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.

The Fas system in the seminiferous epithelium and its possible extra-testicular role.

The Fas system is involved in the control of immune system homeostasis and nonfunctional Fas system leads to autoimmune disease in mice and humans. The Fas system is a mechanism through which cells expressing Fas ligand (FasL) induce apoptosis of Fas expressing cells. In mouse and rat, the testis represents the main source of constitutive FasL in the body. The roles so far proposed for this molecule in the testis, such as maintenance of immunoprivilege and regulation of physiological germ cell apoptosis, need to be reconsidered as both hypotheses are based on an erroneous cellular location of FasL in the seminiferous epithelium. Recently, we demonstrated that in rodents FasL mRNA is present in germ cells and not in Sertoli cells, and that FasL protein is displayed on the surface of spermatozoa. Here we propose that, for the mouse spermatozoa, the FasL may represent a self-defence mechanism against lymphocytes present in the female genital tract. To verify this hypothesis, we performed crossings between males gld, with nonfunctional FasL, and syngenic or nonsyngenic females. We observed a significant decrease of litter size in outbred crossings with gld males compared with wild-type males, suggesting a possible role of FasL in immunoprotection of the sperm in the female genital tract. The possibility that in humans, by analogy with mouse, FasL plays a self-protective role for the spermatozoon cannot be excluded, and awaits experimental information on the expression of FasL on human sperm cells.

E1A stimulates FGF-2 release promoting differentiation of primary endothelial cells.

Basic Fibroblast Growth Factor (FGF-2) is a growth and survival factor and represents one of the most potent differentiation agents of vascular system. In the present study we describe that adenoviral oncoprotein E1A regulates FGF-2 production and determines the acquisition of a pro-angiogenic phenotype in primary bovine aortic endothelial cells (BAEC). Following their transfection, wild type E1A proteins 12S and 13S (wtE1A) stimulated BAEC to differentiate on reconstituted basement membrane matrix (Matrigel). This outcome was paralleled by invasion and migration enhancement in wtE1A-transfected cells. This stimulating effect was absent with the E1A mutant dl646N. Accordingly, zymography and RT - PCR analyses showed that matrix metalloproteinase-9 protein- and mRNA-levels increased following wtE1A transfection. Interestingly, wtE1A-transfected BAEC showed FGF-2 mRNA- and protein-levels higher than controls. Further, FGF-2 neutralization reduced the amount of MMP-9 released in the supernatant of E1A-transfected cells and strongly inhibited BAEC differentiation, thus suggesting that wtE1A activates BAEC by a mechanism, at least partially, dependent on a FGF-2 autocrine/paracrine loop.