Constance E. Brinckerhoff

Signal transduction and cell‐type specific regulation of matrix metalloproteinase gene expression: Can MMPs be good for you?

An abundance of literature over the past several years indicates a growing interest in the role of matrix metalloproteinases (MMPs) in normal physiology and in disease pathology. MMPs were originally defined by their ability to degrade the extracellular matrix, but it is now well documented that their substrates extend far beyond matrix components. Recent reviews discuss the structure and function of the MMP family members, as well as the promoter sequences that control gene expression. Thus, we focus on the signal transduction pathways that confer differential cell‐type expression of MMPs, as well as on some novel non‐matrix degrading functions of MMPs, particularly their intracellular location where they may contribute to apoptosis. In addition, increasing data implicate MMPs as “good guys”, protective agents in some cancers and in helping to resolve acute pathologic conditions. Despite the intricate and complicated roles of MMPs in physiology and pathology, the goal of designing therapeutics that can selectively target MMPs remains a major focus. Developing MMP inhibitors with targeted specificity will be difficult; success will depend on understanding the role of these enzymes in homeostasis and on the careful delineation of mechanisms by which this family of enzymes mediates disease pathology. J. Cell. Physiol. 213: 355–364, 2007.

Modulation by retinoic acid and corticosteroids of collagenase production by rabbit synovial fibroblasts treated with phorbol myristate acetate or poly(ethylene glycol)

Abstract Monolayer cultures of rabbit synovial fibroblasts can be stimulated experimentally to produce large quantities of latent collagenase by treatment with phorbol myristate acetate (PMA) or by formation of multinucleated giant cells with poly(ethylene glycol). We studied the ability of all-trans-retonoic acid and/or corticosteroids to modulate collagenase production induced experimentally. Concomitant addition of PMA along with retinoic acid (10−6 M) inhibited production of collagenase by 60–100%. 10−7 M retinoic acid was partially effective, reducing collagenase by 20%. Addition of dexamethasone (10−7 M) to cultures gave results similar to retinoic acid at 10−6 M. Inhibition of collagenase synthesis was confirmed by SDS-polycrylamide gel electrophoresis of medium from cultures treated with PMA, or with PMA and retinoic or dexamethasone. Addition of both retinoic acid and prednisolone to cultures reduced collagenase production more effectively than either drug alone; both drugs at 10t−10 M for 2 days lowered collagenase levels to 50% of control, while each drug alone at 10−10 M had no effect. Addition of retinoic acid (10−6 M) to cultures containing multinucleated giant cells lowered collagenase production by 70–85%. Removal of the drug resulted in full recovery. Our data indicate that all-trans-retinoic acid inhibited collagenase production induced by PMA or poly(ethylene glucol) and that this inhibition was additive with corticosteroids.

Short Hairpin RNA-Mediated Inhibition of Matrix Metalloproteinase-1 in MDA-231 Cells: Effects on Matrix Destruction and Tumor Growth

Increased matrix metalloproteinase-1 (MMP-1) expression is associated with advanced stages of breast cancer and may be a predictive marker for the development of invasive disease. In this report, we used short hairpin RNA (shRNA) molecules to investigate whether MMP-1 production in MDA-231 breast cancer cells contributed to the degradation of a collagen matrix or tumor formation in nude mice. We created two groups of MDA-231 cell lines. MDA-231 cells containing a vector producing shRNA specific for MMP-1 had a >90% decrease in MMP-1 mRNA and protein compared with cells containing an empty vector, and blocking MMP-1 expression inhibited the in vitro collagenolytic activity of MDA-231 cells. When the cells were injected into the mammary fat pad, there was no difference in the frequency of tumor formation in mice. However, the average tumor size was larger in mice injected with cells containing the empty vector (1,216 +/- 334 mm3) than in mice injected with cells expressing the MMP-1 shRNA (272 +/- 117 mm3; P = 0.027). We conclude that MMP-1 expression is essential for the ability of MDA-231 cells to invade and destroy a collagen matrix and in vivo experiments suggest an important role for MMP-1 in breast tumor growth.

Loss of Heterozygosity on Chromosome 11q22-23 in Melanoma Is Associated with Retention of the Insertion Polymorphism in the Matrix Metalloproteinase-1 Promoter

Matrix metalloproteinase-1 (MMP-1, collagenase-1), which degrades interstitial collagen, is expressed at high levels by some tumor cells and is thought to enhance their invasiveness and metastatic potential. We recently described a common single nucleotide insertion polymorphism (2G allele) at -1,607 bp in the promoter of the MMP-1 gene that creates a binding site for the ETS family of transcription factors, and that is associated with enhanced transcription of this gene and increased enzyme activity. Allelic loss at the MMP-1 locus on chromosome 11 occurs in many tumors including melanoma, an invasive and aggressive cancer. We hypothesized that although loss of either the 1G or 2G allele from 1G/2G heterozygotes is random, retention of the transcriptionally more active 2G allele would favor tumor invasion and metastasis. As a result, a higher proportion of metastases would contain the 2G genotype than the 1G genotype. We report here the development of quantitative methods for assessing allelic loss at the MMP-1 locus, and demonstrate that 83% of the metastatic melanomas with loss of heterozygosity at this locus retained the 2G allele. This supports the hypothesis that retention of the 2G allele favors tumor invasion and metastasis in melanoma.

High levels of MMP‐1 expression in the absence of the 2G single nucleotide polymorphism is mediated by p38 and ERK1/2 mitogen‐activated protein kinases in VMM5 melanoma cells

Matrix metalloproteinase‐1 (MMP‐1) is one of only a few enzymes with the ability to degrade the stromal collagens (types I and III) at neutral pH, and high expression of MMP‐1 has been associated with aggressive and invasive cancers. We recently reported a single nucleotide insertion/deletion polymorphism (SNP) in the collagenase‐1 (MMP‐1) promoter (Rutter et al. [ 1998 ] Can. Res. 58:5321–5325), where the insertion of an extra guanine (G) at −1607 bp creates the sequence, 5′‐GGAA‐3 (2G allele), compared to the sequence 5′‐GAA‐3′ (1G allele). The presence of 2G constitutes a binding site for the ETS family of transcription factors, and increases MMP‐1 transcription in fibroblasts and A2058 melanoma cells cultured in vitro. In addition, the presence of the 2G allele has been linked to several aggressive malignancies as well as to enhanced expression of MMP‐1. In this study, we describe a melanoma cell line, VMM5, that is 1G homozygous, but that is invasive and expresses high levels of MMP‐1 constitutively. The high level of MMP‐1 expression in VMM5 cells is due to the utilization of both the p38 and ERK1/2 transduction pathways. In contrast, in the A2058 cell line, which also expresses MMP‐1 constitutively and which is 2G homozygous, only the ERK pathway is activated. Thus, our data suggest that in the absence of 2G allele and in the presence of the appropriate transcription factors, tumor cells may use alternative signal/transduction pathways and cis‐acting sequences to achieve high levels of MMP‐1 expression, which contribute to the ability of tumor cells to invade, regardless of their genotype.

Dose-dependent suppression by the synthetic retinoid, 4-hydroxyphenyl retinamide, of streptococcal cell wall-induced arthritis in rats

We studied the effects of oral administration of the retinoid, 4-hydroxyphenyl retinamide (4-HPR), on group A streptococcal cell wall-induced polyarthritis in the rat, a model characterized initially by exudative inflammation of peripheral joints followed by chronic proliferative/erosive synovitis. Experimental arthritis was induced in female LEW/N rats by i.p. injection of streptococcal cell walls in saline (15 micrograms/g body weight). Depending upon the experiment, continuous daily oral administration of the retinoid was begun either 14 days prior to induction of the disease, at the time of cell wall administration and/or 11 days and 31 days after cell wall injection. Dosage was either 1 or 2 mmol 4-HPR/kg of chow. During the course of the disease, severity of clinical illness was assessed by determination of clinical severity index, by histological or radiologic examination, and by measurement of production in vitro of collagenase and prostaglandin E2 by excised synovial tissue. In rats fed the retinoid prior to cell wall injection, both the acute and the chronic responses were suppressed. In rats given the retinoid at the time of cell wall injection, the acute inflammatory response was only partially suppressed on the diet containing 2 mmol 4-HPR/kg chow, but the chronic disease was impressively inhibited in a dose dependent manner. Similarly, in animals with established disease, the drug was also effective; however, the more advanced the illness, the less effective the drug. Clinical observations were paralleled by the histological, radiographical and biochemical analyses. Treated animals showed far less synovial proliferation and joint destruction, and synovial tissues taken from these rats produced lesser amounts of collagenase and prostaglandin E2. No significant toxicity of the retinoid was noted. We conclude that oral administration of 4-HPR suppresses, in a dose and time dependent manner, both the acute and chronic stages of streptococcal cell wall-induced arthritis in rats without apparent significant toxicity. Our data suggest that studies of the effects of this retinoid on patients with chronic inflammatory synovitis are warranted.

Effects of all-trans-retinoic acid (retinoic acid) and 4-hydroxyphenylretinamide on synovial cells and articular cartilage

We studied the effects of two retinoids, naturally occurring all-trans-retinoic acid (retinoic acid) and the synthetic 4-hydroxyphenylretinamide (4-OH-PRT) on monolayer cultures of rabbit synovial fibroblasts and on explants of rabbit articular cartilage. Treatment of fibroblasts with phorbol myristate acetate (PMA; 10(-8) M) induced the synthesis and secretion of large amounts of collagenase: this was inhibited if the cells were treated with retinoic acid (10(-6) M) or dexamethasone (10(-7 M). Combined treatment with retinoic acid and the steroid prednisolone, at concentrations as low as 19(-10) M, gave an additive inhibition of collagenase production. Both retinoids inhibited collagenase production, but only 4-OH-PRT prevented the increase in prostaglandin E2 (PGE2) induced by PMA. Levels of plasminogen activator were also increased by treatment with PMA, and concomitant addition of either retinoid further enhanced this stimulation. Possible toxicity was assessed by measuring release of glycosaminoglycans (GAG) from explants of articular cartilage. Treatment with retinoic acid induced release of 80% of the total GAG, whereas treatment with 4-OH-PRT resulted in release of 40% of the total, a finding similar to that seen with untreated samples. 4-OH-PRT inhibited production of collagenase and PGE2 by rabbit synovial fibroblasts but was not toxic to articular cartilage.

Site controlled transgenic mice validating increased expression from human matrix metalloproteinase (MMP-1) promoter due to a naturally occurring SNP.

Matrix metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the bodys most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at -1607 bp, which creates the sequence 5-GGAA-3(2G allele), and which is an ETS binding site. Compared to the 1G allele (5-GAA-3), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity. Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with -4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the lac Z (E.coli ss-galactosidase) gene. We measured the relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ss-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.

Analysis of Matrix Metalloproteinase-1 Gene Polymorphisms and Expression in Benign and Malignant Breast Tumors

A guanine insertion polymorphism in matrix metalloproteinase-1 promoter (MMP-1 2G) is linked to early onset and aggressiveness in cancer. We determined the role of MMP-1 2G on MMP-1 expression and breast cancer severity in patients with breast diseases. We observed no significant difference in genotype distribution among different disease groups. However, MMP-1 expression was significantly higher in atypical ductal hyperplasia than in benign breast disease and in invasive breast cancer compared to in situ breast cancer. MMP-1 2G insertion polymorphism in the invasive group also correlated significantly with the expression of MMP-1 and breast cancer prognostic markers HER2 and P53.

Abstract P4-07-09: The Impact of Matrix Metalloproteinase-1 Promoter 1G/2G Polymorphism on Breast Diseases

Background: Matrix Metalloproteinase-1 (MMP-1) is a ubiquitously expressed interstitial collagenase. Overexpression of MMP-1 has a role in initiating mammary tumorigenesis by degrading stroma and by releasing growth factors. A single guanine insertion polymorphism in the MMP-1 promoter creates the binding site, 59-GGAA-39, for the Ets transcription factor, and increases transcription of MMP-1. The MMP-1 2G polymorphism is linked to early onset, increased risk or aggressiveness of several cancers. Its relationship with other potential markers in invasion and metastasis of breast cancer is unknown. Experimental Design: To study the impact of the 2G polymorphism on breast cancer we analyzed the genotypes of 109 patients (52 invasive breast cancer [IBC], 29 ductal carcinoma in situ [DCIS], 13 atypical ductal hyperplasia [ADH] and 15 benign breast disease). Immunohistochemical (IHC) data for MMP-1, HER2, ER/PR and P53 from these donors were also analyzed. IHC results for MMP-1 were scored as 0 (no expression) or increasing expression of+1, +2 or +3. Data were analyzed using Pearson9s chi-square test to identify statistical significance. Results: A significantly higher number of patients in the IBC group expressed high MMP-1 (+2 and +3; p Conclusions: 1) In the IBC group, the 2G insertion polymorphism contributes to MMP-1 over expression. 2) Increased expression of MMP-1 in ADH and higher 2G allele frequency are consistent with the hypothesis that increased MMP-1 2G polymorphism plays a role in initiation of ADH through up regulation of MMP-1 expression. 3) Earlier studies show prognostic role for the coexistence of increased expression of HER2 and P53 in breast cancer. Our observation of a significant increase in the 2G homozygotes in HER2 and P53 positive patients supports a prognostic role for this polymorphism and suggests its possible association with other breast cancer markers. Thus, the MMP-1 2G polymorphism may both contribute to breast disease onset and serve as a prognostic marker for breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-07-09.