- Email: [email protected]
O4. Osteogenic and adipogenic differentiation in human bone derived cell cultures
114
Abstracts
like constituents but also some possibly novel isoforms of subunits A and B. These subunits confer to the pump a unique sensitivity to vanadate and nitrate as compared with the kidneytype H+ ATPase. In the basolateral membrane of the cell, are found a sodium pump of the kidney type (at/fll) and a bicarbonate/chloride exchanger of the Band 3 family of related peptides, similar to the type 2 anion exchanger (AE2). Although not yet characterized at the molecular level, Na+/H+ antiporters are also demonstrated in the osteoclast. These latter, in conjunction with AE2, serve to maintain the intracellular pH of the osteoclast within physiological limits despite the extrusion of acid at the apical pole. During the differentiation of bone marrow macrophages to osteoclast-like cells, I,25 dihydroxyvitamin D3 induces the concomitant expression of the osteoclast proton-pump subunits, the sodium pumps, carbonic anhydrase and the vitronectin receptor. The effects on the expression of carbonic anhydrase are transcriptional. Calcitonin can up or down regulate the activity of sodium pumps and of the Na+/H+ antiporters in kidney cells as a function of the cell cycle. These effects, as well as the effects on bone resorption and cell retraction by isolated osteoclasts, are G-protein mediated and involve both the CAMP and the PKC pathways.
ORAL COhnUuNI
CATIONS
01. Biochemical charactcrisation of intcgrins membrane8 immunoisolated from an osteoclrstomr S Nesbitt, M Helfrich and M Horton ZCRF, St. Bartholomew’s Hospital, London
in ostcoclast turnour
Technical deficiencies have limited the analysis of osteoclast (00 integrins to immunocytochemistry, but have allowed the expression of vitronectin receptor (VNR) (~~83) and collagen/laminin receptor (a2gl) to be demonstrated. However, the low sensitivity of immunostaining techniques, combined with the cellular heterogeneity exhibited by the bone cell populations used, have created discrepancies in published data. Here, we report a novel and sensitive method enabling the biochemical characterisation of OC antigens to be performed. OC membranes were purified from osteoclastoma tumours by immunoselection for VNR using magnetic beads coated with 13C2 (anti-av) monoclonal antibody. Immunoselected OC membranes were obtained at a high purity, as indicated by the lack of expression of several integrins (eg a3 subunit) associated with the other cell types in the tumour. Purified OC membranes and unselected tumour membranes (the latter comprising membranes from a mixed cell population including OCs) were analysed for integrin expression by immunoprecipitation and western blotting using a sensitive non-radioactive technique incorporating enhanced chemiluminescence. al, a4, ~6, a8, aM, ax, and gpIIb, and 84, p and ~8 (a7 and 87 were not tested) integrin chains were undetectable at a sensitivity limit of 1 ng. ~2, a3, a5, uL, and av, and, gl, 82, 83 and 85 subunits were demonstrated at differing levels of expression in unselected osteoclastoma membranes. From these, only the avb3, a2pl and avpl integrin dimers were found in immunoisolated, purified OC membranes. In conclusion, this procedure of membrane immunoisofation followed by biochemical analysis has clarified which integrins are expressed by OCs, has shown that OCs additionally express the avel integrin dimer and is currently facilitating the isolation of novel osteoclast receptors. 02. Dieintegrins: Useful tools resorption? ROC Oreffo, J Adams, A Houghton Bioscience I, ICI Pharmaceuticals, Cheshire SKI0 4TG Osteoclasts express integrin comprised
to examine
osteoclast
and D Johnstone Alderley Park,
the vitronectin receptor of av and 83 sub-units.
bone
Macclefield,
(VNR), which is an The VNR has been
from the Joint Meeting, September
1992
implicated in the attachment of osteoclasts to bone which precedes bone resorption. Recent evidence has shown that disintegrins block binding of certain integrins to their ligands, thereby preventing cell adhesion. These agents, isolated from venoms of various species of viper are RGD-containing, cysteine-rich proteins. Previous experiments have demonstrated that disintegrins can inhibit osteoclast adhesion in vitro. The objective of this study was to determine whether disintegrins could inhibit osteoclast-mediated bone resorption in mouse calvarial organ cultures. %a++ labelled neonatal mouse calvaria were incubated with PTH (lOOng/ml), IL-l (lo-*0 M) or PGE2 (IO-GM) for 72 hours in the presence or absence of eristostatin, echistatin, agkistrostatin or rhodostomin/kistrin. Inhibition of bone resorption was measured by a reduction in 45ca++ released into the media. All four disintegrins completely blocked stimulated, but not basal bone resorption at lo-6 M concentration. The effect of echistatin was dose-dependent with an IC50 of IOOnM. Preliminary studies also indicate that echistatin inhibits osteoclast formation and recruitment in this model. These observations suggest that these agents can be used as tools to determine the role of integrins in osteoclast bone resorption. 03. Rabbit marrow stromal cell lines established by incorporation of the temperature-sensitive simian virus 40 large T antigen ME Nuttall, A Kocialkowski, A Darby, KA Hart’, D Johnstone* and BA Ashton Department of Rheumatology, R.j.6A.H. Orthopaedic Hospital, Oswestry, Shropshire, SYlO 7AG and ‘I.C.1. Pharmaceuticals, Alder@ Park, Macclesfield, Cheshire, SKI0 4TG Immortalization of cells at various stages within the osteoblast lineage would enable detailed examination of the genes critical in controlling the differentiation pathway. Freshly isolated rabbit femoral bone-marrow cells (5xlO6ceUs) were electroporated with a plasmid containing the temperature-sensitive SV40 large T antigen and neomycin resistence genes (tSA58lJ19). Two clones, RCl and RC2 survived selection in 5OOuM G418 for 4 weeks at the permissive temperatie (33 Oc), where the large T antigen is functional, driving the proliferation of cells with differentiation arrested. RCI and RC2 at passage 33 and 35 respectively (equivalent to 132 and 140 population doublings) were shown to have retained the large T antigen by Northern and Western analysis. After switching to 370 C the clones were examined for osteoblastic features by enzyme assay, PCR and Northern analysis and tested for osteogenic potential by implantation into athymic mice. PCR analysis showed that osteonectin was expressed constitutively by both clones and that matrix-gla protein message was induced in both by lo-9M 1,25dihydroxyvitamin D Wit D3). Neither RCl or RC2 expressed alkaline phosphatase activity even after dexamethasone or Vit D3. Both clones exprssed a range of cytokines (TGFa and p, EGF and PDGF) but only RC2 expressed ILlb, IL-6 and IGF-2. Expression of IL-6 was induced in RCl by Vit D3 treatment. Histological analysis showed the presence of bone in intramuscular implants of both clones after 14 days in vivo. RCI and RC2 may represent, therefore, two immortalized early osteogenic precursor ceils.
04. Odeogenic and adipogenic differentiation in human bone derived cell culhues R Gundle, MJO Francis, SE Graves and JN Beresford’ Nuffield Department of Orthopaedic Surgery and ‘M.R.C. Bone Xesearch Laboratory, Nufield Orthopaedic Centre, Oxford. OX3 7LD Ascorbate and glucocorticoids promote osteogenic differentiation in animal bone cell cultures. Here, explants of adult human trabecular bone were cultured for up to 11 weeks in the presence of 1OOpM L-ascorbate 2-phosphate (A-2-P; a long acting derivative of L-ascorbate) in the absence (-) or presence (+) of 1OnM
Abstracts
from the Joint Meeting,
September
1992
115
dexamethasone (Dex). After about 4 weeks, a dense cell multilayer was present with much extracellular matrix in all cultures. After about 6 weeks large, lipid laden cells resembling adipocytes appeared in (+) cultures only. These cells appeared to migrate out from the trabecular explants and spread over the underlying cell layer. When these cultures were passaged lipid laden cells were initially absent but after about 2 weeks, corresponding to the time taken for cultures to attain confluence, their numbers increased rapidly. When passaged early (about 4 weeks) or late (1 6 weeks) and grown to confluence, (+) but not (-) cultures mineralised extensively in medium supplemented with either 5mM inorganic phosphate or 10 mM p-glycerophosphate. The reappearance of lipid laden ceils in late passaged (+) cultures was prevented under these conditions. In summary: 1) A-2-P and Dex when present continuously promote osteogenic and adipogenic differentiation in this human bone ceU culture system 2) The differentiation of adipocytes is a late event, requiring culture for extended periods post-confluence. 3) Factors which promote mineralisation in passaged (+) culture inhibit adipogenesis. 4) Identification of the factors which regulate the differentiation of adipogenic and osteogenic cells in this culture system should enhance our understanding of the aetiology of osteoporosis, in which loss of bone is associated with an increase in the volume of the marrow adipose tissue.
05. Structural similarities between cartilage aggrecan and link protein PJ Neame and CN Young Shriners Hospital for Crippled Children,
shark
Tampa,
and
Articular cartilage is comprised of a small number of cells embedded within a matrix primarily composed of collagen and proteoglycan (PC). The functional integrity of the tissue is highly dependent on the maintenance of matrix structure, which, in turn is controlled by the chondrocytes. In normal tissue there is a slow but steady turnover of matrix components such that their levels remain constant. In certain diseased states the equilibrium is upset resulting in a net loss of matrix components. The object of the present study was to upset artificially the synthetic/loss equilibrium by enzymic depletion and assess the ability of chondrocytes to respond by increasing PG synthesis. Bovine articular cartilage explants were depleted using enzymes as follows; 10 unit/ml Streptomyces hyaluronidase (induced a 30% loss of PC), 2000 unit/ml testicular hyaluronidase (50% loss of PC) and 100 unit/ml collagenase (35% loss of PG) and control (6% loss of PG). Collagenase also induced a 50% loss of collagen. Collagenase treatment induced a 50% stimulation of PG synthesis above control levels. Elevated synthesis levels were maintained for 9 days. Testicular hyaluronidase induced a brief elevation in PC synthesis on day 3 of culture. Streptomyces hyaluronidase treatment did not cause an alteration in the rate of PG synthesis above control levels. Alterations in the size and degradation profile of the PG synthesised by couagenase treated explants were observed using composite agarose/acrylimide gel electrophoresis. It appears therefore, that collagen and matrix organisation is more important than PG levels in the control of PG synthesis in articular cartilage explant cultures.
mammalian
Fhidn,
USA
The structures of the globular domains of mammalian, and even avian cartilage aggrecans are conserved. The same is found with the link protein, which stabilizes the hyaluronate-aggrecan complex. To identify residues which vary over large evolutionary distances, we have been examining the structures of aggrecan and link protein from shark cartilage. Cartilage from the neural arch of a reef shark (Carcharhinus springeri) was extracted with dissociative buffers in the presence of protease inhibitors. The extract was “spiked” with 5 mg of rooster coombe hyaluronic acid, and the proteoglycan aggregate re-associated by dialysis. Sequential cesium chloride gradients (associative and dissociative) yielded both aggrecan and link protein, which were purified by gel filtration. Proteolytic digestion and Edman degradation of aggrecan and link protein have enabled us to determine approx 70% of the sequence of the hyaluronatebinding regions of both molecules. In comparing shark link protein to other known link protein sequences, we have found that the region, around the disulfide bond in the IgC-like domain are very conserved, while the rest of this domain is less conserved. In the Proteoglycan Tandem Repeat domain (PTR), the most conserved regions are around the 8A4 epitope in loop B. This is the “apex” of the loops, defined by the 4th and 5th and 8th and 9th cysteines in link protein. In shark aggrecan similar conservation is seen. At no point in our analyses of shark aggrecan have we found peptides which derive from the G3 (Cterminal, lectin-like) domain. Shark aggrecan may thus either not have this domain or it may have been removed at an earlier stage of protein maturation. This work will enable site-directed mutagenesis experiments designed to analyze the structurally important features of aggrecan link protein.
06. The effect of matrix depletion on proteoglycan ryntheais in articular cartilage explants D Lee, G Bentley and lC Archer IRC in Bimnedical Materials, Institute of Orthopuedics, Brocklq Hill, Stanmore, Middlesex ond ‘Department of Anatomy, Uniuersity College of Wales, Cardiff, UK
07. Changes in proteoglycan content and rulphation endochondml ossification C Farquharson, CC Whitehead and N Loveridge Institute of Animal Physiology and Genetics Research, Edinburgh, and Rawett Research Institute, Aberdeen
during
Roslin,
In tibia1 dyschondroplasia (TD) chondrocyte maturation is arrested at the transitional zone. We have used this model to investigate the role of proteoglycan sulphation (Alcian Blue) and content (immunocytochemistry) in chondrocyte maturation. Sections of growth plate of normal and TD chicks were either stained with Alcian Blue (CEC method) or immunolabelled with MAb’s to chondroitin 4 (C4S) & 6 (C6S) -sulphate and keratan sulphate (KS). Peroxidase and Alcian Blue staining were quantified by microdensitometry. In the proliferating (PZ) and transitional (TZ) zones of normals, C4 & 6s and KS immunostaining were similar whereas in the hypertrophic (HZ) zone, C4 6~ 6.S were slightly lower (12.8%; and 18.1% respectively) and KS was markedly lower (57.6%). In TD, C4S and KS in the PZ and TZ were similar to normals. Alcian Blue staining (0.5M MgCl2) in normals decreased between the PZ and TZ (44.64b)‘but rose again in the HZ. In TD, there was a higher level of staining in the TZ (38.8%). In normals, Alcian Blue staining (0.7M MgCl2) was lower in the TZ (41.8%) and HZ (39.9%) than in the PZ. In TD, levels in the TZ were 29.546, higher than in normals. These results suggest that in the transitional zone it is the sulphation of proteoglycans rather than the amount that is important in chondrocyte maturation.
08. Vitamin D receptor mutations and their stereochemical consequence* AR Rut, M Hewison, K Kristjansson, B Luisi, RE Walker MR Hughes and JLH O’Riordan The Middlesex Hospital, London, Baylor College of Medicine, Houston TX 77030 and Glasgow University, Glasgow
,
Hereditary vitamin D resistant rickets (HVDRR) is due to tissue resistance to the action of 1,25-dihydroxyvitamin D3 [1,25(0H)2D$ Six mutations have been described in the gene the vitamin D receptor (VDR). These have caused changes in either
Abstracts
like constituents but also some possibly novel isoforms of subunits A and B. These subunits confer to the pump a unique sensitivity to vanadate and nitrate as compared with the kidneytype H+ ATPase. In the basolateral membrane of the cell, are found a sodium pump of the kidney type (at/fll) and a bicarbonate/chloride exchanger of the Band 3 family of related peptides, similar to the type 2 anion exchanger (AE2). Although not yet characterized at the molecular level, Na+/H+ antiporters are also demonstrated in the osteoclast. These latter, in conjunction with AE2, serve to maintain the intracellular pH of the osteoclast within physiological limits despite the extrusion of acid at the apical pole. During the differentiation of bone marrow macrophages to osteoclast-like cells, I,25 dihydroxyvitamin D3 induces the concomitant expression of the osteoclast proton-pump subunits, the sodium pumps, carbonic anhydrase and the vitronectin receptor. The effects on the expression of carbonic anhydrase are transcriptional. Calcitonin can up or down regulate the activity of sodium pumps and of the Na+/H+ antiporters in kidney cells as a function of the cell cycle. These effects, as well as the effects on bone resorption and cell retraction by isolated osteoclasts, are G-protein mediated and involve both the CAMP and the PKC pathways.
ORAL COhnUuNI
CATIONS
01. Biochemical charactcrisation of intcgrins membrane8 immunoisolated from an osteoclrstomr S Nesbitt, M Helfrich and M Horton ZCRF, St. Bartholomew’s Hospital, London
in ostcoclast turnour
Technical deficiencies have limited the analysis of osteoclast (00 integrins to immunocytochemistry, but have allowed the expression of vitronectin receptor (VNR) (~~83) and collagen/laminin receptor (a2gl) to be demonstrated. However, the low sensitivity of immunostaining techniques, combined with the cellular heterogeneity exhibited by the bone cell populations used, have created discrepancies in published data. Here, we report a novel and sensitive method enabling the biochemical characterisation of OC antigens to be performed. OC membranes were purified from osteoclastoma tumours by immunoselection for VNR using magnetic beads coated with 13C2 (anti-av) monoclonal antibody. Immunoselected OC membranes were obtained at a high purity, as indicated by the lack of expression of several integrins (eg a3 subunit) associated with the other cell types in the tumour. Purified OC membranes and unselected tumour membranes (the latter comprising membranes from a mixed cell population including OCs) were analysed for integrin expression by immunoprecipitation and western blotting using a sensitive non-radioactive technique incorporating enhanced chemiluminescence. al, a4, ~6, a8, aM, ax, and gpIIb, and 84, p and ~8 (a7 and 87 were not tested) integrin chains were undetectable at a sensitivity limit of 1 ng. ~2, a3, a5, uL, and av, and, gl, 82, 83 and 85 subunits were demonstrated at differing levels of expression in unselected osteoclastoma membranes. From these, only the avb3, a2pl and avpl integrin dimers were found in immunoisolated, purified OC membranes. In conclusion, this procedure of membrane immunoisofation followed by biochemical analysis has clarified which integrins are expressed by OCs, has shown that OCs additionally express the avel integrin dimer and is currently facilitating the isolation of novel osteoclast receptors. 02. Dieintegrins: Useful tools resorption? ROC Oreffo, J Adams, A Houghton Bioscience I, ICI Pharmaceuticals, Cheshire SKI0 4TG Osteoclasts express integrin comprised
to examine
osteoclast
and D Johnstone Alderley Park,
the vitronectin receptor of av and 83 sub-units.
bone
Macclefield,
(VNR), which is an The VNR has been
from the Joint Meeting, September
1992
implicated in the attachment of osteoclasts to bone which precedes bone resorption. Recent evidence has shown that disintegrins block binding of certain integrins to their ligands, thereby preventing cell adhesion. These agents, isolated from venoms of various species of viper are RGD-containing, cysteine-rich proteins. Previous experiments have demonstrated that disintegrins can inhibit osteoclast adhesion in vitro. The objective of this study was to determine whether disintegrins could inhibit osteoclast-mediated bone resorption in mouse calvarial organ cultures. %a++ labelled neonatal mouse calvaria were incubated with PTH (lOOng/ml), IL-l (lo-*0 M) or PGE2 (IO-GM) for 72 hours in the presence or absence of eristostatin, echistatin, agkistrostatin or rhodostomin/kistrin. Inhibition of bone resorption was measured by a reduction in 45ca++ released into the media. All four disintegrins completely blocked stimulated, but not basal bone resorption at lo-6 M concentration. The effect of echistatin was dose-dependent with an IC50 of IOOnM. Preliminary studies also indicate that echistatin inhibits osteoclast formation and recruitment in this model. These observations suggest that these agents can be used as tools to determine the role of integrins in osteoclast bone resorption. 03. Rabbit marrow stromal cell lines established by incorporation of the temperature-sensitive simian virus 40 large T antigen ME Nuttall, A Kocialkowski, A Darby, KA Hart’, D Johnstone* and BA Ashton Department of Rheumatology, R.j.6A.H. Orthopaedic Hospital, Oswestry, Shropshire, SYlO 7AG and ‘I.C.1. Pharmaceuticals, Alder@ Park, Macclesfield, Cheshire, SKI0 4TG Immortalization of cells at various stages within the osteoblast lineage would enable detailed examination of the genes critical in controlling the differentiation pathway. Freshly isolated rabbit femoral bone-marrow cells (5xlO6ceUs) were electroporated with a plasmid containing the temperature-sensitive SV40 large T antigen and neomycin resistence genes (tSA58lJ19). Two clones, RCl and RC2 survived selection in 5OOuM G418 for 4 weeks at the permissive temperatie (33 Oc), where the large T antigen is functional, driving the proliferation of cells with differentiation arrested. RCI and RC2 at passage 33 and 35 respectively (equivalent to 132 and 140 population doublings) were shown to have retained the large T antigen by Northern and Western analysis. After switching to 370 C the clones were examined for osteoblastic features by enzyme assay, PCR and Northern analysis and tested for osteogenic potential by implantation into athymic mice. PCR analysis showed that osteonectin was expressed constitutively by both clones and that matrix-gla protein message was induced in both by lo-9M 1,25dihydroxyvitamin D Wit D3). Neither RCl or RC2 expressed alkaline phosphatase activity even after dexamethasone or Vit D3. Both clones exprssed a range of cytokines (TGFa and p, EGF and PDGF) but only RC2 expressed ILlb, IL-6 and IGF-2. Expression of IL-6 was induced in RCl by Vit D3 treatment. Histological analysis showed the presence of bone in intramuscular implants of both clones after 14 days in vivo. RCI and RC2 may represent, therefore, two immortalized early osteogenic precursor ceils.
04. Odeogenic and adipogenic differentiation in human bone derived cell culhues R Gundle, MJO Francis, SE Graves and JN Beresford’ Nuffield Department of Orthopaedic Surgery and ‘M.R.C. Bone Xesearch Laboratory, Nufield Orthopaedic Centre, Oxford. OX3 7LD Ascorbate and glucocorticoids promote osteogenic differentiation in animal bone cell cultures. Here, explants of adult human trabecular bone were cultured for up to 11 weeks in the presence of 1OOpM L-ascorbate 2-phosphate (A-2-P; a long acting derivative of L-ascorbate) in the absence (-) or presence (+) of 1OnM
Abstracts
from the Joint Meeting,
September
1992
115
dexamethasone (Dex). After about 4 weeks, a dense cell multilayer was present with much extracellular matrix in all cultures. After about 6 weeks large, lipid laden cells resembling adipocytes appeared in (+) cultures only. These cells appeared to migrate out from the trabecular explants and spread over the underlying cell layer. When these cultures were passaged lipid laden cells were initially absent but after about 2 weeks, corresponding to the time taken for cultures to attain confluence, their numbers increased rapidly. When passaged early (about 4 weeks) or late (1 6 weeks) and grown to confluence, (+) but not (-) cultures mineralised extensively in medium supplemented with either 5mM inorganic phosphate or 10 mM p-glycerophosphate. The reappearance of lipid laden ceils in late passaged (+) cultures was prevented under these conditions. In summary: 1) A-2-P and Dex when present continuously promote osteogenic and adipogenic differentiation in this human bone ceU culture system 2) The differentiation of adipocytes is a late event, requiring culture for extended periods post-confluence. 3) Factors which promote mineralisation in passaged (+) culture inhibit adipogenesis. 4) Identification of the factors which regulate the differentiation of adipogenic and osteogenic cells in this culture system should enhance our understanding of the aetiology of osteoporosis, in which loss of bone is associated with an increase in the volume of the marrow adipose tissue.
05. Structural similarities between cartilage aggrecan and link protein PJ Neame and CN Young Shriners Hospital for Crippled Children,
shark
Tampa,
and
Articular cartilage is comprised of a small number of cells embedded within a matrix primarily composed of collagen and proteoglycan (PC). The functional integrity of the tissue is highly dependent on the maintenance of matrix structure, which, in turn is controlled by the chondrocytes. In normal tissue there is a slow but steady turnover of matrix components such that their levels remain constant. In certain diseased states the equilibrium is upset resulting in a net loss of matrix components. The object of the present study was to upset artificially the synthetic/loss equilibrium by enzymic depletion and assess the ability of chondrocytes to respond by increasing PG synthesis. Bovine articular cartilage explants were depleted using enzymes as follows; 10 unit/ml Streptomyces hyaluronidase (induced a 30% loss of PC), 2000 unit/ml testicular hyaluronidase (50% loss of PC) and 100 unit/ml collagenase (35% loss of PG) and control (6% loss of PG). Collagenase also induced a 50% loss of collagen. Collagenase treatment induced a 50% stimulation of PG synthesis above control levels. Elevated synthesis levels were maintained for 9 days. Testicular hyaluronidase induced a brief elevation in PC synthesis on day 3 of culture. Streptomyces hyaluronidase treatment did not cause an alteration in the rate of PG synthesis above control levels. Alterations in the size and degradation profile of the PG synthesised by couagenase treated explants were observed using composite agarose/acrylimide gel electrophoresis. It appears therefore, that collagen and matrix organisation is more important than PG levels in the control of PG synthesis in articular cartilage explant cultures.
mammalian
Fhidn,
USA
The structures of the globular domains of mammalian, and even avian cartilage aggrecans are conserved. The same is found with the link protein, which stabilizes the hyaluronate-aggrecan complex. To identify residues which vary over large evolutionary distances, we have been examining the structures of aggrecan and link protein from shark cartilage. Cartilage from the neural arch of a reef shark (Carcharhinus springeri) was extracted with dissociative buffers in the presence of protease inhibitors. The extract was “spiked” with 5 mg of rooster coombe hyaluronic acid, and the proteoglycan aggregate re-associated by dialysis. Sequential cesium chloride gradients (associative and dissociative) yielded both aggrecan and link protein, which were purified by gel filtration. Proteolytic digestion and Edman degradation of aggrecan and link protein have enabled us to determine approx 70% of the sequence of the hyaluronatebinding regions of both molecules. In comparing shark link protein to other known link protein sequences, we have found that the region, around the disulfide bond in the IgC-like domain are very conserved, while the rest of this domain is less conserved. In the Proteoglycan Tandem Repeat domain (PTR), the most conserved regions are around the 8A4 epitope in loop B. This is the “apex” of the loops, defined by the 4th and 5th and 8th and 9th cysteines in link protein. In shark aggrecan similar conservation is seen. At no point in our analyses of shark aggrecan have we found peptides which derive from the G3 (Cterminal, lectin-like) domain. Shark aggrecan may thus either not have this domain or it may have been removed at an earlier stage of protein maturation. This work will enable site-directed mutagenesis experiments designed to analyze the structurally important features of aggrecan link protein.
06. The effect of matrix depletion on proteoglycan ryntheais in articular cartilage explants D Lee, G Bentley and lC Archer IRC in Bimnedical Materials, Institute of Orthopuedics, Brocklq Hill, Stanmore, Middlesex ond ‘Department of Anatomy, Uniuersity College of Wales, Cardiff, UK
07. Changes in proteoglycan content and rulphation endochondml ossification C Farquharson, CC Whitehead and N Loveridge Institute of Animal Physiology and Genetics Research, Edinburgh, and Rawett Research Institute, Aberdeen
during
Roslin,
In tibia1 dyschondroplasia (TD) chondrocyte maturation is arrested at the transitional zone. We have used this model to investigate the role of proteoglycan sulphation (Alcian Blue) and content (immunocytochemistry) in chondrocyte maturation. Sections of growth plate of normal and TD chicks were either stained with Alcian Blue (CEC method) or immunolabelled with MAb’s to chondroitin 4 (C4S) & 6 (C6S) -sulphate and keratan sulphate (KS). Peroxidase and Alcian Blue staining were quantified by microdensitometry. In the proliferating (PZ) and transitional (TZ) zones of normals, C4 & 6s and KS immunostaining were similar whereas in the hypertrophic (HZ) zone, C4 6~ 6.S were slightly lower (12.8%; and 18.1% respectively) and KS was markedly lower (57.6%). In TD, C4S and KS in the PZ and TZ were similar to normals. Alcian Blue staining (0.5M MgCl2) in normals decreased between the PZ and TZ (44.64b)‘but rose again in the HZ. In TD, there was a higher level of staining in the TZ (38.8%). In normals, Alcian Blue staining (0.7M MgCl2) was lower in the TZ (41.8%) and HZ (39.9%) than in the PZ. In TD, levels in the TZ were 29.546, higher than in normals. These results suggest that in the transitional zone it is the sulphation of proteoglycans rather than the amount that is important in chondrocyte maturation.
08. Vitamin D receptor mutations and their stereochemical consequence* AR Rut, M Hewison, K Kristjansson, B Luisi, RE Walker MR Hughes and JLH O’Riordan The Middlesex Hospital, London, Baylor College of Medicine, Houston TX 77030 and Glasgow University, Glasgow
,
Hereditary vitamin D resistant rickets (HVDRR) is due to tissue resistance to the action of 1,25-dihydroxyvitamin D3 [1,25(0H)2D$ Six mutations have been described in the gene the vitamin D receptor (VDR). These have caused changes in either