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Regulation of extracellular matrix metabolism by growth factors in human skin fibroblasts
388
389
BASEMENT MEMBRANEBIOSYNTHESIS IN DEVELOPMENT. J.H. Fessler, A.G. Campbell, T. Strecker, C. Buzin, K. Garrison, R. Sterne and L . I . Fessler, Molecular Biology Institute, UCLA, Los Angeles, CA 90024 Cross-adsorbed antibodies were made against purified Drosophila basement membrane collagen, laminin, proteoglycan and an entactin-like molecule. Antigenic determinants for all these reagents appeared at approximately the same time, during embryonic organogenesis, and colocated by immunofluorescence to basement membranes. When cel] dispersions of early, undifferentiated embryos were cultured, some individual cells stained with these antibodies at equivalent times to those in whole embryos. These cell cultures also incorporated [35S]-methionine into a collagenase-sensitive polypeptide of the size of the Drosophila collagen antigen. We conclude that Drosophila embryos coordinately regulate the start of synthesis of the components of basement membranes that encompass whole groups of cells in an organ, yet that the clock which determines this continues to run in individual cells, disrupted from their normal cell neighbors.
AUTORADIOGRAPHIC STUDY OF THE ORIGIN OF BASEMENT MEMBRANE (BM) COMPONENTS IN THE CHICKEN EMBRYO. F. Harrisson. Department of Anatomy and Embryology, University of Antwerp, Belgium. The contribution of hypoblast to the assembly of the BM of the epiblast was investigated in chimaeric blastoderms resulting from the transplantation of quail hypoblast labeled for 2 hr with 3H-glucosamine or with 3H-fucose, into chicken embryos deprived
of their own
hypoblast. After culture for 5 hr, the autoradiograms obtained after fixation and routine processing did not only show silver grains over the graft, but also at the basal side of the host epiblast, where a BM is known to be present. Chase experiments revealed that unprocessed, tritiated molecules are not transferred. Enzyme treatment with hyaluronidases or with chondroitinase ABC prior to dipping of sections of chimaerae labeled with 3H-glucosamine did not influence the final autoradiographic labeling of the BM. The present results suggest that the assembly of the BM involves, at least, the transfer of glycoproteins from the underlying tissue to the epiblast, and emphasizes on its dual origin.
890
391
REGULATION OF EXTRACELLULAR MATRIX METABOLISM BY GROWTH FACTORS IN HUMAN SKIN FIBROBLASTS. R. HATA, K. ARAI, H. SUNADA and Y. NAGAI. Dept. Tissue Physiol. , Med. Res. Inst., Tokyo Med. & Dent. Univ., Tokyo i01, Japan. To investigate the regulation mechanisms of extracellular matrix metabolism in human skin fibroblasts, the celis were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as standard conditions. Presence of epidermal growth factor(EGF,2-5Ong/ml) or sodium ascorbate (O. 05-1mM ) in the culture medium for 4-5 days stimulated growth and synthesis of non-collagenous proteins of the cells. And these factors acted synergistically when both factors were present in the medium. Collagen synthesis of the cells was also activated by the presence of ascorbate, while it was reduced by the presence of EGF. On the other hand, production of acidic glyeosaminoglycan was activated only by the presence of EGF. These results show that both growth factors regulate extracellular matrix metabolism of human skin fibroblasts quite differently.
EVIDENCE OF GLYCOSAMINOGLYCANS(GAG) ROLEIN AVZANLUNGMORPHOGENESIS IN VITRO. P.Carinci,E.Becchetti,G.Stabellini,R. Evangeiisti and A.Pagliarini. Institute of Histology and general Embryology,University oF Ferrara, Via Fossato di Mortara 64, 44100 Ferrara, Italy. 8-7 day chick embryo lung explants were maintained for 48 hours at 37% according to method oF WolFF and i~aFFen (1962) in Gibco medium 199 alone or added with a) exogenousGAG (hyaluronic acid,HA; chondroitin 4-6 sulphate+dermatan sulphate , CS+OS; HA+CS+OS,at the 142 pg/ml concentration); b) chondroitinase ABC, 0.32 U/ml; c) testicular hyaluronidase, 285 Ng/ml, Cultures were then removed,photographedunder stereomicroscope,Fixed in Bouin Fluid and serial sections were cut at 4-7p and stained £or morphological and histochemical examination. Morphogenesiswas evaluated by counting the 2nd and 3rd subdivisions of bronchial branching. CULTURES 2na+3rdSUBDIVISIONS TREATMENI NUMBER /CULTURES 199 (controls)
27
P
16.5 ± 1.g
HA
12
14.2 C 1.0
> 0.01
CS+DS
12
18.2 ± 1.8
> 0.02
CS.JIS+HA
12
18.1 ~ 1.8
)
Chondroitlnase ABC 16
12.5 ! 2.1
> 0.001
Hyaluronidase
12.6 - 1.5
0.05
+
8
> 0.001
Morphogenesis is significantlyalteredFollowingthe indu-
ced change oF mesenchymal GAG pattern (marked decreasedoF sulphated GAG when bronchial branching is reduced).
160S
389
BASEMENT MEMBRANEBIOSYNTHESIS IN DEVELOPMENT. J.H. Fessler, A.G. Campbell, T. Strecker, C. Buzin, K. Garrison, R. Sterne and L . I . Fessler, Molecular Biology Institute, UCLA, Los Angeles, CA 90024 Cross-adsorbed antibodies were made against purified Drosophila basement membrane collagen, laminin, proteoglycan and an entactin-like molecule. Antigenic determinants for all these reagents appeared at approximately the same time, during embryonic organogenesis, and colocated by immunofluorescence to basement membranes. When cel] dispersions of early, undifferentiated embryos were cultured, some individual cells stained with these antibodies at equivalent times to those in whole embryos. These cell cultures also incorporated [35S]-methionine into a collagenase-sensitive polypeptide of the size of the Drosophila collagen antigen. We conclude that Drosophila embryos coordinately regulate the start of synthesis of the components of basement membranes that encompass whole groups of cells in an organ, yet that the clock which determines this continues to run in individual cells, disrupted from their normal cell neighbors.
AUTORADIOGRAPHIC STUDY OF THE ORIGIN OF BASEMENT MEMBRANE (BM) COMPONENTS IN THE CHICKEN EMBRYO. F. Harrisson. Department of Anatomy and Embryology, University of Antwerp, Belgium. The contribution of hypoblast to the assembly of the BM of the epiblast was investigated in chimaeric blastoderms resulting from the transplantation of quail hypoblast labeled for 2 hr with 3H-glucosamine or with 3H-fucose, into chicken embryos deprived
of their own
hypoblast. After culture for 5 hr, the autoradiograms obtained after fixation and routine processing did not only show silver grains over the graft, but also at the basal side of the host epiblast, where a BM is known to be present. Chase experiments revealed that unprocessed, tritiated molecules are not transferred. Enzyme treatment with hyaluronidases or with chondroitinase ABC prior to dipping of sections of chimaerae labeled with 3H-glucosamine did not influence the final autoradiographic labeling of the BM. The present results suggest that the assembly of the BM involves, at least, the transfer of glycoproteins from the underlying tissue to the epiblast, and emphasizes on its dual origin.
890
391
REGULATION OF EXTRACELLULAR MATRIX METABOLISM BY GROWTH FACTORS IN HUMAN SKIN FIBROBLASTS. R. HATA, K. ARAI, H. SUNADA and Y. NAGAI. Dept. Tissue Physiol. , Med. Res. Inst., Tokyo Med. & Dent. Univ., Tokyo i01, Japan. To investigate the regulation mechanisms of extracellular matrix metabolism in human skin fibroblasts, the celis were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as standard conditions. Presence of epidermal growth factor(EGF,2-5Ong/ml) or sodium ascorbate (O. 05-1mM ) in the culture medium for 4-5 days stimulated growth and synthesis of non-collagenous proteins of the cells. And these factors acted synergistically when both factors were present in the medium. Collagen synthesis of the cells was also activated by the presence of ascorbate, while it was reduced by the presence of EGF. On the other hand, production of acidic glyeosaminoglycan was activated only by the presence of EGF. These results show that both growth factors regulate extracellular matrix metabolism of human skin fibroblasts quite differently.
EVIDENCE OF GLYCOSAMINOGLYCANS(GAG) ROLEIN AVZANLUNGMORPHOGENESIS IN VITRO. P.Carinci,E.Becchetti,G.Stabellini,R. Evangeiisti and A.Pagliarini. Institute of Histology and general Embryology,University oF Ferrara, Via Fossato di Mortara 64, 44100 Ferrara, Italy. 8-7 day chick embryo lung explants were maintained for 48 hours at 37% according to method oF WolFF and i~aFFen (1962) in Gibco medium 199 alone or added with a) exogenousGAG (hyaluronic acid,HA; chondroitin 4-6 sulphate+dermatan sulphate , CS+OS; HA+CS+OS,at the 142 pg/ml concentration); b) chondroitinase ABC, 0.32 U/ml; c) testicular hyaluronidase, 285 Ng/ml, Cultures were then removed,photographedunder stereomicroscope,Fixed in Bouin Fluid and serial sections were cut at 4-7p and stained £or morphological and histochemical examination. Morphogenesiswas evaluated by counting the 2nd and 3rd subdivisions of bronchial branching. CULTURES 2na+3rdSUBDIVISIONS TREATMENI NUMBER /CULTURES 199 (controls)
27
P
16.5 ± 1.g
HA
12
14.2 C 1.0
> 0.01
CS+DS
12
18.2 ± 1.8
> 0.02
CS.JIS+HA
12
18.1 ~ 1.8
)
Chondroitlnase ABC 16
12.5 ! 2.1
> 0.001
Hyaluronidase
12.6 - 1.5
0.05
+
8
> 0.001
Morphogenesis is significantlyalteredFollowingthe indu-
ced change oF mesenchymal GAG pattern (marked decreasedoF sulphated GAG when bronchial branching is reduced).
160S