prolonged recovery of ultraviolet b-irradiated skin in neuropsin (klk8)-deficient mice

Cutaneous Biology

Prolonged recovery of ultraviolet B-irradiated skin in neuropsin(KLK8)-deficient mice

T . K I R I H A R A , K . M A T S U M O T O - M I Y A I , Y . N A K A M U R A , T . S A D A Y A M A ,

S . Y O S H I D A * A N D S . S H I O S A K A

Division of Structural Cell Biology, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101, Japan

*Department of Anatomy, Asahikawa Medical College, Asahikawa, Japan

Accepted for publication 25 November 2002

Summary Background Neuropsin (KLK8), a serine protease of the kallikrein family, is thought to be involved

in the function of keratinocytes, i.e. migration, differentiation and desquamation. However, how

neuropsin participates is still unknown.

Objective To observe the epidermal function of serine protease in neuropsin-deficient mice.

Methods We irradiated the skin of neuropsin-deficient mice with ultraviolet light to induce acute

inflammation and compared the morphology with that of wild-type mice.

Results We observed a phenotypic change in the epidermis. An acute inflammatory dose of

ultraviolet light induced a marked increase in neuropsin mRNA expression in the skin. The signal

intensity of the mRNA expression was highest on day 2–3 after irradiation, when keratinocytes

were aligned irregularly in the recovery period. Morphological comparison between neuropsin – ⁄ –and + ⁄ + mice revealed that an irregular alignment of cells in the thickened epidermis was obvious

on day 2 after irradiation in the wild-type mice, whereas it was prolonged for at least 2 days in the

neuropsin-deficient mice. The stratum corneum of neuropsin-deficient mice was remarkably thicker

than that of the wild-type mice at 5, 14 and 21 days after irradiation. The increase, as a response to

this stimulus, in involucrin immunoreactivity, a marker for cell envelope assembly, was delayed in

the mutant mice.

Conclusions Thus, neuropsin might be involved early in the process of differentiation, such as in

the assembly of the cell envelope, but not in migration and desquamation.

Key words: epidermis, kallikrein family, keratinocyte, neuropsin, serine protease

The kallikreins constitute a subfamily of secreted serine

proteases of about 230 amino acid residues. There is a

large kallikrein multigene containing 13 different

kallikrein genes in rats,1,2 26 in mice,3,4 and at least

15 in humans.5,6 Some members of the kallikrein

family have been postulated to be involved in the

processing of precursor protein for physiologically

active proteins or peptides.4 However, recent studies

suggest that members of the family are involved not

only in the activation and processing of such precursor

proteins, but also in a variety of cellular events such as

neural plasticity,7 the terminal differentiation of kera-

tinocytes,8 and the regulation of myelin turnover.9

Neuropsin (KLK8 in mouse and human; also known

as human TADG-14 ⁄ ovasin and rat BSP-1) was first

cloned from the mouse brain by us in 199510 and since

has been shown to belong to the kallikrein multigene

family11,12 and to be involved in a variety of functions

in the brain, epidermis and uterus.7,8,13–16 Previous

studies suggested that epidermal neuropsin was impli-

cated in cornification or desquamation, because it is

related to the pathogenesis of hyperkeratosis upon

inflammation,8 and is seen in hyperkeratotic epidermal

diseases, such as psoriasis vulgaris, seborrhoeic kera-

tosis, lichen planus and squamous cell carcinoma.15

This notion is fully consistent with the observation thatCorrespondence: Dr Sadao Shiosaka.

E-mail: [email protected]

British Journal of Dermatology 2003; 149: 700–706.

700 � 2003 British Association of Dermatologists

there was no or only a weak expression of neuropsin

mRNA in the skin of individuals with basal cell

carcinoma.15 To examine neuropsin’s still unknown

function, we produced neuropsin-deficient mice and

studied them by irradiating their skin with ultraviolet

(UV) B light to induce acute inflammation. We then

compared the morphology with that of wild-type mice.

Here we observed a phenotypic change in the epidermis

of neuropsin-deficient mice and found that neuropsin is

involved in an earlier process of the terminal differen-

tiation rather than following cornification and desqu-

amation.

Materials and methods

Generation of neuropsin-deficient mice

Neuropsin-deficient mice were produced as described

elsewhere.17 Briefly, the complementary DNA sequence

of neuropsin was used to isolate genomic clones from a

kFIXII mouse 129 ⁄ Sv genomic library (Stratagene, La

Jolla, CA, U.S.A.). The targeting vector was constructed

by replacing the genomic sequences encoding exons

1–3 with a neomycin-resistance cassette. The ES cells

(1 · 107) were transfected by electroporation (Bio-Rad

Gene Pulsar; 230 V, 500 lF, 0Æ4 cm cuvette, Biorad,

Hercules, CA, U.S.A.) with a NotI-linearized targeting

construct, placed on feeder layers and then subjected to

positive–negative selection for 8 days in 400 lg mL)1

of G418 (Geneticin, Life Technologies Inc., Rockville,

MD, U.S.A.) and 2 lmol L)1 ganciclovir (a gift from

Syntex Incorporated, Tokyo, Japan). The generation of

chimeras and mutant mice was performed essentially

as described previously. The initial chimeric mice were

mated with C57BL ⁄ 6J. Three clones yielded heterozy-

gotes. Heterozygous F1 animals from 129 ⁄ Sv (foun-

der) · C57BL ⁄ 6J have been backcrossed to C57BL ⁄ 6J

animals through more than seven generations. All

animals used in the present study were treated in

accordance with the institutional guidelines for animal

welfare (Animal Centre Guidelines of the Nara Institute

of Science and Technology).

In situ hybridization histochemistry

Forty-two C57BL ⁄ 6J mice (three mice for each time

point; 7-week-old; Japan SLC, Shizuoka, Japan)

were used for in situ hybridization histochemistry to

detect neuropsin mRNA. The histochemistry was

performed according to Chen et al.10,18 and Kitayoshi

et al.8 Briefly, the sections were fixed for 20 min with

4% formaldehyde in 0Æ1 mol L)1 phosphate buffer

(pH 7Æ4), washed twice in phosphate buffer, and then

treated with 10 mg mL)1 of protease K (Roche Diag-

nostic GmbH, Mannheim, Germany) in 50 mmol L)1

Tris–HCl (pH 7Æ4) and 5 mmol L)1 ethylenediamine

tetraacetic acid. The sections were fixed again for

20 min as above and acetylated with 0Æ25% acetic

anhydride in 0Æ1 mol L)1 triethanolamine for 10 min.

After dehydration through an ascending alcohol

series, the sections were hybridized with 35S-labelled

cRNA probes10 at 55 �C overnight. The sections were

washed at 65 �C in 50% formamide, 2 · saline

sodium citrate buffer, and 5 mmol L)1 dithiothreitol.

Subsequently, they were treated with RNase A then

washed again at high stringency. After dehydration

through an ascending alcohol series, they were

immersed in Kodak NTB2 emulsion and exposed for

3 weeks.

Quantification of hybridization signals

Hybridization signals were quantified with randomly

selected areas that contain 10 consecutive cells of the

stratum granulosum under a bright-field microscope.

Forty-two areas in total (three sections; one section

per animal) were counted for each time point. The

specificity of the hybridization signals was checked

with a sense hybridization probe. The average grain

density of the background in the stratum granulosum

was 0Æ44 ± 0Æ13 grains per cell and the value was

almost identical to the silver grains on the glass slide

using an anti-sense hybridization probe (Fig. 1a-

0–21). Areas with a grain density at least four times

that of the background were counted as positive. The

number of silver grains per cell was indicated

(Fig. 2).

Ultraviolet B irradiation and histology

Each of 24 neuropsin – ⁄ – and + ⁄ + mice (three mice for

each time point; 8–11 weeks old) was used to examine

the histology of UVB-irradiated skin. A bank of six

fluorescent sunlamps (ATTO, DT-20CMP, Tokyo,

Japan), which emit rays of between 280 and 320 nm

with a peak at 312 nm, was used. After the dorsal skin

was shaved, an acute (54 s) single exposure of UVB

(360 J cm)2) was irradiated on to the dorsal skin of the

mice according to several studies.19–21 Nonirradiated

(control ¼ 0 days) or UVB-irradiated skin from mice

which had survived 1, 2, 3, 5, 7, 15 and 21 days after

irradiation, was cryostat-sectioned 10 lm thick,

N E U R O P S I N ( K L K 8 ) I N U V B - I R R A D I A T E D S K I N 7 0 1

� 2003 British Association of Dermatologists, British Journal of Dermatology, 149, 700–706

Figure 1. Time course of the changes in neuropsin mRNA expression and epidermal morphology after ultraviolet (UV) irradiation. UV irradiation

induced a marked increase in hybridization signals for neuropsin mRNA in the skin of C57BL ⁄ 6J (C57) mice. Skin samples were prepared at 0

(nonirradiated control ¼ a-0) and 1, 2, 3, 5, 7 or 21 days after UV irradiation (a-1, a-2, a-3, a-5, a-7 or a-21, respectively). UV induced

morphological changes were compared between wild-type [NP + ⁄ +; b-0–21] and neuropsin-deficient [NP – ⁄ –; c-0–21] mice over the same period.

Healing was apparently delayed in the NP – ⁄ – mouse skin. Scale bar ¼ 50 lm. White lines represent the surface of the epidermis. Abbreviations:

d, dermis; ep, epidermis; sb, stratum basale; sc, stratum corneum; sg, stratum granulosum; ss, stratum spinosum.

7 0 2 T . K I R I H A R A et al.


mounted on glass slides, and stored at )80 �C until use.

Two sets of serial tissue sections were produced, one

was subjected to haematoxylin–eosin staining for

morphological observation and the other to immunoh-

istochemistry for involucrin, a marker protein of

cornified envelope.

Immunohistochemistry for involucrin and neuropsin

The immunofluorescent techniques used for involucrin

and neuropsin were in accord with Ishida-Yamamoto

and Iizuka22 and Inoue et al., respectively.14 Six

micrometre thick cryostat sections of frozen samples

were air-dried and fixed with acetone at 4 �C for

10 min. After preincubation with 5% bovine serum

albumin in phosphate-buffered saline (PBS) for 1 h at

room temperature, the sections were incubated with

anti-involucrin antibody (Berkeley Antibody Company,

Richmond, CA, U.S.A.; 1 : 1000) or antineuropsin

monoclonal antibody (MabB5, MBL Co., Nagoya,

Japan; 1 : 1000) diluted with 5% bovine serum albu-

min in PBS overnight at 4 �C. After a wash, the

sections were incubated with fluorescein isothiocya-

nate (FITC)-labelled antibody (TAGO, Camarillo, CA,

U.S.A.) diluted with PBS (1 : 1500) overnight at 4 �C.

Fluorescent images were acquired by computer

through a charged couple device (CCD) camera

(Hamamatsu Photonics, Hamamatsu, Japan) and ana-

lysed using WinRoof v3.3 image analysis software

(Mitani Co., Fukui, Japan).

Data analyses

The unpaired t-test was performed for statistical ana-

lyses using StatViewJ-5Æ0 software (SAS institute, Cary,

NC, U.S.A.).

Results

Brief irradiation of shaved dorsal skin with UVB light

induced a transient increase in neuropsin mRNA

expression in the epidermis of the C57BL ⁄ 6J mouse

(Fig. 1a-1–7). The expression of the hybridization

signal was still low at 1 day after irradiation (Fig. 1a-1)

but became prominent at 2 days (Fig. 1a-2), when the

keratinocytes aligned irregularly, and peaked at 3 days

(Fig. 1a-3). Thereafter, the signal gradually faded

(Fig. 1a-5,7) with a return to the control level 21 days

after irradiation (Fig. 1a-21, cf. Fig. 1a-0). A low level

of neuropsin mRNA is constitutively expressed in

normal skin (Fig. 1a-0).8,14 There was no change of

signal expression in the skin of the control group of

mice which were shaved and survived without irradi-

ation (control group).

The silver grains per cell in the stratum granulosum

were counted (Fig. 2). The increase in silver grains per

cell was significant at 2, 3 (peak; 2Æ5 times of control),

5 and 7 days after irradiation (P < 0Æ0001; 2, 3, 5 and

7 days after irradiation vs. control, n ¼ 30 each). The

data showed that neuropsin mRNA levels rapidly rose

at day 2–3 after irradiation in the thick epidermis

composed of loosely aligned keratinocytes. Therefore,

we suggest that neuropsin functions in the transient

period, when keratinocytes are highly activated.

The loss of the neuropsin gene in the neuropsin-

deficient mice was checked by polymerase chain

reaction and confirmed by Southern and Northern blot

analyses,17 and by immunohistochemistry for neurop-

sin (Fig. 3). Neuropsin-deficient mice showed no

apparent difference from the wild-type either in appear-

ance on visual inspection or in the growth curves of

body weight up to 120 weeks of age (data not shown).

There was also no apparent difference in the morphol-

ogy of the skin and structures of the epidermal

sublayers among homozygous, heterozygous and wild

genotypes under nonpathological conditions (data not

shown).

To explore whether the morphology of the highly

activated skin is changed in the neuropsin-deficient

mice, we compared the time course of epidermal

morphological change after acute UV irradiation

between wild-type and mutant mice. UV irradiation

7

6

5

4

3

2

1

0

No.

of s

ilver

gra

in/c

ell i

n st

r.gr

anul

osum

0 1 2 3 5 7 14 21(days)

Shaved control

UV-irradiated group*

* * *

Figure 2. Time course of the changes in neuropsin mRNA expres-

sion. Ultraviolet (UV) irradiation induced a marked increase in silver

grains per keratinocyte in the stratum granulosum of C57BL ⁄ 6J

mice. Silver grains were counted on micrographs. Asterisks show the

significance between the UV-irradiated group (filled square) and

shaved control (open square) (P < 0Æ0001). Error bars indicate the

mean ± SEM, n ¼ 42 for each point.



induced evident morphological changes in the wild-

type mice (Fig. 1b-0–21) consistent with the findings

previously published.23,24 Proliferated cells were

loosely and irregularly aligned in the stratum spinosum

and granulosum 2 days after UV irradiation (Fig. 1b-2)

and parakeratosis was also extensive in the stratum

corneum in this period (Fig. 1b-2, white arrows). The

epidermis became thickest 3 days after irradiation and

the parakeratosis disappeared (Figs 1b-3 and 3a). The

prickle and granular cells were aligned in an orderly

manner in close contact with adjacent cells in this

period. However, neuropsin-deficient mice exhibited

different morphological changes during UV-induced

skin injury and recovery (Fig. 1c-2–5). The acute

morphological changes prior to 2 days after irradi-

ation were identical in wild-type and mutant mice

(Fig. 1b-1,2, 1c-1,2). However, the period in which

keratinocytes were aligned loosely was evidently pro-

longed in the neuropsin-deficient mice (Fig. 1c-2,3).

Parakeratosis was not only more severe, but also lasted

much longer in the mutant than the wild-type mice

(Fig. 1c-2,3, white arrows, cf. Fig. 1b-2,3). Thus, the

results strongly suggested that neuropsin is involved in

the dynamic period, when cells are actively aligning

and assembling prior to cornification.

To examine this issue further, the thickness of the

entire epidermis (stratum basale, stratum granulosum,

stratum spinosum and stratum corneum) was meas-

ured and compared between wild-type and mutant

mice (Fig. 4a; cf. Figs 1b-0–21 and 1c-0–21). The

highest values for epidermal thickness both in wild-type

and in neuropsin-deficient mice were obtained at

3 days after irradiation (Fig. 4a). The decrease there-

after was significantly faster at 5–7 days postirradia-

tion in the mutant mice (Fig. 4a, day 5–7; P < 0Æ02 at

day 7 and 14; wild vs. mutant). The thickness of the

stratum corneum differed significantly between the two

(Fig. 4b). The stratum corneum of neuropsin-deficient

mice was remarkably thicker at 5, 14 and 21 days

after the irradiation (Fig. 4b, P < 0Æ001; P < 0Æ001,

P < 0Æ02, respectively; cf. Fig. 1b-5 and 1c-5, 1b-21,

1c-21; wild vs. mutant). The remaining three layers

(stratum basale, stratum granulosum and stratum

spinosum) did not change significantly except on

day 7 after the irradiation, when they were thinner

(P < 0Æ005). This may have been caused by a delay of

differentiation in these layers. Therefore, desquamation

in the mutant might be completely impaired at least up

to day 21.

To analyse the function of neuropsin further, invo-

lucrin, a marker for the initiation of cell envelope

assembly,25 was immunostained to compare mutant

with wild-type mouse skin (Fig. 4c). Involucrin immu-

noreactivity transiently increased and reached a peak

3 days after irradiation in the wild-type mice (Fig. 4c, a

black arrow). However, the peak of the transient

increase of immunoreactivity was markedly delayed

in the mutant mice (Fig. 4c, grey arrow; P < 0Æ05 at

day 3 and P < 0Æ01 at day 7, wild type vs. mutant).

Discussion

This study clearly demonstrated that the skin of

neuropsin-deficient mice was more vulnerable to an

acute inflammatory dose of UVB than that of wild-type

mice. A deficiency of neuropsin caused marked pro-

longation of the recovery period after UVB irradiation,

i.e. thickening of the entire epidermis, thickening of the

Figure 3. Confirmation of deficiency of neuropsin. The deficiency of

neuropsin was confirmed with immunohistochemistry using

antineuropsin antibody. No immunofluorescence was seen in back

skin of the neuropsin (NP) – ⁄ – mouse (a), although clear fluores-

cence (white arrow) appeared in the epidermis of the NP + ⁄ + mouse

(b). Asterisks in (a) and (b) indicate auto fluorescence of the hair.

Scale bar ¼ 50 lm, ep; epidermis.



stratum corneum and transient irregular alignment of

cells. The increase in involucrin immunoreactivity as a

response to stimulus was delayed in the mutant. As

involucrin is an essential protein for initiating assembly

of the cornified cell envelope, this delay in the increase

in immunoreactive involucrin after UVB irradiation

represents the prolongation of epidermal differentiation

in the mutant. Therefore, neuropsin might be involved

early in the process of differentiation, such as during

the assembly of the cell envelope in the stratum

spinosum and granulosum, by cleaving matrix or cell

adhesion protein(s) that organize the epidermal struc-

ture.26,27 In contrast, neuropsin might not be involved

in the proliferation and migration of keratinocytes,

because there was no or only a weak expression of

neuropsin mRNA in the skin of an individual with

basal cell carcinoma15 and the migrating epithelial

tongue during incisional wounding.8 All together,

neuropsin might be involved in the early process of

differentiation, such as in the assembly of the cell

envelope, but not in migration and desquamation.

Acknowledgments

The authors thank Dr A.Ishida-Yamamoto (Depart-

ment of Dermatology, Asahikawa Medical College) for

critical reading of the manuscript. This work was

supported in part by a grant from the Ministry of

Science, Sport, Culture and Technology.

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Figure 4. Time course of epidermal morphological change after UV

irradiation. (a) Comparison of changes in the thickness of the epi-

dermis (lm) between neuropsin (NP) + ⁄ + and NP – ⁄ – mice. In the

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prolonged recovery of ultraviolet b-irradiated skin in neuropsin (klk8)-deficient mice

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