Permeability Barrier Disruption Coordinately Regulates mRNA
Levels for Key Enzymes of Cholesterol, Fatty Acid, and
Ceramide Synthesis in the Epidermis
Ian R. Harris, Angela M. Farrell, Carl Grunfeld, Walter M. Holleran, Peter M. Elias, and Kenneth R . Feingold
Dermatology and Medi cal (Metabolism) Services, Department of Veterans Aft:, irs Medi cal Cem er and Departments of Dermatology and Medicin e, U niversity of
Ca liforn ia, Sa.11 Francisco, Cali fornia, U .S.A.
The extracellular lipids of the stratum corneunt, which
are comprised mainly of cholesterol, fatty acids, and
ceramides, are essential for epidern1al penneability
barrier function. Moreover, disruption of the permeability
barrier results in an increased cholesterol, fatty acid, and
ceranude synthesis in the underlying epidermis. Tlus
increase in lipid synthesis has been shown previously to
be due to increased activities of HMG-CoA reductase,
acetyl-CoA carboxylase, fatty acid synthase and serine
pah11itoyl transferase, key enzytnes of cholesterol, fatty
acid, and ceramide synthesis, respectively. In the present
study, we detennined whether the tnRNA levels for the
key enzymes required for synthesis of these three classes
of lipids increase coordinately during barrier recovery.
By northern blotting, the steady-state mRNA levels for
HMG-CoA reductase, HMG-CoA synthase, farnesyl pyrophosphate synthase, and squalene synthase, key enzytnes
for cholesterol synthesis, all increased significantly after
barrier disruption by either acetone or tape stripping.
Additionally, the steady-state mRNA levels of acetylCoA carboxylase and fatty acid synthase, required for
fatty acid synthesis, as well as serine palmitoyl transferase,
the rate-limiting enzyme of de novo ceratnide synthesis,
also increased. Furthennore, artificial restoration of the
permeability barrier b y occlusion after barrier disruption
prevented the increase in n1RNA levels for all of these
enzymes, except farnesyl pyrophosphate synthase, indicating a specific link of the increase in mRNA levels to
barrier requirements. The parallel increase in epidermal
mRNA levels for the enzymes required for cholesterol,
fatty acid, and ceramide synthesis may be due to one or
n1ore transcription factors that regulate lipid requirements for permeability barrier function in keratinocytes.
Ke)l words: acetyl-CoA caJ'boxylase/HMG-CoA reductase/
saine palmitO)'l tmnsferase/SREBP. J Invest Dermatol
109:783-787, 1997
T
these lipid synth etic pathwa ys h;1ve in barri er homeostasis (Feingold
eta/, 1990; Holleran et a/, 1991a, b; Mao-Qiang cc a/, 1993). T he
increase in cholesterol synth esis is attrib uted to an in crease in th e
activity of 3-hydro:-..-y-3-nJ ethylglutatyi- Coe nzy me A (HMG-CoA)
red uctase, an important enzym e for choles terol synth esis, explicable
both by an increase in the proportion of enzyme th at is activated by
dephosphmy lation (Proksc b ct a/, 1990) and by an increase in the
steady-state mRNA levels of HM G-CoA redu ctase Qackso n eta/,
1992), resultin g in an in crease in enzyme mass Qackso n et a/, 1992) .
Likewise, the increase in furry ac id synth esis is attributed to increased
activities of acetyl-CoA ca rboxylase (ACC) and fatty ac id synth ase
(FAS), key enzymes of £my acid synth esis (Ottey er al, 1995). Fi nally,
th e increase in ceramide sy nth esis is associated w ith increased serine
palmi toyl transfera se (SPT) acti vity, th e rate- linu ting enzyme of de
novo eramide synth esis (H a lleran eta /, 19913). W hether barrier
disrupti on stimul ates ACC, FAS, and SPT activi ty by in creasing mRNA
levels is not known. N either is it known w hether mRNA levels for
oth er key enzymes in the cholesterol sy nthetic pathway are stimulated.
M any of the proteins .important for choles terol metabolism , such as
the low-density lipoprotei.n receptor, HMG-CoA reductase, HMGCoA synthase, fa rnesyl pyrophosphate synthetase (FPPS), and squ alene
synth ase, are regulated coo rdinately (Goldstei n and Brown, 1990). T his
coo rdinate regul atio n has been shown to be due to transctiption facto rs
called sterol regulatmy element bindi ng proteins (SREBP) (Yokoyama
el a/, 1993; Shim ano et a/, 1996) . The regulation of f.my acid synthesis
proteins, ACC, and FAS has rece ntly also been shown to be regulated
he extracellular lipids oF tbe stratum co rn eum , within
wluch the lipid-depleted co m eocytes are emb edded, are
essential For a co mpetent penn eabili ty barrier (Grubauer
eta/, 1989). These lipids differ from th e mainly polar
membrane lipids of th e nucleated ceLl layers in that th ey
consist mainl y of choles terol, fi·ee ta tty ac ids, and ceramides (Lampe
et a/, 1983a, b). Acute disruption of the barrie r ca n be ac hi eved by
seve ral treatments w hi ch remove th e lipid, including solve nts such as
aceto ne and detergents such as sodium. dodecyl sul fate (SDS), or by
repeated tap e stripping (Menon eta/, 1985a; Proksch et al, 1990).
Barrier disrupti on indu ces metaboli c changes in th e und erlying
epidernus that resul t in th e rapid return oflipid to the stratum co rn eum
in tersti ces leading to barrier recovery. These changes include stimulation
of epidermal cholesterol, f:my acid, and ceramid e synth esis (Meno n et a/,
1985a; Grubau er eta/, 1987; Holleran et a/, 199"la). Most importan tly,
inhibition of eith er cholesterol, f:ttty acid, or ce ra nud e synth esis res ults
in abn orm al permeabili ty barri er homeostasis indica ting the key ro le
Manuscript received May 3, 1997; revised july 24 , 1997; accepted for
pubJi cation August I 5, "1997.
ll..ep rint requ ests to: Dr. Kenn eth 1<... Fein gold , Metabo lism Section (11 J F),
VA M edi cal Center, 4150 C lem ent Street, San !'rancisco, CA 94 12 1.
Abbrev iatio ns: ACC, acety i-CoA carbo,;ylase; FAS, (m y acid synthase; FPPS,
f.1n1esy l pyro phosphate synthase; HMG -CoA, 3-hydr0:\)'-3-methylglu taryiCoenzyme A; SPT, serin e palmitoyl tnnsferase; SlliB P, sterol regul atory
element binding pl·otein ; TEWL, transe pidennal water loss .
0022-202X/ 97 / $10.50 · Copyri ght
1997 by T he Society fo r Investigative Dermato logy, lnc.
783
784
H AIU<.IS ET I lL
by SREBP und er so m e co nd itio ns (Bennett et al, 1995; Lo pez er a/,
1996); however, o ur laboratory and others have sh own that several
phys io logic an d patho ph ysiologic co nditions ca n result in the d isco rda nt
regulati on of ge nes invo lved in cho lestero l m etabo lis m (Fein go ld
et a/, 1995). Fo r exampl e, fo Uowing lipo polysaccharide or cytokin e
adm.i nistrati o n (TNFa , IL-1, TNFa + TL-1), HM G-CoA redu ctase
mRNA levels in the li ver of Syrian ham sters i n crease m o re th an 10fo ld w itho u t a signifi cant in crease in mRNA levels fo r th e lowde nsity lip op ro tein recepto r, H M G-Co A synthase, or FPPS (Feingold
eta /, 1995).
T he p urpose of thi s stud y was to determin e (i) w hether there is a
coo rdinate in crease in th e ep iderm al mR .NA levels of enzym es, other
than H M G-CoA redu ctase, in volved i n cb olestero.l sy nthesis fo!J ow ing
acute barrier disrup tio n , an d (ii) w he ther th e inc rease in activities of
ACC, FAS, and SPT are attti buted to in creas es io the m l:tNA levels
for th ese enzymes.
M ATERIALS AND M ETH O D S
Animals and reagents Hai rl ess male mice (Crl :SKH I- hrBR.) were purchased
from C harles R iver (Wi.lmington, MA). Molec ular B iology grade chemi cals
were pu rchased fro m Sigma (St. Lo uis, MO) and Fischer (Fai rlawn, NJ). [a.32P] dCT P (3000 C i pe r mmol, 10 mCi per ml) was p urc h :~se d fro m New
.England N uclea r (Boston, MA). T he Mu ltiprime Labelli ng System was pu rchased fro m Amcrsham (A mersham , U.K.). Mini-spi n co lu mns (G-50) we re
purchased fi:om Worthin gton (Freehold , NJ). O ligo(dT)-ce iJu lose, type 77 F,
was purchased fro m Pharmacia (Uppsala , Swede n). Nytran Plus membrane was
purchased fi·om Schleicher and Schu ell (Keene, N H). pin-X centrifuge fi lters
were purchased fi·o m Co rning Costar (Cambridge, MA) . eDNA fo r HMGCoA ;:eductase (p H R.ed-102, ATCC no. 57042) was purchased fi·o m the
Am erica n Type Tissue C ul ture Co ll ection (R.ockviHe, MD). eDNA fo r rat
1-IM G-CoA syuthase (LA LI A) (R.osser cl a/, 1989) and FPPS (CR.39) (As hby
and Edwards, 1989) were kin dly provided by Dr. P.A. Edwards (U ni versity of
Cal iforni a Los An geles) . eDNA fo r rat squalene synthase Qiang eta /, 1993) was
kin dly prov ided by Dr. J. Shechter (Unifo rmed Services, University of H.ealth
Se1vices, Beth esda , DC). eDNA fo r rat ACC (Lopez-Casillas cr a/, 1989) was
ki ndly provided by Dr. K- H. Kim (Purdue University, Laf:1yette, IN). eDN A
fo r rat FAS was kin dly provid ed by Drs C.M. Amy and S. Smith (Oaklan d
Childrens Hosp ita l, C A). eDNA fo r mo use SPT (LCB2) (Nagiec ct a/, 1996)
was ki nc!Jy provided by Dr. R.C. Dickso n (U ni versity of Kentucky, KY).
eDNA fo r rat cyclophilin eDNA (pCD1 5:8-1) was kin dly provided by D r. G.
Strewler (Harvard Medica.! School, Boston , MA). Fuj i R.X fdm was used fo r
autorad iograph y (Fischer Scientific, Fairlawn, NJ).
A cute barri er disruption Followin g Nembu ta l anesthetic the torso skin of
6-8-wk-o ld ma le hairl ess mi ce was treated by gentl y appl yin g acetone-soaked
cotton balls tor 5-10 min as described in previous publicati ons (Menon cl a/,
1985a) or by applying and removing cell op hane tap e (Tesa Tuck, New Rochell e,
NY) 3-5 times successively. Controls fo r acetone perturbatio n of the ba rri er
were trea ted with cotton balls soaked in 0. 9% (wt/vol) sodium chloride.
U ntreated ani mals se rved as controls fo r tape stri p barrier perturbation. T he
transep idem1al water loss (TEWL) was measured immedia.tely after treatment
using a Meece electrolytic water analyzer, as describ ed prev iously (Menon ct a/,
1985a; Holl eran el a/, 1991a). Animals with a TEWL rate greater than 6 mg
per cm2 per b (normal < 0.3 mg pe r cm2 per h) after barrier disru ption were
incl uded in this study. Occl usio n of tape stripped mice was achi eved by
immediately inserting the mo use into a thu mb of a powdcdess Latex glove as
deseti bed previously (G ru ba uer eta /, I 989).
Isolation of the epidermis l' our h fo ll ow ing ba rri er disru ption, the animals
were killed by lsofl ura ne anesthesia (A bbot, C hi cago, IL) and the ski n excised.
T he subcutaneous f:1t was removed by sc rap in g with a sca lpe l bla de and the
skin was then placed in 1.0 mM ethylenediamine tt tra acetic acid in calcium and
magnesium free phosph ate- buiie red so lu tio n pH 7.4 fo r 35 min at 37°C Qackson
el. a/, 1992) . T he ski n was blotted dry and the epiderm is was . removed by
scrap in g with a sca lpel bla de . T he epi dermis was then snap fi·ozen in li qu id
nitrogen and stored below - 70°C.
Isolation of epid ermal mR.NA and northern blotting Tota.l R.NA was
prepared by a va ri ation of th e guan idinium thiocya nate method (C homczynski
and Sacchi, 1987) . Briefl y, epidermis (0.2- 0.4 g fi·om th e entire skin of two
mi.ce) was homogenized in 4 ml guanidin ium thiocyanate solution (4 M
guanidinium thiocyanate, 25 mM sodiu m citrate, pJ--J 7.0, 0.5%, wt/vol, Nlauroylsarcosin e, 0. 1 M 2-mercaptoe than.ol) usin g a ground glass hand- held
homogeni zer and so nicated twice for 50 s at 80% du ty cycl e o utp ut 2 (Son.if1 er
cell disrupte1·, VWR, San Fra ncisco, C A) sta ndi ng o n ice between bursts . Tota.l
TH E JOURNA L O F INVESTl GATIVE DE R MATOLOGY
J:tNA was pu rifi ed and added to oli go(dT)-cellul ose to obta in poly (A)+ RNA.
Q uantitation was determin ed by measuri ng the absorbance at 260 nm. Eq ual
amounts, 4- 10 ~l g, po ly (A) + RNA were appli ed to l % (wt/vol) agarose-6%
(vol!vol) fo rmalde hyde gels and electrop horesed. T he poly (A)+ R.NA was
tra nsfe rred el ectrop horeticall y onto Nytran plus membrane and fix ed by bakin g.
Hyb1id iza ti on was carri ed out ove mi ght at 65°C in 0.75 M sod ium chlorid e,
75 mM sodi um citrate, 2% (wt/vol) SDS, 10% (wt/vol) Dextran sulf.1te,
2X Denhart's solu tion, and lOO mg per ml sheared salm on sperm DNA.
Membranes were washed in 30 mM sodium chlori de , 3 mM sodium citrate,
0. 11){, (wt/vol) SDS at 65°C fo r 30 min . M embran es were exposed to x-ray
fi lm at -70°C with intensify in g screens. Q uantitation of ftlm , exposed in the
lin ear range of sensitivity, was achi eved using a Biorad (Hercules, CA)
densitom eter. T he densitometry guantitation was adjusted for cyclop hilin levels
of the sam e sampl e detemti ned on the sa me blot. Data in eac h experiment are
exp ressed as fo ld of control, where th e co ntrol equ als one.
Statistics Statisti cal signifi cance was determin ed usi ng a two-ta il ed unpaired
Student's t test, a one-tail ed un pa ired Stu dent's t test was used where indi ca ted.
R esul ts are expressed as mean :!:: SEM .
R ES U LTS
Effect of tape stripping on lipid synthetic enzyme mRNA
levels To determ.i ne w h eth er th e increase in cholesterol synthesis
after b arrier disruptio n is d ue to an increase in o nly HM G - CoA
redu ctase or to a coo rdinate increase in seve ral e nzym es in ch olesterol
synth eti c pathway, w e m easured mRN A levels o f HMG-CoA re du ctase, HM G-CoA syn thase, F PPS, and sq ualene sy nthase . Fo ur h aft er
ta pe stri pping th ere was a signifi ca nt in crease in the m RNA fo r all of
these gen es, in dicating coo rd inate upregulati o n of ch olesterol sy ntheti c
en zym es foll owi11 g b<uTi er disrupti o n (Fig 1).
Previo us studi es had demo nstrated an in crease in ACC, FAS, and
SPT activity fo ll owin g barri er dism pti o n . We next determin ed w h eth er
th e in crease in A CC, FAS, an d SPT activ ities after tap e strippin g is
acco mp anied by an increase in the levels of their respective rnR N A .
T he m R..NA levels of th e en zy m.es of £~tty acid synth esis, ACC , and
FAS increased signi fica ntl y, by 178% and 220%, resp ectively (Fig 2).
L ikewise, the stea dy- state mRNA levels o f th e ra te-Jim.itin g en zym e
of de novo ce ramid e sy nth esis, SPT (LC B 2, 2 .3 kb transcrip t), increase d
by 3 15% (Fig 3). T hu s, barri er d isrupti o n by tape strippin g res ul ts in
a parallel in crease in the stea dy- state mRNA levels for several key
en zym es of ch ol es terol, £~ t ty acid, and ce ramid e sy nth esi .
Effect of acetone treatm.ent on cpidennallipid synthesis enzyrne
mRNA levels T o determ.ine w hether the in crease in mRNA levels
is a sp ecific respo nse to barri er di sruptio n , rath er than a respo nse to
tap e strippin g, we next used th e orga ni c solve nt, aceto ne , to disrup t
th e pen nea biJi ty bar-rier. W h en com p:m:~d with tape strippin g, aceton e
trea tm ent res ulted in a vir tuall y identi cal increase in th e mRNA levels
fo r all of th e key enzy m es assessed (Fig 4). T hese res ults show th at
th e in crease in th e mRNA levels is not uniqu e for th e m eth od o f
barrier disrup tio n , but occurs regardl ess of th e rneth o d used fo r bani er
disru ptio n .
Effect of occlusion on mRNA levels following tape
stripping To determin e w heth er the chan ges in mRNA levels are
du e to barri er disrup tio n rath er th an injury, we next provided an
artifi cial barri er by La tex occlusio n imm edi ately fo Uowing barri er
perturbati on . In prev io us stu d ies, occl usio n prevented th e in crease in
lipid syn th esis and th e in creases in acti viti es of th e lipid sy nth eti c
enzym es. As sh ow n in Fig 5 , occlusio n also dim.inished th e increase
in mRNA leve ls fo r HM G-CoA redu ctase, HMG-CoA synth ase, and
squ alene sy nth ase . ln con tras t, FPPS ml<...N A lev els we re not effec ted
b y o cclusio n (Fig 5). T h e in creased mRNA levels fo r key en zym es
for f<lt ty acid and cerami de synth esis also were r edu ce d by occlusio n
(Fig 5). T hese results suggest th at, w ith th e excep ti o n of FPPS, the
increase in mR NA levels tha t occurs foll owin g barri er disrupti o n is
du e to ba rrier per:turbati.o n per se.
DISCUSS ION
Prev io us studi es have dem.o nstrated th at th e extracellul ar lipids o f th e
stratum co rn eum are esse ntial for a c m pe tent epiderm al perm eabili ty
ba rrier (Grubaue r et a/, 1987; Fein gold et a/, 1990). Follow ing ac ute
VO L. 109. NO. 6
BA I"\.R. I ER. IUGU LAT 10 N O F LIP11) SY NTH ETI C ENZYMES
I) ECE MllE1\. 1997
a
C
C TS
TS
C TS
C
b
TS
P<O.OS
785
rZ1D Con\rot
~Tap os tr ip
p<0.001
p-<0,001
HMH-CoA HMG-CoA
reductase
FPPS
synthase
Squalene
synthase
Figure 1. mRNA levels for enzymes required for cholesterol synthesis increase following tape stripping. Mi ce were tape stripped 3-5 times successively
until a T EW L va lu e of 6 mg per cm2 per h was achi eved. Poly (A)+ RNA was isolated 4 h after barrier disruption and northern blottin g was perfonned as in
Mmcrials and M ctl10ds . (tt) R.epresentative northern blots for cholesterol sy nth esis enzymes. C, control; TS, tape stripped. (b) Northern bl ot data representative of
lllul tipl e experiments are presented as mea n :!: SE M and expressed versns fo ld of th e animals co ntrol (i.e. , l.O) after correcting for loadin g using cyd ophili11 mRNA
levels. Co ntrol n = 4-5 , ta pe stripped 11 = 4- 5, except FPPS co ntrol n = I 0, ta pe stripp ed 11 = l 0.
c
TS
C
TS
b
l2:'ra Control
5iSZ52!3 Tape strip
a
P<0.05
c0
i
2
~
ACC
0
FAS
:!!
g
&
:cQ.
0
l
1
.
$
.li
z
a:
E
FAS
ACC
Figure 2. n-.RNA levels for enzymes required for fatty acid synthesis increase following tape stripping. Mice were tape stripped 3-5 tim es successively
until a TE W L valu e of 6 n1 g !Jer cm2 per h svas achi eved. Po ly (A) + RNA was iso lated 4 h after barrier disrupti on and north ern blotting was performed as in
Ma tcri<tls mtd J\lfc tlwds. (tt) R.eprescn ta ti ve north em bl ots for 1\CC and FAS enzymes. C, co ntrol; TS, tape stri!Jped. (b) North ern blot data rep resentative of multi ple
L'XpeJim ents are !-' resented as mea n :!: SE M and expressed 11C"r.< 1ts fold of the co mro\ animals after co rrecting for cycl ophilin. Co ntrol n = 4-5, tape stripped n = 4-5.
di srupti o n o f th e pe rm ea bility barri er th ere is a return o f lipid to th e
extra cellul ar space th at resto res barri e r functi o n. This ho m eos ta tic
repair response includ es a rapid sec retion of a pool o f pre fo rm ed
lamell ar bo di es fi·o m th e o utermost granular cells, an increase in lipid
sy nth esis in the nuclea ted cdl b ye rs o f th e epide rmis, an d n ew lam ellar
bo dy fo rmati o n in the granular cell layers (P roksc h eta/, 1993) .
M o reove r, th e ac ti viti es of HM G-Co A redu ctase, ACC, FAS, and
SPT, enzym es required fo r de novo cholesterol, fu tty acids, and
cera mid e sy nth esis, <lis o in crease sh ortly after barri er disrupti on (Proksch
eta/, 1990; H o ll eran ct a/, 199 l a; O ttey ct a/, 1995) . The in crease in
th e ac ti vity o f HMG -Co A redu ctase , ACC, FAS, and SP T fo!Jowing
barrier disruptio n can also be prevented b y pmvision o f an artifi cial
barrier, indi ca tin g that th e in crease in th ese enzy m es is no t a gen era.!
injury resp o nse but is regulated by penn e:1bili ty b:1rri er fun c ti o n.
Inhibiti o n of HM G-CoA redu ctase, ACC, and SPT afte r barri er
di sruptio n res ults in impaired barrier ho m eostas is (Pro ksc h et a/, 1990;
H oll eran et a/, 199 1b; M ao- Qian g et a/, J 993) .
In th e prese nt stud y we ha ve d em o nstrated that fo llow ing disrupti on
o f th e perm ea bility barri er th ere is a coo rdinate in crease in several
enzy m es required fo r cho lestero l sy nth es is (HM G-Co A redu ctase,
HM G - CoA sy nth ase, FPPS, and squ alen e sy nthase). M o reove r, the
in crease in mRNA levels is independ ent of th e m eth o d use d to p erturb
the barri er and , w ith th e excepti o n of FPPS, ca n be dimini shed by
providin g an artifi cial perm ea bi lity barri er. Additi o nall y, we ha ve also
dem o nstrated th at mRNA fo r ACC and FAS, th e two rate-limitin g
enzy m es fo r fu tty acid sy nth es is, and fo r S PT, the rate- limitin g e nzym e
that catalyzes th e first step fo r ce ramid e sy nth esis, in crease in th e
epid ermi s aft er barri er disrupti o n. B eca use th e in crease in A CC, FAS,
a
c
TS
b
e::z:zZJ Control
= rape Strip
4·
P<0.001
'5
:0
c
0
SPT
~ 3
>
0
•
"D
g
c
~ 2
a.
.!2
g"
•
>
.!!
~ l
a:
E
SPT
Figure 3. mRNA levels for SPT required for ceratnide synthesis increase
foll owing tape stripping. Mi ce were tape stripped 3-5 tim es successively
until a TEW L valu e of 6 mg per cm2 per h was achi eved . Poly (A)+ R NA
was isolated 4 h aft er barri er disruption and northern bl ottin g was perlo nn ed
as in Mrlfcrirds 11 11rl Metlwrls . (rt) Rq:> resentati ve northern blot for SPT. C. control;
TS, rape stripped . (b) Northern blot dar" representative of muJti ple experim ems
are prese nted as mean :!: SEM :md expressed IJCt~<t-ts fo ld of the control animals
after co rrecting fo r cycl ophilin. Co ntrol n = 5, tape stripped n = 5.
786
T H E JOU I,NAL O F I NVESTIGAT IVE D EIUvtAT OLOGY
HA R.RJS ET A L
lmll Control
limlllil
5
Aceton e
4
3
2
0
HMG-CoA
HMG-CoA
reductase
synthase
FPPS
Squa lene
ACC
FAS
SPT
synthase
Figure 4. mRNA levels for enzymes required for cholesterol, fatty acid,
and ceramide synthesis increase following acetone treatment. Mi ce we re
wip ed with ace tone soak ed cotton swabs until a TEWL valu e of 6 1ng per cm2
per h was achieved. Poly (A)+ RNA was iso lated 4 h after ba rri er disru ption
and northern blotting was performed as in 1\!fatcria/s a111i Methods. Northern blot
da ta representati ve of mul tipl e experim ents are prese nted as mean ± SEM and
expressed versus fo ld of the control anim als after correc tin g fo r cyclo philin .
Contro l n = 5, acetone n = 5. *p < 0.05 two- tail ed Student's t test, :fp < 0.05
one-tail ed Stu de nt's t test.
1mml
1.5
Tape strip
~ Occ lusion
"
:Ec
2
.E c
1.0
Q.
(J
0
g~
~ ~
.!!~
~a:
g 0.5
E
0.0
HMG-CoA HMG-CoA
reductase aynthaso
FPPS
Squa lene
ACC
FAS
SPT
synthase
Figure 5. Effect of occlusion on mRNA levels for enzymes required
for cholesterol, fatty acid, and ceramide synthesis. Mi ce were tape stripp ed
3-5 tim es successively until a TEW L va lu e of6 mg per cm2 per h was achi eved
and occluded with Latex. Poly (A)+ RN A was isola ted 4 h after batTier
disru pti on and north ern blottin g was perform ed as in l'vlnlerials nnd Mctllods.
Data are representative of multipl e experiments and are presented as mea n
± SEM and exp ressed IJcrsns fo ld of th e co ntrol animals after correctin g fo r
cycl op hilin . Tape stri pped control n = 5-6, tape stri pped occl uded n = 5-6 .
*p < 0.05 two-tailed Student's t test, t p < 0.05 one- tail ed Student's t test.
and SPT mRN A levels co uld be preve nted by pro vision of an artifi cial
permeabili ty barri er, these changes are du e to th e perm eabili ty barri er
req uirem ents rath er than a no nspecifi c respo nse to injury.
T he coo rdin ate in crease i_n the mRNA levels fo ll owin g di srupti o n
of th e perm eabili ty barri er indi ca tes that th ere co uld be regulati o n of
th ese genes by transctipti on fucto rs that bind to a shared regul ato ry
elem ent in the prom o ters of th ese genes . Sterol regulato ry elem ent
binding pro teins - 1 and -2 (SIU BP) a.re transcription facto rs th at
regulate th e transcripti o n of genes co ntai ning a fun cti o nal sterol
respo n e elem ent in their pr01noter sequ ence (Yo koya ma eta /, 1993;
Hua et a/, 1993). T hese include genes in vo lved in: (i) choleste rol
ho m eostasis, such as the low-density lipo prote.in receptor, w hi ch we
have shown previo usly in creases after barrier p erturbatiOti Qackso n et
al, 1992); (ii) cholestero l synth esis, such as .1-:IM G- CoA redu ctase,
HM G-CoA synth ase, FPPS, and squ alene sy nth ase; and (iii) fa tty acid
sy nth es is, i.e ., ACC and FAS (Yokoyama et a/, 1993; Jiang ct a/, 1993;
Beru1 ett et al, 1995; Vall ett et al, 1996; Lo pez er a/, 1996; E ri csso n et a/,
1996) . Precursor SREBP are =1 25- kDa p ro teins loca ted in th e
m embrane of the endo plasmic re ti culum and nu cleus. Wh en ste rol
levels decrease, SREBP are cleaved by two pro teases, releasing a matu re
=68-kDa N -termin al fragm ent that is th en free to enter th e nucl eus
and bind to th e sterol respo nse elem ent and activate transcription
(W an g et al, 199 4) . Acute disrupti o n of th e barri er may res ult in a
de rease in sterol levels in keratin ocy tes becau se of an increased
utili zati o n o f cholesterol to fo rm extra cellul ar lame!Jar m embrane
sb:uctures. T I-lls dec rease in cho lesterol co ncentrati o n wo uld increase
the pro teolyti c cleavage of precurso r SR EBP to fo rm mature SREBP,
w lu ch would then in crease th e mRNA levels fo r £1 tty acid and
cholestero l synth eti c enzym es . At the present tim e assays to determin e
th e acti va tion of SR..EBP-2 in murin e epidermi s are not available,
beca use mmine SlliBP-2 is no t recogni zed by currently available
(hum an) antib odi es . In additi o n to Sll iBP, cholesterol and fatty acid
sy nth eti c enzym es are also regul ated by o ther transcripti o n fac tors such
as AP-1, SP-1 , NF- Y, and R ed25 (O sbo rn e et a!, 1992; Vall ett and
Osborne, 1994; J ac kso n et a/, 1995; Yi e h et a/, 1995). It remain s to be
detem1in ed w heth er SREBP or o the r transcripti o n fac tors are activa ted
fo ll owing acute disrupti o n.
It is not known w hich transcription facto rs regulate SPT transCliptio n ;
howev er, SREBP- 2 is unlik ely to be a transcripti on factor for SP T.
Preliminaty studi es fi·om o ur Iabo rato ty have dem onstrated that changes
in tl1 e endoge no us ste rol levels in th e epidermis res ult in a coo rdin ate
change in mRNA levels for SREBP- 2 and cholestero l and futty acid
sy ntheti c enzy mes; howeve r, SPT mRNA levels were not affec ted by
changes in stero l le vels1 Furthermore, foll owin g barrier di srupti o n ,
th e in crease in SPT acti vity is delayed relati ve to the in crease in f.1 tty
acid synth eti c enzy mes, ACC and FAS, and th e cho lesterol sy nthetic
enzym e, HM G- CoA redu ctase . This would suggest th at ceramid e
sy nth es is is n o t regulated by the same f.1Ctors as cho les terol and fa tty
ac id synthesis.
T he exac t molecul ar me chanism s by w hi ch th e epi de rmal penneability barri er requirem ents regulate th e expressio n of th ese lipid sy nth etic
ge nes are unknow n . A gradi ent in th e co ncentrati o n of cakium io ns
has been describ ed in th e epidem-ll s, w ith th e hi ghes t co nce ntrati o n of
calcium io ns in th e nucl ea ted b yers of the o uter epiderm.is and the
lowest in th e basal la ye r (M eno n cl a/, 1985b). Foll owing barri er
disrupti o n, the calcium gradi ent is abo lished, retun-lln g in parallel w ith
th e fo rm ati o n o f a functi o na.l barrier (M eno n et a/, 199 4). In creasin g
the extern al co nce ntra tio n o f calcium and po tass ium ions fo ll owin g
barri er disrupti o n prevents th e in crease in HM G- CoA redu ctase
acti vity, redu ces th e return oflipid to th e stratum co rneum , and inhibits
barri er recove ry (Lee et a/, 199 2); howeve r, it remains unkn own
w hether calcium io ns inAu ence th e expression o f lipid sy nth etic ge nes
and /o r the ac ti vatio n o f tran scripti o n f.1c tors such as SREBP in the
epidermis.
In th e prese nt study, FPPS mR NA levels in crease after barri er
disrupti o n, as do o th er cholesterol sy nth eti c genes; ho weve r, th e
increase in FPPS mRNA levels .is n o t diminished by occlusio n fo llo win g
barri er perturb ati o n . Thus, under these circumstan ces, FPPS m.ay be
regulated by f.1c tors in additio n to those controlled directly by th e
perm ea bility barri er. Th e expressio n of FPPS in oth er tiss ues is also
not alwa ys coordin ately regul ated w ith HM G-Co A redu ctase and
HM G-CoA synthase (Feingold cr a/, 1995). Sterol m edi ated suppression
of a human leukemia cell lin e THP-1 can be reversed by th e additio n
of a calcium io no ph ore A23 187, w hi ch results in a rapid in crease in
mRNA of up to 40-fold fo r HMG-CoA redu ctase and 15-fold for
HMG- Co A sy nth ase w ith little o r no change in FPPS ml~NA levels
(Wilkin and Ed wa rds, 1992). In addition to providin g substrate for
cholesterol synth es is, FPPS is a key enzym e fo r the sy nthesis of
mevalo nate metab olites required fo r th e pre nylati on of proteins,
ubiquin o ne sy nth esis, and th e glycosylati o n of pro teins via doli chols.
Prenylated proteins include proto oncoge nes and lamin that control cell
di visio n and may be in volved in the in creased ce!J division th at occ urs
following barri er perturbation , a chan ge that is also o nly partially
prevented by o cclusio n (Proksc h eta/, 1991). R ecent studi es by o ur
labo rato ty have show n th at th e prenylati o n of proteins is in creased
foll owing barri er di sruption , and th at this increase is no t blocked by
1Harris IR, Fa rrell AM , Grunfeld C, et nl: Identifi ca ti on of sterol regulotory
element binding pro tein in epidermis: modul ati on in parallel to changes in key
enzymes of cholesterol and fatty acid synth esis. J It west Dem111tof 108 :553,
1997 (abstr.) .
VO L. 109, NO. 6
DEC EMBER 1997
occl usion (Denda er a/, 1997). O ne could spec ulate that in creased FPPS
acti vity is requi red to provide substrate fo r the prenylati on of proteins
required fo r cells to progress through the G 1 phase of the cell cycle
or to undergo DNA synth esis. T his w o ul d acco unt for the in crease in
activity prior to the onset of D NA synthesis, which w e have p reviously
shown to occur 18- 24 h after barrier disru ptio n (Proksc h er a/, 1991).
In summary, followin g acute disrup ti on of th e epiderm al pem1 eabili ty
barri er th ere is a coo rdin ate increase in the m.I~ N A leve ls for cholesterol
synth esis enzym es; HM G- CoA redu ctase, HM G-C oA synth ase, FPPS,
squalene synth ase . T here is also an in crease in the mJ~A levels fo r
fatty acid synth esis enzymes, ACC an d FAS , and SPT, th e rate-limi ting
em yme of de novo ceramid e synth esis. Furthermo re, with th e exception
of FPPS, we have dem onstrated th at th e expression of these genes in
the epidermis is regul ated by epidermal permeabili ty ba rrier fun cti on.
T he parallel increase in m.RNA levels for these li pid sy nthetic enzym es
suggests coo rdinate regulati on by specifi c transcriptio n facto rs that are
acti va ted fo llowi ng barrier disruptio n.
This work ""IS SII]J]JOrlerl by gm11ISjm 111 t.he ational fn stitllles of Health (A R. 3963 9,
A R. 19098) anrl fn> lll the R.esemrh Sewice qf I he Dcparlment 4 Veterans Affair.<.
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