Protein kinase C inhibitors stimulate arachidonic and docosahexaenoic acids release from uterine stromal cells through a Ca2+-independent pathway1Enzymes: Phospholipase A2 (EC 3.1.1.4).1

AbstractThe mechanisms underlying arachidonic acid (AA) release by uterine stromal (UIII) cells were studied. Stimulation of AA release by calcium ionophore and PMA are inhibited by various PKC inhibitors and by calcium deprivation. These results suggest the involvement of an AA-specific cPLA2 as the release of docosahexaenoic acid (DHA) from prelabelled cells is much lower than the release of AA. The results also show a more original stimulation of AA and DHA release induced by PKC inhibitors, which is insensitive to calcium deprivation. This stimulation is not due to acyltransferase inhibition, suggesting the participation of a Ca2+-independent PLA2 (iPLA2). However, iPLA2 activity measured in UIII cells is inhibited by the specific iPLA2 inhibitor, BEL, and is not stimulated by PKC inhibitors, in contrast with the AA and DHA release. It seems therefore that this iPLA2 cannot be involved in this mechanism. The participation of another iPLA2, BEL-insensitive, is discussed

N-Cadherin Mediates Neuronal Cell Survival through Bim Down-Regulation

<div><p>N-cadherin is a major adhesion molecule involved in the development and plasticity of the nervous system. N-cadherin-mediated cell adhesion regulates neuroepithelial cell polarity, neuronal precursor migration, growth cone migration and synaptic plasticity. In vitro, it has been involved in signaling events regulating processes such as cell mobility, proliferation and differentiation. N-cadherin has also been implicated in adhesion-dependent protection against apoptosis in non-neuronal cells. In this study, we investigated if the engagement of N-cadherin participates to the control of neuronal cells survival/death balance. We observed that plating either primary mouse spinal cord neurons or primary rat hippocampal neurons on N-cadherin recombinant substrate greatly enhances their survival compared to non-specific adhesion on poly-L-lysine. We show that N-cadherin engagement, in the absence of other survival factors (cell-matrix interactions and serum), protects GT1-7 neuronal cells against apoptosis. Using this cell line, we then searched for the signaling pathways involved in the survival effect of N-cadherin engagement. The PI3-kinase/Akt survival pathway and its downstream effector Bad are not involved, as no phosphorylation of Akt or Bad proteins in response to N-cadherin engagement was observed. In contrast, N-cadherin engagement activated the Erk1/2 MAP kinase pathway. Moreover, N-cadherin ligation mediated a 2-fold decrease in the level of the pro-apoptotic protein Bim-EL whereas the level of the anti-apoptotic protein Bcl-2 was unchanged. Inhibition of Mek1/2 kinases with U0126, and the resulting inhibition of Erk1/2 phosphorylation, induced the increase of both the level of Bim-EL and apoptosis of cells seeded on the N-cadherin substrate, suggesting that Erk phosphorylation is necessary for cell survival. Finally, the overexpression of a phosphorylation defective form of Bim-EL prevented N-cadherin-engagement induced cell survival. In conclusion, our results show that N-cadherin engagement mediates neuronal cell survival by enhancing the MAP kinase pathway and down-regulating the pro-apoptotic protein Bim-EL.</p> </div

Overexpression of Bim-EL S69G prevents N-cadherin induced cell survival.

<p>Expression vectors coding for wild type Bim-EL and Bim-EL S69G were lipofected in 24 hours starved GT1-7 cells together with pEGFP at a 2/1 ratio. Cell lysates of equivalent protein content were analyzed by western blotting at 8 hours and 26 hours post-transfection for Bim and GFP expression; actin was analyzed as loading control (A). Two hours after lipofection, cells were harvested from transfected cultures and seeded on N-cadherin. Preparations were fixed 6 hours and 24 hours later (corresponding to 8 hours and 26 hours post-transfection) and nuclei stained. The percentage of GFP positive cells with condensed nuclei was then determined for each condition in duplicates (≥100 GFP⁺ cells counted each time). Represented data are the mean of two independent experiments. Asterisks indicate a significant difference (P<0.05) as compared with GFP expression alone.</p

Erk1/2 phosphorylation is up-regulated by N-cadherin engagement.

<p>Serum-starved GT1-7 cells were seeded at low density on PL or N-cad and grown for the indicated time in serum-free medium (A); alternatively confluent GT1-7 cell cultures were subjected to a calcium switch (B). Equivalent amount of proteins were blotted onto nitrocellulose membranes that were incubated either with anti-phospho Erk1/2, anti-total Erk1/2, or anti-α-tubulin. The relative densities of phospho-Erk1/2 were normalized to total Erk1/2 (A) or to α-tubulin (B). Histograms present the data of three independent experiments (mean ± SD).</p

Hypothetical model for N-cadherin mediated cell survival signaling in neurons.

<p>Hypothetical model for N-cadherin mediated cell survival signaling in neurons.</p

N-cadherin engagement sustains spinal cord and hippocampal neuronal cell survival.

<p>(A) In 24 hours hippocampal cultures, the number of βIII tubulin-positive cells per µm² were counted on each substrate and expressed as a percentage of that on N-cad substrates, arbitrarily fixed to 100%. (B) The numbers of βIII tubulin-positive cells with condensed (white bars) or non-condensed nuclei (dark bars) were determined in ventral spinal cords cultures after 24 hours on each substrate. The number of βIII tubulin-positive cells per µm² was expressed as a percentage of the number of βIII tubulin-positive cells per µm² on N-cad substrates, arbitrarily fixed to 100%. Results are expressed as the mean ± SD of two independent experiments.</p

Mek 1/2 inhibitor induces apoptosis on cells seeded on N-cadherin.

<p>Serum-starved GT1-7 cells were pretreated with 20 µM U0126 or vehicle for 1 hour, then seeded at medium density on N-cad with or without 20 µM U0126 for 6 hours (A). Apoptosis was evaluated by determination of nuclei condensation following DAPI staining and cytochrome c release following immunostaining. Arrows point toward cells showing condensed nuclei and absence of cytochrome c staining while asterisks indicate a cell with healthy nuclei and cytochrome c staining. Scale bar: 20 µm. (B, C). The percentage of cells with condensed nuclei and diffuse/absent cytochrome c labeling was quantified at 6 and 24 hours in treated and non-treated cells seeded on PL or N-cad. Results are expressed as the mean ± SD of four independent experiments. Asterisks indicate a significant difference (P<0.05) as compared with PL.</p

Inhibition of calcium-dependent cell-cell adhesion induces apoptosis of GT1-7 cells in suspension.

<p>Serum-starved GT1-7 cells were cultured in suspension in agarose-coated dishes in the presence of calcium (1.8 mM) or without calcium (2.5 mM EDTA). (A) Phase-contrast photomicrographs were taken at time 0 and 24 hr of incubation. (B) Cells lysates were then analyzed by western blotting for PARP cleavage. Western blot representative of two independent experiments.</p