Tài liệu Báo cáo Y học: Regulated expression and intracellular localization of cystatin F in human U937 cells pptx

Regulated expression and intracellular localization of cystatin F
in human U937 cells
Carl-Michael Nathanson
1
, Johan Wasse
´
lius
2
, Hanna Wallin
1
and Magnus Abrahamson
1
1
Department of Clinical Chemistry, Institute of Laboratory Medicine, and
2
Department of Ophtalmology, University of Lund,
University Hospital, Lund, Sweden
Cystatin F is a cysteine peptidase inhibitor recently discov-
ered in haematopoietic cells by cDNA cloning. To further
investigate the expression, distribution and properties of the
native human inhibitor the promyeloid cell line U937 has
been studied. The cells expressed relatively large quantities of
cystatin F, which was found both secreted and intracellu-
larly. The intracellular levels were unusually high for a
secreted cystatin ( 25% of the cystatin F in 2- or 4-day
culture medium). By contrast, U937 cells contained only
3–4% of the related inhibitor, cystatin C. Cystatin F purified
from lysates of U937 cells showed three major forms car-
rying two, one or no carbohydrate chains. Immunocyto-
chemistry demonstrated a marked cytoplasmic cystatin F
staining in a granular pattern. Double staining with a marker

for endoplasmic reticulum revealed no colocalization for
cystatin F. Analysis of the promoter region of the cystatin F
gene (CST7) showed that it, like that of the cystatin C gene
(CST3), is devoid of typical TATA- and CAAT-box ele-
ments. In contrast to the cystatin C promoter, it does not
contain multiple Sp1 binding sites, but has a unique site for
C/EBPa, possibly explaining the restricted expression of the
cystatin F gene. Cells stimulated with all-trans retinoic acid
to differentiate them towards a granulocytic pathway,
showed a strong ( 18-fold) down-regulation of intracellu-
lar cystatin F and almost abolished secreted levels of the
inhibitor. Stimulation with tetradecanoyl phorbol acetate,
causing monocytic differentiation, also resulted in down-
regulation (two fold to threefold) of cystatin F expression,
whereas the cystatin C expression was essentially unaltered
in both experiments. The results suggest that cystatin F as an
intracellular cysteine peptidase inhibitor with readily regu-
lated expression, may be a candidate to control the cysteine
peptidase activity known to be essential for antigen presen-
tation in different blood cell lineages.
Keywords: cysteine protease; cysteine protease inhibitor;
cystatin F; cystatin C; expression pattern.
Mammalian papain-like (family C1) peptidases, such as
cathepsins B, H, L, S and K, can be regulated by natural
inhibitors belonging to the cystatin superfamily. These
inhibitors are of three major types [1]. Those of type 1,
cystatins A and B (also called stefins), are approximately
100 amino-acid long polypeptides lacking signal peptides
and disulfide bridges. Type 2 cystatins, synthesized with
signal peptides, are approximately 120 amino acids long and

contain at least two disulfide bridges. Type 3 cystatins, the
kininogens, are larger proteins containing three tandemly
repeated type 2-like cystatin domains. Type 2 cystatins
identified in higher animals include cystatins C, D, S, SA,
and SN [2], but also the recently reported cystatin E/M [3,4]
and cystatin F [5–7].
Human cystatin F is synthesized as a 145-amino-acid
residue preprotein, with the first 19 residues theoretically
constituting a signal peptide. Secretion of a 126-residue
mature protein has been verified for recombinant cystatin F
produced in insect cells [5]. The mature sequence shows 29–
34% identity when compared to the sequences of other
human type 2 cystatins. It has two possible N-glycosylation
motifs at positions 36–38 and 88–90 (cystatin C numbering).
Recombinant cystatin F expressed in Sf9 insect cells is
indeed glycosylated [5].
Compared to other cystatins, cystatin F is an unusually
specific inhibitor. Among the family C1 cysteine peptidases
studied, cystatin F binds papain and cathepsin L with high
affinity (K
i
0.1–1 n
M
), but does not inhibit cathepsin B
(K
i
> 1000 n
M
) [5,6]. Cathepsin L is a peptidase involved in
the normal lysosomal turnover of proteins. However, more

specialized functions have also been attributed to the
enzyme. So has cathepsin L been shown to be involved in
the loading of MHC II complex at antigen presentation. In
cortical thymic epithelial cells from mice devoid of the
cathepsin L gene, CLIP 22 and CLIP 10, two intermediate
processing products of the invariant chain accumulate [8].
This indicates that the amount of cysteine peptidase activity
in the processing compartment for the invariant chain is
crucial for antigen presentation and, moreover, that this
activity may be regulated by a cystatin [9].
Besides the inhibitory site for family C1 peptidases, cyst-
atin F carries a second site for inhibition of the family C13
enzyme, mammalian legumain or asparginyl endopeptidase
Correspondence to M. Abrahamson, Department of Clinical
Chemistry, Institute of Laboratory Medicine, University Hospital,
S-221 85 Lund, Sweden. Fax: + 46 46 130064, Tel.: + 46 46 173445
E-mail:
Abbreviations: ATRA, all-trans retinoic acid; TPA, tetradecanoyl
phorbol acetate; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
Note: web page available at />Note: nucleotide sequences are available in the DDBJ/EMBL/
GenBank databases under accession nos AJ51067, AJ51068, AJ51069
and AJ51070.
(Received 12 June 2002, revised 12 August 2002,
accepted 11 September 2002)
Eur. J. Biochem. 269, 5502–5511 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03252.x
(K
i
for the pig enzyme, 10 n
M

) [10]. Like cathepsin L,
mammalian legumain has been implicated in antigen pres-
entation, but through processing of the antigen rather than
the MHC part of the complex. Manoury et al.[11]studied
the processing of tetanus toxin in disrupted lysosomes from
an EBV-transformed B-cell line. By using a competitive
substrate they demonstrated that the degradation of tetanus
toxin was due largely to the activity of human legumain,
however, common inhibitors of family C1 cysteine peptid-
ases or aspartic proteases did not affect the degradation.
Thus, cystatin F has the potential to regulate two different
enzyme activities relevant for antigen presentation. In this
context, it is intriguing that the cystatin F expression seems
restricted to haematopoietic cells [5,6]. By Northern blotting
of RNA from human tissues, the highest cystatin F mRNA
levels were seen in peripheral blood cells and spleen [5]. At
cDNA library Southern blotting of 61 human cell types,
cystatin F was observed mainly in resting T-cells, premono-
cytic cells, activated dendritic cellsand some natural killer cell
clones [6]. This is in agreement with analysis of cystatin F
EST clones in a collection of > 650 cDNA libraries, showing
that 54 cystatin F clones were present in 20 of the libraries, all
of which were derived from immune cells (mainly primary
dendritic and T cells) [5]. Further analysis of 10 human
immune cell lines showed the highest secretion levels from the
premyeloid cell line, U937, low secretion levels from T-cell
lines and no secretion from B-cell lines [5].
The present investigation was undertaken to further
study cystatin F, through analysis of the expression, distri-
bution and properties of the native human inhibitor in U937

cells.
MATERIALS AND METHODS
In silico
cloning and sequencing of the human
cystatin F gene
A full-length cystatin F sequence from cDNA clone
HCUDE60 [5] was run in a
FASTA
search with the GCG
package at the Karolinska Institute Sequence Analysis
Computer (KISAC, http://130.237.126.207; Karolinska
Institutet, Stockholm, Sweden) to identify a full length
human genomic clone containing the cystatin F gene. A 106-
kb fragment (AL035661) was acquired which contained the
entire gene including the promoter region. To verify the
in silico cloned cystatin F gene sequence, the four exons
including  100 bp of 5¢-and 3¢ flanking sequences as well as
the promoter region were amplified by PCR using primers
listed in Table 1. Both strands of the fragments were
sequenced with an ABI Prism 310 Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA) using the Big Dye
Terminator Cycle Sequencing Kit (Applied Biosystems) and
either of the oligonucleotides used for PCR as sequencing
primer. The sequencing reactions were performed according
to the manufacturer’s advice. The cystatin F gene sequences
determined have been deposited in the GenBank/EMBL
database under accession nos AJ51067, AJ51068, AJ51069
and AJ51070.
Promoter analysis
To study the promoter region of the cystatin F and C genes,

the nucleotides )1to)600 gene segments were analysed
with the MOTIF search engine (the vertebrate database),
Bioinformatics Centre, Institute for Chemical Research,
Kyoto University, Japan ( />using a cut-off score of 85.
Cell culture
The human promyleoid cell line U937 (ATCC no. CRL-
2367) was cultured in RMPI medium (Life Technologies
Ltd, Paisley, UK) supplemented with 5% (v/v) foetal calf
serum (Life Technologies) and antibiotics (10 UÆmL
)1
penicillin and 10 lgÆmL
)1
streptomycin; Life Technologies)
at 37 °C and an atmosphere with 5% CO
2
, in 75 cm
2
culture flasks (Costar, Cambridge, MA, USA). For differ-
entiation experiments, cells (1 · 10
6
mL
)1
) were seeded and
cultured in the presence of 1 l
M
ATRA for 4 days or in the
presence of 0.13 l
M
TPA for 2 days. After incubation for 4
and 2 days, respectively, the cells were set to 10

6
cellsÆmL
)1
in fresh medium. ATRA or TPA was added at the same
concentrations as above. The cells were incubated for an
additional 24-h period. Following the second incubation the
cells were counted and separated from the media. One
portion of the cells was lysed with 1 mL Triton-X100 in
NaCl/P
i
containing 1 · inhibitor cocktail (final concentra-
tions of 5 m
M
benzamidinium hydrochloride, 15 m
M
NaN
3
and 10 m
M
EDTA) per 6 · 10
7
cells, for analysis in ELISA
and Western blot experiments. Another portion of the cells
was spun down to object glasses and used for immunocyto-
chemical staining, and from the rest of the cells RNA was
Table 1. Primers used for PCR amplification, sequencing and probe amplification. Standard amplification reactions were accomplished in a Perkin
Elmer GeneAmp 2400 thermal cycler (Perkin Elmer, Foster City, CA, USA), using 0.6 l
M
primers and reagents from Perkin Elmer. The PCR
cycles repeated 30 times were (denaturation, 94 °C, 30 s – annealing at specified temperature, 30 s – extension, 72 °C, 60 s). The sequencing

reactions were performed in the same instrument using standard reaction conditions.
Gene part
Upstream primer
(5¢-to 3¢)
Downstream primer
(5¢-to 3¢)
Annealing
temp. (°C)
Seg. size
(bp)
Upper promoter
TGA AGC TGG AAA CCA TCA TTC AAA ACA TTA GCA GGA ATT TTC 52 343
Lower promoter GGA GTT CTG CCA GGG AAC CAC GAC AGG GGA GAA CGC CAC TTA 57 587
Exon 1 GTG CTG CCT GAG AAG GAT TG GAA AGT GCC CTG GGG AAG ACC 62 333
Exon 2 TGA AGG CCC CAC TAA CAT CAG TAT ATC CGC CCT GCT CTC CTA 56 314
Exon 3 GAG GCC CTG CTT CCT AGT GGA TGC GTT AGA GAC GTG GTG ACG 58 289
Exon 4 CCG CAG GGA AAG TCT AAG CTC ACA TCT CTG CTG ATT ATT CAG 55 497
Cystatin F probe CTT CTG CTG CCT GGT CTT GA GCA CTT CAC CCG CTC ACT CGT CA 57 542
Cystatin C probe CGG CGA GTA CAA CAA AGC CA GGA GGT GTG CAT AAG AGG TG 57 320
Ó FEBS 2002 Cystatin F in U937 cells (Eur. J. Biochem. 269) 5503
extracted. The culture media were supplemented with 1·
inhibitor cocktail and saved at )20 °C until analysed.
Western blotting
To 1 mL of cell extract prepared as described above, 10 lL
of Cm-papain coupled to Sepharose-4B resin (with capacity
to bind  10 lg cystatin) [12] was added. The solution was
incubated for 4 days at 4 °C on a rocking table. Following
incubation the Sepharose gel from 1 mL of mixture was
allowed to sink and the supernatant was discarded. Sample
buffer containing SDS and reducing agent was added and

proteins were separated electrophoretically on 16% SDS-
polyacrylamide gels and the buffer system of Laemmli [13].
The gels were electro-blotted using poly(vinylidene difluo-
ride) membranes (Millipore, Bedford, MA, USA), the filters
were subsequently incubated with a polyclonal rabbit anti-
(human cystatin F) IgG (anti-cysF) [5] and visualized by
chemoluminescence (ECL Plus kit; Amersham, Bucking-
hamshire, UK).
Immunocytochemical staining
Approximately 80 000 cells were spun down to object glasses
and fixed in 3.7% (v/v) formaldehyde (Merck KGaA,
Darmstadt, Germany) in NaCl/P
i
. Unspecific binding sites
were blocked with 0.2% (w/v) bovine serum albumin. Anti-
cysF, diluted 1 : 10 000 in NaCl/P
i
for the absorption and
double staining experiments and 1 : 1000 for the regulation
experiments, was applied in the presence of 0.1% (w/v)
saponin. Following incubation with the primary antibody
thecellswherewashedandincubatedwithanOregonGreen
labelled goat anti-rabbit IgG secondary antibody (Molecular
Probes, Inc., Eugene, OR, USA) for 2 h. Finally, the cells
were washed and mounted in PVA-DABCO (2.5% w/v 1.4-
diazacyclo [2,2,2]octane, 13% w/v polyvinlyalcohol, 33%
w/v glycerol, and 0.13
M
Tris/HCl, pH 8).
To visualize the cells a Bio-Rad MRC1024UV confocal

laser-scanning microscope was used and the images taken
were processed using Adobe
PHOTOSHOP
(Adobe Systems,
Mountain View, CA, USA).
For double staining of cystatin F and ER the staining
was performed on cells cultured for 2 days. Cystatin F was
stained as described above and after the last washing step
20 lgÆmL
)1
Texas Red conjugated concanavalin A
(Molecular Probes) was applied and subsequently incubated
for 30 min at room temperature. The cells were then washed
and mounted as above.
Quantitative protein assays
Cell lysates and culture media were analysed by ELISA to
determine the concentrations of cystatin F [5] and cystatin C
[14,15]. The total protein content in cell lysates was
determined with the Coomassie Protein Assay Reagent
(Pierce, Rockford, IL, USA) according to the manufac-
turer’s Micro Method.
RNA extraction and Northern blot
RNA was extracted from approximately 2 · 10
7
cells
according to Chomczynski and Sacchi [16]. Twenty lgof
RNA was separated electrophoretically on 1% agarose gels
and transferred to Hybond-N nylon membranes (Amer-
sham). The filters were prehybridized in Clontech Express
Hybridization Solution (Clontech Laboratories, Palo Alto,

CA, USA) for 30 min at 65 °C. A [a-
32
P]dCTP labelled
cystatin F specific probe was added to 5 mL hybridization
solution to give a specific activity of 10
6
cpm/mL. Following
hybridization over night the filters were rinsed in 2 · NaCl/
Cit and washed in 2 · NaCl/Cit and 0.1% SDS for 45 min
at room temperature; 0.2 · NaCl/Cit and 0.5% SDS for
45 min at room temperature; 0.2 · NaCl/Cit and 0.5%
SDS for 30 min at 50 °C. After exposure the filters were
stripped and the procedure was repeated with cystatin C and
GAPDH specific probes.
The cystatin F specific probe used was a 542-bp PCR
fragment amplified from the cystatin F cDNA clone,
HCUDE60 (Table 1). The cystatin C specific probe was a
320-bp PCR fragment amplified from the full-length human
cystatin C cDNA clone, C6a [17] (Table 1) and the
GAPDH specific probe used was a human GAPDH cDNA
(Clonetech). Probes were labelled with [a-
32
P]dCTP
(Amersham) according to the user manual in the Multi-
prime DNA labelling system kit (Amersham).
Subcellular fractionation
U937 cells (10
7
) were pelleted and resuspended in 1.5 mL
homogenizing buffer (0.25

M
sucrose, 10 m
M
tricine/NaOH,
pH 7.4, 1 m
M
EDTA) and homogenized in a glass homog-
eniser. The homogenate was treated in a series of centrif-
ugation steps. After each centrifugation step the pellet was
redissolved in lysis buffer (0.2% (v/v) Triton-X100 in water)
and the supernatant was transferred to the next step. Each
lysed pellet formed one fraction. The last supernatant
formed the supernatant fraction. The centrifugation steps
were as follows: debris fraction: 10 min, 600 g;heavy
fraction: 10 min, 2000 g; light fraction: 10 min, 15 000 g.
Cystatin C, cystatin F and total protein were measured as
above. The activity of b-hexosaminidase was measured
according to Hultberg et al.[18].
The heavy and the light fractions contained elevated
levels of cystatin F and were therefore selected for an
ultracentrifugation experiment. Cells (1.4 · 10
8
) were pel-
leted and treated as above with the exception that the heavy
and light fraction pellets were not lysed in lysis buffer but
resolved in homogenizing buffer. Optiprep (Axis-Shield
PoC AS, Oslo, Norway) was diluted to 20% in Optiprep
dilutent (8% sucrose, 1 m
M
EDTA, 20 m

M
Tricine/NaOH,
pH 7.8). Five mL Optiprep solution was overlaid with
400 lL sample and centrifuged at 180 000 g,3h,4°Cina
vertical angle rotor (Vti80, Beckman Instruments, Inc., Palo
Alto, CA, USA) in a Beckman L8-M80 ultracentrifuge
(Beckman Instruments). Finally the heavy fraction was
divided in 9, and the light fraction in 11, subfractions by
puncturing the plastic centrifuge tube with a syringe
connected to a peristaltic pump and a fraction collector.
Materials
Oligonucleotides were purchased from DNA Technology
A/S (Aarhus, Denmark). U937 cells were a kind gift of
U. Gullberg, Department of Haematology, Lund University,
Sweden. Recombinant human cystatin C was expressed in
an Escherichia coli system and purified as described [19].
5504 C M. Nathanson et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Recombinant human cystatin F was produced in a bacu-
lovirus expression system [5]. If nothing else stated chemicals
were from Sigma (St Louis, MO, USA).
RESULTS
Native cystatin F in human U937 cells
Earlier reported characteristics of cystatin F have been
based on studies of recombinant protein [5,6]. To extend our
knowledge about the inhibitor, we wanted to study the
properties and distribution of the native protein from a
human cell source. The promyelocytic cell line, U937, was
chosen as starting material because it showed the highest
secretion of cystatin F among 10 cell lines previously
analysed [5]. It was not possible to detect cystatin F directly

in culture media or cell lysates by immunoblotting. How-
ever, by absorption on Sepharose-bound Cm-papain fol-
lowed by Western blot analysis, the cell lysate-derived
inhibitor could readily be detected. Cystatin F present in
U937 cells shows a four-band pattern at SDS/PAGE
(Fig. 1). At comparison with insect cell produced recom-
binant cystatin F (Fig. 1, lanes 1 and 2), which appears as
one mono- (band B) and one di-glycosylated (band A)
species at approx. equal proportions [5], it was evident that
native human cystatin F exists in these two glycosylated
forms, but also as one most likely unglycosylated species
(lane 3, band C), with mobility exactly as recombinant
cystatin F after deglycosylation with PNGase F [5]. The
antiserum also recognized a weaker additional band (lane 3,
band a)withM
r
approximately 3200 Da higher than the
diglycosylated cystatin F species. The nature of this band is
presently unknown and is discussed below.
In order to study the production of cystatin F compared
to the ubiquitous inhibitor, cystatin C, we measured the
cystatin contents in cell lysates and culture media of cells
grown for 2 and 4 days with cystatin F and cystatin C
specific ELISAs. Generally, the total cystatin F amounts
were approximately 10 times lower than those of cystatin C
(Fig. 2A,B) but the proportional distribution of localization
of the two proteins differ. The genes for both cystatins
encode a signal peptide and the secreted amounts of the
proteins are higher than the intracellular ones. However,
while cystatin C displays 24–30 times higher secreted levels

(Fig. 2B), cystatin F only shows approximately three times
higher extracellular protein levels (Fig. 2A). Thus, a signi-
ficant part of the cystatin F produced by U937 cells is
retained intracellularly or is taken up by the cells immedi-
ately following secretion. Accordingly, the relatively low
amount of cystatin F produced by U937 cells reported
earlier [5] is an underestimate of the real production of
cystatin F in U937 cells.
The high portion of intracellular cystatin F led us to
fractionate cells to determine the localization of the
inhibitor. Initially cells were fractionated in a debris fraction
(containing nuclei and cell debris), a heavy and a light
fraction containing mitochondria and various cell organ-
elles, and a supernatant containing the smallest cellular
vesicles. Cystatin F, cystatin C and protein contents in the
fractions were measured. The highest cystatin F level was
seen in the debris fraction (Fig. 3A) but the ratio between
cystatin F and protein (Fig. 3B) revealed that cystatin F
was enriched in both the heavy and the light fractions
compared to the total (with a factor of 2) and supernatant
(with a factor of 20) fractions. Cystatin C levels were below
the detection limit in all fractions.
α
C
A
B
3
1
2
Fig. 1. Western blotting of native human cystatin F. A cystatin F spe-

cific polyclonal antiserum was used for immunoblotting, after cysta-
tin F in a U937 cell extract had been partially purified by affinity
chromatography on immobilized Cm-papain. Lanes 1 and 2 contain
30 and 10 ng insect cell-derived recombinant cystatin F, respectively.
The two bands seen represent cystatin F with one (B) and two (A)
carbohydrate side chains [5]. Lane 3 shows cystatin F produced by
U937 cells. The mono- and diglycosylated forms (A and B) can be seen
but also a nonglycosylated form (C). The band marked a is discussed
in the main text.
0
1
2
3
4
5
6
7
8
2
4
B
0
10
20
30
40
50
60
70
80

24
Incubation time (days)
Cystatin F (ng/culture)
A
Cystatin C (ng/culture)
Fig. 2. Cystatin F and C produced by U937 cells. (A) Cystatin F was
quantified in cell extracts and media by ELISA. In the representative
experiment shown (one out of three with the same trend of results),
intracellular levels (black bars) of cystatin F were approximately 3-fold
lower than the extracellular levels (grey bars) both after 2 and 4 days of
incubation. (B) Similarly measured by a specific ELISA, the cystatin C
levels were 24- to 30-fold higher extracellularly than intracellularly.
The total cystatin C amount was  10-fold higher than the total
cystatin F amount. Measurements were performed in duplicate and
results are presented as mean values with error bars denoting SD.
Ó FEBS 2002 Cystatin F in U937 cells (Eur. J. Biochem. 269) 5505
The heavy and the light cellular fractions were subfract-
ionated by ultracentrifugation to further localize cystatin F.
We also measured the activity of the mainly lysosomal
enzyme, b-hexosaminidase, to assess if cystatin F is located
in lysosome-like organelles. In both the heavy (Fig. 4A,
subfraction 8) and the light (Fig. 4B, subfraction 9)
fractions cystatin F coeluted with the peak of b-hexos-
aminidase activity.
To further investigate the intracellular localization of
cystatin F we stained cells with the polyclonal antiserum
and visualized the immunoreaction with a FITC labelled
secondary antibody in a confocal microscope. Cystatin F
showed a vesicular staining pattern (Fig. 5A), whereas after
preincubation with recombinant cystatin F the signal was

absorbed completely (Fig. 5B). The latter demonstrates that
cystatin F is specifically stained by the antiserum at
immunohistochemistry. The vesicular staining of cystatin F
agrees with the results from the fractionation experiments
and taken together, this strongly indicates that the major
portion of intracellular cystatin F is present in smaller
vesicles.
In mouse fibroblasts overproducing human cystatin C,
intracellular cystatin C routed for direct secretion is
detectable and it shows costaining with markers for the
ER [20]. This prompted us to examine a possible costaining
of intracellular cystatin F (Fig. 6A) with Texas Red labelled
concanavalin A as an ER-specific marker (Fig. 6B). A
colocalization of cystatin F with the ER marker would give
a yellow colour at overlays, but no such costaining could
be observed (Fig. 6C). Thus, the intracellular cystatin F
detected by the antiserum is likely not protein detected on
the direct transport route to secretion, but rather protein
temporarily stored in granules or found in vesicles following
uptake from the medium.
Fig. 3. Cystatin F concentrations in main subcellular fractions of U937.
Cells were mechanically homogenized and fractioned in four fractions
by stepwise centrifugation, as detailed in the Materials and methods
section. Cystatin F concentrations were measured by ELISA and
protein concentrations by a Coomassie binding assay. (A) The debris
fraction showed the highest cystatin F concentration and the super-
natant the lowest. (B) The cystatin F/protein ratio is > twofold higher
in the heavy and light fractions than in the debris fraction or in total
U937 cellular extract and > 20-fold higher than in the supernatant
fraction. Measurements were performed in duplicate and results are

presentedasmeanvalueswitherrorbarsdenotingSD.
Fig. 4. Cystatin F in subfractions of the heavy and light subcellular U937
fractions. Cystatin F concentrations were measured with a specific
ELISA, total protein concentration was measured with Coomassie
binding assay and b-hexosaminidase activity was measured with a
colourimetric assay, as detailed in the Materials and methods section.
(A) Subfractions of the heavy subcellular fraction obtained by ultra-
centrifugation in Optiprep. The total protein concentration (closed
triangles and short dashed line, scale on right y-axis) was elevated in
subfractions 3, 7, 8 and 9 with the highest concentration in fraction 8.
The concentration of cystatin F (closed rectangles and line, scale on
left y-axis) and b-hexosaminidase activity (open circles with long
dashed line, scale on left y-axis) were elevated in subfractions 7, 8 and 9
with the highest concentrations in fraction 8. (B) Subfractions of the
light subcellular fraction obtained by ultracentrifugation. Cystatin F
and b-hexosaminidase were elevated in fraction 9.
5506 C M. Nathanson et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Regulation of cystatin F expression
To shed light on the regulation of the cystatin F gene, we
examined the sequence of the human gene, CST7.Atan
initial search of the High Throughput Genome database at
Karolinska Institutet (Stockholm, Sweden) by FASTA with
the cDNA for cystatin F as probe, a 106 000 bp contig
from chromosome 20 was found. Aligning putative exons
from the cDNA showed that the contig contained the whole
gene plus a long flanking stretch upstream of the cDNA
start position. The human CST7 gene consists of four exons
[21] as the mouse counterpart [6]. The three introns, 7711 bp
in the found contig (7872 bp in Morita et al. [21]), 1468 bp
and 590 bp, respectively, are localized in the same pattern as

in the mouse gene. The second and third introns are
localized in exactly the same positions, taking amino-acid
homology match in account, as those of the other six known
human type 2 cystatins, strongly indicating that the genes
originate from a common ancestor. To verify the exon/
intron junctions and coding segments, primers were con-
structed  100 bp 5¢ and 3¢ of the exons in the flanking
intron segments. Direct sequencing of PCR products
derived from genomic DNA of a normal Caucasian
individual was performed and the sequences were compared
with the contig. The obtained sequences showed no
differences to the database contig sequence. The exon/
intron junctions were all in agreement with the GT/AG
consensus sequence.
The 5¢ flanking promoter region of the human cystatin F
gene does not have an unusually high GC content (over
nucleotides )1to)1000,  50%; nucleotides )1to)3000,
 45%) and comparing CpG to GpC gives a ratio of  1/5.
By contrast, a ratio of  1, indicating a continuously
expressed gene [22], is found in the promoter region of the
human cystatin C gene [23]. The first 600 bp of the cystatin
C(CST3)andF(CST7) promoter regions (calculated from
the starting ATG) differs in some obvious manners (Fig. 7).
Cap signals are found in the vicinity of the start ATG
codons but further upstream CST7 contains a second one
138 bp 5¢- to an alternative start codon [6]. The a-band in
Fig. 1 possibly derives from a transcript initiated by this
second initiation site. The promoter of the ubiquitously
expressed cystatin C gene, CST3, contains eight Sp1 binding
sites while CST7 only has one (at )314). Two GC-box

elements can be found in CST3 while CST7 has one at
)312. At position )500 a CAATT/enhancer binding protein
a (C/EBPa) site is found in CST7, with no counterpart in
CST3.
Expression of cystatin F in differentiated U937 cells
Radomska et al. [24] reported that TPA differentiates U937
cells in a monocytic direction and gives a down-regulation of
C/EBPa. ATRA gives in contrast a granulocytic differen-
tiation of U937 cells and a subsequent up-regulation of C/
EBPa, so we decided to test if the expression of cystatin F is
regulated through differentiation.
Stimulation by TPA. U937 cells were grown in the
presence of 0.13 l
M
TPA for 2 days. A 2.5-fold down-
regulation of cystatin F both in cell lysates and in culture
media could be seen (Fig. 8A). Cystatin C was slightly
A
B
Fig. 5. Immunocytochemical staining of cysta-
tin F. U937 cells were spun down and fixed on
object glasses. (A) The cells were incubated
with a specific polyclonal antiserum against
recombinant cystatin F and subsequently
hybridized with a secondary FITC labelled
antibody against rabbit IgG. A vesicular
staining pattern can be seen. (B) After prein-
cubation of the primary antibody with an
excess of recombinant cystatin F [5] a total
absorption of the signal was apparent.

Fig. 6. Double staining of cystatin F and ER.
(A) Cystatin F was stained with a human
cystatin F specific polyclonal antiserum and a
FITC labelled secondary antibody (green sig-
nal). (B) Following the cystatin F staining, the
ER was stained with Texas Red conjugated
concanavalin A (red signal). (C) Overlay of
images in (A) and (B), where a yellow colour
would indicate colocalization. Arrows indicate
distinct ÔholesÕ in the ER staining where cyst-
atin F stains.
Ó FEBS 2002 Cystatin F in U937 cells (Eur. J. Biochem. 269) 5507
down-regulated by TPA, with a factor of  1.6 (Fig. 8B).
To investigate whether the regulation was transcriptional or
translational we extracted RNA from the cells. The results
from Northern blotting of the RNA (Fig. 8C) demonstrate
a strong down-regulation of cystatin F expression at the
mRNA level while cystatin C and control GAPDH mRNA
levels were virtually unchanged. The down-regulation of
cystatin F was also visualized through immunostaining of
cells grown in presence or absence of TPA. A less
pronounced staining could be seen in the TPA stimulated
cells than in the unstimulated (Fig. 8D). Thus, TPA
markedly down-regulates the cystatin F gene expression.
This down-regulation would fit with C/EBPa as one
regulator of the cystatin F expression in U937 cells.
Stimulation by ATRA. The results were similar but even
more pronounced when U937 cells were incubated with
ATRA (Fig. 9). The intracellular cystatin F levels measured
by ELISA were 18-fold lower after ATRA treatment and

9-fold lower in cell culture media (Fig. 9A). Furthermore
Fig. 8. Regulation of cystatin expression in
TPA stimulated U937 cells. Cells were incu-
bated for 2 days in the presence of 0.13 l
M
TPA. The results from one representative
experiment out of three performed are shown.
(A) Cystatin F measurement, showing a
2.5-fold down-regulation upon TPA-stimula-
ted differentiation (unstimulated, grey bars;
stimulated, black bars). (B) Cystatin C meas-
urement, demonstrating a 1.6-fold down-
regulation (unstimulated, grey bars; stimula-
ted, black bars). (C) Northern blot of RNA
derived from unstimulated (lane 1) and sti-
mulated (lane2) U937 cells, using specific
cDNA probes for cystatin F (top), cystatin C
(middle) and GAPDH (bottom). (D) Immu-
nostaining of cystatin F in unstimulated (left)
and TPA-stimulated (right) cells.
Fig. 7. Structure of the cystatin F gene and promoter. The structures of the human cystatin F (CST7)andcystatinC(CST3) genes are illustrated
schematically at the top. In the upstream region of the CST7 gene, the ATG (arrowhead) at position 0 is the most probable initiation codon for
translation. Another ATG and possible initiation site is found at )66. Both possible mRNAs have corresponding cap signals (diamond). Only one
Sp1 element (pyramid) can be found in the )600 to )1 segment whereas in the CST3 promoter 7 elements are found. Among other possible
transcription factor sites found at a motif search, a unique C/EBPa element in the cystatin F promoter (star) could be of relevance for expression in
U937 and native haematopoietic cells.
5508 C M. Nathanson et al. (Eur. J. Biochem. 269) Ó FEBS 2002
were mRNA signals almost completely lost in the treated
cells (Fig. 9C). When comparing the immunostained cells
before and after treatment the cystatin F signals were more

or less extinct after stimulation (Fig. 9D). Such strong
evidence of down-regulation could not be seen for cystatin C.
The already low intracellular levels did not change and in
the culture media only a 1.3-fold decrease was seen
(Fig. 9B). Furthermore was the mRNA expression
unchanged after ATRA treatment (Fig. 9C) and, thus, no
indication of regulation by differentiation was at hand for
cystatin C. In contrast to the TPA experiment, the strong
down-regulation of cystatin F expression by ATRA con-
tradictthatC/EBPa is a major regulator of cystatin F
expression in U937 cells.
DISCUSSION
Cystatin F is a potent inhibitor of several important cysteine
peptidases [5,10], but the physiological role of the inhibitor
is still unknown. The immune cell restricted expression of
cystatin F indicates that the inhibitor is involved in regu-
lation of proteolytic events specific to such cells. Regulation
of antigen presentation could be one such event. This agrees
with results from cDNA libraries [5,6], demonstrating that
dendritic cell subpopulations of haematopoietic cells are
among the most abundant cystatin F producers. The in vitro
properties of recombinant cystatin F show that it is a potent
inhibitor of cathepsin L [5,6], as well as mammalian
legumain [10]. These enzymes are known to be involved in
invariant chain [8] and antigen [11] processing, respectively.
Our present results showing a distinct intracellular localiza-
tion of cystatin F are interesting in this context. Although a
detailed study of the cystatin F transport route was beyond
the scope of the present investigation, the fact that
cystatin F is present in significant quantities intracellularly

supports a possible intracellular function of cystatin F.
Furthermore, the apparent distribution to smaller vesicles/
granules in the U937 cells indicates that cystatin F is at least
partially localized to organelles resembling the fused lyso-
somes/endosomes where antigen and invariant chain pro-
cessing take place in antigen presenting cells [9]. Thus, as
shown by our present comparisons with the distribution and
regulation of cystatin C, cystatin F appears to be a better
candidate for regulation of antigen presentation than
cystatin C. Although control of critical cathepsin S activity
in mouse dendritic cells [9] has been contributed to cystatin
C it is a distinct possibility that cystatin F also is involved in
such regulation, possibly in co-operation with cystatin C. It
should, however, be stressed that this suggested function of
cystatin F is hypothetical. Although most cystatins inhi-
biting cathepsin L also inhibit cathepsin S, an inhibitory
activity of cystatin F against cathepsin S has not yet been
reported.
The expression of the cystatin F (CST7)andC(CST3)
genes differ considerably, both with respect to the overall
levels and with respect to tissue/cell-specific expression
pattern. CST3 expression is generally higher than that of
CST7, as stressed by our present results for U937 cells.
Moreover, CST3 is ubiquitously expressed [23], whereas
cystatin F transcripts are found exclusively in immune cells
[5,6]. Possible explanations to these differing expression
patterns can be found in the comparison of the CST3 and
Fig. 9. Regulation of cystatin expression in
ATRA stimulated U937 cells. Cells were incu-
bated for 4 days in the presence of 1 l

M
ATRA. The results from one representative
experiment out of three performed are shown.
(A) Cystatin F showed an 18-fold down-
regulation in cell extract and a 9-fold down-
regulation in medium (unstimulated, grey
bars; stimulated, black bars). (B) The cystatin
C concentration was barely detectable but
unchanged in cell lysate; a slight down-regu-
lation could be seen in culture medium. (C)
Northern blot of RNA derived from unstim-
ulated (lane 1) and stimulated (lane2) U937
cells, using specific cDNA probes for cysta-
tin F (top), cystatin C (middle) and GAPDH
(bottom). (D) Immunostaining of cystatin F
in unstimulated (left) and ATRA-stimulated
(right) cells.
Ó FEBS 2002 Cystatin F in U937 cells (Eur. J. Biochem. 269) 5509
CST7 promoters presented here (Fig. 7). The CST3 pro-
moter contains elements typical for a house-keeping gene
(multiple Sp1 and GC-box elements), has an extremely high
GC content and a CpG to GpC ratio close to unity, which
agrees with its relatively high and unspecific expression
pattern. The CST7 promoter, in contrast, lacks most of
these CST3 promoter features. This agrees with the
relatively low level of cystatin F expression in those few
cells where it is expressed at all, such as U937. The
expression of CST7 is readily regulated in contrast to that of
CST3, as demonstrated in the present study. This difference
must clearly be due to other differences in the two

promoters. The unique C/EBPa binding site found in
CST7 may at least partially explain the restricted and
regulated cystatin F expression.
The reasons why cystatin F in U937 cells is transported in
a different way than the major type 2 cystatin of mammalian
tissues, cystatin C, are unknown and certainly merit further
studies. The cystatin F and C genes both encode preproteins
with typical signal peptides, and the gene products are
readily secreted in mammalian [20] and insect [5] cells,
respectively. Cystatin F is an unusual type 2 cystatin in that
it is a glycoprotein [5,6], and it is known that many proteins
found in intracellular vesicular compartments are targeted
through their carbohydrate side chains [25]. The comobility
of the cystatin F bands from U937 cells with those of
recombinant cystatin F in the Western blot experiment of
the present study, strongly suggests that the native human
inhibitor is glycosylated as predicted. Native cystatin F
appears as three major bands when detected in U937 cell
homogenates, which correlate exactly with the mobilities of
di-, mono- and unglycosylated forms of recombinant
cystatin F previously characterized by enzymatic removal
of N-linked carbohydrate and carbohydrate composition
analysis [5]. It is also noteworthy that all three forms were
partially purified by their affinity to Cm-papain, strongly
indicating that the carbohydrate side chains do not affect the
enzyme-binding properties of cystatin F and should, thus,
not affect the inhibition of papain-like family C1 peptidases.
The antiserum we used for detection of intracellular
cystatin F detected an additional band at Western blotting
(Fig. 1). The nature of this band is unknown, but given the

high specificity of the antiserum [5] and the absorption step
on Cm-papain used, it is most likely an additional form of
cystatin F. The band possibly represents a gene product of
the alternative ATG start codon (Fig. 7). If transcription
starts at the alternative start site then the protein would gain
41 in-frame N-terminal amino acids with no resemblance to
a signal peptide but rather to a transmembrane segment [6].
This theoretical cystatin F variant has a calculated M
r
of
23 000 Da. A calculation taken from the Western blot
(Fig. 1) gives a M
r
of 22 500 Da for the additional band
detected. The possibility that the alternative ATG start
codon is partially used in human cells is not contradicted by
previous results, as previous recombinant cystatin F studies
used constructs lacking the upstream ATG codon [5,6].
An alternative suggestion for a physiological function of
cystatin F can be speculated from our present results. The
expression of cystatin F in unstimulated U937 cells, but not
in more differentiated cells of the same lineage resembles the
expression pattern of proteins known to be substitutents of
secretory granules of granulocytes, such as cathepsin G [26].
Perhaps cystatin F is involved in inflammatory reactions
promoting granulocyte migration and release of granule
content to combat exogenous threats. Cystatin F could, e.g.
inactivate family C1 target enzymes from bacteria or
protozoan parasites such as the virulence factor of Chagas’
disease, cruzipain, in Trypanosoma cruzi infections. Cystatin

F inhibits cruzipain as strongly as it inhibits cathepsin L
in vitro (M. Abrahamson and J. Scharfstein, unpublished
results). Clearly, a more detailed study of the subcellular
localization and transport of cystatin F in model U937 cells
would be valuable in understanding the role of cystatin F in
haematopoietic cells. Further work to localize subfractions
of native haematopoetic cells expressing cystatin F should
provide additional clues to the function of cystatin F in
health and disease.
Since acceptance of this manuscript for publication, we
have become aware of two recent publications that contain
results of relevance for the present work. In a study aiming
at characterization of the proteome of lysosomes in U937
cells, cystatin F was found as one of 15 proteins binding to
immobilised mannose-6-phosphate receptor [27], indicating
that either cystatin F contains mannose-6-phosphate or is
co-localized with, and binding to, a mannose-6-phosphate
containing protein. This favours our suggestion that intra-
cellular cystatin F is localized in lysosomes, rather than
being present in secretory granules. In a paper describing
changes in general gene expression in mature activated
dendritic cells compared to immature dendritic cells, the
cystatin F gene was among the top 50 of those up-regulated
upon maturation of the cells [28]. This supports our
conclusion that cystatin F is readily regulated and present
in cells of relevance for antigen presentation and, thus, may
be a candidate for control of the cysteine peptidase activity
essential for this process.
ACKNOWLEDGEMENTS
We wish to thank Drs Klaudia Brix (Bonn) and Arne Egesten (Malmo

¨
)
for helpful technical discussions. This study was supported by grants
from the Swedish Medical Research Council (no. 09915), the Medical
faculty at Lund University, the A. O
¨
sterlund Foundation and the
Crafoord Foundation.
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