Stress-Activated Signaling Pathways Mediate the Stimulation of Pregnancy-Associated Plasma Protein-A Expression in Cultured Human
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《内分泌学杂志》
Endocrine Research Unit (Z.T.R., L.K.B., C.A.C.), Division of Endocrinology, Metabolism
Nutrition, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Department of Molecular Biology (C.O.), University of Aarhus, DK-8000 Aarhus C, Denmark
Abstract
Pregnancy-associated plasma protein-A (PAPP-A) is an IGF binding protein protease that appears to function as a posttranslational modulator of IGF bioavailability in response to injury. A previous study indicated that the proinflammatory cytokines, TNF and IL-1, were potent stimulators of PAPP-A expression in cultured human fibroblasts. In this study, we investigated the intracellular signaling pathways mediating cytokine-stimulated PAPP-A expression. Treatment of human fibroblasts with TNF and IL-1 (1 nM) had little or no effect on phosphatidylinositol 3-kinase and Erk1/2 activation, pathways commonly associated with proliferation. On the other hand, TNF and IL-1 induced p38, c-Jun N-terminal kinase (JNK), and nuclear factor (NF)B activation, pathways more closely related to stress response. An inhibitor of p38 activation (SB203580) had no effect on TNF- or IL-1-stimulated PAPP-A expression. The JNK inhibitor, SP600125, had no effect on IL-1- or TNF-stimulated PAPP-A mRNA expression. However, SP600125 effectively inhibited IL-1-induced PAPP-A protein expression. MG-132, a proteasome inhibitor that blocked degradation of the intrinsic NFB inhibitor, IB, and thereby prevented NFB activation, was a potent inhibitor of both TNF- and IL-1-stimulated PAPP-A mRNA and protein expression and IGF binding protein-4 protease activity. MG-132 had no effect on JNK phosphorylation or p38 activation, and SB203580 and SP600125 had no effect on IB degradation, documenting inhibitor specificity. BAY11–7082, another inhibitor of NFB activation, also inhibited TNF- and IL-1-stimulated PAPP-A expression and IGF binding protein-4 protease activity. These data indicate that NFB activation is the primary mediator of cytokine-stimulated PAPP-A expression in human fibroblasts.
Introduction
PREGNANCY-ASSOCIATED plasma protein-A (PAPP-A) is an IGF binding protein (IGFBP) protease expressed by a variety of cell types including normal human fibroblasts, osteoblasts, and vascular smooth muscle cells (1, 2, 3). In vitro, PAPP-A functions to cleave IGFBP-4, an inhibitory IGFBP, consequently increasing bioavailable IGF for receptor activation (2, 4). Several recent studies suggest a similar role for PAPP-A in modulating site- and event-specific IGF signaling during injury repair responses in vivo. Thus, PAPP-A expression is increased in healing human skin after a first intention wound in association with activated fibroblasts and macrophages (5). PAPP-A is also up-regulated in unstable atherosclerotic plaque and smooth muscle cells in response to vascular injury (3, 6), suggesting a role for PAPP-A in the normal response to injury in multiple tissues.
Little is known about the signaling mechanisms regulating PAPP-A expression. We have recently shown that PAPP-A gene and protein expression in cultured human fibroblasts is stimulated by proinflammatory cytokines involved in injury repair responses, namely TNF and IL-1 (7). The aim of this study was to determine the specific intracellular signaling pathways mediating cytokine-regulated PAPP-A expression. The focus was on the major common pathways associated with cytokine stimulation in various cell types including phosphatidylinositol 3-kinase (P13K), MAPK, and nuclear factor (NF)B pathways (8, 9, 10, 11, 12, 13). Herein we show that activation of NFB is the primary mediator of TNF- and IL-1-stimulated PAPP-A expression in human fibroblasts.
Materials and Methods
Materials
TNF and IL-1 were purchased from Research Diagnostics Inc. (Flanders, NJ), and RIA-grade BSA was from Sigma Chemical Corp. (St. Louis, MO). Antibodies against phosphorylated and total Akt, Erk1/2, p38, and c-Jun N-terminal kinase (JNK) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against NFB, inhibitory B (IB)-, and phospho-Hsp27 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against IB was a kind gift from Dr. David McKean (Mayo Clinic, Rochester, MN). The specific inhibitors MG-132, SB203580, and PD98059 were purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA), and SP600125 and BAY11–7082 were obtained from Calbiochem (San Diego, CA). Tissue culture supplements and fetal bovine serum were obtained from Life Technologies (Grand Island, NY). Reagents for SDS-PAGE were purchased from Bio-Rad Laboratories (Richmond, CA).
Cell cultures
Primary cultures of adult human dermal fibroblasts were purchased from the Human Genetic Mutant Cell Repository (Camden, NJ) and cultured as reported previously (1, 7, 14, 15). For all experiments, cells were washed twice and incubated in serum-free medium containing 0.1% BSA overnight before experimental treatment. The cells were again washed and changed to serum-free medium plus experimental additions for the indicated times. For some studies, cells were pretreated for 60 min with specific inhibitors before the addition of cytokines. Dose-response experiments were initially performed for each inhibitor to determine the minimum effective concentration. At the end of the incubation, conditioned media were collected, centrifuged to remove any debris and stored at –70 C. Cell numbers were determined at the time of media collection using a Coulter counter (Coulter Electronics, Hialeah, FL).
RNA isolation and cDNA synthesis
Total RNA was extracted from cells using the RNeasy minikit (QIAGEN, Valencia, CA) and treated with deoxyribonuclease (DNA-free, Ambion, Inc., Austin, TX). Four hundred nanograms of RNA were reverse transcribed using TaqMan reverse transcription reagents (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions.
Real-time PCR
Real-time quantitative PCR analyses were performed using the ABI PRISM 7700 sequence detection system and software (PE Applied Biosystems). Primer and probe sequences for specific detection and amplification of PAPP-A and 28S as well as assay validations were described previously (7, 15).
PAPP-A ELISA
PAPP-A levels in cell-conditioned media were measured using an ultra-sensitive ELISA kit kindly provided by Diagnostic Systems Laboratories, Inc. (Webster, TX). Minimum sensitivity is 0.24 mIU/liter with intra- and interassay coefficients of variation of 4.7 and 4.2%, respectively.
Western immunoblotting
After treatment, cells were lysed, subjected to SDS-PAGE, and transferred to polyvinyl difluoride, as described previously (16). Filters were blocked with 5% nonfat dry milk in Tris-buffered saline/0.1% Tween and probed with primary antibody at the supplier’s recommended dilution. After reaction with the appropriate secondary antibody conjugated with horseradish peroxidase, blots were visualized using enhanced chemiluminescence reagents (Amersham Biosciences Inc., Piscataway, NJ) and autoradiography.
NFB nuclear translocation
Human fibroblasts were pretreated ± inhibitors for 60 min and then stimulated with vehicle or 1 nM TNF or IL-1 for 10 min. After stimulation, cells were washed three times with cold PBS, lysed, and nuclear-specific proteins isolated using the protocol included with NucBuster (Novagen, Madison, WI). Detection of NFB in the nucleus was quantitated using the NoShift (Novagen) transcription factor assay kit. Briefly, 20 μg of nuclear protein was incubated with biotin-labeled wild-type DNA (10 pmol) alone or in combination with 10-fold molar excess biotin-labeled mutant DNA or cold wild-type DNA. Reactions were added to streptavidin-coated plates and incubated for an hour. Wells are washed three times and treated with primary antibody (anti-p65 subunit) for an hour and washed three times and exposed with the antimouse secondary antibody. After five washes, 3', 5, 5'-tetramethyl benzidine substrate was added and the reaction stopped after 15 min by addition of 1 N HCL. The absorbance was read at 450 nm (EL808, BIO-Tek Instruments, Inc., Highland Park, VT) to quantify nuclear NFB levels.
IGFBP-4 protease assay
Cell-free IGFBP-4 proteolysis was assayed as previously described (1, 2, 3, 15). Conditioned medium was incubated at 37 C for 2 h with 125I-IGFBP-4 and 5 nM IGF-II, without and with neutralizing PAPP-A antibody (1). IGF-II binds IGFBP-4 and increases its susceptibility to cleavage by PAPP-A in vitro (17). Reaction products were separated by SDS-PAGE and visualized by autoradiography. Extent of proteolysis, i.e. loss of intact and generation of 18-kDa radiolabeled fragments, was determined using enhanced laser densitometry (Ultroscan XL; Pharmacia LKB Biotechnology, Piscataway, NJ).
Promoter scan
Using nt 1–180 of the sequence encoding preproPAPP-A (accession no. BC078657), an expressed sequence tag sequence (accession no. CB988079), which extended approximately 400 bp upstream, was identified. The chromosomal sequence stretch further 1000 bp upstream (stretch between position 115994630 and 115995630 on the human chromosome 9) was analyzed for the presence of potential NFB binding sites using the Transcription Element Search System (http://www.cbil/upenn.edu/cgi-bin/tess/tess).
Statistical analysis
Results are expressed as mean ± SEM for the indicated number of experiments. Statistical analyses were performed using ANOVA and Dunnett for comparisons to control and Scheffe for multiple group comparisons. Results were considered statistically significant at P < 0.05.
Results
TNF- and IL-1-stimulated signaling pathways
In the first set of experiments, we determined the effect of TNF and IL-1 on major common pathways associated with cytokine stimulation including PI3K, MAPK, and NFB pathways in cultured human fibroblasts. To examine the activation of PI3K, Western blot analyses were performed using antibodies specific for phosphorylated Akt, a downstream substrate of PI3K (8, 16). Treatment of human fibroblasts with TNF or IL-1 for 10, 30, 60, or 120 min did not result in detectable Akt phosphorylation at Ser473 or Thr308 sites (data not shown). Reprobing with antibody against total Akt indicated that the protein was present and unaltered by treatment, and a positive control for stimulation of Akt phosphorylation (IGF-I) demonstrated that the signaling pathway was functional in these cells.
The MAPK family has three main subgoupings: Erk1(/2), JNK, and p38 kinase (9). The Erk1/2 pathway is most commonly linked to the regulation of cell proliferation, whereas p38 and JNK pathways are more closely related to stress (10). As shown in Fig. 1, stimulation with TNF and IL-1 had little or no effect on Erk1/2 phosphorylation, which appears to be constitutive in these cells. On the other hand, cytokine-mediated p38 phosphorylation was induced at 10 and 30 min with a decrease toward basal at 60 and 120 min. Endogenous JNK phosphorylation (46 and 54 kDa) was likewise transiently induced 10 and 30 min after cytokine treatment and undetectable again at 60 min. IL-1 appeared to be more effective than TNF in activating JNK, which was confirmed in further dose-response experiments (not shown). These data indicate that the PI3K and Erk1/2 pathways are not activated in human fibroblasts by TNF and IL-1. However, these cytokines do induce the protein kinases, p38 and JNK.
The NFB pathway is the predominant mediator of prooxidant stimuli, such as inflammatory cytokines (11). The principal mechanism by which NFB is activated is through phosphorylation of intrinsic inhibitors, the IBs (11). Normally, IB binds NFB and sequesters it in the cytoplasm. Phosphorylation of IB identifies it for ubiquitination and subsequent proteasome-mediated degradation, freeing NFB to translocate into the nucleus in which it regulates gene expression. IB (37 kDa) and IB (46 kDa) are prototypes of the inhibitory IB family. Western blotting of lysates of cells after stimulation with TNF or IL-1 indicated a rapid loss of IB within 10 min, which returned to detectable levels by 60 min (Fig. 2). TNF and IL-1 had little or no effect on IB. NFB translocation was confirmed using a transcription factor assay kit to detect nuclear NFB. As indicated in Table 1, TNF and IL-1 treatment increased levels of nuclear NFB 2- to 4-fold.
Inhibitors of intracellular signaling and PAPP-A expression
As shown in Fig. 3 and as reported previously (7), treatment of human fibroblasts with 1 nM TNF or IL-1 significantly increased PAPP-A levels 3- to 4-fold in 24-h conditioned medium. Although TNF and IL-1 stimulated p38 activity, SB203580 had no significant effect on PAPP-A expression when used at concentrations shown to inhibit p38 activation, as monitored by Hsp27 phosphorylation (see Fig. 5). Likewise, PD98059, an inhibitor of Erk1/2 activation, had no effect on TNF- and IL-1-induced PAPP-A. However, MG-132, a proteasome inhibitor that blocks degradation of IB and thus NFB activation, was a potent inhibitor of both TNF- and IL-1-induced PAPP-A expression. The JNK inhibitor, SP600125, effectively inhibited IL-1-stimulated PAPP-A levels in conditioned medium but had no effect on TNF-induced PAPP-A. To confirm the role of NFB activation, BAY11–7082, which inhibits inducible phosphorylation of IB, effectively inhibited TNF- and IL-1-stimulated PAPP-A expression (Fig. 4).
To document the specificity of the inhibitors as well as determine possible signaling cross-talk, we assessed the effect of MG-132, SB203580, and SP600125 on the NFB, p38, and JNK pathways. Western immunoblotting confirmed that MG-132 blocked the degradation of IB induced by TNF (not shown) and IL-1 (Figs. 5 and 6). The slower migrating form of IB with IL-1 and MG-132 treatment was confirmed by Western blot as phosphorylated IB, which is not altered by MG-132 (data not shown). The apparent lower level of IB in cells treated with IL-1 and MG-132 may be due to a reduced ability of the IB antibody to recognize the phosphorylated form of IB. MG-132 inhibits phosphorylated IB degradation in the proteasome, which results in inhibition of NFB activation (Table 1). BAY11–7082 similarly inhibited NFB activation (Table 1). On the other hand, MG-132 had no effect on p38 activity, as measured by phosphorylation of its downstream substrate, Hsp27 (12, 13), or JNK activation after treatment with IL-1 (Fig. 5). Likewise, SB203580 had no effect on IL-1-induced IB degradation or JNK phosphorylation (Fig. 5). SP600125 effectively inhibited JNK phosphorylation by IL-1 without affecting IB (Fig. 6) or NFB activation (Table 1).
To determine whether MG-132 and SP600125 inhibit cytokine-stimulated PAPP-A gene expression, real-time RT-PCR was performed on RNA isolated from human fibroblasts preincubated with MG-132 or SP600125 and then stimulated with TNF and IL-1 for 4 h. As shown in Fig. 7, MG-132 was able to prevent TNF- and IL-1-stimulated PAPP-A gene expression without altering basal expression. On the other hand, SP600125 had no effect on TNF- or IL-1-induced PAPP-A mRNA expression. Western blots done in parallel indicated that SP600125 inhibited IL-1-stimulated JNK phosphorylation as in Fig. 6, so lack of an effect on PAPP-A mRNA expression was not due to problems with the reagent.
Functional consequence of regulated PAPP-A expression through NFB activation
PAPP-A functions as an IGFBP protease with subsequent alteration in IGF bioactivity (2, 4, 14). To determine whether changes in functional activity of PAPP-A reflect expression data, cell-conditioned medium after treatment with IL-1 and TNF without and with MG-132 or BAY11–7082 was subjected to the 125I-IGFBP-4 protease assay (1, 2, 3, 15). As shown in Figs. 8 and 9, the IGFBP-4 remained intact during incubation in unconditioned media. Control conditioned media, with low levels of PAPP-A (Fig. 3), had low levels of proteolytic activity as indicated by the appearance of radiolabeled fragments. Treatment with IL-1 or TNF clearly enhanced proteolysis of IGFBP-4 over control. In samples from three separate experiments, this enhancement ranged from 2- to 4-fold. MG-132 completely inhibited TNF- and IL-1-stimulated proteolysis, with little apparent affect on basal proteolysis (Fig. 8). That this proteolytic activity was due to PAPP-A was confirmed using an inhibitory PAPP-A antibody (+ lanes). Figure 9 confirms the role of NFB activation using the alternative inhibitor, BAY11–7082. Again, IL-1 and TNF treatment enhanced IGFBP-4 proteolysis, and this increase was completely prevented with BAY11–7082. Direct addition of MG-132 or BAY11–7082 to the cell-free assay had no effect on basal or cytokine-stimulated IGFBP-4 proteolysis (data not shown).
Discussion
These data indicate that activation of NFB is the main determinant of TNF- and IL-1-induced PAPP-A gene expression and function in cultured human fibroblasts.
NFB is activated in response to prooxidative stimuli, such as inflammatory cytokines, and is associated with a variety of injury responses in vivo (11, 18, 19, 20, 21). In this study, the addition of TNF or IL-1 to human fibroblasts resulted in rapid degradation of IB and translocation of NFB to the nucleus. Treatment of cells with the proteasome inhibitor, MG-132, blocked TNF- and IL-1-induced IB degradation and NFB activation. This was associated with an inhibition of PAPP-A mRNA and protein expression and proteolytic activity. Similar effects were seen with the inhibitor of cytokine-induced IB phosphorylation, BAY11–1084. TNF and IL-1 had no effect on IB, suggesting that regulation of IB is responsible for cytokine regulation of NFB activation. Although it was reported that IL-1 induced IB and IB degradation, whereas TNF regulated only IB degradation (22), this was not observed in our study. One of the genes activated by NFB is the IB gene (23, 24), possibly explaining the transient nature of cytokine-induced degradation of IB. The NFB/IB autoregulatory system ensures that the induction of NFB is transient and that the activated cell returns to a quiescent state, important in a well-controlled stress response. Data so far are consistent with PAPP-A being a part of this well-controlled stress response. The concept of NFB as a transcription factor for PAPP-A gene expression awaits identification of the promoter region. Searching untranscribed genomic sequence 1000 bp upstream to an expressed sequence tag sequence, which aligned with sequence encoding the very N-terminal of PAPP-A and continued approximately 400 bp further upstream, revealed the presence of three potential NFB binding sites. However, it will require further experiments to determine whether any of those candidate transcription factor binding sites are active.
TNF and IL-1 also activated JNK in human fibroblasts. Unlike NFB activation, IL-1 was more effective than TNF in stimulating JNK. Furthermore, the recent availability of a specific inhibitor of JNK, SP600125 (25), allowed direct demonstration of whether JNK was important for cytokine stimulation of PAPP-A. This selective JNK inhibitor was previously used to demonstrate that JNK is critical for IL-1-induced expression of another metalloproteinase, collagenase, in synoviocytes (26). SP600125 inhibited IL-1- but not TNF-stimulated accumulation of PAPP-A protein in cell-conditioned medium. SP6000125 also inhibited IL-1- but not TNF-stimulated IGFBP-4 proteolysis (data not shown). However, SP600125 did not inhibit IL-1- (or TNF)-induced PAPP-A mRNA expression. NFB and JNK are activated simultaneously under a variety of stress conditions, and there are several levels of cross-talk, including competition and collaboration, between the two (27). Our data suggest that these two pathways may be involved in mediating IL-1-mediated increases in PAPP-A expression but that NFB activation is the main determinant of TNF-induced PAPP-A expression in human fibroblasts. An interaction between NFB and JNK is not surprising because signal transduction after cytokine stimulation has been shown to result in activation of parallel kinase cascades regulating activator protein-1 (via JNK) and NFB (28, 29). However, this interaction is not the same for PAPP-A. Because JNK can also be involved in promoting stability of mRNA transcripts (30) and PAPP-A mRNA is not affected by SP6000125, the interaction with NFB might be complementary rather than cooperative.
Wound healing is a multiphase process characterized by an acute inflammatory response followed by an extended proliferative stage (31). During the acute response to injury, inflammatory cytokines including IL-1 and TNF are produced by infiltrating immune cells (32). Cytokines stimulate collagen accumulation via the regulation of growth factors produced by fibroblasts at the site of injury, including IGF-I. During the proliferative phase, IGF-I expression is increased (33). However, because IGF-I bioavailability is regulated by IGFBPs, the regulation of IGFBP protease expression becomes physiologically important. Therefore, cytokine regulation of PAPP-A expression and elevated free IGF-I may be important to the normal healing process. Increases in PAPP-A have been observed in several systems of oxidative stress and response to injury (3, 4, 5, 6). These data provide a possible mechanistic link between stress and PAPP-A expression through stress-activated intracellular signaling pathways and suggest a novel target for controlling IGF availability via PAPP-A in undesirable stress responses such as fibrosis, vascular restenosis, and atherosclerosis.
Footnotes
This work was supported by National Institutes of Health Grant R01 HL074871 (to C.A.C.), American Heart Association Grant 02255432 (to Z.T.R.), and the Mayo Foundation.
Current address for Z.T.R.: Department of Internal Medicine, Division of Endocrinology, University of Missouri, D1110A Diabetes Center, One Hospital Drive, Columbia, Missouri 65211.
The authors have no conflict of interest.
First Published Online November 3, 2005
Abbreviations: IB, Inhibitory B; IGFBP, IGF binding protein; JNK, c-Jun N-terminal kinase; NF, nuclear factor; PAPP-A, pregnancy-associated plasma protein-A.
Accepted for publication October 25, 2005.
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Nutrition, Mayo Clinic College of Medicine, Rochester, Minnesota 55905
Department of Molecular Biology (C.O.), University of Aarhus, DK-8000 Aarhus C, Denmark
Abstract
Pregnancy-associated plasma protein-A (PAPP-A) is an IGF binding protein protease that appears to function as a posttranslational modulator of IGF bioavailability in response to injury. A previous study indicated that the proinflammatory cytokines, TNF and IL-1, were potent stimulators of PAPP-A expression in cultured human fibroblasts. In this study, we investigated the intracellular signaling pathways mediating cytokine-stimulated PAPP-A expression. Treatment of human fibroblasts with TNF and IL-1 (1 nM) had little or no effect on phosphatidylinositol 3-kinase and Erk1/2 activation, pathways commonly associated with proliferation. On the other hand, TNF and IL-1 induced p38, c-Jun N-terminal kinase (JNK), and nuclear factor (NF)B activation, pathways more closely related to stress response. An inhibitor of p38 activation (SB203580) had no effect on TNF- or IL-1-stimulated PAPP-A expression. The JNK inhibitor, SP600125, had no effect on IL-1- or TNF-stimulated PAPP-A mRNA expression. However, SP600125 effectively inhibited IL-1-induced PAPP-A protein expression. MG-132, a proteasome inhibitor that blocked degradation of the intrinsic NFB inhibitor, IB, and thereby prevented NFB activation, was a potent inhibitor of both TNF- and IL-1-stimulated PAPP-A mRNA and protein expression and IGF binding protein-4 protease activity. MG-132 had no effect on JNK phosphorylation or p38 activation, and SB203580 and SP600125 had no effect on IB degradation, documenting inhibitor specificity. BAY11–7082, another inhibitor of NFB activation, also inhibited TNF- and IL-1-stimulated PAPP-A expression and IGF binding protein-4 protease activity. These data indicate that NFB activation is the primary mediator of cytokine-stimulated PAPP-A expression in human fibroblasts.
Introduction
PREGNANCY-ASSOCIATED plasma protein-A (PAPP-A) is an IGF binding protein (IGFBP) protease expressed by a variety of cell types including normal human fibroblasts, osteoblasts, and vascular smooth muscle cells (1, 2, 3). In vitro, PAPP-A functions to cleave IGFBP-4, an inhibitory IGFBP, consequently increasing bioavailable IGF for receptor activation (2, 4). Several recent studies suggest a similar role for PAPP-A in modulating site- and event-specific IGF signaling during injury repair responses in vivo. Thus, PAPP-A expression is increased in healing human skin after a first intention wound in association with activated fibroblasts and macrophages (5). PAPP-A is also up-regulated in unstable atherosclerotic plaque and smooth muscle cells in response to vascular injury (3, 6), suggesting a role for PAPP-A in the normal response to injury in multiple tissues.
Little is known about the signaling mechanisms regulating PAPP-A expression. We have recently shown that PAPP-A gene and protein expression in cultured human fibroblasts is stimulated by proinflammatory cytokines involved in injury repair responses, namely TNF and IL-1 (7). The aim of this study was to determine the specific intracellular signaling pathways mediating cytokine-regulated PAPP-A expression. The focus was on the major common pathways associated with cytokine stimulation in various cell types including phosphatidylinositol 3-kinase (P13K), MAPK, and nuclear factor (NF)B pathways (8, 9, 10, 11, 12, 13). Herein we show that activation of NFB is the primary mediator of TNF- and IL-1-stimulated PAPP-A expression in human fibroblasts.
Materials and Methods
Materials
TNF and IL-1 were purchased from Research Diagnostics Inc. (Flanders, NJ), and RIA-grade BSA was from Sigma Chemical Corp. (St. Louis, MO). Antibodies against phosphorylated and total Akt, Erk1/2, p38, and c-Jun N-terminal kinase (JNK) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against NFB, inhibitory B (IB)-, and phospho-Hsp27 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody against IB was a kind gift from Dr. David McKean (Mayo Clinic, Rochester, MN). The specific inhibitors MG-132, SB203580, and PD98059 were purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA), and SP600125 and BAY11–7082 were obtained from Calbiochem (San Diego, CA). Tissue culture supplements and fetal bovine serum were obtained from Life Technologies (Grand Island, NY). Reagents for SDS-PAGE were purchased from Bio-Rad Laboratories (Richmond, CA).
Cell cultures
Primary cultures of adult human dermal fibroblasts were purchased from the Human Genetic Mutant Cell Repository (Camden, NJ) and cultured as reported previously (1, 7, 14, 15). For all experiments, cells were washed twice and incubated in serum-free medium containing 0.1% BSA overnight before experimental treatment. The cells were again washed and changed to serum-free medium plus experimental additions for the indicated times. For some studies, cells were pretreated for 60 min with specific inhibitors before the addition of cytokines. Dose-response experiments were initially performed for each inhibitor to determine the minimum effective concentration. At the end of the incubation, conditioned media were collected, centrifuged to remove any debris and stored at –70 C. Cell numbers were determined at the time of media collection using a Coulter counter (Coulter Electronics, Hialeah, FL).
RNA isolation and cDNA synthesis
Total RNA was extracted from cells using the RNeasy minikit (QIAGEN, Valencia, CA) and treated with deoxyribonuclease (DNA-free, Ambion, Inc., Austin, TX). Four hundred nanograms of RNA were reverse transcribed using TaqMan reverse transcription reagents (PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions.
Real-time PCR
Real-time quantitative PCR analyses were performed using the ABI PRISM 7700 sequence detection system and software (PE Applied Biosystems). Primer and probe sequences for specific detection and amplification of PAPP-A and 28S as well as assay validations were described previously (7, 15).
PAPP-A ELISA
PAPP-A levels in cell-conditioned media were measured using an ultra-sensitive ELISA kit kindly provided by Diagnostic Systems Laboratories, Inc. (Webster, TX). Minimum sensitivity is 0.24 mIU/liter with intra- and interassay coefficients of variation of 4.7 and 4.2%, respectively.
Western immunoblotting
After treatment, cells were lysed, subjected to SDS-PAGE, and transferred to polyvinyl difluoride, as described previously (16). Filters were blocked with 5% nonfat dry milk in Tris-buffered saline/0.1% Tween and probed with primary antibody at the supplier’s recommended dilution. After reaction with the appropriate secondary antibody conjugated with horseradish peroxidase, blots were visualized using enhanced chemiluminescence reagents (Amersham Biosciences Inc., Piscataway, NJ) and autoradiography.
NFB nuclear translocation
Human fibroblasts were pretreated ± inhibitors for 60 min and then stimulated with vehicle or 1 nM TNF or IL-1 for 10 min. After stimulation, cells were washed three times with cold PBS, lysed, and nuclear-specific proteins isolated using the protocol included with NucBuster (Novagen, Madison, WI). Detection of NFB in the nucleus was quantitated using the NoShift (Novagen) transcription factor assay kit. Briefly, 20 μg of nuclear protein was incubated with biotin-labeled wild-type DNA (10 pmol) alone or in combination with 10-fold molar excess biotin-labeled mutant DNA or cold wild-type DNA. Reactions were added to streptavidin-coated plates and incubated for an hour. Wells are washed three times and treated with primary antibody (anti-p65 subunit) for an hour and washed three times and exposed with the antimouse secondary antibody. After five washes, 3', 5, 5'-tetramethyl benzidine substrate was added and the reaction stopped after 15 min by addition of 1 N HCL. The absorbance was read at 450 nm (EL808, BIO-Tek Instruments, Inc., Highland Park, VT) to quantify nuclear NFB levels.
IGFBP-4 protease assay
Cell-free IGFBP-4 proteolysis was assayed as previously described (1, 2, 3, 15). Conditioned medium was incubated at 37 C for 2 h with 125I-IGFBP-4 and 5 nM IGF-II, without and with neutralizing PAPP-A antibody (1). IGF-II binds IGFBP-4 and increases its susceptibility to cleavage by PAPP-A in vitro (17). Reaction products were separated by SDS-PAGE and visualized by autoradiography. Extent of proteolysis, i.e. loss of intact and generation of 18-kDa radiolabeled fragments, was determined using enhanced laser densitometry (Ultroscan XL; Pharmacia LKB Biotechnology, Piscataway, NJ).
Promoter scan
Using nt 1–180 of the sequence encoding preproPAPP-A (accession no. BC078657), an expressed sequence tag sequence (accession no. CB988079), which extended approximately 400 bp upstream, was identified. The chromosomal sequence stretch further 1000 bp upstream (stretch between position 115994630 and 115995630 on the human chromosome 9) was analyzed for the presence of potential NFB binding sites using the Transcription Element Search System (http://www.cbil/upenn.edu/cgi-bin/tess/tess).
Statistical analysis
Results are expressed as mean ± SEM for the indicated number of experiments. Statistical analyses were performed using ANOVA and Dunnett for comparisons to control and Scheffe for multiple group comparisons. Results were considered statistically significant at P < 0.05.
Results
TNF- and IL-1-stimulated signaling pathways
In the first set of experiments, we determined the effect of TNF and IL-1 on major common pathways associated with cytokine stimulation including PI3K, MAPK, and NFB pathways in cultured human fibroblasts. To examine the activation of PI3K, Western blot analyses were performed using antibodies specific for phosphorylated Akt, a downstream substrate of PI3K (8, 16). Treatment of human fibroblasts with TNF or IL-1 for 10, 30, 60, or 120 min did not result in detectable Akt phosphorylation at Ser473 or Thr308 sites (data not shown). Reprobing with antibody against total Akt indicated that the protein was present and unaltered by treatment, and a positive control for stimulation of Akt phosphorylation (IGF-I) demonstrated that the signaling pathway was functional in these cells.
The MAPK family has three main subgoupings: Erk1(/2), JNK, and p38 kinase (9). The Erk1/2 pathway is most commonly linked to the regulation of cell proliferation, whereas p38 and JNK pathways are more closely related to stress (10). As shown in Fig. 1, stimulation with TNF and IL-1 had little or no effect on Erk1/2 phosphorylation, which appears to be constitutive in these cells. On the other hand, cytokine-mediated p38 phosphorylation was induced at 10 and 30 min with a decrease toward basal at 60 and 120 min. Endogenous JNK phosphorylation (46 and 54 kDa) was likewise transiently induced 10 and 30 min after cytokine treatment and undetectable again at 60 min. IL-1 appeared to be more effective than TNF in activating JNK, which was confirmed in further dose-response experiments (not shown). These data indicate that the PI3K and Erk1/2 pathways are not activated in human fibroblasts by TNF and IL-1. However, these cytokines do induce the protein kinases, p38 and JNK.
The NFB pathway is the predominant mediator of prooxidant stimuli, such as inflammatory cytokines (11). The principal mechanism by which NFB is activated is through phosphorylation of intrinsic inhibitors, the IBs (11). Normally, IB binds NFB and sequesters it in the cytoplasm. Phosphorylation of IB identifies it for ubiquitination and subsequent proteasome-mediated degradation, freeing NFB to translocate into the nucleus in which it regulates gene expression. IB (37 kDa) and IB (46 kDa) are prototypes of the inhibitory IB family. Western blotting of lysates of cells after stimulation with TNF or IL-1 indicated a rapid loss of IB within 10 min, which returned to detectable levels by 60 min (Fig. 2). TNF and IL-1 had little or no effect on IB. NFB translocation was confirmed using a transcription factor assay kit to detect nuclear NFB. As indicated in Table 1, TNF and IL-1 treatment increased levels of nuclear NFB 2- to 4-fold.
Inhibitors of intracellular signaling and PAPP-A expression
As shown in Fig. 3 and as reported previously (7), treatment of human fibroblasts with 1 nM TNF or IL-1 significantly increased PAPP-A levels 3- to 4-fold in 24-h conditioned medium. Although TNF and IL-1 stimulated p38 activity, SB203580 had no significant effect on PAPP-A expression when used at concentrations shown to inhibit p38 activation, as monitored by Hsp27 phosphorylation (see Fig. 5). Likewise, PD98059, an inhibitor of Erk1/2 activation, had no effect on TNF- and IL-1-induced PAPP-A. However, MG-132, a proteasome inhibitor that blocks degradation of IB and thus NFB activation, was a potent inhibitor of both TNF- and IL-1-induced PAPP-A expression. The JNK inhibitor, SP600125, effectively inhibited IL-1-stimulated PAPP-A levels in conditioned medium but had no effect on TNF-induced PAPP-A. To confirm the role of NFB activation, BAY11–7082, which inhibits inducible phosphorylation of IB, effectively inhibited TNF- and IL-1-stimulated PAPP-A expression (Fig. 4).
To document the specificity of the inhibitors as well as determine possible signaling cross-talk, we assessed the effect of MG-132, SB203580, and SP600125 on the NFB, p38, and JNK pathways. Western immunoblotting confirmed that MG-132 blocked the degradation of IB induced by TNF (not shown) and IL-1 (Figs. 5 and 6). The slower migrating form of IB with IL-1 and MG-132 treatment was confirmed by Western blot as phosphorylated IB, which is not altered by MG-132 (data not shown). The apparent lower level of IB in cells treated with IL-1 and MG-132 may be due to a reduced ability of the IB antibody to recognize the phosphorylated form of IB. MG-132 inhibits phosphorylated IB degradation in the proteasome, which results in inhibition of NFB activation (Table 1). BAY11–7082 similarly inhibited NFB activation (Table 1). On the other hand, MG-132 had no effect on p38 activity, as measured by phosphorylation of its downstream substrate, Hsp27 (12, 13), or JNK activation after treatment with IL-1 (Fig. 5). Likewise, SB203580 had no effect on IL-1-induced IB degradation or JNK phosphorylation (Fig. 5). SP600125 effectively inhibited JNK phosphorylation by IL-1 without affecting IB (Fig. 6) or NFB activation (Table 1).
To determine whether MG-132 and SP600125 inhibit cytokine-stimulated PAPP-A gene expression, real-time RT-PCR was performed on RNA isolated from human fibroblasts preincubated with MG-132 or SP600125 and then stimulated with TNF and IL-1 for 4 h. As shown in Fig. 7, MG-132 was able to prevent TNF- and IL-1-stimulated PAPP-A gene expression without altering basal expression. On the other hand, SP600125 had no effect on TNF- or IL-1-induced PAPP-A mRNA expression. Western blots done in parallel indicated that SP600125 inhibited IL-1-stimulated JNK phosphorylation as in Fig. 6, so lack of an effect on PAPP-A mRNA expression was not due to problems with the reagent.
Functional consequence of regulated PAPP-A expression through NFB activation
PAPP-A functions as an IGFBP protease with subsequent alteration in IGF bioactivity (2, 4, 14). To determine whether changes in functional activity of PAPP-A reflect expression data, cell-conditioned medium after treatment with IL-1 and TNF without and with MG-132 or BAY11–7082 was subjected to the 125I-IGFBP-4 protease assay (1, 2, 3, 15). As shown in Figs. 8 and 9, the IGFBP-4 remained intact during incubation in unconditioned media. Control conditioned media, with low levels of PAPP-A (Fig. 3), had low levels of proteolytic activity as indicated by the appearance of radiolabeled fragments. Treatment with IL-1 or TNF clearly enhanced proteolysis of IGFBP-4 over control. In samples from three separate experiments, this enhancement ranged from 2- to 4-fold. MG-132 completely inhibited TNF- and IL-1-stimulated proteolysis, with little apparent affect on basal proteolysis (Fig. 8). That this proteolytic activity was due to PAPP-A was confirmed using an inhibitory PAPP-A antibody (+ lanes). Figure 9 confirms the role of NFB activation using the alternative inhibitor, BAY11–7082. Again, IL-1 and TNF treatment enhanced IGFBP-4 proteolysis, and this increase was completely prevented with BAY11–7082. Direct addition of MG-132 or BAY11–7082 to the cell-free assay had no effect on basal or cytokine-stimulated IGFBP-4 proteolysis (data not shown).
Discussion
These data indicate that activation of NFB is the main determinant of TNF- and IL-1-induced PAPP-A gene expression and function in cultured human fibroblasts.
NFB is activated in response to prooxidative stimuli, such as inflammatory cytokines, and is associated with a variety of injury responses in vivo (11, 18, 19, 20, 21). In this study, the addition of TNF or IL-1 to human fibroblasts resulted in rapid degradation of IB and translocation of NFB to the nucleus. Treatment of cells with the proteasome inhibitor, MG-132, blocked TNF- and IL-1-induced IB degradation and NFB activation. This was associated with an inhibition of PAPP-A mRNA and protein expression and proteolytic activity. Similar effects were seen with the inhibitor of cytokine-induced IB phosphorylation, BAY11–1084. TNF and IL-1 had no effect on IB, suggesting that regulation of IB is responsible for cytokine regulation of NFB activation. Although it was reported that IL-1 induced IB and IB degradation, whereas TNF regulated only IB degradation (22), this was not observed in our study. One of the genes activated by NFB is the IB gene (23, 24), possibly explaining the transient nature of cytokine-induced degradation of IB. The NFB/IB autoregulatory system ensures that the induction of NFB is transient and that the activated cell returns to a quiescent state, important in a well-controlled stress response. Data so far are consistent with PAPP-A being a part of this well-controlled stress response. The concept of NFB as a transcription factor for PAPP-A gene expression awaits identification of the promoter region. Searching untranscribed genomic sequence 1000 bp upstream to an expressed sequence tag sequence, which aligned with sequence encoding the very N-terminal of PAPP-A and continued approximately 400 bp further upstream, revealed the presence of three potential NFB binding sites. However, it will require further experiments to determine whether any of those candidate transcription factor binding sites are active.
TNF and IL-1 also activated JNK in human fibroblasts. Unlike NFB activation, IL-1 was more effective than TNF in stimulating JNK. Furthermore, the recent availability of a specific inhibitor of JNK, SP600125 (25), allowed direct demonstration of whether JNK was important for cytokine stimulation of PAPP-A. This selective JNK inhibitor was previously used to demonstrate that JNK is critical for IL-1-induced expression of another metalloproteinase, collagenase, in synoviocytes (26). SP600125 inhibited IL-1- but not TNF-stimulated accumulation of PAPP-A protein in cell-conditioned medium. SP6000125 also inhibited IL-1- but not TNF-stimulated IGFBP-4 proteolysis (data not shown). However, SP600125 did not inhibit IL-1- (or TNF)-induced PAPP-A mRNA expression. NFB and JNK are activated simultaneously under a variety of stress conditions, and there are several levels of cross-talk, including competition and collaboration, between the two (27). Our data suggest that these two pathways may be involved in mediating IL-1-mediated increases in PAPP-A expression but that NFB activation is the main determinant of TNF-induced PAPP-A expression in human fibroblasts. An interaction between NFB and JNK is not surprising because signal transduction after cytokine stimulation has been shown to result in activation of parallel kinase cascades regulating activator protein-1 (via JNK) and NFB (28, 29). However, this interaction is not the same for PAPP-A. Because JNK can also be involved in promoting stability of mRNA transcripts (30) and PAPP-A mRNA is not affected by SP6000125, the interaction with NFB might be complementary rather than cooperative.
Wound healing is a multiphase process characterized by an acute inflammatory response followed by an extended proliferative stage (31). During the acute response to injury, inflammatory cytokines including IL-1 and TNF are produced by infiltrating immune cells (32). Cytokines stimulate collagen accumulation via the regulation of growth factors produced by fibroblasts at the site of injury, including IGF-I. During the proliferative phase, IGF-I expression is increased (33). However, because IGF-I bioavailability is regulated by IGFBPs, the regulation of IGFBP protease expression becomes physiologically important. Therefore, cytokine regulation of PAPP-A expression and elevated free IGF-I may be important to the normal healing process. Increases in PAPP-A have been observed in several systems of oxidative stress and response to injury (3, 4, 5, 6). These data provide a possible mechanistic link between stress and PAPP-A expression through stress-activated intracellular signaling pathways and suggest a novel target for controlling IGF availability via PAPP-A in undesirable stress responses such as fibrosis, vascular restenosis, and atherosclerosis.
Footnotes
This work was supported by National Institutes of Health Grant R01 HL074871 (to C.A.C.), American Heart Association Grant 02255432 (to Z.T.R.), and the Mayo Foundation.
Current address for Z.T.R.: Department of Internal Medicine, Division of Endocrinology, University of Missouri, D1110A Diabetes Center, One Hospital Drive, Columbia, Missouri 65211.
The authors have no conflict of interest.
First Published Online November 3, 2005
Abbreviations: IB, Inhibitory B; IGFBP, IGF binding protein; JNK, c-Jun N-terminal kinase; NF, nuclear factor; PAPP-A, pregnancy-associated plasma protein-A.
Accepted for publication October 25, 2005.
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