TIG3 Tumor Suppressor-Dependent Organelle Redistribution and Apoptosis in Skin Cancer Cells

TIG3 is a tumor suppressor protein that limits keratinocyte survival during normal differentiation. It is also important in cancer, as TIG3 level is reduced in tumors and in skin cancer cell lines, suggesting that loss of expression may be required for cancer cell survival. An important goal is identifying how TIG3 limits cell survival. In the present study we show that TIG3 expression in epidermal squamous cell carcinoma SCC-13 cells reduces cell proliferation and promotes morphological and biochemical apoptosis. To identify the mechanism that drives these changes, we demonstrate that TIG3 localizes near the centrosome and that pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization and organelle distribution. Organelle accumulation at the centrosome is a hallmark of apoptosis and we demonstrate that TIG3 promotes pericentrosomal organelle accumulation. These changes are associated with reduced cyclin D1, cyclin E and cyclin A, and increased p21 level. In addition, Bax level is increased and Bcl-XL level is reduced, and cleavage of procaspase 3, procaspase 9 and PARP is enhanced. We propose that pericentrosomal localization of TIG3 is a key event that results in microtubule and microfilament redistribution and pericentrosomal organelle clustering and that leads to cancer cell apoptosis

Etude du rôle des lysosomes et du cholestérol au cours de la différenciation des kératinocytes épidermiques

Etude du rôle des lysosomes et du cholestérol au cours de la différenciation des kératinocytes épidermiques (par Ralph Jans) La majorité des rôles protecteurs de l'épiderme, couche superficielle de la peau, sont garantis par les kératinocytes qui se différencient de manière progressive et terminale dans les couches suprabasales de ce tissu, dont le renouvellement est assuré par la prolifération de cellules-souches dans la couche basale. Toute perturbation des mécanismes qui contrôlent la prolifération et/ou la différenciation des kératinocytes conduit à des dysfonctionnements. Comprendre ces mécanismes reste donc un défi majeur pour la biologie cutanée. Plusieurs types cellulaires subissent l'exocytose de lysosomes quand il y a entrée d'ions calcium dans les cellules. Puisque les kératinocytes subissent une entrée de calcium au cours de leur différenciation, ces cellules pourraient exocyter des lysosomes dans ces circonstances. Un traitement de kératinocytes avec un ionophore induit en effet une sécrétion de la forme mature lysosomale de la cathepsine D, une libération de l'activité des enzymes lysosomales solubles cathepsine C et β-galactosidase, ainsi que l'apparition des protéines lysosomales membranaires Lamp-1 et Lamp-2 au niveau de la membrane plasmique. L'exocytose de lysosomes peut donc faire partie de la différenciation des kératinocytes, mais permet aussi à ces cellules de réparer des ruptures de la membrane plasmique. Puisque le cholestérol pourrait contrôler certaines voies de signalisation au cours de la différenciation des kératinocytes, nous avons analysé les effets d'une déplétion en cholestérol induite par un traitement avec la méthyl-β-cyclodextrine sur le phénotype et la signalisation du kératinocyte. Cette déplétion induit une augmentation de l'expression de l'involucrine, marqueur de différenciation tardive, et une répression de l'expression de la kératine 10, marqueur de la différenciation précoce, et de la kératine 14, marqueur des k??ratinocytes non-différenciés. Ce traitement active le récepteur de l'EGF et HER2, ainsi que la MAP kinase p38 dont l'activation (en particulier l'activation de p38α) est responsable de l'augmentation de l'expression de l'involucrine. En résumé, nos observations suggèrent un nouveau rôle pour les lysosomes au cours de la différenciation des kératinocytes épidermiques, mais suggèrent surtout un rôle critique du cholestérol dans la régulation de ce processus.Role of lysosomes and cholesterol during the differentiation process of epidermal keratinocytes (by Ralph Jans) A major part of the protective role of the epidermis, superficial layer of the skin, is guaranteed by the keratinocytes that differentiate progressively and terminally in the suprabasal layers of this tissue. The renewal of the epidermis is performed by proliferating stem cells in the basal layer. Perturbation of the mechanisms that regulate the proliferation and/or the differentiation of keratinocytes leads to an invalid barrier function. Therefore, elucidating these mechanisms is a major challenge for skin researchers. In several cell types, lysosomes undergo exocytosis upon entry of calcium into the cells. Since keratinocytes are subjected to an entry of calcium during their differentiation in vivo, these cells could exhibit an exocytosis of lysosomes under these circumstances. The results presented in this work show that an incubation of keratinocytes with the calcium ionophore ionomycin triggers the secretion of the enzymatic activities of the lysosomal enzymes cathepsin C and β-galactosidase as well as the release of the lysosomal form of cathepsin D. This treatment also induces the appearance of the lysosomal membrane proteins Lamp-1 and Lamp-2 at the plasma membrane of keratinocytes. Exocytosis of lysosomes could be part of the keratinocyte differentiation process, but could also allow these cells to repair their plasma membrane upon disruption due to mechanical stresses. Since cholesterol could be involved in the regulation of several signal transduction pathways during keratinocyte differentiation, we have investigated the effects of a depletion of cholesterol on the keratinocyte phenotype and on selected signaling pathways. Cholesterol depletion was induced by incubating the cells with methyl-β-cyclodextrin. This treatment, followed by an inhibition of cholesterol neosynthesis using lovastatin, triggers an upregulation of the expression of involucrin, a late differentiation marker, and a downregulation of keratin 10 and keratin 14, which, respectively, are markers of early-differentiating and undifferentiated keratinocytes. Cholesterol depletion activates the membrane receptors EGFR and HER2 and the MAP kinase p38. Using the specific inhibitor PD169316, we demonstrate that the p38α isoform is responsible for the upregulation of involucrin during cholesterol depletion. In summary, our observations suggest a novel role for lysosomes during keratinocyte differentiation and indicate a critical role for cholesterol in the regulation of this process.(DOCMED00)--FUNDP, 200

Cholesterol depletion upregulates involucrin expression in epidermal keratinocytes through activation of p38

Cholesterol has been recently suggested to regulate the early steps of keratinocyte differentiation through lipid rafts. In many cell types, depletion of cholesterol activates signaling proteins like epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or extracellular signal-regulated kinase (ERK) known to affect cell differentiation. In this study, we explored the effects of cholesterol depletion on the phenotype of cultured keratinocytes, using a treatment with methyl-β-cyclodextrin (MβCD) to extract cholesterol and a treatment with lovastatin to inhibit cholesterol neosynthesis. Analysis of the expression of differentiation marker genes in early differentiating confluent cultures reveals that cholesterol depletion induces downregulation of keratin 14 (K14) and keratin 10 (K10) and upregulation of involucrin. MβCD treatment induces phosphorylation of EGFR, HER2, and ERK, but not HER3. Inhibition of EGFR with PD153035 impairs the MβCD-induced phosphorylation of EGFR, HER2, and ERK, but does not impair the alteration of K14, K10, or involucrin gene expression, indicating that other signaling proteins regulate this phenomenon. p38 has been suggested to regulate the expression of involucrin during keratinocyte differentiation. We found that MβCD treatment induces a prolonged phosphorylation of p38 in general and p38α in particular. An inhibition of p38 with PD169316 impairs the upregulation of involucrin mRNAs by a treatment with MβCD, but not by a p38δ-activating TPA treatment, which might suggest that cholesterol depletion alters involucrin gene expression through activation of p38α/β