PHF6 mutations in adult acute myeloid leukemia.

Loss of function mutations and deletions encompassing the plant homeodomain finger 6 (PHF6) gene are present in about 20% of T-cell acute lymphoblastic leukemias (ALLs). Here, we report the identification of recurrent mutations in PHF6 in 10/353 adult acute myeloid leukemias (AMLs). Genetic lesions in PHF6 found in AMLs are frameshift and nonsense mutations distributed through the gene or point mutations involving the second plant homeodomain (PHD)-like domain of the protein. As in the case of T-ALL, where PHF6 alterations are found almost exclusively in males, mutations in PHF6 were seven times more prevalent in males than in females with AML. Overall, these results identify PHF6 as a tumor suppressor gene mutated in AML and extend the role of this X-linked tumor suppressor gene in the pathogenesis of hematologic tumors.

CUTLL1, a novel human T-cell lymphoma cell line with t(7;9) rearrangement, aberrant NOTCH1 activation and high sensitivity to gamma-secretase inhibitors.

Activating mutations in NOTCH1 are present in over 50% of human T-cell lymphoblastic leukemia (T-ALL) samples and inhibition of NOTCH1 signaling with gamma-secretase inhibitors (GSI) has emerged as a potential therapeutic strategy for the treatment of this disease. Here, we report a new human T-cell lymphoma line CUTLL1, which expresses high levels of activated NOTCH1 and is extremely sensitive to gamma-secretase inhibitors treatment. CUTLL1 cells harbor a t(7;9)(q34;q34) translocation which induces the expression of a TCRB-NOTCH1 fusion transcript encoding a membrane-bound truncated form of the NOTCH1 receptor. GSI treatment of CUTLL1 cells blocked NOTCH1 processing and caused rapid clearance of activated intracellular NOTCH1. Loss of NOTCH1 activity induced a gene expression signature characterized by the downregulation of NOTCH1 target genes such as HES1 and NOTCH3. In contrast with most human T-ALL cell lines with activating mutations in NOTCH1, CUTLL1 cells showed a robust cellular phenotype upon GSI treatment characterized by G1 cell cycle arrest and increased apoptosis. These results show that the CUTLL1 cell line has a strong dependence on NOTCH1 signaling for proliferation and survival and supports that T-ALL patients whose tumors harbor t(7;9) should be included in clinical trials testing the therapeutic efficacy NOTCH1 inhibition with GSIs.

Effects of pregnancy on insulin receptor in liver, skeletal muscle and adipose tissue of rats.

The mechanism responsible for insulin resistance during pregnancy remains unclear. Considerable evidence indicates that the insulin receptor plays an important role in insulin sensitivity. It seems possible that the hormonal milieu during gestation could have an effect on the insulin receptor. In the present study, measurements of tyrosine phosphorylation and protein content of the insulin receptor and expression of its gene in liver, skeletal muscle and adipose tissue indicate that during pregnancy significant changes occur in these parameters. We found that at the end of early gestation (day 10), muscle and adipose tissue are very sensitive to insulin action because the amount, phosphorylation and gene expression of insulin receptor is higher than in late gestation (days 15-20), while the tissue which is most sensitive to insulin action in late gestation is the liver. Our hypothesis is that these results are connected with changes in the concentrations of estradiol and progesterone observed during pregnancy. In conclusion, our previous and present findings seem to demonstrate that the different concentrations of gestational hormones play an important role in insulin sensitivity in this period and that each tissue responds in the most appropriate manner to guarantee the gestation in its entirety.

A regulatory cascade involving retinoic acid, Cbfa1, and matrix metalloproteinases is coupled to the development of a process of perichondrial invasion and osteogenic differentiation during bone formation.

Tissue-remodeling processes are largely mediated by members of the matrix metalloproteinase (MMP) family of endopeptidases whose expression is strictly controlled both spatially and temporally. In this article, we have examined the molecular mechanisms that could contribute to modulate the expression of MMPs like collagenase-3 and MT1-MMP during bone formation. We have found that all-trans retinoic acid (RA), which usually downregulates MMPs, strongly induces collagenase-3 expression in cultures of embryonic metatarsal cartilage rudiments and in chondrocytic cells. This effect is dose and time dependent, requires the de novo synthesis of proteins, and is mediated by RAR-RXR heterodimers. Analysis of the signal transduction mechanisms underlying the upregulating effect of RA on collagenase-3 expression demonstrated that this factor acts through a signaling pathway involving p38 mitogen-activated protein kinase. RA treatment of chondrocytic cells also induces the production of MT1-MMP, a membrane-bound metalloproteinase essential for skeletal formation, which participates in a proteolytic cascade with collagenase-3. The production of these MMPs is concomitant with the development of an RA-induced differentiation program characterized by formation of a mineralized bone matrix, downregulation of chondrocyte markers like type II collagen, and upregulation of osteoblastic markers such as osteocalcin. These effects are attenuated in metatarsal rudiments in which RA induces the invasion of perichondrial osteogenic cells from the perichondrium into the cartilage rudiment. RA treatment also resulted in the upregulation of Cbfa1, a transcription factor responsible for collagenase-3 and osteocalcin induction in osteoblastic cells. The dynamics of Cbfa1, MMPs, and osteocalcin expression is consistent with the fact that these genes could be part of a regulatory cascade initiated by RA and leading to the induction of Cbfa1, which in turn would upregulate the expression of some of their target genes like collagenase-3 and osteocalcin.

Collagenase-3 expression in breast myofibroblasts as a molecular marker of transition of ductal carcinoma in situ lesions to invasive ductal carcinomas.

Collagenase-3 (matrix metalloproteinase 13; MMP-13), a protease originally identified in breast carcinoma, is characterized by a potent degrading activity against a wide spectrum of extracellular matrix proteins. The aims of this study were to localize and identify the MMP-13-expressing cells in invasive human breast carcinoma and to evaluate the role of MMP-13 in transition to invasive lesions by studying ductal carcinoma in situ (DCIS). We found expression of MMP-13 in stromal fibroblast-like cells in all 21 invasive ductal carcinomas studied and in 4 of 9 invasive lobular carcinomas. In most carcinomas, expression of MMP-13 was limited to small stromal foci in the tumor area. Combined in situ hybridization and immunohistochemistry showed coexpression of alpha-smooth muscle actin immunoreactivity and MMP-13 mRNA in myofibroblasts. In contrast, cytokeratin-positive cancer cells, alpha-smooth muscle actin-positive vascular smooth muscle cells, CD68-positive macrophages, and CD31-positive endothelial cells were all MMP-13 mRNA negative. In situ hybridization for MMP-13 in 17 DCIS lesions revealed expression in 10 cases. Immunohistochemical analysis of all DCIS cases identified microinvasion in 8 of the 17 lesions. Seven of the eight lesions with microinvasion were MMP-13 positive. Further analysis showed that MMP-13 expression was often associated with the microinvasive events. This particular expression pattern was unique for MMP-13 among other MMPs analyzed, including MMP-2, -11, and -14. We conclude that MMP-13 is primarily expressed by myofibroblasts in human breast carcinoma and that expression in DCIS lesions often is associated with microinvasive events. On the basis of these data, we propose that MMP-13 may play an essential role during transition of DCIS lesions to invasive ductal carcinomas.

Collagen metabolism is markedly altered in the hypertrophic cartilage of growth plates from rats with growth impairment secondary to chronic renal failure.

Skeletal growth depends on growth plate cartilage activity, in which matrix synthesis by chondrocytes is one of the major processes contributing to the final length of a bone. On this basis, the present work was undertaken to ascertain if growth impairment secondary to chronic renal insufficiency is associated with disturbances of the extracellular matrix (ECM) of the growth plate. By combining stereological and in situ hybridization techniques, we examined the expression patterns of types II and X collagens and collagenase-3 in tibial growth plates of rats made uremic by subtotal nephrectomy (NX) in comparison with those of sham-operated rats fed ad libitum (SAL) and sham-operated rats pair-fed with NX (SPF). NX rats were severely uremic, as shown by markedly elevated serum concentrations of urea nitrogen, and growth retarded, as shown by significantly decreased longitudinal bone growth rates. NX rats showed disturbances in the normal pattern of chondrocyte differentiation and in the rates and degree of substitution of hypertrophic cartilage with bone, which resulted in accumulation of cartilage at the hypertrophic zone. These changes were associated with an overall decrease in the expression of types II and X collagens, which was especially marked in the abnormally extended zone of the hypertrophic cartilage. Unlike collagen, the expression of collagenase-3 was not disturbed severely. Electron microscopic analysis proved that changes in gene expression were coupled to alterations in the mineralization as well as in the collagen fibril architecture at the hypertrophic cartilage. Because the composition and structure of the ECM have a critical role in regulating the behavior of the growth plate chondrocytes, results obtained are consistent with the hypothesis that alteration of collagen metabolism in these cells could be a key process underlying growth retardation in uremia.

Identification and enzymatic characterization of two diverging murine counterparts of human interstitial collagenase (MMP-1) expressed at sites of embryo implantation.

Remodeling of fibrillar collagen in mouse tissues has been widely attributed to the activity of collagenase-3 (matrix metalloproteinase-13 (MMP-13)), the main collagenase identified in this species. This proposal has been largely based on the repeatedly unproductive attempts to detect the presence in murine tissues of interstitial collagenase (MMP-1), a major collagenase in many species, including humans. In this work, we have performed an extensive screening of murine genomic and cDNA libraries using as probe the full-length cDNA for human MMP-1. We report the identification of two novel members of the MMP gene family which are contained within the cluster of MMP genes located at murine chromosome 9. The isolated cDNAs contain open reading frames of 464 and 463 amino acids and are 82% identical, displaying all structural features characteristic of archetypal MMPs. Comparison for sequence similarities revealed that the highest percentage of identities was found with human interstitial collagenase (MMP-1). The new proteins were tentatively called Mcol-A and Mcol-B (Murine collagenase-like A and B). Analysis of the enzymatic activity of the recombinant proteins revealed that both are catalytically autoactivable but only Mcol-A is able to degrade synthetic peptides and type I and II fibrillar collagen. Both Mcol-A and Mcol-B genes are located in the A1-A2 region of mouse chromosome 9, Mcol-A occupying a position syntenic to the human MMP-1 locus at 11q22. Analysis of the expression of these novel MMPs in murine tissues revealed their predominant presence during mouse embryogenesis, particularly in mouse trophoblast giant cells. According to their structural and functional characteristics, we propose that at least one of these novel members of the MMP family, Mcol-A, may play roles as interstitial collagenase in murine tissues and could represent a true orthologue of human MMP-1.

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.

Different bone growth rates are associated with changes in the expression pattern of types II and X collagens and collagenase 3 in proximal growth plates of the rat tibia.

Skeletal growth depends on endochondral ossification in growth plate cartilage, where proliferation of chondrocytes, matrix synthesis, and increases in chondrocyte size all contribute to the final length of a bone. To learn more about the potential role of matrix synthesis/degradation dynamics in the determination of bone growth rate, we investigated the expression of matrix collagens and collagenase 3 in tibial growth plates in three age groups of rats (21, 35, and 80 days after birth), each characterized by specific growth rates. By combining stereological and in situ hybridization techniques, it was found that the expression of matrix collagens and collagenase 3 was specifically turned on or off at specific stages of the chondrocyte-differentiation cycle, and these changes occurred as a temporal sequence that varied depending of animal growth rate. Furthermore, the expression of these matrix proteins by a growth plate chondrocyte was found to be sped up or slowed down depending of the growth rate. In addition to expression of types II and X collagen, collagenase-3 expression was found to constitute a constant event in the series of changes in gene expression that takes place during the chondrocyte-differentiation process. Collagenase-3 expression was found to show a biphasic pattern: it was intermittently expressed at the proliferative phase and uniformly expressed at the hypertrophic stage. An intimate relationship between morphological and kinetic changes associated with chondrocyte hypertrophy and changes in the expression pattern of matrix collagens and collagenase 3 was observed. Present data prove that the matrix synthesis/degradation dynamics of the growth plate cartilage varied depending on growth rate; these results support the hypothesis that changes in matrix degradation and synthesis are a critical link in the sequence of tightly regulated events that lead to chondrocytic differentiation.

Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation.

Collagenase 3 (MMP-13) is a recently identified member of the matrix metalloproteinase (MMP) gene family that is expressed at high levels in diverse human carcinomas and in articular cartilage from arthritic patients. In addition to its expression in pathological conditions, collagenase 3 has been detected in osteoblasts and hypertrophic chondrocytes during fetal ossification. In this work, we have evaluated the possibility that Cbfa1 (core binding factor 1), a transcription factor playing a major role in the expression of osteoblastic specific genes, is involved in the expression of collagenase 3 during bone formation. We have functionally characterized a Cbfa motif present in the promoter region of collagenase 3 gene and demonstrated, by cotransfection experiments and gel mobility shift assays, that this element is involved in the inducibility of the collagenase 3 promoter by Cbfa1 in osteoblastic and chondrocytic cells. Furthermore, overexpression of Cbfa1 in osteoblastic cells unable to produce collagenase 3 leads to the expression of this gene after stimulation with transforming growth factor beta. Finally, we show that mutant mice deficient in Cbfa1, lacking mature osteoblasts but containing hypertrophic chondrocytes which are also a major source of collagenase 3, do not express this protease during fetal development. These results provide in vivo evidence that collagenase 3 is a target of the transcriptional activator Cbfa1 in these cells. On the basis of these transcriptional regulation studies, together with the potent proteolytic activity of collagenase 3 on diverse collagenous and noncollagenous bone and cartilage components, we proposed that this enzyme may play a key role in the process of bone formation and remodeling.

Hepatic expression of growth hormone receptor/binding protein and insulin-like growth factor I genes in uremic rats. Influence of nutritional deficit.

The molecular basis for GH resistance in chronic renal failure is unknown. It may partly reside in a decreased number of hepatic GH receptors and subsequently reduced IGF-I synthesis. To investigate the hepatic expression of GH receptor/binding protein (GHBP) and IGF-I genes in chronic renal failure, mRNA levels and the concentrations of its splicing variants were measured by Northern Blot in male 5/6 nephrectomized rats (NX, n = 9), aged 26 +/- 1 days, and three groups of sham-operated rats: (1) fed ad libitum (SAL, n = 9); (2) pair-fed with NX (SPF, n = 7); and (3) pair-fed with NX in terms of protein ingestion but calorically supplemented up to the intake of SAL (SPF+, n = 8). NX rats had severe renal failure, serum urea nitrogen 106 +/- 11 mg/dl (mean +/- SEM), and were growth retarded. GH receptor/GHBP gene expression was detected as two bands of 4.7 and 1.2 kb, respectively. The amount of mRNA was lower (P < 0.0001) in NX than SAL, either when both bands were considered together or separately. No differences were found between NX, SPF, and SPF+. Serum GHBP concentrations were higher (P < 0.01) in NX rats than the other groups. For the IGF-I gene, two bands of 7.5 and 1.8-0.8 kb were identified. Expression of IGF-I gene was reduced (P < 0.05) in NX in comparison with SAL, this reduction being more marked for the 7.5 kb transcript (amount of mRNA equal to 56.6 +/- 2.6 vs 84.8 +/- 6.2% of values found in SAL rats). There were no differences between NX and SPF. Normalization of caloric intake in SPF+ resulted in partial recovery of the 7.5-kb band and did not modify the 1.8-0.8 kb mRNAs. Circulating IGF-I levels were no different among the four groups. These data confirm that expression of liver GH receptor/GHBP and IGF-I genes is markedly decreased in uremic rats. Nutritional deficiency and not uremia itself seems to be the main causal factor, with protein deficit playing a major role.

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.

Nucleotide sequences specific for nonnominal immunoglobulin allotypes in rheumatoid arthritis patients and in normal individuals and their expression in synovial tissue of rheumatoid arthritis patients.

The production of antibodies against nonnominal immunoglobulin allotypes in rheumatoid arthritis (RA) patients suggests that the immune system of these patients has been exposed to such foreign allotypes. The presence of nonnominal allotypes is, however, a genetic enigma. We searched for nucleotide sequences specific for nonnominal G3mg and G3mb copies in individuals homozygous for these alleles. Using a sensitive and specific nested polymerase chain reaction (PCR) method with genomic DNA from blood of 18 RA patients and 5 normal controls, we found G3mg sequences in 18 of 18 tested G3mb homozygous persons. The allele specificity of the PCR fragments was confirmed by sequencing and RFLP analysis. The PCR products contained genomic nonspliced parts of the nonnominal sequences. An analysis of cDNA from inflammatory tissue of 5 RA patients detected nonnominal G3mb sequences in 1 of 3 tested G3mg homozygotes and G3mg sequences in 2 of 2 tested G3mb homozygotes. The cDNA-derived PCR products contained sequences from normally spliced nonnominal Ig fragments. The results also showed that the nonnominal Ig sequences were present in very low copy numbers, lower than the Mendelian 1-2 copies per cell. The origin of such a low copy number of Ig gene fragments may be explained by a virus-mediated capture and transfer mechanism of Ig gene fragments generated by the normal Ig switch-associated gene excision process.

Collagenase 2 (MMP-8) expression in murine tissue-remodeling processes. Analysis of its potential role in postpartum involution of the uterus.

Neutrophil collagenase or collagenase 2 (MMP-8) is unique among the family of matrix metalloproteinases (MMPs) because of its exclusive pattern of expression in inflammatory conditions. At present, no evidence of the occurrence of this enzyme in tissues other than human has been reported. In this work, we have cloned the murine homologue of human collagenase 2. The isolated cDNA contains an open reading frame coding for a polypeptide of 465 amino acids, which is 74% identical to its human counterpart. The mouse collagenase 2 exhibits the domain structure characteristic of several MMPs, including a signal sequence, a prodomain with the cysteine residue essential for enzyme latency, an activation locus with the Zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin. It also contains the three conserved residues (Tyr-209, Asp-230, and Gly-232) located around the Zinc-binding site and are distinctive of the collagenase subfamily. Northern blot analysis of RNAs isolated from a variety of mouse tissues revealed that collagenase 2 is expressed at late stages during mouse embryogenesis, coinciding with the appearance of hematopoietic cells. In addition, collagenase 2 was highly expressed in the postpartum uterus starting at 1 day postpartum and extending up to 5 days. Enzymatic analysis revealed that matrilysin, another MMP overexpressed in uterine tissue, is able to activate murine procollagenase 2. These data suggest that both enzymes could form an activation cascade resulting in the generation of the collagenolytic activity required during the process of massive connective tissue resumption occurring in the involuting uterus.

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.

Collagenase-3 expression is associated with advanced local invasion in human squamous cell carcinomas of the larynx.

Collagenase-3 (MMP-13) is a matrix metalloproteinase recently identified on the basis of differential expression in normal breast tissues and in breast carcinoma. To date, collagenase-3 expression has been reported only in breast carcinomas and in articular cartilage of arthritic patients; the presence and possible implication of this enzyme in the progression of other malignant tumours are unknown. In this study collagenase-3 mRNA expression has been analysed by northern blot in a series of 35 matched squamous cell carcinomas of the larynx and the corresponding adjacent non-neoplastic tissues. In addition, mRNA expression of membrane type 1-matrix metalloproteinase (MT1-MMP) and gelatinase A, two matrix metalloproteinases which have the ability to activate collagenase-3 in vitro, was also examined in the same cases. No collagenase-3 expression was detected in any of the 35 normal mucosae, but collagenase-3 mRNA was observed in 20 of the 35 carcinomas (57 per cent). Western blot analysis revealed 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 mRNA expression, or in any of the normal tissues. The protein was localized predominantly in tumour epithelial cells. Collagenase-3 expression correlated significantly with better histological differentiation of the tumours (p = 0.026), as well as with advanced local invasion (p = 0.026). Collagenase-3 upregulation was also significantly associated with MT1-MMP and gelatinase A overexpression. These findings suggest that collagenase-3 expression may contribute to the progression of a significant subset of squamous cell carcinomas of the larynx and that its coordinate overexpression with MT1-MMP and gelatinase A may have a cooperative effect in the progression of the tumours.

Structural analysis and promoter characterization of the human collagenase-3 gene (MMP13).

Human collagenase-3 (MMP13) is a recently identified member of the matrix metalloproteinase (MMP) family that is expressed in breast carcinomas and in articular cartilage from arthritic patients. In this work we have isolated and characterized genomic clones coding for human collagenase-3. This gene is composed of 10 exons and 9 introns and spans over 12.5 kb. The overall organization of the collagenase-3 gene is similar to that of other MMP genes clustered at chromosome 11q22, including fibroblast collagenase (MMP-1), matrilysin (MMP-7), and macrophage metalloelastase (MMP-12), but is more distantly related to genes coding for stromelysin-3 (MMP-11), gelatinase-A (MMP-2), and gelatinase-B (MMP-9), which map outside of this gene cluster. Nucleotide sequence analysis of about 1 kb of the 5'-flanking region of the collagenase-3 gene revealed the presence of a TATA box, an AP-1 motif, a PEA-3 consensus sequence, an osteoblast specific element (OSE-2), and a TGF-beta inhibitory element. Transient transfection experiments in HeLa and COS-1 cells with chloramphenicol acetyltransferase (CAT)-containing constructs showed that the AP-1 site is functional and responsible for the observed inducibility of the reporter gene by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). However, and in contrast to other MMP genes, no significative synergistic effect on CAT activity between the AP-1 and PEA-3 elements found in the collagenase-3 gene promoter was found. DNA binding analysis with nuclear extracts from HeLa cells revealed the formation of specific complexes between collagenase-3 promoter sequences containing the AP-1 site and nuclear proteins. The presence of this AP-1 functional site, which is able to confer responsiveness to a variety of tumor promoters and oncogene products, amy contribute to explaining the high-level expression of collagenase-3 in breast carcinomas and degenerative joint diseases.