Published online before print December 10, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
-induced genes in bone cells
,1,2
* Department of Oral Biology, School of Dental Medicine, and
Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York
2 Correspondence: Department of Oral Biology, University at Buffalo, SUNY, 36 Foster Hall, 3435 Main Street, Buffalo, NY 14214. E-mail: sgaffen{at}buffalo.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(TNF-) in the preosteoblast cell line MC3T3-E1. Using Affymetrix microarrays, 80–90 genes were up-regulated, and 19–50 genes were down-regulated with IL-17 and TNF- as compared with TNF- alone. These included proinflammatory chemokines and cytokines, inflammatory genes, transcriptional regulators, bone-remodeling genes, signal transducers, cytoskeletal genes, genes involved in apoptosis, and several unknown or unclassified genes. The CXC family chemokines were most dramatically induced by IL-17 and TNF-, confirming the role of IL-17 as a potent mediator of inflammation and neutrophil recruitment. Several transcription factor-related genes involved in inflammatory gene expression were also enhanced, including molecule possessing ankyrin repeats induced by lipopolysaccharide/inhibitor of 
B
(MAIL/B), CCAAT/enhancer-binding protein 
(C/EBP), and C/EBPß. We also identified the acute-phase gene lipocalin-2 (LCN2/24p3) as a novel IL-17 target, which is regulated synergistically by TNF- and IL-17 at the level of its promoter. A similar but not identical pattern of genes was induced by IL-17 and TNF- in ST2 bone marrow stromal cells and murine embryonic fibroblasts. This study provides a profile of genes regulated by IL-17 and TNF- in osteoblasts and suggests that in bone, the major function of IL-17 is to cooperate and/or synergize with other cytokines to amplify inflammation.
Key Words: synergy • chemokine • lipocalin
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The current understanding of bone diseases such as rheumatoid arthritis (RA) and periodontal disease supports a model in which inflammation is a key step in promoting bone destruction [4
, 5
]. In particular, osteoblasts are highly responsive to immune-derived cytokines. They respond to these by promoting osteoclast development and activity and by amplifying the inflammatory state further through the secretion of numerous inflammatory effectors. Many bone-acting cytokines have been identified, including tumor necrosis factor (TNF-), interleukin (IL)-6, and IL-1ß, as well as the receptor activator of nuclear factor (NF)-B (RANK)/RANK ligand (RANKL)/osteoprotegrin (OPG) family of TNF receptor (TNFR)-related cytokines (reviewed in refs. [4
, 6
]).
Recently, the T cell-derived cytokine IL-17 has also been shown to be an important mediator of inflammatory arthritis and other diseases affecting bone [7 , 8 ]. For example, IL-17 is highly elevated in synovial fluid from RA and osteoarthritis patients and has also been implicated in periodontal disease [9 10 11 ]. In addition, IL-17 has been implicated in animal models of RA. IL-17-deficient mice are resistant to collagen-induced arthritis (CIA); blocking IL-17 in a mouse model of RA reduces disease symptoms; and excess IL-17 exacerbates disease [12 13 14 15 16 ]. Furthermore, mice deficient in the T cell costimulatory molecule-inducible costimulator are resistant to CIA, and a major defect in these mice is a reduction in levels of IL-17 [17 ]. Thus, it is clear that IL-17 plays a significant role in inflammatory bone disease and may be an attractive, therapeutic target.
IL-17 is particularly intriguing, as the cytokine and its receptor are the founding members of a novel family of cytokines, whose biological functions and mechanisms of signaling remain poorly understood (reviewed in ref. [18
]). Unlike typical inflammatory cytokines produced by the innate-immune system, IL-17 is secreted largely by memory T cells, primarily in response to IL-23 and T cell receptor signals [19
20
21
22
]. The IL-17 receptor (IL-17R) is expressed ubiquitously, and consequently, nearly all cells are potential targets of IL-17 [23
, 24
]. However, very little is currently known about the signaling mechanisms induced by this unique receptor. Early reports indicated that like many inflammatory cytokines, IL-17 activates the transcription factor NF-B, probably through the adaptor TNFR-associated factor 6 [23
, 25
]. However, NF-B activation is weak in many cell types (e.g., the osteoblast cell line MC3T3-E1) [26
]. Several mitogen-activated protein kinase pathways and the Janus tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) pathway have also been implicated in IL-17 signaling (reviewed in ref. [18
]), but it is likely that additional signaling pathways exist.
The endpoint of most signaling cascades is specific gene expression. Therefore, to gain a better understanding of IL-17-mediated signaling pathways, it is valuable to obtain a comprehensive picture of genes activated by this cytokine. To date, some gene targets of IL-17 have been defined in various cellular backgrounds, most of which are inflammatory in nature (e.g., cytokines, chemokines, prostaglandins, and nitric oxide; reviewed in refs. [8
, 27
]). However, given the ubiquitous expression of the IL-17R, it is highly likely that many IL-17 target genes have not yet been discovered and also, that there is cell-type specificity in signaling. It is important that in inflammatory settings such as RA, IL-17 is just one component in a highly complex cytokine network. In this regard, it has been well documented that a primary function of IL-17 is to act cooperatively with other cytokines, such as TNF-, IL-1ß, interferon-
, and IL-4 [28
29
30
31
32
33
]. Accordingly, it is also important to determine the influence of other cytokines on IL-17 function, particularly with respect to gene expression.
To provide further information regarding the physiological and pathological functions of IL-17 and to identify novel IL-17 target genes that will ultimately help dissect IL-17 signaling pathways, we used a microarray technique to examine the spectrum of genes regulated by IL-17. Because of the clear role for IL-17 and TNF- in RA and the penchant of IL-17 to synergize with TNF-, we examined genes induced by IL-17 in cooperation with TNF- in a preosteoblast cell background. Most genes identified herein were linked directly to inflammatory processes. One novel IL-17 gene target found in this study is lipocalin-2 (LCN2/24p3), an acute-phase protein involved in regulating apoptosis. In addition, several transcription factor-related genes involved in regulation of inflammatory genes were revealed, as well as genes involved in bone remodeling, cytoskeleton, signal transduction, and regulation of apoptosis.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-minimum essential medium (-MEM; Sigma Chemical Co., St. Louis, MO), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bioproducts, Woodland, CA), penicillin, streptomycin, and L-glutamine (Gibco/Invitrogen, Carlsbad, CA). Recombinant human IL-17 and TNF- were obtained from R&D Systems (Minneapolis, MN). For microarray analysis, MC3T3-E1 cells were grown to confluence in T175 flasks (
107 cells per sample). They were incubated for 16 h in -MEM/0.3% FBS and stimulated with TNF- (2 ng/ml) or together with IL-17 (200 ng/ml) for 2 h. For other stimulations, cells were seeded onto 10 cm dishes and grown to confluence in -MEM/10% FBS. Following attachment, cells were washed twice in phosphate-buffered saline (PBS), incubated in -MEM/0.3% FBS overnight, and stimulated with the indicated cytokines for the designated time periods. MEF and ST2 cells were not serum-starved prior to stimulation.
Microarray analysis
Total RNA from MC3T3-E1 cells was prepared using the RNeasy kit (Qiagen, Valencia, CA). Relative mRNA levels were assessed using the Affymetrix mouse genome gene chip U74v2, processed and performed by the Roswell Park Cancer Institute (RPCI) Gene Expression Core Facility (Buffalo, NY) [26
]. The experiment was repeated twice with separately prepared mRNA, and four arrays were generated. Affymetrix Microarray Suite Version 5.0 managed the raw data. The raw absolute expression signals were exported to dChip Version 1.3 (http://www.dchip.org), GeneSpring Version 6.2 (Silicon Genetics, Redwood City, CA), and GeneTraffic Version 3.0 (Iobion Informatics, La Jolla, CA) for further analysis. Expression signals were transformed and normalized by algorithms implemented in different software packages. Changes in gene expression were considered significant if the detection P value was less than 0.05 (termed "present" in Affymetrix Microarray Suite) in at least three of four arrays, and the magnitude of change was at least 2.0.
Real-time polymerase chain reaction (PCR)
Total RNA was isolated using the RNeasy kit (Qiagen) with on-column DNase digestion to eliminate DNA contamination. Single-stranded cDNA was synthesized using reverse transcriptase (RT; murine Moloney leukemia virus (MuMLV); Fermentas, Hanover, MD) and random hexamer primers. Real-time PCR was performed in triplicate using the iCycler iQ Real-Time PCR detection system and iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), according to the manufacturer’s protocol. For the negative control, MuMLV was omitted, and a control cDNA dilution series was created for each gene to establish a relative standard curve. PCR reactions consisted of one cycle at 95°C for 360 s, followed by 40 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 20 s. Each reaction was subjected to melting temperature analysis to confirm single-amplified products. For quantification, target genes were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPD) controls. Primer pair sequences were as follows: CXC chemokine ligand 1 (CXCL1), 5'-CAC CCA AAC CGA AGT CAT AG-3' and 5'-AAG CCA GCG TTC ACC AGA-3'; CXCL2, 5'-CGC CCA GAC AG AAG TCA TAG-3' and 5'-TCC TCC TTT CCA GGT CAG TTA-3'; CXCL5, 5'-GGT CCA CAG TGC CCT ACG-3' and 5' GCG AGT GCA TTC CGC TTA-3'; CC chemokine ligand 2 (CCL2), 5'-GCC TGC TGT TCA CAG TTG C-3' and 5'-TGT ATG TCT GGA CCC ATT CCT-3'; CCL5, 5'-CAC CAC TCC CTG CTG CTT-3' and 5'-ACA CTT GGC GGT TCC TTC-3'; LCN2/24p3, 5'-CAG CTT TCA GAT GTA CAG CAC C-3' and 5'-CAT GGC GAA CTG GTT GTA GTC-3'; molecule possessing ankyrin repeats induced by lipopolysaccharide (LPS; MAIL)/inhibitor of B (IB), 5'-TGA CAT CAC CGC AAA CGC-3' and 5'-GAA ATC CTG GCA CTG GTC TC-3'; cyclooxygenase-2 (COX2), 5'-TGG AAA AGG TTC TTC TAC GGA G-3' and 5'-TGA ACC CAG GTC CTC GCT-3'; epiregulin (EREG), 5'-TGC CTC TTG GGT CTT GAC G-3' and 5'-ACT TTG TAA TCT GCA CTT GAG CC-3'; CCAAT/enhancer-binding protein (C/EBP), 5'-TGC CAT GTA CGA CGA CGA G-3' and 5'-GCC GCT TTG TGG TTG CTG-3'; C/EBPß, 5'-CGC ACC ACG ACT TCC TCT-3' and 5'-CGA GGC TCA CGT AAC CGT-3'; oncostatin M receptor (OSMR), 5'-TAT TTC TTG GGA GCC CGT AT-3' and 5'-TCT GAA GTT GTA ACG GAC GC-3'; suppressor of cytokine signaling 3 (SOCS3), 5'-AGC TCC AAA AGC GAG TAC CAG-3' and 5'-CTC ACA CTG GAT GCG TAG GTT-3; FAS (Fas or CD95), 5'-GAA ATC GCC TAT GGT TGT TG-3' and 5'-ATG GTT TCA CGA CTG GAG G-3'; pleckstrin homology, Sec7, and coiled-coil domains 3 (PSCD3), 5'-GCG CTG GTT CAT CCT CAC AG-3' and 5'-CAT CGG CCT CCG TCT TGC-3'; GAPD, 5'-GAC AAC TTT GGC ATT GTG G-3' and 5'-ATG CAG GGA TGA TGT TCT G-3'; matrix metalloprotinease 13 (MMP-13), 5'-TGA TGC CAT TAC CAG TCT CC-3' and 5'-AAT GTC ATA ACC ATT CAG AGC C-3'; tissue inhibitors of metalloproteinases 2 (TIMP2), 5'-CAC GCT TAG CAT CAC CCA-3' and 5'-TCC ATC CAG AGG CAC TCA T-3'; RANKL, 5'-GAT GAA AGG AGG GAG CAC G-3' and 5'-AAG GGT TGG ACA CCT GAA TG-3'; OPG, 5'-CTT CGT GCC TTG ATG GAG AG-3' and 5'-GGA AAG TGG GAT GTT TTC AA-3'; cortactin (CTTN), 5'-AGG TGC CAT CTG CCT ATC A-3' and 5'-TCT CGG CTT CTG CCT TCC-3'; coactosine-like 1 (COLT1), 5'-GGC TGT TCG CCT TTG TGC-3' and 5'-CTC CTT CCG GTC GCT GAT-3'; 14-3-3-, 5'-TGG CGC TCA ACT ACT CGG-3' and 5'-TCT TGC TGG TCG CTC GTC-3'.
Flow cytometry
Cells were trypsinized and resuspended in 1 ml staining buffer (PBS/2% FBS) containing 200 ng propidium iodide (PI) and 200 ng RNase. After incubation at 37°C for 20 min, cells were analyzed on a FACSCalibur using Cellquest software (BD Biosciences, San Jose, CA).
Luciferase assays
The mouse LCN2/24p3 promoter was PCR-amplified from mouse genomic DNA using Taq DNA polymerase (Fermentas). The primers used were 5'-GGG ACG CGT CAG GAG CAG CAA AC-3' and 5'-GAC CTC GAG GGC CAT GGT TTC CAC-3'. The amplification product was subcloned into the promoterless luciferase reporter vector pGL3-Basic (Promega, Madison, WI) and confirmed by sequencing. For luciferase assays, 1 x 105 MC3T3-E1 cells were seeded on 12-well plates and cotransfected with 0.5 µg of the 24p3 luciferase reporter plasmid and 10 ng Renilla luciferase plasmid (provided by Dr. Xin Lin, University at Buffalo, State University of New York) as an internal standard. Where applicable, a tenfold excess of the plasmid pCMV5-C/EBP (a kind gift of Dr. Lynn Vales, University of Medicine and Dentistry of New Jersey, Piscataway, NJ) or NF-B p65 (provided by Dr. Xin Lin, MD Anderson Cancer Center, Houston, TX) was added to the transfection reaction. Cells were stimulated with indicated cytokines for 6 h and lysed, and supernatants were analyzed for luciferase activity using an Orion MPL2 luminometer (Berthold Detection Systems, Oak Ridge, TN).
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. To this end, MC3T3-E1 cells were stimulated for 2 h with a suboptimal dose of TNF- (2 ng/ml), alone or together with a high dose of IL-17 (200 ng/ml). We chose this rapid time-point, as IL-17 triggers production of chemokines and other secreted factors, and we wished to reduce confounding secondary effects as much as possible. In addition, this is the earliest time-point at which the hallmark IL-17 target gene IL-6 is up-regulated [26
]. Total RNA was isolated and applied to Affymetrix mouse genome U74Av2 microarrays, which include over 12,488 probe sets interrogating 12,000 full-length mouse genes and expressed sequence tag clusters from the UniGene database. Depending on the software used for analysis, 80–90 genes were up-regulated, and 19–50 genes were down-regulated in cells treated with TNF- + IL-17 compared with TNF- alone. Table 1
lists genes that were induced or reduced more than twofold, as determined by at least two out of three microarray analysis software packages. Genes were categorized according to function and included chemokines and cytokines, immune response/inflammatory genes, transcriptional regulators, bone-remodeling genes, cytoskeleton-related genes, and signal transducers, as well as various unknown or uncategorized genes. |
View this table: [in a new window] |
Table 1. Genes Regulated by TNF- or TNF- + IL-17 in MC3T3-E1 Cells
|
, or IL-17 + TNF-, and total RNA was analyzed by real-time PCR. A relative standard curve was created for each gene to calibrate the PCR efficiency of different primer pairs (data not shown). The majority of up-regulated genes identified by microarray analysis was confirmed successfully by real-time PCR (Table 2
). As expected, the fold changes determined by real-time PCR were often greater than those obtained in the microarray analysis, as Affymetrix microarrays are known to underestimate differences among samples. For example, CXCL5 was induced 4.5-fold in the microarray but 13.4-fold by real-time PCR (comparing TNF- with TNF-+IL-17). Similarly, CXCL2 was enhanced only twofold via microarray but 8.3-fold via real-time PCR. |
View this table: [in a new window] |
Table 2. Quantitative Analysis of Selected Genes Regulated by IL-17 and/or TNF- in MC3T3-E1 Cells
|
and IL-17 (Table 1)
, which were tested by real-time PCR (Table 2)
. In particular, TIMP2 and COTL1 were confirmed to be down-regulated by magnitudes similar to that obtained in the microarray analysis (–1.3-fold and –1.5-fold, respectively, Table 2
). In contrast, CTTN, PSCD3, and 14-3-3- were not down-regulated when tested by real-time PCR. These varying results highlight the critical importance of performing independent validation of microarray data.
Importantly, the real-time PCR analysis also enabled us to examine whether genes identified in the microarrays were induced by IL-17 alone. We found that many but not all genes up-regulated by IL-17 and TNF- were also enhanced by IL-17 alone in MC3T3-E1 cells. For example, CXCL5/LIX mRNA was increased 72.8-fold by IL-17 compared with unstimulated cells (Table 1
and ref. [36
]), and LCN2/24p3 was increased 2.3-fold by IL-17. In contrast, MAIL/IB was not induced significantly by IL-17 alone but was induced 3.5-fold by TNF- + IL-17 compared with TNF-. Among all the genes tested in real-time PCR analysis, none were exclusively induced by IL-17 without also being enhanced cooperatively (synergistically or additively) by TNF-. These data confirm that IL-17 exerts widespread cooperativity with TNF- in enhancing gene expression.
It should be noted that Affymetrix microarrays are known to have a high false-negative rate, making it likely that not all target genes will be identified in any given screen. Therefore, we tested several genes that did not meet the stringent exclusion criteria applied to our microarray data, including CCL2, COX2, C/EBPß, OSMR, RANKL, OPG, CASP1, and FAS (Table 2)
. Some were chosen because they appeared to be induced weakly on the microarrays and represented potentially intriguing biological targets of IL-17 (i.e., FAS, OSMR, and C/EBPß); others have been linked to IL-17 signaling in published literature (e.g., COX2, RANKL, and CCL2) [37
38
39
40
]. We found that almost all of these genes were enhanced at least mildly by IL-17 and/or TNF- treatment.
Genes induced by IL-17 in other cell types
The IL-17R is expressed ubiquitously, and it is possible that this cytokine may exhibit cell-type specificity with respect to its gene targets. Therefore, we examined ST2 cells and MEF cells for their responsiveness to IL-17 and TNF-. ST2 is a bone marrow stromal cell line with osteogenic potential [41
]. Similarly, osteoblasts are closely related to fibroblasts; both are of mesenchymal origin, and they respond in a similar manner to stimulation by various cytokines and chemokines [3
]. Accordingly, we examined the ability of IL-17 to up-regulate a subset of the more strongly enhanced genes in ST2 and MEF cells by real-time PCR analysis. As shown in Tables 3
and 4
, many genes were indeed enhanced strongly by IL-17 in these cell backgrounds. For example, CXCL1, LCN2/24p3, MAIL/IB, and FAS were induced much more potently by IL-17 alone in MEF cells than MC3T3-E1 cells (ranging from 7.3-fold for FAS to 476-fold for LCN2/24p3). In addition, these genes were enhanced cooperatively with TNF-. ST2 cells were also responsive to IL-17 alone, in a manner similar to MEF cells, and most genes were further enhanced by TNF- treatment. As expected, there was evidence for cell-type specificity in expression of certain genes. For example, the chemokine CCL2 was strongly induced by IL-17 in ST2 cells (93-fold). It is not surprising that the bone-related gene RANKL was only induced significantly in MC3T3-E1 cells.
|
View this table: [in a new window] |
Table 3. Quantitative Analysis of Selected Genes Regulated by IL-17 and/or TNF- in MEF Cells
|
|
View this table: [in a new window] |
Table 4. Quantitative Analysis of Selected Genes Regulated by IL-17 and/or TNF- in ST2 Cells
|
in all cell lines tested. Induction of 24p3 is a rapid event, as semiquantitative RT-PCR analysis showed the induction occurred as early as 30 min after stimulation in MC3T3-E1 cells (data not shown). In addition, 24p3 is enhanced even more strongly in MEF and ST2 cells, particularly by IL-17 alone (Tables 3
and 4) and thus, is not unique to osteoblasts.
To explore the mechanism of 24p3 up-regulation, we tested whether IL-17 and TNF- control activation of the 24p3 gene promoter. As this promoter has not been characterized in detail [42
, 43
], we obtained 1.4 kb genomic sequence upstream of the 24p3 gene by PCR using primers designed from sequence data from public databases. This putative promoter was subcloned upstream of the luciferase reporter gene in the promoterless pGL3-basic vector (termed 24p3-Luc). MC3T3-E1 cells were transiently transfected with this plasmid and stimulated with the indicated cytokines, and luciferase activity in lysates was assessed after 24 h. As shown in Figure 1A
, the relative reporter activities of 24p3-Luc were significantly enhanced (22.6-fold) by IL-17 and TNF- compared with IL-17 (1.7-fold) or TNF- (2.8-fold) alone. It is notable that this is one of the few genes known so far to exhibit synergy at the level of the promoter (e.g., compare with the IL-6 promoter; ref. [26
]). Thus, the 24p3 gene is controlled by IL-17 and TNF- primarily by initiation of transcription through its proximal promoter. Computer analysis of this region identified many putative transcription factor-binding sites (Fig. 1)
. We were particularly intrigued by the presence of several C/EBP and NF-B elements, as both of these transcription factors have been implicated in IL-17 and TNF- synergy [23
, 26
], and C/EBP is expressed inducibly by IL-17 and TNF- in these cells (Tables 1
and 2)
. Upon overexpression of C/EBP or the NF-B p65 subunit, the basal 24p3 promoter was strongly enhanced, suggesting that these (or related) transcription factors can transactivate the 24p3 gene (Fig. 1B)
.
 View larger version (25K): [in a new window] |
Figure 1. The LCN2/24p3 promoter is activated synergistically by IL-17 and TNF- in MC3T3-E1 cells. (A) MC3T3-E1 cells were transfected in triplicate with a luciferase reporter plasmid containing the 24p3 putative promoter (fragment –1404 to +34), together with an internal control Renilla luciferase plasmid. Twenty-four hours after transfection, cells were stimulated with IL-17 (200 ng/ml) and/or TNF- (2 ng/ml) and lysed, and luciferase activity in cell lysates was measured. Data are represented as the mean ± SD of luciferase activity relative to the Renilla luciferase activity. Results are representative of at least three independent experiments. ***, P < 0.001, versus untreated. (B) Cells were transfected with the 24p3 promoter together with a tenfold excess of a control pCMV4 vector or expression plasmids encoding C/EBP or NF-B p65 and analyzed as in A. Schematic diagram of the proximal 24p3 promoter and predicted C/EBP and NF-B sites are shown.
|
treatment. Although TNF- is a potent inducer of apoptosis in many cells, MC3T3-E1 cells did not undergo significant apoptosis in response to TNF- or IL-17, even after 72 h of treatment (data not shown). As apoptosis was not affected, we examined the effects of IL-17 and TNF- on cell cycle (Fig. 2
and Table 5
). After stimulation for 12 h or 24 h, TNF- treatment alone slightly reduced the portion of cells in G0/G1 phase, and it increased the portion in S and G2/M phase. However, IL-17 alone did not alter cell cycle detectably nor did it reverse the effect of TNF-. In addition, IL-17 did not alter cell growth kinetics and doubling time (not shown). Thus, neither 24p3 nor any other genes regulated by IL-17 and TNF- appear to influence cellular survival or proliferation.
 View larger version (15K): [in a new window] |
Figure 2. Regulation of cell cycle by IL-17 and/or TNF- in MC3T3-E1 cells, which were stimulated for 24 h without cytokines (Untreated) or with TNF- (2 ng/ml) and/or IL-17 (200 ng/ml). Cells were stained with PI and RNaseA and analyzed by flow cytometry. Results were analyzed by CellQuest software. See also Table 5
.
|
|
View this table: [in a new window] |
Table 5. Regulation of Cell Cycle by IL-17 and/or TNF-
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
[26
, 34
35
36
, 46
]. To date, surprisingly little is known about IL-17-mediated signaling events. To define signaling pathways in molecular detail, it is critical to identify genes whose expression is a direct result of signal transduction. Thus, we performed microarrays to identify genes induced rapidly in bone following IL-17 and TNF- stimulation, which can ultimately be used to dissect biochemical signaling pathways. We designed this study to identify genes induced by IL-17 alone and in conjunction with TNF-. As expected, many genes up-regulated in MC3T3-E1 cells were associated with inflammatory responses. In addition, genes involved in bone remodeling, transcriptional regulation, and signal transduction were identified, many of which have never before been linked to IL-17. The most prominent of these are discussed in detail below.
Chemokines
Numerous studies have linked IL-17 to chemokine gene regulation and the consequent effects on neutrophil recruitment and activation [31
, 36
, 47
48
49
50
51
52
]. Further support for the physiological importance of IL-17 in chemokine production came from studies in IL-17R-deficient (IL-17R–/–) mice [53
]. We identified four CXC- and CC-family chemokine genes strongly up-regulated by IL-17 and/or TNF-: CXCL1 [growth-related oncogene (GRO)/keratinocyte (KC), CXCL2 (macrophage-inflammatory protein-2/GROß), CXCL5 (LIX), and CCL5 regulated on activation, normal T expressed and secreted (RANTES). In addition, we found that CCL2 (MCP-1 monocyte chemoattractant protein-1) is up-regulated by these cytokines by real-time PCR, although its cooperative regulation was not strong enough to score on the Affymetrix microarrays. These chemokines are functional, as conditioned media from stimulated MC3T3-E1 cells enhanced neutrophil migration [36
]. The present study revealed a more complete spectrum of chemokines that are induced rapidly by IL-17 and TNF-, and results are consistent with the model that IL-17 activation of bone cells influences neutrophil recruitment and subsequent inflammation in vivo.
LCNs and apoptosis
One of the most strongly up-regulated genes identified in this study is the lipocalin 24p3. The 24p3 protein is an acute phase protein involved in multiple apoptotic events, including "passive" apoptosis induced by IL-3 withdrawal from cytokine-dependent cells [44
, 45
]. Despite the clear role of 24p3 in regulating apoptosis, we did not detect any significant effects of IL-17 or TNF- on cellular proliferation, survival, or apoptosis (Table 5 , Fig. 2
, and data not shown). It is interesting that 24p3 induces apoptosis in hematopoietic cells but not in fibroblastic cells [45
]. It is possible that after IL-17-induced chemokine expression and subsequent neutrophil recruitment, 24p3 causes apoptosis of cells in the inflammatory environment and thereby modulates inflammatory events. However, more experiments are required to prove this hypothesis.
Transcriptional regulators
Many proinflammatory effectors activate the transcription factor NF-B. IL-17 induces NF-B in numerous cell types, including fibroblasts [23
]. In addition, most IL-17-regulated genes contain crucial NF-B sites in their promoters, such as IL-6 [54
], CXCL5/LIX [55
], and CXCL1/Gro [56
]. However, we have found that IL-17 induces NF-B activation only weakly, if at all, in MC3T3-E1 cells [26
]. Thus, NF-B may play an important but poorly understood role in controlling IL-17 target genes. To date, little is known about the mechanisms underlying IL-17-induced NF-B activation, but a clue in this regard comes from our finding that the gene encoding MAIL/IB was induced by IL-17 and TNF-. The IB family of NF-B inhibitors is induced transcriptionally following NF-B activation, normally providing negative-feedback signaling in response to NF-B activation. However, IB appears to act in a positive manner to enhance target gene expression, particularly IL-6 [57
]. We found that IB was up-regulated by IL-17 and/or TNF- in all cell lines tested. Furthermore, it was reported recently that IL-1ß and LPS but not TNF- induced IB expression [58
, 59
]. This is consistent with our results, as TNF- alone does not induce IB significantly. Thus, enhancement of IB may be a mechanism by which IL-17 and TNF- control target gene expression via the NF-B pathway.
The C/EBP (also known as NF-IL6) family is another transcription factor family identified in these studies (Table 1)
. We previously reported that induction of C/EBP is partly responsible for cooperative enhancement of the IL-6 promoter by IL-17 and TNF- [26
]. Like NF-B, C/EBP proteins regulate numerous inflammatory genes. Their DNA binding sites are highly conserved, and thus, they exhibit considerable redundancy in regulating target genes. In the present study, we found that that C/EBPß is also modestly up-regulated by IL-17 and TNF- in all cell lines tested. It is interesting that a number of the IL-17-induced genes identified in this study are known to be controlled by C/EBP family members, such as COX2 and IL-6 [60
]. In addition, 24p3 has been reported to be up-regulated by ectopic C/EBPß expression, and our data indicate it also appears to be a target of C/EBP [43
] (Fig. 1)
. Thus, the C/EBP family probably plays a key role in regulating IL-17 responses.
Genes that regulate bone
One mechanism of IL-17-induced bone loss may be its ability to induce MMP, which degrade extracellular matrix during bone remodeling and tumor metastasis. In MC3T3-E1 cells, MMP-13 is up-regulated by IL-17, which is consistent with previous reports [61
, 62
]. Although no other MMPs (such as MMP-1 and MMP-3) were identified in this study, a tissue inhibitor of MMPs, TIMP-2, was mildly down-regulated by the combination of IL-17 and TNF- in MC3T3-E1 and MEF cells (Tables 2
and 3)
. Presumably, suppression of TIMP-2 could enhance activities of other MMPs, providing another mechanism by which IL-17 may control bone turnover.
The RANK/RANKL/OPG system is a critically important regulatory system for bone turnover. Osteoblasts express RANKL on the cell surface, which engages its counter-receptor RANK on osteoclast precursors and helps trigger their maturation (reviewed in ref. [63
]). OPG is a soluble decoy receptor, which prevents signaling from RANK. The balance between OPG and RANKL dictates the degree of osteoclastogenesis and hence, bone destruction. Several reports have implicated IL-17 in regulating OPG and RANKL [64
, 65
]. As the RANKL system is so central to bone remodeling and inflammation, we used real-time PCR to examine RANKL and OPG specifically, although neither gene was identified on the microarray. Expression of OPG and RANKL in all cell lines was low, as only the lowest dilutions of mRNA amplified detectable message (data not shown). At lower dilutions, RANKL message was detected and was mildly induced by TNF- and TNF- + IL-17 in MC3T3-E1 cells. However, RANKL was not induced significantly in MEF cells, and it was only induced very weakly in ST2 cells, consistent with the bone-specific nature of this gene. OPG expression did not change significantly in any cell lines tested. Consistent with these data, no ligands to date have been found to stimulate significant RANKL expression in differentiated osteoblast models such as MC3T3-E1 (Keith L. Kirkwood, University of Michigan, personal communication). Furthermore, at least one report indicates that the effects of IL-17 and TNF- on osteoclastic bone resorption are separable from RANK signaling [35
]. Thus, the effects of IL-17 on bone may occur through mechanisms independent of the RANK/RANKL/OPG axis.
Signal transduction
Several genes potentially involved in signal transduction were regulated by IL-17 and TNF- (Table 1)
. Of these, only SOCS3 and PSCD3 were validated by real-time PCR, and their enhancement was modest in MC3T3-E1 cells (less than twofold, Table 2
). The fact that neither TNF- nor IL-17 appears to regulate gene expression of signaling intermediates is not surprising, as most signal transducers tend to be controlled post-transcriptionally by post-translational modifications such as phosphorylation or cellular localization. Understanding how IL-17 impacts such biochemical signaling events will probably require proteomic rather than genomic approaches.
Summary and perspectives
Although IL-17 is a T cell-restricted cytokine, it exhibits properties of a proinflammatory cytokine, acting in conjunction with classic inflammatory cytokines such as TNF- and IL-1ß. In osteoblasts, genes that were highly induced by IL-17, TNF-, or both fell into the categories of chemokines and cytokines, immune response regulators, transcriptional regulators, bone-remodeling genes, signal transducers, cytoskeleton-related genes, and unknown or unclassified genes. Although some of these have been linked previously to IL-17, such as IL-6 and chemokines, we also identified genes that have been functionally connected to IL-17 for the first time, such as 24p3 and IB. This is the first reported large-scale study addressing the role of IL-17 in gene expression in bone cells. This global view of genes implicates IL-17 as a potent mediator of inflammation and neutrophil recruitment.
Received September 2, 2004; revised November 10, 2004; accepted November 19, 2004.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
/IL-1 interactions in bone resorption pathophysiology Cytokine Growth Factor Rev. 15,49-60[CrossRef][Medline]B ligand in vitro Oral Microbiol. Immunol. 18,30-36[CrossRef][Medline] is mediated by CCAAT/enhancer-binding protein family members J. Biol. Chem. 279,2559-2567, and interleukin-17 synergistically up-regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci Arthritis Rheum. 44,2078-2083[CrossRef][Medline] and IL-17 leads to enhanced expression and additional recruitment of AP-1 family members, Egr-1 and NF-B in osteoblast-like cells Cytokine 26,169-177[CrossRef][Medline] on chemokine secretion in human fetal intestinal epithelial cells Clin. Exp. Immunol. 125,56-63[CrossRef][Medline]-induced RANTES gene expression in human colonic subepithelial myofibroblasts J. Immunol. 169,1683-1687-induced stabilization of interleukin-6 mRNA in human pancreatic myofibroblasts Biochim. Biophys. Acta 1591,69-74[Medline]B and receptor activator of nuclear factor B (RANK)/RANK ligand signaling? Bone 28,378-386[Medline]B J. Biol. Chem. 273,27467-27473 and TNF- J. Immunol. 162,494-502 B-inducing kinase is a common mediator of IL-17-, TNF--, and IL-1 ß-induced chemokine promoter activation in intestinal epithelial cells J. Immunol. 162,5337-5344 chemokine from mesothelial cells J. Immunol. 165,5814-5821 B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages J. Immunol. 155,3593-3600[Abstract]B Nature 430,218-222[CrossRef][Medline]B-, a new anti-inflammatory nuclear protein induced by lipopolysaccharide, is a negative regulator for nuclear factor-B J. Endotoxin Res. 9,187-191[CrossRef] B ligand/osteoprotegerin balance J. Immunol. 170,2655-2662B ligand: modulation of the expression by osteotropic factors and cytokines Biochem. Biophys. Res. Commun. 275,768-775[CrossRef][Medline]This article has been cited by other articles:
 |
|
 |
|
  Y. R. Chan, J. S. Liu, D. A. Pociask, M. Zheng, T. A. Mietzner, T. Berger, T. W. Mak, M. C. Clifton, R. K. Strong, P. Ray, et al. Lipocalin 2 Is Required for Pulmonary Host Defense against Klebsiella Infection J. Immunol., April 15, 2009; 182(8): 4947 - 4956. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Shen, N. Li, P. Gade, D. V. Kalvakolanu, T. Weibley, B. Doble, J. R. Woodgett, T. D. Wood, and S. L. Gaffen IL-17 Receptor Signaling Inhibits C/EBP{beta} by Sequential Phosphorylation of the Regulatory 2 Domain Sci. Signal., February 24, 2009; 2(59): ra8 - ra8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. R. Conti, F. Shen, N. Nayyar, E. Stocum, J. N. Sun, M. J. Lindemann, A. W. Ho, J. H. Hai, J. J. Yu, J. W. Jung, et al. Th17 cells and IL-17 receptor signaling are essential for mucosal host defense against oral candidiasis J. Exp. Med., February 16, 2009; 206(2): 299 - 311. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hartupee, C. Liu, M. Novotny, D. Sun, X. Li, and T. A. Hamilton IL-17 Signaling for mRNA Stabilization Does Not Require TNF Receptor-Associated Factor 6 J. Immunol., February 1, 2009; 182(3): 1660 - 1666. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Guttman-Yassky, M. A. Lowes, J. Fuentes-Duculan, L. C. Zaba, I. Cardinale, K. E. Nograles, A. Khatcherian, I. Novitskaya, J. A. Carucci, R. Bergman, et al. Low Expression of the IL-23/Th17 Pathway in Atopic Dermatitis Compared to Psoriasis J. Immunol., November 15, 2008; 181(10): 7420 - 7427. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.L. Gaffen and G. Hajishengallis A New Inflammatory Cytokine on the Block: Re-thinking Periodontal Disease and the Th1/Th2 Paradigm in the Context of Th17 Cells and IL-17 Journal of Dental Research, September 1, 2008; 87(9): 817 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Yu, M. J. Ruddy, H. R. Conti, K. Boonanantanasarn, and S. L. Gaffen The Interleukin-17 Receptor Plays a Gender-Dependent Role in Host Protection against Porphyromonas gingivalis-Induced Periodontal Bone Loss Infect. Immun., September 1, 2008; 76(9): 4206 - 4213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. C. Owen, S. J. Roberts, S. F. Ahmed, and C. Farquharson Dexamethasone-induced expression of the glucocorticoid response gene lipocalin 2 in chondrocytes Am J Physiol Endocrinol Metab, June 1, 2008; 294(6): E1023 - E1034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Lindemann, Z. Hu, M. Benczik, K. D. Liu, and S. L. Gaffen Differential Regulation of the IL-17 Receptor by {gamma}c Cytokines: INHIBITORY SIGNALING BY THE PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY J. Biol. Chem., May 16, 2008; 283(20): 14100 - 14108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Godinez, T. Haneda, M. Raffatellu, M. D. George, T. A. Paixao, H. G. Rolan, R. L. Santos, S. Dandekar, R. M. Tsolis, and A. J. Baumler T Cells Help To Amplify Inflammatory Responses Induced by Salmonella enterica Serotype Typhimurium in the Intestinal Mucosa Infect. Immun., May 1, 2008; 76(5): 2008 - 2017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Haider, M. A. Lowes, M. Suarez-Farinas, L. C. Zaba, I. Cardinale, A. Khatcherian, I. Novitskaya, K. M. Wittkowski, and J. G. Krueger Identification of Cellular Pathways of "Type 1," Th17 T Cells, and TNF- and Inducible Nitric Oxide Synthase-Producing Dendritic Cells in Autoimmune Inflammation through Pharmacogenomic Study of Cyclosporine A in Psoriasis J. Immunol., February 1, 2008; 180(3): 1913 - 1920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Kramer, W. Hanel, F. Shen, N. Isik, J. P. Malone, A. Maitra, W. Sigurdson, D. Swart, J. Tocker, T. Jin, et al. Cutting Edge: Identification of a Pre-Ligand Assembly Domain (PLAD) and Ligand Binding Site in the IL-17 Receptor J. Immunol., November 15, 2007; 179(10): 6379 - 6383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Hartupee, C. Liu, M. Novotny, X. Li, and T. Hamilton IL-17 Enhances Chemokine Gene Expression through mRNA Stabilization J. Immunol., September 15, 2007; 179(6): 4135 - 4141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. N. Patel, C. A. King, S. R. Bailey, J. W. Holt, K. Venkatachalam, A. Agrawal, A. J. Valente, and B. Chandrasekar Interleukin-17 Stimulates C-reactive Protein Expression in Hepatocytes and Smooth Muscle Cells via p38 MAPK and ERK1/2-dependent NF-{kappa}B and C/EBPbeta Activation J. Biol. Chem., September 14, 2007; 282(37): 27229 - 27238. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Maitra, F. Shen, W. Hanel, K. Mossman, J. Tocker, D. Swart, and S. L. Gaffen Distinct functional motifs within the IL-17 receptor regulate signal transduction and target gene expression PNAS, May 1, 2007; 104(18): 7506 - 7511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. J. Yu, M. J. Ruddy, G. C. Wong, C. Sfintescu, P. J. Baker, J. B. Smith, R. T. Evans, and S. L. Gaffen An essential role for IL-17 in preventing pathogen-initiated bone destruction: recruitment of neutrophils to inflamed bone requires IL-17 receptor-dependent signals Blood, May 1, 2007; 109(9): 3794 - 3802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. H. Chang, H. Park, and C. Dong Act1 Adaptor Protein Is an Immediate and Essential Signaling Component of Interleukin-17 Receptor J. Biol. Chem., November 24, 2006; 281(47): 35603 - 35607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Shen, Z. Hu, J. Goswami, and S. L. Gaffen Identification of Common Transcriptional Regulatory Elements in Interleukin-17 Target Genes J. Biol. Chem., August 25, 2006; 281(34): 24138 - 24148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Koenders, E. Lubberts, F. A. J. van de Loo, B. Oppers-Walgreen, L. van den Bersselaar, M. M. Helsen, J. K. Kolls, F. E. Di Padova, L. A. B. Joosten, and W. B. van den Berg Interleukin-17 Acts Independently of TNF-{alpha} under Arthritic Conditions J. Immunol., May 15, 2006; 176(10): 6262 - 6269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Cowland, T. Muta, and N. Borregaard IL-1beta-Specific Up-Regulation of Neutrophil Gelatinase-Associated Lipocalin Is Controlled by I{kappa}B-{zeta} J. Immunol., May 1, 2006; 176(9): 5559 - 5566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Kramer, L. Yi, F. Shen, A. Maitra, X. Jiao, T. Jin, and S. L. Gaffen Cutting Edge: Evidence for Ligand-Independent Multimerization of the IL-17 Receptor J. Immunol., January 15, 2006; 176(2): 711 - 715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M T Bardella, M L Bianchi, and A Teti Chronic inflammatory intestinal diseases and bone loss Gut, October 1, 2005; 54(10): 1508 - 1508. [Full Text] [PDF]
|
|
|
|
|
|
F. McAllister, A. Henry, J. L. Kreindler, P. J. Dubin, L. Ulrich, C. Steele, J. D. Finder, J. M. Pilewski, B. M. Carreno, S. J. Goldman, et al. Role of IL-17A, IL-17F, and the IL-17 Receptor in Regulating Growth-Related Oncogene-{alpha} and Granulocyte Colony-Stimulating Factor in Bronchial Epithelium: Implications for Airway Inflammation in Cystic Fibrosis J. Immunol., July 1, 2005; 175(1): 404 - 412. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
