TRAF family proteins interact with the common neurotrophin receptor and modulate apoptosis induction.

The common neurotrophin receptor, p75(NTR), has been shown to signal in the absence of Trk tyrosine kinase receptors, including induction of neural apoptosis and activation of NF-kappaB. However, the mechanisms by which p75(NTR) initiates these intracellular signal transduction pathways are unknown. Here we report interactions between p75(NTR) and the six members of TRAF (tumor necrosis factor receptor-associated factors) family proteins. The binding of different TRAF proteins to p75(NTR) was mapped to distinct regions in p75(NTR). Furthermore, TRAF4 interacted with dimeric p75(NTR), whereas TRAF2 interacted preferentially with monomeric p75(NTR). TRAF2-p75(NTR), TRAF4-p75(NTR), and TRAF6-p75(NTR) interactions modulated p75(NTR)-induced cell death and NF-kappaB activation with contrasting effects. Coexpression of TRAF2 with p75(NTR) enhanced cell death, whereas coexpression of TRAF6 was cytoprotective. Furthermore, overexpression of TRAF4 abrogated the ability of dimerization to prevent the induction of apoptosis normally mediated by monomeric p75(NTR). TRAF4 also inhibited the NF-kappaB response, whereas TRAF2 and TRAF6 enhanced p75(NTR)-induced NF-kappaB activation. These results demonstrate that TRAF family proteins interact with p75(NTR) and differentially modulate its NF-kappaB activation and cell death induction.     

Autocrine and paracrine regulation of tissue inhibitor of metalloproteinases, transin, and urokinase gene expression in metastatic and nonmetastatic mammary carcinoma cells.

Acquisition of metastatic competence by tumor cells is frequently accompanied by increased expression of extracellular proteases capable of degrading basement membrane and extracellular matrix. However, very little is known about how the genes encoding these enzymes and their inhibitor proteins are regulated in metastatic versus nonmetastatic cells. In this report, we have compared autocrine and paracrine regulation of tissue inhibitor of metalloproteinases (TIMP), transin, and urokinase plasminogen activator (uPA) genes in genetically related nonmetastatic SP1 and metastatic A3a cell lines. Compared to SP1 cells, metastatic A3a cells showed 15-20-fold higher transin, 3-5-fold less TIMP mRNA, and comparable levels of uPA mRNA. A qualitatively similar shift in expression of these genes was rapidly (i.e., 4-8 h) induced in nonmetastatic SP1 cells following the addition of conditioned medium from A3a cells. The gene-regulating activity present in A3a conditioned medium was heat-labile, suggesting that it was protein in nature. The responsiveness of SP1 cells to the factor(s) secreted by A3a conditioned medium was inhibited by cycloheximide. Basic fibroblast growth factor mimicked the effect of the A3a conditioned medium as an inducer of transin expression in the tumor cells. Although medium conditioned by the tumor cells did not affect uPA expression, addition of epidermal growth factor to the tumor cells transiently induced expression of uPA with a biphasic response that differed in SP1 and A3a cells. Initial induction of uPA at 2-4 h was similar for both cell lines, but after 24 h of exposure to epidermal growth factor, SP1 cells showed a net reduction in uPA, whereas metastatic cells returned to the unstimulated levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor necrosis factor receptor-associated death domain protein is involved in the neurotrophin receptor-mediated antiapoptotic activity of nerve growth factor in breast cancer cells

The common neurotrophin receptor p75(NTR) has been shown to initiate intracellular signaling that leads either to cell survival or to apoptosis depending on the cell type examined; however, the mechanism by which p75(NTR) initiates its intracellular transduction remains unclear. We show here that the tumor necrosis factor receptor-associated death domain protein (TRADD) interacts with p75(NTR) upon nerve growth factor (NGF) stimulation. TRADD could be immunodetected after p75(NTR) immunoprecipitation from MCF-7 breast cancer cells stimulated by nerve growth factor. In addition, confocal microscopy indicated that NGF stimulation induced the plasma membrane localization of TRADD. Using a dominant negative form of TRADD, we also show that interactions between p75(NTR) and TRADD are dependent on the death domain of TRADD, thus demonstrating its requirement for binding. Furthermore, the p75(NTR)-mediated activation of NF-kappaB was inhibited by transfection with a dominant negative TRADD, resulting in an inhibition of NGF antiapoptotic activity. These results thus demonstrate that TRADD is involved in the p75(NTR)-mediated antiapoptotic activity of NGF in breast cancer cells.     

Suppression of IL‐8‐Src signalling axis by 17β‐estradiol inhibits human mesenchymal stem cells‐mediated gastric cancer invasion

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs‐mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin‐8 (IL‐8) protein. Administration of IL‐8 specific neutralizing antibody significantly inhibits HBMMSCs‐mediated gastric cancer motility. Treatment of recombinant IL‐8 soluble protein confirmed the role of IL‐8 in mediating HBMMSCs‐up‐regulated cell motility. IL‐8 up‐regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β ‐estradiol inhibit HBMMSCS‐induced cell motility via suppressing activation of IL8‐Src signalling in human gastric cancer cells. 17β‐estradiol inhibits IL8‐up‐regulated Src downstream target proteins including p‐Cas, p‐paxillin, p‐ERK1/2, p‐JNK1/2, MMP9, tPA and uPA. These results suggest that 17β‐estradiol significantly inhibits HBMMSCS‐induced invasive motility through suppressing IL8‐Src signalling axis in human gastric cancer cells.     

Microenvironment-induced TIMP2 loss by cancer-secreted exosomal miR-4443 promotes liver metastasis of breast cancer

We aimed to investigate the role of exosomal miR-4443 in metastasis of breast cancer (BCa). In vitro wound-healing assay and transwell invasion assay were used to investigate effect of miR-4443 on BCa cells. Animal experiments were performed to confirm its effects in vivo. miR-4443 promotes the metastasis of BCa cells through downregulating tissue inhibitors of metalloproteinase 2 (TIMP2) and upregulating matrix metalloproteinases (MMPs). Highly invasive BCa cells have a higher expression of miR-4443 in both cells and exosomes. The exosomes derived from highly invasive BCa cells mainly gather in the primary tumor and liver. In vivo, overexpression of miR-4443 in noninvasive BCa cells induces liver metastasis, accompanied with downregulated TIMP2, and upregulated MMP-2 in both the primary tumor and liver. When we armed MCF-10A exosomes with miR-4443 inhibitors to treat mice bearing high-miR-4443 tumors, exosomes accumulated in the primary tumor, and liver following the upregulation of TIMP2 and downregulation of MMP2, and the metastasis was inhibited. Highly invasive BCa cells destroy natural barriers against metastasis by delivering exosomal miR-4443 to stromal cells of the primary tumor and impairing TIMP2, consequently activating MMP; circulating exosomal miR-4443 might promote BCa cells lodging in future metastatic sites through the similar mechanisms.     

Functional interaction of Fas-associated phosphatase-1 (FAP-1) with p75(NTR) and their effect on NF-kappaB activation.

The common neurotrophin receptor p75(NTR), a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP-1 (Fas-associated phosphatase-1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas-mediated apoptosis. Here we have shown by co-immunoprecipitation that FAP-1 also binds to the p75(NTR) cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP-1 and C-terminal Ser-Pro-Val residues of p75(NTR). Furthermore, cells expressing a FAP-1/green fluorescent protein showed intracellular co-localization of FAP-1 and p75(NTR) at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor-associated factor 6)-mediated NF-kappaB activation and tamoxifen-induced apoptosis in 293T cells expressing p75(NTR). The results revealed that TRAF6-mediated NF-kappaB activation was suppressed by p75(NTR) and that the p75(NTR)-mediated NF-kappaB suppression was reduced by FAP-1 expression. Interestingly, a mutant of the p75(NTR) intracellular domain with a single substitution of a Met for Val in its C-terminus, which cannot interact with FAP-1, displayed enhanced pro-apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP-1 may be involved in suppressing p75(NTR)-mediated pro-apoptotic signaling through its interaction with three C-terminal amino acids (tSPV). Thus, FAP-1 may regulate p75(NTR)-mediated signal transduction by physiological interaction through its third PDZ domain.     

Quantitative secretome analysis reveals that COL6A1 is a metastasis-associated protein using stacking gel-aided purification combined with iTRAQ labeling

In cancer metastasis, secreted proteins play an important role in promoting cancer cell migration and invasion and thus also in the increase of cancer metastasis in the extracellular microenvironment. In this study, we developed a strategy that combined a simple gel-aided protein purification with iTRAQ labeling to quantify and discover the metastasis-associated proteins in the lung cancer cell secretome. Secreted proteins associated with lung cancer metastasis were produced using CL1-0 and CL1-5 cells with different metastatic abilities. Quantitative secretomics analysis identified a total of 353 proteins, 7 of which were considered to be metastasis-associated proteins. These included TIMP1, COL6A1, uPA, and AAT, all of which were higher in CL1-5, and AL1A1, PRDX1, and NID1, which were higher in CL1-0. Six of these metastasis-associated proteins were validated with Western blot analysis. In addition, pathway analysis was performed in building the interaction network between the identified metastasis-associated proteins. Further functional analysis of COL6A1 on the metastatic abilities of CL1 cells was also carried out. An RNA interference-based knock-down of COL6A1 suppressed the metastatic ability of CL1-5 cells; in contrast, a plasmid-transfected overexpression of COL6A1 increased the metastatic ability of CL1-0 cells. This study describes a simple and high throughput sample purification method that can be used for the quantitative secretomics analysis of metastasis-associated proteins.     

Tissue inhibitor of metalloproteinases‐1‐induced scattered liver metastasis is mediated by host‐derived urokinase‐type plasminogen activator

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases‐1 (TIMP‐1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP‐1‐induced modulation of a pro‐metastatic microenvironment in the liver. Indeed, in livers of uPA‐ablated mice elevated TIMP‐1 levels did not trigger HGF signalling and did not promote metastasis of a murine T‐lymphoma cell line. In contrast, lack of tumour cell‐derived uPA induced by gene silencing did not interfere with this pro‐metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP‐1 levels. This newly identified co‐operation between TIMP‐1 and host uPA suggests that therapies, simultaneously interfering with pro‐ and anti‐proteolytic pathways may be beneficial for patients with metastatic disease.     

NSAID inhibition of prostate cancer cell migration is mediated by Nag-1 Induction via the p38 MAPK-p75(NTR) pathway

The nonsteroidal anti-inflammatory drugs (NSAID) R-flurbiprofen and ibuprofen have been shown to induce expression of p75(NTR) (neurotrophin receptor) in prostate cancer cell lines. p75(NTR), a tumor necrosis factor receptor superfamily member, is a proapoptotic protein that functions as a tumor suppressor in the human prostate. Expression of p75(NTR) is lost as prostate cancer progresses and is minimal in several metastatic prostate cancer cell lines. NSAIDs induce p75(NTR) through activation of the p38 mitogen-activated protein kinase (MAPK) pathway, with a concomitant decrease in cell survival. Here, we show that treatment with R-flurbiprofen and ibuprofen induces expression of the NSAID-activated gene-1 (Nag-1) protein, a divergent member of the TGF beta (TGF-β) family, in PC-3 cells. Using the selective pharmacologic inhibitor of p38 MAPK, SB202190, and p38 MAPK-specific siRNA (small interfering RNA), we show that Nag-1 induction following NSAID treatment is mediated by the p38 MAPK pathway. p75(NTR)-specific siRNA pretreatment shows that Nag-1 induction by NSAIDs is downstream of p75(NTR) induction. Decreased survival of NSAID-treated cells is rescued by p75(NTR)-specific siRNA but not by Nag-1 siRNA. Transwell chamber and in vitro wound healing assays demonstrate decreased cell migration upon NSAID treatment. Pretreatment of PC-3 cells with p75(NTR) and Nag-1-specific siRNA shows that NSAID inhibition of cell migration is mediated by Nag-1 and p75(NTR). These results demonstrate a role for Nag-1 in NSAID inhibition of cell migration, but not survival.     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.     

Thioredoxin alters the matrix metalloproteinase/tissue inhibitors of metalloproteinase balance and stimulates human SK-N-SH neuroblastoma cell invasion.

Thioredoxin (Trx) inhibited tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 activity with an approximate IC50 of 0.3 microM, matrix metalloproteinase (MMP)-2 activity with an approximate IC50 of 2 microM but did not inhibit MMP-9 activity. This differential capacity of Trx to inhibit TIMP and MMP activity resulted in the promotion of MMP-2 and MMP-9 activity in the presence of molar TIMP excess. Inhibition of TIMP and MMP-2 activity by Trx was dependent upon thioredoxin reductase (TrxR), was abolished by Trx catalytic site mutation and did not result from TIMP or MMP-2 degradation. HepG2 hepatocellular carcinoma cells induced to secrete Trx inhibited TIMP activity in the presence of TrxR. SK-N-SH neuroblastoma cells secreted TrxR, which inhibited TIMP and MMP-2 activity in the presence of Trx. Trx stimulated SK-N-SH invasive capacity in vitro in the absence of exogenous TrxR. This study therefore identifies a novel extracellular role for the thioredoxin/thioredoxin reductase redox system in the differential inhibition of TIMP and MMP activity and provides a novel mechanism for altering the TIMP/MMP balance that is of potential relevance to tumor invasion.     

Comparison of metalloproteinase protein and activity profiling

Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The current study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer cell lines and 1 normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media (in both amount and variety). TIMP-1 was produced in all cell lines. MMP activity assays with three different fluorescence resonance energy transfer (FRET) substrates were then used to determine whether protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was confirmed only in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays.     

Functional proteomics study reveals that N-Acetylglucosaminyltransferase V reinforces the invasive/metastatic potential of colon cancer through aberrant glycosylation on tissue inhibitor of metalloproteinase-1

N-Acetylglucosaminyltransferase-V (GnT-V) has been reported to be up-regulated in invasive/metastatic cancer cells, but a comprehensive understanding of how the transferase correlates with the invasive/metastatic potential is not currently available. Through a glycomics approach, we identified 30 proteins, including tissue inhibitor of metalloproteinase-1 (TIMP-1), as a target protein for GnT-V in human colon cancer cell WiDr. TIMP-1 was aberrantly glycosylated as characterized by the addition of beta1,6-N-acetylglucosamine, polylactosaminylation, and sialylation in GnT-V-overexpressing WiDr cells. Compared with normal TIMP-1, the aberrantly glycosylated TIMP-1 showed the weaker inhibition on both matrix metalloproteinase (MMP)-2 and MMP-9, and this aberrancy was closely associated with cancer cell invasion and metastasis in vivo as well as in vitro. Integrated data, both of TIMP-1 expression level and aberrant glycosylation, could provide important information to aid to improve the clinical outcome of colon cancer patients.     

uPA and MMP‐2 were involved in self‐assembled network formation in a two dimensional co‐culture model of bone marrow stromal cells and endothelial cells

Two dimensional (2D) co‐cultures of human bone marrow stromal cells (HBMSCs) and human umbilical vein endothelial cells (HUVECs) stimulate osteoblastic differentiation of HBMSCs, induce the formation of self‐assembled network and cell interactions between the two cell types involving many vascular molecules. Because of their strong activities on angiogenesis and tissue remodeling, urokinase plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), matrix metalloproteinase‐2 (MMP‐2) as well tissue inhibitors of matrix metalloproteinase‐2 (TIMP‐2) were investigated in this 2D co‐culture model. We found that the expression of uPA, MMP‐2 in the co‐cultured cells was significantly higher than those in mono‐cultured cells. In opposite, PAI‐1, expressed only by HUVECs is not regulated in the co‐culture. Inhibition assays confirm that uPA played a critical role in the formation of self‐assembled network as neutralization of uPA disturbed this network. In the same context, inhibition of MMP‐2 prevented the formation of self‐assembled network, while the inhibition of uPA abolished the over expression and the activity of MMP‐2. This upregulation could initiate the uPA expression and proteolysis processes through the MMP‐2 activity, and may contribute to endothelial cell migration and the formation of this self‐assembled network observed in these 2D co‐cultured cells. J. Cell. Biochem. 114: 650–657, 2013. © 2012 Wiley Periodicals, Inc.     

Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.

The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.     

人组织型基质金属蛋白酶抑制剂-1在Pichia pastoris酵母中重组表达研究

 为研究组织型基质金属蛋白酶抑制剂 (TIMPs)的分子作用机制 ,探讨了在 Pichia pastoris酵母中高效表达分泌型人组织型基质金属蛋白酶抑制剂 - 1 (TIMP- 1 )的技术路线 ,并对产物性质进行初步研究 .通过 PCR从含有 TIMP- 1基因的 p BS质粒获得了该基因的全长序列 ,构建了 p PIC9/T1表达载体 ,电击法转化酵母 ,通过表型筛选和 PCR鉴定证实了目的基因已稳定整合入 Pichiapastoris酵母基因组中 .SDS- PAGE表明表达量高达 40 mg/L培养上清 .用免疫印迹法确定了产物的正确性 ;同时 ,反向明胶酶谱法证明了重组蛋白具有抑制基质金属蛋白酶的活性 .