In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: DeltaFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of DeltaFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, DeltaFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that DeltaFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.

Osteoblasts and adipocytes may develop from common bone marrow mesenchymal precursors. Transgenic mice overexpressing ⌬FosB, an AP-1 transcription factor, under the control of the neuron-specific enolase (NSE) promoter show both markedly increased bone formation and decreased adipogenesis. To determine whether the two phenotypes were linked, we targeted overexpression of ⌬FosB in mice to the osteoblast by using the osteocalcin (OG2) promoter. OG2-⌬FosB mice demonstrated increased osteoblast numbers and an osteosclerotic phenotype but normal adipocyte differentiation. This result firmly establishes that the skeletal phenotype is cell autonomous to the osteoblast lineage and independent of adipocyte formation. It also strongly suggests that the decreased fat phenotype of NSE-⌬FosB mice is independent of the changes in the osteoblast lineage. In vitro, overexpression of ⌬FosB in the preadipocytic 3T3-L1 cell line had little effect on adipocyte differentiation, whereas it prevented the induction of adipogenic transcription factors in the multipotential stromal cell line ST2. Also, ⌬FosB isoforms bound to and altered the DNA-binding capacity of C/EBP␤. Thus, the inhibitory effect of ⌬FosB on adipocyte differentiation appears to occur at early stages of stem cell commitment, affecting C/EBP␤ functions. It is concluded that the changes in osteoblast and adipocyte differentiation in ⌬FosB transgenic mice result from independent cell-autonomous mechanisms.Although osteoblasts and adipocytes represent two morphologically and functionally distinct cell types, it has been proposed that they may develop from a common mesenchymal precursor in the bone marrow (36,40,43). Indeed, an inverse relationship between adipocyte and osteoblast differentiation has been suggested, as exemplified by increased bone marrow adipocytes in age-related bone loss (4,6,29,32,60) or after treatment with glucocorticoids (57). Several regulatory factors involved in osteoblast and adipocyte differentiation have been identified (27,42,45). However, the identities of the factors that control commitment at the branching point between the osteoblast and adipocyte lineages and the degree of plasticity between the two cell types are still uncertain (33,38).We have recently reported that transgenic mice overexpressing ⌬FosB, a member of the activator protein 1 (AP-1) family of transcription factors, under the control of the neuron-specific enolase (NSE) promoter develop not only a severe and progressive osteosclerotic phenotype, characterized as increased bone formation, but also a pronounced decrease in adipogenesis and fat levels (25, 47, 53). The AP-1 family of basic leucine zipper transcription factors comprises various combinations of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) proteins, which upon dimer formation regulate gene transcription by binding to consensus response elements present in the promoter region of target genes (23). Several studies have demonstrated an important regulatory role of AP-1 factors, especially the ...

ABSTRACT:Introduction: Activator protein (AP)-1 family members play important roles in the development and maintenance of the adult skeleton. Transgenic mice that overexpress the naturally occurring ⌬FosB splice variant of FosB develop severe osteosclerosis. Translation of ⌬fosb mRNA produces both ⌬FosB and a further truncated isoform (⌬2⌬FosB) that lacks known transactivation domains but, like ⌬FosB, induces increased expression of osteoblast marker genes. Materials and Methods:To test ⌬2⌬FosB's ability to induce bone formation in vivo, we generated transgenic mice that overexpress only ⌬2⌬FosB using the enolase 2 (ENO2) promoter-driven bitransgenic Tet-Off system. Results: Despite ⌬2⌬FosB's failure to induce transcription of an AP-1 reporter gene, the transgenic mice exhibited both the bone and the fat phenotypes seen in the ENO2-⌬FosB mice. Both ⌬FosB and ⌬2⌬FosB activated the BMP-responsive Xvent-luc reporter gene and increased Smad1 expression. ⌬2⌬FosB enhanced BMP-induced Smad1 phosphorylation and the translocation of phospho-Smad1 (pSmad1) to the nucleus more efficiently than ⌬FosB and showed a reduced induction of inhibitory Smad6 expression. Conclusions: ⌬FosB's AP-1 transactivating function is not needed to induce increased bone formation, and ⌬2⌬FosB may act, at least in part, by increasing Smad1 expression, phosphorylation, and translocation to the nucleus.

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Transgenic mice overexpressing deltaFosB, a naturally occurring splice variant of FosB, develop an osteosclerotic phenotype. The increased bone formation has been shown to be due, at least in part, to autonomous effects of deltaFosB isoforms on cells of the osteoblast lineage. However, abdominal fat and marrow adipocytes are also markedly decreased in deltaFosB mice, leading to low serum leptin levels. Increased bone mass has been linked to the absence of leptin and leptin receptor signaling in ob/ob and db/db mice. Thus, in addition to affecting directly osteoblastogenesis and bone formation, deltaFosB isoforms might increase bone mass indirectly via a decrease in leptin. To test this hypothesis, we restored normal circulating levels of leptin in deltaFosB mice via sc implanted osmotic pumps. Complete histomorphometric analysis demonstrated that trabecular bone volume as well as dynamic parameters of bone formation was unchanged by this treatment in both deltaFosB transgenic mice and control littermates. This demonstration that restoring circulating levels of leptin in deltaFosB transgenic mice failed to rescue the bone phenotype further indicates that the marked increase in bone formation is autonomous to the osteoblast lineage.