Therapeutic Effect of Anti-Inflammatory Tripeptide Cream in Hand-Foot Syndrome/Skin Reaction Related to Anticancer Drugs: a Randomized, Double-Blind, Placebo-Controlled Pilot Trial.

Purpose: Hand-foot syndrome (HFS) and hand-foot skin reaction (HFSR) are relatively common toxicities that interfere with the quality of life (QoL) of patients with cancer. Anti-inflammatory tripeptide cream (ATPC) is a complex formulation of anti-inflammatory tripeptides, the CD99-agonist BinterinTM and the Wnt-antagonist WinhibinTM. The present study aimed to assess the therapeutic effects of ATPC in HFS/HFSR associated with anticancer drugs. Materials and Methods: This was a single-center, randomized, double-blind, placebo-controlled trial. Patients who developed grade 1 HFS/HFSR after systemic anticancer treatments were enrolled, and randomly assigned to receive either ATPC or placebo cream (PC) and followed up at 3-week intervals for up to nine weeks. Primary endpoint was the development of grade ≥ 2 HFS/HFSR. Results: Between April 2019 and July 2022, 60 patients (31 in the ATPC and 29 in the PC group) completed the study. The incidence of grade ≥ 2 HFS/HFSR was significantly lower in the ATPC than in the PC group (25.8% vs. 51.7%, p=0.039). The ATPC showed trends towards a better QoL score, assessed by a HFSR and QoL questionnaire at 9 weeks (26.0 vs. 29.9, p=0.574), and a lower frequency of discontinuation, interruption, or dose reduction of anticancer drugs (51.6% vs. 58.6%, p=0.586) than the PC group over 9 weeks, though without statistical significance. Conclusion: Our results showed that ATPC significantly decreased the development of grade ≥ 2 HFS/HFSR in patients already with HFS/HFSR. Therefore, ATPC may be an effective treatment for HFS/HFSR associated with anticancer drugs.

A Basic Domain-Derived Tripeptide Inhibits MITF Activity by Reducing its Binding to the Promoter of Target Genes.

The keratinocytes in UV-irradiated skin produce and secrete α-melanocyte-stimulating hormone. α-Melanocyte-stimulating hormone upregulates the expression of MITF in melanocytes through the cAMP‒protein kinase A‒CREB signaling pathway. Thereafter, MITF induces the expression of melanogenic genes, including the tyrosinase gene TYR and TYRP-1 and TYRP-2 genes, which leads to the synthesis and accumulation of melanin. In this study, we examined whether MITF basic region-derived tripeptides can bind to the DNA-binding domain of MITF and inhibit MITF-induced melanogenesis through the inhibition of MITF‒DNA binding. MITF-KGR, a representative MITF-derived tripeptide, suppressed the transcriptional activity of MITF by disrupting its binding to the promoter region of the target genes, which resulted in the inhibition of skin epidermis thickness and melanin synthesis in vivo and in vitro. Our results indicate that MITF-KGR exerts an inhibitory effect on melanogenesis by targeting MITF.

CD99-PTPN12 Axis Suppresses Actin Cytoskeleton-Mediated Dimerization of Epidermal Growth Factor Receptor.

The epidermal growth factor receptor (EGFR), a member of ErbB receptor tyrosine kinase (RTK) family, is activated through growth factor-induced reorganization of the actin cytoskeleton and subsequent dimerization. We herein explored the molecular mechanism underlying the suppression of ligand-induced EGFR dimerization by CD99 agonists and its relevance to tumor growth in vivo. Epidermal growth factor (EGF) activated the formation of c-Src/focal adhesion kinase (FAK)-mediated intracellular complex and subsequently induced RhoA-and Rac1-mediated actin remodeling, resulting in EGFR dimerization and endocytosis. In contrast, CD99 agonist facilitated FAK dephosphorylation through the HRAS/ERK/PTPN12 signaling pathway, leading to inhibition of actin cytoskeletal reorganization via inactivation of the RhoA and Rac1 signaling pathways. Moreover, CD99 agonist significantly suppressed tumor growth in a BALB/c mouse model injected with MDA-MB-231 human breast cancer cells. Taken together, these results indicate that CD99-derived agonist ligand inhibits epidermal growth factor (EGF)-induced EGFR dimerization through impairment of cytoskeletal reorganization by PTPN12-dependent c-Src/FAK inactivation, thereby suppressing breast cancer growth.

Alleviation of Ultraviolet-B Radiation-Induced Photoaging by a TNFR Antagonistic Peptide, TNFR2-SKE.

Ultraviolet (UV) radiation of the sunlight, especially UVA and UVB, is the primary environmental cause of skin damage, including topical inflammation, premature skin aging, and skin cancer. Previous reports show that activation of nuclear factor-κB (NF-κB) in human skin fibroblasts and keratinocytes after UV exposure induces the expression and release of proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), and subsequently leads to the production of matrix metalloproteases (MMPs) and growth factor basic fibroblast growth factor (bFGF). Here, we demonstrated that TNFR2-SKEE and TNFR2-SKE, oligopeptides from TNF receptor-associated factor 2 (TRAF2)-binding site of TNF receptor 2 (TNFR2), strongly inhibited the interaction of TNFR1 as well as TNFR2 with TRAF2. In particular, TNFR2-SKE suppressed UVB- or TNF-α-induced nuclear translocalization of activated NF-κB in mouse fibroblasts. It decreased the expression of bFGF, MMPs, and COX2, which were upregulated by TNF-α, and increased procollagen production, which was reduced by TNF-α. Furthermore, TNFR2-SKE inhibited the UVB-induced proliferation of keratinocytes and melanocytes in the mouse skin and the infiltration of immune cells into inflamed tissues. These results suggest that TNFR2-SKE may possess the clinical potency to alleviate UV-induced photoaging in human skin.

CD99-Derived Agonist Ligands Inhibit Fibronectin-Induced Activation of ß1 Integrin through the Protein Kinase A/SHP2/Extracellular Signal-Regulated Kinase/PTPN12/Focal Adhesion Kinase Signaling Pathway.

The human CD99 protein is a 32-kDa glycosylated transmembrane protein that regulates various cellular responses, including cell adhesion and leukocyte extravasation. We previously reported that CD99 activation suppresses ß1 integrin activity through dephosphorylation of focal adhesion kinase (FAK) at Y397. We explored a molecular mechanism underlying the suppression of ß1 integrin activity by CD99 agonists and its relevance to tumor growth in vivo CD99-Fc fusion proteins or a series of CD99-derived peptides suppressed ß1 integrin activity by specifically interacting with three conserved motifs of the CD99 extracellular domain. CD99CRIII3, a representative CD99-derived 3-mer peptide, facilitated protein kinase A-SHP2 interaction and subsequent activation of the HRAS/RAF1/MEK/ERK signaling pathway. Subsequently, CD99CRIII3 induced FAK phosphorylation at S910, which led to the recruitment of PTPN12 and PIN1 to FAK, followed by FAK dephosphorylation at Y397. Taken together, these results indicate that CD99-derived agonist ligands inhibit fibronectin-mediated ß1 integrin activation through the SHP2/ERK/PTPN12/FAK signaling pathway.

Paired Ig-Like Type 2 Receptor-Derived Agonist Ligands Ameliorate Inflammatory Reactions by Downregulating ß1 Integrin Activity.

The paired immunoglobulin-like type 2 receptor (PILR) family consists of two functionally opposite members, inhibitory PILRα and activating PILRß receptors. PILRs are widely expressed in various immune cells and interact with their ligands, especially CD99 expressed on activated T cells, to participate in immune responses. Here we investigated whether PILR-derived agonists inhibit ß1 integrin activity as ligands for CD99. PILR-derived peptides as well as PILR-Fc fusion proteins prevented cell adhesion to fibronectin through the regulation of ß1 integrin activity. Especially, PILRpep3, a representative 3-mer peptide covering the conserved motifs of the PILR extracellular domain, prevented the clustering and activation of ß1 integrin by dephosphorylating FAK and vinculin, which are major components of focal adhesion. In addition, PILRpep3 inhibited transendothelial migration of monocytes as well as endothelial cell tube formation. Furthermore, upon intraperitoneal injection of PILRpep3 into mice with collagen-induced arthritis, the inflammatory response of rheumatoid arthritis was strongly suppressed. Taken together, these results suggest that PILR-derived agonist ligands may prevent the inflammatory reactions of rheumatoid arthritis by activating CD99.

CD99 inhibits CD98-mediated ß1 integrin signaling through SHP2-mediated FAK dephosphorylation.

The human CD99 protein is a 32-kDa type I transmembrane glycoprotein, while CD98 is a disulfide-linked 125-kDa heterodimeric type II transmembrane glycoprotein. It has been previously shown that CD99 and CD98 oppositely regulate ß1 integrin signaling, though the mechanisms by which this regulation occurs are not known. Our results revealed that antibody-mediated crosslinking of CD98 induced FAK phosphorylation at Y397 and facilitated the formation of the protein kinase Cα (PKCα)-syntenin-focal adhesion kinase (FAK), focal adhesions (FAs), and IPP-Akt1-syntenin complex, which mediates ß1 integrin signaling. In contrast, crosslinking of CD99 disrupted the formation of the PKCα-syntenin-FAK complex as well as FA via FAK dephosphorylation. The CD99-induced dephosphorylation of FAK was apparently mediated by the recruitment of Src homology region 2 domain-containing phosphatase-2 (SHP2) to the plasma membrane and subsequent activation of its phosphatase activity. Further consequences of the activation of SHP2 included the disruption of FAK-talin and talin-ß1 integrin interactions and attenuation in the formation of the IPP-Akt1-syntenin complex at the plasma membrane, which resulted in reduced cell-ECM adhesion. This report uncovers the molecular mechanisms underlying the inverse regulation of ß1 integrin signaling by CD99 and CD98 and may provide a novel therapeutic approach to treat inflammation and cancer.

YAC tripeptide of epidermal growth factor promotes the proliferation of HaCaT keratinocytes through activation of EGFR.

Epidermal growth factor (EGF) is known to play key roles in skin regeneration and wound-healing. Here, we demonstrate that Pep2-YAC, a tripeptide covering residues 29-31 in the B loop of EGF, promotes the proliferation of HaCaT keratinocytes with activity comparable to EGF. The treatment of HaCaT cells with Pep2-YAC induced phosphorylation, internalization, and degradation of EGFR and organization of signaling complexes, which consist of Grb2, Gab1, SHP2, and PI3K. In addition, it stimulated the phosphorylation of ERK1/2 at Thr 202/Tyr 204 and of Akt1 at Ser 473 and the nuclear translocation of EGFR, STAT3, c-Jun, and c-Fos. These results suggest that Pep2-YAC may be useful as a therapeutic agent for skin regeneration and wound-healing as an EGFR agonist.

Hyaluronic acid promotes angiogenesis by inducing RHAMM-TGFß receptor interaction via CD44-PKCδ.

Hyaluronic acid (HA) has been shown to promote angiogenesis. However, the mechanism behind this effect remains largely unknown. Therefore, in this study, the mechanism of HA-induced angiogenesis was examined. CD44 and PKCδ were shown to be necessary for induction of the receptor for HA-mediated cell motility (RHAMM), a HA-binding protein. RHAMM was necessary for HA-promoted cellular invasion and endothelial cell tube formation. Cytokine arrays showed that HA induced the expression of plasminogen activator-inhibitor-1 (PAI), a downstream target of TGFß receptor signaling. The induction of PAI-1 was dependent on CD44 and PKCδ. HA also induced an interaction between RHAMM and TGFß receptor I, and induction of PAI-1 was dependent on RHAMM and TGFß receptor I. Histone deacetylase 3 (HDAC3), which is decreased by HA via rac1, reduced induction of plasminogen activator inhibitor-1 (PAI-1) by HA. ERK, which interacts with RHAMM, was necessary for induction of PAI-1 by HA. Snail, a downstream target of TGFß signaling, was also necessary for induction of PAI-1. The down regulation of PAI-1 prevented HA from enhancing endothelial cell tube formation and from inducing expression of angiogenic factors, such as ICAM-1, VCAM-1 and MMP-2. HDAC3 also exerted reduced expression of MMP-2. In this study, we provide a novel mechanism of HA-promoted angiogenesis, which involved RHAMM-TGFßRI signaling necessary for induction of PAI-1.

The activation of CD99 inhibits cell-extracellular matrix adhesion by suppressing ß(1) integrin affinity.

CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with Mn(2+) or by ß(1) integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated ß(1) integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the ß(1) stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matrix adhesion by suppressing ß(1) integrin affinity. [BMB reports 2012; 45(3): 159-164].

Integrin α(5) interacts with EGFR, is necessary for FcɛRI signaling and is necessary for allergic inflammation in relation with angiogenesis.

Recent reports have suggested role for epidermal growth factor receptor (EGFR) in asthma and skin inflammation. Integrin(s) are known to be necessary for the transactivation of EGFR. The roles of EGFR and integrin(s) in allergic inflammation were investigated. Antigen stimulation induced activation of EGFR and interaction between EGFR and integrin α(5) in Rat Basophilic Leukemia (RBL2H3) cells and bone marrow-derived mouse mast cells (BMMCs). Flow cytometry revealed increased phosphorylation of EGFR on cell surfaces. Antigen stimulation induced interaction between EGFR and FcɛRI in both RBL2H3 cells and BMMCs. Blocking of EGFR or integrin α exerted negative effects on rac1 activity and secretion of ß-hexosaminidase in both RBL2H3 cells and BMMCs. EGFR and integrin α(5) were found to be necessary for IgE-dependent cutaneous anaphylaxis. FAK (focal adhesion kinase), interacted with EGFR and with FcɛRI upon antigen stimulation, and it was necessary for the increased secretion of ß-hexosaminidase in both RBL2H3 cells and BMMCs. EGFR and integrin α(5) were necessary for interactions between activated RBL2H3 cells, BMMCs and rat aortic endothelial cells (RAECs). Conditioned medium of antigen-stimulated RBL2H3 cells promoted RAECs tube formation, rat aortic ring formation and blood vessel formation. Conditioned medium of antigen-stimulated BMMCs also had the same effects on RAECs. This enhanced angiogenic potential of RAECs was dependent on EGFR and integrin α(5). In conclusion, EGFR, via interaction with FcɛRI and integrin α(5), is necessary for allergic inflammation associated with cellular interaction.

Xclaudin 1 is required for the proper gastrulation in Xenopus laevis.

Claudin 1 is one of the tight junctional proteins involved in the tight sealing of the cellular sheets and plays a crucial role in the maintenance of cell polarity. Although its structure and physiological function in intercellular adhesion is relatively well understood, we have little information about its possible involvement in early development of vertebrates. We found Xclaudin 1 is expressed maternally in the oocyte of Xenopus laevis and the zygotic expression initiates stage 9 in the animal hemisphere but not in the vegetal hemisphere, limited on the ectoderm and mesoderm until the end of gastrulation. We have investigated a potential role for claudin 1 at gastrulation by gain and loss-of-function studies. Over-expression of Xclaudin 1 resulted in gastrulation defect in a dose-dependent manner. Knockdown of Xclaudin 1 by antisense morpholino oligonucleotides (MOs) blocked convergent extension, whereas ectopic expression of Xclaudin 1-myc mRNA rescued these defects. However, altered expression of Xclaudin 1 did not inhibit mesodermal gene expression. Taken together, our results suggest that Xclaudin 1 is required for proper convergent extension movement during Xenopus gastrulation.

Cancer/testis antigen CAGE exerts negative regulation on p53 expression through HDAC2 and confers resistance to anti-cancer drugs.

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M(R)) and the human hepatic cancer cell line SNU387 (SNU387(R)) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387(R) cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M(R) cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.

Celastrol binds to ERK and inhibits FcepsilonRI signaling to exert an anti-allergic effect.

The role of celastrol, a triterpene extracted from the Chinese "Thunder of God Vine," in allergic inflammation was investigated. Celastrol decreased the secretion of beta-hexosaminidase, decreased the release of histamine, decreased the expression of Th2 cytokines and decreased calcium influx and cell adhesion in antigen-stimulated RBL2H3 cells. Exposure to celastrol decreased the phosphorylation of extracellular regulated kinase (ERK) and the ERK kinase activity was decreased in RBL2H3 cells. A molecular dynamics simulation showed binding of celastrol to a large pocket in ERK2, which serves as the ATP-binding site. Exposure to celastrol inhibited the interaction between immunoglobulin Fc epsilon receptor I (FcepsilonRIgamma) and ERK and inhibited interaction between FcepsilonRIgamma and protein kinase C delta (PKCdelta). Antigen stimulation induced an interaction between Rac1 and ERK as well as an interaction between Rac1 and PKCdelta. Inhibition of ERK decreased Rac1 activity and inhibition of Rac1 decreased ERK activity in antigen-stimulated RBL2H3 cells. Celastrol regulated the expression of epithelial-mesenchymal transition (EMT)-related proteins through inhibition of PKCalpha, PKCdelta, and Rac1 in antigen-stimulated RBL2H3 cells. Exposure to celatrol inhibited PKCdelta activity in antigen-stimulated RBL2H3 cells. Celastrol exerted a negative effect on FcepsilonRIbeta signaling by inhibiting the interaction between heat shock protein 90 (hsp90) and proteins, such as, FcepsilonRIbeta, Akt and PKCalpha. Celastrol exerted a negative effect on in vivo atopic dermatitis induced by 2, 4-dinitrofluorobenzene (DNFB), which requires ERK. Celastrol also showed an inhibitory effect on skin inflammation induced by phorbol myristate acetate (PMA) in Balb/c mice. In summary, celastrol binds to ERK and inhibits FcepsilonRI signaling to exert an anti-inflammatory effect.

Increased tissue transglutaminase expression in human atherosclerotic coronary arteries.

BACKGROUND AND OBJECTIVE: Transglutaminase 2 (TGase 2) is a calcium-dependent cross-linking enzyme that catalyzes a covalent iso-peptide bond between two proteins. Interestingly, this catalysis can activate the nuclear factor-kappaB (NF-kappaB) through the polymerization of the inhibitory protein of NF-kappaB (I-kappaB). The objective of the present study was to investigate the expression of TGase 2 in the human atherosclerotic human coronary artery, and the possible roles of TGase 2 in NF-kappaB activation. METHODS AND RESULTS: We explored whether expressions of TGase 2 and NF-kappaB are associated in atherosclerosis. Using human samples, we found that TGase 2 was markedly higher than normal in the neointimal tissue of atherosclerotic coronary arteries with atherosclerosis progression. TGase 2 activity was also increased approximately two-fold in the atherosclerotic vascular wall. In immunofluorescence analysis, NF-kappaB, COX-2, and TNF-alpha were co-localized at TGase 2-positive neointimal smooth muscle cells. A promoter assay test showed that NF-kappaB activity increased in both the human monocyte and human breast carcinoma cell by TGase 2, and that TGase 2-mediated NF-kappaB activation was reversed by TGase 2 siRNA. CONCLUSION: According to these results, we suggest that TGase 2 may function as an activator in the NF-kappaB pathway; this effect may occur in the atherosclerotic vessel wall.

Tissue transglutaminase-induced down-regulation of matrix metalloproteinase-9.

Tissue transglutaminase (TGase 2) has been reported to have multiple functions in addition to its function as a biological adhesive. To identify its roles, we investigated the effects of TGase 2 on gelatinase activity. The MMP-9 activity of certain cell lines was significantly inhibited with retinoic acid treatment, and this effect was reversed in the presence of a TGase 2 inhibitor. Furthermore, TGase 2 overexpression reduced the MMP-9 protein expression levels and inhibited its activity in both culture media and cell lysate. The decreased mRNA levels of MMP-9 and the results of a promoter assay revealed that TGase 2 may be involved in MMP-9 transcription. Further, data obtained in an immunoprecipitation assay and an electrophoretic mobility shift assay demonstrated that TGase 2 binds to c-Jun and suppresses its binding activity toward AP-1. These results suggest that TGase 2 inhibits MMP-9 via downregulation of MMP-9 transcription activity by blocking the binding of the Jun-fos complex to an AP-1 site.

Hyaluronic acid targets CD44 and inhibits FcepsilonRI signaling involving PKCdelta, Rac1, ROS, and MAPK to exert anti-allergic effect.

Effects of hyaluronic acid (HA) on allergic inflammation were investigated. HA exerted negative effects on beta-hexoaminidase secretion and histamine release in antigen-stimulated rat basophilic leukemia (RBL2H3) cells. HA inhibited interaction between IgE and FcepsilonRI and between FcepsilonRI and PKCdelta. HA inhibited CD44 interaction with PKCalpha, indicating that HA targets CD44. PKCalpha and -delta were responsible for increased Rac1 activity and expression of p47(phox), p67(phox). HA inhibited phosphorylation of PKCalpha and -delta. Rac1 was responsible for increased ROS, and NADPH oxidase was the main source for ROS. The inhibition of PKC prevented antigen from increasing phosphorylation of ERK and p38 MAPK. ERK, p38 MAPK, and ROS, were responsible for secretion of beta-hexosaminidase, histamine release, and induction of chemokines. HA suppressed induction of chemokines, such as MIP-2 and Sprr-2a. CD44 mediated effect of antigen on phosphorylation of ERK, p38MAPK, ROS production, secretion of beta-hexosaminidase, and histamine release. GPCR did not mediate allergic function of antigen or affect anti-allergic function of HA. In vivo anti-allergic effect of HA was investigated using Nc/Nga mice model of DNFB-induced atopic dermatitis. HA reduced skin lesions in Nc/Nga mice treated with DNFB, decreased expression levels of MIP-2, Sprr-2a, and serum IgE level. In conclusion, hyaluronic acid exerts negative effect on allergic inflammation by targeting CD44 and inhibiting FcepsilonRI signaling.

The anti-inflammatory activity of Phellinus linteus (Berk. & M.A. Curt.) is mediated through the PKCdelta/Nrf2/ARE signaling to up-regulation of heme oxygenase-1.

It has been reported that heme oxygenase-1 (HO-1) mediates the anti-inflammatory activity of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. This continuing work aimed to elucidate the signaling pathway to the up-regulation of HO-1 by PL. In RAW264.7 macrophage cells, PL was able to enhance phosphorylation of protein kinase Cdelta (PKCdelta), but not PKCalpha/betaII, in a time-dependent manner. PL-induced HO-1 expression was dramatically released by GF109203X, a general inhibitor of PKC, and rottlerin, a specific PKCdelta inhibitor but not by Gö6976, a selective inhibitor for PKCalpha/beta. Additionally, PL treatment resulted in a marked increase in antioxidant response element (ARE)-driven transcriptional activity, which was dependent on PKCdelta but not PKCalpha. An increase by PL treatment in the ARE-driven transcriptional activity was further enhanced by Nrf2, whereas it was diminished by Keap1. Furthermore, pretreatment of rottlerin and overexpression of PKCdelta (K376R), a kinase-inactive form of PKCdelta, partly blocked the suppression by PL of nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and iNOS promoter activity, which were elevated in the lypopolysaccharide (LPS)-activated macrophages. Similarly, expression of matrix metalloproteinase-9 (MMP-9) and its promoter activity were suppressed by PL, which were dependent upon PKCdelta. The present findings indicate that Phellinus linteus gives rise to an anti-inflammatory activity though the PKCdelta/Nrf2/ARE signaling to the up-regulation of HO-1 in an in vitro inflammation model.

A splice variant of CD99 increases motility and MMP-9 expression of human breast cancer cells through the AKT-, ERK-, and JNK-dependent AP-1 activation signaling pathways.

The CD99 gene encodes two distinct transmembrane proteins by alternative splicing of its transcript. To examine the effects of two CD99 isoforms on the invasive phenotypes of breast cancer cells, MDA-MB-231 and MCF-7 human breast cancer cell lines were stably transfected with CD99 cDNAs encoding the major wild-type form (type I) or a minor splice variant (type II). As a result, expression of CD99 type II, but not type I, markedly elevated the motility, binding to fibronectin, MMP-9 expression, and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. In MDA-MB-435 breast cancer cells expressing both CD99 type I and type II, invasion-related cellular activities were inhibited by the transfection of small interfering RNA (siRNA) targeted to CD99 type II. Meanwhile, CD99 type II-induced MMP-9 expression in MDA-MB-231 cells was shown to be mediated by the binding of AP-1 factors to the MMP-9 gene promoter. Gel shift assay revealed that ligation of CD99 type II with antibody resulted in the binding of JunD to the AP-1 site of the MMP-9 promoter region. Initiation of CD99 type II signaling by antibody ligation increased expression of JunD and FosB AP-1 factors, along with phosphorylation of Src, Akt, p38 MAPK, ERK, and JNK. Knockdown of JunD and FosB by siRNA transfection abolished the positive effects of CD99 type II on the motility and MMP-9 expression of MDA-MB-231 cells. Increased expression of JunD and FosB as well as elevated cell motility and MMP-9 expression by CD99 type II ligation were also abrogated by inhibitors, dominant-negative forms, and siRNAs for Akt1, ERK1/2, and JNK1 but not for p38 MAPK. These results suggest that expression of a splice variant of CD99 contributes to the invasive ability of human breast cancer cells by up-regulating AP-1-mediated gene expression through the Akt-dependent ERK and JNK signaling pathways.