Emmanuelle Boisvieux-Ulrich

Development and functions of the cytoskeleton during ciliogenesis in metazoa

Summary— The different steps of ciliogenesis occurring in quail oviduct were compared to the ciliogenesis pattern described in other metazoan species.

Inhibition of caspase-dependent mitochondrial permeability transition protects airway epithelial cells against mustard-induced apoptosis

In the present study, the toxicity of yperite, SM, and its structural analogue mechlorethamine, HN2, was investigated in a human bronchial epithelial cell line 16HBE. Cell detachment was initiated by caspase-2 activation, down-regulation of Bcl-2 and loss of mitochondrial membrane potential. Only in detached cells, mustards induced apoptosis associated with increase in p53 expression, Bax activation, decrease in Bcl-2 expression, opening of the mitochondrial permeability transition pore, release of cytochrome c, caspase-2, -3, -8, -9 and -13 activation and DNA fragmentation. Apoptosis, occurring only in detached cells, could be recognized as anoikis and the mitochondrion, involved both in cell detachment and subsequent cell death, appears to be a crucial checkpoint. Based on our understanding of the apoptotic pathway triggered by mustards, we demonstrated that inhibition of the mitochondrial pathway by ebselen, melatonin and cyclosporine A markedly prevented mustard-induced anoikis, pointing to these drugs as interesting candidates for the treatment of mustard-induced airway epithelial lesions.

Detection of plasma membrane cholesterol by filipin during microvillogenesis and ciliogenesis in quail oviduct.

Using filipin as a probe for the presence of membrane cholesterol, the evolution of cholesterol distribution in the apical plasma membrane was studied during estrogen-induced ciliogenesis in quail oviduct and compared with the distribution of intramembrane particles (IMPs). Ciliary growth is preceded by the first step of microvillus differentiation. Microvilli emerge in membrane domains rich in IMPs and devoid of filipin-cholesterol (f-c) complexes. However growing microvillus membrane shows f-c complexes. During ciliary growth, microvilli lengthen from 0.5 to 2 microns, indicating that the microvillar membrane is not a membrane reservoir for ciliogenesis. During ciliary growth, the characteristic ciliary necklace IMP rows appear progressively at the base of cilia. The first IMP row is organized in a membrane circlet lacking of f-c complexes, whereas the new shaft membrane in the middle of the circlet exhibits numerous complexes. These two different domains of the cilia keep their specificity during ciliary growth. Only the ciliary tip shows fewer complexes than the shaft membrane. The apical membrane of differentiated ciliated cells is thus composed of various domains, the ciliary shaft full of f-c complexes and poor in IMPs, the ciliary necklace is devoid of f-c complexes and rich in IMPs, the microvilli membrane is rich in both IMPs and f-c complexes, and the interciliary membrane is poor in both f-c complexes and IMPs, whereas the undifferentiated cells exhibit an apical membrane in which f-c complexes and IMPs are distributed homogeneously.

Extracellular matrix-dependent differentiation of rabbit tracheal epithelial cells in primary culture

SummaryThe differentiation of tracheal epithelial cells in primary culture was investigated according to the nature of the extracellular matrix used. Cultures obtained by the explant technique were realized on a type I collagen substratum either as a thin, dried coating or as a thick, hydrated gel supplemented with culture medium and serum. These two types of substratum induced distinct cell morphology and cytokeratin expression in the explant derived cells. Where cells are less proliferating (from Day 7 to 10 of culture), differentiation was evaluated by morphologic ultrastructural observations, immunocytochemical detection of cytokeratins, and determination of cytokeratin pattern by biochemical analysis.The epithelium obtained on gel was multilayered, with small, round basal cells under large, flattened upper cells. The determination of the keratin pattern expressed by cells grown on gel revealed an expression of keratin 13, already considered as a specific marker of squamous metaplasia, that diminished with retinoic acid treatment. Present results demonstrated by confocal microscopy that K13-positive cells were large upper cells with a dense keratin network, whereas lower cells were positively stained with a specific monoclonal antibody to basal cells (KB37). Moreover, keratin neosynthesis analysis pointed out a higher expression of K6, a marker of hyperproliferation, on gel than on coating. All these data suggest a differentiation of rabbit tracheal epithelial cells grown on gel toward squamous metaplasia. By contrast, the epithelium observed on coating is nearly a monolayer of very large and spread out cells. No K13-positive cells were observed, but an increase in the synthesis of simple epithelium marker (K18) was detected. These two substrata, similar in composition and different in structure, induce separate differentiation and appear as good tools to explore the mechanisms of differentiation of epithelial tracheal cells.

Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells

Abstract. The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARα (CD336), RARβ (CD2019), RARγ (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARβ/γ (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARα and β agonists or RAR panagonist, but not the RARγ agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARα/β and RXR agonists. The RARγ agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARβγ antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1–S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARα/β ligands are effective inhibitors of squamous differentiation. On the contrary, RARγ ligand appears to be inefficient in metaplasia inhibition, but the selective RARγ agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.

Interactions du benzoate d'oestradiol et de la progesterone sur le development de l'oviducte de caille (Coturnix coturnix japonica): I. Etude pondèrale et histologique

Abstract The effects of estradiol benzoate (O.) and progesterone (P.) alone and in combination was investigated in quail (Coturnix coturnix japonica) oviduct growth. At physiological doses, O. induced the differentiation of only two distinct cell types of the undifferentiated epithelium: ciliated cells and tubular gland cells. When injected with O., P. inhibited epithelial cell multiplication, cilia formation and tubular gland differentiation. But P. induced the differentiation of the goblet cells and played the most important part in the formation of secretory granules. Thus, after the activation of undifferentiated epithelial cells by estrogen, O. and P. were necessary at every stage to arrive at a functional unit.

Mechanisms of doxycycline-induced cytotoxicity on human bronchial epithelial cells

Doxycycline (DOX), a synthetic tetracycline, may have potential utility in the management of cancers and in the treatment of chronic inflammatory diseases due to its role in growth, invasion and metastasis of many tumors, on cell proliferation and as inducer of apoptosis. Some studies established its role in the treatment of lesions induced by mustards, warfare agents causing severe damage with blistering and tissue detachment in exposed areas of the body. In the present study, the effect of Dox was investigated in a human bronchial epithelial cell line. Dox induced a time- and concentration-dependent cell proliferation inhibition, associated with a cell cycle arrest in S phase, a decrease in viability due to apoptosis and necrosis, and cell detachment. This latter was partly correlated with early activation of caspase-3 before detachment, and with mitochondrial alteration. Cell transfection with a Bcl-2 encoding vector showed a decrease both in mitochondrial depolarization and cell detachment. Dox-induced apoptosis included decrease in Bcl-2 expression, increase in Bak expression and caspase-3 and -9 activation but appeared to be p53- and Bax-independent. A better comprehension of the Dox-induced apoptotic pathway could allow to abolish its toxic effects, improving the therapeutic efficiency of Dox.

Structure and Assembly of the Oviduct Ciliary Membrane

For species whose fertilization is internal, the oviduct is a part of the female genital tract which is the site for a number of different steps required for reproduction, including gamete transport, fertilization, and early embryonic development.

INDUCTION OF CILIOGENESIS IN OVIDUCT EPITHELIUM

Publisher Summary The purpose of this chapter is to describe a method for inducing ciliogenesis in a model of arrested avian oviduct development. In the developing oviduct, estrogens influence both epithelial proliferation and differentiation. During the differentiation phase, the cellular composition of the luminal epithelium is modified in response to hormonal influence. Initially, the epithelium is characterized by a predominance of ciliated cells that are found in the female genital tract of all vertebrates. In a second step, the characteristic oviduct epithelium consists of ciliated and secretory cells, forming, in most cases, a mucociliary epithelium as in airways of the respiratory tract. In birds, only the left ovary and oviduct develop. The luminal epithelium is composed of a single layer of cuboidal cells that are undifferentiated with a primary cilium. Ciliary beat is oriented from the infundibulum to the uterus, and the determination of ciliary polarity is established before differentiation. Ciliogenesis occurs in the same basic pattern in oviduct cells of mammals and birds. Centrioles move under the cellular surface and attach to the apical plasma membrane that has previously developed microvilli. Centrioles are then called basal bodies, from which the cilia grow at the cell surface by lengthening of the axonemal microtubules. According to the nature and dose of hormones and the duration of treatment, development is oriented toward either the ciliogenic or the secretory path of epithelial differentiation. Estrogen treatment induces ciliogenesis; undifferentiated epithelium turns into both undifferentiated cells and cells undergoing ciliogenesis. Progesterone hyperstimulation in association with estrogen treatment strongly induces the secretory process and inhibits the differentiation of ciliated cells. The balance between estrogen and progesterone is critical for harmonious development of the oviduct. The chapter details the methods, ovariectomy, and hormonal treatment.

Mitochondrial inside-out signalling during alkylating agent-induced anoikis.

Exposure of epithelial respiratory cells to the alkylating agent, mechlorethamine (HN2), induces anoikis initiated by mitochondrial depolarization and caspase-2 activation. The mechanisms of disruption of cell interactions were investigated and expression of integrins, E-cadherin, and actin were therefore studied after HN2 treatment. In the adherent cells, an early disruption of F-actin occurred associated with cell rounding. Inhibitors of caspase-2 resulted in attenuating of the decline of adhesion proteins and microfilaments. HN2-induced down-regulation of beta1 integrin, E-cadherin expression and F-actin pattern occurred in detached cells but were efficiently prevented by inhibitors of mitochondrial permeabilization. Moreover, inhibiting mitochondrial depolarization improved significantly both cell survival and capacity of detached cells to re-adhere. These findings confirm the pro-survival integrins and E-cadherin mediated signalling pathway. The central role of mitochondria in HN2-induced cell detachment is reinforced, suggesting that mitochondria acts as a key executor of reduced cell adherence during anoikis and could be responsible of an inside-out signalling. Present data support the potential of these therapeutics, generated via the inhibition of mitochondrial depolarization, as protectors against the alkylating agent lesions.