The interaction of the second Kunitz-type domain (KD2) of TFPI-2 with a novel interaction partner, prosaposin, mediates the inhibition of the invasion and migration of human fibrosarcoma cells.

TFPI-2 (tissue factor pathway inhibitor-2) has recently been recognized as a new tumour suppressor gene. Low expression of this protein in several types of cancers allows for enhanced tumour growth, invasion and metastasis. To investigate the molecular mechanism responsible for the tumour-suppressor effects of TFPI-2, we performed yeast two-hybrid analysis and identified PSAP (prosaposin) as a TFPI-2-interacting partner. This interaction was confirmed by co-immunoprecipitation and immunofluorescence. The region of TFPI-2 that interacts with PSAP is located in the KD2 (Kunitz-type domain 2). Further study showed that PSAP does not affect the function of TFPI-2 as a serine proteinase inhibitor, but that TFPI-2 could inhibit the invasion-promoting effects of PSAP in human HT1080 fibrosarcoma cells. The results of the present study revealed that TFPI-2 interacts with PSAP, which may play an important role in the physiology and pathology of diseases such as cancer.

Comparative transcriptome analysis provides insights into molecular pathway and genes associated with head-type formation and phenotypic divergence in Chinese cabbage.

Background: The heading type of Chinese cabbage is a significant commercial trait with high economic value. At present, research on the phenotypic divergence and formation mechanism of heading type is limited. Results: Through comparative-transcriptome analysis, the formation and phenotypic divergence mechanism of the leafy head of diploid overlapping type cabbage, diploid outward-curling type cabbage, tetraploid overlapping type cabbage, and tetraploid outward-curling type cabbage were systematically and comprehensively investigated, and the phenotype-specific genes of four varieties were revealed. These phenotype-specific differentially expressed genes (DEGs) were considered crucial for cabbage heading type through WGCNA. Some transcription factors have been predicted as significant genes for phenotypic divergence, including the members of the bHLH, AP2/ERF-ERF, WRKY, MYB, NAC, and C2CH2 families. Phytohormone-related genes, including abscisic acid/auxin hormone, may play an important role in the phenotypic divergence of head type in cabbage. Conclusion: Comparative-transcriptome analysis supports a role for phytohormone-related genes and some transcription factors in head-type formation and divergence for four cultivars. These findings increase our understanding of the molecular basis for pattern formation and divergence of the leafy heads of Chinese cabbage and will contribute to developing more desirable leafy head patterns.

The interaction between ubiquitin C-terminal hydrolase 37 and glucose-regulated protein 78 in hepatocellular carcinoma.

The ubiquitin C-terminal hydrolase (UCH) is a subfamily of deubiquitinating enzymes, which consists of four members: UCH-L1, UCH-L3, UCH37, and BRCA1-associated protein-1. Although there is growing evidence that UCH enzymes and human malignancies are closely correlated, there have been few studies on UCH37, especially on its interactions with other proteins. In the current study, a functional proteomic analysis was performed to screen UCH37-interacting proteins in hepatocellular carcinoma (HCC), and glucose-regulated protein 78 was identified as one interacting with UCH37, which was confirmed by co-immunoprecipitation and confocal laser scanning microscopy analysis, suggesting that their interaction could provide a new insight into the mechanism of HCC.

Expression and characterization of Kunitz domain 3 and C-terminal of human tissue factor pathway inhibitor-2.

Human tissue factor pathway inhibitor-2 (hTFPI-2) is a serine protease inhibitor and its inhibitory activity is enhanced by heparin. The Kunitz domain 3 and Cterminal of hTFPI-2 (hTFPI-2/KD3C), which has the activity toward heparin calcium, have been successfully expressed in Pichia pastoris and purified by SPSepharose and heparin-Sepharose chromatography. The Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, and circular dichroism (CD) experiment results implied that hTFPI-2/KD3C contained small contents of alpha-helix and beta-strand, but large amounts of random coil and two kinds of disulfide bonds, gauche-gauche-gauche (ggg) and trans-gauchetrans (tgt). The interaction of hTFPI-2/KD3C with heparin calcium was investigated by CD. It was found that heparin calcium induced b-strands in hTFPI-2/ KD3C to different extents depending on the ratio of hTFPI-2/KD3C and heparin calcium.

Effects of tissue factor pathway inhibitor-2 expression on biological behavior of BeWo and JEG-3 cell lines.

OBJECTIVES: To investigate the effect of tissue factor pathway inhibitor-2 (TFPI-2) expression on biological behavior of BeWo and JEG-3 cell lines. MATERIAL AND METHODS: The expression of TFPI-2 in BeWo and JEG-3 cells was upregulated by pEGFP-N3-TFPI-2 and downregulated by small interference RNA transfection, confirmed by Western blotting assay and real-time polymerase chain reaction (RT-PCR). Boyden chamber, Cell Counting Kit-8 (CCK-8), and Hoechst 33258/terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assays were used for migration, invasion, and proliferation/apoptosis analysis, respectively. RESULTS: In Western blotting and RT-PCR assay, protein and messenger RNA (mRNA) expression of TFPI-2 in transfected BeWo and JEG-3 cells were confirmed. Expression of TFPI-2 inhibited BeWo and downregulated JEG-3 cell migration, invasion, proliferation, and induced apoptosis (P < .05) in Boyden chamber, CCK-8, Hoechst 33258, and TUNEL detection, respectively. CONCLUSIONS: TFPI-2 expression caused invasion and proliferation impair and induced apoptosis in TFPI-2 regulated BeWo and JEG-3 cells. It provides a clue for potential role of TFPI-2 in trophoblast.

Recombinant TFPI-2 enhances macrophage apoptosis through upregulation of Fas/FasL.

Tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine proteinase inhibitor with inhibitory activity toward activated factor XI, plasma kallikrein, plasmin, certain matrix metalloproteinases, and the tissue factor-activated factor VII complex. In addition, TFPI-2 has other functions such as promoting cell migration and inducing apoptosis. In the present study, we investigated if TFPI-2 induced apoptosis in cultured U937-derived macrophages and the possible signal pathways that involved in the apoptotic process. Apoptotic DNA fragment detection and caspase-3,9 activity measurements indicated that rTFPI-2 promoted U937-derived macrophage apoptosis. Hoechst 33342 assay and flow cytometry further showed that rTFPI-2 induced apoptosis in cultured macrophages in a dose-dependent manner. Because death receptors of the TNF family such as Fas are the best-understood death pathways that recruit Fas-associated death domain (FADD) and procaspase-8 to the receptor in macrophages, we investigated the expression of Fas and its ligand (FasL) and downstream signal caspase-8 by Western blot analysis. The results indicated that the process of apoptosis triggered by rTFPI-2 was, at least in part, actively conducted by U937-derived macrophages possibly through Fas/FasL signal pathway. In brief, rTFPI-2 may have the potential usefulness in inducing macrophages apoptosis, which suggest TFPI-2 might have antiatherogenic effects.

Peptide corresponding to the C terminus of tissue factor pathway inhibitor inhibits mesangial cell proliferation and activation in vivo.

Mesangial cells (MsCs) are one of the resident cell types in the glomerulus and are important with respect to its function and structure. The activation and proliferation of MsCs occur in several types of glomerulonephritis, particularly proliferative glomerulonephritis, producing a series of protein factors and matrix components that impair the normal structure and function of the glomerulus. To inhibit proliferation or induction of apoptosis is considered to be one mechanism that can be used to treat these diseases. In previous studies, we found that the tissue factor pathway inhibitor (TFPI) induces the apoptosis of cultured rat MsCs. Here, we expressed a series of TFPI fragments as fusion proteins to maltose binding protein (MBP-TFPI(162-188), MBP-TFPI(187-241), MBP-TFPI(240-276), MBP-TFPI(162-241), MBP-TFPI(187-276) and MBP-TFPI(162-276)) and applied them to cultured rat mesangial cells. The C terminus of TFPI, a peptide corresponding to residues 240-276 of TFPI, was confirmed to induce apoptosis of MsCs in vitro. To observe the effect of this peptide on MsCs in vivo, we performed intramuscular gene transfer treatment on a rat model of proliferative glomerulonephritis with a plasmid containing the gene for the C terminus of TFPI. This revealed that the C terminus of TFPI exhibited suppressive effects on the activation and proliferation of MsCs and, thereby, improved renal function. Our data indicate that the C terminus of TFPI could be used in the treatment of proliferative glomerulonephritis.