Tissue Inhibitor of Metalloproteinase–3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells

Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain

Myc Cooperates with Ras by Programming Inflammation and Immune Suppression.

The two oncogenes KRas and Myc cooperate to drive tumorigenesis, but the mechanism underlying this remains unclear. In a mouse lung model of KRasG12D-driven adenomas, we find that co-activation of Myc drives the immediate transition to highly proliferative and invasive adenocarcinomas marked by highly inflammatory, angiogenic, and immune-suppressed stroma. We identify epithelial-derived signaling molecules CCL9 and IL-23 as the principal instructing signals for stromal reprogramming. CCL9 mediates recruitment of macrophages, angiogenesis, and PD-L1-dependent expulsion of T and B cells. IL-23 orchestrates exclusion of adaptive T and B cells and innate immune NK cells. Co-blockade of both CCL9 and IL-23 abrogates Myc-induced tumor progression. Subsequent deactivation of Myc in established adenocarcinomas triggers immediate reversal of all stromal changes and tumor regression, which are independent of CD4+CD8+ T cells but substantially dependent on returning NK cells. We show that Myc extensively programs an immune suppressive stroma that is obligatory for tumor progression

Reactivation of Myc transcription in the mouse heart unlocks its proliferative capacity

Abstract: It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies

Effects of DNA damage in smooth muscle cells in atherosclerosis

Rationale: DNA damage and the DNA damage response (DDR) have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double strand breaks (DSBs) are hypothesized to promote plaque progression and instability, in part by promoting cell senescence, apoptosis and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. Objective: To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. Methods and Results: Human atherosclerotic plaque VSMCs showed increased expression of multiple DDR proteins in vitro and in vivo, particularly the MRN complex (MRE11, RAD50, NBS1) that senses DSB repair. Oxidative stress-induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated two novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with ApoE-/- mice. SM22α-NBS1/ApoE-/- VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/ApoE-/- VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. Conclusions: Human atherosclerotic plaque VSMCs show increased DNA damage including DSBs and DDR activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability

ADAM17 depletion in hVSMCs regulates FAS shedding and increases sensitisation to TIMP-3-dependent apoptosis.

<p>A. Western blot for ADAM17 (A17) or FAS in hVSMCs (Cont), infected with lentivirus conferring puromycin resistance alone (Puro), control non-targeting shRNA (shCont) or shRNA targeting ADAM17 (A17-3 and A17-4). ADAM17 is the middle band, flanked by non-specific bands. B. Soluble FAS levels measured by ELISA in cell medium after 72 h from cells in A. Data are means ± SEM, n = 3. *** = P < 0.001, NS = Not Significant. C. Caspase-3 activity in hVSMC lysates from cell transduced with lentivirus expressing control shRNA or ADAM17 targeting shRNA (A17-3 and A17-4) infected with 300 pfu cell^-1 RAd60 or RAdT3-expressing adenovirus. Data are means ± SEM, n = 3. * = P < 0.05, ** = P < 0.01, NS = Not Significant. D. Quantification of surface FAS by single cell image analysis, data shows the mean number of spot-like FAS surface structures per cell, data are means ± SEM, n = 3. * = P < 0.05, *** = P < 0.001.</p