ISSN:0884-0431    

DOI:10.1002/jbmr.5650110602

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110603

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractWe studied the endochondral ossification that occurs during the transition of soft to hard callus during fracture healing in the rabbit. During this process, parts of the cartilaginous soft callus are invaded by capillaries, and new bone is laid down onto the central unresorbed cartilage struts. We found that the chondrocytes within these cartilage struts changed phenotype and became bone‐forming cells which directly replaced the central cartilage core with bone matrix. We have termed this bone “lacunar” bone to distinguish it from the “vascular” bone laid down by osteoblasts. With time the lacunar bone spread beyond the confines of the lacunae and gradually replaced all the cartilage matrix that was originally present in the early endochondral spicules. The lacunar bone could still be distinguished from the vascular bone as follows: (1) it was woven bone, whereas vascular bone was lamellar bone; (2) it contained acid phosphatase activity, whereas vascular bone did not; and (3) it had strong antigenicity for bone sialoprotein, whereas this noncollagenous protein was undetectable in vascular bone. Eventually the hard callus was resorbed and remodeled, but at an interim period of endochondral ossification the direct replacement of cartilaginous callus by the formation of lacunar bone is a rapid mechanism by which the mechanical strength of fracture callus is

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110604

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractThe aim of this study was to investigate the mechanism by which translation of parathyroid hormone (PTH) mRNA is regulated with regard to the subcellular distribution of PTH mRNA and RNA:protein interactions. Sucrose density ultracentrifugation of RNA from bovine parathyroid cells indicated that there was no evidence for a pool of nonribosomal PTH mRNA, and the extracellular calcium concentration had no effect on polysome size. UV cross‐linking studies revealed two proteins in parathyroid cell cytosol which bound specifically to the 5′‐untranslated region (UTR) of PTH mRNA with molecular masses of 66 and 68 kD while proteins with apparent molecular masses of 48 and 70 kD bound to the 3′‐UTR. In vitro translation assays indicated that parathyroid cell cytosol contains factors that inhibit translation of PTH mRNA. Fractionation of cytosol revealed that this effect was associated with proteins within the molecular mass range 30–90 kD. To determine which sequences in PTH mRNA mediate translational regulation, RNA was synthesized from luciferase gene constructs containing the 5′‐and/or 3′‐UTR of PTH mRNA, and translated in vitro. Addition of parathyroid cell cytosol reduced the translation of RNA containing the 5′‐ and 3′‐UTR of PTH mRNA by 44 + 7% but had no effect on the translation of RNA containing only the luciferase coding region. Translation of RNA containing only the 5′‐UTR of PTH mRNA was unchanged; however, cytosol reduced the translation of RNA containing the 3′‐UTR by 31 + 9%. These data demonstrate a role for RNA:protein interactions in the regulation of PTH synthesis and that translational control is mediated primarily through inter

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110605

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractWe have previously reported that parathyroid hormone (PTH) and PTH related protein (PTHrP) stimulate expression of interleukin‐6 (IL‐6) and leukemia inhibitory factor (LIF) in osteoblasts in vitro. In the current study, we have developed a model of hormone injection into the subcutaneous space overlying mouse parietal bones to demonstrate that similar processes occur in osteoblasts in vivo. Specifically, PTH and PTHrP rapidly and transiently induce expression of the mRNAs encoding IL‐6 and LIF. The effects are dose‐dependent, with a maximal stimulation of approximately 50‐fold for each cytokine. Although PTH and PTHrP activate both adenyl cyclase and phospholipase C‐dependent signal transduction pathways, stimulation of IL‐6 and LIF depends on adenyl cyclase since it is not reproduced by PTH(3–34), a partial agonist that only activates phospholipase C. These results confirm our previous in vitro studies and support the hypothesis that IL‐6 and/or LIF are physiologically important mediators of at least some of the actions

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110606

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractWe determined whether basic fibroblast growth factor (bFGF) regulated the expression of IL‐6 and NFIL‐6 in osteoblasts. In mouse osteoblastic MC3T3‐E1 cells, bFGF (10−8M) increased NFIL‐6 mRNA 2‐fold at 30 minutes and 3‐fold at 2 h. IL‐6 mRNA was increased by bFGF 10−8M after 1 h. IL‐6 protein was detectable in control cultures but was significantly increased by bFGF (10−8M) at 4 h. Immunofluorescence analysis of MC3T3‐E1 cells showed primarily cytoplasmic and perinuclear NFIL‐6 staining in control cultures while bFGF‐treated cells showed increased NFIL‐6 staining at 2 and 4 h. Western blotting revealed that bFGF increased NFIL‐6 protein at 2 h. In calvarial mouse osteoblasts, bFGF 10−8M induced IL‐6 mRNA as early as 1 h and significantly increased IL‐6 protein levels as early as 2 h. In conclusion, bFGF stimulates IL‐6 and NFIL‐6 mRNA in osteoblasts. The increase in NFIL‐6 mRNA was associated with increased NFIL‐6 protein. The increase in IL‐6 mRNA was also associated with increased IL‐6 protein. We propose that activations of NFIL‐6 and IL‐6 may be imp

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110607

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractFollowing their terminal differentiation, highly specialized cells, ameloblasts, odontoblasts, and osteoblasts sequentially elaborate mineralized tissues. While the developmental expression pattern of matrix proteins has been studied extensively, less attention has been paid to the molecules involved in calcium handling, such as calcium‐binding proteins. This shortcoming, as well as previous conflicting data, led us to conduct studies on calbindin‐D9kand calbindin‐D28kin rat mandibular bone and incisor based on several methods established on rat ameloblasts in vivo. Radioimmunoassays showed that calbindin‐D28kaccounts for approximately 0.1% of cytosolic proteins in the ectomesenchymal fraction and 1% in the epithelial fraction of the rat incisor and is 100‐fold more concentrated than calbindin‐D9kin both tissue types. Western blot analysis confirmed that the anticalbindin‐D28kreactive species corresponded to the well characterized renal calbindin‐D28kin the ectomesenchyme. In this tissue, calbindin‐D28kwas ultrastructurally immunolocalized in the odontoblasts. Quantitative immunocytochemistry showed that labeling was distributed throughout their nucleus and cytoplasm. The similar cytoplasmic distribution of both calbindin‐D proteins and mRNAs suggests that their expression is regulated at the subcellular level. In particular, immunoreactive calbindin‐D28kappeared to be associated with rough endoplasmic reticulum. Calbindin‐D9kantisense probe showed negligible labeling in odontoblasts, in parallel with the protein quantities measured (∼10 ng/mg of total protein). Finally, in situ hybridization showed transcripts for both calbindins‐D in ameloblasts and also in osteoblasts. In summary, the present results support the concept that an elevated expression of these vitamin D–dependent calcium‐binding proteins may characterize the phenotype of cells directly involved in the elaboration of mineralized tis

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110608

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractTwelve‐week‐old female rats were ovariectomized (OVX) and compared with sham‐operated control rats at 3, 5, 7, 10, 14, 30, and 60 days postoperation with respect to the expression of type I collagen and osteopontin mRNAs, as well as bone structure and the number of osteoclasts. The trabecular number and separation were significantly decreased and increased, respectively, in the metaphyseal trabecular bones of OVX rat femurs. The number of osteoclasts was significantly increased in the same region of OVX rats at 3 and 5 days postoperation. Type I collagen mRNA was expressed in osteoblasts, and osteopontin mRNA was expressed in some osteoclasts, in mononuclear cells on the bone resorption surface, and in osteocytes near the resorption surface. In the metaphyseal trabecular bone, type I collagen and osteopontin mRNA expression levels in individual cells was initially increased in OVX rats from 7 to 10 days postoperation, and this was sustained for 60 days. The number of osteopontin mRNA‐expressing osteocytes was also significantly increased at 10 days postoperation, which lasted until 60 days. In the epiphysis, an increase in type I collagen mRNA expression was initially observed in OVX rats at 14 days postoperation, which lasted until 60 days. The number of osteopontin mRNA‐expressing osteocytes was virtually identical until 30 days postoperation in the epiphysis. These findings indicated that the biological activities of osteoblasts and osteocytes are stimulated in bones of the OVX rat and that the response for OVX differs between the metaphysis and epiphysis. Furthermore, the number of osteopontin mRNA‐expressing osteocytes was increased only in bones that tended to be resorbed after OVX. This indicates that some osteocytes were stimulated to express osteopontin mRNA by estrogen deficiency and suggests that these osteopontin mRNA‐expressing osteocytes may be involved in regulation of bo

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110609

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractExtracellular cations have paradoxical trophic and toxic effects on osteoblast function. In an effort to explain these divergent actions, we investigated in MC3T3‐E1 osteoblasts if polyvalent cations differentially modulate the agonist‐stimulated cyclic adenosine monophosphate (cAMP) pathway, an important regulator of osteoblastic function. We found that a panel of cations, including gadolinium, aluminum, calcium, and neomycin, inhibited prostaglandin E1(PGE)‐stimulated cAMP accumulation but paradoxically potentiated parathyroid hormone (PTH)‐stimulated cAMP production. In contrast, these cations had no effect on forskolin‐ or cholera toxin–induced increases in cAMP, suggesting actions proximal to adenylate cyclase and possible modulation of receptor interactions with G proteins. Phorbol 12‐myristate 13‐acetated (PMA) mimicked the effects of cations on PGE1‐and PTH‐stimulated cAMP accumulation in MC3T3‐E1 cells, respectively, diminishing and augmenting the responses. Moreover, down‐regulation of protein kinase C (PKC) by overnight treatment with PMA prevented gadolinium (Gd3+) from attenuating PGE1‐ and enhancing PTH‐stimulated cAMP production, indicating involvement of PKC‐dependent pathways. Cations, however, activated signal transduction pathways not coupled to phosphatidylinositol‐specific phospholipase C (PI‐PLC), since there was no corresponding increase in inositol phosphate formation or intracellular calcium concentrations. In addition, pertussis toxin treatment failed to prevent Gd3+‐mediated suppression of PGE1‐Stimulated cAMP, suggesting actions independent of Gαi. Thus, polyvalent cations may either stimulate or inhibit hormone‐mediated cAMP accumulation in osteoblasts. These differential actions provide a potential explanation for the paradoxical trophic and toxic effects of cations on osteoblast function that occur in

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110610

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY

摘要:

AbstractOsteopenia due to deficient extracellular matrix synthesis is a hallmark of osteogenesis imperfecta (OI). Previous studies carried out within 72 h of osteoblast subculture, at an early stage of matrix synthesis, indicated that for osteoblasts derived from human OI patients the total amounts of collagen, osteonectin, and three proteoglycans were significantly reduced, while total amounts of thrombospondin, fibronectin, and matrix hyaluronan were elevated compared with age‐matched controls. The current study was undertaken to follow OI osteoblast matrix metabolism as that matrix is synthesized, deposited, and matured. Steady‐state metabolic radiolabeling was used to follow the metabolism of collagen, hyaluronan, and total proteoglycan by OI and normal osteoblasts for up to 5 weeks. Trabecular osteoblasts from non‐OI controls showed an increase in total and matrix‐associated collagen synthesis during the first and second week, reaching steady‐state levels by week 4. In contrast, cultured OI osteoblasts did not increase either the total (medium + matrix‐associated) or matrix‐associated collagen during the entire 5‐week period. Proteoglycan synthesis exhibited a pattern similar to that for collagen. OI‐derived proteoglycans differed from controls in that levels in OI cultures did not reflect the normal time‐dependent increase in total proteoglycan and proteoglycan matrix deposition. OI osteoblast hyaluronan synthesis was increased when compared with age‐matched controls during 4 weeks of culture. In contrast, the ratios of calcium to phosphorus solublized from control and the OI extracellular matrix were not statistically different. Thus, with respect to the synthesis of collagen, proteoglycans, and hyaluronan, OI osteoblasts fail to parallel controls in depositing and elaborating extracellular matrix duri

ISSN:0884-0431    

DOI:10.1002/jbmr.5650110611

出版商:John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR)    

年代:1996

数据来源: WILEY