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Extracellular matrix synthesis during mesenchymal cell condensation and chondrogenesis
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GENE STRUCTURE FOR A NOVELHUMANLOW MOLECULAR WEIGHT COLLAGEN. L. Tikka a,b, T. Plhlajan/emi c, D.J. Prockop b and K.Tryggvason a. Departmentsof aBiochemistry and CMedical Biochemistry, University of Oulu, Oulu, Finland, bjefferson Institute of Molecular Medicine, Thomas Jefferson
CELLULAR FORM OF FIBRONECTIN IN DEVELOPING AND REACTIVE TISSUES. T. Vartio, L. Laitin e n , O . N~rv~nen, L-E. Thornell and I. Virtanen. University of Helsinki, Helsinki, Finland, and University of UmeA, Ume~, Sweden. A monoclonal mouse hybridoma antibody 52 DH] (DH) which reacted exclusively with the cellular but not with the plasma fibronectin was characterized and used to study the expression of the cellular form of the protein (cFn). CFn had similar distribution in the periceIIular matrix of embryonic and adult fibroblasts and it could also be revealed in a variety of other cultured normal and malignant human cells. In embryonic tissues cFn was abundant in developing basement membranes. In adult tissues it was confined only to endothelia of large blood vessels and was absent in basement membranes. The protein was detected also in glomerular mesangium in both embryonic and adult kidneys. CFn was prominent in the reactive stroma of malignant tumors as well as in activated endometrial stromal tissue. The results suggest that cFn is the form of the protein which may have a particular role in developing and reactive tissues in embryos and adults.
University, Philadelphia, USA Overlapping cDNA-clones coding for a part of the conagenousdomain, a short noncollagenousC-terminal domain and the 3'.untranslated region for a new type of human collagen have recently been characterized (T. Pihlajaniemi et aL , Proc. Natl. Acad. Sci, USA, in press). One of the clones has a deletion of 36 bp in the region of overlap without disturbing the continuous collagenous sequence. By St-nuclease protection experiments the clones were shown to originate from two different mRNA:s suggesting the possibility of alternative splicing ot the primary transcript. We have isolated genomic clones for the respective gene and sequenced the 8 most 3' exons.One of the exonsencodes the 36bp deleted from one of the cDNA:s, which demonstrates that the deletion can result from alternative splicing of this exon, an event that has not previously been reported for collagen genes. The size pattern of sequenced exons (27-, 81-, 36-, 54-, 54-, 36-, 87-, 39-bp + 3' -untranslated
region ) differs from that of other collagen genes except for the two 54 bp exons typically found in fibrillar collagen genes. Each of the exons studied begins with an intact codon for glycine. The 8 exons sequenced so far have 414 bp of coding sequences which are spread over about 20 kb in the gene. Since these sequences (including3'-untranslatedregion) cover only about 24% of the 3 kb long mRNA, it is likely that this new type of low molecular weight collagen is encoded by a very large gene. 79
E x t r a c e l l u l a r m a t r i x s y n t h e s i s d u r i n g mesenchymal c e l l condensation and chondrogenesis. N.S. Vasan and H. Bost, D e p t . o f Anatomy, New Jersey Medical School, Newark, NJ., USA. Changes in t h e c o m p o s i t i o n o f ECM occur d u r i n g limb mesenchymal c e l l ~ i g r a t i o n , cond e n s a t i o n and d i f f e r e n t i a t i o n . Our s t u d i e s show t h a t in c h i c k embryos between stages 17-21, the c e l l s m i g r a t e through a m a t r i x r i c h in HA. During prechondrogenic condensat i o n ( S t a g e s 22-24) t h e r e is an increase in the HS l e v e l in ECM; the HA l e v e l d e c l i n e s due t o an i n c r e a s e in h y a l u r o n i d a s e degradation. Labelling s t u d i e s i n d i c a t e t h a t the changes in the ECM are due t o the a l t e r ations in the m e t a b o l i c machinery o f the c e l l s . Subsequent t o c o n d e n s a t i o n ( s t a g e 2 5 ) , the c e n t r a l core c e l l s begin t o s y n t h e s i z e a m a t r i x r i c h in c h o n d r o i t i n s u l f a t e p r o t e o glycans (CS-PG). When mesenchymal c e l l s from the core r e g i o n were p l a t e d in v i t r o , cells t h a t formed an aggregate in ~ h accumulated a HS r i c h m a t r i x , and by day 3 began to synt h e s i z e CS-PG. A m a j o r p r o p o r t i o n o f CS-PG monomers were a b l e t o i n t e r a c t w i t h exogenous HA. The r e s u l t s suggest t h a t p r e c h o n d r o qenic condensation o f mesenchymal c e l l s is critical in limb chondrogenesis, and a m i c r o e n v i r o n m e n t r i c h in HS f a c i l i t a t e s this process. ( Supported by a g r a n t from NIH HD 16147 and Foundation o f UMDNJ).
DIFFERENTIAL EXPRESSION OF mRNA'S FOR TYPES I, II AND III COLLAGENS IN DEVELOPING CALLUS TISSUE IN RATS. E. Vuorio~ P. Multim~ki~ M.Sandberg~ H.Aro and H. Aho. Departments of Medical Chemistry and Pathology, Univ. of Turku, SF-20520 Turku, Finland. An experimental fracture healing model in the rat tibio-fibular bone was employed to study the appearance of mRNAs for types I, if and III collagens during endochondral fracture repair. Total RNA was extracted from normal bone and from callus tissue 5, 7, i0, 14 and 28 days after the fracture. Iissue samples were taken at the same time for histologial staining and for in situ nybridization. The results show that during the first week of fracture repair type III collagen ' mRNA is increased rapidly, followed by cartilage-specific II collagen mRNA during the second week. The 28-day callus resembles bone by containing mainly type I collagen mRNAs and very little type II or iIl collagen mRNA. In situ hybridization of callus sections localized type II collagen in cells which were surrounded by cartilage matrix exhibiting alcianophilia at pH 2.5 as well as at pH 0.5, but reduced or abolished alcianophilia after hyaluronidase treatment. Osteoblasts producing actively type I collagen were identified similarily. 26S
78
GENE STRUCTURE FOR A NOVELHUMANLOW MOLECULAR WEIGHT COLLAGEN. L. Tikka a,b, T. Plhlajan/emi c, D.J. Prockop b and K.Tryggvason a. Departmentsof aBiochemistry and CMedical Biochemistry, University of Oulu, Oulu, Finland, bjefferson Institute of Molecular Medicine, Thomas Jefferson
CELLULAR FORM OF FIBRONECTIN IN DEVELOPING AND REACTIVE TISSUES. T. Vartio, L. Laitin e n , O . N~rv~nen, L-E. Thornell and I. Virtanen. University of Helsinki, Helsinki, Finland, and University of UmeA, Ume~, Sweden. A monoclonal mouse hybridoma antibody 52 DH] (DH) which reacted exclusively with the cellular but not with the plasma fibronectin was characterized and used to study the expression of the cellular form of the protein (cFn). CFn had similar distribution in the periceIIular matrix of embryonic and adult fibroblasts and it could also be revealed in a variety of other cultured normal and malignant human cells. In embryonic tissues cFn was abundant in developing basement membranes. In adult tissues it was confined only to endothelia of large blood vessels and was absent in basement membranes. The protein was detected also in glomerular mesangium in both embryonic and adult kidneys. CFn was prominent in the reactive stroma of malignant tumors as well as in activated endometrial stromal tissue. The results suggest that cFn is the form of the protein which may have a particular role in developing and reactive tissues in embryos and adults.
University, Philadelphia, USA Overlapping cDNA-clones coding for a part of the conagenousdomain, a short noncollagenousC-terminal domain and the 3'.untranslated region for a new type of human collagen have recently been characterized (T. Pihlajaniemi et aL , Proc. Natl. Acad. Sci, USA, in press). One of the clones has a deletion of 36 bp in the region of overlap without disturbing the continuous collagenous sequence. By St-nuclease protection experiments the clones were shown to originate from two different mRNA:s suggesting the possibility of alternative splicing ot the primary transcript. We have isolated genomic clones for the respective gene and sequenced the 8 most 3' exons.One of the exonsencodes the 36bp deleted from one of the cDNA:s, which demonstrates that the deletion can result from alternative splicing of this exon, an event that has not previously been reported for collagen genes. The size pattern of sequenced exons (27-, 81-, 36-, 54-, 54-, 36-, 87-, 39-bp + 3' -untranslated
region ) differs from that of other collagen genes except for the two 54 bp exons typically found in fibrillar collagen genes. Each of the exons studied begins with an intact codon for glycine. The 8 exons sequenced so far have 414 bp of coding sequences which are spread over about 20 kb in the gene. Since these sequences (including3'-untranslatedregion) cover only about 24% of the 3 kb long mRNA, it is likely that this new type of low molecular weight collagen is encoded by a very large gene. 79
E x t r a c e l l u l a r m a t r i x s y n t h e s i s d u r i n g mesenchymal c e l l condensation and chondrogenesis. N.S. Vasan and H. Bost, D e p t . o f Anatomy, New Jersey Medical School, Newark, NJ., USA. Changes in t h e c o m p o s i t i o n o f ECM occur d u r i n g limb mesenchymal c e l l ~ i g r a t i o n , cond e n s a t i o n and d i f f e r e n t i a t i o n . Our s t u d i e s show t h a t in c h i c k embryos between stages 17-21, the c e l l s m i g r a t e through a m a t r i x r i c h in HA. During prechondrogenic condensat i o n ( S t a g e s 22-24) t h e r e is an increase in the HS l e v e l in ECM; the HA l e v e l d e c l i n e s due t o an i n c r e a s e in h y a l u r o n i d a s e degradation. Labelling s t u d i e s i n d i c a t e t h a t the changes in the ECM are due t o the a l t e r ations in the m e t a b o l i c machinery o f the c e l l s . Subsequent t o c o n d e n s a t i o n ( s t a g e 2 5 ) , the c e n t r a l core c e l l s begin t o s y n t h e s i z e a m a t r i x r i c h in c h o n d r o i t i n s u l f a t e p r o t e o glycans (CS-PG). When mesenchymal c e l l s from the core r e g i o n were p l a t e d in v i t r o , cells t h a t formed an aggregate in ~ h accumulated a HS r i c h m a t r i x , and by day 3 began to synt h e s i z e CS-PG. A m a j o r p r o p o r t i o n o f CS-PG monomers were a b l e t o i n t e r a c t w i t h exogenous HA. The r e s u l t s suggest t h a t p r e c h o n d r o qenic condensation o f mesenchymal c e l l s is critical in limb chondrogenesis, and a m i c r o e n v i r o n m e n t r i c h in HS f a c i l i t a t e s this process. ( Supported by a g r a n t from NIH HD 16147 and Foundation o f UMDNJ).
DIFFERENTIAL EXPRESSION OF mRNA'S FOR TYPES I, II AND III COLLAGENS IN DEVELOPING CALLUS TISSUE IN RATS. E. Vuorio~ P. Multim~ki~ M.Sandberg~ H.Aro and H. Aho. Departments of Medical Chemistry and Pathology, Univ. of Turku, SF-20520 Turku, Finland. An experimental fracture healing model in the rat tibio-fibular bone was employed to study the appearance of mRNAs for types I, if and III collagens during endochondral fracture repair. Total RNA was extracted from normal bone and from callus tissue 5, 7, i0, 14 and 28 days after the fracture. Iissue samples were taken at the same time for histologial staining and for in situ nybridization. The results show that during the first week of fracture repair type III collagen ' mRNA is increased rapidly, followed by cartilage-specific II collagen mRNA during the second week. The 28-day callus resembles bone by containing mainly type I collagen mRNAs and very little type II or iIl collagen mRNA. In situ hybridization of callus sections localized type II collagen in cells which were surrounded by cartilage matrix exhibiting alcianophilia at pH 2.5 as well as at pH 0.5, but reduced or abolished alcianophilia after hyaluronidase treatment. Osteoblasts producing actively type I collagen were identified similarily. 26S