Borrelia burgdorferi infection regulates CD1 expression in human cells and tissues via IL1-β

The appearance of group 1 CD1 proteins (CD1a, CD1b and CD1c) on maturing myeloid DC is a key event that converts myeloid DC to effective lipid APC. Here, we show that Borrelia burgdorferi, the causative agent of Lyme disease, triggers appearance of group 1 CD1 proteins at high density on the surface of human myeloid DC during infection. Within human skin, CD1b and CD1c expression was low or absent prior to infection, but increased significantly after experimental infections and in erythema migrans lesions from Lyme disease patients. The induction of CD1 was initiated by borrelial lipids acting through TLR-2 within minutes, but required 3 days for maximum effect. The delay in CD1 protein appearance involved a multi-step process whereby TLR-2 stimulated cells release soluble factors, which are sufficient to transfer the CD1-inducing effect in trans to other cells. Analysis of these soluble factors identified IL-1β as a previously unknown pathway leading to group 1 CD1 protein function. This study establishes that upregulation of group 1 CD1 proteins is an early event in B. burgdorferi infection and suggests a stepwise mechanism whereby bacterial cell walls, TLR activation and cytokine release cause DC precursors to express group 1 CD1 proteins.     

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK)—endogenous negative regulators of Src-family protein kinases

C-terminal Src kinase (CSK) and CSK-homologous kinase (CHK) are endogenous inhibitors of the Src-family protein tyrosine kinases (SFKs). Since constitutive activation of SFKs contributes to cancer formation and progression, to prevent excessive activation of SFKs, their activity in normal cells is kept at the basal level by CSK and CHK. CSK and CHK inactivate SFKs by specifically phosphorylating a consensus tyrosine (called Y_T) near their C-termini. Upon phosphorylation, the phospho-Y_T engages in intramolecular interactions that lock the SFK molecule in an inactive conformation. SFKs are anchored to the plasma membrane, while CSK and CHK are localized predominantly in the cytosol. To inhibit SFKs, CSK and CHK need to translocate to the plasma membrane. Recruitment of CSK and CHK to the plasma membrane is mediated by the binding of their SH2, SH3 and/or kinase domains to specific transmembrane proteins, G-proteins and adaptor proteins located near the plasma membrane. For CSK, membrane recruitment often accompanies activation. CSK and CHK employ two types of direct interactions with SFKs to achieve efficient Y_T phosphorylation: (i) short-range interactions involving binding of the active sites of CSK and CHK to specific residues near Y_T, (ii) long-range non-catalytic interactions involving binding of SFKs to motifs located distally from the active sites of CSK and CHK. The interactions between CSK and SFKs are transient in nature. Unlike CSK, CHK binds tightly to SFKs to form stable protein complexes. The binding is non-catalytic as it is independent of Y_T. More importantly, the tight binding alone is sufficient to completely inhibit SFKs. This non-catalytic inhibitory binding represents a novel mechanism employed by CHK to inhibit SFKs. Given that SFKs are implicated in cancer development, compounds mimicking the non-catalytic inhibitory mechanism of CHK are potential anti-cancer therapeutics.     

Differential regulation of monocyte cytokine release by αV and β(2) integrins that bind CD23

The human soluble CD23 (sCD23) protein displays highly pleiotropic cytokine-like activity. Monocytic cells express the sCD23-binding integrins αVβ(3), αVβ(5), αMβ(2) and αXβ(2), but it is unclear which of these four integrins most acutely regulates sCD23-driven cytokine release. The hypothesis that ligation of different sCD23-binding integrins promoted release of distinct subsets of cytokines was tested. Lipopolysaccharide (LPS) and sCD23 promoted release of distinct groups of cytokines from the THP-1 model cell line. The sCD23-driven cytokine release signature was characterized by elevated amounts of RANTES (CCL5) and a striking increase in interleukin-8 (IL-8; CXCL8) secretion, but little release of macrophage inflammatory protein 1β (MIP-1β; CCL4). Antibodies to αVβ(3) or αXβ(2) both promoted IL-8 release, consistent with the sCD23-driven pattern, but both also evoked strong MIP-1β secretion; simultaneous ligation of these two integrins further increased cytokine secretion but did not alter the pattern of cytokine output. In both model cell lines and primary tissue, integrin-mediated cytokine release was more pronounced in immature monocyte cells than in mature cells. The capacity of anti-integrin monoclonal antibodies to elicit a cytokine release response is epitope-dependent and also reflects the differentiation state of the cell. Although a pattern of cytokine release identical to that provoked by sCD23 could not be elicited with any individual anti-integrin monoclonal antibody, αXβ(2) and αVβ(3) appear to regulate IL-8 release, a hallmark feature of sCD23-driven cytokine secretion, more acutely than αMβ(2) or αVβ(5).     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Low-intensity pulsed ultrasound stimulates matrix metabolism of human annulus fibrosus cells mediated by transforming growth factor β1 and extracellular signal-regulated kinase pathway

Purpose: There are limited strategies to restore the damaged annulus fibrosus (AF) of the intervertebral disc. Low-intensity pulsed ultrasound (LIPUS) has positive effects on the proliferation of several types of cells and the repair of damage tissue in vivo. However, scientific evidence of therapeutic effects of LIPUS on AF cells remains limited. The purpose of this study is to evaluate the feasibility of applying LIPUS to the repair of the AF.

Materials and methods: We used an in vitro model of human AF cells subjected to LIPUS stimulation to examine its effects on cell proliferation and matrix metabolism. Cell viability, synthesis of collagen and glycosaminoglycan (GAG), expression of matrix metalloproteinases (MMPs) and transforming growth factor β1 and pathways involving mitogen-activated protein kinases (MAPKs) were investigated.

Results: LIPUS significantly enhanced proliferation of AF cells after 5 days of treatment. LIPUS with an intensity of 0.5?W/cm² increased the collagen and GAG synthesis and decreased the expressions of MMP-1 and -3 of human AF cells. Real-time polymerase chain reactions and western blotting analysis revealed that LIPUS could increase transforming growth factor β1 (TGF-β1) and activate extracellular signal-regulated kinase (ERK) pathway. In addition, TGF-β receptor kinase inhibitor could suppress the ultrasound-induced alterations in cell viability and matrix metabolism.

Conclusions: The findings suggested that LIPUS could be useful as a physical stimulation of cell metabolism for the repair of the AF.     

Differential expression of β1, β3 and β4 integrins in sarcomas of the small,round, blue cell category

Integrins are a large and complex family of membrane spanning heterodimeric cell surface glycoproteins mediating cell/cell and cell/matrix interactions. Small, round, blue cell sarcomas (SRBCS) are a group of poorly differentiated tumours of various and in part uncertain histogenesis displaying similar cytomorphology. Among them are rhabdomyosarcomas (RMS), ganglioneuroblastomas [(G)NB], primitive peripheral neuroectodermal tumours (pPNET) and Ewing's sarcomas (ES). Thirty-two SRBCS were studied immunohistochemically for the distribution of 1, 3 and 4 integrins in situ. We found complex and to some extent differential patterns of 1, 3 and 4 integrin subunit expression in different types of SRBCS: all of the sarcomas studied were consistently 1⁺, 4^–, 2^–. Four of nine RMS were completely negative for all other integrin subunits studied while one RMS was 5⁺ throughout and three RMS were focally 5⁺. Three RMS expressed the 6 and v chains. In contrast to RMS, pPNET and ES, all of which were 1^–, 3^–, (G)NB were 3⁺ and frequently co-expressed 1. The eight pPNET and seven ES studied showed a similarily restricted integrin profile that was limited to the expression of 1 and 5 in nearly all cases. In summary, RMS were 1⁺, 1^–, 3^– and heterogeneously expressed 5 and 6. (G)NB were generally 1⁺, 1⁺, 3⁺, 5^–, 6^–. pPNET and ES were 1⁺, 1^–, 3^–, 5⁺, 6^–. The data illustrate a complex expression pattern of various integrins in SRBCS, a differential expression pattern of some of the integrin subunits among different types of SRBCS and almost identical integrin profiles in pPNET and ES.This paper is dedicated to Prof. Dr. Dres. h.c. Wilhelm Doerr on the occasion of his 80^th birthday     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Transforming growth factor β1 acts as an inducer of matrix metalloproteinase expression and activity in human bone-metastasizing cancer cells

Bone metastases are a common complication in prostate and breast cancer patients. It leads to extensive morbidity and eventually mortality. Matrix metalloproteinases (MMPs) are known to be involved in the metastatic process. MMP activity can be down-regulated by transforming growth factor 1 (TGF-1), a growth-modulating factor, found in high concentrations in the bone. TGF-1 acts through the TGF-1 inhibitory element (TIE) element, a cis-acting element found in the promoter region of most MMP genes, with the exception of MMP-2. We used three human cell lines relevant for bone metastases, namely prostate adenocarcinoma PC-3, breast adenocarcinoma MDA-MB-231, and adenocarcinoma cells of unknown origin, Hs696, and one human osteosarcoma cell line, SAOS-2, and showed that in these cell lines TGF-1 partially lost its repressing action on MMP expression. TGF-1 was able to induce MMP-9 activity and protein expression in all three bone-metastatic tumour cell types, whereas MMP-9 protein levels were repressed in SAOS-2 cells. In PC-3 cells, TGF-1 repressed MMP-1 expression, whereas in MDA-MB-231 and SAOS-2 cells, an increase in the expression of MMP-1 protein was detected. Additionally, an increase in MMP-3 expression was observed in Hs696 cells. Expression and activity of the tissue inhibitors of matrix metalloproteinases, TIMP-1 and TIMP-2, were found increased in both PC-3 and MDA-MB-231 cells. With respect to cell proliferation, TGF-1 was able to induce a dose-dependent growth inhibition of up to 50% in primary human mammary epithelial cells. However, in none of the tumour cell lines was TGF-1 able to suppress growth substantially. Data presented in this paper support the hypothesis that TGF-1 can potentially disrupt the balance existing between osteoclast- and osteoblast-derived MMP activity by inducing altered expression of matrix metalloproteinases and their tissue inhibitors derived from bone-metastasizing cancer cells. This could eventually lead to skeletal destruction in patients with advanced metastatic disease.     

Western gray squirrel (Rodentia: Sciuridae): a primary reservoir host of Borrelia burgdorferi in Californian oak woodlands?

In California, dense woodlands have been recognized as important biotopes where humans are exposed to the nymphal stage of the western blacklegged tick, Ixodes pacificus Cooley & Kohls, the primary vector of the Lyme disease spirochete Borrelia burgdorferi sensu stricto (s.s.), in the far-western United States. To identify the principal reservoir host(s) of this spirochete, and of closely related spirochetes in the B. burgdorferi sensu lato (s.l.) complex, in dense woodlands in Mendocino County, California, approximately 50 species of birds and mammals, including wood rats and kangaroo rats, were evaluated as potential hosts for vector ticks and borreliae in 2002 and 2003. Although polymerase chain reaction (PCR) and sequencing analyses revealed that many vertebrate species had been exposed to one or more members of the B. burgdorferi s.l. spirochetal complex, only the western gray squirrel, Sciurus griseus, fulfilled the major criteria for a reservoir host of B. burgdorferi s.s. Ear-punch biopsies from eight of 10 squirrels collected from five separate woodlands were PCR-positive for B. burgdorferi s.s., 47% of I. pacificus larvae (n = 64) and 31% of nymphs (n = 49) removed from squirrels contained B. burgdorferi s.l., and the engorgement status of I. pacificus larvae was associated positively with acquisition of spirochetes. Overall, 83 and 100% of the amplicons sequenced from PCR-positive I. pacificus larvae and nymphs, respectively, were identified as B. burgdorferi s.s, Among the five remaining positive I. pacificus larvae, three contained B. bissettii and two had uncharacterized B. burgdorferi s.l. Borrelia burgdorferi s.s. was detected in one of five larvae and zero of two nymphs of the Pacific Coast tick, Dermacentor occidentalis Marx, that likewise had been removed from squirrels. The rickettsial agent of human anaplasmosis, Anaplasma phagocytophilum, was detected in the blood or ear biopsies of two squirrels and in one (1.6%) of 64 I. pacificus larvae and two (4.1%) of 49 nymphs obtained from squirrels. The one rickettsial-positive larva was coinfected with B. burgdorferi s.s. The apparently high reservoir potential of S. griseus for B. burgdorferi s.s., plus the fact that the geographic distribution of this squirrel coincides well with that of most reported human cases of Lyme disease in this region, indicated that it may be essential for maintaining foci of B. burgdorferi s.s. in certain types of woodlands. The findings with respect to A. phagocytophilum, although of less certain significance, suggest that S. griseus could serve as a secondary host of this rickettsia.     

Cytokines (IL-4 and IFN-γ) and antibodies (IgE and IgG2a) produced in mice infected with Borrelia burgdorferi sensu stricto via nymphs of Ixodes ricinus ticks or syringe inoculations

Mice were tolerant to tick bites during three infestations with nymphs of Ixodes ricinus infected with Borrelia burgdorferi sensu stricto. To determine whether tick bites influence the immune response against B. burgdorferi, we examined the production of cytokines IL-4 and IFN-γ by lymph node cells of BALB/c mice and IL-4 deficient BALB/c mice after tick inoculation versus syringe inoculation of B. burgdorferi. We also measured IgG2a anti-borrelial antibodies and total IgE in these mice. Results showed that BALB/c mice developed a Th2 immune response against B. burgdorferi after tick inoculation and a mixed Th1/Th2 response after syringe inoculation of B. burgdorferi. IL-4 deficient mice produced a Th1 immune response in both cases. IL-4 produced following tick bites greatly decreased the production of anti-borrelial IgG2a antibodies by comparison with the production of anti-borrelial IgG2a antibodies produced following syringe injection of B. burgdorferi. Received: 23 November 1999 / Accepted: 9 December 1999     

Resveratrol: Antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and β-thalassemia patients

Thalassemia and sickle-cell anemia (SCA) present a major public health problem in countries where the number of carriers and affected individuals is high. As a result of the abnormalities in hemoglobin production, cells of thalassemia and SCA patients exhibit oxidative stress, which ultimately is responsible for the chronic anemia observed. Therefore, identification of compounds exhibiting both antioxidant and hemoglobin-inducing activities is highly needed. Our results demonstrate resveratrol to be such a compound. This was shown both in the human K562 cell line, as well as in erythroid precursors derived from normal donors and β-thalassemia patients. Resveratrol was shown to exhibit antioxidant activity and to stimulate the expression of the γ-globin genes and the accumulation of fetal hemoglobin (HbF). To the best of our knowledge, this is the first report pointing to such a double effect of resveratrol. Since this natural product is already marketed as an antioxidant, future investigations should concentrate on demonstrating its potential to augment HbF production in experimental animal models (e.g., thalassemia and SCA mice) as well as in patients. We believe that the potential of clinical use of resveratrol as an antioxidant and HbF stimulator may offer a simple and inexpensive treatment to patients.     

Reversal of diabetes by βTC3 cells encapsulated in alginate beads generated by emulsion and internal gelation

Encapsulation of insulin-producing cells in alginate beads could improve the treatment of type 1 diabetes by reducing or eliminating the need for immunosuppression. We have recently adapted an emulsion and internal gelation process to β-cell encapsulation. This process has the advantages of being well suited for m(3)/h production rates and allowing the use of increased alginate concentrations. Compared with 1.5% alginate beads generated by a standard extrusion process, 5% alginate emulsion-generated beads demonstrated greater in vitro stability and greater volumetric exclusion of antibody-sized pullulan. When βTC3 cells were transplanted into streptozotocin-induced allogeneic diabetic mice, a significant decrease in the blood glucose levels was seen within 2 days with the 5% emulsion-generated beads but not until >16 days with the 1.5% extrusion-generated beads. This was correlated with higher cell survival and lower graft-specific plasma immunoglobulin levels. These results suggest that higher-concentration alginate beads generated by emulsion and internal gelation have improved graft immunoprotection. The emulsion process is a promising and scalable technology for cellular therapies requiring immune isolation.     

Site-specific regulation of CAV2.2 channels by protein kinase C isozymes βII and ε

Ca_v2.2 high voltage-gated calcium channels are regulated by phorbol-12-myristae, 13-acetate (PMA) via Ser/Thr protein kinase C (PKC) phosphorylation sites in the I–II linker and C-terminus of the α₁ 2.2 subunit. Here we show that PMA enhancement of Ca_v2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC βII or PKC ε isozymes, as shown previously for Ca_v2.3 currents, and that microinjection of PKC βII or PKC ε isozymes in the oocytes expressing the WT Ca_v2.2 channels increases the basal barium current (I_Ba). The I–V plot shows a large increase in current amplitude with PKC βII and PKC ε isozymes with only a small shift in the peak I_Ba in the hyperpolarizing direction. The potentiation of Ca_v2.2 currents by microinjection of PKC βII and PKC ε isozymes was not altered by the inhibition of G proteins with GDPβS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of α₁ 2.2 subunit revealed that PKC βII or PKC ε isozymes (but not PKC α or δ) can provide full enhancement through the stimulatory site (Thr-422) in the I–II linker but that PKC ε is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC βII or ε at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC βII also enhances Ca_v2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC ε provides lesser enhancement with C-terminal sites and only with Ser-2108 and Ser-2132. Sites Ser-1757 and Ser-2132, but not Ser-2108, are dominant over the inhibitory site Ser-425. Collectively, these results reveal a hierarchy of regulatory sites in Ca_v2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.     

Expansion of human trophoblastic spheroids is promoted by decidualized endometrial stromal cells and enhanced by heparin-binding epidermal growth factor-like growth factor and interleukin-1 β

Successful pregnancy in humans depends on deep invasion of the maternal decidua by extravillous trophoblast cells (EVTs), a process regulated by autocrine and paracrine signals in the decidual-trophoblast microenvironment. Here we examined whether trophoblast invasion is affected by decidual differentiation of endometrial stromal cells (ESC) and modulated locally by cytokines and growth factors. Trophoblast spheroids were generated from the EVT-derived cell line AC-1M88 and placed onto monolayers of either undifferentiated or decidualized ESC, or directly onto tissue culture surface. Co-cultures were treated with epidermal growth factor (EGF), hepatocyte growth factor, heparin-binding EGF-like growth factor (HB-EGF), interleukin-1β (IL-1β) and leukaemia inhibitory factor (LIF). Expansion of spheroids over 2-3 days was significantly enhanced by a monolayer of undifferentiated ESC compared with tissue culture surface and further increased if ESC had been decidualized. HB-EGF and IL-1β, alone or in combination with LIF, stimulated spheroid expansion but only on undifferentiated ESC. CEACAM1, an adhesion molecule implicated in trophoblast invasion, was up-regulated in AC-1M88 cells by conditioned medium from decidualized ESC, and by HB-EGF, IL-1β and LIF in combination. Treatment of ESC with HB-EGF or IL-1β increased the level of the tetraspanin CD82, a metastasis suppressor found in decidual cells at the implantation site. We suggest that decidualized ESC support trophoblast invasion by paracrine signals that may include HB-EGF, IL-1β and LIF.     

Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-β and TNF-α in 3D culture

Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C–C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p = 0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-β and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p < 0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p < 0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.     

Induction of β-catenin by the suppression of signal regulatory protein α1 in K562 cells

The signal regulatory protein (SIRP) α1 is a cell surface receptor expressed predominantly in monocytes, granulocytes, dendritic cells, as well as hematopoietic stem cells. In contrast, SIRPα1 expression is significantly reduced in the majority of myeloid malignancies. SIRPα1 is a negative regulator of signaling and its reduced expression is considered to play a role in the pathogenesis of these diseases through aberrant signaling. To identify SIRPα1 downstream target genes, we established SIRP α1-knockdown chronic myeloid leukemia K562 (K562SIRPα1KD) cells expressing reduced levels of SIRPα1 by stably transfecting SIRPα1 siRNAs. Microarray analysis demonstrated that several genes, including β-catenin, were significantly induced in K562SIRPα1KD cells. Real-time PCR and Western blot analyses, confirmed the induction of this gene. Phosphorylation of Ser9 of glycogen synthesis kinase (GSK) -3β, results in the inactivation of GSK-3β, leading to the induction of β-catenin. We found significant phosphorylation of extracellular signal-regulated kinase (ERK), Akt, as well as of GSK-3β-Ser9, which may play a role in the up-regulation of β-catenin in K562SIRPα1KD cells. To our knowledge, this is a first report demonstrating the relationships between SIRPα1 and β-catenin in leukemia cells.     

Inhibition of c-Jun N-terminal kinase and nuclear factor κ B pathways mediates fisetin-exerted anti-inflammatory activity in lipopolysccharide-treated RAW264.7 cells

Although fisetin, a natural flavonoid, was known to inhibit proliferation, carcinogenesis and inflammation, the underlying anti-inflammatory mechanism of fistein still remains unclear. Thus, in the present study, the anti-inflammatory mechanism of fisetin was investigated in association with mitogen-activated protein kinase (MAPK) and nuclear factor κ B (NF-κB) pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophages. We found that fisetin significantly reduced the nitrate oxide (NO) production and also inhibited the expression of pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) at protein and mRNA levels in LPS-stimulated cells. Consistently, fisetin significantly reduced the LPS-stimulated secretion of proinflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor α (TNF-α). Furthermore, fisetin suppressed the activation of nuclear factor κ B (NF-κB) and the phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal regulated kinase (ERK) and p38 MAPK in LPS-treated RAW264.7 cells. Overall, our findings demonstrate that fisetin exerted anti-inflammatory activity via inactivation of JNK and NF-κB in LPS-stimulated macrophage cells.     

cAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic β-cells

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.     

Inhibition of mitochondrial respiration and rapid depletion of mitochondrial glutathione by β-phenethyl isothiocyanate: mechanisms for anti-leukemia activity

AIMS: β-Phenethyl isothiocyanate (PEITC) is a natural product with potent anticancer activity against human leukemia cells including drug-resistant primary leukemia cells from patients. This study aimed at investigating the key mechanisms that contribute to the potent anti-leukemia activity of PEITC and at evaluating its therapeutic potential. RESULTS: Our study showed that PEITC caused a rapid depletion of mitochondrial glutathione (GSH) and a significant elevation of reactive oxygen species (ROS) and nitric oxide, and induced a disruption of the mitochondrial electron transport complex I manifested by an early degradation of NADH dehydrogenase Fe-S protein-3 and a significant suppression of mitochondrial respiration. Using biochemical and pharmacological approaches, we further showed that inhibition of mitochondrial respiration alone by rotenone caused only a moderate cytotoxicity in leukemia cells, whereas a combination of respiratory inhibition and an ROS-generating agent exhibited a synergistic effect against leukemia and lymphoma cells. INNOVATION AND CONCLUSION: Although PEITC is a reactive compound and might have multiple mechanisms of action, we showed that a rapid depletion of GSH and inhibition of mitochondrial respiration are two important early events that induced synergistic cytotoxicity in leukemia cells. These findings not only suggest that PEITC is a promising compound for potential use in leukemia treatment, but also provide a basis for developing new therapeutic strategies to effectively kill leukemia cells by using a novel combination to modulate ROS and inhibit mitochondrial respiration.