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Bone formation and resorption are tightly coupled under normal conditions, and the interaction of osteoclast precursors with cells of the osteoblast lineage is a prerequisite for osteoclast formation. Cbfa1 is an osteoblast-specific transcription factor that is essential for osteoblast differentiation and bone formation. At present, it is not known whether Cbfa1 regulates any of the osteoblast-derived factors involved in the bone resorption pathway. Osteoprotegerin (OPG) is an osteoblastsecreted glycoprotein that functions as a potent inhibitor of osteoclast differentiation and bone resorption. Cloning and computer analysis of a 5.9-kilobase human OPG promoter sequence revealed the presence of 12 putative Cbfa1 binding elements (osteoblast-specific element 2 (OSE 2 )), suggesting a possible regulation of OPG by Cbfa1. We cloned the promoter upstream of the ␤-galactosidase reporter gene (pOPG5.9␤gal) and evaluated whether Cbfa1 could regulate its expression in transient transfection assays. The 5.9-kilobase promoter directed increased levels of reporter gene expression, reminiscent of OPG protein levels in osteoblastic cell lines (BALC and U2OS) as compared with the nonosteoblastic cell line COS1. Cotransfection of a Cbfa1 expression construct along with pOPG5.9␤gal reporter construct led to 39-, 7-, and 16-fold increases in ␤-galactosidase activity in COS1, BALC, and U2OS cells, respectively. Removal of all the putative OSE 2 elements led to an almost complete loss of transactivation. Mutational analysis demonstrated that the proximal OSE 2 element contributes to a majority of the effects of Cbfa1, and Cbfa1 bound to the proximal element in a sequencespecific manner. Further, overexpression of Cbfa1 led to a 54% increase in OPG protein levels in U2OS cells. These results indicate that Cbfa1 regulates the expression of OPG, thereby further contributing to a molecular link between bone formation and resorption.Bone growth, development, and maintenance in mammals is a highly regulated process. The level of bone mass is dependent on the balance between bone formation and resorption. At the cellular level, this balance involves the coordinate regulation and interaction of the component cell types: bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are derived from mesenchymal stem cells, and their formation and function are under the control of an osteoblast-specific transcription factor known as core binding factor a1 (Cbfa1) or osteoblast specific factor 2 (Osf2) 1 (1-3). Cbfa1 has been shown to regulate the expression of genes that characterize the osteoblast phenotype, including osteocalcin, osteopontin, type I collagen, bone sialoprotein, and collagenase-3, by binding to DNA sequence elements called OSE 2 that are present in the control regions of these genes (1, 4 -6). Cbfa1 knock out animals completely lack bones because of a maturational arrest of osteoblasts and die soon after delivery (2, 3). Further, haploinsufficiency of the Cbfa1 gene product in mice and humans heterozygous for the Cbfa...

SummaryThe immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Igh 6 scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the V, gene repertoire of the GC B cells was strongly influenced by the source of the CD4 § T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies dedined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germline-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.

Continuous infusion of PTH in vivo results in active bone resorption. To investigate the molecular basis of the catabolic effect of PTH in vivo, we evaluated the role of OPG and RANKL, which are known to influence osteoclast formation and function. Weanling rats fed a calcium-free diet were parathyroidectomized and infused with PTH via an Alzet pump to examine: 1) the changes of serum-ionized calcium and osteoclast number, 2) the expression of OPG/RANKL mRNA and protein, and 3) the expression of osteoblast phenotype bone formation-associated genes such as osteoblast specific transcription factor, osteocalcin, bone sialoprotein, and type I collagen. PTH (1--38) (0.01--20 microg/100 g) continuous infusion for 1--24 h resulted in a dose-dependent increase in serum-ionized calcium in parathyroidectomized rats and a corresponding dose-dependent increase in osteoclast number, indicating an increased bone resorption. At 20 microg/100 g PTH dose level, serum-ionized calcium was 2.1-fold of the vehicle control and not different from the Sham-parathyroidectomized rats, and osteoclast number was 3-fold of the vehicle control and 1.7-fold of the Sham-parathyroidectomized rats. In the distal femur, RANKL mRNA expression was increased (27-fold) and OPG mRNA expression was decreased (4.6-fold). The changes in RANKL and OPG mRNA levels were rapid (as early as 1 h), dose dependent, and sustained over a 24-h period that was examined. Immunohistochemical evaluation of bone sections confirmed that OPG level was reduced in proximal tibial metaphysis upon PTH infusion. Circulating OPG protein level was also decreased by 32% when compared with the parathyroidectomized control. The expression of genes that mark the osteoblast phenotype was significantly decreased [osteoblast specific transcription factor (2.3-fold), osteocalcin (3-fold), bone sialoprotein (2.8-fold), and type I collagen (5-fold)]. These results suggest that the catabolic effect of PTH infusion in vivo in this well-established resorption model is associated with a reciprocal expression of OPG/RANKL and a co-ordinate decrease in the expression of bone formation-related genes. We propose that the rapid and sustained increase in RANKL and decrease in OPG initiate maintain and favor the cascade of events in the differentiation/recruitment and activation of osteoclasts.