Search

Murine osteoclast precursors and osteoblasts express the integrin alpha(v)beta(5), the appearance of which on the cell surface is controlled by the beta(5), and not the alpha(v), subunit. Here, we show that a 173-base pair proximal region of the beta(5) promoter mediates beta(5) basal transcription in macrophage (osteoclast precursor)-like and osteoblast-like cells. DNase I footprinting reveal four regions (FP1-FP4) within the 173-base pair region, protected by macrophage nuclear extracts. In contrast, osteoblast nuclear extracts protect only FP1, FP2, and FP3. FP1, FP2, and FP3 bind Sp1 and Sp3 from both macrophage and osteoblast nuclear extracts. FP4 does not bind osteoblast proteins but binds PU.1 from macrophages. Transfection studies show that FP1 and FP2 Sp1/Sp3 sites act as enhancers in both MC3T3-E1 (osteoblast-like) and J774 (macrophage-like) cell lines, whereas the FP3 Sp1/Sp3 site serves as a silencer. Mutation of the FP2 Sp1/Sp3 site totally abolishes promoter activity in J774 cells, with only partial reduction in MC3T3-E1 cells. Finally, we demonstrate that PU.1 acts as a beta(5) silencer in J774 cells but plays no role in MC3T3-E1 cells. Thus, three Sp1/Sp3 sites regulate beta(5) gene expression in macrophages and osteoblast-like cells, with each element exhibiting cell-type and/or activation-suppression specificity.

Tumor necrosis factor-α (TNF) exerts its transcriptional effects via activation of nuclear transcription factor-κ B (NF-κB). NF-κB is sequestered in the cytosol by IκBα and, in most cells, released upon serine phosphorylation of this inhibitory protein which then undergoes rapid, ubiquitin-dependent degradation. In contrast, we find TNF induction of NF-κB in murine bone marrow macrophages (BMMs), is mediated, by c-Src, in a cell, and cytokine specific manner. The non-receptor tyrosine kinase is rapidly mobilized and activated upon TNF exposure. Within the same time frame, TNF induced c-Src associates with IκBα in a long lived complex. The proto-oncogene, when associated with IκBα phosphorylates the inhibitory protein on tyrosine 42. Consistent with the pivotal role played by c-Src in TNF-induced IκBα tyrosine phosphorylation, NF-κB activation, by the cytokine, is markedly delayed and reduced in c-src−/− BMMs. Underscoring the physiological significance of c-Src activation of NF-κB, TNF induction of IL-6, which is an NF-κB mediated event, is substantially diminished in c-src−/− BMMs.

We have identified a novel steroid hormone response element in the avian beta3 integrin promoter. This sequence, comprising three hexameric direct repeat half-sites separated by nine and three nucleotides binds vitamin D receptor (VDR)-retinoid X receptor (RXR) and retinoic acid receptor (RAR)-RXR heterodimers. VDR-RXR binds direct repeats separated by three base pairs, and RAR-RXR recognizes half-sites separated by nine bases, whereas the central half-site interacts with both heterodimers. Retinoic acid and 1, 25-dihydroxyvitamin D3 activate both a genomic fragment including the transcriptional start site and an oligonucleotide containing the three repeats, linked to a heterologous promoter. Co-addition of the steroids produces neither synergy nor an additive effect; rather the result equals that for retinoic acid alone. Scatchard analysis demonstrates that RAR-RXR has greater affinity than VDR-RXR for the composite element. Based on these findings we propose a model in which there is specific, polarity-defined binding of VDR-RXR and RAR-RXR to three half-sites, which form two overlapping steroid response elements, with the central half-site common to both. Our results identify a novel mechanism by which one steroid hormone can modulate the activity of a second, by competing for a shared half-site in a composite response element.

Osteoclastic bone resorption is dependent upon cell-matrix recognition. This process is mediated by the integrin αvβ3 whose expression is enhanced, in avian osteoclast precursors, by bone-seeking steroids. The purpose of this study was to determine if bone-modulating cytokines impact on αvβ3 expression by mouse marrow macrophages (BMMs), known to differentiate into osteoclasts. Of the cytokines tested. Interleukin-4 (IL-4) is most effective in increasing β3 mRNA levels by a mechanism involving transactivation of the β3 gene. Moreover, IL-4 augmented β3 mRNA is mirrored by plasma membrane appearance of αvβ3. As IL-4 induces β3 and not αv mRNA, the β3 chain appears to regulate surface expression of the heterodimer. The functional significance of IL-4-induced αvβ3 is underscored by the fact that, while attachment to fibronectin is unaltered, treatment of BMMs with the cytokine enhances αvβ3-mediated binding to vitronectin 5-fold. Expression of this heterodimer by BMMs driven along a non-osteoclastic lineage suggests αvβ3 may play a role in the inflammatory response of macrophages.