Effect of cyclic AMP on the intracellular degradation of newly synthesized collagen

Prostaglandin E1 and cholera toxin increased the intracellular levels of cyclic AMP of human lung fibroblasts. With prostaglandin E1, the increase in cyclic AMP occurred within 10 min followed by a decline to less than one-half of peak values in 6 h. With cholera toxin, the increase occurred within 60 min but the level of cyclic AMP remained increased for 6 h. Both agents caused a decrease in collagen production as expressed as the proportion of newly synthesized protein represented by collagen. The increase in cyclic AMP levels was accompanied by a marked increase in the proportion of newly synthesized collagen which was degraded intracellularly prior to secretion. Analysis of the degraded collagen showed it to be predominantly less than 1000 daltons in molecular mass, but still in peptide linkage. The data are consistent with the hypothesis that cyclic AMP levels in diploid fibroblasts regulate the amount of collagen produced by fibroblasts, at least in part, by modulating the level of intracellular collagen degradation.     

Modulation of collagen production by fibroblasts. Effects of chronic exposure to agonists that increase intracellular cyclic AMP.

Cultured human lung fibroblasts were evaluated for their responsiveness to isoprenaline (isoproterenol) or prostaglandin E2 before and after chronic incubation with the agonist. Cells incubated for 6 h with either agonist were suppressed in terms of collagen production and exhibited increased intracellular cyclic AMP. Cells incubated for 72 h with the agonist and then re-challenged for 6 h with the same agonist did not demonstrate suppressed collagen production or increased cyclic AMP. Cells incubated for 72 h with isoprenaline still responded to prostaglandin E2 when challenged for 6 h; however, when the order of agonist exposure was reversed, cells incubated with prostaglandin E2 did not respond to a challenge by isoprenaline. If cells were allowed to recover for 48 h without the agonist after a 72 h chronic incubation, they recovered their responsiveness to the agonist. The results indicate that, although cultured fibroblasts may become desensitized to one agonist, they may retain their sensitivity to a second agonist and chronic suppression of collagen production may be achieved by alternate exposure to isoprenaline and prostaglandin E2.     

Cyclic AMP-induced inhibition of collagen lattice contraction by fibroblasts may be attenuated by both cyclic AMP dependent and independent mechanisms

The contraction of collagen lattices made with forskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracelluar cyclic AMP raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-β (TGF-β). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-β, but not by aFGF and insulin, when added simultaneously. Thus, TGF-β may attenuate CT's inhibition on collagen lattice contraction by attenuating CTinduced intracellualr cyclic AMP increse, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMPindependent mechannism. © 1993 Wiley-Liss, Inc.     

Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.     

Modulation of human dermal fibroblast extracellular matrix metabolism by the lymphokine leukoregulin

The effect of leukoregulin, a 50-kD lymphokine with unique antitumor properties, was studied in vitro on several fibroblast functions. Leukoregulin did not inhibit fibroblast proliferation, as measured by cell enumeration and [3H]thymidine incorporation, and had no cytotoxic effect in terms of increased membrane permeability detected by trypan blue exclusion, two of the major leukoregulin actions on tumor cells. Leukoregulin induced a dose-dependent decrease in collagen synthesis, demonstrated by decreased [3H]proline incorporation into collagenase-digestible protein, as early as 6 h after the addition of the lymphokine to human fibroblasts. Leukoregulin inhibited the synthesis of both type I and type III collagen, as measured by SDS-PAGE and by specific radioimmunoassay. Neutralizing antibodies to interleukin-1 alpha, interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma failed to alter the effect of leukoregulin on collagen synthesis, attesting that leukoregulin action was not due to contamination by these cytokines. Inhibition of collagen synthesis occurred concomitantly with increased secretion of prostaglandin E2 and a transient rise in intracellular cyclic AMP content, peaking at 6 h. However, blocking prostaglandin synthesis with indomethacin did not counteract inhibition of collagen synthesis by leukoregulin, demonstrating independence of this action of leukoregulin from cyclooxygenase metabolites. Leukoregulin also stimulated glycosaminoglycan production in a dose-dependent manner, affecting the synthesis of hyaluronic acid as the major fibroblast-derived extracellular glycosaminoglycan. In addition, secretion of neutral proteases (collagenase, elastase, caseinase) was increased. These observations indicate that leukoregulin is able to regulate synthesis of molecules critical to the deposition of the extracellular matrix by nontransformed nonmalignant fibroblasts.     

Synthesis of mucous glycoproteins by rabbit tracheal cells in vitro. Modulation by substratum, retinoids and cyclic AMP.

One function of airway epithelium is the secretion of mucins, which comprise an important component of the mucous lining layer. We demonstrate that rabbit tracheal epithelial cells grown in primary culture incorporate [3H]glucosamine into material released into the medium which is characterized as mucin by the following criteria: high Mr, monosaccharide composition, ion-exchange behaviour different from that of glycosaminoglycans and oligosaccharides attached via N-acetylgalactosamine. The production of mucin by the cells requires growth on a substratum of collagen gel and is enhanced by retinoids in the extracellular medium. In the presence of retinoids, 8-bromo cyclic AMP and factors present in medium from 3T3 fibroblasts each further stimulate mucin production. These results indicate that an isolated epithelial-cell culture system, in the absence of nervous, mesenchymal or other tissue types, can be used to answer questions about the regulation of mucin production at the cellular level.     

Export of cyclic AMP by mammalian reticulocytes

Suspensions of reticulocyte-enriched red cells produce and extrude cyclic AMP in proportion to their reticulocyte content. When reticulocytes are treated with the beta-adrenergic agonist isoproterenol, up to 3.5 pmoles of cyclic AMP/min/mg protein appear in the extracellular medium. Extruded cyclic AMP can occur against apparent concentration gradients and is inhibited by agents (iodoacetate, dinitrophenol) that deplete cellular ATP, as well as by probenecid and prostaglandin A1. Extrusion of cyclic AMP depends on the availability of intracellular cyclic AMP but is not obligatorily coupled to adenylate cyclase activity: extrusion continues following termination of a pulse of stimulation and exhibits a temperature dependence that differs from that for cyclic AMP production in response to isoproterenol. Erythropoietin affects neither production nor extrusion of cyclic AMP by reticulocytes. In whole blood, cyclic AMP extruded by reticulocytes may be a significant source of plasma cyclic nucleotide.     

Effects of adenosine 3'':5''-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts.

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.     

Pattern of collagen synthesis and chemotactic response of fibroblasts derived from mucopolysaccharidosis patients

For comparative studies on the migratory potential we screened fibroblast strains derived from mucopolysaccharidosis (MPS) patients regarding their differential response to chemotactic stimuli and analysed their production of extracellular matrix components. Indirect immunofluorescence staining of MPS-fibroblasts showed the same distribution of type I and type III collagen and of fibronectin as in controls. Biochemical quantification of type I and type III collagen demonstrated an unaltered ratio of these collagen types, although the total amount of newly synthesized collagens was slightly reduced in fibroblasts from MPS patients. Whereas the synthesis of major extracellular matrix components was close to normal, the response of the MPS cells to chemotactic stimuli was greatly affected. Chemotactic migration was improved when fibroblasts were pretreated with medium conditioned by normal fibroblasts, although they never reached normal levels.     

Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts.

It has been previously shown that transforming growth factor beta (TGF beta) is capable of stimulating fibroblast collagen and fibronectin biosynthesis. The purpose of this study was to examine the mechanisms involved in TGF beta stimulation of fibroblast biosynthetic activity. Our results indicate that TGF beta causes a marked enhancement of the production of types I and III collagens and fibronectin by cultured normal human dermal fibroblasts. The rate of collagen production by fibroblasts exposed to TGF beta was 2-3-fold greater than that of control cells. These effects were associated with a 2-3-fold increase in the steady-state amounts of types I and III collagen mRNAs and a 5-8-fold increase in the amounts of fibronectin mRNAs as determined by dot-blot hybridization with specific cloned cDNA probes. In addition, the increased production of collagen and fibronectin and the increased amounts of their corresponding mRNAs remained elevated for at least 72 h after removal of TGF beta. These findings suggest that TGF beta may play a major role in the normal regulation of extracellular matrix production in vivo and may contribute to the development of pathological states of fibrosis.     

Modulation of heart fibroblast migration and collagen gel contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor-beta, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

Role of Extracellular Adenosine in Ethanol-Induced Desensitization of Cyclic AMP Production

Abstract: The decrease in receptor-stimulated cyclic AMP production after chronic ethanol exposure was suggested previously to be secondary to an ethanol-induced increase in extracellular adenosine. The present study was undertaken to ascertain whether a similar mechanism was responsible for the ethanol-induced desensitization of cyclic AMP production in PC12 pheochromocytoma cells. The acute addition of ethanol in vitro significantly increased both basal cyclic AMP content and extracellular levels of adenosine. A 4-day exposure to ethanol decreased basal as well as 2-chloroadenosine- and forskolin-stimulated cyclic AMP contents. No change in cyclic AMP content was observed after a 2-day exposure of PC12 cells to ethanol. Inclusion of adenosine deaminase during the chronic ethanol treatment significantly decreased extracellular levels of adenosine, yet the percentage decrease in 2-chloroadenosine- and forskolin-stimulated cyclic AMP levels after chronic ethanol exposure was not changed by the inclusion of the adenosine deaminase. Similar results were obtained when the chronic treatment was carried out with serum-free defined media. The ethanol-induced desensitization could not be mimicked by chronic exposure of PC12 cells to adenosine analogues. A 24-h exposure of PC12 cells to 2-chloroadenosine resulted in a decrease in the subsequent ability of this adenosine analogue to stimulate cyclic AMP content, but basal and forskolin-stimulated cyclic AMP levels were increased. Similar results were obtained after a 4-day exposure of PC12 cells to 2-chloroadenosine or 5'- N -ethylcarboxamido-adenosine. The present results indicate that the ethanol-induced decrease in receptor-stimulated cyclic AMP content in PC12 cells is not due to an increase in extracellular adenosine.     

Modulation of Heart Fibroblast Migration and Collagen Gel Contraction by IGF-I

Dynamic interactions between cells and the extracellular matrix are essential in the regulation of a number of cellular processes including migration, adhesion, proliferation and differentiation. A variety of factors have been identified which modulate these interactions including transforming growth factor+, platelet-derived growth factor and others. Insulin-like growth factors have been shown to regulate collagen production by heart fibroblasts; however, the effects of this growth factor on the interactions of heart fibroblasts with the extracellular matrix have not been examined. The present studies were carried out to determine the effects of IGF-I on the ability of fibroblasts to interact with the extracellular matrix and to begin to determine the mechanisms of this response. These experiments illustrate that IGF-I treatment results in increased migration, collagen reorganization and gel contraction by heart fibroblasts. IGF-I has been shown to activate both the mitogen-activated protein kinase and phophatidylinositol-3 kinase pathways in isolated cells. Experiments with pharmacological antagonists of these pathways indicate that the mitogen-activated protein kinase pathway is essential for IGF-I stimulated collagen gel contraction by fibroblasts. These studies illustrate that IGF-I modulates the ability of fibroblasts to interact with the collagen matrix and that activation of multiple signaling pathways by IGF-I may produce distinct downstream responses in these cells.     

Hyperoxic-induced alterations in lung cell structure and function. Effects on cellular cyclic AMP content and comparison to alterations produced by exogenous cyclic AMP

Hyperoxic exposure in vitro of two lung-derived cell types (the epithelial-derived L2 cells and WI-38 fibroblasts) inhibits cellular replication, produces striking morphologic changes and may result in cell death; these effects have been observed consistently in other cell types. Hyperoxic exposure of L2 cells is associated with an increase in cellular cyclic AMP content (cellular cyclic AMP content 454 +/- 115 fmol/micrograms DNA in cells exposed to pO2 677 Torr for 96 h compared to 136 +/- 17 fmol/microgram DNA in air-grown cells). Hyperoxic exposure of WI-38 fibroblasts is not associated with increased cyclic AMP content. Although cultivation of L2 cells in the presence of exogenous dibutyryl cyclic AMP does inhibit replication and produce morphologic alterations, similar effects are produced by sodium butyrate alone. Hyperoxic exposure alters cyclic AMP metabolism in some cell types, but the structural and functional alterations observed in L2 cells and WI-38 fibroblasts following hyperoxic exposure are not produced by changes in cellular cyclic AMP content.     

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa of the adrenal cortex.

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa (the capsular fraction) from the rat adrenal cortex have been studied. Although high concentrations of extracellular calcium potentiated the stimulatory action of ACTH on cyclic AMP and aldosterone output, tetracaine or verapamil inhibited aldosterone output but not cyclic AMP production during ACTH-stimulation. Lanthanum reduced both aldosterone and cyclic AMP accumulation induced by ACTH. These results suggest that an extracellular calcium would be essential in stimulating the capsular steroidogenesis without involvement of the cyclic AMP system.     

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.     

Possible involvement of Ca2+-calmodulin system in cyclic AMP action in cholesterol ester hydrolytic response to ACTH in bovine adrenocortical cells

In order to elucidate the relationship between cyclic AMP and the Ca2+-calmodulin system in the steroidogenic response to adrenocorticotropic hormone (ACTH), the effects of calmodulin inhibitors (trifluoperazine and W-7) on cortisol production and cellular cholesterol ester hydrolysis induced by ACTH or dibutyryl cyclic AMP in bovine adrenocortical cells were examined in the absence of extracellular Ca2+. These calmodulin inhibitors inhibited not only the cortisol production and the cholesterol ester hydrolysis induced by ACTH in the absence of extracellular Ca2+, but also inhibited the dibutyryl cyclic AMP-induced cortisol production and the cholesterol ester hydrolysis in the absence of extracellular Ca2+. These results suggested the possibility that cyclic AMP action was mediated by the Ca2+-calmodulin system in the activation process of cellular cholesterol ester hydrolysis in the steroidogenic response to ACTH.     

Multiple Signalling Pathways Interact in the Regulation of Nerve Growth Factor Production in L929 Fibroblasts

Fibroblasts are one of several cell types producing nerve growth factor (NGF) in neuronal targets. In previous studies we found that NGF production is up-regulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) and serum, down-regulated by corticosterone, and unaffected by dibutyryl-cyclic AMP (db-cyclic AMP) in fibroblasts. As fibroblasts in vivo are likely to be exposed to regulatory effects by more than one of these agents at any given time, we examined the effects of combinations of them on NGF production using L929 fibroblasts as a model system. TPA and serum together stimulated NGF production 10-fold more than either agent alone. Corticosterone reduced NGF mRNA and NGF production to less than 10% of basal levels whether or not TPA or serum, or both, were present but not in the presence of the glucocorticoid antagonist RU486. Corticosterone did not increase the rate of NGF mRNA degradation. Forskolin and db-cyclic AMP prevented NGF mRNA induction by TPA and serum without changing basal levels. TPA induced c-fos and junB mRNAs transiently and preceding NGF mRNA induction but c-jun mRNA remained undetectable. Forskolin enhanced the induction of both junB and c-fos mRNA whereas corticosterone prolonged junB mRNA induction. Thus, TPA induction of NGF mRNA is modulated differentially by corticosterone and cyclic AMP. c-fos and junB may play a role in the underlying mechanisms.