The endogenous caspase-8 inhibitor c-FLIPL regulates ER morphology and crosstalk with mitochondria.

Components of the death receptor-mediated pathways like caspase-8 have been identified in complexes at intracellular membranes to spatially restrict the processing of local targets. In this study, we report that the long isoform of the cellular FLICE-inhibitory protein (c-FLIP(L)), a well-known inhibitor of the extrinsic cell death initiator caspase-8, localizes at the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs). ER morphology was disrupted and ER Ca(2+)-release as well as ER-mitochondria tethering was decreased in c-FLIP(-/-) mouse embryonic fibroblasts (MEFs). Mechanistically, c-FLIP ablation resulted in enhanced basal caspase-8 activation and in caspase-mediated processing of the ER-shaping protein reticulon-4 (RTN4) that was corrected by re-introduction of c-FLIP(L) and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIP(L) emerges as a component of the MAMs signaling platforms, where caspases appear to regulate ER morphology and ER-mitochondria crosstalk by impinging on ER-shaping proteins like the RTN4.

c-Flip overexpression affects satellite cell proliferation and promotes skeletal muscle aging.

This study shows that forcing c-Flip overexpression in undifferentiated skeletal myogenic cells in vivo results in early aging muscle phenotype. In the transgenic mice, adult muscle histology, histochemistry and biochemistry show strong alterations: reduction of fibers size and muscle mass, mitochondrial abnormalities, increase in protein oxidation and apoptosis markers and reduced AKT/GSK3ß phosphorylation. In the infant, higher levels of Pax-7, PCNA, P-ERK and active-caspase-3 were observed, indicating enhanced proliferation and concomitant apoptosis of myogenic precursors. Increased proliferation correlated with NF-κB activation, detected as p65 phosphorylation, and with high levels of embryonic myosin heavy chain. Reduced regenerative potential after muscle damage in the adult and impaired fiber growth associated with reduced NFATc2 activation in the infant were also observed, indicating that the satellite cell pool is prematurely compromised. Altogether, these data show a role for c-Flip in modulating skeletal muscle phenotype by affecting the proliferative potential of undifferentiated cells. This finding indicates a novel additional mechanism through which c-Flip might possibly control tissue remodeling.

FLIP is expressed in mouse testis and protects germ cells from apoptosis.

Apoptosis control in adult testis is crucial to achieve normal spermatogenesis. In this study c-FLIP, an apoptosis-modulating protein, was investigated. In Western blot and immunohistochemical analyses, the 55 KDa c-FLIP long isoform (c-FLIP(L)) was found to be expressed strongly in spermatocytes and spermatids, at low levels in spermatogonia and at almost undetectable levels in Sertoli cells. This expression pattern was confirmed by Northern blot analyses. Further experiments carried out on GC-1spg germ cell line revealed that reducing c-FLIP(L) expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIP(L) expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIP(L) expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIP(L) expression. Finally, TUNEL staining of seminiferous tubules incubated with anti-Fas antibody showed that apoptosis occurs mostly in basally located germ cells, indicating that such cells, expressing low levels of c-FLIP(L), are sensitive to Fas-mediated apoptosis. These data indicate for the first time that c-FLIP(L) might control germ cell apoptosis and caspase activity in the adult testis.

Adenosine potentiates forskolin-induced delay of meiotic resumption by mouse denuded oocytes: evidence for an oocyte surface site of adenosine action.

Adenosine is present in the mouse follicular fluid and has been shown to interfere with oocyte maturation in vitro. To clarify the mechanism of adenosine action on meiotic arrest, we have characterized the synergistic action of this purine with forskolin on the meiotic resumption of mouse denuded oocytes. Forskolin delays meiotic resumption by approximately 1 hour; adenosine at concentrations ranging between 30-750 microM has no significant effect. Conversely, adenosine treatment together with forskolin produces a further delay in the resumption of meiosis. This adenosine effect is dose-dependent and mimicked by adenosine analogs like N6-phenylisopropyl adenosine (PIA), 2-chloroadensoine (2-CLA), 5'-N-ethylcarboxamide (NECA). Dipyridamole, which inhibits adenosine transport, does not prevent the meiosis-arresting synergistic effect of adenosine with forskolin. Adenosine causes a 50% increase of adenosine triphosphate (ATP) content in the oocyte. However, this increase is not directly responsible for the observed delay in oocyte maturation for the following reasons: (1) the dose response of inhibition of meiotic resumption does not correlate with the doses of adenosine producing an increase in ATP; (2) dipyridamole blocks the increase in intracellular ATP, but it has no effect on the adenosine inhibition of maturation; (3) adenosine analogs inhibit oocyte maturation but do not affect intracellular ATP levels. These results suggest that the synergism of adenosine with forskolin on meiotic arrest does not require uptake of the nucleoside nor its conversion to ATP and that the adenosine effects are exerted at the level of the oocyte plasma membrane.

Effect of follicle-stimulating hormone on cyclic adenosine monophosphate level and on meiotic maturation in mouse cumulus cell-enclosed oocytes cultured in vitro.

We have reported that in vitro treatment with follicle-stimulating hormone (FSH) delays by about 3 h spontaneous meiotic resumption in cumulus cell-enclosed mouse oocytes. In the present paper we show that the temporary meiotic block is accompanied by a transient increase of cAMP concentration in the oocyte. In cumulus cell-oocyte complexes stimulated with 1 microgram/ml FSH, cAMP significantly increases within 1 h both in the whole complex (from a basal value of 1.9 +/- 0.2 to 169 +/- 13 fmol) and in the enclosed oocyte (from 0.9 +/- 0.2 to 2.4 +/- 0.2 fmol), then progressively decreases to basal values. Stimulation by FSH does not cause any cAMP increase in denuded oocytes. As the concentration of cAMP in the cells decreases, the percentage of oocytes escaping the meiotic block imposed by FSH increases. If the complexes are cultured in the presence of 1 microgram/ml FSH plus 1 mM isobutyl-1-methylxanthine (1BMX), cAMP concentration increases approximately 250-fold in the complex, and 10-fold in the enclosed oocyte; the level of cAMP in the oocyte drops very rapidly (50% degradation in less than 2 min) if the oocyte is then transferred to IBMX-free medium. The data are discussed in terms of the possible role of cAMP transfer from cumulus cells to the oocyte in the regulation of meiotic progression in mouse oocytes.

Granulosa cells stimulate in vitro the expansion of isolated mouse cumuli oophori: involvement of prostaglandin E2.

Granulosa cells (2 X 10(6) per ml) obtained from pregnant mare's serum gonadotropin (PMSG)-primed mice induce within 24 h the expansion in vitro of cocultured mouse cumuli oophori. Experiments with conditioned media showed that the expansion-promoting action of granulosa cells is due to diffusible factor(s) released into the culture medium. Studies with prostaglandin synthetase inhibitors and direct measurements of prostaglandin E2 (PGE2) released by granulosa cells in the culture medium have also been performed. The results strongly suggest that the cumulus oophorus expansion-promoting action of granulosa cells is mediated by PGE2, and support the hypothesis (Downs and Longo, 1983) that granulosa cells might play a similar role in the mechanism of cumulus expansion in vivo. The suggestion is advanced that coculture with granulosa cells might be of help to allow physiologic expansion in culture of immature cumuli obtained from preovulatory follicles in in vitro fertilization programs.