Mesenchymal stem cells (MSCs) that overexpress secreted frizzled-related protein 2 (sFRP2) exhibit an enhanced reparative phenotype. The secretomes of sFRP2-overexpressing MSCs and vector control-MSCs were compared through liquid chromatography tandem mass spectrometry. Proteomic profiling revealed that connective tissue growth factor (CTGF; CCN2) was overrepresented in the conditioned media of sFRP2-overexpressing MSCs and MSC-derived CTGF could thus be an important paracrine effector. Subcutaneously implanted, MSC-loaded polyvinyl alcohol (PVA) sponges and stented excisional wounds were used as wound models to study the dynamics of CTGF expression. Granulation tissue generated within the sponges and full-thickness skin wounds showed transient upregulation of CTGF expression by MSCs and fibroblasts, implying a role for this molecule in early tissue repair. Although collagen and COL1A2 mRNA were not increased when recombinant CTGF was administered to sponges during the early phase (day 1–6) of tissue repair, prolonged administration (>15 days) of exogenous CTGF into PVA sponges resulted in fibroblast proliferation and increased deposition of collagen within the experimental granulation tissue. In support of its physiological role, CTGF immunoinhibition during early repair (days 0–7) reduced the quantity, organizational quality and vascularity of experimental granulation tissue in the sponge model. However, CTGF haploinsufficiency was not enough to reduce collagen deposition in excisional wounds. Similar to acute murine wound models, CTGF was transiently present in the early phase of human acute burn wound healing. Together, these results further support a physiological role for CTGF in wound repair and demonstrate that when CTGF expression is confined to early tissue repair, it serves a pro-reparative role. These data also further illustrate the potential of MSC-derived paracrine modulators to enhance tissue repair.

TNFα, produced by most malignant cells, orchestrates the interplay between malignant cells and myeloid cells, which have been linked to tumor growth and metastasis. Although TNFα can exist as one of two isoforms, a 26-kDa membrane tethered form (mTNFα) or a soluble 17-kDa cytokine (sTNFα), the vast majority of published studies have only investigated the biological effects of the soluble form. We demonstrate for the first time that membrane and soluble isoforms have diametrically opposing effects on both tumor growth and myeloid content. Mouse lung and melanoma tumor lines expressing mTNFα, generated small tumors devoid of monocytes versus respective control lines or lines expressing sTNFα. The lack of myeloid cells was due to a direct effect of mTNFα on myeloid survival via induction of cell necrosis by increasing reactive oxygen species. Human NSCLCs expressed varying levels of both soluble and membrane TNFα, and gene expression patterns favoring mTNFα were predictive of improved lung cancer survival. These data suggest that there are significant differences in the role of different TNFα isoforms in tumor progression and the bioavailability of each isoform may distinctly regulate tumor progression. This insight is critical for effective intervention in cancer therapy with the available TNFα inhibitors, which can block both TNFα isoforms.

BackgroundProgrammed necrosis is a form of caspase-independent cell death whose molecular regulation is poorly understood. While tumor necrosis factor-alpha (TNF-α) has been identified as an activator of programmed necrosis, the specific context under which this can happen is unclear. Recently we reported that TNF-α can be expressed by human tumor cells as both a membrane tethered (mTNF-α) and a soluble (sTNF-α) form. Whereas low level, tumor-derived sTNF-α acts as a tumor promoter, tumor cell expression of mTNF-α significantly delays tumor growth in mice, in large part by induction of programmed necrosis of tumor associated myeloid cells. In this study we sought to determine the molecular mechanism involved in mTNF-α oxidative stress-induced cell death by evaluating the known pathways involved in TNF receptor-induced programmed necrosis.MethodsThe source of Reactive Oxygen Species (ROS) in mTNF-α treated cells was determined by coculturing RAW 264.7 monocytic and L929 fibroblasts cells with fixed B16F10 control or mTNF-α expressing-melanoma cells in the presence of inhibitors of NADPH and mitochondria ROS. To identify the down-stream effector of TNF-a receptors (TNFR), level of phospho-RIP-1 and ceramide activity were evaluated.To determine whether mTNF-mediated cell death was dependent on a specific TNFR, cell death was measured in primary CD11b myeloid cells isolated from wild-type or TNFR-1, TNFR-2, TNFR-1 and TNFR-2 double knockout mice, cocultured with various TNF-α isoform.ResultsTumor derived-mTNF-α increased ROS-mediated cytotoxicity, independent of caspase-3 activity. Although TNFR on target cells were required for this effect, we observed that mTNF-induced cell death could be mediated through both TNFR-1 and the death domain-lacking TNFR-2. ROS generation and cytotoxicity were inhibited by a mitochondrial respiratory chain inhibitor but not by an inhibitor of NADPH oxidase. mTNF-α mediated cytotoxicity was independent of RIP-1, a serine/threonine kinase that serves as a main adaptor protein of sTNF-α induced programmed necrosis. Instead, mTNF-α-induced ROS and cell death was prohibited by the ceramide-activated protein kinase (CAPK) inhibitor.ConclusionThese findings demonstrate that the mTNF-α isoform is an effective inducer of programmed necrosis through a caspase independent, ceramide-related pathway. Interestingly, unlike sTNFα, mTNF-induced programmed necrosis is not dependent on the presence of TNFR1.