Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors

Nonspecific cytotoxic cells (NCC) are the teleost equivalent of mammalian lymphokine-activated natural killer cells. The cytotoxic activities of NCC are enhanced by stress-activated serum factors (SASF) present in tilapia acute-phase serum. In the present study purified NCC and xenogeneic target HL-60 tumor cells and nuclei were distinguishable in mixtures determined by flow cytometry. NCC activated by target HL-60 cells undergo activation-induced programmed cell death (AIPCD) during 12- to 16-h killing assays as shown by Annexin-V binding and nuclear DNA fragmentation results. Annexin-V binding studies also demonstrated that NCC kill HL-60 cells by an apoptotic mechanism. NCC are protected from AIPCD by 4-h preincubation in 50% SASF. Pretreatment also produced more than a fourfold increase in NCC cytotoxicity (effector/target (E:T) ratio = 100:1). In the absence of SASF preincubation, the percentage of apoptotic NCC increased from 8 to 91% at E:T ratios of 1:0 and 1:1, respectively. Kinetic studies (E:T = 10:1) demonstrated that the percentage of NCC exhibiting HL-60-dependent AIPCD increased between 0.1 and 12 h and then decreased inversely with total cell necrosis over the next 60 h. Preincubation of NCC with SASF protected NCC from AIPCD for over 72 h. Crosslinkage of the NCCRP-1 receptor with monoclonal antibody (mab) 5C6 produced AIPCD between 1 and 100 microg/mL mab concentrations. Preincubation with SASF completely protected NCC from mab 5C6-dependent AIPCD. SASF-mediated protection of NCC from AIPCD was dependent upon divalent cations, as demonstrated by increases in DNA hypoploidy of 38, 67, and 88% following preincubation in the presence of 10, 100, and 1000 microM EDTA, respectively. SASF also protected NCC from glucocorticoid- (i. e., dexamethasone) induced apoptosis. Combined, these results demonstrated that NCC activity is down-regulated by AIPCD. Release of SASF into the peripheral circulation may prevent negative regulation of NCC by AIPCD by increasing recycling capacity. Results are discussed in the context of the effects of acute stressors on innate immunity.     

In vivo activation of tilapia nonspecific cytotoxic cells by Streptococcus iniae and amplification with apoptosis regulatory factor(s)

An important component of immediate innate responses of tilapia to stress is the release within minutes of soluble cytokine-like substances into the peripheral circulation. These cytokine-like stress factors bind nonspecific cytotoxic cells (NCC) and produce 3-4-fold increased cytotoxicity. In the present study, the in vivo responses of tilapia NCC following injection with different isolates of intact killed Streptococcus iniae was investigated. Activated cytotoxicity of NCC in the peripheral blood (PB) was produced by increased specific activity of resident cells rather than increased numbers. Tilapia injected intravenously (i.v.) with killed S. iniae produced different cytotoxicity responses compared to fish injected intraperitoneally (i.p.). In the spleen (S) and anterior kidney (AK), there was no correlation between S. iniae isolate and cytotoxicity response at 4, 8 or 24 h following i.p. injection. The NCC response following i.v. injection of killed bacteria was different. Within minutes following i.v. injection, NCC cytotoxicity from the PB increased 100% compared to naive controls. The existence of subsets of differentiated NCC in the PB was suggested because i.v. injection had no amplification effects on NCC from the AK or S. Likewise, NCC from the PB only appeared to exhibit a degree of antigen specificity. S. iniae strain #173 produced activation of cytotoxicity compared to isolates #164 and ATCC. Evidence for soluble factor (cytokine?) involvement in increased cytotoxicity was obtained by passive activation of NCC with serum from #173 (i.v.) injected fish. Incubation of this serum with control (na?ve) NCC produced large increases in the cytotoxicity of labelled HL-60 target cells. Similarly obtained serum from fish injected with ATCC and #164 isolates had no amplification activity. Studies were also performed to study the mechanism(s) of passive activation. Flow cytometric analysis revealed that NCC from the S, AK and PB constitutively expressed cytosolic (not membrane) FasL. Stress serum treated NCC obtained from the peripheral blood produced an increase in the expression of FasL, CAS and FADD by Western blot examination. These data indicated that cytokine like factors in the serum of stressed tilapia activate increased NCC cytotoxicity (possibly) by stimulating the expression of proteins involved in activation of programmed cell death.     

Mechanisms of nonspecific cytotoxic cell regulation of apoptosis: cytokine-like activity of Fas ligand

The role of FasL/FasR pathways of immunoregulation of programmed cell death in teleost cytotoxic innate immunity has not been previously examined. In the present study, constitutive cytosolic soluble FasL (sFasL) was detected in anterior kidney (AK), peripheral blood (PBL) and liver NCC obtained from tilapia. Ligation of NCC by tumour cells caused the release of sFasL that was associated with lysis of HL-60 targets in 14 h killing assays. Evidence that sFasL mediated this activity was that anti-(human) FasL inhibited tilapia and catfish (cf.) NCC lysis of FasR+ HL-60 tumour cells. Inhibition was concentration dependent. Lysis of IM-9 targets (12% positive for FasR) by (cf.) anterior kidney and PBL NCC was only partially inhibited by anti-FasL mab. Activated NCC from both species were negative for the expression of membrane FasL and FasR. These data confirmed that NCC lyse sensitive tumour cells by multiple effector pathways. Pretreatment of (FasR+) HL-60 cells with anti-FasR mab completely inhibited cf. cytotoxicity at low (100:1) E:T ratios. Anti-FasR mab did not inhibit the lysis of IM-9 targets by cf. NCC. This study demonstrated that for catfish and tilapia, initial target cell conjugate formation was required; however, the terminal killing mechanism depended on at least two different pathways of cytotoxicity. One pathway depended on the release of preformed soluble FasL by activated NCC in the presence of FasR positive target cells. A second pathway has yet to be determined.     

Cross-linkage of a receptor on nonspecific cytotoxic cells or treatment with calcium ionophore triggers increased phosphorylation of serine residues

Cross-linkage of a putative receptor protein on nonspecific cytotoxic cells (NCC) with monoclonal antibody (mab) 5C6 produces: activation of cytotoxicity; increased release of free cytosolic calcium; increased levels of IP3 and IP4; and increased DNA synthesis. Mab-binding also caused increased expression of various proto-oncogene kinases and increased expression of tyrosine phosphorylated proteins. In the present study NCC triggering by mab 5C6 was linked to serine specific kinase/phosphatase action. Concomitant with mab 5C6 activation, two proteins of 55-60 and 25-30 kDa were hyperphosphorylated on serine residues (15-30 min post-activation) determined by immunoprecipitation with an anti-phosphoserine specific mab. These proteins appeared to be components of a large macromolecular protein complex (> 170 kDa) determined by resolution in reducing and non-reducing SDS-PAGE. Sequential immunoprecipitation experiments revealed that these proteins were not PKC or p56^lck. Activation of NCC with the calcium ionophore A23187 caused the expression of this same complex of serine phosphorylated proteins. These data indicate that hyperphosphorylation of serine residues is associated with increased NCC cytotoxicity. Activation may be associated with receptor cross-linkage or calcium ionophore treatment, both events producing the phosphorylation of the same substrates.     

Flow cytometry-based assay for determination of teleost cytotoxic cell lysis of target cells.

BACKGROUND: The nonradiometric assays previously developed to detect cellular cytotoxic activity have been hindered by many difficulties. Among the problems are the requirement for expensive commercial kits and the use of techniques that produce high background noise and decreased sensitivity. In addition, these assays did not account for bidirectional apoptosis (activation-induced cell death [AICD]). Most attempts to derive cytometry-based cytotoxicity assays have been unsuccessful because individual effectors and targets could not be identified (i.e., "separated") using gating techniques. METHODS: In the present study, teleost nonspecific cytotoxic (NCC) and mammalian target cells were each sufficiently different in size to identify them by flow cytometry (FCM). Using appropriate gating and discriminator techniques, these two cell populations were differentiated based on scatter properties and propidium iodide (PI) binding. Total capacity for PI binding was obtained by permeabilization of the targets with ice-cold acetone. Spontaneous PI binding was relatively low. This technique detected cytotoxicity at effector-to-target ratios (E:T) of 1:1 and after only 30 min cocultivation. RESULTS: Tilapia NCC from peripheral blood kill human transformed target cells by necrosis and apoptosis as identified by PI binding. Maximum killing of HL-60 targets (approximately 100%) occurred by 180 min cocultivation. For the same time, the killing of IM-9 did not exceed 60%. Almost 90% of IM-9 targets are lysed following 14 h of cocultivation. The maximum killing of both HL-60 and IM-9 targets was observed at a 25:1 E:T ratio after 14 h. Comparisons of the chromium(>51) release assay with flow detection of cytotoxicity revealed that FCM detected 55% lysis of the target cells compared with 2% cytotoxicity by chromium release, after a cocultivation time of 240 min. DISCUSSION: FCM detection of (teleost) NCC lysis of target cells using PI uptake is more sensitive than standard chromium release assays. This level of sensitivity was observed because NCC and targets were sufficiently different in size such that they could be resolved by scatter plots. Using FCM, cytotoxicity was detected earlier and at lower E:T ratios than previously reported for chromium release assays. Although tilapia were reported previously to be not capable of lysing IM-9 targets by chromium release detection, the more sensitive method of FCM detected cytotoxicity using PI uptake. HL-60 lysis by tilapia NCC exhibited saturable kinetics but occurred at different times post-cocultivation.     

Molecular cloning of cellular apoptosis susceptibility (CAS) gene in Oreochromis niloticus and its proposed role in regulation of non-specific cytotoxic cell (NCC) functions

Cellular apoptosis susceptibility (CAS) gene is a homologue of the chromosome segregation gene (CSE) in yeast, involved in multiple cellular mechanisms associated with cell proliferation as well as cell death. CAS is highly expressed in proliferating cells but at a lower level in quiescent cells and tissues. Therefore it appears that CAS may play an important role in cancer development. We have previously identified CAS in tilapia non-specific cytotoxic cells (NCC) with a cross-reacting monoclonal antibody. Its expression was up-regulated in NCC in response to apoptosis regulatory factors. In the present report, the molecular cloning and expression of CAS in NCC is described, suggesting the importance of this protein in regulation of teleost immune functions. Furthermore, CAS expression is proposed as one of the mechanisms of regulation of activation induced programmed cell death (AIPCD) in these cytotoxic cells. As CAS expression is ubiquitous, we expect that these studies will help identify proliferating cells protected from apoptosis in additional tissues.     

Identification of a scavenger receptor homologue on nonspecific cytotoxic cells and evidence for binding to oligodeoxyguanosine

In mammals scavenger receptors (SR) are expressed by monocytic-macrophage lineage cells and B-cells. Studies of various teleost species have indirectly demonstrated the presence of SR receptors on phagocytic or endothelial cells by showing the uptake of SR ligands (i.e. derivatised (acetylated) lipoproteins) by these cells. In the present study, nonspecific cytotoxic cells (NCC) were examined for membrane expression of an SR-like protein. Approximately 15-25% of purified NCC expressed scavenger receptor class A (SR-A) demonstrated by binding by a monoclonal (2F8) specific for mouse SR-A (types I, II). Flow cytometric analysis determined that SR binding cells had the same size and 'side scatter' characteristics as NCC. Two colour flow analysis of NCC demonstrated that only a subset of NCC expressed the SR-A-like protein and non-NCC were SR-A negative. Membrane expression of SR on NCC was confirmed by fluorescence microscopy. Analysis of the tissue distribution of SR bearing cells demonstrated that in both catfish and tilapia, SR-A was expressed by NCC in the peripheral blood, spleen and anterior kidney. Experiments were also done to determine if the ligands known to bind mammalian SR-A had a similar specificity for the teleost receptor. Cold competition binding experiments determined that anti-SR-A antibody competed with and reduced biotinylated polyguanosine 20-mer binding to NCC by approximately 40%. Two other types of ligands known to bind (mammalian) SR-A (i.e. polyvinyl sulphate and dextran sulphate) likewise decreased anti-SR-A antibody binding to NCC by 40%. These studies for the first time demonstrated that NCC express the teleost orthologue of mammalian SR-A, suggesting that NCC may participate in physiologic regulation of lipid metabolism in addition to functions of innate immunity.     

Natural cytotoxic cells of gilthead seabream: maximum percentage of lysis

The maximum percentage of lysis of head-kidney non-specific cytotoxic cells (NCC) against mammalian tumour cells (L1210 and K562) in the marine teleost gilthead seabream (Sparus aurata L.) was studied. The present data indicate the short period of time necessary for gilthead seabream NCC to form conjugates and deliver a lethal hit. The maximum percentage of lysis observed demonstrates that seabream NCC activity against L1210 tumour cells is faster than against K562 tumour cells. This kinetic parameter suggests that fish NCC show a less efficient cytotoxic activity than their mammalian counterparts. The possibility of applying theoretical treatments to systems consisting of lower vertebrate non-specific cytotoxic cells and tumour targets, similar to those applied to mammals, is considered, and the phylogenetic implications of our findings are discussed.     

Nonspecific cytotoxic cells as effectors of immunity in fish

Nonspecific cytotoxic cells (NCC) may be the teleost fish equivalent of mammalian natural killer (NK) cells. Although significant differences exist between species regarding many characteristics of these cells, both NCC and NK cells share similarities: in the types of target cells sensitive to lysis; in mechanisms of target cell recognition; in the requirements for a competent lytic cycle; and both types of effectors participate in mediating the lysis of infectious microorganisms. A putative antigen binding receptor obtained from catfish NCC has now been characterized using monoclonal antibodies (mabs). This receptor is a vimentin-like protein. Preliminary studies indicate that NCC recognize a 40 kD protein on the membranes of susceptible target cells. Solubilized target cell protein can specifically bind to NCC and inhibit killing.Similar to NK cells, NCC require cell contact with the target cell to deliver the lethal cytotoxic hit. NCC appear to be the more potent cytotoxic cells because fewer are required to kill an individual target cell and less time is required for this action to occur than for NK cells. Unlike NK cells, NCC do not recycle under experimental conditions. Preliminary studies were also reviewed to characterize signal transduction responses. Monoclonal antibody against the vimentin-like protein receptor activates NCC cytotoxicity, initiates the production of significant increased levels of free cytoplasmic calcium, and causes the production of inositol lipid intermediates (specifically phosphotidylinositol 1, 4–5 trisphosphate). NCC may be important effectors of anti-parasite immunity. Although these cells probably do not elicit memory responses, data suggest that they do recognize antigen and can be activated and recruited into peripheral tissue where they mediate cytolytic responses.     

Src-dependent Syk activation controls CD69-mediated signaling and function on human NK cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cgamma2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.     

Hyperosmotic stress activates the insulin receptor in CHO cells

Stress factors, such as osmotic stress and genotoxic agents, activate stress kinases, whereas growth factors preferentially stimulate the structurally homologous mitogen-activated protein kinases, ERK1/2. Hyperosmolarity also has insulin-mimicking action as reflected by ERK1/2 activation and by the stimulation of glucose uptake in adipocytes. We examined to what extent hyperosmolarity activates components of the insulin receptor (IR) signalling pathway. CHO cells expressing the human IR were treated with 500 mM NaCl or 700 mM sorbitol and the activation of insulin signalling intermediates was studied. Hyperosmolarity induced tyrosine phosphorylation of the IR beta-subunit, and the adaptor proteins p52-Shc, p66-Shc, and IRS1. Furthermore, the stress kinases JNK and p38 were activated. When CHO cells were transfected with a kinase-dead IR (K1030R) mutant, hyperosmolarity did not induce tyrosine phosphorylation of the IR, indicating that hyperosmolarity induced IR autophosphorylation directly, rather than inducing phosphorylation by an exogenous tyrosine kinase. A partially purified and detergent-solubilized IR was not phosphorylated in response to hyperosmolarity, suggesting that hyperosmolarity activates the receptor only when present in the plasma membrane. In cells stably expressing the kinase-dead IR, IRS1 and Shc Tyr phosphorylation was abrogated, indicating that the hyperosmolarity signalling was dependent on an active IR tyrosine kinase. In contrast, the stress kinases p38 and JNK were normally activated by hyperosmolarity in the IR-K1030R mutant. We conclude that, at least in CHO cells, hyperosmolarity signals partially through IR autophosphorylation and subsequent activation of the IR downstream targets. This may be responsible for some of the insulin-mimicking effects of hyperosmolarity. The activation of stress kinases by hyperosmolarity occurs independent of the IR.     

Antiprotozoan activity of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus)

Nonspecific cytotoxic cells (NCC) obtained from channel catfish (Ictalurus punctatus) kill Tetrahymena pyriformis, an opportunistic parasite in fish. Based upon this fact, a new mechanism for nonspecific cellular anti-parasitic immunity in fish is proposed. Optimum in vitro conditions for NCC killing of deciliated T. pyriformis were first obtained. Lysis of T. pyriformis by NCC occurred by 10 hr of cocultivation of effector and target cells. During this time period, 50 to 60% cytotoxicity occurred. Fish anti-T. pyriformis serum enhanced NCC killing of T. pyriformis either by prolonging immobilization (after the cilia regeneration period) or by delaying cilia regeneration. Shared antigenic determinants between T. pyriformis, Ichthyophthirius multifiliis, and NC-37 target cells were demonstrated by binding-depletion experiments. For these studies, NCC were depleted from anterior kidney cells (the hemopoetic organ in fish) by preincubating formalin-treated T. pyriformis, I. multifiliis, or viable NC-37 target cells with NCC for 3 hr. Conjugates of effector and target cells were removed by overlaying on fetal bovine serum. Unconjugated fish anterior kidney cells were tested for cytotoxic activity against NC-37 or T. pyriformis target cells. Cold target inhibition experiments by using a 4-hr 51chromium cytotoxicity assay also demonstrated these shared antigenic determinants. Target-specific antisera, used to mediate the killing of T. pyriformis by NCC, were required only for immobilizing the targets, and did not function in an antibody-dependent cell-mediated (ADCC)-like mechanism. Scanning electron micrographs of NCC-T. pyriformis conjugates additionally demonstrated NCC binding to both cilia and cell surface determinants.     

Src-family tyrosine kinases in activation of ERK-1 and p85/p110-phosphatidylinositol 3-kinase by G/CCKB receptors.

We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.     

The mixed-lineage kinase DLK undergoes Src-dependent tyrosine phosphorylation and activation in cells exposed to vanadate or platelet-derived growth factor (PDGF)

Some data in the literature suggest that serine/threonine phosphorylation is required for activation of the mixed-lineage kinases (MLKs), a subgroup of mitogen-activated protein kinase kinase kinases (MAPKKKs). In this report, we demonstrate that the MLK family member DLK is activated and concurrently tyrosine-phosphorylated in cells exposed to the protein tyrosine phosphatase inhibitor vanadate. Tyrosine phosphorylation appears crucial for activation as incubation of vanadate-activated DLK molecules with a tyrosine phosphatase substantially reduced DLK enzymatic activity. Interestingly, the effects of vanadate on DLK are completely blocked by treatment with a Src family kinase inhibitor, PP2, or the expression of short hairpin RNA (shRNA) directed against Src. DLK also fails to undergo vanadate-stimulated tyrosine phosphorylation and activation in fibroblasts which lack expression of Src, Yes and Fyn, but reintroduction of wild-type Src or Fyn followed by vanadate treatment restores this response. In addition to vanadate, stimulation of cells with platelet-derived growth factor (PDGF) also induces tyrosine phosphorylation and activation of DLK by a Src-dependent mechanism. DLK seems important for PDGF signaling because its depletion by RNA interference substantially reduces PDGF-stimulated ERK and Akt kinase activation. Thus, our findings suggest that Src-dependent tyrosine phosphorylation of DLK may be important for regulation of its activity, and they support a role for DLK in PDGF signaling.     

Role of protein kinase activation in the induction of B cell adhesion by MHC class II ligands.

Engagement of MHC class II (Ia) molecules on B cells induces tyrosine phosphorylation, phosphoinositide turnover, elevation of intracellular calcium concentrations, and a rise in cAMP levels. However, a role for these biochemical signals in mediating functional responses induced by Ia ligands remains largely undefined. In this study, we utilized the induction of B cell adhesion by Ia ligands to demonstrate a role for signals transduced via Ia molecules in the generation of a functional response. Ia ligands that induced B cell aggregation induced tyrosine phosphorylation, whereas Ia ligands that did not induce B cell aggregation failed to induce any detectable tyrosine phosphorylation. Ia-induced B cell aggregation and tyrosine phosphorylation were inhibited by genistein and by herbimycin A, inhibitors of tyrosine kinases (PTK). Sphingosine and calphostin C, inhibitors of protein kinase C (PKC), also inhibited Ia-induced adhesion whereas HA1004, an inhibitor of cyclic nucleotide-dependent kinases, did not. Ia ligands induced both LFA-1-dependent and LFA-1-independent B cell adhesion. These two pathways of cell adhesion differed in their requirement for activation signals. PKC activation was sufficient for LFA-1-dependent adhesion, whereas LFA-1-independent adhesion required independent phosphorylation events mediated by PKC and by PTK. These results provide functional relevance for biochemical signals transduced via Ia molecules by demonstrating that Ia-induced B cell adhesion is mediated by the activation of PKC and by one or more PTK.     

Identification of the non-specific cytotoxic cell receptor protein 1 (NCCRP1) in regenerating axolotl limbs

The teleost non-specific cytotoxic cells (NCC) are evolutionary precursors of the mammalian natural killer (NK) cells and an important element of innate immunity. The non-specific cytotoxic cell receptor protein (NCCRP1) is a characteristic cell surface protein with main functions in target cell recognition and cytotoxicity with sequence information available for many species of fish. We have isolated a cDNA encoding the Axolotl homologue of fish NCCRP1 out of limb regeneration blastema and analysed its expression by RT-PCR. Sequence analysis revealed a high degree of homology with teleost NCCRP1 on nucleotide and deduced amino acid levels. NCCRP1 contains a conserved C-terminal F-box-associated domain (FBA) and proline-rich motifs (PRM) characteristic for this protein family. NCCRP1 is expressed in multiple tissues with high levels in limb regeneration blastema. The present work describes for the first time the cloning of the NCCRP1 gene in a tetrapod vertebrate providing a valuable link between fish and higher vertebrates. Our findings suggest the existence of NCC in axolotl and a role of the innate immune system in the processes of limb regeneration.     

Hyaluronan constitutively regulates activation of multiple receptor tyrosine kinases in epithelial and carcinoma cells

Hyaluronan (HA) is enriched in the pericellular matrices of many malignant human tumors, and manipulations of HA interactions have strong effects on tumor progression in animal models. Increased HA production stimulates ERBB2 activation, leading to increased cell survival activities and several malignant cell properties. On the other hand, inhibition of constitutive HA-tumor cell interactions in malignant cells inhibits these properties. We have now investigated the role of HA in activation of several additional receptor tyrosine kinases (RTKs), i.e. IGF1R-beta, PDGFR-beta, EGFR and c-MET, in colon, prostate, and breast carcinoma cells. In each case we show that antagonists of endogenous HA interactions inhibit their tyrosine phosphorylation, i.e. activation. On the other hand, we show that these RTKs are activated in phenotypically normal or relatively benign tumor cells by experimentally increasing HA production. We also investigated the role of HA in constitutive versus ligand-induced activation of RTKs. In HCA7 colon and C4-2 prostate carcinoma cells, ERBB2 is constitutively activated in a ligand-independent manner, whereas IGF1R-beta and PDGFR-beta require ligand interaction for activation. We show that both constitutive activation of ERBB2 and ligand-mediated activation of IGF1R-beta and PDGFR-beta are reversed by co-treatment of the cells with a HA antagonist. We conclude that HA serves a general function in RTK activation.     

Protein kinase activation and protein phosphorylation in Naegleria fowleri amebae in response to normal human serum

Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [32P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.