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Fibroblasts, which are widely distributed and play a key part in tissue fibrosis, are phenotypically and functionally heterogeneous. Recent studies reported that bone marrow can be a source of tissue fibroblast. In the study reported here, we investigated in vivo characterization of bone marrowderived fibroblasts recruited into various fibrotic lesions. Mice were engrafted with bone marrow isolated from transgenic mice expressing green fluorescent protein (GFP), and fibrotic lesions were induced by cancer implantation (skin), excisional wounding (skin), and bleomycin administration (lung). A small population of GFP + fibroblast was found even in nonfibrotic skin (8.7% ± 4.6%) and lung (8.9% ± 2.5%). The proportion of GFP + fibroblasts was significantly increased after cancer implantation (59.7% ± 16.3%) and excisional wounding (32.2% ± 4.8%), whereas it was not elevated after bleomycin administration (7.1% ± 2.4%). Almost all GFP + fibroblasts in fibrotic lesions expressed type I collagen, suggesting that bone marrow-derived fibroblasts would contribute to tissue fibrosis. GFP + fibroblasts expressed CD45, Thy-1, and α-smooth muscle actin at various proportions. Our results suggested that bone marrow-derived fibroblasts expressed several fibroblastic markers in vivo and could be efficiently recruited into fibrotic lesions in response to injurious stimuli; however, the degree of recruitment frequency might depend on the tissue microenvironment. Stem Cells 2005;23:699-706

The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. Here we examined the effect of ascochlorin, a prenyl-phenol anti-tumor compound from the fungus Ascochyta viciae, on the regulation of signaling pathways that control MMP-9 expression in human renal carcinoma (Caki-1) cells. Ascochlorin reduced the invasive activity of Caki-1 cells and inhibited phorbol 12-myristate 13-acetate-induced increases in MMP-9 expression and activity in a dose-dependent manner. Reporter gene, electrophoretic mobility shift, kinase inhibitor assays, and in vitro kinase assay showed that ascochlorin inhibits MMP-9 gene expression by suppressing activation of the nuclear transcription factor activator protein-1 (AP-1) via the extracellular signalregulated kinase 1 and 2 pathway. The AP-1 family member most specifically affected by ascochlorin was Fra-1. Ascochlorin did not affect the activation of the c-Jun N-terminal or p38 kinase pathways. Moreover, transfection of Caki-1 cells with AP-1 decoy oligodeoxynucleotides resulted in the suppression of phorbol 12-myristate 13-acetate-induced MMP-9 expression and invasion. In conclusion, ascochlorin represents a unique natural anti-tumor compound that specifically inhibits MMP-9 activity through suppression of AP-1-dependent induction of MMP-9 gene expression.Ascochlorin (see Fig. 1A), a prenyl-phenol compound isolated from the fungus, Ascochyta viciae, was originally found to have antiviral antibiotic activity (1). In addition to its antiviral and anti-fungal activity, natural and synthetic derivatives of ascochlorin reduce serum cholesterol and triglyceride levels suppress hypertension and tumor development and ameliorate type I and II diabetes (2-9). Moreover, several ascochlorin derivatives have been reported to be potent agonists of nuclear hormone receptors, including peroxisome proliferator-activated receptor ␥, suggesting that the structure of ascochlorin would be useful in designing modulators of nuclear receptors (6, 10, 11, 13).The most well known extracellular matrix (ECM) 1 -degrading enzymes are the matrix metalloproteinases (MMPs). MMPs are a family of zinc-dependent endoproteinases that are capable of degrading all the components of the ECM. MMPs are structurally and functionally homologous proteins, with at least 20 known members. MMPs can be divided into four families based on structure and substrate specificity: collagenases, gelatinases, stromelysins, and membrane-associated MMPs. Among the previously reported human MMPs, gelatinase-A (MMP-2) and gelatinase-B (MMP-9) are key enzymes that degrade type IV collagen (14, 15). These two gelatinases share structural and catalytic similarities, but transcription of the MMP-2 and MMP-9 genes is independently regulated due to distinct arrays of regulatory elements in the gene promoters. Regulated expression of MMP-9 has been implicated in renal development, macrophage differentiation, atherosclerosis, inflammation, rheumatoid arthritis, and tumor invasion (16 -17). I...

The expression of matrix metalloproteinase-9 (MMP-9) has been implicated in the invasion and metastasis of cancer cells. Here, we found that an antitumor antibiotic, ascofuranone, inhibits invasion and MMP-9 induction induced by phorbol myristate acetate (PMA) in human cell lines. Ascofuranone also inhibits the protein expression and transcription of MMP-9 induced by tumor necrosis factor-alpha. The inhibition of MMP-9 induction was observed in human cancer cell lines as well as primary rat mesangial cells. Furthermore, as evidenced by MMP-9 promoter and electrophoretic mobility shift assays, ascofuranone specifically inhibited MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, ascofuranone suppressed PMA-induced phosphorylation of Ras, Raf, MEK and extracellular signal-regulated kinase (ERK), upstream factors involved in AP-1activation, whereas the phosphorylation of p38 and JNK/mitogen-activated protein kinase was not affected by ascofuranone, suggesting that the primary target of ascofuranone for suppression of the AP-1 induction is present in upstream of ERK signaling pathway. These results suggest that the suppression of MMP-9 expression, at least in part, contributes to the antitumor activity of ascofuranone.

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