Skeletal unloading induced by hindlimb suspension in rats reduces bone formation and induces osteopenia, but its effect on adipogenesis is unknown. We assessed the effects of unloading and transforming growth factor (TGF) ␤2 on bone marrow stromal cell adipocyte differentiation in relation with osteoblast differentiation. Skeletal unloading rapidly (4 -7 days) decreased osteoblast transcription factor Runx2, osteocalcin (OC), and type I collagen messenger RNA (mRNA) levels and reduced bone formation in the long bone metaphysis. Conversely, unloading increased expression of the adipocyte transcription factor peroxisome proliferatoractivated receptor ␥2 (PPAR␥2) at 4 days and increased expression of the adipocyte differentiation genes lipoprotein lipase (LPL) and aP2 in the bone marrow stroma at 7 days. Consistently, unloading increased the number and volume of adipocytes in the bone marrow stroma. Continuous (0 -7 days) and late (4 -7 days) treatments with TGF-␤2 corrected the abnormal expression of Cbfa1/Runx2, OC, and type I collagen mRNAs and normalized bone formation in unloaded metaphyseal bone. Moreover, both TGF-␤2 treatments decreased PPAR␥2 and C/EBP␣ mRNA levels at 4 days and normalized aP2 and LPL expression and adipocyte number and volume at 7 days. These results show that skeletal unloading increases adipocyte differentiation concomitantly with inhibition of osteoblast differentiation. These abnormalities are prevented and reversed by TGF-␤2, suggesting a role for TGF-␤ in the control of adipogenic differentiation in the bone marrow stroma. (J Bone Miner Res 2002;17:668 -677)

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.