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 Fin human U937 cells Carl-Michael Nathanson1, Johan Wasse´lius2, Hanna Wallin1and Magnus Abrahamson1 1 Department of Clini

Regulated expression and intracellular localization of cystatin F

in human U937 cells

Carl-Michael Nathanson1, Johan Wasse´lius2, Hanna Wallin1and Magnus Abrahamson1

1

Department of Clinical Chemistry, Institute of Laboratory Medicine, and2Department 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 (Ki 0.1–1 nM),but does not inhibit cathepsin B (Ki> 1000 nM) [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: Magnus.Abrahamson@klinkem.lu.se

Abbreviations: ATRA,all-trans retinoic acid; TPA,tetradecanoyl

phorbol acetate; GAPDH,glyceraldehyde-3-phosphate

dehydrogenase.

Note: web page available at http://www.klinkem.lu.se/E/abrahamson

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)

(Ki for the pig enzyme,10 nM) [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 protepeptid-ases 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 cells and 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

M A T E R I A L S A N D M E T H O D S

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 aFASTAsearch 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 )1 to )600 gene segments were analysed with the MOTIF search engine (the vertebrate database), Bioinformatics Centre,Institute for Chemical Research, Kyoto University,Japan (http://www.motif.genome.ad.jp/) 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)1streptomycin; Life Technologies)

at 37C and an atmosphere with 5% CO2,in 75 cm2 culture flasks (Costar,Cambridge,MA,USA) For differ-entiation experiments,cells (1· 106mL)1) were seeded and cultured in the presence of 1 lMATRA for 4 days or in the presence of 0.13 lMTPA for 2 days After incubation for 4 and 2 days,respectively,the cells were set to 106cellsÆmL)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/Picontaining 1· inhibitor cocktail (final concentra-tions of 5 mMbenzamidinium hydrochloride,15 mMNaN3 and 10 mMEDTA) per 6· 107cells,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

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

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 4C 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/Pi Unspecific binding sites

were blocked with 0.2% (w/v) bovine serum albumin

Anti-cysF,diluted 1 : 10 000 in NaCl/Pifor 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

the cells where washed and incubated with an Oregon Green

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 0.13MTris/HCl,pH 8)

To visualize the cells a Bio-Rad MRC1024UV confocal

laser-scanning microscope was used and the images taken

were processed using AdobePHOTOSHOP(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· 107 cells

according to Chomczynski and Sacchi [16] Twenty lg of

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 65C A [a-32P]dCTP labelled cystatin F specific probe was added to 5 mL hybridization solution to give a specific activity of 106cpm/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 50C 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-32P]dCTP (Amersham) according to the user manual in the Multi-prime DNA labelling system kit (Amersham)

Subcellular fractionation U937 cells (107) were pelleted and resuspended in 1.5 mL homogenizing buffer (0.25Msucrose,10 mMtricine/NaOH,

pH 7.4,1 mMEDTA) and homogenized in a glass homog-eniser The homogenate was treated in a series of centrif-ugation steps After each centrifcentrif-ugation 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· 108) 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 mMEDTA,20 mMTricine/NaOH,

pH 7.8) Five mL Optiprep solution was overlaid with

400 lL sample and centrifuged at 180 000 g, 3 h, 4C in a 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]

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)

R E S U L T S

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) with Mrapproximately 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

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

B

0 10 20 30 40 50 60 70 80

Incubation time (days)

A

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.

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

presented as mean values with error bars denoting SD.

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.

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 At an

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)1 to )1000,  50%; nucleotides )1 to )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) and F (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 lM 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

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 holes in the ER staining where

cyst-atin F stains.

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 (unstimulaTPA-stimula-ted,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) and cystatin C (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.

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-tradict that C/EBPa is a major regulator of cystatin F

expression in U937 cells

D I S C U S S I O N

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 Reguregu-lation

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) and C (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

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 stimunstim-ulated (lane2) U937

cells,using specific cDNA probes for

cysta-tin F (top),cystacysta-tin C (middle) and GAPDH

(bottom) (D) Immunostaining of cystatin F

in unstimulated (left) and ATRA-stimulated

(right) cells.

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 Mrof

23 000 Da A calculation taken from the Western blot

(Fig 1) gives a Mrof 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

A C K N O W L E D G E M E N T S

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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