CCAAT/enhancer binding proteins (C/EBPs) are expressed by osteoblasts and adipocytes during differentiation. C/EBPβ is critical for adipogenesis; however, its role in osteoblastogenesis is unclear, and its function in the postnatal skeleton is not known. To study C/EBPβ in osteoblasts in vivo, we created transgenic mice expressing full length C/EBPβ under the control of a 3.8 kb fragment of the human osteocalcin promoter. Two transgenic lines were established in a friend leukemia virus strain B genetic background, and compared with wild type littermate controls. Both C/EBPβ transgenic lines exhibited osteopenia, with a 30% decrease in bone volume, due to a decrease in trabecular number. The number of osteoblasts and osteoclasts per bone perimeter was not changed. Bone marrow stromal cells from C/EBPβ transgenics showed reduced mineralization, and reduced alkaline phosphatase mRNA levels. Calvarial osteoblasts from C/EBPβ transgenics displayed reduced alkaline phosphatase activity. To determine the consequences of the Cebpb deletion in vivo, the phenotype of Cebpb null mice was compared with that of wild type controls of identical genetic composition. Cebpb null mice exhibited reduced weight, body fat, and bone mineral density, and decreased bone volume, due to a decrease in trabecular number. The number of osteoblasts and osteoclasts per bone perimeter was not changed. C/EBPβ downregulation by RNA interference in calvarial osteoblasts had no effect on osteoblast differentiation/function. The phenotype of the Cebpb inactivation may be secondary to systemic indirect effects, and to direct effects of C/EBPβ in osteoblasts. In conclusion, C/EBPβ plays a role in mesenchymal cell differentiation and its misexpression in vivo causes osteopenia.

The role of circulating IGF-I in skeletal acquisition and the anabolic response to PTH is not well understood. We generated IGF-I-deficient mice by gene deletions of IGF ternary complex components including: (1) liver-specific deletion of the IGF-I gene (LID), (2) global deletion of the acid-labile (ALS) gene (ALSKO), and (3) both liver IGF-I and ALS inactivated genes (LA). Twelve-week-old male control (CTL), LID, ALSKO, and LA mice were treated with vehicle (VEH) or human PTH(1–34) for 4 weeks. VEH-treated IGF-I-deficient mice (i.e. LID, ALSKO and LA mice) exhibited reduced cortical cross-sectional area (P = 0.001) compared with CTL mice; in contrast, femoral trabecular bone volume fractions (BV/TV) of the IGF-I-deficient mice were consistently greater than CTL (P<0.01). ALSKO mice exhibited markedly reduced osteoblast number and surface (P<0.05), as well as mineral apposition rate compared with other IGF-I-deficient and CTL mice. Adherent bone marrow stromal cells, cultured in β-glycerol phosphate and ascorbic acid, showed no strain differences in secreted IGF-I. In response to PTH, there were both compartment- and strain-specific effects. Cortical bone area was increased by PTH in CTL and ALSKO mice, but not in LID or LA mice. In the trabecular compartment, PTH increased femoral and vertebral BV/TV in LID, but not in ALSKO or LA mice. In conclusion, we demonstrated that the presentation of IGF-I as a circulating complex is essential for skeletal remodeling and the anabolic response to PTH. We postulate that the ternary complex itself, rather than IGF-I alone, influences bone acquisition in a compartment-specific manner (i.e. cortical vs trabecular bone).

A spontaneous mouse mutant, designated ‘small’ (sml), was recognized by reduced body size suggesting a defect in the IGF1/GH axis. The mutation was mapped to the chromosome 1 region containing Irs1, a viable candidate gene whose sequence revealed a single nucleotide deletion resulting in a premature stop codon. Despite normal mRNA levels in mutant and control littermate livers, western blot analysis revealed no detectable protein in mutant liver lysates. When compared with the control littermates, Irs1 ^sml /Irs1 ^sml (Irs1 ^sml/sml ) mice were small, lean, hearing impaired; had 20% less serum IGF1; were hyperinsulinemic; and were mildly insulin resistant. Irs1 ^sml/sml mice had low bone mineral density, reduced trabecular and cortical thicknesses, and low bone formation rates, while osteoblast and osteoclast numbers were increased in the females but not different in the males compared with the Irs1 ^+/+ controls. In vitro, Irs1 ^sml/sml bone marrow stromal cell cultures showed decreased alkaline phosphatase-positive colony forming units (pre-osteoblasts; CFU-AP+) and normal numbers of tartrate-resistant acid phosphatase-positive osteoclasts. Irs1 ^sml/sml stromal cells treated with IGF1 exhibited a 50% decrease in AKT phosphorylation, indicative of defective downstream signaling. Similarities between engineered knockouts and the spontaneous mutation of Irs1 ^sml were identified as well as significant differences with respect to heterozygosity and gender. In sum, we have identified a spontaneous mutation in the Irs1 gene associated with a major skeletal phenotype. Changes in the heterozygous Irs1 ^{+ /sml} mice raise the possibility that similar mutations in humans are associated with short stature or osteoporosis.