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Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81-127, is highly amyloidogenic in vitro , even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81-127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo , and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81-127-based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis., (© 2019 Ueda et al.)

Sirtuin 1 (SIRT1), an NAD(+)-dependent histone deacetylase, plays crucial roles in various biological processes including longevity, stress response, and cell survival. Endoplasmic reticulum (ER) stress is caused by dysfunction of ER homeostasis and exacerbates various diseases including diabetes, fatty liver, and chronic obstructive pulmonary disease. Although several reports have shown that SIRT1 negatively regulates ER stress and ER stress-induced responses in vitro and in vivo, the effect of ER stress on SIRT1 is less explored. In this study, we showed that ER stress induced SIRT1 expression in vitro and in vivo. We further determined the molecular mechanisms of how ER stress induces SIRT1 expression. Surprisingly, the conventional ER stress-activated transcription factors XBP1, ATF4, and ATF6 seem to be dispensable for SIRT1 induction. Based on inhibitor screening experiments with SIRT1 promoter, we found that the PI3K-Akt-GSK3β signaling pathway is required for SIRT1 induction by ER stress. Moreover, we showed that pharmacological inhibition of SIRT1 by EX527 inhibited the ER stress-induced cellular death in vitro and severe hepatocellular injury in vivo, indicating a detrimental role of SIRT1 in ER stress-induced damage responses. Collectively, these data suggest that SIRT1 expression is up-regulated by ER stress and contributes to ER stress-induced cellular damage., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Myeloid Elf-1-like factor (MEF) or Elf4 is an ETS transcription factor that activates innate immunity-associated genes such as lysozyme (LYZ), human β-defensin 2 (HβD2), and interleukin-8 (IL-8) in epithelial cells and is also known to influence cell cycle progression. MEF is transcriptionally activated by E2F1, but the E2F1-mediated transcriptional activation is inhibited by p53 through E2F1-p53 protein interaction. Although the transcriptional activation of MEF has been investigated in depth, its post-translational regulation is not well explored. By overexpressing MEF cDNA in human cell lines, here we show that MEF protein expression is suppressed by p53. By screening a number of E3 ligases regulated by p53, we found that MDM2 is involved in the effect of p53 on MEF. MDM2 is transcriptionally activated by p53 and interacts with MEF protein to enhance MEF degradation. MDM2 reduces MEF protein expression, as well as stability and function of MEF as transcriptional activator. Furthermore, MDM2 was able to down-regulate MEF in the absence of p53, indicating a p53-independent effect on MEF. Notably, MEF transcriptionally activates MDM2, which was previously demonstrated to be the mechanism by which MEF suppresses the p53 protein. These results reveal that in addition to the potential of MEF to down-regulate p53 by transcriptionally activating E3 ligase MDM2, MEF participates with MDM2 in a novel autoregulatory feedback loop to regulate itself. Taken together with the findings on the effect of p53 on MEF, these data provide evidence that the p53-MDM2-MEF axis is a feedback mechanism that exquisitely controls the balance of these transcriptional regulators., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53(-/-) cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

CD163, a scavenger receptor that is expressed at high levels in the monocyte-macrophage system, is a critical factor for the efficient extracellular hemoglobin (Hb) clearance during hemolysis. Because of the enormous detrimental effect of liberated Hb on our body by its ability to induce pro-inflammatory signals and tissue damage, an understanding of the molecular mechanisms associated with CD163 expression during the acute phase response is a central issue. We report here that α(1)-acid glycoprotein (AGP), an acute phase protein, the serum concentration of which is elevated under various inflammatory conditions, including hemolysis, up-regulates CD163 expression in both macrophage-like differentiated THP-1 (dTHP-1) cells and peripheral blood mononuclear cells in a time- and concentration-dependent manner. Moreover, the subsequent induction of Hb uptake was also observed in AGP-treated dTHP-1 cells. Among representative acute phase proteins such as AGP, α(1)-antitrypsin, C-reactive protein, and haptoglobin, only AGP increased CD163 expression, suggesting that AGP plays a specific role in the regulation of CD163. Consistently, the physiological concentrations of AGP induced CD163, and the subsequent induction of Hb uptake as well as the reduction of oxidative stress in plasma were observed in phenylhydrazine-induced hemolytic model mice, confirming the in vivo role of AGP. Finally, AGP signaling through the toll-like receptor-4 (TLR4) and CD14, the common innate immune receptor complex that normally recognizes bacterial components, was identified as a crucial stimulus that induces the autocrine regulatory loops of IL-6 and/or IL-10 via NF-κB, p38, and JNK pathways, which leads to an enhancement in CD163 expression. These findings provide possible insights into how AGP exerts anti-inflammatory properties against hemolysis-induced oxidative stress.

Otitis media (OM) is the most common childhood bacterial infection and the major cause of conductive hearing loss in children. Mucus overproduction is a hallmark of OM. Streptococcus pneumoniae is the most common gram-positive bacterial pathogen causing OM. Among many mucin genes, MUC5AC has been found to be greatly up-regulated in the middle ear mucosa of human patients with OM. We previously reported that S. pneumoniae up-regulates MUC5AC expression in a MAPK ERK-dependent manner. We also found that MAPK phosphatase-1 (MKP-1) negatively regulates S. pneumoniae-induced ERK-dependent MUC5AC up-regulation. Therapeutic strategies for up-regulating the expression of negative regulators such as MKP-1 may have significant therapeutic potential for treating mucus overproduction in OM. However, the underlying molecular mechanism by which MKP-1 expression is negatively regulated during S. pneumoniae infection is unknown. In this study we show that phosphodiesterase 4B (PDE4B) mediates S. pneumoniae-induced MUC5AC up-regulation by inhibiting the expression of a negative regulator MKP-1, which in turn leads to enhanced MAPK ERK activation and subsequent up-regulation of MUC5AC. PDE4B inhibits MKP-1 expression in a cAMP-PKA-dependent manner. PDE4-specific inhibitor rolipram inhibits S. pneumoniae-induced MUC5AC up-regulation both in vitro and in vivo. Moreover, we show that PDE4B plays a critical role in MUC5AC induction. Finally, topical and post-infection administration of rolipram into the middle ear potently inhibited S. pneumoniae-induced MUC5AC up-regulation. Collectively, these data demonstrate that PDE4B mediates ERK-dependent up-regulation of mucin MUC5AC by S. pneumoniae by inhibiting cAMP-PKA-dependent MKP-1 pathway. This study may lead to novel therapeutic strategy for inhibiting mucus overproduction.

Amyloid fibril formation of mutant transthyretin (TTR) that causes familial amyloid polyneuropathy occurs in the extracellular space. Thus, secretion of TTR variants contributes to the pathogenesis of amyloidosis. However, the molecular mechanisms underlying the endoplasmic reticulum (ER) exit or retention and subsequent degradation of TTR variants remain unclear. Here, we demonstrated that the nonsecreted TTR variants, such as D18G TTR and amyloidogenic TTRs with introduced monomeric mutation (M-TTRs), stably interact with the ER chaperone BiP in mammalian cells. These proteins were co-secreted with the secreted form of BiP in which the KDEL signal was removed, indicating that BiP partially contributes to the ER retention of nonsecreted TTR variants. More interestingly, the degradation efficiency of nonsecreted TTRs was increased when BiP was down-regulated by small interfering RNA. Thus, BiP protects the TTR variants from immediate degradation. Additionally, we showed that the stability of nonsecreted TTR variants is not disturbed in the coat complex II-deficient conditions, which are enough to inhibit the ER export of secreted TTR variants, including wild-type TTR. Therefore, the post-ER retrieval mechanism might not contribute to the ER-associated degradation of nonsecreted TTR variants. These findings suggest that the affinity to the ER-resident protein BiP regulates the fate of TTR variants in the ER.

Gram-positive bacterium Streptococcus pneumoniae is an important human pathogen that colonizes the upper respiratory tract and is also the major cause of morbidity and mortality worldwide. S. pneumoniae causes invasive diseases such as pneumonia, meningitis, and otitis media. Despite the importance of pneumococcal diseases, little is known about the molecular mechanisms by which S. pneumoniae-induced inflammation is regulated, especially the negative regulatory mechanisms. Here we show that S. pneumoniae activates nuclear factor of activated T cells (NFAT) signaling pathway and the subsequent up-regulation of inflammatory mediators via a key pneumococcal virulence factor, pneumolysin. We also demonstrate that S. pneumoniae activates NFAT transcription factor independently of Toll-like receptors 2 and 4. Moreover, S. pneumoniae induces NFAT activation via both Ca(2+)-calcineurin and transforming growth factor-beta-activated kinase 1 (TAK1)-mitogen-activated protein kinase kinase (MKK) 3/6-p38alpha/beta-dependent signaling pathways. Interestingly, we found for the first time that tumor suppressor cylindromatosis (CYLD) acts as a negative regulator for S. pneumoniae-induced NFAT signaling pathway via a deubiquitination-dependent mechanism. Finally, we showed that CYLD interacts with and deubiquitinates TAK1 to negatively regulate the activation of the downstream MKK3/6-p38alpha/beta pathway. Our studies thus bring new insights into the molecular pathogenesis of S. pneumoniae infections through the NFAT-dependent mechanism and further identify CYLD as a negative regulator for NFAT signaling, thereby opening up new therapeutic targets for these diseases.

The transforming growth factor beta (TGF-beta) pathway represents an important signaling pathway involved in the regulation of diverse biological processes, including cell proliferation, differentiation, and apoptosis. Despite the known role of TGF-betaR-mediated signaling in suppressing immune response, its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here, we show for the first time that TGF-betaR-Smad3/4 signaling pathway acts as a positive regulator for TLR2 induction by bacterium nontypeable Hemophilus influenzae (NTHi) in vitro and in vivo. The positive regulation of TLR2 induction by TGF-betaR is mediated via a dual mechanism involving distinct signaling pathways. One mechanism involves functional cooperation between the TGF-betaR-Smad3/4 pathway and NF-kappaB pathway. Another involves MAP kinase phosphatase 1 (MKP-1)-dependent inhibition of p38 MAPK, a known negative regulator for TLR2 induction. Moreover, we showed that TbetaR-mediated signaling is probably activated by NTHi-derived TGF-beta mimicry molecule via an autocrine-independent mechanism. Thus, our study provides new insights into the role of TGF-beta signaling in positively regulating host defense response by tightly controlling the expression level of TLR2 during bacterial infections and may lead to new therapeutic strategies for modulating host defense and inflammatory response.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl- channel at the plasma membrane, and its malfunction results in cystic fibrosis, the most common lethal genetic disease in Caucasians. Quality control of CFTR is strictly regulated by several molecular chaperones. Here we show that calreticulin (CRT), which is a lectin-like chaperone in the endoplasmic reticulum (ER), negatively regulates the cell surface CFTR. RNA interference-based CRT knockdown induced the increase of CFTR expression. Consistently, this effect was observed in vivo. CRT heterozygous (CRT+/-) mice had a higher endogenous expression of CFTR than the wild-type mice. Moreover, CRT overexpression induced cell surface expression of CRT, and it significantly decreased the cell surface expression and function of CFTR. CRT overexpression destabilized the cell surface CFTR by enhancing endocytosis, leading to proteasomal degradation. Deletion of the carboxyl domain of CRT, which results in its ER export, increased the negative effect and enhanced the interaction with CFTR. Thus, CRT in the post-ER compartments may act as a negative regulator of the cell surface CFTR.

Toll-like receptor 2 (TLR2) plays an important role in host defense against bacterial pathogens. Activation of TLR2 signaling not only induces the activation of innate immunity and instructs the development of the acquired immunity but also leads to the detrimental inflammatory responses in inflammatory and infectious diseases. To avoid detrimental inflammatory responses, TLR2 signaling must be tightly regulated. In contrast to the relative known positive regulation of TLR2 signaling, its negative regulation, however, is largely unknown. In addition the distal signaling components that link TLR2 to its downstream signaling pathways have yet to be further defined. In the present study we have provided direct evidence for the negative regulation of TLR2 signaling by the tumor suppressor cylindromatosis (CYLD). We showed that activation of TLR2 signaling by TLR2 ligands including peptidoglycan (PGN), MALP-2, and Pam3CSK4 induces activation of IKKs-IkappaBalpha and MKK3/6-p38 pathways not only by TRAF6 but also by TRAF7, a recently identified TRAF family member. The activation of both pathways leads to the transcription of TNF-alpha, IL-1beta, and IL-8 as well as CYLD. CYLD in turn leads to the inhibition of TRAF6 and TRAF7 likely via a deubiquitination-dependent mechanism. The present studies thus unveil a novel autoregulatory feedback mechanism that negatively controls TLR2-IKKs-IkappaBalpha/MKK3/6-p38-NF-kappaB-dependent induction of immune and inflammatory responses via negatively cross-talking with both TRAF6 and TRAF7. These findings provide novel insights into autoregulation and negative regulation of TLR signaling.

Epidermal growth factor receptor (EGFR) has been shown to play important roles in regulating diverse biological processes, including cell growth, differentiation, apoptosis, adhesion, and migration. Its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here we show for the first time that EGFR acts as a negative regulator for TLR2 induction by the bacterium nontypeable Haemophilus influenzae (NTHi) in vitro and in vivo. The negative regulation of TLR2 induction by EGFR is mediated via an Src-MKK3/6-p38 alpha/beta MAP kinase-dependent mechanism. Moreover, direct activation of EGFR signaling by the bacterium NTHi-derived EGF-like factor appears to be responsible for triggering the downstream Src-MKK3/6-p38 MAPK signaling, which in turn leads to the negative regulation of TLR2 induction. Finally, exogenous EGF increases NTHi invasion of host epithelial cells, thereby demonstrating the biological significance of TLR2 regulation by EGFR signaling. The evidence we provided in the present study may suggest a novel strategy utilized by bacteria to attenuate host defensive and immune response by negatively regulating the expression of host defense receptor TLR2. These studies may bring new insight for fully understanding the important role of EGFR signaling in regulating host defense and immune response by tightly controlling TLR2 induction during bacterial infections.

Myeloid elf-1-like factor (MEF) or Elf4, which is a member of the ETS transcription factor family, up-regulates the basal expression of lysozyme gene in epithelial cells and is constitutively localized in the nucleus. The mammalian cell nucleus is organized into distinct nuclear domains or compartments that are essential for diverse physiological processes. Promyelocytic leukemia (PML) nuclear body or nuclear domain 10 is one of the nuclear domains and is involved in tumor suppression and regulation of transcription. Here, we investigate the role of PML nuclear body in MEF transactivation. We show that PML, but not Sp100, induced the accumulation of MEF in PML nuclear bodies and that MEF and PML physically interacted. This interaction stimulated MEF transcriptional activity, resulting in the up-regulation of endogenous lysozyme expression. Amino acids 348-517 of MEF were required for the accumulation of MEF in PML nuclear bodies and up-regulation of lysozyme transcription, which is enhanced by PML. Moreover, the C-terminal region of MEF spanning amino acids 477-517 was the putative region required for interaction between MEF and PML as determined with the use of the mammalian two-hybrid system. In addition, heat-shock treatment induced the accumulation of MEF in endogenous PML nuclear bodies and enhanced MEF transactivation of lysozyme gene. Thus, the recruitment of MEF to PML nuclear bodies may partly regulate lysozyme transcription in epithelial cells.

In contrast to the extensive studies on the role of transforming growth factor-beta (TGF-beta) in regulating cell proliferation, differentiation, and apoptosis over the past decade, relatively little is known about the exact role of TGF-beta signaling in regulating host response in infectious diseases. Most of the recent studies have suggested that TGF-beta inhibits macrophage activation during infections with pathogens such as Trypanosoma cruzi and Leishmania, thereby favoring virulence. In certain situations, however, there is also evidence that TGF-beta has been correlated with enhanced resistance to microbes such as Candida albicans, thus benefiting the host. Despite these distinct observations that mainly focused on macrophages, little is known about how TGF-beta regulates host primary innate defensive responses, such as up-regulation of mucin, in the airway epithelial cells. Moreover, how the TGF-beta-Smad signaling pathway negatively regulates p38 mitogen-activated protein kinase (MAPK), a key pathway mediating host response to bacteria, still remains largely unknown. Here we show that nontypeable Haemophilus influenzae, a major human bacterial pathogen of otitis media and chronic obstructive pulmonary diseases, strongly induces up-regulation of MUC5AC mucin via activation of the Toll-like receptor 2-MyD88-dependent p38 path-way. Activation of TGF-beta-Smad signaling, however, leads to down-regulation of p38 by inducing MAPK phophatase-1, thereby acting as a negative regulator for MUC5AC induction. These studies may bring new insights into the novel role of TGF-beta signaling in attenuating host primary innate defensive responses and enhance our understanding of the signaling mechanism underlying the cross-talk between TGF-beta-Smad signaling pathway and the p38 MAPK pathway.

Despite the importance of glucocorticoids in suppressing immune and inflammatory responses, their role in enhancing host immune and defense response against invading bacteria is poorly understood. We have demonstrated recently that glucocorticoids synergistically enhance nontypeable Haemophilus influenzae (NTHi)-induced expression of Toll-like receptor 2 (TLR2), an important TLR family member that has been shown to play a critical role in host immune and defense response. However, the molecular mechanisms underlying the glucocorticoid-mediated enhancement of TLR2 induction still remain unknown. Here we show that glucocorticoids synergistically enhance NTHi-induced TLR2 expression via specific up-regulation of the MAPK phosphatase-1 (MKP-1) that, in turn, leads to dephosphorylation and inactivation of p38 MAPK, the negative regulator for TLR2 expression. Moreover, increased expression of TLR2 in epithelial cells greatly enhances the NTHi-induced expression of several key cytokines, including tumor necrosis factor-alpha and interleukins 1beta and 8, thereby contributing significantly to host immune and defense response. These studies may bring new insights into the novel role of glucocorticoids in orchestrating and optimizing host immune and defense responses during bacterial infections and enhance our understanding of the signaling mechanisms underlying the glucocorticoid-mediated attenuation of MAPKs.

Transforming growth factor-beta (TGF-beta) and related factors are multifunctional cytokines that regulate diverse cellular processes, including proliferation, differentiation, apoptosis, and immune response. The involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin, a primary innate defensive response for mammalian airways, however, still remains unknown. Here, we report that the bacterium nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, utilizes the TGF-beta-Smad signaling pathway together with the TLR2-MyD88-TAK1-NIK-IKKbeta/gamma-IkappaBalpha pathway to mediate NF-kappaB-dependent MUC2 mucin transcription. The NTHi-induced TGF-beta receptor Type II phosphorylation occurred at as early as 5 min. Pretreatment of NTHi with TGF-beta neutralization antibody reduced up-regulation of MUC2 transcription. Moreover, functional cooperation of NF-kappaB p65/p50 with Smad3/4 appears to positively mediate NF-kappaB-dependent MUC2 transcription. These data are the first to demonstrate the involvement of TGF-beta receptor-mediated signaling in bacteria-induced up-regulation of mucin transcription, bring insights into the novel role of TGF-beta signaling in bacterial pathogenesis, and may lead to new therapeutic intervention of NTHi infections.

The recognition of invading microbes followed by the induction of effective innate immune response is crucial for host survival. Human surface epithelial cells are situated at host-environment boundaries and thus act as the first line of host defense against invading microbes. They recognize the microbial ligands via Toll-like receptors (TLRs) expressed on the surface of epithelial cells. TLR2 has gained importance as a major receptor for a variety of microbial ligands. In contrast to its high expression in lymphoid tissues, TLR2 is expressed at low level in epithelial cells. Thus, it remains unclear whether the low amount of TLR2 expressed in epithelial cells is sufficient for mediating bacteria-induced host defense and immune response and whether TLR2 expression can be up-regulated by bacteria during infection. Here, we show that TLR2, although expressed at very low level in unstimulated human epithelial cells, is greatly up-regulated by nontypeable Hemophilus influenzae (NTHi), an important human bacterial pathogen causing otitis media and chronic obstructive pulmonary diseases. Activation of an IKKbeta-IkappaBalpha-dependent NF-kappaB pathway is required for TLR2 induction, whereas inhibition of the MKK3/6-p38alpha/beta pathway leads to enhancement of NTHi-induced TLR2 up-regulation. Surprisingly, glucocorticoids, well known potent anti-inflammatory agents, synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the p38 MAP kinase pathway. These studies may bring new insights into the role of bacteria and glucocorticoids in regulating host defense and immune response and lead to novel therapeutic strategies for modulating innate immune and inflammatory responses for otitis media and chronic obstructive pulmonary diseases.

Lysozyme is an important component of innate immunity against common pathogens at mucosal surfaces. We previously cloned and characterized the bovine lysozyme 5A (lys5A) promoter with the purpose of determining cis- and trans-acting elements controlling airway epithelial cell-specific expression. We found that such expression is controlled by protein binding to an ETS consensus sequence located approximately at -46 to -40 bp from the transcription start site. The identity of the ETS-related protein responsible for gene transactivation was unknown. In this study, we screened six ETS-related proteins by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, the myeloid Elf-1-like factor (MEF) was the most potent. Gel shift analysis of epithelial cell nuclear extracts using a lys5A probe including the ETS-binding site (-50/-31) yielded a single band with retarded mobility. This band was supershifted by an antibody directed against MEF. Supporting the possibility that MEF is responsible for functional transactivation of lysozyme in epithelial cells, we found that antisense MEF mRNA decreased lys5A promoter activity and that MEF overexpression in stably transfected cells increased lysozyme mRNA and protein expression. We conclude that MEF is required for epithelial cell transactivation of lysozyme.

GRK5, a recently cloned member of the G protein-coupled receptor kinase family, has been shown to phosphorylate and participate in the desensitization of angiotensin II (Ang II) type 1A (AT1A) receptors. In this study, the effect of angiotensin II on GRK5 expression was examined in cultured vascular smooth muscle cells and aortas of Ang II-infused hypertensive rats. In vascular smooth muscle cells, Ang II (100 nM) up-regulated GRK5 mRNA as early as 1 h, with a peak at 16 h. This up-regulation was dose- and calcium-dependent. The increase in GRK5 mRNA was reflected in a smaller increase in protein expression, which nonetheless had functional significance since AT1 receptor phosphorylation was increased and phospholipase C activation was decreased following prolonged incubation with Ang II. In aortas of Ang II-infused hypertensive rats, both GRK5 mRNA and protein levels increased approximately 3-fold compared with sham-operated rats at 5 and 7 days, respectively. This up-regulation was blocked either by losartan or by the nonspecific vasodilator hydralazine. Since a subpressor dose of Ang II did not increase GRK5 mRNA levels and norepinephrine infusion also increased GRK5 mRNA expression, we conclude that Ang II-induced GRK5 up-regulation in rat aortas may be due to hypertension per se. Hormone- and hemodynamic stress-induced GRK5 regulation may provide a novel molecular basis for long-term regulation of agonist sensitivity of vascular cells.

To obtain cDNAs for analysis of mucin gene transcription in rat models of human disease, we screened a rat intestinal cDNA library in lambda ZAPII using an upstream non-tandem repeat cDNA fragment of the human MUC 2 gene (Gum, J., Hicks, J., Toribara, N., Rothe, E., Lagace, R., and Y., K. (1992) J. Biol. Chem. 267, 21375-21383). Three cDNAs, 1-1, 8-1, and 21-1, were isolated. A translation start site was found in cDNA 21-1. Combined nucleotide sequence for the three cDNAs contained an open reading frame spanning 4546 base pairs. This amino-terminal sequence contains a non-tandem repeat domain enriched in cysteine (1391 residues) followed by an irregular tandem repeat domain (122 residues). Identity with the human gene is about 80% in the non-tandem repeat domain and about 38% in the irregular tandem repeat domain. Primer extension and S1 nuclease protection analysis indicate a transcription start site at 28 base pairs upstream of translation initiation. Northern analysis showed expression of cognate RNA in the intestine and airway but not heart and spleen. The cDNAs have been used to isolate the gene promoter, the structure of which should yield clues to the regulation of mucin expression in rat models of human disease.

The principal role of lysozyme is to prevent bacterial invasion at body surfaces. We are interested in how lysozyme is regulated at the surface of the respiratory tract, where the serous gland cell is regarded as the primary cellular source of this enzyme. Since the cow genome contains at least 10 lysozyme-like genes, our objective was to determine which of them are expressed in the cow tracheal gland serous cell. By screening tracheal cDNA libraries with a probe constructed from the cDNA encoding stomach lysozyme 2, we obtained 3 lysozyme cDNAs: 5a (1023 base pairs (bp)), 7a (1060 bp), and 14d (1249 bp). cDNA 7a corresponds to a previously reported gene (showing sequence identity to the stomach 2 lysozyme gene), whereas cDNAs 5a and 14d correspond to lysozyme genes not previously reported. Northern blot analysis of cow tracheal RNA showed lysozyme mRNAs of three distinct lengths. Based on hybridization with probes specific for each cDNA, we determined that the longest transcript corresponded to cDNA 5a, the shortest to 7a, and the intermediate-length transcript to 14d. Cultured cow tracheal gland serous cell RNA, reverse transcribed and amplified by the polymerase chain reaction with primers common to all three cDNAs, yielded a product that hybridized to oligonucleotide probes specific for all three cDNAs but most strongly to that for 5a. These results indicate that multiple lysozyme mRNAs are expressed in the cow trachea and that the lysozyme encoded by cDNA 5a is the major form expressed in the tracheal gland serous cell. This serous cell lysozyme is predicted to differ importantly in structure from both 7a and 14d lysozymes, with an arginine:lysine ratio almost 10-fold higher. The sequence differences may underlie functional differences, including variable resistance to proteolysis and variable affinity for large polyanions (e.g. mucins) found in the respiratory tract lumen.