Matti Ahonen

Tissue inhibitor of metalloproteinases-3 induces apoptosis in melanoma cells by stabilization of death receptors

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin (ADAM) activity. We have previously shown that adenovirally expressed tissue inhibitor of metalloproteinases-3 (TIMP-3) induces apoptosis in melanoma cells and inhibits growth of human melanoma xenografts. Here, we have studied the role of death receptors in apoptosis of melanoma cells induced by TIMP-3. Our results show, that the exposure of three metastatic melanoma cell lines (A2058, SK-Mel-5, and WM-266-4) to recombinant TIMP-3, N-terminal MMP inhibitory domain of TIMP-3, as well as to adenovirally expressed TIMP-3 results in stabilization of tumor necrosis factor receptor-1 (TNF-RI), FAS, and TNF-related apoptosis inducing ligand receptor-1 (TRAIL-RI) on melanoma cell surface and sensitizes these cells to apoptosis induced by TNF-α, anti-Fas-antibody and TRAIL. Stabilization of death receptors by TIMP-3 results in activation of caspase-8 and caspase-3, and subsequent apoptosis is blocked by specific caspase-8 inhibitor (Z-IETD-FMK) and by pan-caspase inhibitor (Z-DEVD-FMK). Adenovirus-mediated expression of TIMP-3 in human melanoma xenografts in vivo resulted in increased immunostaining for TNF-RI, FAS, and cleaved caspase-3, and in apoptosis of melanoma cells. Taken together, these results show that TIMP-3 promotes apoptosis in melanoma cells through stabilization of three distinct death receptors and activation of their apoptotic signaling cascade through caspase-8.

Targeted inhibition of human collagenase-3 (MMP-13) expression inhibits squamous cell carcinoma growth in vivo.

Squamous cell carcinomas (SCCs) of the head and neck are characterized by a high tendency for local invasion and metastasis to lymph nodes. Collagenase-3 (MMP-13) is specifically expressed by tumor cells in SCCs of the head and neck and its expression correlates with their invasion capacity. To specifically examine the role of MMP-13 in the growth and invasion of SCC, we constructed a hammerhead ribozyme targeted against human MMP-13 mRNA. The anti-MMP-13 ribozyme effectively cleaved MMP-13 transcripts in vitro. Adenoviral delivery of the anti-MMP-13 ribozyme to cutaneous metastatic SCC cells in culture resulted in potent and specific inhibition of the production of proMMP-13 and markedly suppressed invasion of SCC cells through Matrigel. In addition, adenoviral delivery of anti-MMP-13 ribozyme promoted apoptosis in SCC cells within 72 h. Intratumoral injection of anti-MMP-13 ribozyme coding adenovirus into human SCC xenografts established in SCID mice potently suppressed tumor growth, inhibited MMP-13 expression and gelatinolytic activity and reduced the number of proliferating cells within the tumors. These results provide evidence for an important role for MMP-13 in SCC growth and invasion and identify MMP-13 as a promising target for ribozyme-based therapy of SCC in vivo.

Accelerated up-regulation of L-Sox5, Sox6, and Sox9 by BMP-2 gene transfer during murine fracture healing.

Fracture repair is the best‐characterized situation in which activation of chondrogenesis takes place in an adult organism. To better understand the mechanisms that regulate chondrogenic differentiation of mesenchymal progenitor cells during fracture repair, we have investigated the participation of transcription factors L‐Sox5, Sox6, and Sox9 in this process. Marked up‐regulation of L‐Sox5 and Sox9 messenger RNA (mRNA) and smaller changes in Sox6 mRNA levels were observed in RNAse protection assays during early stages of callus formation, followed by up‐regulation of type II collagen production. During cartilage expansion, the colocalization of L‐Sox5, Sox6, and Sox9 by immunohistochemistry and type II collagen transcripts by in situ hybridization confirmed a close relationship of these transcription factors with the chondrocyte phenotype and cartilage production. On chondrocyte hypertrophy, production of L‐Sox5, Sox9 and type II collagen were down‐regulated markedly and that of type X collagen was up‐regulated. Finally, using adenovirus mediated bone morphogenetic protein 2 (BMP‐2) gene transfer into fracture site we showed accelerated up‐regulation of the genes for all three Sox proteins and type II collagen in fractures treated with BMP‐2 when compared with control fractures. These data suggest that L‐Sox5, Sox6, and Sox9 are involved in the activation and maintenance of chondrogenesis during fracture healing and that enhancement of chondrogenesis by BMP‐2 is mediated via an L‐Sox5/Sox6/Sox9‐dependent pathway.

Human TIMP-3 Is Expressed During Fetal Development, Hair Growth Cycle, and Cancer Progression

We studied the expression and regulation of TIMP-3, a recently cloned member of the tissue inhibitor of the metalloproteinase family, during human fetal development and in various human tissues, with emphasis on epithelial structures. Expression of TIMP-3 mRNA was detected by in situ hybridization in developing bone, kidney, and various mesenchymal structures. At 16 weeks of gestation, ectoderm-derived cells of hair germs expressed TIMP-3 mRNA, and beginning from the twentieth week consistent expression was detected in epithelial outer root sheath cells of growing hair follicles. In normal adult human skin, expression of TIMP-3 mRNA was limited to hair follicles, starting at the early anagen (growing) phase and vanishing at the catagen (regressing) phase. TIMP-3 mRNA was not detected in benign hair follicle-derived tumors but was present in tumor cells of infiltrative basal cell carcinomas and in surrounding stromal cells in squamous cell carcinomas. Human primary keratinocytes in culture expressed TIMP-3 mRNAs, the levels of which were upregulated by transforming growth factor-β (TGF-β), whereas interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) had no effect. Our results suggest a role for TIMP-3 in connective tissue remodeling during fetal development, hair growth cycle, and cancer progression.

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human chondrosarcomasNote

The aim of the present study was to characterise the ability of malignant chondrosarcomas to invade normal bone by analysing their production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). For this purpose 12 chondrosarcomas were investigated for the expression of mRNAs for several MMPs and all 4 TIMPs by Northern hybridisation, and for immunohistochemical localisation of the proteins. A characteristic finding of these analyses was increased expression of MMP‐13, MMP‐14 and TIMP‐2 mRNAs in chondrosarcomas when compared with nonmalignant control samples. Individual chondrosarcomas also exhibited elevated levels of MMP‐1, MMP‐7 and MMP‐9 mRNAs. The results of Northern hybridisations were supported by immunohistochemical stainings of the corresponding tumour areas for MMP‐2, MMP‐14 and TIMP‐2, further suggesting that these may have prognostic value for determining whether individual chondrosarcomas are locally aggressive or have a probability of recurrence. Another finding of the present study was a marked heterogeneity in histologic appearance and gene expression of the chondrosarcomas, emphasising the importance of analysing several areas of these tumours to get representative results. These findings suggest that analysis of MMPs could be a useful diagnostic indicator in patients with cartilaginous tumours and could help in differentiating between a low‐grade malignant chondrosarcoma and a benign growing enchondroma.

Potential applications of tissue inhibitor of metalloproteinase (TIMP) overexpression for cancer gene therapy.

Invasion and metastasis of malignant cells is a multistep process which involves detachment of cells from primary tumor, degradation of structural barriers such as basement membrane and collagenous extracellular matrix (ECM), as well as migration of cells through the degraded matrix (see MacDougal and Matrisian, 1995; Johnsen et al., 1998). Tumor invasion in vivo involves interplay between tumor cells, stromal cells and inflammatory cells, all of which can express a variable set of proteolytic enzymes and contribute to degradation of peritumoral stromal matrix. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all components of the ECM (see Birkedal-Hansen et al., 1993). Upregulation of MMP expression has been documented in a number of malignant tumors and has been associated with invasive capacity of tumors. In addition, MMPs apparently play a role in tumor-induced angiogenesis. The activity of MMPs is inhibited specifically by tissue inhibitors of metalloproteinases (TIMPs). Here we discuss the role of MMPs

Induction of periosteal callus formation by bone morphogenetic protein-2 employing adenovirus-mediated gene delivery.

Although the chondrogenic response of periosteum is well established in healing fractures, the mechanisms mediating the proliferation and differentiation of periosteal chondroprogenitor cells are poorly understood. In the present study we demonstrate that bone morphogenetic protein-2 (BMP-2), introduced by adenovirus-mediated gene transfer, alone is capable of inducing callus formation at the site of periosteal injection. Both immunohistochemistry and Northern analysis demonstrated activation of type II collagen production between days 4 and 7 after the injection, followed by activation of type X collagen expression. The activation of chondrogenesis was associated with increased expression of L-Sox5 and Sox9, suggesting that the BMP-2 effect is mediated via Sox proteins. This capacity of adenovirus-mediated overproduction of BMP-2 to induce chondrogenesis (and subsequent endochondral ossification) should be useful for tissue engineering of cartilage and bone.