Human macrophage metalloelastase (MMP-12) expression is induced in chondrocytes during fetal development and malignant transformation.

Fetal development and tumor progression both require a complex system of extracellular matrix (ECM) synthesis and breakdown, which is mediated by, for instance, the matrix metalloproteinases (MMPs). Human metalloelastase (MMP-12) is an MMP, the expression of which has so far been documented in macrophages associated with atherosclerosis, wound repair, and certain cancers. In this study we first examined the expression of MMP-12 during human fetal development. By in situ hybridization MMP-12 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column, in ribs, and in extremities undergoing ossification, beginning at the gestational age of 8 weeks. Also, periosteal cells expressed MMP-12 at 11 weeks. No expression of MMP-12 mRNA could be noted in other fetal tissues, including the skin, lungs, intestine, kidney, and liver. Expression of MMP-12 mRNA could not be detected in adult normal cartilage or osteosarcomas, but in chondrosarcomas both macrophages (8 of 19 samples) (identified by CD68 immunostaining) and chondrosarcoma cells (8 of 19) were positive. MMP-12 was also demonstrated in the tumors by western blotting and it was expressed in the same regions as MMP-13 mRNA. By immunostaining, MMP-12 mRNA colocalized with the protein in both fetal and chondrosarcoma specimens. Unlike basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha) induced MMP-12 mRNA production in chondrosarcoma-derived HTB-94 cells. Our results suggest that MMP-12 plays an important role in ECM remodeling during fetal bone development and is induced when chondrocytes undergo malignant transformation.

Matrilysin-2, a new matrix metalloproteinase expressed in human tumors and showing the minimal domain organization required for secretion, latency, and activity.

We have identified a human placenta cDNA coding for a new member of the matrix metalloproteinase (MMP) family. The isolated cDNA encodes a polypeptide of 261 amino acids, the smallest MMP identified to date, which contains several structural features of MMPs including the signal sequence, the prodomain involved in enzyme latency, and the catalytic domain with the zinc-binding site. However, it lacks the hinge region and hemopexin-domain present in most MMPs. According to these structural characteristics, the human MMP described herein has been called matrilysin-2 (MMP-26), because it exclusively shares with matrilysin this minimal domain organization required for secretion, latency, and activity. The amino acid sequence of matrilysin-2 also contains a threonine residue adjacent to the Zn-binding site that has been defined as a specific feature of matrilysin. Chromosomal location of the matrilysin-2 gene showed that it maps to the short arm of chromosome 11, a location distinct to that of other MMP genes. Matrilysin-2 was expressed in Escherichia coli, and, after purification and refolding, the recombinant protein was found to degrade synthetic substrates commonly used for assaying MMPs. Furthermore, this protein hydrolyzed type IV collagen, fibronectin, fibrinogen, and gelatin, which indicated that matrilysin-2 is a potent enzyme with a wide substrate specificity. In addition, it was found that matrilysin-2 is able to activate progelatinase B. Proteolytic activity of matrilysin-2 against all of these substrates was abolished by synthetic inhibitors and by tissue inhibitors of metalloproteinases. Expression analysis revealed that matrilysin-2 is detected not only in placenta and uterus but is widely expressed in malignant tumors from different sources as well as in diverse tumor cell lines. These data together with its broad spectrum of proteolytic activity, suggest that matrilysin-2 may play a role in some of the tissue-remodeling events associated with tumor progression.

An overview of collagenase-3 expression in malignant tumors and analysis of its potential value as a target in antitumor therapies.

Collagenase-3 (MMP-13) is a member of the matrix metalloproteinase family of endopeptidases that is characterized by a potent degrading activity against a wide spectrum of substrates. This enzyme was first detected in breast carcinomas but it is also overexpressed in a variety of malignant tumors including head and neck carcinomas, chondrosarcomas, skin carcinomas, and carcinomas of the female genital tract. Clinical studies have revealed that in all these tumors collagenase-3 expression is associated with invasive and metastatic tumors. Analysis of the molecular mechanisms underlying its marked overexpression in malignant tumors has allowed to identify different cytokines, growth factors and tumor promoters with ability to up-regulate collagenase-3 expression in tumor cells, or in stromal fibroblasts surrounding epithelial tumor cells. The first strategies designed to target this enzyme are being developed, and are mainly directed to prepare synthetic inhibitors with ability to selectively block the collagenase-3 proteolytic activity. Alternatively, inhibitors of the signal transduction pathways mediating the expression of this enzyme by tumor cells may also be useful for collagenase-3 targeting. These studies together with those performed on other enzymes associated with tumor processes may lead to the development of novel therapeutic strategies to control the progression and metastatic capacity of neoplastic cells.

Phenotypic switching in Candida albicans is controlled by a SIR2 gene.

We report the cloning of a gene from the human fungal pathogen Candida albicans with sequence and functional similarity to the Saccharomyces cerevisiae SIR2 gene. Deletion of the gene in C. albicans produces a dramatic phenotype: variant colony morphologies arise at frequencies as high as 1 in 10. The morphologies resemble those described previously as part of a phenotypic switching system proposed to contribute to pathogenesis. Deletion of SIR2 also produces a high frequency of karyotypic changes. These and other results are consistent with a model whereby Sir2 controls phenotypic switching and chromosome stability in C.albicans by organizing chromatin structure.

Expression and regulation of collagenase-3 (MMP-13) in human malignant tumors.

Human collagenase-3 (MMP-13) is a matrix metalloproteinase originally identified in breast carcinomas. Recent studies have revealed that this enzyme is also produced by a variety of malignant tumors including head and neck carcinomas, chondrosarcomas and basal cell carcinomas of the skin. In all cases, the expression of collagenase-3 is associated with aggressive tumors. Different cytokines, growth factors and tumor promoters are able to up-regulate collagenase-3 expression in tumor cells or in stromal cells surrounding epithelial tumor cells. Functional analysis of the collagenase-3 gene promoter has allowed the identification of AP-1 and OSE-2 elements mediating, at least in part, its expression in both normal and pathological conditions.

Matrix metalloproteinases and their expression in mammary gland.

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events. The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation, and is controlled by growth factors, cytokines, hormones, as well as interactions with the ECM macromolecules. Furthermore, the activity of the MMPs is regulated by their natural endogenous inhibitors, which are members of the tissue inhibitor of metalloproteinases (TIMP) family. In the normal mammary gland, MMPs are expressed during ductal development, lobulo-alveolar development in pregnancy and involution after lactation. Under pathological conditions, such as tumorigenesis, the dysregulated expression of MMPs play a role in tumor initiation, progression and malignant conversion as well as facilitating invasion and metastasis of malignant cells through degradation of the ECM and basement membranes.

Collagenase-3 (MMP-13) expression in chondrosarcoma cells and its regulation by basic fibroblast growth factor.

Human collagenase-3 (MMP-13) is a member of the matrix metalloproteinase family of enzymes that was originally identified in breast carcinomas and subsequently detected during fetal ossification and in arthritic processes. In this work, we have found that collagenase-3 is produced by HCS-2/8 human chondrosarcoma cells. An analysis of the ability of different cytokines and growth factors to induce the expression of collagenase-3 in these cells revealed that basic fibroblast growth factor (bFGF or FGF-2) strongly up-regulated the expression of this gene. By contrast, other factors, including interleukin-1beta and transforming growth factor-beta, previously found to induce collagenase-3 expression in other cell types, did not exhibit any effect on the expression of this gene in chondrosarcoma cells. Further analysis of the bFGF-induced expression of collagenase-3 in human chondrosarcoma cells revealed that its effect was time and dose dependent, but independent of the de novo synthesis of proteins. Western blot analysis revealed that the up-regulatory effect of bFGF on collagenase-3 was also reflected at the protein level as demonstrated by the increase of immunoreactive protein in the conditioned medium of HCS-2/8 cells treated with bFGF. Immunohistochemical analysis of the presence of collagenase-3 in a series of 8 benign and 16 malignant cartilage-forming neoplasms revealed that all analyzed malignant chondrosarcomas stained positively for collagenase-3, whereas only 2 of 8 benign lesions produced this protease. In addition, the finding that bFGF was detected in all analyzed chondrosarcomas, together with the above in vitro studies on HCS-2/8 cells, suggest that this growth factor may be an in vivo modulator of collagenase-3 expression in these malignant tumors. These results extend the pattern of tumor types with ability to produce this matrix metalloproteinase and suggest that collagenase-3 upregulation may contribute to the progression of human chondrosarcomas.

Differential effects of transforming growth factor-beta on the expression of collagenase-1 and collagenase-3 in human fibroblasts.

Collagenase-3 (MMP-13) is a matrix metalloproteinase (MMP) originally identified in breast carcinomas which is also produced at significant levels during fetal ossification and in arthritic processes. In this work, we have found that transforming growth factor beta1 (TGF-beta1), a growth factor widely assumed to be inhibitory for MMPs, strongly induces collagenase-3 expression in human KMST fibroblasts. In contrast, this growth factor down-regulated the expression in these cells of collagenase-1 (MMP-1), an enzyme highly related to collagenase-3 in terms of structure and enzymatic properties. The positive effect of TGF-beta1 on collagenase-3 expression was dose- and time-dependent, but independent of the effects of this growth factor on cell proliferation rate. Analysis of the signal transduction mechanisms underlying the up-regulating effect of TGF-beta1 on collagenase-3 expression demonstrated that this growth factor acts through a signaling pathway involving protein kinase C and tyrosine kinase activities. Functional analysis of the collagenase-3 gene promoter region revealed that the inductive effect of TGF-beta1 is partially mediated by an AP-1 site. Comparative analysis with the promoter region of the collagenase-1 gene which contains an AP-1 site at equivalent position, confirmed that TGF-beta1 did not have any effect on CAT activity levels of this promoter. Finally, by using electrophoretic mobility shift assays and antibody supershift analysis, we propose that c-Fos, c-Jun, and JunD may play major roles in the collagenase-3 activation by TGF-beta1 in human fibroblasts.

Regulation of collagenase-3 expression in human breast carcinomas is mediated by stromal-epithelial cell interactions.

Collagenase-3 (MMP-13) is a recently identified member of the human matrix metalloproteinase gene family that is expressed in breast carcinomas and in articular cartilage from arthritic patients. Here, we have studied the cellular origin of this enzyme in breast carcinomas by in situ RNA hybridization, and we found that collagenase-3 is expressed by stromal cells immediately adjacent to epithelial tumor cells but not by the tumor cells themselves; nor is it expressed by the normal breast glandular epithelium. Consistent with this observation, coculture experiments using human fibroblasts and MCF-7 breast cancer cells revealed that conditioned medium from breast cancer cells stimulated the fibroblastic expression of collagenase-3 mRNA. In contrast, no stimulatory effect was observed when medium from fibroblast cells was added to breast cancer cells. These results strongly suggest that transcription of collagenase-3 in stromal cells is activated by diffusible factors released from epithelial breast cancer cells. A survey of a series of cytokines and growth factors known for their ability to induce collagenase-3 expression in human fibroblasts identified interleukin-1alpha and interleukin-1beta as potential candidates for inducing the expression of this MMP gene in breast carcinomas. According to these results, collagenase-3 should be included among the molecular factors that are detected during the stromal reaction to invasive breast cancer and that, by concerted action, may be essential for tumor growth and progression.

Alternative splicing gives rise to two novel long isoforms of Zn-alpha 2-glycoprotein, a member of the immunoglobulin superfamily.

We have isolated, from a rat liver cDNA library, two cDNAs encoding novel long isoforms of Zn-alpha2-glycoprotein (Zn-alpha2-gp), a member of the immunoglobulin superfamily with a high degree of sequence similarity to class-I major histocompatibility complex (MHC) antigens. Nucleotide (nt) sequence analysis of these two novel cDNAs has revealed that they contain insertions of 138 and 123 nt between the second and third exons of Zn-alpha2-gp, resulting in in-frame insertions of 46 and 41 amino acids (aa), respectively. Analysis of the mechanism of generation of both isoforms, named Zn-alpha2-gpA and Zn-alpha2-gpB, has shown that they result from a series of alternative splicing events, including alternative use of two additional exons, and of two different 3'-splice sites present in the first of these novel exons. The occurrence of these alternative splicing events in Zn-alpha2-gp could contribute to increasing the diversity of this nonpolymorphic and soluble class-I MHC antigen.

Cloning and expression analysis of the cDNA encoding rat Zn-alpha 2-glycoprotein.

We report here the nucleotide (nt) sequence of a rat liver cDNA encoding Zn-alpha 2-glycoprotein (Zn-alpha 2-gp), a plasma protein with a high degree of sequence similarity to class-I major histocompatibility complex (MHC) antigens. The deduced amino acid (aa) sequence contains the coding information for 293 aa residues and shows 60% identity with the aa sequence of human Zn-alpha 2-gp and 35% identity with that corresponding to the extracellular domains of RT1, a rat class-I MHC antigen. Northern blot analysis showed that rat Zn-alpha 2-gp is expressed in liver, but not in a wide number of tissues, including prostate, mammary gland, kidney, intestine, lung, pancreas, ovary, uterus, thyroid, placenta, spleen, brain and heart.

Structure and expression in breast tumors of human TIMP-3, a new member of the metalloproteinase inhibitor family.

A new member of the metalloproteinase inhibitor family of proteins has been cloned from a complementary DNA library derived from a human breast tumor. The isolated complementary DNA contains an open reading frame 633 base pairs long, encoding a polypeptide of 211 amino acids, which has been called tissue inhibitor of metalloproteinase 3 (TIMP-3). This protein displays low sequence similarity to the previously known human TIMPs but shows a high degree of similarity with chicken inhibitor of metalloproteinase 3, a recently described metalloproteinase inhibitor stimulated during oncogenic transformation of chicken fibroblasts and with the ability to promote some phenotypic properties of transformed cells. Northern blot analysis of RNA from human tissues revealed that the TIMP-3 gene is expressed in placenta and uterus but not in liver and ovary. In addition, TIMP-3 transcripts were detected in all breast carcinomas examined. On the basis of these expression data in breast tumors, together with its high degree of structural homology with chicken inhibitor of metalloproteinase 3, a possible role for human TIMP-3 in the regulation of connective tissue turnover and remodeling is proposed.

Mapping of the human Zn-alpha 2-glycoprotein gene (AZGP1) to chromosome 7q22 by in situ hybridization.

The gene coding for Zn-alpha 2-glycoprotein (AZGP1), a human protein with a high degree of similarity to class I major histocompatibility complex (MHC) antigens, was mapped by fluorescent in situ hybridization to chromosome 7q22, a common breakpoint in myelodysplastic syndromes. Since classical MHC genes map on chromosome 6, this assignment indicates that besides duplication of the putative common ancestor gene, transposition events to different chromosomes have also been involved in the evolutionary diversification of this gene family.

Human Zn-alpha 2-glycoprotein: complete genomic sequence, identification of a related pseudogene and relationship to class I major histocompatibility complex genes.

The human gene (AZGP1) encoding Zn-alpha 2-glycoprotein (Zn-alpha 2-gp), a protein present in several biological fluids and produced by a subtype of breast carcinomas, has been cloned and its complete nucleotide sequence determined. The gene spans over 9.7 kb, and its overall organization and nucleotide sequence are very similar to those of the first four exons of class I MHC genes. However, the Zn-alpha 2-gp gene differs from these genes in several significant ways. It lacks the coding information for the transmembrane and cytoplasmic domains typical of MHC genes, which is consistent with its presence as a soluble protein in different physiological and pathological fluids. In addition, it contains a high density of repetitive sequences, including Alu, MER, and MIR elements, which are not present at equivalent positions in class I MHC genes. Finally, its 5'-flanking region lacks the class I MHC regulatory complex and the interferon consensus sequence characteristic of class I MHC genes. These findings may explain the different expression pattern of Zn-alpha 2-gp and class I MHC genes in human tissues. Southern blot hybridization of DNA from several species with a cDNA probe indicated that Zn-alpha 2-gp genes are present in a wide variety of animal species, including monkey, rat, mouse, dog, cow, and rabbit. The human genome also contains a putative Zn-alpha 2-gp pseudogene that has been isolated and partially characterized. This pseudogene has an intron-exon organization identical to that of the functional gene, but it presents two deleterious mutations in the third exon that lead to the appearance of premature stop codons. Finally, considering the lack of polymorphism in the Zn-alpha 2-gp gene in comparison with MHC genes, putative roles for this human glycoprotein in the transport of nonpolymorphic substances or in intercellular recognition processes are proposed.