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REGULAR. ARTICLES 



Cholesterol Sulfate Inhibits Proteases that are Involved in 
Desquamation of Stratum Corneum 

Junko Sato, Mitsuhiro Denda, Jotaro Nakanishi, Junko Nomura, and Junichi Koyama 



We previously reported that desmosomes play a key role 
in the adhesion of corneocytes, and their digestion by 
two types of serine proteases leads to desquamation. 
Patients with recessive X-linked ichthyosis show hyper- 
keratosis attributable to desmosomes, associated with an 
increased content of cholesterol sulfate (CS) and an 
increased thickness of stratum corneum. In this study, 
therefore, we examined the possibility that CS provokes 
the abnormal desquamation, acting as a protease 
inhibitor. Scaling was induced on mice after topical 
application of chymostatin and leupeptin. Visible scale 
was also observed on mice after topical application of 
CS. We found that the stratum corneum thickness of CS- 
treated mice was increased in comparison with that of 
vehicle-treated mice. The thickness of the epidermis and 
the labeling index with proliferating cell nuclear antigen 



from CS-treated mice was almost the same as that from 
vehicle-treated mice. Moreover, in the stratum corneum 
of CS-treated mice, the content of desmosomes was 
higher than that in vehicle-treated mice. CS also 
inhibited the protease-induced cell dissociation of 
human stratum corneum sheets. In vitro, CS competi- 
tively inhibited both types of serine protease: the Kj for 
trypsin was 5.5 X 10" 6 M and that for chymotrypsin was 
2.1 X 1IT 6 M. These results indicate that CS retards 
desquamation by acting as a protease inhibitor. Thus, 
accumulation of stratum corneum in recessive X-linked 
ichthyosis may be a result of the inhibition by excessive CS 
of proteases involved in the dissolution of desmosomes, 
required for desquamation of the stratum corneum. Key 
words: chymotrypsin /desmosome /ichthyosis /trypsin. J Invest 
Dermatol 111:189-193, 1998 



Recessive X-linked ichthyosis, which is caused by a 
deficiency of steroid sulfatase, is a disease exhibit- 
ing hyperkeratosis accompanied by accumulation of 
cholesterol sulfate (CS). It has long been thought 
that lipids, especially CS, act as comeocyte cohesion 
elements. Cholesterol-lowering substances are known to induce scaling 
disorder (Williams and Elias, 1987; Williams et al, 1987), whereas 
topical application of CS induced scaling without acanthosis or increased 
labeling index (Maloney et al, 1984; Elias et al, 1984). The highly 
cohesive ungulate hoof is particularly rich in CS (Wertz and Downing, 
1984); however, no significant differences in CS content were found 
between tightly cohesive stratum corneum of the palm, and the loosely 
cohesive stratum corneum of the upper arm (Serizawa et al, 1992). 
Thus, the relationship between CS and accumulated stratum corneum 
is unclear. Williams suggested the possibility that the failure to 
desquamate in recessive X-linked ichthyosis may be due to CS- 
mediated inhibition of desmosome proteolysis (Williams, 1991). We 
have investigated the mechanism of desquamation in stratum corneum, 
showing that desmosomes play a key role in the adhesion of corneocytes, 
and that their digestion by two types of serine proteases leads to 
desquamation (Suzuki et al, 1993, 1994). Lundstrom and Egelnrd also 
reported that chymotrypsin-like enzyme activity in the stratum comeum 
may play a role in the desquamation process (Lundstrom and Egelrud, 
1991; Sondell et al, 1995). It was reported that numerous desmosomal 



Manuscript received July 1, 1997; revised Febr 
publication February 26, 1998. 

Reprint requests to: Dr. Junko Sato, Shiscido 
Fukuura, Kanazawa-ku, Yokohama 236-8643, Jap; 



structures remained in the outermost layers of the stra 
recessive X-linked ichthyosis (Anton-lamprecht, 1974; Bazex et al, 
1978; Mesquita-Guimaraes, 1981). Because CS inhibited the serine 
protease, acrosin, required for normal sperm capacitation (Burck and 
Zimmerman, 1980), in this study we examined the possibility that CS 
acts as a protease inhibitor, retarding desquamation of stratum comeum. 

MATERIALS AND METHODS 

Animals and topical application Cholesterol 3-Sulfate (CS, Sigma, St. 
Louis, MO) and chymostatin (Peptide Institute, Osaka, Japan) were dissolved 
in dimethyl sulfoxide (DMSO, Wako, Japan) to prepare a 10 raM solution. 
Leupetin (Peptide Institute) was dissolved in distilled water to prepare a 10 mM 
solution. Eighty microliters of CS solution, chymostatin and leupeptin, DMSO 
and distilled water, or DMSO alone was applied to the backs of hairless mice 

from Hoshino (Saitama, Japan). After 3 d. biopsy and collection of stratum 
comeum by tape-stripping was undertaken. 

Histologic observations Biopsy samples were fixed in 10% fonnalin and 
embedded in paraffin. The sections were stained with hematoxylin and eosin. 
Measurements of the thickness of the epidermis were made with a light 
microscope equipped with a CCD camera and image analysis system (Olympus 



in an Epon-cposy in 
croscope (H7100, 
attend uranyl aceta 



0022-202X/98/S10.50 • Copyright © 1998 by The Society for Investigative Dermatology, Inc. 
189 



190 SATO ETAL 



THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 



0.1 M Tris HC1 pH 9, 9 M urea, 2% sodium dodccyl sulfate, and 1% 
mercaptoethanol (200 [0.1 of buffer per 2 mg stratum comeum) for IS h at 37°C 
(Lundstrom and Egelrud, 1990). The extracts were prepared by mixing them 
with a Laemmli's sample buffer (Laemmli, 1970), followed by heating on a 
boiling water bath for 10 min. After centrifugarion, the supernatant was analyzed 

Electophoretic transfer of proteins from the gel to PVDF membrane (Applied 
Biosystems, CA) was followed by immunostating with a Promega Protoblot 
Western Blot AP System (Promega, Madison, WI). Monoclonal antibody, anti- 
DG I, obtained by Boehringer (Mannheim, Germany), was used for detection 

sheet was obtained from the back of a volunteer 5 d after sun exposure. One 

(8 mM DMDAO and 2 mM sodium dodecyl sulfate) with CS, phosphatidyl- 
choline (Sigma, St. Louis, MO), palmitic acid (Sigma), taurocholic acid (Sigma), 
or each vehicle, containing 60 |ig kanamycin at 37°C for 24 h (Takahashi el at, 
1987). The number of released cells in the detegent mixture was counted using 
a Burker-Turk hemocytometer. 

Assay for inhibitory behavior of cholesterol sulfate Crystalline porcin 
pancreatic trypsin (Wako, Osaka, Japan) and crystalline bovine pancreatic 

Ser^Vrg-MCA^piOT-vT o/'suc-Leu-Leu-Val-Tyr-MCA (3120-V) (Peptide 
Institute), respectively, as the substrate. Inhibitory activities of phosphatidyl- 
choline, palmitic acid, cholesterol, and taurocholic acid were also examined. 
All assays were performed at 37°C in 0.1 M Tris HC1 (pH 8.0). 

Statistics The significance of difference was tested using the unpaired 
Student's t test. 

RESULTS 

Application of CS increases the stratum comeum thickness and 
induces abnormal scaling Visible scales were observed on the 
backs of mice 3 d after topical application of CS (Fig 1). Biopsy taken 
at this point showed increased thickness of the stratum comeum layer 
from mice treated with CS in comparison with that from vehicle- 
treated mice (Fig 2). Moreover, quantitative studies showed an 
increased number of stratum comeum layers of CS- versus vehicle- 
treated skin (Fig 3). Furthermore, the thickness of the living part of 
the epidermis from CS-treated mice was the same as that from vehicle- 
treated mice (data not shown). Finally, there were no differences 
between CS-treated and vehicle-treated skin in the labeling index with 
proliferating cell nuclear antigen, a measure of proliferative acitivity 
(data not shown). These results show the effect of CS on increasing 
the number of cell layers, the thickness, and the abnormal scaling of 
the stratum comeum. To examine whether CS acts as a detergent to 
disrupt enzyme function, we also applied phosphatidylcholine as 
amphopatic lipid to the backs of mice. Both daily treatment of 4.88 mg 
CS per ml (10 mM) and daily treatment of 0.488 mg CS per ml 
(1 mM) induced scales on the backs of mice after topical application; 
however, topical application of 4.88 mg phosphatidylcholine per ml 
did not show an increase in the number of scales compared with 
vehicle treatment (data not shown). 

The increased content of desmosomal protein in the stratum 
comeum of CS-treated mice The stratum comeum of mice 
treated with CS or vehicle for 3 d was obtained by tape-stripping and 
the extracted proteins were subjected to sodium dodecyl sulfate - 
polyacrylamide gel electrophoresis. The separated proteins were trans- 
ferred to PVDF membranes and reacted with anti-DG I. As seen in 
Fig 4, the tendency of the increased content of desmoglein I was 
noted in the stratum comeum of CS-treated mice (p = 0.0503, n = 
3). These results suggest that degradation of desmosomes was inhibited 
by the topical application of CS. 

CS inhibited cell dissociation from the stratum cornucm 
sheet CS inhibited cell dissociation in this assay in a concentration- 
dependent manner (Fig 5). One hundred micromoles and 1 mM of 
CS inhibited cell dissociation to 64.0% and 56.9% of the 100% controls, 
respectively. Other amphopathic lipids, 1 mM phosphatidylcholine, 




Figure 1. Increasing the abnormal scales on the backs of mice after 
topical application of CS. Skin surface appearance of a normal vehicle- 
treated control mouse (a) versus a cholesterol sulfate-treated mouse (f>). Animals 
were treated with 80 pj daily for 3 d. 



a ■ b 




Figure 2. Increased thickness of the stratum comeum layer from mice 
treated with CS. (a) The stratum comeum of vehicle-treated control, (b) The 
stratum comeum from animals treated with 80 ul cholesterol sulfate (10 mM) 
daily for 3 d. Scale bars: 5 Urn. 



1 mM palmitic acid, and 1 mM taurocholic acid did not inhibit cell 
dissociation from the stratum comeum sheet (Table I). These results 
show that CS acts directly on cell shedding in the stratum comeum 
and the effect is not simply a detergent effect. 

Inhibition of trypsin and chymotrypsin by CS Phospha- 
tidylcholine, palmitic acid, cholesterol, and taurocholic acid did not 
show inhibitory activity in the assay using trypsin (Fig 6). CS was 
found to be a potent inhibitor of trypsin and chymotrypsin in vitro. 



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CHOLESTEROL SULFATE INHIBITS PROTEASES 191 




192 SATO ET AL 



THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 



in vivo after topical application, the possibility that CS acts as an 
inhibitor in the stratum corneum is indicated. 

DISCUSSION 

The mechanism of regulation of desquamation in the stratum comeum 
is still unknown. We have shown previously that two types of serine 
protease are involved, and that degradation of desmosomes leads to 
desquamation (Suzuki et al, 1993, 1994). Lundstrom and Egelrud also 
reported chymotrypsin-like enzyme activity in the stratum comeum 
(Lundstrom and Egelrud, 1991; Sondell et al, 1995). The disorder of 
cornification, recessive X-linked ichthyosis, exhibits retention hyper- 
keratosis accompanied by accumulation of CS. Because CS inhibits 
the serine protease, acrosin (Burck and Zimmerman, 1980), we decided 
to examine the possibility that CS also plays a role as an inhibitor in 

Increased thickness of the stratum corneum reportedly is induced 
by topical application of CS without acanthosis, increased labeling 
index, or dermal inflammation (Maloney et al, 1984; Elias et al, 1984). 
We also observed scaling on the backs of hairless mice after topical 
applications of CS, and confirmed that there was no difference between 
CS-treated and vehicle-treated mice in the thickness of the epidermis 
and the labeling index with proliferating cell nuclear antigen, an index 
of proliferative activity. In contrast, the number of stratum corneum 
layers and the content of desmoglein I in the stratum comeum of CS- 
treated mice were higher than those in vehicle-treated mice. These 
results suggest that digestion of desmosomal proteins is inhibited by 
CS treatment. 




1/S(1/mol 3120-V xlO ) Cholesterol sulfate (xlO^M) 



Figure 8. Cholesterol sulfate is a competitive inhibitor of chymotrypsin. 

(a) Lineweaver-Burk plot of the inhibition of chymotrypsin by cholesterol 
sulfate (A, 2.98 X 1CT 6 M; □, 2.23 X 1CT 6 M); O, no cholesterol sulfate, (b) 
Dixon plot of chymotrypsin by cholesterol sulfate (O, 1.21 X lCT 1 M 3120-V; 
A, 6.06 X 1CT 5 M 3120-V; □, 3.03 X 10" 5 M 3120-V). 



It has been reported that CS also acts as a second messenger for 
protein kinase C, hence the effects of CS on stratum comeum retention 
might be mediated indirecdy (Chida et al, 1995). To avoid such effects 
of CS on living keratinocytes, we examined the effect of CS in the 
stratum corneum sheets. We found that the cell dissociation from 
the stratum comeum sheet was inhibited by CS, suggesting that CS 
acts directly on the cell shedding in the stratum comeum. 

Finally, we examined the extent of the inhibitory properties of CS 
using commercially available crystallized trypsin and chymotrypsin as 
model enzymes, because enzymes in the stratum comeum were shown 
to be trypsin-like and chymotrypsin-like proteases (Suzuki et al, 1993, 
1994). CS competitively inhibited both trypsin and chymotrypsin with 
K"; values of 5.5 X lCT 6 M and 2.1 X 10" 6 M, respectively. Thus, 
in vitro experiments for inhibitory properties of CS on model enzymes 
showed that values had micromolar concentrations. We used 
1-10 mM CS in the stratum comeum sheet assay and topical application, 
because there may be some difficulties with CS penetration into the 
stratum comeum. Maloney et al (1984) also reported that =5-10 mM 
of CS was needed to induce abnormal scales. 

Although leupeptin and chymostatin showed more potent inhibitors 
of cell dissociation than CS in the stratum comeum sheet assay (data 
not shown), application of CS results in more obvious stratum comeum 
scaling compared with the mixture of leupeptin and chymostatin. This 
may reflect a high affinity of CS for the stratum comeum intercellular 
lipids, and for the physicochemical properties of CS itself (Williams, 
1991). 

To examine whether CS acts as a detergent to disrupt enzyme 
function, amphopatic lipids were tested in looking for scaling in vivo, 
the cell dissociation assay, and the enzyme assay in vitro. Topical 
application of phosphatidylcholine did not induce scales. The cell 
dissociation from the stratum corneum was not inhibited by phospha- 
tidylcholine, palmitic acid, and taurocholic acid. In the enzyme assay 
system in vitro, phosphatidylcholine, palmitic acid, cholesterol, and 
taurocholic acid did not show inhibitory activity. These results show 
the effect is not simply a detergent effect. 

The above results indicate that CS influences desquamation by acting 
as a serine protease inhibitor. Further, the accumulated stratum corneum 
in recessive X-linked ichthyosis may be caused by the inhibition of 
trypsin-like and chymotrypsin-like proteases by excessive CS. This 
could account for the abnormal persistence of numerous desmosomal 
structures in the outermost layers of the stratum comeum in this disease 
(Anton-Lamprecht, 1974; Bazex et al, 1978; Mesquita-Guimaraes, 
1981). Our results suggest a possible new role of CS as an inhibitor 
in desquamation. In addition, quantitative lipid analysis of porcine 



VOL. Ill, NO. 2 AUGUST 1998 



CHOLESTEROL SULFATE INHIBITS PROTEASES 193 



epidermal strata revealed that CS exhibited its concentration in the 
deeper stratum corneum and then abrupdy decreased in the surface 
layer (Cox and Squier, 1986). Thus, CS might regulate not only 
desquamation in pathologic stratum corneum, but also normal 
desquamation; the content of CS in the stratum corneum might 
influence the desquamation process in the normal stratum corneum. 



to Pre/. DrP. M. Elias for helpful suggest. 



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