K. M. Faisal Khan

Targeting Prostaglandin E2 Receptors as an Alternative Strategy to Block Cyclooxygenase-2-dependent Extracellular Matrix-induced Matrix Metalloproteinase-9 Expression by Macrophages

COX-2-dependent prostaglandin (PG) E2 synthesis regulates macrophage MMP expression, which is thought to destabilize atherosclerotic plaques. However, the administration of selective COX-2 inhibitors paradoxically increases the frequency of adverse cardiovascular events potentially through the loss of anti-inflammatory prostanoids and/or disturbance in the balance of pro- and anti-thrombotic prostanoids. To avoid these collateral effects of COX-2 inhibition, a strategy to identify and block specific prostanoid-receptor interactions may be required. We previously reported that macrophage engagement of vascular extracellular matrix (ECM) triggers proteinase expression through a MAPKerk1/2-dependent increase in COX-2 expression and PGE2 synthesis. Here we demonstrate that elicited macrophages express the PGE2 receptors EP1–4. When plated on ECM, their expression of EP2 and EP4, receptors linked to PGE2-induced activation of adenylyl cyclase, is strongly stimulated. Forskolin and dibutryl cyclic-AMP stimulate macrophage matrix metalloproteinase (MMP)-9 expression in a dose-dependent manner. However, an EP2 agonist (butaprost) has no effect on MMP-9 expression, and macrophages from EP2 null mice exhibited enhanced COX-2 and MMP-9 expression when plated on ECM. In contrast, the EP4 agonist (PGE1-OH) stimulated macrophage MMP-9 expression, which was inhibited by the EP4 antagonist ONO-AE3-208. When compared with COX-2 silencing by small interfering RNA or inhibition by celecoxib, the EP4 antagonist was as effective in inhibiting ECM-induced proteinase expression. In addition, ECM-induced MMP-9 expression was blocked in macrophages in which EP4 was silenced by small interfering RNA. Thus, COX-2-dependent ECM-induced proteinase expression is effectively blocked by selective inhibition of EP4, a member of the PGE2 family of receptors.

Matrix Metalloproteinase (MMP)-1 and MMP-3 Induce Macrophage MMP-9: Evidence for the Role of TNF-α and Cyclooxygenase-2

Matrix metalloproteinase (MMP)-9 (gelatinase B) participates in a variety of diverse physiologic and pathologic processes. We recently characterized a cyclooxygenase-2 (COX-2)→PGE2→EP4 receptor axis that regulates macrophage MMP-9 expression. In the present studies, we determined whether MMPs, commonly found in inflamed and neoplastic tissues, regulate this prostanoid-EP receptor axis leading to enhanced MMP-9 expression. Results demonstrate that exposure of murine peritoneal macrophages and RAW264.7 macrophages to MMP-1 (collagenase-1) or MMP-3 (stromelysin-1) lead to a marked increase in COX-2 expression, PGE2 secretion, and subsequent induction of MMP-9 expression. Proteinase-induced MMP-9 expression was blocked in macrophages preincubated with the selective COX-2 inhibitor celecoxib or transfected with COX-2 small interfering RNA (siRNA). Likewise, proteinase-induced MMP-9 was blocked in macrophages preincubated with the EP4 antagonist ONO-AE3-208 or transfected with EP4 siRNA. Exposure of macrophages to MMP-1 and MMP-3 triggered the rapid release of TNF-α, which was blocked by MMP inhibitors. Furthermore, both COX-2 and MMP-9 expression were inhibited in macrophages preincubated with anti-TNF-α IgG or transfected with TNF-α siRNA. Thus, proteinase-induced MMP-9 expression by macrophages is dependent on the release of TNF-α, induction of COX-2 expression, and PGE2 engagement of EP4. The ability of MMP-1 and MMP-3 to regulate macrophage secretion of PGE2 and expression of MMP-9 defines a nexus between MMPs and prostanoids that is likely to play a role in the pathogenesis of chronic inflammatory diseases and cancer. These data also suggest that this nexus is targetable utilizing anti-TNF-α therapies and/or selective EP4 antagonists.

Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression.

Chronic inflammatory diseases are characterized by the persistent presence of macrophages and other mononuclear cells, tissue destruction, cell proliferation, and the deposition of extracellular matrix (ECM). The tissue degradation is mediated, in part, by enhanced proteinase expression by macrophages. It has been demonstrated recently that macrophage proteinase expression can be stimulated or inhibited by purified ECM components. However, in an intact ECM the biologically active domains of matrix components may be masked either by tertiary conformation or by complex association with other matrix molecules. In an effort to determine whether a complex ECM produced by vascular smooth muscle cells (SMC) regulates macrophage degradative phenotype, we prepared insoluble SMC matrices and examined their ability to regulate proteinase expression by RAW264.7 and thioglycollate-elicited peritoneal macrophages. Here we demonstrate that macrophage engagement of SMC-ECM triggers PKC-dependent activation of MAPKerk1/2 leading to increased expression of cyclooxygenase (COX)-2 and prostaglandin (PG) E2 synthesis. The addition of PGE2 to macrophage cultures stimulates their expression of both urokinase-type plasminogen activator and MMP-9, and the selective COX-2 inhibitor NS-398 blocks ECM-induced proteinase expression. Moreover, ECM-induced PGE2 and MMP-9 expression by elicited COX-2–/– macrophages is markedly reduced when compared with the response of either COX-2+/– or COX-2+/+ macrophages. These data clearly demonstrate that SMC-ECM exerts a regulatory role on the degradative phenotype of macrophages via enhanced urokinase-type plasminogen activator and MMP-9 expression, and identify COX-2 as a targetable component of the signaling pathway leading to increased proteinase expression.

IL-6–Mediated Induction of Matrix Metalloproteinase-9 Is Modulated by JAK-Dependent IL-10 Expression in Macrophages

The mechanisms by which IL-6 contributes to the pathogenesis of chronic inflammatory diseases and cancer are not fully understood. We previously reported that cyclooxygenase-2 (Cox-2)–dependent PGE2 synthesis regulates macrophage matrix metalloproteinase (MMP)-9 expression, an endopeptidase that participates in diverse pathologic processes. In these studies, we determined whether IL-6 regulates the Cox-2→PGE2→MMP-9 pathway in murine macrophages. IL-6 coinduced Cox-2 and microsomal PGE synthase-1, and inhibited the expression of 15-hydroxyprostaglandin dehydrogenase, leading to increased levels of PGE2. In addition, IL-6 induced MMP-9 expression, suggesting that the observed proteinase expression was regulated by the synthesis of PGE2. However, inhibition of PGE2 synthesis partially suppressed IL-6–mediated induction of MMP-9. In the canonical model of IL-6–induced signaling, JAK activation triggers STAT and MAPKerk1/2-signaling pathways. Therefore, the ability of structurally diverse JAK inhibitors to block IL-6–induced MMP-9 expression was examined. Inhibition of JAK blocked IL-6–induced phosphorylation of STAT3, but failed to block the phosphorylation of MAPKerk1/2, and unexpectedly enhanced MMP-9 expression. In contrast, MEK-1 inhibition blocked IL-6–induced phosphorylation of MAPKerk1/2 and MMP-9 expression without affecting the phosphorylation of STAT3. Thus, IL-6–induced MMP-9 expression is dependent on the activation of MAPKerk1/2 and is restrained by a JAK-dependent gene product. Using pharmacologic and genetic approaches, we identified JAK-dependent induction of IL-10 as a potent feedback mechanism controlling IL-6–induced MMP-9 expression. Together, these data reveal that IL-6 induces MMP-9 expression in macrophages via Cox-2–dependent and –independent mechanisms, and identifies a potential mechanism linking IL-6 to the pathogenesis of chronic inflammatory diseases and cancer.

Selective Activation of MAPKerk1/2 by Laminin-1 Peptide α1:Ser2091–Arg2108 Regulates Macrophage Degradative Phenotype

Components of the extracellular matrix contain cryptic domains, which are exposed by proteolysis and elicit biological responses distinct from intact molecules. The disparate cellular response to extracellular matrix fragments and parent intact molecules suggests differential recognition and signaling pathways. In experiments reported here, we demonstrate that urokinase and matrix metalloproteinase-9 expression by RAW264.7 macrophages is stimulated by a synthetic laminin peptide derived from the α1-chain (SRARKQAASIKVAVSADR), whereas intact laminin-1 has no effect on proteinase expression by macrophages. Incubation of macrophages with α1:SRARKQAASIKVAVSADR stimulates tyrosine phosphorylation of several proteins including mitogen-activated protein kinase (MAPK)erk1/2. In contrast, neither intact laminin-1 nor the β1-chain peptide CDPGYIGSR stimulated protein tyrosine phosphorylation in these cells. Inhibition of tyrosine kinases or protein kinase C blocked α1-chain peptide-induced phosphorylation of MAPKerk1/2 and the up-regulation of steady state levels of urokinase mRNA and matrix metalloproteinase-9 activity. A MAPK kinase inhibitor blocked α1-chain-induced phosphorylation of MAPKerk1/2 and the induction of proteinase expression. Intact laminin-1, which was unable to induce macrophage proteinase expression, failed to stimulate the phosphorylation of MAPKerk1/2. These data demonstrate that incubation of macrophages with α1:SRARKQAASIKVAVSADR, but not intact laminin-1, triggers protein kinase C-dependent activation of MAPKerk1/2, leading to the up-regulation of proteinase expression.

Tobacco Smoke Induces Urokinase-Type Plasminogen Activator and Cell Invasiveness: Evidence for an Epidermal Growth Factor Receptor–Dependent Mechanism

Multiple tobacco smoke-related premalignant and malignant lesions develop synchronously or metachronously in various organ sites, including the oral cavity. Both field cancerization and clonal migration seem to contribute to the occurrence of multiple tumors. Although the importance of endogenous factors (e.g., oncogenes) in regulating clonal migration is well established, little is known about the role of exogenous factors. Hence, the main objective of this study was to elucidate the mechanism by which tobacco smoke stimulated the migration of cells through extracellular matrix (ECM). Treatment of MSK-Leuk1 cells with a saline extract of tobacco smoke induced the migration of cells through ECM. Tobacco smoke induced the expression of urokinase-type plasminogen activator (uPA), resulting in plasmin-dependent degradation of ECM and increased cell migration. AG1478, a small-molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, a neutralizing antibody to EGFR, or an antibody to amphiregulin, an EGFR ligand, also blocked tobacco smoke-mediated induction of uPA and cell migration through ECM. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase activity, caused similar inhibitory effects. Taken together, these results suggest that tobacco smoke activated the EGFR-->extracellular signal-regulated kinase 1/2 MAPK pathway, causing induction of uPA. This led, in turn, to increased plasmin-dependent degradation of matrix proteins and enhanced cell migration through ECM. These data strongly suggest that chemicals in tobacco smoke can mimic the effects of oncogenes in regulating uPA-dependent cell invasion through ECM. These findings also strengthen the rationale for determining whether inhibitors of EGFR tyrosine kinase reduce the risk of tobacco smoke-related second primary tumors.

Matrix Metalloproteinase-Dependent Microsomal Prostaglandin E Synthase-1 Expression in Macrophages: Role of TNF-α and the EP4 Prostanoid Receptor

Matrix metalloproteinase (MMP)-9 contributes to the pathogenesis of chronic inflammatory diseases and cancer. Thus, identifying targetable components of signaling pathways that regulate MMP-9 expression may have broad therapeutic implications. Our previous studies revealed a nexus between metalloproteinases and prostanoids whereby MMP-1 and MMP-3, commonly found in inflammatory and neoplastic foci, stimulate macrophage MMP-9 expression via the release of TNF-α and subsequent induction of cyclooxygenase-2 and PGE2 engagement of EP4 receptor. In the current study, we determined whether MMP-induced cyclooxygenase-2 expression was coupled to the expression of prostaglandin E synthase family members. We found that MMP-1– and MMP-3–dependent release of TNF-α induced rapid and transient expression of early growth response protein 1 in macrophages followed by sustained elevation in microsomal prostaglandin synthase 1 (mPGES-1) expression. Metalloproteinase-induced PGE2 levels and MMP-9 expression were markedly attenuated in macrophages in which mPGES-1 was silenced, thereby identifying mPGES-1 as a therapeutic target in the regulation of MMP-9 expression. Finally, the induction of mPGES-1 was regulated, in part, through a positive feedback loop dependent on PGE2 binding to EP4. Thus, in addition to inhibiting macrophage MMP-9 expression, EP4 antagonists emerge as potential therapy to reduce mPGES-1 expression and PGE2 levels in inflammatory and neoplastic settings.