Matthew P. Vincenti

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.

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.

A synthetic triterpenoid selectively inhibits the induction of matrix metalloproteinases 1 and 13 by inflammatory cytokines

OBJECTIVE To address the effects of a novel synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), on the induction of matrix metalloproteinases 1 and 13 (MMP-1, MMP-13) by inflammatory cytokines. METHODS Human chondrosarcoma cells stimulated with inflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha) were used to study the effects of CDDO on the induction of MMPs and the invasion of cells through a collagen matrix. RESULTS CDDO selectively reduced the induction of MMP-1 and MMP-13 at the levels of messenger RNA and protein. Treatment with CDDO prior to cytokine stimulation enhanced this inhibition, and we demonstrated that CDDO functions at the level of transcription. Additionally, CDDO reduced IL-1beta-mediated invasion of cells through a collagen matrix. CONCLUSION This study demonstrates that CDDO is a novel inhibitor of MMP-1 and MMP-13 gene expression mediated by inflammatory cytokines. Thus, CDDO may have therapeutic potential for the inhibition of joint degradation in osteoarthritis.

The potential of signal transduction inhibitors for the treatment of arthritis: is it all just JNK?

Rheumatoid arthritis (RA) is a connective tissue disease that affects more than 1,000,000 people in the US (1). This autoimmune disorder is driven largely by the recruitment of activated immune cells (T and B cells) and macrophages to the afflicted joints. There, the cytokines IL-1 and TNF, which are produced by these cells, mediate the irreversible joint destruction seen in RA (2). The downstream genes activated by these cytokines encode both inflammatory molecules and secreted proteinases of the matrix metalloproteinase (MMP) family (3). The interstitial collagenases (collagenase-1, MMP-1; collagenase-2, MMP-8; collagenase-3, MMP-13; MT1-MMP, MMP-14) act at a committed step in the progression of RA, degrading type II collagen in cartilage (1, 3). Of these enzymes, particular attention has focused on MMP-1 and MMP-13 because they are induced in response to IL-1 and TNF and are found at elevated levels in RA (4). Therefore, inhibition of cytokine-induced MMP-1 and MMP-13 gene expression has been a prime target for the development of new arthritis drugs. Earlier studies identified sequences in the promoters of these two MMP genes that mediate the increase in gene expression, including a proximal AP-1 site that binds members of the Fos and Jun families of transcription factors (5, 6), and several upstream sequences such as an NF-κB–like element (7) and a Cbfa1/OSF2/RUNX-2 (RUNX-2) binding site (8). Mutational and deletional analysis has indicated that these upstream sites cooperate with the proximal AP-1 site in order to drive transcription (7, 9, 10). Further, although both MMP-1 and MMP-13 expression increases with cytokine stimulation, this increase is somewhat cell type–restricted and differs somewhat between the two genes. The restricted expression of MMP-13 in osteoblasts and chondrocytes may reflect the predominant expression in these cells of RUNX-2, which activates the MMP13 but not the MMP1 promoter (8, 11) (J.A. Mengshol and C.E. Brinckerhoff, unpublished work). In contrast, cytokines induce MMP-1 expression in virtually all human connective tissue cells (4), clearly indicating that the mechanisms controlling expression of these two collagenases differ. Recent studies have begun to analyze signal transduction pathways for their possible role in the transcriptional activation of MMP-1 and MMP-13 in cells associated with arthritic disease. The enzymes that facilitate these pathways are termed the mitogen-activated protein kinases (MAPKs) (12) and include the c-Jun N-terminal kinases (JNKs), the extracellular signal–regulated kinases (ERKs), and the p38 kinases. The availability of chemical compounds that can specifically block one or another signal transduction pathway has greatly aided these studies (Figure ​(Figure1).1). In a recent issue of the JCI, Han et al. (13) in the laboratory of Gary Firestein describe the ability of a new inhibitor, SP600125, to specifically block JNK, thus providing a tool to investigate the role of this kinase in MMP gene expression and in the pathophysiology of RA. They show that SP600125 completely blocks IL-1–induced expression of c-Jun and collagenase mRNAs and prevents the accumulation of phospho-Jun in cultured synovial cells. The in vitro binding of nuclear proteins to an AP-1–containing DNA fragment is likewise severely inhibited, consistent with evidence that this cis-acting sequence is a major target of IL-1 induction, apparently as a result of JNK activation. Figure 1 IL-1 activation of MAPK pathways. IL-1 binds to its cellular receptor and activates receptor-associated proteins (red), such as IRAK, TRAF6, and TAK1. The result is activation of several MAPK kinases (MKK6, MKK4, MKK7, and MEK1; green), which phosphorylate ... These results may suggest a central role for the pathway, at least in cultured cells, but the data are slightly less clear-cut in the animal studies, since the authors observe incomplete silencing of collagenase mRNA induction and only a partial inhibition of joint destruction in SP600125-treated rats. Other pathways may contribute to this pathological process. Alternatively, other cells, in addition to synovial fibroblasts, or other proteinases, in addition to the well studied MMPs, may be involved joint destruction.

Interleukin-1 beta induction of matrix metalloproteinase-1 transcription in chondrocytes requires ERK-dependent activation of CCAAT enhancer-binding protein-beta

Interleukin‐1 beta (IL‐1β) is a central mediator of inflammation and connective tissue destruction in rheumatoid arthritis. IL‐1β activates articular chondrocytes to produce matrix metalloproteinase‐1 (MMP‐1), an enzyme capable of dismantling the collagen scaffold of articular cartilage. To define the transcription factors and signaling intermediates that activate MMP‐1 transcription in chondrocytes, we performed transient transfection of MMP‐1 promoter constructs followed by reporter assays. These studies identified an IL‐1β‐responsive region of the human MMP‐1 promoter that contains a consensus CCAAT enhancer‐binding protein (C/EBP) binding site. Deletion of this site reduced overall transcriptional activity of the MMP‐1 promoter, as well as decreased fold induction by IL‐1β. IL‐1β stimulation of chondrocytes increased binding of C/EBP‐β to the MMP‐1 C/EBP site. Extracellular signal regulated kinase (ERK) pathway‐dependent phosphorylation of C/EBP‐β on threonine 235 activates this transcription factor. Here we show that IL‐1β stimulation of chondrocytes induced phosphorylation of C/EBP‐β on threonine 235, and that the ERK pathway inhibitor PD98059 reduced this phosphorylation. We further show that PD98059 reduces IL‐1β‐induced MMP‐1 mRNA expression in chondrocytes. Moreover, inhibition of the ERK pathway by expression of dominant‐negative forms of ERK1 and ERK2 impaired the ability of IL‐1β to transactivate the MMP‐1 promoter. Our findings demonstrate a novel role for C/EBP‐β in IL‐1β‐induced connective tissue disease and define a new nuclear target for the ERK pathway in MMP‐1 gene activation. J. Cell. Physiol.

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.

CCAAT Enhancer Binding Protein-β Regulates Matrix Metalloproteinase-1 Expression in Interleukin-1β–Stimulated A549 Lung Carcinoma Cells

Matrix metalloproteinase-1 (MMP-1) is an inflammation-inducible neutral protease that mediates extracellular matrix remodeling and promotes tumor invasion. In this study, we examined the activation of MMP-1 gene expression in A549 lung carcinoma cells stimulated with the inflammatory cytokine interleukin-1β (IL-1β). We found that MMP-1 mRNA levels were maximal following 16 hours of IL-1β stimulation and that this correlated with the expression of the transcription factor CCAAT enhancer-binding protein-β (CEBPB). Knockdown of CEBPB expression with short hairpin RNA abrogated the expression of MMP-1, MMP-3, and MMP-10 in IL-1β–stimulated A549 cells. An established CEBP element in the MMP-1 promoter was found to be required for basal and IL-1β–induced transcription. Electrophoresis mobility shift assays showed that CEBPB binds to this promoter element maximally 16 hours after IL-1β stimulation. DNA affinity chromatography studies showed that the LAP1, LAP2, and LIP isoforms of CEBPB bind to the IL-1β–responsive CEBPB site in the MMP-1 promoter. Exogenous expression of the LAP1 and LAP2 isoforms stimulated the MMP-1 promoter, whereas LIP had no effect. Phosphorylation of CEBPB at Thr235 peaked at 16 hours in IL-1β–stimulated cells. The MEK inhibitor U0126 inhibited this phosphorylation and reduced MMP-1 gene induction. These studies establish CEBPB as an important mediator of metalloproteinase gene activation during inflammatory responses in lung cancer cells and highlight the different regulatory roles of CEBPB isoforms. (Mol Cancer Res 2009;7(9):1517–24)

Interleukin-1 beta and transforming growth factor-beta 3 cooperate to activate matrix metalloproteinase expression and invasiveness in A549 lung adenocarcinoma cells

Cytokines present in the tumor microenvironment can promote the invasiveness and metastatic potential of cancer cells. We therefore investigated the effects of interleukin-1 beta (IL-1B) and transforming growth factor beta-3 (TGFB3) on the non-small cell lung carcinoma (NSCLC) cell line A549. We found that these cytokines synergistically activated matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10 gene expression in these cells through mitogen-activated protein kinase (MAPK)-dependent pathways. Consistent with this, both cytokines stimulated epithelial to mesenchymal transition and MAPK-dependent invasion through Matrigel™. These studies identify IL-1B and TGFB3 as pro-invasive factors in NSCLC and potential therapeutic targets for tumor progression.

Interleukin‐1β mediates metalloproteinase‐dependent renal cell carcinoma tumor cell invasion through the activation of CCAAT enhancer binding protein β

Effective treatment of metastatic renal cell carcinoma (RCC) remains a major medical concern, as these tumors are refractory to standard therapies and prognosis is poor. Although molecularly targeted therapies have shown some promise in the treatment of this disease, advanced RCC tumors often develop resistance to these drugs. Dissecting the molecular mechanisms underlying the progression to advanced disease is necessary to design alternative and improved treatment strategies. Tumor‐associated macrophages (TAMs) found in aggressive RCC tumors produce a variety of inflammatory cytokines, including interleukin‐1β (IL‐1β). Moreover, the presence of TAMs and high serum levels of IL‐1β in RCC patients correlate with advanced disease. We hypothesized that IL‐1β in the tumor microenvironment promotes the development of aggressive RCC tumors by directing affecting tumor epithelial cells. To address this, we investigated the role of IL‐1β in mediating RCC tumor cell invasion as a measure of tumor progression. We report that IL‐1β induced tumor cell invasion of RCC cells through a process that was dependent on the activity of matrix metalloproteinases (MMPs) and was independent of migration rate. Specifically, IL‐1β induced the expression of MMP‐1, MMP‐3, MMP‐10, and MT1‐MMP in a mechanism dependent on IL‐1β activation of the transcription factor CCAAT enhancer binding protein β (CEBPβ). Consistent with its role in MMP gene expression, CEBPβ knockdown significantly reduced invasion, but not migration, of RCC tumor cells. These results identify the IL‐1β /CEBPβ/MMP pathway as a putative target in the design of anti‐metastatic therapies for the treatment of advanced RCC.