Lucrezia Manente

Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC–MEF2 complexes

Histone deacetylase (HDAC) inhibitors are promising new epi‐drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II‐specific HDAC inhibitor MC1568, we separated class I‐ and class II‐dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4–HDAC3–MEF2D complex, and (iii) paradoxically, by inhibiting differentiation‐induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue‐selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2–HDAC complexes in a repressed state. Our results suggest that HDAC class II‐selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.

Distribution of Notch protein members in normal and preeclampsia-complicated placentas

Notch proteins are a transmembrane receptor family that is structurally and functionally conserved from worms to humans. The mammalian family of Notch proteins consists of several genes encoding Notch receptors and related Notch ligands. Notch signaling is involved in different aspects of the cell-fate decision tree: differentiation, proliferation, and apoptosis. These three processes are finely regulated in human placenta in order to allow a successful pregnancy and correct fetal growth. Notch and its ligands also participate in vascular remodeling and stabilization. Vasculogenesis and blood regulation are of importance in the human placenta for normal fetal development and growth; any disorder of these systems leads to preeclampsia. Drawing on this background, we have investigated the expression of Notch-1, Notch-4, and Jagged-1, together with two members related to the Notch pathway in angiogenesis: VEGF and p21. Normal and preeclamptic human placentas have been evaluated by immunohistochemistry. In preeclamptic samples, a down-regulation of Notch pathway members occurs with a weak/moderate expression of the Notch protein members in all components of placenta compared with physiological placentas that, at term, exhibit the strong expression of Jagged-1 and a moderate expression of both Notch-1 and Notch-4 in all compartments of the placental villi. Moreover, preeclamptic samples also reveal a down-regulation of VEGF expression, together with a moderate nuclear expression of p21Cip1 in the syncytiotrophoblast, cytotrophoblast, and endothelial cells. This down-regulation of VEGF in preeclamptic placentas, in turn, probably decreases Notch protein expression in placental compartments and in endothelial cells and could offer an ethiopathogenetic explanation for the onset of this pathology.

The Helicobacter pylori's protein VacA has direct effects on the regulation of cell cycle and apoptosis in gastric epithelial cells

In this study, we have evaluated the effects on cell cycle regulation of VacA alone and in combination with other two Helicobacter pylori proteins, cytotoxin‐associated protein (CagA) and HspB, using the human gastric epithelial cells (AGS). Our results indicate that VacA alone was able to inhibit the G1 to S progression of the cell cycle. The VacA capacity of inhibiting cell progression from G1 to S phase was also observed when cells were co‐transfected with CagA or HspB. Moreover, VacA over‐expression caused apoptosis in AGS cells through activation of caspase 8 and even more of caspase 9, thus indicating an involvement of both the receptor‐mediated and the mitochondrial pathways of apoptosis. Indeed, the two pathways probably can co‐operate to execute cell death with a prevalence of the mitochondrial pathways. Our data taken together provide additional information to further enhance our understanding of the molecular mechanism by which H. pylori proteins alter the growth status of human gastric epithelial cells. J. Cell. Physiol. 214: 582–587, 2008.

The pattern of expression of Notch protein members in normal and pathological endometrium.

The objective of this study was to investigate the pattern of expression and the localization of Notch‐1, Notch‐4 and Jagged‐1 in physiological and pathological human endometrium and to evaluate the expression levels of two major regulators of the G1 checkpoint, namely cyclin D1 and p21. Sixty samples of physiological endometrium and 60 samples of pathological endometrium were used for the study. Evaluation of the expression level and the distribution of Notch pathway members and cell‐cycle proteins was performed by immunohistochemistry. In the physiological endometrium we observed an increase of Notch‐1 and Jagged‐1 from proliferative to secretory phase and an opposite trend for Notch‐4. In menopause, the level of expression of all three members of the Notch pathway decreased. We also observed a cyclin D1 increase from proliferative to secretory phase. By contrast, p21 showed a slight increase from proliferative to secretory phase. In the pathological endometrium, we observed an increase of Notch‐1 expression from polyps to carcinoma and decrease for Notch‐4 and Jagged‐1. Moreover, we observed a higher expression of cyclin D1 in all the endometrial pathologies. By contrast, the expression level of p21 slightly increased from polyps to carcinoma. We concluded that in human endometrium Notch‐4 seems to be more involved in controlling proliferation, whereas Notch‐1 seems to be more involved in differentiation programming. Deregulation of these functions may induce the onset of several endometrial pathologies from polyps to cancer.

The serine protease HtrA1 specifically interacts and degrades the tuberous sclerosis complex 2 protein.

Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway. Mol Cancer Res; 8(9); 1248–60. ©2010 AACR.

Human papillomavirus-16 E7 interacts with siva-1 and modulates apoptosis in HaCaT human immortalized keratinocytes†

The viral factor E7 plays a key role in the well‐established association between “high‐risk” Human Papillomavirus (HPV) infection and the development of epithelial malignant tumors, as uterine cervix and ano‐genital cancer. To delve into the molecular mechanisms of HPV‐mediated cell transformation, we searched for novel potential cellular targets of the HPV‐16 E7 oncoprotein, by means of the yeast two‐hybrid technique, identifying a protein–protein interaction between HPV‐16 E7 and the pro‐apoptotic cellular factor Siva‐1. Using co‐precipitation assays and the “PepSets” technique, we confirmed this physical interaction and mapped accurately, for both proteins, the amino acid residues involved. Additionally, we found that HPV‐16 E7 competed in vitro with the binding of the Bcl‐XL anti‐apoptotic factor to Siva‐1, an interaction that has a major inference in UV radiation‐induced apoptosis. In HaCaT immortalized human keratinocytes, forced HPV‐16 E7 expression by retroviral infection caused Siva‐1 transcript up‐regulation, detected by cDNA macroarray hybridization and real‐time quantitative PCR, paralleled by an increased amount of protein. Confirming the anti‐apoptotic role of HPV‐16 E7 in the HaCaT cellular model, evaluated by nuclear morphology, we also found that Siva‐1 expression produced a significant increase of the apoptotic rate in UV radiation‐exposed HaCaT cells, and that this effect resulted explicitly counteracted by HPV‐16 E7. Being apoptosis a key physiological process for the elimination of irreversibly injured cells, the anti‐apoptotic role of HPV‐16 E7, performed at least by its interference with Siva‐1, can be considered an additional mechanism for the survival of damaged, potentially transforming, cell clones. J. Cell. Physiol. 212: 118–125, 2007.

The Helicobacter pylori protein HspB interferes with Nrf2/Keap1 pathway altering the antioxidant response of Ags cells.

Helicobacter pylori infection causes chronic oxidative stress on gastric mucosa, thereby causing mucosal damage and increasing the risk of gastric adenocarcinoma. Nrf2 is an important transcription factor, regulating the antioxidant response in the cells. Nrf2 signaling is repressed by Keap1 at basal condition and induced by oxidative stress. The aim of our study was to analyze whether the H. pylori proteins interfered in the Nrf2/Keap1 pathway.

3- O -methylfunicone, a metabolite of Penicillium pinophilum , inhibits proliferation of human melanoma cells by causing G 2 + M arrest and inducing apoptosis

Objectives:  Melanoma cells take advantage of impaired ability to undergo programmed cell death in response to different external stimuli and chemotherapeutic drugs; this makes prevention of tumour progression very difficult. The aim of this study was to demonstrate whether 3‐O‐methylfunicone (OMF), a metabolite of Penicillium pinophilum, has the ability to arrest cell population growth and to induce apoptosis in A375P (parental) and A375M (metastasis derivatived) melanoma cell lines.

Role of NEDD8 in HIV-associated lipodystrophy

The pathogenetic bases of HAART-associated lipodystrophy are still poorly known, even if it is clear that adipose tissue and its metabolism are sensitive to antiretroviral therapy alone and/or in combination with HIV infection. The NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression, morphogenesis and tumorigenesis. We investigated the possible involvement of NEED8 in adipogenesis and, consequently, in HIV-related lipodystrophy. One hundred HIV-1-infected patients were included in the study. Using an in vitro model of adipogenesis we evaluated the effects on adipogenesis of the forced expression of NEDD8 together with efavirenz, stavudine, saquinavir, amprenavir and indinavir, belonging to the three main classes of anti-HIV medications. We showed that NEDD8 expression level is higher in the peripheral blood of HIV patients developing lipodystrophy. Coherently, forced expression of NEDD8 in an in vitro model of adipogenesis was able to perturb expression of some key proteins involved in adipogenesis, such as C/EBPalpha and PPARgamma, possibly acting throughout the NEDD8/p27/beta-catenin pathway. Moreover, three out of five evaluated drugs were able to affect adipocyte differentiation: efavirenz, stavudine and saquinavir. Finally, we have shown that NEDD8 was expressed in the fat tissue of lipodystrophic patients, being significantly higher in the lipodystrophic patients with respect to the controls, thus further confirming the altered NEDD8 expression in the fat tissue of HIV-infected patients affected by lipodystrophy. Taken together, our data support the hypothesis of an implication of NEDD8 through p27 and beta-catenin pathways in the disruption of adipogenesis and consequent lipodystrophy in patients affected by HIV infection under HAART therapy with qualitative and quantitative differences according to diverse antiretroviral treatments. These evidences indicate the NEDD8/beta-catenin/p27 pathway as a possible molecular target for prevention of lipodystrophy development in patients under HAART therapy.

Pkn is a novel partner of cyclin T2a in muscle differentiation.

With the aim to find novel partners of human Cyclin T2a, we performed a two‐hybrid screening in yeast using the full‐length cDNA of this cyclin as bait, and a human heart cDNA library as preys source. Upon several interesting genes selected, our attention has been focused on the cDNA coding for PKNα, a fatty acid‐ and Rho‐activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. Co‐immunoprecipitation and in vitro pull‐down assays independently confirmed the interaction between the two proteins. Luciferase assays, performed on NIH3T3 cell extracts after transfection with a MyoD‐responsive promoter, pointed out that PKNα was able to enhance MyoD‐dependent transcription, and that this effect was further increased when cyclin T2a was co‐overexpressed. Finally, overexpression of both Cyclin T2a and PKNα in C2C12 cells strongly enhanced the expression of myogenic differentiation markers, such as Myogenin and Myosin Heavy Chain, during starvation‐induced differentiation. Taken together, our data strengthen the hypothesis that Cyclin T2a plays a role in muscle differentiation, and propose PKNα as a novel partner of Cyclin T2a in this process. J. Cell. Physiol. 207: 232–237, 2006.