2 Department of Internal Medicine, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112
3 Department of Pathology, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112
4 Program in Human Molecular Biology and Genetics, Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112
5 Department of Internal Medicine III, Martin Luther University, 40 06097 Halle-Wittenburg, Germany
Address correspondence to Guy A. Zimmerman, Program in Human Molecular Biology and Genetics, University of Utah, 15 North 2030 East Bldg. 533, Rm. 4220, Salt Lake City, UT 84112. Tel.: (801) 585-0727; Fax: (801) 585-0701; email: guy.zimmerman{at}hmbg.utah.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: inflammation innate immunity gene expression translation transcription
Abbreviations used in this paper: mRNA, messenger RNA; mTOR, mammalian target of rapamycin; PAF, platelet activating factor; PMN, polymorphonuclear leukocyte; RACE, rapid amplification of cDNA ends; RAR, retinoic acid receptor; RARE, retinoic acid response element; RXR, retinoid X receptor; UTR, untranslated region.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Besides acute responses, there is now evidence that PMNs mediate inflammatory events over hours and can express new proteins and polypeptides, including chemokines (2). Soluble agonists, receptor-mediated phagocytosis, and integrin engagement dramatically alter levels of messenger RNA (mRNA) transcripts in human PMNs, and many of these gene products are under transcriptional control (25). New evidence also indicates that transcription is important in the regulation of PMN apoptosis and, thus, the duration and intensity of inflammation (1, 6). Although control of this process in activated PMNs is incompletely understood, key transcription factors are clearly central modulators. For example, constitutively expressed NF-B translocates from the cytoplasm to the nucleus in agoinist-stimulated PMNs where it binds to promoter motifs and regulates transcription of specific genes in regulated and dysregulated inflammation (2, 7, 8).
Here, we report a previously unrecognized mechanism of gene regulation in activated PMNs that involves unique interplay at posttranscriptional and transcriptional checkpoints. Critical posttranscriptional pathways are currently recognized with increasing frequency as analysis of the human genome proceeds, and there is now clear evidence that posttranscriptional dysregulation is a mechanism of disease (911). Posttranscriptional control at translational checkpoints may be particularly important in biologic systems in which factors with potent activities are precisely synthesized with respect to timing and magnitude and in a cell-specific fashion (911). These features characterize synthesis of chemokines, cytokines, and other factors by activated PMNs (18). In the studies reported here, we found that, in response to cellular activation, PMNs rapidly translate constitutive mRNA encoding the retinoic acid receptor (RAR)-, a nuclear hormone receptor and transcription factor that regulates the expression of multiple downstream genes in a variety of cells (12). In PMNs stimulated with platelet activating factor (PAF), translation of RAR-
mRNA is controlled by the intracellular signaling kinase mammalian target of rapamycin (mTOR), a specialized regulator of translation that we have found recently to respond to outside-in signals in human PMNs (13). The newly synthesized RAR-
protein then modulates expression of the chemokine IL-8. These observations identify a previously unrecognized mechanism in the inflammatory portfolio used by PMNs in which they modulate synthetic outcomes by rapid, regulated translation of a critical transcription factor.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polymorphonuclear Leukocyte Isolation.
PMNs were isolated from healthy adult donor heparinized whole blood (75 U heparin/10 ml) using techniques described previously by our laboratory (14). The procedures were approved by the University of Utah Institutional Review Board. Isolated PMNs were resuspended in HBSS (BioWhittaker, Walkersville) with 0.5% human serum albumin (Baxter Healthcare Inc.) to a concentration of 1520 x 106 cells/ml. Purity of the human PMNs was >95%.
HL-60 Cell Culture and Differentiation.
Cells of the human promyelocytic leukemia HL-60 cell line were obtained from American Type Culture Collection and grown in Iscove's Modified Dulbecco's Medium (BioWhittaker) supplemented with 20% FBS (Hyclone), penicillin G, and Streptomycin (BioWhittaker). Amphotericin B (Sigma-Aldrich) was added to the culture medium (2.5 µg/ml). The cell cultures were passed biweekly to maintain logarithmic growth. For these experiments, 2 x 106 HL-60 cells were resuspended in fresh media and were then differentiated along the PMN (granulocytic) lineage with 1.3% DMSO (American Type Culture Collection) for 6 d (15) (also see Selection for Differentiated HL-60 Cells and Assessment of HL-60 Differentiation). Viability was assessed by exclusion of 0.2% Trypan blue (Sigma-Aldrich) and was routinely >80% after differentiation.
Selection for Differentiated HL-60 Cells.
The purity of differentiated HL-60 surrogate PMNs was increased by negative selection in which cells that expressed the transferrin receptor (CD71) were removed (16). HL-60 cells treated with DMSO as above were incubated with the mouse antihuman CD71 (transferrin receptor) monoclonal antibody (1 µg/ml) for 30 min. The CD71-positive cells were complexed to magnetic microbeads coated with goat antimouse IgG (Miltenyi Biotec) and subsequently removed by passage through a magnetized column.
Assessment of HL-60 Differentiation.
Differentiation of HL-60 cells to surrogate PMNs (HL-60 PMNs) (15) was assessed by several criteria including nuclear segmentation, loss of transferrin receptor expression (16), and gain of responsivity to PAF (17). Immunocytochemical and flow cytometric analyses were used to detect transferrin receptor expression. Immunocytochemical experiments were done according to our previously published protocols (18), and the cells were examined with a Bio-Rad Laboratories confocal microscope (Hercules). Flow cytometry was performed on a Becton Dickinson FACScanner using minor modifications of previously published protocols (16, 19). PAF-induced cytosolic calcium influxes were assayed using a Shimadzu spectrofluorophotometer after incubation with the calcium sensitive fluorescent dye Fura-2 (Molecular Probes) (20).
Isolation of Polyribosomal RNA from Differentiated HL-60 Surrogate PMNs for mRNA Expression Array Analysis.
For polyribosomal profiling experiments, we used differentiated HL-60 surrogate PMNs rather than primary PMNs to minimize difficulties in maintaining integrity of leukocyte RNA during cell fractionation and transcript analysis (21). Differentiated HL-60 PMNs (25 x 106 cells/sample) were left quiescent or stimulated with PAF (108 M) for 1 h at 37°C. The cells were lysed, and ribosomal profiling was conducted using minor modifications of procedures that we have described elsewhere (22). Total RNA was isolated from the polyribosomal fractions from each sample and used to generate 32P-dATP labeled cDNA, which was then employed as a template in expression analysis (Atlas Human Cancer cDNA Expression Array, 77421; CLONTECH Laboratories, Inc.).
RAR- Protein Expression.
Differentiated HL-60 PMNs or freshly isolated primary human PMNs (2.5 x 106) were resuspended in M-199 media (37°C) and left quiescent or activated with PAF in indicated concentrations at 37°C in 5% CO2. In selected experiments, the cells were pretreated (30 min) before activation with specific translational inhibitors (puromycin, cycloheximide, and rapamycin). At the indicated time points, the cells were incubated with ProteaseArrest/EDTA (100x) for 5 min at room temperature to prevent protein degradation. The cells were pelleted and then lysed in 200 µl of 1x Laemmli buffer (0.025 M Tris, pH 6.8, 4% glycerol, 2% SDS, 1% ß-mercaptoethanol, 0.25% bromophenyl blue). Samples were stored frozen at 20°C until SDS-PAGE and Western analysis was conducted as described previously (23).
RAR- and IL-8 mRNA Expression.
Quiescent or PAF-stimulated human PMNs were lysed in Trizol reagent, and total RNA was isolated via a modification of previously described methods (24). SuperScript II or M-MLV reverse transcriptase (Invitrogen) was then used to generate cDNA. The cDNA was used for a PCR reaction designed to amplify a 233-bp product generated using primers against the RAR- gene sequence (sense 5'-GGAGCTCATTGAGAAGGTGC-3'; antisense 5'-TTGAGGAGGGTGATCTGGTC-3'). Primers designed to amplify a 530-bp sequence of the GAPDH gene were used as an internal control. In selected experiments, primers specifically constructed based on the RAR-
1 (5'-CTGTCTGCCTCCCTTCTGA-3') and RAR-
2 (5'-CCACCCCTAATCCCTTCCT-3') isoforms (25) were used with a primer common to the coding region of RAR-
1 and RAR-
2 (see Characterization of the 5'-Untranslated Region of the RAR-
mRNA Using Rapid Amplification of cDNA Ends). For real time PCR measurements, we purchased premixed PCR primers against RAR-
(Hs00230907_m1) and IL-8 (Hs00174103) and separate primers for GAPDH (GAPDH Probe [designated "Joe"], GAPDH forward, GAPDH reverse). The Bio-Rad Laboratories iCycler IQ (Hercules), the MJ Research, Inc. Opticon 2, and Applied Biosystems 7900 HT real-time PCR analysis systems were used to generate quantitative measures of the expression of RAR-
, IL-8, and GAPDH mRNAs.
Characterization of the 5'-Untranslated Region of the RAR- mRNA Using Rapid Amplification of cDNA Ends.
Total RNA was isolated from unstimulated human PMNs and used to generate first-strand rapid amplification of cDNA ends (RACE)ready cDNA using reagents purchased from CLONTECH Laboratories, Inc. with the Smart RACE cDNA Amplification kit. The sequence for the 5'-untranslated region (UTR) of the RAR- mRNA was amplified by PCR using the nested universal primer supplied by CLONTECH Laboratories, Inc. and a specific primer directed at the conserved regions of the RAR-
gene (5'-CCCCATAGTGGTAGCCTGAG-3'). The RAR-
5'-UTR fragments were ligated into a pCR 2.1 vector and cloned into INF' Escherichia coli bacteria supplied with the TA Cloning kit (pCR II, pCR 2.1) purchased from Invitrogen. Plasmid DNA was extracted and prepared for sequencing using a QIAprep Spin DNA Miniprep kit with supportive reagents purchased from QIAGEN. An Applied Biosystems ABI 3700 96-capillary sequencer operated by the DNA Sequencing Core Facility at the University of Utah was used to obtain sequences from the 5'-UTR containing plasmid DNA. DNASis software (MiraiBio) was used for analyzing the secondary structure of the 5'-UTR of the RAR-
mRNA.
Detection of IL-8 Protein.
Freshly isolated human PMNs were incubated with PAF (108 M), AM-580 (106 M), or the combination of both reagents at 37°C in 5% CO2 for various times. For other experiments, we used the same conditions with and without rapamycin pretreatment (10 nM) for 1 h. The supernatants were collected and frozen at 20°C until analysis by ELISA. IL-8 protein levels were quantitatively assessed using the human IL-8 DuoSet ELISA system from R&D Systems according to their protocols. Standard 96-well ELISA plates with samples were analyzed using SOFTmax for Macintosh Elisa software (Molecular Devices) and a Molecular Devices THERMOmax Microplate Reader.
Online Supplemental Material.
In Fig. S1, HL-60 cells were cultured, differentiated, and characterized as described in preceding sections. In Fig. S2, PMNs were isolated, stimulated with PAF, and IL-8 mRNA and protein were analyzed as outlined above. Figs. S1 and S2 are available at http://www.jem.org/cgi/content/full/jem.20040224/DC1.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unstimulated HL-60 PMNs displayed typical ribosomal profiles (22, 28) consisting of 40S and 60S ribosomal subunit peaks and an intact 80S single ribosome peak, with smaller polysome peaks in the gradient (Fig. 1 A). When we stimulated these cells with PAF, a potent inflammatory phospholipid that rapidly activates PMNs (13, 29, 30), the heights of the 40S and 60S ribosomal subunit peaks decreased, and in parallel the height of the 80S monosome peak increased (Fig. 1 A) indicating translation initiation (10). We then examined patterns of mRNAs associated with polysomes in resting and activated surrogate PMNs by cDNA microarray analysis (22) to identify transcripts undergoing active translation (28). We found 28 mRNAs that were differentially associated with polysomes in PAF-stimulated cells compared with unstimulated HL-60 PMNs (Table I). One transcript of particular interest was RAR-, a nuclear hormone receptor that binds retinoic acid (12). The RAR-
gene is best known as the target of chromosomal rearrangement in acute promyelocytic leukemia (31). Although RAR-
has not been identified in terminally differentiated mature PMNs, it modulates the commitment of pluripotent hematopoietic progenitors to the neutrophil lineage (32). Because the RARs directly and indirectly regulate transcription of multiple gene products (12), appearance of the mRNA encoding RAR-
in polysomes in response to activation of HL-60 surrogate PMNs suggested that it may represent a key checkpoint in PMN synthetic pathways.
|
|
RAR- mRNA Is Constitutively Expressed in Primary Human PMNs and Is Translated into Protein in a Signal-dependent Fashion.
Although differentiated HL-60 PMNs exhibit features of circulating human neutrophils (see The mRNA for RAR- Is Found in Polyribosomes of PAF-stimulated Surrogate PMNs and Fig. S1) and are a useful surrogate model, the biological features of cell lines are often substantially different when compared with those observed in the corresponding primary human cell. Therefore, we characterized RAR-
mRNA and protein expression in freshly isolated human PMNs and in PMNs activated with PAF. Like HL-60 surrogate PMNs, quiescent primary PMNs express mRNA for RAR-
at baseline (Fig. 2 A). Levels of the RAR-
transcript did not increase and, in fact, decreased after stimulation with PAF (Fig. 2 A). Little or no RAR-
protein was present in resting PMNs, but in contrast it was dramatically induced after cellular activation with PAF (Fig. 2 A, bottom and not depicted). Recombinant PAF acetylhydrolase, an enzyme that selectively degrades PAF (30), blocked expression of RAR-
protein under these conditions indicating specificity of the agonist effect (not depicted). This same pattern of RAR-
expression was seen in an experiment using neonatal PMNs isolated from umbilical cord blood at the time of term delivery and stimulated with PAF under conditions similar to those used for PMNs from adult donors (not depicted). In addition to PAF, TNF
, lipopolysaccharide, and n-formyl-methionyl-leucyl-phenylalanine also triggered rapid synthesis of RAR-
(not depicted). Thus, outside-in signals delivered by other classes of receptors also induce this response, in addition to the PAF signaling pathway.
|
Synthesis of RAR- Protein is Regulated at Translational Checkpoints in Activated PMNs.
Although there is differential expression of RARs in a variety of cells, most studies have focused on mechanisms by which RAR- regulates the activity of target promoters (12, 31), and pathways that control synthesis of the protein are not well understood. Experiments in Fig. 2 A indicated that RAR-
protein is translated from constitutive mRNA in activated neutrophils. When we shortened the time course, we found that RAR-
protein could be expressed within intervals as short as 5 min in PAF-stimulated PMNs (Fig. 2 B). This, together with the discrepancy between RAR-
mRNA and protein levels in resting PMNs (Fig. 2, A and B), is a strong indication that RAR-
is under translational control (10, 13, 22, 23, 28). Actinomycin D, a transcriptional inhibitor, did not block synthesis of RAR-
protein under these conditions, indicating that its production is controlled post-transcriptionally (Fig. 2 C). In contrast, actinomycin D completely inhibited expression of the mRNA for IL-8 and synthesis of the IL-8 protein (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20040224/DC1), consistent with previous observations that transcriptional regulation is critical in its production by human PMNs (2). Using similar experimental protocols, actinomycin D also selectively inhibited production of other protein products of stimulated neutrophils (13). Studies with puromycin and cycloheximide, which are translational inhibitors, confirmed that accumulation of RAR-
protein requires new synthesis in neutrophils activated with PAF (Fig. 3 A).
|
The 5'-UTR of the RAR-1 mRNA in PMNs exceeds 500 nucleotides, is very GC rich, and computer folding indicates that it contains numerous hairpins and significant secondary structure (predicted
G = 393.4 kcal/mol) (Fig. 3 B). Transcripts with 5'-UTR secondary structures that require energy to unwind exceeding 60 kcal/mol are often repressed at the translational level (9, 10, 13, 23, 24, 33). Previously, it was noted that sequence features of the 5'-UTRs of mRNAs coding for RAR isoforms predict translational control (9, 11), but this mode of regulated expression of their proteins has not been reported in human leukocytes.
In other cell types, mTOR regulates events that unfold 5'-UTR secondary structure, relieving repression and initiating translation of a subset of key mRNAs that are under tight synthetic control (13, 23, 24, 33). In parallel studies, we found that mTOR and its downstream targets (p70S6 kinase and eukaryotic initiation factor-4E binding protein-1 [4E-BP1]) are present and active in stimulated PMNs (13), suggesting the possibility that mTOR controls RAR- synthesis in response to PAF stimulation. Rapamycin, an immunosuppressant macrolide, specifically blocks mTOR-dependent signaling in myeloid leukocytes (13, 23) in addition to other cell types (33). We found that rapamycin potently inhibits the synthesis of RAR-
protein in PAF-stimulated PMNs (Fig. 3 C). In contrast, it had no effect on the level of actin (Fig. 3 C), a gene product that is independent of signal-dependent translational control (23).
Rapamycin is used clinically to prevent rejection after organ transplantation and to reduce the incidence of vascular restenosis after interventional procedures in atherosclerotic vascular syndromes (34). It is generally believed that its therapeutic effects largely involve blocking proliferation of dividing cells (34, 35). Inhibition of expression of RAR- (Fig. 3 C) and a subgroup of other key gene products in PMNs (13), which are terminally differentiated cells that do not divide, indicates that rapamycin also modulates other cellular responses and may have previously unrecognized effects on inflammation. Inhibition of RAR-
protein expression in activated PMNs by rapamycin, which is a critical molecular probe for the mTOR pathway (33, 36), also provides new insights into the interaction between specialized translation control pathways and transcriptional regulation (9) in this cell type. Although it has been observed that homologues of mTOR influence transcriptional activity in response to nutrient stress in yeast (36) and that mTOR regulates expression of Bcl-3, a member of the NF-
B family of transcriptional regulators, in other human cells (24, 37) selective translational control of a transcription factor in activated myeloid leukocytes has not been reported previously.
RAR- Regulates the Expression of IL-8 by Activated PMNs.
One mechanism by which RAR- modulates transcription is by binding to promoter sequences that contain retinoic acid response elements (RAREs) (12). RAR-
binds consensus elements more efficiently in the presence of a second group of retinoic acid receptors, the retinoid X receptors (RXRs) (12). We found that RXR-
protein is constitutively present in resting PMNs (Fig. 4 A), indicating that it is available to form RXR-RAR heterodimers and that RXR
and RAR-
1 are differentially expressed in this cell type. IL-8 is one of a relatively small number of RAR-responsive products known to by synthesized by activated PMNs among the large group of genes whose expression is regulated by retinoic acid (12). We found that mRNA for IL-8 increased after stimulation of PMNs with PAF (Fig. S2 and Fig. 4 B), consistent with induced synthesis of this chemokine in response to stimulation (2). We then examined the effect of AM-580, a synthetic retinoid agonist with specificity for RAR-
(38), under conditions in which expression of RAR-
and IL-8 is induced. AM-580 significantly attenuated IL-8 transcript accumulation after 60 min of stimulation with PAF, whereas treatment of unactivated PMNs with AM-580 had no effect (Fig. 4 B). In parallel, we found that PAF-stimulated PMNs synthesize significant amounts of IL-8 protein. Treatment with AM-580 markedly attenuated PAF-induced IL-8 accumulation as measured by ELISA (Fig. 4 C). A similar effect on PAF-induced IL-8 protein synthesis was noted with all-trans retinoic acid treatment (not depicted). AM-580 (Fig. 4 C) or all-trans retinoic acid (not depicted) alone did not significantly alter IL-8 synthesis. We then pretreated PMNs with rapamycin before stimulation with PAF (Fig. 4 D) to inhibit the induction of RAR-
protein (Fig. 3 C). AM-580 failed to attenuate synthesis of IL-8 under these conditions (Fig. 4 D). Together these results indicate that engagement of newly expressed RAR-
1 by ligands can alter IL-8 transcription and synthesis of IL-8 protein in stimulated human PMNs and that mTOR regulates this sequence of events by controlling RAR-
translation. Furthermore, they indicate that RAR-
has activities beyond granulopoiesis (39) in this cell lineage. It is also possible that unliganded RAR-
1 regulates expression of IL-8 in PMNs activated with PAF (40). The PAF and retinoid systems have previously been reported to synergistically alter expression of cyclooxygenase 2 in neural cells and fibroblasts (41), but convergence of these signaling and regulatory mechanisms has not been observed in myeloid leukocytes.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Acknowledgments |
---|
This work was supported in part by the Children's Health Research Center of the University of Utah and the Primary Children's Medical Center Foundation (awards to C.C. Yost and F.J. Rubner), a Physician-Scientist Training Award from the American Diabetes Association (American Diabetes Association no. PID2206052 to M.M. Denis), the National Institutes of Health (HL-44525 to G.A. Zimmerman, HL-66277 to A.S. Weyrich, HL-44513 to T.M. McIntyre, and P50 HL-50153 to T.M. McIntyre and G.A. Zimmerman), and Established Investigator Award from the American Heart Association (to A.S. Weyrich). C.C. Yost is a member of the Fellowship-to-Faculty Transition Program at the University of Utah, which is supported in part by the Howard Hughes Medical Institute Biomedical Research Program for Medical Schools.
The authors have no conflicting financial interests.
Submitted: 4 February 2004
Accepted: 27 July 2004
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|