Titanium dioxide and carbon black nanoparticles disrupt neuronal homeostasis via excessive activation of cellular prion protein signaling.

BACKGROUND: Epidemiological emerging evidence shows that human exposure to some nanosized materials present in the environment would contribute to the onset and/or progression of Alzheimer's disease (AD). The cellular and molecular mechanisms whereby nanoparticles would exert some adverse effects towards neurons and take part in AD pathology are nevertheless unknown. RESULTS: Here, we provide the prime evidence that titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) bind the cellular form of the prion protein (PrPC), a plasma membrane protein well known for its implication in prion diseases and prion-like diseases, such as AD. The interaction between TiO2- or CB-NPs and PrPC at the surface of neuronal cells grown in culture corrupts PrPC signaling function. This triggers PrPC-dependent activation of NADPH oxidase and subsequent production of reactive oxygen species (ROS) that alters redox equilibrium. Through PrPC interaction, NPs also promote the activation of 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn provokes the internalization of the neuroprotective TACE α-secretase. This diverts TACE cleavage activity away from (i) TNFα receptors (TNFR), whose accumulation at the plasma membrane augments the vulnerability of NP-exposed neuronal cells to TNFα -associated inflammation, and (ii) the amyloid precursor protein APP, leading to overproduction of neurotoxic amyloid Aß40/42 peptides. The silencing of PrPC or the pharmacological inhibition of PDK1 protects neuronal cells from TiO2- and CB-NPs effects regarding ROS production, TNFα hypersensitivity, and Aß rise. Finally, we show that dysregulation of the PrPC-PDK1-TACE pathway likely occurs in the brain of mice injected with TiO2-NPs by the intra-cerebro-ventricular route as we monitor a rise of TNFR at the cell surface of several groups of neurons located in distinct brain areas. CONCLUSION: Our in vitro and in vivo study thus posits for the first time normal cellular prion protein PrPC as being a neuronal receptor of TiO2- and CB-NPs and identifies PrPC-coupled signaling pathways by which those nanoparticles alter redox equilibrium, augment the intrinsic sensitivity of neurons to neuroinflammation, and provoke a rise of Aß peptides. By identifying signaling cascades dysregulated by TiO2- and CB-NPs in neurons, our data shed light on how human exposure to some NPs might be related to AD.

Production of seedable Amyloid-ß peptides in model of prion diseases upon PrPSc-induced PDK1 overactivation.

The presence of amyloid beta (Aß) plaques in the brain of some individuals with Creutzfeldt-Jakob or Gertsmann-Straussler-Scheinker diseases suggests that pathogenic prions (PrPSc) would have stimulated the production and deposition of Aß peptides. We here show in prion-infected neurons and mice that deregulation of the PDK1-TACE α-secretase pathway reduces the Amyloid Precursor Protein (APP) α-cleavage in favor of APP ß-processing, leading to Aß40/42 accumulation. Aß predominates as monomers, but is also found as trimers and tetramers. Prion-induced Aß peptides do not affect prion replication and infectivity, but display seedable properties as they can deposit in the mouse brain only when seeds of Aß trimers are co-transmitted with PrPSc. Importantly, brain Aß deposition accelerates death of prion-infected mice. Our data stress that PrPSc, through deregulation of the PDK1-TACE-APP pathway, provokes the accumulation of Aß, a prerequisite for the onset of an Aß seeds-induced Aß pathology within a prion-infectious context.

Cerebellar compartmentation of prion pathogenesis.

In prion diseases, the brain lesion profile is influenced by the prion "strain" properties, the invasion route to the brain, and still unknown host cell-specific parameters. To gain insight into those endogenous factors, we analyzed the histopathological alterations induced by distinct prion strains in the mouse cerebellum. We show that 22L and ME7 scrapie prion proteins (PrP22L , PrPME7 ), but not bovine spongiform encephalopathy PrP6PB1 , accumulate in a reproducible parasagittal banding pattern in the cerebellar cortex of infected mice. Such banding pattern of PrP22L aggregation did not depend on the neuroinvasion route, but coincided with the parasagittal compartmentation of the cerebellum mostly defined by the expression of zebrins, such as aldolase C and the excitatory amino acid transporter 4, in Purkinje cells. We provide evidence that Purkinje cells display a differential, subtype-specific vulnerability to 22L prions with zebrin-expressing Purkinje cells being more resistant to prion toxicity, while in stripes where PrP22L accumulated most zebrin-deficient Purkinje cells are lost and spongiosis accentuated. In addition, in PrP22L stripes, enhanced reactive astrocyte processes associated with microglia activation support interdependent events between the topographic pattern of Purkinje cell death, reactive gliosis and PrP22L accumulation. Finally, we find that in preclinically-ill mice prion infection promotes at the membrane of astrocytes enveloping Purkinje cell excitatory synapses, upregulation of tumor necrosis factor-α receptor type 1 (TNFR1), a key mediator of the neuroinflammation process. These overall data show that Purkinje cell sensitivity to prion insult is locally restricted by the parasagittal compartmentation of the cerebellum, and that perisynaptic astrocytes may contribute to prion pathogenesis through prion-induced TNFR1 upregulation.

Protective role of cellular prion protein against TNFα-mediated inflammation through TACE α-secretase.

Although cellular prion protein PrPC is well known for its implication in Transmissible Spongiform Encephalopathies, its functions remain elusive. Combining in vitro and in vivo approaches, we here show that PrPC displays the intrinsic capacity to protect neuronal cells from a pro-inflammatory TNFα noxious insult. Mechanistically, PrPC coupling to the NADPH oxidase-TACE α-secretase signaling pathway promotes TACE-mediated cleavage of transmembrane TNFα receptors (TNFRs) and the release of soluble TNFR, which limits the sensitivity of recipient cells to TNFα. We further show that PrPC expression is necessary for TACE α-secretase to stay at the plasma membrane in an active state for TNFR shedding. Such PrPC control of TACE localization depends on PrPC modulation of ß1 integrin signaling and downstream activation of ROCK-I and PDK1 kinases. Loss of PrPC provokes TACE internalization, which in turn cancels TACE-mediated cleavage of TNFR and renders PrPC-depleted neuronal cells as well as PrPC knockout mice highly vulnerable to pro-inflammatory TNFα insult. Our work provides the prime evidence that in an inflammatory context PrPC adjusts the response of neuronal cells targeted by TNFα through TACE α-secretase. Our data also support the view that abnormal TACE trafficking and activity in prion diseases originate from a-loss-of-PrPC cytoprotective function.

Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.

Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.

Pulp stem cells: implication in reparative dentin formation.

Many dental pulp stem cells are neural crest derivatives essential for lifelong maintenance of tooth functions and homeostasis as well as tooth repair. These cells may be directly implicated in the healing process or indirectly involved in cell-to-cell diffusion of paracrine messages to resident (pulpoblasts) or nonresident cells (migrating mesenchymal cells). The identity of the pulp progenitors and the mechanisms sustaining their regenerative capacity remain largely unknown. Taking advantage of the A4 cell line, a multipotent stem cell derived from the molar pulp of mouse embryo, we investigated the capacity of these pulp-derived precursors to induce in vivo the formation of a reparative dentin-like structure upon implantation within the pulp of a rodent incisor or a first maxillary molar after surgical exposure. One month after the pulp injury alone, a nonmineralized fibrous matrix filled the mesial part of the coronal pulp chamber. Upon A4 cell implantation, a mineralized osteodentin was formed in the implantation site without affecting the structure and vitality of the residual pulp in the central and distal parts of the pulp chamber. These results show that dental pulp stem cells can induce the formation of reparative dentin and therefore constitute a useful tool for pulp therapies. Finally, reparative dentin was also built up when A4 progenitors were performed by alginate beads, suggesting that alginate is a suitable carrier for cell implantation in teeth.

PDK1 decreases TACE-mediated α-secretase activity and promotes disease progression in prion and Alzheimer's diseases.

α-secretase-mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-ß (Aß) peptides, and α-cleavage of cellular prion protein (PrP(C)) prevents its conversion into misfolded, pathogenic prions (PrP(Sc)). The mechanisms leading to decreased α-secretase activity in Alzheimer's and prion disease remain unclear. Here, we find that tumor necrosis factor-α-converting enzyme (TACE)-mediated α-secretase activity is impaired at the surface of neurons infected with PrP(Sc) or isolated from APP-transgenic mice with amyloid pathology. 3-phosphoinositide-dependent kinase-1 (PDK1) activity is increased in neurons infected with prions or affected by Aß deposition and in the brains of individuals with Alzheimer's disease. PDK1 induces phosphorylation and caveolin-1-mediated internalization of TACE. This dysregulation of TACE increases PrP(Sc) and Aß accumulation and reduces shedding of TNF-α receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent α-secretase activity and cleavage of APP, PrP(C) and TNFR1, and attenuates PrP(Sc)- and Aß-induced neurotoxicity. In mice, inhibition or siRNA-mediated silencing of PDK1 extends survival and reduces motor impairment following PrP(Sc) infection and in APP-transgenic mice reduces Alzheimer's disease-like pathology and memory impairment.

Understanding the neurospecificity of Prion protein signaling.

The cellular prion protein PrP(C) is the normal counterpart of the scrapie prion protein PrP(Sc), the main component of the infectious agent of transmissible spongiform encephalopathies (TSEs). It is a ubiquitous cell-surface glycoprotein, abundantly expressed in neurons, which constitute the targets of TSE pathogenesis. The presence of PrP(C) at the surface of neurons is an absolute requirement for the development of prion diseases and corruption of PrP(C) function(s) within an infectious context emerges as a proximal cause for PrP(Sc)-induced neurodegeneration. Experimental evidence gained over the past decade indicates that PrP(C) has the capacity to mobilize promiscuous signal transduction cascades that, notably, contribute to cell homeostasis. Beyond ubiquitous effectors, much data converge onto a neurospecificity of PrP(C) signaling, which may be the clue to neuronal cell demise in prion disorders. In this article, we highlight the requirement of PrP(C) for TSEs-associated neurodegeneration and review the current knowledge of PrP(C)-dependent signal transduction in neuronal cells and its implications for PrP(Sc)-mediated neurotoxicity.

Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C.

In previous studies, we observed that mice knocked out for the serotonin-2B receptor (5-HT(2B)R) show defects in bone homeostasis. The present work focuses on the downstream targets relaying the anabolic function of this receptor in osteoblasts. A functional link between the 5-HT(2B)R and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) is established using the C1 osteoprogenitor cell line. During C1 osteogenic differentiation, both 5-HT(2B)R and TNAP mRNA translations are delayed with respect to extracellular matrix deposition. Once the receptor is expressed, it constitutively controls TNAP activity at a post-translational level along the overall period of mineral deposition. Indeed, pharmacological inhibition of the 5-HT(2B)R intrinsic activity or shRNA-mediated 5-HT(2B)R knockdown prevents TNAP activation, but not its mRNA translation. In contrast, agonist stimulation of the receptor further increases TNAP activity during the initial mineralization phase. Building upon our previous observations that the 5-HT(2B)R couples with the phospholipase A2 pathway and prostaglandin production at the beginning of mineral deposition, we show that the 5-HT(2B)R controls leukotriene synthesis via phospholipase A2 at the terminal stages of C1 differentiation. These two 5-HT(2B)R-dependent eicosanoid productions delineate distinct time windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes are shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from 5-HT(2B)R-null mice exhibit defects in TNAP activity.

Production of reactive oxygen species is turned on and rapidly shut down in epithelial cells infected with Chlamydia trachomatis.

Reactive oxygen species (ROS) are many-faceted compounds involved in cell defense against pathogens, as well as in cell signaling. Their involvement in the response to infection in epithelial cells remains poorly documented. Here, we investigated the production of ROS during infection with Chlamydia trachomatis, a strict intracellular pathogen, in HeLa cells. C. trachomatis induced a transient increase in the ROS level within a few hours, followed by a return to basal level 9 hours after infection. At this time point, the host enzyme dedicated to ROS production, NADPH oxidase, could no longer be activated by external stimuli, such as interleukin-1beta. In addition, Rac, a regulatory subunit of the NADPH oxidase complex, was relocated to the membrane of the compartment in which the bacteria develop, the inclusion, while other subunits were not. Altogether, these results indicate that C. trachomatis infection elicits the production of ROS and that the bacteria rapidly target the activity of NADPH oxidase to shut it down. Prevention of ROS production at the onset of the bacterial developmental cycle might delay the host response to infection.

Cellular prion protein coupling to TACE-dependent TNF-alpha shedding controls neurotransmitter catabolism in neuronal cells.

Despite considerable efforts to unravel the role of cellular prion protein (PrP(C)) in neuronal functions, the mechanisms by which PrP(C) takes part in the homeostasis of a defined neuronal phenotype remain poorly characterized. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) neurons, we assessed the contribution of PrP(C) to bioaminergic cell functions. We established that in 1C11-derived neuronal cells antibody-mediated PrP(C) ligation triggered tumor necrosis factor (TNF)-alpha release, through recruitment of the metalloproteinase TNF-alpha converting enzyme (TACE). TNF-alpha shed in response to PrP(C) acts as a second message signal, eliciting serotonin (5-HT) or norepinephrine (NE) degradation in 1C11(5-HT) or 1C11(NE) cells, respectively. Our data thus introduced TNF-alpha as a PrP(C)-dependent modulator of neuronal metabolism. Of note, we previously reported on a control of neurotransmitter catabolism by 5-HT(2B) or alpha(1D) autoreceptors in 1C11 bioaminergic neurons, via the same TACE/TNF-alpha pathway (Ann. N Y Acad. Sci. 1091, 123). Here, we show that combined stimulation of PrP(C) and these two bioaminergic receptors add their effects on neurotransmitter degradation. Overall, these observations unveil a novel contribution of PrP(C) to the control of neuronal functions and may have implications regarding dysfunction of the bioaminergic systems in prion diseases.

Overstimulation of PrPC signaling pathways by prion peptide 106-126 causes oxidative injury of bioaminergic neuronal cells.

Transmissible spongiform encephalopathies, also called prion diseases, are characterized by neuronal loss linked to the accumulation of PrP(Sc), a pathologic variant of the cellular prion protein (PrP(C)). Although the molecular and cellular bases of PrP(Sc)-induced neuropathogenesis are not yet fully understood, increasing evidence supports the view that PrP(Sc) accumulation interferes with PrP(C) normal function(s) in neurons. In the present work, we exploit the properties of PrP-(106-126), a synthetic peptide encompassing residues 106-126 of PrP, to investigate into the mechanisms sustaining prion-associated neuronal damage. This peptide shares many physicochemical properties with PrP(Sc) and is neurotoxic in vitro and in vivo. We examined the impact of PrP-(106-126) exposure on 1C11 neuroepithelial cells, their neuronal progenies, and GT1-7 hypothalamic cells. This peptide triggers reactive oxygen species overflow, mitogen-activated protein kinase (ERK1/2), and SAPK (p38 and JNK1/2) sustained activation, and apoptotic signals in 1C11-derived serotonergic and noradrenergic neuronal cells, while having no effect on 1C11 precursor and GT1-7 cells. The neurotoxic action of PrP-(106-126) relies on cell surface expression of PrP(C), recruitment of a PrP(C)-Caveolin-Fyn signaling platform, and overstimulation of NADPH-oxidase activity. Altogether, these findings provide actual evidence that PrP-(106-126)-induced neuronal injury is caused by an amplification of PrP(C)-associated signaling responses, which notably promotes oxidative stress conditions. Distorsion of PrP(C) signaling in neuronal cells could hence represent a causal event in transmissible spongiform encephalopathy pathogenesis.

Early in vivo and in vitro effects of amelogenin gene splice products on pulp cells.

Recombinant amelogenin gene splice products A+4 and A-4, implanted in the pulp, induce the recruitment, proliferation, and differentiation of reparative cells. Our aim was to investigate the precocious events occurring in the pulp 1 d and 3 d after implantation of agarose beads alone or loaded with A+4 or A-4. Proliferation and cell recruitment towards an odonto/osteogenic phenotype were visualized by detection of the proliferation cell nuclear antigen (PCNA) and RP59. After implantation of beads alone or loaded with A+4, at day 3, pulp cells were moderately immunopositive for osteopontin (OP), whereas labeling was strongly positive upon treatment with A-4. Dentin sialoprotein (DSP) labeling was not detectable. Parallel in vitro studies were carried out on odontoblastic and mesenchymal progenitor cells in order to evaluate the effect of the amelogenin peptides on the expression of a series of marker genes involved in the odontoblastic/osteogenic/chondrogenic differentiation pathways. Altogether, our results suggest that the 'signaling' effects of the amelogenin peptides A+4 and A-4 may differ according to the type of target cells, their stage of differentiation, the time of treatment, and the type of amelogenin peptide (A+4 or A-4).

Control of bioamine metabolism by 5-HT2B and alpha 1D autoreceptors through reactive oxygen species and tumor necrosis factor-alpha signaling in neuronal cells.

Homeostasis of the central nervous system relies on the proper integration of cell-signaling pathways recruited by a variety of neuronal and non-neuronal factors, with the aim of tightly controlling neurotransmitter metabolism, storage, and transport. We took advantage of the 1C11 neuroectodermal cell line, endowed with the capacity to selectively differentiate into serotonergic (1C11(5-HT)) or noradrenergic (1C11(NE)) neurons, to identify functional targets of serotonin (5-hydroxytryptamine [5-HT]) and norepinephrine (NE) autoreceptors possibly involved in the control of neuronal functions. We demonstrate that 5-HT(2B) and adreno alpha(1D) receptors are coupled to reactive oxygen species (ROS) production through NADPH oxidase activation in 1C11(5-HT) and 1C11(NE) neuronal cells, respectively. In the signaling cascade linking 5-HT(2B) receptors to NADPH oxidase, phospholipase A2-mediated arachidonic acid production is required for ROS synthesis. ROS, in turn, act as second message signals and control the activation of TACE (TNF-alpha converting enzyme), a member of a disintegrin and metalloproteinase family. 5-HT(2B) and alpha(1D) receptor stimulation triggers TACE-dependent TNF-alpha shedding in the surrounding milieu of 1C11(5-HT) and 1C11(NE) cells. In these cells, shed TNF-alpha triggers degradation of 5-HT and NE into 5-HIAA and MHPG, respectively. Finally, we observe that 5-HT(2B) and alpha(1D) receptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete neuronal phenotype. Altogether, our data indicate that couplings of 5-HT(2B) and alpha(1D) autoreceptors to ROS and TNF-alpha signaling control neurotransmitter metabolism in 1C11-derived neuronal cells. Eventually, we might explain the origin of oxidative stress and high level of TNF-alpha in neurodegenerative diseases as a consequence of deviation of normal signaling pathways coupled to neurotransmitters.

Reactive oxygen species-dependent TNF-alpha converting enzyme activation through stimulation of 5-HT2B and alpha1D autoreceptors in neuronal cells.

A major determinant of neuronal homeostasis is the proper integration of cell signaling pathways recruited by a variety of neuronal and non-neuronal factors. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C115-HT) or noradrenergic (1C11NE) neurons, we identified serotonin (5-hydroxytryptamine, 5-HT)- and norepinephrine (NE)-dependent signaling cascades possibly involved in neuronal functions. First, we establish that 5-HT2B receptors and 1D adrenoceptors are functionally coupled to reactive oxygen species (ROS) synthesis through NADPH oxidase activation in 1C115-HT and 1C11NE cells. This observation constitutes the prime evidence that bioaminergic autoreceptors take part in the control of the cellular redox equilibrium in a neuronal context. Second, our data identify TACE (TNF- Converting Enzyme), a member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the 5-HT2B and 1D receptor-NADPH oxidase signaling pathways. Upon 5-HT2B or 1D receptor stimulation, ROS fully govern TNF- shedding in the surrounding milieu of 1C115-HT or 1C11NE cells. Third, 5-HT2B and 1Dreceptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete serotonergic or noradrenergic phenotype. Overall, these observations suggest that 5-HT2B and 1D autoreceptors may play a role in the maintenance of neuron- and neurotransmitter-associated functions. Eventually, our study may have implications regarding the origin of oxidative stress as well as up-regulated expression of proinflammatory cytokines in neurodegenerative disorders, which may relate to the deviation of normal signaling pathways.

Modulation of serotonergic receptor signaling and cross-talk by prion protein.

The inducible serotonergic 1C115-HT cell line expresses a defined set of serotonergic receptors of the 5-HT2B, 5-HT1B/D, and 5-HT2A subtypes, which sustain a regulation of serotonergic associated functions through G-protein-dependent signaling. 1C115-HT cells have been instrumental to assign a signaling function to the cellular prion protein PrPC. Here, we establish that antibody-mediated ligation of PrPC concomitant to agonist stimulation of 5-HT receptors modulates the couplings of all three serotonergic receptors present on 1C115-HT cells. Specific impacts of PrP antibodies were monitored depending on the receptor and pathway considered. PrPC ligation selectively cancels the 5-HT2A-PLC response, decreases the 5-HT1B/D negative coupling to adenylate cyclase, and potentiates the 5-HT2B-PLA2 coupling. As a result, PrPC ligation disturbs the functional interactions occurring between the signaling pathways of the three receptor subtypes. In 1C115-HT cells, antagonizing cross-talks arising from 5-HT2B and 5-HT2A receptors control the 5-HT1B/D function. PrPC ligation reinforces the negative regulation exerted by 5-HT2B on 5-HT1B/D receptors. On the other hand it abrogates the blocking action of 5-HT2A on the regulatory loop linking 5-HT1B/D receptors. We propose that the ligation of PrPC affects the potency or dynamics of G-protein activation by agonist-bound serotonergic receptors. Finally, the PrPC-dependent modulation of 5-HT receptor couplings is restricted to 1C115-HT cells expressing a complete serotonergic phenotype. It critically involves a PrPC-caveolin platform implemented on the neurites of 1C115-HT cells during differentiation. Our findings define PrPC as a modulator of 5-HT receptor coupling to G-proteins and thereby as a protagonist contributing to the homeostasis of serotonergic neurons. They provide a foundation for uncovering the impact of prion infection on serotonergic functions.

Noggin inhibits chondrogenic but not osteogenic differentiation in mesodermal stem cell line C1 and skeletal cells.

Osteoblasts and chondroblasts are derived from common mesenchymal progenitors. Although bone morphogenetic protein induces mesenchymal differentiation into both osteogenic and chodrogenic lineage cells in vitro, its inhibitor, Noggin, is expressed exclusively during chondrogenic but not osteogenic differentiation in an embryonal carcinoma-derived mesodermal cell line, C1. We hypothesized that Noggin may regulate cell differentiation in a lineage-specific manner. To test this hypothesis, Noggin was overexpressed using recombinant adenovirus (Ad/Noggin) in mesodermal C1 cells to examine whether Noggin specifically inhibits chondrogenic differentiation. Noggin overexpression by recombinant adenovirus infection reduced Sox9, patched, Ihh, and type II, X, and XI collagen mRNA expression levels in C1 cell aggregates that were induced to differentiate into chondrocyte lineage by culturing in differentiation medium. In contrast, Noggin overexpression did not affect osteogenic differentiation in C1 cells because osteoblast phenotypic markers such as osteocalcin and alkaline phosphatase mRNA levels were not altered. We further examined whether Noggin also differentially affects chondrogenesis and osteogenesis in limb development by using organ cultures of long bone. Ad/Noggin infection into 15.5 d post conception limb skeletal rudiments that were cultured on filter membrane in vitro or on the chorioallantoic membranes in ovo inhibited the levels of chondrogenesis, which were evaluated based on alcian blue staining. These results suggest that Noggin specifically blocks chondrogenic differentiation, rather than osteogenic differentiation, in mesodermal stem cell line C1 and skeletal cells.

Paracrine and autocrine signals promoting full chondrogenic differentiation of a mesoblastic cell line.

UNLABELLED: The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. INTRODUCTION: Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. MATERIALS AND METHODS: C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10(-7) M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. RESULTS AND CONCLUSIONS: Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.