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1,2 by Human Glioblastoma Cells Involves Cytoplasmic and Secreted Furin-Like Proteases1
*
Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tübingen, Tübingen, Germany; and
Institute of Virology, University of Marburg, Marburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is a putative mediator of immunosuppression associated with
malignant glioma and other types of cancer. Subtilisin-like proprotein
convertases such as furin are thought to mediate TGF- processing.
Here we report that human malignant glioma cell lines express furin
mRNA and protein, exhibit furin-like protease (FLP) activity, and
release active furin into the cell culture supernatant. FLP activity is
not modulated by exogenous TGF- or neutralizing TGF- Abs.
Exposure of LN-18 and T98G glioma cell lines to the furin inhibitor,
decanoyl-Arg-Val-Lys-Arg-chloromethylketone, inhibits processing of the
TGF-1 and TGF-2 precursor molecules and, consequently, the
release of mature bioactive TGF- molecules. Ectopic expression of
PDX, a synthetic antitrypsin analog with antifurin activity, in the
glioma cells inhibits FLP activity, TGF- processing, and TGF-
release. Thus, subtilisin-like proprotein convertases may represent a
novel target for the immunotherapy of malignant glioma and other
cancers or pathological conditions characterized by enhanced TGF-
bioactivity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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type 2
(2, 3, 4). Consistent with a pivotal role of TGF- in the
growth of malignant gliomas, expression of TGF-2 antisense mRNA in
9L (5) or in C6 rat gliomas (6) induced their
regression. Similarly, gene transfer-mediated ectopic expression of the
functional TGF- antagonist, decorin, promoted the regression of rat
C6 gliomas (7).
TGF- is synthesized as a pre-pro-TGF- polypeptide that contains a
signaling peptide (pre; residues 1–29), the pro region (residues
30–278), and the mature TGF- moiety (residues 279–390)
(8). Activation to the mature 12.5-kDa TGF-1, which
needs to dimerize to exert its biological effects, depends on the
action of subtilisin-like proprotein convertases such as furin, as
shown for purified proteins obtained from cell culture supernatants in
a cell-free system (9). In fact, it has been suggested
that TGF- promotes furin gene expression as part of an amplification
cascade of its own activation in fibroblasts and rat hepatocytes
(10, 11). The subtilisin-like proprotein convertases of
mammalian cells constitute a family of proprotein convertases related
to bacterial subtilisins and yeast Kex2p that process multiple protein
precursors, including growth factors, proteases of the coagulation and
complement cascades, glycoproteins of viral envelopes and bacterial
exotoxins at multibasic recognition sites (12, 13, 14). Furin
seems to be universally expressed in mammalian cells and localizes
mainly to the trans-Golgi network.
In the present study, we examined the role of furin-like protease
(FLP)3 in the
processing of TGF- in malignant glioma cells. Because TGF-2 is
the predominant isoform of TGF- secreted by human malignant glioma
cells and because TGF-2 has the same cleavable consensus site for
furin (R-X-R/K-R) as TGF-1, we were specifically interested in
examining whether human glioma cells express furin and whether
inhibition of furin may result in the inhibition of the processing of
both isotypes of TGF- in intact glioma cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The synthetic furin inhibitor,
decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk), as well as
the furin substrate for the fluorescence assays,
N-t-butoxycarbonyl-Arg-Val-Arg-Arg-7-amido-4-methylcoumarine
(boc-RVRR-amc), were purchased from Bachem (Heidelberg, Germany).
[methyl-3H]Thymidine and 5'
[
-32P]dCTP were obtained from Amersham
(Braunschweig, Germany). The following Abs were purchased: rabbit
polyclonal Ab to TGF-1 from Promega (Mannheim, Germany) and rabbit
polyclonal Ab to TGF-2 from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-furin antiserum was raised as previously described
(15). Human rTGF-1 and rTGF-2 were purchased from
Roche (Mannheim, Germany). ELISAs for total TGF-1 and TGF-2 were
obtained from R&D Systems (Minneapolis, MN).
Cell culture
Sv-FHAS is an SV40 large T-Ag immortalized fetal human astrocytic cell line that was provided by A. Muruganandam and D. Stanimirovic (Institute of Biological Sciences, National Research Council of Canada, Ottawa, Canada). The human glioma cell lines were provided by Dr. N. de Tribolet (Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland) and maintained in DMEM supplemented with 10% FCS and penicillin (100 IU/ml)/streptomycin (100 µg/ml) (16). Transfections were conducted by electroporation (7), using a PDX expression plasmid (17) and a human furin expression plasmid provided by J. Creemers (Center for Human Genetics, University of Leuven, Belgium) (18).
TGF- bioassay
Levels of bioactive TGF- were determined using a modification
of the CCL64 bioassay (7). Briefly, 5000 CCL64 cells were
adhered to 96-well plates for 24 h. After removal of regular
medium, the cells were exposed to cell culture supernatants diluted in
serum-free medium for 56 h and then labeled with
[methyl-3H]thymidine (1 µCi/well)
for additional 16 h. Cell culture supernatants were obtained by
seeding 106 cells in a
25-cm2 culture flask. After 24 h, the cells
were washed with PBS and then incubated with serum-free medium. After
an additional 48 h, the conditioned medium was harvested, cell
debris was removed by centrifugation, and the supernatant was stored at
-20°C. Cell counts were obtained at the end of supernatant
generation were used to normalize the supernatants for cell culture
density. Latent TGF- was activated by heating of the supernatants to
85°C for 5 min (19).
RT-PCR
RT-PCR for the detection of furin mRNA expression was performed
according to standard procedures. The following primer sequences were
used: furin sense (nucleotides corresponding to NotI site
are underlined, plus nt -7 to 11 containing the ATG),
5'-TTTTTTGCGGCCGCCCCCCCATGGAGCTGAA-3';
furin antisense (nucleotides EcoRI are underlined, plus nt
428–409), 5'-TTTTGAATTCGTGTAGCCCTGCGCCCAGGC-3' (35
cycles of 40 s at 94°C, 60 s at 56°C, and 60 s at
72°C); -actin sense (nt 409–429), 5'-TGTTTGAGACCTTCAACACCC-3';
-actin antisense (nt 937–918), 5'-AGCACTGTGTTGGCGTACAG-3' (35
cycles of 40 s at 94°C, 60 s at 53°C, and 60 s at
72°C); PDX sense (nt 552–571 of the
1-antitrypsin mRNA), 5'-CGTGGAGAAGGGTACT
CAAG-3'; PDX antisense (nt 1164–1145 of the
1-antitrypsin mRNA, but with the last two
nucleotides complementary to the changes made to generate PDX,
underlined) (17), 5'-GACCTCGGGGGGGATAGATC-3'
(30 cycles of 45 s at 94°C, 45 s at 60°C, and 60 s
at 72°C).
Northern blot analysis
Total RNA was isolated using the RNeasy kit (Qiagen, Hilden,
Germany). Four micrograms of total RNA per lane was separated on 1.2%
agarose gels and blotted onto nylon membranes (Amersham). The filters
were hybridized according to standard procedures with
32P-labeled full-length human cDNA probes for
furin (20), provided by W. J. van de Ven (Center for Human
Genetics, University of Leuven and Flanders Interuniversity Institute
for Biotechnology, Leuven, Belgium), TGF-1 (21) and
TGF-2 (22), gifts from A. Fontana (University
Hospital Zurich, Zurich, Switzerland). All blots were rehybridized with
a -actin probe (23). Autoradiography signals were
quantified with a phosphor imager (Fuji BasReader 1500; Raytest,
Staubenhardt, Germany), and the signals of interest were normalized
against -actin expression.
Immunoblot analysis
The levels of TGF- and furin protein expression were assessed
by immunoblot analysis. The general procedure has been described
previously (24). Briefly, cells were lysed in 50 mM
Tris-HCl (pH 8) containing 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40,
2 µg/ml aprotinin, 10 µg/ml leupeptin, and 100 µg/ml PMSF. For
the detection of TGF- in the supernatant, serum-free supernatants
were concentrated (because of the low protein production of some of the
cell lines) with the Centriplus centrifugal filter device YM-3 (3000-Da
cut-off; Millipore, Eschborn, Germany). This concentrated supernatant
was also used for the immunoblot detection of furin in the supernatant.
The furin antiserum was used at a dilution of 1/1000 in 10 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 0.1% Tween 20, 5% skim milk, 2% BSA, and
0.01% sodium azide; the Abs to TGF-1 and TGF-2 were used at
1/2000 and 1/3000. Anti-rabbit IgG (Santa Cruz Biotechnology) at a
dilution of 1/3500 in PBS-0.05% Tween 20–1.3% skim milk was used as
a secondary Ab to detect TGF-, and anti-mouse IgG (Amersham)
diluted 1/2000 was used to detect furin. Labeling was visualized using
enhanced chemiluminescence (ECL system; Amersham). Equal protein
loading was ascertained by Ponceau S staining for all blots.
FLP activity
To determine cellular FLP activity, 15,000 cells were adhered to 96-well plates for 24 h. Growth medium was replaced by 50 µl DMEM without phenol red, but containing 0.25% Triton X-100 for permeabilization and boc-RVRR-amc (100 µM) as a substrate. Because of the stable FLP activity over a range of 48 h in that assay, the data shown were measured at 4 h after substrate addition. Fluorescence was measured at 380 nm excitation and 460 nm emission wavelengths. The data were normalized to cell density by parallel staining of another 96-well plate with crystal violet. For determination of FLP activity in the supernatant, cells were grown in a 75-cm2 culture flask. At nearly confluent stage, normal growth medium was changed to 7 ml DMEM without phenol red. The supernatant was harvested 48 h later, cleared of cell debris by centrifugation, and stored until further use at -20°C. The assay was performed with 0.25% Triton X-100 and 100 µM substrate in a total volume of 100 µl. The data were normalized to the protein concentration in the supernatant.
Statistical analysis
All experiments reported herein were performed at least three times with similar results. A correlation between various sets of data was examined by linear correlation analysis as outlined below.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess whether glioma cells express furin, RNA was prepared
from 12 different human malignant glioma cell lines and analyzed for
furin mRNA by Northern blot (Fig. 1
A). A 4.2-kb furin mRNA
species was detected in all cell lines. The highest levels of furin
mRNA were detected in U138 MG, LN-428, U373 MG, and LN-308 cells. Low
levels were detected in U87 MG, T98G, and LN-319 cells. RT-PCR
confirmed that all glioma cell lines expressed furin mRNA (data not
shown).
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B). Overall, mRNA
and protein levels appeared to correlate. For instance, LN-308, the
cell line showing the highest mRNA expression, also yielded the
strongest protein signal on the immunoblot.
FLP activity was determined by measuring conversion of a fluorogenic
substrate, boc-RVRR-amc (Fig. 1C). Overall, there was little
variation in FLP activity among the cell lines. FLP activity correlated
less well with mRNA and protein levels, suggesting that the enzyme
assay not only detects furin activity but also the activity of related
FLPs that are probably expressed by glioma cells. Of note, the
astrocytic cell line sv-FHAS exhibited lower FLP activity than any of
the glioma cell lines, consistent with the low levels of furin protein
(Fig. 1B).
Because furin has been reported to be regulated by TGF- in
fibroblasts and rat hepatocytes (10, 11), we examined
whether exogenous TGF-1 or TGF-2 modulate FLP activity. LN-18 or
T98G exposed to either isoform of TGF- for 4 or 24 h failed to
exhibit a significant change in FLP activity. Moreover, neutralizing
TGF- Abs failed to modulate FLP activity in these cells (data not
shown), suggesting that endogenous TGF- does not modulate FLP
activity.
TGF- synthesis and release by glioma cells
TGF-1 and TGF-2 mRNA and protein expression and TGF-
bioactivity were assessed in the same panel of glioma cell lines.
Northern blot analysis revealed that most glioma cell lines expressed a
5.1-kb mRNA for TGF-1 and TGF-2 (Fig. 2A). Immunoblot analysis was
performed with total cellular soluble protein lysates and with
supernatant protein (Fig. 2, B and C). Cellular
lysates contained comparable levels of 55-kDa TGF-1, but greatly
differing levels of 55-kDa TGF-2. The 12.5-kDa active fragment was
not detected in the cellular lysate (Fig. 2, B and
C). Both the 55- and 12.5-kDa fragments for TGF-1 and
TGF-2 were detected in the supernatant of both cell lines. These
data suggest that TGF- is mainly released as the 55-kDa form and
further processed following release. Appropriate control experiments
were performed to assure that the TGF-1 Ab did not recognize human
recombinant TGF-2, and vice versa (data not shown). The levels of
TGF-1 and TGF-2 in the supernatant were also quantified by ELISA
(Fig. 2D) and by CCL64 bioassay, which determines both
TGF-1 and TGF-2, but allows differentiation of latent and active
TGF- (Fig. 2E). Overall, there was fairly good
correlation between the levels of mRNA and protein expression
determined by the different methods. For instance, U87 MG, D247 MG, and
LN-229 cells, which showed very low TGF-2 mRNA expression on
Northern blot analysis, also showed correspondingly low levels of
TGF-2 protein on immunoblot analysis or in the ELISA. Statistical
analysis revealed that the levels of active TGF- determined by
bioassay correlated to the sum of TGF-1 and TGF-2 levels in the
ELISA (r = 0.716; p = 0.009), and the
levels of total TGF- in the bioassay correlated to the sum of
TGF-1 and TGF-2 levels in the ELISA as well (r =
0.675; p = 0.016). There was no clear-cut correlation
between furin mRNA and protein levels (Fig. 1) and the activation of
TGF- (Fig. 2), suggesting that furin is not the only mediator of
TGF- processing in glioma cells. However, some of the data fit
rather well, e.g., the very efficient processing of TGF-1 and
TGF-2 in LN-308 cells that are strongly furin positive.
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Because 1) furin is thought to be required for the processing of
55-kDa TGF- to active 12.5-kDa TGF-, 2) the 12.5-kDa TGF- is
not detected in the cell lysate, but in the supernatant, and 3) high
levels of 55-kDa TGF- are released into the cell culture supernatant
where 12.5-kDa TGF- is also detected, we concluded that significant
TGF- processing must take place in the cell culture supernatant or
at the extracellular aspect of the cell membrane. First, to examine
whether FLP activity localizes to the extracellular aspect of the cell
membrane, we performed the fluorescent FLP activity assay in parallel
with (standard) and without cellular permeabilization (see
Materials and Methods). These experiments revealed that the
FLP activity of nonpermeabilized cells did not exceed 10% of the
activity measured in permeabilized cells, consistent with previous
reports that some furin is exposed at the cell surface
(26, 27, 28). Yet, the residual 10% may also originate from
minor penetration during the assay of the substrate into the cells or
from furin release into the supernatant (see below).
Thus, we tested the hypothesis that furin is released into the
supernatant to convert 55-kDa TGF- to 12.5-kDa TGF-. Fig. 3A shows that immunoreactive
furin can be detected in the supernatant of most glioma cell lines.
Particularly high levels were detected in LN-308 cells, corresponding
to the high intracellular levels of furin in these cells (Fig. 1B). Interestingly, there was very little release of 60-kDa
furin into the cell culture supernatant. Moreover, glioma cell
supernatants exhibited significant FLP activity (Fig. 3B).
As observed for cellular furin and cellular FLP (Fig. 1), there was no
strong correlation of furin levels and FLP activity in the supernatant
(Fig. 3).
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in glioma
cells
To assess whether furin-like enzymes mediate TGF- processing in
glioma cells, we monitored changes in TGF- release induced by an
inhibitor of furin, dec-RVKR-cmk. Fig. 4A shows that dec-RVKR-cmk
inhibited the formation of active (12.5-kDa) TGF-1 and TGF-2 in a
concentration-dependent manner. The loss of active TGF- after
exposure to the furin inhibitor was confirmed at the level of ELISA
(Fig. 4, B and C) and bioassay (Fig. 4D). In parallel, there was an increase in the 55-kDa pro
form of TGF-1 and TGF-2 in the supernatant (Fig. 4A).
In contrast, the levels of intracellular 55-kDa pro-TGF- were
unaffected by inhibition of FLP activity (data not shown), confirming
that most TGF- conversion by furin takes place extracellularly. The
inhibitor was equally effective in inhibiting TGF- synthesis in the
glioma cell lines that express both isoforms of TGF- (Fig. 4) and in
sv-FHAS astrocytes that express only TGF-1 (data not shown).
Together these observations, notably the loss of 12.5-kDa TGF- and
the relative increase in 55-kDa TGF-, support the hypothesis that
released soluble furin plays a prominent role in the processing of
TGF- by glioma cells.
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bioactivity
PDX is a synthetic serine protease inhibitor (serpin) designed to
selectively inhibit furin (17). The LN-18 and T98G glioma
cell lines were transfected with a PDX expression plasmid
(17). PDX transgene expression was verified by RT-PCR
(Fig. 5A). PDX-transfected
glioma cell sublines exhibited a minor, but significant, reduction in
cytoplasmic FLP activity (Fig. 5B). However, although the
reduction of FLP activity did not exceed 20%, there was strong
inhibition of TGF- processing, as assessed by the levels of 12.5-kDa
TGF- in the supernatant (Fig. 5C). Quantification of the
immunoblots for 12.5-kDa TGF- revealed a reduction of TGF-1 to
<60% in T98G cells and of TGF-2 to <70% in LN-18 and to <40%
in T98G cells (Fig. 5D). PDX transgene expression was not
very prominent in the glioma cell lines and was lost within a few
passages in vitro, suggesting strong selection pressure against furin
inhibition in vitro (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a cytokine that is released by glioma cells in large
quantities in vitro and in vivo and has been considered central to the
malignant progression of glial tumors and to the immune dysfunction in
human patients with glioblastoma (4). This is because
TGF- promotes tumor angiogenesis, enhances migration and invasion,
and inhibits T cell-mediated immune responses. Experimental therapeutic
efforts to neutralize TGF- activity associated with glioblastoma
include antisense strategies (5, 6, 29, 30) and antagonism
by decorin (7, 31).
Here we examined the biochemical pathways of TGF- synthesis and
processing in malignant glioma cells and asked whether proteases of the
subtilisin-like proprotein convertase family such as furin are involved
in TGF- maturation and might therefore become a target for
immunotherapy in these tumors. We find that glioma cells express furin
mRNA and protein and exhibit FLP activity in a fluorometric enzyme
assay. Cellular FLP activity does not correlate well with furin protein
levels, indicating that this enzyme assay detects the activity of other
furin-related proteases as well.
As indicated above, there is an increasing number of subtilisin-like
proprotein convertases, which are now designated SPC1–8 and are
candidate enzymes for TGF- processing (14). There were
also significant differences in the levels of released furin and the
levels of FLP activity in the supernatant among the cell lines (Fig. 3). A definite physiological role of released furin has not been
characterized. However, previous studies have postulated a role for
furin release in the processing of cellular substrates
(32). The present study suggests that the release of furin
may play a crucial role in creating an immunosuppressive
microenvironment in human gliomas via enhanced TGF- processing.
We confirm that the synthesis and release of large levels of TGF-1
and TGF-2 are a feature of cultured glioma cell lines (Fig. 2).
Numerous previous studies have confirmed that glioblastoma cells
produce TGF- in vivo too (reviewed in Ref. 4). Here, we
identify one or more FLPs as the enzyme(s) responsible for TGF-
activation in glioma cells (Fig. 4). Furin has to date been
characterized as a TGF-1-processing enzyme in cell-free systems
(9). For the first time ever, we show that furin
inhibitors abrogate TGF-1 processing in intact cells and extend
these observations to the processing of TGF-2.
The present study does not allow firm conclusions about whether FLP
activity is pathologically elevated in glioma cells and thus primarily
responsible for the high level of TGF- release from human gliomas in
vivo. In contrast to other cell types (10, 11), TGF-
appears not to promote furin mRNA expression or activity to enhance its
own release in a positive autocrine loop in glioma cells (data not
shown).
At present, there is little information on the expression and activity
of FLPs in other cell types in vitro and in vivo (33). Our
study indicates that differences in furin expression or FLP activity
alone do not account for most of the variation in the levels of
TGF-1 or TGF-2 that are processed and released in a panel of
human glioma cell lines. Furthermore, the phenotype obtained after
targeted disruption of the furin gene in mice revealed that furin is
probably not the only pathway that regulates the processing of TGF-
family members (33). Yet, the effects of the synthetic
pseudosubstrate furin inhibitor, dec-RVKR-cmk (Fig. 4), and the
antitrypsin derivative, PDX (Fig. 5), demonstrate that furin may be a
suitable target to reduce the TGF- bioactivity associated with
glioma cells. Of note, because subtilisin-like proprotein convertases
other than furin may be chiefly responsible for the inhibition of
TGF- processing observed here, inhibition of furin alone may not be
sufficient for successful immunotherapy of malignant glioma. Thus, the
present study defines a novel strategy for the pharmacotherapy or
somatic gene therapy (using PDX) of conditions associated with
pathological levels of TGF- bioactivity, including glioblastoma.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Weller, Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tübingen, School of Medicine, Hoppe Seyler Strasse 3, 72076 Tübingen, Germany. E-mail address: michael.weller{at}uni-tuebingen.de
3 Abbreviations used in this paper: FLP, furin-like protease; dec-RVKR-cmk, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; boc-RVRR-amc, N-t-butoxycarbonyl- Arg-Val-Arg-Arg-7-amido-4-methylcoumarine.
Received for publication August 8, 2000. Accepted for publication April 12, 2001.
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K. Sasaki, K. Sato, Y. Akiyama, K. Yanagihara, M. Oka, and K. Yamaguchi Peptidomics-based Approach Reveals the Secretion of the 29-Residue COOH-Terminal Fragment of the Putative Tumor Suppressor Protein DMBT1 from Pancreatic Adenocarcinoma Cell Lines Cancer Res., September 1, 2002; 62(17): 4894 - 4898. [Abstract] [Full Text] [PDF]
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 J. Mercapide, R. Lopez De Cicco, D. E. Bassi, J. S. Castresana, G. Thomas, and A. J. P. Klein-Szanto Inhibition of Furin-mediated Processing Results in Suppression of Astrocytoma Cell Growth and Invasiveness Clin. Cancer Res., June 1, 2002; 8(6): 1740 - 1746. [Abstract] [Full Text] [PDF]
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 A.-M. Khatib, G. Siegfried, M. Chretien, P. Metrakos, and N. G. Seidah Proprotein Convertases in Tumor Progression and Malignancy : Novel Targets in Cancer Therapy Am. J. Pathol., June 1, 2002; 160(6): 1921 - 1935. [Abstract] [Full Text] [PDF]
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 T. Fernandez, S. Amoroso, S. Sharpe, G. M. Jones, V. Bliskovski, A. Kovalchuk, L. M. Wakefield, S.-J. Kim, M. Potter, and J. J. Letterio Disruption of Transforming Growth Factor {beta} Signaling by a Novel Ligand-dependent Mechanism J. Exp. Med., May 20, 2002; 195(10): 1247 - 1255. [Abstract] [Full Text] [PDF]
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J. Wischhusen, G. Jung, I. Radovanovic, C. Beier, J. P. Steinbach, A. Rimner, H. Huang, J. B. Schulz, H. Ohgaki, A. Aguzzi, et al. Identification of CD70-mediated Apoptosis of Immune Effector Cells as a Novel Immune Escape Pathway of Human Glioblastoma Cancer Res., May 1, 2002; 62(9): 2592 - 2599. [Abstract] [Full Text] [PDF]
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) and TGF-
)
were measured by ELISA. Data are expressed as picograms per milliliter
(mean of duplicate values) per 106 cells released in 48
h. E, Levels of active (
