Marco Marchisio

Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-ζ translocation to the nucleus of NGF-treated PC12 cells

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738–29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non‐canonical 3‐phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC‐ζ nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3‐kinase (PI 3‐K) activityand its product, phosphatidylinositol 3,4,5‐trisphosphate [PtdIns(3,4,5)P3]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3‐K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3‐K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P3. Maximal translocation of PKC‐ζ from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG‐sensitive, PKC‐α. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1‐O‐octadeyl‐2‐O‐methyl‐sn‐glycero‐3‐phosphocholine, was unable to abrogate nuclear translocation of the DAG‐insensitive PKC‐ζ. These data suggest that a nuclear increase in PI 3‐K activity and PtdIns(3,4,5)P3 production are necessary for the subsequent nuclear translocation of PKC‐ζ. Furthermore, they point to the likelihood that PKC‐ζ is a putative nuclear downstream target of PI 3‐K during NGF‐promoted neural differentiation.—Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3‐kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC‐ζ translocation to the nucleus of NGF‐treated PC12 cells. FASEB J. 13, 2299–2310 (1999)

Expression profile of the embryonic markers nanog, OCT-4, SSEA-1, SSEA-4, and frizzled-9 receptor in human periodontal ligament mesenchymal stem cells

Mesenchymal stem cells (MSCs) are self‐renewing cells with the ability to differentiate into various mesodermal‐derived tissues. Recently, we have identified in adult human periodontal ligament (PDL) a population of stem cells (PDL‐MSCs) with the ability to differentiate into osteoblasts and adipocytes. The aim of the present work was to further characterize this population and the expression profile of its cells. To achieve our objective we have used flow cytometry, magnetic cell sorting, cytokine antibody array, and light and electron microscope immunostaining. Our results show that the PDL‐MSCs contain a subpopulation of frizzled‐9 (CD349) positive cells expressing a panel of key mesenchymal and embryonic markers including CD10, CD26, CD29, CD44, CD73, CD90, CD105, CD166, SSEA‐1, and SSEA‐4. They are additionally positive for nanog and Oct‐4; two critical transcription factors directing self‐renewal and pluripotency of embryonic stem cells, and they also express the cytokines EGF and IP‐10. The presence of nanog, Oct‐4, SSEA‐1, and SSEA‐4 suggests that PDL‐MSCs are less differentiated than bone marrow‐derived MSCs. Taken together, these data indicate the presence of immature MSCs in PDL and suggest that the frizzled‐9/Wnt pathway plays an important role in regulating proliferation and differentiation of these cells. J. Cell. Physiol. 225: 123–131, 2010.

Nuclear association of tyrosine-phosphorylated Vav to phospholipase C-γ1 and phosphoinositide 3-kinase during granulocytic differentiation of HL-60 cells

The granulocytic differentiation of HL‐60 cells induced by all‐trans retinoic acid was accompanied by a progressive tyrosine phosphorylation of specific proteins in either cells or isolated nuclei. Among these phosphoproteins, we identified the Vav adaptor in whole cells as well as in the inner nuclear compartment, where the increase in its tyrosine phosphorylation level was more conspicuous. We also demonstrated the differentiation‐dependent association of nuclear phosphorylated Vav to phospholipase C‐γ1 and to the p85 regulatory subunit of phosphoinositide 3‐kinase. The role of the Vav/phospholipase C‐γ1/phosphoinositide 3‐kinase phosphoprotein complexes in the nuclei of HL‐60 induced to differentiate along the granulocytic lineage is discussed.

Discrete subcellular localization of phosphoinositidase C β, γ and δ in PC12 rat pheochromocytoma cells

Phosphoinositidase C activity was revealed in nuclei isolated from PC12 rat pheochromocytoma cells incubated with tritiated phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Phosphoinositide breakdown was found to be optimal at neutral pH and Ca ++ concentrations ranging from endogenous levels to millimolar values. To characterize the enzymes involved, three monoclonal antibodies directed against the β, γ and δ phosphoinositidase C isoforms were employed. A combination of Western blot immunochemical analysis on cytoplasmic and nuclear fractions and of in situ immunocytochemistry on intact cells and isolated nuclei indicated that phosphoinositidase C γ, though predominantly cytoplasmic, was present in both cell compartments. On the contrary, phosphoinositidase C β was exclusively localized in the nucleus, whereas phosphoinositidase C δ was restricted to the cytoplasm. These data suggest that inositol lipid breakdown is controlled by different phosphoinositidase C isozymes in the various cell compartments, and support the notion that a separate phosphoinositide signalling system is located in the nucleus.

Stromal derived factor-1α (SDF-1α) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway

Stromal‐derived factor‐1α (SDF‐1α), the high‐affinity ligand of CXC‐chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF‐1α‐mediated Jurkat cell apoptosis was observed in serum‐free or serum‐containing cultures, peaked at SDF‐1α concentrations of 10–100 ng/ml, required 3 days to take place, and was completely blocked by the z‐VAD‐fmk tripeptide caspase inhibitor. Although SDF‐1α did not modify the expression of TNF‐α or that of TNF‐RI and TNF‐RII, it increased the expression of surface Fas/APO‐1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF‐1α to induce apoptosis was inhibited by an anti‐CD95 Fab′ neutralizing antibody. These findings suggest a role for SDF‐1α in the homeostatic control of CD4+ T‐cell survival/apoptosis mediated by the CD95‐CD95L pathway.

Wnt Signaling Behaves as a “Master Regulator” in the Osteogenic and Adipogenic Commitment of Human Amniotic Fluid Mesenchymal Stem Cells

AbstractHuman amniotic fluid mesenchymal stem cells (huAFMSCs) are emerging as a promising therapeutic option in regenerative medicine. Here, we characterized huAFMSC phenotype and multipotentiality. When cultured in osteogenic medium, huAFMSC displayed a significant increase in: Alkaline Phosphatase (ALP) activity and mRNA expression, Alizarin Red S staining and Runx2 mRNA expression; whereas maintaining these cells in an adipogenic culture medium gave a time-dependent increase in PPARγ and FABP4 mRNA expression, glycerol-3-phosphate dehydrogenase (GPDH) activity and positivity to Oil Red Oil staining. These results confirm that huAFMSCs can differentiate toward osteogenic and adipogenic phenotypes. The canonical Wnt/ßcatenin signaling pathway appears to trigger huAFMSC osteoblastogenesis, since during early phases of osteogenic differentiation, the expression of Dishevelled-2 (Dvl-2), of the non-phosphorylated form of ß-catenin, and the phosphorylation of glycogen synthase kinase-3ß (GSK3ß) at serine 9 were upregulated. On the contrary, during adipogenic differentiation Dvl-2 expression decreased, whereas that of ß-catenin remained unchanged. This was associated with a late increase in GSK3ß phosphorylation. Consistent with this scenario, huAFMSCs exposure to Dickkopf-1, a selective inhibitor of the Wnt signaling, abolished Runx2 and ALP mRNA upregulation during huAFMSC osteogenic differentiation, whereas it enhanced FABP4 expression in adipocyte-differentiating cells. Taken together, these results unravel novel molecular determinants of huAFMSC commitment towards osteoblastogenesis, which may represent potential targets for directing the differentiation of these cells and improving their use in regenerative medicine. FigureSchematic representation of Wnt pathway involved in the osteogenic and adipogenic differentiation of huAFMSCs. Our paper demonstrates that osteogenic commitment of these cells is linked to the stimulation of Wnt signal leading to the final transcriptional activation of early osteogenic markers such as RUNX-2 and ALP, mediated by β-catenin. DKK1 is a secreted Wnt antagonist that may be used as a drug to inhibit Wnt signal. In contrast, adipogenic commitment involves early inhibition of Wnt pathway leading to ubiquitination/degradation of β-catenin. This results in the transcription of PPARγ and FABP4, considered as the main initiators of adipogenesis. APC, adenomatous polyposis coli; βcat, β-catenin; CK1, casein kinase 1; DKK1, dickkopf 1; Dvl, Dishevelled; GSK3β, glycogen synthase kinase 3β; LRP5/6, low density lipoprotein receptor-related protein 5/6

Proteome of human stem cells from periodontal ligament and dental pulp.

Background Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro. Methodology/Principal Findings The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4–7 and 6–9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin. Conclusion/Significance This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.

Influence of the human immunodeficiency virus type 1 Tat protein on the proliferation and differentiation of PC12 rat pheochromocytoma cells

Rat pheochromocytoma PC12 cells were permanently transfected with a plasmid vector, containing the tat gene of human immunodeficiency virus type 1 (HIV-1). Various clones were obtained showing the production of different levels of bioactive Tat protein (Tat) after transient cotransfection with an HIV-1 long terminal repeat-chloramphenicol acetyltransferase reporter plasmid. Under conditions of serum starvation, tat-positive PC12 clones expressing high levels of Tat showed a significantly (P < 0.05) higher proliferation rate with respect to both mock-transfected PC12 cells and tat-positive PC12 cells expressing lower levels of Tat. Moreover, all tat-positive PC12 cell clones showed a partial morphological differentiation into sympathetic-like neurons, when seeded in low density (5 x 10(3) cells/cm2) cultures. On the other hand, mock-transfected PC12 cells showed the round shaped morphology typical of untreated PC12 cells and displayed signs of neuronal differentiation only after treatment with 100 ng/ml of nerve growth factor. The addition of 5 micrograms/ml of anti-Tat monoclonal antibody to the culture medium of tat-positive PC12 cell clones almost completely blocked their increased proliferation rate (P < 0.05), but did not affect neuronal differentiation. A significant (P < 0.05) increase in cell proliferation was consistently observed in PC12 cells supplemented with low concentrations of Tat (5 to 25 ng/ml), whereas neuronal differentiation was hardly affected by exogenous Tat. Our data strongly suggest that Tat exerts a complex influence on the proliferation and differentiation of PC12 cells, and this might help in increasing understanding of the pathogenesis of the frequent neurological disorders observed in AIDS patients.

Proteome analysis of human Wharton's jelly cells during in vitro expansion

BackgroundThe human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Whartons jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine.ResultsTo better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time.ConclusionsOur work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.

Changes of Nuclear PI-PLC γ1 During Rat Liver Regeneration

Abstract We have previously demonstrated that rat liver nuclei contain PI-PLC β 1 and γ 1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669–678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP 2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (β 1 , γ 1 , δ 1 ) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC β 1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear γ 1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC δ 1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC γ 1 that paralleled the in situ observation whereas the β 1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP 2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP 2 , obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the γ 1 isoform of PI-PLC.