Lipopolysaccharide-Induced Expression of Matrix Metalloproteinases in Human Monocytes Is Suppressed by IFN-γ via Superinduction of ATF-3 and Suppression of AP-1¹ Free

Since the discovery of the first matrix metalloproteinase (MMP)³ in the regressing tadpole tail by Gross and Lapiere in 1962 (1), the list of functions assigned to various MMPs has been expanding rapidly. MMPs were initially thought to be a group of enzymes with the sole function of regulating extracellular matrix (ECM) composition. However, intense research in recent years has shown that ECM degradation is not the sole and possibly not the main function of these proteinases. Indeed, plenty of evidence supports roles of MMPs in physiological processes such as embryogenesis and wound healing/tissue remodeling and also in pathological processes that include arthritis, pulmonary diseases, cardiovascular ailments, and cancer (2, 3, 4, 5, 6, 7). In addition to ECM remodeling MMPs regulate signal transduction and growth factor function, and recent findings also indicate that MMPs are immunoregulators by acting on inflammatory cytokines, chemokines, and other immune proteins (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18).

Not surprisingly, the activity of MMPs is tightly regulated at multiple levels that include gene expression, compartmentalization, proenzyme activation, and enzyme inactivation, catabolism, and clearance (3, 18). Expression of many MMPs is normally low or absent in healthy tissues, but they are induced when tissue remodeling is required, during inflammation, and in disease states such as rheumatoid arthritis and cancer in which high levels of MMPs may signify a poor prognosis in human patients (19). MMP gene expression is mainly regulated at the transcriptional level in response to hormones, growth factors, cytokines, and cell-cell/cell-matrix interactions (3, 5, 20). Though the expression of MMPs in diseased states is intended to restore normal homeostasis, in a setting such as inflammation the up-regulation of various MMPs may lead to more damage than repair (5).

Rheumatoid arthritis and atherosclerosis are disorders of chronic inflammation, and macrophages are central players in both of these pathological conditions (21, 22, 23). Though the importance of macrophage-derived MMPs in cartilage and/or bone destruction in rheumatoid arthritis has not been fully elucidated, their role in atherosclerosis progression is well documented (21, 24). In atherosclerosis, macrophages accumulate lipid to form foam cells. The accumulation of foam cells in an atheroma is reversible and does not in itself cause clinical consequences. However, the secretion of MMPs by the atheromatous plaque-associated macrophages can cause plaque destabilization, leading to acute coronary events with high morbidity and mortality (22, 23, 25). Among the MMPs produced by macrophages, MMP-1 (collagenase-1), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-7 (matrilysin-1), MMP-9 (gelatinase B), MMP-12 (metalloelastase), and MMP-13 (metalloproteinase-13) have been associated with atherosclerotic plaque instability and rupture (22, 23, 25, 26, 27). Therefore, understanding the regulation of MMPs in inflammatory conditions is crucial in formulating a therapeutic intervention to prevent disease progression.

Most studies on regulation of MMP expression have been performed using fibroblasts, chondrocytes, osteoblasts, and cell lines such as HEK293, HeLa and NIH-3T3 cells (7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45), with very few studies using monocytes/macrophages (46, 47, 48). Because MMP expression is regulated differently in the various cell types and cell lines studied to date, we were interested in understanding MMP regulation in primary human monocytes and macrophages that are relevant for inflammatory diseases, and defining mechanisms that inhibit MMP expression. In this study, we found that in primary human macrophages IFN-γ globally inhibits induction of MMPs by Toll-like receptors, innate immune receptors that play a key role in activation of macrophages by microbial products and endogenous ligands that include ECM degradation products. Further analysis of the regulation of MMP-1 showed that the mechanism of IFN-γ-mediated inhibition involves induction of the transcriptional repressor activating transcription factor (ATF)-3 and suppression of DNA binding by AP-1 proteins. Prostaglandin E₂ (PGE₂) also down-regulated LPS-induced MMP-1 expression, and suppression by PGE₂ and IFN-γ converged on inhibition of AP-1 proteins and regulation of AP-1 family member Fra-1. These results define mechanisms that inhibit MMP expression in primary human macrophages.

PBMC were obtained from whole blood from disease-free volunteers by density gradient centrifugation with Ficoll (Invitrogen). CD14⁺ monocytes were purified from fresh PBMCs with anti-CD14 magnetic beads (Miltenyi Biotec), as recommended by the manufacturer. Purity of monocytes was >97% as verified by FACS, and freshly isolated monocytes were used in many experiments, as noted in text and figure legends. Some experiments were performed using macrophages, which were derived by culturing monocytes in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (HyClone) in the presence of 10 ng/ml of human M-CSF (PeproTech). THP-1 human monocytic cells were obtained from ATCC and cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (HyClone). LPS (100 ng/ml), PMA (100 ng/ml), LiCl (20 mM), cycloheximide (20 μg/ml), and SB216763 (10 μM) were purchased from Sigma-Aldrich. PGE₂ was obtained from Sigma-Aldrich and Calbiochem.

A lentivirus-based vector expressing the human STAT1 or Fra-1 cDNA driven by a human phospho-glycerol kinase promoter was used to generate recombinant lentiviral particles as described (49). A construct that contained a transcription cassette encoding enhanced GFP (eGFP) driven by the human phospho-glycerol kinase promoter was used to generate control viral particles for STAT1 and Fra-1 expression experiments. RNAi in the THP-1 monocytic cell line was performed using lentiviral transduction to stably express shRNAs that targeted STAT1 or ATF-3, or DSRed2 as a control. For STAT1 RNA interference, oligonucleotides encoding several different short hairpin RNAs (shRNAs) that target human STAT1 were cloned into the lentivirus-based RNAi vector pLL3.7 that also contains a transcription cassette encoding eGFP driven by a CMV promoter (49). Lentivirus-based RNAi vectors against ATF3 were purchased from Sigma-Aldrich. Constructs that were effective in suppressing STAT1 or ATF-3 expression were identified using transient cotransfection of HEK 293T cells with expression plasmids encoding the respective genes. The constructs that were most effective in HEK 293T cells (containing the shRNA sequences 5′-GCGTAATCTTCAGGATAAT-3′ or 5′-ACCTTGCAGAACAGAGAAC-3′ for human STAT1 and Sigma-Aldrich’s TRC Numbers of 0000013570 and 0000013572 for human ATF-3) were used to generate recombinant lentiviral particles. Lentiviral particles encoding interfering shRNA against red fluorescence protein DSRed2 (5′-GTGGGAGCGCGTGATGAAC-3′) were used as a control for STAT1/ATF3 RNAi experiments. THP-1 cells were incubated overnight with recombinant lentiviral particles at a ratio of 1:50 in the presence of 4 μg/ml polybrene. The efficiency of transduction was evaluated using flow cytometry and fluorescence microscopy to monitor eGFP expression and was typically >90%. For RNAi experiments with primary human monocytes, we used nucleofection of short interfering RNA duplexes (siRNA) that targeted GSK3β or Fra-1, or control siRNAs. GSK3β- and Fra-1-specific prevalidated siRNAs and nontargeting control siRNAs were purchased from Dharmacon. siRNAs were transfected into primary human macrophages using the Amaxa Nucleofector device set to program Y-001 with the Human Monocyte Nucleofector Kit (Amaxa).

For real-time PCR, total RNA was extracted using an RNeasy mini kit and 1 μg of total RNA was reverse transcribed using a First Strand cDNA Synthesis kit (Fermentas). Real-time, quantitative PCR was performed using iQ SYBR-Green Supermix and iCycler iQ thermal cycler (Bio-Rad) following the manufacturer’s protocols. Triplicate reactions were run for each sample and mRNA levels were normalized relative to β-actin. The generation of only the correct size amplification products was confirmed using agarose gel electrophoresis.

ChIP assays were performed using the ChIP-IT Express Kit (Active Motif) according to the manufacturer’s instructions. Chromatin was prepared from 2 × 10⁷ primary human monocytes per condition and was sheared to an average size of 500 bp using a Missonics Sonicator 3000. Rabbit polyclonal Abs against ATF-3 (sc-188) or control rabbit IgG (sc-2027) (Santa Cruz Biotechnology) were used for immunoprecipitation; each immunoprecipitation contained chromatin derived from 2 × 10⁶ monocytes. Immunoprecipitates were subjected to PCR with primers that span the ATF site located 2017 bp upstream of the transcription start site of the MMP1 gene. Primer sequences are: upstream, 5′-AGGGATTTTGTTTAAGTAAAGG-3′; downstream, 5′-GCATGTGACCATCTGTGATT-3′.

Whole cell and nuclear extracts were obtained, and protein levels quantitated using the Bradford assay (Bio-Rad), as previously described (50). For immunoblotting, 30 μg of THP-1 whole cell lysates were fractionated on 7.5% polyacrylamide gels using SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore), and ECL was used for detection. For EMSA, 5 μg of nuclear extracts from primary monocytes were incubated for 15 min at room temperature with 0.5 ng of ³²P-labeled double stranded AP-1 (5′- AAA GCA TGA GTC AGA CAC - 3′ and 5′- GTG TCT GAC TCA TGC TTT −3′) or CRE (5′- TTC CAG GGT GAC GTC TTA GGC- 3′ and 5′-GCC TAA GAC GTC ACC CTG GAA-3′) oligonucleotides derived from the human MMP-1 promoter in a 15 μl binding reaction containing 40 mM NaCl and 2 μg of poly-dI-dC (Pharmacia), and complexes were resolved on nondenaturing 4.5% polyacrylamide gels. For supershift assays, nuclear extracts were incubated with Abs on ice for 60 min before the addition of radiolabeled oligonucleotides as previously described (51). All supershift Abs used were purchased from Santa Cruz Biotechnology.

We wished to identify physiological mechanisms that suppress MMP expression in primary human myeloid cells. We first determined which MMPs are induced in monocytes and macrophages by inflammatory stimuli. Primary monocytes were treated with inflammatory factors for 3 h or overnight (12 h) and MMP mRNA levels were measured using real-time PCR. The TLR4 ligand LPS was the most potent inducer of MMP expression (data not shown), and activated expression of at least seven MMP genes more than 5-fold at either an early time point (3 h) (MMP-1, MMP-3, MMP-10, and MMP-12; Fig. 1,A), or at a later time point after LPS stimulation (12 h) (MMP-2, MMP-7, and MMP-9; Fig. 1,B). Expression of many MMPs, with certain exceptions such as MMP-2, is regulated by AP-1 transcription factors and we and others had previously shown that IFN-γ can suppress AP-1-mediated gene expression (52). Therefore, we tested whether IFN-γ could suppress LPS-induced MMP expression. Pretreatment of monocytes with IFN-γ for 3 h strikingly decreased LPS-induced expression of the MMPs that were studied (Fig. 1, A and B); similar results were obtained with primary M-CSF-differentiated macrophages (data not shown). As IFN-γ typically synergizes with LPS to superinduce expression of inflammatory genes such as COX-2 and IL-6, we measured COX-2 and IL-6 mRNA to rule out nonspecific suppressive effects of IFN-γ on gene expression. As expected, IFN-γ increased LPS-induced accumulation of COX-2 and IL-6 mRNA (Fig. 1,C). In addition, IFN-γ augmented LPS-induced expression of ADAM-17, a cell surface MMP (Fig. 1,C). These results show that IFN-γ specifically and strongly suppressed LPS-induced expression of multiple secreted MMPs that have AP-1 binding sequences (also known as phorbol ester-responsive elements or TPA-responsive elements) in their promoters and are known to be positively regulated by AP-1 proteins (3, 53, 54, 55). The phorbol ester PMA is a strong activator of AP-1-mediated transcription, and IFN-γ also suppressed PMA-induced expression of MMPs, but not of TNF-α (Fig. 1,D). PMA was a stronger inducer of MMP expression than TLR ligands, which correlated with stronger induction of AP-1 proteins by PMA (data not shown). These results show that IFN-γ strongly suppresses MMP expression in primary human monocytes. The dependence of expression of many MMP genes on AP-1, together with the previously reported inhibition of AP-1 proteins by IFN-γ, suggested that IFN-γ may suppress MMP expression at least in part by inhibiting AP-1. However, IFN-γ-mediated inhibition of MMP-2 (Fig. 1 B), which is not regulated by AP-1, suggests IFN-γ inhibits MMP expression by several mechanisms.

Previously we (52) and others (56) have shown that IFN-γ inhibits TLR-induced IL-10 production by enhancing glycogen synthase kinase 3 (GSK-3) activity and that inhibition of GSK-3 reverses IFN-γ suppression of IL-10 production. This prompted us to investigate whether the same mechanism was also applicable to IFN-γ regulation of MMPs. However, GSK inhibitors did not have any effect on IFN-γ-mediated suppression of MMP expression (Fig. 2,A); reversal of IFN-γ-mediated suppression of IL-10 expression confirmed the efficacy of GSK-3 inhibition (data not shown). In addition, we used siRNA to knockdown GSK-3β expression in primary monocytes to ∼30% of control at both mRNA and protein levels (Fig. 2, B and D), a level sufficiently low to attenuate IFN-γ-mediated inhibition of IL-10 expression (Ref.52 and data not shown). However, reduced GSK-3β expression had no discernable effect on IFN-γ suppression of TLR4-induced MMP expression (Fig. 2,B), and similar results were obtained when PMA was used to induce MMP expression (Fig. 2 C). Our results suggest that the mechanisms by which IFN-γ suppresses LPS induction of MMP genes differ from mechanisms used to suppress induction of the potent anti-inflammatory factor IL-10.

Activation of gene expression by IFN-γ is mediated by STAT1, but mechanisms by which IFN-γ inhibits gene expression are not well understood (57). Therefore, we investigated whether IFN-γ-mediated suppression of MMP expression was dependent on STAT1. STAT1 protein expression was too stable to achieve successful knockdown of expression in primary monocytes, so instead we used THP-1 monocytic cells in which STAT1 expression was stably diminished secondary to transduction with lentiviral vectors that express shRNAs targeting STAT1 mRNA (58). IFN-γ effectively suppressed TLR4-induced MMP-1 and MMP-10 expression in control THP-1 cells that expressed shRNA that targets the irrelevant DSRed2 mRNA, but IFN-γ-mediated suppression of MMP induction was completely abrogated in THP-1 cells expressing low STAT1 levels (Fig. 3). Effects of IFN-γ alone on MMP expression were not consistently observed (data not shown). Thus, suppression of MMP expression by IFN-γ is dependent on STAT1.

STAT1 could suppress MMP expression directly by binding to MMP promoters and recruiting transcriptional corepressors, although there is minimal precedent for such a direct repressive mechanism. Alternatively, STAT1 could activate expression of genes that encode transcriptional repressors that target MMP genes. To differentiate between these two possibilities, we tested whether the inhibitory effect of IFN-γ on MMP expression was dependent on new protein synthesis. Primary monocytes were stimulated with LPS and IFN-γ in the presence of cycloheximide (added 15 min before IFN-γ or LPS) to block protein synthesis. As shown in Fig. 4,A, LPS induced MMP-1, MMP-3 and MMP-10 mRNAs in the absence of protein synthesis, indicating that LPS increases their expression at least in part by inducing posttranslational modification of pre-existing proteins. However, cycloheximide abolished the inhibition of MMP-1 and MMP-3 expression by IFN-γ, whereas suppression of MMP-10 expression remained intact (Fig. 4,A). These results suggest that IFN-γ induction of a repressor is required for suppression of MMP-1 and MMP-3 expression, whereas inhibition of MMP-10 expression is more direct. This notion was further supported by a kinetic analysis of IFN-γ-mediated inhibition. Down-regulation of LPS-induced MMP-3 expression required greater than 1 h of pretreatment with IFN-γ, consistent with the requirement for synthesis of an inhibitor, whereas MMP-10 expression was suppressed even when IFN-γ was added simultaneously with LPS (Fig. 4 B).

The results suggesting that MMP-1 and MMP-3 expression is suppressed by an IFN-γ-induced inhibitor prompted us to use microarray analysis to identify transcriptional repressors that were induced by IFN-γ in TLR-stimulated monocytes. Interestingly, IFN-γ superinduced the expression of the repressor ATF-3 in LPS-stimulated monocytes, and this result was confirmed by real-time PCR in monocytes and THP-1 cells (Fig. 5,A and data not shown). The induction of ATF-3 expression by IFN-γ was STAT1-dependent (Fig. 5,A), consistent with the notion that MMP-1 expression is suppressed by an IFN-γ-induced STAT1-dependent protein. ATF-3 has been described as a TLR-induced feedback inhibitor that suppresses TLR-induced gene expression by binding to CRE/ATF promoter elements (59). Because CREs have been implicated in regulation of MMP-1 expression (48), we investigated the role of ATF-3 in regulation of MMP-1 expression in our system. EMSA assays detected an LPS-induced complex that bound to an oligonucleotide corresponding to a functional CRE sequence located upstream of the MMP-1 proximal promoter (−2017 relative to transcription start site) and was further superinduced by IFN-γ (Fig. 5, B and C). DNA binding by this IFN-γ-superinduced complex was disrupted by ATF-3 Abs but not by control Abs (Fig. 5,C). Furthermore, treatment of monocytes with LPS plus IFN-γ induced recruitment of ATF-3 to the endogenous MMP-1 promoter in the vicinity of the −2017 ATF site (Fig. 5,D). Taken together with the real-time PCR results (Fig. 5,A), these results suggest that IFN-γ induces DNA binding of the ATF-3 transcriptional repressor that can potentially suppress MMP-1 expression. The role of ATF-3 in regulating MMP-1 expression was investigated by using lentivirus-mediated transduction of THP-1 monocytes to create two stable cell lines that express different shRNAs targeting ATF-3. In those ATF3-RNAi THP-1 cells, the synergistic LPS/IFN-γ induction of ATF-3 was reduced to ∼40% that of control (Fig. 5,E), and decreased ATF-3 expression resulted in reversal of IFN-γ-mediated suppression of LPS-induced MMP-1 gene expression (Fig. 5 F). Attempts to test the effects of ATF-3 overexpression were not successful as increased expression of ATF-3 induced cell death (data not shown). These results implicate IFN-γ-induced ATF-3 in mediating the suppressive effects of IFN-γ on MMP expression.

The AP-1 element located between base pairs −72 and −66 of the MMP-1 promoter is crucial for MMP-1 gene expression (41, 55, 60, 61, 62) and we and others have shown that IFN-γ suppresses AP-1-dependent gene expression. Thus, despite the negative data on the role of GSK-3 (Fig. 2), we further investigated the effects of IFN-γ on LPS-induced AP-1 proteins. EMSA experiments using the MMP-1 promoter AP-1 site showed an LPS-induced DNA-binding complex (Fig. 6,A). Interestingly, LPS-induced binding to the AP-1 oligonucleotide was suppressed by IFN-γ (Fig. 6,A), suggesting suppression of AP-1 proteins as a complementary mechanism by which IFN-γ suppresses MMP-1 expression. Supershift experiments showed that the LPS-induced complex was reactive with Fra-1 Abs, but not with Fos or Jun Abs (Fig. 6,B). Because the MMP-1 promoter-derived AP-1 oligonucleotide contains an overlapping Ets site, we also investigated whether the LPS-induced complexes contained Ets proteins, but supershift experiments with PEA-3, PU.1, ETS-1, ETS-2, and ERG 1/2/3 showed no reactivity (Fig. 6,B and data not shown). These results suggested a role for Fra-1 in LPS and IFN-γ regulation of MMP-1 expression, and this was further investigated using RNA interference and overexpression of Fra-1. Indeed, knockdown of Fra-1 expression resulted in diminished LPS-induced MMP-1 expression (Fig. 6,C), and conversely overexpression of Fra-1 increased MMP-1 expression (Fig. 6,D), thus implicating Fra-1 in induction of MMP-1 expression by LPS. We did not detect regulation of Fra-1 expression by IFN-γ (data not shown), but, interestingly, PGE₂ which suppressed MMP-1 expression in monocytes (Fig. 7,A) also suppressed Fra-1 expression (Fig. 7,B). EMSA using the MMP-1 promoter AP-1 site demonstrated that in the presence of PGE₂, the composition of the LPS-induced DNA binding complex was altered such that the complex was no longer shifted by Fra-1 Abs (Fig. 7 C). These results support the notion that PGE₂ inhibits LPS-induced MMP-1 gene expression by suppressing expression of Fra-1 that contributes to induction of MMP-1 expression through the proximal promoter AP-1 site. Overall, our findings support a role for Fra-1 in the positive and negative regulation of MMP-1 expression, and suggest that IFN-γ inhibits MMP-1 transcription in part by suppressing DNA binding by Fra-1. Although IFN-γ also suppresses other AP-1 proteins such as Fos and Jun (52), we were not able to link regulation of Fos and Jun with regulation of MMP-1 expression in our system.

MMP expression needs to be tightly regulated during inflammation to allow necessary angiogenesis and tissue remodeling but to avoid excessive tissue damage. In this study, we found that IFN-γ, a potent macrophage activating cytokine, globally inhibits MMP expression at the same time that it synergistically enhances the production of proinflammatory cytokines. Thus, as the intensity of inflammation increases in response to IFN-γ there is a built-in mechanism to restrain associated tissue damage. We have also identified two mechanisms by which IFN-γ inhibits MMP expression, by inducing expression of the transcriptional repressor ATF-3 and simultaneously suppressing the function of AP-1 transcriptional activators.

The regulation of MMP gene expression has been extensively studied, although much of this work has utilized reporter gene approaches in cell lines and nonphysiological activators such as PMA (7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). This previous work has revealed the importance of proximal promoter sequences, especially a composite AP-1/Ets site, in basal and inducible MMP-1 and MMP-3 expression. More recently, the importance of promoter and enhancer sequences outside MMP proximal promoters is becoming increasingly apparent, including upstream CRE sites (48, 63). Surprisingly little is known about MMP regulation in primary cells, especially hematopoietic cells such as monocytes and macrophages. In addition, although important MMP promoter elements have been extensively analyzed, less is known about transcription factors that regulate MMP expression. Our work implicates ATF-3 in the regulation of MMP-1 expression. Because ATF-3 is induced by TLR stimulation alone, it participates in feedback inhibition of TLR-induced gene expression, as has been previously reported in other systems (59). In addition, superinduction of ATF-3 by IFN-γ contributes to IFN-γ-mediated suppression of MMP-1 gene expression. Transfection of primary monocytes or THP-1 cells with MMP promoter-driven reporter plasmids resulted in cell activation, presumably secondary to an innate immune response to exogenous DNA, and rapid extinction of reporter gene expression (H. Ho, unpublished data). Thus, the reporter gene approach was not informative in studying LPS and IFN-γ regulation of MMP expression.

A consistent finding in multiple systems and cell types has been the important role of the proximal promoter AP-1 site in the expression of MMP-1 and MMP-3 (3, 41, 55, 60, 61, 62). Therefore, given our previous findings that IFN-γ can suppress AP-1-mediated IL-10 transcription by a GSK-3-dependent mechanism, we investigated a potential role for regulation of AP-1 proteins by IFN-γ in suppression of MMP expression. LPS induced a Fra-1-containing complex that bound to the MMP-1 promoter AP-1 site, and the binding of this complex was suppressed by IFN-γ. Fra-1 has been previously implicated in promoting MMP expression (64, 65), although its regulation by LPS and IFN-γ has not been previously appreciated. We confirmed the importance of Fra-1 in MMP-1 gene expression using both knockdown and overexpression approaches and implicated Fra-1 in mediating PGE₂ regulation of MMP-1 expression. In contrast to PGE₂, IFN-γ did not inhibit Fra-1 expression, suggesting that IFN-γ may suppress DNA binding of Fra-1 by posttranslational modifications or indirectly by regulating other members of the AP-1 family. Overall, the data show that IFN-γ regulated MMP-1 expression by at least two mechanisms, induction of ATF-3 and suppression of DNA binding by a Fra-1-containing AP-1 complex. Of the MMPs studied whose expression was inhibited by IFN-γ (Fig. 1), all contained AP-1 sites with the exception of MMP-2 (66). Thus, although suppression of AP-1 may contribute to diminished expression of several MMPs, it cannot explain inhibition of MMP-2 by IFN-γ. Interestingly, ATF-3 has been implicated in negative regulation of MMP-2 (59), suggesting a role for ATF-3 in regulation of MMP gene expression beyond the regulation of MMP-1 expression that was investigated in this study. Collectively, our data and previous reports suggest that IFN-γ can suppress MMP expression by different mechanisms. This notion is further supported by our results suggesting that MMP-10 expression was suppressed by IFN-γ by a mechanism that differed from suppression of MMP-1 because inhibition of MMP-10 did not require new protein synthesis, and IFN-γ inhibits MMP-9 expression by modifying chromatin and transcriptional coactivator recruitment (28, 30, 35, 67, 68). These findings highlight that IFN-γ suppresses MMP expression by several mechanisms and provide insights into how IFN-γ down-regulates gene expression.

Prior studies from several laboratories including ours have described antagonistic functions for STAT1 and STAT3 in the regulation of cell growth/survival and inflammatory gene expression (58, 69). It appears that this opposition of STAT1 and STAT3 also extends to the regulation of MMP expression. STAT3 has been shown to be an important and strong inducer of MMPs such as MMP-1 and MMP-10 (70, 71), and was found to bind directly to a STAT-binding element located between base pairs −53 and −45 upstream in the MMP-1 proximal promoter after stimulation with EGF (71). We have shown that inhibition of MMP-1 expression by IFN-γ is dependent on STAT1, but likely occurs indirectly via regulation of ATF-3 and AP-1 proteins. Consistent with these results, we were not able to detect binding of STAT1 to the MMP-1 promoter AP-1 site or the previously described STAT-biding element that binds STAT3 (H. Ho, unpublished observations), suggesting that STAT1 does not directly compete with STAT3 for binding to the MMP-1 promoter. On the other hand, it is possible that STAT-1 inhibits MMP-10 expression by a direct mechanism, as has been shown for IFN-γ suppression of the Perlecan gene (72), because inhibition of MMP-10 expression by IFN-γ was STAT1-dependent and rapid and independent of new protein synthesis.

In summary, our results demonstrate that IFN-γ differentially regulates gene expression in primary human monocytes and macrophages by augmenting the production of inflammatory cytokines such as IL-6 and TNF-α, but at the same time globally suppressing MMP production. The suppression of MMPs by IFN-γ may represent a self-protective mechanism to limit undesirable damage to tissues during intense immune and inflammatory reactions that are required to eradicate infectious pathogens. The results also reveal that IFN-γ modulates MMP expression by regulating the expression and function of ATF-3, Fra-1 and potentially other AP-1 proteins. Understanding the mechanisms by which IFN-γ regulates these genes and transcription factors may yield useful insights into formulating future approaches to enhancing immune clearance of invading pathogens and yet limiting damage to tissues and organ systems.

We thank Janice Chen for helpful discussions, Kyung-Kyun Park-Min for review of the manuscript, and Inez Rogatsky and Barbara Nikolajczyk for advice concerning ChIP assays.

The authors have no financial conflict of interest.