Retinoic acid regulates,in vitro,the two normal pathways of differentiation of human laryngeal keratinocytes

Summary We have investigated the regulation of the two normal differentiation pathways followed by laryngeal epithelium. Using a tissue culture system that permits growth of cells at the air-liquid interface in serum-free medium, we found that modulating the concentration of retinoic acid is sufficient to determine which pathway is used. At 10⁻⁸ M retinoic acid, the cells form a stratified squamous epithelium which expresses the differentiation-specific keratin K13. At 10⁻⁷ M retinoic acid, the cells form a ciliated pseudostratified epithelium, with no expression of K13. These results are distinct from those seen with foreskin keratinocytes, which have only a single pathway of normal differentiation. This work was supported by grant 3 P01 DC00203 from the National Institute on Deafness and Other Communication Disorders, Bethesda, MD (B.M.S., A.L.A. and T.P.D.) and grants from the Otolaryngology Foundation, Capt. Leo Berger, and the Morris S. and Florence H. Bender Foundation (M.G.M.).     

Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro

The human embryonal carcinoma cell lines $\text{NT2}\text{D1}$ and $\text{NT2}\text{B9}$, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits differentiation of normal diploid rat osteoblasts in vitro

The influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent halogenated aromatic hydrocarbon, on the development of bone tissue-like organization in primary cultures of normal diploid calvarial-derived rat osteoblasts was examined. Initially, when placed in culture, these cells actively proliferate while expressing genes associated with biosynthesis of the bone extracellular matrix. Then, post-proliferatively, genes are expressed that render the osteoblast competent for extracellular matrix mineralization and maintenance of structural as well as functional properties of the mature bone-cell phenotype. Our results indicate that, in the presence of TCDD, proliferation of osteoblasts was not inhibited but post-confluent formation of multicellular nodules that develop bone tissue-like organization was dramatically suppressed. Consistent with TCDD-mediated abrogation of bone nodule formation, expression of alkaline phosphatase and osteocalcin was not upregulated post-proliferatively. These findings are discussed within the context of TCDD effects on estrogens and vitamin D-responsive developmental gene expression during osteoblast differentiation and, from a broader biological perspective, on steroid hormone control of differentiation.     

Retinoic acid alters epithelial differentiation during palatogenesis.

Retinoids are teratogenic in humans and animals, producing a syndrome of craniofacial malformations that includes cleft palate. This study investigates the mechanism through which retinoic acid induces cleft palate. Murine palatogenesis after exposure to retinoic acid in utero is compared to normal development and to alterations observed after exposure in organ culture to retinoic acid or epidermal growth factor (EGF). Human embryonic palatal shelves were placed in the organ culture system and the responses to retinoic acid and EGF were compared to those of the murine palatal shelves. Growth factors play a role in normal development and are found in the embryonic palate. In other cell culture systems, retinoids alter the expression of EGF receptors. Our results suggest that in the medial epithelial cells of the palate, retinoic acid sustains the expression of the EGF receptor and the binding of EGF at a time when the expression in control medial cells has declined, and these control cells subsequently undergo programmed cell death. The continued DNA synthesis, proliferation, survival, and shift in phenotype of the medial cells is believed to interfere with the adhesion and fusion of opposing palatal shelves, resulting in cleft palate.     

Retinoic acid and the differentiation of lymphohaemopoietic stem cells

The study of haemopoiesis enables us to address one of the central questions of developmental biology, concerning the molecular mechanisms by which a multipotent cell develops into distinct differentiated progeny. Recent work⁽¹⁾ suggests specific roles for retinoic acid receptors at two distinct stages of haemopoiesis. Continuous cell lines of lymphohaemopoietic progenitors were established by infection with a retrovirus containing a dominant negative retinoic acid receptor. The cell lines depend on stem cell factor for their proliferation and can be induced to diffentiate into B-lymphocytes, erythrocytes, neutrophils, monocytes, mast cells and megakaryocytes. Since lymphohaemopoietic progenitors represent less than 0.01% of nucleated marrow cells, immortalised progenitors provide a valuable system with which to study haemopoiesis on a molecular level.     

Cyclic AMP induces ultrastructural differentiation of normal adult human adrenocortical cells cultured in vitro.

Electron microscopic studies have revealed that cyclic AMP, like ACTH, induces structural differentiation of adult human adreno-cortical cells cultured in vitro. These findings support the hypothesis that cyclic AMP can function as an intracellular mediator of the action of ACTH on the human adrenal gland.     

Retinoic acid respecifies limb bud cells in vitro.

Retinoic acid (RA) is known to have dramatic effects on limb pattern formation and has been shown to exert its effects on limbs by converting anterior limb bud cells into cells with posterior positional properties. In this study we find that dissociated posterior limb bud cells from chick and mouse embryos cultured at high density (micromass cultures) are able to stimulate the formation of supernumerary digits when grafted into developing wing buds and that the positional identity of both chick and mouse limb bud cells can be maintained for finite periods of time in vitro. Furthermore, using this assay system we have tested whether anterior cells from mouse and chick limb buds can be converted into cells with posterior identity by exposure to RA in vitro. We find that anterior limb bud cells acquire posterior properties after culture in the presence of RA.     

Retinoic acid differentiation of HL-60 cells promotes cytoskeletal polarization

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of alpha-tubulin does not significantly change; but the centrosomal-microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome-MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.     

Retinoic acid induces myogenin synthesis and myogenic differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C

The F3 molecule is a member of the immunoglobulin superfamily anchored to membranes by a glycane-phosphatidylinositol, and is predominantly expressed on subsets of axons of the central and peripheral nervous system. In a previous paper (Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991. Neuron. 6:595-606), we have established that F3 fulfills the operational definition of a cell adhesion molecule and that it stimulates neurite outgrowth when presented to sensory neurons as a surface component of transfected CHO cells. In the present study the question as to whether soluble forms of F3 would be functionally active was addressed in vitro on cultures of mouse dorsal root ganglion neurons. We observed that preparations enriched in soluble F3 had no effect on neuron attachment but enhanced neurite initiation and neurite outgrowth in a dose-dependent manner. By contrast, soluble NCAM-120 does not have any measurable effect on these phenomena. Addition of anti-F3 monovalent antibodies reduced the number of process-bearing neurons and the neuritic output per neuron to control values. Addition of cerebrospinal fluid, a natural source of soluble F3, also stimulated neurite extension, and this effect was partially blocked by anti-F3 antibodies. Our results suggest that the soluble forms of adhesive proteins with neurite outgrowth-promoting properties could act at a distance from their site of release in a way reminiscent of growth and trophic factors.     

Retinoic acid modulates the retinoblastoma protein during adipocyte terminal differentiation

Terminal differentiation is characterized by a permanent withdrawal of cells from the cell cycle. Retinoblastoma protein (RB) has been involved in cell cycle progression. Accumulating evidence also implicates RB in the promotion of differentiation of many cell types. We present new insights into the role of RB and other cell cycle regulatory proteins in adipocyte differentiation and on the role of retinoic acid (RA) in the regulation of the latter process. It is shown that RA reduces RB expression and enhances RB phosphorylation by a mechanism that involves down-regulation of the cyclin-dependent kinase inhibitor (CKI) p21(Cip1), having this fact as important consequences for both the cell cycle progression and the adipocyte differentiation process. The effects of RA result in the blockage of adipogenesis, but may also favor the retention of a pool of adipose cells able to re-enter the cell cycle, which may be important for the developmental dynamics of adipose tissue in vivo. In addition, these results reinforce the idea that there is a cross-talk between the cell cycle machinery and the adipocyte differentiation machinery that can be modulated by external signals, including nutrients.     

Retinoic acid inhibits dendritic cell differentiation driven by interleukin-4

All-trans-retinoic acid (atRA) appears to affect Th1-Th2 differentiation and its effects on immune responses might also be mediated by dendritic cell (DC). Nonetheless, studies have been showing contradictory results since was observed either induction or inhibition of DC differentiation. Our aim was to investigate atRA action on human monocyte derived DC differentiation. For this purpose we tested pharmacological and physiological doses of atRA with or without cytokines. Cell phenotypes were analyzed by flow cytometry and function was investigated by phagocytosis and respiratory burst. DC, positive control group, was differentiated with GM-CSF and IL-4 and maturated with TNF-α. We demonstrated that atRA effects depend on the dose used as pharmacological doses inhibited expression of all phenotypic markers tested while a physiological dose caused cell differentiation. However, atRA combined or not with cytokines did not promote DC differentiation. In fact, atRA was detrimental on IL-4 property as a DC inductor.     

Retinoic acid, hemin and hexamethylen bisacetamide interference with "in vitro" differentiation of chick embryo chondrocytes.

We have investigated the effect of all-trans Retinoic acid, and of substances (Hemine and Hexamethylene bisacetamide) which interfere with "in vitro" differentiation of mesenchyme derived cell lineages on the expression of specific markers of hyperthrophy in "in vitro" differentiating chick embryo chondrocytes. (Castagnola P., et al., 1986). Continuous treatment of chondrogenic cells in conditions allowing differentiation "in vitro" with Retinoic acid resulted in persistence of type I collagen synthesis and in lack of type X collagen and Ch 21 protein expression. Hemin treated cells secreted a reduced amount of type X collagen. HMBA treatment inhibited type X collagen expression and caused reduction of the ratio between type II collagen and Ch 21 synthesized. The data indicate an independent regulation of these markers during chondrocyte differentiation.     

Retinoic acid increases CD15 expression in immortalized rat astrocytes

Summary We have studied the expression of the CD15 (3-fucosyl-N-acetyl-lactosamine) epitope on immortalized astroglial cells derived from embryonic (E 19/20) rat brain. Immortalization was achieved by pulse-treatment of primary culture with 5-azacytidine. Seventy-three permanent cell lines were established by repeated cell cloning. Clones expressing GFAP, A2B5, and vimentin were regarded as immature astrocytes. One of these clones expressing CD15 was selected for manipulation studies. Monoclonal antibody was used for immunocytochemical detection of CD15 epitope and in immunoblot analysis. CD15 expression was visible in about 20% of the cells and was associated with a special morphological appearance. In the presence of retinoic acid the proportion of CD15-positive cells increased in a time-dependent manner, reaching about 90% within four days. Again, this expression was associated with the formation of distinct morphological features, including immunoreactive perinuclear granula, tips of processes and contact sites. After treatment with neuraminidase, all cells showed CD15-positive immunoreaction, revealing the presence of the epitope masked by sialylation. Immunoblot patterns of glycoproteins from trypsinized and mechanically detached cell preparations suggest that proteins, carrying sialylated CD15, might represent intracellular precursors of extracellularly active molecules.     

大鼠海马神经干细胞体外培养分化过程中钾电流的变化

目的:探讨新生大鼠海马神经干细胞体外培养分化后的神经元样细胞钾电流的变化.方法:神经干细胞体外扩增培养并传代后,撤除有丝分裂原并加血清诱导分化,应用全细胞电压钳技术检测分化后培养1 d、7 d、14 d、21 d细胞的电压依赖性钾电流.结果:分化后培养1 d的细胞,未检测出钾电流;分化后培养7 d、14 d、21 d的细胞,在 50 mV电压水平下的钾电流幅值分别为(18.077±2.789)pA/pF, (13.099±2.742)pA/pF, (34.045±8.067)pA/pF.该电流为两种电流的混合,分别能被TEA和4-AP所阻断,可能为缓慢失活的延迟整流钾电流(IK)和快速失活的瞬时外向钾电流(IA).结论:新生大鼠海马神经干细胞诱导分化后,随着体外培养时间的延长,钾离子通道的功能逐渐成熟.