Download Non-redundant roles of cathepsins L, B and S in CD1a+ dendritic

MINERVA BIOTEC 2008;20:59-67
Non-redundant roles of cathepsins L, B
and S in CD1a+ dendritic cells knocked-down
for cathepsin S by RNA interference
R. TIRIBUZI 1, S. MARTINO 1, E. CIRACI 2, F. D’ANGELO 1, I. DI GIROLAMO,
A. DATTI 1, G. F. BOTTAZZO 2, A. C. BERARDI 2, A. ORLACCHIO 1
Aim. The ability to modulate the functional properties of
dendritic cells (DCs) with chemical drugs or via RNAbased technologies may lead to significant therapeutic
applications. In light of their relevance to the biology of
DCs, particularly within the antigen-presentation pathway, cysteine cathepsins L, B and S were investigated
with the long-term objective of assessing their value as
molecular target.
Methods. Cathepsin expression was monitored via a
cell-based model in which human, CD34+hematopoietic stem cells (HSCs) were induced to differentiate into
CD1a+DCs. Time-course analyses were performed via
Real time RT-PCR and Western blotting. The same experiments were conducted, in parallel, using HSCs subjected to cathepsin S knockdown by RNA interference.
Results. Processing of cathepsins L, B and S is subjected
to temporal patterns of expression throughout DC differentiation (a 14 day process). The mature form of
cathepsin S appeared in the lysosomal fraction on day
7, while mature cathepsin L and B proteins displayed
such localization only upon completion of differentiation (day 14). The non-redundant roles of these cathepsins were evident on day 7, as CatS-RNAi-mediated knockdown cells were found to show a marked decrease of
HLA-DR expression.
Fundings.—This work is supported by grants from FIRB Idea
Progettuale #RBIP06FH7J 002, Consorzio Inter-Universitario di
Biotecnologie INNB 2005-2007, Ministero della Salute RF-UMB-2006339457 Italy, Fondazione Cassa Risparmio Perugia #2007.0149.02 and
Consorzio INBB to A.O. and by grant from Ministero della Salute Italy to
A.C.B.
Acknowledgements.—We thank Dr. M. Di Ianni and Dr. L. Moretti
(University of Perugia) for assisting with Real Time PCR setup.
Received on April 21, 2008.
Accepted for publication on May 28, 2008.
Address reprint requests to: A. Orlacchio, Full Professor of Biochemistry,
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione
di Biochimica e Biologia Molecolare, Università degli Studi di Perugia, Via
del Giochetto, 06126 Perugia, Italy. E-mail: [email protected].
Vol. 20 - No. 2
1Section of Molecular Biology and Biochemistry
Department of Experimental Medicine and Biochemical
Sciences, University of Perugia, Perugia, Italy
2Stem Cells Research Laboratory, IRCCS
Bambino Gesù Pediatric Hospital, Rome, Italy
Conclusion. Cathepsins L, B and S are subjected to a temporal regulation of expression that is apparently associated with the progress of the differentiation process.
CD1a+DCs knocked-down for cathepsin S show the nonredundant roles of cathepsin L, B and S within the DC
maturation pathway and highlight the potential of
cathepsin S as a molecular target for drug discovery
research.
Key words: Hematopoietic stem cells - Dendritic cells Cysteine endopeptidases - RNA interference.
D
endritic cells (DCs) are professional antigen-presenting cells that efficiently link innate and adaptive immune systems 1-3 and maintain tolerance to self
proteins.4, 5 DCs originate from hematopoietic stem
cells (HSCs), and appear as immature cells prior to
migration into peripheral tissues, where they differentiate and acquire the capacity to process internalized
antigens through an endosomal MHC-II-restricted
pathway. Following antigen capture, DCs migrate to
the draining lymphoid tissue and mature phenotypically, a process that is concomitant with the up-regulation of CD40, CD80, CD86, MHC-II molecules and
CC-chemokine receptor 7 (CCR7). In the draining
lymphoid tissue, functionally mature DCs present the
peptide-MHC-II complexes on cell surface and inter-
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TIRIBUZI NON-REDUNTANT ROLES OF CATHEPSINS L, B AND S IN CD1A+ DENDRITIC CELLS KNOCKED-DOWN FOR CATHEPSIN S BY RNA INTERFERENCE
act with antigen-specific lymphocytes. At this stage,
DCs activate lymphocytes and produce proinflammatory cytokines, such as interleukin-12 and TNF-α.
Recently, an active role of DCs within the central
nervous system (CNS) was demonstrated in both aging
and neurodegenerative diseases.6-9 Thus, therapeutic
approaches for these disorders could be designed
based on the development of chemical drugs or RNA
interference (RNAi),10 which target molecular entities
affecting the functionality of DCs within the CNS.
Lysosomal proteases such as cysteine cathepsins
L, B and S mediate proteolytic events integral to
immuno-response biology.11-15 The engagement of
these enzymes materializes at two interrelated events,
namely, the processing of the invariant chain (Ii), a
chaperone molecule critical to MHC-II assembly and
transport, and the degradation of protein antigens.
Cathepsins L, B and S are active in different types of
antigens presenting cells (APCs). In this regard, a
large body of literature has attempted to ascertain the
specific role and the potential redundant activity of
each cathepsin through the use of knockout mice 1621 and enzyme inhibitors.22-24 Emerging data indicate
a defined expression pattern of cathepsins L, B and S
in different types of APCs such as DCs, but are insufficient to clarify the exclusive role of each enzyme.
In this study, we used a previously established cell
model, based on the differentiation in vitro of CD34+
hematopoietic stem cells into CD1a+DCs,25 to perform
a comparative analysis of cysteine cathepsins L (EC.
3.4.22.15 CatL), B (EC 3.4.22.1, CatB), and S (EC
3.4.22.27, CatS) in order to determine whether these
enzymes may be considered valid molecular targets for
the modulation of DC functionality within the CNS.
Here, we show that cathepsins L, B and S are subjected to a temporal regulation of expression that is
associated with the DC differentiation process. In addition, using interference RNA technology to generate
knockdown CTSS-CD1a+DCs, we demonstrate a nonredundant, physiological impact of cathepsins L, B and
S within the DC differentiation pathway and, in particular, a major role uniquely played by cathepsin S.
Materials and methods
Generation of DCs from CD34+ hematopoietic stem
cells
Following a previously established protocol,25 CD34enriched cells were transferred to 25 cm2 flasks and
cultured at a density of 105 cells/mL for 14 days. Cells
were maintained in GIBCO® RPMI1640 medium
(Invitrogen) containing 10% heat-inactivated fetal calf
serum (Euroclone) in the presence of a cytokine cocktail composed of human recombinant SCF (10 ng/mL),
Flt3L (50 ng/mL), GM-CSF (50 ng/mL), IL-4 (10 ng/mL),
and TNF-α (2.5 ng/mL) (PeproTech EC). Every third
day, half of the culture medium was replaced by fresh
medium supplemented with the same cytokine cocktail. The generation of DCs peaked on day 14.
Immunophenotypic analyses
Cells were washed and resuspended in phosphatebuffered saline (PBS), supplemented with 1% bovine
serum albumin and incubated with either FITC- or
PE-conjugated monoclonal antibodies (mAbs) for 30
min at 4 °C in the dark. The mAbs used were the following: anti -CD1a, -CD4, -CD14, -CD15, -CD19, CD34, -CD40, -CD56, -CD80, -CD83, -CD86, -CD235a
(glycophorin A), -HLA-ABC, -HLA-DR (BD
Biosciences). Negative controls were isotype-matched,
irrelevant mAbs. Cells were analyzed by flow cytometry using a FACScan and the CellQuest software (BD
Biosciences) for data management. Cells were electronically gated according to light-scattering properties to discriminate cell debris.
T cell proliferation assay MLR
Isolation of human CD34+ hematopoietic stem cells
Blood samples were collected from 86 healthy volunteer subjects. Approval for these studies was
60
obtained from the Ospedale Pediatrico Bambino Gesù
review board. Human peripheral blood mononuclear
cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Paque™ PLUS (GE Healthcare).
CD34+cells were purified by immunomagnetic selection using the mini-magnetic-activated cell sorter
(MACS) system (Miltenyi Biotec) according to the
manufacturer’s instructions. Mean purity of CD34enriched cells, determined by flow cytometry using a
FACScan (BD Biosciences), was 94.8%, with a median value of 92 throughout an 86-98.5 range.
CD1a+DCs (15×103) were co-cultured in 96-well
plates, in triplicate, with 3×105 allogenic, monocytedepleted PBMCs for 5 days. During the last 6 h of
culture 20 µM of 5-bromo-2’-deoxyuridine (BrdU)
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NON-REDUNTANT ROLES OF CATHEPSINS L, B AND S IN CD1A+ DENDRITIC CELLS KNOCKED-DOWN FOR CATHEPSIN S BY RNA INTERFERENCE TIRIBUZI
(BrdU Flow Kits, BD Biosciences) was added in each
well and lymphocyte proliferation was assessed
through BrdU incorporation measured by flow cytometry.26 Results are expressed as the percentage of proliferating BrdU+lymphocytes.
Real Time RT-PCR
Total RNA from: 1) untreated, peripheral blood
CD34+HSCs (1×106cells); 2) HSCs treated for 7 days
with the cytokine cocktail (1×106 CD34-CD1a+ cells);
3) differentiated DCs (1×106 CD1a+DCs) was isolated
using an RNeasy Mini Kit (Qiagen, Hilden, Germany).
Reverse transcription was carried out using 1 µg of
total RNA in the presence of 200 U of Super Script IITM
Reverse Transcriptase and 10 ng/µL of random hexamers as the reverse primers (Invitrogen). Real Time RTPCR was performed using 5 ng of cDNA, the AssayOn-DemandTM for human CTSL (Hs00266474_m1),
CTSB (Hs00157194_m1), CTSS (Hs00175407_m1) and
18S rRNA (Hs99999901_s1) genes, and the TaqMan
Universal PCR Master Mix No AmpErase UNG (Applied
Biosystems, Foster City, CA in an ABI PRISM 5700
Sequence Detector System (Applied Biosystems).
Relative quantifications of mRNA were determined
via a standard curve method using 18S rRNA as the
endogenous control and a comparative 2-ΔΔCT method
based on normalization of the target to 18S rRNA.
The ΔCt was determined by subtracting the Ct of 18S
rRNA from the Ct of the target.27
Generation of CTSS knockdown CD1a+DCs by RNA
interference
Freshly isolated CD34+cells were resuspended in
RPMI1640 supplemented with cytokines used to generate DCs, and immediately subjected to transfection
using predesigned siRNAs targeting the CatS gene
(CTSS: ID-113084-113085, Ambion, UK).
As controls, cells were subjected to mock transfection or transfection using a scrambled siRNA (ID46183G, Ambion) under the same experimental conditions. The procedure was carried out in triplicate
using a 24-well plate. Each well contained 2×105 cells
in a volume of 500 µL. The siRNAs, at a concentration
of 150 nM, were combined with DOTAP® Liposomal
Transfection Reagent (Roche Diagnostics, Italy) and
maintained for 30 min at room temperature to form
complexes. The mixture (50 µL was then overlaid
dropwise on the cell cultures. Following 4 h incuba-
Vol. 20 - No. 2
tion, 1.2 mL of RPMI+10% FCS and cytokines was
added to each well. On day 3 and day 9 of culture,
cells were centrifuged, resuspended in 500 µL of
cytokine-enriched culture medium and exposed to a
second and third transfection round. On days 7 and
14, control, mock-, mock-scrambled siRNA- and CTSS
siRNA-transfected cells were harvested and employed
for biochemical assays and phenotypic analyses. Visual
inspection and routine flow cytometry tests indicated
that this methodological approach did not affect cell
morphology, viability or proliferation rate.
Western blotting
To obtain cell extracts, cells were harvested, washed
in PBS, lysed for 1 h in 10 mM sodium phosphate
buffer, pH6.0, containing 0.1% (v/v) Nonidet NP-40,
and subjected to sonication. These steps were performed at 4 ºC.28 Protein content in each sample was
measured using a protein assay kit from Bio-Rad
Laboratories. After boiling for 5 min in loading buffer,
samples containing 15 µg protein were separated
through a 12% gel under reducing conditions.
Precursors and mature forms of cathepsins L, B and
S were analyzed by Western blotting, that was performed as previously described 29 using polyclonals
from Santa Cruz Biotechnology as the primary antibodies. Immunodetection was carried out by employing the enhanced, chemiluminescent Amersham ECL
Plus™ kit (GE Healthcare). For each blot, several time
exposures were performed to ensure that the results
were obtained in the linear response range of the
film. Additionally, densitometric scans via Image J
software (National Institute of Health) showed that
the intensity of the bands were proportional to protein content. β-actin was employed for normalization
purposes.
Subcellular fractionation
Crude lysosomal fractions were isolated by differential centrifugation. Cells were washed twice with icecold PBS and resuspended in 0.25M ice-cold sucrose
to a concentration of 8×105 cells/mL. Cells were repeatedly passed through a Wheaton homogenizer to disrupt plasma membranes, after which the homogenate
was centrifuged (3 000×g for 10 min, 4 ºC) to remove
cellular debris, plasma membrane and nuclei. The
resulting supernatant was spun at 11 000×g for 20
min at 4 ºC to obtain a lysosomal-enriched preparation
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TIRIBUZI NON-REDUNTANT ROLES OF CATHEPSINS L, B AND S IN CD1A+ DENDRITIC CELLS KNOCKED-DOWN FOR CATHEPSIN S BY RNA INTERFERENCE
60
Day 0
60
45
45
Counts
30
15
0
30
15
Day 3
0
45
30
B
0 10 1 10 2 10 3 10 4
HLA-DR
0
Day 7
45
Day 14
30
15
0
A
0 10 1 10 2 10 3 10 4 0 10 1 10 2 10 3 10 4
CD34
CD1a
C
20
0
Figure 1.—Generation of CD1a+DCs from CD34+HSCs. A) CD34 and
CD1a expression during the differentiation of CD1a+DCs from
CD34+HSCs (Day 0,3,7,14). Cells were incubated with different FITCor PE-conjugated monoclonal antibodies (mAbs) for 30 min at 4°C in
the dark. Negative controls were isotype-matched, irrelevant mAbs.
Cells were collected and analyzed using a FACScan (Becton Dickinson)
flow cytometer. Data analysis was performed via the CellQuest software (Becton Dickinson). Values are reported as the means of four
experiments. Control cells (❏), stained cells (■). B) HLA-DR expression in CD1a+DCs stimulated with LPS. The analysis was performed
as described in the Materials and methods. Control cells (❏), stained
cells (■). C) MLR assay. 3×105 CD1a+DCs were co-cultured in 96well plates with a similar number of CD4+lymphocytes. Results were
expressed as percentage of proliferating (BrdU+) lymphocytes. The
assay is described in the Materials and methods. PBMCs= CD4+T
cells (❏); CD1a+DCs (14 day differentiation) (■); LPS= LPS-treated DCs
(14 day differentiation) (■).
in the pellet (L fraction) and a microsomal component
(M fraction) in the supernatant. Presence of Lamp-2,
assessed by immunoblot, and the activity of β-hexosaminidase, measured through a fluorometric assay,
were used as internal controls for the L fraction.28, 29
Immunocytochemistry
CD34+HSCs, CD1a- and CD1a+DCs were resuspended in PBS and centrifuged using a Heraeus-Christ
DIGIFUGE GL cytospin at 700 rpm for 7 min. Cells
were fixed in 4% paraformaldehyde for 30 min,
washed in PBS, treated with blocking solution (i.e. 10%
FBS, 0.1% Triton X-100 in PBS) for 30 min at room temperature, and incubated overnight at 4°C with either
62
Results
LPS
0
45
80
40
CD1a+DCs
15
100
PBMCs
30
% Brdu+CD4+ cells
Counts
15
an anti-human CatS polyclonal antibody (Santa Cruz
Biotechnology, sc-6503) or a FITC-conjugated, antihuman-CD74 (invariant chain) monoclonal antibody
(cat. N-11-0749-71, clone LN2). Antibodies were routinely diluted in PBS buffer containing 1% FBS and
0.01% Triton X-100.
After being extensively washed, cells were incubated with ALEXA Fluor-, TRICT-conjugated secondary
antibodies. After mounting with VECTASHIELD® medium, cells were observed using a Nikon Eclipse TE
2000 S fluorescence microscope and subjected to
analysis through the Cell F software (Olympus, Japan).
Generation of CD1a+DCs from hematopoietic stem
cells
CD34+ hematopoietic stem cells (CD34+HSCs), purified from the peripheral blood of 86 healthy subjects,
were induced to proliferate and differentiate into DCs
in the presence of a cytokine cocktail composed of
human Flt3L, GM-CSF, IL-4, and TNF-α. The differentiation process was monitored through the expression of CD34 and CD1a antigens.25, 30 As shown in
Figure 1A, >90% of progenitor cells were CD34+ and
lacked CD1a. This pattern, however, began to reverse
starting on day 3. Following 7 days of cytokine treatment, cultures displayed CD1a (45.2% cell population, mean fluorescence intensity [MFI] =93.7±10.5)
and HLA-DR (35.1%, MFI=400±30), while CD34
expression was markedly decreased (14.5%,
MFI=5.6±1.1), thereby revealing a fate specification
stage toward DC lineage commitment. At this stage,
cells became CD1a+ lineage restricted (CD34CD1a+cells).
On day 14, the expression of CD1a+ increased to
73.5% (MFI=285±3), consistent with a DC phenotype.25 Similarly, cells displayed significantly higher levels of other DC markers, such as the co-stimulatory
molecules CD80, CD86, CD40, CD83 (data not
showed). Further, the differentiation into CD1a+DCs
was confirmed by the expression of HLA-DR molecules (83.2%, MFI=1562±20).
The functionality of CD1a+DCs was shown by their
ability to respond to 100 ng/mL LPS, which triggered
a marked increase of HLA-DR molecules (MFI=
2757±21.3) (Figure 1B) and stimulated heterogeneous
CD4+ lymphocytes in MLR tests (Figure 1C).
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Neither CD1a nor other DC-specific differentiation
markers, such as CD14, CD15, CD19, CD56, glycophorin A, were expressed in HSCs cultured for 3, 7
and 14 days in the presence of the single cytokines
used in the DC differentiation cocktail (data not
shown).
CatL
KDa
62
45
36
26
CatB
Expression of cathepsins L, B and S during CD34+HSC
differentiation into CD1a+DCs
CatB
CatS
49
49
29
29
CatS
66
ENZYME PROCESSING
66
26
26
SUBCELLULAR LOCALIZATION
Cysteine proteases are translated as latent precursors and subsequently converted into mature, enzymatically active forms. Since limited proteolysis of
the proenzymes and intracellular trafficking may control the activities of cathepsins and their corresponding physiological roles,12, 15 we analysed the subcellular
distribution of CatL, CatB and CatS in CD34+HSCs,
CD34-CD1a+ cells and CD1a+DCs.
Figure 2B shows that in CD34+HSCs, the precursors of both CatL and CatB are restricted to the microsomal fraction, unlike that of CatS which was found
to be equally distributed between microsomal (M)
L M L M L M
CD34+ CD34 CD1+
HSCs CD1a DCs
cells
CD
34 +
CD HS
34 Cs
ce CD
ll 1
CD s a
1+
DC
s
β actin
A
Expression level
Cathepsins were analyzed by Western blotting using
polyclonal antibodies raised against both mature and
precursor enzyme forms.
Figure 2A shows the patterns of cathepsin expression in freshly isolated peripheral blood HSCs
(CD34+HSCs) and in HSCs induced to differentiate
into CD1a+DCs. Mature forms of cathepsins L, B and
S were undetectable in untreated HSCs which, instead,
exhibited bands that corresponded to enzyme precursors with apparent molecular weights of 62KDa
(CatL), 49KDa (CatB) and 66KDa (CatS). On day 7,
CD34-CD1a+cells showed the same profile for both
CatL and CatB, while CatS could be detected as a
26KDa band attributable to the mature enzyme protein. Mature CatL (26KDa) and CatB (29KDa) appeared
in differentiated CD1a+DCs (day 14), whereas the 62
and 66KDa precursor bands of CatL and CatS, respectively, were barely detectable.
Notably, these results were consistent throughout
the entire collection of samples (N=86) obtained from
healthy subjects and suggest that cathepsins L, B and
S undergo a time-controlled regulation of expression
that appears to be correlated with the DC differentiation process.
Vol. 20 - No. 2
KDa
62
45
36
26
CatL
C
8
6
4
2
0
6
4
2
0
6
4
2
0
B
CTSL
CTSB
CTSB
CD34+ CD34 CD1+
HSCs CD1a DCs
cells
Lamp 2
3
2
1
0
KDa
120
L M
CD34+
HSCs
L M
CD34+
CD1a+
L M
CD1+
DCs
D
Figure 2.—Cathepsin processing in CD34+HSCs and CD1a+DCs at
different differentiation stages. A) Identical amounts of cell proteins
(15 µg) were loaded into a 12% polyacrylamide gel, separated by
SDS-PAGE and subjected to Western blotting as previously described.29
Immunodetection was performed using polyclonals as the primary antibodies for cathepsins. β-actin is shown for normalization purposes. B)
Cells were subjected to sub-cellular fractionation by differential centrifugation to separate the lysosomal fraction. Subcellular fractions
were analysed for protease content by SDS-PAGE and Western blotting as previously described. C) Fractions were tested for the presence
of the lysosomal protein Lamp 2 (by Western blotting analysis) and the
lysosomal glycohydrolase β-hexosaminidase (by fluorometric assay
determination). L: lysosomal fraction; M: microsomal fraction. RSA
indicates Relative Specific Activity, namely the ratio between the percent of total β-hexosaminidase activity and the percent of total protein content observed in a subcellular fraction. D) Real-time analysis
of CTSL, CTSB, CTSS gene expression. Transcriptional rates measured
in CD34-CD1a+ cells and CD1a+DCs are relative to those observed in
freshly isolated CD34+HSCs.
and lysosomal (L) compartments. On day 7, CD34CD1a+cells showed the mature form of CatS in the L
fraction, while a single band of comparable intensity
and corresponding to the precursor form was detected in the M compartment. On day 7, CatL exhibited
equal distribution of the precursor form in the M and
L compartments, while no changes were observed
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TIRIBUZI NON-REDUNTANT ROLES OF CATHEPSINS L, B AND S IN CD1A+ DENDRITIC CELLS KNOCKED-DOWN FOR CATHEPSIN S BY RNA INTERFERENCE
for CatB. CatS distribution did not change upon DC differentiation (day 14); by contrast, CatL and CatB were
present in the L fraction as a number of different precursors co-localized with the correspondent mature
forms. Only precursor forms of CatB and CatS were
detected in the M fraction of differentiated cells. In all
instances, β-Hexosaminidase activity and Lamp-2
detection were employed as markers of the lysosomal
compartment (Figure 2C).
Together, these results point to spatially- and temporally-controlled mechanism(s) that differentially
affect the expression of the three cathepsins in relation to the status of the DC differentiation process.
TRANSCRIPTIONAL RATES
Western blotting analyses were supported, in parallel, by the quantification of gene transcripts via Real
Time RT-PCR. At day 7, CatL and CatB mRNAs displayed a 7- and 2-fold increase, respectively, followed
by either a sharp reversal to day 0 levels (CatL) or a
further, slight increase toward completion of cell differentiation (CatB) (Figure 2D). On the other hand, the
expression of CatS mRNA was uniform during the
entire course of the experiment. These results suggest that expression of cathepsins L and B are controlled, at least partially, at the transcriptional level
during the differentiation process.
CatL and CatB in CD1a+DCs knocked-down for CatS
The physiological roles of CatL, CatB and CatS
were investigated in differentiating cells knockeddown for CatS by RNA interference (Figure 3). After
7 day of culture, no forms of CatS could be detected
in siRNA-transfected cells, (Figure 3A; lane: CTSS
siRNA 7D) in contrast to what was observed in both
control and mock-transfected cells (Figure 3A, lanes:
Control 14D; Mock 14D). In CatS knockdown cells the
expression of CD1a was unaffected (Figure 3B).
Conversely, the surface marker HLA-DR showed a
71% reduction (Figure 3C) and the invariant (Ii) chain
was significantly decreased (95%) as compared to
controls (Figure 3D). In all instances, RNAi-transfected, mock-transfected and control cells exhibited
identical viability and proliferation rates. The same
data were confirmed in knockdown DCs (14 days)
(Figure 3A-D).
Notably, CatS knockdown did not affect the expression of either CatL and CatB. Both proteases were
64
present as precursor form on day 7 (Figure 3E, F)
and as mature forms at day 14 (Figure 3G, H).
Together, these results indicate that, in the in vitro
system employed, CatL and CatB do not play the same
role of CatS within the physiological pathway that
underlies intracellular trafficking and maturation of
the MHC-II (Figure 3C, D).
Discussion
This study shows that cathepsins L, B, and S do
not play redundant roles in CD1a+DCs and that their
expression is controlled at the protein level by discrete
mechanisms associated with the progression of HSC
differentiation into DCs.
In human HSCs treated with a basal cocktail composed of GM-CSF, IL-4, TNF-α, SCF and Flt3L, differential patterns of enzyme expression were evident
throughout a two-week culture. Only precursor
cathepsin forms were present in HSCs, consistent with
the virtually ubiquitous nature of these enzymes and
the hypothetical existence of proteolytic reservoirs
susceptible to specific activation signaling from the surrounding environment.
The expression of the mature forms of CatL and
CatB could be detected within the lysosomal fraction
upon completion of cell differentiation (day 14),
whereas the mature form of cathepsin S became evident in the lysosomal subcellular fraction on day 7,
concurrently with the emergence of CD1a. Protein
level and localization of CatS did not change afterwards, thereby confirming a physiological relevance
of this enzyme in functionally mature DCs.19-21 This
was further demonstrated by CatS gene silencing
which was followed by a 71% reduction of surface
HLA-DR and a 95% decrease of the invariant (Ii) chain
expression.
In CatS knockdown cells induced to differentiate,
both CatL and CatB were normally processed. Upon
DC differentiation, these enzymes appeared as mature
proteins which, however, did not show the capacity
to replace the role of CatS in the control of MHC II
expression.
It was previously reported that both CatB and, to a
larger extent, CatL, may share with CatS a functional
role in MHC-II processing.17, 31 CatS is indeed involved
in the processing of the invariant chain (Ii), which is
degraded by the enzyme to its smallest class II-binding fragment (CLIP).20 Ii is a membrane protein that
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NON-REDUNTANT ROLES OF CATHEPSINS L, B AND S IN CD1A+ DENDRITIC CELLS KNOCKED-DOWN FOR CATHEPSIN S BY RNA INTERFERENCE TIRIBUZI
14D
300
1200
7D
400
0
0
Co
ntr
ol
CT
SS mock
siR
NA
Co
ntr
ol
CT
SS moc
siR k
NA
B
C
7D
KDa
KDa
CatL
75
7D
50
25
0
Co
ntr
ol
CT
SS moc
siR k
NA
100
26
A
800
Co
ntr
ol
CT
SS mock
siR
NA
200
M. F. I.
M. F. I.
66
14D
100
HLA-DR
D
Co
ntr
ol
CT
SS mock
siR
NA
KDa
CatS
1600
14D
Co
ntr
ol
CT
SS moc
siR k
NA
CD1a
% li positive cells
400
β-actin
KDa
CatB
KDa
CatL
CatB
62
26
29
ol
M
oc
k
SS
siR
NA
49
Co
ntr
ol
M
oc
CT
k
SS
siR
NA
G
CT
F
45
36
ntr
Co
ntr
ol
M
o
ck
CT
SS
siR
NA
E
Co
Co
ntr
ol
M
oc
CT
k
SS
siR
NA
49
H
Figure 3.—Cathepsin S knockdown. A) Cathepsins S processing following siRNA transfection. HSCs treated with the cytokine cocktail were
lysed after 7 and 14 days of culture, as described in the Materials and methods. Cell protein (15 µg) was loaded into a 12% polyacrylamide
gel, separated by SDS-PAGE and subjected to Western blotting analysis. Immunodetection was carried out using a polyclonal antibody
against CatS. β-actin is shown for normalization purposes. Control: untransfected cells after 7 and 14 days of culture; mock: scrambled
siRNA-transfected cells after 7 day and 14 days of culture; CTSS siRNA 7D: cells subjected to two rounds of transfection with CatS siRNA (on
days 1 and 3); CTSS siRNA 14D: cells subjected to three rounds of transfection with CatS siRNA (on days 1, 3 and 9). B, C, D) CD1a, HLADR and Ii levels following siRNA transfection. CTSS siRNA-transfected cells, mock-transfected cells and control CD34-CD1a+DCs were treated for 7 days (7D) with cytokine cocktail prior to incubation with different FITC- or PE-conjugated monoclonal antibodies (mAbs) for 30 min
at 4 °C in the dark. The same procedure was performed using the same cell sets after 14 days (14D) of treatment. Negative controls were isotype-matched irrelevant mAbs. B) CD1a MIF expression detected by FACS analysis. C) HLA-DR MIF expression detected by FACS analysis.
D) Percent of Ii chain-positive cells observed through a fluorescence microscope and subjected to analysis via Cell F software (Olympus, Japan).
E, F, G, H) Cathepsins L and B processing following CatS siRNA transfection. Cell protein (15 µg) was loaded into a 12% polyacrylamide gel
for SDS-PAGE prior to Western blotting analysis. Immunodetection was carried out using polyclonal antibodies. Control: control cells after
7 and 14 days of culture; mock: mock-transfected cells after 7 and 14 days of culture; CTSS siRNA 7D: cells subjected to two rounds of transfection with CatS siRNA (on days 1 and 3); CTSS siRNA 14D: cells subjected to three rounds of transfection with CatS siRNA (on days 1, 3 and
9). E) CatL and F) CatB expression in CTSS siRNA 7D cells (treated with cytokine cocktail for 7 days). G) CatL and H) CatB expression in CTSS
siRNA 14D cells (treated with cytokine cocktail for 14 days).
promotes MHC-II antigen processing and presentation.32 In the absence of Ii, MHC-II α and β chains bind
to unfolded ER polypeptides and are largely contained in aggregates. Conversely, Ii binds to the peptide binding cleft of αβ heterodimers and stabilizes the
conformation of MHC-II molecules.33
CatS-/- and CatL-/- mice revealed that both enzymes
are required for Ii degradation.17-19, 34 However, other
studies have demonstrated that these two proteases
have distinct, non-redundant roles in antigen presentation. In this respect, CatS was found to mediate
the final step of Ii proteolysis into αβ-CLIP in both B
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cells and DCs, whereas CatL performs the same function in cortical thymic epithelial cells.17-19 Further,
studies carried out in knockout mice, ruled out the
impact of cathepsin B, notwithstanding its relatively
high levels, on both Ii processing and MHC II antigen
presentation,35 despite earlier reports suggesting the
contrary based on biochemical studies.31, 36, 37
Our results indicate that Cat L, B and S have non
redundant role at list toward the final step of the
MHC-II processing in CD1a+DCs and suggest that this
could be a result of a tight mechanism of regulation
that control the processing of these enzymes during
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TIRIBUZI NON-REDUNTANT ROLES OF CATHEPSINS L, B AND S IN CD1A+ DENDRITIC CELLS KNOCKED-DOWN FOR CATHEPSIN S BY RNA INTERFERENCE
the differentiation of CD34+HSCs to CD1a+DCs.
Furthermore, this study show the defined expression
pattern of cathepsin L, B and S in DCs and confirm
these enzymes as important players within the immune
system.38-41 This, in turn, suggests that these enzymes
have potential roles as immuno-modulators which
may be relevant for the study of neurodegenerative
diseases in which inflammation emerges as major
causal event and cathepsins have already be shown
to be implicated.10, 42, 43
Conclusions
The in vitro cell model employed in this study clearly showed that the lysosomal cysteine proteases CatL
CatB and CatS have distinct, non-redundant roles in
DCs. This is particularly evident in CatS knockdown
DCs, which confirmed the physiological role of this
enzyme within the antigen presentation pathway. The
use of RNA interference technology clearly revealed
CatS as a possible molecular target for immunotherapy applications in DCs.
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