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Differential effects of TGF β and BMP4 on the gene expression and differentiated function of preosteoblasts
ATTACHMENT OF FLG 29.1 P
BIOSYNTHETIC P
OCUSTIC CELLS TO
LLS
IN
DIFFERENTIAL EFFECTS OF TGF l3ANC 3MP4 ON THE GENE EXPRESSION AND ;;FFERENTIATED FUNCTION OF PRE-
TI-IE
OSTEOBL&5TIC LIN of Dental Research an Universitv of Florence. Cells within the osteoblastic linea e express numerous proteins that contain Asp-Gly-Arg (RGD), t ce sequence that binds to cell membrane associated mtegrins, and secrete them at particular sta es during the maturational sequence both LZ wivo and in vitro. 8 ubsequent to serving their as yet unidentified roles in osteoblastic metabolism, these proteins are entombed within mineralized matrix, but most likely still function in the recruitment and maturation of osteoclasts and their recursors. FLG ?9._l cells, cloned from a patient with acute leu r:emia, fuse ayd exhlblt many osteoclastic traits followin treatment with lr M 12-O. tetradecanoylphorbol I3-acetate 8-P A). In this study, FLG 29.1 cell attachment to several RGD-containing bone matrix proteins (serum-free conditioned medium from untreated adult human p:eosteoblasts SFCM), thrombos ondin (TSP), fibronectin (FN) vitronectin ( &i ), osteopontin (Of) and bone sialoprotein (BSP) all at 0.2 IBM dotted onto bacteriological plastic) was investigated after 48 hrs of incubation with and wlthout TPA. With TPA, there was a marked increase in cell attachment (BSl:, OP > FN, VN > TSP, SFCM) compared to tissue culture plastic; however, fusion % of total attached cells) was not markedly enhanced. ‘Without 4=FA, the cells coptinued to proliferate and there was virtually no attachment or f&Ion on plastic or SFCM. IJ-Iowever, cells adhered to TSP. and ap eared to Initiate fusionon other proteins (BSP. QP > FN, VN). Pnterestingly, SFCM, which contains TSP, FN and VN and strongly supports bone-formin cell attachment, is considerably less active in mediating FL 6 29.1 cell attachment than the individual roteins. This dab suggests the secretion of a potential inhibitor Ps) by preosteoblasts, which had not been stimulated by factors known to increase bone resorption. These studies indicate that in this preostaoclast model system, products of cells at various sta es of maturation in the osteoblastic lineage support attachment. a owever, fusion events may be mediated by products expressed at late stages of osteoblastic differentiation.
OSTBOBLASTS. I-l. OU.*R.G. Hammonds Jnr. D.M.Findlnv, T.J Martin and K.W. Ng. Depanment of Medicine, The University of Melbourne, St. Vincent’s Hospital. Melbourne, Australia and *Genentech Inc.. San Francisco, CA, USA TGFp and BMP4 share the ability fo induce endochondral bone formation in vivo. This study examines the effects of recombinant TGFp and BMP4 in a retinoic acid (RA) responsive rat clonal.preosteoblast ceil line, UMR 201. These cells express themRNA for BMP 1.2and 3 as well as u, p and ‘I RA receptors. They respond to lo-6M RA with a significant increase in the steady state levels of mRNA for pro-al(I) collagen, osteopontin (OP). osteonectin (ON), matrix gla protein (MGP) and alkaline phosphatase (ALP). Pra al(I) collagen mRNA was increased by TGFp (1 @ml) and BMW (50 @ml). The addition of either BMP4 or TGF p to RA resulted in a further increase in pro-al(l) collagen mRNA levels. Nuclear run-on experiments showedthat RA and TGF 0 but not BMP4 increased the transcriptional rate of the pro-al(l) collagen gene. Both TGFp and BMW reduced the constitutive as well as RA-induced
36 DIFFERENTIATION-DEPENDENT EFFECTS OF DEXAMETHASONE ON OSTEOPONTIN mRNA EXPRESSION IN BONE-DERIVED CELLS. S.B. Rodan, E.E. Ooas, S. Harada and G.A. Rodan. Merck Sharp & Dohme Res. Labs., West Point, PA USA. bone cell stimulate Glucocorticoids vitro but inhibit bone differentiation in formation in vivo. Osteopontin (Sppl, 2ar. pp69) is a cell attachment protein highly expressed in differentiated osteoblasts (OB). The object of this study was to examine the effect of dexamethasone (dex) in cell lines which express 08 features to different degrees. In ROS 17/2.8 cells, which express many osteoblastic features, In calvariadex inhibited Sppl expression. derived RCT3 cells, which express many but not all 08 features (lack osteocslcin for example), both dex and 1,25(OHjzD, (VP) increased Sppl- mRNA levels and had additive effects. In calvariaderived RCTl cells, which have no 08 features unless treated with retinoic acid (RA), dex and VD alone had verv small effects but tooether they increased Sppl &NA synergistically. -TRAB-11 is an immortalized clonal bone-derived cell line from tibia of adult rats with high levels of type I collagen (COLI) mRNA, low levels of Sppl mRNA and RA inducible alkaline phosphatase (API. Dex increased Sppl mRNAin these cells SO-100 fold in In the time and dose-dependent zresence of dex (30 nM) in se~%n~~plete medium the cells became cuboidal and had well-defined refractile borders. Dex had no effect on AP or COL I mRNA, while RA, phorbol esters and VD had no effect on Sppl mRNA in these cells. These findings, which-iuggestdifferentiation-dependent effects of dex on osteoblastic cells, could reconcile the differences between in vitro and in vivo effects. They also illustrate the need for multiple agents (RA, VD, glucocorticoids) for full expression of the osteoblastic phenotype.
39 OSTEOCLAST-MATRIX INTERACTIONS Br, AND 8, INTEGRIN CHAINS ARE COEXPRES&D ON FOCAL ADHESION OVER asp11 AND FIBRONECTIN COATED M.Grano, s.c01ucci. F.Zigrino, COVERSLIPS. M.Serra, K..Scotlandi. G.Zambonin. P.C.Marchisi.0, A.Teti, A.Zambonin Zallone. Isti!.utodi Anatomia Umana, Universita' di Elari . Human osteoclast-like cells harvested from giant cell tumours of bsne (GCT), ehardcterized on the basis of multinucleation. TRAP activity, bone resorbing capability and response to calciotropic hormones have been utilized to' study adhesion and integrin expression on Several matrix proteins. GCT cells, detached from culture dishes, were plated for 6 hrs in absence of serum onto coverslips coated with specific proteins: BSPII, Fibronectin (FN), collagen and osteocalcin; serum and BSA were the positive and negative controls. The adhesion percentage was as high as serum on BSPII and FN; was slightly reduced on osteocalcin and limited to around 507. of cells on collagen. Focal adhesions, absent on osteocalcin, were present on all the other RGD sequence containing substrata . 0% integrin chain was expressed in focal adhesion on collagen. FN and BSPII. while l3~chain was diffuse in cells plated on collagen and colocalized with 13, in focal adhesion on FN and BSPII. In all the conditions tested ctzwas absent and a. strongly stained a granular perinuclear area. Thus the identity of the o chain linked to the 13~ has not identified. The presence of two integriKEtinbEEg same focal adhesion has already been described in endothelial and melanoma cells and can be linked to the peculiar migration capability of osteoclasts. Preliminary observations confirmed these data also in podosomes of non tumoral human osteoclasts.
expression of OP mRNA. RA also increased the transcriptional rate of
the OP gene but rhis rise was opposed by the addition of TGFp or BMP4. TGFD increased ON and MGP mRNA exnression but no additive effeci wasobserved when addedtoRA. In’contrast, BMP3 alone had no influence on ON or MGP mRNA exoression but it significantly enhanced theRA induction of MGP mRN’A. Both TGFp and BMP4 opposed the RA induced increase in ALP activity which correlated with a fall in ALP mRNA levels. Measurement of mRNA stability showed thnt BMW selectively increased thedegmdiltion of ALP butnot OP mRNA. Inconclusion, the study has demonstrated differences between the actions of TGFD and BMW in UMR 201 cells. This lends suooort to rhe concept that coordinated expression of members of th;‘TGFP superfamily may be necessary to control the progression of specific cell types through their differentiation pathways in an autocrine orparacrine manner.
79
BIOSYNTHETIC P
OCUSTIC CELLS TO
LLS
IN
DIFFERENTIAL EFFECTS OF TGF l3ANC 3MP4 ON THE GENE EXPRESSION AND ;;FFERENTIATED FUNCTION OF PRE-
TI-IE
OSTEOBL&5TIC LIN of Dental Research an Universitv of Florence. Cells within the osteoblastic linea e express numerous proteins that contain Asp-Gly-Arg (RGD), t ce sequence that binds to cell membrane associated mtegrins, and secrete them at particular sta es during the maturational sequence both LZ wivo and in vitro. 8 ubsequent to serving their as yet unidentified roles in osteoblastic metabolism, these proteins are entombed within mineralized matrix, but most likely still function in the recruitment and maturation of osteoclasts and their recursors. FLG ?9._l cells, cloned from a patient with acute leu r:emia, fuse ayd exhlblt many osteoclastic traits followin treatment with lr M 12-O. tetradecanoylphorbol I3-acetate 8-P A). In this study, FLG 29.1 cell attachment to several RGD-containing bone matrix proteins (serum-free conditioned medium from untreated adult human p:eosteoblasts SFCM), thrombos ondin (TSP), fibronectin (FN) vitronectin ( &i ), osteopontin (Of) and bone sialoprotein (BSP) all at 0.2 IBM dotted onto bacteriological plastic) was investigated after 48 hrs of incubation with and wlthout TPA. With TPA, there was a marked increase in cell attachment (BSl:, OP > FN, VN > TSP, SFCM) compared to tissue culture plastic; however, fusion % of total attached cells) was not markedly enhanced. ‘Without 4=FA, the cells coptinued to proliferate and there was virtually no attachment or f&Ion on plastic or SFCM. IJ-Iowever, cells adhered to TSP. and ap eared to Initiate fusionon other proteins (BSP. QP > FN, VN). Pnterestingly, SFCM, which contains TSP, FN and VN and strongly supports bone-formin cell attachment, is considerably less active in mediating FL 6 29.1 cell attachment than the individual roteins. This dab suggests the secretion of a potential inhibitor Ps) by preosteoblasts, which had not been stimulated by factors known to increase bone resorption. These studies indicate that in this preostaoclast model system, products of cells at various sta es of maturation in the osteoblastic lineage support attachment. a owever, fusion events may be mediated by products expressed at late stages of osteoblastic differentiation.
OSTBOBLASTS. I-l. OU.*R.G. Hammonds Jnr. D.M.Findlnv, T.J Martin and K.W. Ng. Depanment of Medicine, The University of Melbourne, St. Vincent’s Hospital. Melbourne, Australia and *Genentech Inc.. San Francisco, CA, USA TGFp and BMP4 share the ability fo induce endochondral bone formation in vivo. This study examines the effects of recombinant TGFp and BMP4 in a retinoic acid (RA) responsive rat clonal.preosteoblast ceil line, UMR 201. These cells express themRNA for BMP 1.2and 3 as well as u, p and ‘I RA receptors. They respond to lo-6M RA with a significant increase in the steady state levels of mRNA for pro-al(I) collagen, osteopontin (OP). osteonectin (ON), matrix gla protein (MGP) and alkaline phosphatase (ALP). Pra al(I) collagen mRNA was increased by TGFp (1 @ml) and BMW (50 @ml). The addition of either BMP4 or TGF p to RA resulted in a further increase in pro-al(l) collagen mRNA levels. Nuclear run-on experiments showedthat RA and TGF 0 but not BMP4 increased the transcriptional rate of the pro-al(l) collagen gene. Both TGFp and BMW reduced the constitutive as well as RA-induced
36 DIFFERENTIATION-DEPENDENT EFFECTS OF DEXAMETHASONE ON OSTEOPONTIN mRNA EXPRESSION IN BONE-DERIVED CELLS. S.B. Rodan, E.E. Ooas, S. Harada and G.A. Rodan. Merck Sharp & Dohme Res. Labs., West Point, PA USA. bone cell stimulate Glucocorticoids vitro but inhibit bone differentiation in formation in vivo. Osteopontin (Sppl, 2ar. pp69) is a cell attachment protein highly expressed in differentiated osteoblasts (OB). The object of this study was to examine the effect of dexamethasone (dex) in cell lines which express 08 features to different degrees. In ROS 17/2.8 cells, which express many osteoblastic features, In calvariadex inhibited Sppl expression. derived RCT3 cells, which express many but not all 08 features (lack osteocslcin for example), both dex and 1,25(OHjzD, (VP) increased Sppl- mRNA levels and had additive effects. In calvariaderived RCTl cells, which have no 08 features unless treated with retinoic acid (RA), dex and VD alone had verv small effects but tooether they increased Sppl &NA synergistically. -TRAB-11 is an immortalized clonal bone-derived cell line from tibia of adult rats with high levels of type I collagen (COLI) mRNA, low levels of Sppl mRNA and RA inducible alkaline phosphatase (API. Dex increased Sppl mRNAin these cells SO-100 fold in In the time and dose-dependent zresence of dex (30 nM) in se~%n~~plete medium the cells became cuboidal and had well-defined refractile borders. Dex had no effect on AP or COL I mRNA, while RA, phorbol esters and VD had no effect on Sppl mRNA in these cells. These findings, which-iuggestdifferentiation-dependent effects of dex on osteoblastic cells, could reconcile the differences between in vitro and in vivo effects. They also illustrate the need for multiple agents (RA, VD, glucocorticoids) for full expression of the osteoblastic phenotype.
39 OSTEOCLAST-MATRIX INTERACTIONS Br, AND 8, INTEGRIN CHAINS ARE COEXPRES&D ON FOCAL ADHESION OVER asp11 AND FIBRONECTIN COATED M.Grano, s.c01ucci. F.Zigrino, COVERSLIPS. M.Serra, K..Scotlandi. G.Zambonin. P.C.Marchisi.0, A.Teti, A.Zambonin Zallone. Isti!.utodi Anatomia Umana, Universita' di Elari . Human osteoclast-like cells harvested from giant cell tumours of bsne (GCT), ehardcterized on the basis of multinucleation. TRAP activity, bone resorbing capability and response to calciotropic hormones have been utilized to' study adhesion and integrin expression on Several matrix proteins. GCT cells, detached from culture dishes, were plated for 6 hrs in absence of serum onto coverslips coated with specific proteins: BSPII, Fibronectin (FN), collagen and osteocalcin; serum and BSA were the positive and negative controls. The adhesion percentage was as high as serum on BSPII and FN; was slightly reduced on osteocalcin and limited to around 507. of cells on collagen. Focal adhesions, absent on osteocalcin, were present on all the other RGD sequence containing substrata . 0% integrin chain was expressed in focal adhesion on collagen. FN and BSPII. while l3~chain was diffuse in cells plated on collagen and colocalized with 13, in focal adhesion on FN and BSPII. In all the conditions tested ctzwas absent and a. strongly stained a granular perinuclear area. Thus the identity of the o chain linked to the 13~ has not identified. The presence of two integriKEtinbEEg same focal adhesion has already been described in endothelial and melanoma cells and can be linked to the peculiar migration capability of osteoclasts. Preliminary observations confirmed these data also in podosomes of non tumoral human osteoclasts.
expression of OP mRNA. RA also increased the transcriptional rate of
the OP gene but rhis rise was opposed by the addition of TGFp or BMP4. TGFD increased ON and MGP mRNA exnression but no additive effeci wasobserved when addedtoRA. In’contrast, BMP3 alone had no influence on ON or MGP mRNA exoression but it significantly enhanced theRA induction of MGP mRN’A. Both TGFp and BMP4 opposed the RA induced increase in ALP activity which correlated with a fall in ALP mRNA levels. Measurement of mRNA stability showed thnt BMW selectively increased thedegmdiltion of ALP butnot OP mRNA. Inconclusion, the study has demonstrated differences between the actions of TGFD and BMW in UMR 201 cells. This lends suooort to rhe concept that coordinated expression of members of th;‘TGFP superfamily may be necessary to control the progression of specific cell types through their differentiation pathways in an autocrine orparacrine manner.
79