Lupus-related transcript homologous to Abelson-murine leukemia virus

Vo1.155, No. 1,1988

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Pages 295-299

August 30, 1988

LUPUS-RELATED TRANSCRIPT HOMOLOGOUSTO ABELSON-MURINE LEUKEMIA VIRUS

Yasuhiro Deguchi, Shigeru Negoro and Susumu Kishimoto The Third Department of Internal Medicine, Osaka U n i v e r s i t y School of Medicine, Fukushima-ku, Osaka 553, Japan Received June 22, 1988

SUMMARY: In the p r e s e n t study, we found the e x p r e s s i o n of t r a n s c r i p t s homologous to Abelson murnie leukemia virus (A-MuLV) in lupus-prone mice and nonautoimmune mice. One of them, as the l u p u s - r e l a t e d t r a n s c r i p t , is e x p r e s s e d in o n l y lupus-prone mice and i n d u c e d by b a c t e r i a l l i p o p o l y s a c c h a r i d e (LSP). We c o u l d not induce the t r a n s c r i p t by LSP in nonautoimmune mice t h a t we examined. The s i g n i f i c a n c e of the t r a n s c r i p t autoimmune events in lupus-prone mice is discussed. ®1988Ac~demiopr~s~,~nc.

In

systemic

erythematosus(SLE),

autoimmune

diseases

such

connecitve tissue inflammation

are the most prominent p a t h o l o g i c

changes,

causes of autoimmune disorders are s t i l l

as

lupus

and microvascular damage

but most of the fundamental

unknown. I n i t i a l

models of SLE have provided evidence for

systemic

studies of murine

immune complexes containing

the

major e n v e l o p e g l y c o p r o t e i n ,

gp70, of endogenous r e t r o v i r u s e s

corresponding antibody(1).

is c l e a r that mice i n h e r i t determinants for

their

It

endogenous r e t r o v i r u s e s

as chromosomal genes(2).

segments seem to be expressed s e l e c t i v e l y integration

i n t o chromosomal

differentiated

Certain v i r a l

depending on t h e i r

in which i n d u c t i o n

is

gene

site

of

l i n k e d to the

state of the c e l l s ( 3 ) .

Abelson murine retrovirus

loci,

and the

leukemia v i r u s ( A - M u L V )

and induces B c e l l

both lymphoid and f i b r o b l a s t i c

lymphomas in v i v o and is a b l e to t r a n s f o r m cells

in v i t r o ( 4 ) .

markedly increases in autoimmune mice(5), events in B and/or T c e l l e x p r e s s i o n of t r a n s c r i p t s

is a r e p l i c a t i o n - d e f e c t i v e

activation.

C-myc RNA e x p r e s s i o n

which may i n t e r a c t

with various

In the present study, we studied the

homologous to A-MuLV in l u p u s - p r o n e mice and

295

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nonautoimmune mice using cloned probes s p e c i f i c the A-MuLV genome, and the induction by b a c t e r i a l transcripts

lipopolysaccharide(LPS).

in r e l a t i o n

The significance

of these

to autoimmune events and c-myc oncogene expression

in lupus-prone mice is discussed.

MATERIALS AND METHODS Mice: Mice used in t h i s study were 2 day-16 weeks old. BXSB male f o r lupus-prone mice, BXSB female, C57BL/6 and BALB/c mice for control mice were maintained in our laboratory. RNA i s o l a t i o n and northern b l o t h y b r i d i z a t i o n : Total RNA from spleen c e l l s was prepared by the g u a n i d i n i u m - t h i o c y a n a t e - c e s i u m c h l o r i d e procedure, denatured in g l y o x a l , and e l e c t r o p h o r e s e d in 1.4% agarose g e l s ( 2 0 ~ g per sample)(6). RNA was transferred to nylon membranes(Biodyne, Pall Inc. New York). The f i l t e r s were h y b r i d i z e d ( 7 ) in a s o l u t i o n i n c l u d i n g 50% formamide, 0.75M sodium c h l o r i d e , and 10% d e x t r a n s u l f a t e at 42 °C w i t h n i c k - t r a n s l a t e d 32p subcloned DNA probes from A-MuLV genome, which was a g i f t f~om JCRB(8) and v-myc DNA(Takara Chemical I n c . ) ( s p e c i f i c a c t i v i t y , 1.5xlO ° cpm per mg). The f i n a l washes were done under c o n d i t i o n s w i t h O.3xSSC(15 mM sodium c h l o r i d e and 1,5 mM sodium c i t r a t e , pH7.2) at 45 °C f o r 1 hr each, before autoradiography. Lipopolysaccharide(LPS) stimulation: The LPS preparation(Lipopolysaccharide E. C o l i 055, D i f c o Inc.) was d i l u t e d to the d e s i r e d c o n c e n t r a t i o n s w i t h sterile s a l i n e and used at a f i n a l volume of O.4ml to i n j e c t the mice intraperitoneally(i,p.).

RESULTS AND DISCUSSION Expression of the t r a n s c r i p t s homologous to A-MuLV We f i r s t

detected the 6.5 and 5 kb t r a n s c r i p t s

both lupus-prone mice and control

mice,

and the 8 kb t r a n s c r i p t

to A-MuLV in only lupus-prone mice(Fig.l), w i l d type t r a n s c r i p t

homologous to A-MuLV in homologous

which is a same size as neither

of A-MuLV(v-abl) or c e l l u l a r

homolog t r a n s c r i p t of A-

MuLV(c-abl). Induction of the lupus-related t r a n s c r i p t with LPS stimulation Recent data demonstrates that b a c t e r i a l in

cultured

investigated

spleen

cells

from

the in v i v o e f f e c t

various

LPS activates xenotropic virus strains

of

mice(9).

We next

of LPS on the e x p r e s s i o n of the l u p u s -

r e l a t e d t r a n s c r i p t homologous to A-MuLV genome in r e l a t i o n to the a c t i v a t i o n of x e n o t r o p i c v i r u s . w i t h LPS i n j e c t i o n

In f i g . 2 , we showed the i n d u c t i o n of the t r a n s c r i p t in o n l y B×SB male f o r the l u p u s - p r o n e mice, but not in

BXSB female for normal control mice.

First, 296

2-month-old BXSB male mice with

Vol. 155, No. 1, 1988

1

2

KB 8-

3

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4

~ " ;

"~

6

~.1 1 0 0 :> LU ,-I Z 0 U) U) LU n. O. X l¢l

5=

10

®

(9

0

LPS 0

4

12

24

INJECTION

TIME

48

7 2 HR

AFTER INJECTION

Fig,l:

Expression of the transcripts homologous to A-MuLV genome in mice, Lane I: BXSB male; 2, BXSB female; 3, C57BL/6; 4, BALB/c. Each mice strains were 2-month-old.

Fig.2:

Response of the expression of lupus-related transcript homologous to A-MuLV genome after a single i,p. injection of 25 ~g of LPS and saline in 2-month-old B×SB mice, Open c i r c l e : BXSB male mice; closed c i r c l e , BXSB female mice, Each point represents the mean value for three mice,

relatively

high expression of l u p u s - r e l a t e d t r a n s c r i p t homologous to A-MuLV,

BXSB female and other c o n t r o l

mice w i t h o u t i t s expression were i n j e c t e d

with 25 ug of LPS. Compared with p r e i n j e c t i o n of the t r a n s c r i p t

increased s i g n i f i c a n t l y

levels,

i.p.

amounts of expression

4 hr a f t e r the i n j e c t i o n

in both

mice(fig.2),

The amounts peaked between 12 and 24 hr and r e t u r n e d t o t h e

pretreatment

levels

level

within

72 hr,

At the peak response, the e x p r e s s i o n

were about I0 times higher than those before i n j e c t i o n of LPS,

At l a s t we examined the r e l a t i o n disease signs,

such as p r o t e i n u r i a ,

et al and we reported t h e r e l a t i o n

to the expression of the t r a n s c r i p t and lymphoadenopathy and so on(lO),

to c-myc oncogene expression and disease

s i g n s in l u p u s - p r o n e mice and SLE p a t i e n t s ( 5 , the

similar

expression

expression during

pattern

Mountz

of

the

II),

transcript

In t a b l e as

the disease process and the t r a n s c r i p t

could be r e l a t e d to the disease a c t i v i t y 297

I, we showed

c-myc

oncogene

expression

in lupus-prone mice,

level

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Table I : Correlation of lupus-related transcript, c-myc transcript and lupus manifestations

age lupus-related transcipt(8kb),c-myc transcript,disease* 1 day + + 1 month + + + 3 month +++ +++ +++ 6 month +++ +++ +++ * Disease manifestations are glomerulonephritis, moderate lymphoproliferation, degenerative coronary artery disease, and some kinds of autoantibodies.

A-MuLV transforming functions are mediated by 120 kd protein(pl20). protein is a hybrid molecule containing Moloney-murine MuLV) gag gene s t r u c t u r a l

This

leukemia v i r u s ( M -

p r o t e i n s p15, p12, and a small

p a r t of p30, as

w e l l as a M u L V - u n r e l a t e d component encoded by abl sequence(12).

Many of

n a t u r a l l y occurring A-MuLV variants have also been isolated(13).

Variants

t h a t express p r o t e i n s as small activity

for

including

A-MuLV have been shown to possess c l o s e l y

activity

with

fibroblasts(14).

as 90 kd was shown to r e t a i n t r a n s f o r m i n g

specifiity

A number of

transforming

retroviruses

associated

for tyrosine phosphorylation(15).

It

kinase

is possible

that the phosphorylation change by l u p u s - r e l a t e d t r a n s c r i p t homologous to AMuLV genome could e f f e c t on unknown gene a c t i v a t i o n which is important for pathogenesis of lupus. the l u p u s - r e l a t e d

We f u r t h e r study the cloning and characterization of

t r a n s c r i p t which might have key r o l e for pathogenesis of

the disease.

ACKNOWLEDGEMENT This work is supported in g r a n t s from the M i n i s t r y Science, and the M i n i s t r y of Welfare and Health in Japan.

of Education and

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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