MIA/CD-RAP Regulates MMP13 and Is a Potential New Disease-Modifying Target for Osteoarthritis Therapy.

Melanoma inhibitory activity/cartilage-derived retinoicacid-sensitive protein (MIA/CD-RAP) is a protein expressed and secreted by chondrocytes and cartilaginous tissues. MIA/CD-RAP-deficient mice develop milder osteoarthritis than wildtype mice. In this study, we investigated MIA/CD-RAP downstream targets to explain this reduced disease development. As a possible mediator, we could detect matrix metalloproteinase 13 (MMP13), and the influence of MIA/CD-RAP on MMP13 regulation was analyzed in vitro using SW1353 chondrosarcoma cells and primary chondrocytes. The femoral head cartilage of WT and MIA/CD-RAP -/- mice were cultured ex vivo to further investigate MMP13 activity. Finally, osteoarthritis was surgically induced via DMM in C57BL/6 mice, and the animals were treated with an MIA/CD-RAP inhibitory peptide by subcutaneously implanted pellets. MMP13 was regulated by MIA/CD-RAP in SW1353 cells, and MIA/CD-RAP -/- murine chondrocytes showed less expression of MMP13. Further, IL-1ß-treated MIA/CD-RAP -/- chondrocytes displayed less MMP13 expression and activity. Additionally, MIA/CD-RAP-deficient ex vivo cultured cartilage explants showed less MMP13 activity as well as reduced cartilage degradation. The mice treated with the MIA/CD-RAP inhibitory peptide showed less osteoarthritis development. Our findings revealed MIA/CD-RAP as a new regulator of MMP13 and highlighted its role as a potential new target for osteoarthritis therapy.

Multimolecular analysis of stable immunological synapses reveals sustained recruitment and sequential assembly of signaling clusters.

The formation of the immunological synapse between T cells and antigen-presenting cells (APC) begins within minutes of contact and can take hours for full T-cell activation. Although early phases of the synapse have been extensively studied for a select number of proteins, later phases have not yet been examined in detail. We studied the signaling network in stable synapses by measuring the simultaneous localization of 25 signaling and structural molecules over 2 h at the level of individual synapses using multi-epitope ligand cartography (MELC). Signaling proteins including phospho(p)ZAP70, pSLP76, pCD3ζ, and pLAT, along with proteins that influence synapse structure such as F-actin, tubulin, CD45, and ICAM-1, were localized in images of synapses and revealed the multidimensional construction of a mature synapse. The construction of the stable synapse included intense early TCR signaling, a phase of recruitment of structural proteins, and a sustained increase in signaling molecules and colocalization of TCR and pLAT signaling clusters in the center of the synapse. Consolidation of TCR and associated proteins resulted in formation of a small number of discrete synaptic microclusters. Development of synapses and cSMAC composition was greatly affected by the absence of Vav1, with an associated loss in PLCγ1 recruitment, pSLP76, and increased CXCR4. Together, these data demonstrate the use of multi-epitope ligand cartography to quantitatively analyze synapse formation and reveal successive recruitment of structural and signaling proteins and sustained phosphorylation at the mature synapse.

PAG/Cbp suppression reveals a contribution of CTLA-4 to setting the activation threshold in T cells.

BACKGROUND: PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation. RESULTS: PAG-depletion enhanced Src kinase activity and augmented proximal T-cell receptor signaling; exactly the phenotype expected for loss of this negative regulator. Surprisingly, rather than becoming hyper-proliferative, PAG-suppressed T cells became unresponsive. This was mediated by a Fyn-dependent hyper-phosphorylation of the inhibitory receptor CTLA-4, which recruited the protein tyrosine phosphatase Shp-1 to lipid rafts. Co-suppression of CTLA-4 abrogates this inhibition and restores proliferation to T cells. CONCLUSION: We have identified a fail-safe mechanism as well as a novel contribution of CTLA-4 to setting the activation threshold in T cells.

Integrating signals from the T-cell receptor and the interleukin-2 receptor.

T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR) signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R) signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells.