pronounced reduction in the amount of NGAL mRNA and NGAL protein synthesized in cells treated with small interfering RNA against IκB-ζ and a total lack of activation of an LCN2 promoter construct with a mutated NF-κB site. As IL-17 stimulation stabilizes the IκB-ζ transcript, we propose a model where TNF-α induces activation and binding of NF-κB to the promoters of both NFKBIZ and LCN2 genes but induce only transcription of IκB-ζ. Co-stimulation with IL-17 leads to accumulation of IκB-ζ mRNA and IκB-ζ protein, which can bind to NF-κB on the LCN2 promoter and thus induce NGAL expression.

IκBζ expression is regulated by miR-124a

IκBζ belongs to the nuclear members of the IκB protein family. Its function in regulating the activity of the transcription factor NFκB is poorly understood. Here, we demonstrate that human IκBζ is posttranscriptionally regulated by microRNA (miR)-124a. In HepG2 cells miR-124a was not endogenously expressed, but upon enforced expression dramatically inhibited the interleukin-1β-induced protein expression of IκBζ. The predicted binding site for miR-124a in the 3’UTR of the IκBζ mRNA revealed an imperfect match resulting in miR-124a-mediated suppression of IκBζ expression through translational repression. Reporter gene analyses revealed that miR-124a targets IκBζ mRNA through base pairing to the partially complementary sequence in the 3’UTR that was predicted as a binding site by in silico analysis. Furthermore, we demonstrate that the 7mer seed match is sufficient for recognition of the IκBζ mRNA. Together, our data identify IκBζ as a target of miR-124a that might be involved in the fine-tuning of NFκB-mediated gene expression.

Gene-specific Requirement of a Nuclear Protein, IκB-ζ, for Promoter Association of Inflammatory Transcription Regulators

Expression of many inflammatory genes is induced through activation of the transcription factor NF-kappaB. In contrast to the advanced understanding of cytoplasmic control of NF-kappaB activation, its regulation in the nucleus has not been fully understood despite its importance in selective gene expression. We previously identified an inducible nuclear protein, IkappaB-zeta, and demonstrated that this molecule is indispensable for the expression of a group of NF-kappaB-regulated genes. In this study, we established a unique gene induction system, in which IkappaB-zeta is expressed independently of inflammatory stimuli, to specifically investigate the molecular basis underlying IkappaB-zeta-mediated gene activation. We show that in the presence of IkappaB-zeta other primary response genes are dispensable for the expression of the target secondary response genes. ChIP analyses revealed that IkappaB-zeta is required for stimulus-induced recruitment of NF-kappaB onto the target promoter in a gene-specific manner. Surprisingly, IkappaB-zeta is also necessary for the gene-selective promoter recruitment of another inflammatory transcription factor, C/EBPbeta, and the chromatin remodeling factor Brg1. We propose a new gene regulatory mechanism underlying the selective expression of inflammatory genes.

Class-specific Regulation of Pro-inflammatory Genes by MyD88 Pathways and IκBζ

Toll-like receptors trigger the induction of primary response genes via MyD88-mediated activation of NF-kappaB and other transcription factors. These factors then act in concert with primary response gene products to induce secondary response genes. Although the MyD88 pathway is important for the expression of both primary and secondary response genes, we show that the recruitment of NF-kappaB, RNA polymerase, and the TATA-binding protein is MyD88-dependent only at secondary response genes. This selective dependence correlates with the fact that MyD88 is required for nucleosome remodeling and histone H3K4 trimethylation at secondary response promoters, whereas rapidly induced primary response promoters are assembled into poised MyD88-independent chromatin structures. At a subset of secondary response promoters, IkappaBzeta was identified as a selective regulator of H3K4 trimethylation and preinitiation complex assembly after nucleosome remodeling. These mechanistic distinctions advance our understanding of the diverse molecular cascades that underlie the differential regulation of pro-inflammatory genes.

Anti-inflammatory activity of probiotic Bifidobacterium: Enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells

To determine the anti-inflammatory activity of probiotic Bifidobacteria in Bifidobacteria-fermented milk (BFM) which is effective against active ulcerative colitis (UC) and exacerbations of UC, and to explore the immunoregulatory mechanisms. Peripheral blood mononuclear cells (PBMNC) from UC patients or HT-29 cells were co-cultured with heat-killed probiotic bacteria or culture supernatant of Bifidobacterium breve strain Yakult (BbrY) or Bifidobacterium bifidum strain Yakult (BbiY) to estimate the amount of IL-10 or IL-8 secreted. Both strains of probiotic Bifidobacteria contained in the BFM induced IL-10 production in PBMNC from UC patients, though BbrY was more effective than BbiY. Conditioned medium (CM) and DNA of both strains inhibited IL-8 secretion in HT-29 cells stimulated with TNF-alpha, whereas no such effect was observed with heat-killed bacteria. The inhibitory effect of CM derived from BbiY was greater than that of CM derived from BbrY. DNAs of the two strains had a comparable inhibitory activity against the secretion of IL-8. CM of BbiY induced a repression of IL-8 gene expression with a higher expression of IkappaB-zeta mRNA 4 h after culture of HT-29 cells compared to that in the absence of CM. Probiotic Bifidobacterium strains in BFM enhance IL-10 production in PBMNC and inhibit IL-8 secretion in intestinal epithelial cells, suggesting that BFM has anti-inflammatory effects against ulcerative colitis.

A cis-element in the 3′-untranslated region of IκB-ζ mRNA governs its stimulus-specific expression

IκB-ζ, an essential transcriptional regulator in inflammatory reactions, is induced by microbial substances that stimulate Toll-like receptors and interleukin (IL)-1β but not by tumor necrosis factor (TNF)-α, via specific mRNA stabilization. Here, we attempted to identify a cis-element in IκB-ζ mRNA that confers the specific induction. To evaluate the activities of various fragments in the post-transcriptional regulation, we constructed unique reporter plasmids, in which a fragment was inserted downstream of a destabilized luciferase cDNA transcribed by the SV40 early enhancer/promoter. In NIH3T3 cells, a reporter plasmid harboring IκB-ζ mRNA exhibited elevated luciferase activity following stimulation with lipopolysaccharide or IL-1β, but not TNF-α, indicating the stimulus-specificity. We found that a 165-nucleotide fragment in the 3′-untranslated region conferred the specific induction. Stimulus-specific induction of IκB-ζ was observed by transfection of full-length IκB-ζ mRNA, but not of a mRNA without the fragment. Thus, this sequence is essential for the stimulus-specific induction of IκB-ζ via a post-transcriptional regulatory mechanism.

Induction of the nuclear IκB protein IκB-ζ upon stimulation of B cell antigen receptor

The nuclear IκB protein IκB-ζ is barely detectable in resting cells and is induced in macrophages and fibroblasts following stimulation of innate immunity via Toll-like receptors. The induced IκB-ζ associates with nuclear factor (NF)-κB in the nucleus and plays crucial roles in its transcriptional regulation. Here, we examined the induction of IκB-ζ in B lymphocytes, one of the major players in adaptive immunity. Upon crosslinking of the surface immunoglobulin complex, IκB-ζ mRNA was robustly induced in murine B-lymphoma cell line A20 cells. While the crosslinking activated NF-κB and induced its target gene, IκB-α, co-crosslinking of Fcγ receptor IIB to the surface immunoglobulin complex inhibited NF-κB activation and the induction of IκB-ζ and IκB-α, suggesting critical roles for NF-κB in the induction. These results indicate that IκB-ζ is also induced by stimulation of B cell antigen receptor, suggesting that IκB-ζ is involved in the regulation of adaptive immune responses.

IL-1β-Specific Up-Regulation of Neutrophil Gelatinase-Associated Lipocalin Is Controlled by IκB-ζ

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding protein that exerts a bacteriostatic effect by sequestering iron. Strong induction of NGAL synthesis has been observed in inflamed epithelium of the lungs and colon. Expression of NGAL is up-regulated in the lung epithelial cell line A549 by IL-1beta, but not by TNF-alpha, despite an induction of NF-kappaB binding to the NGAL promoter by both cytokines. In this study, we present evidence that the IL-1beta specificity is caused by a requirement of the NGAL promoter for the NF-kappaB-binding cofactor IkappaB-zeta for transcriptional activation. Up-regulation of NGAL expression in A549 cells following IL-1beta stimulation was dependent on de novo protein synthesis and was greatly diminished by a small interfering against IkappaB-zeta mRNA. Cotransfection of A549 cells with a plasmid expressing IkappaB-zeta made TNF-alpha capable of inducing NGAL transcription, indicating that IkappaB-zeta induction is the only factor discriminating between IL-1beta and TNF-alpha in their ability to induce NGAL expression. Coexpression of the cofactor Bcl-3, which is closely related to IkappaB-zeta, did not enable TNF-alpha to induce NGAL transcription. A functional NF-kappaB site of the NGAL promoter was required for IkappaB-zeta to exert its effect. The human beta defensin 2 gene also required IkappaB-zeta for its IL-1beta-specific induction in A549 cells. Our findings indicate that a common regulatory mechanism has evolved to control expression of a subset of antimicrobial proteins expressed in epithelial cells.

Spontaneous Ocular Surface Inflammation and Goblet Cell Disappearance in IκBζ Gene-Disrupted Mice

The ocular surface epithelium is part of the mucosal defense system. Because transcription factor NF-kappa B in mucosal epithelial cells plays a central role in regulating the genes that govern the onset of mucosal inflammatory responses, we examined the role of a regulator of NF-kappa B, I kappa B zeta, in murine ocular surface inflammation. The eyes of I kappa B zeta(-/-) mice were analyzed biomicroscopically and histologically. I kappa B zeta expression in normal mouse cornea and conjunctiva was examined by RT-PCR. The results were compared with those obtained in other tissues by real-time PCR. I kappa B zeta mRNA on the ocular surface and in other mucosal tissues was localized by in situ hybridization. I kappa B zeta(-/-) mice manifested chronic inflammation, specifically in the ocular surface, but not in other tissues. In normal mice, I kappa B zeta was expressed in a variety of mucosal tissues. The I kappa B zeta transcript was predominantly distributed in the epithelia of these tissues. As inflammatory symptoms progressed on the ocular surface of I kappa B zeta(-/-) mice, inflammatory cells, mainly CD45R/B220(+) and CD4(+) cells, intensely infiltrated the submucosa of the conjunctival epithelia. This infiltration was accompanied by an almost complete loss of goblet cells in the conjunctival epithelia. The authors postulate that I kappa B zeta in the ocular surface epithelia negatively regulates the pathologic progression of ocular surface inflammation.