- Research article
- Open Access
Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1
© Zhou et al; licensee BioMed Central Ltd. 2009
Received: 26 August 2008
Accepted: 19 March 2009
Published: 19 March 2009
Expressed in embryonic development, matrix metalloprotein-9 (MMP-9) is absent in most of developed adult tissues, but recurs in inflammation during tissue injury, wound healing, tumor formation and metastasis. Expression of MMP-9 is tightly controlled by extracellular cues including pro-inflammatory cytokines and extracellular matrix (ECM). While the pathologic functions of MMP-9 are evident, the intracellular signaling pathways to control its expression are not fully understood. In this study we investigated mechanism of cytokine induced MMP-9 with particular emphasis on the role of p21-activated-kinase-1 (PAK1) and the down stream signaling.
In response to TNF-alpha or IL-1alpha, PAK1 was promptly activated, as characterized by a sequential phosphorylation, initiated at threonine-212 followed by at threonine-423 in the activation loop of the kinase, in human skin keratinocytes, dermal fibroblasts, and rat hepatic stellate cells. Ectopic expression of PAK1 variants, but not p38 MAP kinase, impaired the TNF-alpha-induced MMP-9 expression, while other MMPs such as MMP-2, -3 and -14 were not affected. Activation of Jun N-terminal kinase (JNK) and NF-kappaB has been demonstrated to be essential for MMP-9 expression. Expression of inactive PAK1 variants impaired JNK but not NF-kappaB activation, which consequently suppressed the 5'-promoter activities of the MMP-9 gene. After the cytokine-induced phosphorylation, both ectopically expressed and endogenous PAK1 proteins were promptly accumulated even in the condition of suppressing protein synthesis, suggesting the PAK1 protein is stabilized upon TNF-alpha stimulation. Stabilization of PAK1 protein by TNF-alpha treatment is independent of the kinase catalytic activity and p21 GTPase binding capacities. In contrast to epithelial cells, mesenchymal cells require 3-dimensional type-I collagen in response to TNF-alpha to massively express MMP-9. The collagen effect is mediated, in part, by boost JNK activation in a way to cooperate the cytokine signaling.
We identified a novel mechanism for MMP-9 expression in response to injury signals, which is mediated by PAK1 activation and stabilization leading JNK activation.
Degradation of extracellular matrix (ECM), as mediated by matrix metalloproteinases (MMPs) and antagonized by tissue inhibitors of matrix metalloproteinases (TIMPs), is critical for embryonic development and adult tissue homeostasis [1, 2]. Conversely, uncontrolled ECM degradation occurs in many degenerative diseases [3, 4]. For instance, excessive presence of MMPs has been well documented in cancer dissemination, arthritis development, and chronic wound progression . Among the twenty-some members of the MMP family in either the human or murine genome the proteinases can be classified not only based on their structural conservation, but also by the means of their expression. Of our interest for many years is the regulation of MMP-9, a type-IV collagenase in tissue injury and repair. Not expressed in most of adult developed tissues, MMP-9 is promptly expressed in response to tissue damages under the control of pro-inflammatory cytokines [6, 7]. Nascent proMMP-9 is maintained in latency through interaction between a conserved cysteine in the pro-peptide domain and a zinc atom in the catalytic domain. Furthermore, TNF-α-induced maturation of proMMP-9 by skin cells is mediated by down regulation of TIMP-1, while the converting enzymes are seemingly constitutively present . Recently we uncovered alpha-1-antichymotrypsin (alpha-ACT) as a novel pathological inhibitor that directly antagonizes the proMMP-9 converting enzyme in skin tissues . Thus, generation of active MMP-9 within tissues is tightly monitored at the levels of gene expression, protein processing, and antagonization by inhibitors targeting at either proMMP-9 (by TIMP-1) or converting enzyme (by alpha-ACT).
Innate immunity and its produced waves of inflammatory cytokines are the initial signals to trigger expression of MMPs in tissue injury phase. Compelling evidence of in vitro experiments also demonstrates the critical roles of inflammatory cytokines in control of MMP expression and activation. Intracellular signals governing the expression of MMPs have been extensively studied; among them, Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-kB) signaling pathways are essential to induce many MMPs [10, 11]. Still outstanding is that how other extracellular cue, such as ECM, in cooperation with pro-inflammatory cytokines control MMP expression, of which is particularly critical for mesenchymal cells. For instance, in addition to TNF-α or IL-1, the 3-dimentional type-I collagen is also required to maximally induce MMP-9 by either human dermal fibrobaslts or rat hepatic stellate cells [6, 7].
p21-activatd kinase (PAK), a family of serine/threonine kinases conserved from yeast to human, is important for regulation of cytoskeleton, cell migration, and cell cycle progression . PAK1 was originally identified through its binding to p21 GTPase [13, 14]. Like many other protein kinases, PAK1 retains inactive by its own pseudo-substrate like domain, and is activated by binding GTP-charged proteins, which consequently leads conformational changes and auto-phosphorylation. Non-canonically, PAK1 can be phosphorylated and activated by PDK1 which phosphorylates a conserved threonine-423 within the active loop . In addition to regulating cytoskeletal proteins, PAK1 also controls MAP kinases, such as JNK and p38 MAP kinases [16, 17]. Furthermore, PAK1 also modulate NF-κB activity [18, 19]. Although the dynamics of cytoskeleton and the formation of stress filaments are critical for cell-matrix interactions in inflammation, very little is known if and how inflammatory cytokines regulate PAK1 in control of MMP expression.
In this study we demonstrated an unrecognized mechanism by which TNF-α activates PAK1 by a sequential phosphorylation from threonine-212 to threonine-423 in human epithelial cells and fibroblasts. Similarly, IL-1α also promotes PAK1 activation in rat hepatic stellate cells. Moreover, TNF-α treatment promptly results in accumulation of PAK1 protein, but not p38 MAP kinase, in part by stabilization of the former. Although the details are unknown to date, TNF-α-mediated stabilization of PAK1 is independent of its catalytic activity and p21 GTPase binding capacities. Ectopic expression of PAK1 variants impairs JNK but not NF-κB pathway, which in turn suppresses the promoter activation and transcription of MMP-9. Expression of other MMPs such as MMP-2, MMP-3, and MMP-14 as well as TIMP-1 is, in contrast, not affected by PAK1. We also characterized the differences of MMP-9 expression between human keratinocytes and dermal fibroblasts. For keratinocytes, TNF-α or IL-1 is sufficient to induce MMP-9, while mesenchymal cells such as dermal fibroblasts and hepatic stellate cells require type-I collagen as an additional factor which boosts JNK activity to maximally induce MMP-9.
Cytokine-induced expression of MMP-9 by human skin is partially reconstituted by primary keratinocytes and dermal fibroblasts
Primary human keratinocytes produced minimal level of proMMP-9 in response to TNF-α, and the expression was also additionally enhanced in concert with TGF-β (Fig. 1D). Of note are two clear distinguished features between the dermal fibroblasts and keratinocytes in terms of MMP-9 expression. First, ECM has a profound role to induce MMP-9 expression by dermal fibroblasts but not keratinocytes (data not shown). Second, MMP-2, a mesenchymal MMP is largely absent in keratinocytes. Taken together, both the keratinocytes and dermal fibroblasts of human skin may contribute to the massive expression of MMP-9 under the stimulation of inflammatory cytokines in inflammation.
PAK1 mediates TNF-α-induced expression of MMP-9 but not MMP-2
TNF-α-induced stabilization of the ectopically expressed PAK1 protein
We then addressed the nature of TNF-α regulation of PAK1. First, we analyzed the cell lines constitutively expressing PAK1 driven the viral promoter. After 3 days of TNF-α exposure the protein level of the ectopically expressed PAK1 was unexpectedly elevated, indicating a possible mechanism by which TNF-α signaling results in stabilization of PAK1 protein (Fig. 2D). Importantly, the TNF-α induced accumulation of PAK1 protein is independent of the kinase activities and p21 GTPase binding, as measured by the similar elevation of the variants of K299R and the triple mutant in response to TNF-α. Measurement at fine time points demonstrated again the prompt mode of elevation of the ectopically expressed PAK1 after TNF-α treatment (Fig. 2E). Because the ectopically expressed PAK1 is driven by constitutively active viral promoter, the TNF-α induced elevation of the kinase is, therefore, likely mediated by stabilization of the protein. To strengthen the notion we treated the dermal fibroblasts with cycloheximide to block protein synthesis and to monitor the degradation of the kinase. Under 20 μg/ml of cycloheximide, which sufficiently suppresses many protein syntheses, the PAK1 protein was still elevated under TNF-α treatment, demonstrating that the cytokine-dependent elevation of PAK1 is mediated by stabilization (Fig. 2E).
Expression of MMP-9, but not MMP-2, -3, -14, is specifically controlled by PAK1
JNK, but neither p38 MAP kinase nor PI3 kinase, is downstreamof PAK1 to promote MMP-9 expression
Not regulated by PAK1, NF-κB signaling is essentialfor MMP-9 expression
Phosphorylation, activation, and stabilization of endogenous PAK1 in response to TNF-α/IL-1α
Contribution of collagen to TNF-α-induced MMP-9 expression by fibroblasts is associated with persistent activation ofJNK
Expressed in early embryonic development, MMP-9 is largely silent in many adult tissues; while MMP-2, another member of the gelatinase family, is constitutively expressed in health tissues. However, upon injury such as mechanical trauma, thermal burn, and viral infection, MMP-9 is promptly elevated, indicating its role in wound healing by activating the stromal cells, releasing growth factors, and initiating cell migration. On the other hand, persistent presence of large amount of MMPs is often associated with, if not results in, many inflammatory diseases such as chronic wounds, arthritis, and cancer metastasis. Despite large body of efforts to discover the intracellular signal pathways to control MMPs in variety of cells, little is known as to how ECM in cooperating with the cytokine signals regulates MMP expression, particularly by the mesenchymal cells. In ECM scaffolds the mesenchymal cells are intensified by the intracellular cytoskeletal networks which are essential for cell anchoring, migration, and proliferation in the waves of cytokines during wound healing processes. Organization of intracellular cytoskeletal framework is largely orchestrated by small GTPases and their down stream effectors. To these regards, we investigated the role of PAK1 in regulation of MMPs by three cell types from msenchymal to epithelial cells. In summary, we have found (1) TNF-α triggers a sequential phosphorylation events in PAK1, starting at threonine-212 followed by threonine-423, which may confers the kinase into active state; (2) TNF-α promptly elevates PAK1 protein level, presumably through stabilization, which is independent on the kinase activity and p21 GTPase binding capacity; (3) TNF-α induced expression of MMP-9 is mediated by PAK1/JNK pathway which controls transcriptional initiation of MMP-9 promoter.
Originally identified as targets by GTP-loaded p21 protein, PAK family with 6 members has been demonstrated to serve as important regulators in cytoskeletal dynamic and cell motility, presumably through phosphorylation of the downstream substrates such as Lim kinase and myosin light chain kinase . Like many protein kinases, PAK1 activity is restrained in latency by formation of homodimer through the interaction between the kinase inhibitory domain and the catalytic activation loop, which prevents the kinase from access the substrates . The first step to provoke activation of PAK1 is believed to disrupt such trans-inhibition interaction, which can be conducted by variant ways such as binding of p21 GTPase, partial cleavage by caspase, association with sphingolipids, and phosphorylation . The second step is to maintain the "opened" status by auto-phosphorylation at theronine-423 of the active loop in catalytic domain [29, 30]. On the other hand, it is unknown whether inflammatory cytokines regulate PAKs, and how such regulation controls MMP in inflammation process.
In this study we first assessed the contribution of dermal fibroblasts and keratinocytes to the massive expression of MMP-9 by non-healing skin tissues. Although both dermal fibroblasts and keratinocytes produce MMP-9 in response to TNF-α/IL-1, dermal fibroblasts require type-I collagen as additional factor to maximally induce the proteinase, which makes pathophysiological sense as a paradigm between an enzyme and its substrate in a mutual demanding in order to maintain tissue homeostasis (Fig. 1). Still unknown is how TGF-β in concert with TNF-α enhances MMP-9 expression by fibroblasts of human and rodent skin, while TGF-β alone is not sufficient to induce the proteinase . According to present results, TGF-β is unable to cooperate with TNF-α to activate JNK or NF-κB pathway (Fig. 5B and data not shown). However, the magnified activation of JNK by the dermal fibroblasts cultured on type-I collagen as shown in Fig. 10 may explain, at least in part, the role of TGF-β through the capacious capacity to induce of type-I collagen (data not shown). The role of TGF-β is surely cell-type specific and depends on the context of other signaling, as we found TGF-β suppresses the IL-1-induced expression of MMPs by hepatic stellate cells [7, 26].
Still intriguing is how MMP-9, but not MMP-3, is specifically subjected to PAK1 control under the same TNF-α treatment (Fig. 2 and 4). The suppression of MMP-9 expression by PAK1 can be attributed, in part, to the transcription activation, since the ectopically expressed PAK1 mutant impairs the proximal 5'-promoter activities of MMP-9, and completely suppresses the accumulation of the mRNA and protein of MMP-9. Such suppression is in line with the impaired activation of JNK, but not NF-κB (Fig. 5 and 8). This notion is further reinforced by our inhibition experiment, by which JNK inhibitor thoroughly disrupts expression of MMP-9 (Fig. 5). Clues of PAK1 in regulates MAP kinase come from the work on yeast mating as well as reconstitution experiments [16, 17, 31, 32]. In addition to JNK, PAK1 has been shown to induce activation of p38 MAP kinase pathways [33, 34]. In our study, neither expression of p38 MAP kinase variants nor inhibition with specific inhibitor for the kinase does affect the MMP-9 at all (Fig. 4 and 7).
It is unknown, to date, how TNF-α promotes activation of PAK1 protein . Phosphorylation of thronine-423 in the active loop has been demonstrated as an indicator for activation of PAK1 [15, 24]. In addition to p21 GTPase binding and thereafter induced auto-phosphorylation of PAK1, PDK was also found to phosphorylate thronine-423 of PAK1 . In this study we found TNF-α exerts sequential phosphorylation of PAK1, started at thronine-212 (10-min) and followed by thronine-423 phosphorylation (20-min). Following PAK1 activation is the accumulation of the protein of the kinase per se. We concluded that TNF-α induced accumulation of PAK1 is regulated at post-transcriptional level. Such conclusion is based on the following evidences: (1) Cycloheximide, which suppresses protein synthesis in general, does not affect PAK1 protein under TNF-α stimulation; (2) The constitutively expressed PAK1 under viral promoter is also up regulated by TNF-α, and as a control the p38 MAP kinase is not altered at all, which strongly supports the mechanism of protein stabilization; (3) Up regulation of PAK1 by TNF-α or IL-1 seems ubiquitous, since it is detected by human dermal fibroblasts, and keratinocytes, as well as rat hepatic stellate cells; (4) TNF-α induced accumulation of 66-kDa PAK1 is inversely correlated with the loss of the degradation band at 25-kDa (Fig. 8A). All these indicate that TNF-α may somehow attenuate the degradation of PAK1 protein. Clearly, the TNF-α induced stabilization of PAK1 is independent of its intrinsic kinase and p21 GTPase-binding capacity. Most of PAK1 is compartmented in cytosolic pool, of which is promptly induced by TNF-α; while the kinase in the membrane fractions is not regulated (data not shown). To date, it is unknown how TNF-α regulates PAK1 stability.
We identified a novel mechanism of MMP-9 expression controlled by TNF-α through stabilization and activation of PAK1 which in turn activates JNK pathway leading the transcription of MMP-9.
Materials and reagents
Cytokines were purchased from R&D Systems. Antibodies against PAK1, p38 MAP kinase, I-kappaB, MMP-3 and MMP-9 were purchased from Santa Cruz Biotechnologies. Antibodies for JNK, phospho-JNK (Phospho-SAPK/JNK (Thr183/Tyr185), phospho-PAK1 (Thr423)/PAK2 (Thr402) were from Cell Signaling Technology. Antibodies for phosphor-212-threonine-PAK1 were from Sigma. Antibodies for GAPDH were from Chemicon. SuperSignal West Fermto Maximum Sensitivity Substrate was from PIERCE. The 5'-670 bp promoter of human MMP-9 was cloned into plasmid pGL2/firefly luciferase (pGL-5'670-MMP9). The Dual-Luciferase® Reporter (DLR) Assay System was from Promega. Lentivirus encoding PAK1 variants including wild type, triple mutant (H83L, H86L and K299R) and K299R were kindly provided by G. Bokoch (The Scripps Research Institute, La Jolla, CA). Collagenase, inhibitors for IKK (#401486), JNK inhibitors (SP600125), and p38 MAP kinases (SB 239063) were from Calbiochem.
Clinical biopsies including normal skin and chronic wounds were collected according to the protocol approved by Internal Review Board at the University of Southern California and are consent by patients. The 6-mm punch biopsies were placed in 2-ml DMEM with antibiotics (200 U/ml penicillin G sodium, 200 U/ml streptomycin sulfate and 0.5 μg/ml amphotericin B). To accumulate secreted factors the biopsies were incubated in the medium for 6 hours with supply of 5% CO2 at 37°C. The conditioned medium was cleared of debris by centrifugation at 5,000 g prior to zymography or Western blot analysis.
Cell culture, lentiviral transduction, and luciferase assay
To isolate human dermal fibroblasts, full thickness skin was treated by 20 mM EDTA in DMEM at 37°C for 3 hrs. After removal of epithelial sheets the dermal tissue was incubated in collagenase (2 mg/ml) in DMEM at 37°C for 16 hrs. The resultant cells were seeded on dishes and cultured in DMEM with 10% FBS. The second passage of cells was used for experiments. Immortalized human keratinocytes were kindly provided by Dr. David Woodley at USC. Primary rat hepatic stellate cells were supplied from the USC Research Center for Alcoholic and Pancreatic Diseases, and cultured in DMEM with 10% FBS. Kratinocytes and human dermal fibroblasts were transduced by lentivirus prepared in 293T cells. To ensure the high transducing efficiency the cells were infected twice. Efficiency of transduction was measured by immunostaining with anti-PAK1 as well by expression of green fluorescent protein as an indicator. To create 3-dimensional culture, fibroblasts were embedded in type-I collagen as previously described . To measure the promoter activities the dermal fibrobalsts were transfected by a reporter plasmid together with pGL/CMV-renilla luciferase as a reference. Luciferase activities were measured by the dual luciferase assay kit.
Cell treatment and real-time RT-PCR
Cells on plastic or 3D ECM were treated with TNF-α (10 ng/ml), IL-1α (10 ng/ml), TGF-β1 (1 ng/ml) in DMEM with 1% FBS for 16 hrs. Total RNA was extracted using TRIZOL reagent according to the manufacturer's instructions (Invitrogen Life Technologies). First-strand cDNA was produced using First-Strand cDNA Synthesis by SuperScript II Reverse Transcriptase with random primers. Two micrograms of total RNA was used for each reverse transcription reaction mixture (20 μl). Real-time PCR was carried out using an ABI Prism 7900 HT (Applied Biosystems). 10 μl reactions were set up in 384-well PCR plate using the following final concentrations: 1 μmole each of forward and reverse primers, 1× SYBR Green master mix (qPCR Mastermix Plus for SYBR Green I, Eurogentec), and 5 ng of cDNA. For each condition three duplicates were used to minimize the variation. Cycling conditions were as follows: initial step (50°C for 2 min), hot activation (95°C for 10 min), amplification (95°C for 15 s, 60°C for 1 min) repeated 40 times, and quantification with a single fluorescence measurement. Data were analyzed using ABI Prism SDS 2.1 software. The relative gene expression was calculated by the ΔCt method. Briefly, the resultant mRNA was normalized to its own GAPDH. Final results were expressed as n-fold difference in gene expression relative to GAPDH mRNA and calibrator as follows: n-fold = 2-(ΔCt sample-ΔCt GAPDH), where ΔCt values of the sample and the calibrator were determined by subtracting the average Ct value of the transcript under investigation from the average Ct value of the GAPDH gene for each sample. For human MMP-9 the forward primer has the sequence GGG AGA CGC CCA TTT CG and the reverse primer is CGC GCC ATC TGC GTT T. For GAPDH, the forward primer, GAA GGT GAA GGT CGG AGT 3', and backward primer GAA GAT GGT GAT GGG ATT TC 3' 20 mer. For MMP-14, the primers are TGG AGG AGA CAC CCA CTT TGA, and GCC ACC AGG AAG ATG TCA TTT C. For MMP-2, the primers are GAG AAC CAA AGT CTG AAG AG, and GGA GTG AGA ATG CTG ATT AG. For MMP-3, the primers are GCT GCA AGG GGT GAG GAC AC, and GAT GCC AGG AAA GGT TCT GAA GTG. The primers for TIMP-1 are TCT GGC ATC CTC TTG TTG CTA T, and CCA CAG CGT CGA ATC CTT.
This work was supported by grants from National Institutes of Health (R01 grants, AR051558 to YPH and DK069418 to YPH, GM50967 to WLG), and the Wright Foundation (YPH). We acknowledge the NIAAA-supported Non-parenchymal Liver Cell Core (R24 AA12885) for hepatic stellate cells. We thank Wes Grimm for proof reading.
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