Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca++ medium (0.09 mM) as measured by incorporation of [3H]thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC50 of about 10 microM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 microM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number over control at 10 microM. These results are of importance since they suggest Ca++ may influence the effect of retinoids on keratinocytes.     

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Altered Patterns of Protein Phosphorylation and Synthesis Caused by Methyl Mercury in Cerebellar Granule Cell Culture

In the preceding report we demonstrated a dose-dependent increase in 32P-phosphoprotein labeling after 24-h exposure of cultured cerebellar granule neurons to methyl mercury (MeHg), a response that was not observed in glial cultures. In the present study we have examined 32P-labeled phosphoproteins by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. At concentrations of 0.5 and 1 microM, which were not extensively cytotoxic, MeHg enhanced phosphorylation of numerous acidic proteins, particularly a cluster of proteins with Mr approximately 28,000 and pI approximately 5.7-5.9 (pp 28/5.7-5.9) and a protein with Mr approximately 58,000 and pI approximately 5.6. The pp28 cluster displayed considerable two-dimensional pattern variability from one experiment to the next, suggesting susceptibility to subtle structural modifications. Time course studies revealed that increased 32P phospholabeling of pp28/5.7-5.9 was detectable after 12-h exposure to 3 microM MeHg and reached values of 300-500% of control by 24 h. These studies also showed that among the 21 proteins analyzed by two-dimensional densitometry, 32P phospholabeling of four proteins increased by 20-50% and of two proteins decreased by 20-50% after 24-h treatment. However, exposure to 10 microM MeHg produced stimulation of pp28/5.7-5.9 32P phospholabeling within 2 h. Under these conditions a relatively high stimulation (sevenfold) of pp28/5.7 phospholabeling occurred, while pp28/5.9 32P phospholabeling was only moderately (5-20%) enhanced. 35S and 32P double-label analysis of cells treated with 0, 0.5, and 1 microM MeHg indicated specific stimulation of 32P phospholabeling of these proteins without increased polypeptide synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

Regulation of mouse preimplantation development: inhibitory effect of genistein, an inhibitor of tyrosine protein phosphorylation, on cleavage of one-cell embryos

We investigated the effects of genistein, an inhibitor of tyrosine protein phosphorylation, on mouse 1-cell embryos, since in response to mitogenic stimuli tyrosine protein phosphorylation in somatic cells is implicated in initiation of DNA synthesis. Genistein inhibits cleavage of 1-cell embryos in a concentration-dependent and reversible manner; biochanin A, which is a less potent inhibitor of tyrosine protein phosphorylation, is a less potent inhibitor of cell cleavage. Genistein does not inhibit [35S]methionine incorporation, but does inhibit [3H]thymidine incorporation. Consistent with genistein's ability to inhibit cleavage by inhibiting DNA synthesis is that the loss of genistein's ability to inhibit cleavage corresponds with exit of the 1-cell embryos from S phase. Genistein is likely to inhibit tyrosine protein phosphorylation in situ, since it reduces by 80% the relative amount of [32P]phosphotyrosine present in 1-cell embryos; genistein does not inhibit either [32P]orthophosphate uptake or incorporation. As anticipated, genistein has little effect on inhibiting changes in the pattern of phosphoprotein synthesis during the first cell cycle, since tyrosine protein phosphorylation constitutes a small percentage of total protein phosphorylation. Alkalai treatment of [32P]radiolabeled phosphoproteins transferred to Immobilon reveals a base-resistant set of phosphoproteins of Mr = 32,000 that displays cell-cycle changes in phosphorylation. Although these properties suggest that these phosphoproteins may be related to the p34cdc2 protein kinase, phosphoamino acid analysis of [32P]radiolabeled phosphoproteins reveals that they are not enriched for phosphotyrosine; the inactive for p34cdc2 protein kinase contains a high level of phosphotyrosine. Results of these experiments suggest that tyrosine protein phosphorylation in response to the fertilizing sperm may be involved in initiating DNA synthesis in the 1-cell embryo, as well as converting a meiotic cell cycle to a mitotic one.     

Effect of retinoic acid on the phosphorylation of endogenous proteins in HL-60 cells

HL-60 cells were incubated with [32P]-Pi in order to label endogenous phosphoproteins in situ. These were then resolved via two-dimensional electrophoresis and autoradiograms were made from the gels. A comparison of autoradiograms made from retinoic-acid-differentiated cells with those made from control cells revealed a small number of phosphoproteins whose labeling was enhanced in differentiated cells. Incubating HL-60 cells with [35S]-methionine revealed that RA induced the synthesis of one of these proteins, (53 kDa, pl 4.8) although not to a degree sufficient to account for the increased phosphate labeling observed in differentiated cells. Difluoromethylornithine (DFMO), which arrests HL-60 cell proliferation without inducing differentiation, had no effect on endogenous protein phosphorylation. Treatment of DFMO-arrested cells with retinoic acid, however, increased the phosphorylation of the proteins and resulted in differentiation of the cells. Densitometric analysis of autoradiograms made from two-dimensional gels revealed that the phosphorylation of the 53-kDa, pl 4.8 protein was significantly elevated in cells exposed to RA for as little as 12 hours, i.e., before the cells were committed to differentiate and before a significant increase in the number of phenotypically mature cells was observed. It therefore appears that this protein may be an intermediate in the retinoic-acid-induced differentiation process.     

The 27,000 daltons stress proteins are phosphorylated by protein kinase C during the tumor promoter-mediated growth inhibition of human mammary carcinoma cells

Phorbol-12-myristate-13-acetate (PMA) inhibited growth of human mammary carcinoma cell lines and increased mainly the phosphorylation of two cytosolic phosphoproteins (pp) of 27 kD with isoelectric points of 5.5 (pp27a) and 5.0 (pp27b). The time course of pp27 phosphorylation closely paralleled the rapid PMA-induced subcellular redistribution of protein kinase C (PKC) activity and its subsequent down regulation. Addition of phospholipase C and fetal calf serum to intact cells or purified PKC to a cell free system enhanced the phosphorylation of both pp27 suggesting that the two polypeptides are specific substrates for PKC. Exposure of human mammary carcinoma cells to stress inducers such as arsenite or cadmium increased the 32P incorporation of both pp27 to an extent comparable to PMA. The increased phosphorus content following stress was rather due to a higher rate of synthesis of both pp27 than to a higher phosphorylation state of these polypeptides as determined by [3H]-leucine labeling. These results indicate that the major substrates of PKC, phosphorylated during the PMA-induced growth inhibition of human mammary carcinoma cells, are members of the stress protein family, suggesting a new possible function for these proteins.     

Co-ordinated changes in the cyclic AMP signalling system and the phosphorylation of two nuclear proteins of Mr 130,000 and 110,000 during proliferative stimulation of the rat parotid gland by isoprenaline. Possible identity of the two proteins with pp135 and nucleolin.

Parotid glands were stimulated to growth by repeated injection of the beta-agonist isoprenaline into rats. Incubation of intact parotid-gland lobules with [32P]Pi and subsequent analysis of nuclear proteins revealed in the stimulated glands an increased 32P incorporation into two acid-soluble non-histone proteins with apparent Mr values of 110,000 and 130,000 (p110 and p130). After a single injection of isoprenaline, leading to a biphasic increase in DNA synthesis (maximum at 24 h), the same two proteins showed a transiently increased 32P incorporation at 17 h after injection. At this time point at the onset of DNA synthesis the total activity of soluble cyclic AMP-dependent protein kinase decreased. No change in p110/p130 phosphorylation was observed at 0.3 h after stimulation, a time of maximal stimulation of secretion. Administration of the beta-antagonist propranolol 8 h after the injection of isoprenaline suppressed the increase in DNA synthesis, the preceding changes in the concentration of cyclic AMP and in the activity of cyclic AMP-dependent protein kinase, as well as the increased phosphorylation of p110 and p130. Cross-reactivity of p110 and p130 with specific antisera against two nucleolar phosphoproteins of similar molecular mass (nucleolin and pp135), as well as their localization in a nucleolar cell fraction, indicated a possible identity of p110 and p130 with these two proteins. Our results suggest that nucleolin and pp135 are nuclear target proteins of cyclic AMP in the cyclic AMP-influenced regulation of the transition of cells from the G1 to the S phase.     

Protein Phosphotyrosine in Mouse Brain: Developmental Changes and Regulation by Epidermal Growth Factor, Type I Insulin-Like Growth Factor, and Insulin

Using antiphosphotyrosine antibodies, we have investigated protein phosphorylation in mouse brain during development in intact animals and in reaggregated cerebral cultures. Under basal conditions, in vivo and in vitro, the levels of two main phosphoproteins, of Mr 120,000 and 180,000 (pp180), increased with development, reaching a maximum in the early postnatal period and decreasing thereafter. In adult forebrain, pp180 was still highly phosphorylated, but it was not detected in cerebellum or in peripheral tissues. In reaggregated cortical cultures, epidermal growth factor (EGF), type I insulin-like growth factor (IGF-I), and insulin enhanced protein tyrosine phosphorylation of several proteins, which were specific for EGF or IGF-I/insulin. In highly enriched neuronal or astrocytic monolayer cultures, some proteins phosphorylated in basal conditions, or in response to EGF and IGF-I, were found in both types of culture, whereas others appeared cell type specific. In addition, in each cell type, some proteins were phosphorylated under the action of both growth factors. These results indicate that tyrosine protein phosphorylation is maximal in mouse brain during development and is regulated by growth factors in neurons as well as in astrocytes.     

Phosphorylations of the subcellular matrix in cells transformed by Rous' sarcoma virus

The transforming protein of Rous' sarcoma virus (RSV) is a phosphoprotein of Mr 60 000 (pp60src) which displays protein kinase activity specific for tyrosine residues; pp60src is associated with the plasma membrane and is recovered in the detergent-insoluble material which represents the subcellular matrix of the cell. After phosphorylation of this material of RSV-transformed cells with [gamma-32P]ATP, five phosphoproteins have been detected which are not seen in normal cells. These proteins (Mr = 135 000, 125 000, 75 000, 70 000, 60 000) contain phosphotyrosine. Their phosphorylation is strongly inhibited by anti-pp60src antibodies. In cells transformed by a temperature-sensitive mutant of RSV, these phosphoproteins, present at the permissive temperature, are no longer detected at the non-permissive temperature. It is concluded that these phosphorylations are mediated by pp60src protein kinase activity. This supports a possible role of the phosphorylation of cytoskeletal proteins in the transformation process.     

Diverse mitogenic agents induce rapid phosphorylation of a common set of cellular proteins at tyrosine in quiescent mammalian cells

Protein phosphorylation of quiescent human skin fibroblasts was analyzed following stimulation by epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, serum, or 12-O-tetradecanoyl-phorbol-13-acetate. In mitogen-treated cells, a markedly increased phosphorylation of two Mr = 43,000 proteins and two Mr = 41,000 proteins was always detected by two-dimensional gel electrophoresis. More acidic forms were the dominant species and contained phosphotyrosine, phosphoserine, and phosphothreonine, while the basic forms contained phosphotyrosine and phosphoserine. The two Mr = 41,000 proteins were structurally related to each other. All mitogens seemed to stimulate the phosphorylation of each protein with the same site specificity. Induction of the same set of phosphoproteins was observed in mitogen-stimulated rat and mouse fibroblasts as well. These stimulated phosphorylations occurred rapidly, were maximal 5 min after exposure of cells to mitogens, and diminished gradually after 30 min. Mitogen-induced phosphorylation of these proteins was correlated to the extent of mitogen-stimulated DNA synthesis. In addition, such increased protein phosphorylation was not observed in exponentially growing cells, nor in Rous sarcoma virus-transformed rat cells. Thus, phosphorylation of the Mr = 43,000 and 41,000 proteins, which represents a common and specific response of cells to mitogens, could constitute an early event involved in the control of cellular G0----G1 transition.     

Photoisomerization of retinoic acid and its influence on regulation of human keratinocyte growth and differentiation

Retinoic acid constantly undergoes structural inter-conversions among the geometrical isomers (all-trans-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid and 9-13-di-cis-retinoic acid) by photoisomerization under natural light. Geometric isomers of retinoic acid thus formed showed different effects on human epidermal keratinocyte growth and differentiation. The ability of the isomers to inhibit the synthesis of cornified envelope (terminal event in the keratinocyte differentiation program) changed rapidly when illuminated by white fluorescent light. The 11-cis-retinoic acid had a 3-fold stronger activity to inhibit the growth of keratinocytes than the other geometric isomers. On the other hand, all-trans-retinoic acid, 9-cis-retinoic acid and 9-13-di-cis-retinoic acid exhibited a 3-fold greater ability to inhibit synthesis of involucrin, transglutaminase and the cornified envelopes. The regulation of keratin expression by the geometric isomers of retinoic acids was extremely complex. Level of keratin-1 (K1) mRNA was increased by 11-cis-retinoic acid and 13-cis-retinoic acid, but suppressed by 9,13-di-cis-retinoic acids while all-trans-retinoic acid and 9-cis-retinoic acid had no effect. Level of keratin-10 (K10) mRNA was strongly inhibited by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid as compared to 13-cis-retinoic acid and 9,13-di-cis-retinoic acids. The mRNA level of keratin-14 (K14) was suppressed by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid but not influenced by 13-cis-retinoic acid and 9,13-di-cis-retinoic acid. Natural light induced structural inter-conversions among the geometric isomers of retinoic acids in tissues-especially the skin, might play a crucial role in the regulation of growth and differentiation of keratinocytes.     

Planarian regeneration: in vivo and in vitro effects of calcium and calmodulin on DNA synthesis

Regeneration does not occur when planarians are grown in Ca2+-free medium. The possible effect of calcium upon DNA synthesis was therefore studied using cultured planarian cells and regenerating planarian fragments. In the cultures, DNA synthesis was Ca2+-dependent and required a minimum of 10(-6) M Ca2+ in the medium. It was gradually decreased in cells grown in Ca2+-free medium. Addition of Ca2+ to these cultures raised DNA synthesis. The time lag between addition of Ca2+ and stimulation of DNA synthesis varied with culture age. The triggering effect of Ca2+ was amplified by ionophore A 23187. A calcium binding protein, ram testis calmodulin, intensified the stimulatory effect of calcium, but EGTA blocked this effect. In the presence of trifluoperazine (TFP), DNA synthesis was not stimulated by Ca2+. This inhibition by TFP was overcome by adding calmodulin to the medium. Ca2+ therefore triggered DNA synthesis in vitro, and this role might have been potentiated by calmodulin. In vivo, DNA synthesis was shown to be dependent on the Ca2+ concentration in the medium in which intact or regenerating planarians were grown. In 12-h regenerates, the Ca2+ concentration in the medium was no longer critical. Total calcium content decreased just after sectioning until completion of healing (at 6 h) and then rose significantly to a peak at 12 h which coincided with the first peak of DNA synthesis. The calmodulin content gradually diminished during the first 6 h after sectioning. After a transient rise at 12 h, calmodulin content further decreased until 48 h. The results demonstrate the crucial role of Ca2+ in triggering DNA synthesis in planarian cells in vitro and in regenerating fragments. Calmodulin, whose concentration is very low in planarians compared to vertebrates, might help to induce the first peak of DNA synthesis at 12 h after sectioning, but is probably not the main Ca2+-binding protein involved in the regeneration process.     

Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland

Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.     

Effect of retinoic acid on proliferation and differentiation of cultured chondrocytes in terminal differentiation

We obtained terminally differentiated chondrocytes in monolayer culture from chick embryonal growth plates, and examined the effect of retinoic acid on these cells. The cells treated with retinoic acid ceased type X collagen synthesis and showed decreased calcium incorporation into cell layers. Retinoic acid tended to stimulate proliferation of the cultured chondrocytes. It also increased DNA accumulation dose-dependently in the range from 1 nM to 1 microM. DNA synthesis in the growth phase and confluency was stimulated within 10 h after addition of 0.1 microM retinoic acid. [3H]Retinoic acid binding, which was inhibited by simultaneous addition of excess unlabeled retinoic acid, was detected in both the cytosolic and nuclear fractions of the chondrocytes. The retinoic acid binding capacity of the nuclear fraction was increased by pretreating the cells with retinoic acid. These results indicate that retinoic acid binds to both the cytosolic and nuclear fractions of cultured chondrocytes, and induces their proliferation and dedifferentiation.     

Differential effects of physiological concentrations of retinoic acid in vitro on chondrogenesis and myogenesis in chick craniofacial mesenchyme

Recent studies have shown that in the developing limb bud retinoic acid is a skeletal morphogen at physiological levels, but a potent teratogen at higher levels. Retinoic acid has also been shown to be teratogenic during facial development, but very low levels may have an as yet unspecified role in normal development. In the present study the effects of retinoic acid on chondrogenesis and myogenesis by craniofacial cells grown in micromass cell culture were investigated. Retinoic acid, at concentrations of 0.01-100 ng/ml, was supplied to cells derived from day-4 (H.H stage 23/24) chick embryo mandibular, maxillary and frontonasal processes, grown in micromass cultures for 4 days in both serum-containing and defined media. Based on Alcian-blue-staining, concentrations of retinoic acid of 0.1-1 ng/ml were found to enhance chondrogenesis by mandibular cells grown in defined medium, while greater concentrations up to 100 ng/ml inhibited chondrogenesis. By contrast, chondrogenesis was generally retarded by all concentrations of retinoic acid applied to frontonasal cells grown in defined medium and when applied to both mandibular and frontonasal cells when grown in serum-containing medium. Cells from stage-23/24 maxillae did not display any significant chondrogenic activity in either medium under these culture conditions. Unlike chondrogenesis, myogenesis in mandibular, frontonasal and maxillary cultures was greater in defined than serum-containing medium, based on the appearance of immunologically detectable muscle myosin, and was reduced considerably less in defined medium by all concentrations of retinoic acid tested. In the presence of serum however, myogenesis was retarded with increasing concentrations of retinoic acid beyond 1 ng/ml in micromass cultures from all three facial regions.(ABSTRACT TRUNCATED AT 250 WORDS)