Journal of Dermatological Science, 4 (1992) 0
1992 Elsevier
DESC
Science
Publishers
1 l- 17
B.V. All rights
reserved.
09231811/92/$05.00
11
00146
Determination of the action spectrum for UV-induced plasminogen activator synthesis in mouse keratinocytes in vitro Akira
Takashima,
Shoshi
Yasuda
and Nobuyuki
Mizuno
Department of Dermatology, Nagoya City University Medical School, Nagoya, Japan (Received
Key words:
15 March
1990; accepted
16 March
Plasminogen
activator;
UV light; Keratinocyte;
1992)
Action
spectrum
Abstract Mouse
epidermal
plasminogen to a broad response
keratinocyte-derived
activator spectrum study
(PA) activity
Pam
212 cells were irradiated
by measuring
of light in the UVB range
revealed
that
a maximal
the capacity induced
enhancement,
with UV light, and the culture
to convert
a significant 15-fold
exogenous
elevation
higher
than
plasminogen
of PA activity
non-irradiated
media
into plasmin.
were examined Exposure
at 16 h after irradiation.
controls,
was induced
for
of cells A dose-
at a sublethal
UVB dose of 100 J/m’. which significantly inhibited cell proliferation without affecting cell viability. Addition of 5 ug/ml of cycloheximide lowered the UV-induced elevation of PA activity, suggesting that protein synthesis is required for this phenomenon.
Action
spectra
for PA synthesis
and the data
demonstrated
that
sults suggest
that UV exposure
were obtained
the action
by irradiating
spectrum
is an important
was 250-320
physiological
Introduction Plasminogen activator (PA) is a highly specific serine protease which converts the inactive zymogen plasminogen into plasmin, a general protease of trypsin-like specificity. At least two types of PAS have been identified and characterized, urokinase-type PA (uPA) and tissue-type PA (tPA). Keratinocytes have been shown to express mRNA for, and to synthesize and secrete, PA of both types in damaged skin as well as in culture Correspondence
to: Akira
Takashima.
Present
address:
De-
partment of Dermatology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd.. Dallas, TX 75235-9069, USA.
trigger
cells with monochromatic
light ranging
nm in length
between
for modulating
with a peak
PA synthesis
from 250 to 360 mm,
260 and 280 nm. The re-
in the epidermis.
[l-7]. Recent studies have suggested that PA plays an important role in several physiological and pathological processes in the epidermis, including keratinocyte migration [ 3,8,9], terminal differentiation of keratinocytes [ 2,8, lo], acantholysis induced by pemphigus IgG [ 1,4,6,7,11], and development or maintenance of psoriatic epiderma1 changes [6,7,12,13]. Previous studies using cultures of various cell types have shown that PA production can be modulated by many biological effecters, including tumor promoters [ 5,141, retinoids [ 151, steroid hormones [ 151, immunological cytokines [ 15,161 and cyclic nucleotides [ 171. Rather limited information is available, however, concerning the biological stimuli for regulating PA production in
12
keratinocytes [ 1,2,8,18]. One of the likely physiological modulators in keratinocyte PA synthesis is UV light, and we have suggested the involvement of the PA/plasmin system in the experimentally induced sunburn reaction [ 191. Although UV light has been reported to enhance PA synthesis in fibroblasts [20-231, no one has examined the effects of UV exposure on keratinocyte PA production. Therefore, we undertook in vitro studies to reveal whether UV light stimulates PA synthesis in keratinocytes and to determine the action spectrum for this photoreaction. Materials and Methods Cell culture
Mouse epidermal keratinocyte-derived cell line, Pam 2 12 [ 241, was used in this study. Cells were cultured in RPM1 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco), 10 mM HEPES buffer, 100 U/ml penicillin and 100 ug/ml streptomycin. Cultures were maintained at 37 “C in an atmosphere of 5 % CO, in air and 100 % relative humidity. In UV exposure experiments, cells were trypsinized and 5 x lo4 cells were cultured on a 35 mm tissue culture dish (Falcon, Oxnard, CA) in the above media for 24 h. Subsequently, cells were washed 3 times with 0.15 M phosphate buffered saline (PBS), pH 7.4, and were irradiated with UV light in the presence of 1 ml PBS. Cell viability was assessed by trypan blue exclusion and the cell number determined using a hemocytometer. Light sources and UV irradiation Cells adherent to culture dishes were exposed to UV light through a quartz lid. The light sources used in time course and dose-dependency studies (Figs. 1 and 2) were UVB fluorescence lamps, FL20S.E-30 (Toshiba, Tokyo), which emitted wavelengths ranging from 280 to 370 nm with a peak at 305 nm. The irradiance on the dish bottom was 4.2 W/m2, as measured with a UV radiometer (UVR 305/365.D, Eisai-Toshiba, Tokyo). In other studies (Table I and Fig. 3), cells were irradiated with an irradiation monochromator
(CRM-FM, Japan Spectroscopic Co., Tokyo), equipped with a 5 kW xenon arc lamp and a plane grating with 600 lines/mm*. A uniform irradiation system with a 4 x 4 cm irradiating area was set at the exit slit, and the irradiance uniformity measured at 280 nm was 100 k 16%. The half-band width was 10.4 nm. The irradiance was measured with a calibrated quartz vacuum thermopile (Japan Spectroscopic Co.). Assay of plasminogen activator activity Immediately after the UV exposure, cells were washed with PBS, fed with 1 ml of assay medium, serum-free RPM1 1640 supplemented with HEPES and antibiotics, and cultured for an additional 3-48 h. the culture medium was centrifuged at 1500 x g for 15 min and the supernatant was assayed for PA activity. An aliquot (400 ~1) of culture supernatant was added to a mixture of 450 ug/ml of plasminogen (Sigma Chemical Co., St. Louis, MO) (10 ul), 0.5 M Tris-HCl, pH 8.0 (100 ~1) and distilled water (400 ~1). An incubation for 45 min at 37 “C was performed to convert plasminogen into plasmin. Subsequently, the reaction mixture was incubated for 15 min at 37 “C in the presence of 0.2 mM Boc-Val-LeuLys-MCA (Peptide Institute, Osaka) (50 ul), a synthetic fluorogenic peptide substrate for plasmin [25]. The reaction was terminated by adding 2 ml of 0.1 N acetic acid. PA activity was assessed by measuring the liberated AMC with a fluorescence spectrophotometer (Hitachi 65010s) (excitation at 380 nm, emission at 460 nm), and was expressed in IU/ml using serial dilutions of purified urokinase (Green Cross, Osaka) as standards. Results Effects of UVB irradiation on secretion of plasminogen activator activity Initial experiments were carried out to examine whether irradiation of Pam 212 cells with a broad spectra of UVB enhance the secretion of PA activity. Fig. 1 shows the typical time course of PA activity in the culture medium following
l-
-40
0
400 UVB fluence (Jim’)
Fig. 2. UV dose-response of PA activity, cell number and cell viability in Pam 212 cells. Cells (5 x 10” cells/dish) were cultured for 24 h in serum-supplemented media, then irradiated with the indicated doses of UVB and cultured for an additional 24 h. Data shown are the mean k SD of PA activity (0) and the mean number of cells (0) and cell viability (A) from duplicate cultures determined at 24 h after exposure. PA activity is plotted on Y-axis in logarithmic scale. The cell number immediately after UV exposure was 2.2 & 0.2 ( x 10’ cells/ dish).
Culture period after UVB exposure
(h)
Fig. 1. Kinetics of induction of PA activity after UV irradiation. Cells were irradiated with 80 J/m’ of UVB (0), and incubated thereafter in serum-free media. Control nonirradiated cells (A) were treated in parallel. At the indicated times. conditioned medium was collected and tested for PA activity. PA activity is plotted on Y-axis in logarithmic scale. Data shown are the mean k SD from triplicate cultures. The broken line indicates the UV-induced PA activity, which is the difference in the PA level between UV-irradiated and non-irradiated cells.
UVB irradiation. In the absence of UV exposure, PA activity was first detected after 16 h of culture. Subsequently, the activity increased rapidly and it reached 0.06 and 0.47 IU/ml at 24 and 48 h, respectively. Irradiation of cells with 80 J/ m2 of UVB induced a significant (P
1992 Elsevier
DESC
Science
Publishers
1 l- 17
B.V. All rights
reserved.
09231811/92/$05.00
11
00146
Determination of the action spectrum for UV-induced plasminogen activator synthesis in mouse keratinocytes in vitro Akira
Takashima,
Shoshi
Yasuda
and Nobuyuki
Mizuno
Department of Dermatology, Nagoya City University Medical School, Nagoya, Japan (Received
Key words:
15 March
1990; accepted
16 March
Plasminogen
activator;
UV light; Keratinocyte;
1992)
Action
spectrum
Abstract Mouse
epidermal
plasminogen to a broad response
keratinocyte-derived
activator spectrum study
(PA) activity
Pam
212 cells were irradiated
by measuring
of light in the UVB range
revealed
that
a maximal
the capacity induced
enhancement,
with UV light, and the culture
to convert
a significant 15-fold
exogenous
elevation
higher
than
plasminogen
of PA activity
non-irradiated
media
into plasmin.
were examined Exposure
at 16 h after irradiation.
controls,
was induced
for
of cells A dose-
at a sublethal
UVB dose of 100 J/m’. which significantly inhibited cell proliferation without affecting cell viability. Addition of 5 ug/ml of cycloheximide lowered the UV-induced elevation of PA activity, suggesting that protein synthesis is required for this phenomenon.
Action
spectra
for PA synthesis
and the data
demonstrated
that
sults suggest
that UV exposure
were obtained
the action
by irradiating
spectrum
is an important
was 250-320
physiological
Introduction Plasminogen activator (PA) is a highly specific serine protease which converts the inactive zymogen plasminogen into plasmin, a general protease of trypsin-like specificity. At least two types of PAS have been identified and characterized, urokinase-type PA (uPA) and tissue-type PA (tPA). Keratinocytes have been shown to express mRNA for, and to synthesize and secrete, PA of both types in damaged skin as well as in culture Correspondence
to: Akira
Takashima.
Present
address:
De-
partment of Dermatology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd.. Dallas, TX 75235-9069, USA.
trigger
cells with monochromatic
light ranging
nm in length
between
for modulating
with a peak
PA synthesis
from 250 to 360 mm,
260 and 280 nm. The re-
in the epidermis.
[l-7]. Recent studies have suggested that PA plays an important role in several physiological and pathological processes in the epidermis, including keratinocyte migration [ 3,8,9], terminal differentiation of keratinocytes [ 2,8, lo], acantholysis induced by pemphigus IgG [ 1,4,6,7,11], and development or maintenance of psoriatic epiderma1 changes [6,7,12,13]. Previous studies using cultures of various cell types have shown that PA production can be modulated by many biological effecters, including tumor promoters [ 5,141, retinoids [ 151, steroid hormones [ 151, immunological cytokines [ 15,161 and cyclic nucleotides [ 171. Rather limited information is available, however, concerning the biological stimuli for regulating PA production in
12
keratinocytes [ 1,2,8,18]. One of the likely physiological modulators in keratinocyte PA synthesis is UV light, and we have suggested the involvement of the PA/plasmin system in the experimentally induced sunburn reaction [ 191. Although UV light has been reported to enhance PA synthesis in fibroblasts [20-231, no one has examined the effects of UV exposure on keratinocyte PA production. Therefore, we undertook in vitro studies to reveal whether UV light stimulates PA synthesis in keratinocytes and to determine the action spectrum for this photoreaction. Materials and Methods Cell culture
Mouse epidermal keratinocyte-derived cell line, Pam 2 12 [ 241, was used in this study. Cells were cultured in RPM1 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Gibco), 10 mM HEPES buffer, 100 U/ml penicillin and 100 ug/ml streptomycin. Cultures were maintained at 37 “C in an atmosphere of 5 % CO, in air and 100 % relative humidity. In UV exposure experiments, cells were trypsinized and 5 x lo4 cells were cultured on a 35 mm tissue culture dish (Falcon, Oxnard, CA) in the above media for 24 h. Subsequently, cells were washed 3 times with 0.15 M phosphate buffered saline (PBS), pH 7.4, and were irradiated with UV light in the presence of 1 ml PBS. Cell viability was assessed by trypan blue exclusion and the cell number determined using a hemocytometer. Light sources and UV irradiation Cells adherent to culture dishes were exposed to UV light through a quartz lid. The light sources used in time course and dose-dependency studies (Figs. 1 and 2) were UVB fluorescence lamps, FL20S.E-30 (Toshiba, Tokyo), which emitted wavelengths ranging from 280 to 370 nm with a peak at 305 nm. The irradiance on the dish bottom was 4.2 W/m2, as measured with a UV radiometer (UVR 305/365.D, Eisai-Toshiba, Tokyo). In other studies (Table I and Fig. 3), cells were irradiated with an irradiation monochromator
(CRM-FM, Japan Spectroscopic Co., Tokyo), equipped with a 5 kW xenon arc lamp and a plane grating with 600 lines/mm*. A uniform irradiation system with a 4 x 4 cm irradiating area was set at the exit slit, and the irradiance uniformity measured at 280 nm was 100 k 16%. The half-band width was 10.4 nm. The irradiance was measured with a calibrated quartz vacuum thermopile (Japan Spectroscopic Co.). Assay of plasminogen activator activity Immediately after the UV exposure, cells were washed with PBS, fed with 1 ml of assay medium, serum-free RPM1 1640 supplemented with HEPES and antibiotics, and cultured for an additional 3-48 h. the culture medium was centrifuged at 1500 x g for 15 min and the supernatant was assayed for PA activity. An aliquot (400 ~1) of culture supernatant was added to a mixture of 450 ug/ml of plasminogen (Sigma Chemical Co., St. Louis, MO) (10 ul), 0.5 M Tris-HCl, pH 8.0 (100 ~1) and distilled water (400 ~1). An incubation for 45 min at 37 “C was performed to convert plasminogen into plasmin. Subsequently, the reaction mixture was incubated for 15 min at 37 “C in the presence of 0.2 mM Boc-Val-LeuLys-MCA (Peptide Institute, Osaka) (50 ul), a synthetic fluorogenic peptide substrate for plasmin [25]. The reaction was terminated by adding 2 ml of 0.1 N acetic acid. PA activity was assessed by measuring the liberated AMC with a fluorescence spectrophotometer (Hitachi 65010s) (excitation at 380 nm, emission at 460 nm), and was expressed in IU/ml using serial dilutions of purified urokinase (Green Cross, Osaka) as standards. Results Effects of UVB irradiation on secretion of plasminogen activator activity Initial experiments were carried out to examine whether irradiation of Pam 212 cells with a broad spectra of UVB enhance the secretion of PA activity. Fig. 1 shows the typical time course of PA activity in the culture medium following
l-
-40
0
400 UVB fluence (Jim’)
Fig. 2. UV dose-response of PA activity, cell number and cell viability in Pam 212 cells. Cells (5 x 10” cells/dish) were cultured for 24 h in serum-supplemented media, then irradiated with the indicated doses of UVB and cultured for an additional 24 h. Data shown are the mean k SD of PA activity (0) and the mean number of cells (0) and cell viability (A) from duplicate cultures determined at 24 h after exposure. PA activity is plotted on Y-axis in logarithmic scale. The cell number immediately after UV exposure was 2.2 & 0.2 ( x 10’ cells/ dish).
Culture period after UVB exposure
(h)
Fig. 1. Kinetics of induction of PA activity after UV irradiation. Cells were irradiated with 80 J/m’ of UVB (0), and incubated thereafter in serum-free media. Control nonirradiated cells (A) were treated in parallel. At the indicated times. conditioned medium was collected and tested for PA activity. PA activity is plotted on Y-axis in logarithmic scale. Data shown are the mean k SD from triplicate cultures. The broken line indicates the UV-induced PA activity, which is the difference in the PA level between UV-irradiated and non-irradiated cells.
UVB irradiation. In the absence of UV exposure, PA activity was first detected after 16 h of culture. Subsequently, the activity increased rapidly and it reached 0.06 and 0.47 IU/ml at 24 and 48 h, respectively. Irradiation of cells with 80 J/ m2 of UVB induced a significant (P
