Yuko Tsutsumi-Ishii

Modulation of Human β-Defensin-2 Transcription in Pulmonary Epithelial Cells by Lipopolysaccharide-Stimulated Mononuclear Phagocytes Via Proinflammatory Cytokine Production

Human β-defensin (hBD)-2, a cationic antimicrobial peptide primarily induced in epithelial cells in response to inflammatory stimuli, plays an important role in host defense. To elucidate the expression mechanism of hBD-2 in the lung, we investigated the modulation of hBD-2 transcription in pulmonary epithelial cells by mononuclear phagocytes stimulated with LPS. Coculture of A549 pulmonary epithelial cells with Mono-Mac-6 monocytic cells in the presence of Escherichia coli LPS markedly up-regulated hBD-2 promoter activity, whereas A549 alone did not respond to LPS to activate the hBD-2 promoter. Furthermore, IL-1β and TNF-α in the culture supernatants from LPS-stimulated monocytic cells activated the hBD-2 promoter in A549 cells. Of note, IL-1β was more potent than TNF-α in this effect. In addition, a mutation of the NF-κB site at −200 (pκB1 site) completely abolished this IL-1β- and TNF-α-induced hBD-2 promoter activation, whereas NF-κB inhibitors (MG-132 and helenalin) strongly suppressed it. Moreover, electrophoretic mobility shift assay suggested that NF-κB, consisting of p65-p50 heterodimer, could bind to the pκB1 site in cytokine-stimulated A549 cells. Interestingly, flow cytometric analysis revealed that A549 cells expressed CD14 but lacked Toll-like receptor 4, which may account for the hyporesponsiveness of A549 cells to LPS. Taken together, these results suggest that hBD-2 expression in pulmonary epithelial cells is modulated by NF-κB via the actions of IL-1β and TNF-α produced by LPS-stimulated mononuclear phagocytes.

Activation of Integrin-Linked Kinase Is a Critical Prosurvival Pathway Induced in Leukemic Cells by Bone Marrow–Derived Stromal Cells

Integrin-linked kinase (ILK) directly interacts with beta integrins and phosphorylates Akt in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. In this study, we examined the functional role of ILK activation in leukemic and bone marrow stromal cells on their direct contact. Coculture of leukemic NB4 cells with bone marrow-derived stromal mesenchymal stem cells (MSC) resulted in robust activation of multiple signaling pathways, including ILK/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducers and activators of transcription 3 (STAT3), and Notch1/Hes. Blockade of PI3K or ILK signaling with pharmacologic inhibitors LY294002 or QLT0267 specifically inhibited stroma-induced phosphorylation of Akt and glycogen synthase kinase 3beta, suppressed STAT3 and ERK1/2 activation, and decreased Notch1 and Hes1 expression in leukemic cells. This resulted in induction of apoptosis in both leukemic cell lines and in primary acute myelogenous leukemia samples that was not abrogated by MSC coculture. In turn, leukemic cells growing in direct contact with bone marrow stromal elements induce activation of Akt, ERK1/2, and STAT3 signaling in MSC, accompanied by significant increase in Hes1 and Bcl-2 proteins, which were all suppressed by QLT0267 and LY294002. In summary, our results indicate reciprocal activation of ILK/Akt in both leukemic and bone marrow stromal cells. We propose that ILK/Akt is a proximal signaling pathway critical for survival of leukemic cells within the bone marrow microenvironment. Hence, disruption of these interactions by ILK inhibitors represents a potential novel therapeutic strategy to eradicate leukemia in the bone marrow microenvironment by simultaneous targeting of both leukemic cells and activated bone marrow stromal cells.

NF-κB-mediated transcriptional regulation of human β-defensin-2 gene following lipopolysaccharide stimulation

β‐Defensins are cationic peptides with broad‐spectrum antimicrobialactivities that contribute to innate host defense. Among humanβ‐defensins (hBDs), hBD‐2 is up‐regulated in epithelial tissues andmononuclear phagocytes in response to bacterial infection andproinflammatory cytokines. However, little is known about the molecularmechanism of hBD‐2 gene regulation. Here, we investigatedlipopolysaccharide (LPS)‐mediated transcriptional regulation of thehBD‐2 gene by focusing on the roles of NF‐κB, STAT, and NF‐IL‐6 sitesin mononuclear phagocytes using RAW264.7 cells, which are sensitive toLPS. Luciferase reporter analyses demonstrated that two NF‐κB siteswere essential for full LPS responsiveness of the hBD‐2 gene. Further, both NF‐κB sites were also crucial for basal transcriptionalactivity. In contrast, neither the NF‐IL‐6 nor STAT binding site wasrequired for LPS‐induced hBD‐2 transcription. Electrophoretic mobilityshift assay indicated that in unstimulated cells, NF‐κB p50 homodimerbound to both NF‐κB sites, whereas the p65‐p50 heterodimer formedcomplexes with these sites following LPS stimulation. Together, theseobservations indicate that NF‐κB plays an important role in theregulation of hBD‐2 gene expression in response to LPS.

Isolation of cDNA Encoding Guinea Pig Neutrophil Cationic Antibacterial Polypeptide of 11 kDa (CAP11) and Evaluation of CAP11 mRNA Expression during Neutrophil Maturation

Neutrophils contain various antibacterial polypeptides and proteins in the granules that contribute to the killing of microorganisms. Recently, we have purified a cationic antibacterial polypeptide of 11 kDa (CAP11) from guinea pig neutrophil granules. CAP11 is a homodimer of G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43joined with one disulfide bond. In this study, to understand the regulation of CAP11 expression, we isolated and analyzed cDNA encoding CAP11. Furthermore, we investigated the expression of CAP11 mRNA during neutrophil maturation and localization of CAP11 among neutrophil granule subsets. Sequence analysis of CAP11 cDNA isolated from guinea pig bone marrow cells using rapid amplification of cDNA ends technique indicated that CAP11 is synthesized as a precursor comprising 178 amino acid residues, which is composed of a signal peptide (N-terminal 29 residues), a propeptide (106 residues), and a C-terminal mature peptide (43 residues). Interestingly, the predicted CAP11 precursor displayed the characteristic features of cathelicidins, a novel protein family of antibacterial polypeptides with a conserved cathelin-like pro-region and a variable C-terminal antibacterial domain. Northern blot and Western blot analyses using neutrophils, macrophages, eosinophils, mononuclear cells, and bone marrow cells revealed that only neutrophils and bone marrow cells expressed CAP11 mRNA and contained CAP11, suggesting that expression of CAP11 is neutrophil lineage-specific. Furthermore, Northern blot analysis using bone marrow cells separated according to their maturation stages showed that CAP11 mRNA was predominantly expressed in the cells at later stages of neutrophil maturation. Consistent with this, in situ hybridization using CAP11-specific cRNA probe demonstrated that CAP11 mRNA was primarily expressed at metamyelocyte stage. In addition, extracellular release assay revealed that CAP11 was readily released from neutrophils accompanied with gelatinase by low concentrations ofN-formyl-Met-Leu-Phe without release of specific and azurophil granule components, and CAP11 was found to be exclusively present in the fraction containing gelatinase granules, prepared by Percoll density gradient centrifugation. Together these observations indicate that CAP11 is a member of cathelicidin family and its mRNA is preferentially expressed at the later stage of neutrophil maturation (i.e. metamyelocyte stage). Furthermore, CAP11 may be stored in the granule subset, possibly the gelatinase granule.

Role of CCAAT/Enhancer-Binding Protein Site in Transcription of Human Neutrophil Peptide-1 and -3 Defensin Genes

The human neutrophil defensins (human neutrophil peptides (HNPs)), major components of azurophilic granules, contribute to innate and acquired host immunities through their potent antimicrobial activities and ability to activate T cells. Despite being encoded by nearly identical genes, HNP-1 is more abundant in the granules than HNP-3. We investigated the regulation of HNP-1 and HNP-3 expression at the transcriptional level using a promyelocytic HL-60 cell line. Luciferase analysis showed that transcriptional levels of HNP-1 and HNP-3 promoters were equivalent and that an ∼200-bp region identical between promoters was sufficient for transcriptional activity. Furthermore, overlapping CCAAT/enhancer-binding protein (C/EBP) and c-Myb sites in the region were found to be required for efficient transcription. Gel mobility shift assay demonstrated that C/EBPα predominantly bound to the C/EBP/c-Myb sites using HL-60 nuclear extracts. No specific binding to C/EBP/c-Myb sites was observed in nuclear extracts from mature neutrophils, which expressed neither C/EBPα protein nor HNP mRNAs. Taken together, these findings suggest that the difference in the amounts of HNP-1 and HNP-3 peptides in neutrophils is caused by posttranscriptional regulation and that C/EBPα plays an important role in the transcription of HNP genes in immature myeloid cells.

Evaluation of the effect of human β-defensins on neutrophil apoptosis

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Antimicrobial human beta-defensins (hBDs) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria but also function as immunomodulatory molecules by inducing cytokine and chemokine production and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, to further evaluate the role of hBDs in innate immunity, we investigated the action of hBD-1 to -4 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, hBD-3 most potently suppressed neutrophil apoptosis among hBD-1 to -4, accompanied with the down-regulation of truncated Bid (a pro-apoptotic protein), up-regulation of Bcl-x(L) (an anti-apoptotic protein) and inhibition of mitochondrial membrane potential change and caspase 3 activity. Furthermore, we revealed that neutrophils expressed CC chemokine receptor (CCR) 6, and the action of hBD-3 was completely abrogated by a neutralizing anti-CCR6 mAb. Collectively, these observations suggest that hBDs, especially hBD-3, can not only kill bacteria but also modulate (suppress) neutrophil apoptosis via the action on CCR6. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion.

Evaluation of the expression of human CAP18 gene during neutrophil maturation in the bone marrow

To understand the gene expression of CAP18 (18‐kDa cationic antibacterial protein), a member of cathelicidins, we evaluated mRNA and protein expression of CAP18 using human bone marrow cells and mature neutrophils. Northern blot analysis revealed that CAP18 mRNA was expressed more abundantly in bone marrow cells than mature neutrophils, whereas Western blot analysis indicated that CAP18 protein was more abundant in mature neutrophils than bone marrow cells. Consistent with this, in situ hybridization using bone marrow cells demonstrated that the expression of CAP18 mRNA was neutrophil lineage‐specific and was observed primarily in myelocytes (>95%) with limited expression in more immature cells (promyelocytes) and mature cells (metamyelocytes, band cells, and segmented neutrophils). Furthermore, immunohistochemical study indicated that, coincident with the increase of CAP18 mRNA levels, CAP18‐positive cells increased markedly at myelocyte stage, and the increased levels remained almost constant (>95%) in metamyelocytes, band cells, and segmented neutrophils, although the mRNA levels were remarkably reduced in these cells. Together these observations indicate that CAP18 gene transcription likely occurs lineage‐ and stage‐specifically at the myelocyte stage of neutrophil maturation in the bone marrow and results in the synthesis and cytoplasmic accumulation of CAP18, which is present in the subsequent stages of neutrophil maturation. J. Leukoc. Biol. 64:845–852; 1998.

Evaluation of the effect of human beta-defensins on neutrophil apoptosis.

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Antimicrobial human beta-defensins (hBDs) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria but also function as immunomodulatory molecules by inducing cytokine and chemokine production and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, to further evaluate the role of hBDs in innate immunity, we investigated the action of hBD-1 to -4 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, hBD-3 most potently suppressed neutrophil apoptosis among hBD-1 to -4, accompanied with the down-regulation of truncated Bid (a pro-apoptotic protein), up-regulation of Bcl-x(L) (an anti-apoptotic protein) and inhibition of mitochondrial membrane potential change and caspase 3 activity. Furthermore, we revealed that neutrophils expressed CC chemokine receptor (CCR) 6, and the action of hBD-3 was completely abrogated by a neutralizing anti-CCR6 mAb. Collectively, these observations suggest that hBDs, especially hBD-3, can not only kill bacteria but also modulate (suppress) neutrophil apoptosis via the action on CCR6. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion.

PPARgamma-Active triterpenoid CDDO enhances ATRA-induced differentiation in APL

Acute promyelocytic leukemia (APL) is associated with oncogenic PML-RARα that acts as a dominant negative transcriptional repressor of retinoic acid (RA) receptor target genes by recruiting histone deacetylase (HDAC). The peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor family that forms heterodimers with retinoid X receptor (RXR). In addition to RAR targets, PML-RARα silence a wide range of nuclear receptor target genes including PPARγ targets. All-trans-retinoic acid (ATRA), a ligand for the RA receptor (RAR), restores normal retinoid signaling and induces terminal differentiation of APL cells; however, APL cells can develop resistance to ATRA. Using ATRA sensitive NB4 and ATRA-resistant derivative NB4MR2 cell lines, we demonstrate that PPARγ ligand 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) enhances pro-apoptotic and differentiating effects of ATRA in ATRA-sensitive NB4 cells and partially reverses ATRA resistance in NB4MR2 cells. The CDDO/ATRA combination synergistically induces RARβ2 expression both in ATRA-sensitive and -resistant APL cells. RARβ2 mRNA induction by CDDO/ATRA was mediated in part by enhanced H3-Lys9 acetylation in the RARβ2 promoter which in turn increased the affinity of RARβ for βRARE. PPARγ specific inhibitor T007 and silencing of PPARγ by siRNA diminished CDDO-induced maturation and RARβ2 mRNA along with PPARγ induction indicating that PPARγ activation is at least partially responsible for the RARβ2 transcription and maturation induction. In an in vivo mouse model of APL, CDDO derivative CDDO-methyl ester markedly enhanced ATRA-induced maturation and extended the survival of mice. In summary, these results provide rationale for the combined targeting of RAR and PPARγ nuclear receptors in the therapy of APL.

PML-RARα and AML1–ETO translocations are rarely associated with methylation of the RARβ2 promoter

The acute promyelocytic leukemia-specific PML–RARα fusion protein is a dominant-negative transcriptional repressor of retinoic acid receptor (RAR) target genes, which recruits HDAC and corepressor proteins and inhibits coactivators. Another oncogenic transcription factor, AML1–ETO, was proposed to cause an HDAC-dependent repression of RAR target genes. The RAR target RARβ2 gene has been reported to be frequently silenced by hypermethylation in many types of cancer cells. We examined the methylation status of the RARβ2 and asked if demethylation could reverse ATRA resistance in ATRA-resistant PML–RARα and AML1–ETO-positive cells. PML–RARα positive NB4 and its ATRA-resistant subvariant MR2 and AML1–ETO expressing Kasumi-1 cells had heterozygous methylation of RARβ2. Although DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine partially reversed RARβ2 CpG methylation in these cells, it did not significantly enhance ATRA-induced RARβ2 mRNA expression and induction of maturation. However, the histone acetylase inhibitor SAHA combined with ATRA significantly reactivated RARβ2 mRNA both in NB4 and MR2 cells with degradation of PML–RARα, which was associated with maturation. In contrast, SAHA did not affect AML1–ETO levels and failed to induce RARβ2 expression and maturation in Kasumi-1 cells. In primary AML samples, RARβ2 expression was uniformly low; however, no specific correlation was observed between the methylation of the RARβ2 gene and expression of the fusion proteins, PML–RARα, and AML1–ETO. These results demonstrate that oncogenic PML–RARα and AML1–ETO translocations are rarely associated with RARβ2 promoter methylation in primary AML samples.