BackgroundPatients suffering from osteoporosis show an increased number of adipocytes in their bone marrow, concomitant with a reduction in the pool of human mesenchymal stem cells (hMSCs) that are able to differentiate into osteoblasts, thus leading to suppressed osteogenesis.MethodsIn order to be able to interfere with this process, we have investigated in-vitro culture conditions whereby adipogenic differentiation of hMSCs is impaired and osteogenic differentiation is promoted. By means of gene expression microarray analysis, we have investigated genes which are potential targets for prevention of fat cell differentiation.ResultsOur data show that BMP2 promotes both adipogenic and osteogenic differentiation of hMSCs, while transforming growth factor beta (TGFβ) inhibits differentiation into both lineages. However, when cells are cultured under adipogenic differentiation conditions, which contain cAMP-enhancing agents such as IBMX of PGE2, TGFβ promotes osteogenic differentiation, while at the same time inhibiting adipogenic differentiation. Gene expression and immunoblot analysis indicated that IBMX-induced suppression of HDAC5 levels plays an important role in the inhibitory effect of TGFβ on osteogenic differentiation. By means of gene expression microarray analysis, we have investigated genes which are downregulated by TGFβ under adipogenic differentiation conditions and may therefore be potential targets for prevention of fat cell differentiation. We thus identified nine genes for which FDA-approved drugs are available. Our results show that drugs directed against the nuclear hormone receptor PPARG, the metalloproteinase ADAMTS5, and the aldo-keto reductase AKR1B10 inhibit adipogenic differentiation in a dose-dependent manner, although in contrast to TGFβ they do not appear to promote osteogenic differentiation.ConclusionsThe approach chosen in this study has resulted in the identification of new targets for inhibition of fat cell differentiation, which may not only be relevant for prevention of osteoporosis, but also of obesity.

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EGF-like growth factors activate their ErbB receptorsby promoting receptor-mediated homodimerization or, alternatively, by the formation of heterodimers with the orphan ErbB-2 through an as yet unknown mechanism. To investigate the selectivity in dimer formation by ligands, we have applied the phage display approach to obtain ligands with modified C-terminal residues that discriminate between ErbB-2 and ErbB-3 as dimerization partners. We used the epidermal growth factor/ transforming growth factor ␣ chimera T1E as the template molecule because it binds to ErbB-3 homodimers with low affinity and to ErbB-2/ErbB-3 heterodimers with high affinity. Many phage variants were selected with enhanced binding affinity for ErbB-3 homodimers, indicating that C-terminal residues contribute to the interaction with ErbB-3. These variants were also potent ligands for ErbB-2/ErbB-3 heterodimers despite negative selection for such heterodimers. In contrast, phage variants positively selected for binding to ErbB-2/ErbB-3 heterodimers but negatively selected for binding to ErbB-3 homodimers can be considered as "second best" ErbB-3 binders, which require ErbB-2 heterodimerization for stable complex formation. Our findings imply that epidermal growth factor-like ligands bind ErbB-3 through a multi-domain interaction involving at least both linear endings of the ligand. Apparently the ErbB-3 affinity of a ligand determines whether it can form only ErbB-2/ErbB-3 complexes or also ErbB-3 homodimers. Because no separate binding domain for ErbB-2 could be identified, our data support a model in which ErbB heterodimerization occurs through a receptor-mediated mechanism and not through bivalent ligands.The recent determination of the crystal structure of the extracellular domain of ErbB-1 in complex with its ligands epidermal growth factor (EGF) 1 or transforming growth factor ␣ (TGF-␣) has provided evidence for the formation of homodimeric ErbB-1 complexes through a receptor-mediated dimerization mechanism (1, 2). Ligand binding to both domains I and III of the extracellular domain of the receptor involves the transition of ErbB-1 from a "closed" to an "open" state, which then permits dimerization with another liganded ErbB-1 through interaction of domain II residues within these receptors. Most likely this mechanism can serve as a paradigm for homodimerization of other liganded ErbB receptors, such as ErbB-3 and ErbB-4. However, it is well established that EGFlike growth factors preferentially signal through heterodimers of their cognate receptor with the orphan ErbB-2. The mechanism by which EGF-like growth factors bind their receptors in heterodimeric receptor complexes remains an open question.Dimerization of ligand-bound receptor tyrosine kinases is a mechanism that is thought to activate the intrinsic kinase domain followed by transphosphorylation and subsequent docking of cellular signal transducing proteins. As a consequence, ligand binding serves as a potential site for regulation of cell proliferation in diseases where ErbB rece...

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