Metastasis of malignant tumor cells involves cell-cell and cellmatrix interactions, which regulate the expression and localization of proteolytic enzymes. In the present study, we investigated the expression and localization of the lysosomal cysteine proteinase cathepsin B and its natural inhibitors cystatin A, B and C in high-(MV3), intermediate-(SKmel28) and low-invasive (SKmel23, WM164) human melanoma cell lines grown on plastic or in contact with monomeric or fibrillar collagen type I. Neither the transcript levels of cathepsin B nor those of the natural inhibitors, cystatin B and C, were altered by the interaction of melanoma cells with collagen type I. However, protein expression and cellular localization of cathepsin B and its inhibitors were markedly affected. In contrast to low-invasive cells, high-invasive cells constitutively released procathepsin B when cultured on plastic. In addition, contact of invasive cells with fibrillar collagen type I resulted in the release of both mature forms of the protease. Perturbation studies using inhibitory antibodies against the b1 subunit of the integrin receptor indicated a role for the b1 integrin receptor family in the regulation of cathepsin B release. Cystatin B protein expression was much lower in high-invasive cells in both culture conditions, when compared to low-invasive cells. Cystatin C expression was comparable in all cells, but cell contact to fibrillar collagen type I induced its expression. These results strongly implicate a pivotal role of cell-matrix interactions for the regulation of cathepsin B localization and activity in melanoma cells. ' 2005 Wiley-Liss, Inc.Key words: cathepsin B; extracellular matrix; melanoma; cystatins Tumor invasion and metastasis are multistep processes, which include tumor-host interactions mediated by cell-cell and cellmatrix contacts. 1 To invade adjacent tissue and metastasize, tumor cells have to cross various extracellular barriers like extracellular matrix (ECM), basement membranes and interstitial stroma. Degradation of ECM components and remodeling of connective tissue and basement membranes requires the concerted action of various classes of intra-and/or extracellular proteases. 2 These include serine proteases (plasmin, tissue-type plasminogen activator, urokinase-type plasminogen activator and Cat G), matrix metalloproteases (MMP-2, -9, -1, -7, -3 and MT1-MMP), aspartic proteases (Cat D) and cysteine proteases (Cat B, H and L). [3][4][5][6][7][8] Cat B (EC 3.4.22.16) is a lysosomal cysteine protease of the papain family of proteases, 9 which is also involved in a number of disease processes, such as osteoarthritis 10,11 and rheumatoid arthritis. 12 This enzyme has also been shown to be involved in tumor invasion and metastasis. Increase in Cat B activity or upregulation of transcripts/protein expression was found in various types of carcinomas, like breast, 13 lung, 14 gastrointestinal 15 and urogenital 16,17 carcinomas. In addition, Cat B has also been shown to be overexpressed in chondrosarcomas, 18 glioma...
UVA radiation is increasingly used to treat fibrotic skin disorders. However, the mechanisms underlying the therapeutic effects of UVA for these disorders are only partially understood. Cathepsin L is a lysosomal cysteine protease, which has been shown to degrade various matrix proteins thus contributing to extracellular remodeling. Therefore, we investigated whether UVA irradiation regulates the expression and release of cathepsin L in human dermal fibroblasts. No alterations were found after single irradiation; however, a significantly increased extracellular release of cathepsin L was observed after repeated irradiation up to four times. The transcript levels of cathepsin L were elevated after repetitive irradiation, leading to increased amounts of total cathepsin L protein. Furthermore, higher amounts of extracellular cathepsin L were associated with a significant reduction of intracellular processed cathepsin L and an accumulation of unprocessed procathepsin L. The use of specific inhibitors elucidated mannose phosphate-independent sorting pathways of cathepsin L leading to enhanced secretion and reduced intracellular processing. This is the first study which demonstrates that alternate trafficking mechanisms mediate the extracellular release of a cysteine protease induced by repetitive UVA irradiation.
Human papillomavirus type 8 (HPV8) is associated with the development of non-melanoma skin cancer. In the past we already delved into the mechanisms involved in keratinocyte invasion, showing that the viral E7 oncoprotein is a key player that drives invasion of basal keratinocytes controlled by the extracellular protein fibronectin. To unravel further downstream effects in E7 expressing keratinocytes we now aimed at characterizing gene and protein/phosphoprotein alterations to narrow down on key cellular targets of HPV8-E7. We now show that gene expression of GADD34 and GDF15 are strongly activated in the presence of E7 in primary human keratinocytes. Further analyses of fibronectin-associated factors led to the identification of the Src kinase family members Fyn and Lyn being aberrantly activated in the presence of HPV8-E7. Phospho-proteomics further revealed that E7 not only targets cell polarity and cytoskeletal organization, but also deregulates the phosphorylation status of nuclear proteins involved in DNA damage repair and replication. Many of these differentially phosphorylated proteins turned out to be targets of Fyn and Lyn. Taken together, by using unbiased experimental approaches we have now arrived at a deeper understanding on how fibronectin may affect the signaling cascades in HPV8 positive keratinocytes, which may be key for skin tumorigenesis and that may also aid in the development of novel therapeutic approaches for betaHPV-mediated cancers.
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