Interrelationship between cyclic AMP level and chondrogenesis in vitro

A consistent chondrogenesis takes place in micro-mass cultures of stage 23-24 chicken limb bud mesenchymal cells. In these cultures a short, marked elevation of cAMP level was detected at the time of the onset of cartilage phenotype expression. On the other hand, exogeneous glycosaminoglycans which inhibited chondrogenesis caused a reduction in the cAMP level of the cells. These correlations between cAMP level and phenotypic characteristics suggest that, among other things required in chondrogenesis, cAMP level may be a prominent factor.     

Effects of cyclic AMP on limb bud chondrogenesis in low cell density culture

In a previous paper, it was shown that the limb bud mesodermal cells differentiated into cartilage even at low cell density by lowering the serum content in the culture medium (Hattori & Ide, Exp cell res 150 (1984) 338) [20]. The present paper describes the effects of cAMP on limb bud chondrogenesis at low cell density. cAMP promoted chondrogenesis at low cell density in cultures with various concentrations of serum. The limb bud cells differentiated into cartilage cells without forming aggregates. cAMP inhibited the loss of chondrogenic capability in serum-rich medium. The relationship between cAMP level and serum content is also discussed.     

Somite chondrogenesis: alterations in cyclic AMP levels and proteoglycan synthesis

Cyclic AMP (cAMP) levels have been shown to have a positive influence on chondrogenesis in limb buds and pelvic cartilage. In the present study the level of cAMP was measured during somite chondrogenesis in vitro and found to decrease from 1.38 pmol/micrograms DNA on day 0 to 0.9 pmol/micrograms DNA on day 6. Inclusion of notochord with somites caused a marked reduction, with levels decreasing from 1.41 pmol/micrograms DNA on day 0 to 0.36 pmol/micrograms DNA on day 6. Concurrently, the incorporation of radioactive sulfate into sulfated glycosaminoglycans increased from day 3 to day 6 by 38% in somite and 77% in somite-notochord explants. The aggregation of proteoglycans was analyzed by gel chromatography and found to increase with a corresponding decrease in cAMP levels. The results indicate that a decrease in cAMP levels may be necessary for chondrogenic expression in somites.     

The effect of avian retroviruses on limb bud chondrogenesis in vitro

Mesenchymal cells isolated from stage 24 embryonic chicken limb buds were infected with the temperature-sensitive transformation mutants of Rous sarcoma virus tsNY68, tsNY10 and tsLA25 at the nonpermissive temperature for transformation (41 degrees C). Virus infection greatly inhibited subsequent limb bud chondrogenesis under nontransforming conditions, as indicated by a reduction in the rate of 35SO4 incorporation into cell-associated proteoglycans. The inhibition of chondrogenesis was directly related to the percentage of cells infected with tsNY68 at 41 degrees C. The observed inhibition of chondrogenesis was independent of src gene expression since this effect was also caused by many viruses which lack the src gene, including the leukosis viruses RAV-1, RAV-2 and MAV-2(0); the src deletion mutant RSVtd107; and the reticuloendotheliosis viruses REV-T and SNV. Infection of mesenchymal cells with tsNY68 under nontransforming conditions did not cause changes in parameters such as the rate of thymidine incorporation, total cell DNA and total cell protein. Infection with tsNY68 at 41 degrees C resulted in altered kinetics of 35SO4 incorporation into cell-associated proteoglycans and a corresponding reduction in 35SO4-labeled proteoglycans extracted from the cell layer. There were no apparent quantitative effects on the rate of accumulation of proteoglycans in the culture medium. The proteoglycans extracted from the cells and the collected medium of tsNY68-infected cultures were smaller than those of uninfected cultures, as shown by agarose gel chromatography.     

Caffeine effects on cyclic AMP levels in the mouse embryonic limb and palate in vitro

Caffeine is a teratogen that causes limb and palate malformations in rodents. Since the ability to raise cyclic nucleotide levels is a known biological action of caffeine, cyclic AMP levels were measured in CD-1 mouse embryonic forelimb from whole embryo culture and embryonic limb and palate cells grown in primary culture following treatment with various concentrations of caffeine (0, 1, 3, or 10 mM). In forelimb buds from whole embryo culture, a dose-dependent response was observed. Caffeine at 1 mM concentration stimulated cyclic AMP levels to 151% of control value at 60 min. Even greater stimulation of cyclic AMP occurred at higher caffeine concentrations. A dose-dependent response was seen in both limb and palate cell culture. In limb cell culture, all caffeine concentrations significantly stimulated cyclic AMP after 10 min compared to control. In palate cell culture, there was a twofold increase in cyclic AMP at the 1-mM caffeine concentration. At higher caffeine concentrations, cyclic AMP was significantly increased after 60 min. In addition, stimulation of cyclic AMP in cultured limb and palate cells by isoproterenol, a beta-adrenergic agonist, was used as a positive control. Isoproterenol stimulated a 2.5-fold greater response in the palate cells than in the limb bud cells at isoproterenol levels of 10(-5) or 10(-4) M. The increase of cyclic AMP may be influential in the process of abnormal limb or palate development.     

Evidence for histogenic interactions during in vitro limb chondrogenesis

The requirement for homotypic cell interaction was studied by making chimeric micromass cultures containing various proportions of chick and quail limb mesenchyme. Cultures made from limb mesenchyme from embryos of Hamburger and Hamilton stages 23–24 produce large clumps of cartilage cells, identified by the accumulation of an extracellular matrix which stains with alcian blue at pH 1 and by the ability of cells to take up ³⁵SO₄ rapidly, as demonstrated autoradiographically. Dissociated mesenchyme from stage 19 embryos did not produce cartilage in micromass cultures, but only precartilage cell aggregates. Micromass cultures prepared from mixtures of mesenchyme cells obtained from stage 19 and stages 23–24 embryos contained decreasing numbers of cartilage nodules as the proportion of stage 19-derived mesenchyme increased. At the same time the number of aggregates was not affected. When the ratio of stage 19- to stage 24-derived cells was 3:1 or greater, no nodules were detected. The actual number of cells from each stage was verified by using mixtures of quail and chick cells, which are microscopically distinguishable. Additional evidence suggests that the stage 19-derived mesenchyme inhibits chondrogenesis by passively preventing stage 24-derived cells from interacting. The results presented are consistent with the suggestions that (1) homotypic cell interaction plays a role in limb chondrogenesis and (2) the capacity to interact in the required manner is acquired after the embryos have reached stage 19. These phenomena might be involved in the normal histogenesis of cartilage tissue.     

Inhibition of chondrogenesis by retinoic acid in limb mesenchymal cells in vitro: Effects on PGE2 and cyclic AMP concentrations

Effects of retinoic acid (RA) on prostaglandin E₂ (PGE₂) and cyclic AMP (cAMP) concentrations were investigated in high density, micromass cultures of mesenchymal cells derived from chick limb buds. Exposure of cells during the initial 24 h of culture to RA concentrations between 0.05–1.0 μg/ml inhibited chondrogenesis in a dose-dependent manner with 1.0 μg/ml totally inhibiting cartilage formation. Concentrations of PGE₂ and cAMP increased during the prechondrogenic period in control cells in a closely related way and remained elevated throughout the six-day period examined. Addition of RA (0.05 and 0.5 μg/ml) did not significantly alter cAMP concentrations at any time point, but significantly elevated PGE₂ levels relative to control cells in six-day cultures in a concentration-dependent manner. Addition of dibutyryl cAMP enhanced chondrogenesis in control cells between days 3 and 4, but failed to alter the inhibitory effect of RA on chondrogenesis. The results indicate that while PGE₂ and cAMP are important signals in cartilage differentiation, the inhibitory effects of RA on this process are mediated through some other mechanism.     

Exogenous glycosaminoglycans modulate chondrogenesis, cyclic AMP level and cell growth in limb bud mesenchyme cultures

Effects of hyaluronate, heparin and chondroitin-6-sulfate were studied on micromass cultures of chick limb bud mesenchyme (Hamburger and Hamilton stages 23-24). Histochemical, electron microscopical, biochemical and radiochemical investigations of day 4 cultures revealed dose-dependent inhibitory effects of these glycosaminoglycans on chondrogenesis, cyclic AMP level and growth of cells. In addition, hyaluronate with 100 micrograms/ml dose caused a displacement of newly formed proteoglycan from cultures into the medium. It is supposed that exogenous glycosaminoglycans influence ionic equilibrium in the immediate vicinity of cells and disturb the organization of the prechondrogenic extracellular matrix resulting in alterations of cell membrane--cytoskeleton associations. These alterations may provoke a reduction in cyclic AMP level and DNA synthesis. It is suggested that a reduction in cyclic AMP level preceding the expression of cartilage phenotype results in the inhibition of chondrogenesis.     

Changes in cyclic AMP levels during development in Myxococcus xanthus.

Cyclic AMP levels doubled in Myxococcus xanthus under conditions in which cells aggregate and form fruiting bodies. In liquid medium, glycerol- or dimethyl sulfoxide-induced sporulating cultures exhibited a sharp but transient rise in cyclic AMP concentration after 45 min.     

Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMP1

Effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on chondrogenesis and concentrations of prostaglandin E2 (PGE2) and cyclic AMP (cAMP) were investigated in micromass cultures of chick limb mesenchyme derived from the distal tip of stage 25 limb buds. TPA completely inhibited chondrogenesis during the first 4 days of culture; however, a few small cartilage nodules formed by day 6. Relative to control cultures, both PGE2 and cAMP concentrations were altered by TPA treatment during the 6-day period of cell culture. Concentrations of both compounds increased in control cells during the first 24 h of culture and then declined during the remaining 5 days. In TPA-treated cells both PGE2 and cAMP levels increased progressively during the 6 days of days of cell culture, each being elevated at day 6 by twofold over control cells. The results suggest the presence of regulatory pathways important in chondrogenesis which occur independent of those initiated by PGE2 and the cAMP system.     

Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMP

Effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on chondrogenesis and concentrations of prostaglandin E₂ (PGE₂) and cyclic AMP (cAMP) were investigated in micromass cultures of chick limb mesenchyme derived from the distal tip of stage 25 limb buds. TPA completely inhibited Chondrogenesis during the first 4 days of culture; however, a few small cartilage nodules formed by day 6. Relative to control cultures, both PGE₂ and cAMP concentrations were altered by TPA treatment during the 6-day period of cell culture. Concentrations of both compounds increased in control cells during the first 24 h of culture and then declined during the remaining 5 days. In TPA-treated cells both PGE₂ and cAMP levels increased progressively during the 6 days of cell culture, each being elevated at day 6 by twofold over control cells. The results suggest the presence of regulatory pathways important in Chondrogenesis which occur independent of those initiated by PGE₂ and the cAMP system.     

Increase in cyclic AMP levels by relaxin in newborn rhesus monkey uterus cell culture

Summary A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus (NRMU). NRMU cells respond to relaxin, in the presence of 1 μM forskolin, by producing intracellular adenosine 3′, 5′-cyclic monophosphate (cAMP). The increase in cAMP levels is dose, time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1×10⁵ cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however, 200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones, growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin, oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin E₂ resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying relaxin-mediated biological responsesin vitro as well as functioning as the primary component of a relaxinin vitro bioassay. Editor's statement This paper details a smooth muscle cell line that is responsive to relaxin and provides a useful assay system for the hormone, as well as providing a model system for the study of the mechanisms of relaxin action.     

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

Abstract. We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.     

Chondrogenesis in mouse limb buds in vitro: Effects of dibutyryl cyclic AMP treatment

We studied the effects of dibutyryl cyclic AMP (dbcAMP) on mouse limb-bud chondrogenesis at three stages of embryonic development. After 24 h of culture, limb buds with or without a covering of ectoderm were treated with 1 mM dbcAMP for 48 h and were then compared with untreated cultured limb buds. Treatment with dbcAMP enhanced cartilaginous differentiation in organ cultures of stage-17 and -19 (according to Theiler's) limb buds, although the presence of ectoderm reduced the level of dbcAMP stimulation. By stage 20, treatment with dbcAMP irreversibly inhibited cartilaginous differentiation. These results suggest that the responsiveness of mesenchymal limb-bud cells to dbcAMP is stage related. The results of histological studies as well as of analyses of DNA content and sulphated glycosaminoglycan accumulation supported the hypothesis that dbcAMP treatment induces recruitment of initially non-chondrogenic cells whose commitment explains the enhancement of cartilaginous differentiation. Limb-bud competence for chondrogenesis throughout the three developmental stages studied is also discussed.     

Effect of prostaglandin E2 on cyclic AMP levels in limb cells of mouse mutant brachypodism.

Mouse embryo limb cells carrying either the brachypodism (bpH/bpH) mutation or its wild-type (+/+) allele were tested for their ability to accumulate cyclic AMP in response to prostaglandin E2 (PGE2) between Embryonic Days E12 and E14. Mutant cells exhibited a precocious increase in cyclic AMP. In the absence of PGE2 but in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX), the brachypodism cells accumulated a significantly lower amount of cyclic AMP by Day E14. Limb cells carrying the bpH mutation may provide a useful experimental system to study the PGE2-cyclic AMP-cartilage differentiation interrelationship.     

Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis

When the subridge mesoderm of the embryonic chick limb bud is cultured in the absence of the apical ectodermal ridge and adjacent ectoderm, the cells rapidly progress through the various stages of chondrogenesis. During the first day of culture, the cells initiate condensation, and during subsequent days, deposit a cartilage matrix. In the present study, we show that early in the first day there is a progressive 2-fold increase in cell surface galactosyltransferase activity towards endogenous acceptors. Later in the first day, although the cells are still in condensation, endogenous galactosyltransferase activity has decreased, suggesting in situ galactosylation of surface acceptors. During subsequent development, when cartilage matrix is being deposited, surface galactosyltransferase activity remains low. All controls have been performed to insure cell surface localization of enzyme activity. Two other surface glycosyltransferases show very low levels of activity, which do not change significantly during culture. We suggest that during cellular condensation, an interaction between surface galactosyltransferases and acceptors on adjacent cells occurs, and this interaction may be causally related to subsequent chondrogenic differentiation.     

Inhibition of "spontaneous," notochord-induced, and collagen-induced in vitro somite chondrogenesis by cyclic AMP derivatives and theophylline.

The present study represents a first step in investigating the possible involvement of cyclic AMP in the stimulation of somite chondrogenesis elicited by extracellular matrix components produced by the embryonic notochord. Dibutyryl cyclic AMP (db-cAMP) at 1.0 mM severely impairs “spontaneous” somite chondrogenesis, i.e., inhibits the formation of the small amount of cartilaginous matrix normally formed by embryonic somites in vitro in the absence of inducing tissues. This inhibition of cartilaginous matrix formation is reflected in a 30–40% reduction in sulfated glycosaminoglycan (GAG) accumulation. 8-Bromo-cyclic AMP also severely inhibits cartilage formation and sulfated GAG accumulation by somite explants. This impairment is limited to cyclic AMP derivatives; dibutyryl cyclic GMP, 5′-AMP, and 2′,3′-AMP have no effect. The inhibitory effect of cyclic AMP derivatives is mimicked by the cyclic AMP-phosphodiesterase inhibitor, theophylline, and potentiated by the addition of both db-cAMP and theophylline. Dibutyryl cyclic AMP and/or theophylline also inhibit the stimulation of cartilaginous matrix formation and sulfated GAG accumulation normally elicited by the embryonic notochord, reducing accumulation to a level similar to that found in somite explants without notochord. The inhibition of chondrogenesis by cyclic AMP in notochord-somite explants appears to result from an inability of somites to respond and not from an effect on the inductive capacity of the notochord, since db-cAMP has no detectable effect on the synthesis of molecules (sulfated GAG and collagen) by the notochord that have been implicated in its inductive activity. Finally, db-cAMP and/or theophylline inhibit the stimulation of somite chondrogenesis normally elicited by purified Type I collagen substrates. Dibutyryl cyclic AMP and theophylline reduce sulfated GAG accumulation by somites cultured on collagen to a level even below that accumulated by somites cultured in the absence of collagen, i.e., on Millipore filters.     

Genetic manipulation of cyclic AMP levels in Drosophila melanogaster.

Cyclic AMP levels in $\text{Drosophila}$,$\text{melanogaster}$ adults can be altered genetically by changing the number of doses of chromomere 3D4 contained in the genome, a chromomere previously shown to control the activity of cyclic AMP phosphodiesterase in a dose-dependent manner. Flies completely deficient for chromomere 3D4 have 2–7 times the cyclic AMP level of flies with one or two doses of chromomere 3D4. Cyclic AMP levels are significantly depressed in flies carrying three doses of 3D4. Cyclic GMP levels are not influenced in a dose-dependent manner by chromomere 3D4. The effect on cyclic AMP levels may provide a useful system for investigating physiological and developmental consequences of aberrant cyclic AMP levels in the intact organism.