A Novel Role for the TRPV1
Channel in UV-Induced Matrix
Metalloproteinase (MMP)-1
Expression in HaCaT Cells
YOUNG MEE LEE,1 YEON KYUNG KIM,1 KYU HAN KIM,1* SU JUNG PARK,2
SUNG JOON KIM,2** AND JIN HO CHUNG1
Department of Dermatology, Seoul National University College of Medicine, Laboratory of Cutaneous Aging Research, Clinical
Research Institute, Seoul National University Hospital, Institute of Dermatological Science, Seoul National University, Seoul, Korea
Department of Physiology and Biophysics, Seoul National University College of Medicine, Seoul, Korea
Transient receptor potential vanilloid type 1 (TRPV1) is a molecular sensor for detecting adverse stimuli, such as capsaicin, heat, and acid.
TRPV1 has been localized in keratinocytes and is suggested to be a mediator of heat-induced matrix metalloproteinase-1 (MMP-1). With
regard to the multimodal activation of TRPV1, we hypothesize that TRPV1 might also mediate UV-induced MMP-1 in keratinocytes. In
HaCaT, a human keratinocyte cell line, we initially confirmed capsaicin-induced membrane current and Ca2þ influx. UV irradiation induced
slow and persistent calcium influx and increased membrane current, which was inhibited by TRPV1 inhibitors (capsazepine and ruthenium
red). The UV-induced MMP-1 expression in HaCaT was also decreased by TRPV1 inhibitors and was facilitated by capsaicin. Knock-down
of TRPV1 using siRNA transfection also decreased MMP-1 expression, as well as UV-induced Ca2þ influx in HaCaT. UV failed to induce
MMP-1 expression in HaCaT cells cultured in Ca2þ-free media. Both the UV-induced increase in [Ca2þ]i and MMP-1 were suppressed by
Go¨6976 (a calcium-dependent PKC inhibitor), but not by rottlerin (a calcium-independent PKC inhibitor). In addition to a plausible role of
TRPV1 in UV-induced MMP-1 expression, we showed that UV increased TRPV1 expression in both HaCaT cells and human skin in vivo.
From these results, we suggest that UV-induced MMP-1 expression might be mediated in part by PKC-dependent activation of TRPV1 and
subsequent Ca2þ-influx in human keratinocytes.
J. Cell. Physiol. 219: 766–775, 2009.
2009 Wiley-Liss, Inc.
Skin aging can be attributed to extrinsic and intrinsic
(chronologic) processes that are commonly related to
increased wrinkling, sagging, and laxity (Jenkins, 2002). Extrinsic
aging is generally referred to as photoaging since it is most
commonly caused by repeated exposure to ultraviolet (UV)
light. Whereas naturally aged skin is smooth, pale, and finely
wrinkled, photoaged-skin is coarsely wrinkled and is associated
with dyspigmentation and telangiectasia (Gilchrest, 1989).
Alterations in collagen, which is the major structural
component of the skin, have been considered to be a cause of
skin aging and have been observed in both naturally aged and
photoaged-skin (Fisher et al., 1997; Varani et al., 2000).
However, the mechanisms of collagen destruction in aged skin
have not been fully clarified.
Collagen destruction is partly related to the induction of
matrix metalloproteinase (MMP), which is secreted by
epidermal keratinocytes and dermal fibroblasts. MMPs are a
family of structurally related matrix-degrading enzymes that
play important roles in various destructive processes, including
inflammation (Vincenti and Brinckerhoff, 2002), tumor invasion
(Mignatti et al., 1986; Mignatti and Rifkin, 1993), and skin aging
(Fisher et al., 1997, 2002; Varani et al., 2000). The levels of MMP
are increased by various stimuli, such as UV light, oxidative
stress, and cytokines. Although the signaling mechanism is still
unclear, UV radiation triggers DNA binding of activator
protein-1 (AP-1), which induces MMPs; collagenase (MMP-1),
stromelysin (MMP-3), and gelatinase (MMP-9) (Fisher et al.,
1996). Once collagen is cleaved by MMP-1, it is further degraded
by MMP-3 and MMP-9, which are also induced by exposure to
UV light (Sternlicht and Werb, 2001).
Recently, it has been suggested that calcium can regulate the
expression or activation of MMPs. The combined actions
of Ca2þ, EGF, and MMP-9 regulate the contributions of
extracellular-regulated kinase and chemotactic migration of
keratinocytes (Morris and Chan, 2007). Ca2þ may be involved
in controlling the activity of MMP-12 (Gossas and Danielson,
2006). Increased extracellular calcium levels induce the MMP-9
gene expression in human keratinocytes (Kobayashi et al., 2001;
Mukhopadhyay et al., 2004), and the inhibition of calcium influx
decreases the level of MMP-1 mRNA (Kohn et al., 1994).
Modulation of intracellular calcium levels can change the
secretion of MMP-1 from migrating keratinocytes (Sudbeck
et al., 1997). In our previous study, it was suggested that calcium
influx through transient receptor potential vanilloid type 1
Abbreviations: TRPV1, transient receptor potential vanilloid-1;
HaCaT, immortalized human keratinocyte cell line; CPZ,
capsazepine; RR, ruthenium red; UV, ultraviolet; MMP-1, matrix
metalloproteinase-1.
Contract grant sponsor: Korea Health 21 R&D Project, Ministry of
Health & Welfare, Republic of Korea;
Contract grant number: A060180.
*Correspondence to: Kyu Han Kim, Department of Dermatology,
Seoul National University Hospital, 28, Yeongeon-Dong, Chongno￾Gu, Seoul 110-744, Korea. E-mail: [email protected]
**Correspondence to: Sung Joon Kim, Department of Physiology
and Biophysics, Seoul National University College of Medicine, 28,
Yeongeon-Dong, Chongno-Gu, Seoul 110-744, Korea.
E-mail: [email protected]
Received 2 October 2008; Accepted 8 January 2009
Published online in Wiley InterScience
(www.interscience.wiley.com.), 10 February 2009.
DOI: 10.1002/jcp.21729
ORIGINAL ARTICLE 766
Journal of Journal of
Cellular
Physiology
Cellular

2009 WILEY-LISS, INC.
(TRPV1) is critical in heat shock-induced MMP-1 expression in
HaCaT cells (Li et al., 2007).
TRPV1 is a member of the non-selective cationic channel
family. Activation of TRPV1 induces Ca2þ influx, which is
inhibited by a specific antagonist, capsazepine (Wood et al.,
1988; Bevan et al., 1992; Oh et al., 1996). Also, TRPV1 is directly
activated by exposure to heat or protons (reduced pH)
(Tominaga et al., 1998). Such activating conditions implicate
TRPV1 as a primary biological sensor to thermo-chemical
stimulation and tissue injury. Apart from the pain sensory nerve
(Oh et al., 1996), TRPV1 has been reported to be present in
other tissues, such as brain (Sasamura et al., 1998; Mezey et al.,
2000), kidney (Mezey et al., 2000), bronchial epithelial cells
(Veronesi et al., 1999), and epidermal keratinocytes (Denda
et al., 2001). Furthermore, stimulation with capsaicin increases
cytoplasmic Ca2þ concentration ([Ca2þ]c) of keratinocytes,
and this is blocked by capsazepine (Inoue et al., 2002).
We have recently reported the role of TRPV1 in heat￾induced MMP-1 expression of keratinocytes (Li et al., 2007). In
general, UV light is a more critical factor inducing MMP
expression and skin aging. In the present study, therefore, we
investigated whether UV light activates TRPV1 signals, and
whether UV-induced MMP-1 expression is affected by TRPV1
agonists and antagonists, including siRNA for TRPV1. Also, the
putative signaling pathways related to TRPV1 (e.g., Ca2þ and
protein kinase C) were investigated. Finally, we also examined
whether exposure to UV changed the expression of TRPV1 in
HaCaT cells and human skin in vivo.
Materials and Methods
Materials
Capsaicin, capsazepine, ruthenium red, rottlerin, and
staurosporine were purchased from Sigma (St. Louis, MO).
Go¨6976 was obtained from Calbiochem (San Diego, CA). Anti￾human MMP-1 antibody was acquired from Lab Frontier (Seoul,
Korea). Rabbit polyclonal antibody against TRPV1 was supplied by
Chemicon (Temecula, CA). The cell culture media, antibiotics, and
Trizol reagent were purchased from Life Technologies (Rockville,
MD). Fetal bovine serum (FBS) was obtained from Hyclone (Logan,
UT). The calcium-sensitive indicator, Fluo-4 AM, was obtained
from Molecular Probes (Carlsbad, CA).
Cell culture and treatments
The immortalized human keratinocytes cell line, HaCaT, was
cultured in Dulbecco’s modified Eagle’s media (DMEM)
supplemented with glutamine (2 mM), penicillin (400 U/ml),
streptomycin (50 mg/ml), and 10% FBS at 378C in a humidified
atmosphere containing 5% CO2. For treatment, the cells were
cultured to 80% confluence and then maintained in culture media
without FBS for 24 h. The cells were washed with phosphate￾buffered saline (PBS) and irradiated with UV. After UV treatment,
the culture medium was replaced with fresh medium without FBS,
and the cells were further incubated for the indicated times. When
required, TRPV1 antagonists (capsazepine or ruthenium red) and
PKC inhibitors (rottlerin, staurosporine, or Go¨6976) were added
30 min before UV treatment, and incubated for the indicated times.
The role of extracellular calcium in the UV experiment was
examined by serum-starving the HaCaT cells for 24 h and culturing
the HaCaT cells in either calcium-free DMEM (Invitrogen,
Carlsbad, CA) or calcium-containing DMEM for 30 min before UV
treatment. Fresh culture medium was added, and the cells were
further incubated for the indicated times.
UV irradiation
For immunofluorescence staining, buttock skin was irradiated with
a Waldmann UV-800 (Waldmann, Villingen-Schwenningen,
Germany) phototherapy device and a F75/85W/UV21 fluorescent
lamp with an emission spectrum between 285 and 350 nm (peak,
310–315 nm), as described previously (Seo et al., 2001). The
strength of UV irradiation at the skin surface was measured using a
Waldmann UV meter (model 585100). The buttock skin was
irradiated with UV light filtered through a Kodacel filter (TA401/
407; Kodak, Rochester, NY), and the minimal erythema dose
(MED) was determined 24 h after irradiation. The MED usually
ranges between 70 and 90 mJ/cm2 for the brown skin of Koreans.
This study used 2 MED. The irradiated and non-irradiated buttock
skin samples were obtained from each subject using a punch biopsy.
This study was approved by the Institutional Review Board at the
Seoul National University Hospital, and all subjects provided
written informed consent.
In the Ca2þ measuring and patch clamp experiment, irradiation
was accomplished using a handle lamp UV (model VL-6.LM; Vilber
Lourmat, Torcy, France). The spectral output was 300–320 nm,
with a peak at 311 nm. The UV strength was measured using a
Waldmann UV meter (model 585100). With the handle lamp, 2 min
of exposure to UV delivered 36 mJ/cm2 to the glass slides.
In the Western blot, real-time RT-PCR experiment, the HaCaT
cells were irradiated with a Philips TL 20W/12 RS fluorescent sun
lamp with an emission spectrum ranging between 275 and 380 nm
(peak, 310–315 nm) (Seo et al., 2003). A Kodacel filter (TA401/407;
Kodak) was used to block UVC, which has wavelengths of
<290 nm. The UV strength was measured using a Waldmann UV
meter (model 585100).
Western blot analysis
In order to determine the amounts of MMP-1 secreted into the
culture media, equal aliquots of conditioned culture media from an
equal number of cells were fractionated by 10% SDS–PAGE,
transferred to a Hybond ECL membrane (Amersham Biosciences,
Buckinghamshire, England), and analyzed by Western blotting with
a rabbit monoclonal antibody against MMP-1 (Lab Frontier) by
enhanced chemiluminescence (Amersham Biosciences). In order
to analyze the activation of TRPV1, the cells were lysed with a lysis
buffer (50 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA,
1 mM EGTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 5 mM
phenylmethylsulfonyl fluoride [PMSF], and 1 mM DTT) containing
1% Triton X-100. Insoluble debris was removed by centrifugation
at 13,000 rpm for 10 min, and the protein content was determined
using the Bradford reagent (Bio-Rad, Hercules, CA). Equal amounts
(50 mg) of the protein samples were fractionated and transferred,
as described above, and analyzed by Western blotting using rabbit
polyclonal antibody against TRPV1 (Chemicon). As controls, the
levels of the corresponding b-actin were determined in the same
cell lysates using the antibodies for b-actin (Santa Cruz
Biotechnology, Santa Cruz, CA). The signal strengths were
quantified using a densitometric program (TINA; Raytest
Isotopenme b gerate, Straubenhardt, Germany).
RNA analysis
Total RNA was prepared from HaCaT cells using the Trizol
method according to the manufacturer’s protocol (Life
Technologies). The isolated RNA samples were electrophoresed
in 1% agarose gels to assess the quality and quantity. One
microgram of the total RNA was used in a 20-ml reaction volume
for first-strand cDNA synthesis using a first-strand cDNA
synthesis kit for RT-PCR, according to the manufacturer’s
instructions (MBI Fermentas, Vilnius, Lithuania). Semi-quantitative
RT-PCR was performed using 1 ml of the first-strand cDNA
product using the following primers for the human genes: 36B4
(cDNA for human acidic ribosomal phosphoprotein PO), which did
not change with UV irradiation (Cho et al., 2008); (forward, 50
TGG GCT CCA AGC TGC-30
; reverse, 50
-GGC TTC GCT GGC
TCC CAC-30
), MMP-1 (forward, 50
-ATT CTA CTG ATA TCG
GGG CTT TGA-30
; reverse, 50
-ATG TCC TTG GGG TAT CCG
TGT AG-30
), TRPV1 (forward, 50
-TGT GCC GTT TCA TGT TT-30
JOURNAL OF CELLULAR PHYSIOLOGY
TRPV1 MEDIATES UV-INDUCED MMP-1 EXPRESSION 767
reverse, 50
-TGC ACC TTC CAG ATG TT-30
). The following PCR
conditions were used: one cycle of initial denaturation (5 min at
948C), 21 cycles (36B4) or 28 cycles
(MMP-1 and TRPV1) of amplification (1 min at 948C, 1 min at 608C,
and 1 min at 728C), and 1 cycle of final extension (10 min at 728C).
The PCR amplifications were carried out in a cycle number
corresponding to the logarithmic amplification phase. The
reaction products were electrophoresed in 2.0% agarose gels
and visualized with ethidium bromide. The signal strengths were
quantified using a densitometric program (TINA). No PCR
products were obtained in the control reactions with the
reverse transcriptase omitted. After normalizing versus the
intensity of 36B4, the percentage increases or decreases
were determined. Each experiment was repeated at least three
times.
The mRNAs for TRPV1 and MMP-1 were also measured using a
real-time TaqMan quantitative reverse transcription polymerase
chain reaction (RT-PCR) with an ABI PRISM 7500 Sequence
Detection System (Applied Biosystems, Foster City, CA). The
technique is based on the ability to detect the RT-PCR product
directly with no downstream processing. This was accomplished by
monitoring the increase in fluorescence of a dye-labeled DNA
probe specific for each factor under study, plus a probe specific for
the 36B4 gene, which is used as an endogenous control for the
assay. TaqMan probes and primers for TRPV1 and MMP-1 were the
Assay-on-Demand gene expression products (Applied
Biosystems). The 36B4 sequences of the probes, forward and
reverse primers, which were designed by Applied Biosystems were
as follows: VIC-labeled probe 50
-CGC GGG AAG GCT GTG GTG
CT-30
, forward 50
-ATG CAG CAG ATC CGC ATG T-30
, reverse
50
-TTG CGC ATC ATG GTG TTC TT-30
. The PCR reaction was
carried out according to the manufacturer’s instructions. As a
negative control, the PCR reactions without the template cDNA
were added to the reaction wells. All the samples were run in
triplicate.
Immunofluorescence
For immunofluorescence staining, the biopsy samples were fixed in
10% buffered formaldehyde for 24 h, and embedded in paraffin wax.
The samples were deparaffinized with xylene and then rehydrated
through a descending gradient of ethanol. After several washes in
PBS, the endogenous peroxidase activity was quenched using
3% hydrogen peroxide for 6 min. The sections were then blocked
with a blocking solution (Zymed, San Francisco, CA) for 30 min, and
washed and incubated with the primary antibody; a rabbit
polyclonal antibody against TRPV1 (Chemicon) in a humidified
chamber at 48C for 18 h. After washing in PBS, the sections were
incubated with the secondary TRITC-conjugated goat anti-rabbit
IgG (Zymed) antibody for 1 h at room temperature. The nuclei
were counterstained by DAPI staining. All the sections were
examined immediately and photographed with a Zeiss LSM 510
META confocal laser-scanning microscope (Zeiss, Yena,
Germany).
Transfection with siRNA
The gene silencing of human TRPV1 was performed with the
sequence-specific siRNA reagents designed by the BLOCK-iT
Fig. 1. Capsaicin-induced changes in [Ca2R]i and membrane current in HaCaT cells. A: Fluorescence signals of Fluo-4 (Ffluo-4) loaded in HaCaT
cells. Data from 12 cells marked as different regions of interest (ROIs, see inset). Ten micromolars capsaicin (CAPS) and 7 mM ionomycin (IONO)
were applied, as indicated, in the trace. a.u., arbitrary units of Fflou-4. B: Summary of peak fluorescence intensities normalized to the control Ffluo-4
P < 0.05); #
P < 0.05 versus 10 mM capsaicin-treated group in 2 mM [Ca2R]o. C: Capsaicin (1 mM)-activated membrane current in HaCaT cells.
Current–voltage relationships (I-V curves) were obtained by ramp pulses from 120 to 100 mV under the whole-cell clamp conditions. The
capsaicin-induced current was blocked by the addition of capsazepine (10 mM). D: Summary of the capsaicin- and capsazepine-sensitive currents
(n U 6). Membrane currents normalized against the control current of each cell measured at 120 and 100 mV were averaged. M
P < 0.05 versus
capsaicin-untreated control group; #
P < 0.05 versus capsaicin-treated group.
JOURNAL OF CELLULAR PHYSIOLOGY
768 LEE ET AL.
RNAi program (Invitrogen). The human TRPV1 siRNA sequence
was as follows: 50
-GGA TTG CCC TCA CGA GGA A-30
Silencer1 Negative Control #1 (Ambion, Cambridge, UK) was
used as a scramble control siRNAs (SCR). HaCaT cells were
transfected with TRPV1 siRNA or SCR using OligofectAMINE
(Invitrogen), as recommended by the manufacturer.
HaCaT cells were transfected with silencing of the same
sequence for the human TRPV1 siRNA tagged Alexa Fluor 647 and
thereby the transfected cells could be identified during the
measurement of [Ca2þ]i
.
Ca2R-imaging experiments
HaCaT cells were cultured on cover glasses, then loaded with 4mM
Fluo-4 AM (Molecular Probes) in serum-free medium at room
temperature for 45 min After washing three times with serum-free
medium, cells on the cover glasses were transferred to
custom-built observation chambers, and allowed to accommodate
for 20 min.
Capsaicin was used at a final concentration of 1 or 10 mM in
Tyrode’s buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM
CaCl2, 10 mM glucose, and 10 mM HEPES [pH 7.2]). The
fluorescence intensity was measured using a confocal laser￾scanning microscope (LSM 510 META, Zeiss) fitted with
appropriate filters and a PL Fluotar objective (200, 0.5 NA) that
was controlled by SCAN Ware 5.10 software (Zeiss). The
experiments were performed at 378C in a humidified chamber.
During the Ca2þ measuring procedure, UV was irradiated by using
a handle UV lamp (model VL-6.LM; Vilber Lourmat). With the
handle lamp, two min of exposure to UV directly delivered 36 mJ/
cm2 to the glass slides. The measurements lasted for 20 min, with
images taken every 1 or 4 sec.
Electrophysiology
HaCaT cells were transferred into a bath mounted on the stage of
an inverted microscope (IX-70; Olympus, Osaka, Japan). The bath
(0.15 ml) was superfused at 5 ml/min, and voltage clamp
experiments were performed at room temperature (22–258C).
Patch pipettes with a free-tip resistance of about 2.5 MV were
connected to the head stage of a patch-clamp amplifier (Axopatch-
1D; Axon Instruments, Foster City, CA). Liquid junction potentials
were corrected with an offset circuit before each experiment. A
conventional whole-cell clamp was achieved by rupturing the
patch membrane after making a giga-seal. pCLAMP software
(version 7.0; Axon Instruments) and Digidata-1322A (Axon
Instruments) were used for the acquisition of data and the
application of command pulses. Voltage and current data were
low-pass filtered (5 kHz), stored in a Pentium-grade computer, and
analyzed using pCLAMP software (version 9.0; Microcal
Software, Inc., Northampton, MA) and Origin (version 6.1;
Microcal Software, Inc.). Cells were held at 60 mV. The
voltage protocol was composed of ramp pulses from 120 to
100 mV for 1 sec. The normal bath solution for the whole-cell
patch clamp contained (in mM) 140 NaCl, 5 CsCl, 1 MgCl2, 2 CaCl2,
10 glucose, and 10 HEPES at a pH of 7.4 (titrated with NaOH).
The pipette solution contained (in mM) 140 CsCl, 1 MgCl2, 4
MgATP, 10 EGTA, and 10 HEPES at a pH of 7.2 (titrated with
CsOH).
Statistics
Statistical significance was determined by Student’s t-test. Results
are presented as the means  SEM. All reported P values are two￾tailed and significance was accepted when the P-value was <0.05.
Fig. 2. UV-induced changes in [Ca2R]i and membrane currents in HaCaT cells. A: UV irradiation (36 mJ/cm2 for 2 min) slowly increased Fflou-4 of
HaCaT cells. The increase in [Ca2R]i continued after irradiation, then slowly recovered. B: Summary of the UV-induced [Ca2R]i(Fflou-4) increase
and its inhibition by TRPV1 blockers.The Fflou-4of each ROI (see inset) was normalized against the initial controlat the time points indicated (1 and
2 min, and the peak after irradiation). Pretreatment of HaCaT cells with 10 mM ruthenium red (RR), 10 mM capsazepine (CPZ), or Ca2R-free
conditions attenuated the UV-induced Fflou-4 signals. C: UV-activated membrane current in HaCaT cells. The I-V curves were obtained before and
afterUVirradiationfortwomin.D:TheUV-inducedcurrentwaslargelysuppressedbypretreatmentwithcapsazepine(10mM).E:Summaryofthe
UV- and capsazepine-sensitive currents (n U 6). The normalized membrane currents (pA/pF) measured at 120 and 100 mV were averaged. M
P < 0.05, versus UV-untreated control group; #
P < 0.05, versus UV-treated peak group.
JOURNAL OF CELLULAR PHYSIOLOGY
TRPV1 MEDIATES UV-INDUCED MMP-1 EXPRESSION 769
Results
Activation of TRPV1-like current and Ca2R influx
by UV irradiation
To confirm the functional expression of TRPV1 in HaCaT cells,
the capsaicin-induced increase in intracellular Ca2þ
concentration ([Ca2þ]i
) was measured. The fluorescence
intensity of Fluo-4 (Ffluo4) was increased by capsaicin (Fig. 1A).
Pretreatment with capsazepine (10 mM) inhibited the capsaicin￾induced increase in [Ca2þ]i (data not shown). Capsaicin, at 1
and 10 mM, increased the Ffluo4 to 129.9  2.1% and
140.8  2.1% of control values, respectively (Fig. 1B). In
comparison, the application of 7 mM ionomycin increased
the Ffluo4 to 193.7  5.7% (n ¼ 14). The capsaicin-induced
increase in [Ca2þ]i was not observed in the absence of
extracellular Ca2þ (Fig. 1B, n ¼ 9). The whole-cell patch clamp
study showed that capsaicin treatment increased an outwardly
rectifying conductance. The capsaicin-induced current was
blocked by pretreatment with capsazepine (Fig. 1C,D, n ¼ 6).
UV irradiation for two min (36 mJ/cm2
) slowly increased
the Ffluo4, reflecting an increase in [Ca2þ]i (D[Ca2þ]i
) of HaCaT
cells (264.2  15.9%; Fig. 2A,B, n ¼ 13). TheD[Ca2þ]i continued
for 40–60 sec after the end of UV exposure, and reversed
slowly thereafter (Fig. 2A). A similar D[Ca2þ]i was induced by
72 mJ/cm2 of UV irradiation that approximated the level used in
the Western blot study (data not shown). The UV-induced
D[Ca2þ]i was inhibited by pretreatment with either ruthenium
red (10 mM) or capsazepine (10 mM; Fig. 2B, n ¼ 13). Also, the
omission of Ca2þ in the bath solution inhibited the UV-induced
D[Ca2þ]i (Fig. 2B, n ¼ 10). Under the whole-cell patch clamp of
HaCaT cells, UV irradiation (36 mJ/cm2
) also increased the
membrane conductance with a weak outwardly rectifying
voltage-dependence (Fig. 2C, n ¼ 9). The UV-induced current
usually remained elevated after the cessation of irradiation.
Pretreatment with capsazepine (10 mM, 5 min) suppressed the
UV-induced currents (Fig. 2D,E, n ¼ 6).
TRPV1 antagonists inhibited UV-induced MMP-1
expression
Next, we investigated whether the UV-induced MMP-1
expression was affected by TRPV1 antagonists. For this
purpose, HaCaT cells were exposed to UV in 75 mJ/cm2
, and
the level of secreted MMP-1 was measured in culture media
72 h after UV-irradiation. Consistent with previous reports
(Kim et al., 2005, 2006), UV irradiation increased the level of
MMP-1 protein expression (215.6  22.9%, n ¼ 4).
Pretreatment of HaCaT cells with ruthenium red decreased
the UV-induced MMP-1 protein expression compared with the
UV-treated control group (152.6  2.33%, 80.3  2.2%,
56.3  9.3% by 1, 5, and 10 mM, respectively; Fig. 3A, n ¼ 4).
Real-time RT-PCR analysis performed 72 h after UV irradiation
showed an increase in MMP-1 mRNA (471.0  103.8% of the
UV-untreated control group, n ¼ 4), which was also suppressed
by 10 mM ruthenium red (175.1  13.8% of the UV-treated
control; Fig. 3C, n ¼ 4).
Fig. 3. InhibitionofUV-inducedMMP-1expressionbyTRPV1antagonists.Afterpretreatmentwithrutheniumred(RR)orcapsazepine(CPZ)for
30 min, the cultured HaCaT cells were treated with UV irradiation (75 mJ/cm2
). Fresh media containing ruthenium red were added, and the cells
were further incubated for 72 h. A,B: The amounts of MMP-1 protein released into the culture media were determined by Western blotting and
quantified by densitometry. Each level of MMP-1 protein expression was normalized to that of the corresponding b-actin, and the mean values are
shown as bar graphs (lower parts of a and b). C,D: Real-time RT-PCR analysis of MMP-1 mRNA from HaCaT cells treated with UV and TRPV1
blockers (RR and CPZ). M
P < 0.05 versus UV-untreated control group; #
P < 0.05 versus UV-treated control group.
JOURNAL OF CELLULAR PHYSIOLOGY
770 LEE ET AL.
We also tested the effect of capsazepine, a more specific
blocker of TRPV1, on UV-induced MMP-1 protein expression.
Compared with the control response to UV irradiation
(395.7  69.9%, n ¼ 4), the UV-induced increase in MMP-1
protein expression was reduced to 328.7  69.3%,
248.3  43.6%, and 194.5  25.1% by 1, 2, and 5 mM of
capsazepine, respectively (Fig. 3B, n ¼ 4). By real-time RT-PCR
analysis, we also confirmed that capsazepine inhibited the UV￾induced increase of MMP-1 mRNA expression (Fig. 3D, n ¼ 4).
TRPV1 agonist-induced MMP-1 expression
As was expected from the above results, capsaicin treatment
alone increased the expression of MMP-1 proteins at 72 h in
HaCaT cells (308.0  4.0%, 353.5  5.5%, 486.5  8.5% by 0.2,
0.5, and 1 mM, respectively, versus the UV-untreated control
group, n ¼ 3; P < 0.05). Interestingly, pretreatment with
capsaicin significantly augmented the UV-induced MMP-1
expression level. Capsaicin at 0.2, 0.5, and 1 mM increased the
level of UV-induced MMP-1 protein expression to
1446  10.1%, 2020  17.9%, and 2300  167.1%, respectively
(Fig. 4A, n ¼ 3). Real-time PCR analysis also showed that
capsaicin increased the mRNA level of MMP-1 and the
UV-induced expression of mRNA for MMP-1 (Fig. 4B, n ¼ 3).
TRPV1 knock-down inhibited UV-induced
MMP-1 expression
In addition to the pharmacologic evidence shown above, the
role of TRPV1 in UV-induced MMP-1 production was further
investigated by genetic silencing of mRNA for TRPV1. HaCaT
cells transfected with siRNA were identified from the co￾labeled Alexa Fluor 647 (red). Then, we loaded the transfected
HaCaT cells with Ca2þ indicator Fluo-4 AM (green) for the
measurement of [Ca2þ]i (Fig. 5A).
The functional downregulation of TRPV1 by TRPV1-specific
siRNA was confirmed by the [Ca2þ]i measurements. As shown
in Figure 5B, the UV-induced D[Ca2þ]i was largely abolished in
the HaCaT cells treated with TRPV1-specific siRNA
(107.9  2.7% vs. the negative control siRNA group [NC],
n ¼ 12; #
P < 0.05). siRNA treatment also reduced the capsaicin￾induced D[Ca2þ]i (Fig. 5B, n ¼ 12). HaCaT cells transfected
with NC siRNA labeled with Cy5 consistently showed UV￾induced D[Ca2þ]i and capsaicin-induced D[Ca2þ]i
. Transfection
with TRPV1-siRNA decreased the mRNA and protein levels of
TRPV1 in HaCaT cells (Fig. 5C). UV-induced MMP-1 was also
suppressed by TRPV1-siRNA transfection, but not by NC
siRNA (Fig. 5C–E). In these experiments, we also found that
UV irradiation increased the expression of TRPV1, as well as
MMP-1 (Fig. 5C–E), and this result was further investigated
below (Fig. 7).
Role of PKC in UV-induced MMP-1 expression
The above results suggest that UV-activated TRPV1 and the
subsequent increase in [Ca2þ]i induced MMP-1 in human
keratinocytes. To evaluate the role of extracellular Ca2þ, the
HaCaT cells were irradiated with UV in Ca2þ-free media. The
UV-induced MMP-1 protein expression was abolished in
the Ca2þ-free media, and add-back of Ca2þ recovered MMP-1
expression in a dose-dependent manner (Fig. 6A).
As a candidate signaling step distal to the D[Ca2þ], we
investigated whether PKC is involved in UV-induced MMP-1
expression. For this purpose, HaCaT cells were pretreated
with the following PKC inhibitors for 30 min before UV
irradiation: staurosporine (ST; a broad-spectrum PKC
inhibitor), Go¨6976 (a calcium-dependent PKCa/b inhibitor),
and rottlerin (ROT; a calcium-independent PKCd inhibitor).
Staurosporine and Go¨6976 inhibited UV-induced expression of
MMP-1 in a dose-dependent manner. In contrast, rottlerin did
not inhibit UV-induced expression of MMP-1 (Fig. 6B).
Although we initially thought that PKC might be activated by
increased [Ca2þ]i
, it was also possible that PKC could trigger
TRPV1 activation (Premkumar and Ahern, 2000). Consistent
with this assumption, pretreatment with staurosporine (50 nM)
or Go¨6976 (0.1 mM) decreased UV-induced Ca2þ influx in
HaCaT cells (120.5  6.9% and 118.1  4.2%, respectively,
versus the UV-treated control group, n ¼ 9; #
P < 0.05), while
rottlerin (1 mM) had no such effect (Fig. 6C).
UV increased TRPV1 expression in HaCaT cells and
human skin in vivo
From the results shown in Figure 5C,E, we speculated that UV
irradiation not only activated TRPV1, but also increased the
expression of TRPV1 in keratinocytes. The increase in TRPV1
expression after UV irradiation (75 mJ/cm2
) was confirmed in
HaCaT (128.4  6.3% and 178.7  2.9% at 24 and 48 h, post￾Fig. 4. Augmentation of UV-induced MMP-1 expression by capsaicin
in HaCaT cells. HaCaT cells were irradiated with UV light (75 mJ/cm2
)
after 30 min of pretreatment with capsaicin (0.2–1 mM). Fresh media
containing capsaicin was added, and the cells were further incubated
for 72 h. A: The amounts of MMP-1 protein released into the culture
media were analyzed 72 h post-treatment by Western blotting.
MMP-1 signals were normalized to the corresponding b-actin level,
and the mean values are shown as bar graphs (n U 3, lower part).
B:The level of MMP-1 mRNA was measured by real-time RT-PCR.
P < 0.05 versus UV-untreated control group; #
P < 0.05 versus UV￾treated control group (n U 3).
JOURNAL OF CELLULAR PHYSIOLOGY
TRPV1 MEDIATES UV-INDUCED MMP-1 EXPRESSION 771
treatment, respectively, vs. the UV-untreated control group,
n ¼ 4; 
P < 0.05; Fig. 7).
To investigate the pattern of TRPV1 expression in vivo and
the changes induced by UV irradiation, we performed
immunofluorescence analysis in human skin biopsy
samples. Intense cytoplasmic expression of the human skin in
the control group was observed in the granular and
spinous layers of the epidermis. Consistent with the in vitro
results, UV irradiation increased the level of TRPV1 expression
in human skin, particularly 24 h post-UV irradiation (Fig. 8,
n ¼ 5).
Discussion
Our present study using HaCaT cells can be summarized as
follows: (1) UV activated Ca2þ influx and non-selective cationic
current, which were suppressed by TRPV1 antagonist; (2) the
UV-induced expression of MMP-1 was suppressed by TRPV1
antagonists and TRPV1 siRNA, while the UV-induced
expression of MMP-1 was augmented by capsaicin; (3) Ca2þ
influx and PKC mediated UV-induced expression of MMP-1;
and (4) UV increased the expression of TRPV1 in human
keratinocytes both in vivo and in vitro (Fig. 9).
The phenomenon of UV-induced D[Ca2þ]i was first
described in human epidermal keratinocytes (Nakagaki et al.,
1990). The positive effects of UV light on [Ca2þ]i and membrane
conductance have been reported in other types of cells, such as
peripheral lymphocytes (Spielberg et al., 1991; Schieven et al.,
1993), Jurkat T cells (Spielberg et al., 1991), and in several types
of mammalian cells (Mendez and Penner, 1998). However,
these previous studies neither identified the UV-induced ion
channels nor investigated the implications for the photoaging
of skin, the primary tissue that encounters UV light. TRPV1 is
well-recognized to be activated by highly diverse modalities of
physicochemical stimuli, including heat, protons, and vanilloid
compounds (Caterina et al., 1997; Tominaga et al., 1998;
Veronesi et al., 1999). Thus, our present study suggests for
the first time that UV light, a component of the
electromagnetic wave spectrum, can also activate TRPV1 in
keratinocytes.
The inhibition of UV-induced MMP-1 expression by TRPV1
antagonists, TRPV1 siRNA transfection and Ca2þ-free media,
suggests that the Ca2þ influx associated with TRPV1 might play
a role in the signaling pathway of photoaging. Moreover, the
involvement of the calcium-dependent PKC pathway was
suspected from the inhibitory effects of pharmacologic
inhibitors on the UV-induced D[Ca2þ]i
, as well as MMP-1
secretion (Fig. 6C). Since previous studies have shown that PKC
signaling could transduce a variety of stimuli to activate TRPV1
(Premkumar and Ahern, 2000; Vellani et al., 2001), it was
suggested that UV-activated PKC might be necessary for the
activation of TRPV1 (Fig. 9). Previous studies involving human
keratinocytes have indicated roles of PKC in the transduction of
UV light stimuli to cellular responses (Matsui et al., 1994, 1996;
He et al., 2004; Grandjean-Laquerriere et al., 2005). However, it
was also demonstrated that a specific subtype of PKC (PKCd)
was downregualted by photoaging stimuli, while PKCa was
upregulated by the chronologic aging process in skin fibroblasts
Fig. 5. Inhibition of UV-induced increase in [Ca2R]i and MMP-1 expression by si-TRPV1 transfection. A: Transfected HaCaT cells were identified
by the punctuated Alexa Flour 647 fluorescence (see the cells with red spots in the inset). In those cells, UV-induced [Ca2R]i increases were not
observed (left part). B: The summary ofUV-induced Ca2R influx and capsaicin-induced Ca2R influx in control, si-TRPV1,or negative controlsiRNA
(NC) transfected HaCaT cells. C–E: After incubation at 37-C for 24 h, cells were irradiated with UV light (75 mJ/cm2
). Fresh culture medium was
added and the cells were further incubated for 48 h. (C) RT-PCR (upper parts) and Western blot analysis (lower parts) of TRPV1 and MMP-1
mRNAs. The expression of 36B4 mRNA and b-actin are presented as a negative control in RT-PCR and Western blot, respectively. Each mRNA
expressionlevelwasnormalizedversusthatofthecorresponding36B4,whichdidnotchangewithUVirradiation(upperpart).D,E:Summaryofthe
real-time RT-PCR analysis of MMP-1 (D) and TRPV1 (E) in control and siRNA-treated groups. M
P < 0.05 versus the UV-untreated control group; #
P < 0.05 versus UV-treated negative control group.
JOURNAL OF CELLULAR PHYSIOLOGY
772 LEE ET AL.
(Bossi et al., 2008), Therefore, we could not exclude the
possibility that PKC might also play a role distal to D[Ca2þ]i for
the MMP-1 secretion. The long delay between the early Ca2þ
signal and MMP-1 secretion indicated that there remains much
to be elucidated for understanding the responses of
keratinocytes to UV light.
Since UV light has a multitude of effects, it is difficult to
identify a specific mechanism for channel activation. It appears
that ion channels other than TRPV1 are also activated by UV
light in HaCaT cells because the inhibition of Ca2þ influx by
capsazepine was incomplete, and was smaller than the inhibition
by ruthenium red, a non-specific blocker for various TRPV
channels. These results may indicate that Ca2þ-permeable
TRPV channels other than TRPV1 might also play a role in
mediating UV-induced MMP-1 expression. According to the
literature, both TRPV3 and TRPV4 have been proposed as
warmth-activating or volume-activating cation channels in
keratinocytes (Chung et al., 2003, 2004).
The molecular mechanisms of UV-induced photoaging in
epidermal keratinocytes have been extensively investigated and
differential mechanisms have been suggested depending on the
spectrum of wavelengths. Among the putative mechanisms, the
role of reactive oxygen species (ROS) generation, the activation
of transcription factors (NFkB, AP-1, and AP-2), and
proinflammatory cytokines have been commonly proposed
(Berneburg et al., 2000; Fisher et al., 2002; Rabe et al., 2006).
Although previous studies have not directly demonstrated
intracellular Ca2þ signaling as a critical trigger factor for
photoaging, various studies have suggested a role for Ca2þ
signaling in the expression or activation of MMPs. Increasing
extracellular calcium levels induce the expression of the MMP-9
gene in human keratinocytes (Kobayashi et al., 2001;
Mukhopadhyay et al., 2004), and the inhibition of calcium influx
decreases the level of MMP-1 mRNA expression (Kohn et al.,
1994). Changes in the intracellular calcium levels can regulate
the secretion of MMP-1 by migrating keratinocytes (Sudbeck
et al., 1997). Our previous report (Li et al., 2007) and the
present findings together suggest a critical role for Ca2þ influx
Fig. 7. UV-induced changes of TRPV1 expression in HaCaT cells.
After UV irradiation (75 mJ/cm2
), HaCaT cells were further incubated
for the time indicated above each lane. Levels of TRPV1 were
determined by Western blotting. The summary of normalized
TRPV1 against b-actin are shown as bar graphs (lower part). M
P < 0.05,
versus UV-untreated control group.
Fig. 6. UV-induced MMP-1 expression through calcium-dependent PKC signaling in HaCaT cells. A: The culture media for HaCaT cells were
changed to serum-free DMEM, or calcium and serum-free DMEM for 30 min, or (B) pretreated with PKC inhibitors (Staurosporine, Go¨6976, or
rottlerin) for 30 min before UV irradiation. The amounts of MMP-1 protein released into culture media were analyzed 72 h post-treatment by
Western blotting. The data shown are representative of three independent experiments. C: Summary of the UV-induced D[Ca2R]i(DFfluo-4) and
effects of PKC blockers. M
P < 0.05, versus UV-untreated control group. #
P < 0.05, versus UV-treated control group.
JOURNAL OF CELLULAR PHYSIOLOGY
TRPV1 MEDIATES UV-INDUCED MMP-1 EXPRESSION 773
through TRPV1 in MMP-1 expression. In our present data,
UV-induced MMP-1 expression was decreased in calcium-free
media, and addition of extracellular Ca2þ recovered MMP-1
expression in a dose-dependent manner. It is suggested that
UV-activated TRPV1 and the subsequent increase in [Ca2þ]i
induced MMP-1 in human keratinocytes. However, the large
interval of time between D[Ca2þ]i and MMP-1 expression
suggest that highly complex signaling steps are involved
between the two events, which remain to be investigated.
The abnormal level of [Ca2þ]i and the deranged signaling
cause various human diseases (Missiaen et al., 2000). The
profound influence of Ca2þ signaling on the growth and
differentiation of keratinocytes is well-recognized (Hennings
et al., 1980; Bodo et al., 2005). One of the most striking
examples is Darrier’s disease, which is induced by defective
SERCA in epidermal cells (Dhitavat et al., 2004). In this regard,
the activation of TRPV1 and subsequent Ca2þ influx might have
multiple effects on keratinocytes. Actually, it has been reported
that activation of epidermal TRPV1 induces the expression of
COX-2 and the release of proinflammatory mediators (Southall
et al., 2003). Also, a role of TRPV1 in hair growth has been
suggested (Bodo et al., 2005). In this context, our present
results suggest a new role for TRPV1 and Ca2þ signaling in the
process of photoaging of skin.
Also, the upregulation of TRPV1 by UV in human skin and
HaCaT cells was a novel finding which might imply a
feed-forward mechanism, in which the stimulant could
upregulate a biological signal transformer, that is, TRPV1.
Therefore, exposure of human skin to UV light might cause
amplified aging and inflammatory responses mediated by
TRPV1.
Taken together, our findings suggest that calcium influx
through TRPV1 is critical for UV-induced MMP-1 expression in
HaCaT cells and a calcium-dependent PKC is involved in the
signaling pathway. The above results support the notion that
epidermal TRPV1 may function as a sensor for a wide variety of
Fig. 9. A model of the role of TRPV1 in UV-induced MMP-1
expression. This scheme suggests signaling steps identified in the
present study. UV light activates TRPV1 via a PKC-dependent
pathway. Ca2R influx and PKC mediate the UV induced expression of
MMP-1. The expression of TRPV induced by UV light (dotted line)
might augment this signaling mechanisms in response to exposure to
sunlight. It is suggested that it improves the knowledge about the role
of TRPV1 receptor in skin photoaging.
Fig. 8. Immunofluorescence staining of TRPV1 in UV-treated human skin in vivo. The buttock skin of human subjects was UV-treated, as
described in Materials and Methods Section. Immunofluorescence staining was performed using a rabbit polyclonal antibody against TRPV1.
TRITC-conjugated goat anti-rabbit IgG secondary antibody was used for visualization. Nuclei were counterstained with DAPI. The figures shown
areTRPV1-immunoreactivityincontrolhumanskin,and2,4,8,24,48,and72hpost-UV-irradiation.Thenegativecontrol(NC)wasincubatedwith
goat anti-rabbit IgG.
JOURNAL OF CELLULAR PHYSIOLOGY
774 LEE ET AL.
noxious stimuli, such as UV. It follows that TRPV1 might be a
target for preventing skin photoaging, which is most commonly
caused by exposure to UV light.
Acknowledgments
This study was supported by a grant of the Korea Health 21
R&D Project, Ministry of Health & Welfare, Republic of Korea
(A060180) and by a research agreement with the Amore-Pacific
Corporation.
Literature Cited
Berneburg M, Plettenberg H, Krutmann J. 2000. Photoaging of human skin. Photodermatol
Photoimmunol Photomed 16:239–244.
Bevan S, Hothi S, Hughes G, James IF, Rang HP, Shah K, Walpole CS, Yeats JC. 1992.
Capsazepine: A competitive antagonist of the sensory neurone excitant capsaicin. Br J
Pharmacol 107:544–552.
Bodo E, Biro T, Telek A, Czifra G, Griger Z, Toth BI, Mescalchin A, Ito T, Bettermann A,
Kovacs L, Paus R. 2005. A hot new twist to hair biology: Involvement of vanilloid receptor-
1 (VR1/TRPV1) signaling in human hair growth control. Am J Pathol 166:985–998.
Bossi O, Gartsbein M, Leitges M, Kuroki T, Grossman S, Tennenbaum T. 2008.
UV irradiation increases ROS production via PKCdelta signaling in primary murine
fibroblasts. J Cell Biochem 105:194–207.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. 1997. The
capsaicin receptor: A heat-activated ion channel in the pain pathway. Nature 389:816–824.
Cho S, Kim HH, Lee MJ, Lee S, Park CS, Nam SJ, Han JJ, Kim JW, Chung JH. 2008.
Phosphatidylserine prevents UV-induced decrease of type I procollagen and increase of
MMP-1 in dermal fibroblasts and human skin in vivo. J Lipid Res 49:1235–1245.
Chung MK, Lee H, Caterina MJ. 2003. Warm temperatures activate TRPV4 in mouse 308
keratinocytes. J Biol Chem 278:32037–32046.
Chung MK, Lee H, Mizuno A, Suzuki M, Caterina MJ. 2004. TRPV3 and TRPV4 mediate
warmth-evoked currents in primary mouse keratinocytes. J Biol Chem 279:21569–21575.
Denda M, Fuziwara S, Inoue K, Denda S, Akamatsu H, Tomitaka A, Matsunaga K. 2001.
Immunoreactivity of VR1 on epidermal keratinocyte of human skin. Biochem Biophys Res
Commun 285:1250–1252.
Dhitavat J, Fairclough RJ, Hovnanian A, Burge SM. 2004. Calcium pumps and keratinocytes:
Lessons from Darier’s disease and Hailey-Hailey disease. Br J Dermatol 150:821–828.
Fisher GJ, Datta SC, Talwar HS, Wang ZQ, Varani J, Kang S, Voorhees JJ. 1996. Molecular
basis of sun-induced premature skin ageing and retinoid antagonism. Nature 379:335–339.
Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, Voorhees JJ. 2002. Mechanisms
of photoaging and chronological skin aging. Arch Dermatol 138:1462–1470.
Fisher GJ, Wang ZQ, Datta SC, Varani J, Kang S, Voorhees JJ. 1997. Pathophysiology of
premature skin aging induced by ultraviolet light. N Engl J Med 337:1419–1428.
Gilchrest BA. 1989. Skin aging and photoaging: An overview. J Am Acad Dermatol 21:610–
613.
Gossas T, Danielson UH. 2006. Characterization of Ca2þ interactions with matrix
metallopeptidase-12: Implications for matrix metallopeptidase regulation. Biochem J
398:393–398.
Grandjean-Laquerriere A, Le Naour R, Gangloff SC, Guenounou M. 2005. Contribution of
protein kinase A and protein kinase C pathways in ultraviolet B-induced IL-8 expression by
human keratinocytes. Cytokine 29:197–207.
He YY, Huang JL, Chignell CF. 2004. Delayed and sustained activation of extracellular signal￾regulated kinase in human keratinocytes by UVA: Implications in carcinogenesis. J Biol
Chem 279:53867–53874.
Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH. 1980. Calcium
regulation of growth and differentiation of mouse epidermal cells in culture. Cell 19:245–
254.
Inoue K, Koizumi S, Fuziwara S, Denda S, Inoue K, Denda M. 2002. Functional vanilloid
receptors in cultured normal human epidermal keratinocytes. Biochem Biophys Res
Commun 291:124–129.
Jenkins G. 2002. Molecular mechanisms of skin ageing. Mech Ageing Dev 123:801–810.
Kim HH, Cho S, Lee S, Kim KH, Cho KH, Eun HC, Chung JH. 2006. Photoprotective and
anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo. J Lipid Res 47:921–
930.
Kim HH, Shin CM, Park CH, Kim KH, Cho KH, Eun HC, Chung JH. 2005. Eicosapentaenoic
acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts. J Lipid Res
46:1712–1720.
Kobayashi T, Kishimoto J, Ge Y, Jin W, Hudson DL, Ouahes N, Ehama R, Shinkai H,
Burgeson RE. 2001. A novel mechanism of matrix metalloproteinase-9 gene expression
implies a role for keratinization. EMBO Rep 2:604–608.
Kohn EC, Jacobs W, Kim YS, Alessandro R, Stetler-Stevenson WG, Liotta LA. 1994.
Calcium influx modulates expression of matrix metalloproteinase-2 (72-kDa type IV
collagenase, gelatinase A). J Biol Chem 269:21505–21511.
Li WH, Lee YM, Kim JY, Kang S, Kim S, Kim KH, Park CH, Chung JH. 2007. Transient
receptor potential vanilloid-1 mediates heat-shock-induced matrix
metalloproteinase-1 expression in human epidermal keratinocytes. J Invest Dermatol
127:2328–2335.
Matsui MS, Wang N, DeLeo VA. 1996. Ultraviolet radiation B induces differentiation and
protein kinase C in normal human epidermal keratinocytes. Photodermatol
Photoimmunol Photomed 12:103–108.
Matsui MS, Wang N, MacFarlane D, DeLeo VA. 1994. Long-wave ultraviolet radiation
induces protein kinase C in normal human keratinocytes. Photochem Photobiol
59:53–57.
Mendez F, Penner R. 1998. Near-visible ultraviolet light induces a novel ubiquitous calcium￾permeable cation current in mammalian cell lines. J Physiol 507:365–377.
Mezey E, Toth ZE, Cortright DN, Arzubi MK, Krause JE, Elde R, Guo A, Blumberg PM,
Szallasi A. 2000. Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1-
like immunoreactivity, in the central nervous system of the rat and human. Proc Natl Acad
Sci USA 97:3655–3660.
Mignatti P, Rifkin DB. 1993. Biology and biochemistry of proteinases in tumor invasion.
Physiol Rev 73:161–195.
Mignatti P, Robbins E, Rifkin DB. 1986. Tumor invasion through the human amniotic
membrane: Requirement for a proteinase cascade. Cell 47:487–498.
Missiaen L, Robberecht W, van den Bosch L, Callewaert G, Parys JB, Wuytack F,
Raeymaekers L, Nilius B, Eggermont J, De Smedt H. 2000. Abnormal intracellular
ca(2þ)homeostasis and disease. Cell Calcium 28:1–21.
Morris VL, Chan BM. 2007. Interaction of epidermal growth factor, Ca2þ, and matrix
metalloproteinase-9 in primary keratinocyte migration. Wound Repair Regen 15:907–915.
Mukhopadhyay S, Munshi HG, Kambhampati S, Sassano A, Platanias LC, Stack MS. 2004.
Calcium-induced matrix metalloproteinase 9 gene expression is differentially regulated by
ERK1/2 and p38 MAPK in oral keratinocytes and oral squamous cell carcinoma. J Biol Chem
279:33139–33146.
Nakagaki T, Oda J, Koizumi H, Fukaya T, Yasui C, Ueda T. 1990. Ultraviolet action
spectrum for intracellular free Ca2þ increase in human epidermal keratinocytes. Cell
Struct Funct 15:175–179.
Oh U, Hwang SW, Kim D. 1996. Capsaicin activates a nonselectivecation channel in cultured
neonatal rat dorsal root ganglion neurons. J Neurosci 16:1659–1667.
Premkumar LS, Ahern GP. 2000. Induction of vanilloid receptor channel activity by protein
kinase C. Nature 408:985–990.
Rabe JH, Mamelak AJ, McElgunn PJ, Morison WL, Sauder DN. 2006. Photoaging:
Mechanisms and repair. J Am Acad Dermatol 55:1–19.
Sasamura T, Sasaki M, Tohda C, Kuraishi Y. 1998. Existence of capsaicin-sensitive
glutamatergic terminals in rat hypothalamus. Neuroreport 9:2045–2048.
Schieven GL, Kirihara JM, Gilliland LK, Uckun FM, Ledbetter JA. 1993. Ultraviolet radiation
rapidly induces tyrosine phosphorylation and calcium signaling in lymphocytes. Mol Biol
Cell 4:523–530.
Seo JY, Kim EK, Lee SH, Park KC, Kim KH, Eun HC, Chung JH. 2003. Enhanced expression
of cylooxygenase-2 by UV in aged human skin in vivo. Mech Ageing Dev
124:903–910.
Seo JY, Lee SH, Youn CS, Choi HR, Rhie GE, Cho KH, Kim KH, Park KC, Eun HC, Chung
JH. 2001. Ultraviolet radiation increases tropoelastin mRNA expression in the epidermis
of human skin in vivo. J Invest Dermatol 116:915–919.
Southall MD, Li T, Gharibova LS, Pei Y, Nicol GD, Travers JB. 2003. Activation of epidermal
vanilloid receptor-1 induces release of proinflammatory mediators in human
keratinocytes. J Pharmacol Exp Ther 304:217–222.
Spielberg H, June CH, Blair OC, Nystrom-Rosander C, Cereb N, Deeg HJ. 1991.
UV irradiation of lymphocytes triggers an increase in intracellular Ca2þ and prevents lectin￾stimulated Ca2þ mobilization: Evidence for UV- and nifedipine-sensitive Ca2þ channels.
Exp Hematol 19:742–748.
Sternlicht MD, Werb Z. 2001. How matrix metalloproteinases regulate cell behavior. Annu
Rev Cell Dev Biol 17:463–516.
Sudbeck BD, Pilcher BK, Pentland AP, Parks WC. 1997. Modulation of intracellular calcium
levels inhibits secretion of collagenase 1 by migrating keratinocytes. Mol Biol Cell 8:811–
824.
Tominaga M, Caterina MJ, Malmberg AB, Rosen TA, Gilbert H, Skinner K, Raumann BE,
Basbaum AI, Julius D. 1998. The cloned capsaicin receptor integrates multiple pain￾producing stimuli. Neuron 21:531–543.
Varani J, Warner RL, Gharaee-Kermani M, Phan SH, Kang S, Chung JH, Wang ZQ, Datta
SC, Fisher GJ, Voorhees JJ. 2000. Vitamin A antagonizes decreased cell growth and
elevated collagen-degrading matrix metalloproteinases and stimulates collagen
accumulation in naturally aged human skin. J Invest Dermatol 114:480–486.
Vellani V, Mapplebeck S, Moriondo A, Davis JB, McNaughton PA. 2001. Protein kinase
activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and
anandamide. J Physiol 534:813–825.
Veronesi B, Oortgiesen M, Carter JD, Devlin RB. 1999. Particulate matter initiates Go6976
inflammatory cytokine release by activation of capsaicin and acid receptors in a human
bronchial epithelial cell line. Toxicol Appl Pharmacol 154:106–115.
Vincenti MP, Brinckerhoff CE. 2002. Transcriptional regulation of collagenase (MMP-1,
MMP-13) genes in arthritis: Integration of complex signaling pathways for the recruitment
of gene-specific transcription factors. Arthritis Res 4:157–164.
Wood JN, Winter J, James IF, Rang HP, Yeats J, Bevan S. 1988. Capsaicin-induced ion fluxes
in dorsal root ganglion cells in culture. J Neurosci 8:3208–3220.
JOURNAL OF CELLULAR PHYSIOLOGY
TRPV1 MEDIATES UV-INDUCED MMP-1 EXPRESSION 775