Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention Cancer Health Disparities Conference 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, W.
Right arrow Articles by Strongin, A. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, W.
Right arrow Articles by Strongin, A. Y.
[Cancer Research 64, 8657-8665, December 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Matrix Metalloproteinase-26 Is Associated with Estrogen-Dependent Malignancies and Targets {alpha}1-Antitrypsin Serpin

Wei Li1,3, Alexei Y. Savinov1, Dmitri V. Rozanov1, Vladislav S. Golubkov1, Hirad Hedayat2, Tatiana I. Postnova1, Natalia V. Golubkova1, Yu Linli3, Stanislaw Krajewski2 and Alex Y. Strongin1

1 Cell Adhesion and Extracellular Matrix Biology Program, and 2 Apoptosis and Cell Death Research Program, Cancer Research Center, The Burnham Institute, La Jolla, California; and 3 The Key Laboratory of Pathobiology, Ministry of Education, Jilin University, Jilin, China


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteases exert control over cell behavior and affect many biological processes by making proteolytic modification of regulatory proteins. The purpose of this paper is to describe novel, important functions of matrix metalloproteinase (MMP)-26. {alpha}1-Antitrypsin (AAT) is a serpin, the primary function of which is to regulate the activity of neutrophil/leukocyte elastase. Insufficient antiprotease activity because of AAT deficiency in the lungs is a contributing factor to early-onset emphysema. We recently discovered that AAT is efficiently cleaved by a novel metalloproteinase, MMP-26, which exhibits an unconventional PH81CGVPD Cys switch motif and is autocatalytically activated in cells and tissues. An elevated expression of MMP-26 in macrophages and polymorphonuclear leukocytes supports the functional role of MMP-26 in the AAT cleavage and inflammation. We have demonstrated a direct functional link of MMP-26 expression with an estrogen dependency and confirmed the presence of the estrogen-response element in the MMP-26 promoter. Immunostaining of tumor cell lines and biopsy specimen microarrays confirmed the existence of the inverse correlations of MMP-26 and AAT in cells/tissues. An expression of MMP-26 in the estrogen-dependent neoplasms is likely to contribute to the inactivation of AAT, to the follow-up liberation of the Ser protease activity, and because of these biochemical events, to promote matrix destruction and malignant progression. In summary, we hypothesize that MMP-26, by cleaving and inactivating the AAT serpin, operates as a unique functional link that regulates a coordinated interplay between Ser and metalloproteinases in estrogen-dependent neoplasms.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The family of human matrix metalloproteinases (MMPs) currently comprises 25 zinc metalloproteinases (1) . Synthesized as inactive precursors, MMPs require proteolytic activation to express enzymatic activity. Once activated, MMPs can be inhibited by tissue inhibitors of proteinases (TIMPs). Activation, whether occurring intracellularly or extracellularly, requires the cleavage of a prodomain located downstream of a signal peptide. The latency of MMP zymogens is maintained by the coordination of the active site zinc by an unpaired Cys sulfhydryl group within a PRCG(V/N)PD conserved sequence motif of the propeptide.

MMP-26 is a recently discovered and only partially characterized human proteinase (2, 3, 4) , and it is distinguished from all other known mammalian MMPs in several ways. In contrast to all other individual MMPs, the MMP-26 gene does not exist in the murine genome. We suspect that MMP-26 recently evolved and in the process developed an unconventional, incompletely understood function in humans. In length, MMP-26 is closest to MMP-7, which is the shortest MMP. The catalytic domain of MMP-26 has a low degree of similarity with other individual MMPs and is only about 50, 46, 55, and 50% identical with that of MMP-3, -7, -12, and -13, respectively. The conserved PRCGXXD Cys switch involved in the latency of other MMPs is replaced in MMP-26 by the unique PH81CGVPD sequence (5 , 6) . The presence of the unique PH81CGVPD Cys switch motif in the MMP-26 sequence, in addition to other atypical structural features, leads to the unorthodox, autolytic mechanisms of the MMP-26 zymogen activation and contributes to the unusual physiologic role of the protease in cells and tissues (7 , 8) .

It also appears that the variables of substrate specificity, transcription regulation, mechanisms of activation, cellular compartmentalization, and functional roles of MMP-26 and MMP-7 are distinct from each other (7 , 9, 10, 11, 12, 13) . Several independent studies have demonstrated that MMP-26 is generally associated with the cell compartment rather than with the extracellular milieu despite the presence of the signal peptide in the proenzyme of MMP-26 peptide sequence and in contrast with several other secretory MMPs including MMP-7 (2, 3, 4 , 7 , 8 , 14) .

Recent data, which are subject to a range of interpretations, suggest that MMP-26 is expressed in normal cells of epithelial origin as well as in specific carcinomas including endometrium, breast, and prostate carcinomas (2 , 3 , 7 , 8 , 10 , 12 , 13 , 15) . On the other hand, there is a well substantiated report that states that MMP-26 is largely associated with a cycling human endometrium and that the expression of the protease disappears or is completely obscured in endometrial carcinomas (14) . Additional studies are clearly required to gain a better understanding of the role, regulation and function of MMP-26 in various physiologic conditions, including neoplasms.

Here, we report a novel, physiologically relevant and important function of MMP-26. MMP-26 is highly efficient in cleaving the ubiquitous serpin (Ser proteinase inhibitor) a1-antitrypsin (AAT). A deficiency of AAT is directly associated with an increased risk of developing a chronic, obstructive pulmonary disease (16) . The inactivation of the AAT function not only releases the activity of inflammatory Ser proteinases, especially neutrophil elastase, but it also is associated with the inflammation and proteolytic destruction of the lower respiratory tract. The MMP-26 proteolysis of AAT sheds much needed light on the biological control mechanisms that regulate the pericellular proteolysis by the enzymes from the Ser and metalloproteinases superfamilies. Consistent with the pro-inflammatory role of MMP-26, our studies have shown an elevated expression of MMP-26 in inflammatory cells including macrophages, and polymorphonuclear leukocytes. In addition, our data suggest an estrogen dependence of MMP-26 expression and demonstrate the specific association of MMP-26 with estrogen-dependent breast, endometrium, and ovarian carcinomas. Taken together, our data suggest that hormone-regulated MMP-26 functions as a novel pro-inflammatory proteinase. The results of this study provide us with a more complete and better understanding of the role, regulation, and function of MMP-26 in inflammation and in neoplasms.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Reagents were obtained from Sigma (St. Louis, MO), unless otherwise indicated. Human AAT was from Calbiochem (San Diego, CA). TIMP-1, TIMP-2, and the hydroxamate inhibitor GM6001 came from Chemicon International (Temecula, CA). A hydroxamate inhibitor of MMPs, AG3340, was kindly provided by Dr. P. Baciu (Allergan, Irvine, CA). The fluorogenic peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 was from Bachem (King of Prussia, PA). Pfx-polymerase and cell culture media were from Invitrogen (Carlsbad, CA). Phycoerythrin-conjugated goat antirabbit immunoglobulin G (IgG) was from Jackson ImmunoResearch (King of Prussia, PA). The rabbit polyclonal antibody to AAT was from DAKO (Carpinteria, CA). The recombinant catalytic domains of MMP-26, membrane type-1 MMP (MT1-MMP), and MMP-2 were prepared as described previously (17 , 18) . The recombinant proMMP-9 (19) was kindly provided by Dr. Jeffrey W. Smith (The Burnham Institute). The purified catalytic domain of MMP-26 was used to raise antibodies in rabbits. These antibodies were purified on a protein A column. The catalytic domain of MT1-MMP was expressed in Escherichia coli, isolated, and refolded to restore the native conformation as reported earlier (18) .

Expression and Purification of Proenzyme of Matrix Metalloproteinase-26.
cDNA coding for the MMP-26 proenzyme was subcloned into the modified pET21a(+) expression vector under the control of regulatory elements of a tac-promoter. The resulting plasmid was transformed into E. coli BL21 (DE3) cells. Expression of proenzyme of MMP-26 was induced in E. coli with 2.5 mmol/L isopropyl-1-thio-ß-D-galactopyranoside. E. coli cells (from 250 mL of the medium) were lysed in 10 mL of a B-PER bacterial protein extraction reagent (Pierce, Rockford, IL). The insoluble pellet was collected by centrifugation at 27,000 x g and again resuspended in 10 mL of the B-PER reagent supplemented with lysozyme (200 µg/mL). The inclusion bodies largely represented by proenzyme of MMP-26 were collected by centrifugation and dissolved in 4 mL of 20 mmol/L Tris-HCl buffer (pH 8.0) supplemented with 8 mol/L urea and 10 mmol/L dithiothreitol. Proenzyme of MMP-26 was purified from the solubilized material by fast protein liquid chromatography on a MonoQ column. Proenzyme of MMP-26 was eluted from the column with a linear 0 to 500 mmol/L NaCl gradient. Fractions were analyzed for the presence of proenzyme of MMP-26 by SDS-PAGE and gelatin zymography in 15% acrylamide gels containing 1 mg/mL gelatin.

Refolding and Activation of Proenzyme of Matrix Metalloproteinase-26.
We developed a new method to achieve more efficient refolding of proenzyme of MMP-26. Unless this new, optimized protocol, described below, is implemented, the yield of refolded MMP-26 will be low either because of the protein precipitation during dialysis or because of the incomplete activation of proenzyme of MMP-26. Specifically, fractions containing the purified material were pooled and diluted with 20 mmol/L Tris-HCl buffer (pH 8.0), containing 8 mol/L urea and 10 mmol/L dithiothreitol to the final protein concentration of 0.1 mg/mL. Refolding of proenzyme of MMP-26 accompanied by its autoactivation and conversion into the highly active MMP-26 enzyme was accomplished by dilution dialysis of the 0.1 mg samples. To refold and activate proenzyme of MMP-26, the diluted sample was dialyzed twice for 24 hours by dilution dialysis against 50 mmol/L HEPES buffer (pH 7.5), containing 200 mmol/L NaCl, 10 mmol/L CaCl2, 20 mmol/L ZnCl2, and 0.01% Brij-35 to stepwise decrease the concentration of urea from 8 to 1 mol/L and then from 1 to 0.1 mol/L. The 0.1 mol/L urea sample was dialyzed against 50 mmol/L HEPES buffer (pH 7.5), containing 200 mmol/L NaCl, 10 mmol/L CaCl2, 20 mmol/L ZnCl2, and 0.01% Brij-35 to remove traces of urea.

Enzyme Assays.
MMP-26 enzymatic activity was measured using the quenched fluorescent peptide substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (3) . The kinetic experiments were conducted in 50 mmol/L HEPES buffer (pH 6.8), containing 200 mmol/L NaCl, 10 mmol/L CaCl2, 20 µmol/L ZnCl2, 10 mmol/L MgCl2, and 0.01% Briji-35. To identify the inhibitory potency of AG3340 and GM6001, the apparent inhibitor dissociation constant (Kiapp) was calculated by fitting the data to Morrison’s equation. The assays were performed with a peptide substrate (5 mmol/L) and the inhibitor concentrations ranging from 2 to 50 nmol/L. The fluorescence was monitored at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. The total concentrations of the MMP-26 proenzyme, the catalytic domain of MMP-26, and the catalytic domain of MT1-MMP were measured by absorption at 280 nm and calculated using a molar extinction coefficient of 47,000, 39,000 and 57,000 mol/L/cm, respectively. The proteases were titrated with either AG3340 or GM6001 to determine the concentration of catalytically potent enzymes. The results were found to be in the range of 15, 15, and 15 to 30% from the total concentration of the MMP-26 proenzyme, the catalytic domain of MMP-26, and the catalytic domain of MT1-MMP, respectively.

Immunohistochemistry.
For the characterization of MMP-26 expression in normal tissues, we constructed tissue microarrays each containing 130 specimens, representing 1-mm-diameter cylindrical cores acquired from paraffin blocks of normal human tissues, fixed with buffered formalin and/or Bouin’s fixative (20) . The following tissues and organs were represented in human normal tissue array: skin, skeletal muscle and smooth muscle, heart, aorta and lung from musculo-respiratory and cardiovascular systems, esophagus, stomach, small intestine, jejunum, colon, appendix, liver, pancreas from alimentary tract, spleen, thymus, tonsil, lymph nodes, bone marrow from hematolymphoid systems, kidney and bladder from urinary tract, breast, fallopian tube, cervix with endometrium, uterus with myometrium and peritoneum, placenta and prostate, testis from female and male reproductive organs respectively, cerebral cortex and cerebellum from the central nervous system and thyroid, and adrenal gland with medulla and adrenal cortex as an example of endocrine organs.

We also used the similarly designed gastric adenocarcinoma array that represented tumor tissue and matching normal tissue biopsies. We also used the NCI 60 tumor cell line panel (20 , 21) plus 14 additional cancer cell lines maintained in our laboratory: seven breast cancer cell lines (231, BT474, HS574, A1N4, 10A, 468, and ZR751), five prostate cancer lines (PPC1, ALVA31, JCA1, LNCap, and TSU-PRL), the lymphoma line RS11846, and the endometrium carcinoma line Ishikawa.

We also used the similarly designed arrays representing 0.6-mm-diameter cylindrical cores. These arrays included the tumor samples from bladder, breast, cervix, colon, endometrium, esophagus, kidney, larynx, liver, lung, muscle, ovary, pancreas, prostate, skin, testis, and uterus; biopsies of lymphomas, sarcomas, mesothelioma, leiomyosarcomas, squamous, small cell and non-small cell carcinomas; and the samples of cirrhotic and normal liver and normal breast, cervix, colon, esophagus, heart, jejunum, kidney, larynx, lung, muscle, pancreas, prostate, skin, stomach, thymus, and thyroid.

Arrays were stained with the antibody to the catalytic domain of MMP-26. Where indicated, arrays were also stained for AAT. Staining with the primary antibody was followed by a diaminobenzidine-based detection method employing horseradish peroxidase system (20 , 22) . For all tissues examined, the immunostaining procedure was performed in parallel using either preimmune serum or antiserum depleted by incubation with recombinant protein immunogen to verify specificity of the results.

The immunostaining results were scored according to intensity as 0, negative; 1+, weak; 2+, moderate; and 3+, strong. The scoring of immunostaining for tumors and tumor cell lines was calculated by multiplying the percentage of immunopositive cells (0 to 100) by the staining intensity score (0, 1, 2, or 3), yielding arithmetic scores ranging from 0 to 300.

Reverse Transcription-Polymerase Chain Reaction.
Endometrium carcinoma Ishikawa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)-10% fetal calf serum in the presence of phenol red. In parallel, cells were grown under estrogen-free conditions in DMEM supplemented with estrogen-deficient, charcoal-stripped fetal calf serum (Gemini Bio-products, Woodland, CA) without phenol red. To stimulate MMP-26 synthesis, 17-ß-estradiol (1 x 10–9 mol/L) was then added to the medium, and cells were cultivated for an additional 24 hours. Expression of MMP-26 and estrogen receptor was analyzed by reverse transcription-PCR using the total RNA samples isolated from the cells. The 5'-TGACATGCAGATGCATGCTCTGC-3' and 5'-CTAGGGTCGTGATACCAGTAAGTG-3' primers were used to give rise to the 500-bp fragment of MMP-26. The 5'-GAGAGGTGATGTCTGTGTTAGC-3' and 5'-CAATAGGCATCTGGATTAGTGC-3' primers were used to amplify the 560-bp fragment of estrogen receptor. Amplification of ß-actin was used to ascertain the equal amount of cDNA in each reaction.

Cleavage of {alpha}1-Antitrypsin.
AAT (400 ng) was coincubated for 2 hours at 37°C with the indicated amounts of the proteases in 20 µL of 50 mmol/L HEPES buffer (pH 6.8) containing 200 mmol/L NaCl, 10 mmol/L CaCl2, 20 µmol/L ZnCl2, 10 mmol/L MgCl2, and 0.01% Briji-35. The reactions were stopped by adding 2% SDS and analyzed by SDS-PAGE.

To prepare the material for the NH2-terminal sequence analyses of the cleavage fragments, the reactions (40 µL) contained 10 µg of AAT and 10 ng of MMP-26. The proteolytic fragments were separated by 4 to 20% gradient SDS-PAGE and then transferred onto a membrane. The Coomassie blue-stained protein bands were subjected to the NH2-terminal microsequencing.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Characterization of Recombinant Matrix Metalloproteinase-26.
Previous research from our and other laboratories demonstrated that the recombinant MMP-26 expressed in E. coli, purified and refolded from the inclusion bodies, was capable of cleaving the ECM proteins (2, 3, 4, 5) . The efficiency of this cleavage was, however, relatively low compared with that of other MMPs. These results suggested that we were dealing either with the intrinsically low proteolytic activity of the MMP-26 molecule or with an insufficient yield of the properly folded recombinant protease because of the imperfect refolding protocols. To determine which was the case, we redesigned and optimized our method of isolating MMP-26 from E. coli inclusion bodies, and we retested the multiple refolding protocols of the isolated proenzyme of MMP-26. Our efforts resulted in establishing strongly improved protocols that provided us with sufficient amounts of the highly active MMP-26 enzyme (see Materials and Methods). In brief, after solubilization of the purified inclusions in 8 mol/L urea, proenzyme of MMP-26 was purified under denaturing conditions to give a band at the molecular mass of 29 kDa (Fig. 1)Citation . Unless the optimized refolding protocols were used, the yield of refolded MMP-26 was low, either because of the protein precipitation during dialysis or because of the incomplete activation of the 29-kDa proenzyme of MMP-26. Our data confirmed that proenzyme of proenzyme of MMP-26 is capable of autocatalytic activation. Consistent with our earlier data and the results of other groups (5 , 6 , 8) , refolding of the purified 29-kDa zymogen was accompanied by its quantitative autocatalytic conversion into the proteolytically potent and mature 19.5-kDa MMP-26 enzyme (Fig. 1)Citation . The presence of the alternatively processed 21-kDa form frequently characterized the partially refolded samples of MMP-26 with low proteolytic activity. The catalytic domain construct that represented the Thr90-Pro261 sequence of the MMP-26 polypeptide chain was lower in its apparent molecular mass because this construct was shorter from its NH2 terminus relative to the enzyme species of MMP-26 generated by autocatalytic activation. The refolded samples of active MMP-26 did not lose activity over time and were not subjected to any additional autoproteolytic degradation. These features favorably distinguished our current MMP-26 samples from those generated in the earlier work (2 , 3 , 5 , 8) .



View larger version (54K):
[in this window]
[in a new window]
  		Fig. 1. Expression of proenzyme of MMP-26 (proMMP-26) in E. coli, purification from the inclusion bodies and refolding of the purified enzyme. The total cell lysate, the purified inclusion bodies, and the purified denatured MMP-26 proenzyme were analyzed by SDS-PAGE and stained with Coomassie blue (left panel). The purified samples of denatured proenzyme of MMP-26 were subjected to refolding, as described in Materials and Methods, to generate the properly folded MMP-26 enzyme (refolded MMP-26) or were refolded by alternative methods (incompletely refolded MMP-26) and analyzed by SDS-PAGE (middle panel) and gelatin zymography (right panel). The isolated catalytic domain of MMP-26 (catMMP-26), isolated as described previously (6) , was tested in parallel. Molecular masses of the MMP-26 forms are shown on the right.

 
Microsequencing confirmed the expected NH2 termini of the MMP-26 proenzyme with one extra initiation codon coded for Met residue (MV18PVPP). Proteolytic activity of the 29-kDa proenzyme and the 19.5-kDa enzyme of MMP-26 was verified by analyzing the protein samples in gelatin gels (Fig. 1)Citation . The COOH-terminal autocatalytic truncation identified and characterized in our earlier work explains the presence of two gelatinolytically active 18-kDa (full-length) and 14-kDa (C-end truncated) species of the samples of the MMP-26 catalytic domain.

The active MMP-26 concentration was determined by active site titration with either AG3340 or GM6001 hydroxamate inhibitors using the fluorogenic substrate MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2. Consistent with the results by other groups (3 , 5) , both inhibitors were highly potent against MMP-26 and exhibited a Kiapp in the low nanomolar range (GM6001, 0.8 nmol/L; AG3340, 1.5 nmol/L; Fig. 2ACitation ). The active site titration showed the concentrations of refolded MMP-26 to be approximately 15% of the total enzyme concentration. These levels of refolding substantially exceeded those reported earlier (3 , 5) .



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 2. MMP-26 cleaves AAT in vitro. A, inhibition kinetics of MMP-26 by a hydroxamate inhibitor AG3340. Increasing concentrations of AG3340 were added to 13 nmol/L (1 mg/mL) refolded MMP-26 in 50 mmol/L HEPES buffer (pH 6.8) containing 200 mmol/L NaCl, 10 mmol/L CaCl2, 20 µmol/L ZnCl2, 10 mmol/L MgCl2, and 0.01% Briji-35. After 30 minutes, the residual enzymatic activity was measured using the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 substrate. Each data point represents the mean value of a triple measurement. The data were fitted to Morrison’s equation to calculate the Kiapp value. The inhibitory data were also used to determine the concentration of catalytically active MMP-26, which normally was in the range of 15% from the total concentration of the MMP-26 proenzyme. RFU, relative fluorescence unit. B. MMP-26 cleaves AAT with high efficiency. AAT (400 ng) was coincubated for 2 hours with the indicated amounts of MMP-26. The digest samples were analyzed by SDS-10% PAGE. C, inhibition of MMP-26 by hydroxamates and TIMPs. AAT (400 ng) was coincubated with MMP-26 (2 ng) for 2 hours either alone or in the presence of TIMP-1 (8 ng; 3-fold molar excess), TIMP-2 (8 ng; 4-fold molar excess), GM6001, or AG3340 (both at 1 mmol/L). Arrow, the 55-kDa cleavage fragment of AAT. D, activation of MMP-2 and MMP-9. The zymogens of MMP-2 and MMP-9 were activated with p-aminophenylmercuric acetate (2 mmol/L). The recombinant 55-kDa proenzyme of MMP-9 (proMMP-9) used in our studies was lacking the COOH-terminal hemopexin domain (19) . proMMP-2, proenzyme of MMP-2. Quantitative activation of MMP-2 and MMP-9 and their conversion into the respective enzymes were verified by gelatin zymography. The AAT cleavage efficiency of the activated MMP-2 and MMP-9 was tested in E. E, AAT cleavage by the individual MMPs. AAT (400 ng) was incubated with increasing amounts of refolded MMP-26, the catalytic domain of MMP-26 (catMMP-26), the catalytic domain of MT1-MMP (catMT1-MMP), and activated MMP-2 and MMP-9. Arrow, the 55-kDa cleavage fragment of AAT. Under our experimental conditions, the molar amounts of catMT1-MMP, MMP-26, catMMP-26, MMP-2, and MMP-9 that were required to accomplish a 50% conversion of AAT into the 55-kDa cleavage product were related as 20, 18, 5, 2.5, and 1, respectively. The concentrations of catalytically active MMP-26, catMMP-26, and catMT1-MMP were determined by active site titration with either AG3340 or GM6001. The concentrations of MMP-2 and MMP-9 were measured by absorption at 280 nm and calculated using a molar extinction coefficient of 59,000 and 67,000 mol/L/cm, respectively.

 
{alpha}1-Antitrypsin Is a Target of Matrix Metalloproteinase-26 Proteolysis.
AAT serpin is a clinically relevant protein target of proteolysis by MMPs such as MMP-9 in vivo (23) as well as a common and convenient substrate for testing the functional activity of the individual MMPs in vitro. MMP-26 cleaved 61-kDa AAT near the COOH terminus and generated the 55-kDa NH2-terminal fragment as well as a COOH-terminal fragment of approximately 6 kDa (Fig. 2BCitation ; the 6-kDa fragment is not shown). The catalytic amounts of MMP-26 fully proteolyzed AAT (the enzyme:substrate molar ratio at a range of 1:125) in 2 hours, suggesting a superior activity of MMP-26 relative to the earlier reports (refs. 3 and 5 ; Fig. 2BCitation ). Hydroxamates AG3340 and GM6001 as well as TIMPs, such as TIMP-1 and TIMP-2, inhibited the functional activity of MMP-26 and blocked proteolysis of AAT by this protease (Fig. 2C)Citation .

The very high activity of MMP-26 against the serpin prompted us to compare the proteolytic potency of MMP-26 with other individual MMPs. For these comparative analyses, we used the individual catalytic domain of MT1-MMP, MMP-2, and MMP-9. All of these individual MMPs, and especially the catalytic domain of the MT1-MMP, are known to be highly efficient in cleaving protein substrata (18 , 24) . Intriguingly, the COOH-terminal PEX domain is a negative regulator of the catalytic potency of the active site of the MT1-MMP, and as a result, the full-length MT1-MMP is less efficient catalytically than its individual catalytic domain (25) . In addition, MT1-MMP is a membrane-tethered protease that can barely access the soluble AAT, thereby suggesting no physiologic relevance of MT1-MMP proteolysis to AAT.

Before the AAT cleavage, MMP-2 and MMP-9 were each activated by p-aminophenylmercuric acetate (Fig. 2D)Citation . Gelatin zymography confirmed the full conversion of the proenzyme of MMP-2 and MMP-9 into the respective enzyme (Fig. 2E)Citation . The cleavage assay demonstrated that the efficiency of MMP-26 was far superior to that of MMP-2 or MMP-9 in cleaving AAT and that it was similar to the catalytic domain of MT1-MMP, which was, in our experience, the most potent protease species in terms of its protein cleaving capabilities. The enzyme of MMP-26 generated via the refolding and autocatalytic activation of recombinant proenzyme was approximately three times more efficient in our cleavage test than the individual catalytic domain of MMP-26.

We also asked ourselves the question of whether or not MMP-26 proteolysis inactivates AAT. According to Park et al. (5) , MMP-26 cleaved the 61-kDa AAT near the COOH terminus and generated the 55-kDa NH2-terminal fragment as well as the COOH-terminal 6-kDa fragments commencing at Leu377 and Met382 (Fig. 3)Citation . These cleavages, which take place in the immediate proximity of the Met382-Ser383 active site of AAT, unavoidably inactivate the serpin. The MMP-26 cleavage sites in the AAT molecule were identified by determining the molecular mass of the proteolytic fragments and deducing the resultant peptide sequence (5) rather than by directly identifying the NH2-terminal sequence of the cleavage products. To confirm and extend the cleavage sequence data, we subjected AAT to an exhaustive proteolysis by MMP-26 and then identified the peptide sequence by cleavage fragments by NH2-terminal microsequencing (Fig. 3A)Citation . Our studies identified the two additional MMP-26 cleavage sites of AAT and allowed us to reconstruct the entire cleavage map of this serpin (Fig. 3B)Citation . Thus, we identified the cleavage fragments of AAT with apparent molecular masses of 55, 44, 38, 31, 25, 18, 13, and 6 kDa (Fig. 3A)Citation . According to the NH2-terminal sequence analyses, 55-, 33-, and 18-kDa fragments exhibited an NH2 terminus (Glu1-Asp-Pro-Gln) of intact AAT. Both 40- and 13-kDa fragments had the NH2 terminus commencing at Leu136-Thr-Thr-Gly. The NH2-terminal sequence of the 25-kDa fragment was Val224-Lys-Asp-Thr, whereas the 6-kDa fragment represented the digest sequences commencing from Leu377 and Met382. The Phe376-Leu377 and Pro381-Met382 cleavage sites are localized within the extended loop that exposes the Met382-Ser383 active site of the serpin. Two other scissile bonds (Gln135-Leu136 and Glu223-Val224) are close to the 136 to 145 and 228 to 233 ß-strand regions of the AAT molecule, respectively. It appears that the cleavage of either Gln135-Leu136 or Glu223-Val224 scissile bond will affect the system of antiparallel ß-strands, the interactions of which are essential for the spatial structure of AAT (Fig. 3C)Citation .



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3. MMP-26 cleaves the functionally essential sequence regions of AAT. A, proteolysis of AAT by MMP-26. AAT (10 ng) was subjected to proteolysis by MMP-26 (DIGEST). The cleavage sample was separated by SDS-PAGE. After transfer to a membrane, the NH2-terminal sequence of the bands was determined by NH2-terminal microsequencing. The molecular mass markers are shown on the right. The molecular mass of the cleavage fragment is shown on the left. B, cleavage fragments of AAT. Numbering of AAT starts from the NH2-terminal Met residue of the signal peptide. The scissile bonds are indicated by arrows. C, spatial model of AAT. Arrows, the positions of the Gln135{downarrow}Leu136, Glu223{downarrow}Val224, Phe376{downarrow}Leu377, and Pro381{downarrow}Met382 cleavage sites in the AAT molecule.

 
We infer from these data that MMP-26, rather than other individual MMPs, is likely to have been uniquely designed to target AAT in physiologic and pathophysiologic conditions in vivo. To corroborate this hypothesis, we evaluated pro-inflammatory cells for the presence of MMP-26.

Matrix Metalloproteinase-26 Is Associated with Macrophages and Polymorphonuclear Leukocytes.
An antibody raised against the catalytic domain was highly specific to MMP-26 and did not interact with either MT1-MMP or MMP-2 (Fig. 4A)Citation . Using this antibody to MMP-26, the in vivo pattern of expression of MMP-26 was examined in normal human tissue arrays as well as malignant tissue arrays by immunohistochemistry. Consistent with the role of MMP-26 in the cleavage of AAT, we found a strong association of MMP-26 expression with pro-inflammatory cells such as macrophages, and polymorphonuclear leukocytes (Fig. 4B)Citation . Thus, alveolar macrophages demonstrated strong MMP-26 immunoreactivity. Similarly, neutrophils existing in a tonsil lymphoid follicule were strongly positive, whereas peripheral lymphocytes exhibited no immunoreactivity. In agreement with these findings, MMP-26 was up-regulated in infiltrating polymorphonuclear leukocytes in gastric adenocarcinoma. Staining was highly specific for these cell types. No background staining was observed in tumor cells and other interstitial cell types.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 4. Immunohistochemical analysis of MMP-26 expression in normal human tissues and in tumor cell lines. A. An antibody to MMP-26 does not cross-react with other MMPs. The samples of MMP-26, catMMP-26, MMP-2, and the catalytic domain of MT1-MMP (250 ng each) were tested by Western blotting with the rabbit antibody against the catalytic domain of MMP-26. B. MMP-26 is expressed in macrophages (LUNG) and polymorphonuclear leukocytes in normal tissue (TONSIL) and in gastric adenocarcinoma biopsy. C, immunohistochemical analysis of tumor cell lines. Representative MMP-26 immunostaining results are presented in the microarray of the NCI 60 tumor cell line panel and 14 additional cancer cell lines maintained in our laboratories (20) . Left and right panels, MMP-26–positive and MMP-26–negative cell lines, respectively. Examples of MMP-26 immunostaining are presented at the original magnification of x40.

 
Matrix Metalloproteinase-26 Is a Novel Marker of Melanomas and Estrogen-Dependent Carcinomas.
The expression of MMP-26 was examined and scored in 74 tumor cell lines of the NCI panel. Representative examples of immunostaining are presented in Fig. 4CCitation . In the NCI panel, MMP-26 expression correlated positively with estrogen dependence. This correlation was especially evident in breast cancer cell lines, in which estrogen-dependent MCF7, ZR-751, and T-47D cells showed strong MMP-26 immunoreactivity. In turn, estrogen-independent HS578T, BT-549, and NCI/ADR-RES cells were clearly MMP-26 negative. Similarly, estrogen dependency was associated with MMP-26 immunoreactivity in ovarian carcinoma cell lines. Estrogen-dependent endometrial carcinoma Ishikawa cells were also found to be MMP-26 positive. In addition, MMP-26 immunoreactivity was identified in lymphoma SR cells and renal carcinoma A498 cells. Five cell lines (UACC-62, UACC-257, MALME-3M, SK-MEL-5, SK-MEL-28) from seven melanoma cell lines were highly positive, and two (LOX IMVI and SK-MEL-2) were negative (Table 1)Citation . These findings, especially the association of MMP-26 immunoreactivity with the estrogen status of the cells and the presence of MMP-26 in melanomas, call for additional investigation.


View this table:
[in this window]
[in a new window]
  		Table 1 MMP-26 expression in tumor cell lines correlates with estrogen dependency

 
The hormone-activated estrogen receptor complex binds to the specific estrogen-response element located in the promoter region of estrogen-regulated genes. We speculate that it is highly likely that MMP-26 is regulated by estrogen through an estrogen-response element. The MatInspector4 and Promoter5 software packages were used for extensive re-evaluation of the MMP-26 gene promoter sequence. We looked for the presence of estrogen-response element–like motifs (GGTCANNNTGACC) in the MMP-26 gene. Indeed, an estrogen-response element–like motif was found in the promoter region of the MMP-26 gene (nucleotides –129/–117; 5'-GGTCACTCTTGCCC-3'), having the characteristic of the estrogen-response element, a 13-bp palindromic element consisting of two 5-bp arms separated by a 3-bp spacer. The MMP-26 estrogen-response element has one arm of the palindromic element sequences with perfect nucleotide homology to the described estrogen-response element and a second incomplete set. The two arms of the palindrome are separated by the exact spacing (3-bp) which is essential for estrogen receptor action (Fig. 5A)Citation . The relative positions of the estrogen-response element and other transcription factor-binding sites in the MMP-26 promoter are shown in Fig. 5ACitation .



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 5. The expression of the human MMP-26 gene is regulated by estrogen. A, Regulatory elements of the promoter region and exon–intron structure of MMP-7 (matrilysin) and MMP-26 (matrilysin-2). The exons (numbered from the 5' end of the gene) are shown as boxes, with their sizes in nucleotides given in parentheses. {square}, noncoding regions. The domain structure is shown in the top of the figure (SP, signal peptide; PRO, prodomain; CAT, catalytic domain; CT, COOH-terminal tail). Transcription factor binding sites localized within an approximately 1-kb region upstream of the transcription initiation site are indicated within boxes. Transcription start is indicated with a bent arrow. The positions of the binding sites located in the sense and antisense strands are given above and below the boxes, respectively. Note the similar exon–intron structure of MMP-7 and MMP-26 and the different structure of their promoter regions as well as the presence of the estrogen-response element in MMP-26. TATA, TATA-box; AP-1, activator protein-1; PEA3, polyoma virus enhancer A-binding protein-3; TIE, TGF-ß inhibitory element; Tcf-4, T-cell factor-4; CCAAT, CCAAT-binding proteins; CIZ, Cas-interacting zinc finger protein; PA, the highly unusual poly(A) site in the 5' region of the MMP-26 promoter. B. Estrogen up-regulates MMP-26 in estrogen receptor–positive endometrium carcinoma Ishikawa cells. The transcriptional activity of the MMP-26 promoter and the estrogen receptor (ER) promoter were determined by reverse transcription-PCR using RNA isolated from cells grown in DMEM-10% fetal calf serum, estrogen-depleted cells, and estrogen-replenished cells (Lanes 1, 2, and 3, respectively). Reverse transcription-PCR was followed by agarose gel electrophoresis of the reaction aliquots. Amplification of ß-actin was used to ascertain the equal amount of cDNA in each reaction. C, immunofluorescence staining of Ishikawa cells with an antibody against MMP-26. Cells were stained with an antibody against MMP-26 followed by phycoerythrin-conjugated antirabbit IgG. Note the cytoplasmic staining of MMP-26 (red). There was no background staining of the cells with control rabbit IgG. 4',6-diamidino-2-phenylindole (blue) was used for nuclear staining.

 
To confirm directly the role of estrogen in regulating the transcriptional activity of the MMP-26 promoter we used endometrial carcinoma Ishikawa cells. We specifically selected these estrogen-dependent cells because they demonstrated MMP-26 immunoreactivity in our immunostaining studies.

Estrogen Stimulates Matrix Metalloproteinase-26 Expression via an Estrogen-Response Element of the Matrix Metalloproteinase-26 Promoter.
We analyzed the expression of MMP-26 in Ishikawa cells treated with estrogen. For these purposes, cells were grown with and without estrogen and then stimulated with estrogen to up-regulate the synthesis of MMP-26. To obtain evidence that MMP-26 and the estrogen receptor are expressed in the cell samples, we performed semiquantitative PCR amplification of cDNAs generated by the reverse transcription of mRNA. The total mRNA pool was isolated from Ishikawa cells and subjected to reverse transcription-PCR amplification. Fig. 5BCitation shows that estrogen depletion reduced the expression of the MMP-26 gene, whereas cells replenished with estrogen restored the transcriptional activity of the MMP-26 gene. The presence of MMP-26 in Ishikawa cells was also demonstrated by immunofluorescence staining (Fig. 5C)Citation . Accordingly, we concluded that estrogen regulates the MMP-26 gene expression via the estrogen receptor and the estrogen-response element motif of the MMP-26 promoter.

Inverse Correlations of Matrix Metalloproteinase-26 with {alpha}1-Antitrypsin in Cells and Tissues.
The redundancy of the MMPs as well as the low levels of MMP-26 expression in cells complicates the identification of AAT cleavage by MMP-26 in vivo. The available broad-spectrum hydroxamate inhibitors cannot specifically target and discriminate MMP-26 from the other structurally similar redundant MMPs. To overcome these experimental difficulties and to support our hypothesis that MMP-26 cleaves AAT in vivo, we used a different approach. We analyzed the immunoreactivity of MMP-26 and AAT in cell and tissue specimens. For these purposes, we used the NCI tumor cell line microarrays (Fig. 6A)Citation and the 308-specimen tumor/normal tissue microarrays derived from individual patients (Fig. 6B)Citation . Breast and colon carcinomas and especially melanomas were shown to synthesize sufficient quantities of AAT (26, 27, 28) . Thus, Pernick et al. (27) reported that AAT immunostains were positive almost as frequently as traditional melanoma markers.



View larger version (39K):
[in this window]
[in a new window]
  		Fig. 6. Inverse correlations of MMP-26 with AAT in tumor cell lines and in tumor and normal tissue biopsy samples. A, inverse correlations of MMP-26 with AAT in tumor cell lines. Representative MMP-26 and AAT immunostaining of the individual cell lines from the NCI tumor cell line microarray. The four top panels represent the MMP-26–positive, AAT-deficient tumor cell lines. The AAT-positive, MMP-26–deficient tumor cell lines are shown in the two bottom panels. The samples are shown at the original magnification of x40. B. Tissue microarrays each containing 308 specimens, representing 1-mm (diameter) cylindrical cores acquired from paraffin blocks of normal and tumor human tissues were stained for MMP-26, AAT, and with a control rabbit IgG (top, middle, and bottom panels, respectively). Specimens (1, normal liver; 2, cirrhotic liver; 3, normal liver;4, striated muscles) that expressed high and low levels of MMP-26 and AAT, respectively, are shown within the squares. Specimens (I, normal kidney; II, liver infiltrated by pancreatic adenocarcinoma; III, small cell and non-small cell lung carcinomas) that exhibited high and low levels of AAT and MMP-26 are in the ovals.

 
Immunostaining of the NCI array demonstrated the inverse correlations of MMP-26 with AAT in the renal carcinoma A498, melanoma MALME-3M, ovarian carcinoma SK-OV-3, breast carcinoma T-47D, colon carcinoma SW-620, and breast carcinoma MDA-MB-435 cells. Thus, the levels of MMP-26 immunoreactivity were high in the A498, the MALME-3M, the SK-OV-3, and the T-47D cells. In contrast, the SW-620 and the MDA-MB-435 cells, which both express high levels of AAT, were deficient in MMP-26 immunoreactivity (Fig. 6A)Citation .

In agreement with these results, there was an inverse correlation of MMP-26 with AAT in biopsy samples of normal and cirrhotic liver as well as in specimens of normal kidney, of liver infiltrated by pancreatic adenocarcinoma, and in small cell and non-small cell lung carcinomas. Because of MMP-26 proteolysis of AAT, there should be an AAT-deficiency in certain cells/tissues with high levels of MMP-26. Conversely, high levels of AAT in specimens should correlate with low levels MMP-26. The findings of both of these characteristic patterns in the cell/tissue samples support our hypothesis that MMP-26 proteolysis contributes to the regulation of AAT in vivo.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
AAT is one of the major serpins controlling proteinases, especially neutrophil elastase, in many biological pathways. Inadequate antiprotease activity in the lungs because of an AAT deficiency is a major factor in early-onset emphysema. In addition to emphysema, the imbalance between neurophil elastase and AAT is generally thought to cause tissue damage, which could create a favorable tissue environment for malignant progression. A deficiency in AAT is associated with increased risk of liver cancer, bladder cancer, gallbladder cancer, malignant lymphoma, and lung cancer (29) .

Several individual MMPs including MMP-1, MMP-3, MMP-7, and MMP-9 have been reported to cleave AAT and to destroy its serpin activity (23 , 30, 31, 32) . It has been suggested that AAT is a critical target of MMP-9 proteolysis (23) . Excess neutrophil elastase was found to produce lesions in MMP-9 knockout mice that were deficient in cleaving AAT (23) . There is evidence that MMP-9 is activated in cells/tissues by MMP-26 (8 , 23) , a recently identified and partially characterized novel, unconventional protease (2, 3, 4 , 6 , 7 , 10 , 11) . Our current data make it evident that MMP-26 itself is unusually highly potent in cleaving AAT. Thus, MMP-26 is 10 to 20 times more efficient in the AAT cleaving activity compared with MMP-9 and MMP-2, the most common soluble MMPs. The earlier data also suggested that MMP-26 was capable of degrading AAT but with relative inefficiency. The reason for this, as our studies demonstrated, was poor refolding of the recombinant MMP-26 enzyme. Our optimized refolding protocols provided a high-quality enzyme with unexpectedly high proteolytic potency against AAT. Our data imply that the likely important function of MMP-26 in vivo is the cleavage of AAT. Our additional investigation (in collaboration with Dr. Jeff W. Smith’s laboratory, The Burnham Institute), which used the substrate phage cleavage libraries of approximately 1 x 1010 random peptide hexamers for identification of the cleavage preferences of MMP-26, resulted in finding the preferred cleavage motif of MMP-26 (data not shown). Consistent with our hypothesis that AAT is a physiologically relevant cleavage target for MMP-26, this motif was identical to the major cleavage site of AAT (Fig. 3)Citation .

The ability of MMP-26 to target AAT correlated with the expression of protease in circulating, infiltrating blood cells such as macrophages and polymorphonuclear leukocytes. Reverse transcription-PCR also supported the expression of MMP-26 in T cells (data not shown) as well as in B cells (9) . It is highly likely that MMP-26 functions specifically in these cell types to promote inflammation by cleaving AAT and by liberating the activity of neutrophil elastase to promote inflammation. We suggest that MMP-26, by cleaving AAT, contributes to the coordinated interplay of the two distinct superfamilies of proteinases, Ser proteinases and metalloproteinases, thereby functioning to promote these proteinases to work in concert.

Our studies also demonstrated that the promoter of the MMP-26 gene represents the estrogen-response element. The presence of the estrogen-response element explains the expression of MMP-26 in hormone-regulated carcinomas. Our analysis of the NCI 60 tumor cell line panel (20) and 14 additional cancer cell lines provided direct evidence linking MMP-26 to the estrogen-positive breast, endometrial, and ovarian carcinomas. The functional link of MMP-26 with estrogen explains the association of MMP-26 with menstrual period and cycling endometrium (15 , 33, 34, 35) . Thus, endometrial expression of MMP-26 comes to a maximum in the early secretory phase and then decreases to nondetectable levels in the late secretory and menstrual phases. Overall, our data suggest the involvement of MMP-26 in reproductive processes as well as the association of MMP-26 expression with estrogen-dependent tumors. These data also suggest that MMP-26, which does not exist in rodents because they do not exhibit the menstrual cycle, evolved to acquire a novel, albeit poorly understood, function in regulating reproductive processes in humans. Furthermore, mice express at least seven AAT isoforms encoded by a family of genes, whereas there is only a single gene of AAT in humans (36 , 37) . These events complicate the proteolytic regulation of AAT in mice and explain why no MMP-26 is required in rodents. The most recent publications also link MMP-26 to estrogen receptor and estrogen and support the observations presented in this study (38 , 39) .

We have also identified the expression of MMP-26 in several melanoma cell lines represented in the NCI tumor cell line panel. In addition to the estrogen-response element, the MMP-26 gene promoter includes several other transcription factor-binding sites such as T-cell factor-4 (Tcf-4) and activator protein-1 (AP1), the transcriptional efficiency of which has already been directly confirmed in our earlier work (7 , 10) . We believe Jun/Fos, through the AP1 site, and ß-catenin, through the Tcf-4 site, are involved in the regulation of MMP-26 expression in melanomas.

The inverse correlations of MMP-26 with AAT detected in the tumor cell lines from the NCI tumor cell array as well as in the specimens of normal and tumor biopsies support our hypothesis that the MMP-26 activity appears to significantly contribute to the AAT proteolysis in vivo. Taken together, our results and the other works imply that MMP-26 is an important factor that regulates the proteolytic activity in estrogen-dependent hyperplastic and malignant tissues (7 , 8 , 10 , 13, 14, 15 , 33, 34, 35 , 38 , 40) . The precise molecular mechanisms and an accurate estimate of the relative contribution of MMP-26 to the AAT proteolysis in vivo remain to be elucidated.

The mechanisms documented in this study suggest the involvement of MMP-26 in inflammation and malignant progression of estrogen-dependent tumors and brings us several steps closer to understanding the functional role of the unconventional MMP-26 enzyme in physiologic and pathologic processes.


   ACKNOWLEDGMENTS
 
We thank Xiakun Xiao for skillful histologic assistance in tissue microarray technology and Xianshu Huang for excellent immunocytochemistry.


   FOOTNOTES
 
Grant support: NIH grants CA83017 and CA77470 (A. Strongin).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Alex Y. Strongin, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-3100; Fax: 858-646-3192; E-mail: strongin{at}burnham.org

4 Internet address: http://genomatix.gsf.de/cgi-bin/matinspector/matinspector.pl. Back

5 Internet address: http://www.cbs.dtu.dk/services/promoter/ Back

Received 8/23/04. Revised 9/ 9/04. Accepted 9/29/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Egeblad M, Werb Z New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2:161-74.[Medline]
  2. Marchenko GN, Ratnikov BI, Rozanov DV, Godzik A, Deryugina EI, Strongin AY Characterization of matrix metalloproteinase-26, a novel metalloproteinase widely expressed in cancer cells of epithelial origin. Biochem J 2001;356:705-18.[CrossRef][Medline]
  3. Park HI, Ni J, Gerkema FE, Liu D, Belozerov VE, Sang QX Identification and characterization of human endometase (matrix metalloproteinase-26) from endometrial tumor. J Biol Chem 2000;275:20540-4.[Abstract/Free Full Text]
  4. Uria JA, Lopez-Otin C Matrilysin-2, a new matrix metalloproteinase expressed in human tumors and showing the minimal domain organization required for secretion, latency, and activity. Cancer Res 2000;60:4745-51.[Abstract/Free Full Text]
  5. Park HI, Turk BE, Gerkema FE, Cantley LC, Sang QX Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26. J Biol Chem 2002;277:35168-75.[Abstract/Free Full Text]
  6. Marchenko ND, Marchenko GN, Strongin AY Unconventional activation mechanisms of MMP-26, a human matrix metalloproteinase with a unique PHCGXXD cysteine-switch motif. J Biol Chem 2002;277:18967-72.[Abstract/Free Full Text]
  7. Marchenko ND, Marchenko GN, Weinreb RN, et al ß-Catenin regulates the gene of MMP-26, a novel metalloproteinase expressed both in carcinomas and normal epithelial cells. Int J Biochem Cell Biol 2004;36:942-56.[CrossRef][Medline]
  8. Zhao YG, Xiao AZ, Newcomer RG, et al Activation of pro-gelatinase B by endometase/matrilysin-2 promotes invasion of human prostate cancer cells. J Biol Chem 2003;278:15056-64.[Abstract/Free Full Text]
  9. Bar-Or A, Nuttall RK, Duddy M, et al Analyses of all matrix metalloproteinase members in leukocytes emphasize monocytes as major inflammatory mediators in multiple sclerosis. Brain 2003;126:2738-49.[Abstract/Free Full Text]
  10. Marchenko GN, Marchenko ND, Leng J, Strongin AY Promoter characterization of the novel human matrix metalloproteinase-26 gene: regulation by the T-cell factor-4 implies specific expression of the gene in cancer cells of epithelial origin. Biochem J 2002;363:253-62.[CrossRef][Medline]
  11. Park HI, Jin Y, Hurst DR, et al The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling. J Biol Chem 2003;278:51646-53.[Abstract/Free Full Text]
  12. Zhang J, Cao YJ, Zhao YG, Sang QX, Duan EK Expression of matrix metalloproteinase-26 and tissue inhibitor of metalloproteinase-4 in human normal cytotrophoblast cells and a choriocarcinoma cell line, JEG-3. Mol Hum Reprod 2002;8:659-66.[Abstract/Free Full Text]
  13. Zhao YG, Xiao AZ, Park HI, et al Endometase/matrilysin-2 in human breast ductal carcinoma in situ and its inhibition by tissue inhibitors of metalloproteinases-2 and -4: a putative role in the initiation of breast cancer invasion. Cancer Res 2004;64:590-8.[Abstract/Free Full Text]
  14. Isaka K, Nishi H, Nakai H, et al Matrix metalloproteinase-26 is expressed in human endometrium but not in endometrial carcinoma. Cancer 2003;97:79-89.[CrossRef][Medline]
  15. Tunuguntla R, Ripley D, Sang QX, Chegini N Expression of matrix metalloproteinase-26 and tissue inhibitors of metalloproteinases TIMP-3 and -4 in benign endometrium and endometrial cancer. Gynecol Oncol 2003;89:453-9.[CrossRef][Medline]
  16. Coakley RJ, Taggart C, O’Neill S, McElvaney NG {alpha}1-Antitrypsin deficiency: biological answers to clinical questions. Am J Med Sci 2001;321:33-41.[CrossRef][Medline]
  17. Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 1995;270:5331-8.[Abstract/Free Full Text]
  18. Ratnikov B, Deryugina E, Leng J, Marchenko G, Dembrow D, Strongin A Determination of matrix metalloproteinase activity using biotinylated gelatin. Anal Biochem 2000;286:149-55.[CrossRef][Medline]
  19. Kridel SJ, Chen E, Kotra LP, Howard EW, Mobashery S, Smith JW Substrate hydrolysis by matrix metalloproteinase-9. J Biol Chem 2001;276:20572-8.[Abstract/Free Full Text]
  20. Krajewska M, Zapata JM, Meinhold-Heerlein I, et al Expression of Bcl-2 family member Bid in normal and malignant tissues. Neoplasia 2002;4:129-40.[CrossRef][Medline]
  21. Weinstein JN, Myers TG, O’Connor PM, et al An information-intensive approach to the molecular pharmacology of cancer. Science 1997;275:343-9.[Abstract/Free Full Text]
  22. Krajewski S, Krajewska M, Shabaik A, Miyashita T, Wang HG, Reed JC Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2. Am J Pathol 1994;145:1323-36.[Abstract]
  23. Liu Z, Zhou X, Shapiro SD, et al The serpin {alpha}1-proteinase inhibitor is a critical substrate for gelatinase B/MMP-9 in vivo. Cell 2000;102:647-55.[CrossRef][Medline]
  24. Will H, Atkinson SJ, Butler GS, Smith B, Murphy G The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3. J Biol Chem 1996;271:17119-23.[Abstract/Free Full Text]
  25. Tam EM, Wu YI, Butler GS, Stack MS, Overall CM Collagen binding properties of the membrane type-1 matrix metalloproteinase (MT1-MMP) hemopexin C domain: the ectodomain of the 44-kDa autocatalytic product of MT1-MMP inhibits cell invasion by disrupting native type I collagen cleavage. J Biol Chem 2002;277:39005-14.[Abstract/Free Full Text]
  26. Keppler D, Markert M, Carnal B, Berdoz J, Bamat J, Sordat B Human colon carcinoma cells synthesize and secrete {alpha}1-proteinase inhibitor. Biol Chem Hoppe Seyler 1996;377:301-11.[Medline]
  27. Pernick NL, DaSilva M, Gangi MD, Crissman J, Adsay V "Histiocytic markers" in melanoma. Mod Pathol 1999;12:1072-7.[Medline]
  28. Somiari RI, Sullivan A, Russell S, et al High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast. Proteomics 2003;3:1863-73.[CrossRef][Medline]
  29. Sun Z, Yang P Role of imbalance between neutrophil elastase and {alpha}1-antitrypsin in cancer development and progression. Lancet Oncol 2004;5:182-90.[CrossRef][Medline]
  30. Mast AE, Enghild JJ, Nagase H, Suzuki K, Pizzo SV, Salvesen G Kinetics and physiologic relevance of the inactivation of {alpha}1-proteinase inhibitor, {alpha}1-antichymotrypsin, and antithrombin III by matrix metalloproteinases-1 (tissue collagenase), -2 (72-kDa gelatinase/type IV collagenase), and -3 (stromelysin). J Biol Chem 1991;266:15810-6.[Abstract/Free Full Text]
  31. Sires UI, Murphy G, Baragi VM, Fliszar CJ, Welgus HG, Senior RM Matrilysin is much more efficient than other matrix metalloproteinases in the proteolytic inactivation of {alpha}1-antitrypsin. Biochem Biophys Res Commun 1994;204:613-20.[CrossRef][Medline]
  32. Zhang Z, Winyard PG, Chidwick K, et al Proteolysis of human native and oxidised {alpha}1-proteinase inhibitor by matrilysin and stromelysin. Biochim Biophys Acta 1994;1199:224-8.[Medline]
  33. Goffin F, Munaut C, Frankenne F, et al Expression pattern of metalloproteinases and tissue inhibitors of matrix-metalloproteinases in cycling human endometrium. Biol Reprod 2003;69:976-84.[Abstract/Free Full Text]
  34. Li Q, Wang H, Zhao Y, Lin H, Sang QA, Zhu C Identification and specific expression of matrix metalloproteinase-26 in rhesus monkey endometrium during early pregnancy. Mol Hum Reprod 2002;8:934-40.[Abstract/Free Full Text]
  35. Pilka R, Whatling C, Domanski H, Hansson S, Eriksson P, Casslen B Epithelial expression of matrix metalloproteinase-26 is elevated at mid-cycle in the human endometrium. Mol Hum Reprod 2003;9:271-7.[Abstract/Free Full Text]
  36. Barbour KW, Goodwin RL, Guillonneau F, Wang Y, Baumann H, Berger FG Functional diversification during evolution of the murine {alpha}(1)-proteinase inhibitor family: role of the hypervariable reactive center loop. Mol Biol Evol 2002;19:718-27.[Abstract/Free Full Text]
  37. Forsyth S, Horvath A, Coughlin P A review and comparison of the murine {alpha}1-antitrypsin and {alpha}1-antichymotrypsin multigene clusters with the human clade A serpins. Genomics 2003;81:336-45.[CrossRef][Medline]
  38. Pilka R, Domanski H, Hansson S, Eriksson P, Casslen B Endometrial TIMP-4 mRNA is high at midcycle and in hyperplasia, but down-regulated in malignant tumours: coordinated expression with MMP-26. Mol Hum Reprod 2004;10:641-50.[Abstract/Free Full Text]
  39. Pilka R, Norata GD, Domanski H, et al Matrix metalloproteinase-26 (matrilysin-2) expression is high in endometrial hyperplasia and decreases with loss of histological differentiation in endometrial cancer. Gynecol Oncol 2004;94:661-70.[CrossRef][Medline]
  40. Impola U, Uitto VJ, Hietanen J, et al Differential expression of matrilysin-1 (MMP-7), 92 kD gelatinase (MMP-9), and metalloelastase (MMP-12) in oral verrucous and squamous cell cancer. J Pathol 2004;202:14-22.[CrossRef][Medline]
  41. van Dijk MA, Floore AN, Kloppenborg KI, van ’t Veer LJ A functional assay in yeast for the human estrogen receptor displays wild-type and variant estrogen receptor messenger RNAs present in breast carcinoma. Cancer Res 1997;57:3478-85.[Abstract/Free Full Text]
  42. Couillard S, Gutman M, Labrie C, Belanger A, Candas B, Labrie F Comparison of the effects of the antiestrogens EM-800 and tamoxifen on the growth of human breast ZR-75–1 cancer xenografts in nude mice. Cancer Res 1998;58:60-4.[Abstract/Free Full Text]
  43. Liu ZJ, Zhu BT Concentration-dependent mitogenic and antiproliferative actions of 2-methoxyestradiol in estrogen receptor-positive human breast cancer cells. J Steroid Biochem Mol Biol 2004;88:265-75.[CrossRef][Medline]
  44. Keen JC, Yan L, Mack KM, et al A novel histone deacetylase inhibitor, scriptaid, enhances expression of functional estrogen receptor {alpha} (ER) in ER negative human breast cancer cells in combination with 5-aza 2'-deoxycytidine. Breast Cancer Res Treat 2003;81:177-86.[CrossRef][Medline]
  45. Jang ER, Lim SJ, Lee ES, et al The histone deacetylase inhibitor trichostatin A sensitizes estrogen receptor {alpha}-negative breast cancer cells to tamoxifen. Oncogene 2004;23:1724-36.[CrossRef][Medline]
  46. Vladusic EA, Hornby AE, Guerra-Vladusic FK, Lakins J, Lupu R Expression and regulation of estrogen receptor ß in human breast tumors and cell lines. Oncol Rep 2000;7:157-67.[Medline]
  47. Weinstein-Oppenheimer CR, Burrows C, Steelman LS, McCubrey JA The effects of ß-estradiol on Raf activity, cell cycle progression and growth factor synthesis in the MCF-7 breast cancer cell line. Cancer Biol Ther 2002;1:256-62.[Medline]
  48. Bramlett KS, Burris TP Target specificity of selective estrogen receptor modulators within human endometrial cancer cells. J Steroid Biochem Mol Biol 2003;86:27-34.[CrossRef][Medline]
  49. Kammerer U, Thanner F, Kapp M, Dietl J, Sutterlin M Expression of tumor markers on breast and ovarian cancer cell lines. Anticancer Res 2003;23:1051-5.[Medline]
  50. Kang SK, Choi KC, Tai CJ, Auersperg N, Leung PC Estradiol regulates gonadotropin-releasing hormone (GnRH) and its receptor gene expression and antagonizes the growth inhibitory effects of GnRH in human ovarian surface epithelial and ovarian cancer cells. Endocrinology 2001;142:580-8.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Hum ReprodHome page
R. Pilka, I. Oborna, V. Lichnovsky, P. Havelka, H. Fingerova, P. Eriksson, S. Hansson, and B. Casslen
Endometrial expression of the estrogen-sensitive genes MMP-26 and TIMP-4 is altered by a substitution protocol without down-regulation in IVF patients
Hum. Reprod., December 1, 2006; 21(12): 3146 - 3156.
[Abstract] [Full Text] [PDF]

Home page
 Ann OncolHome page
O. Graesslin, A. Cortez, R. Fauvet, M. Lorenzato, P. Birembaut, and E. Darai
Metalloproteinase-2, -7 and -9 and tissue inhibitor of metalloproteinase-1 and -2 expression in normal, hyperplastic and neoplastic endometrium: a clinical-pathological correlation study
Ann. Onc., April 1, 2006; 17(4): 637 - 645.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
A. Y. Savinov, A. G. Remacle, V. S. Golubkov, M. Krajewska, S. Kennedy, M. J. Duffy, D. V. Rozanov, S. Krajewski, and A. Y. Strongin
Matrix Metalloproteinase 26 Proteolysis of the NH2-Terminal Domain of the Estrogen Receptor {beta} Correlates with the Survival of Breast Cancer Patients.
Cancer Res., March 1, 2006; 66(5): 2716 - 2724.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted