Simonetta Petrungaro

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

AbstractApoptosis 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-FLIPL) 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-FLIPL expression increases Fas-dependent apoptosis. Conversely, restoring c-FLIPL expression reduces this response to control levels. Caspase-10 expression was found to match c-FLIPL expression pattern; further, caspase-10 activation upon anti-Fas treatment inversely correlated with c-FLIPL 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-FLIPL, are sensitive to Fas-mediated apoptosis.These data indicate for the first time that c-FLIPL might control germ cell apoptosis and caspase activity in the adult testis.

Necroptosis: Molecular Signalling and Translational Implications

Necroptosis is a form of programmed necrosis whose molecular players are partially shared with apoptotic cell death. Here we summarize what is known about molecular signalling of necroptosis, particularly focusing on fine tuning of FLIP and IAP proteins in the apoptosis/necroptosis balance. We also emphasize necroptosis involvement in physiological and pathological conditions, particularly in the regulation of immune homeostasis.

Characterization of signaling pathways leading to Fas expression induced by TNF-alpha: pivotal role of NF-kappaB.

TNF‐α is known to induce a strong up‐regulation of Fas expression in mouse Sertoli cell cultures, leading to their apoptosis triggered by effector FasL‐bearing cells. These data suggest that increased Fas expression on the cell surface might be a key event in the pathogenesis of autoimmune orchitis, by inducing a leakage of the blood‐tubular barrier as a consequence of Sertoli cell apoptosis. In the present paper, we have investigated the signal transduction mechanisms involved in the regulation of Fas expression induced by TNF‐α in mouse Sertoli cells. We studied the role of the transcription factor NF‐κB and of MAPKs in regulating Fas expression. By using Sertoli cells transfected with a NF‐κB Luc reporter gene, we proved that TNF‐α activates the IκB/NF‐κB system. Moreover, the use of the proteasome inhibitor lactacystin led us to demonstrate that NF‐κB is required for TNF‐α mediated Fas expression. By using specific inhibitors for each MAPK, we confirmed the pivotal role of the IκB/NF‐κB system by demonstrating that ERKs, p38, and JNK are not involved in Fas up‐regulation by TNF‐α. The comprehension of these pathways could be relevant to the knowledge of the pathogenesis of autoimmune disorders in immune privileged districts of the body.

Cancer Microenvironment and Endoplasmic Reticulum Stress Response

Different stressful conditions such as hypoxia, nutrient deprivation, pH changes, or reduced vascularization, potentially able to act as growth-limiting factors for tumor cells, activate the unfolded protein response (UPR). UPR is therefore involved in tumor growth and adaptation to severe environments and is generally cytoprotective in cancer. The present review describes the molecular mechanisms underlying UPR and able to promote survival and proliferation in cancer. The critical role of UPR activation in tumor growth promotion is discussed in detail for a few paradigmatic tumors such as prostate cancer and melanoma.

c-Flip overexpression reduces cardiac hypertrophy in response to pressure overload.

Objective Activation of Fas signaling has been associated with the development of cardiomyocyte hypertrophy. In the present study, we investigated the effects of increased expression of c-Flip, a natural modulator of Fas receptor signaling, in a mouse model of cardiac growth response to pressure overload. Methods A transgenic mouse overexpressing c-Flip in the heart was generated in FVB/N strain. Echocardiographic, hemodynamic, histological and molecular analyses were carried out under basal conditions and after transverse aortic constriction (TAC)-induced pressure overload. Results Overexpression of c-Flip in ventricular heart tissue was functionally silent under basal conditions affecting neither cardiac morphology nor basal cardiac function. Transgenic mice were then subjected to pressure overload by TAC procedure. Under such conditions, c-Flip transgenic mice showed normal left ventricular function with a significantly reduced left ventricular hypertrophy compared with wild-type mice and reduced induction of the cardiac “fetal” gene programme. Further, analysis of intracellular signaling pathways indicated that c-Flip overexpression reduced phosphorylation of both the glycogen synthase kinase 3β (GSK3β) and Akt as compared with controls. Finally, the reduction of the TAC-induced hypertrophy was not accompanied by significant apoptosis increase. Conclusion Altogether, these findings indicate c-Flip as a key regulator of the cardiac response to ventricular pressure overload.

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/GSK3β 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.

Autophagy in Prostate Cancer and Androgen Suppression Therapy

The role of autophagy is known to be highly complex and context-dependent, leading to both cancer suppression and progression in several tumors including melanoma, breast and prostate cancer. In the present review, recent advances in an understanding of the involvement of autophagy in prostate cancer treatment are described. The regulatory effects of androgens on prostate cancer cell autophagy are particularly discussed in order to highlight the effects of autophagy modulation during androgen deprivation. A critical evaluation of the studies examined in the present review suggests the attractive possibility of autophagy inhibition combined with hormonal therapy as a promising approach for prostate cancer treatment.

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-FLIPL), 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 Ca2+-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-FLIPL and caspase inhibition, resulting in the recovery of a normal ER morphology and ER-mitochondria juxtaposition. Thus, the caspase-8 inhibitor c-FLIPL 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 expression and function in fetal mouse gonocytes

Apoptosis is a key mechanism in spermatogenesis, and in testis, most gonocytes degenerate at fetal and postnatal ages to select a cell subset committed to become germ stem cells. The aim of the present study is to investigate mechanisms controlling the massive apoptosis of fetal gonocytes. We evaluated the expression and function of c‐Flip, an apoptosis inhibitor known to interfere with the proapoptotic Fas‐signaling pathway in a variety of cell types, but never investigated in fetal testis. Expression of c‐Flip long isoform (c‐FlipL) within fetal testis was localized in gonocytes at 16.5 and 18.5 days post coitum (dpc), both at the mRNA and protein level, while it was weakly expressed or undetectable at earlier stages. Moreover, Fas protein was found in fetal testes at 13.5, 16.5, and 18.5 dpc. Testes at 18.5 dpc, expressing high levels of c‐FlipL, were resistant to Fas‐induced apoptosis while they became highly sensitive when c‐FlipL was inhibited by antisense c‐Flip oligos. In addition, there was an inverse relation between gonocyte spontaneous apoptosis sensitivity and c‐FlipL levels. Furthermore, caspase‐10 activity was inversely related with c‐FlipL expression, suggesting that caspase‐10 might be a target of c‐FlipL. These data represent the first evidence demonstrating c‐Flip expression in fetal testes and its role in protecting gonocytes from Fas‐dependent apoptosis.

Testis atrophy and reduced sperm motility in transgenic mice overexpressing c-FLIPL

OBJECTIVE To study the effect of c-FLIP overexpression in testicular germ cells. DESIGN A novel transgenic mouse model overexpressing the apoptotic modulator c-FLIP in the testis was generated. SETTING Animal facility and university research laboratory. ANIMAL(S) Transgenic mice overexpressing the long isoform of c-FLIP (c-FLIP(L)) under the transcriptional control of a 400 bp long regulatory region of the Stra8 promoter. INTERVENTION(S) Spermatozoa motility and testis histological, immunohistochemical, and Western blot analyses were carried out in transgenic and control derived specimens. MAIN OUTCOME MEASURE(S) Testis morphology, sperm motility, and germ cell apoptosis were assayed. RESULTS Stra8 promoter was found to activate the ectopic overexpression of c-FLIP(L) in round and elongated spermatids. As a consequence of such overexpression, a dramatic loss of germ cells was observed, resulting in testicular atrophy associated with reduced sperm motility. CONCLUSION(S) The data show that c-FLIP(L) forced expression in haploid male germ cells has detrimental effects on spermatogenesis and sperm quality and reveal a possible mechanism underlying the onset of testicular atrophy.