The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma.

Osteopontin (SPP1, a secreted phosphoprotein 1) is primarily involved in immune responses, tissue remodelling and biomineralization. However, it is also overexpressed in many cancers and regulates tumour progression by increasing migration, invasion and cancer stem cell self-renewal. Mechanisms of SPP1 overexpression in gliomas are poorly understood. We demonstrate overexpression of two out of five SPP1 isoforms in glioblastoma (GBM) and differential isoform expression in glioma cell lines. Up-regulated SPP1 expression is associated with binding of the GLI1 transcription factor to the promoter and OCT4 (octamer-binding transcription factor 4) to the first SPP1 intron of the SPP1 gene in human glioma cells but not in non-transformed astrocytes. GLI1 knockdown reduced SPP1 mRNA and protein levels in glioma cells. GLI1 and OCT4 are known regulators of stem cell pluripotency. GBMs contain rare cells that express stem cell markers and display ability to self-renew. We reveal that SPP1 is overexpressed in glioma initiating cells defined by high rhodamine 123 efflux, sphere forming capacity and stemness marker expression. Forced differentiation of human glioma spheres reduced SPP1 expression. Knockdown of SPP1, GLI1 or CD44 with siRNAs diminished sphere formation. C6 glioma cells stably depleted of Spp1 displayed reduced sphere forming capacity and downregulated stemness marker expression. Overexpression of the wild type Spp1, but not Spp1 lacking a Cd44 binding domain, rescued cell ability to form spheres. Our findings show re-activation of the embryonic-type transcriptional regulation of SPP1 in malignant gliomas and point to the importance of SPP1-CD44 interactions in self-renewal and pluripotency glioma initiating cells.

Melanoma dormancy in a mouse model is linked to GILZ/FOXO3A-dependent quiescence of disseminated stem-like cells.

Metastatic cancer relapses following the reactivation of dormant, disseminated tumour cells; however, the cells and factors involved in this reactivation are just beginning to be identified. Using an immunotherapy-based syngeneic model of melanoma dormancy and GFP-labelled dormant cell-derived cell lines, we determined that vaccination against melanoma prevented tumour growth but did not prevent tumour cell dissemination or eliminate all tumour cells. The persistent disseminated melanoma tumour cells were quiescent and asymptomatic for one year. The quiescence/activation of these cells in vitro and the dormancy of melanoma in vivo appeared to be regulated by glucocorticoid-induced leucine zipper (GILZ)-mediated immunosuppression. GILZ expression was low in dormant cell-derived cultures, and re-expression of GILZ inactivated FOXO3A and its downstream target, p21CIP1. The ability of dormancy-competent cells to re-enter the cell cycle increased after a second round of cellular dormancy in vivo in association with shortened tumour dormancy period and faster and more aggressive melanoma relapse. Our data indicate that future cancer treatments should be adjusted according to the stage of disease progression.

Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.

Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.

Nerve growth factor and proNGF simultaneously promote symmetric self-renewal, quiescence, and epithelial to mesenchymal transition to enlarge the breast cancer stem cell compartment.

The discovery of cancer stem cells (CSCs) fundamentally advanced our understanding of the mechanisms governing breast cancer development. However, the stimuli that control breast CSC self-renewal and differentiation have still not been fully detailed. We previously showed that nerve growth factor (NGF) and its precursor proNGF can stimulate breast cancer cell growth and invasion in an autocrine manner. In this study, we investigated the effects of NGF and proNGF on the breast CSC compartment and found that NGF or proNGF enrich for CSCs in several breast cancer cell lines. This enrichment appeared to be achieved by increasing the number of symmetric divisions of quiescent/slow-proliferating CSCs. Interestingly, in vitro NGF pretreatment of MCF-7 luminal breast cancer cells promoted epithelial to mesenchymal transition in tumors of severe combined immunodeficient mice. Furthermore, p75(NTR), the common receptor for both neurotrophins and proneurotrophins, mediated breast CSC self-renewal by regulating the expression of pluripotency transcription factors. Our data indicate, for the first time, that the NGF/proNGF/p75(NTR) axis plays a critical role in regulating breast CSC self-renewal and plasticity.

Transient TNF regulates the self-renewing capacity of stem-like label-retaining cells in sphere and skin equivalent models of melanoma.

BACKGROUND: It is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse. RESULTS: Using an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal. CONCLUSIONS: We conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor.

Role of p75 neurotrophin receptor in stem cell biology: more than just a marker.

p75(NTR), the common receptor for both neurotrophins and proneurotrophins, has been widely studied because of its role in many tissues, including the nervous system. More recently, a close relationship between p75(NTR) expression and pluripotency has been described. p75(NTR) was shown to be expressed in various types of stem cells and has been used to prospectively isolate stem cells with different degrees of potency. Here, we give an overview of the current knowledge on p75(NTR) in stem cells, ranging from embryonic to adult stem cells, and cancer stem cells. In an attempt to address its potential role in the control of stem cell biology, the molecular mechanisms underlying p75(NTR) signaling in different models are also highlighted. p75(NTR)-mediated functions include survival, apoptosis, migration, and differentiation, and depend on cell type, (pro)neurotrophin binding, interacting transmembrane co-receptors expression, intracellular adaptor molecule availability, and post-translational modifications, such as regulated proteolytic processing. It is therefore conceivable that p75(NTR) can modulate cell-fate decisions through its highly ramified signaling pathways. Thus, elucidating the potential implications of p75(NTR) activity as well as the underlying molecular mechanisms of p75(NTR) will shed new light on the biology of both normal and cancer stem cells.

Insulin-like growth factor 1 receptor and p38 mitogen-activated protein kinase signals inversely regulate signal transducer and activator of transcription 3 activity to control human dental pulp stem cell quiescence, propagation, and differentiation.

Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration.

Colon cancer cells escape 5FU chemotherapy-induced cell death by entering stemness and quiescence associated with the c-Yes/YAP axis.

PURPOSE: Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. RESULTS: We show that a clonal 5F31 cell population, resistant to 1 µmol/L 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). CONCLUSIONS: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123(low)) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123(high) progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123(low) cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123(low) cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123(high) cells to the Rh123(low) phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123(low) cells. Moreover, the Rh123(low) cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123(low) melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123(low) G0 cells promotes the exit of cycling cells from the stem cell compartment.

Inactivation of the HIF-1α/PDK3 signaling axis drives melanoma toward mitochondrial oxidative metabolism and potentiates the therapeutic activity of pro-oxidants.

Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.

Interleukin-7 regulates adipose tissue mass and insulin sensitivity in high-fat diet-fed mice through lymphocyte-dependent and independent mechanisms.

Although interleukin (IL)-7 is mostly known as a key regulator of lymphocyte homeostasis, we recently demonstrated that it also contributes to body weight regulation through a hypothalamic control. Previous studies have shown that IL-7 is produced by the human obese white adipose tissue (WAT) yet its potential role on WAT development and function in obesity remains unknown. Here, we first show that transgenic mice overexpressing IL-7 have reduced adipose tissue mass associated with glucose and insulin resistance. Moreover, in the high-fat diet (HFD)-induced obesity model, a single administration of IL-7 to C57BL/6 mice is sufficient to prevent HFD-induced WAT mass increase and glucose intolerance. This metabolic protective effect is accompanied by a significant decreased inflammation in WAT. In lymphocyte-deficient HFD-fed SCID mice, IL-7 injection still protects from WAT mass gain. However, IL-7-triggered resistance against WAT inflammation and glucose intolerance is lost in SCID mice. These results suggest that IL-7 regulates adipose tissue mass through a lymphocyte-independent mechanism while its protective role on glucose homeostasis would be relayed by immune cells that participate to WAT inflammation. Our observations establish a key role for IL-7 in the complex mechanisms by which immune mediators modulate metabolic functions.

Expression of CD34 in hematopoietic cancer cell lines reflects tightly regulated stem/progenitor-like state.

Hematopoietic cancer stem cells preserve cellular hierarchy in a manner similar to normal stem cells, yet the underlying regulatory mechanisms are poorly understood. It is known that both normal and malignant stem/progenitor cells express CD34. Here, we demonstrate that several cell lines (HL-60, U266) derived from hematopoietic malignancies contain not only CD34(-) but also CD34(+) subpopulations. The CD34(+) cells displayed a stem/progenitor-like phenotype since, in contrast to CD34(-) cells, they frequently underwent cellular division and rapidly formed colonies in methylcellulose-based medium. Strikingly, a constant fraction of the CD34(+) and CD34(-) cell subpopulations, when separated, rapidly switched their phenotype. Consequently, both separated fractions could generate tumors in immunocompromised NOD/LtSz-scid/scid mice. Cultures in vitro showed that the proportion of CD34(+) stem/progenitor-like cells in the population was decreased by cell-cell contact and increased by soluble factors secreted by the cells. Using cytokine arrays, we identified some of these factors, notably thymopoietin that was able to increase the proportion of CD34(+) cells and overall colony-forming capacity in tested cell lines. This action of thymopoietin was conserved in mononuclear cells from bone marrow. Therefore, we propose that hematopoietic cancer cell lines containing subpopulations of CD34(+) cells can provide an in vitro model for studies of cancer stem/progenitor cells.

Human umbilical cord blood cells form epidermis in the skin equivalent model.

Recently, human embryonic stem cells have been differentiated in vitro into functional epidermal keratinocytes. Here, we demonstrated that these cells can be generated also from non-embryonic, human umbilical cord blood (hUCB) cells that have the potential to differentiate into cells of non-hematopoietic lineage. Human UCB mono-nucleated cells were cultivated in monolayer and in three-dimensional skin equivalent cultures and assayed for the presence of phenotype-specific markers. Our results determined that after one month of culturing in serum containing medium, the hUCB cells produced morphologically homogeneous colonies of epithelial cells expressing keratinocyte-specific markers. They also formed stratified epidermis in organ cultures that contained sporadic CD1a-positive cells within the accurate strata. We concluded that hUCB cells have the capacity to differentiate into functional epidermal keratinocytes and may serve as a source of high-quality keratinocytes for clinical applications.

A subpopulation of malignant CD34+CD138+B7-H1+ plasma cells is present in multiple myeloma patients.

OBJECTIVE: It is generally assumed that plasma cells from multiple myeloma (MM) patients do not express the stem cell marker CD34. This assumption has led to several clinical trials based on autologous CD34(+) cell transplantation. However, the results of these trials have been disappointing. MATERIALS AND METHODS: We investigated the presence of CD34(+) cell populations in RPMI 8226, KARPAS 417, and U266 MM cell lines in vitro and during their growth as plasmacytoma tumors in nonobese diabetic severe combined immunodeficient mice, and in plasma cells isolated from the bone marrow of 38 MM patients. RESULTS: We showed that in both patients and cell lines, a small population of plasma cells expresses CD34. These cells display morphological characteristics of MM plasma cells, are CD19-negative, and express B7-H1 (PD-L1), a T-cell inhibitory molecule. In patients, CD34(+)CD138(+) cells expressed Ki67, a marker for proliferation. Moreover, when cells from the human myeloma cell line U266 were injected into nonobese diabetic severe combined immunodeficient mice, the U266-derived plasmacytoma tumors showed a large CD34(+)CD138(+) Ki67(+) cell population, indicating that these cells were not quiescent in vivo. CONCLUSIONS: MM patients carry a small subpopulation of cycling CD34(+)CD138(+)B7-H1(+) plasma cells. Their presence may limit the clinical benefits of autologous CD34(+) cell transplantation.

Asymmetric distribution of epidermal growth factor receptor directs the fate of normal and cancer keratinocytes in vitro.

Cancer cells are unequal in a tumor mass and in established cultures. This is attributable to cancer stem cells with the unique ability to self-renew and to generate differentiating progeny. This ability is controlled at the level of asymmetric division by mechanisms that are yet not well defined. We found that normal and cancer keratinocyte fate was linked to the asymmetric distribution of epidermal growth factor receptor (EGFR) during mitosis. Although essential for epithelial cell proliferation, differentiation, and survival, this receptor was not present on the surface of cells satisfying criteria for stem cells such as quiescence, competence to produce functionally distinct daughters, high proliferative and clonogenic potential, sphere formation ability, and expression of stem cell markers. In contrast, keratinocytes displaying EGFR acquired a more differentiated phenotype, suggesting that EGFR may be involved in a switch from stem to transient amplifying cell fate. This switch was associated with changes in the expression profile of cell cycle, survival, and mitochondria controlling proteins that varied between normal and cancer cells. In conclusion, it appears that an unequal distribution of EGFR at mitosis controls keratinocyte fate by balancing quiescence and cycling of EGFR(-) cells, clearly malfunctioning in cancer. We believe that our findings provide mechanistic insights into the development of resistance to anti-EGFR therapies.

Human dental pulp stem cells differentiate into neural crest-derived melanocytes and have label-retaining and sphere-forming abilities.

Adult tissues contain highly proliferative, clonogenic cells that meet criteria of multipotent stem cells and are potential sources for autologous reparative and reconstructive medicine. We demonstrated that human dental pulp contains self renewing human dental pulp stem cells (hDPSCs) capable of differentiating into mesenchymal-derived odontoblasts, osteoblasts, adipocytes, and chondrocytes and striated muscle, and interestingly, also into non-mesenchymal melanocytes. Furthermore, we showed that hDPSC cultures include cells with the label-retaining and sphere-forming abilities, traits attributed to multipotent stem cells, and provide evidence that these may be multipotent neural crest stem cells.

Variable Bax antigenicity is linked to keratinocyte position within epidermal strata and UV-induced apoptosis.

Pro- and anti-apoptotic members of the Bcl-2 family are fundamental in the control of apoptosis. Among them, Bax plays a key role in apoptosis induction by mediating the release of apoptogenic factors from mitochondria to the cytosol. In this report, we investigated, by immunohistofluorescence, the in vivo distribution of Bax in normal human epidermis before and 24 h after exposure to solar-simulated radiation. Bax expression was evaluated with three different, Western blot pretested, anti-Bax antibodies (Ab) and correlated with markers of keratinocyte differentiation and apoptosis using anti-beta(1) integrin and anti-active caspase-3 Abs respectively. Using anti-Bax N20 and A-3533 polyclonal Ab, we found that, whereas undifferentiated keratinocytes of the basal proliferative compartment contained Bax in the cytosol, the differentiated suprabasal cells had Bax mainly in the nucleus. This immunoreactivity pattern was not modified by skin irradiation. Interestingly, the well known apoptosis-related Bax redistribution to mitochondria in response to a cell death signal, could be detected only with yet another, the 2D2 monoclonal Ab. This relocalization occurred specifically in apoptotic, active caspase-3 positive cells of irradiated epidermis. Our data highlight the differentiation- and apoptosis-associated changes in the pattern of Bax subcellular and cellular distribution as uncovered by different anti-Bax Abs and suggest that Bax undergoes successive activation that progresses in parallel with keratinocyte differentiation and apoptosis.

TRPC channels determine human keratinocyte differentiation: new insight into basal cell carcinoma.

Aberrant keratinocyte differentiation is considered to be a key mechanism in the onset of hyperproliferative dermatological diseases, including basal cell carcinoma (BCC). It is, therefore, vital to understand what drives keratinocytes to develop such pathological phenotypes. The role of calcium in keratinocyte differentiation is uncontested but the mechanisms controlling calcium-induced differentiation have yet to be completely elucidated. This study was designed to investigate the role of calcium-permeable TRPC channels in human keratinocyte differentiation and BCC, using a combination of molecular and cell biology approaches, involving electrophysiology and Ca(2+)-imaging, on the HaCaT cell line, primary cultures of normal human keratinocytes, and BCC cells. We demonstrated that TRPC1/TRPC4 channel expression was important for keratinocyte differentiation, as knocking out these channels (by siRNA strategy) prevented the induction of Ca(2+)-induced differentiation. TRPC1/TRPC4-mediated calcium entry and endoplasmic reticulum Ca(2+) content increased significantly in differentiated keratinocytes. However, the failure of BCC cells to differentiate was related to a lack of TRPC channel expression and calcium entry. In summary, our data demonstrate that TRPC1 and TRPC4 channels are key elements in keratinocyte Ca(2+) homeostasis and differentiation and may therefore be responsible for skin pathologies.

TRPV6 is a Ca2+ entry channel essential for Ca2+-induced differentiation of human keratinocytes.

Ca(2+) is an essential factor inducing keratinocyte differentiation due to the natural Ca(2+) gradient in the skin. However, the membrane mechanisms that mediate calcium entry and trigger keratinocyte differentiation had not previously been elucidated. In this study we demonstrate that Ca(2+)-induced differentiation up-regulates both mRNA and protein expression of a transient receptor potential highly Ca(2+)-selective channel, TRPV6. The latter mediates Ca(2+) uptake and accounts for the basal [Ca(2+)](i) in human keratinocytes. Our results show that TRPV6 is a prerequisite for keratinocyte entry into differentiation, because the silencing of TRPV6 in human primary keratinocytes led to the development of impaired differentiated phenotype triggered by Ca(2+). The expression of such differentiation markers as involucrin, transglutaminase-1, and cytokeratin-10 was significantly inhibited by small interfering RNA-TRPV6 as compared with differentiated control cells. TRPV6 silencing affected cell morphology and the development of intercellular contacts, as well as the ability of cells to stratify. 1,25-Dihydroxyvitamin D3, a cofactor of differentiation, dose-dependently increased TRPV6 mRNA and protein expression in human keratinocytes. This TRPV6 up-regulation led to a significant increase in Ca(2+) uptake in both undifferentiated and differentiated keratinocytes. We conclude that TRPV6 mediates, at least in part, the pro-differentiating effects of 1,25-dihydroxyvitamin D3 by increasing Ca(2+) entry, thereby promoting differentiation. Taken together, these data suggest that the TRPV6 channel is a key element in Ca(2+)/1,25-dihydroxyvitamin D3-induced differentiation of human keratinocytes.

Down-regulation of human RNA/DNA helicase SUV3 induces apoptosis by a caspase- and AIF-dependent pathway.

BACKGROUND INFORMATION: The nuclear gene hSUV3 (human SUV3) encodes an ATP-dependent DNA/RNA helicase. In the yeast Saccharomyces cerevisiae the orthologous Suv3 protein is localized in mitochondria, and is a subunit of the degradosome complex which regulates RNA surveillance and turnover. In contrast, the functions of human SUV3 are not known to date. RESULTS: In the present study, we show that a fraction of human SUV3 helicase is localized in the nucleus. Using small interfering RNA gene silencing in HeLa cells, we demonstrate that down-regulation of hSUV3 results in cell cycle perturbations and in apoptosis, which is both AIF- and caspase-dependent, and proceeds with the induction of p53. CONCLUSIONS: In addition to its mitochondrial localization, human SUV3 plays an important role in the nucleus and is probably involved in chromatin maintenance.