The nature and tissue distribution of non-collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and beta-glycerophosphate (beta-GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX-treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP-1), type I collagen, SPARC, and alkaline phosphatase was stimulated 6-, 5-, 3-, and 2.5-told, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP-1 and BSP) were mostly secreted into the culture medium in the non-mineralizing (-beta-GP) cultures, but were the predominant non-collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+ beta-GP). Extraction of the tissue matrix with 4 M GuHCl and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non-covalently and covalently with the collagenous matrix. SPP-1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de-mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP-1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP-1 incorporating significantly more [32PO4] than the 55 kDa SPP-1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non-collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.

Abstract. To determine the effects of transforming growth factor-~ (TGF-[3) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-I~ did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two-to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-13, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-~ stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-13, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-13 on bone cell populations are likely to be important in bone remodeling and fracture repair.T RANSFORMING growth factor-I] (TGF-l]) ~ is a 25-kD dimeric polypeptide that can affect the growth, differentiation, and activity of a variety of different cell types (17,32). Although present in most normal and transformed cells studied, TGF-l] is stored in relatively high amounts in platelets, suggesting a role for this growth factor in wound healing (1). TGF-[3 is also concentrated in bone, where it was originally identified as a cartilage-inducing factor (29) that exists in two forms, CIF-A and CIF-B. Recently, two major forms and a minor form of porcine TGF-l] have been isolated (5). The major forms are distinct homodimers, whereas the minor form is a heterodimer. Amino acid sequence data indicate that the major forms of TGF-l] correspond to CIF-A and CIF-B (30).TGF-l] promotes a wound healing response in vivo that is characterized by increased cellular proliferation and protein synthesis (25,31). Studies of connective tissue cells in vitro have demonstrated that TGF-[3 stimulates the synthesis of matrix protein including collagen and fibronectin (7, 9, 25, 1. Abbreviations used in this paper: SPARC, secreted protein acidic rich in cysteine; TGF-13, transforming growth factor-13. 39). In studies of bone, TGF-l] has been shown to stimulate bone resorption (...

To characterize the bone-like tissue produced by rat bone marrow cells (RBMC) from young adult femurs, the synthesis of bone proteins and the expression of their mRNA were studied in vitro. RBMC plated at a density of 5 x 10(3) cells/cm2 and grown in the presence of 10(-8) M dexamethasone (Dex) and 10 mM beta-glycerophosphate (beta-GP) produced mineralized bone nodules, which were first evident at day 3 and increased markedly to day 13. However, in the absence of dexamethasone, few mineralized nodules were observed. The formation of mineralized nodules was reflected by the uptake of 45Ca, which also increased markedly to day 13. Analysis of bone protein expression by Northern and slot-blot hybridizations revealed an increase in mRNA levels of collagen type I (Col I), osteonectin/SPARC (ON), alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OC) during the formation of mineralized nodules. Whereas the Col I, ON, ALP, and OPN mRNAs were expressed before the formation of mineralized nodules was evident and were also expressed at various levels in the absence of Dex, the expression of BSP and OC mRNA was induced in the bone-forming cultures. The expression of BSP mRNA was correlated temporally with bone tissue formation, reaching maximal levels on day 16. In contrast, OC mRNA was expressed later and, following induction, increased over the 28 day culture period. Production of matrix proteins during the rapid formation of the bone tissue appeared to reflect the levels of the respective mRNAs. However, whereas some of the collagen and almost all of the SPARC were secreted into the culture medium, virtually all of the OPN and most of the BSP were extracted from the mineralized tissue matrix with EDTA. Some OPN and BSP were present in the medium, especially early in the culture, and a significant amount of BSP was also found associated with the collagenous tissue matrix. These studies point to the importance of Col I, ALP, OPN, and BSP, but not ON or OC, in the initial formation of bone tissue.

Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine-HCl (GuHCl) and 4 M GuHCl - 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS-PAGE with a relative mass approximately 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented approximately 0.5% of the total labelled proteins secreted. It comigrated on SDS-PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.

Previous studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982]. Several of these clonal populations have been used to study the effects of long-term culture and inter- and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively alpha 1(I)-trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1-2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long-term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter- and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.

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A number of bone cell clones isolated from rat calvaria have been maintained in culture for more than 3 years. Several of these clones have undergone dramatic changes in phenotype. One of these clones, RCB 2.2, was observed originally to have a fibroblastic morphology in culture and to respond to parathyroid hormone (PTH), but not prostaglandin E2 (PGE2), with an increase in intracellular cAMP. Throughout several passages in early subcultures, these cells synthesized mostly type I collagen, with small amounts of type III and type V collagens. Whereas PTH had no detectable effect on collagen synthesis, PGE2 decreased the amount of total cell layer collagen, with the greatest effect on type III collagen, while increasing the proportion of type V collagen. Subsequent studies on these cells during 3 years in culture have indicated changes in their phenotype including a progressive change in morphology to a more cuboidal shape and a change in collagen synthesis, the cells producing large amounts of the "embryonic" collagen, alpha 1(I) trimer. The reason(s) for the change in collagen expression is unknown, but may be the result of a change in which gene(s) is being expressed.

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