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Delivery of bioactive molecules into the cell: the Trojan horse approach

Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin.

Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia.

Osteoclasts, the sole bone-resorbing cells, arise by fusion and differentiation of monocyte/macrophage precursors. Matrix degradation requires adhesion of the osteoclast to bone, an integrin alphavbeta3-mediated event that also stimulates signals which polarize the cell and secrete resorptive molecules such as hydrochloric acid and acidic proteases. Two cytokines are necessary and sufficient for osteoclastogenesis, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), both produced by mesenchymal cells in the bone marrow environment. M-CSF promotes survival and proliferation of osteoclast precursors. It also contributes to their differentiation and regulates the cytoskeletal changes that accompany bone resorption. Binding of M-CSF to c-Fms, its receptor, recruits adapter proteins and cytosolic kinases, thereby activating a variety of intracellular signals. We herein review how alphavbeta3 and M-CSF, alone and in concert, impact production, survival, and function of the osteoclast, thereby controlling skeletal mass. Signals from alphavbeta3 and/or c-Fms activate Syk and Vav3, originally defined by their function in lymphoid cells. Genetic depletion of either protein generates a strong bone phenotype, underscoring the promise of osteoimmunobiology.

αvβ3 and macrophage colony‐stimulating factor: partners in osteoclast biology

Cytokine responses against periodontal infection: protective and destructive roles.

Publisher Summary This chapter describes the methodology to generate and transduce full-length Tat fusion proteins that can be applied to a broad spectrum of proteins independent of size or function. Bacterially expressed N'-terminal in-frame Tat fusion proteins are isolated from bacteria by sonication in 8 M urea. The use of 8 M urea achieves two goals. First, the majority of recombinant proteins in bacteria are present in inclusion bodies as denatured insoluble proteins, especially full-length proteins. Sonication in urea solubilizes this material, thus allowing for its isolation. Second, denatured Tat fusion proteins have an enhanced potential to elicit biological responses. The denatured Tat fusion proteins are made soluble in an aqueous buffer and added directly to the medium of cells in tissue culture. To date, this strategy is used to generate and transduce more than 60 full-length proteins and domains from 15 to 120 kDa from a variety of classes, suggesting that many proteins may be transduced into cells. Transduction of full-length Tat fusion proteins directly into ∼100% of primary or transformed cells has broad implications for manipulating intracellular processes in both experimental in vitro tissue culture systems and animal models.

Protein transduction: delivery of Tat-GTPase fusion proteins into mammalian cells.

The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1‐induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1‐induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the avb3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1‐induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF1‐induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata. (J Bone Miner Res 1998;13:50‐58)

Meena A. Chellaiah

Papers

Protein transduction: delivery of Tat-GTPase fusion proteins into mammalian cells.

Colony stimulating factor-1-induced osteoclast spreading depends on substrate and requires the vitronectin receptor and the c-src proto-oncogene.