Alternative translation of osteopontin generates intracellular and secreted isoforms that mediate distinct biological activities in dendritic cells.

Abstract

Osteopontin (Opn) contributes to diverse biological processes that include immune responses, vascularization, and bone formation. Until recently, studies describing the activities of Opn have focused on the cytokine-like properties of the secreted protein. Here, we show that alternative translation of a single Opn mRNA species generates a secreted and intracellular isoform. Utilization of a 5' canonical translation start site generates a protein that includes an N-terminal signal sequence allowing targeting to secretory vesicles and cytokine secretion, whereas usage of a downstream start site generates a shortened protein that lacks the N-terminal signal sequence and localizes mainly to cytoplasm. The coordinated action of these Opn gene products regulates the functional phenotype of subsets of dendritic cells.

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Alternative translation of osteopontin generates
intracellular and secreted isoforms that mediate
distinct biological activities in dendritic cells
Mari L. Shinohara*†, Hye-Jung Kim*†, June-Ho Kim*, Virgilio A. Garcia*, and Harvey Cantor*†‡
*Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute, and†Department of Pathology, Harvard Medical School, 44 Binney Street,
Boston MA 02115
Contributed by Harvey Cantor, March 17, 2008 (sent for review January 25, 2008)
Osteopontin (Opn) contributes to diverse biological processes that
include immune responses, vascularization, and bone formation.
Untilrecently,studiesdescribingtheactivitiesofOpnhavefocused
on the cytokine-like properties of the secreted protein. Here, we
show that alternative translation of a single Opn mRNA species
generates a secreted and intracellular isoform. Utilization of a 5?
canonical translation start site generates a protein that includes an
N-terminalsignalsequenceallowingtargetingtosecretoryvesicles
and cytokine secretion, whereas usage of a downstream start site
generates a shortened protein that lacks the N-terminal signal
sequence and localizes mainly to cytoplasm. The coordinated
action of these Opn gene products regulates the functional phe-
notype of subsets of dendritic cells.
signal sequence ? podosome
T
and cell regeneration, through interactions with mononuclear
and endothelial cells. Virtually all of these activities have been
attributed to an interaction between secreted Opn (Opn-s) and
its receptors on target cells (1, 2). For example, an interaction
between Opn-s with ?v?3and CD44 receptors on macrophages
that enhances IL-12 and inhibits IL-10 responses (3) promotes
Th1 development (4) and cellular resistance to apoptosis (5–7).
However, a nonsecreted species of Opn has been implicated in
a growing number of cellular processes, including migration,
fusion, and motility (8–11). Recent studies have revealed that an
intracellular form of Opn (Opn-i) in plasmacytoid dendritic cells
(pDC) interacts with IRF7 to induce IFN? expression (12),
whereas expression of Opn-i in conventional DC (cDC) pro-
motes differentiation of IL-17-producing T helper cells (Th17
cells) (13).
These findings prompted us to define the genetic and molec-
ular origin of Opn-i and Opn-s in DC. Possibly, a weakly
hydrophobic signal sequence may allow a single Opn protein to
migrate into both secretory vesicles and cytoplasm, secondary to
ERleakageintocytosol,asdemonstratedforprionproteins(14).
Alternatively,Opnmayspecifytwodistinctproteinisoformswith
distinct cellular locations and biologic function.
Here, we demonstrate that Opn-i and Opn-s represent alter-
native translational products of a single full-length Opn mRNA.
TranslationofOpn-sisinitiatedfromthe5?canonicalAUGstart
site, whereas translation of the Opn-i isoform is initiated from a
downstream non-AUG codon. Downstream translation of Opn-i
is accompanied by deletion of the N-terminal 16-aa signal
sequence, allowing the shortened protein product to localize in
cytoplasm but not secretory vesicles. The shortened Opn trans-
lation isoform activates ifna4 gene expression and podosome
formation in pDC, thus contributing to the characteristic bio-
logical activities of this DC subset. These findings indicate that
factors that alter the translational balance of Opn in favor of
either Opn-i or Opn-s may contribute to the phenotype of
activated DC.
he osteopontin (Opn) glycoprotein regulates diverse biolog-
ical processes, including immune responses, vascularization,
Results
DCsExpressTwoOpnIsoforms.Full-lengthOpn(Opn-FL)contains
a signal peptide that targets nascent protein to secretory vesicles,
however, the mechanism allowing intracellular retention of Opn
is unknown. To determine whether both Opn isoforms were
present in DC, we performed Western blot analysis of whole cell
lysates. We detected two bands (75 kDa/70 kDa) in both pDC
(Fig. 1A) and cDC (data not shown) using the mAb (O-17)
specific for Opn sequence immediately downstream of 16-aa
signal sequence (Fig. 1B). This observation opened the possi-
bility that the difference in size (?5 kDa) reflected deletion of
the 16-aa signal sequence. We then analyzed proteins translated
in vitro from full-length Opn mRNA and an Opn mRNA
truncation mutant (?48Koz) that lacked the first 48 nucleotides
encoding the Opn signal sequence (amino acid 1–16). An
artificial AUG/Kozak sequence was introduced to initiate trans-
lation (Fig. 1 B and C). In vitro translation (IVT) of full-length
Opn mRNA gave rise to a doublet of similar size to that
identifiedbyWesternblotanalysisofpDC(Fig.1A),whereasthe
?48Kozconstructgeneratedasinglebandthatmigratedwiththe
lower band of the doublet (Fig. 1C). These results suggest that
the doublet represented Opn-FL protein (upper band) and a
shortened protein (lower band) that may correspond to intra-
cellular Opn, provisionally designated Opn-i.
The Opn-i Isoform Is Not Generated from Alternative Opn Transcrip-
tion Initiation or Splicing. We identified transcription initiation
sites using RLM-RACE (RNA ligase-mediated RACE) analysis
(15). Although a single band was amplified in total cDNA from
unactivated bone marrow (BM)-derived DC (BM-DC) (Fig. 2A,
band A), multiple bands appeared after CpG activation of DC
(Fig. 2A, bands A–C). Cloning and sequencing revealed that
bandAwastranscribedfromtheadenineresidueatposition?90
in exon 1 (90 nucleotides upstream of the ATG translation start
codon) [Fig. 2B and supporting information (SI) Fig. S1 and SI
Text]. Alternative transcription start sites in exon 4 (band B) and
exon 6 (band C) were too far downstream to account for the
shorter Opn peptide shown in Fig. 1.
We next amplified by PCR the region between exons 1 and 4
of Opn cDNA to ask whether alternative splicing might produce
Author contributions: M.L.S. and H.C. designed research; M.L.S., H.-J.K., J.-H.K., and V.A.G.
performed research; M.L.S., H.-J.K., and H.C. analyzed data; and M.L.S. and H.C. wrote the
paper.
The authors declare no conflict of interest.
‡To whom correspondence should be addressed. E-mail: harvey?cantor@dfci.harvard.edu.
§ExaminationofCUGcodons[anothercommonnon-AUGtranslationinitiationcodon(16)]
furtherdownstream(atposition?100and?127;Fig.S3A)bymutagenesisdidnotchange
the pattern from Opn WT mRNA (‘‘Full’’) (Fig. S3B) nor did mutating GUG (Val) to GAC
(Asp) affect translation (Fig. S3C).
This article contains supporting information online at www.pnas.org/cgi/content/full/
0802301105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA
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the shorter Opn-i product using DC and T cells. Although no
shorter bands were detected, some samples showed a larger band
than expected, i.e., alternative splicing might provide an addi-
tional mRNA sequence rather than reducing the size of mRNA
(Fig. S2A). Although sequencing of the additional band revealed
that alternative splice added a 52-nt extension at the 3?-end of
exon 1 (Fig. S1), this did not account for Opn-i, because Opn
mRNA containing the 52-nt extension failed to generate an
additional Opn peptide product compared to canonical Opn
mRNA (Fig. S2 B and C).
Identification of a Nucleotide Sequence Required for Alternative
Translation Initiation of Opn-i. Although the GUG codon is pre-
ferred for non-AUG translation initiation (16), GUG codons
immediately downstream of the signal peptide-encoding se-
quence did not initiate translation: GUG3 GUC mutations at
positions ?55 and ?61 (Fig. S3A) did not impair generation of
Opn-FL and Opn-i peptides (Fig. S3B)§.
We next tested series of Opn deletion mutants with no AUG
codon to define sequences essential for production of the Opn-i
isoform. Expression of full-length Opn cDNA construct was
compared to Opn nucleotide deletion constructs in transfected
293FT cells by immunoblotting (Fig. 3A). Opn plasmid ?1–39,
whichlacksthefirst39nucleotides(Fig.3A),generatedthelower
but not the upper Opn band (Fig. 3B), suggesting that the Opn-i
protein did not require the canonical AUG codon but was
initiated by a non-AUG codon downstream of position ?39.
Additional deletion to position ?48 (?1–48) completely elimi-
nated alternative translation of the shortened protein (Fig. 3B),
suggesting that the 9-nt sequence between positions ?40 and
?48 (GCC TCC TCC) encoding Asp-Ser-Ser was essential for
translation of the Opn-i isoform.
TheOpn-iIsoformLocalizesOutsideSecretoryVesicles.Weexamined
subcellular localization of Opn in 293FT cells transfected with
GFP-fusion constructs of full-length Opn (Opn-fl) or a ?48Koz
Opn deletion mutant. Opn-fl-GFP protein colocalized with
Golgi and cytoplasm, whereas Opn?48Koz-GFP appeared to
reside mainly in cytoplasm (Fig. 4A). To further examine dis-
tribution of Opn isoforms expressed without an added Kozak
sequence, we cotransfected 293FT cells with Opn-fl and
Opn?1–39 (see Fig. 3A) tagged with FLAG and V5 epitopes,
respectively. Proteins generated from deletion mutant
Opn?1–39 appeared to localize in cytoplasm, whereas proteins
encoded by the Opn-fl construct localized to perinuclear secre-
tory vesicles (Fig. 4B). The latter observation was confirmed
using V5-tagged Opn-fl constructs that displayed colocalization
of Opn-FL with Golgi marker GOLPH4 (Fig. 4C). Occasional
colocalization of Opn-FL FLAG-tagged products with Opn?39
Fig. 1.
species. (A) Opn detection from pDC lysate by immunoblotting with O-17 Ab.
(B) Transcripts from ‘‘Full’’ (Opn-fl) and ‘‘Opn?48Koz.’’ Triangles (‚) denote
AUGcodons,includingthesecondendogenousAUGatposition84aa.O-17Ab
recognizes amino acids spanning position 17–32 aa. (C) IVT of two Opn
constructs shown in B. Peptide products were phosphatase-treated. Negative
control of IVT was performed without RNA template (‘‘no RNA’’).
Two peptides of different size translated from a single Opn mRNA
Fig. 2.
gel shows nested PCR (‘‘o,’’ outer PCR; ‘‘i,’’ inner). Bands corresponding to products from Opn transcripts generated by RLM-RACE are labeled A–C. The position
of the Opn specific inner primer is shown in Fig. S1. (B) Schematic of transcription start sites A–C on Opn.
RLM-RACE of Opn mRNA and structure of the Opn gene. (A) Opn WT and KO DC were activated with or without CpG-1668 (0.2 ?g/ml) ? 3 h. Agarose
Fig. 3.
nativetranslation.(A)SchematicofOpndeletionmutantstoassessalternative
translation. (B) 293FT cells were transfected with Opn expression constructs:
Opn-fl,Opn?1–39,Opn?1–48,GFP(negativecontrol);Opn-FLandOpn-iwere
detected by immunoblotting with O-17 Ab in 293FT cell lysates.
Codons encoding aa sequence Ala-Ser-Ser participate in Opn alter-
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V5-taggedproductswasconsistentwithalternativetranslationof
Opn-fl mRNA (Fig. 4B).
We analyzed subcellular localization of endogenous Opn in
BM-derived DC using Opn 2A1 Ab that detects both Opn-FL
and Opn-i isoforms. Although there was almost no detectable
colocalizationofOpnwiththeGOLPH4Golgimarker(Fig.4D),
Opn localized extensively in the cytoplasm (Fig. 4E) and was also
detected in dendritic processes (Fig. 4F).
To confirm localization of Opn-FL, we fractionated 293FT
lysates by density gradient. Golgi content (detected by Syntaxin 6
and Vti1b Ab) was found at the top of the gradient (fractions 5–7)
(Fig. 5). A single band was detected mainly in fraction 7 of the
Opn-fl transfectants (Fig. 5 Upper), whereas no band was detected
in any Percoll fraction derived from the Opn?1–39 transfectants
(Fig. 5 Lower), suggesting that Opn-FL, but not Opn-i, localized in
Golgi.Becausethesegradientfractionsdidnotincludecytoplasmic
material, as judged from the absence of GAPDH markers, a
fraction (P1) containing total cell lysate was analyzed. Opn-fl
transfectants displayed two bands in P1, whereas Opn?1–39 trans-
fectants gave rise to a single band in this fraction. These findings
indicatethattheshortisoformofOpnisfoundmainlyincytoplasm,
rather than secretory vesicles and Golgi.
An Opn Isoform Lacking the Signal Sequence Restores Biological
Activity of DC. We measured Opn-mediated IFN-? production
using a reporter assay (12). Transfection of 293FT cells with the
Opn?1–39 or Opn?48Koz construct gave 4–8-fold more ifna4
promoter activity than transfection of the full Opn construct (Fig.
6A).ExpressionofOpn-ibytheOpn?48Kozconstructalsoinduced
a15-foldincreaseinformationofpodosomes(17)bypDC(Fig.6B).
Without stimulation, both Opn WT and Opn-deficient pDC were
spherical (Fig. S4 A and B). After stimulation with CpG, Opn WT
butnotOpn-deficientpDCdisplayedefficientpodosomeformation
(Fig. S4 C and D), and the Opn-deficient defect was particularly
evident in their failure to form long podosomes (Fig. 6B). Expres-
sion of lenti-Opn-fl or lenti-Opn?48Koz in Opn-deficient DC
rescued podosome formation (Fig. S4 E and F), whereas lentiviral
expression of GFP did not (Fig. S4G). Results are quantitatively
analyzed in Fig. 6B. These data suggest that Opn-i is required for
IFN-? production and podosome formation and that it is function-
ally distinct from the Opn-s isoform.
Discussion
Although previous studies have suggested that an intracellular
form of Opn (Opn-i) may mediate some of its biological activ-
ities, its genetic origin has not been established. Here, we show
that Opn-i is generated from alternative translation of a non-
AUG site downstream of the canonical AUG sequence. This
mechanism, which does not involve alternative mRNA transcrip-
tion initiation or splicing, generates a full-length secreted Opn
Fig.4.
constructs were transfected into 293FT cells and counterstained with Golgi
marker GM130. ‘‘Opn-fl-GFP’’ includes WT Opn with the canonical Opn ATG
translation start codon. ‘‘Opn?48Koz-GFP’’ lacks a signal sequence and an
artificial ATG codon with the Kozak sequence. (B) Cotransfection of ‘‘Opn-fl-
FLAG’’and‘‘Opn?1–39-V5’’constructsinto293FT.(C)Usingasystemsimilarto
(B), Opn-fl-V5 construct was transfected into 293FT and detected with V5 Ab
and counterstained with GOLPH4 Ab. BM-DC stained with Opn 2A1 Ab and
counterstainedwithGOLPH-4Ab(D)orGAPDHAb(E).(F)DendritesofBM-DC
stainedwithOpn2A1Ab.NucleusstainingwascarriedoutwithDRAQ5(A)or
TOPRO3 (B–E).
SubcellularlocalizationofOpn-FLandOpn-i.(A)TwoOpn-GFPfusion
Fig. 5.
gradient analysis of Opn in 293FT cells transfected with Opn-fl or Opn?1–39
expression constructs. Opn was detected with O-17 Ab.
Biochemical detection of Opn-FL targeted to Golgi vesicles. Percoll
Fig. 6.
and podosome formation. (A) Opn expression and ifna4 activation in 293FT
cellstransientlytransfectedwithincreasingconcentrationsofOpnexpression
vectorsalongwithIRF7andMyD88,asdescribed(12).(B)OpnWTandKOpDC
were not infected or were infected with the indicated Opn lentiviral con-
structs. Proportions of pDC with long (length of podosome ? cell diameter;
gray) and short (length of podosome ? cell diameter; black) podosomes were
calculated from total 120 cells per sample.
Functional analyses of the Opn-i isoform: ifna4 promoter activation
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protein (Opn-s) and a smaller intracellular product (Opn-i) from
a single full-length mRNA species.
Analysis of the mammalian genome revealed that unexpect-
edly large numbers of proteins are produced from a relatively
small number of genes using several posttranscriptional mech-
anisms. Alternative translation, which allows generation of mul-
tiple proteins from a single mRNA species, is used by Opn to
expand the biological range of the products through a transla-
tional mechanism that allows differential expression of the
N-terminal signal sequence. The two proteins generated by this
mechanism –Opn-s and Opn-i– localize to characteristic intra-
cellular sites and mediate distinct functions in DC and T cells.
More than a dozen instances of mammalian genes that pro-
duce isoforms by in-frame alternative translation initiation have
been reported. A striking feature of these genes is that most
encode regulatory proteins, including transcription factors, pro-
tooncogenes, kinases, and growth factors (reviewed in ref. 16).
Alternative translation initiation of non-AUG codons can also
regulatethelocalizationofproteins,includingFgf2(18,19),Fgf3
(Int-2) (20), hck (21), and VEGF (22). In the case reported here,
the alternative Opn translation initiation site is downstream of
the canonical AUG start codon and therefore differs from
previously reported instances of alternative translation in which
codons locate upstream of the first in-frame AUG codon. In this
case, the downstream location of the alternative translation site
allows generation of either full-length or truncated protein
products that are secreted or retained by the cell.
Selection of the downstream initiation codon can occur by
leaky scanning of ribosome, internal ribosome entry or ribo-
somal shunting (16, 23, 24). In the case of Opn alternative
translation, leaky scanning of ribosome is most likely. Entry
through an internal ribosome entry site (IRES) is unlikely
because the 40-nt stretch between the AUG codon, and the
alternative translation region is probably not sufficiently long to
form an IRES. Ribosomal shunting is also unlikely, because
ribosomes are not likely to skip the canonical AUG, which is
surrounded by a sequence (ACGACCAUGA) that closely re-
sembles the Kozak translation initiation consensus sequence
(GCCRCCAUGG: R ? purine) and probably accounts for the
consistent generation of Opn-FL from Opn-fl mRNA templates.
Ribosomes that escape the canonical Opn AUG start codon
may continue to scan downstream mRNA sequences until the
ribosome encounters another Kozak-like sequence and/or a
stable mRNA hairpin secondary structure (16, 24, 25) that stalls
the ribosome and allows recognition of an alternative translation
initiation site (26, 27). Folding prediction analysis of Opn mRNA
suggests that extremely stable secondary structures (kcal/mol)
can be formed, and their hairpin stability is greater (?14.7 and
?11.4 kcal/ml) than the hairpin formed immediately 3? to the
canonical AUG codon (?11.1 kcal) (Fig. S5).
The size of Opn-i generated in 293FT cells from the 5? deletion
mutant Opn?1–39 was similar to the isoform generated from a
non-AUG codon residing downstream of the nucleotide sequence
that encodes the signal peptide. Analysis of subcellular localization
ofOpn-ibyconfocalmicroscopyandbiochemicalanalysesrevealed
that alternatively translated Opn-i localized mainly to the cyto-
plasm. We did not detect Opn-i in Golgi/secretory granules, pre-
sumablyreflectingtheabsenceofasignalsequenceanddirectentry
of the nascent protein into cytoplasm after synthesis. These obser-
vations explain the contribution of Opn-i to intracellular signaling
pathways, including IRF7 activation after association with MyD88
in pDC (12), and its association with ezrin (8) and the cytoplasmic
portion of CD44 (9, 10). The impact of Opn-i on podosome
formationmayreflectitscontributiontoactinpolymerizationatthe
leading edge of migration, consistent with its association with the
ERM (ezrin/radixin/moesin) complex that couples cell surface
adhesion molecules with actin filaments (8). Ongoing studies are
intendedtodefinethenon-AUGcodonwithinpositions40–51that
initiates alternative translation and to delineate the mechanism of
this process.
We have shown that, although APC, including DC and macro-
phages, are rich in Opn-i, T cells tend to secrete rather than retain
Opn (4). Cell type-specific translation of Opn from the canonical
and alternative sites defined here may account for the dominance
of Opn-s expression in activated T cells compared with the domi-
nance of Opn-i in DC (4). Tissue specific regulation of translation
of Fgf2 results in kidney and skeletal muscle expression of the
AUG-initiated isoform, whereas production of the alternative
CUG-initiated translational isoforms predominate in brain and
liver. Cell type-specific factors expressed by T cells and DC that
determine the relative expression levels of Opn-i and Opn-s after
cellular activation may account for the impact of Opn gene expres-
sion on the functional phenotype of these cells.
Methods
Cells and Reagents. DC and T cells were prepared as described (12). Rabbit
polyclonal mouse Opn O-17 Ab was raised against a synthetic peptide
(LPVKVTDSGSSEEKLY) (IBL America) immediately downstream of the Opn
signal peptide (29). Protein samples for IVT and Western blot analysis were
treated with calf intestine alkaline phosphatase (CIP, New England Biolabs).
Mouse Opn 2A1 Ab (not signal sequence; Santa Cruz) was used for immuno-
fluorescence. Additional Abs include: mouse anti-GM130, mouse anti-
syntaxin 6, mouse anti-Vti1b (BD PharMingen), and rabbit anti-GOLPH4 (Ab-
cam) (Golgi markers), rabbit anti-GAPDH (Abcam) (cytoplasmic marker),
mouse anti-V5 (Invitrogen), rabbit anti-FLAG (Sigma), Alexa 488-conjugated
anti-mouseIgG,andAlexa546-conjugatedanti-rabbitIgG(MolecularProbes).
OpnExpressionConstructs.LentiviralexpressionconstructspMLS3(Opn-fl)and
pMLS5 (no signal sequence, Opn?49Koz) were described (12). Constructs for
IVT and Opn expression in 293FT cells contain backbone expression vector
pcDNA5/FRT/V5-His TOPO TA (Invitrogen). Point mutations in the Opn-fl
sequence were introduced by site-directed PCR mutagenesis using Opn-fl
constructs as templates with QuikChange II XL Site-Directed Mutagenesis Kit
(Stratagene).N-terminalFLAG-taggedOpn-FLconstructswereproducedwith
pCMV-Tag4 vector (Stratagene). Opn-GFP fusion expression vector was pro-
duced by inserting EGFP downstream of Opn cDNA in-frame.
5? RLM-RACE to Determine Transcription Initiation Sites. RLM-RACE was per-
formedusingFirstChoiceRLM-RACEkit(Ambion).Outerprimerannealingsite
wasinExon7,innerprimerbindingregionisshowninFig.S1.Amplifiedbands
were excised from an agarose gel, cloned into vectors, and the 5?-end of Opn
transcripts determined by sequencing.
IVT. Transcription was carried out with T7 RNA polymerase (Promega) from
linearized Opn expression constructs. IVT was performed in rabbit reticulocyte
lysates (Promega) with [35S] Met at 30°C for 90 min. Samples were phosphatase-
treated.
Immunofluorescence Analysis by Confocal Microscopy. Opn-expressing 293FT
cells and BM-DC were harvested 24 h after transfection and replating, respec-
tively. Images were obtained with a Nikon TE2000-U inverted Microscope
equipped with C1 Plus Confocal Laser Scanning system.
Ifna4 Promoter Reporter Assay. The assay was performed as described (12) by
using QUANTI-Blue system (InvivoGen) with a slight modification using back-
bone vector pCMV4 for Opn expression. Total amounts of DNA were kept
constant by supplementation with empty pCMV4 control vector.
Isolation of Secretory Vesicles. Secretory granules of 293FT cells were isolated
byPercolldensitygradientcentrifugationasdescribed(28,31).Homogenates
of Opn-transfected cells were centrifuged at 150 ? g for 5 min (P1 ? the
pellet),andsupernatantwascentrifugedat1,500?gfor10min.Theresulting
pellet was suspended in homogenizing buffer containing 40% Percoll and
centrifuged at 20,000 ? g for 20 min to obtain seven fractions.
ACKNOWLEDGMENTS. We thank C. D. Novina and B. Wang for expertise and
criticaldiscussionsandD.Laznikfortechnicalassistance.Thisworkwassupported
bytheNationalInstitutesofHealth(GrantsAI12184andAI48125,toH.C.)andby
National Research Service Award Fellowship T32CA70083 (to M.L.S.).
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