EXPERIMENTAL AND THERAPEUTIC MEDICINE 5: 438-442, 2013
Abstract. This study tested the hypothesis that membrane-teth-
ered type-1 matrix metalloproteinase (MT1-MMP)-induced
proteolysis of T cell CD44 is important for defining the
migration and function of autoreactive T cells, including
diabetogenic, insulin-specific and Kd-restricted IS-CD8+
cells. To confirm the importance of MT1-MMP proteolysis
of CD44 in type 1 diabetes (T1D), the anti-diabetic effects
of three MMP inhibitors (3(S)-2,2-dimethyl-4[4-pyridin-4-
hydroxamate [AG3340], 2-(4-phenoxyphenylsulfonylmethyl)
thiirane [SB-3CT] and epigallocatechin-3-gallate [EGCG])
were compared using an adoptive diabetes transfer model in
non-obese diabetic (NOD) mice. Only AG3340 was capable
of inhibiting both the activity of MT1-MMP and the shedding
of CD44 in T cells; and the transendothelial migration and
homing of IS-CD8+ T cells into the pancreatic islets. SB-3CT
and EGCG were incapable of inhibiting T cell MT1-MMP
efficiently. As a result, AG3340 alone, but not SB-3CT or
EGCG, delayed the onset of transferred diabetes in NOD
mice. In summary, the results of the present study emphasize
that the MT1-MMP-CD44 axis has a unique involvement
in T1D development. Accordingly, we suggest that a potent
small-molecule MT1-MMP antagonist is required for the
design of novel therapies for T1D.
The pathogenesis of type 1 diabetes (T1D) involves the activa-
tion of autoimmune T killer cells within the pancreas-draining
lymph nodes. Activated autoimmune T cells then leave the
regional lymphatics, enter the bloodstream and gradually
transmigrate from the bloodstream through the pancreatic
endothelium and into the islets of Langerhans where they
destroy insulin-producing β cells (1). The dynamic interaction
of T cell CD44 with its endothelial ligand (a non-sulfated linear
hyaluronan glycosaminoglycan) is essential for accomplishing
the firm adhesion of T cells to the pancreatic endothelium and
then for the transendothelial migration and subsequent homing
of the adherent T cells into the islets (2-4).
Our previous work and the studies of others have suggested
that the invasion-promoting membrane type-1 matrix metal-
loproteinase (MT1-MMP) (5) dynamically regulates the
functionality of the cell surface-associated signaling and
adhesion receptor CD44 in cancer cells and diabetogenic
T cells (6-9). By means of the regulatory proteolysis of CD44,
MT1-MMP mediates the transition from T cell adhesion to
endothelial cells to T cell transmigration. When combined,
these cellular processes result in the sustained homing of
autoreactive T cells into the pancreatic islets. As a result, the
efficiency of T cell homing in the islets is directly propor-
tional to the severity of the diabetic disease. The inhibition
of MT1-MMP proteolysis of CD44 drastically reduced the
diabetogenic efficiency of T cells, immobilized T cells on the
endothelium, repressed the homing of diabetogenic T cells into
the pancreatic islets, reduced insulitis and mononuclear cell
infiltration and promoted the recovery of the insulin-producing
β cells in non-obese diabetic (NOD) mice with freshly devel-
oped T1D. The importance of the MT1-MMP-CD44 axis in
T1D has thus been identified in a diabetes transfer model with
NOD mice and in freshly diabetic NOD mice (Savinov, 2005
A highly potent MMP inhibitor, 3(S)-2,2-dimethyl-4[4-
acid hydroxamate (AG3340), has been used previously to effi-
ciently control T cell MT1-MMP activity (6,9). The Ki values
of AG3340 against MMP-2, MMP-3, MMP-13 and MT1-MMP
are ~100, 300, 40 and 200 pM, respectively. Other individual
Targeting the T‑cell membrane type‑1 matrix
metalloproteinase‑CD44 axis in a transferred
type 1 diabetes model in NOD mice
ALEXEI Y. SAVINOV1 and ALEX Y. STRONGIN2
1Sanford Research, University of South Dakota, Sioux Falls, SD 57105;
2Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
Received September 18, 2012; Accepted October 25, 2012
Correspondence to: Dr Alex Strongin, Sanford-Burnham Medical
Research Institute, 10901 North Torrey Pines Road, La Jolla, CA
Abbreviations: AG3340, 3(S)-2,2-dimethyl-4[4-pyridin-4-yloxy-
benzenesulfonyl]-thiomorpholine-3-carboxylic acid hydroxamate;
DiI, didodecyl-tetramethylindocarbocyanine perchlorate; EGCG,
epigallocatechin-3-gallate; MMP-2, matrix metalloproteinase-2;
MMP-9, matrix metalloproteinase-9; MT1-MMP, membrane type-1
matrix metalloproteinase; NOD, non-obese diabetic; SB-3CT,
2-(4-phenoxyphenylsulfonylmethyl)thiirane; T1D, type 1 diabetes
Key words: CD44, proteolysis, type 1 diabetes, metalloproteinases,
migration, T cell homing
SAVINOV and STRONGIN: MT1-MMP PROTEOLYSIS OF CD44 IN TYPE 1 DIABETES
MMPs are significantly less sensitive to AG3340 inhibition
(e.g. the Ki values for MMP-1 and MMP-7 are 10 and 55 nM,
respectively). AG3340 was used as an oral anti-angiogenic
drug in phase I-III clinical trials in humans with advanced
non-small cell lung cancer and prostate cancer. The trials were
halted due to the drug's lack of effectiveness in patients with
the late-stage disease (10).
To shed additional light on the physiological significance
of the MT1-MMP-CD44 axis in the homing of diabetogenic
T cells and also on the importance of the specific T cell
MT1-MMP-dependent targeting of CD44, the anti-diabetic
potencies of two broad-range non-hydroxamate MMP inhibi-
tors [2-(4-phenoxyphenylsulfonylmethyl)thiirane (SB-3CT)
and epigallocatechin-3-gallate (EGCG)] were tested using a
transferred diabetes model in NOD mice. SB-3CT and EGCG,
however, do not inhibit MT1-MMP efficiently. SB-3CT
exhibits a dithiolate moiety that chelates the active-site zinc.
While SB-3CT is an effective and selective MMP-2/MMP-9
gelatinase inhibitor, it either does not inhibit or poorly inhibits
other MMPs and the closely related metalloprotease TACE
(tumor necrosis factor α-converting enzyme) (11,12). EGCG,
a major catechin of green tea, also exhibits inhibitory, albeit
largely non-specific, effects on MMPs (13-18). Due to their
proven ability to transfer diabetes to NOD mice effectively
and rapidly (6,19,20), highly diabetogenic, insulin-specific,
CD8-positive, Kd-restricted T cells of the TGNFC8 clone
(IS-CD8+ T cells) were used in the present study. The results
demonstrated that the MT1-MMP-targeting inhibitor AG3340,
but not SBC3T and EGCG (despite their potency against MMPs
distinct from MT1-MMP), exhibited a significant anti-diabetic
action. The specific effect of AG3340 demonstrates the impor-
tance of the MT1-MMP-CD44 axis in diabetogenesis, thus
making T cell MT1-MMP a promising drug design target for
Materials and methods
General reagents. Reagents were from Sigma (St. Louis, MO,
USA) unless indicated otherwise. AG3340 was a gift of Dr
Peter Baciu (Allergan, Irvine, CA, USA). SB-3CT (an inhibitor
of MMP-2 and MMP-9) and α1-antitrypsin were purchased
from Calbiochem (La Jolla, CA, USA).
Mice and cells. NOD/LtJ mice were from the Jackson Laboratory
(Bar Harbor, ME, USA). IS-CD8+ cells (insulin-specific,
CD8-positive, Kd-restricted T cells of the TGNFC8 clone from
the NOD mouse pancreas) (20) were maintained in Click's
medium supplemented with 5% fetal calf serum, 2x105 M
β-mercaptoethanol, 20 mM penicillin-streptomycin, 3 mg/ml
L-glutamine and 5 U/ml murine interleukin-2. Every 3 weeks,
the IS-CD8+ cells were stimulated with irradiated NOD
splenocytes (2000 Rad) loaded with the L15YLVCGERG23
insulin B chain peptide (10 µg/ml) (19).
Induction of diabetes. Mice received AG3340 (1 mg/kg),
SB-3CT or EGCG (10 or 100 mg/kg) or PBS IP. After 30 min,
IS-CD8+ cells (1x107) in PBS were injected IV into the irradi-
ated (725 Rad, 24 h in advance) 5-8-week-old male recipient
mice (5-6 animals/group). Afterwards, the mice received one
injection of their respective inhibitor every other day until
they developed diabetes (1-2 weeks). The onset of diabetes
was monitored by measuring urine and blood glucose levels
with Diastix strips and a glucose meter (Fisher Scientific,
Hampton, NH, USA), respectively. Mice with urine glucose
levels ≥300 mg/dl for 3 consecutive days were considered to
be diabetic. The animal treatment protocols were approved by
the institutional Animal Care Committee.
Fluorescent labeling of IS-CD8+ cells. IS-CD8+ cells
(1x107/ml) were labeled with a fluorescent didodecyl-tetra-
methylindocarbocyanine perchlorate (DiI) dye. DiI-labeled
cells (1x107) were injected IV into irradiated (725 Rad,
24 h in advance) 5-8-week-old mice (4-5 mice/group). The
mice received AG3340 (1 mg/kg), SB-3CT or EGCG (10 or
100 mg/kg) or PBS IP 30 min prior to the cell injection. After
24 h, the pancreata were removed from euthanized mice, fixed
in 0.1 M periodate-lysine-paraformaldehyde phosphate buffer,
sucrose-saturated and freeze-molded in OCT compound
(Sakura Finetek, Torrance, CA, USA). Each pancreas was cryo-
sectioned into 7 µm-sections separated by a 60 µm-interval.
DiI-labeled cells were counted by a blinded observer using a
fluorescence microscope and the cell positions relative to the
islet boundary were recorded. The DiI-cells localized within
the islet boundary were considered to be ‘inside’. The DiI-cells
adjacent to an islet but outside of the islet boundary were
considered to be ‘at the entrance’ of the islet (9,19).
Cell biotinylation. IS-CD8+ cells were surface biotinylated
with sulfo-NHS-LC-biotin (Pierce, Rockford, IL, USA) (9),
re-suspended in serum-free Click's medium supplemented
with AG3340 (50 µM), SB-3CT (100 µM) or EGCG (50, 100
and 500 µM) and allowed to adhere for 4 h to plastic coated
with 2% type I collagen. The cells were then lysed using
50 mM N-octyl-β-D-glucopyranoside (9). Biotin-labeled
CD44 was captured on streptavidine-agarose beads from both
the cell lysate and medium samples. The captured samples
were examined by western blotting with the CD44 antibody
(clone IM7.8.1; BD Biosciences, Franklin Lakes, NJ, USA).
MMP-2 activation. IS-CD8+ cells (1x106) in serum-free Click's
medium were supplemented with purified MMP-2 (20 ng)
and allowed to either adhere for 18 h to plastic coated with
2% type I collagen or remain in solution. AG3340 (50 µM),
SB-3CT (100 µM) or EGCG (50, 100 and 500 µM) were
added to the cells. After 18 h, 30 µl samples of medium were
withdrawn and analyzed by gelatin zymography to identify the
Cleavage of α1-antitrypsin. α1-antitrypsin (250 ng) was
co-incubated for 2 h at 37˚C with p-aminophenylmercuric
acetate-activated MMP-2 (7 ng) (21,22). The reactions were
stopped using 2% SDS and analyzed using 10% gel electro-
phoresis followed by Coomassie staining.
Results and discussion
MT1-MMP sheds CD44 in T cells. To demonstrate MT1-MMP
proteolysis of T cell CD44, IS-CD8+ T cells were surface
biotinylated with membrane-impermeable biotin. The labeled
cells were then allowed to either adhere to a gelatin-coated
EXPERIMENTAL AND THERAPEUTIC MEDICINE 5: 438-442, 2013
plastic or were kept in solution. The cells were then lysed
and biotin-labeled CD44 was captured from the cell lysate
and medium samples using streptavidine-agarose beads. The
captured samples were examined by western blotting with the
CD44 antibody to measure the level of the released, soluble
CD44 ectodomain and the residual, membrane-anchored,
cellular CD44 in the medium and the cell lysates, respec-
tively. In addition, media samples were analyzed by gelatin
zymography to determine the activation status of MMP-2.
The soluble MMP-2 proenzyme is known to be directly
activated by cellular MT1-MMP (21,23). To inhibit cellular
MT1-MMP and, as a result, to repress the conversion of the
MMP-2 proenzyme into the enzyme, the IS-CD8+ T cells,
where indicated, were supplemented with AG3340, SB-3CT
or EGCG (Fig. 1).
Consistent with previous observations (6,9), endogenous
MT1-MMP was latent in non-adherent T cells, while the
adhesion of T cells induced the activation of MT1-MMP.
MT1-MMP activation resulted in the subsequent activation
of exogenous MMP-2 and the cleavage of T cell CD44. By
contrast, non-adherent T cells did not activate MMP-2 and
shed cell CD44 inefficiently. AG3340 fully blocked the activa-
tion of MMP-2 and shedding of CD44 in adherent IS-CD8+
T cells. SB-3CT (an inefficient inhibitor of MT1-MMP) had no
significant effect on either MMP-2 activation or CD44 shed-
ding, while only an exceedingly high (500 mM) concentration
of EGCG demonstrated a partial inhibition of MMP-2 activa-
tion without any significant effect on CD44 proteolysis.
SB-3CT was highly potent at inhibiting MMP-2 proteolysis
of α1-antitrypsin (a sensitive and readily available protein
substrate of MMPs) (24-26). In the absence of SB-3CT, MMP-2
proteolysis led to conversion of the 61 kDa α1-antitrypsin
serpin into the 55 kDa degradation fragment that represented
the N-terminal portion of the α1-antitrypsin molecule. In
turn, a nanomolar range of concentrations of SB-3CT totally
blocked the cleavage of α1-antitrypsin in vitro (Fig. 1).
AG3340 inhibits the intra-islet homing of IS-CD8+ cells in
NOD mice. To determine the anti-diabetic potential of the
SB-3CT and EGCG non-MT1-MMP inhibitors relative to that
of AG3340, NOD mice received an IP injection of the indicated
concentrations of SB-3CT, EGCG or AG3340. DiI-labeled
IS-CD8+ cells were then injected IV into the NOD mice. After
24 h, labeled IS-CD8+ cells were counted at the periphery and
inside the islets (Fig. 2). In the absence of AG3340, T cells
efficiently transmigrated into the islets. By contrast, in the
presence of AG3340 T cells were detected at the islet entrance.
A few cells were found inside the islets. SB-3CT and EGCG,
which were used at a much higher concentration than AG3340,
did not affect the homing of IS-CD8+ cells into the pancreatic
islet (Fig. 3).
MT1-MMP inhibitor delays development of transferred
diabetes in NOD mice. To corroborate the results further,
IS-CD8+ cells were injected in NOD mice. Prior to the cell
injection (30 min), the mice received either the inhibitors or
Figure 1. AG3340 inhibits MT1-MMP and the shedding of CD44 in IS-CD8+ T cells. (Upper panel) Gelatin zymography of MMP-2. To analyze the activation
of MMP-2 by cellular MT1-MMP, adherent (A) and non-adherent (NA) IS-CD8+ cells were each incubated for 18 h in serum-free medium. Purified MMP-2
(20 ng) was added to the cells. The activation of MMP-2 was analyzed by gelatin zymography of the medium aliquots to observe the conversion of the
68 kDa MMP-2 proenzyme into the 62 kDa MMP-2 mature enzyme. Where indicated, AG3340, SB-3CT or EGCG were added to the cells for 18 h. (Middle
panel) Western blotting of CD44. IS-CD8+ cells were surface-biotinylated and were then either allowed to adhere, in serum-free medium, to plastic coated
with type I collagen/gelatin (adherent, A) or remained in suspension (non-adherent, NA). Where indicated, AG3340, SB-3CT or EGCG were added to the cells.
Cell lysate and medium samples were captured with streptavidin-agarose beads. CD44 was analyzed in the captured sample aliquots (50 mg total protein
each) by western blotting with an antibody to the CD44 ectodomain. (Bottom panel) MMP-2 is inhibited by low concentrations of SB-3CT. α1-Antitrypsin
was incubated with MMP-2. The digest samples were analyzed by reducing SDS-gel electrophoresis. Where indicated, SB-3CT was added to the samples.
AG3340, 3(S)-2,2-dimethyl-4[4-pyridin-4-yloxy-benzenesulfonyl]-thiomorpholine-3-carboxylic acid hydroxamate; MT1-MMP, membrane type-1 matrix
metalloproteinase; MMP-2, matrix metalloproteinase-2; SB-3CT, 2-(4-phenoxyphenylsulfonylmethyl)thiirane; EGCG, epigallocatechin-3-gallate.
SAVINOV and STRONGIN: MT1-MMP PROTEOLYSIS OF CD44 IN TYPE 1 DIABETES
PBS (control) IP. The inhibitor injections continued every
other day until the mice developed diabetes. AG3340 at a
concentration as low as 1 mg/kg delayed the onset of diabetes
approximately 2-fold compared with the control (Fig. 3). By
contrast, there was no delay of the transferred diabetes onset
in mice which received SB-3CT and EGCG, which are potent
inhibitors of MMPs other than MT1-MMP.
As has been shown previously in the context of a type
2 diabetes rat model, MMP-2, MMP-12 and MT1-MMP
are upregulated in diabetic males and high-fat-fed
female Zucker diabetic fatty rats as compared with their
non-diabetic lean counterparts (27). PD166793 [(S)-2-
(4'-bromo-biphenyl-4-sulfonylamino)-3-methyl butyric acid; a
broad-range inhibitor with EC50 values of 6100, 47, 12, 7200,
7900, 8 and 240 nM against MMP-1, MMP-2, MMP-3, MMP-7,
MMP-9, MMP-13 and MT1-MMP, respectively] (28,29)
preserved β cell mass, presumably, by affecting the turnover of
certain extracellular matrix molecules in the islets. Despite the
fact that the mechanisms of the protective effects and relative
importance of the individual targets of the MMP inhibitors
in T1D and in type 2 diabetes are not completely understood,
it is clear that in a transfer diabetes model in NOD mice only
AG3340, the antagonist of MT1-MMP, delivered clinically
relevant effects. Due to the wide-range specificity of the MMP
inhibitors, only a simultaneous assessment of AG3340, SB-3CT
and EGCG permitted us to conclude that T cell MT1-MMP is
predominant in T1D. Based on these data, it is likely that the
combined effect of the individual MMPs, including MMP-2
Figure 3. AG3340 inhibits transendothelial migration of IS-CD8+ T cells and delays the onset of transferred diabetes in NOD mice. (A) AG3340 inhibits the
transmigration of IS-CD8+ cells into the pancreatic islets. Mice received AG3340, SB-3CT, EGCG or PBS 30 min prior to the injection of the cells. IS-CD8+
cells were labeled with DiI and then injected in NOD mice. In 24 h, the labeled cells with their intra-islet location were counted in the cryostat sections of the
entire pancreas. (B) AG3340 delays the onset of adoptively transferred diabetes in NOD mice. IS-CD8+ cells were injected in NOD mice. Mice received AG3340,
SB-3CT,EGCG or PBS by one injection every other day until they developed diabetes (approximately 1-2 weeks). The onset of diabetes was monitored daily
by measuring urine glucose levels with Diastix reagent strips. Mice with urine glucose levels of ≥300 mg/dl for 3 consecutive days were considered diabetic.
* P=0.02, **P=0.015 by Fisher's test. AG3340, 3(S)-2,2-dimethyl-4[4-pyridin-4-yloxy-benzenesulfonyl]-thiomorpholine-3-carboxylic acid hydroxamate; NOD,
non-obese diabetic; SB-3CT, 2-(4-phenoxyphenylsulfonylmethyl)thiirane; EGCG, epigallocatechin-3-gallate; DiI, didodecyl-tetramethylindocarbocyanine
Figure 2. AG3340 inhibits the intra-islet homing of IS-CD8+ T cells. NOD mice were treated with AG3340, SB-3CT or EGCG by injection. In 30 min, this
injection was followed by the injection of DiI-labeled IS-CD8+ T cells. After 24 h, the cryo-sections of the pancreata were examined using a fluorescence
microscope. The DiI-labeled cells were ascribed their position, either at the entrance of the islet or inside the pancreatic islets, and counted. At least 100
islets per mouse (4-5 mice/group) were examined. The islets are easily recognized by their morphological characteristics including lower fluorescence and a
compact, dense, structure. Representative images of the pancreatic islets from NOD mice that received an injection of DiI-labeled cells are shown. AG3340,
3(S)-2,2-dimethyl-4[4-pyridin-4-yloxy-benzenesulfonyl]-thiomorpholine-3-carboxylic acid hydroxamate; SB-3CT, 2-(4-phenoxyphenylsulfonylmethyl)thi-
irane; EGCG, epigallocatechin-3-gallate; NOD, non-obese diabetic; DiI, didodecyl-tetramethylindocarbocyanine perchlorate.
EXPERIMENTAL AND THERAPEUTIC MEDICINE 5: 438-442, 2013 Download full-text
and MMP-9, which are distinct from MT1-MMP and effi-
ciently inhibited by SB-3CT, is less important. We conclude
that MT1-MMP antagonists would be efficient in delaying
T1D transfer into NOD mice. These results demonstrate
the functional importance of the MT1-MMP-CD44 axis in
mediating the efficiency of transendothelial migration and the
homing of diabetogenic T cells into the pancreatic islets (30).
These current findings, particularly when combined with
our prior results (6,9), provide a working hypothesis for the
novel, anti-diabetic, application of the sharply focused,
specific inhibitors of MT1-MMP. The data suggest that the
inhibition of T cell MT1-MMP is a step forward in the design
of novel and effective therapies for T1D. It is now likely that
the pharmacological inhibition of MT1-MMP by specific
antagonists will diminish the homing of T killer cells into
the islets. Consequently, is possible that this favorable event
would stimulate the regeneration of insulin-producing β cells
in the islets (9), leading to a more positive outcome for T1D
This study was supported by National Institutes of Health
grants CA83017, CA77470 and RR020843 (Strongin AY) and
JDRF grant 262008-276 (Savinov AY).
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