Charles I. Coon

Interleukin‐1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c‐jun N‐terminal kinase, and nuclear factor κB: Differential regulation of collagenase 1 and collagenase 3

OBJECTIVE To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. METHODS The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. RESULTS We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. CONCLUSION These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

Nuclear factor κB/p50 activates an element in the distal matrix metalloproteinase 1 promoter in interleukin-1β-stimulated synovial fibroblasts

OBJECTIVE To determine how interleukin-1 (IL-1), through activation of collagenase 1 (matrix metalloproteinase 1 [MMP-1]) transcription in synovial fibroblasts, contributes to cartilage degradation in rheumatoid arthritis. METHODS Primary rabbit synovial fibroblasts were transiently transfected with MMP-1 promoter/ luciferase constructs, and promoter activity in response to IL-1 was assessed. A minimal IL-1-response element was defined and used to evaluate DNA binding proteins by electrophoretic mobility shift assay and in situ ultraviolet crosslinking assay. RESULTS Transcriptional activation of the MMP-1 gene by IL-1 in rabbit synovial fibroblasts required a dorsal-like element, which was located at nucleotide (nt) -3,029, as well as an activator protein 1 site at nt -77. Importantly, an IL-1-induced DNA binding activity that was specific for the dorsal-like element contained the p50 subunit of nuclear factor kappaB (NF-kappaB). CONCLUSION These studies demonstrate, for the first time, a role for NF-kappaB in the induction of MMP-1, and suggest a mechanism of NF-kappaB-mediated cartilage degradation in rheumatoid arthritis.

Cell‐type specific regulation of human interstitial collagenase‐1 gene expression by interleukin‐1β (IL‐1β) in human fibroblasts and BC‐8701 breast cancer cells

Interleukin‐1β (IL‐1β) is a potent cytokine that stimulates interstitial collagenase‐1 (matrix metalloproteinase‐1; MMP‐1). In this study, we compared the mechanism(s) by which IL‐1β induces collagenase gene expression in two very different cells, normal human foreskin fibroblasts (HFFs) and an aggressive breast cancer cell line, BC‐8701 cells. Northern analysis showed that the time course of collagenase induction was distinct in the two cells: although both cells expressed low levels of MMP‐1 constitutively, addition of IL‐1β increased MMP‐1 mRNA in HFFs by 1 h and levels remained high over a 24‐h period. In contrast, MMP‐1 levels in IL‐1β‐treated BC‐8701 cells did not increase until 4 h, peaked by 12 h and then declined. To analyze the transcriptional response, we cloned and sequenced more than 4,300 bp of the human MMP‐1 promoter, and from this promoter clone, we prepared a series of 5′‐deletion constructs linked to the luciferase reporter and transiently transfected these constructs into both cell types to measure both basal and IL‐1β induced transcription. When both cell types were uninduced, promoter fragments containing less than 2,900 bp gave only a minimal transcriptional response, while larger fragments showed increased transcriptional activity. With IL‐1β treatment, significant responsiveness (P < 0.001) in HFFs was seen only with the larger fragments, while in the BC‐8701 cells, all fragments were significantly induced with IL‐1β. Finally, we found that IL‐1β stabilized MMP‐1 mRNA in normal fibroblasts, but not in BC‐8701 breast cancer cells. We conclude that both the transcriptional and post‐transcriptional regulation of MMP‐1 gene expression by IL‐1β is controlled by cell‐type specific mechanisms, and we suggest that IL‐1 induced MMP‐1 expression in tumor cells and in neighboring stromal cells may amplify the invasive ability of tumor cells. J. Cell. Biochem. 66:322–336, 1977.

RNA Interference Inhibition of Matrix Metalloproteinase-1 Prevents Melanoma Metastasis by Reducing Tumor Collagenase Activity and Angiogenesis

Melanoma incidence is increasing worldwide, and metastatic melanoma is almost completely resistant to every known therapy. New approaches to treating melanoma are urgently needed, and a greater understanding of the biology of melanoma invasion and metastasis will aid in their creation. A high proportion of invasive melanomas have a constitutively active Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascade; however, the downstream effectors of ERK signaling that contribute to melanoma invasion and metastasis are unknown. ERK signaling drives the production of the interstitial collagenase matrix metalloproteinase-1 (MMP-1), which is expressed specifically by invasive melanomas. Using short hairpin RNAs (shRNA) to knock down MMP-1 expression in a human melanoma cell line, we investigated the role of MMP-1 in melanoma metastasis in a xenograft model. Knockdown of MMP-1 had no effect on primary tumor growth, but reduction of MMP-1 expression significantly decreased the ability of the melanoma to metastasize from the orthotopic site in the dermis to the lung. Mechanistically, tumor cells expressing MMP-1 shRNAs had diminished collagenase activity, which is required for tumor cell invasion. Additionally, attenuation of MMP-1 expression reduced angiogenesis. These results show, for the first time, that targeted inhibition of MMP-1, a single effector of the Raf/MEK/ERK signaling cascade, prevents the progression of melanoma from a primary to metastatic tumor and, as such, may represent a useful therapeutic tool in controlling this disease.

A Matrix Metalloproteinase-1/Protease Activated Receptor-1 signaling axis promotes melanoma invasion and metastasis

Hallmarks of malignant melanoma are its propensity to metastasize and its resistance to treatment, giving patients with advanced disease a poor prognosis. The transition of melanoma from non-invasive radial growth phase (RGP) to invasive and metastatically competent vertical growth phase (VGP) is a major step in tumor progression, yet the mechanisms governing this transformation are unknown. Matrix metalloproteinase-1 (MMP-1) is highly expressed by VGP melanomas, and is thought to contribute to melanoma progression by degrading type I collagen within the skin to facilitate melanoma invasion. Protease activated receptor-1 (PAR-1) is activated by MMP-1, and is also expressed by VGP melanomas. However, the effects of MMP-1 signaling through PAR-1 have not been examined in melanoma. Here, we demonstrate that an MMP-1/PAR-1 signaling axis exists in VGP melanoma, and is necessary for melanoma invasion. Introduction of MMP-1 into RGP melanoma cells induced gene expression associated with tumor progression and promoted invasion in vitro, and enhanced tumor growth and conferred metastatic capability in vivo. This study demonstrates that both the type I collagenase and PAR-1 activating functions of MMP-1 are required for melanoma progression, and suggests that MMP-1 may be a major contributor to the transformation of melanoma from non-invasive to malignant disease.

The 2G Single Nucleotide Polymorphism (SNP) in the MMP-1 Promoter Contributes to High Levels of MMP-1 Transcription in MCF-7/ADR Breast Cancer Cells

Degradation of stromal collagens in the extracellular matrix is mediated largely by matrix metalloproteinase-1 (MMP-1; collagenase-1), and high constitutive levels of MMP-1 in breast cancer correlate with a poor prognosis and invasive disease. MMP-1 expression is, in part, controlledby the mitogen-activated protein kinase (MAPK) pathway(s), which may target several activator protein-1 (AP-1) and polyoma enhancing activity-3/E26 virus (PEA3/ETS) sites within the promoter. An additional ETS site in the MMP-1 promoter is conferred by a single nucleotide polymorphism (SNP) at −1607 bp, when two guanines (5‘-GGAT-3’; ‘2G allele/SNP’) are present instead of one guanine (5‘-GAT-3’; ‘1G allele/SNP’). This SNP is adjacent to an AP-1 site at −1602 bp, and in the presence of the 2G allele (ETS site), these sites cooperate to induce higher levels of transcription. ERK 1/2 is one component of the MAPK pathway and is constitutively active in MCF-7/ADR breast cancer cells, which are 1G/2G heterozygotes. This study demonstrates that when these cells are treated with PD098059, an ERK-specific inhibitor, MMP-1 mRNA levels are significantly decreased, suggesting that high constitutive expression of MMP-1 in these cells results from continuous ERK 1/2 activation. Using transient transfection, we determined that this signaling pathway targets different AP-1/ETS sites, depending upon which allele is present. Furthermore, in these cells, the AP-1 site at −1602 bp enhances transcription in the presence of the 2G SNP, but represses transcription from the 1G SNP. Finally, inhibiting ERK signaling and MMP-1 expression blocks type I collagen degradation and reduces the invasive ability of the MCF-7/ADR cells. We conclude that ERK 1/2 signaling and the 2G SNP mediate high levels of MMP-1 expression, which may contribute to the invasive potential of these breast cancer cells.

v-src activation of the collagenase-1 (matrix metalloproteinase-1) promoter through PEA3 and STAT: requirement of extracellular signal-regulated kinases and inhibition by retinoic acid receptors.

Collagenase‐1 (matrix metalloproteinase‐1 (MMP‐1)) degrades the extracellular matrix and enhances the invasive phenotype of tumor cells. v‐src activated MMP‐1 transcription through a series of elements in the proximal promoter, including the E2BP (nt‐172), polyoma virus enhancer A3 (PEA3) (nt‐94), activator protein‐1 (AP‐1) (nt‐72), and signal transducer and activator of transcription (STAT) (nt‐57) consensus sites. Of these sites, PEA3 and STAT contributed specifically to induction by v‐src, whereas the remaining elements were also involved in induction by the phorbol ester phorbol myristate acetate (PMA). However, in contrast to MMP‐1 induction by PMA, an AP‐1 site located at nt‐186 did not contribute to v‐src induction. These results suggest divergence of the tyrosine kinase– and protein kinase C–dependent pathways with respect to MMP‐1 transcription. v‐src induced MMP‐1 through mitogen‐activated protein kinases, with extracellular signal–regulated kinases playing a larger role than c‐jun N‐terminal kinase. Retinoic acid, which inhibits the progression of certain cancers, repressed v‐src–induced MMP‐1 transcription. Constitutive expression of retinoic acid receptors (RARs) α or β, but not γ, or of retinoid X receptor α, repressed v‐src–induced collagenase‐1 transcription. We concluded that oncogenic induction of MMP‐1 by v‐src depends on signaling pathways and cis‐acting sequences that are distinct from those involved in phorbol ester activation. Furthermore, v‐src induction of MMP‐1 may, by acting in concert with other genes, enhance matrix degradation and tumor progression, and retinoic acid and RARs may antagonize this induction in an RAR type–specific manner. Mol. Carcinog. 21:194–204, 1998.

Binding of [14C] azaserine to DNA and protein in the rat and hamster.

The binding of [14C] azaserine or its metabolites to DNA and protein in the organs of rats and hamsters was determined at various time after treatment with [14C] azaserine. The specific activity of 14C labelling of DNA and protein was determined. Rat liver DNA and protein were most extensively labelled at 90 min post-injection, but by 24 h the specific activity decreased to the levels found in pancreas and kidney. Thymus contained negligible amounts of radioactivity at all time-points. DNA and protein from hamster pancreas contained more label than did DNA and protein from rat pancreas. The results suggest that factors other than DNA binding play a role in determining the species and organ specificity of azaserine.