| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
c
shared with IL-2 (2, 3, 4). Due in part to this sharing of receptor
subunits, there is considerable redundancy in the actions of IL-2 and
IL-15. Both cytokines activate the proliferation and differentiation of
T, NK, and B cells (3, 5, 6). Initially, it was not clear why two
cytokines with such similar actions should have evolved and been
retained. However, further analysis revealed dramatic differences
between these two cytokines in terms of their cellular sites of
synthesis and the levels of control of their synthesis and secretion
(7, 8). IL-2 is produced by activated T cells, and its expression is
controlled predominantly at the level of mRNA transcription and message
stabilization. In contrast with the T cell pattern of IL-2 mRNA
expression, IL-15 mRNA expression is widespread. In particular,
Northern blot analysis revealed a broad constitutive expression of
IL-15 mRNA in diverse tissues, including placenta, skeletal muscle,
kidney, lung, heart, fibroblasts, epithelial cells, and activated
monocytes (3, 7). Despite the almost ubiquitous expression of IL-15
mRNA, it has been difficult to demonstrate IL-15 protein in the
supernatants of many cells that express message for this cytokine, one
of the hallmarks of translational regulation. In earlier studies, we
observed that even though LPS/IFN--activated monocytes express high
levels of IL-15 message, the culture supernatants from these cells
contain little or no IL-15 protein as assessed by either an IL-15
specific ELISA or a CTLL-2 proliferation assay (7). This suggested that
normal IL-15 protein production is predominantly regulated
posttranscriptionally. The discordance between IL-15 message expression
and IL-15 protein production led us to examine IL-15 mRNA for
translational impediments. We previously reported that the IL-15
message contains a complex 5'
UTR3 with 10 upstream AUGs
(7). COS cells transfected with an expression construct lacking the 5'
UTR produced four- to fivefold more IL-15 protein than cells
transfected with a construct that retained the 5' UTR. However, the
levels of IL-15 protein synthesized and secreted were still very low
(
3 logs less than those obtained with a comparable IL-2 construct),
suggesting the possibility that additional regulatory elements exist
(7). Here we demonstrate that IL-15 expression is posttranscriptionally
impeded not only by the 5' UTR, but also by the coding sequence and/or
protein sequence of its signal peptide (SP) and mature protein (mp)
carboxyl terminus (C terminus). |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The simian kidney epithelial cell line, COS, and the cytokine-dependent murine cytotoxic T cell line, CTLL-2, were both purchased from the American Type Culture Collection (Rockville, MD). Conditions for CTLL-2 bioassays were previously described (1), and COS transfection conditions are described below.
Abs and cytokines
The rabbit anti-human IL-15 polyclonal Ab used for Western blot analysis was a gift from Harmesh Sharma (Genzyme, Cambridge, MA), and the rabbit anti-human IL-15 polyclonal used for immunoprecipitation was purchased from Serotec. Recombinant human IL-15 was purchased from Peprotech, Inc., and recombinant IL-2 was a gift from Hoffmann-La Roche, Nutley, NJ).
IL-15 and IL-2 expression constructs
The generation of the pEF-Neo/
AUG-IL-15 expression construct
was previously described (7). pEF-Neo/IL-2 CDS was constructed by
amplifying the IL-2 CDS (nucleotides 45–509) using the sense primer
(primer 1) 5'-ATCGAATTCACAATGTACAGGATGCAACTCC-3' and the
antisense primer (primer 2)
5'-GATCGGATCCTCAAGTTAGTGTTGAGATGATGC-3' (underlined
sequences represent nested EcoRI and BamHI
restriction sites, respectively). Amplified material was directionally
subcloned into pEF-Neo, and the sequence was confirmed.
For generation of the chimeric expression constructs, the SP coding sequences of IL-15 (nucleotides 299–460) and IL-2 (nucleotides 45–110) were PCR amplified from existing constructs. Specifically, for IL-15, the sense primer (primer 3) 5'-GATCAAGCTTCCGTGGCTTTGAGTAATGAGA-3' (HindIII) and the antisense primer (primer 4) 5'-GATCGGCGCCGTTGGCTTCTGTTTTAGGAAGC-3' (NarI), and for IL-2, the sense primer 1 and the antisense primer (primer 5) 5'-ATCGGCGCC/TGCACTGTTTGTGACAAGTGC-3' (NarI) were used. The amplified SP coding sequences for both cytokines were then TA cloned into PCRII (Invitrogen, San Diego, CA). Subsequently, the mature IL-15 protein coding sequence (nucleotides 458–802) and the mature IL-2 protein coding sequence (nucleotides 111–518) were also PCR amplified from existing constructs. For IL-15, the sense primer (primer 6) 5'-GATGGCGCCAACTGGGTGAATGTAATAAGTG-3' (NarI) and the antisense primer (primer 7) 5'-GATCGGATCCTCAAGAAGTGTTGATGAACATTTGG-3' (BamHI), and for IL-2, the sense primer (primer 8) 5'-GATCGGCGCCCCTACTTCAAGTTCTACAAAG-3' (NarI) and the antisense primer 2 were used. The amplified mp coding sequences were then digested with NarI and EcoRI and directly subcloned into the PCRII constructs containing the SP coding sequence of the alternative cytokine, e.g., IL-2 SP with the IL-15 mp and vice versa. In the final constructs, the NarI sites behave as linkers between the SP and the mp coding sequences and add a glycine and an alanine residue to the mps. The chimeric constructs were ultimately digested from PCRII and subcloned into pEF-Neo, and sequences were confirmed.
For generation of the IL-15 construct with the C-terminal epitope tag, FLAG (Eastman Kodak, Rochester, NY), an IL-15 fragment was PCR amplified using the same sense primer (primer 3) described above for the SP, and the antisense primer (primer 9) 5'-CGCTATTTGTCATCGTCGTCCTTGTAGTC/AGAAGTGTTGATGAA-3'. The underlined sequence represents the coding region of FLAG in-frame with the last codon (AGA) of IL-15. The material was originally TA cloned into PCR3 and then subcloned into the EcoRI site of pEF-Neo. The IL-15 construct with the combination of the IL-2 SP and FLAG was generated by digesting an IL-2/IL-15 chimeric pSP64poly(A) (Promega, Madison, WI) construct with PvuII and BglII. This left the IL-2 SP coding sequence intact in pSP64PA plus a portion of the IL-15 mp coding sequence, which terminates with an endogenous BglII site. The remaining portion of IL-15’s coding sequence was directly replaced with a BglII, PvuII fragment of IL-15 with C-terminal FLAG (that had been excised from a pSP64PA/IL-15 FLAG construct). The resulting IL-2sp/IL-15 mp/FLAG insert was digested from pSP64PA with EcoRI and cloned into pEF-Neo.
The IL-15 construct with the higher Kozak context was constructed by amplifying the IL-15 CDS with the sense primer (primer 10) 5'-GATGATTCGCCGCCGCCATGAGAATTTCGAAACC-3' (EcoRI) and an antisense primer identical with primer 7, but with a nested EcoRI site. The double underlined sequence in primer 10 represents the improved Kozak context. The amplified material was digested with EcoRI and subcloned into pEF-Neo.
It should be noted that with the exception of 17 nucleotides in the 5' UTR of IL-15 constructs, four nucleotides in the 5' UTR of IL-2 constructs or, if otherwise stated, the natural 5' and 3' UTRs of IL-15 and IL-2 cDNAs have been eliminated in all constructs. Additionally, all constructs use the EcoRI site immediately 5' of the pEF-Neo promoter (elongation factor); based on the position of the EcoRI site, the only sequence contributed by the vector to the 5' end of the mRNA is 5'-GGAATTC-3'
COS cell transfection and lysate and supernatant generation
For CTLL-2 bioassays, COS cells were transfected with 1.5 µg of IL-15 and IL-2 expression constructs following a standard DEAE-dextran protocol (9). For these studies roughly 2 x 105 adherent COS cells/well were transfected in six-well 35-mm plates; after 24 h, the medium was replaced with fresh complete DMEM, and supernatants were harvested 72 h after the transfection for assay. For Western analyses and mRNA stability studies, the DEAE-dextran protocol was scaled up to 1.6 x 106 cells in 75-cm2 flasks. Cellular lysates generated for Western blots were made by adding 1 ml of RIPA buffer/flask (50 mM Tris-HCl (pH 7.4), 1.0% Nonidet P-40, 0.25% NaDOC, 0.15 M NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.1% SDS). For mRNA details, see below.
For time course studies that used IL-2 and IL-15 ELISAs, COS cells were transfected using Lipofectamine (Life Technologies, Grand Island, NY) following the manufacturer’s recommendations scaled up for 1.6 x 106 cells in 75-cm2 flasks. Twenty-four hours after transfection with IL-15 and IL-2 constructs, supernatants were harvested, and the COS cells were trypsinized and washed in PBS. One-fifth of the cells were lysed in RIPA buffer, and the remainder were aliquoted equally into four 60-mm plates with 2.5 ml of fresh complete DMEM. One plate was then harvested (supernatants and lysates) every 24 h for 120 h. To lessen the likelihood of cytokine degradation, supernatants were collected from the remaining plates throughout the time course and replaced with fresh medium.
For IL-15 protein degradation studies, 1 x 107 COS cells (in 400 µl of 1x PBS) were electroporated with 15 µg of the indicated construct (950 µF, 240 V). Electroporated cells were evenly aliquoted into 60-mm plates (five per construct) in 2.5 ml of complete DMEM. At 48 h, each plate was pulsed with 150 µCi of [35S]Cys/[35S]Met (New England Nuclear, Boston, MA, express labeling mix) in 2.5 ml of Cys/Met-free DMEM with 10% dialyzed FCS (cells were not Cys/Met starved before the addition of [35S]Cys/Met). After 1 h in [35S]Cys/Met at 37°C, cells were washed twice in PBS, and then 3 ml of complete DMEM was added as a cold chase. Lysates were made at the indicated time points in 1 ml of RIPA buffer.
Western blotting, ELISAs, and immunoprecipitation
For IL-15 Westerns, RIPA lysates of transfected COS cells were resolved by 4 to 20% gradient SDS-PAGE (Novex, San Diego, CA) and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). Development of the filter with anti-IL-15 polyclonal was performed using the Luminol system (Pierce Chemical, Rockford, IL) and following the manufacturer’s recommendations.
Supernatants and lysates of transfected COS cells were also assessed for IL-2 and IL-15 expression using specific ELISAs (Genzyme), following the manufacturer’s recommendations. However, to compensate for suppression of ELISA sensitivity by RIPA buffer, IL-2 and IL-15 standards were run with a comparable volume of RIPA buffer.
The immunoprecipitation of IL-15 from 35S-labeled pulse/chase lysates used Ultralink immobilized protein A/G (Pierce). Procedures, including incubation times, anti-IL-15 Ab concentrations, and washings, closely followed the manufacturer’s recommendations.
Detection of mRNA and mRNA stability
Total RNA was isolated from transfected COS cells using TRIzol (Life Technologies). RNA samples (5 µg) were assessed by Northern blot analysis for IL-15, IL-2, and ß-actin mRNA following a published protocol (10). For mRNA stability studies, COS cells were replated on 60-mm plates (five per construct) 24 h posttransfection. At 72 h, 5 µg/ml of actinomycin D (Life Technologies) in 3 ml of complete DMEM was added to cells, and total mRNA was isolated at the indicated time points. Relative levels of mRNA expression were determined using a Storm-840 (Molecular Dynamics, Sunnyvale, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To demonstrate that elements in addition to the 5' UTR negatively
influence IL-15 protein production, we compared the translational
efficiencies of IL-2 and IL-15 constructs in a COS cell expression
system. Specifically, IL-15 and IL-2 coding sequences lacking their 5'
and 3' UTRs were subcloned into the expression vector, pEF-Neo, that
uses the strong elongation factor promoter. The IL-15 construct,
pEF-Neo/AUG-IL-15, and the IL-2 construct, pEF-Neo/IL-2 CDS, were
transiently transfected into 2 x 105 COS cells
using DEAE-dextran in 35-mm plates, and supernatants were harvested
72 h later. The quantities of IL-2 and IL-15 protein in the
supernatants were determined by their ability to stimulate CTLL-2 cell
proliferation ([3H]TdR incorporation) compared with that
of IL-15 and IL-2 standards. We determined in this study that the IL-2
transfectants generated roughly 350,000 pg of secreted IL-2 protein,
while the IL-15 transfectants generated only about 360 pg, a 1,000-fold
difference (Fig. 1
). Subsequent studies
assessing IL-2 and IL-15 mRNA expression in transfected COS cells over
time showed virtually equal levels of transcript for the two cytokines
throughout the time course despite the disparity in protein production
observed (Fig. 2). Additionally, IL-15
transcripts generated from pSP64pA/AUG-IL-15 were readily translated
in a wheat-germ lysate, in vitro transcription and translation (TnT)
system, but were poorly translated in a rabbit reticulocyte TnT system.
In contrast, IL-2 transcripts generated using the same vector system
expressed virtually equal quantities of IL-2 protein in the wheat-germ
and rabbit reticulocyte TnT systems (data not shown). Collectively,
these data suggest that in mammalian systems (i.e., COS cells and
rabbit reticulocyte lysates) there are inhibitory/regulatory factors in
addition to those in the 5' UTR that interfere with the efficient
synthesis of IL-15.
|
|
We next examined the IL-15 mRNA for specific elements that might
impede IL-15 expression and focused on the putative IL-15 SP. IL-15 has
been shown to have an unusually long 48-amino acid SP compared with
those of other secreted proteins, whose SPs average 15 to 30 amino
acids in length. Therefore, we considered the possibility that the
IL-15 SP and/or its coding sequence is a negative regulator of IL-15
generation (8). To test this hypothesis, we prepared expression
constructs that exchanged the SP coding sequences of IL-2 and IL-15 so
that they were linked to the alternative mp coding sequence. The
resulting chimeric cDNAs were subcloned into pEF-Neo (i.e., the IL-2 SP
with the IL-15 mp coding region (pEF-Neo/IL-2sp/IL-15 mp) and
reciprocally pEF-Neo/IL-15sp/IL-2 mp) and then transiently transfected
into COS cells. In initial studies, 72 h after transfection, the
levels of secreted IL-15 and IL-2 present in COS supernatants were
assessed by the CTLL-2 bioassay. Supernatants from COS cells
transfected with the pEF-Neo/IL-2sp/IL-15 mp expression construct
contained roughly 20-fold more IL-15 protein than supernatants from COS
cells transfected with pEF-Neo/AUG-IL-15 (Fig. 3A). Reciprocally,
supernatants from COS cells transfected with pEF-Neo/IL-15sp/IL-2 mp
showed roughly a 50-fold drop in secreted IL-2 compared with
supernatants from COS cells transfected with a wild-type IL-2
expression construct (Fig. 3B). These data support
the hypothesis that the IL-15 SP and/or its coding sequence are
important factors in the negative regulation of IL-15 expression.
However, it could be argued that by only assessing COS supernatants for
IL-15 and IL-2 activity we might miss cytokine retained in
intracellular pools or on the surface of the transfected COS cells.
|
). However, at the latter time points
(90 and 120 h), the majority of IL-15 protein produced from either
construct had been secreted. Furthermore, even though the IL-15 SP
contributed to a minor, but meaningful, increase in the percentage of
wild-type IL-15 or chimeric IL-2 (IL-15sp/IL-2 mp) retained in the cell
over time (compare Fig. 4A with 4B and Fig. 4C with 4D), the primary effect of the
IL-15 SP was on the total amount of these cytokines generated. In
particular, at the 72 h time point, roughly 45% of wild-type
IL-15 was retained in the cell, but only a total of 370 pg (cellular
plus secreted) was detected. However, at the same time point, 30% of
the IL-15 chimeric with the IL-2 SP was retained within the cell, but a
total of 6430 pg of IL-15 protein was present in the cell lysates and
secretions, representing a 17-fold increase in total protein
synthesized compared with that observed with wild-type IL-15. Similar
observations were made with wild-type IL-2 vs chimeric IL-2.
Specifically, the IL-15 SP slightly increased the proportion of
chimeric IL-2 protein retained in the cell, but dramatically decreased,
by roughly 30-fold, the total amount of IL-2 produced compared with the
effect of wild-type IL-2. In these studies the ELISA results closely
paralleled those with CTLL-2 bioassays that used the same
constructs. Therefore, it can be concluded that the IL-15 SP and/or its
coding sequence act to impede IL-15 protein expression, but the SP per
se does not lead to an extensive accumulation of IL-15 within
intracellular pools or on the cell surface.
|
AUG-IL-15-FLAG) increased total
IL-15 production (cellular plus secreted) 5- to 10-fold over that
produced by AUG-IL-15 when they were transiently transfected into
COS cells in the pEF-Neo expression system (Fig. 5A). Both these
constructs lack any natural 3' UTR nucleotides, so only the IL-15
coding sequence is affected by FLAG’s coding sequence. Also, the
addition of FLAG to the coding sequence of the IL-2 C terminus had no
influence on its expression (data not shown). Additionally, to the best
of our knowledge, there is no example of the FLAG epitope coding
sequence enhancing the translation of mRNAs tagged with it. These data,
therefore, suggest the existence of additional negative regulatory
elements within the 3' coding sequence or C terminus of IL-15.
Apparently the presence of FLAG disrupts this putative, as yet
undefined, negative regulatory mechanism.
|
AUG-IL-15 (data
not shown), whereas the FLAG only modification, as demonstrated
earlier, enhanced expression by only 5- to 10-fold, and the SP
modification enhanced expression by only 17- to 20-fold. IL-2sp/IL-15
mp/FLAG was also compared with an IL-15 construct with its full
complement of upstream AUGs in a COS cell transfection. The chimeric
transcripts generated >250-fold more protein than the IL-15
transcripts with the upstream AUGs, emphasizing the full inhibitory
potential of these negative elements (Fig. 5
B).
Additionally, lysates from the same transfected COS cells were assessed
by Western blot analysis for IL-15 protein generation. The differences
in intracellular IL-15 protein levels previously noted using the ELISA
technique were also easily visualized by Western blot analysis (Fig. 6A). Importantly, to
control for the possibility of a transcriptional difference between the
various IL-15 constructs, Northern blot analysis for IL-15 was
conducted on the same transfectants (Fig. 6B). There
was virtually no difference between levels of IL-15 message generated
with the alternative constructs, indicating that the protein expression
differences seen with the constructs were not transcriptional, but were
due to posttranscriptional events.
|
IL-15 and IL-2 protein expression differences are not explained by differences in mRNA stability
Since we demonstrated relatively equal levels of IL-15 transcript
expression for the various IL-15 and IL-2 constructs, it seemed
unlikely that differences in mRNA stability could play a major role in
the differences seen in the quantities of IL-15 protein generated.
However, to address this issue more critically, COS cells were
transfected with the above constructs, and after 72 h, the
antibiotic, actinomycin D (5 µg/ml), was added to the cells.
Actinomycin D arrests transcription in eukaryotic cells, allowing for
assessment of mRNA stability. After the addition of actinomycin D,
total RNA was isolated at 0, 2.5, 5.0, 7.5 and 10 h and was
quantitated by Northern blot analysis for IL-15 and ß-actin
transcripts. The levels of IL-15 message were comparable using the
different constructs (Figs. 7 and 8). In
particular, the stability of the IL-15 transcripts was not
significantly altered by SP or FLAG epitope coding sequence
modifications, with the half-life of each of these transcripts
exceeding at least 8 h. As would be predicted, the half-life of
ß-actin transcripts easily exceeded 16 h in these same samples
(Figs. 7 and 8). Additionally, there was
no significant difference between the survival of IL-2 transcripts
linked with the IL-2 or, alternatively, with the IL-15 signal coding
sequence (data not shown). These data eliminate differences in mRNA
stability as the dominant contributor to the disparity in IL-15 and
IL-2 protein expression observed in this system.
|
|
Another possible explanation for the differences in the quantities
of IL-15 and IL-2 protein expressed is that SP and FLAG had an effect
on the proteins at the levels of their intracellular stability and
their rates of breakdown. To address this possibility, AUG-IL-15,
IL-2sp/IL-15 mp pEF-Neo constructs, and IL-15/FLAG were transfected
into COS cells followed at 48 h by a 1-h
[35S]Cys/Met pulse labeling with a subsequent cold
chase. To enhance IL-15 expression in these studies, COS cells were
transfected by electroporation, not DEAE-dextran. Additionally, the
AUG-IL-15 construct was given an initiator codon with a higher Kozak
context; however, this modification did not alter the coding sequence.
Cell lysates using RIPA buffer were prepared 0, 30, 60, 90, and 120 min
after the cold chase. The lysates were subsequently immunoprecipitated
with a rabbit anti-human IL-15 polyclonal and resolved on SDS-PAGE.
These studies indicate that the modifications to IL-15 do not change
the stability of the processed protein compared with that of wild-type
IL-15 protein (Fig. 9). In fact, even 120
min after the cold chase, the level of wild-type IL-15 protein was not
substantially reduced relative to levels of the FLAG- and SP-modified
IL-15 proteins. However, these data clearly demonstrate the presence of
more than one glycosylated species of IL-15; interestingly, wild-type
IL-15 protein and FLAG-modified IL-15 show three species, whereas IL-15
protein modified with IL-2 SP shows only two. These data suggest that
IL-15 SP may be processed unusually, and in fact, this is supported by
recent observations (R. N. Bamford and G. Kurys, unpublished
observations). Nevertheless, the rate of intracellular IL-15 catabolism
does not appear to be decreased by the presence of the IL-2 SP or the
FLAG epitope.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-helix bundle cytokine
family, they require receptor subunits IL-2Rß and
c for their action, and they activate the
proliferation and differentiation of T, NK, and B cells (1, 2, 3).
However, further analysis revealed dramatic differences between these
two cytokines in terms of their cellular sites of synthesis and the
molecular levels of regulation of their expression (7, 8). IL-2 is
predominantly controlled at the level of message transcription and
stabilization. The regulation of IL-15 appears to be much more complex,
with controls at the levels of message transcription, message
translation, and protein secretion. Although IL-15 expression is
regulated in part at the level of transcription. It appears that the
predominant regulation of IL-15 expression is posttranscriptional,
since there is a marked discordance between the high levels of widely
expressed IL-15 message and the little or no IL-15 protein demonstrable
in the cytoplasm or supernatants of cytokine message-expressing cells.
We considered four possible posttranscriptional mechanisms that might
impede the synthesis and secretion of IL-15, including IL-15 mRNA
instability, protein instability, retention of IL-15 in intracellular
pools, and impeded IL-15 mRNA translation. We conclude that the first
three mechanisms are not the predominant factors affecting IL-15
synthesis and secretion. Therefore, we focused on the alternative
possibility that the major impediment to IL-15 synthesis is at the
level of mRNA translation. Control of translation has been observed with a variety of proteins, and this regulation can occur at all levels of translation (e.g., initiation, elongation, and termination). Examples of both the control of global mRNA translation rates and the regulation of translation of individual or small groups of mRNAs have been reported (11). Most mRNA-specific translational regulation has involved cis-acting RNA sequence elements that mediate regulation. Such regulatory sequences in the 5' or 3' UTR of the mRNAs as well as the mature coding sequence (12) have been observed in transcripts for ferritin (13), erythroid 5-aminolevulinate synthase (13), thymidylate synthase (14), and murine p53 (15).
In addition to specific regulatory protein/RNA interactions, there is considerable precedence for an inhibitory effect on translation manifested by upstream AUGs in the 5' UTR upstream of the authentic initiation codon (16, 17). The 5' UTR of effectively translated messages are short, simple, and unencumbered by AUGs upstream of the initiation AUG. In contrast, AUG-burdened 5' UTRs, including those encoding many proto-oncogenes, transcription factors, a variety of receptor proteins, and signal transduction components, are poorly translated (16, 17). It has been suggested that some of these transcripts may not use cap-dependent scanning mechanisms to initiate translation; instead, they may recruit ribosomes to an internal ribosome entry site, bypassing the 5' UTR (18, 19). The 5' UTR of IL-15 message is long and quite complex, and includes multiple upstream AUGs (5 in mice, 10 in humans) (3, 7). We previously reported that the 5' UTR of IL-15, including its 10 upstream AUGs, reduced the efficiency of IL-15 mRNA translation. However, the level of IL-15 protein synthesized and secreted by the construct lacking all upstream AUGs was still very low, markedly less than that observed with a comparable IL-2 construct, suggesting the possibility that additional regulatory elements exist.
To search for additional elements that might impede translation and/or
secretion, we focused on the unusually long (48 amino acids) IL-15 SP.
Data that were first obtained to support the view that the N terminus
is, in fact, a SP include the following: 1) it is processed, yielding
the 114-amino acid cytokine IL-15 (3); 2) IL-15 is secreted by the
cell; and 3) as demonstrated in immunoprecipitation studies in the
presence and the absence of tunicamycin, IL-15 manifests
N-linked glycosylation (R. N. Bamford and G. Kurys,
unpublished observations). Insights into the effect of the IL-15 SP on
the synthesis of IL-15 were gained by generating constructs linking the
mature IL-15 protein coding sequence to the IL-2 SP and reciprocally
linking the IL-15 SP to the IL-2 mp coding sequence. The quantity of
IL-15 generated (sum of cellular and secreted IL-15) increased
approximately 17- to 20-fold when the IL-15 SP was replaced by that of
IL-2. In parallel, the quantity of IL-2 produced was reduced 30- to
50-fold when COS cells were transfected with a construct encoding the
IL-2 mp with the IL-15 SP. We conclude from these studies that a major
factor in the regulation of IL-15 synthesis is a control of its
translation by a phenomenon that involves the IL-15 SP. We reported our
preliminary observations on this issue in abstract form and in a review
article (8, 20). Subsequent reports by others agreed in part with our
observations (21). In particular, Onu and co-workers (21) noted that
various IL-15 constructs were transcribed following transfection, but
this transcription was not associated with efficient secretion of IL-15
protein. Furthermore, after replacing the IL-15 SP with that of the
CD-33 Ag, translation and secretion increased, supporting the view that
IL-15 expression is mainly controlled posttranscriptionally at the
levels of translation and secretion. However, in contrast to our
observations, this group did not note any secretion of IL-15 when IL-15
SP was employed. This difference between the two studies may be due to
the fact that we used a stronger promoter with our constructs and/or
that the CTLL-2 strain they were using was not very IL-15 responsive.
In their studies, although no IL-15 protein was present in the culture
supernatant, IL-15/IgG fusion protein could be identified
intracellularly with an anti-Ig Ab. It was suggested by these
authors that IL-15 protein is stored in the cytoplasm, and fully
synthesized IL-15 may enter the secretory pathway via the cytoplasmic
pool in a regulated manner (21). Our data do not support the presence
of a large cytoplasmic pool of IL-15 protein; however, it is apparent
from microscopy studies that in some transfected cells, a portion of
the synthesized IL-15 protein does not enter the endoplasmic reticulum
(ER) and, in fact, appears cytoplasmic and/or nuclear in nature (G.
Kurys and R. N. Bamford, unpublished observations). Furthermore,
our data strongly suggest that IL-15 protein is trafficked relatively
slowly through the cell once it enters the secretory pathway compared
with IL-2 in the same COS cell system. Importantly, this phenomenon is
only partially dependent on the IL-15 SP, since IL-15 with the IL-2 SP
is also retained within the cells at a higher proportion than wild-type
IL-2 or IL-2 with the IL-15 SP. Nevertheless, when we assessed cultures
from COS cells transfected with the wild-type IL-15 coding sequence
(AUG-IL-15) at 120 h, at least 90% of the IL-15 protein
(though at low levels) could be identified in the culture supernatants,
and <10% was retained within the cell, as evaluated by sensitive
ELISA assays. These data reemphasize that IL-15 protein is a secreted
molecule, but its overall production is very low. Thus, noting the
large discrepancy between the total quantities of IL-15 protein
generated from AUG IL-15 and chimeric IL-15 (IL-2sp/IL-15 mp)
transcripts, it appears that the predominant level of regulation of
IL-15 synthesis mediated by the SP is at the level of translation, with
a lesser component at the level of intracellularly trafficking and
secretion per se.
The SP has been shown to be a participant in a normal phenomenon that involves a transient impediment to translation elongation that affects secreted and cell surface membrane proteins. However, it has not been a major focus of studies on the differential regulatory mechanisms that lead to the induction of specific protein expression. The binding of the signal recognition particle (SRP) to the SP causes signal sequence-dependent and site-specific arrest of chain elongation (22). The SRP binds to the SP as soon as the peptide emerges from the ribosome. This causes a pause in protein synthesis, which presumably gives the ribosome enough time to bind to the ER membrane before synthesis of the polypeptide chain is completed, thereby ensuring that the protein is not released into the cytoplasm. This translational arrest is released when SRP binds its receptor, or docking protein, that is exposed on the cytosolic surface of the rough ER membrane. The binding results in chain completion and transfer into the ER.
The mechanisms underlying the SP coding sequence- and/or protein sequence-mediated regulation of IL-15 translation have not been defined. However, with preliminary in vitro translation studies, we observed that the addition of canine microsomal membranes did not result in IL-15 chain completion and translocation into microsomes in contrast to the situation with the prototypical secretory protein, pre/prolactin, which was fully translocated and processed, (data not shown). Therefore, a number of events or factors may be required for efficient IL-15 mRNA translation/translocation. It is possible that a translational activator(s) for chain elongation and translocation may be needed. Alternatively, a translational repressor or a stable secondary structure in the mRNA may prevent efficient IL-15 mRNA elongation and translocation. Furthermore, inefficient initiation of translation may contribute partially to the low levels of IL-15 protein generated in transfected COS cells. This stems from the observation that the start codon for the IL-15 coding sequence has a weak Kozak context (GTAATGA) (16, 17). In fact, modifying the start codon to a higher context (ACCATGG or GCCGCCATGA) increased IL-15 protein production four- to fivefold in transfected COS cells (data not shown).
In terms of regulation at the level of translation for specific
proteins, 70-kDa heat shock protein mRNA translation in chicken
reticulocytes has been shown to be controlled at the level of
elongation (23). IL-1ß expression is also regulated in part at the
chain elongation termination phases of translation (24). Translational
control has been demonstrated with other cytokines, including TNF-
(25), TGF-ß3 (26), TGF-ß1 (27), and granulocyte-macrophage CSF
(28). Additionally, it has been reported that one of the multiple
levels of insulin biosynthesis regulation includes a glucose-dependent,
signal recognition, particle-mediated translational arrest
(29).
Our studies also reveal that the FLAG epitope modifications made to the C terminus of the IL-15 constructs enhanced the generation of IL-15 protein. As with the SP, the FLAG coding sequence does not appear to substantially affect mRNA stability, nor does the FLAG peptide sequence affect protein secretion or stability, suggesting that the effect is on translation. Noting the position of the FLAG epitope coding sequence, it can be suggested that its presence may enhance the rate or the efficiency of translation termination compared with the situation with wild-type IL-15. Interestingly, it has been reported for the CMV gene, gp48, that an upstream open reading frame in the transcript does not efficiently terminate translation, and ribosomes stack up behind the paused ribosome (30). This inhibitory effect is mediated by the amino acid coding information in the vicinity of the stop codon, and it can be eliminated by adding a single codon to the C terminus of the peptide (31). Although this would be one possible explanation for FLAG’s effect on IL-15 translation, others may include the disruption of a critical secondary structure or protein binding site in IL-15 mRNA by the FLAG coding sequence.
The present studies taken in concert with those we have previously
reported (7, 8) suggest that a major factor in the control of IL-15
expression is multifaceted regulation at the level of IL-15 mRNA
translation. In particular, elements of the 5' UTR, including 10
upstream AUGs, the SP, and the 3' coding sequence of IL-15, participate
in its posttranscriptional regulation. When the above three IL-15
message modifications were combined into a single construct, at least
250-fold more protein was generated than with the wild-type IL-15
construct with an intact 5' UTR. Furthermore, although the total IL-15
production was increased with the modified construct, the ratio of
secreted to retained cytokine was not significantly influenced,
suggesting that the major focus of this regulation is at the level of
translation rather than a dominant effect on protein secretion. These
studies indicate that the translational control of IL-15, like that of
insulin, occurs at multiple distinct levels. The removal of these
negative control mechanisms in an integrated fashion may give rise to a
major increase in IL-15 synthesis. The broad array of negative
regulatory features controlling IL-15 expression may be required due to
the potency of IL-15 in inducing the expression of TNF-, IL-1,
IFN-, and other cytokines involved in the inflammatory response that
if indiscriminately expressed would be associated with serious
disorders, including autoimmune diseases (32). In terms of a more
positive role for IL-15, as a hypothesis, we propose that by
maintaining a pool of translationally inactive IL-15 mRNA, diverse
cells might respond rapidly to an intracellular infection or other
stimuli by transforming IL-15 message into one that can be effectively
translated. The IL-15 protein produced could, in turn, convert T and NK
cells into effective killer cells, which may provide an effective host
response to infectious agents.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Richard N. Bamford, Metabolism Branch, National Cancer Institute, Building 10, Room 4N115, National Institutes of Health, Bethesda, MD 20892-1374.
3 Abbreviations used in this paper: UTR, untranslated region; mp, mature protein; SP, signal peptide; CDS, coding sequence; TnT, transcription and translation; ER, endoplasmic reticulum; SRP, signal recognition particle.
Received for publication August 8, 1997. Accepted for publication January 7, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13:2822.[Medline]
by natural killer cells in vitro. J. Clin. Invest. 96:2578.
production in rheumatoid arthritis. Nat. Med. 3:189.[Medline]
This article has been cited by other articles:
|
|
 |
|
  C. Berger, M. Berger, R. C. Hackman, M. Gough, C. Elliott, M. C. Jensen, and S. R. Riddell Safety and immunologic effects of IL-15 administration in nonhuman primates Blood, September 17, 2009; 114(12): 2417 - 2426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bergamaschi, R. Jalah, V. Kulkarni, M. Rosati, G.-M. Zhang, C. Alicea, A. S. Zolotukhin, B. K. Felber, and G. N. Pavlakis Secretion and Biological Activity of Short Signal Peptide IL-15 Is Chaperoned by IL-15 Receptor Alpha In Vivo J. Immunol., September 1, 2009; 183(5): 3064 - 3072. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rowley, A. Monie, C.-F. Hung, and T.-C. Wu Inhibition of Tumor Growth by NK1.1+ Cells and CD8+ T Cells Activated by IL-15 through Receptor {beta}/Common {gamma} Signaling in trans J. Immunol., December 15, 2008; 181(12): 8237 - 8247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Nielsen, P. Hojman, C. Erikstrup, C. P. Fischer, P. Plomgaard, R. Mounier, O. H. Mortensen, C. Broholm, S. Taudorf, R. Krogh-Madsen, et al. Association between Interleukin-15 and Obesity: Interleukin-15 as a Potential Regulator of Fat Mass J. Clin. Endocrinol. Metab., November 1, 2008; 93(11): 4486 - 4493. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. P. Carroll, V. Paunovic, and M. Gadina Signalling, inflammation and arthritis: Crossed signals: the role of interleukin-15 and -18 in autoimmunity Rheumatology, September 1, 2008; 47(9): 1269 - 1277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Mortier, T. Woo, R. Advincula, S. Gozalo, and A. Ma IL-15R{alpha} chaperones IL-15 to stable dendritic cell membrane complexes that activate NK cells via trans presentation J. Exp. Med., May 12, 2008; 205(5): 1213 - 1225. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Quinn Interleukin-15: A muscle-derived cytokine regulating fat-to-lean body composition J Anim Sci, April 1, 2008; 86(14_suppl): E75 - E83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bergamaschi, M. Rosati, R. Jalah, A. Valentin, V. Kulkarni, C. Alicea, G.-M. Zhang, V. Patel, B. K. Felber, and G. N. Pavlakis Intracellular Interaction of Interleukin-15 with Its Receptor {alpha} during Production Leads to Mutual Stabilization and Increased Bioactivity J. Biol. Chem., February 15, 2008; 283(7): 4189 - 4199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Nielsen, R. Mounier, P. Plomgaard, O. H. Mortensen, M. Penkowa, T. Speerschneider, H. Pilegaard, and B. K. Pedersen Expression of interleukin-15 in human skeletal muscle effect of exercise and muscle fibre type composition J. Physiol., October 1, 2007; 584(1): 305 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Wang, J. W. Holland, A. Carrington, J. Zou, and C. J. Secombes Molecular and Functional Characterization of IL-15 in Rainbow Trout Oncorhynchus mykiss: A Potent Inducer of IFN-{gamma} Expression in Spleen Leukocytes J. Immunol., August 1, 2007; 179(3): 1475 - 1488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zhou, H. Wei, R. Sun, and Z. Tian Recognition of Double-Stranded RNA by TLR3 Induces Severe Small Intestinal Injury in Mice J. Immunol., April 1, 2007; 178(7): 4548 - 4556. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Stoklasek, K. S. Schluns, and L. Lefrancois Combined IL-15/IL-15R{alpha} Immunotherapy Maximizes IL-15 Activity In Vivo J. Immunol., November 1, 2006; 177(9): 6072 - 6080. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Hsu, M. S. Hughes, Z. Zheng, R. B. Bray, S. A. Rosenberg, and R. A. Morgan Primary Human T Lymphocytes Engineered with a Codon-Optimized IL-15 Gene Resist Cytokine Withdrawal-Induced Apoptosis and Persist Long-Term in the Absence of Exogenous Cytokine J. Immunol., December 1, 2005; 175(11): 7226 - 7234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Kutzler, T. M. Robinson, M. A. Chattergoon, D. K. Choo, A. Y. Choo, P. Y. Choe, M. P. Ramanathan, R. Parkinson, S. Kudchodkar, Y. Tamura, et al. Coimmunization with an Optimized IL-15 Plasmid Results in Enhanced Function and Longevity of CD8 T Cells That Are Partially Independent of CD4 T Cell Help J. Immunol., July 1, 2005; 175(1): 112 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kitaya, T. Yamaguchi, and H. Honjo Central Role of Interleukin-15 in Postovulatory Recruitment of Peripheral Blood CD16(-) Natural Killer Cells into Human Endometrium J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2932 - 2940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kobayashi, S. Dubois, N. Sato, H. Sabzevari, Y. Sakai, T. A. Waldmann, and Y. Tagaya Role of trans-cellular IL-15 presentation in the activation of NK cell-mediated killing, which leads to enhanced tumor immunosurveillance Blood, January 15, 2005; 105(2): 721 - 727. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Blaser, S. Roychowdhury, D. J. Kim, N. R. Schwind, D. Bhatt, W. Yuan, D. F. Kusewitt, A. K. Ferketich, M. A. Caligiuri, and M. Guimond Donor-derived IL-15 is critical for acute allogeneic graft-versus-host disease Blood, January 15, 2005; 105(2): 894 - 901. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Nishimura, A. Fujimoto, N. Tamura, T. Yajima, W. Wajjwalku, and Y. Yoshikai A novel autoregulatory mechanism for transcriptional activation of the IL-15 gene by a nonsecretable isoform of IL-15 generated by alternative splicing FASEB J, January 1, 2005; 19(1): 19 - 28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Schluns, K. D. Klonowski, and L. Lefrancois Transregulation of memory CD8 T-cell proliferation by IL-15R{alpha}+ bone marrow-derived cells Blood, February 1, 2004; 103(3): 988 - 994. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Toomey, F. Gays, D. Foster, and C. G. Brooks Cytokine requirements for the growth and development of mouse NK cells in vitro J. Leukoc. Biol., August 1, 2003; 74(2): 233 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Parrish-Novak, D. C. Foster, R. D. Holly, and C. H. Clegg Interleukin-21 and the IL-21 receptor: novel effectors of NK and T cell responses J. Leukoc. Biol., November 1, 2002; 72(5): 856 - 863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Ohta, T. Hiroi, M.-N. Kweon, N. Kinoshita, M. H. Jang, T. Mashimo, J.-I. Miyazaki, and H. Kiyono IL-15-Dependent Activation-Induced Cell Death-Resistant Th1 Type CD8{alpha}{beta}+NK1.1+ T Cells for the Development of Small Intestinal Inflammation J. Immunol., July 1, 2002; 169(1): 460 - 468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. De Creus, K. Van Beneden, F. Stevenaert, V. Debacker, J. Plum, and G. Leclercq Developmental and Functional Defects of Thymic and Epidermal V{gamma}3 Cells in IL-15-Deficient and IFN Regulatory Factor-1-Deficient Mice J. Immunol., June 15, 2002; 168(12): 6486 - 6493. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Yajima, H. Nishimura, R. Ishimitsu, T. Watase, D. H. Busch, E. G. Pamer, H. Kuwano, and Y. Yoshikai Overexpression of IL-15 In Vivo Increases Antigen-Driven Memory CD8+ T Cells Following a Microbe Exposure J. Immunol., February 1, 2002; 168(3): 1198 - 1203. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Neely, S. M. Robbins, E. K. Amankwah, S. Epelman, H. Wong, J. C. L. Spurrell, K. K. Jandu, W. Zhu, D. K. Fogg, C. B. Brown, et al. Lipopolysaccharide-Stimulated or Granulocyte-Macrophage Colony-Stimulating Factor-Stimulated Monocytes Rapidly Express Biologically Active IL-15 on Their Cell Surface Independent of New Protein Synthesis J. Immunol., November 1, 2001; 167(9): 5011 - 5017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Suzuki, H. Nakazato, H. Matsui, M. Hasumi, Y. Shibata, K. Ito, Y. Fukabori, K. Kurokawa, and H. Yamanaka NK cell-mediated anti-tumor immune response to human prostate cancer cell, PC-3: immunogene therapy using a highly secretable form of interleukin-15 gene transfer J. Leukoc. Biol., April 1, 2001; 69(4): 531 - 537. [Abstract] [Full Text]
|
|
|
|
|
|
G. Brewer Misregulated Posttranscriptional Checkpoints: Inflammation and Tumorigenesis J. Exp. Med., January 15, 2001; 193(2): f1 - f4. [Full Text] [PDF]
|
|
|
|
|
|
T. A. Fehniger, K. Suzuki, A. Ponnappan, J. B. VanDeusen, M. A. Cooper, S. M. Florea, A. G. Freud, M. L. Robinson, J. Durbin, and M. A. Caligiuri Fatal Leukemia in Interleukin 15 Transgenic Mice Follows Early Expansions in Natural Killer and Memory Phenotype Cd8+ T Cells J. Exp. Med., January 15, 2001; 193(2): 219 - 232. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Fehniger and M. A. Caligiuri Interleukin 15: biology and relevance to human disease Blood, January 1, 2001; 97(1): 14 - 32. [Full Text] [PDF]
|
|
|
|
 |
|
 K. Kitaya, J. Yasuda, I. Yagi, Y. Tada, S. Fushiki, and H. Honjo IL-15 Expression at Human Endometrium and Decidua Biol Reprod, September 1, 2000; 63(3): 683 - 687. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. C. Ku, M. Murakami, A. Sakamoto, J. Kappler, and P. Marrack Control of Homeostasis of CD8+ Memory T Cells by Opposing Cytokines Science, April 28, 2000; 288(5466): 675 - 678. [Abstract] [Full Text]
|
|
|
|
|
|
H. Nishimura, T. Yajima, Y. Naiki, H. Tsunobuchi, M. Umemura, K. Itano, T. Matsuguchi, M. Suzuki, P. S. Ohashi, and Y. Yoshikai Differential Roles of Interleukin 15 mRNA Isoforms Generated by Alternative Splicing in Immune Responses in Vivo J. Exp. Med., January 3, 2000; 191(1): 157 - 170. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Kurys, Y. Tagaya, R. Bamford, J. A. Hanover, and T. A. Waldmann The Long Signal Peptide Isoform and Its Alternative Processing Direct the Intracellular Trafficking of Interleukin-15 J. Biol. Chem., September 22, 2000; 275(39): 30653 - 30659. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Marks-Konczalik, S. Dubois, J. M. Losi, H. Sabzevari, N. Yamada, L. Feigenbaum, T. A. Waldmann, and Y. Tagaya IL-2-induced activation-induced cell death is inhibited in IL-15 transgenic mice PNAS, October 10, 2000; 97(21): 11445 - 11450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Azimi, M. Nagai, S. Jacobson, and T. A. Waldmann IL-15 plays a major role in the persistence of Tax-specific CD8 cells in HAM/TSP patients PNAS, December 4, 2001; 98(25): 14559 - 14564. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
