Abstract. The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle ceils and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells, rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of lot,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type UI in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-fll inhibited myogenic differentiation. However, unlike BMP-2, TGF-fll decreased ALP activity in both C26 and C20 cells. TGF-/$1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.CTOPIC bone formation is elicited at intramuscular sites by implantation of bone inducing factors contained in demineralized bone matrix (28,35,36). This indicates that cells of the osteoblast lineage have a close relationship with those of the muscular lineage in their ontogeny, and the development of the two cell lineages may be mutually regulated by some factor(s) stored in bone matrix. The components and the action mechanism of these bone inducing factors had long remained obscure. This is mainly due to the difficulties of purification of these factors and the lack of suitable in vitro bioassay systems.Recently, a number of laboratories have isolated bioactive proteins which induce cartilage and/or bone formation at the sites implanted (1,13,30,39,40). Human cDNAs for seven different bone morphogenetic proteins (BMPs), t BMP-1, BMP-5, BMP-6 (Vgr-1 [141), and BMP-7 (OP-1125, 30]) have been cloned (41,42). The sequences deduced from these cDNAs have indicated that BMP-2 through BMP-7 are members of transforming growth factorfl (TGF-fl) superfamily (14,25,41,42). Furthermore, active recombinant human bone mor...

Accumulation of advanced glycation end products (AGEs) on nucleotides, lipids, and peptides/proteins are an inevitable component of the aging process in all eukaryotic organisms, including humans. To date, a substantial body of evidence shows that AGEs and their functionally compromised adducts are linked to and perhaps responsible for changes seen during aging and for the development of many age-related morbidities. However, much remains to be learned about the biology of AGE formation, causal nature of these associations, and whether new interventions might be developed that will prevent or reduce the negative impact of AGEs-related damage. To facilitate achieving these latter ends, we show how invertebrate models, notably Drosophila melanogaster and Caenorhabditis elegans, can be used to explore AGE-related pathways in depth and to identify and assess drugs that will mitigate against the detrimental effects of AGE-adduct development.

It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and increases bone mass in rats when injected in vivo. Immunoreactive OGP in high abundance is present physiologically in the serum, mainly in the form of an OGP‐OGP binding protein complex. A marked increase in serum bound and unbound OGP accompanies the osteogenic phase of post‐ablation marrow regeneration and associated systemic osteogenic response. Authentic OGP is identical to the C‐terminus of histone H4 and shares a five residue motif with a T‐cell receptor beta‐chain V‐region and the Bacillus subtilis outB locus. Since these latter proteins have not been implicated previously in the control of cell proliferation or differentiation, OGP may belong to a novel, heretofore unrecognized family of regulatory peptides. Perhaps more importantly, OGP appears to represent a new class of molecules involved in the systemic control of osteoblast proliferation and differentiation.

No abstract

No abstract

HL-60, a cell line established from a patient with promyelocytic leukaemia, responds to a variety of inducing agents by ceasing division and acquiring some of the characteristics of either granulocytes or monocytes. Among the agents so far tested, only a comparative few occur naturally in vertebrates and would appear to have significant clinical potential in the treatment of leukaemic patients. One of the most promising of these is the dihydroxymetabolite of vitamin D3, 1,25(OH)2D3. This compound circulates in normal man and has a major role in calcium homeostasis. Moreover, it has recently been reported that 1,25(OH)2D3 increases the survival time of mice injected with myeloid leukaemia cells. We and McCarthy et al. have previously shown that HL-60 cells respond to near physiological levels of 1,25(OH)2D3 by rapidly acquiring a number of monocyte-like features. Here we document that these phenotypic changes are preceded by a marked decrement in the expression of the c-myc oncogene. In fact, the diminution in the level of c-myc mRNA parallels the dose dependency and metabolite specificity shown by the various other indicators of phenotypic change. In addition, we demonstrate that removal of vitamin D3, after the onset of maturational change, results in the reappearance of elevated myc mRNA levels. We believe this to be the first demonstration of a sequential relationship between the application of an exogenous inducing agent, a reduction in myc mRNA levels and the development of characteristics associated with normal cell maturation.

1,25-Dihydroxyvitamin D3 [1,25(0H)2D3] stimulates bone resorption in man and other vertebrates, in part, by increasing the number of osteoclasts, the principal resorbing cells of bone. Because osteoclasts are very likely derived from a member(s) of the mononuclear phagocyte family, we determined if 1,25(OH)2D3 promotes maturation of these cells by studying its effects on the human promyelocytic leukemia cell line HL-60. Of the vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10-10 to 10-7 M, induces the differentiation of HL60 into mono-and multinucleated macrophage-like cells. Phenotypic change is evident within 24 hr and reaches a plateau between 72 and 96 hr of incubation. The changes are metabolite-specific and include (i) adherence to substrate, (ii) acquisition of the morphological features of mature monocytes, (iii) a 4-to 6-fold enhancement in lysozyme synthesis and secretion, (iv) increase in the fraction of a-naphthyl acetate esterase-positive cells from approximately 2% to 100% of the population, and (v) the acquisition of several monocyte-associated cell surface antigens. More importantly, treated HL-60 cells acquire the capacity to bind and degrade bone matrix, two of the essential, functional characteristics of osteoclasts and related boneresorbing cells. These results, considered together with the reported action of 1,25(OH)2D3 on nontransformed mononuclear cells, are consistent with the view that vitamin D3 enhances bone resorption and osteoclastogenesis in vivo by promoting the differentiation of precursor cells.Of the several circulating factors known to affect bone resorption, one of the most potent is 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. This compound, when administered in picomolar to nanomolar concentrations, markedly stimulates resorptive activity (1) and promotes a readily measurable increase in the number of osteoclasts, the principal resorbing cells of bone (2). It is generally assumed that the appearance of increased numbers of osteoclasts is responsible for enhanced bone resorption. However, the mechanism(s) by which 1,25(OH)2D3 alters the size of the osteoclast population, and therefore resorptive activity, is presently unknown.Osteoclasts originate by fusion of circulating mononuclear precursor cells and almost certainly represent one of the endstage cells of mononuclear phagocyte differentiation (3, 4). Like osteoclasts, other mature, nonproliferative members of this family-e.g., monocytes and macrophages (Mos)-possess the capacity to attach to and degrade bone matrix (5, 6), and therefore they serve as useful models with which to study the mechanisms of bone resorption in tissue culture. In a previous study, we showed that Mos isolated from vitamin D-deficient animals exhibit, in vitro, the same bone resorptive dysfunction characteristically observed in the intact, calciferol-deprived animal (7). This dysfunction is not corrected by the direct addition of 1,25(OH)2D3 to M4 cultures, but administration of the metabolite to vitamin D-depleted animals for several days ...

A five-month-old girl with autosomal-recessive osteopetrosis received a bone-marrow transplant from her five-year-old HLA-MLC-identical brother after preparation with cyclophosphamide and modified total-body irradiation. Engraftment was documented by chromosomal analysis. Anemia, thrombocytopenia, and leukoerythroblastosis corrected within 12 weeks of transplantation. Low serum calcium and elevated serum alkaline and acid phosphatase levels became normal. Serial x-ray studies revealed bony remodeling and new nonsclerotic bone formation. A pretransplantation bone biopsy revealed small marrow spaces, rare marrow elements, increased osteoclasts, and no bony resorption. After transplantation, osteoclasts were actively resorbing bone, and medullary cavities contained normal bone marrow. Fluorescent Y-body analysis after transplantation revealed donor (male) osteoclasts and recipient (female) osteoblasts. Monocyte bactericidal activity, markedly decreased before transplantation, became normal. Vision, hearing, growth, and development were progressively improving 16 months after transplantation. Allogeneic bone-marrow transplantation appears to be the treatment of choice in this fatal disorder.