Activated T cells secrete multiple osteoclastogenic cytokines which play a major role in the bone destruction associated with rheumatoid arthritis. While the role of T cells in osteoclastogenesis has received much attention recently, the effect of T cells on osteoblast formation and activity is poorly defined. In this study, we investigated the hypothesis that in chronic inflammation activated T cells contribute to enhanced bone turnover by promoting osteoblastic differentiation. We show that T cells produce soluble factors that induce alkaline phosphatase activity in bone marrow stromal cells and elevated expression of mRNA for Runx2 and osteocalcin. This data indicate that T cell derived factors have the capacity to stimulate the differentiation of bone marrow stromal cells into the osteoblast phenotype. RANKL mRNA was undetectable under any conditions in highly purified bone marrow stromal cells. In contrast, RANKL was constitutively expressed in primary osteoblasts and only moderately up-regulated by activated T cell conditioned medium. Interestingly, both bone marrow stromal cells and osteoblasts expressed mRNA for RANK, which was strongly up-regulated in both cell types by activated T cell conditioned medium. Although, mRNA for the RANKL decoy receptor, osteoprotegerin, was also up-regulated by activated T cell conditioned medium, it's inhibitory effects may be mitigated by a simultaneous rise in the osteoprotegerin competitor TNF-related apoptosis-inducing ligand. Based on our data we propose that during chronic inflammation, T cells regulate bone loss by a dual mechanism involving both direct stimulation of osteoclastogenesis, by production of osteoclastogenic cytokines, and indirectly by induction of osteoblast differentiation and up-regulation of bone turnover via coupling.

Objective. To investigate the role of T cell induction of matrix metalloproteinase 13 (MMP-13) production by human osteoblasts in order to better understand the process of bone loss in rheumatoid arthritis (RA).Methods. Activated T cell-conditioned medium (ACTTCM) was used to mimic the physiologic conditions of inflammation. MMP-13 production by human osteoblasts was assessed using a specific enzyme-linked immunosorbent assay. Specific inhibitors of the p38 mitogen-activated protein (MAP) kinase and the extracellular signal-regulated kinase 1/2 (ERK-1/2) MAP kinase signaling pathways were used to assess their roles in T cell-mediated MMP-13 production. Finally, recombinant cytokines representative of the major components in ACTTCM were assessed for their ability to induce MMP-13.Results. ACTTCM powerfully induced MMP-13 in human osteoblasts. Inhibition of p38 activity abolished, while inhibition of ERK-1/2 activity enhanced, MMP-13 production. We next investigated physiologic levels of the T cell cytokines tumor necrosis factor ␣ (TNF␣), transforming growth factor ␤ (TGF␤), interferon-␥ (IFN␥), and interleukin-17 (IL-17) for their roles in MMP-13 induction. Although individual cytokines had no significant effect, the combination of TNF␣, TGF␤, IFN␥, and IL-17 resulted in a dramatic p38-dependent induction of MMP-13 identical to that produced by ACTTCM.Conclusion. These studies demonstrate for the first time that human osteoblasts produce MMP-13. The results also show that under conditions of chronic inflammation, multiple T cell cytokines synergize to induce high levels of MMP-13 via a mechanism that is dependent on activated p38 MAP kinase and is suppressed by activated ERK-1/2.

Adenovirus early gene 1A (E1A) possesses a potent transcriptional repression function within the first 80 amino acids (E1A 1-80). Our previous analysis of subdomain 1 (residues 1 to 30) revealed strong correlations between residues required for repression and for disruption of TBP-TATA complexes. Here, we report a functional analysis of subdomain 2 (48 to 60) by alanine-scanning mutagenesis. 53Ala, 54Pro, 55Glu, and 56Asp are required for repression in vitro and in vivo and for efficient interaction with p300 but not for disruption of TBP-TATA. These combined results suggest a model for E1A transcription repression. E1A through subdomains 1 and 2 uses coactivators like p300 as scaffolds to access E1A repressible promoters. At the promoter, subdomain 1 interacts with TBP to disrupt TBP-TATA and abort transcription initiation. In further support of this model, we show that E1A 1-80 bound to the p300-binding site retains the ability to interact with TBP.