IL-1beta stimulates rat cardiac fibroblast migration via MAP kinase pathways

The pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) are elevated following acute myocardial infarction (MI) and have been implicated in the pathophysiology of cardiac disease progression. The cardiac fibroblast represents an important effector cell target for cytokine actions. In particular, cytokine-directed cardiac fibroblast migration is likely to impact both myocardial repair following acute MI and pathological myocardial remodeling in the progression to heart failure. In the present study, we examined the migratory response of neonatal rat cardiac fibroblasts to pro-inflammatory cytokines using modified Boyden chamber assays. On the basis of the knowledge of migration in other cell types, we hypothesized that members of the mitogen-activated protein kinase (MAPK) family may regulate this process. This possibility was addressed with the use of immunoblot detection of active phosphorylated MAPK species and pharmacological inhibitors for individual members of the MAPK cascades. IL-1beta stimulated robust and concentration-dependent increases in migration (maximum, 20-fold over control cells). TNF-alpha had lesser effect (fourfold increase over control). IL-6 did not induce migration. Activation of all three MAPK subfamilies (extracellular signal-regulated kinases, c-Jun NH(2)-terminal kinases, and p38) was shown to occur in response to cytokine stimulation. Fibroblast migration was attenuated by pharmacological inhibition of each MAPK subfamily. Understanding the regulation of cardiac fibroblast migration may provide insights in the search for therapies aimed at enhancing the functional nature of the remodeling process.     

Vasoactive intestinal peptide induces IL-8 production in human colonic epithelial cells via MAP kinase-dependent and PKA-independent pathways

Vasoactive intestinal peptide (VIP) has been shown to be a key regulator of intestinal epithelial functions such as mucus and chloride secretion, paracellular permeability, and cell proliferation. However, its regulatory role in intestinal epithelial chemokine production remains unknown. The aim of this study was (1) to determine whether VIP can modulate intestinal epithelial interleukin-8 (IL-8) production and (2) to identify intracellular mediators responsible for this effect. In the human colonic epithelial cell line HT29-Cl.16E, VIP stimulates IL-8 secretion dose-dependently and IL-8 mRNA level at 10(-9) M. The protein kinase A (PKA) inhibitor PKI did not abolish the effect of VIP. However, inhibition of the ERK1/2 and p38 MAPK pathways reduced the VIP-stimulated IL-8 secretion and mRNA level. Together, our results showed that VIP stimulates IL-8 production in intestinal epithelial cells via PKA-independent and MAPK-dependent pathways. These data suggest that VIPergic pathways can play an immunomodulatory role in intestinal epithelial cells, by regulating epithelial IL-8 secretion.     

Probiotic Lactobacillus casei activates innate immunity via NF-kappaB and p38 MAP kinase signaling pathways

Probiotic bacteria are microorganisms that benefit the host through improvement of the balance of intestinal microflora and possibly by augmentation of host defense systems. We examined the mechanisms for the up-regulation of innate immune responses by a probiotic Lactobacillus casei ATCC27139, in vivo. Using mouse models of systemic Listeria monocytogenes infection and MethA fibrosarcoma tumorigenesis in combination with BALB/c and SCID mice, we found that parenteral administration of L. casei ATCC27139 confers a protective effect against L. monocytogenes infection and anti-tumor activity against MethA fibrosarcoma by activation of innate immunity, while L. casei ATCC27139-J1R strains, which are J1 phage-resistant strains that have been selected from MNNG-treated clones, lacked these activities. Substantial differences between ATCC27139 and ATCC27139-J1R strains were observed in the capacity to induce innate cytokines such as TNF-alpha, IL-12, IL-18, and IFN-gamma, and pathogen-associated molecular pattern receptors, TLR2 and Nod2, by spleen cells. In addition, although phosphorylation of NF-kappaB p65 in spleen was equally enhanced in the ATCC27139- and the ATCC27139-J1R-treated groups, phosphorylation of both p38 MAPK and MAPKAPK-2 was significantly induced only by ATCC27139. Furthermore, inhibitors of NF-kappaB (sulfasalazine) and p38 MAPK (SB203580) significantly reduced cytokine production by the spleen cells of the mice treated with L. casei ATCC27139, suggesting that both NF-kappaB and p38 MAPK signaling pathways play important roles in the augmentation of innate immunity by the probiotic L. casei.     

hKSR-2 inhibits MEKK3-activated MAP kinase and NF-kappaB pathways in inflammation

Kinase suppressor of ras (KSR) and MEKK3 (MAP kinase kinase kinase) are integral members of the MAP kinase pathway. We have recently identified a new isoform of the KSR family named human kinase suppressor of ras-2 (hKSR-2), and demonstrated that hKSR-2 negatively regulates Cot, a MAP3K family member which is important in inflammation and oncogenesis [P.L. Channavajhala, L. Wu, J.W. Cuozzo, J.P. Hall, W. Liu, L.L. Lin, Y. Zhang, J. Biol. Chem. 278 (2003) 47089-47097]. In this report, we provide evidence that hKSR-2 also regulates the activity of MEKK3 (another MAP3K family member) in HEK-293T cells. We demonstrate that hKSR-2 is a negative regulator of MEKK3-mediated activation of MAP kinase (specifically ERK and JNK) and NF-kappaB pathways, and concurrently inhibits MEKK3-mediated interleukin-8 production. We find that while hKSR-2 blocks MEKK3 activation, it has little to no effect on other members of the MAP3K family, including MEKK4, TAK1, and Ras-Raf, suggesting that its effects are selective.     

Ghrelin stimulates interleukin-8 gene expression through protein kinase C-mediated NF-kappaB pathway in human colonic epithelial cells

Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone (GH) release and appetite. Although ghrelin is involved in several other responses such as stress and intestinal motility, its potential role in intestinal inflammation is not clear. Here, we show that expression of ghrelin and its receptor mRNA is significantly increased during acute experimental colitis in mice injected intracolonically with trinitrobenzene sulfate (TNBS). We found by PCR that ghrelin receptor mRNA is expressed in non-transformed human colonic epithelial NCM460 cells. Exposure of NCM460 cells stably transfected with ghrelin receptor mRNA to ghrelin, increased IkappaBalpha phosphorylation and its subsequent degradation. In addition, ghrelin stimulated NF-kappaB-binding activity and NF-kappaB p65 subunit phosphorylation, and induced IL-8 promoter activity and IL-8 protein secretion. Furthermore, our data show that ghrelin-induced IkappaBalpha and p65 phosphorylation was markedly reduced by pharmacological inhibitors of intracellular calcium mobilization (BAPTA/AM) and protein kinase C (GF 109203X). Pretreatment with BAPTA/AM or GF109203X also significantly attenuated ghrelin-induced IL-8 production. Together, our results strongly suggest that ghrelin may be a proinflammatory peptide in the colon. Ghrelin may participate in the pathophysiology of colonic inflammation by inducing PKC-dependent NF-kappaB activation and IL-8 production at the colonocyte level.     

Neurotensin stimulates IL-8 expression in human colonic epithelial cells through Rho GTPase-mediated NF-kappa B pathways

Neurotensin (NT), a neuropeptide highlyexpressed in the gastrointestinal tract, participates in thepathophysiology of intestinal inflammation. We recently showed that NTstimulates interleukin-8 (IL-8) expression in NCM460 nontransformedhuman colonic epithelial cells via both mitogen-activating proteinkinase (MAPK)- and NF-B-dependent pathways. However, the molecularmechanism by which NT induces expression of proinflammatory cytokinessuch as IL-8 has not been investigated. In this study we show thatinhibition of endogenous Rho family proteins (RhoA, Rac1, and Cdc42) bytheir respective dominant negative mutants inhibits NT-induced IL-8protein production and promoter activity. Western blot experimentsdemonstrated that NT strongly activated RhoA, Rac1, and Cdc42.Overexpression of the dominant negative mutants of RhoA, Rac1, andCdc42 significantly inhibited NT-induced NF-B-dependent reportergene expression and NF-B DNA binding activity. NT also stimulatedp38 MAPK phosphorylation, and overexpression of dominant negativemutants of RhoA, Rac1, and Cdc42 did not significantly alter p38 andERK1/2 phosphorylation in response to NT. Together, our findingsindicate that NT-stimulated IL-8 expression is mediated via aRho-dependent NF-B-mediated pathway.

     

IL-17 and Th17 cells in human inflammatory diseases

IL-17 was discovered in 1995/96 as a T cell derived cytokine with effects on inflammation and neutrophil activation. In 2006, the precise cell source of IL-17 was identified in the mouse, and these cells were named Th17 cells. They play a role in various human diseases associated with inflammation and destruction such as rheumatoid arthritis, psoriasis, Crohn's disease, multiple sclerosis, where IL-17 can be seen as a therapeutic target.     

A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses

CD4＋ helper T （TH） cells play crucial roles in immune responses. Recently a novel subset of TH cells, termed THIL-17, TH 17 or inflammatory TH （THi）, has been identified as critical mediators of tissue inflammation. These cells produce IL-17 （also called IL-17A） and IL-17F, two most homologous cytokines sharing similar regulations. Here we report that when overexpressed in 293T cells, IL-17 and IL-17F form not only homodimers but also heterodimers, which we name as IL-17A/F. Fully differentiated mouse THi cells also naturally secrete IL-17A/F as well as IL-17 and IL-17F homodimeric cytokines. Recombinant IL-17A/F protein exhibits intermediate levels of potency in inducing IL-6 and KC （CXCL 1） as compared to homodimeric cytokines. IL-17A/F regulation of IL-6 and KC expression is dependent on IL-17RA and TRAF6. Thus, IL-17A/F cytokine represents another mechanism whereby T cells regulate inflammatory responses and may serve as a novel target for treating various immune-mediated diseases.     

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.     

Focal adhesion kinase mediates cell survival via NF-kappaB and ERK signaling pathways

Focal adhesion kinase (FAK) is important to cellular functions such as proliferation, migration, and survival of anchorage-dependent cells. We investigated the role of FAK in modulating normal cellular responses, specifically cell survival in response to inflammatory stimuli and serum withdrawal, using FAK-knockout (FAK^–/–) embryonic fibroblasts. FAK^–/– fibroblasts were more vulnerable to TNF--induced apoptosis, as measured by terminal deoxynucleotidyl transferase positivity. FAK^–/– fibroblasts also demonstrated increased procaspase-3 cleavage to p17 subunit, whereas this was undetectable in FAK^+/+ fibroblasts. Insulin receptor substrate-1 expression was completely abolished and NF-B activity was reduced, with a concomitant decrease in abundance of the anti-apoptotic protein Bcl-x_L in FAK^–/– cells. Upon serum withdrawal, FAK^+/+ cells exhibited marked attenuation of basal ERK phosphorylation, while FAK^–/– cells, in contrast, maintained high basal ERK phosphorylation. Moreover, inhibition of ERK phosphorylation potentiated serum withdrawal-induced caspase-3 activity. This was paralleled by increased insulin receptor substrate (IRS)-2 expression in FAK^–/– cells, although both insulin- and IGF-1-mediated phosphorylation of Akt/PKB and GSK-3 were impaired. This suggests that IRS-2 protects against apoptosis upon serum withdrawal via the ERK signaling pathway. The specific role of FAK to protect cells from apoptosis is regulated by activation and phosphorylation of NF-B and interaction between activated growth factor anti-apoptotic signaling pathways involving both phosphatidylinositol 3-kinase/Akt and MAPK/ERK1/2. We demonstrate that FAK is necessary for upregulation of the anti-apoptotic NF-B response, as well as for normal expression of growth factor signaling proteins. Thus we propose a novel role for FAK in protection from cytokine-mediated apoptosis. apoptosis; ERK1/2; insulin; TNF-; IGF-1     

GLP-2 stimulates colonic growth via KGF, released by subepithelial myofibroblasts with GLP-2 receptors

BACKGROUND: Glucagon-like peptide-2 is thought to act as a growth factor for the gut, but the localization of the GLP-2 receptor and mechanism of action on epithelial growth is unclear. METHODS AND RESULTS: We found glucagon-like peptide-2 (GLP-2) receptors mainly on subepithelial myofibroblasts in rat, mouse, marmoset and human small and large intestine by immunohistochemistry and in situ hybridisation. By double labelling we found that these GLP-2 receptor immunoreactive cells also produce smooth muscle actin and keratinocyte growth factor (KGF). By subcutaneous infusion of either GLP-2 alone, GLP-2 plus KGF antibody, KGF antibody alone or saline in mice, we found that KGF antibody abolished the growth promoting effect of GLP-2 in the large intestine, but not in the small intestine. CONCLUSIONS: Our findings suggest that GLP-2 in the gut acts by activating receptors on the subepithelial myofibroblasts, causing the release of growth factors, which in turn stimulate intestinal growth.     

LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.     

The JNK and P38 MAP kinase signaling pathways in T cell-mediated immune responses

The mitogen-activated protein (MAP) kinase family members, which include the extracellular response kinases (ERK), p38, and c-Jun amino terminal kinases (JNK), play a role in mediating signals triggered by cytokines, growth factors, and environmental stress. JNK and p38 MAP kinases have been involved in inflammatory processes induced by a variety of stimuli, such as oxidative stress. Here, we describe the role of the JNK and p38 MAP kinase signaling pathways in the development of T cells in the thymus, and activation and differentiation of T cells in the peripheral immune system.     

Alpha-1-antitrypsin stimulates fibroblast proliferation and procollagen production and activates classical MAP kinase signalling pathways

Connective tissue formation at sites of tissue repair is regulated by matrix protein synthesis and degradation, which in turn is controlled by the balance between proteases and antiproteases. Recent evidence has suggested that antiproteases may also exert direct effects on cell function, including influencing cell migration and proliferation. The antiprotease, alpha1-antitrypsin, is the major circulating serine protease inhibitor which protects tissues from neutrophil elastase attack. Its deficiency is associated with the destruction of connective tissue in the lung and the development of emphysema, whereas accumulation of mutant alpha1-antitrypsin within hepatocytes often leads to liver fibrosis. In this study, we report that alpha1antitrypsin, at physiologically relevant concentrations, promotes fibroblast proliferation, with maximal stimulatory effects of 118 +/- 2% (n=6, P < 0.02) above media controls for cells exposed to 60 microM. We further show that alpha1antitrypsin also stimulates fibroblast procollagen production, independently of its effects on cell proliferation, with values maximally increased by 34 +/- 3% (n = 6, P < 0.01) above media controls at 30 microM. Finally, mechanistic studies to examine the mechanism by which alpha1-antitrypsin acts, showed that alpha1-antitrypsin induced the rapid activation of p42MAPK and p44MAPK (also known as ERK1/2) and that the specific MEK1 inhibitor PD98059 totally blocked alpha1-antitrypsin's mitogenic effects. These results support the hypothesis that alpha1-antitrypsin may play a role in influencing tissue repair in vivo by directly stimulating fibroblast proliferation and extracellular matrix production via classical mitogen-activated signalling pathways.     

Guggulsterone blocks IL-1beta-mediated inflammatory responses by suppressing NF-kappaB activation in fibroblast-like synoviocytes

Guggulsterone is a plant sterol that is used to treat hyperlipidemia, arthritis, and obesity. Although its anti-inflammatory and anti-hyperlipidemic effects have been well documented, the effect of guggulsterone on fibroblast-like synoviocytes (FLS) has not yet been reported. Therefore, in this study, the effect of guggulsterone on interleukin (IL)-1beta-induced inflammatory responses in the FLS of rheumatic patients was investigated. Treatment of FLS with IL-1beta induced production of chemokines such as RANTES and ENA-78. In addition, Western blot analysis and gelatin zymography revealed that IL-1beta activated matrix metalloproteinase (MMP)-1 and -3 in FLS. However, pre-incubation with guggulsterone completely inhibited the ability of IL-1beta to induce the production of chemokines and to activate MMPs. Although the NF-kappaB binding activity and nuclear p50 and p65 subunit levels, as well as IkappaBalpha degradation in the cytoplasm was greater in cells stimulated with IL-1beta than in unstimulated cells, treatment with guggulsterone abolished all of these increases. Collectively, these results suggest that guggulsterone would be useful as an inhibitor of joint destruction in patients with rheumatoid arthritis.