Basic fibroblast growth factor (bFGF) may be involved in the development and repair of dentine and pulp because bFGF, its related peptides, and FGF receptors are expressed in dental mesenchymal cells. In this study, we examined the effects of bFGF on DNA synthesis, osteonectin/SPARC levels, alkaline phosphatase (ALPase) activity, their mRNA levels, and calcium levels in cultures of human pulp cells. Pulp cells were isolated from three healthy upper wisdom teeth of three patients and maintained separately. These cells produced SPARC, ALPase, and calcified nodules and there was a close correlation between the SPARC-synthetic activity of the cell lines and their levels of ALPase and calcification. The levels of SPARC, ALPase and calcium deposits in the three pulp cell cultures were 10-250 times those of human foreskin fibroblasts. Western blots showed that the pulp cells produced 38-kDa SPARC. Northern blots showed that the pulp cells expressed flg (FGF receptor type 1) transcripts throughout all culture stages, irrespective of the presence or absence of bFGF. The addition of bFGF to the pulp cultures suppressed the increases in ALPase activity, SPARC synthesis, and their mRNA levels, although it increased the incorporation of [3H]thymidine into DNA> 10-fold. The effects of bFGF on ALPase activity and SPARC synthesis were reversible. Furthermore, bFGF abolished the calcification of the extracellular matrix; the calcium content of bFGF-free cultures. These findings suggest that bFGF is a potent mitogen for human pulp cells and that it inhibits the expression of the odontoblast phenotype by the cells at least partly at pretranslational levels.
Objective. To investigate the roles of SPARC (secreted protein, acidic and rich in cysteine) (osteonectin) in arthritis, using cartilage and synovium specimens and synovial fluids (SF) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), and to examine the effects of cytokines, growth factors, and hormones on SPARC synthesis by chondrocytes in culture.Methods. SPARC in cartilage and synovium was immunostained with monoclonal antibodies. SPARC synthesis by cultured chondrocytes was measured by Northern blot analysis, immunoblotting, and sandwich enzyme-linked immunosorbent assay.Results. SPARC was identified in numerous chondrocytes in the superficial and middle zones and in Shigeo Nakamura, DDS, Hisashi Satakeda, DDS, Hiroshi Okamoto, DDS, PhD, Mitsuhide Noshiro, PhD, Yukio Kato, DDS, PhD: Hiroshima University, Hiroshima, Japan; Kyouko Kamihagi, PhD, Masahiko Katayama, PhD: Biotechnology Research Laboratones, Takara Shyuzo, Ohtu, Japan; Haiou Pan, DDS: Drug Discovery Research Laboratories, Hoechst Japan Limited, Kawagoe, Japan; Koichiro Takahashi, PhD: Clinical Pharmacology Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Itabashi-ku, Tokyo, Japan; Yasuo Yoshihara, MD, Masayuki Shimmei, MD: National Defense Medical College, Tokorozawa, Japan; Yasunori Okada, MD, PhD: Cancer Research Institute, Kanazawa University, Kanazawa, Japan.Address reprint requests to Yukio Kato, DDS, PhD, Department of Biochemistry, School of Dentistry, Hiroshima University, 1-2-3, Kasumi, Minamiku, Hiroshima City, 734, Japan.Submitted for publication May 11, 1995; accepted in revised form November 6, 1995.regenerating chondrocytes of RA and OA joints, whereas such staining was absent in these zones of normal cartilage, except For weak signals from a few chondrocytes in the deep zone. In addition, SPARC synthesis was enhanced in synovial cells of RA and OA joints. The average SPARC level in SF was 10-fold higher in the RA than in the OA population. In rabbit articular chondrocyte cultures, administration of transforming growth factor pl (TGFp1) and bone morphogenetic protein 2 increased SPARC levels at 24-48 hours, whereas interleukin-lp (IL-lp), IL-la, tumor necrosis factor a, lipopolysaccharide, phorbol myristate acetate, basic fibroblast growth factor, and dexamethasone decreased SPARC levels at 2&72 hours. TGFP increased SPARC messenger RNA (mRNA) levels at 24 hours, whereas IL-IP caused a marked decrease in SPARC mRNA levels at 24 hours. Furthermore, IL-1 decreased the glycosylation of SPARC. Conclusion. These findings suggest that various growth factors and cytokines, including TGFm and IL-lp, regulate the production of SPARC by chondrocytes at pre-and posttranslational levels, and that SPARC synthesis is markedly enhanced in arthritic joints.SPARC (secreted protein, acidic and rich in cysteine) (osteonectin) (BM-40) is a 3543-kd Ca++-binding glycoprotein, which was initially isolated from 539
A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.
The roles of PTH and PTH-related peptide (PTH-rp) in the expression of matrix metalloproteinases (MMPs) during endochondral bone formation were investigated, using various cartilages obtained from young rabbits and rabbit chondrocyte cultures. Immunohistochemical, immunoblotting, zymographical, and/or Northern blot analyses showed that MMP-2 and -9 levels were much higher in the growth plate than in permanent cartilage in vivo. In growth plate chondrocyte cultures, PTH, PTH-rp, and (Bu)2cAMP increased the amount of MMP-2 present in the culture medium, as revealed by zymograms and immunoblots, whereas the other tested growth factors or cytokines, including bone morphogenetic protein-2 and interleukin-1, did not increase the MMP-2 level. PTH also increased the MMP-2 messenger RNA level within 24 h. In addition, PTH increased MMP-3 and -9 levels in the growth plate chondrocyte cultures. However, in articular chondrocyte cultures, PTH had little effect on the levels of MMP-2, -3, and -9. In contrast to PTH, interleukin-1 induced MMP-3 and -9, but not MMP-2, in growth plate and articular chondrocytes. These findings suggest that in ossifying cartilage, PTH/PTH-rp plays a pivotal role in the induction of various MMPs, including MMP-2 (which is considered to be a constitutive enzyme), and that PTH/PTH-rp is involved in the control of cartilage-matrix degradation during endochondral bone formation.
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