Published online before print February 8, 2007
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B pathway in human THP-1 cellsDepartment of Animal Sciences, Rutgers University, New Brunswick, New Jersey, USA
2 Correspondence: Department of Animal Sciences, Rutgers University, New Brunswick, NJ 08901-8525, USA. E-mail: bagnell{at}aesop.rutgers.edu
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B is important in MMP gene regulation in macrophage cells. Thus, a monocyte/macrophage cell line, THP-1, was used to study the molecular mechanism of RLX action on MMP-2 and MMP-9 expression. After 24 h incubation with porcine RLX (100 ng/ml), conditioned media (CM) and THP-1 cells were collected. Gelatin zymography demonstrated an increase in pro-MMP-9 activity in response to RLX in CM, and no significant change in pro-MMP-2 expression was observed. Immunoblot analysis also revealed an increase in pro-MMP-9 in CM from RLX-treated THP-1 cells. Gel EMSA showed that NF-B DNA-binding activity was elevated in THP-1 cells treated with RLX for 10 min and reached a peak at 30 min. The NF-B DNA complex was supershifted using antibodies against NF-B subunits p50 and p65. Increased expression of the p50 and p65 NF-B subunits was also detected in THP-1 cells after RLX treatment. Incubation with RLX (90 min) reduced THP-1 expression of the NF-B inhibitor protein, IB-
. Using a specific NF-B inhibitor, pyrrolidine dithiocarmate (PDTC) inhibited nuclear binding of NF-B. Pre-exposure to PDTC suppressed pro-MMP-9 activity and protein levels in RLX-treated THP-1 cells. In conclusion, these data suggest that RLX-induced tissue remodeling through increasing MMP-9 expression is dependent on NF-B activation.
Key Words: tissue remodeling • inflammation • macrophage • gelatinase activity
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Monocytes and macrophages exert their effects on the ECM by secreting biologically active substances, such as matrix metalloproteinases (MMPs), at sites of wounding and inflammation [6 , 7 ]. The MMPs are a family of proteolytic enzymes, which degrade ECM components and target multiple regulatory proteins in the ECM, including cytokines, latent growth factors, and cell-matrix adhesion molecules. The gelatinases, MMP-2 and MMP-9, are the primary MMPs responsible for degradation of Type IV collagen, the major component of basement membranes [8 ]. Thus, MMPs produced by monocytes and macrophages may facilitate tissue growth and repair, as well as tumor invasion and metastasis [9 ].
Relaxin (RLX), a member of the insulin superfamily, is a key regulator of tissue remodeling. In rats, RLX promotes widespread collagen reorganization in cervical tissues at term [10 ]. Impaired nipple development in RLX null mice and in RLX-receptor knockout mice is associated with failure of collagen degradation [11 , 12 ]. Moreover, studies in RLX null mice demonstrate an age-related increase in fibrosis in nonreproductive tissues, including the skin [13 ], lung [14 ], heart [15 ], and kidneys [16 ], and this fibrosis is associated with organ dysfunction. Although the mechanisms of RLX action on tissue remodeling are not fully understood, it has been shown that RLX increases MMP expression in vivo [17 ] and in vitro [18 ] to facilitate tissue remodeling.
The human monocyte/macrophage cell line, THP-1, has high affinity for RLX [19 ], and RLX binding triggers intracellular signaling events in THP-1 cells by activating protein kinases [20 21 22 ]. In addition, RLX induces vascular endothelial growth factor expression in THP-1 cells [23 ]. Recently, it was reported that RLX stimulates adhesion and migration in THP-1 cells and in human PBMC [24 ]. Taken together, these data suggest that RLX may play a functional role in regulating monocyte/macrophage activity. However, whether these cells contribute to RLX-induced tissue remodeling is not clear.
The NF-B family of transcription factors regulates several of the tissue-remodeling genes, including MMP-9. NF-B regulates genes that play key roles in the inflammatory response, angiogenesis, tumor invasion, and metastasis [25
]. In addition, there is evidence that activation of NF-B plays an important role in tissue remodeling and wound healing [26
, 27
]. Upon cell stimulation, the IB unit of NF-B is phosphorylated, ubiquitinated, and degraded, allowing free NF-B to be transported to the nucleus, where it activates target genes by binding to cognate DNA regulatory elements. RLX induces NF-B activation in HUVEC and HeLa cells [28
]. Whether NF-B activation is involved in RLX-stimulated tissue remodeling is unknown.
As an extension of work reported earlier [29
] and given the importance of RLX in tissue remodeling and the role of monocytes/macrophages in tissue growth and repair, the objectives of this study were to evaluate the effects of RLX on gelatinases (MMP-2 and MMP-9) in human monocytic THP-1 cells and determine the role of NF-B activation in RLX-induced MMP expression.
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B p50 (sc-1190), and p65 (sc-7151) subunits, IB- (sc-203), and ß-actin (sc-1615) were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat antirabbit or rabbit antigoat IgG-HRP-conjugated secondary antibodies were purchased from Zymed (South San Francisco, CA, USA). Human MMP-9 enzyme from Biogenesis (Kingston, NH, USA) and human MMP-2 enzyme from PanVera Corp. (Madison, WI, USA) were used as positive controls for zymography and immunoblotting. ECL Western blotting reagents and Nick spin columns were obtained from Amersham (Piscataway, NJ, USA). The oligonucleotide NF-B-binding motif was purchased from Promega (Madison, WI, USA). Trypan blue and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-B nuclear translocation, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Unless otherwise indicated, chemicals and reagents were purchased from Sigma Chemical Co. and Invitrogen.
Cell culture
The THP-1 human monocyte cell line was grown in RPMI-1640 medium supplemented with 2% glutamine, 2% penicillin/streptomycin, and 10% FBS in a 5% CO2-humidified atmosphere at 37°C. THP-1 cells were maintained in logarithmic growth (1x105 cells/ml) by passage every 3–4 days. When cells were 80% confluent, the medium was replaced with phenol red-free RPMI medium containing 10% charcoal-stripped FBS. Cells were serum-deprived overnight prior to incubation with porcine RLX (1–300 ng/ml). In some experiments, cells were preincubated with or without the NF-B inhibitor PDTC (10 µM in RPMI-1640 medium) before RLX treatment. THP-1 cell viability was assayed by trypan blue exclusion and was found to be greater than 90% in the presence of PDTC.
Gelatin zymography
Following treatment, THP-1 conditioned medium (CM) was collected and concentrated 10 times using Centricon centrifugal filter devices, YM-30, with a 30-kDa pore diameter cutoff (Millipore, Bedford, MA, USA). Proteins in the concentrated media were quantified using the Bio-Rad dendritic cell protein assay (Bio-Rad, Hercules, CA, USA). Zymography was performed as described previously [17
]. Briefly, samples (20 µg protein) were mixed with equal amounts of SDS sample buffer and loaded under nondenaturing conditions using precast polyacrylamide zymogram gels supplemented with 1% gelatin as the proteinase substrate. Following electrophoresis, gels were washed in renaturing buffer for 30 min to remove the SDS and then incubated in developing buffer overnight at 37°C. Gels were stained with 0.1% Coomassie brilliant blue R 250 followed by destaining. Human MMP-2 and MMP-9 enzyme standards were used as positive controls. Relative gelatinolytic activity was quantified by densitometry.
Immunoblot analysis
THP-1 cell CM was concentrated 10 times using Centricon centrifugal filter devices, and 50 µg protein/lane was subjected to 10% SDS-PAGE. The fractionated proteins were transferred to nitrocellulose membranes (Amersham). Human MMP-9 and MMP-2 enzymes were used as positive controls. Membranes were blocked for 1 h at room temperature in buffer (TBST) containing 10% skimmed milk powder. After washing in TBST, the membranes were incubated overnight at 4°C with one of the following antibodies diluted in 1% TBST: antihuman MMP-2 (1:2000), antihuman MMP-9 (1:2000), anti-NF-B p50 subunit (1:1000), anti-NF-B p65 subunit (1:1000), or anti IB- (1:1000). The membranes were incubated further with HRP-conjugated secondary antibodies (1:5000) in 5% milk-TBST for 1 h at room temperature. Immunoreactive bands were visualized using ECL (Amersham). Membranes were stripped with buffer (100 mM 2-ß-ME, 2% SDS, 62.5 mM Tris-HCl) and reprobed with anti-ß-actin (1:5000) to determine the amount of protein loaded on the gels.
EMSA and supershift assay
Nuclear proteins were extracted after cell treatments as described by Sasaki et al. [30
]. Briefly, THP-1 cells (1x106) treated with RLX for the indicated times were washed with PBS, homogenized in a hypotonic buffer [10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1% Nonidet P-40, and 5% protease inhibitor mixture], and incubated for 10 min on ice. Nuclei were collected by centrifugation at 800 g for 5 min, washed with a hypotonic buffer, and resuspended in a low-salt buffer [20 mM HEPES (pH 7.9), 0.02 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, and 5% protease inhibitor mixture]. An equal volume of high-salt buffer [20 mM HEPES (pH 7.9), 800 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, and 5% protease inhibitor mixture] was added by vortex mixing. Nuclei were incubated for 30 min on ice and centrifuged at 13,000 g for 30 min. The supernatants were collected, and EMSA was performed as described by the manufacturer (Gel Shift Assay Systems, Promega). The double-stranded consensus oligonucleotide containing the NF-B-binding motif (5'-AGTTGAGGGGACTTTCCCAGGC-3') was labeled using T4 polynucleotide kinase and [
-32P] ATP (Amersham; specific activity of 3000 Ci/mmol), and a spin column was used to purify the probe for EMSA. Nuclear proteins (10 µg) were preincubated with the reaction buffer for 15 min at room temperature, followed by incubation with the 32P end-labeled probe at room temperature for 30 min. The DNA–protein complex (20 µl total vol) was resolved on a nondenaturing 6% (v/v) polyacrylamide gel run for 30 min at 250 V in 0.5x Tris-boric acid-EDTA buffer [2.5 mM Tris, 2.5 mM H3BO3, 2 mM EDTA (pH 8.5)]. The gel was then vacuum-dried and exposed to X-ray film (Kodak XAR-5) at –80°C for 24 h. For competition assays, unlabeled excess NF-B probes (10x) were added simultaneously with the labeled probe in the binding buffer for EMSA. Supershift assays were performed by the addition of antibodies (2 µg) against the p50 or p65 subunit of NF-B to the binding reaction mixture.
Statistical analysis
All experiments were performed at least three times, and each treatment group was run in triplicate. Data expressed as the mean ± SEM were analyzed by ANOVA and tested for differences using Fisher’s least significant difference test for multiple comparisons. A statistical probability of P< 0.05 was considered significant.
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Figure 1. Zymographic and immunoblot analysis of pro-MMP-2 and pro-MMP-9 expression in CM from THP-1 cells treated with or without RLX. In the zymograms, clear zones against the dark background indicate gelatinolytic activity, which was quantified by densitometry and graphed in OD units (mean±SEM). Values with different letters are significantly different (P<0.05). (A) Dose-dependent effects of RLX (1–300 ng/ml for 24 h) on pro-MMP-9 and pro-MMP-2 activity. (B) Time-dependent effects of RLX (+; 100 ng/ml) on pro-MMP-9 activity. (C) Pro-MMP-9 protein in CM from THP-1 cells, incubated in the absence (C; control) or presence of RLX (100 ng/ml) for 24 h, was detected by immunoblot analysis.
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Effect of RLX on NF-B DNA-binding activity
NF-B has been implicated in the transcriptional regulation of MMP gene expression. To investigate the role of NF-B activation in RLX-induced, pro-MMP-9 expression in THP-1 cells, EMSA was performed. As shown in Figure 2
, NF-B DNA-binding activity in THP-1 cells increased in response to RLX (100 ng/ml). Time-course studies showed that 10 min of incubation with RLX increased NF-B DNA-binding activity, reaching a peak after 30 min. The specificity of NF-B binding was established in competition experiments, which showed that RLX-induced, NF-B-binding activity (Fig. 3
, Lane 1) was reduced by the addition of excess, unlabeled (cold) NF-B probe (Fig. 3
, Lane 2). To determine which components of the NF-B family were important for DNA binding, a supershift assay using specific antibodies against individual subunits of NF-B was performed. The results showed that addition of p65 (Fig. 3
, Lane 3) or p50 antibodies (Fig. 3
, Lane 4) resulted in supershifting of NF-B DNA-binding complexes, indicating that p65 and p50 subunits were involved.
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Figure 2. Effect of RLX on NF-B DNA-binding activity. THP-1 cells were incubated in the absence (–; control) or presence (+; 100 ng/ml) of RLX for 10, 30, or 60 min. Nuclear extracts (10 µg) were incubated with a 32P-labeled oligonucleotide containing the NF-B-binding motif, and EMSA was performed to determine NF-B DNA-binding activity.
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Figure 3. Specificity of NF-B-binding activity in RLX-treated THP-1 cells. Nuclear extracts from RLX-treated (+; 100 ng/ml for 30 min) THP-1 cells were incubated with a 32P-labeled oligonucleotide containing the NF-B-binding motif and subjected to EMSA (Lane 1). Competition assays were performed by adding unlabeled (cold) NF-B oligonucleotide (10x) to the reaction mixture (Lane 2). In supershift assays, NF-B p65 antibody (Lane 3) or NF-B p50 antibody (Lane 4) was added to the reaction mixture.
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B subunits p65 and p50 and on inhibitor protein IB-B protein subunits p65 and/or p50, THP-1 cells were incubated with RLX (100 ng/ml), and p65 and p50 proteins were measured. Incubation with RLX increased THP-1 cell expression of the p65 (P<0.05; Fig. 4A
) and p50 (P<0.05; Fig. 4B
) subunits. Expression of the p65 and p50 subunit proteins reached a maximum after 3 h of incubation with RLX.
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Figure 4. Effect of RLX on expression of NF-B p65 and p50 subunits and of IB-. THP-1 cells were incubated in the absence (–; control) or presence (+; 100 ng/ml) of RLX for various time periods (30–240 min). NF-B p65 subunit (A), p50 subunit (B), and IB- (C) proteins were detected by immunoblotting. All bands were quantified by densitometry. The NF-B p65 and p50 subunits and IB- protein were expressed relative to ß-actin (mean±SEM). Values with different letters are significantly different (P<0.05).
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B in THP-1 cells could be a result of an effect on IB-, the expression of this NF-B inhibitor protein in response to RLX was evaluated. The results showed that RLX reduced IB- protein in THP-1 cells after 90 min of incubation (P<0.05; Fig. 4C
). This inhibitory effect of RLX on THP-1 cell IB- expression was sustained through 120 min and was followed by the reappearance of IB- protein after 4 h.
NF-B inhibitor PDTC suppresses RLX-induced, pro-MMP-9 expression
To confirm the relationship between NF-B activation and RLX-induced, pro-MMP-9 expression, the effect of a specific NF-B inhibitor, PDTC, was examined. Incubation with PDTC inhibited the nuclear binding of NF-B induced by RLX, as shown by gel shift assay (Fig. 5A
). In addition, gelatin zymography revealed that RLX-induced, pro-MMP-9 activity in media from THP-1 cells was reduced when cells were pretreated with PDTC (Fig. 5B)
. Likewise, immunoblotting studies showed that blocking THP-1 cell NF-B activity with PDTC prevented the RLX-induced increase in latent MMP-9 protein secreted into the media (P<0.05; Fig. 5C
).
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Figure 5. Effect of NF-B inhibitor, PDTC, on RLX-induced, NF-B DNA-binding activity and pro-MMP-9 expression. THP-1 cells were preincubated with PDTC (+; 10 µM) or control media (–) for 30 min, followed by incubation in the absence (–) or presence (+; 100 ng/ml) of RLX for 30 min (EMSA) or 24 h (zymography and immunoblotting). (A) NF-B DNA-binding activity detected by EMSA. (B) Pro-MMP-9 activity in CM was detected by zymography. (C) Pro-MMP-9 protein in CM was detected by immunoblotting, subjected to densitometric analysis, and expressed relative to ß-actin (mean±SEM). Values with different letters are significantly different (P<0.05).
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RLX increases secretion of active MMP-2 and MMP-9 during uterine growth [17
]. Likewise, in MCF-7 breast cancer cells, RLX amplifies MMP-2 and MMP-9 protein secretion and increases invasive activity in vitro [36
]. In the present studies, zymography and immunoblotting revealed that latent forms of MMP-2 (72 kDa) and MMP-9 (92 kDa) were detectable in THP-1 cell media. These data support other studies indicating that THP-1 cells secrete pro-MMP-9 [37
, 38
] and that culture conditions do not favor MMP zymogen activation [39
]. Although pro-MMP-9 can be activated by agents such as p-aminophenylmercuric acetate [40
], secretion of MMPs in latent, zymogen forms provides a means of regulating MMP function to avoid tissue damage as a result of excessive MMP activity. In the studies reported here, MMP-2 and MMP-9 zymogens were secreted constitutively by THP-1 cells. However, only pro-MMP-9 was sensitive to stimulation by RLX. These results are similar to those reported for THP-1 cells and TNF- in that secretion of MMP-9, but not MMP-2, was decreased in THP-1 cells in the presence of neutralizing TNF- antibodies [41
]. The biphasic effects of RLX, with lower doses increasing THP-1 cell pro-MMP-9 expression and higher concentrations having little or no effect, have been reported before for other RLX targets in vitro and in vivo. In THP-1 cells, endotoxin-stimulated cytokine production was suppressed by low doses of RLX (5–10 nM), and higher RLX concentrations were ineffective [28
]. Likewise, RLX elicited a biphasic dose response, in which only low doses of the hormone were effective in dilating renal vasculature [42
], increasing cardiac output, and decreasing arterial load in rats [43
]. Although the explanation for the biphasic effect of RLX is unclear, one possibility is that high concentrations of RLX may down-regulate RLX receptors in target cells and lead to reduced responsiveness [43
].
Although RLX is considered a classical hormone of pregnancy, the pleiotropic roles of RLX in diverse physiological and pathological processes go far beyond its conventional roles in reproduction. It has been shown that RLX interferes with neutrophil activation and/or migration into inflamed tissue [44
]. Moreover, RLX is reported to stimulate leukocyte adhesion and migration through a RLX receptor (LGR7)-dependent mechanism [24
]. In addition, RLX decreased endotoxin-induced TNF- production by THP-1 cells [28
] and reduced joint inflammation in a rodent model of adjuvant-induced arthritis [45
]. Taken together, data suggest that RLX may play a role in regulating immune responses. In addition, RLX protein and gene expression have been identified in normal and neoplastic breast tissues in humans [46
], and elevated concentrations of serum RLX have been linked to high incidence of metastatic disease in breast cancer patients [36
], suggesting a role of RLX in cancer pathology. Given the importance of monocytes and macrophages in immunity, as well as in solid-tumor invasion, it will be of great value to explore RLX-macrophage interaction in immune responsiveness and cancer metastasis.
Activation of NF-B is reported to be important in the induction of MMP-9 gene expression in THP-1 cells [47
, 48
]. In support of this observation, there is a consensus NF-B-binding site in the promoter region of the MMP-9 gene [49
]. RLX activates NF-B in endothelial and epithelial cells [50
]. However, the role of this transcription factor in RLX-induced MMP expression in THP-1 cells is unknown. In this study, we presented several lines of evidence to show that RLX induction of pro-MMP-9 expression in THP-1 cells is dependent, at least in part, on NF-B activation. First, the results from EMSA demonstrated that RLX treatment induces activation of NF-B in THP-1 cells. In addition, immunoblot analysis revealed that RLX increased p50 and p65 subunit proteins of NF-B. Second, the level of IB- was decreased in THP-1 cells treated with RLX. It has been shown that inhibiting IB- protein degradation can block NF-B activation in LPS-stimulated macrophage cells [48
]. Thus, the activation of NF-B by RLX via decreased IB- expression may eventually lead to enhanced MMP-9 expression. The role of NF-B activation in MMP-9 expression was confirmed further by incubating RLX-treated THP-1 cells with PDTC, a chemical that stabilizes the NF-B/IB- complex [51
] and inhibits the nuclear translocation of activated NF-B. The incubation of THP-1 cells with PDTC inhibited the DNA-binding activity of NF-B induced by RLX. These data clearly indicate the importance of NF-B in RLX-induced MMP-9 expression in THP-1 cells; however, the involvement of other transcription factor(s) cannot be ruled out.
In summary, the results presented here indicate that RLX increased MMP-9 expression in THP-1 monocytes, and this effect was time- and dose-dependent. Moreover, we present evidence that RLX increases NF-B activation in these cells by increasing the p65 and p50 subunit proteins and by reducing the expression of the NF-B inhibitor, IB-. Finally, experiments confirmed that RLX-stimulated MMP-9 expression is dependent on NF-B activation in THP-1 cells. The recruitment and activation of macrophage cells are important during normal tissue development [34
] and for tumor invasion [4
], where tissue remodeling is essential. Thus, these findings provide a foundation for further studies to examine the interaction of RLX with macrophage cells in orchestrating tissue remodeling in diverse physiological and pathological processes.
Received September 8, 2006; revised October 5, 2006; accepted January 17, 2007.
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B:linking inflammation and immunity to cancer development and progression Nat. Rev. Immunol. 5,749-759[CrossRef][Medline]B ameliorates myocardial hypertrophy in response to chronic infusion of angiotensin II Cardiovasc. Res. 67,689-698B1 (p50) limits the inflammatory and fibrogenic responses to chronic injury Am. J. Pathol. 166,695-708B in human THP-1 cells Ann. N. Y. Acad. Sci. 1041,314-316[CrossRef][Medline]B p65 (RelA) transcription factor is constitutively activated in human gastric carcinoma tissue Clin. Cancer Res. 7,4136-4142-stimulated matrix metalloproteinase 9 (MMP-9) expression by mitogen-activated protein kinases in THP-1 monocytic cells Biol. Chem. 387,69-78[CrossRef][Medline]B- degradation in LPS-stimulated peritoneal macrophages Cell. Signal. 15,299-306[CrossRef][Medline]B Circ. Res. 92,32-40B transcription factor and HIV-1 EMBO J. 10,2247-2258[Medline]
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