Eco-physiological responses of copepods and pteropods to ocean warming and acidification.

We compare physiological responses of the crustacean copepod Calanus pacificus and pelagic pteropod mollusk Limacina helicina to ocean temperatures and pH by measuring biomarkers of oxidative stress, antioxidant defences, and the activity of the respiratory electron transport system in organisms collected on the 2016 West Coast Ocean Acidification cruise in the California Current System. Copepods and pteropods exhibited strong but divergent responses in the same habitat; copepods had higher oxygen-reactive absorbance capacity, glutathione-S-transferase, and total glutathione content. The ratio between reduced to oxidised glutathione was higher in copepods than in pteropods, indicating lower oxidative stress in copepods. Pteropods showed higher activities of glutathione reductase, catalase, and lipid peroxidation, indicating increased antioxidant defences and oxidative stress. Thus, the antioxidant defence system of the copepods has a greater capacity to respond to oxidative stress, while pteropods already face severe stress and show limited capacity to deal with further changes. The results suggest that copepods have higher adaptive potential, owing to their stronger vertical migration behaviour and efficient glutathione metabolism, whereas pteropods run the risk of oxidative stress and mortality under high CO2 conditions. Our results provide a unique dataset and evidence of stress-inducing mechanisms behind pteropod ocean acidification responses.

Biological responses of midge (Chironomus riparius) and lamprey (Lampetra fluviatilis) larvae in ecotoxicity assessment of PCDD/F-, PCB- and Hg-contaminated river sediments.

We evaluated the utility of chironomid and lamprey larval responses in ecotoxicity assessment of polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/F)-, polychlorinated biphenyls (PCB)- and mercury (Hg)-contaminated river sediments. Sediment samples were collected from the River Kymijoki with a known industrial pollution gradient. Sediment for the controls and lamprey larvae were obtained from an uncontaminated river nearby. Contamination levels were verified with sediment and tissue PCDD/F, PCB and Hg analyses. Behaviour of sediment-exposed chironomid and lamprey larvae were measured with Multispecies Freshwater Biomonitor© utilizing quadrupole impedance conversion technique. In addition, mortality, growth and head capsule deformity incidence of chironomids were used as ecotoxicity indicators. WHOPCDD/F+PCB-TEQ in the R. Kymijoki sediments ranged from the highest upstream 22.36 ng g(-1) dw to the lowest 1.50 ng g(-1) near the river mouth. The sum of PCDD/Fs and PCBs correlated strongly with Hg sediment concentrations, which ranged from <0.01 to 1.15 µg g(-1). Lamprey tissue concentrations of PCDD/Fs were two orders and PCBs one order of magnitude higher in the R. Kymijoki compared to the reference. Chironomid growth decreased in contaminated sediments and was negatively related to sediment ∑PCDD/Fs, WHOPCDD/F+PCB-TEQ and Hg. There were no significant differences in larval mortality or chironomid mentum deformity incidence between the sediment exposures. The distinct behavioural patterns of both species indicate overall applicability of behavioural MFB measurements of these species in sediment toxicity bioassays. Chironomids spent less and lampreys more time in locomotion in the most contaminated sediment compared to the reference, albeit statistically significant differences were not detected. Lamprey larvae had also a greater activity range in some of the contaminated sediments than in the reference. High pollutant levels in lamprey indicate risks for biomagnification in the food webs, with potential health risks to humans consuming fish.

Multi-stressor impacts on fungal diversity and ecosystem functions in streams: natural vs. anthropogenic stress.

Biological assemblages are often subjected to multiple stressors emerging from both anthropogenic activities and naturally stressful conditions, and species' responses to simultaneous stressors may differ from those predicted based on the individual effects of each stressor alone. We studied the influence of land-use disturbance (forest drainage) on fungal decomposer assemblages and leaf decomposition rates in naturally harsh (low pH caused by black-shale dominated geology) vs. circumneutral streams. We used pyrosequencing to determine fungal richness and assemblage structure. Decomposition rates did not differ between circumneutral and naturally acidic reference sites. However, the effect of forest drainage on microbial decomposition was more pronounced in the naturally acidic streams than in circumneutral streams. Single-effect responses of fungal assemblages were mainly related to geology. Community similarity was significantly higher in the naturally acidic disturbed sites than in corresponding reference sites, suggesting that land-use disturbance simplifies fungal assemblages in naturally stressful conditions. Naturally acidic streams supported distinct fungal assemblages with many OTUs (operational taxonomic unit) unique to these streams. Our results indicate that fungal assemblages in streams are sensitive to both structural and functional impairment in response to multiple stressors. Anthropogenic degradation of naturally acidic streams may decrease regional fungal diversity and impair ecosystem functions, and these globally occurring environments therefore deserve special attention in conservation planning.

Nonlinear 3D projection printing of concave hydrogel microstructures for long-term multicellular spheroid and embryoid body culture.

Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propagation. Here, we used a continuous 3D projection printing approach - with an important modification of nonlinear exposure - to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs.

EGFRvIII stimulates glioma growth and invasion through PKA-dependent serine phosphorylation of Dock180.

Glioblastomas (GBMs), the most common and malignant brain tumors, are highly resistant to current therapies. The failure of targeted therapies against aberrantly activated oncogenic signaling, such as that of the EGFR-PI3K/Akt pathway, underscores the urgent need to understand alternative downstream pathways and to identify new molecular targets for the development of more effective treatments for gliomas. Here, we report that EGFRvIII (ΔEGFR/de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in clinical GBM tumors, promotes glioma growth and invasion through protein kinase A (PKA)-dependent phosphorylation of Dock180, a bipartite guanine nucleotide exchange factor (GEF) for Rac1. We demonstrate that EGFRvIII induces serine phosphorylation of Dock180, stimulates Rac1 activation and glioma cell migration. Treatments of glioma cells using the PKA inhibitors H-89 and KT5720, overexpression of a PKA inhibitor (PKI), and in vitro PKA kinase assays show that EGFRvIII induction of serine phosphorylation of Dock180 is PKA-dependent. Significantly, PKA induces phosphorylation of Dock180 at amino acid residue S1250 that resides within its Rac1-activating DHR-2 domain. Expression of the Dock180(S1250L) mutant, but not wild type Dock180(WT), protein in EGFRvIII-expressing glioma cells inhibited receptor-stimulated cell proliferation, survival, migration in vitro and glioma tumor growth and invasion in vivo. Together, our findings describe a novel mechanism by which EGFRvIII drives glioma tumorigenesis and invasion through PKA-dependent phosphorylation of Dock180, thereby suggesting that targeting EGFRvIII-PKA-Dock180-Rac1 signaling axis could provide a novel pathway to develop potential therapeutic strategies for malignant gliomas.

Transcription and redox enzyme activities: comparison of equilibrium and disequilibrium levels in the three-spined stickleback.

Evolutionary and acclimatory responses require functional variability, but in contrast with mRNA and protein abundance data, most physiological measurements cannot be obtained in a high-throughput manner. Consequently, one must either rely on high-throughput transcriptomic or proteomic data with only predicted functional information, or accept the limitation that most physiological measurements can give fewer data than those provided by transcriptomics or proteomics. We evaluated how transcriptional and redox enzyme activity data agreed with regard to population differentiation (i.e. a system in steady state in which any time lag between transcription, translation and post-translational effects would be irrelevant) and in response to an acute 6°C increase in temperature (i.e. a disequilibrium state wherein translation could not have caught up with transcription) in the three-spined stickleback (Gasterosteus aculeatus). Transcriptional and enzyme activity data corresponded well with regard to population differentiation, but less so with regard to acute temperature increase. The data thus suggest that transcriptional and functional measurements can lead to similar conclusions when a biological system is in a steady state. The responses to acute changes must, as has been demonstrated earlier, be based on changes in cellular conditions or properties of existing proteins without significant de novo synthesis of new gene products.

Stock-specific variation of trophic position, diet and environmental stress markers in Atlantic salmon Salmo salar during feeding migrations in the Baltic Sea.

This study investigated stock-specific variation in selected ecophysiological variables during the feeding migrations of Atlantic salmon Salmo salar in the Baltic Sea. Oxidative stress biomarkers and EROD (ethoxyresorufin-O-deethylase, Cyp1A enzyme) activity were used as indicators of possible environmental stress and stable isotopes as determinants of diet and trophic position. Latvian S. salar stocks Daugava and Gauja had distinct stable-isotope signatures compared to the other stocks, indicating differences in migration patterns, residency or arrival times, or dietary specialization among stocks. Salmo salar originating from Daugava and Gauja also had lower catalase enzyme activity than the other stocks. Post-smolts originating from rivers of the Gulf of Finland had elevated EROD activities compared to fish of the same age from Bothnian Bay rivers, which could indicate exposure to organochlorine pollutants. No other stock-specific differences in oxidative stress biomarkers were found. The study demonstrates how genetic, oxidative stress biomarker, EROD and stable-isotope data may be combined to study trophic position, prey prevalence and environmental stress of mixed S. salar stocks foraging in the sea.

Use of the Stanford Health Assessment Questionnaire in estimation of long-term productivity costs in patients with recent-onset rheumatoid arthritis.

OBJECTIVE: To evaluate the utility of the Stanford Health Assessment Questionnaire (HAQ) in the estimation of loss of productivity due to early rheumatoid arthritis (RA) and to develop a simple model for analysis of the cost-benefit of therapies. METHODS: In the Finnish Rheumatoid Arthritis Combination Therapy (FIN-RACo) trial, 162 patients with recent-onset RA who were available for the workforce were randomized to receive either a combination of three disease-modifying anti-rheumatic drugs (DMARDs) or a single DMARD for 2 years and were followed up for 5 years. No biological drugs were used. Data on sick leave and RA-related disability pensions came from official register records. Loss of productivity was computed by both the human capital approach (HCA) and the friction cost approach (FCA). Functional capacity was assessed by the HAQ at baseline and at 6 months. RESULTS: Over 5 years, mean loss of productivity per year was EUR 8344 by the HCA and EUR 1928 by the FCA. The level of the HAQ index at 6 months, but not the change in HAQ from baseline, determined productivity costs. With the HCA, a monotonous association between annual loss of productivity and the 6-month HAQ was found: EUR 2087 [95% confidence interval (CI) 1340-2903] per one step (0.13) on the HAQ scale from 0 to 1.88. With the FCA, the increase in loss of productivity was cut at the HAQ level of 0.5 to 0.75 (EUR 17 740 in 5 years). CONCLUSION: The HAQ index at 6 months may serve as a determinant of long-term RA-related indirect costs in economic analyses in early RA.

Analysis of pancreas tissue in a child positive for islet cell antibodies.

AIMS/HYPOTHESIS: Type 1 diabetes is caused by an immune-mediated process, reflected by the appearance of autoantibodies against pancreatic islets in the peripheral circulation. Detection of multiple autoantibodies predicts the development of diabetes, while positivity for a single autoantibody is a poor prognostic marker. The present study assesses whether positivity for a single autoantibody correlates with pathological changes in the pancreas. METHODS: We studied post mortem pancreatic tissue of a child who repeatedly tested positive for islet cell antibodies (ICA) in serial measurements. Paraffin sections were stained with antibodies specific for insulin, glucagon, somatostatin, interferon alpha, CD3, CD68, cyclooxygenase-2 (COX-2), beta-2-microglobulin, coxsackie B and adenovirus receptor (CAR), natural killer and dendritic cells. Apoptosis was detected using Fas-specific antibody and TUNEL assay. Enterovirus was searched for using immunohistochemistry and in situ hybridisation, as well as enterovirus-specific RT-PCR from serum samples. RESULTS: The structure of the pancreas did not differ from normal. The number of beta cells was not reduced and no signs of insulitis were observed. Beta-2-microglobulin and CAR were strongly produced in the islets, but not in the exocrine pancreas. Enterovirus protein was detected selectively in the islets by two enterovirus-specific antibodies, but viral RNA was not found. CONCLUSIONS/INTERPRETATION: These observations suggest that positivity for ICA alone, even when lasting for more than 1 year, is not associated with inflammatory changes in the islets. However, it is most likely that the pancreatic islets were infected by an enterovirus in this child.

Detection of enteroviruses in the intestine of type 1 diabetic patients.

Enterovirus infections have been diagnosed more frequently in type 1 diabetic patients than in the healthy population, and enteroviruses have also been found in the pancreas of diabetic patients. Primary replication of the virus occurs in the gut, but there are no previous studies evaluating possible presence of virus in the intestine of diabetic patients. The purpose of this study was to investigate if enteroviruses can be found in small intestinal tissue of type 1 diabetic patients. Formalin-fixed, paraffin-embedded upper intestinal biopsy samples were analysed for the presence of enterovirus using in situ hybridization and immunohistochemistry. Enterovirus was detected by in situ hybridization in six (50%) of the type 1 diabetic patients (n = 12) but in none of the control subjects (n = 10, P = 0.015). Immunohistochemistry identified enterovirus in nine (75%) of the patients and one (10%) control subject (P = 0.004). The presence of the virus was confirmed by reverse transcription-polymerase chain reaction in one of the four patients from whom a frozen and unfixed sample was available. Intestinal morphology was normal in all study subjects. The results suggest that a substantial proportion of type 1 diabetic patients have an ongoing enterovirus infection in gut mucosa, possibly reflecting persistent enterovirus infection. This observation opens new avenues for further studies on the possible role of enteroviruses in human type 1 diabetes.

Cost of Finnish statutory inpatient rehabilitation and its impact on functional and work capacity of patients with early rheumatoid arthritis: experience from the FIN-RACo trial.

OBJECTIVE: To explore the cost of the statutory inpatient rehabilitation system in Finland and its impact on the functional and work capacity of patients with early rheumatoid arthritis (RA). METHODS: In the Finnish Rheumatoid Arthritis Combination-Therapy trial (FIN-RACo), 195 patients with recent-onset RA, 162 of them available for the work force, were randomly assigned to two different drug treatment strategies for 2 years. Otherwise, the patients received routine multidisciplinary care and, if their functional or work capacity was endangered, were referred to inpatient rehabilitation. After a 5-year follow-up, data on rehabilitation, sick leave, and RA-related disability pensions were obtained from official registers. RESULTS: Of the 162 patients, 49 (30%) underwent inpatient rehabilitation at an average cost of EURO5400. The rehabilitated patients more often worked in white-collar jobs and had more pain and a worse Health Assessment Questionnaire (HAQ) score (1.0 vs. 0.78; p = 0.01) at baseline. Their HAQ scores remained higher throughout follow-up (p<0.001); no change appeared over inpatient periods [mean 0.01; 95% confidence interval (CI) -0.13 to 0.16]. No independent impact of rehabilitation on the HAQ score emerged in an adjusted generalized estimating equations (GEE) model (p = 0.55). Nor did any improvement in work capacity appear: average lost productivity (human capital approach) per patient-year was EURO10 155 (95% CI 6994-14 196) before and EUR 12 839 (95% CI 8589-19 139) after the start of rehabilitation. CONCLUSION: For patients with recent-onset RA, the Finnish statutory inpatient rehabilitation system had no positive impact on either functional or work capacity during the first few years, despite its considerable cost.

Association of tumour necrosis factor a, b and c microsatellite polymorphisms with clinical disease activity and induction of remission in early rheumatoid arthritis.

OBJECTIVE: To study the associations of tumor necrosis factor (TNF) a, b and c microsatellite markers with 1) the clinical disease activity and 2) the induction of remissions in patients with early rheumatoid arthritis (RA) treated with two treatment strategies. METHODS: In the FIN-RACo (FINnish Rheumatoid Arthritis Combination therapy) trial of two years, 195 patients with recent-onset RA were randomly assigned to receive either a combination (COMBI) (sulphasalazine, methotrexate, hydroxychloroquine, and prednisolone) or a single (SINGLE) (initially sulphasalazine with or without prednisolone) disease modifying antirheumatic drug (DMARD) therapy. TNF a, b and c microsatellite and HLA-DRB1 typings were carried out in 165 (79 COMBI; 86 SINGLE) study completers. RESULTS: At baseline the 28 joint disease activity scores (DAS28) of the patients positive for TNFa2, a13 or b1 microsatellite markers were significantly higher than in the other patients. In the SINGLE patients the DAS28 improved comparably in patients with (n = 31) or without (n = 53) the TNFb1 marker (NS), while the DAS28 of the TNFb1-positive COMBI patients (n = 22) improved significantly more than that of the TNFb1-negative cases (n = 57) (p = 0.014). Respective 31.8% (7/22) and 28.1% (16/57) of the COMBI patients with or without TNFb1 allele achieved remission at one year. The corresponding figure in SINGLE patients were 0% (0/31) and 20.8% (11/53) (p = 0.006). At two years the remission frequencies in the TNFb1+/TNFb1- patients in the COMBI and SINGLE were 50.0%/38.6% and 9.7%/22.6%, respectively. CONCLUSION: Early TNFb1+ RA patients have more active disease but respond more favourably to COMBI treatment than the patients without this microsatellite allele. The finding may be of clinical relevance for the choice of DMARDs in early RA.

[Effect of aluminum and iron on lipid metabolism in aquatic invertebrates].

Methods of thin-layer, gas-liquid, and liquid chromatography were applied to the study of the effect of various concentrations of aluminum and iron salts on the contents of phospholipids, cholesterol, and fatty acids in the aquatic invertebrate Hydropsyche contubernalis L. (Trichoptera). It was found that the effect of the metals under study on lipid contents in living organisms depended on the composition of the aqueous medium and concentrations of the metals. Aluminum and iron altered the value of the cholesterol-to-phospholipid molar ratio. In the absence of lethal effects, this was indicative of attempts to switch adaptational biochemical mechanisms to stabilize cellular structures.

Neuroradiological findings (MRS, MRI, SPECT) in infantile neuronal ceroid-lipofuscinosis (infantile CLN1) at different stages of the disease.

Infantile neuronal ceroid-lipofuscinosis (infantile CLN1) is a progressive and uniformly fatal lysosomal storage disease of the nervous system. The purpose of this study was to compare the findings of various radiological examinations of the brain in the course of infantile CLN1 in order to evaluate the relative usefulness of the methods and their potential for monitoring therapeutic interventions. We examined eight infantile CLN1 patients, 51 studies, in various stages of the disease--preclinical to late stage--with proton magnetic resonance spectroscopy (1H-MRS), MRI, and perfusion SPECT, and in addition three benzodiazepine (BZ) receptor ligand SPECT studies. Both 1H-MRS and MRI showed abnormal findings before clinical manifestations of the disease. Cortical hypoperfusion and loss of cortical BZ receptors revealed by SPECT appeared simultaneously with clinical signs. After the age of 4 years MRI and SPECT alterations progressed minimally, whereas 1H-MRS showed progressive deterioration of neurometabolism. Of the four methods used in this study, MRI proved to be the most practicable for diagnosing infantile CLN1; the final diagnosis of infantile CLN1 is confirmed by the characteristic clinical picture and DNA or PPT enzyme analysis. The combination of 1H- MRS and MRI could be most useful for monitoring therapeutic interventions.

Integrin signaling inhibits paclitaxel-induced apoptosis in breast cancer cells.

Inherent or acquired drug resistance is one of the major problems in chemotherapy. The mechanisms by which cancer cells survive and escape the cytotoxic effects of chemotherapeutic agents are essentially unknown. In the present study, we demonstrate that in the MDA-MB-231 and MDA-MB-435 breast cancer cells, ligation of beta1 integrins by their extracellular matrix ligands inhibits significantly apoptosis induced by paclitaxel and vincristine, two microtubule-directed chemotherapeutic agents that are widely used in the therapy of breast cancer. We show that beta1 integrin signaling inhibits drug-induced apoptosis by inhibiting the release of cytochrome c from the mitochondria in response to drug treatment. Further, integrin-mediated protection from drug-induced apoptosis and inhibition of cytochrome c release are dependent on the activation of the PI 3-kinase/Akt pathway. Our results identify beta1 integrin signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells and suggest that activation of this pathway may contribute to the generation of drug resistance.

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis.

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.

Interaction of endostatin with integrins implicated in angiogenesis.

Endostatin, a fragment of collagen XVIII, is a potent antagonist of angiogenesis and inhibitor of tumor growth in mouse models. At present, the mechanism of action of endostatin is unknown. We show here that recombinantly produced human endostatin interacts with alpha(5)- and alpha(v)-integrins on the surface of human endothelial cells. We further demonstrate that the endostatin-integrin interaction is of functional significance in vitro, as we found that immobilized endostatin supports endothelial cell survival and migration in an integrin-dependent manner. Soluble endostatin in turn inhibits integrin-dependent endothelial cell functions, such as cell migration. Taken together, these results implicate integrins as potential targets for endostatin function and support the importance of integrins in endothelial cell biology and angiogenesis.

Proton spectroscopic imaging shows abnormalities in glial and neuronal cell pools in frontal lobe epilepsy.

PURPOSE: Proton magnetic resonance spectroscopic imaging (1H MRSI) can lateralize the epileptogenic frontal lobe by detecting metabolic ratio abnormalities in frontal lobe epilepsy (FLE). We used 1H MRS to lateralize and localize the epileptogenic focus, and we also sought to characterize further the metabolic abnormality in FLE. METHODS: We measured signals from N-acetyl aspartate (NAA), choline-containing compounds (Cho), and creatine + phosphocreatine (Cr) in the supraventricular brain of 14 patients with frontal or frontoparietal epilepsy and their matched controls. The supratentorial brain also was segmented into gray matter, white matter, and cerebrospinal fluid classes. Regional metabolite alterations were compared with localizing and lateralizing results from other examination modalities and with histology from three patients. RESULTS: Spectroscopy lateralized the epileptogenic focus in 10 patients in agreement with video-EEG and functional imaging. In four patients, spectroscopy showed bilateral, focal metabolic abnormality, whereas video-EEG suggested unilateral or midline abnormality. In the epileptogenic focus, Cho and Cr were increased by 23% and 14%, respectively, and NAA was decreased by 11%, suggesting metabolic disturbances both in the glial and in the neuronal cell pools. Two Taylor dysplasia lesions confirmed by histology and one with radiologic diagnosis showed high Cho and low or normal NAA, whereas two dysembryoplastic neurogenic tumors had normal Cho and low NAA. Contralateral hemisphere NAA/(Cho + Cr) was decreased in FLE, indicating diffusely altered brain metabolism. Segmentation of brain tissue did not reveal atrophic changes in FLE. CONCLUSIONS: Spectroscopy is useful in lateralizing frontoparietal epilepsy and shows promise as a "noninvasive biopsy" in epileptogenic lesions.

The proto-oncogene c-Cbl is a positive regulator of Met-induced MAP kinase activation: a role for the adaptor protein Crk.

Hepatocyte growth factor triggers a complex biological program leading to invasive cell growth by activating the c-Met receptor tyrosine kinase. Following activation, Met signaling is elicited via its interactions with SH2-containing proteins, or via the phosphorylation of the docking protein Gab1, and the subsequent interaction of Gab1 with additional SH2-containing effector molecules. We have previously shown that the interaction between phosphorylated Gab1 and the adaptor protein Crk mediates activation of the JNK pathway downstream of Met. We report here that c-Cbl, which is a Gab1-like docking protein, also becomes tyrosine-phosphorylated in response to Met activation and serves as a docking molecule for various SH2-containing molecules, including Crk. We further show that Cbl is similarly capable of enhancing Met-induced JNK activation, and several lines of experimentation suggests that it does so by interacting with Crk. We also show that both Cbl and Gab1 enhance Met-induced activation of another MAP kinase cascade, the ERK pathway, in a Crk-independent manner. Taken together, our studies demonstrate a previously unidentified functional role for Cbl in Met signaling and suggest that Met utilizes at least two docking proteins, Gab1 and Cbl, to activate downstream signaling pathways. Oncogene (2000) 19, 4058 - 4065.

Engagement of the alpha2beta1 integrin inhibits Fas ligand expression and activation-induced cell death in T cells in a focal adhesion kinase-dependent manner.

T-cell receptor (TCR)-mediated apoptosis, also known as activation-induced cell death (AICD), plays an important role in the control of immune response and in the development of T-cell repertoire. Mechanistically, AICD has been largely attributed to the interaction of Fas ligand (Fas-L) with its cell surface receptor Fas in activated T cells. Signal transduction mediated by the integrin family of cell adhesion receptors has been previously shown to modulate apoptosis in a number of different cell types; in T cells, integrin signaling is known to be important in cellular response to antigenic challenge by providing a co-stimulatory signal for TCR. In this study we demonstrate that signaling via the collagen receptor alpha2beta1 integrin specifically inhibits AICD by inhibiting Fas-L expression in activated Jurkat T cells. Engagement of the alpha2beta1 integrin with monoclonal antibodies or with type I collagen, a cognate ligand for alpha2beta1, reduced anti-CD3 and PMA/ionomycin-induced cell death by 30% and 40%, respectively, and the expression of Fas-L mRNA by 50%. Further studies indicated that the alpha2beta1-mediated inhibition of AICD and Fas-L expression required the focal adhesion kinase FAK, a known component in the integrin signaling pathways. These results suggest a role for the alpha2beta1 integrin in the control of homeostasis of immune response and T-cell development. (Blood. 2000;95:2044-2051)