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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 8, 2000 as doi:10.1096/fj.00-0371fje. |
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,§2
* LSUMC Gene Therapy Program,
Department of Pediatrics,
Department of Physiology,
§ Department of Medicine,
|| Department of Surgery,
# Department of Anatomy and Cell Biology, Louisiana State University School of Medicine, New Orleans, Louisiana 70112, USA
2Correspondence: 533 Bolivar St., Room 601, New Orleans, LA, 70112, USA. E-mail: jkolls{at}lsuhsc.edu
SPECIFIC AIMS
In thisstudy, we tested the hypothesis that phorbol myristate acetate (PMA)
activates tumor necrosis factor alpha (TNF-
) -converting enzyme
(TACE)-mediated ectodomain shedding partly via the generation of
reactive oxygen species (ROS), and that H2O2
can directly activate TACE activity. To index TACE activation, we
measured ectodomain shedding of two TACE-dependent substrates, TNF p75
receptor and L-selectin. In addition, we studied ectodomain
shedding in two cell lines—one of which has a mutated, inactive form
of TACE—and tested direct activation of recombinant TACE by ROS by
using an in vitro TACE cleavage assay with and without a
portion of the TACE inhibitory pro-domain.
PRINCIPAL FINDINGS
1. PMA enhances TACE-dependent
ectodomain shedding, which can be attenuated by a ROS
scavenger
Here we used shedding of preformed TNF p75 receptor as a
sensitive marker for TACE activity. We incubated Mono Mac 6 cells, a
human monocytic cell line, with or without PMA for 2 h. Soluble
TNF p75 receptors were measured by enzyme-linked immunoassay (ELISA) in
the culture media of these cells to determine ectodomain shedding
activity. As shown in Fig. 1A
, control cells
exhibited a basal level of spontaneous shedding activity of TNF p75
receptors. PMA significantly increased TNF p75 receptor shedding from
Mono Mac 6 cells. Furthermore, involvement of ROS in PMA-induced TNF
p75 receptor shedding was studied with N-acetyl-L-cysteine (NALC). Mono
Mac 6 cells were incubated with PMA for 2 h in the presence of 100
µM NALC. NALC significantly inhibited PMA-induced TNF p75 receptor
shedding (Fig. 1A
), which suggests that ROS mediate
TNF p75 receptor release induced by PMA. In contrast, treatment of Mono
Mac 6 cells with 100 units/ml of superoxide dismutase (SOD) only
slightly attenuated PMA-induced TNF p75 receptor shedding.
|
To define the role of intracellular ROS in PMA-mediated ectodomain
shedding further, Mono Mac 6 cells were pretreated for 10 min with or
without 2 µM of MnPyP, a cell membrane-permeable SOD mimic that can
specifically eliminate superoxide radicals. These cells were incubated
further with PMA (2 h). As shown in Fig. 1B
, MnPyP did not
affect spontaneous TNF p75 receptor shedding in Mono Mac 6 cells.
However, MnPyP significantly attenuated PMA-induced shedding. These
results suggest that intracellular superoxide molecules play a critical
role in PMA-mediated shedding.
To exclude a transcriptional effect of PMA on TACE-dependent ectodomain
shedding, we performed experiments on PMA-stimulated Jurkat T cells and
measured soluble L-selectin by ELISA on culture
supernatants. PMA induced significant shedding of
L-selectin at 30 min and at 2 h. This shedding was not
blocked by cyclohexamide (CHX, Fig. 1C
).
2. H2O2 enhances
TACE-dependent TNF p75 receptor shedding
in monocytes
To test whether ROS can cause TACE-mediated shedding, we
added H2O2 to cultures of Mono Mac 6 cells with
and without addition of the TACE inhibitor, TAPI.
H2O2 induced TNF p75 receptor shedding, and
this was blocked by inhibition of TACE activity (Fig. 2A
). To define further that ectodomain shedding by ROS is
TACE-dependent, we utilized mouse monocytes (DRM-/-) with a targeted
mutation in the TACE gene encoding an in-frame deletion of the zinc
binding domain. DRM-/- monocytes have been shown to lack the ability to
process TACE-specific substrates. These cell lines express equivalent
amounts of surface TNF p75 receptors, measured by flow cytometry. Both
DRM+/+ and DRM-/- cells were incubated with
H2O2, respectively, for 3 h, and
TNF p75 receptor shedding was measured by ELISA. PMA significantly
increased TNF p75 receptor shedding in DRM+/+ cells. However, no TNF
p75 receptor release was observed in PMA-treated DRM-/- cells.
Similarly, H2O2 failed to elicit any TNF p75
receptor shedding in DRM-/- cells.
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3. H2O2 reverses the
inhibitory effect of pro-domain peptide on TACE activity
To test the ability of ROS to activate TACE by oxidization
of the thiol in the inhibitory portion of the pro-domain, we
constructed an in vitro model of unprocessed TACE by using
recombinant active TACE in combination with a TACE inhibitory peptide
(TIP). Purified TACE without the pro-domain was a gift of Dr. Roy Black
(Immunex Corp., Seattle, Wash.). TACE activity was determined by its
ability to cleave a TACE cleavage peptide (TCP), a 10 amino acid
peptide containing a known TACE cleavage site, SPLAQA*VHLPQP, which
TACE cleaves specifically at alanine/valine. A dinitrophenyl group was
attached to at the N terminus of TCP to enhance absorbance at 350 nm.
TIP contains the consensus cysteine switch motif (PKVCGYLK) of the
prodomain that inhibits TACE activity. Uncleaved TCP migrated as a
single peak that eluted at approximately 17 min (Fig. 2C
). Incubation of TCP with TACE (15 min, 37°C)
resulted in almost complete cleavage of the TCP (95 +
1.2%), with a distinct cleavage product that eluted at approximately
15 min (Fig. 2B
). Pre-incubation of TACE with TIP
significantly decreased the TACE activity to 20 + 2.6%
cleavage of the TCP (Fig. 2C
). Incubating the
TACE/TIP complex with 4 mM H2O2 reversed the
inhibitory effect of TIP, restoring the cleavage activity to 91
+ 3.1%. Moreover, the reversal of TIP inhibition by
H2O2 was specific to TIP, as
H2O2 was ineffective in reversing the
hydroxamate-based TACE inhibitor TAPI (not shown).
CONCLUSIONS
We demonstrated that PMA induced both ROS generation and ectodomain shedding in monocytic cells and T cells. ROS scavengers significantly attenuated PMA-induced ectodomain shedding. Furthermore, exogenous ROS were capable of independently enhancing ectodomain shedding. Taken together, these data suggest that ROS are involved in PMA-induced ectodomain shedding.
Like most metalloproteinases, TACE is synthesized in a latent form. This latency is the result of an intramolecular complex between the cysteine residue in their pro-domain and the zinc atom in the catalytic domain. Milla and colleagues have shown that, in insect cells, removal of the cysteine-rich domain located between the catalytic and transmembrane domains results in the secretion of an inactive form of TACE, which consists of a mature catalytic domain in association with the pro-domain. Functional activity of this complex was restored after dissociation of the complex by treatment with 4-aminophenylmercuric acetate, which suggests that a sulhydral group was involved in the inhibitory complex. Our studies corroborate these findings and further demonstrate that TACE, like other metalloproteinases, could be activated by the dissociation of an inhibitory pro-domain by oxidation of its thiol group that coordinates with and masks the zinc in the catalytic domain. For other metalloproteinases, this activation is referred to as the ‘cysteine switch’ mechanism. Numerous free radicals have been suggested to activate metalloproteinases via oxidizing the cysteine thiol group in the pro-domain.
Consistent with this finding, our in vitro TACE
cleavage assay clearly demonstrates that ROS can directly activate TACE
by oxidizing the inhibitory pro- domain, which explains a potential
mechanism for PMA-induced ectodomain shedding as is illustrated in
Fig. 3
.
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Elevation of ROS and enhanced shedding of TACE-dependent substrates, such as L-selectin and TNF receptors, has been found in numerous physiological and pathological states. We have shown that ROS can activate TACE-mediated ectodomain shedding by a post-transcriptional mechanism. Although this study was conducted in monocytic cells and lymphocytes, the results could be generalized to other cell systems and have potential important biological implications concerning regulation and activation of a variety of cellular membrane metalloproteinases.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0371fje To cite this
article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0371fje 
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