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Differential Calcium Independent Regulation

of Matrix Metalloproteinases and Tissue Inhibitors


of Matrix Metalloproteinases by Interleukin-1␤ and
Transforming Growth Factor-␤ in Peyronie’s Plaque Fibroblasts
Marcello Del Carlo,* Ada A. Cole* and Laurence A. Levine†,‡
From the Departments of Biochemistry and Urology (LAL), Rush University Medical Center, Chicago, Illinois

Purpose: Peyronie’s disease is a fibrotic disorder of the tunica albuginea characterized by the localized formation of an
inelastic plaque. We characterized matrix metalloproteinases and TIMPs (tissue inhibitors of matrix metalloproteinase) in
Peyronie’s disease tissue.
Materials and Methods: Matrix metalloproteinases and TIMPs were investigated in Peyronie’s disease plaque tunica
removed from patients with stable Peyronie’s disease. Immunological methods were used to characterize the matrix
metalloproteinases and TIMPs produced by cell cultures stimulated with transforming growth factor-␤ or interleukin-1␤
(PreproTech, Rocky Hill, New Jersey). Enzyme activity was quantified with a fluorescent substrate and correlated with
mRNA levels using real-time polymerase chain reaction.
Results: Interleukin-1␤ significantly induced (p ⬍0.01) matrix metalloproteinase-1, 3, 10 and 13 protein production,
endogenous matrix metalloproteinase-13 activity (12-fold) and matrix metalloproteinase-13 mRNA expression (11.2-fold)
through a Ca2⫹ independent mechanism in cultured fibroblasts. Transforming growth factor-␤ stimulation failed to induce
any detectable matrix metalloproteinase protein production or activity and conditioned culture medium even had the capacity
to inhibit (p ⬍0.01) the activity of purified recombinant human matrix metalloproteinase-13. Intact Peyronie’s disease
plaques were highly enriched with TIMP-1 to 4 compared to donor matched perilesional tunica.
Conclusions: These data show that, while interleukin-1␤ strongly induces matrix metalloproteinase expression, transform-
ing growth factor-␤ strongly induces TIMP expression without any effect on matrix metalloproteinases and may represent an
important downstream biochemical mechanism that leads to the progression of Peyronie’s disease. The localized accumula-
tion of TIMPs together with decreased matrix metalloproteinase activity in the Peyronie’s disease lesion may be the
biochemical consequence of the transforming growth factor-␤ over expression that has been reported in many fibrotic
disorders, including Peyronie’s disease.

Key Words: penis, penile induration, matrix metalloproteinases, tissue inhibitor of metalloproteinases, microarray analysis

eyronie’s disease predominantly affects men older mote matrix degradation are down-regulated.4 While many

P than 50 years with an estimated prevalence of up to


8.9% with an incidence as high as 21% in patients
diagnosed with Dupuytren’s disease, suggesting a genetic
studies in the literature have focused on the anabolic path-
ways that promote collagen production, to our knowledge no
study has ever focused on the equally important catabolic
predisposition to these fibrotic disorders.1,2 PD is character- activity of MMPs in PD tissue.
ized by the formation of a palpable fibrotic lesion in a local- The types I and III collagen fibers that comprise PD
ized area of the tunica albuginea, consisting mainly of types plaque are extremely resistant to proteolytic degradation.5
I and III collagen.3 A recent report using various DNA To date only the collagenases in the MMP-1, 8 and 13 family
microarrays showed that genes involved in collagen synthe- of mammalian enzymes have been shown to substantially
sis are up-regulated in PD tissue, whereas genes that pro- degrade types I and III fibrillar collagen.6 Human MMP-13
is unique among these 3 collagenases, in that it is also an
extremely efficient gelatinase, which enables it to further
Submitted for publication September 10, 2007. degrade collagen into several smaller and more diffusible
Study received Rush University Medical Center institutional re- peptides.7 Because collagen is the most abundant protein in
view board approval.
Supported by The Dr. Scholl Foundation and The Taylor Founda- the body, MMPs are tightly regulated enzymes.5–7 For ex-
tion. ample, most MMPs are not constitutively expressed and
* Financial interest and/or other relationship with Auxilium. they must be induced by external stimuli, such as growth
† Correspondence: Department of Urology, Rush University Medical
Center, 1725 West Harrison, Suite 352, Chicago, Illinois 60612 (tele- factors, cytokines or physical stress.6,7 MMPs are also ini-
phone: 312-563-5000; FAX: 312-563-5007; e-mail: drlevine@hotmail. tially synthesized as inactive zymogens and they must be
com). activated by the enzymatic removal of a regulatory prodo-
‡ Financial interest and/or other relationship with Pfizer, Auxi-
lium, American Medical Systems, Augusta Medical Systems, Colo- main.6,7 After activation MMP activity can be inhibited by
plast/Mentor and Physiomed. TIMP binding.6 – 8 TIMPs are small proteins approximately

0022-5347/08/1796-2447/0 2447 Vol. 179, 2447-2455, June 2008


THE JOURNAL OF UROLOGY® Printed in U.S.A.
Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION DOI:10.1016/j.juro.2008.01.093
2448 DIFFERENTIAL REGULATION IN PEYRONIE’S PLAQUE FIBROBLASTS

25 kDa in size with a high affinity for MMPs and they also MMP Protein Microarrays and Immunoblot Analysis
represent the principal extracellular regulatory mechanism Conditioned medium from lesional PD fibroblasts were nor-
for MMP activity in mammalian tissue.8 malized for total protein with the BCA™ Protein Assay.
We characterized MMPs and TIMPs in PD tunica. TGF-␤ MMP protein production was analyzed with MMP antibody
has been associated with a wide variety of fibrotic disorders, microarrays 48 hours after stimulation with TGF-␤ or IL-1␤
including PD, and, therefore, it was used to determine its (10 ng/ml) according to the manufacturer protocol (RayBio-
effect on MMP and TIMP production in cultured PD fibro- tech, Atlanta, Georgia). Immunoreactive spots were quanti-
blasts.9 IL-1␤ is an inflammatory cytokine that is well- fied as a function of pixel density and data are expressed as
known for its ability to induce MMP activity and it was used the ratio between cytokine stimulated and baseline MMP
to determine whether collagenase production can be induced production.
in PD fibroblasts derived directly from the PD lesion.10,11 In a separate set of experiments equal amounts of total
protein were separated by sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis on 10% acrylamide gel and then
transferred to nitrocellulose for immunoblot analysis. MMP
MATERIALS AND METHODS
and TIMP proteins were extracted from intact tunica in
Tissue Acquisition and Cell Culture select studies with 4 M guanidinium hydrochloride includ-
Postoperative tunical plaques were obtained from 36 pa- ing protease inhibitors (1 mM ethylenediaminetetraacetic
tients with a mean age of 55.6 years diagnosed with stable acid, 1 mM iodoacetamide, 1 mM phenylmethylsulfonyl flu-
PD (mean history 2.25 years) within 1 hour of surgical oride and 5 ␮g/ml pepstatin A) and 1% CHAPS in 50 mM
excision with the approval of the Rush University Medical sodium acetate (pH 5.8) before extracts were dialyzed
Center institutional review board and informed consent. PD against phosphate buffered saline (pH 7.4) and normalized
plaque fibroblasts were enzymatically liberated with 0.05% for total protein with the BCA protein assay. Urea (0.5 M)
bacterial collagenase in DMEM/F-12 medium supplemented was included to improve protein solubility. Extracts from 2
with 5% fetal bovine serum (Invitrogen™) in the presence of to 8 patients were always included for each immunoblot
penicillin, streptomycin and amphotericin B. Monolayers analyzed. Rabbit anti-human MMP reactive against the
were established in DMEM/Hamm’s F-12 medium supple- hinge region and TIMP antibodies, including MMP-1 (AB813),
mented with 10% fetal bovine serum and maintained in MMP-8 (AB8115), MMP-13 (AB8114), TIMP-1 (AF970),
culture for approximately 7 days before experimental use. TIMP-2 (AF971), TIMP-3 (AF973) and TIMP-4 (AF974)
Confluent monolayers were serum starved in only DMEM- (Chemicon®) were used as previously described.12 Immuno-
F12 for 18 hours overnight with an additional serum-free reactivity for the 2 assays was visualized with enhanced
medium change immediately before stimulation with IL-1␤ chemiluminescence (Amersham, Piscataway, New Jersey)
or TGF-␤1 (10 ng/ml) for 48 hours. The cell permeable Ca2⫹ and quantified using a VersaDoc™ MP Imaging System
chelator BAPTA acetoxymethyl ester was pre-incubated equipped with Quantity One® software.
with the cells for 30 minutes before cytokine stimulation in
select experiments at the maximal sublethal dose of 5 ␮M. MMP-13 Activity Assay
Conditioned medium was stored up to 1 week at 4C with Specific MMP-13 activity was assessed in conditioned me-
0.05% sodium azide. No cell death was observed under any dium using the Fluorokine™ E Human Active MMP-13 Flu-
experimental conditions tested using the LIVE/DEAD® cell orescent Assay according to the manufacturer protocol.
viability assay. Briefly, antibody captured MMP-13 activity was based on
the cleavage of a fluorogenic substrate and enzyme concen-
tration was calculated using a standard curve that was
Tissue Acquisition and In Situ Protein Extraction generated using rhMMP-13.
In a separate set of experiments postoperative tunical
plaques were obtained from 13 patients with a mean age MMP-13 Inhibition Assay
of 52.1 years with PD together with donor matched tunical Purified rhMMP-13 was serially diluted with 1) fresh serum-
grafts taken approximately 1.0 to 1.5 cm away from the free medium, 2) serum-free medium incubated with PD fi-
macroscopically distinct palpable PD lesion. NonPD tuni- broblasts for 48 hours or 3) serum-free medium incubated
cal tissue was also obtained from 6 patients with a mean with cells for 48 hours in the presence and absence of IL-1␤
age of 54.1 years diagnosed with erectile dysfunction with- or TGF-␤ (10 ng/ml). Samples were incubated in a 96-well
out PD who were undergoing prosthesis implantation and plate for 16 hours at 37C with 1 mM of the quenched fluo-
from 2, 21-year-old human donors within 24 hours of rescent peptide Mca-PLGL-Dpa-AR-NH2 before quantifying
death. Total protein was extracted from pulverized tissues MMP activity as a function of signal intensity, as previously
frozen under liquid nitrogen with 4 M guanidinium hydro- described.13
chloride (Sigma®), supplemented with fresh protease inhibi-
tors (1 mM ethylenediaminetetraacetic acid, 1 mM iodoacet- Real-Time PCR
amide, 1 mM phenylmethylsulfonyl fluoride and 5 ␮g/ml Total RNA was isolated using TRIzol® reagent 8 hours after
pepstatin A) and 1% CHAPS (3-[(3-cholamidopropyl)di- cytokine stimulation. Purified RNA was treated with de-
methylammonio]-1-propane-sulfonate) in 50 mM sodium oxyribonuclease I and cDNA was synthesized from 5 ␮g
acetate (pH 5.8) for 24 hours at 4C with end-over-end RNA using SuperScript™ II reverse transcriptase. Real-
agitation. Extracts were then cleared of insoluble debris time PCR was used to quantify specific mRNA expression
with centrifugation, dialyzed against several changes of using available MMP-13 and MMP-14 primer sets (Super-
phosphate buffered saline and stored at ⫺20C. Array Bioscience, Frederick, Maryland) normalized to GAPDH
DIFFERENTIAL REGULATION IN PEYRONIE’S PLAQUE FIBROBLASTS 2449

(glyceraldehyde-3-phosphate dehydrogenase) expression (5=- tectable increase in MMP production, whereas IL-1␤ is a
TGCCATGGGTGGAATCATATTGG-3= forward and 5=-GT- well-known inflammatory cytokine and it strongly up-regu-
CAACGGATTTGGTCGTATTGGG-3= reverse) under certain lated all 3 collagenases. It was also of interest to determine
conditions, including initial hold at 30C for 10 minutes and whether this observed MMP and TIMP production depended
95C for 10 minutes, followed by cycling for 35 cycles at 95C on intracellular Ca because we and others have reported
for 15 seconds, 65C for 20 seconds and 72C for 1 second with moderate clinical success using intralesional injections of
a 7-minute hold at 72C before cooling to the final hold the L-type Ca channel blocker verapamil to treat PD as an
temperature of 4C. All 3 primers were run simultaneously in alternative to surgical correction.14 The intracellular Ca2⫹
duplicate and data were analyzed with SmartCycler®, ver- chelator BAPTA had no detectable effect on MMP or TIMP
sion 2.0. production, thus, suggesting that TGF-␤ or IL-1␤ signaling
proceeds independently of Ca2⫹ during this 48-hour stimu-
Statistical Analysis lation period (fig. 2).
Group means were analyzed with post hoc Bonferroni cor-
rected 1-way ANOVA using Windows® based SPSS®. MMP MMP-13 Activity in PD Plaque Fibroblasts
microarray data were analyzed using the Student t test with Because the presence of MMP protein does not always
p ⬍0.01 considered significant. correlate with enzyme activity,13 a fluorescent peptide
was used to measure MMP-13 activity in the culture me-
RESULTS dium from PD fibroblasts after stimulation with TGF-␤ or
IL-1␤. IL-1␤ stimulation significantly increased MMP-13
MMP Microarray Analysis in PD Plaque Fibroblasts activity and pre-incubation with the Ca2⫹ chelator
Protein microarrays were used to simultaneously analyze BAPTA did not alter this activity (fig. 3). In contrast,
multiple MMP production in cultured cells stimulated TGF-␤ stimulation had no effect on MMP-13 activity
with TGF-␤ or IL-1␤. TGF-␤ did not induce the expression (fig. 3).
of any MMP included on the microarray membrane above
untreated control levels (fig. 1). In contrast, IL-1␤ stimu-
lation significantly increased the production of MMP-1, 3, TIMP Activity in PD Plaque Fibroblasts
10 and 13 by several orders of magnitude under the same Because TGF-␤ and IL-1␤ stimulation induced the secretion
conditions (fig. 1). of TIMP proteins into the culture medium of isolated PD
fibroblasts, we quantified the inhibitory capacity of this na-
scent TIMP production. The enzyme activity of rhMMP-13
Immunoblot Analysis of MMP
was quantified using quenched Mca-PLGL-Dpa-AR-NH2 flu-
and TIMP Production in PD Plaque Fibroblasts
orescent peptide after being serially diluted in 1) fresh se-
Immunoblots were then used to more accurately character-
rum-free medium, 2) serum-free medium incubated with PD
ize the inducible nature of MMP-1, 8 and 13, and TIMP-1 to
fibroblasts for 48 hours (fig. 4, inset, left arrow) or 3) serum-
4 after stimulation with TGF-␤ or IL-1␤. TGF-␤ did not
free medium incubated with cells for 48 hours in the pres-
up-regulate MMP-1, 8 or 13 production at any concentration
ence of IL-1␤ or TGF-␤ (10 ng/ml) (fig. 4, inset, right arrow).
tested, whereas all 4 TIMPs were up-regulated in a dose
Figure 4 shows that rhMMP-13 activity was not signifi-
dependent manner (fig. 2). IL-1␤ up-regulated MMP-1 and 8
cantly inhibited by unstimulated control medium after 48
equally at all concentrations tested, whereas MMP-13 showed
hours of culture with PD fibroblasts, which was almost iden-
a more dose dependent induction (fig. 2). It is interesting to
tical to the result observed using fresh serum-free control
note that TGF-␤ is a well-known profibrotic growth factor
medium. In contrast, TGF-␤ stimulated medium decreased
and it strongly induced TIMP production without any de-
rhMMP-13 activity by approximately 25% at each of the 3
highest concentrations tested (fig. 4).

Real-Time PCR Quantification


of MMP-13 and 14 mRNA in PD Plaque Fibroblasts
The relative specificity of IL-1 and TGF-␤ for inducing sol-
uble MMP-13 mRNA expression was assessed by comparing
it to that shown by the functionally distinct membrane-type
1 MMP (MMP-14) using real-time PCR to quantify mRNA
isolated from confluent PD monolayers.15 While TGF-␤ stim-
ulation significantly up-regulated MMP-13 mRNA expres-
sion approximately 3-fold, IL-1␤ stimulation showed more
than a 10-fold induction of MMP-13 expression, in addition
to MMP-14 mRNA expression, which was significantly
down-regulated by approximately 6-fold below the untreated
control value (fig. 5). Immunoblot revealed that IL-1␤ stim-
ulated MMP-13 protein production correlated with the in-
FIG. 1. Effect of TGF-␤ and IL-1␤ stimulation on MMP protein creased expression of MMP-13 mRNA (fig. 5, inset). Inter-
production using antibody microarrays. Data represents mean of estingly although TGF-␤ stimulation significantly increased
values in 3 experiments using PD plaques from different patients. MMP-13 mRNA, no MMP-13 protein or activity was de-
Values in parentheses represent decreased expression. Vertical ar-
rows indicate up to 3-fold induction. Asterisk indicates statistically tected in culture medium under the same conditions (fig. 5).
significant (Student’s t test p ⬍0.01). This observation suggests that TGF-␤ mediated MMP-13
2450 DIFFERENTIAL REGULATION IN PEYRONIE’S PLAQUE FIBROBLASTS

l) l) l) A
(ng/m (ng/m l) A /m PT )
TGF-β IL-1β ng
/m
0 A
ng
PT BA(5
uM

L
0 (1 B +

O
(1 A + β
β PT F-

TR
-1 F- -1
BA TG

N
C 0.16 0.63 2.5 10 40 C 0.16 0.63 2.5 10 40 IL TG IL

CO
MMP-1 MMP-1 MMP-1
MMP-8 MMP-8 MMP-8

MMP-13 MMP-13 MMP-13

TIMP-1 TIMP-1 TIMP-1


TIMP-2 TIMP-2 TIMP-2

TIMP-3 TIMP-3 TIMP-3

TIMP-4 TIMP-4 TIMP-4

FIG. 2. Effect of TGF-␤ and IL-1␤ (IL-1) stimulation on MMP and TIMP production in conditioned medium from Peyronie’s plaque fibroblasts.
Proteins were immunoblotted with polyclonal antibodies directed against hinge region of MMP-1, 8 or 13 and with polyclonal antibodies
against TIMP-1, 2, 3 or 4. Immunoblots represent typical result in these tissues. C, control.

protein production is substantially regulated at the post- 54.1 years and 2 cadavers at age 21 years were included as
transcriptional level. additional comparators. Protein extracted from all tunical
sources showed positive immunoreactivity for MMP-1, 8
Immunoblot Analysis of MMP and 13, although only tunica from the 21-year-old cadav-
and TIMP Protein From Human Tissue Extracts ers contained full-length MMP-1, 8 and 13 based on the
A separate set of experiments were performed in intact predicted molecular weight of the active enzyme (fig. 6, A).
tunica from 13 men with a mean age of 52.1 years to The PD plaque and perilesional tunica as well as the
determine the relative distribution of the collagenases nonPD prosthesis tunica mostly contained lower molecu-
(MMP-1, 8 and 13) and TIMPs in their native extracellu- lar weight fragments of the respective collagenase (fig. 6,
lar matrix by comparing protein extracts prepared from A). TIMP-1 to 4 were readily detectable in PD tunica,
plaque tunica to the protein extracted from donor matched whereas only TIMP-1 was detected in the adjacent donor
perilesional tunica taken approximately 1.0 to 1.5 cm matched perilesional tunica (fig. 6, B). Tissue availability
away from the macroscopically distinct palpable PD le- unfortunately precluded similar TIMP analysis in nonPD tu-
sion. NonPD tunica from 6 patients with a mean age of nica.

l) l) A
/m /m ) TA PT
ng ng uM P
O
L
0 0 (5 BA BA
(1 (1 A + +
p < 0.001 TR β PT β F-
β
N - 1β F- -1
1 CO IL TG BA IL TG

MMP-13
0.9

0.8

0.7
MMP-13 (ng/ml)

0.6

0.5

0.4

0.3

0.2

0.1

0
Control IL-1 TGF BAPTA BAPTA + BAPTA +
IL-1 TGF
FIG. 3. Effect of TGF-␤ and IL-1␤ (IL-1) stimulation on MMP-13 activity in conditioned medium from Peyronie’s plaque fibroblasts. Antibody
captured MMP-13 activity was based on fluorescent substrate cleavage. Inset, MMP-13 immunoblot reveals relative quantity of protein in
identical conditioned medium also used for this activity assay. Results represent mean ⫾ SEM of 3 separate experiments using cells from
different patients.
DIFFERENTIAL REGULATION IN PEYRONIE’S PLAQUE FIBROBLASTS 2451

18000
Fresh Serum-Free Media
ng/mL TGF-beta

16000 C 0.16 0.63 2.5 10 40

MMP-13
Fluorescent Units

Control Media
14000 TIMP-2

12000 *
* p < 0.01

10000 *
* TGF-beta Media

8000
0 0.25 0.5 1 2 4 8 16 32 64

rhMMP-13 (ng/ml)

IL-1-stimulated conditioned media


18000
ng/mL IL-1 Fresh Serum-Free Media
C 0.16 0.63 2.5 10 40 Control
16000
MMP-13
Fluorescent Units

14000 TIMP-2
*
IL-1 Media
** p < 0.01
12000
** ** ** *
10000
* * p ≥ 0.168

8000
0 0.25 0.5 1 2 4 8 16 32 64

rhMMP-13 (ng/ml)
FIG. 4. Inhibition of rhMMP-13 activity exogenously added to conditioned medium from TGF-␤ or IL-1␤ (IL-1) stimulated Peyronie’s plaque
fibroblasts. Enzyme activity of purified rhMMP-13 was quantified after serially dilution with 1) fresh serum-free medium, 2) serum-free
medium incubated with PD fibroblasts for 48 hours or 3) serum-free medium incubated with cells for 48 hours in presence or absence of IL-1␤
or TGF-␤ at 10 ng/ml using Mca-PLGL-Dpa-AR-NH2 quenched fluorescent peptide at 1 mM. Results represent mean ⫾ SEM of 3 experiments
in cells from different patients.

DISCUSSION MMP-1, 3, 10 and 13 shown by IL-1␤ stimulation, TGF-␤


had the capacity to mimic profibrotic conditions in cultured
The principal feature that characterizes PD is the formation
of an inelastic fibrotic plaque in a localized area of the penis PD fibroblast by strongly inducing TIMP production without
but the underlying biochemical mechanisms are poorly un- any affect on MMP production. Because increased TGF-␤
derstood.1– 4,9 In the current study we focused on the cata- expression has been observed in PD plaque, our data may
bolic activity of the collagenases (MMP-1, 8 and 13) and the represent the downstream biochemical mechanism that ul-
anti-catabolic activity of TIMPs in PD plaque as well as in timately causes the fibrosis seen in PD tissue.
matched perilesional tunica. We found that, while plaque Normal tissue homeostasis is maintained by a highly
and perilesional PD tunica contained fragments of MMP-1, 8 coordinated interplay between the opposing anabolic and
and 13, only plaque tunica contained significantly higher catabolic pathways that are respectively present in virtually
levels of TIMP protein. In contrast to the strong induction of any extracellular matrix.5,6,8 Most basic research in this
2452 DIFFERENTIAL REGULATION IN PEYRONIE’S PLAQUE FIBROBLASTS

FIG. 5. Effect of TGF-␤ and IL-1␤ (IL-1) stimulation on MMP-13 and MMP-14 mRNA expression in Peyronie’s plaque fibroblasts using
real-time PCR. Confluent monolayers were serum starved for 24 hours and mRNA was isolated 8 hours after cytokine stimulation. mRNA
expression was normalized to GAPDH expression and run simultaneously in 1 experiment using 2, 16-well SmartCyclers in tandem. Bar
graph shows MMP-13 mRNA quantification. Inset, MMP-13 immunoblot reveals relative quantity of protein produced by cells treated under
identical conditions. Bar graph shows MMP-14 mRNA quantification. Results represent mean ⫾ SEM of 4 experiments in mRNA from
different patients.

area has focused on the anabolic pathways that promote anism responsible for the progression of fibrotic disorders,
collagen synthesis.3,4 However, recent transgenic animal including PD.
studies have implicated decreased catabolic activity as the Our results seem to fit squarely within this anti-catabolic
principal biochemical mechanism responsible for certain fi- model of fibrosis. Localized accumulation of TIMP proteins
brotic disorders.16,17 For example, a group studying renal decreases net catabolic activity and it may also be sufficient
fibrosis in transgenic mice observed that TIMP-1 mRNA was to promote fibrosis in and of itself. The near absence of
significantly up-regulated (18-fold) in a fibrotic kidney, full-length MMP-1, 8 and 13 together with the highly local-
whereas the expression of types I and III collagen was barely ized accumulation of TIMPs in the PD plaque can theoreti-
above control values (1.6 and 1.9-fold, respectively).16 A cally shift matrix homeostasis toward conditions favoring a
separate group studying cardiac fibrosis in transgenic mice gradual buildup of matrix proteins. Our emphasis on the
reported significantly lower collagenase production, while collagenases (MMP-1, 8 and 13) was based on their unique
TIMP-1, 2 and 4 were increased 2.5, 6 and 2.5-fold, respec- ability to degrade fibrillar types I and III collagen, which are
tively.17 Thus, these studies seem to suggest that inhibiting a logical starting point when investigating matrix homeosta-
catabolic activity in tissue may in fact be the principal mech- sis in tissues rich in fibrillar collagens. However, it should be
DIFFERENTIAL REGULATION IN PEYRONIE’S PLAQUE FIBROBLASTS 2453

A
MMP-1 MMP-8 MMP-13

75 kDa
57 kDa 60 kDa
57 kDa
48 kDa

32kDa
25 kDa 28 kDa
30 kDa

D
e
D
e
D
e

rs

al
rs

al

rs
al

qu
qu
qu

n-P
n-P
n-P

on
ve

on
on
ve

ve

pla
pla
pla

da

es i
da

es i

da
es i

No
No
No

ca
ca

ca

ri-l
PD
ri-l
PD
ri-l
PD

ld
ld

ld

pe
pe
pe

ro
ro

ro

PD
PD
PD

ea
ea

ea
-y
-y

-y
21
21

21
B
Patient 1 of 13 Patient 2 of 13

TIMP-1

TIMP-2

TIMP-3

TIMP-4
al

al
al
al

ion

ion
on
on

esi
esi

Les

Les
ri-l
ri-l

Pe
Pe

FIG. 6. In situ immunoblot analysis of MMPs and TIMPs in tunical tissue. Protein was extracted from intact PD plaque tunica from 13 men
with a mean age of 52.1 years as well as donor matched perilesional tunica taken approximately 1.0 to 1.5 cm away from the macroscopically
distinct palpable PD lesion. NonPD plaques from 6 patients with a mean age of 54.1 years undergoing prosthesis implantation and 2,
21-year-old cadavers were included as additional comparators. A, arrows indicate where latent pro-MMPs should migrate on respective
immunoblots based on predicted molecular weight and purified standards run in parallel, as indicated. B, immunoblot analysis of TIMP-1
to 4 extracted from Peyronie’s plaques and perilesional tunica represent typical result in these tissues.

noted that other MMPs, such as the gelatinases (MMP-2 and BAPTA significantly affected IL-1␤ and TGF-␤ mediated
9), can continue to degrade type I collagen after the triple mRNA expression, we were unable to detect any changes in
helix has been cleaved by a collagenase.5–7 Further studies MMP or TIMP protein production. We emphasize that these
in this regard are clearly warranted. data do not necessarily preclude the involvement of Ca2⫹ in
The nonspecific intracellular Ca2⫹ chelator BAPTA was TGF-␤ mediated tissue fibrosis because significant evidence
used to determine whether Ca2⫹ is required for these sig- in the literature now suggest that TGF-␤ acts early in this
naling events because the L-type Ca2⫹ channel blocker ve- signaling cascade by up-regulating the production of more
rapamil is currently used as a nonsurgical alternative to downstream cytokines, such as connective tissue growth
treat PD.14,18 Interestingly although pretreatment with factor, which has been shown to be more directly associated
2454 DIFFERENTIAL REGULATION IN PEYRONIE’S PLAQUE FIBROBLASTS

with the deposition of matrix proteins in mammalian tis- animal models, including those of PD.4,9,16,17,19 This obser-
sue.19 Thus, while Ca2⫹ does not appear to be necessary for vation provides support for the hypothesis that most mam-
the immediate early signaling events elicited by TGF-␤, malian fibrotic disorders likely proceed through similar sig-
nonetheless it may be required by other delayed primary naling events. Our results highlight the ability of TGF-␤ to
response signaling events that also substantially contribute strongly induce TIMP production, which is markedly inhib-
to the fibrotic progression of PD. itory to rhMMP-13 without any detectable effect on nascent
The near absence of full-length MMP-1, 8 and 13 together MMP production. Because increased TGF-␤ expression has
with the relative abundance of smaller MMP fragments been specifically observed in the PD lesion,4,9 our results
detected in the PD lesion suggest proteolytic inactivation of may represent a biochemical mechanism downstream from
these matrix enzymes. Several studies have now shown TGF-␤ signaling that is ultimately responsible for the pro-
that, while enzymatic removal of the prodomain activates an gression of PD.
inactive MMP, enzymatic removal of the collagen binding
domain conversely inactivates an active MMP.7,20 For exam-
CONCLUSIONS
ple, the C-terminus of MMP-8 is susceptible to proteolytic
activity and it can be cleaved into lower molecular weight Tissue fibrosis is the end biological result of enhanced ana-
fragments with significantly decreased or completely abol- bolic activity and/or decreased catabolic activity sustained
ished collagenolytic activity.20 Plasmin has been identified for a sufficient period. Our data suggest that the PD lesion is
as one of the few enzymes that are capable of removing the likely the result of a localized accumulation of TIMP pro-
prodomain of MMP-13 for initial activation of the zymogen teins, which function to shift normal matrix homeostasis
as well as further removal of the substrate binding domain toward fibrosis by promoting the gradual deposition of ma-
for subsequent inactivation.7 MMP fragmentation generally trix proteins in a discrete tunical area with time.
represents a highly orchestrated regulatory mechanism,
rather than mere nonspecific protein degradation.
An argument that has been posited against comparing
Abbreviations and Acronyms
PD plaque and perilesional tunica with nonPD erectile dys-
function tunica and cadaver tunica is that it has limited BAPTA ⫽ cell permeable Ca2⫹ chelator
significance in terms of defining the biochemical pathways DMEM ⫽ Dulbecco’s modified Eagle’s medium
that underlie PD. The plaque tunica used in the current IL ⫽ interleukin
MMP ⫽ matrix metalloproteinase
study generally represented stable PD from patients under-
PCR ⫽ polymerase chain reaction
going surgical correction. However, nonPD tissues are PD ⫽ Peyronie’s disease
merely presented as additional comparators using tissue PL ⫽ perilesional tunica
that became available during the course of these studies and rh ⫽ recombinant human
they are in no way offered to represent a normal MMP TGF-␤ ⫽ transforming growth factor-␤
profile that is likely present in healthy tunica albuginea. TIMP ⫽ tissue inhibitor of MMP
Normal tunica that is completely free of all known disease
and physical injury is more of a theoretical construct than a
practical reality. We agree with the proposition that the PD REFERENCES
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