Sphingosine kinase 1 and 2 regulate the capacity of mesangial cells to resist apoptotic stimuli in an opposing manner

Abstract Sphingosine kinases (SKs) are key enzymes regulating the production of sphingosine-1-phosphate (S1P), which determines important cell responses including cell growth and death. Here we show that renal mesangial cells isolated from wild-type, SK-1(-/-), and SK-2(-/-) mice show a differential response to apoptotic stimuli. Wild-type mesangial cells responded to staurosporine with increased DNA fragmentation and caspase-3 processing, which was enhanced in SK-1(-/-) cells. In contrast, SK-2(-/-) cells were highly resistant to staurosporine-induced apoptosis. Furthermore, the basal phosphorylation and activity of the anti-apoptotic protein kinase B (PKB) and of its substrate Bad were decreased in SK-1(-/-) but not in SK-2(-/-) cells. Upon staurosporine treatment, phosphorylation of PKB and Bad decreased in wild-type and SK-1(-/-) cells, but remained high in SK-2(-/-) cells. In addition, the anti-apoptotic Bcl-X(L) was significantly upregulated in SK-2(-/-) cells, which may further contribute to the protective state of these cells. In summary, our data show that SK-1 and SK-2 have opposite effects on the capacity of mesangial cells to resist apoptotic stimuli. This is due to differential modulation of the PKB/Bad pathway and of Bcl-X(L) expression. Thus, subtype-selective targeting of SKs will be critical when considering these enzymes as therapeutic targets for the treatment of inflammation or cancer.

Ischemia-induced up-regulation of heme oxygenase-1 protects from apoptotic cell death and tissue necrosis

BACKGROUND: Tissues are endowed with protective mechanisms to counteract chronic ischemia. Previous studies have demonstrated that endogenous heme oxygenase (HO)-1 may protect parenchymal tissue from inflammation- and reoxygenation-induced injury. Nothing is known, however, on whether endogenous HO-1 also plays a role in chronic ischemia to protect from development of tissue necrosis. The aim of this study is, therefore, to evaluate in vivo whether endogenous HO-1 exerts protection on chronically ischemic musculocutaneous tissue, and whether this protection is mediated by an attenuation of the microcirculatory dysfunction. MATERIALS AND METHODS: In C57BL/6-mice, a chronically ischemic flap was elevated and fixed into a dorsal skinfold chamber. In a second group, tin-protoporphyrin-IX was administrated to competitively block the action of HO-1. Animals without flap elevation served as controls. With the use of intravital fluorescence microscopy, microcirculation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day observation period. The time course of HO-1 expression was determined by Western blotting. RESULTS: Chronic ischemia induced an increase of HO-1 expression, particularly at day 1 and 3. This was associated with arteriolar dilation and hyperperfusion, which was capable of maintaining an adequate capillary perfusion density in the critically perfused central part of the flap, demarcating the distal necrosis. Inhibition of endogenous HO-1 by tin-protoporphyrin-IX completely abrogated arteriolar dilation (44.6 +/- 6.2 microm versus untreated flaps: 71.3 +/- 7.3 microm; P < 0.05) and hyperperfusion (3.13 +/- 1.29 nL/s versus 8.55 +/- 3.56 nL/s; P < 0.05). This resulted in a dramatic decrease of functional capillary density (16 +/- 16 cm/cm(2)versus 84 +/- 31 cm/cm(2); P < 0.05) and a significant increase of apoptotic cell death (585 +/- 51 cells/mm(2)versus 365 +/- 53 cells/mm(2); P < 0.05), and tissue necrosis (73% +/- 5% versus 51% +/- 5%; P < 0.001). CONCLUSION: Thus, our results suggest that chronic ischemia-induced endogenous HO-1 protects ischemically endangered tissue, probably by the vasodilatory action of the HO-1-associated carbon monoxide.

Apoptotic cell death in the lactating mammary gland is enhanced by a folding variant of alpha-lactalbumin

Apoptosis is essential to eliminate secretory epithelial cells during the involution of the mammary gland. The environmental regulation of this process is however, poorly understood. This study tested the effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells) on mammary cells. Plastic pellets containing HAMLET were implanted into the fourth inguinal mammary gland of lactating mice for 3 days. Exposure of mammary tissue to HAMLET resulted in morphological changes typical for apoptosis and in a stimulation of caspase-3 activity in alveolar epithelial cells near the HAMLET pellets but not more distant to the pellet or in contralateral glands. The effect was specific for HAMLET and no effects were observed when mammary glands were exposed to native a-lactalbumin or fatty acid alone. HAMLET also induced cell death in vitro in a mouse mammary epithelial cell line. The results suggest that HAMLET can mediate apoptotic cell death in mammary gland tissue.

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Comparison of inorganic nitrogen uptake dynamics following snowmelt and at peak biomass in subalpine grasslands

Subalpine grasslands are highly seasonal environments and likely subject to strong variability in nitrogen (N) dynamics. Plants and microbes typically compete for N acquisition during the growing season and particularly at plant peak biomass. During snowmelt, plants could potentially benefit from a decrease in competition by microbes, leading to greater plant N uptake associated with active growth and freeze-thaw cycles restricting microbial growth. In managed subalpine grasslands, we expect these interactions to be influenced by recent changes in agricultural land use, and associated modifications in plant and microbial communities. At several subalpine grasslands in the French Alps, we added pulses of 15N to the soil at the end of snowmelt, allowing us to compare the dynamics of inorganic N uptake in plants and microbes during this period with that previously reported at the peak biomass in July. In all grasslands, while specific shoot N translocation (per g of biomass) of dissolved inorganic nitrogen (DIN) was two to five times greater at snowmelt than at peak biomass, specific microbial DIN uptakes were similar between the two sampling dates. On an area basis, plant communities took more DIN than microbial communities at the end of snowmelt when aboveground plant biomasses were at least two times lower than at peak biomass. Consequently, inorganic N partitioning after snowmelt switches in favor of plant communities, allowing them to support their growing capacities at this period of the year. Seasonal differences in microbial and plant inorganic N-related dynamics were also affected by past (terraced vs. unterraced) rather than current (mown vs. unmown) land use. In terraced grasslands, microbial biomass N remained similar across seasons, whereas in unterraced grasslands, microbial biomass N was higher and microbial C : N lower at the end of snowmelt as compared to peak biomass. Further investigations on microbial community composition and their organic N uptake dynamics are required to better understand the decrease in microbial DIN uptake.

The apoptotic and proliferation rate of tumour budding cells in colorectal cancer outlines a heterogeneous population of cells with various impacts on clinical outcome

AIMS In colorectal cancer (CRC), tumour buds represent an aggressive cell type at the invasive front with apparently low proliferation. The aim of this study was to determine proliferation and apoptotic rates of buds in comparison to tumour centre, front and mucosa. METHODS AND RESULTS Whole tissue sections from 188 CRC patients underwent immunohistochemistry for Ki67. Ten high-power fields (HPFs) were evaluated in mucosa, tumour centre, tumour front and tumour buds (total = 40 HPFs/case). Caspase-3 and M30 immunohistochemistry were performed on a multipunch tissue microarray from the same cohort. Ki67, caspase-3 and M30 immunoreactivity were correlated with outcome. The average percentage of cells showing Ki67 positivity was 5.2% in mucosa, and was not significantly different between the centre and front of the tumour (38.2% and 34.9%; P < 0.0001); 0.3% of buds showed Ki67 positivity (P < 0.0001). Caspase-3 expression was similar in mucosa, tumour centre and tumour front, but lower in tumour buds (<0.1%; P < 0.0001). M30 staining in buds was decreased (0.01%; P < 0.0001) in comparison to other areas. Ki67 positivity in buds was detrimental to survival in univariate (P = 0.0352) and multivariate (P = 0.0355) analysis. Caspase-3-positive tumours showed better outcome than negative tumours (P = 0.0262); but tumours with caspase-3-positive buds showed a worse outcome than those with caspase-3-negative buds (P = 0.0235). CONCLUSIONS Ki67, caspase-3 and M30 staining is absent in most tumour buds, suggesting decreased proliferation and apoptosis. However, the fact that Ki67 and caspase-3 immunoreactivity was associated with unfavourable prognosis points to a heterogeneous population of tumour buds.

Programmed cell death contributes to postnatal lung development

The rat lung undergoes the phase of maturation of the alveolar septa and of the parenchymal microvascular network mainly during the third postnatal week. Speculating that programmed cell death may contribute to the thinning of the alveolar septa, we searched for the presence of DNA fragmentation in rat lungs between postnatal days 6 and 36 using the TUNEL procedure. The number of positive nuclei was compared at different days. We observed an 8-fold increase of programmed cell death toward the end of the third week as compared to the days before and after this time point. The precise timing of the appearance of the peak depended on the size of the litter. Double-labeling for DNA fragmentation (TUNEL) and for type I and type II epithelial cells (antibodies E11 and MNF-116), as well as morphologic studies at electron microscopic level, revealed that during the peak of programmed cell death mainly fibroblasts and type II epithelial cells were dying. While both dying cell types were TUNEL-positive, nuclear fragments and apoptotic bodies were exclusively observed in the dying fibroblasts. We conclude that programmed cell death is involved in the structural maturation of the lung by reducing the number of fibroblasts and type II epithelial cells in the third postnatal week. We observed that the dying fibroblasts are cleared by neighboring fibroblasts in a later stage of apoptosis, and we hypothesize that type II epithelial cells are cleared by alveolar macrophages in early stages of the programmed cell death process.

Reactive oxygen intermediates contribute to necrotic and apoptotic neuronal injury in an infant rat model of bacterial meningitis due to group B streptococci.

Reactive oxygen intermediates (ROI) contribute to neuronal injury in cerebral ischemia and trauma. In this study we explored the role of ROI in bacterial meningitis. Meningitis caused by group B streptococci in infant rats led to two distinct forms of neuronal injury, areas of necrosis in the cortex and neuronal loss in the dentate gyrus of the hippocampus, the latter showing evidence for apoptosis. Staining of brain sections with diaminobenzidine after perfusion with manganese buffer and measurement of lipid peroxidation products in brain homogenates both provided evidence that meningitis led to the generation of ROI. Treatment with the radical scavenger alpha-phenyl-tert-butyl nitrone (PBN) (100 mg/kg q8h i.p.) beginning at the time of infection completely abolished ROI detection and the increase in lipidperoxidation. Cerebral cortical perfusion was reduced in animals with meningitis to 37.5+/-21.0% of uninfected controls (P < 0.05), and PBN restored cortical perfusion to 72.0+/-8.1% of controls (P < 0.05 vs meningitis). PBN also completely prevented neuronal injury in the cortex and hippocampus, when started at the time of infection (P < 0.02), and significantly reduced both forms of injury, when started 18 h after infection together with antibiotics (P < 0.004 for cortex and P < 0.001 for hippocampus). These data indicate that the generation of ROI is a major contributor to cerebral ischemia and necrotic and apoptotic neuronal injury in this model of neonatal meningitis.

Peak Last Glacial weathering intensity on the North American continent recorded by the authigenic Hf isotope composition of North Atlantic deep-sea sediments

We have retrieved radiogenic hafnium (Hf) isotope compositions (ɛHf) from authigenic Fe–Mn oxyhydroxides of deep northwest Atlantic sediments deposited over the past 26 ka to investigate the oceanic evidence of changes in dissolved weathering inputs from NE America during the last deglaciation. The extraction of seawater-derived Hf isotopic compositions from Fe–Mn oxyhydroxides is not a standard procedure. Comparisons between the Al/Hf ratios and Hf isotopic compositions of the chemically extracted authigenic phase on the one hand, and those of the corresponding detrital fractions on the other, provide evidence that the composition of past seawater has been reliably obtained for most sampled depths with our leaching procedures. This is endorsed most strongly by data for a sediment core from 4250 m water depth at the deeper Blake Ridge, for which consistent replicates were produced throughout. The Hf isotopic composition of the most recent sample in this core also closely matches that of nearby present day central North Atlantic seawater. Comparison with previously published seawater Nd and Pb isotope compositions obtained on the same cores shows that both Hf and Pb were released incongruently during incipient chemical weathering, but responded differently to the deglacial retreat of the Laurentide Ice Sheet. Hafnium was released more congruently during peak glacial conditions of the Last Glacial Maximum (LGM) and changed to typical incongruent interglacial ɛHf signatures either during or shortly after the LGM. This indicates that some zircon-derived Hf was released to seawater during the LGM. Conversely, there is no clear evidence for an increase in the influence of weathering of Lu-rich mineral phases during deglaciation, possibly since relatively unradiogenic Hf contributions from feldspar weathering were superimposed. While the authigenic Pb isotope signal in the same marine sediment samples traced peak chemical weathering rates on continental North America during the transition to the Holocene a similar incongruent excursion is notably absent in the Hf isotope record. The early change towards more radiogenic ɛHf in relation to the LGM may provide direct evidence for the transition from a cold-based to a warm-based Laurentide Ice Sheet on the Atlantic sector of North America.

Plant diversity and ecosystem multifunctionality peak at intermediate levels of woody cover in global drylands

Aim The global spread of woody plants into grasslands is predicted to increase over the coming century. While there is general agreement regarding the anthropogenic causes of this phenomenon, its ecological consequences are less certain. We analysed how woody vegetation of differing cover affects plant diversity (richness and evenness) and the surrogates of multiple ecosystem processes (multifunctionality) in global drylands, and how these change with aridity. Location Two hundred and twenty-four dryland sites from all continents except Antarctica, widely differing in their environmental conditions (from arid to dry-subhumid sites) and relative woody cover (from 0 to 100). Methods Using a standardized field survey, we measured the cover, richness and evenness of perennial vegetation. At each site, we measured 14 soil variables related to fertility and the build-up of nutrient pools. These variables are critical for maintaining ecosystem functioning in drylands. Results Species richness and ecosystem multifunctionality were strongly related to woody vegetation, with both variables peaking at a relative woody cover (RWC) of 41–60. This relationship shifted with aridity. We observed linear positive effects of RWC in dry-subhumid sites. These positive trends shifted to hump-shaped RWC–diversity and multifunctionality relationships under semi-arid environments. Finally, hump-shaped (richness, evenness) or linear negative (multifunctionality) effects of RWC were found under the most arid conditions. Main conclusions Plant diversity and multifunctionality peaked at intermediate levels of woody cover, although this relationship became increasingly positive in wetter environments. This comprehensive study accounts for multiple ecosystem attributes across a range of levels of woody cover and environmental conditions. Our results help us to reconcile contrasting views of woody encroachment found in the current literature and can be used to improve predictions of the likely effects of encroachment on biodiversity and ecosystem services.