Pathological Significance of Human Collagenase3

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To date, human collagenase-3 expression has been associated with two pressing pathological conditions: cancer and arthritic diseases. In fact, collagenase-3 was originally identified as an enzyme produced by mammary carcinomas,1 but further studies have extended its participation to other tumor processes distinct from breast cancer. In addition, the relevance of collagenase-3 as a degradative enzyme in joint diseases has been the focus of notable interest, with a number of papers examining distinct aspects of this connection 9,10,12-15,70

The first studies linking collagenase-3 to tumor processes were performed by Freije et al,1 who detected the expression of collagenase-3 mRNA in some breast carcinomas but not in benign lesions or unaffected normal mammary gland. This expression analysis also revealed three different mRNA species in breast carcinomas which result from the alternative use of different polyadenylation sites in the 3'-flanking region of the collagenase-3 gene. In situ RNA hybridization analysis of collagenase-3 expression in breast carcinomas has shown that collagenase-3 transcripts are detectable in fibroblastic cells surrounding epithelial cells, but not in the carcinoma cells themselves or normal breast glandular epithelium.8 In addition, the fibroblastic cells most positive for collagenase-3 expression were those immediately adjacent to islands of cells present at the invasive edge of the tumor. This pattern of expression resembles that previously noted for other MMPs produced by breast carcinomas, including stromelysin-3, MT1-MMP and gelatinase A, which are also predominantly expressed by fibroblast cells within the tumor stroma adjacent to breast cancer cells.71-73 The stromal expression of collagenase-3 is consistent with coculture experiments using human fibroblasts and MCF-7 breast cancer cells, demonstrating that conditioned medium from these latter cells stimulates the fibroblastic expression of collagenase-3 mRNA. By contrast, no stimulatory effect is observed when medium from fibroblasts is added to epithelial breast cancer cells.8 These results suggest that transcription of the collagenase-3 gene in stromal cells of breast carcinomas is activated by diffusible factors released from epithelial tumor cells. To date, the precise nature of these soluble factors remains unknown, although a survey of cytokines and growth factors in human fibroblasts, has identified IL-1 as a potential candidate to induce collagenase-3 expression in breast carcinomas.8 The expression of col-lagenase-3 in breast carcinomas has been confirmed by Heppner et al,74 although in this work the enzyme was reportedly produced preferentially by tumor cells. These variations in intracellular location have been reported for other MMPs, and stromal expression may play a role in earlier events in tumor progression, whereas epithelial cell expression may be more relevant in late stage carcinomas.75 Although both epithelial and stromal expression of col-lagenase-3 may occur, the presence of this enzyme in breast carcinomas mainly results from stromal expression rather than from synthesis by the tumor cell themselves. This is supported by the finding of collagenase-3 mRNA in different fibroblastic cell lines but not in any of the analyzed breast cancer cells, and by the results of coculture experiments. Conse quently, collagenase-3 should be included among the molecular factors detected during the stromal reaction to invasive breast cancer, whose concerted action may be essential for tumor growth and progression.5 At present, this theory is without direct evidence, although preliminary clinical studies of collagenase-3 expression in these tumors suggest that production of high levels of this enzyme by breast carcinomas is a factor in poor prognosis in breast cancer patients (F. Vizoso, unpublished observations). Further support was provided by Lochter et al76 who have recently provided evidence that collagen invasion by TCL1 mouse mammary carcinoma cells producing collagenase-3 is reduced by half in the presence of collagenase-3 antisense oligonucleotides, but in contrast, invasion of SCg6 cells not expressing collagenase-3 remain unaffected. Treatments that increase collagenase-3 expression such as culture of TCL1 cells in the presence of Matrigel promote collagen invasion, whereas further up-regulation of other MMPs does not cause any additional effect.76 Taken together, these results support the proposal that collagenase-3 expression is one of the proteolytic enzymes relevant to mammary tumor cell progression in the invasive phenotype. Further studies with animal models, including transgenic mice overexpressing collagenase-3 in their mammary glands, will be required to elucidate the precise role of this enzyme in breast cancer.

In addition to these studies implicating collagenase-3 expression in breast carcinomas, more recent studies have revealed that this protein is also produced by other malignant tumors, including laryngeal carcinomas, chondrosarcomas, and ovarian carcinomas.28,77,78 Northern blot analysis of collagenase-3 mRNA in a series of 35 matched squamous cell carcinomas of the larynx and the corresponding non-neoplastic adjacent tissues showed significant levels of expression in 20 of the 35 carcinomas (57%), but not in any of the normal mucosas.28 Western blot analysis confirmed the presence of collagenase-3 protein in those carcinomas with high levels of mRNA expression, whereas no protein was detected in the carcinomas with negative collagenase-3 mRNA expression, or in any of the normal tissues. Immunohistochemical analysis of these carcinomas localized collagenase-3 predominantly in squamous cancer cells. This observation complements recent findings that this enzyme is produced by transformed human epidermal keratinocytes in culture, but not by cultured primary epidermal cells.79 Interestingly, collagenase-3 overexpression in laryngeal carcinomas correlated significantly with advanced local invasion of the tumors, suggesting that this protein may contribute to the progression of a significant subset of squamous cell carcinomas of the larynx.28 On the other hand, and since collagenase-3 has been found in normal chondrocytes and ovarian cells, recent studies have evaluated the possibility that tumors involving these cells also could be a source of collagenase-3 expression. In fact, most chondrosarcomas analyzed produce significant levels of collagenase-3, whereas the protein is either present at low levels or undetectable in benign chondromas.77 The high levels of collagenase-3 present in human chondrosarcomas is of interest in light of previous observations demonstrating that c-/os-transgenic mice develop osteosarcomas and chondrosarcomas which overexpress collagenase-3.78 Similarly, ovarian carcinomas produce collage-nase-3, and furthermore mucinous carcinomas produce even higher levels than other types of ovarian tumors (M.Balbin, unpublished results). Since mucinous carcinomas have a relatively bad prognosis when compared with other ovarian carcinomas,80 these results are consistent with those derived from analysis of other collagenase-3-producing tumors, in which the presence of high levels of this protease seems to be associated with tumor progression and poor clinical outcome.

Besides the evidence linking collagenase-3 to tumor processes, data has been also accumulating that implies a possible role for this enzyme in destructive joint diseases. To this end, Mitchell et al12 demonstrated that collagenase-3 was expressed by chondrocytes in human osteoarthritic cartilage but not in normal articular cartilage. Relative mRNA levels varied significantly between different samples, in some cases showing high levels of collage-nase-3 but not collagenase-1, and vice versa. Shortly after, other groups confirmed and extended these findings, showing that collagenase-3 is not specific to osteoarthritic lesions but also can be found in rheumatoid arthritis.9,10,15 The enzyme mainly localizes to chondrocytes from arthritic cartilage, but its presence has been noted in fibroblasts and macrophage-like cells in both the synovial lining and stroma. Furthermore, a series of proinflammatory cytokines, such as IL-1a, IL-1|, and TNF-a, have been identified as potential mediators of the increased synthesis of collagenase-3 in osteoarthritic cartilage.12-14 The finding of colla-genase-3 in both osteoarthritis and rheumatoid arthritis raised new insights into the potential pathogenetic role of collagenases in the arthritides. According to previous data, collage-nase-1 produced by fibroblasts and macrophage-like cells at the advancing edge of the pannus was believed to be the major enzyme responsible for the degradation of the collagenous structures of the rheumatoid joint. In addition, collagenase-2 released from synovial fluid neutrophils could also contribute to degradation of type II collagen in the inflamed joint. However, none of these enzymes is particularly effective against the major collagen of the articular cartilage. Therefore, the finding of a third type of collagenase very efficient in the cleavage of type II collagen and expressed in the distinct cell types involved in the degradation of articular cartilage strongly suggests that it represents a significant mediator of tissue destruction in the arthritides. The relative contribution of the three human collagenases to these destructive joint events is presently unknown and will likely depend on a number of factors, including their relative expression levels, degree and mechanism of activation, sensitivity to endogenous inhibitors, and relative activity against other cartilage macromol-ecules distinct from type II collagen. Nevertheless, Billinghurst et al,29 through the use of both antibodies reactive to neoepitopes generated by MMP cleavage of type II collagen and a synthetic preferential inhibitor of collagenase-3, have provided evidence that this enzyme plays the major role in cleavage of type II collagen in osteoarthritic cartilage. Therefore, collagenase-3 must be included among the molecular targets for therapeutic intervention designed to restrict the cartilage damage in destructive joint diseases.

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