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

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

) [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

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

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

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,

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

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

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

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

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

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

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

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

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

¨
)
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.
REFERENCES
1. Rawlings, N.D. & Barrett, A.J. (1990) Evolution of proteins of the
cystatin superfamily. J. Mol. Evol. 30, 60–71.
2. Abrahamson, M. (1994) Cystatins. Methods Enzymol. 244, 685–
700.
3. Ni, J., Abrahamson, M., Zhang, M., Fernandez, M.A., Grubb, A.
&Su,J.,YuG.L.,Li,Y.,Parmelee,D.,Xing,L.,Coleman,T.A.,
Gentz, S., Thotakura, R., Nguyen, N., Hesselberg, M. & Gentz,
R. (1997) Cystatin E is a novel human cysteine proteinase inhibitor
with structural resemblance to family 2 cystatins. J. Biol. Chem.
272, 10853–10858.
4. Sotiropoulou, G., Anisowicz, A. & Sager, R. (1997) Identification,
cloning, and characterization of cystatin M, a novel cysteine
proteinase inhibitor, down-regulated in breast cancer. J. Biol.
Chem. 272, 903–910.
5. Ni, J., Fernandez, M.A., Danielsson, L., Chillakuru, R.A., Zhang,
J., Grubb, A., Su, J., Gentz, R. & Abrahamson, M. (1998)
Cystatin F is a glycosylated human low molecular weight cysteine
proteinase inhibitor. J. Biol. Chem. 273, 24797–24804.
6. Halfon,S.,Ford,J.,Foster,J.,Dowling,L.,Lucian,L.,Sterling,
M., Xu, Y., Weiss, M., Ikeda, M., Liggett, D., Helms, A., Caux,
5510 C M. Nathanson et al. (Eur. J. Biochem. 269) Ó FEBS 2002

against the major extracellular human cysteine proteinase
inhibitors cystatin C and kininogen. Scand. J. Clin. Laboratory
Invest 48, 573–582.
15. Bjarnadottir, M., Grubb, A. & Olafsson, I. (1995) Promoter-
mediated, dexamethasone-induced increase in cystatin C produc-
tion by HeLa cells. Scand. J. Clin. Laboratory Invest. 55, 617–623.
16. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162, 156–159.
17. Abrahamson, M., Grubb, A., Olafsson, I. & Lundwall, A. (1987)
Molecular cloning and sequence analysis of cDNA coding for the
precursor of the human cysteine proteinase inhibitor cystatin C.
FEBS Lett. 216, 229–233.
18. Hultberg, B., Lindsten, J. & Sjoblad, S. (1976) Molecular forms
and activities of glycosidases in cultures of amniotic- fluid cells.
Biochem. J. 155, 599–605.
19. Abrahamson, M., Dalboge, H., Olafsson, I., Carlsen, S. & Grubb,
A. (1988) Efficient production of native, biologically active human
cystatin C by Escherichia coli. FEBS Lett. 236, 14–18.
20. Bjarnadottir, M., Wulff, B.S., Sameni, M., Sloane, B.F., Keppler,
D., Grubb, A. & Abrahamson, M. (1998) Intracellular accumu-
lation of the amyloidogenic L68Q variant of human cystatin C in
NIH/3T3 cells. Mol. Pathol. 51, 317–326.
21. Morita, M., Hara, Y., Tamai, Y., Arakawa, H. & Nishimura, S.
(2000) Genomic construct and mapping of the gene for CMAP
(leukocystatin/cystatin F, CST7) and identification of a proximal
novel gene, BSCv (C20orf3). Genomics 67, 87–91.
22. Bird, A.P. (1986) CpG-rich islands and the function of DNA
methylation. Nature 321, 209–213.
23. Abrahamson, M., Olafsson, I., Palsdottir, A., Ulvsback, M.,