The retinol signaling pathway in mouse pluripotent P19 cells

atRA (all-trans-retinoic acid), the active metabolite of retinol (vitamin A), is essential for embryogenesis and maintenance of cellular phenotype in adults. Chemicals that interfere with the metabolism of retinol to atRA, therefore, are a human health concern. During development of a screen for disruptors of this signaling pathway, we investigated whether the mouse pluripotent P19 cell metabolizes retinol to atRA and thus can be used in a cell-based screen for disruptors of the pathway. We found that retinol induced the identical pattern of homeobox gene expression as atRA and its precursor, retinal. Retinol was 160-fold less potent than atRA as an inducer, however. In spite of its lower potency, increased Hoxa1 gene expression was detected 30 min after retinol exposure and increased 40-fold by 2 h. Rdh10 and Aldh1a2/Raldh2, which together convert retinol to atRA in the embryo, were the predominant alcohol and aldehyde dehydrogenases expressed in P19 cells. The cell expressed high mRNA levels of retinol binding proteins, Rbp1 and Rbp4, and the 13,14-dihydroretinol saturase, Retsat. It also expressed all Rar and Rxr isotypes, Crabp1&2, the three Cyp26 genes, and both β-carotene-cleaving genes, Bcmo1 and Bco2. The basal expression levels and retinol responsiveness of 25 pathway-related genes were quantitated by RT-qPCR. A test of the Aldh1a2 inhibitor, citral, showed that the disruption of the pathway was easily detected and quantitated showing that the P19 cell provides an in vitro model system for identifying and exploring the mechanism of action of chemicals that interfere with this critical cellular pathway.     

Ontogeny of insulin binding during chick skeletal myogenesis in vitro.

Our studies show that insulin receptors exist on chicken skeletal muscle cells at all developmental stages in culture. ¹²⁵I-labeled insulin binding at physiological concentrations to mature myotubes demonstrated saturability, binding proportional to cell number, reversibility, and specificity by competition with native hormone which reduced specific binding by 40% with 1 ng/ml and was maximal with 10 μg/ml. Further evidence for specificity was shown by no competition of insulin specific binding with insulin A chain, insulin B chain, growth hormone, and thyrotropin. Two binding sites were detected, with affinity constants of 10¹⁰M^?1 and 2 × 10⁹M^?1. The hormone receptor complex showed rapid dissociation (70% in 30 min) after equilibrium binding. During myogenesis, an increase in insulin receptors occurs from 500 per proliferating myoblast to 3000 per cell equivalent in mature (6 day) myotubes. Since these studies demonstrate that insulin receptors are present and other studies have shown that insulin is present during most of chicken embryogenesis, insulin may regulate muscle development in vivo to a greater degree than previously suspected.     

Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro.

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5'-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.     

A re-evaluation of DNA repair during skeletal myogenesis in vitro

In previous studies on DNA repair during myogenesis, comparisons made of repair in post-replication myoblasts and in myotubes led to the conclusion that the capacity to repair damage in DNA decreased during myoblast differentiation. Using unscheduled DNA synthesis in response to UV-induced damage as an indicator of DNA repair in a myogenic line of rat skeletal muscle, it is demonstrated that nuclei in myotubes possess identical repair capacity as that in proliferating myoblasts. Furthermore, a brief increase in DNA repair capacity was observed to immediately follow the cessation of replicative DNA synthesis. This transient increase in repair capacity is consistent with the data of earlier reports and explains the previous but inappropriate conclusion that repair diminishes during myogenic differentiation. This transient increase in the capacity to repair DNA was not observed in a developmentally defective, non-differentiating line of similar myogenic origin.     

Cell-cell interaction by mouse limb cells during in vitro chondrogenesis: Analysis of the brachypod mutation

This paper contains observations and experiments which collectively demonstrate a requirement for cell-cell interactions among limb bud mesenchyme cells during chondrogenic differentiation. Limb bud cells isolated from brachypodism^H (bp^H) and wild-type mouse embyros between Thieler stage 16–17 and midstage 21 were compared with respect to their abilities to undergo chondrogenic differentiation in high-density micromass cultures. Nodules formed by dissociated Day 12 (stage 20) bp^H limb bud cells have been reported previously to be abnormally reduced in size and number, and delayed in formation. We corroborate these results, but find that bp^H cultures prepared from earlier-stage limb buds (between stages 16–17 and early stage 21) are progressively more like wild-type cultures. Stage 16–17 bp^H cultures at 72 hr actually contain normal numbers of and size nodules, while stage 18 bp^H cultures are intermediate between stages 16–17 and stage 21 in nodule formation. On the other hand, we also find that the initial rate of aggregate formation is normal even in bp^H cultures prepared from stage 20 cultures in which nodule formation is not normal. Preparation of cultures composed primarily of early stage 21 bp^H limb bud cells mixed with small quantities (e.g., 5%) of stage 16–17 wild-type limb bud cells showed significant increases in cartilage nodule formation over control cultures composed only of early stage 21 bp^H cells. Greater proportions of wild-type cells obtained from embryos older than stages 16–17 were required for the same degree of normalization, supporting the hypothesis that a specific cell type, whose proportion decreases normally in the limb bud over time, is required to increase in vitro chondrogenesis by bp^H cells. Additionally, cultures containing stage 23 chick limb cells and early stage 21 bp^H cells at a ratio of 1:20 contained wild-type levels of nodules per square millimeter of culture. Thus, bp^H cells appear to respond to chondrogenic inductive signals from normal limb mesenchyme cells. In order to test for the ability of bp^H limb bud mesenchyme to induce chondrogenesis, stage 16–17 bp^H and wild-type limb bud cells, which form identical numbers of aggregates and nodules in culture, were each mixed with early stage 21 bp^H cells at ratios of 1:20, 1:10, and 1:3. Although low proportions of wild-type stage 17 cells significantly increased the number of aggregates and nodules in these mixed cultures, low proportions of bp^H stage 16–17 cells did not. It is, therefore, concluded that the primary defect of the bp^H mutation is likely to reside in the reduced ability of a specific mesenchyme cell subpopulation to provide an inductive stimulus for chondrogenesis.     

Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.     

Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.     

FGF receptor availability regulates skeletal myogenesis.

Fibroblast growth factors (FGFs) and their receptors are critical participants in embryonic development, including the genesis of skeletal, cardiac, and smooth muscle. FGF signaling is mediated through interactions between multiple FGF ligands and transmembrane tyrosine kinase receptors, resulting in activation of a number of signal transduction pathways. Skeletal myocytes express FGF ligands and receptors in a coordinated fashion, suggesting that these molecules participate in autocrine signaling in the myocyte. Endogenously produced FGF has been shown to inhibit myogenesis, but the role of FGF receptor availability in directing myocyte proliferation and differentiation has not been established. To determine the contribution of receptor availability to the regulation of myogenesis, receptor availability was either increased by expressing a full-length FGF receptor-1 or decreased by expressing a truncated FGF receptor-1 in cultured skeletal myocytes. Constitutive expression of a full-length FGF receptor-1 increased myocyte proliferation and delayed differentiation. Conversely, a reduction in functional FGF receptor signaling by expression of a truncated FGF receptor-1 decreased proliferation and enhanced differentiation of myocytes. These data demonstrate that FGF receptor availability plays a critical regulatory role in skeletal myogenesis.     

Phosphorylation in skeletal myosin light chain modulates the actin--myosin interaction in the presence of regulatory proteins in vitro

The effect of phosphorylation in skeletal myosin light chain (LC2) on the actomyosin and acto-heavymeromyosin (HMM) ATPase activities was investigated in the presence or absence of regulatory proteins (tropomyosin-troponin complex). Phosphorylation in LC2 did not modulate the actin-myosin and actin-HMM interactions over a wide range of KCl concentrations from 30 to 150 mM without regulatory proteins. In the presence of regulatory proteins, phosphorylation in myosin LC2 enhanced the ATPase activity of actomyosin with calcium ions, but the removal of calcium ions made little difference in the ATPase activity between phosphorylated and dephosphorylated myosins. Ca2+-sensitivity of the regulated actomyosin was slightly changed by phosphorylation in myosin LC2. However, both the ATPase activity and Ca2+-sensitivity of the regulated acto-HMM were unaffected by phosphorylation in HMM LC2.     

Cell-cell interaction can influence drug-induced differentiation of murine embryonal carcinoma cells

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.     

Characterization of myogenesis from adult satellite cells cultured in vitro

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.