LaMotte, Robert H.

Lerner, Ethan A.

Reddy, Vemuri B.

Shimada, Steven G.

Sikand, Parul

PubMed

Elsevier - Publisher Connector 

Cathepsin S Elicits Itch and Signals via Protease-Activated Receptors 

suggests that further clarification of the
role of PAX3 expression in non-malig-
nant differentiated melanocytes is now
required. Extensive PAX3 expression in
benign and malignant tissues of the
melanocyte lineage, and absence of
expression in other types of skin cancer,
nevertheless suggests that PAX3 could be
used as an immunohistochemical marker
to differentially diagnose melanoma.
Ethics approval for this study was
obtained from the New Zealand Multi-
Regional Ethics Committee.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
The anti-Pax3 antibody was developed by Dr C.
Ordahl and obtained from the Developmental
Studies Hybridoma Bank developed under the
auspices of the NICHD and maintained by the
University of Iowa, Department of Biological
Sciences, USA. This research was supported by
grants from the Health Research Council of New
Zealand and Dunedin School of Medicine.
Shujie He1, Han-Seung Yoon1,
Bong-Jik Suh2 and Michael R. Eccles1
1Department of Pathology, Dunedin School of
Medicine, University of Otago, Dunedin,
New Zealand and 2Department of Oral
Medicine, School of Dentistry, Chonbuk
National University, Jeonju, Korea
E-mail: shujie.he@stonebow.otago.ac.nz
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online
version of the paper at http://www.nature.com/jid
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J Clin Invest 118:2076–87
Cathepsin S Elicits Itch and Signals via Protease-Activated
Receptors
Journal of Investigative Dermatology (2010) 130, 1468–1470; doi:10.1038/jid.2009.430; published online 14 January 2010
TO THE EDITOR
Cathepsin S is a cysteine protease
linked to inflammatory processes, in-
cluding atherosclerosis and asthma.
The possibility that this or other
cysteine proteases might evoke itch or
be part of a classical ligand-receptor
signaling cascade has not been con-
sidered previously. We show here that
human cathepsin S evokes itch and
activates human protease-activated
receptors (PARs) 2 and 4.
The sensation of itch is mediated by
two distinct non-overlapping popula-
tions of cutaneous nerve fibers that
evoke comparable degrees of itch
(Johanek et al., 2008; Namer et al.,
2008). One set of fibers, the mechano-
insensitive population, is more respon-
sive to histamine than to cowhage. The
other set is mechanosensitive and is
more responsive to cowhage than to
histamine (Johanek et al., 2008; Namer
et al., 2008). Histamine is a classical
mediator of itch and is associated with
a wheal and flare. As most clinical
itches do not have a wheal or flare and
do not respond to antihistamines,
histamine is not thought to contribute
to most itches (Ikoma et al., 2006).
Cowhage refers to a tropical legume or,
in this case, the loose hairs that cover
the pods of Mucuna pruriens and evoke
itch. The active component of cowhage
is mucunain, a cysteine protease that
serves as a ligand for PARs 2 and 4
(Reddy et al., 2008). The identification
of an endogenous mediator with prop-
erties similar to cowhage could lead to
insights into non-histamine-mediated
itch. We focused on human cathepsin
S because it shares active site sequence
homology with mucunain and is selec-
tively up-regulated in human keratino-
cytes upon stimulation with interferon-
gamma, consistent with a possible
pruritic role in inflammatory skin dis-
ease (Schwarz et al., 2002).
There are 15 human cathepsins,
including 11 cysteine, 2 aspartic, and
2 serine proteases. Cathepsins were
traditionally considered lysosomal pro-
teases. It is now recognized that the
broad expression and range of pH
dependence of some cathepsins reveal
that they have many functional roles,
including tissue remodeling, metastasis
and inflammation. Examples of cysteine
cathepsin activities include cleavage of
collagen by cathepsin L to generate
endostatin (Felbor et al. 2000), an
endogenous inhibitor of angiogenesis,
and cleavage of the invariant chain in
antigen-presenting cells by cathepsin S
(Nakagawa et al., 1999) as part of the
inflammatory cascade.
There are four PARs and they are
members of the G-protein-coupled
receptor family. Their identified endo-
genous activators are all serine pro-
teases. These proteases trigger the
activation of PARs by unmasking
‘tethered ligand’ sequences near the
N-termini of the receptors. Certain
kallikrein-related peptidases and mastAbbreviation: PAR, protease-activated receptor
1468 Journal of Investigative Dermatology (2010), Volume 130
VB Reddy et al.
Cathepsin S, Itch, and Protease-Activated Receptors
cell tryptase, which are serine pro-
teases, can activate PAR2 in vitro
(Oikonomopoulou et al., 2006, Stefansson
et al., 2008), but they have not been
demonstrated to be endogenous pruritic
agents. Serine proteases and PAR2 have
also been linked to barrier function
(Hachem et al., 2006). The presence of
PAR2 on free nerve endings in the skin,
keratinocytes and dorsal root ganglia link
this receptor to itch and pain (Steinhoff
et al., 2000, 2003; Shpacovitch et al.,
2008). Data on PAR4 are more limited,
revealing that it is expressed in rat dorsal
root ganglia and its activation, at least in
this species, appears to be anti-nocicep-
tive (Asfaha et al., 2007). A PAR4
hexapeptide agonist has recently been
reported to cause scratching in mice
(Tsujii et al., 2008).
Cathepsin S and mucunain are
endogenous and exogenous cysteine
proteases, evoke similar itch and noci-
ceptive sensations, and serve to activate
PARs 2 and 4 (Figures 1 and 2; Reddy
et al., 2008). The relative contribution
of PAR2 versus PAR4 activation in
cathepsin S-induced sensations is not
addressed by these data. Cathepsin S
has been implicated in neuropathic
pain, manifested by increased gene
expression in rat dorsal root ganglia
following peripheral nerve injury. Such
pain was lessened by a cathepsin
S inhibitor (Barclay et al., 2007). These
observations suggest that cathepsin S
may have an excitatory effect on
neurons. As keratinocytes have been
demonstrated to express cathepsin
S (Schwarz et al., 2002), a role for this
protease in keratinocyte-neuronal com-
munication may be expected. Cathe-
psin S may contribute to the pruritus of
inflammatory skin conditions, including
atopic dermatitis and psoriasis, and
could have a role in barrier function.
Cathepsin S and other endogenous or
exogenous cysteine proteases may acti-
vate PARs as a part of additional
inflammatory processes. For example,
Der p1, a mite cysteine protease as-
sociated with asthma, activates PAR2
(Asokananthan et al., 2002). Taken
together, we suggest that exogenous
and endogenous cysteine proteases
interact with PARs as a part of signaling
cascades in homeostasis and disease.
CONFLICT OF INTEREST
The authors state no conflict of interest.
ACKNOWLEDGMENTS
This work was supported in part by a Clinical and
Translational Science Award, National Center for
Research Resources (NCRR) Grant UL1
RR0249139 (a component of the National Insti-
tutes of Health (NIH)), NIH Grant P01 NS047300
(RHL, PI), and an agreement between Massachu-
setts General Hospital and Shiseido (Tokyo,
Japan). Psychophysical experiments were ap-
proved by the Yale University Human Investiga-
tion Committee. The study was conducted
according to the Declaration of Helsinki Princi-
ples. Participants gave their written informed
consent. We thank Aurel Iuga and Barry Green
for their input and technical assistance.
Vemuri B. Reddy1, Steven G.
Shimada2, Parul Sikand2, Robert H.
LaMotte2 and Ethan A. Lerner1
1Cutaneous Biology Research Center,
Massachusetts General Hospital, Charlestown,
Massachusetts, USA and 2Department of
Anesthesiology, Yale University School of
Medicine, New Haven, Connecticut, USA
Correspondence: Ethan A. Lerner, Cutaneous
Biology Research Center, Massachusetts Gen-
eral Hospital-East, Building 149, 13th Street,
Charlestown, Massachusetts 02129, USA.
E-mail: elerner@partners.org
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online
version of the paper at http://www.nature.com/jid
REFERENCES
Asfaha S, Cenac N, Houle S et al. (2007) Protease-
activated receptor-4: a novel mechanism of
inflammatory pain modulation. Br J Pharma-
col 150:176–85
Pe
rc
e
ive
d 
in
te
ns
ity
25a b
20
15
10
5
0
Pe
rc
e
ive
d 
in
te
ns
ity
25
20
15
10
5
0
0 5 10 15 20
Time after application (minutes)
0 5 10 15 20
Time after application (minutes)
Itch
Prick
Burn
Moderate
Weak
Barely
detectable
Itch
Prick
Burn
Figure 1. Recombinant human cathepsin S evokes itch. (a) Mean perceived intensity of itch, pricking/
stinging, and burning sensations evoked by a single inactivated spicule reconstituted with cathepsin S.
Briefly, spicules were inactivated by autoclaving and then soaked in a solution containing 4mgml1
cathepsin S for 1 hour, washed, and dried. The spicules were applied to the skin, and nine, healthy,
unmedicated volunteers (four female, five male, 18 years or older) rated the evoked sensations,
according to similar methods described (Reddy et al., 2008). The mean rating of each sensory quality,
obtained every 30 seconds, is accompanied by an SE that is plotted every 5minutes starting at
the peak magnitude of the sensory quality. (b) Mean perceived intensity of sensations evoked by
a single spicule of native cowhage (the same nine subjects).
34
0:
38
0 
Ra
tio
0 60 120 180
Time (seconds)
0.001 0.01 0.1 1 10
Cathepsin S (μM)
PAR4
PAR2
SS
4a b
3
2
1
0
34
0:
38
0 
Ra
tio
4
3
2
1
0
Cathepsin S
PAR4
PAR2
Controls
Figure 2. Recombinant human cathepsin S activates human protease-activated receptors (PARs).
(a) Single-cell imaging of cathepsin S (2 mM) induced responses in HeLa cells transfected with either
PAR 2 or PAR 4 as measured by ratiometric calcium imaging in cells loaded with fura-2 as in Reddy et al.
(2008). The responses to PARs 2 and 4 were blocked by the protease inhibitor E64 (10 mM) as a control.
Other controls included vector alone and PAR1. Cathepsin S was added as indicated. (b) Concentration–
effect responses of cathepsin S in HeLa cells transiently transfected with PARs 2 and 4 as determined
by ratiometric imaging of 20 cells per data point. Salmon sperm (SS) DNA as control. For both (a) and (b),
the averages from at least three separate experiments were then combined±SD (see Supplementary
material online).
www.jidonline.org 1469
VB Reddy et al.
Cathepsin S, Itch, and Protease-Activated Receptors
Asokananthan N, Graham PT, Stewart DJ
et al. (2002) House dust mite allergens
induce pro-inflammatory cytokines from
respiratory epithelial cells: the cysteine
protease allergen, Der p 1, activates
protease-activated receptor (PAR)-2
and inactivates PAR-1. J Immunol 169:
4572–8
Barclay J, Clark AK, Ganju P et al. (2007) Role of
the cysteine protease cathepsin S in neuro-
pathic hyperalgesia. Pain 130:225–34
Felbor U, Dreier L, Bryant RA et al. (2000)
Secreted cathepsin L generates endostatin
from collagen XVIII. EMBO J 19:1187–94
Hachem JP, Houben E, Crumrine D et al. (2006)
Serine protease signaling of epidermal per-
meability barrier homeostasis. J Invest Der-
matol 126:2074–86
Ikoma A, Steinhoff M, Sta¨nder S et al. (2006) The
neurobiology of itch. Nat Rev Neurosci
7:535–47
Johanek LM, Meyer RA, Friedman RM et al.
(2008) A role for polymodal C-fiber afferents
in nonhistaminergic itch. J Neurosci 28:
7659–69
Nakagawa TY, Brissette WH, Lira PD et al. (1999)
Impaired invariant chain degradation and
antigen presentation and diminished col-
lagen-induced arthritis in cathepsin S null
mice. Immunity 10:207–17
Namer B, Carr R, Johanek LM et al. (2008)
Separate peripheral pathways for pruritus in
man. J Neurophysiol 100:2062–9
Oikonomopoulou K, Hansen KK, Saifeddine M
et al. (2006) Kallikrein-mediated cell signal-
ling: targeting proteinase-activated receptors
(PARs). Biol Chem 387:817–24
Reddy VB, Iuga AO, Shimada S et al. (2008)
Cowhage evoked itch is mediated by a novel
cysteine protease — a ligand of protease
activated receptors. J Neurosci 28:4331–5
Schwarz G, Boehncke WH, Braun M et al. (2002)
Cathepsin S activity is detectable in human
keratinocytes and is selectively upregulated
upon stimulation with interferon-g. J Invest
Dermatol 119:44–9
Shpacovitch V, Feld M, Hollenberg MD
et al. (2008) Role of protease-activated
receptors in inflammatory responses, innate
and adaptive immunity. J Leukoc Biol 83:
1309–22
Stefansson K, Brattsand M, Roosterman D
et al. (2008) Activation of proteinase-
activated receptor-2 by human kallikrein-
related peptidases. J Invest Dermatol
128:18–25
Steinhoff M, Vergnolle N, Young SH et al. (2000)
Agonists of proteinase-activated receptor 2
induce inflammation by a neurogenic
mechnism. Nat Med 6:151–8
Steinhoff M, Neisius U, Ikoma A et al. (2003)
Proteinase-activated receptor 2 mediates
itch: a novel pathway for pruritus in human
skin. J Neurosci 23:6176–80
Tsujii K, Andoh T, Lee JB et al. (2008) Activa-
tion of proteinase-activated receptors induces
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dependent and independent pathways in
mice. J Pharmacol Sci 108:385–8
IRF4 Expression without IRF4 Rearrangement Is a General
Feature of Primary Cutaneous Diffuse Large B-Cell
Lymphoma, Leg Type
Journal of Investigative Dermatology (2010) 130, 1470–1472; doi:10.1038/jid.2009.418; published online 7 January 2010
TO THE EDITOR
The involvement of the interferon
regulatory factor 4 gene (IRF4), also
known as multiple myeloma antigen 1
(MUM1), in balanced rearrangement or
translocation has been recently obser-
ved in a subset of cutaneous T-cell
lymphomas (CTCLs), such as cutaneous
anaplastic large-cell lymphoma (C-ALCL)
and transformed mycosis fungoides
(Feldman et al., 2009; Pham-Ledard
et al., 2009). IRF4 expression reaches a
high level in differentiated plasma cells
and is also detectable by immunostaining
in some activated T cells and melano-
cytes, with the latter providing internal
positive controls on skin sections (Falini
et al., 2000; Lu, 2008). Despite such a
restricted immunostaining pattern, IRF4
is an essential regulator at multiple steps
of B-cell differentiation, such as pre-B-
cell differentiation, germinal center for-
mation, immunoglobulin class switch
recombination, and terminal differentia-
tion of B cells to plasma cells, as shown
in IRF4-deficient mice (reviewed in
Shaffer et al., 2009). IRF4 is also essential
for T-helper (Th) cell differentiation and is
required for either Th2 or Th17 cell
development (Brustle et al., 2007; Zheng
et al., 2009). An oncogenic role of IRF4
has first been supported by the identifica-
tion of IRF4 involvement in the
t(6;14)(p25;q32) translocation in some
cases of multiple myeloma (MM) (Iida
et al., 1997). In t(6;14)(p25;q32), IRF4 is
juxtaposed with the immunoglobulin
heavy-chain gene locus leading to IRF4
deregulated expression (Iida et al., 1997;
Yoshida et al., 1999; Shaffer et al., 2008).
Alternatively, IRF4 rearrangements in
peripheral T-cell lymphoma do not
commonly involve either the TCRB or
the TCRA gene locus, as shown in eight
C-ALCL and two systemic T-cell lympho-
mas (Feldman et al., 2009).
Among primary cutaneous B-cell
lymphomas, primary cutaneous diffuse
large B-cell lymphoma, leg type
(PCLBCL, leg type) is an original entity
with poor prognosis that was first
reported in 1996 and that mostly affects
the leg(s) in elderly but may also arise at
other sites in approximately 10% of
cases (Vermeer et al., 1996; Willemze
et al., 2005; Meijer et al., 2008).
PCLBCL, leg type, differs from primary
cutaneous follicle center lymphoma by
the presence of confluent sheets of
centroblasts and immunoblasts, many
with a peculiar round cell morphology,
which strongly express B-cell CLL/
lymphoma 2 (BCL2), IRF4, and fork-
head box P1 (FOXP1) (Willemze et al.,
2005; Meijer et al., 2008). Interestingly,
round cell morphology, strong BCL2
Abbreviations: FISH, fluorescence in situ hybridization; IRF4, interferon regulatory factor 4; MM, multiple
myeloma; MUM1, multiple myeloma antigen 1; PCLBCL, leg type, primary cutaneous diffuse large B-cell
lymphoma, leg type
1470 Journal of Investigative Dermatology (2010), Volume 130
A Pham-Ledard et al.
IRF4 Locus in Cutaneous Large B-Cell Lymphoma


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Cathepsin S Elicits Itch and Signals via Protease-Activated Receptors

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