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O Kellermann

Publications and source records attributed to O Kellermann.

At least 19 recordsLinked to original sources
PubMed2006

Noxp20 and Noxp70, two new markers of early neuronal differentiation, detected in teratocarcinoma-derived neuroectodermic precursor cells.

The murine 1C11 cell line, derived from F9 pluripotent teratocarcinoma cells, exhibits features of a bipotential neuronal precursor as it converts into serotonergic or catecholaminergic neurons under appropriate induction. In order to point out molecular markers expressed in this early neuroectodermic commitment, we used a cDNA subtractive hybridization method. The 105 different isolated cDNAs represented 75 known genes, expressed sequence tags (EST) or genomic fragments. A majority of known proteins encoded by these sequences are involved in cellular mobility or migration. We characterized two sequences showing identities with ESTs and we called them Noxp20 and Noxp70. The Noxp20 transcript encodes a putative protein with a predicted caspase recruitment domain and the Noxp70 transcript encodes a putative protein displaying a Zn-finger domain. Consistent with their roles in neuronal cell development, in situ hybridization showed that Noxp20 and Noxp70 are over-expressed in brain. At embryonic days 12 and 15, Noxp20 is strongly expressed in the ventricular and intermediate zones of the brain and of the spinal cord. At embryonic day 15, Noxp70 was found to be strongly expressed in the ventricular zone around the telencephalic ventricle, and to a lower extent in the thalamus and hypothalamus. At post-natal day 10, Noxp20 mRNA was detected in the dentate gyrus, the hippocampus, the cerebellum and the olfactory bulb.

Amino Acid Sequence↗
PubMed2005

Stepwise control of osteogenic differentiation by 5-HT(2B) receptor signaling: nitric oxide production and phospholipase A2 activation.

During development, antagonists of 5-HT(2) receptor subtypes cause morphological defects of mesodermal and neural crest derivatives including the craniofacial skeleton. We used an inducible mesoblastic cell line, C1, able to fully convert into osteocytes within 12 days, to assess the involvement of 5-HT(2) receptors during osteogenic differentiation. On day 5 of the osteogenic program, immediately before matrix mineralization, the cells selectively implement 5-HT(2B) receptors (5-HT(2B)R) which remain functional until terminal differentiation. In 5-HT-depleted medium, the receptor exhibits a constitutive activity leading to basal nitric oxide (NO) release and phospholipase A2 (PLA2)-dependent arachidonic acid (AA) production. Blockade of this intrinsic activity affects the efficiency of mineralization by decreasing calcium incorporation within the matrix by 40%. Optimal bone matrix mineralization involves both NO and PLA2 signaling pathways. Moreover, between day 5 and day 10, at the beginning of mineral deposition, the 5-HT(2B)R promotes prostaglandin E2 production through AA-dependent cyclooxygenase (COX) activation. From day 10 onwards, when C1 osteoblasts undergo conversion into osteocyte-like cells, COX activity is quenched. Altogether these observations indicate that the 5-HT(2B)R contributes in an autocrine manner to osteogenic differentiation and highlight a switch in the downstream targets of the receptor at the terminal stage of the program. Finally, in addition to its autocrine function, the 5-HT(2B)R responds to 5-HT by increasing NO production and AA release. These findings raise concern regarding the use of 5-HT(2B)R-related drugs that may interfere with bone metabolism in physiological or pathological situations.

Animals↗
PubMed2004

New cellular models for tracking the odontoblast phenotype.

Odontoblasts and osteoblasts differ functionally and histologically. Because of their close relationship, mesenchymal cells derived from teeth and bone are difficult to distinguish ex vivo. Indeed, the main non-collagenous components of the odontoblastic extracellular matrix, dentin sialoprotein (DSP) or dentin matrix protein 1 (DMP1), have also been detected in osteoblasts. The need to develop cellular models of odontoblast differentiation and to identify markers specific for the odontoblast lineage, has led us to establish clonal cell lines from tooth germs of day 18 mouse embryos transgenic for an adenovirus-SV40 recombinant plasmid. In this study, we analyzed the phenotypes of three independent clones by RT-PCR and Western blot. These clones synthesised DSP, DMP1 and other extracellular matrix proteins typical of the odontoblast and are therefore likely to be derived from the pulp. Transcripts encoding a set of homeobox proteins involved in craniofacial development, such as Pax9, Msx1, Cbfa1, Dlx2 and 5 were also expressed albeit at a different level. These features of the pulpal clones are shared by the C1 mesodermal cells that are capable of differentiating along osteogenic, chondrogenic or adipogenic lineages In contrast, transcripts for two LIM-domain homeobox family genes (Lhx6 and Lhx7) were only detected in the dental clones. Since these genes are preferentially expressed in the mesenchyme of the developing tooth, this suggests that our transgenic-derived cell lines retain intrinsic properties of odontoblastic cells. They may help to characterise genes specifying the odontoblast phenotype and the signalling pathways underlying odontoblast differentiation.

Adenoviridae↗
PubMed2001

Overexpression of murine small heat shock protein HSP25 interferes with chondrocyte differentiation and decreases cell adhesion.

Although multiple functions for the small heat shock protein HSP25 have been proposed, its specific role during developmental and differentiation processes is not known. Cartilage is one of the tissues in which HSP25 is specifically and highly expressed during development. C1 cells, able to form aggregates in vitro, can be induced to differentiate into chondrocytes. In this study, we generated two stable transfected clones overexpressing HSP25 at two different levels. Cell morphology and growth rate were modified in both clones, although the actin content and distribution did not seem to be altered. Overexpressing clones had more difficulties in coalescing, leading to smaller aggregates and they did not differentiate into chondrocytes. Subsequently, these aggregates tended to dissociate into loose masses of dying cells. The strength of all these effects was directly correlated to the level of HSP25 overexpression. These data suggest that overexpressing HSP25 decreases cellular adhesion and interferes with chondrocyte differentiation.

Actin Cytoskeleton↗
PubMed2001

NHE3 activity and trafficking depend on the state of actin organization in proximal tubule.

The present study was addressed to define the contribution of cytoskeleton elements in the kidney proximal tubule Na+/H+ exchanger 3 (NHE3) activity under basal conditions. We used luminal membrane vesicles (LMV) isolated from suspensions of rat cortical tubules pretreated with either colchicine (Colch) or cytochalasin D (Cyto D). Colch pretreatment of suspensions (200 microM for 60 min) moderately decreased LMV NHE3 activity. Cyto D pretreatment (1 microM for 60 min) elicited an increase in LMV NHE3 transport activity but did not increase Na-glucose cotransport activity. Cyto D pretreatment of suspensions did not change the apparent affinity of NHE3 for internal H+. In contrast, after Cyto D pretreatment of the suspensions, NHE3 protein abundance was increased in LMV and remained unchanged in cortical cell homogenates. The effect of Cyto D on NHE3 was further assessed with cultures of murine cortical cells. The amount of surface biotinylated NHE3 increased on Cyto D treatment, whereas NHE3 protein abundance was unchanged in cell homogenates. In conclusion, under basal conditions NHE3 activity depends on the state of actin organization possibly involved in trafficking processes between luminal membrane and intracellular compartment.

Actins↗
PubMed2001

Bone morphogenetic protein regulation of forkhead/winged helix transcription factor Foxc2 (Mfh1) in a murine mesodermal cell line C1 and in skeletal precursor cells.

Mfh1/Foxc2 is a member of forkhead/winged helix transcription factor family in which its members serve as key regulators in embryogenesis and cell differentiation in various species. Mutant mice null for Mfh1 show defects in axial and cranial skeletogenesis, suggesting requirement of Mfh1 for skeletal tissue development. However, the roles of Mfh1 and its regulation during early skeletogenesis have not been understood fully yet. In this study, we investigated developmental regulation of Mfh1 expression during embryonic skeletogenesis in vivo and in vitro chondrogenic cell differentiation using a mesodermal progenitor-like cell line C1. We first examined expression patterns of Mfh1 in relation to the cartilage phenotype-related molecules including bone morphogenetic proteins (BMPs) during mouse embryogenesis by in situ hybridization. In 10.5 days postcoitum (dpc) mouse limb, Mfh1 messenger RNA (mRNA) was expressed in the mesenchymal cells in the tissues that later give rise to skeleton. In 11.5 dpc embryos, Mfh1 transcripts were expressed in the cell condensation of skeletal blastemas. BMP2 transcripts were expressed in the cell condensation proximal to the Mfh1-expressing cells in the limbs and those of BMP-7 were expressed in the mesenchymal tissue surrounding the Mfh1-positive cell condensation. In 12.5 dpc and 13.5 dpc embryos, the expression of Mfh1 was localized to the perichondrium, which surrounds cells that express noggin and SOX9 mRNA. BMP-2 expression was overlapped with that of Mfh1 in the peripheral layer of 12.5 dpc and 13.5 dpc limb skeletal blastemas. Mfh1 expression persisted in the perichondrium of 15.5 dpc embryos though its level was reduced. We then examined the expression of Mfh1 in the mouse mesodermal cell line C1 that differentiates into chondrocytes in vitro. Mfh1 mRNA was expressed constitutively at low levels in C1 cells before the induction of its differentiation. On the differentiation of C1 cells into chondrocytes by the treatment with dexamethasone (Dex), Mfh1 expression was increased and peaked on day 4 of Dex treatment. Treatment with BMP-4/7 and BMP-7 protein also enhanced Mfh1 expression in C1 cells. To further examine the causative relationship between BMP and Mfh1 in mesenchymal tissue, we performed a mouse limb bud organ culture to implant BMP proteins with carriers into the mesenchymal tissue of the limb bud. Implantation of BMP-7 protein in the limb bud of 11.5 dpc embryos induced Mfh1 expression, suggesting that BMP regulates Mfh1 expression in limb mesenchyme. These results indicate that Mfh1 expression is associated with the early stage of chondrogenic differentiation both in vivo and in vitro and that BMPs regulate Mfh1 expression in skeletal precursor cells.

Animals↗
PubMed2000

Signal transduction through prion protein.

The cellular prion protein PrPc is a glycosylphosphatidylinositol-anchored cell-surface protein whose biological function is unclear. We used the murine 1C11 neuronal differentiation model to search for PrPc-dependent signal transduction through antibody-mediated cross-linking. A caveolin-1-dependent coupling of PrPc to the tyrosine kinase Fyn was observed. Clathrin might also contribute to this coupling. The ability of the 1C11 cell line to trigger PrPc-dependent Fyn activation was restricted to its fully differentiated serotonergic or noradrenergic progenies. Moreover, the signaling activity of PrPc occurred mainly at neurites. Thus, PrPc may be a signal transduction protein.

Animals↗
PubMed2000

Regulation by neurotransmitter receptors of serotonergic or catecholaminergic neuronal cell differentiation.

The murine F9-derived 1C11 clone exhibits a stable epithelial morphology, expresses nestin, an early neuroectodermal marker, and expresses genes involved in neuroectodermal cell fate. Upon appropriate induction, 100% of 1C11 precursor cells develop neurite extensions and acquire neuronal markers (N-CAM, synaptophysin, gammagamma-enolase, and neurofilament) as well as the general functions of either serotonergic (1C11*(/5HT)) (5HT, 5-hydroxytryptamine) or noradrenergic (1C11**(/NE)) (NE, norepinephrine) neurons. The two programs are shown to be mutually exclusive. 1C11 thus behaves as a neuroepithelial cell line with a dual bioaminergic fate. 1C11*(/5HT) cells implement a functional 5-HT transporter and thereby a complete serotonergic phenotype within 4 days, whereas 5-HT(1B/D), 5-HT(2B), and 5-HT(2A) receptors are sequentially induced. The accurate time schedule of catecholaminergic differentiation was defined. Catecholamine synthesis, storage, and catabolism are acquired within 4 days; the noradrenergic phenotype is complete at day 12 and includes a functional norepinephrine transporter and an alpha(1D)-adrenoreceptor (day 8). The time-dependent onset of neurotransmitter-associated functions proper to either program is similar to in vivo observations. Along each pathway, the selective induction of serotonergic or adrenergic receptors is shown to be an essential part of the differentiation program, since they promote an autoregulation of the corresponding phenotype.

Animals↗
PubMed2000

PDZ-dependent activation of nitric-oxide synthases by the serotonin 2B receptor.

Taking advantage of three cellular systems, we established that 5-HT(2B) receptors are coupled with NO signaling pathways. In the 1C11 serotonergic cell line and Mastomys natalensis carcinoid cells, which naturally express the 5-HT(2B) receptor, as well as in transfected LMTK(-) fibroblasts, stimulation of the 5-HT(2B) receptor triggers intracellular cGMP production through dual activation of constitutive nitric-oxide synthase (cNOS) and inducible NOS (iNOS). The group I PDZ motif at the C terminus of the 5-HT(2B) receptor is required for recruitment of the cNOS and iNOS transduction pathways. Indeed, the 5-HT(2B) receptor-mediated NO coupling is abolished not only upon introduction of a competitor C-terminal 5-HT(2B) peptide in the three cell types but also in LMTK(-) fibroblasts expressing a receptor C-terminally truncated or harboring a point mutation within the PDZ domain. The occurrence of a direct functional coupling between the receptor and cNOS activity is supported by highly significant correlations between the binding constants of drugs on the receptor and their effects on cNOS activity. The 5-HT(2B)/iNOS coupling mechanisms appear more complex because neutralization of endogenous Galpha(13) by specific antibodies cancels the cellular iNOS response while not interfering with cNOS activities. These findings may shed light on physiological links between the 5-HT(2B) receptor and NO and constitute the first demonstration that PDZ interactions participate in downstream transductional pathways of a G protein-coupled receptor.

Amino Acid Sequence↗
PubMed1999

Lineage-dependent collagen expression and assembly during osteogenic or chondrogenic differentiation of a mesoblastic cell line.

The mesoblastic clone, C1, behaves as a tripotential progenitor able to self-renew and to differentiate toward osteogenesis, chondrogenesis, or adipogenesis in response to specific inducers. In this study, expression and deposition by the C1 cells of essential components of the extracellular matrix, collagens type I, II, III, V, XI, VI, IX, and X were followed along the osteogenic and chondrogenic pathways, through biochemical, immunochemical, and electron microscopy analyses. Implementation of each program involves profiles of collagen synthesis and matrix assembly close to those documented in vivo. Depending on the applied inducers, cells adopt a defined identity and, controls acting at transcriptional and posttranslational levels adapt the set of deposited collagens to one particular cell fate. Osteogenic C1 cells selectively build a type I collagen matrix also containing type III, V, and XI collagens but selectively exclude type II collagen. Chondrogenic C1 cells first elaborate a type II collagen network and then acquire hypertrophic chondrocyte properties while assembling a type X collagen matrix as in the growth plate. This study provides an example of how a mesoblastic cell line can develop, in vitro, each of its genetic programs up to terminal differentiation. Intrinsic factors and time-dependent cell-matrix interactions might, as in vivo, underline the implementation of an entire differentiation program.

Cell Differentiation↗
PubMed1999

Noggin expression in a mesodermal pluripotent cell line C1 and its regulation by BMP.

Osteoblasts and chondrocytes are derived from mesodermal stem cells and their differentiation is under the control of coordinated interaction among signaling molecules. Noggin is one of the signaling molecules which bind to and inactivate BMPs to induce neural tissues and dorsal mesoderm in Xenopus. However, its expression and regulation in mammalian cells has not been known. In this study, we investigated expression of noggin in murine pluripotent mesodermal cell line, C1. Noggin expression was very low in these C1 cells before they were induced to differentiate. When C1 cells were induced to differentiate into chondrocytes in aggregate cultures in the presence of dexamethasone(dex), noggin expression was significantly increased. In a sharp contrast, when the C1 cells were induced to differentiate into osteoblastic cells by the treatment with beta glycerophosphate (betaGP) and ascorbic acid (AA), noggin mRNA expression remained to be barely detectable. Noggin expression was also observed in the developing cartilage of vertebrae in 15.5 dpc mouse embryos. The noggin mRNA level in C1 cells in monolayer cultures was enhanced significantly by the treatment with BMP4/7 in a dose-dependent manner with a maximal effect at 100 ng/ml. The BMP4/7 effect on noggin expression was time dependent starting within 12 h and peaked at 24 h. These results indicate that noggin is expressed in the pluripotent mesodermal cell line C1 and that its expression is regulated by BMP.

Animals↗
PubMed1999

Prion protein and neuronal differentiation: quantitative analysis of prnp gene expression in a murine inducible neuroectodermal progenitor.

The biological function of the cellular prion protein, PrP(c), is currently unknown. The presence of PrP(c) transcripts in the developing neural tube from embryonic day 13.5 and the predominant expression of PrP(c) in the adult brain is suggestive of a role in the onset and/or modulation of neuronal functions. We took advantage of the bipotential neuroectodermal 1C11 cell line to monitor PrP(c) expression during its bioaminergic differentiations. The F9-derived 1C11 precursor cell line displays a stable and immature phenotype in the absence of extracellular signal and, upon induction, has the capacity to acquire a complete serotonergic or noradrenergic phenotype, the two pathways being mutually exclusive. A real-time quantitative PCR assay was developed to assess PrP(c) gene expression at definite times of the two programs that correspond to sequential acquisition of neurotransmitter-specific functions. 1C11 cells and their differentiated progenies express significant amounts of PrP transcripts and of the corresponding protein. A unique decrease in prnp gene expression is observed upon entry into the serotonergic pathway, correlating with a downregulation at the protein level. Moreover, nerve growth factor (NGF) is shown to induce a decrease in the level of prnp gene expression along the serotonergic - but not the noradrenergic - pathway. Our study accurately establishes that prnp gene expression (i) is strongly upregulated concomitantly with cell fate restriction of multipotential cells towards the neural lineage; (ii) is differentially regulated along the serotonergic versus noradrenergic differentiation program of a unique neuroectodermal progenitor. The 1C11 cell line may provide a new tool for studying prion infectivity in a well-defined neuronal context.

Amyloid↗
PubMed1999

Kidney cortex cells derived from SV40 transgenic mice retain intrinsic properties of polarized proximal tubule cells.

BACKGROUND: We have developed a nontransformed immortalized mice kidney cortex epithelial cell (MKCC) culture from a mouse transgenic for a recombinant plasmid adeno-SV40 (PK4). Methods and Results. After 12 months in culture, the immortalized cells had a stable homogeneous epithelial-like phenotype, expressed simian virus 40 (SV40) T-antigen, but failed to induce tumors after injection in nude mice. Epithelium exhibited polarity with an apical domain bearing many microvilli separated from lateral domains by junctional complexes with ZO1 protein. The transepithelial resistance was low. A Na-dependent glucose uptake sensitive to phlorizin and a Na-dependent phosphate uptake sensitive to arsenate were present. Western blot analysis of membrane fractions showed that anti-Na-Pi antiserum reacted with a 87 kD protein. The Na/H antiporters NHE-1, NHE-2, and NHE-3 mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR). The corresponding proteins with molecular weights of 111, 81, and 75 kD, respectively, could be detected by Western blot and were shown to be functional. Parathyroid hormone (PTH) induced a tenfold increase in cAMP and reduced the Na-dependent phosphate uptake and NHE-3 activity, as observed in proximal tubule cells. Isoforms alpha, delta, epsilon, and zeta of protein kinase C (PKC) were present in the cells. Angiotensin II (Ang II) elicited a translocation of the PKC-alpha toward the basolateral and apical domains. CONCLUSION: Thus, the MKCC culture retains the structural and functional properties of proximal tubular cells. To our knowledge, it is the first cell culture obtained from transgenic mice that exhibits the NHE-3 antiporter and type II Na-Pi cotransporter. MKCCs also display functional receptors for PTH and Ang II. Thus, MKCCs offer a powerful in vitro system to study the cellular mechanisms of ion transport regulation in proximal epithelium.

Angiotensin II↗
PubMed1998

Mouse 5-HT2B receptor-mediated serotonin trophic functions.

5-HT2B receptors, in addition to phospholipase C stimulation, are able to trigger activation of the proto-oncogene product p21ras. During mouse embryogenesis, a peak of 5-HT2B receptor expression is detected at the neurulation stage; we localized the 5-HT2B expression in neural crest cells, heart myocardium, and somites. The requirement for functional 5-HT2B receptors shortly after gastrulation, is supported by culture of embryos exposed to 5-HT2B-high affinity antagonist such as ritanserin, which induces morphological defects in the cephalic region, heart and neural tube. Functional 5-HT2B receptors are also expressed during the serotonergic differentiation of the mouse F9 teratocarcinoma-derived clonal cell line 1C11. Upon 2 days of induction by cAMP, 5-HT2B receptors become functional, and on day 4, the appearance of 5-HT2A receptors coincides with the onset of active serotonin transporter by these cells. Active serotonin uptake is modulated by serotonin suggesting autoreceptor functions for 5-HT2B receptors.

Animals↗