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P52. Collagens of porcine growth plate
Abstracts
from the Joint Meeting,
The Fh4 isoforrns present examined. It was found amounts of each isoform, extraction of any isoform
September
1992
in the 1M and 2M extracts have been that each extract ccntained similar demonstrating that there no selective
PSZ. Collagen0 of porcine growth plate J Wardale Muscle and Collagen Research Group, Department of Veterinary Medicine, Uniwrsity of Bristol, Langford, Bristol BS 18 7YD Osteochondrosis, a major cause of leg weakness in pigs, mainly affects the growth plate and articular cartilages. In our preliminary studies we have concentrated on characterising the collagena in these tissues from apparently healthy animals aged 4-5 months. The collagenous amponents have bean sequentially extracted by a variety of methods and quantitated by SDSPAGE and scanning densitometry. Collagen typea I, II, VI, IX and XI are present in both types of cartilage, type X collagen only being found in growth plate. Growth plate cartilage comprised 18% type I, 63% type II, 1% type IX, 0.3% type X and 17% type Xl collagens. Articular cartilage comprised 12% type 1,76% type II, 4% type IX and 9% type XI collagens. The solubilities of some collagen types also varied. 26% type I, 56% type II and no type XI was extracted from growth plate compared to 41% type I, 88% type II and 28% type XI from articular cartilage. The mature collagen cross-link, hydroxylysyl-pyridinoline, was identified in both tissues using HPLC with 0.42 mol/mol collagen detected in articular cartilage but only 0.27 moI/mol in growth plate. Immunolocalisation studies revealed a similar distribution of collagen types II, LX and XI in both tissues although staining for type XI appeared stronger in articular cartilage. Staining for type I collagen was predominantly at the articular surface compared to a weak stain throughout the growth plate.
P53. TGF_B, A marker of differentiation of chick growth plate chondrocytcs in oioo BH Thorp and SB Jakowlew AFRC Inslitute of Animal Physiology and Genetics, Roslin, Midlothian. Laboratory of Chemopreuention, Narional Cancer Institute, Bethesda, Maryland TGF+s regulate chondrocyte proliferation and differentiation in vitro (Centrella et al, J.Biol. Chem 2869, 1987). TGF-Bs are localized to transitional and hypertrophied cells in normal chick growth plates and are rarely seen in proliferating chondrocytes. TGF-@ localization is reduced in the transitional chondrocytes of dyschondroplastic growth plates, and TGF+s reappear in areas of repair where differentiation has recommenced. This study used antibodies to localize TGF-fis in proximal tibiotarsi of normal chicks and in those fed phosphate and calcium deficient diets. In hypocalcaemic and hypophosphataemic rickets, there are accumulations of proliferating and hypertrophic chondrocytes respectively. In accumulated hypertrophic chondrocytes of hypophosphataemic rickets there is a reduction in TGF-fl staining but transitional chondrocytes stain intensely. In hypocalcaemic rickets, accumulated, proliferating chondrocytes contained little TGF-8, but the hypertrophying chondrccytes are positively stained. These studies show that TGF-B localization in growth plate chondrocytes indicates active differentiation in vivo and suggest the presence of TGF+ nay be a prerequisite for differentiation. In addition, there is frequently little TGF-B in from resorbing accumulated matrix chondroclasts hypophosphataemic and dyschondroplastic growth plates, providing further evidence that the TGF+s normally seen in chonclroclasts are mainly matrix derived.
129
P54. Collagen gene expression in the bovine growth plate A Marriott, JA. Hoyland’, AJ Freemont’ , S Ayad and ME Grant Department of Biochemistry and Molecular Biology, and “Rheumatology, University of Manchester, Oxford Road, Manchester Ml3 9PT Endochondral bone formation takes place at cartilaginous growth plates. The growth-plate chondrocytes pass through a series of stages of differentiation resulting in morphological distinct regions: the resting or reserve zone through zones of proliferation, maturation and finally hypertrophy. There is evidence to suggest that these different populations of chondrocyte are also functionally distinct. It is well established that chondrocyte hypertrophy in the chick is concomitant with the synthesis of type X collagen. However, there are marked differences the size and organisation of the mammalian growth plate compared to that of the developing chick. Using in situ hybridisation the expression of type X collagen mRNA was compared to that of other cartilage-specific collagens. The mRNA levels were also correlated with immunofluorescent localisation of the collagens deposited in the matrix. The results indicate that in the bovine growth plate collagen gene expression is regulated as a function of chondrocyte maturation.
P55. Evidence for alternative 5’ untranslated sequences in bovine type X collagen JT Thomas, CJ Cresswell, ME Grant, and RP Boot-Handford Department of Biochemistry and Molecular Biology, University of Manchester, Manchester We have previously described the isolation and characterization of overlapping cDNAs encoding the complete sequence of bovine type X collagen. However, the 5’-untranslated region (UTR) was deduced from a single clone. Subsequent m-screening of the bovine hypertrophic chondrocyte cDNA library with a PCR product designed to encode the N-terminal noncollagenous domain has yielded 12 independent clones extending into the S-UTR which can be divided into three subgroups. Each of the 12 clones contains 48bp of common sequence immediately 5’ of the ATG start site followed by one of three alternative sequences extending a further 55-100bp in the 5’ direction. Screening a bovine genomic library with the PCR product-described above resulted in the isolation of 4 clones. Initial mapping and sequencing analyses have demonstrated the presence of a 169bp exon equivalent to exon 2 in the human and chick genes. We are currently sequencing restriction fragments soecificallv recoenised bv the common and different 5’-UTR’s in n , order to deter;ine their organization and possible function within the type X collagen gene.
P56. Chondrocyte gene expression: factors influencing collagen synthesis S Manning, ME Grant and JT Thomas Department of Biochemistry and Molecular Biology, University Manchester, Stopford Building, Oxford Rd., Manchester Ml3 9PT During the process of endochondral ossification, involving the differentiation and hypertrophy of chondrocytes, the short chain collagen type X is selectively expressed. The growth plate is largely an avascular tissue, therefore, factors capable of influencing chondrocyte maturation are either autocrine in nature, or exocrine imported by the invading vasculature at the mineralization front. Chondrocytes from regions of permanent cartilage were cultured in serum free medium under a variety of conditions to determine the factors responsible for the induction of the developmentally regulated type X collagen. The exocrine factor EGF was shown to promote type X collagen synthesis, over a period of 13 days in culture, at a concentration of long/ml. The transcription of a gene is partially dependent upon the degree of methylation of certain regulatory elements. Methylated
from the Joint Meeting,
The Fh4 isoforrns present examined. It was found amounts of each isoform, extraction of any isoform
September
1992
in the 1M and 2M extracts have been that each extract ccntained similar demonstrating that there no selective
PSZ. Collagen0 of porcine growth plate J Wardale Muscle and Collagen Research Group, Department of Veterinary Medicine, Uniwrsity of Bristol, Langford, Bristol BS 18 7YD Osteochondrosis, a major cause of leg weakness in pigs, mainly affects the growth plate and articular cartilages. In our preliminary studies we have concentrated on characterising the collagena in these tissues from apparently healthy animals aged 4-5 months. The collagenous amponents have bean sequentially extracted by a variety of methods and quantitated by SDSPAGE and scanning densitometry. Collagen typea I, II, VI, IX and XI are present in both types of cartilage, type X collagen only being found in growth plate. Growth plate cartilage comprised 18% type I, 63% type II, 1% type IX, 0.3% type X and 17% type Xl collagens. Articular cartilage comprised 12% type 1,76% type II, 4% type IX and 9% type XI collagens. The solubilities of some collagen types also varied. 26% type I, 56% type II and no type XI was extracted from growth plate compared to 41% type I, 88% type II and 28% type XI from articular cartilage. The mature collagen cross-link, hydroxylysyl-pyridinoline, was identified in both tissues using HPLC with 0.42 mol/mol collagen detected in articular cartilage but only 0.27 moI/mol in growth plate. Immunolocalisation studies revealed a similar distribution of collagen types II, LX and XI in both tissues although staining for type XI appeared stronger in articular cartilage. Staining for type I collagen was predominantly at the articular surface compared to a weak stain throughout the growth plate.
P53. TGF_B, A marker of differentiation of chick growth plate chondrocytcs in oioo BH Thorp and SB Jakowlew AFRC Inslitute of Animal Physiology and Genetics, Roslin, Midlothian. Laboratory of Chemopreuention, Narional Cancer Institute, Bethesda, Maryland TGF+s regulate chondrocyte proliferation and differentiation in vitro (Centrella et al, J.Biol. Chem 2869, 1987). TGF-Bs are localized to transitional and hypertrophied cells in normal chick growth plates and are rarely seen in proliferating chondrocytes. TGF-@ localization is reduced in the transitional chondrocytes of dyschondroplastic growth plates, and TGF+s reappear in areas of repair where differentiation has recommenced. This study used antibodies to localize TGF-fis in proximal tibiotarsi of normal chicks and in those fed phosphate and calcium deficient diets. In hypocalcaemic and hypophosphataemic rickets, there are accumulations of proliferating and hypertrophic chondrocytes respectively. In accumulated hypertrophic chondrocytes of hypophosphataemic rickets there is a reduction in TGF-fl staining but transitional chondrocytes stain intensely. In hypocalcaemic rickets, accumulated, proliferating chondrocytes contained little TGF-8, but the hypertrophying chondrccytes are positively stained. These studies show that TGF-B localization in growth plate chondrocytes indicates active differentiation in vivo and suggest the presence of TGF+ nay be a prerequisite for differentiation. In addition, there is frequently little TGF-B in from resorbing accumulated matrix chondroclasts hypophosphataemic and dyschondroplastic growth plates, providing further evidence that the TGF+s normally seen in chonclroclasts are mainly matrix derived.
129
P54. Collagen gene expression in the bovine growth plate A Marriott, JA. Hoyland’, AJ Freemont’ , S Ayad and ME Grant Department of Biochemistry and Molecular Biology, and “Rheumatology, University of Manchester, Oxford Road, Manchester Ml3 9PT Endochondral bone formation takes place at cartilaginous growth plates. The growth-plate chondrocytes pass through a series of stages of differentiation resulting in morphological distinct regions: the resting or reserve zone through zones of proliferation, maturation and finally hypertrophy. There is evidence to suggest that these different populations of chondrocyte are also functionally distinct. It is well established that chondrocyte hypertrophy in the chick is concomitant with the synthesis of type X collagen. However, there are marked differences the size and organisation of the mammalian growth plate compared to that of the developing chick. Using in situ hybridisation the expression of type X collagen mRNA was compared to that of other cartilage-specific collagens. The mRNA levels were also correlated with immunofluorescent localisation of the collagens deposited in the matrix. The results indicate that in the bovine growth plate collagen gene expression is regulated as a function of chondrocyte maturation.
P55. Evidence for alternative 5’ untranslated sequences in bovine type X collagen JT Thomas, CJ Cresswell, ME Grant, and RP Boot-Handford Department of Biochemistry and Molecular Biology, University of Manchester, Manchester We have previously described the isolation and characterization of overlapping cDNAs encoding the complete sequence of bovine type X collagen. However, the 5’-untranslated region (UTR) was deduced from a single clone. Subsequent m-screening of the bovine hypertrophic chondrocyte cDNA library with a PCR product designed to encode the N-terminal noncollagenous domain has yielded 12 independent clones extending into the S-UTR which can be divided into three subgroups. Each of the 12 clones contains 48bp of common sequence immediately 5’ of the ATG start site followed by one of three alternative sequences extending a further 55-100bp in the 5’ direction. Screening a bovine genomic library with the PCR product-described above resulted in the isolation of 4 clones. Initial mapping and sequencing analyses have demonstrated the presence of a 169bp exon equivalent to exon 2 in the human and chick genes. We are currently sequencing restriction fragments soecificallv recoenised bv the common and different 5’-UTR’s in n , order to deter;ine their organization and possible function within the type X collagen gene.
P56. Chondrocyte gene expression: factors influencing collagen synthesis S Manning, ME Grant and JT Thomas Department of Biochemistry and Molecular Biology, University Manchester, Stopford Building, Oxford Rd., Manchester Ml3 9PT During the process of endochondral ossification, involving the differentiation and hypertrophy of chondrocytes, the short chain collagen type X is selectively expressed. The growth plate is largely an avascular tissue, therefore, factors capable of influencing chondrocyte maturation are either autocrine in nature, or exocrine imported by the invading vasculature at the mineralization front. Chondrocytes from regions of permanent cartilage were cultured in serum free medium under a variety of conditions to determine the factors responsible for the induction of the developmentally regulated type X collagen. The exocrine factor EGF was shown to promote type X collagen synthesis, over a period of 13 days in culture, at a concentration of long/ml. The transcription of a gene is partially dependent upon the degree of methylation of certain regulatory elements. Methylated