Tumor tissue inhibitor of metalloproteinases-1 (TIMP-1) in hormone-independent breast cancer might originate in stromal cells, and improves stratification of prognosis together with nodal status.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is shown to be a potential marker for poor prognosis in breast cancer, but the biology of TIMP-1 is only partially understood. In this study, TIMP-1 production was studied in a co-culture model of hormone-independent breast cancer cell lines and mesenchymal stem cells mimicking the stromal components of the tumor. In addition, the prognostic value of TIMP-1 was histologically evaluated in a clinical material of 168 patients with hormone-independent breast tumors. The hormone-independent breast cancer (BC) cell lines MDA-MB-231, M4A4 and NM2C5 did not produce TIMP-1 protein in measureable quantities. Six tested primary mesenchymal stem cell lines all produced TIMP-1. Co-culturing of mesenchymal stem cells and breast cancer cells resulted in positive immunocytochemical diffuse staining for TIMP-1 for both cell types. Culturing breast cancer cells with MSC-conditioned media resulted in a positive cytoplasmic immunoreactivity for TIMP-1, and TIMP-1 protein concentration in cell lysates increased 2.7-fold (range 1.1-4.7). The TIMP-1 mRNA levels remained unaffected in BC cells. This might suggest that breast cancer cells can take up TIMP-1 produced by stromal cells and are thus displaying cellular immunoreactivity. In addition, TIMP-1 was shown to improve stratification of prognosis in clinical material.

Increased expression of glucocorticoid receptor beta in lymphocytes of patients with severe atopic dermatitis unresponsive to topical corticosteroid.

BACKGROUND: Variable response to topical glucocorticoid therapy occurs in the treatment of severe atopic dermatitis (AD). Glucocorticoid receptor (GR)-beta does not bind glucocorticoids but antagonizes the activity of the classic GRalpha, and could thus account for glucocorticoid insensitivity. OBJECTIVES: To investigate GRalpha and GRbeta mRNA and protein expression in lymphocytes of patients with AD before and after treatment with topical corticosteroids. METHODS: Blood was collected from 11 healthy donors, 10 patients with mild AD and 13 patients with severe AD. mRNA was isolated from peripheral blood mononuclear cells. Expression of GRalpha and GRbeta mRNA was determined by reverse transcriptase-polymerase chain reaction and quantitated. Expression of the GRs was confirmed by Western blot analysis. RESULT: The expression of GRalpha mRNA was detected in all subjects. GRbeta mRNA was detected in four out of 11 healthy volunteers, five out of 10 patients with mild AD and 11 out of 13 patients with severe AD. The incidence of GRbeta mRNA expression was higher in patients with severe AD (85%) than in patients with mild AD (50%), and significantly higher than in healthy volunteers (36%, P = 0.033). Four of the 13 patients with severe AD showed a 3.3-13.2-fold increase in the expression of GRbeta mRNA during a 2-week treatment with topical corticosteroids. In these patients the response to topical corticosteroids was poor. CONCLUSIONS: Expression of GRbeta is increased during topical corticosteroid treatment in the lymphocytes of patients with AD and, in particular, glucocorticoid-insensitive AD is associated with increased expression of GRbeta.

ADAM-TS5, ADAM-TS6, and ADAM-TS7, novel members of a new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family.

We report the primary structure of three novel, putative zinc metalloproteases designated ADAM-TS5, ADAM-TS6, and ADAM-TS7. All have a similar domain organization, comprising a preproregion, a reprolysin-type catalytic domain, a disintegrin-like domain, a thrombospondin type-1 (TS) module, a cysteine-rich domain, a spacer domain without cysteine residues, and a COOH-terminal TS module. These genes are differentially regulated during mouse embryogenesis and in adult tissues, with Adamts5 highly expressed in the peri-implantation period in embryo and trophoblast. These proteins are similar to four other cognate gene products, defining a distinct family of human reprolysin-like metalloproteases, the ADAM-TS family. The other members of the family are ADAM-TS1, an inflammation-induced gene, the procollagen I/II amino-propeptide processing enzyme (PCINP, ADAM-TS2), and proteins predicted by the KIAA0366 and KIAA0688 genes (ADAM-TS3 and ADAM-TS4). Individual ADAM-TS members differ in the number of COOH-terminal TS modules, and some have unique COOH-terminal domains. The ADAM-TS genes are dispersed in human and mouse genomes.

Expression of matrix metalloproteinases (MMP-1 and -2) and their inhibitors (TIMP-1, -2 and -3) in oral lichen planus, dysplasia, squamous cell carcinoma and lymph node metastasis.

Although matrix metalloproteinases (MMPs) are among the potential key mediators of cancer invasion, their involvement in premalignant lesions and conditions is not clarified. Therefore, we studied, using in situ hybridization, immunohistochemistry and zymography the expression and distribution of MMP-1 and -2, and their tissue inhibitors (TIMPs -1, -2 and -3) in oral squamous cell carcinomas (SCC) and lymph node metastases as well as in oral lichen planus, epithelial dysplasias and normal buccal mucosa. In oral SCC and lymph node metastasis, MMP-1 mRNA was detected in fibroblastic cells of tumoral stroma. In two out of ten carcinomas studied, the peripheral cells of neoplastic islands were also positive. MMP-2 mRNA expression was noted in fibroblasts surrounding the carcinoma cells, and no signal in carcinoma cells was detected. A clear TIMP-3 mRNA expression was seen in stromal cells surrounding the neoplastic islands in all SCCs and lymph node metastases studied. TIMP-1 mRNA was detected in some stromal cells surrounding the neoplastic islands, whereas the mRNA expression for TIMP-2 was negligible. On the other hand, expression of MMPs and TIMPs was consistently low in oral epithelial dysplasias, lichen planus and normal mucosa. In certain epithelial dysplasias and lichen planus, MMP-1 and -2 mRNA expressions were detected in few fibroblasts under the basement membrane zone, but normal mucosa was completely negative. In SCC and lymph node metastasis, a detectable immunostaining for MMP-1 in stromal cells and in some carcinoma cells was observed. MMP-2 immunoreactivity was detected in the peripheral cell layer in neoplastic islands and in some fibroblast-like cells of tumoral stroma. Immunostaining for TIMP-3 was detected in stromal cells surrounding the neoplastic islands. A weak positive staining for TIMP-1 was located in tumoral stroma, whereas the immunostaining for TIMP-2 was negative. Using zymography, elevated levels of MMP-2 and MMP-9 were observed in carcinoma samples in comparison with lichen planus or normal oral mucosa. Our results indicate that the studied MMPs and TIMPs are clearly up-regulated during invasion in oral SCC. However, there was also a clear, although weak, up-regulation of the expression of the MMPs but not TIMPs in some of the lichen planus and dysplastic lesions.

Production of membrane-type matrix metalloproteinase-1 (MT-MMP-1) in early human placenta. A possible role in placental implantation?

The extracellular matrix proteolytic machinery is known to play a major role in trophoblast invasion, a process that shares similar features with the pathology of tumor invasion. In this study we investigated the expression of the recently described membrane-type matrix metalloproteinase-1 (MT-MMP-1; MMP-14) in early human placenta and decidual membrane to determine whether it might play a role in invasion. With in situ hybridization, the cytotrophoblasts of trophoblastic columns and the infiltrating intermediate trophoblasts in the decidual membrane were found to be the main producers of MT-MMP-1 mRNA. Gene expression was also seen in the villous double-layered trophoblastic epithelium and in the decidual cells of the decidual membrane. In endothelial and fibroblastic cells, however, the hybridization signal was either very weak or nonexistent. Immunohistochemical analysis and immunoelectron microscopy correlated well with the in situ hybridization findings. The most significant exception to this consisted of pericytes of spiral arteries, which appeared to lack MT-MMP-1 mRNA but showed intensive intracytoplasmic staining for the antigen. Our results show that MT-MMP-1 mRNA production is highly characteristic of intermediate trophoblasts, and MT-MMP-1 may have general importance in the tissue organization of early human placenta. We propose that MT-MMP-1 could be one of the key enzymes in the process of trophoblast invasion, acting alone or as a cell-surface activator of other proteinases.

mRNA expressions of TIMP-1, -2, and -3 and 92-KD type IV collagenase in early human placenta and decidual membrane as studied by in situ hybridization.

Cytotrophoblasts of early placenta invade the decidual membrane, gestational endometrium, and spiral arteries during early pregnancy. Unlike tumor invasion, this physiological invasion is well controlled, although its molecular mechanisms are largely unknown. We have previously shown that cytotrophoblasts synthesize significant mRNAs for 72-KD Type IV collagenase, laminin, and Type IV collagen, proteins implicated in extracellular matrix turnover and migration. In this study we used in situ hybridization and immunohistochemistry to investigate the mRNA expression pattern of 92-KD Type IV collagenase and the matix metalloproteinase inhibitors TIMP-1, TIMP-2, and TIMP-3 in early human placenta and decidual membrane. mRNAs for 92-KD Type IV collagenase, TIMP-1, TIMP-2, and TIMP-3 were found in the cells of cytotrophoblastic columns, the endothelial and fibroblastic stromal cells of villi, and the large decidualized cells of decidual membrane. TIMP-1 expression was notably accentuated in the fibroblasts of fibrotic villi. In the decidual membrane, the signals for 92-KD Type IV collagenase and TIMP-1 mRNA were particularly strong around the glandular structures. The trophoblastic epithelium of villi and the epithelial cells of decidual glands showed a signal for 92-KD Type IV collagenase and TIMP-2, but not for TIMP-1 or TIMP-3. The coincidental expression of the proteolytic 92-KD Type IV collagenase and inhibitors TIMP-1, TIMP-2, and TIMP-3 generally in the same cells suggests that the activity of 92-KD Type IV collagenase, which is regulated by TIMPs, plays an important role in placental tissue organization and in the invasion of trophoblastic cells into the uterine wall.

Expression of the tissue metalloproteinase inhibitors TIMP-1 and TIMP-2 in malignant fibrous histiocytomas and dermatofibromas as studied by in situ hybridization and immunohistochemistry.

The expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was studied in eight malignant fibrous histiocytomas (MFH) and in eight dermatofibromas (DF) using in situ hybridization methods (ISH). Immunohistochemical stainings were also performed using corresponding antibodies to TIMP-1 and TIMP-2. In ISH the neoplastic cells of MFHs showed a high level of expression for both TIMP-1 and TIMP-2 mRNAs. The cells usually expressed similarly both TIMPs, except for osteoclast-like giant cells, which showed a distinct signal for TIMP-2 but not for TIMP-1. A distinctly lower level of both TIMP-1 and TIMP-2 mRNAs was seen in DFs. Immunohistochemical stainings were concordant with the results obtained by ISH. The findings suggest that the behavior of MFHs and DFs is not directly or solely dependent on the quantity of type IV collagenase inhibitors. The increased TIMP synthesis in MFHs might represent a chaotic response of malignant cells to increased matrix degradation. Alternatively, it may reflect a deranged communication between type IV collagenases and TIMPs in malignant tissues.

72 KD and 92 KD type IV collagenase, type IV collagen, and laminin mRNAs in breast cancer: a study by in situ hybridization.

It is widely accepted that basement membrane (BM) components are synthesized by epithelial cells and that production of BM-degrading proteases by cancer cells is necessary for invasive growth. In this study we used nucleic acid in situ hybridization (ISH) to investigate the presence of mRNAs for 72 KD and 92 KD Type IV collagenase, alpha 1 (IV) chain of Type IV collagen, and laminin B1 chain in 20 breast carcinomas of various histological types. The mRNA signals for 72 KD Type IV collagenase, Type IV collagen, and laminin were much more abundant in stromal fibroblasts and endothelial cells than in carcinoma cells. The signal for 92 KD Type IV collagenase mRNA was strong in carcinoma cells and considerably weaker in stromal fibroblasts and endothelial cells. Labeling for 72 KD and 92 KD Type IV collagenase mRNA was also found in benign fibroadenomas and for 92 KD Type IV collagenase in non-neoplastic ducts and acini. The results indicate that stromal cells have a more important role in the synthesis and degradation of BMs in breast carcinomas than previously thought and that production of these enzymes is not restricted to malignancy.

Expression of 72 kilodalton type IV collagenase (gelatinase A) in benign and malignant ovarian tumors.

BACKGROUND: 72 Kilodalton (kd) type IV collagenase is a matrix metalloproteinase that specifically cleaves type IV collagen molecules. The enzyme has been postulated to have an important role in the invasion and spread of malignant tumors. EXPERIMENTAL DESIGN: In situ hybridization was used to study the expression of the 72 kd type IV collagenase mRNA in 24 benign, 2 semimalignant, and 15 malignant ovarian tumors and in 5 metastases of ovarian serous adenocarcinomas. The results were correlated with the expression of the mRNA for the alpha 1(IV) chain of type IV collagen and with the corresponding immunohistochemical distribution of the enzyme. RESULTS: The results showed that the more malignant an ovarian tumor was, the more clearly mRNA expressions for both 72 kd type IV collagenase and the alpha 1(IV) chain could be detected in tumor cells. The expression of both types of mRNAs was localized within the cells of tumor stroma and occurred mainly in fibroblasts and vascular endothelial cells. Epithelial tumor cells only rarely expressed these mRNAs. Immunohistochemical stainings localized the 72 kd collagenase as well to the stromal cells as to the epithelial cells of both benign and malignant tumors. CONCLUSIONS: The findings indicate that genes for the 72 kd type IV collagenase and for its substrate are simultaneously active in the same cells of the tumor stroma. The difference in the in situ hybridization and immunohistochemical findings could be explained by a possible variation in the metabolic balance between synthesis and accumulation of the protein in different cell types. It can also be proposed that the activity of the 72 kd type IV collagenase would be mediated through a receptor-like mechanism present on epithelial cells which could bind the 72 kd type IV collagenase synthesized elsewhere. There is also a possibility that the gelatinolytic activity of the mesenchymally synthesized 72 kd type IV collagenase would be consumed to degrade extracellular matrix proteins other than basement membranes.

Simultaneous expression of 70 kilodalton type IV collagenase and type IV collagen alpha 1 (IV) chain genes by cells of early human placenta and gestational endometrium.

BACKGROUND: In this study we used in situ hybridization to investigate the expression of the genes 70 kilodalton (kd) collagenase and the alpha 1(IV) collagen chain of type IV collagen in cells of early human placenta and gestational endometrium. EXPERIMENTAL DESIGN: The aim was to study the spatial distribution of these gene expressions within a developing tissue which possesses physiologic invasive potential. The results obtained for the 70 kd type IV collagenase mRNA expression were also compared with the immunohistochemical distribution of the corresponding antigen. RESULTS: Expression of mRNAs for these proteins was found in cells of trophoblastic columns, stromal cells of villi and in cells of decidua and endometrial stroma. The only differences between the expressions was the lower level of signals for 70 kd type IV collagenase in fibroblastic stromal cells and endothelial cells of villi and in the pericytic cells of spiral arteries. Otherwise the results for both types of mRNA were comparable. We also studied the immunohistochemical distribution of the 70 kd type IV collagenase using specific monoclonal antibodies against the enzyme. Immunohistochemistry supported well the findings obtained by in situ hybridization. CONCLUSIONS: The results indicate that the genes for the 70 kd type IV collagenase and for the alpha 1(IV) collagen chain are simultaneously active in cells of placenta and gestational endometrium and the same cells which produce type IV collagen also can produce the cleaving enzyme, the 70 kd type IV collagenase. The results also show that the cytotrophoblastic cells, which during early pregnancy invade the extracellular matrix and spiral arteries of uterine wall contain significant amount of mRNA for the 70 kd type IV collagenase. This finding supports the concept that the 70 kd type IV collagenase would be important for invasion, and in the case of this study, also for the physiologic invasion of placental cytotrophoblasts.

Molecular cloning of murine 72-kDa type IV collagenase and its expression during mouse development.

We report the isolation of a cDNA clone providing the first and complete sequence of mouse 72-kDa type IV collagenase. The clone contains 2800 nucleotides with a 1986-nucleotide open reading frame coding for 662 amino acids. The amino acid sequence includes a 29-residue signal peptide, an 80-residue propeptide, and a 553-residue enzyme proper. The sequence identity between the mouse and human enzymes is 96% with all cysteine residues conserved. The carboxyl-terminal domain of the mouse enzyme contains two more residues than the human enzyme. Northern hybridization analysis revealed considerable expression of the enzyme gene in newborn mouse lung, heart, kidney, and psoas muscle tissues, whereas only weak or no signals were observed in liver, spleen, and brain. Expression of the gene was substantially reduced in the same tissues of 3-month-old mice. In situ hybridization analysis of 72-kDa type IV collagenase expression in 10-15-day-old mouse embryos showed that the gene was intensely expressed in mesenchymal cells. Brain and surface ectoderm were completely negative. The epithelial tissue component of developing organs was negative with the exception of salivary gland. Although the expression varied somewhat between different mesenchymal tissues, no temporal or spatial changes could be associated with the advancement of epithelial branching morphogenesis. These findings together with our previous data on the expression of 72-kDa type IV collagenase in human tumors indicate that this enzyme has some very specific roles both in the physiological and pathological degradation of extracellular matrix. Furthermore, it has become clear that the closely related 92-kDa type IV collagenase differs completely with respect to expression pattern as well as gene regulation. The mouse cDNA clones reported in this study may provide important tools unraveling the actual roles of these enzymes in vivo.

Localization of messenger RNA for Mr 72,000 and 92,000 type IV collagenases in human skin cancers by in situ hybridization.

We have examined the expression of 2 type IV collagen degrading enzymes (Mr 72,000 and 92,000 type IV collagenases) in human skin cancer by in situ hybridization. In all cases of infiltrating carcinomas of squamous cell (9 of 9) and basal cell (5 of 5) types, messenger RNA for the Mr 72,000 type IV collagenase was present in numerous fibroblasts. These were especially abundant in the stroma adjacent to the invasive tumor nodules. Malignant cells were negative for mRNA for the Mr 72,000 enzyme in all cases as were all other epithelial as well as endothelial cells. mRNA for the Mr 92,000 type IV collagenase was present in all 9 squamous cell and in 3 of the 5 basal cell carcinomas. In all these cases, a subpopulation of tissue macrophages was found to be positive, while malignant cells showed a signal for Mr 92,000 type IV collagenase in 6 of the squamous cell carcinomas but in none of the basal cell carcinomas. In all cases, the signal for this mRNA was confined to cells located at the tumoral/stromal interface or in the close vicinity of tumor nodules. No mRNA for any of the 2 collagenases was detected in 3 biopsies of normal skin. In vitro studies have indicated that collagenases are involved in the degradation of the extracellular matrix during cancer invasion. The present findings are consistent with such a role of the Mr 72,000 and 92,000 type IV collagenases in squamous and basal cell carcinomas in situ. The findings also demonstrate that degradative enzymes are not necessarily produced by the malignant cells themselves but may be generated by induction or recruitment of nonmalignant stromal cells.

70 K type IV collagenase (gelatinase).

Type IV collagenase (gelatinase) is a 70,000 dalton neutral metalloproteinase that specifically cleaves type IV collagen in addition to degrading denatured collagen (gelatin). It is secreted in a latent proenzyme form that is converted proteolytically in the extracellular space to a 62,000 dalton active enzyme. The primary structure, enzymatic properties as well as gene structure, demonstrate that type IV collagenase is closely related with the other well characterized metalloproteinases, interstitial collagenase and stromelysin. However, the structure of type IV collagenase differs from the others in that it is larger and contains three internal repeats that resemble the type II domains of fibronectin. Also, initial characterization of the promoter region of the gene indicates that its regulation differs from the other proteinase genes. Type IV collagenase is presumably required for the normal turnover of basement membranes. Augmented activity is linked with the invasive potential of tumor cells and the enzyme is believed to play a major role in the penetration of basement membranes by metastatic cells. Measurements of enzyme activity and mRNA levels as well as immunostaining of a variety of tumor cells and tissues suggest that assays for the enzyme may have value in the follow-up of malignant growth.

Collagen biosynthesis in lichen sclerosus et atrophicus studied by biochemical and in situ hybridization techniques.

Collagen was studied by biochemical and immunohistochemical techniques and by in situ hybridization in four patients with lichen sclerosus et atrophicus (LSA). The solubility of collagen in acetic acid and pepsin was increased in the lesional skin of the LSA patients when compared with their non-affected skin or samples from control subjects, indicating that a marked proportion of the collagen was newly synthesized. Collagen synthesis was slightly increased in fibroblasts derived from the lesional skin of the LSA patients. In situ hybridization with human sequence-specific cDNAs to type I procollagen demonstrated active fibroblasts in lesional skin of LSA patients, further confirming a high level of collagen synthesis in LSA. Remarkably, active fibroblasts were not associated with inflammatory cell infiltrates noted in LSA skin nor did they express transforming growth factor beta(TGF beta 1). Our results indicate that despite the degeneration of connective tissue, there is an active regeneration process in LSA with significant collagen synthesis.