Elsevier Editorial System(tm) for Biochemical and Biophysical Research Communications Manuscript Draft Manuscript Number: Title: Coupled expression of Dipeptidyl peptidase-IV and Fibroblast activation protein-α in transformed astrocytic cells Article Type: Regular Article Keywords: DASH; dipeptidyl peptidase-IV; fibroblast activation protein-α; glioblastoma. Corresponding Author: Mr Aleksi Sedo, Corresponding Author's Institution: 1st Faculty of Medicine of the Charles University in Prague First Author: Eva Balaziova, MD Order of Authors: Eva Balaziova, MD; Petr Busek, MD, PhD; Jarmila Stremenova, PhD; Lucie Sromova, MS; Evzen Krepela, MD, PhD; Libuse Lizcova, MS; Aleksi Sedo

JOINT LABORATORY OF CANCER CELL BIOLOGY OF THE 1ST

FACULTY OF MEDICINE,

CHARLES UNIVERSITY AND THE INSTITUTE OF PHYSIOLOGY, ACADEMY OF SCIENCES

OF THE CZECH REPUBLIC

Professor Aleksi Šedo, MD, PhD, DSc., Institute of Biochemistry and Experimental Oncology,

1st Faculty of Medicine, Charles University, U Nemocnice 5, Prague 2, 128 53, Czech Republic Tel/Fax.: -420-2-2496 5826, Mobile:-420-728 748 692, E-mail: Aleksi@cesnet.cz, www.lf1.cuni.cz/lbnb

Cover Letter

Dear Sir/Madam 12.8.2010

We would like to submit our MS “Coupled expression of Dipeptidyl peptidase-IV and Fibroblast

activation protein-α in transformed astrocytic cells” for consideration to be published in Biochemical and

Biophysical Research Communications as a Short Communication.

The MS has not been published and is not submitted elsewhere. There is neither link with commercial sources

nor other financial interest or conflict of interests associated with the MS.

¨

All authors agree with submission of the MS and its eventual publication in BBRC.

Sincerely yours,

Prof. Aleksi Sedo, MD, PhD

For authors

Cover Letter

Reasearch Highlights

Correlation of DPP-IV and FAP expression in glioblastoma primary cultures

Coupled dynamics of DPP-IV and FAP expression in astrocytoma cell lines

DPP-IV and FAP probably coregulated on the transcriptional level

*Research Highlights

Coupled expression of Dipeptidyl peptidase-IV and Fibroblast activation protein-α in

transformed astrocytic cells

Eva Balaziova 1, Petr Busek

1, Jarmila Stremenova

1, Lucie Sromova

1, Evzen Krepela

1,3,

Libuse Lizcova 2, Aleksi Sedo

*,1

1Institute of Biochemistry and Experimental Oncology of the 1st Faculty of Medicine,

Charles University in Prague, U Nemocnice 5, Prague 2, 128 53, Czech Republic,

2Institute of Clinical Biochemistry and Laboratory Diagnostics of the 1st Faculty of Medicine,

Charles University in Prague and General University Hospital in Prague, U Nemocnice 2,

Prague 2, 128 01, Czech Republic

3Laboratories of Molecular and Cell Biology, Department of Pneumology and Thoracic

Surgery, University Hospital Bulovka, Budinova 2, Prague 8, 180 81, Czech Republic

eva.balaziova@lf1.cuni.cz (E. Balaziova), busekpetr@seznam.cz (P. Busek),

jarmila.stremenova@lf1.cuni.cz (J. Stremenova), lsrom@lf1.cuni.cz (L. Sromova),

libuse.lizcova@vfn.cz (L. Lizcova), enkre@seznam.cz (E. Krepela), aleksi@cesnet.cz (A.

Sedo)

* Corresponding author, phone: +420 224 96 5826, Fax.: +420 224 965 742,

aleksi@cesnet.cz

*ManuscriptClick here to view linked References

Abstract

Dipeptidyl peptidase-IV (DPP-IV) and fibroblast activation protein (FAP) are speculated

to participate in the regulation of multiple biological processes, due to their unique enzymatic

activity as well as by non-hydrolytic molecular interactions. Currently, the role of DPP-IV

and FAP in the development and progression of various types of tumours, including

glioblastoma, is intensively studied and their functional crosstalk is hypothesized. Here we

describe the correlative expression of DPP-IV and FAP mRNA in primary cell cultures

derived from human glioblastoma and associated expression dynamics of both molecules in

astrocytoma cell lines depending on culture conditions. Uncoupled expression of DPP-IV

transgene and the endogenous FAP argues for the co-regulation of DPP-IV and FAP genes

expression rather on the transcriptional than posttranscriptional level. Understanding of the

expressional and functional coordination of DPP-IV and FAP may help clarify the

mechanisms of biological roles of both molecules in transformed astrocytic cells.

Key words: DASH; dipeptidyl peptidase-IV; fibroblast activation protein-α; glioblastoma.

Abbreviations: DPP: dipeptidyl peptidase; FAP: fibroblast activation protein; DMEM:

Dulbecco’s modified Eagle’s medium; FCS: foetal calf serum; DASH: Dipeptidyl peptidase-

IV activity and/or structure homologues

1. Introduction

The plasma membrane-bound serine protease dipeptidyl peptidase-IV (DPP-IV; EC

3.4.14.5) was discovered by Hopsu-Havu [1] on the basis of its unique substrate specificity as

a protease cleaving X-Pro dipeptides from the amino-terminus of peptides and proteins.

However, a number of molecules constituting a group of “Dipeptidyl peptidase-IV Activity

and/or Structure Homologues” (DASH) was discovered subsequently. In addition to the

canonical DPP-IV, this group comprises another dominantly plasma membrane-bound

protease, fibroblast activation protein-/seprase (EC 3.4.21.-; FAP), and the intracellularly

localized DPP8, DPP9, quiescent cell proline dipeptidase (QPP, formerly known as DPP-II or

DPP7), thymus-specific serine protease and DPP IV-β [2].

Due to its capacity to process and thus functionally regulate a number of biologically

active peptides, involved in the processes of cell growth, migration, invasion and

neovascularization, DPP-IV is believed to be a significant player in cancer pathogenesis [3].

FAP, in addition to the DPP-IV-like exopeptidase activity, also possesses gelatinolytic

endopeptidase activity, and was thus suggested to participate in degradation of structural

proteins of the extracellular matrix during tissue remodelling and cancer cell invasion [4].

Moreover, some biological roles of DPP-IV as well as FAP seem to be independent on their

enzymatic activity [5; 6].

Since the DPP-IV and FAP proteins share 54% amino acid sequence identity and both

genes are localized close to each other on chromosome 2 [7], some authors suggest them to be

a product of gene duplication [8]. Coexpression of DPP-IV and FAP was described in the

cancer of the oesophagus and skin, and in multiple transformed cell lines [9; 10; 11; 12; 13].

Recently we described correlation between the expression of DPP-IV and FAP in the

human glioblastoma tissues [14]. Moreover, heterooligomeric complexes, containing both

DPP-IV and FAP, were observed on the surface of fibroblasts and endothelial cells,

determining their migratory and invasive potential [9; 15; 16]. The mixed complexes possess

both exopeptidase as well as endopeptidase proline-specific enzymatic activity, the latter

being even more potent than that of the homodimeric FAP/seprase, were shown to participate

in gelatine binding and degradation [9].

In the present study, we investigated the association of DPP-IV and FAP expression in

transformed glial cells.

2. Materials and Methods

2.1. Cell lines, primary cell cultures and sample preparation

Cell lines U138MG and U87MG (ATCC, Middlesex, UK) and the DPP-IV-transfected

U87MG cells, using a DPP-IV cDNA vector (see below) and the Mifepristone inducible Gene

Switch System (Invitrogen, Prague, Czech Republic) [3], were cultured in the Dulbecco’s

modified Eagle’s medium (DMEM; Sigma, Prague, Czech Republic) supplemented with 10%

foetal calf serum (FCS; Sigma). In some experiments, a serum-free DMEM was used to

model growth factors deficiency conditions, inducing adaptive cell differentiation [17].

Primary cell cultures were derived from biopsies of human high grade astrocytic

tumours. The study was approved by the Institutional ethic committee and was conducted in

accordance with the Declaration of Helsinki. All patients signed informed consent before

operation.

A fresh tissue sample was cut into small pieces and cultured in DMEM, supplemented

with 20% FCS (Sigma), and 100 µg/ml Streptomycin and 100 U/ml Penicillin G (Sigma).

Between the 5th - 7th day of explantation, when outgrowths were observed, the explants were

removed and the medium was replaced with fresh DMEM supplemented with 10% FBS and

the antibiotics.

Cells lysates (cca 20 x 106 cells/ml) were prepared on ice in a lysis buffer (10 mM

Tris-HCl pH 7.5, containing 1 mM EGTA, 1 mM Na2EDTA, 1% Triton X-100, 0.1% SDS,

and 10% glycerol) supplemented with a mixture of protease inhibitors (pepstatin A 25 µM,

AEBSF 200 µM, E-64 50 µM) and cleared by centrifugation at 27000 g and 4°C for 30 min.

For separation of the soluble and membrane fractions, the cells were first disrupted by

sonication (three times at ice cold bath for 15 s in the total volume of 7 ml) and the

homogenates were centrifuged at 250 g and 4°C for 10 min. The resulting pellets were

dispersed in the lysis buffer, 10-times passed through a 25 G needle and then ultracentrifuged

at 136000 g and 4°C for 30 min. The supernatant, i.e. the soluble fraction, was stored at -74°C

until further analysis. The pellet was dispersed in the lysis buffer, passed through the needle

and ultracentrifuged as described above. The resulting supernatant, i.e. the solubilized

membrane fraction, was loaded onto a gel filtration column (see below).

2.2. Construction of a DPP-IV cDNA vector and cell transfection

U87MG cells were transfected with human DPP-IV using the Mifepristone inducible

Gene Switch System (Invitrogen). Glioma cells were transfected with the regulatory vector

and a pGene vector containing full-length cDNA of human DPP-IV using the

LipofectamineTM2000 (Invitrogen) according to the manufacturer’s instructions. Stable

clones were selected with Zeocin (Invitrogen) and Hygromycin (Invitrogen). Mifepristone

(Invitrogen) in concentrations of 0.25 nM was used to induce DPP-IV expression in

transfected cells; cells not treated by Mifepristone were used in control experiments.

2.3. Fluorescent in situ hybridisation (FISH)

FISH analyses were carried out on the suspensions of fixed cells. To identify

chromosome 2, a commercially available centromeric DNA probe CEP 2 (D2Z1) Spectrum

Orange and a differently labelled DNA probe CEP 18 (D18Z1) Spectrum Green, serving as a

control, were used according to the manufacturer’s recommendations (Abbott Molecular, Des

Plaines, USA). To localize the DPP-IV and FAP DNA sequences, respectively, commercially

available BAC probes were used (FAP - RP11-576I16 and DPP-IV - RP11-178A14,

Pentagen, Kolin, Czech Republic).

2.4. Real-time reverse transcription-polymerase chain reaction (RT-PCR)

To determine the expression levels of DPP-IV and FAP- mRNAs, a coupled RT-

PCR was used. The isolation of total RNA from cells, real-time RT-PCR quantification of the

investigated transcripts and -actin mRNA (an internal reference transcript), and evaluation of

the transcript expression data were performed as described previously [3; 14].

2.5. Gel filtration chromatography and enzymatic activity assays

To separate and identify the DPP-IV-like exopeptidase and the endopeptidase

enzymatic activities present in the soluble and solubilized membrane cell fractions, gel

filtration chromatography on a column of Sephacryl S-300 (Pharmacia, Uppsala, Sweden) as

described previously was used [18].

The exopeptidase and endopeptidase enzymatic activities in individual collected

fractions were determined with the fluorogenic substrates glycyl-L-prolyl-7-

amidomethylcoumarine (H-Gly-Pro-AMC) and N-benzyloxycarbonyl-glycyl-L-prolyl-7-

amidomethylcoumarine (Z-Gly-Pro-AMC) (both from Bachem, Bubendorf, Switzerland),

respectively, in a final concentration of 50 M. Cell surface and the total cellular enzymatic

activities as well as activities in fractions after the gel filtration were determined using the

continuous rate fluorimetric assay as described in detail previously [17; 19].

2.6. Enzyme-Linked ImmunoSorbent Assay (ELISA)

The levels of DPP-IV and FAP proteins were determined using the Clear Microplate

kits (R&D Systems, Abingdon, UK) according to the manufacturer’s recommendations.

Recombinant human FAP and DPP-IV proteins were used as negative controls in the DPP-IV

and FAP assays, respectively. The microplate reader Sunrise (Tecan, Malmedorf,

Switzerland) was used to measure the absorbance of samples at 450 nm. The measured

absorbance values were corrected by subtracting the absorbance values obtained at a second

wavelength of 570 nm. The resulting differential absorbance values were used for

constructing the calibration curves and data evaluation.

2.7. Immunocytochemistry

Cells were cultured on glass coverslips and then fixed with 4% paraformaldehyde at a

room temperature for 5 min. After the preincubation in 3% of heat-inactivated FCS for 20

min, the immunodetection of DPP-IV and FAP was performed by an overnight incubation at 4

°C with the respective monoclonal primary antibodies: mouse anti-human CD26 (clone M-

A261, at a dilution of 1:100; Abcam, Cambridge, UK) and rat anti-human FAP-α (clone D28,

at a dilution of 1:200; Vitatex, New York, USA). This step was followed by incubation with

the Alexa Fluor 488-labeled goat anti-mouse IgG (at a dilution of 1:1000, Molecular

Probes/Invitrogene) and the Alexa Fluor 546-labeled goat anti-rat IgG (at a dilution of 1:600,

Molecular Probes/Invitrogene), respectively. In controls, the primary antibodies were omitted

from the immunostaining procedure. The coverslips with cells were mounted in the anti-

fading Gel/Mount medium (Biomeda Corp., Foster City, USA), observed and photographed

using laser scanning microscope FV300 (Olympus, Prague, Czech Republic).

2.8. Flow cytometry analysis

Cells were detached by 0.02% Na2EDTA in PBS pH 7.4, fixed in 2%

paraformaldehyde for 20 min at a room temperature, and then washed twice in PBS for CD26

immunodetection or in the 1x Permeabilization Buffer (eBiosciences, San Diego, USA) for

the FAP immunodetection. Staining by primary antibodies was performed in the dark at room

temperature for 30 min, using a phycoerythrin conjugated monoclonal anti-CD26 antibody

(clone 222113, at a dilution of 1:20; R&D Systems) or unconjugated rat monoclonal anti-FAP

antibody (clone D28, at a dilution of 1:50; Vitatex). For FAP detection, the cells were washed

twice with the 1x Permeabilization Buffer and were stained with the Alexa Fluor 546-labeled

goat anti-rat IgG secondary antibody (at a dilution of 1:50; Molecular Probes). Samples were

analysed on a flow cytometer FACS Canto (BD Biosciences, Franklin Lakes, USA),

following with data evaluation using the FlowJo software (TreeStar Inc., Ashland, USA).

2.9. Statistical analysis

The Mann-Whitney test was used to evaluate the differences between the investigated

groups. Correlation of quantitative variables was assessed by computing the Spearman’s

correlation coefficient. For calculations, software package Statistica 8.0 (StatSoft, Tulsa,

USA) was used.

3. Results and Discussion

3.1. DPP-IV and FAP coexpression in primary astrocytic cell cultures

Similarly as in the bioptic samples of glioblastoma multiforme [14], we observed

coexpression and a tight correlation of expression of DPP-IV and FAP mRNA transcripts in

the established primary cell cultures derived from the human glioblastoma multiforme tissue

explants. Flow cytometry analysis confirmed the presence of DPP-IV and FAP proteins in the

obtained primary cell cultures (data not shown). Expression of DPP-IV and FAP templates

correlated with both cell surface and the total cellular DPP-IV-like hydrolytic activity (Table

1), suggesting presence of canonical DPP-IV and FAP in the plasma membrane as well as

intracellular compartments of glioblastoma primary cell cultures.

3.2. DPP-IV and FAP coexpression during experimentally induced changes of DPP-IV-like

enzymatic activity

To assess the dynamic association of DPP-IV and FAP expression, permanent

glioblastoma cell lines were cultured under conditions of growth factors deficiency, i.e. in the

serum-free DMEM, which substantially increase their DPP-IV-like enzymatic activity [17;

20]. To this end, U87MG and U138MG cell lines, both coexpressing DPP-IV and FAP and

exhibiting a presence of the respective gene loci in the proper localization on the chromosome

2 (Figs. 1, 2), were used. In these experiments, the upregulation of biochemically assayed

DPP-IV-like enzymatic activity was associated with increased transcription of both DPP-IV

and FAP mRNAs as well as with the increase of DPP-IV and FAP proteins as determined by

ELISA (Fig. 3, Table 2).

To further distinguish DPP-IV and FAP activities within the whole DPP-IV-like

enzymatic activity, the proline-specific endopeptidase enzymatic activity exhibited by FAP,

but not by the canonical DPP-IV, was measured using Z-Gly-Pro-AMC as a substrate [21].

Since the prolyl endopeptidase (PEP; EC 3.4.21.26) differing from FAP by the molecular

mass (Mr) is responsible for substantial part of the cellular proline-specific endopeptidase

activity, gel filtration chromatography was utilized before the biochemical assay in order to

separate FAP and PEP.

In U87MG cells cultured in serum free media, both the DPP-IV-like exopeptidase (H-

Gly-Pro-AMC as a substrate) and the proline-specific endopeptidase (Z-Gly-Pro-AMC as

substrate) enzymatic activities assayed in fractions after gel filtration were increased in the

Mr-region of about 410-610 kDa (Fig. 4). Although both DPP-IV and FAP typically occur as

homodimers with Mr of 180 - 220 kDa [2] their higher oligomeric complexes having Mr of

480 - 900 kDa are known to occur in some cells as well [9; 15].

Another proline-specific endopeptidase activity peak (Fig. 4) present in the elution

profiles of the soluble but not plasma membrane subcellular fractions and showing Mr of

about 80 kDa corresponds well with the cytosolic PEP [22].

Contrary to U87MG cells, the proline-specific endopeptidase activity attributable to

FAP was not detectable in U138MG cells, either under the growth factors proficiency or

deficiency culture conditions, while the DPP-IV exopeptidase activity in U138MG cells rose

similarly as in U87MG cells (data not shown). An abundant expression of FAP transcript

accompanied by a high FAP protein concentration in context with the absence of

characteristic proline-specific endopeptidase activity may argue for the expression of

enzymatically inactive seprase/FAP alternative spliced variant(s) in U138MG cells. There are

at least eleven known alternative spliced variants of the seprase/FAP primary transcript, some

of them encoding FAP protein isoforms devoid of enzymatic activity [23; 24; 25]. Taken

together, the associated dynamics of DPP-IV and FAP expression on the transcriptional and

translational levels in glioblastoma cell lines further argues for their putative joint

involvement in certain biological processes, not necessarily executed by or dependent on their

proteolytic activity.

The changes in DPP-IV and FAP expression induced by cultivation under growth

factors deficiency conditions were reverted by addition of 10% foetal calf serum into the

culture media.

3.3. Expression of FAP in DPP-IV transfected cells

In experiments with the DPP-IV transfected U87MG cells, neither the upregulation of

FAP- mRNA and protein expression nor the increase of relevant endopeptidase enzymatic

activity were observed after the induction of DPP-IV overexpression (Figs 3,4). Uncoupling

of the exogenous (recombinant) DPP-IV and the endogenous FAP expression in the

transfected cells, where the DPP-IV transgene is inserted away from its physiological

genomic context, suggests that the correlated expression of the endogenous DPPIV and FAP,

seen under the conditions of growth factors deficiency or proficiency, is more likely a result

of a joint control of DPP-IV and FAP genes expression than a consequence of an indirect

reciprocal regulation involving changes of mRNA and/or protein expression levels of DPP-IV

and FAP (Fig. 3, Table 2).

4. Conclusions

In the last decade, the role of DPP-IV and FAP in multiple physiological as well as

pathological processes, including regulation of growth, migration and invasion of transformed

cells, has been revealed. Their functional crosstalk has been hypothesized. Here we describe

for the first time the correlative expression of DPP-IV and FAP in glioblastoma-derived

primary cell cultures and their associated expression dynamics in the permanent glioblastoma

cell lines. These results argue for a contextual role of both molecules in certain biological

processes.

Acknowledgment: This work was supported by the Grant Agency of Charles University in

Prague, Czech Republic, grants GAUK 99208 and GAUK 89607 and by Ministry of

Education, Youth and Sports of the Czech Republic project MSMT 0021620808.

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FIGURE LEGENDS

Figure 1

Immunodetection of DPP-IV (green) and FAP (red) in U87MG and U138MG cell lines.

Original magnification 600x.

Figure 2

Genomic localization of DPP-IV (green) and FAP (rouge) loci on the chromosome 2 detected

by FISH in U87 and U138 glioma cell lines. Centromeric probe in red.

Figure 3

Relative increment of DPP-IV (empty bars) and FAP (full bars) mRNA expression and

plasma membrane DPP-IV-like enzymatic activity (full line). (A) U87MG and (B) U138MG

cells cultured in serum free DMEM compared to the ones grown in 10% foetal calf serum

supplemented DMEM. (C) DPP-IV transfected U87MG cells treated by 0.25 nM

Mifepristone to upregulate transgenic DPP-IV compared to the control untreated counterparts.

Figure 4

Elution profile of DPP-IV-like exopeptidase (full line) and prolyl endopeptidase (dotted line)

enzymatic activity in U87MG cell line cultured (A) in 10% foetal calf serum supplemented

DMEM, (B) serum free DMEM, (C) DPP-IV transfected U87MG control untreated cells and

(D) DPP-IV transfected U87MG cell treated by 0.25 nM Mifepristone to upregulate

transgenic DPP-IV expression.

Figure 1

Figure 2

Figure 3

Figure 4

TABLES

Table 1. Correlations of DPP-IV and FAP transcripts and DPP-IV-like enzymatic activity in

primary cell cultures derived from human glioblastoma

FAP transcript DPP-IV-like cell DPP-IV-like total surface activity cellular activity DPP-IV transcript R=0.73; p=0.0001 R=0.87; p=0.0001 R=0.88; p=0.0001 FAP transcript n.a. R=0.58; p=0.006 R=0.59; p=0.006

n = 21, R - Spearman´s correlation coefficient, p = significance of correlation; n.a.: not

applicable

Table 2. DPP-IV and FAP immunopositivity (ELISA) in U87MG, U138MG and DPP-IV

transfected U87MG glioma cell lines.

Culture conditions DPP-IV FAP

Mean + S.D. [ηg/μg protein]

U87MG SFM 56.1 + 0.5 90.5 +12.3

FCS 13.5 + 6.7 23.3 + 1.2

U138MG SFM 66.4 + 1.2 108.7 + 0.7

FCS 36.6 + 3.3 88.4 + 1.8

Transfected U87MG Mifepristone 105.4 + 1.7 5.4 + 1.7

Controls 3.8 + 0.7 8.4 + 1.8

SFM: seum free media; FCS: DMEM supplemented with 10% foetal calf serum;

Mifepristone: DPP-IV transfected U87MG cells stimulated to express transgene DPP-IV by

0,25 nM Mifepristone; Controls: Untreated DPP-IV transfected U87MG cells.

Tables 1 and 2