Chick Skin Fibroblasts Research Articles

TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.

ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

Tracing the pathway between mutation and phenotype in osteogenesis imperfecta: isolation of mineralization-specific genes.

The brittleness of bone in people with lethal (type II) osteogenesis imperfecta, a heritable disorder caused by mutations in the type I collagen genes, arises from the deposition of abnormal collagen in the bone matrix. The inability of the abnormal collagen to participate in mineralization may be caused by its failure to interact with other bone proteins. Here, we have designed a strategy to isolate the genes important for mineralization of collagen during bone formation. Cells isolated from 16-day embryonic chick calvaria and seeded post-confluence in culture deposited a mineralized matrix over a period of 2 weeks. Chick skin fibroblasts seeded and cultured under the same conditions did not mineralize. Using RT-PCR, we prepared short cDNAs (approximately 300 bp) corresponding to the 3' ends of mRNA from fibroblasts and separately from the mineralizing calvarial cells. Subtractive cDNA hybridization generated a pool of cDNAs that were specific to mineralizing calvarial cells but not to fibroblasts. Screening of 100,000 plaques of a chick bone ZAP Express cDNA library with this pool of mineralizing-specific cDNAs identified ten clones which comprised full-length cDNAs for the bone proteins osteopontin (eight of the ten positives), bone sialoprotein II (one of the ten positives), and cystatin (one of the ten positives). cDNAs for type I collagen, fibronectin, alkaline phosphatase, house-keeping genes, and other genes expressed in fibroblasts were not identified in this preliminary screen. The pool of short cDNAs is likely to comprise cDNAs for further bone-specific genes and will be used to screen the entire bone cDNA library of 4.2 million clones.

Ascorbic acid reduces tropoelastin levels in culture medium of chick skin fibroblasts

Ascorbic acid reduced tropoelastin levels in the culture medium of embryonic chick skin fibroblasts to one-third or one-fourth of their original value in a dose- and exposure time-dependent manner under conditions in which collagen synthesis was stimulated 1.8-fold. The steady-state level of elastin mRNA remained essentially constant, whereas the collagen mRNA content increased in proportion to the increase in collagen synthesis with ascorbic acid treatment. These results suggest that the reduction of tropoelastin levels is not controlled by the elastin mRNA level but, rather, is related to the posttranslational modification of the tropoelastin molecule.

Stimulation of elastin expression by minoxidil in chick skin fibroblasts.

Minoxidil inhibited the proliferation of embryonic skin fibroblasts during the growth phase but not during the stationary phase. Minoxidil stimulated elastin synthesis two-fold in a dose-dependent manner at a concentration of 1 mM during the stationary phase. The stimulation of elastin synthesis paralleled a comparable increase in elastin mRNA level. These results suggest that the stimulation of elastin expression by minoxidil in skin fibroblasts was controlled at the elastin mRNA level and also suggest that its elastin-stimulating effect is not related to the suppressive effect on cell proliferation. Minoxidil appears to be a potent stimulator for elastin expression in skin fibroblasts.

Partial characterization of a unique 84-kDa polypeptide stimulated by ascorbic acid in skin fibroblasts.

Ascorbic acid stimulated the synthesis of a noncollagenous 84 kDa polypeptide by up to 3.5-fold in primary cultures of chick skin fibroblasts. Stimulation was observed with concentrations ranging 0.4 to 50 micrograms/ml and was dose-dependent within the concentrations up to 10 micrograms/ml during 24 h treatment under conditions in which procollagen synthesis was increased. This protein was found to be translated with a molecular size of 84 kDa in an in vitro cell-free translation experiment, indicating that the 84 kDa polypeptide was a primary translate. The stimulation was accompanied by a 1.7-fold increase in translational activity of the mRNA for this polypeptide. Its synthesis rapidly reduced when the cells were serially passaged until the third subcultivation, whereas procollagen synthesis remained essentially constant. These results indicate that the stimulation of this unique 84 kDa polypeptide by ascorbic acid was modulated at least in part at a translational level and its synthesis appears to be related to in vitro cellular aging.

Stimuration of elastin expression by minoxidil in chick skin fibroblasts and aortic smooth muscle cells

Stimuration of elastin expression by minoxidil in chick skin fibroblasts and aortic smooth muscle cells

Hyaluronic acid modulates growth, morphology and cytoskeleton in embryonic chick skin fibroblasts.

The action that hyaluronic acid (HA) exerts on cell proliferation was investigated in embryonic chick skin fibroblasts at different ages (7-14 days) and in different cell-cycle stages evaluated by flow cytometry (cells maintained with and without serum). Proliferation was estimated by 3H-thymidine incorporation and cell counting. The results demonstrated hyaluronic acid inhibits cell multiplication in all different environmental conditions examined. The inhibitory effect of HA is more evident in 14-day than 7-day old fibroblasts. The ability of HA to modulate 3H-thymidine incorporation did not involve a change in the time required for cells entering the S phase of the replicating cycle, but is due to a smaller number of cells entering in this phase. As the relationships between components of the extracellular matrix (ECM) and the cytoskeleton are known, parallel studies were carried out on some cytoskeleton proteins. Furthermore, by modifying the capacity of cells to adhere to the substrate, HA induced alterations in cell shape and in cytoskeleton components involved in these processes. We may hypothesize that HA, binding specific membrane receptors, affects cell adhesion and morphology inducing less receptivity of fibroblasts to mitogenic stimuli by transmembrane interactions with cytoskeleton.

Exogenous glycosaminoglycans modulate extracellular and intracellular accumulation of newly synthesized glycosaminoglycans by embryonic chick skin fibroblasts

The effects of exogenous glycosaminoglycans (GAG) on newly synthesized GAG accumulation in intra- and extra-cellular compartments of 17 incubation days chick embryonic skin fibroblasts have been examined. Exogenous GAG are able to act on both total GAG synthesis and secretion by embryonic fibroblasts and on the relative concentration of individual GAG. A decline in hyaluronic acid and an increase in chondroitin sulfate plus dermatan sulfate in the cellular compartment for all GAG administered have been detected. There was also similar behaviour in the extracellular compartment after sulfated GAG administration.

Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro alpha 1(I) and pro alpha 2(I) mRNAs but not beta-actin mRNA. Fibroblasts receiving dexamethasone and [5,6-3H]uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not beta-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, we determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the alpha 2(I) procollagen gene. Nuclear protein blots were probed with the 32P-end-labeled pBR322 vector DNA and 32P-end-labeled alpha 2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the alpha 2(I) procollagen promoter containing DNA were calculated. Three nonhistone DNA-binding proteins of Mr 90,000, 50,000, and 30,000 had altered specificities following dexamethasone treatment.

Glucocorticoids decrease the synthesis of type I procollagen mRNAs

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro.

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.

Modulation of Bleomycin Lung Injury By Short Exposure to Hyperoxia

n e w information about the initiation o f lung fibrosis induced by various fibrogenic agents. Glucocort icoids are used clinically in the t reatment of patients with pulmonary fibrosis. T h e s e corticosteroids have b e e n shown selectively to decrease procollagen synthesis in connect ive tissues and in fibroblasts.6 T h e b leomycin-medi ated increase o f collagen synthesis and collagen accumulation in rat lung is b locked by glucocorticoid t r e a t m e n t . 7 8 To d e t e r m i n e the molecular mechanism by which these drugs regulate collagen synthesis , chick skin and chick lung fibroblasts were treated with b leomycin followed by dexamethasone. This glucocorticoid reversed the b leomycininduced polysomal partit ioning o f type I procollagen m R N A and also reversed the stimulatory effect o f b leomycin on procollagen synthesis. T h e s e data indicate that glucocorticoids regulate col lagen synthesis induced b y b l e o m y c i n in lung and cultured fibroblasts.

Expression of differentiated functions in heterokaryons between skeletal myocytes, adrenal cells, fibroblasts and glial cells

The regulation of both muscle and adrenal functions was examined in heterokaryons formed by fusing differentiated chick skeletal myocytes to Y1 mouse adrenal cells. Mouse fast skeletal myosin light chain one (LC1) synthesis was induced and acetylcholine receptor expression was maintained at muscle control levels. Steroid secretion, although reduced compared with Y1 × Y1 adrenal homokaryon control fusions, was nonetheless maintained at relatively high levels. Steroid secretion in the myocyte × adrenal heterokaryons was constitutively expressed and was not increased by exposure to either adrenocorticotrophic hormone or db-cAMP. The population of heterokaryons was thus simultaneously expressing both muscle and adrenal functions. The steroid secretion in these heterokaryons was compared to that in heterokaryons formed by fusing Y1 adrenal cells to either chick skin fibroblasts or rat C6 glial cells. Both of these sets of heterokaryons exhibited low baseline levels of steroid secretion that were inducible to control values by ACTH. These results extend previous observations showing that heterokaryons are functionally very different than cell hybrids, and exhibit a variety of phenotypic interactions. Although fibroblasts suppress muscle functions in heterokaryons, they are permissive for adrenal functions. C6 glial cells are permissive for both adrenal and muscle functions, and along with several other neurectodermal derivatives contain an inducible skeletal myosin light chain gene. Finally, myocytes and Y1 adrenal cells are mutually permissive for their differentiated functions, and Y1 adrenal cells contain an inducible myosin light chain gene.

Bleomycin treatment of chick fibroblasts causes an increase of polysomal type I procollagen mRNAs. Reversal of the bleomycin effect by dexamethasone.

Bleomycin treatment of primary chick skin fibroblasts and chick lung fibroblasts resulted in a selective dose-dependent increase of cell layer procollagen synthesis. Solid support hybridization of total cellular RNA to 32P-labeled pro-alpha 1(I) and pro-alpha 2(I) cDNAs did not indicate an increase of total cellular procollagen type I mRNAs in bleomycin-treated cells. However, bleomycin treatment of chick skin fibroblasts causes a redistribution of procollagen type I mRNAs within the nuclear, cytoplasmic, and polysomal subcellular fractions. Both the nuclear and cytoplasmic procollagen type I mRNAs are significantly decreased in concentration after bleomycin administration. In contrast, the polysomal procollagen type I mRNAs are significantly increased in both chick skin and lung fibroblasts treated with bleomycin. Administration of dexamethasone to bleomycin-treated fibroblasts resulted in a reversal of the bleomycin-induced increase in cell layer procollagen synthesis. The increased amounts of polysomal procollagen type I mRNAs in bleomycin-treated cells were also reduced by subsequent administration of dexamethasone. These data indicate that bleomycin treatment of chick skin and chick lung fibroblasts results in a specific increase in procollagen synthesis in the cell layer which is mediated by elevated levels of polysomal type I procollagen mRNAs via a repartitioning of these mRNAs within the fibroblast. Furthermore, dexamethasone reverses the bleomycin-induced elevations of both cell layer procollagen synthesis and polysomal type I procollagen mRNAs.

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen.

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast-specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole-like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.

Dexamethasone decreases the amounts of type I procollagen mRNAs in vivo and in fibroblast cell cultures.

Dexamethasone treatment of neonatal chicks resulted in a time- and dose-dependent selective decrease of skin collagen synthesis. Total RNA of chick skin was isolated and hybridized to the cloned cDNAs pCg54 for pro-alpha 1 (I) mRNA and pCg45 for pro-alpha 2(I) mRNA. RNA isolated from the total skin of chicks receiving various doses of dexamethasone had dose-related decreases of pro-alpha 1 (I) and pro-alpha 2(I) mRNAs. The decrease of type I procollagen mRNAs for various doses of dexamethasone were similar to the decreases observed for collagen synthesis in vivo. Dexamethasone treatment of chick skin and chick lung fibroblasts resulted in a selective decrease of procollagen synthesis. A dose-related decrease of procollagen synthesis was observed with chick skin fibroblasts. Dexamethasone-treated chick skin and chick lung fibroblasts had decreased levels of pro-alpha 1 (I) and pro-alpha 2(I) mRNAs as determined by solid support hybridization with pCg54 and pCg45. The dexamethasone-mediated decreases of type I procollagen mRNAs in skin fibroblasts and lung fibroblasts were similar to the decreases observed in procollagen synthesis.

Transferrin Receptor on Chick Fibroblast Cell Surface and the Binding Affinity in Relevance to the Growth Promoting Activity of Transferrin: (transferrin/receptor/cultured fibroblast/molecular recognition/class dependent specificity).

We examined the transferrin (Tf) receptor of chick skin fibroblasts using chick 125 I-Tf. When the cells were incubated with 125 I-Tf on ice, most of the cell-associated 125 I-Tf was found on the cell surface; on the other hand, a large part of it was located inside the cells when incubated at 37°C. By equilibrium binding assay, the number of Tf receptors per cell was determined as 6.7 × 103 . Dissociation constant was estimated to be 2.6 × 10-8 M. The binding of 125 I-Tf was competitively inhibited by the addition of chick unlabeled Tf. Weaker inhibition was observed when bovine Tf was used as a competitor. Horse Tf had no effect on the binding of chick Tf. This agrees well qualitatively with chick cell growth-promoting activites of these Tfs. Removal of Fe from Tf affected the affinity for its receptors. About 5- to 10-fold higher concentrations of chick apo-Tf was needed to achieve the same degree of inhibition of 125 I-Tf binding as that made by chick Fe-Tf.

The effects of antimicrotubule agents on cell motility in fibroblast aggregates

Spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the two aggregates cohered and broadened the area of contact to form a single aggregate with a planar interface between the labelled and unlabelled halves. The motility of cells in the aggregate was determined by measuring the movement of labelled cells across the boundary into the unlabelled half. Exposure to pharmacological doses of antimicrotubule agents resulted in a significant reduction in fibroblast motility in the three-dimensional aggregates.

Cell motility in fibroblast aggregates.

The stable arrangements of cells in tissues and organs require that most tissue cells, whatever their inherent abilities for active pseudopod-directed locomotion, remain confined to the boundaries of their parent tissues and organs for the lifetime of the organism. Contact inhibition of motility represents a potential mechanism for immobilizing cells in tissues and thus in promoting positional stability. In order to assess the effectiveness of contact inhibition in immobilizing cells in 3-dimensional tissue-like masses, spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the 2 aggregates cohere and broaden the area of mutual contact to form a single aggregate with a planar interface between the two. Mobility of cells was detected by the migration of labelled cells across the interface and into the unlabelled aggregate. Based on measurements of distances moved in day-old aggregate pairs, it was found that fibroblasts moved up to 17 cell diameters and up to 36% as far in solid tissue masses as in monolayer culture under optimal conditions for directed migration.

The actin content of fibroblasts.

Cultures of chick skin fibroblasts were dissolved in solutions of sodium dodecyl sulphate, and their entire protein content was examined by gel electrophoresis. The most abundant species migrated in the same position as muscle actin. It gave a similar pattern of iodinated peptides after reaction with radioactive sodium iodide and digestion with proteinases, and contained comparable amounts of Nt-methylhistidine. Its amount was estimated by quantitative densitometry of stained gels with bovine serum albumin as an internal standard, and by radioactive assay of cultures that had been grown in the presence of [35S]methionine. The values obtained ranged from 7 to 14% of the total cellular protein, with an average of 8.5%. A protein band in the position of muscle myosin was also present and accounted for about 2.5% of the total protein. Both this and the actin band increased in relative amount with the age of the cultures.