OBJECTIVEMesenchymal stem cells (MSCs) are known to be capable of suppressing immune responses, but the molecular mechanisms involved and the therapeutic potential of MSCs remain to be clarified.RESEARCH DESIGN AND METHODSWe investigated the molecular mechanisms underlying the immunosuppressive effects of MSCs in vitro and in vivo.RESULTSOur results demonstrate that matrix metalloproteinases (MMPs) secreted by MSCs, in particular MMP-2 and MMP-9, play an important role in the suppressive activity of MSCs by reducing surface expression of CD25 on responding T-cells. Blocking the activity of MMP-2 and MMP-9 in vitro completely abolished the suppression of T-cell proliferation by MSCs and restored T-cell expression of CD25 as well as responsiveness to interleukin-2. In vivo, administration of MSCs significantly reduced delayed-type hypersensitivity responses to allogeneic antigen and profoundly prolonged the survival of fully allogeneic islet grafts in transplant recipients. Significantly, these MSC-mediated protective effects were completely reversed by in vivo inhibition of MMP-2 and MMP-9.CONCLUSIONSWe demonstrate that MSCs can prevent islet allograft rejection leading to stable, long-term normoglycemia. In addition, we provide a novel insight into the mechanism underlying the suppressive effects of MSCs on T-cell responses to alloantigen.

Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been downregulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>؎2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-␤ precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Matrix metalloproteinase-9 (MMP-9) 1 or gelatinase B was originally identified as a secreted enzyme that could degrade collagens, especially collagen IV (1, 2). These early observations have since been refined as it became apparent that denatured collagen I, IV as well as gelatin are efficiently cleaved but that intact collagen I or IV are poor substrates for the enzyme (3, 4). Indeed MMP-9 fails to promote cellular invasion of natural basement membranes (5). Although MMP-9 may not be a determining factor for invasion of collagen IV-rich basement membranes, collagen IV degradation by MMP-9 has been shown to be involved in angiogenesis. For instance, the reduced levels of antiangiogenic peptides derived from collagen IV in mice genetically deficient in MMP-9 reflect the decreased turnover of collagen IV in the absence of MMP-9 (6). Many non-collagenous substrates have also been identified as MMP-9 substrates including cell surface molecules and secreted proteins. These include molecules involved in cell adhesion, cell surface receptors, cytokines, protease inhibitors, angiogenic factors, and growth factors. The cleavage of many of these substrates affects the cellular phenotype and is associated with several pathological conditions in mice (7,8).In cancers MMP-9 produced by the host contributes to squamous cancer progression by activation of the angiogenic 1 The abbreviations used are: MMP, matrix metalloproteinase; PN-1, protease nexin-1; TIMP, tissue inhibitor of metalloproteinases; UPLC, ultraperformance LC; MS E , high/low collision energy MS; MudPIT, ...

The related kinases ATM (ataxia-telangiectasia mutated) and ATR (ataxia-telangiectasia and Rad3-related) phosphorylate a limited number of downstream protein targets in response to DNA damage. Here we report a new pathway in which ATM kinase signals the DNA damage response by targeting the transcriptional cofactor Strap. ATM phosphorylates Strap at a serine residue, stabilizing nuclear Strap and facilitating formation of a stress-responsive co-activator complex. Strap activity enhances p53 acetylation, and augments the response to DNA damage. Strap remains localized in the cytoplasm in cells derived from ataxia telangiectasia individuals with defective ATM, as well as in cells expressing a Strap mutant that cannot be phosphorylated by ATM. Targeting Strap to the nucleus reinstates protein stabilization and activates the DNA damage response. These results indicate that the nuclear accumulation of Strap is a critical regulator in the damage response, and argue that this function can be assigned to ATM through the DNA damage-dependent phosphorylation of Strap.

Matrix metalloproteinase-9 (MMP-9) expression is known to enhance the invasion and metastasis of tumor cells. In previous work based on a proteomic screen, we identified the serpin protease nexin-1 (PN-1) as a potential target of MMP-9. Here, we show that PN-1 is a substrate for MMP-9 and establish a link between PN-1 degradation by MMP-9 and regulation of invasion. PN-1 levels increased in prostate carcinoma cells after downregulation of MMP-9 and in tissues of MMP-9-deficient mice, consistent with PN-1 degradation by MMP-9. We identified three MMP-9 cleavage sites in PN-1 and showed that mutations in those sites made PN-1 more resistant to MMP-9. Urokinase plasminogen activator (uPA) is inhibited by PN-1. MMP-9 augmented uPA activity in the medium of PC3-ML cells by degrading PN-1. Prostate cancer cells, overexpressing PN-1 or treated with MMP-9 shRNA, had reduced cell invasion in Matrigel. PN-1 siRNA restored uPA activity and the invasive capacity. PN-1 mutated in the serpin inhibitory domain, the reactive center loop, failed to inhibit uPA and to reduce Matrigel invasion. This study shows a novel molecular pathway in which MMP-9 regulates uPA activity and tumor cell invasion through cleavage of PN-1. Cancer Res; 70(17); 6988-98. ©2010 AACR.