Coping

and

sickness

absence

Objectives

The

aim

of

this

study

is

to

examine

the

role

of

coping

styles

in

sickness

absence

In

line

with

findings

that

contrast

the

reactive

passive

focused

strategies

problem-solving

strategies

are

generally

associated

with

positive

results

in

terms

of

well-being

and

overall

health

outcomes

our

hypothesis

is

that

such

strategies

are

positively

related

to

a

low

frequency

of

sickness

absence

and

with

short

lengths

-LRB-

total

number

of

days

absent

-RRB-

and

durations

-LRB-

mean

duration

per

spell

-RRB-

Introduction

A

strong

association

exists

between

ill

health

and

sickness

absence

particularly

for

long

absence

spells

-LRB-

Marmot

et

al.

Hensing

et

al.

-RRB-

However

the

decision

of

an

employee

to

go

on

sick

leave

or

to

stay

at

work

is

not

just

the

result

of

his

or

her

-LRB-

ill

-RRB-

health

status

alone

-LRB-

Aronsson

et

al.

Rosvold

and

Bjertness

Sandanger

et

al.

Whitaker

Anonymous

Johansson

and

Lundberg

-RRB-

but

depends

also

on

a

number

of

demographic

social

and

economic

determinants

-LRB-

Johansson

and

Lundberg

Voss

et

al.

Eshoj

et

al.

-RRB-

For

instance

age

-LRB-

Sandanger

et

al.

-RRB-

gender

-LRB-

Evans

and

Steptoe

-RRB-

marriage

-LRB-

Mastekaasa

-RRB-

level

of

education

-LRB-

Eshoj

et

al.

-RRB-

salary

-LRB-

Chevalier

et

al.

-RRB-

and

sickness

absence

history

-LRB-

Landstad

et

al.

-RRB-

are

known

to

be

associated

with

sickness

absence

behaviour

In

addition

the

way

the

individual

deals

with

stressful

situations

-LRB-

at

work

-RRB-

is

likely

to

affect

his

or

her

decision

to

report

ill

In

this

article

we

focus

on

the

role

of

this

kind

of

so-called

employee

coping

behaviour

The

relationship

between

coping

and

illness

behaviour

has

been

a

major

research

focus

over

the

past

two

decades

-LRB-

Somerfield

and

McCrae

-RRB-

A

variety

of

conceptual

coping-frameworks

have

been

proposed

and

numerous

measures

have

been

developed

to

assess

ways

of

coping

-LRB-

McWilliams

et

al.

-RRB-

Pioneering

work

in

the

field

of

coping

has

been

carried

out

by

Folkman

and

Lazarus

-LRB-

-RRB-

who

define

coping

as

the

cognitive

and

behavioral

efforts

made

to

master

tolerate

or

reduce

external

and

internal

demands

and

conflicts

among

them

In

their

opinion

coping

has

to

be

considered

as

a

behaviour

that

is

primarily

determined

by

environmental

demands

that

is

coping

is

an

individual

response

to

a

stressful

environment

In

contrast

other

scholars

-LRB-

Holahan

et

al.

Moos

and

Holahan

-RRB-

consider

coping

primarily

as

a

trait

or

as

a

resource

The

former

refers

to

a

relatively

stable

personal

characteristic

that

is

similar

coping

strategies

are

used

across

a

wide

variety

of

situations

-LRB-

Parker

and

Endler

Carver

and

Scheier

-RRB-

The

latter

refers

to

the

use

of

particular

social

and

personal

characteristics

that

is

personal

resources

on

which

the

individual

may

draw

upon

when

dealing

with

stressful

situations

-LRB-

Pearlin

and

Schooler

-RRB-

This

trait

or

dispositional

approach

of

coping

implies

a

stable

coping

style

or

a

coping

resource

regularly

used

As

early

as

four

decades

ago

Kahn

et

al.

-LRB-

-RRB-

distinguished

between

two

general

coping

strategies

problem-solving

strategies

and

reactive

passive

strategies

Their

idea

of

two

general

coping

strategies

has

been

worked

out

by

Lazarus

and

Folkman

-LRB-

-RRB-

in

what

nowadays

is

probably

the

most

popular

and

widely

accepted

conceptualization

of

coping

behaviour

Problem

solving-coping

refers

to

active

strategies

that

are

directly

targeted

at

solving

the

problem

at

hand

whereas

reactive

passive

focused

coping

refers

to

those

strategies

that

reduce

the

negative

emotions

that

are

evoked

by

the

stressful

situation

-LRB-

Elfering

et

al.

-RRB-

Much

research

on

coping

strategies

reveals

that

both

reactive

passive

strategies

and

avoidance

strategies

result

in

psychological

and

physical

symptoms

-LRB-

Terry

et

al.

Pisarski

et

al.

Penley

et

al.

-RRB-

whereas

active

problem-solving

coping

generally

has

a

positive

impact

on

well-being

and

overall

health

outcomes

-LRB-

Penley

et

al.

-RRB-

However

in

their

recent

review

Austenfeld

and

Stanton

-LRB-

-RRB-

criticized

this

popular

and

almost

generally

accepted

conclusion

They

identified

over

a

hundred

articles

examining

the

relationship

between

reactive

passive

coping

and

adjustment

-LRB-

Stanton

et

al.

-RRB-

and

found

that

hardly

any

of

the

coping

instruments

contained

the

same

set

of

coping

strategies

which

made

it

practically

impossible

to

aggregate

the

findings

Furthermore

the

association

between

reactive

passive

strategies

and

psychological

and

physical

symptoms

appeared

to

be

related

to

the

way

these

strategies

had

been

operationalized

-LRB-

Stanton

et

al.

-RRB-

It

appeared

that

corruption

of

the

original

coping

items

as

well

as

the

use

of

item

formulations

that

include

the

expression

of

emotional

distress

or

self-deprecation

result

in

spurious

correlations

Studies

on

coping

and

sickness

absence

are

scarce

Kristensen

-LRB-

-RRB-

was

among

the

first

to

investigate

this

relationship

and

he

asserted

that

sickness

absence

itself

should

be

regarded

as

coping

behaviour

reflecting

the

individual

's

perception

of

health

or

illness

Sickness

absence

itself

in

his

opinion

is

a

functional

coping

strategy

used

by

employees

to

reduce

work-related

strain

by

avoiding

the

workplace

and

thus

creating

for

themselves

the

opportunity

for

recuperation

Kristensen

was

one

of

the

first

not

to

primarily

focus

on

determinants

of

sickness

absence

but

rather

tried

to

understand

sickness

absence

from

a

coping

perspective

By

doing

so

he

went

beyond

existing

concepts

of

coping

by

considering

sickness

absence

a

type

of

coping

behaviour

-LRB-

Kristensen

-RRB-

As

he

stated

sickness

absence

can

well

be

a

rational

coping

behaviour

seen

in

the

light

of

a

person

's

wish

to

maintain

his/her

health

and

working

capacity

as

such

it

is

the

opposite

of

withdrawal

behaviour

Clearly

this

approach

differs

from

considering

coping

as

a

personality

trait

or

resource

In

the

present

study

coping

is

conceptualized

and

measured

as

a

trait

or

disposition

i.e.

it

is

assumed

that

individuals

tend

to

use

rather

similar

coping

strategies

across

a

wide

variety

of

situations

The

Utrecht

coping

list

-LRB-

UCL

-RRB-

-LRB-

Schreurs

et

al.

-RRB-

was

selected

to

assess

the

employees

coping

style

This

well-validated

self-report

questionnaire

is

the

most

widely

used

coping

inventory

in

the

Netherlands

both

in

research

and

in

practice

-LRB-

Schreurs

et

al.

Schaufeli

and

Van

Dierendonck

Norberg

et

al.

Buitenhuis

et

al.

-RRB-

Like

the

COPE

questionnaire

of

Carver

et

al.

-LRB-

-RRB-

the

UCL

asks

individuals

how

they

deal

with

stressful

situations

that

is

how

often

they

engage

in

various

exertions

encountering

problems

or

unpleasant

occurrences

The

UCL

distinguishes

between

five

coping

styles

that

can

be

grouped

together

into

two

higher-order

coping

styles

active

problem-solving

and

a

reactive

passive

style

-LRB-

Schaufeli

and

Van

Dierendonck

-RRB-

Hence

the

UCL

offers

the

possibility

to

investigate

employees

coping

styles

at

a

more

detailed

level

at

the

same

time

taking

into

account

the

conceptual

distinction

between

problem-solving

and

reactive

passive

coping

Sickness

absence

has

been

measured

in

terms

of

frequency

-LRB-

total

-RRB-

length

of

sickness

absence

-LRB-

mean

-RRB-

duration

of

sickness

absence

spells

as

well

as

by

the

sickness

absence

free

interval

These

sickness

absence

measures

are

defined

in

accordance

with

recommendations

of

Hensing

et

al.

-LRB-

-RRB-

who

pleaded

for

a

more

standardized

international

description

of

sickness

absence

measures

In

their

literature

review

Hensing

et

al.

pointed

out

the

multi-interpretability

of

sick

leave

indicators

and

recommended

basic

measures

to

encompass

the

full

spectrum

of

the

sickness

absence

phenomenon

to

make

studies

more

accessible

for

international

comparisons

Recently

a

study

by

Landstad

et

al.

-LRB-

-RRB-

confirmed

this

line

of

reasoning

by

concluding

that

different

forms

of

absenteeism

need

to

be

studied

simultaneously

in

order

to

distinguish

changes

in

sickness

absence

pattern

correctly

In

summary

then

the

aim

of

the

study

is

to

examine

the

role

of

coping

styles

in

sickness

absence

Based

on

the

fact

that

contrary

to

reactive

passive

strategies

problem-solving

strategies

are

generally

associated

with

positive

results

in

terms

of

well-being

and

overall

health

outcomes

our

hypothesis

is

that

such

strategies

are

positively

related

to

a

low

frequency

of

sickness

absence

and

with

short

lengths

and

durations

Reactive

passive

strategies

on

the

other

hand

are

not

expected

to

be

related

to

sickness

absence

Subjects

and

methods

Study

population

and

participants

Participants

were

employees

of

a

large

Dutch

telecom

company

An

occupational

health

survey

was

sent

to

all

employees

including

an

assessment

of

coping

strategies

-LRB-

response

rate

N

-RRB-

Sickness

absence

of

the

participants

was

followed

up

for

year

after

the

survey

Due

to

missing

sickness

absence

data

the

sample

was

reduced

to

employees

-LSB-

men

-LRB-

mean

age

years

SD

-RRB-

and

women

-LRB-

mean

age

years

SD

-RRB-

-RSB-

A

description

of

the

sample

is

shown

in

Table

During

the

first

quarter

after

the

start

of

the

study

of

the

participants

have

not

been

absent

because

of

sickness

whereas

of

the

participants

have

been

absent

for

more

than

days

-LRB-

length

-RRB-

Table

and

absenteeism

of

participants

Variable

Age

mean

-LRB-

SD

-RRB-

-LRB-

min

max

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Marital

status

Married

or

Divorced

or

Educational

level

Lower

vocational

Intermediate

vocational

Higher

vocational

education

and

Missing/something

Working

years

present

job

Sickness

absence

first

quarter

days

Function

Blue

collar

-LRB-

executive

-RRB-

Office

workers

-LRB-

administrative

-RRB-

Managerial

Compared

to

non-participants

participants

were

predominantly

male

older

better

paid

and

were

less

absent

for

sickness

-LRB-

see

Table

-RRB-

Table

and

absenteeism

of

participants

and

non-participantsParticipants

-LRB-

n

-RRB-

Non-participants

-LRB-

n

-RRB-

T

Test

-LRB-

women

-RRB-

Age

mean

-LRB-

SD

-RRB-

in

-LRB-

-RRB-

-LRB-

-RRB-

Salary

-LRB-

-RRB-

Absenteeism

Length

mean

-LRB-

SD

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Frequency

-LRB-

-RRB-

-LRB-

-RRB-

Measures

Coping

style

We

assessed

the

coping

strategy

of

the

participants

using

the

shortened

version

of

the

original

Utrecht

Coping

List

-LRB-

UCL

-RRB-

-LRB-

Schreurs

et

al.

-RRB-

This

questionnaire

was

designed

to

measure

the

coping

strategies

people

use

in

stressful

situations

either

life

events

or

daily

hassles

Each

item

is

rated

on

a

four-point

Likert

scale

ranging

from

one

-LRB-

never

-RRB-

to

four

-LRB-

very

often

-RRB-

The

UCL

includes

five

dimensions

-LRB-

-RRB-

active

problem-focusing

-LRB-

five

items

e.g.

thinking

of

different

possibilities

to

solve

a

problem

-RRB-

-LRB-

-RRB-

seeking

social

support

-LRB-

five

items

e.g.

seeking

comfort

and

sympathy

-RRB-

-LRB-

-RRB-

palliative

reaction

pattern

-LRB-

four

items

e.g.

looking

for

distraction

-RRB-

-LRB-

-RRB-

avoidance

behavior

-LRB-

three

items

e.g.

complying

to

avoid

problematic

situations

-RRB-

and

-LRB-

-RRB-

expression

of

emotions

-LRB-

two

items

e.g.

showing

frustrations

-RRB-

The

first

three

coping

styles

were

found

to

cluster

into

a

second-order

active

problem-solving

factor

whereas

both

final

styles

clustered

into

a

reactive

passive

factor

-LRB-

Schaufeli

and

Van

Dierendonck

-RRB-

According

to

the

test

manual

the

internal

consistencies

as

well

as

the

test

retest

reliability

are

satisfactory

-LRB-

Schreurs

et

al.

-RRB-

In

order

to

assess

the

factorial

validity

of

the

shortened

UCL

in

our

employee

sample

a

confirmative

factor

analysis

was

carried

out

Sickness

absence

Sickness

absence

data

were

taken

from

the

sickness

absence

records

of

the

employees

filed

in

the

database

of

ArboNed

an

occupational

health

service

-LRB-

OHS

-RRB-

serving

the

telecom

company

All

spells

of

absence

for

medical

reasons

were

centrally

reported

and

registered

by

the

executive

manager

of

the

company

Absence

spells

longer

than

weeks

were

verified

by

an

occupational

physician

by

inviting

the

employee

on

sick

leave

for

an

interview

Therefore

the

validity

of

the

absence

data

is

assumed

to

be

high

Measures

used

are

-LRB-

-RRB-

-LRB-

total

-RRB-

length

of

sickness

absence

in

current

and

new

spells

during

the

study

period

-LRB-

year

-RRB-

per

sick

listed

person

-LRB-

i.e.

total

number

of

days

absent

-RRB-

-LRB-

-RRB-

frequency

of

sickness

absence

-LRB-

new

sick-leave

spells

during

the

study

period

-LRB-

year

-RRB-

and

-LRB-

-RRB-

-LRB-

mean

-RRB-

duration

of

sickness

absence

-LRB-

sick-leave

days

in

new

spells

during

the

study

period

-LRB-

year

-RRB-

per

spell

-RRB-

The

duration

of

sickness

absence

is

classified

into

more

or

less

than

days

In

our

sample

short-term

sickness

-LRB-

less

than

days

-RRB-

accounts

for

of

the

absences

and

mainly

represents

minor

ailments

Finally

we

assessed

the

median

time

before

the

onset

of

a

new

sick

leave

period

after

the

occupational

health

survey

Statistical

analysis

Confirmatory

factor

analysis

-LRB-

CFA

-RRB-

using

the

AMOS

software

program

-LRB-

Arbuckle

-RRB-

was

used

to

test

the

fit

of

two

competing

models

that

assumes

that

all

items

load

on

one

general

coping

factor

and

that

assumes

that

the

items

load

on

the

five

hypothesized

correlated

factors

Maximum

likelihood

estimation

methods

were

used

and

the

input

for

each

analysis

was

the

covariance

matrix

of

the

items

The

goodness-of-fit

of

both

models

was

evaluated

using

the

χ2

goodness-of-fit

statistic

and

the

root

mean

square

error

of

approximation

-LRB-

RMSEA

-RRB-

However

χ2

is

sensitive

to

sample

size

so

that

the

probability

of

rejecting

a

hypothesized

model

increases

when

sample

size

increases

even

if

the

difference

between

the

fitted

model

and

the

true

underlying

model

is

very

small

To

overcome

this

problem

the

computation

of

relative

goodness-of-fit

indices

is

strongly

recommended

-LRB-

Bentler

-RRB-

Three

relative

goodness-of-fit

indices

were

computed

the

normed

fit

index

-LRB-

NFI

-RRB-

the

non-normed

fit

index

-LRB-

NNFI

-RRB-

and

the

comparative

fit

index

-LRB-

CFI

-RRB-

The

latter

is

particularly

recommended

for

model

comparison

purposes

-LRB-

Goffin

-RRB-

For

all

relative

fit-indices

as

a

rule

of

thumb

values

greater

than

are

considered

as

indicating

a

good

fit

-LRB-

Byrne

pp.

-RRB-

whereas

values

smaller

than

for

RMSEA

indicate

acceptable

fit

-LRB-

Cudeck

and

Browne

-RRB-

Next

Cronbach

alphas

were

calculated

for

the

UCL-subscales

In

a

next

step

scale

scores

for

different

coping

strategies

were

calculated

and

transformed

into

scale

scores

ranging

from

to

Finally

tertiles

of

the

distribution

of

the

scale

scores

were

used

to

distinguish

between

low

medium

and

high

levels

of

the

coping

strategies

To

examine

the

relationship

between

coping

and

sickness

absence

odds

ratios

and

corresponding

confidence

intervals

were

calculated

using

logistic

regression

analysis

Stepwise

multiple

logistic

regression

analysis

was

used

to

study

the

-LRB-

confounding

-RRB-

influence

of

sociodemographic

factors

and

other

determinants

on

the

relationship

between

coping

and

sickness

absence

The

magnitude

of

the

-LRB-

confounding

-RRB-

effects

was

assessed

by

calculating

the

proportion

of

the

excess

risk

-LRB-

OR

minus

-RRB-

explained

when

fitting

these

terms

in

the

model

Finally

the

period

between

the

health

surveillance

and

the

onset

of

a

new

period

of

absenteeism

was

evaluated

using

survival

analysis

Since

we

wish

to

estimate

the

probability

of

absenteeism

at

a

designated

time

interval

-LRB-

conditional

probability

-RRB-

the

Kaplan

Meier

methodology

-LRB-

Kaplan

and

Meier

-RRB-

has

been

applied

With

this

statistical

technique

means

medians

and

confidence

intervals

of

the

survival

-LRB-

in

this

study

the

onset

of

absenteeism

-RRB-

are

calculated

without

making

assumptions

about

the

survival

distribution

Results

UCL

factor

structure

As

can

be

seen

in

Table

confirmatory

factor

analysis

-LRB-

EFA

-RRB-

corroborated

the

underlying

five-factor

structure

of

the

short

form

of

the

UCL

More

particularly

all

fit-indices

of

the

hypothesized

model

with

five

correlated

factors

sufficed

their

respective

criteria

except

NNFI

that

approached

its

criterion

of

The

mean

correlation

between

the

five

factors

was

ranging

from

to

Moreover

the

fit

of

was

superior

to

that

of

that

assumed

that

all

items

load

on

one

undifferentiated

coping

factor

-LRB-

Δχ2

df

P

-RRB-

Hence

the

factorial

validity

of

the

was

demonstrated

Table

indices

of

one-factor

-LRB-

-RRB-

and

two-factor

-LRB-

-RRB-

models

of

coping

-LRB-

-RRB-

GFI

goodness

of

fit

index

AGFI

adjusted

goodness

of

fit

index

RMSEA

root

mean

square

estimate

of

approximation

NFI

normed

fit

index

NNFI

non-normed

fit

index

CFI

comparative

fit

index

all

χ2

P

The

Cronbach

alphas

for

the

subscales

avoidance

behaviour

expression

of

emotions

seeking

social

support

active

problem-focusing

and

palliative

reaction

in

this

study

were

and

respectively

Although

some

values

are

slightly

below

which

is

recommended

for

established

scales

all

values

are

well

above

which

is

deemed

satisfactory

for

newly

developed

scales

-LRB-

Nunnaly

and

Bernstein

-RRB-

Sickness

absence

and

demographics

As

can

be

seen

from

Table

length

-LRB-

total

number

of

days

absent

-RRB-

and

duration

-LRB-

mean

duration

per

spell

-RRB-

of

sickness

absence

are

associated

with

gender

-LRB-

i.e.

women

-RRB-

being

divorced

or

single

having

an

intermediate

or

lower

education

a

shorter

period

working

in

the

present

-LRB-

current

-RRB-

job

lower

salary

higher

age

and

a

history

of

sickness

absence

both

for

length

and

frequency

Likewise

a

higher

frequency

of

sickness

absence

was

associated

with

gender

-LRB-

i.e.

women

-RRB-

being

divorced

an

intermediate

salary

and

a

history

of

sickness

absence

both

for

length

and

frequency

In

our

sample

there

is

no

association

between

absence

frequency

and

level

of

education

the

period

working

in

the

current

job

or

age

Table

of

demographics

and

sickness

absenceSickness

absenceLength

daysDuration

daysFrequency

CIGender

Married

Education

Higher

vocational

Interm

vocational

Lower

vocational

Present

-LRB-

current

-RRB-

job

Salary

Low

Intermediate

High

Age

History

sickness

absence

in

days

-LRB-

length

-RRB-

year

before

History

frequency

-LRB-

year

before

-RRB-

n

ranges

between

due

to

missing

values

Sickness

absence

and

ways

of

coping

As

displayed

in

Table

a

greater

length

-LRB-

total

number

of

days

-RRB-

of

sickness

absence

is

predicted

by

low

or

medium-active

problem-focusing

avoidance

behaviour

and

a

medium

or

high

palliative

reaction

The

frequency

and

the

duration

of

sickness

absence

are

associated

in

a

similar

way

however

the

latter

showing

a

relation

with

low

seeking

social

support

rather

than

a

palliative

reaction

Table

summarizes

the

significant

associations

between

various

sickness

absence

measures

and

ways

of

coping

-LRB-

Table

-RRB-

It

can

be

seen

from

this

table

that

the

crude

ORs

of

the

active

and

avoidant

coping

styles

show

the

most

consistent

patterns

of

associations

across

all

sickness

absence

measures

Table

of

coping

and

sickness

absenceSickness

absenceLength

daysDuration

daysFrequency

CIProblem-solvingActive

problem-focusing

Seeking

social

support

Palliative

reaction

Reactive

passiveAvoidance

behaviour

Expression

of

emotions

Low

Summary

table

absenceLengthDurationFrequencyProblem-solvingActive

problem-focusingXXXSeeking

social

support

X

Palliative

reactionX

XReactive

passiveAvoidance

behaviourXXXExpression

of

emotions

Sickness

absence

and

demographics

and

ways

of

coping

Of

course

the

question

arises

whether

or

not

the

association

between

coping

and

sickness

absence

could

be

explained

by

previous

sickness

absence

and

by

demographics

Therefore

Table

displays

the

ORs

for

the

three

sickness

absence

measures

with

coping

strategies

after

adjustment

for

previous

sickness

absence

and

the

demographics

mentioned

in

Table

Table

ratios

-LRB-

CI

-RRB-

for

sickness

absence

-LRB-

length

days

frequency

duration

days

-RRB-

associated

with

different

coping

styles

measured

at

the

start

of

a

follow-up

study

-LRB-

n

-RRB-

AdjustmentsLength

daysDuration

daysFrequency

CIProblem-solvingActive

problem

focusingNo

adjustments

-LRB-

crude

OR

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

-LRB-

female

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

marital

status

-LRB-

married

-RRB-

-LRB-

-RRB-

-RRB-

-LRB-

-RRB-

Seeking

social

supportNo

adjustments

-LRB-

crude

OR

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

-LRB-

female

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

marital

status

-LRB-

married

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Palliative

reactionNo

adjustments

-LRB-

crude

OR

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

-LRB-

female

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

marital

status

-LRB-

married

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Reactive

passiveAvoidance

behaviourNo

adjustments

-LRB-

crude

OR

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

-LRB-

female

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

marital

status

-LRB-

married

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Expression

of

emotionsNo

adjustments

-LRB-

crude

OR

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

-LRB-

female

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

-LRB-

high

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

History

sickness

absence

length

gender

salary

education

marital

status

-LRB-

married

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

All

odds

ratios

are

based

each

time

on

the

same

employees

without

missing

values

on

each

variable

in

the

model

Length

Adjustment

for

sickness

absence

history

increases

the

excess

risk

to

be

absent

for

more

than

days

in

one

year

by

for

active

problem-focusing

-LRB-

thus

sickness

absence

history

reduces

the

effect

of

active

coping

-RRB-

while

reducing

it

by

and

for

palliative

reaction

and

avoidance

coping

respectively

After

adjustment

for

gender

and

sickness

absence

history

the

excess

risk

for

length

in

addition

to

palliative

reaction

and

sickness

absence

history

decreases

by

The

excess

risk

for

length

adjusted

for

salary

in

addition

to

active

problem-focusing

sickness

absence

history

and

gender

increases

by

In

summary

adjusted

for

several

confounding

variables

the

length

of

sickness

absence

is

effectively

influenced

by

active

problem-focusing

and

palliative

reaction

Frequency

Adjustment

for

sickness

absence

history

barely

minimizes

the

risk

for

frequency

by

coping

considering

active

problem-focusing

and

avoidance

behaviour

For

palliative

coping

the

reduction

for

the

excess

risk

amounts

to

When

adjusted

for

gender

in

addition

to

sickness

absence

history

the

risk

of

high

frequency

in

association

with

palliative

reaction

reduces

by

another

In

sum

adjusted

for

several

confounding

variables

the

frequency

of

sickness

absence

is

effectively

influenced

by

active

problem-focusing

avoidance

behaviour

and

expression

of

emotions

Duration

Adjustment

for

sickness

absence

history

reduces

the

excess

risk

of

active

problem

focusing

by

of

seeking

social

support

and

palliative

reaction

by

and

of

avoidance

behaviour

by

Adjustment

for

gender

in

association

with

seeking

social

support

affects

the

excess

risk

of

duration

by

In

summary

adjusted

for

several

confounding

variables

the

duration

of

sickness

absence

is

effectively

influenced

by

active

problem

focusing

avoidance

behaviour

and

seeking

social

support

Effects

on

the

onset

of

a

new

period

of

absenteeism

During

the

first

year

the

median

time

before

the

onset

of

a

new

episode

of

absenteeism

is

significantly

shorter

for

those

low

in

active

problem-focusing

high

in

avoidance

and

high

in

a

palliative

response

For

the

two

remaining

coping

styles

no

significant

results

were

found

-LRB-

Table

-RRB-

This

means

that

employees

who

are

used

to

solving

problems

actively

instead

of

avoiding

problems

or

engaging

in

alternative

behaviours

enter

sick

leave

later

the

next

time

Table

Meier

the

relation

between

different

coping

styles

and

the

onset

of

absenteeism

in

the

year

after

coping

assessmentLog

rankMedian

-LRB-

days

-RRB-

CIStatdfSignProblem-solvingActive

problem-focusing

Seeking

social

support

Palliative

reaction

Reactive

passiveAvoidance

behaviour

Expression

of

emotion

Discussion

In

accordance

with

our

hypothesis

and

after

adjustment

for

potential

confounders

employees

with

an

active

problem-solving

coping

strategy

are

less

likely

to

drop

out

because

of

sickness

absence

in

terms

of

frequency

length

-LRB-

total

number

of

days

absent

longer

than

days

-RRB-

and

duration

-LRB-

mean

duration

per

spell

more

than

days

-RRB-

of

sickness

absence

This

positive

effect

is

observed

in

the

case

of

seeking

social

support

only

for

duration

of

sickness

absence

and

in

the

case

of

palliative

reaction

only

for

length

and

frequency

of

sickness

absence

In

contrast

an

avoidant

coping

style

representing

a

reactive

passive

strategy

significantly

increases

the

likelihood

of

frequent

absences

as

well

as

the

duration

of

sickness

absence

Expression

of

emotions

representing

another

reactive

passive

strategy

has

no

effect

on

sickness

absence

The

median

time

before

the

onset

of

a

new

episode

of

absenteeism

finally

is

significantly

extended

for

active

problem-solving

and

reduced

for

avoidance

and

for

a

palliative

response

In

summary

we

conclude

that

in

accordance

with

our

hypothesis

a

problem-solving

coping

strategy

in

contrast

to

a

reactive

passive

coping

strategy

significantly

reduces

sickness

absence

This

result

seems

to

corroborate

other

research

findings

that

showed

that

problem-solving

coping

is

associated

with

well

being

and

overall

health

outcomes

-LRB-

Kohn

-RRB-

On

the

other

hand

our

results

are

at

odds

with

research

findings

that

document

a

positive

relationship

between

reactive

passive

coping

and

health

-LRB-

Austenfeld

and

Statton

Coyne

and

Racioppo

-RRB-

Austenfeld

and

Statton

-LRB-

-RRB-

have

argued

that

the

negative

effect

of

reactive

passive

coping

on

health

may

partly

be

attributed

to

the

operationalizaton

of

this

construct

and

therefore

recommended

a

clear

description

of

the

reactive

passive

coping

items

used

The

idea

is

that

reactive

passive

coping

can

be

separated

into

two

factors

namely

emotional

expression

and

emotional

processing

-LRB-

Lazarus

-RRB-

The

former

factor

is

an

active

attempt

to

acknowledge

explore

meanings

or

come

to

an

understanding

of

one

's

emotions

Items

measuring

emotional

processing

however

focus

on

the

acknowledgement

of

emotions

the

validness

and

importance

of

feelings

the

delving

into

the

feelings

Especially

emotional

processing

has

a

positive

association

with

health

although

how

the

influencing

occurs

is

still

unclear

The

items

that

tap

reactive

passive

coping

in

the

UCL

refer

to

the

expression

of

emotions

and

not

to

their

processing

This

probably

explains

the

indifferent

and

negative

effect

on

sickness

absence

by

emotional

expression

and

avoidance

behaviour

respectively

A

second

possible

explanation

can

be

that

reactive

passive

strategies

have

a

positive

relationship

to

health

but

not

necessarily

with

sickness

absence

Our

study

partly

refutes

the

assumption

of

Kristensen

-LRB-

-RRB-

that

sickness

absence

is

a

coping

strategy

by

itself

Kristensen

claimed

that

employees

who

use

sickness

absence

as

a

coping

strategy

would

experience

less

work-related

strain

especially

in

jobs

with

poor

decision

latitude

Accordingly

because

they

are

no

longer

exposed

to

their

stressful

jobs

employees

would

recuperate

during

sickness

absence

especially

in

the

case

of

psychosomatic

symptoms

In

our

study

sickness

absence

history

that

can

be

considered

a

proxy

of

the

coping

strategy

of

sickness

absence

had

only

a

minor

impact

on

sickness

absence

given

a

general

coping

style

And

although

the

effect

is

less

strong

the

measured

coping

strategies

of

the

UCL

still

have

an

effect

on

sickness

absence

The

favourable

outcome

of

problem-solving

coping

in

relation

to

sickness

absence

can

be

attributed

to

being

engaged

in

active

transactions

between

person

and

environment

with

the

aim

of

alleviating

stress-inducing

situations

-LRB-

Lazarus

Huizink

et

al.

Roesch

and

Weiner

-RRB-

Efforts

to

remove

the

stressor

gathering

information

and

finding

possible

solutions

for

the

problems

are

a

few

examples

In

general

these

strategies

are

associated

with

self-confidence

and

perceived

control

and

are

observed

in

individuals

who

are

persistent

and

assertive

self-efficacious

and

less

anxious

and

depressed

-LRB-

Heppner

Heppner

and

Baker

-RRB-

Two

factors

in

the

evaluation

of

problem-solving

coping

should

be

commented

upon

Men

are

believed

to

be

more

likely

to

confront

a

problem

with

active

coping

whereas

women

are

believed

to

exhibit

a

more

reactive

passive

response

-LRB-

Pearlin

and

Schooler

Hamilton

and

Fagot

-RRB-

For

instance

a

meta-analysis

of

Tamres

et

al.

-LRB-

-RRB-

showed

that

compared

to

men

women

are

more

likely

to

use

indirect

strategies

that

involve

verbal

expression

or

to

seek

emotional

support

Huizink

et

al.

-LRB-

-RRB-

however

argue

that

the

presumed

effectiveness

of

problem-solving

strategies

is

based

on

the

assumption

that

male-gender

role

behaviour

is

superior

She

suggests

that

studies

as

a

result

of

gender

bias

have

failed

to

identify

other

styles

of

coping

as

potentially

effective

In

our

study

however

considering

several

styles

the

adjustment

for

gender

barely

affects

the

influence

of

coping

on

sickness

absence

measures

Another

complicating

factor

in

the

evaluation

of

the

effectiveness

of

problem-solving

coping

may

be

that

reviewers

group

several

distinct

coping

behaviours

under

this

one

single

coping

category

in

an

effort

to

simplify

the

findings

-LRB-

Tamres

et

al.

-RRB-

For

instance

problem-solving

coping

may

be

composed

of

different

behaviours

This

is

underscored

by

our

finding

that

different

problem-solving

strategies

have

different

outcomes

on

sickness

absence

Seeking

social

support

for

example

affects

only

duration

-LRB-

marginally

-RRB-

whereas

active

problem-focusing

affects

length

duration

and

frequency

The

difference

in

outcome

of

different

sickness

absence

measures

in

the

case

of

seeking

social

support

may

be

clarified

by

Stansfeld

et

al.

-LRB-

-RRB-

who

argues

that

social

support

may

influence

absence-related

behaviour

and

encourage

a

person

to

take

absence

at

a

time

of

illness

Contrarily

one

may

postulate

that

social

support

also

shortens

sickness

absence

Both

postulations

may

result

in

the

absence

of

a

substantial

effect

To

the

authors

knowledge

this

is

the

first

study

with

four

sick

leave

outcome

measures

in

relation

to

coping

that

reveals

a

more

comprehensive

picture

of

changes

in

the

sick

leave

pattern

In

line

with

Isacsson

et

al.

-LRB-

-RRB-

we

can

conclude

that

adding

more

measures

gives

a

more

comprehensive

picture

of

sickness

absenteeism

and

of

differences

between

groups

For

instance

the

present

study

demonstrates

a

relation

between

a

palliative

coping

reaction

and

length

of

sickness

absence

in

contrast

with

the

duration

of

sick

leave

Without

the

differential

pattern

for

sickness

absence

the

differential

effects

of

several

coping

strategies

would

remain

invisible

Another

and

perhaps

even

more

important

argument

to

use

different

measures

of

sickness

absence

is

the

accessibility

of

this

study

for

international

comparisons

in

future

research

Finally

the

multi-factorial

aetiology

of

sickness

absence

requires

discussion

Alexanderson

-LRB-

-RRB-

pointed

out

that

different

disciplines

and

scientific

traditions

deal

in

different

ways

with

absenteeism

In

medical

science

for

instance

the

focus

of

research

is

on

occurrence

etiology

and

intervention

whereas

the

focus

in

medical

sociology

is

on

interacting

factors

within

a

pre-circumscribed

model

She

and

other

authors

-LRB-

Whitaker

Alexanderson

-RRB-

therefore

categorized

the

many

factors

of

sickness

absence

in

three

levels

macro/national

level

-LRB-

Alexanderson

-RRB-

-LRB-

e.g.

insurance

systems

-RRB-

organizational

level

-LRB-

Jeurissen

and

Nyklicek

Vahtera

et

al.

-RRB-

-LRB-

e.g.

job

demands

resources

-RRB-

and

individual

level

-LRB-

e.g.

gender

education

-RRB-

Recognizing

this

phenomenon

our

analyses

were

adjusted

for

several

known

risk

factors

at

the

level

of

the

individual

Since

the

present

study

was

conducted

in

one

Dutch

company

the

influence

of

organizational

and

socioeconomic

factors

was

equally

present

in

all

groups

and

in

this

sense

controlled

for

A

strong

point

of

our

study

is

the

detailed

way

in

which

sickness

absence

is

assessed

using

objective

archival

data

Thus

far

relatively

little

attention

has

been

paid

to

the

implications

of

different

quantitative

measures

of

sickness

absence

Moreover

a

prospective

design

was

used

that

allowed

for

predicting

future

sickness

absenteeism

A

limitation

of

the

study

is

the

non-recurring

measurement

of

coping

in

our

study

Therefore

we

can

not

rule

out

the

possibility

that

sickness

absence

might

influence

the

way

employees

cope

with

stressful

situations

Although

coping

styles

as

measured

with

the

UCL

have

proven

to

be

relatively

stable

in

time

-LRB-

Norberg

et

al.

-RRB-

reversed

causation

can

not

be

ruled

out

A

second

limitation

could

be

the

Cronbach

's

alpha

of

some

subscales

of

the

UCL

-LRB-

slightly

below

the

-RRB-

However

the

criterion

of

is

an

arbitrary

value

that

is

not

universally

accepted

as

the

minimum

level

of

acceptability

As

an

example

of

the

arbitrariness

of

this

criterion

Nunnally

-LRB-

-RRB-

mentioned

that

αs

ranging

from

would

be

acceptable

but

in

the

second

edition

of

that

book

he

suggests

that

is

the

minimally

acceptable

value

without

further

justification

-LRB-

Nunnally

-RRB-

Moreover

the

minimally

required

degree

of

reliability

is

a

function

of

the

research

purpose

for

individual-level

diagnostic

research

α

should

be

much

higher

than

for

the

basic

group-level

research

reported

in

our

study

-LRB-

Peterson

-RRB-

Hence

we

used

a

minimum

threshold

for

coefficient

α

of

as

was

recently

proposed

by

De

Vellis

-LRB-

-RRB-

In

spite

of

these

limitations

the

results

of

the

present

study

support

the

notion

that

problem-solving

coping

and

reactive

passive

strategies

are

inextricably

connected

with

frequency

duration

length

and

onset

of

sickness

absence

Especially

active

problem-focusing

decreases

the

chance

of

future

sickness

absence

B O B I O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O B O O O O O O O O O O O O B O O B I O O O O B O O O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O B O O O O B O B I O O O O B O B I O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I B I O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O B O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O B O O O O O B O B I O O O O O O O O O O O O O O O O O O O B I O O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B O O O O O O O O O O O O B O B I O O O O O B I O O O O O O O O O O O O O O B I O O B O O O O O B I O O O O O O O O O O O O O O O O O B I O B O O O O B O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O 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The RhoA transcriptional program in pre-T cells The GTPase RhoA is essential for the development of pre-T cells in the thymus . To investigate the mechanisms used by RhoA to control thymocyte development we have used Affymetrix gene profiling to identify RhoA regulated genes in T cell progenitors . The data show that RhoA plays a specific and essential role in pre-T cells because it is required for the expression of transcription factors of the Egr-1 and AP-1 families that have critical functions in thymocyte development . Loss of RhoA function in T cell progenitors causes a developmental block that pheno-copies the consequence of losing pre-TCR expression in Recombinase gene 2 -LRB- Rag2 -RRB- null mice . Transcriptional profiling reveals both common and unique gene targets for RhoA and the pre-TCR indicating that RhoA participates in the pre-TCR induced transcriptional program but also mediates pre-TCR independent gene transcription . 1 Introduction The T cell antigen receptor -LRB- TCR -RRB- complex comprises variable α/β subunits that recognise peptide/major histocompatibility -LRB- MHC -RRB- complexes and invariant signal transduction subunits of the CD3 antigen . A key stage in T cell development in the thymus is the selection of cells that have successfully rearranged their TCR-β locus . This occurs in T cell precursors , which do not express of the MHC receptors , CD4 and CD8 -LRB- double negative -LRB- DN -RRB- thymocytes -RRB- -LSB- 1 -RSB- . The DN stages of intrathymic differentiation can be followed by the sequential pattern of expression of CD44 and CD25 . T cell progenitors enter the thymus as CD44 + CD25 − cells -LRB- termed DN1 -RRB- ; these then express CD25 -LRB- DN2 -RRB- and begin to rearrange T-cell receptor β loci . Cells then lose CD44 expression and continue β chain rearrangements to completion -LRB- DN3 -RRB- . Cells that successfully rearrange their TCR-β locus will express a functional receptor complex known as the pre-TCR comprising a TCR-β chain , the p-Tα subunit and the signalling subunits of the CD3 antigen -LSB- 2 -- 4 -RSB- . When the pre-TCR is expressed at the cell membrane it promotes cell survival and entry into the cell cycle -LSB- 5 -RSB- ; cells downregulate CD25 and transit to the DN4 pre-T cell subset . DN4 T cells undergo proliferative expansion and differentiate to CD4 + CD8 + double positive -LRB- DP -RRB- cells . These cells then undergo TCR α-chain gene rearrangements and upon expression of a functional α/β TCR complex are subjected to positive and negative selection to generate CD4 + or CD8 + single positive -LRB- SP -RRB- thymocytes -LSB- 6 -- 8 -RSB- . Normal pre-T cell development requires the coordination of a complex program of gene transcription by signal transduction networks mediated by the pre-TCR complex and cytokine and stromal signals -LSB- 9,5 -RSB- . Mice deficient for the recombinase activating -LRB- RAG -RRB- genes which are unable to rearrange TCR-β subunits and express a pre-TCR are unable to progress beyond the DN3 stage of thymocyte development -LSB- 10 -RSB- . Similarly , the absence of pre-Tα or CD3 subunits blocks thymocyte development at the DN3 stage -LSB- 11,3 -RSB- . The transition of thymocytes beyond the pre-T cell stage of thymocyte differentiation is also dependent on signal transduction networks mediated by tyrosine and serine kinases -LSB- 3,12,13 -RSB- . In this context , crucial responses are mediated by Rho family guanine nucleotide binding proteins such as RhoA , Rac-1 and CDC42 -LSB- 14 -- 17 -RSB- . The importance of RhoA for thymocyte development has been demonstrated by studies of transgenic mice that express Clostridium botulinum C3-transferase under the control of T cell specific promoters such as the p56lck and CD2 promoters -LSB- 17,15,18 -RSB- . This toxin selectively ADP-ribosylates RhoA within its effector-binding domain and abolishes its biological function . Transgenic mice that express C3-transferase under the control of the p56Lck promoter have a small thymus and severely reduced numbers of peripheral T cells -LSB- 15,17 -RSB- . This phenotype is caused by survival defects in DN2 and DN3 thymocytes that lack Rho function -LSB- 15 -RSB- . During embryogenesis no cells progress beyond the DN1 stage but in adult mice a few T cell progenitors survive and develop to DN4s and beyond -LSB- 15 -RSB- . This ` leakiness ' either reflects selection of cells that compensate for loss of Rho function in DN2/3 thymocytes or reflects heterogeneous and asynchronous expression of the lck promoter in adult DN thymocytes . The few DP thymocytes and mature T cells found in adult Lck-C3 transgenic mice have numerous defects including reduced survival , proliferation , integrin mediated cell adhesion and defective cell motility -LSB- 15,19 -RSB- . A complementary strategy to probe Rho function in the thymus used the CD2 locus control region -LRB- LCR -RRB- to target C3 transferase to T cell progenitors -LRB- CD2 -- C3 mice -RRB- -LSB- 18 -RSB- . The inhibition of Rho function at the DN2/3 stage in CD2 -- C3 mice is not leaky and causes T cells to become blocked in differentiation at the DN3 stage of thymus development . CD2 -- C3 mice thus have a thymic phenotype indistinguishable from the phenotype of Recombinase gene null mice or mice lacking key structural or signaling components of preTCR complex -LSB- 10,18,20,21 -RSB- : thymocyte development is blocked at the pre-T cell/DN3 stage -LSB- 18 -RSB- . The basis for the failure of pre-T cell differentiation in CD2 -- C3 transferase mice is not known . One way to address this issue is to use microarray gene expression profiling to determine the impact of losing Rho function on transcriptional responses in pre-T cells . Previous studies in transformed cell lines have identified a role for RhoA in regulating activating protein-1 -LRB- AP1 -RRB- family of transcription factors -LSB- 22 -- 24 -RSB- but transcriptional targets for RhoA signal transduction in primary non-transformed cells have not been explored . The present data show that RhoA regulates expression of genes encoding members of the Fos/Jun and early growth response -LRB- Egr -RRB- family of transcription factors but also has an impact on expression of genes regulating diverse biological functions including serine/threonine kinases , protein phosphatases , enzymes that regulate protein biosynthesis and proteins that regulate nuclear structure and function . 2 Materials and methods 2.1 Mice Mice were bred and maintained under specific pathogen-free conditions in the transgenic animal unit . RAG2 − / − and C3 transgenic mice , which selectively express the bacterial toxin C3-transferase under the control of CD2 promoter and locus control region in the thymus , have been described in detail elsewhere -LSB- 10,18 -RSB- . 2.2 Flow cytometric analysis Fluorescein isothiocyanate -LRB- FITC -RRB- , phycoerythrin -LRB- PE -RRB- , allophycocyanin -LRB- APC -RRB- , and biotin-conjugated antibodies were obtained from Pharmingen -LRB- San Diego , CA -RRB- . Tricolour -LRB- PE/Cy5 and APC/Alexa750 -RRB- conjugated antibodies and fluorophore-streptavidin conjugates were from Caltag -LRB- Burlingame , CA -RRB- . Thymocytes were stained for cell surface markers and analysed on a FACS Calibur -LRB- Becton Dickinson Immunocytometry Systems Franklin Lakes CA -RRB- . Data were analysed using CellQuest BDIS or FlowJo -LRB- Treestar Inc , Ashland OR -RRB- software . CD4 − CD8 − double negative thymocyte subsets were analysed for CD44 and CD25 expression following lineage exclusion of mature DP and SP cells as well as non-T cell lineage cells using a cocktail of biotinylated antibodies -LRB- CD4 , CD8 , CD3 B220 , Mac-1 , NK , Gr-1 , and γδ -RRB- revealed with streptavidin-tricolour and costained with CD25-FITC , CD44-PE and Thy1.2-APC . Intracellular phospho-S6 and TCR-β intracellular staining was carried out on thymocytes pre stained to identify DN3 and DN4 subpopulations . Cells were subsequently fixed in 1 % paraformaldehyde for 10 min at room temperature , washed in PBS and permeabilized with saponin buffer -LRB- 0.5 % saponin , 5 % FBS , 10 mM HEPES -LSB- pH 7.4 -RSB- in PBS -RRB- for 10 min at room temperature . Permeabilized cells were incubated with PE-conjugated TCRβ antibody and phospho S6 antibody for 45 min at room temperature , washed in saponin buffer and subsequently stained with FITC conjugated donkey anti rabbit IgG -LRB- Jackson Immunoresearch , West Grove PA -RRB- . Cells were analysed on an LSR Flow Cytometer -LRB- BDIS -RRB- . 2.3 RNA extraction Thymocytes were isolated from 4 - to 6-week-old RAG2 − / − , CD2 -- C3 and C57BL/6 control mice . DN3 pre-T cells were purified firstly by removing CD4 , CD8 DP cells using MACS CD4 + and - MACS CD8 + T cell isolation kits -LRB- Miltenyi Biotec , Ltd. -RRB- followed by magnetic autoMACS separation . CD4/CD8 depleted thymocytes were then labelled with Thy1.2-APC , PE conjugated CD4 , CD8 , CD44 and TCRγδ antibodies , FITC conjugated CD25 antibodies and the DN3 subpopulation -LRB- Thy1 + CD4 − / CD8 − / CD44 − / CD25 + -RRB- was purified by cell sorting on a FACS Vantage -LRB- BDIS -RRB- using Cell Quest software -LRB- BDIS -RRB- . All sorted cells were analysed by flow cytometry and only used for experiments if the purity was 95 -- 98 % . Total RNA was extracted from sorted cells using the Qiagen RNeasy kit according to manufacturers instructions . Total RNA was quantified using the RNA 6000 Nano LabChip ® kit and analyzed on an Agilent 2100 Bioanalyzer -LRB- Agilent Technologies -RRB- . Total RNA from up to 10 thymi were pooled for microarray analysis and quantitative PCR . 2.4 Microarray Microarray analysis was carried out by the Finnish DNA Microarray Centre at the Centre for Biotechnology , Turku , Finland . For Affymetrix sample preparation , 100 -- 1000 ng of total RNA was used as the starting material to synthesise target cRNA using the GeneChip ® Eukaryotic Small Sample Target Labeling Assay Version II according to manufacturers ' instructions -LRB- Affymetrix , Santa Clara , CA -RRB- . The cRNA target sample was hybridised to the Affymetrix Mouse Genome 430 2.0 Array -LRB- 45,101 probes sets and expressed sequence tag -LRB- EST -RRB- clusters -RRB- . Data was analysed with Affymetrix Gene Chip Operating Software -LRB- GCOS -RRB- version 1.1 and filtered according to recommendations of the manufacturer . Comparison analysis was used to compare the expression profiles from two arrays -- results were obtained by analyzing the experimental array in comparison to the baseline array . The global method of scaling was used for normalizing hybridization intensity between arrays . The statistical algorithm used by GCOS defined the expression status of each gene-specific probe set as not changed , increased , marginally increased , decreased or marginally decreased . Genes that were absent or unchanged between comparisons were excluded from the results . Comparison analysis defines a gene as up-regulated if the signal log ratio between the baseline sample and the experimental sample is larger than 1 -LRB- 2-fold -RRB- and the experimental sample is present . Similarly changed genes were further analyzed/classified using the gene ontology tool -LRB- www.Affymetrix.com -RRB- and represented by biological function . Gene lists were complied and sorted by genes that were either commonly or uniquely regulated in CD2-C3 and RAG2 − / − thymocytes . 2.5 Real-time RT-PCR analysis Purified total RNA -LRB- 200 ng -RRB- was reverse transcribed using the iScript ™ cDNA synthesis kit -LRB- BioRad -RRB- . Real time RT-PCR was performed in a 96-well plate using iQ ™ SYBR Green based detection -LRB- BioRad -RRB- on a BioRad iCycler in 20 μl reaction volume containing 1 μl cDNA -LRB- 20 ng -RRB- , 0.8 μl 10μM sense and antisense primers , 10 μl iQ ™ SYBR Green supermix , and 4 μl nuclease free water . Each reaction was performed in duplicate and each experiment repeated in triplicate . 18S rRNA levels were used to normalise RNA concentrations between samples and the relative mRNA levels were calculated using the equation : where E is the efficiency of PCR , ct is the threshold cycle , u is the mRNA of interest , r is the reference gene -LRB- 18S rRNA -RRB- , s is the sample and c is the control sample . Primers used for RT-PCR were designed using Beacon Designer 2 software . Primer sequences are : Egr1 forward 5 ′ - ACAGAAGGACAAGAAAGCAGAC-3 , reverse 5-CCAGGAGAGGAGTAGGAAGTG-3 ′ , Fos forward 5 ′ - CTACTGTGTTCCTGGCAATAGC-3 ′ , reverse 5 ′ - AACATTGACGCTGAAGGACTAC-3 ′ , Egr3 forward 5 ′ - TGACCAACGAGAAGCCCAATC-3 ′ , reverse 5 ′ - GCTAATGATGTTGTCCTGGCAC-3 ′ , Jun forward 5 ′ - CGCCTCGTTCCTCCAGTC-3 ′ reverse 5 ′ - ACGTGAGAAGGTCCGAGTTC-3 ′ , JunB forward 5 ′ - CTTCTACGACGATGCCCTCAAC-3 ′ , reverse 5 ′ - GTTCAAGGTCATGCTCTGTTTTAGG-3 ′ , Nur77 forward 5 ′ - CCTGTTGCTAGAGTCTGCCTTC-3 ′ , reverse 5 ′ - CAATCCAACACCAAAGCCACG-3 ′ , 18S RNA forward 5 ′ - GTAACCCGTTGAACCCCATT-3 ′ , reverse 5 ′ - CCATCCAATCGTAGTA-3 ′ . 3 Results and discussion Mice expressing the RhoA inhibitor Clostridium Botulinum C3 transferase under the control of the CD2 locus control region -LRB- LCR -RRB- have been described previously -LSB- 18 -RSB- . Loss of RhoA function in CD2-C3 mice results in loss of CD4/CD8 double positive thymocytes -LRB- Fig. 1A -RRB- with a block in thymocyte development at the DN3 stage -LRB- Fig. 1B -RRB- . Rag2 − / − mice also block thymus development at the DN3 stage -LRB- Figs. 1A and B -RRB- due to failed expression of the pre-TCR complex . Despite this superficial similarity , loss of Rho function does not prevent TCR beta locus rearrangements although there is a reduced frequency of DN3 cells expressing intracellular TCR-β subunits -LRB- Fig. 1C -RRB- . Ectopic expression of transgenic TCR complexes can not reverse the developmental block in CD2 -- C3 thymocytes demonstrating that failed expression of TCR complexes does not explain the developmental defects caused by loss of Rho function -LSB- 18 -RSB- . Rho thus appears necessary for pre-TCR function although these results do not discriminate between a direct role for RhoA in pre-TCR signalling or a role for this GTPase in the cytokine/stromal signalling pathways that synergise with pre-TCR signals to control pre-T cell differentiation -LSB- 25 -- 27 -RSB- . CD2 -- C3 transferase mice provide a good model system to probe the immediate transcriptional consequences of losing RhoA function in pre-T cells since the CD2-LCR initiates expression of transgenes in T cell progenitors as they transit from the DN2 to DN3 stage of thymocyte development -LSB- 18 -RSB- . Accordingly , it is possible to isolate DN3 thymocytes ex vivo that have only just switched on expression of C3 transferase and prepare RNA for DNA microarray analysis . The Affymetrix Mouse Genome 430 2.0 array , representing 39 000 murine gene transcripts , was used to transcriptionally profile DN3 pre-T cells purified from the thymi of CD3 -- C3 transgenic mice . In these experiments DN3 thymocytes from wild type control mice were transcriptionally profiled and used for comparisons . The microarray data were analyzed with the Affymetrix Gene Chip Operating Software -LRB- GCOS , version 1.1 -RRB- by comparing the gene expression profiles of CD2 -- C3 pre-T cells to the profile of normal cells to reveal changes above or below wild type levels . DN3 pre-T cells expressed approximately 18 000 genes and there were only small changes in the transcriptional profile of CD2 -- C3 DN3 thymocytes compared to control wild type DN3s . We focused our analysis on ⩾ 2-fold changes , which were further analyzed using the Affymetrix gene ontology tool . A full list of the genes regulated 2-fold or greater by loss of RhoA function in DN3 thymocytes is shown in Supplementary Fig. 1 . There were approximately 18 000 genes expressed in wild type DN3 thymocytes : loss of RhoA function caused a ⩾ 2-fold decrease in expression of 383 genes and a ⩾ 2-fold increase in expression of 190 genes . RhoA regulated genes in DN3 thymocytes encode proteins with diverse biological functions including serine/threonine kinases , protein phosphatases , enzymes that regulate protein biosynthesis . This point is illustrated in Fig. 2A , which shows the genes whose expression is regulated more than 10-fold . The largest decrease was in expression of the gene encoding FosL2 , a Fos family transcription factor -LRB- 630-fold decrease -RRB- but other large changes included a subunit of eukaryotic translation initiation factor 2 , Protein Kinase C beta and the membrane receptor CD69 . There was also a loss of expression of genes encoding proteins that regulate nuclear structure and function such as genes encoding histones that are involved in nucleosome assembly . In a similar category there was reduced expression of mRNA encoding special AT-rich sequence binding protein 1 -LRB- SATB1 -RRB- in CD2-C3 DN3s -LRB- Supplementary Fig. 1 -RRB- . SATB1 acts as a scaffold for chromatin modifiers and is known to regulate higher order chromatin structure and to regulate gene expression by acting as a `` docking site '' for several chromatin remodeling enzymes -LSB- 28 -- 30 -RSB- . Of 55 genes whose expression was decreased ⩾ 5-fold , 23 encoded transcription factors ; of 384 genes decreased ⩾ 2-fold , 63 were transcription factors -LRB- Fig. 2B -RRB- . Transcription factors downregulated in CD2-C3 DN3 thymocytes included Kruppel family transcription factors KLF4 , KLF6 and KLF9 . There was also loss of expression of members of the immediate early gene family Egr1 and Egr3 , AP-1 family members Fos , FosL2 , FosB , Jun , JunB and downregulated expression of genes encoding Ets1 and Nurr77 . To validate the microarray analysis , quantitative real-time PCR was used to compare expression of a selection of these different transcription factors in wild type versus CD2-C3 thymocytes -LRB- Fig. 2C -RRB- . The experiments confirmed the reduction in Fos , Jun , JunB , Egr1 and Egr3 and NURR77 in CD2 -- C3 DN3s compared to wild type controls . A number of gene targets for Egr transcription factors have been described -LSB- 31 -RSB- and in this context , RhoA inactivation caused loss of expression of multiple members of the MAP kinase phosphatase family DUSP 1 , 2 , 4 , 6 , 16 . The latter are induced in negative feedback mechanisms that modulate MAP kinase activity and have been described as targets for Egr family transcription factors -LSB- 31 -- 33 -RSB- . The loss of DUSP expression is thus almost certainly a secondary consequence of failed expression of Egr molecules . Egr1 and Egr3 are well characterised pre-TCR induced genes -LSB- 34 -- 38 -RSB- . To verify this we compared the transcriptional profile of CD2-C3 DN3s with Recombinase gene 2 − / − -LRB- RAG2 − / − -RRB- DN3s which lack expression of the pre-TCR complex because they fail to rearrange their TCR beta locus . Of the 23 transcription factors downregulated ⩾ 5-fold in CD2 -- C3 thymocytes 18 were also downregulated in Rag2 − / − DN3s -LRB- Fig. 2D -RRB- and these included Egr1 , Egr3 , Fos , Fosl2 , FosB , Ets1 , Nurr77 . These microarray analyses were confirmed by quantitative real-time PCR analysis of wild type versus Rag2 − / − thymocytes -LRB- data not shown -RRB- . The common loss of expression of members of the Egr family in CD2 -- C3 and Rag2 − / − pre-T cells is thus consistent with a RhoA requirement for the pre-TCR induced gene transcription . However , comparisons of the total number of genes regulated uniquely in CD2 -- C3 and Rag2 − / − DN3s -LRB- Fig. 3 -RRB- -LRB- VENN diagrams -RRB- indicate that there are a number of gene changes in CD2 -- C3 cells that are not seen in Rag2 − / − DN3s . The full list of genes that were commonly and uniquely regulated 2-fold and greater in CD2 C3 versus Rag2 − / − pre-T cells is shown in Supplementary Fig. 2 . In terms of ⩾ 2-fold , gene repression , CD2-C3 and Rag2 − / − DN3s had 180 gene targets in common with 202 genes being downregulated uniquely in CD2 -- C3 pre-T cells and 120 genes downregulated uniquely in Rag2 − / − cells . In terms of ⩾ 2-fold gene increases , CD2 -- C3 and Rag2 − / − DN3s had 65 gene targets in common , 129 genes that were increased uniquely in CD2-C3 DN3s and 182 increased uniquely in Rag2 − / − cells -LRB- Fig. 3 -RRB- . The common transcriptional changes caused by loss of the pre-TCR in Rag2 − / − DN3s or loss of Rho function in CD2 -- C3 mice indicates that Rho function is necessary for some pre-TCR induced transcriptional responses . However , there are genes downregulated in DN3s lacking RhoA function that are unchanged in Rag2 − / − DN3s -LRB- Supplementary Fig. 3 -RRB- indicating that RhoA is not just required to mediate the preTCR induced gene program but may also participate in cytokine/stromal cell-initiated signalling pathways that control pre-T cell differentiation . The genes uniquely lost in CD3 -- C3 DN3 thymocytes include serine kinases -LRB- Protein Kinase C beta and Map3k14 -RRB- ; transcription factors -LRB- SpiB and Dlx1 -RRB- and regulators of protein biosynthesis -LRB- Eif2s3y -RRB- -LRB- Supplementary Fig. 3 -RRB- . One gene of interest lost in CD2 -- C3 DN3s but not Rag2 null DN3s was Hes-1 which is induced by Notch receptor-ligand interactions . Notch signalling is required throughout DN to DP stage of thymocyte development to support T cell metabolism and survival -LSB- 39 -- 42 -RSB- . A RhoA requirement for Notch signalling has not been described but it is known that RhoA is necessary for integrin mediated cell adhesion and thymocyte cell migration -LSB- 19 -RSB- . Accordingly , decreased Hes-1 expression could reflect that pre-T cells lacking Rho function can not make normal contacts with stromal cells that express the Notch ligands -LSB- 43,42,44,45 -RSB- . There were also unique transcriptional changes seen in Rag2 − / − cells that were not found in the CD2 -- C3 DN3s -LRB- Supplementary Fig. 4 -RRB- which indicates that the pre-TCR can regulate a program of gene transcription via RhoA independent pathways The unique changes in Rag2 − / − cells not surprisingly included loss of expression of rearranged TCR-β subunits . One other gene downregulated in Rag2 − / − DN3s but not CD2 -- C3 DN3s was RPS6 , which encodes the ribosomal S6 subunit . In this respect , we recently described high basal levels of S6 phosphorylation in ex vivo β selected DN3s and no detectable S6 phosphorylation in ex vivo Rag2 − / − DN3s -LSB- 13 -RSB- . The data in Fig. 4 quantify S6 phosphorylation in CD2 -- C3 DN3s compared to wild type cells . Normal DN3s are heterogeneous for phosphoS6 with the majority of cells being phosphoS6low but a significant percentage of cells are phosphoS6high . The DN3 thymocyte subpopulation can be subdivided into cells that have not yet completed TCR-β locus rearrangements and those that express a functional TCR-β subunit that allows surface expression and signalling of the pre-TCR complex . Analysis of TCRβ expression by intracellular staining revealed that DN3s that express icTCR-β chains are generally phosphoS6high whereas DN3s that are icTCR-β null are uniformly phosphoS6low . In CD2 -- C3 DN3s there were also high levels of phosphoS6 in TCR-β high cells . The presence of normal S6 phosphorylation in β selected DN3s lacking Rho function is a further indication that Rho is necessary for a subset of responses in DN3 thymocytes but does not globally block signalling . One important question is whether the transcriptional changes in CD2 -- C3 DN3s explain why loss of RhoA function results in failed thymocyte differentiation ? In this respect , microarray analysis of Rag2 − / − pre-T cells revealed decreased expression of TCR-β subunits as a major defect -LRB- Supplementary Fig. 4 -RRB- and failed expression of the TCR-β subunit is indeed responsible for the DN3 developmental block in Rag2 − / − mice . In CD2 -- C3 DN3s there were several gene defects that would explain why RhoA function is essential for pre-T cell development . For example , RhoA is required for expression of Egr3 and Ets1 and loss of either of these transcription factors inhibits pre-T cell proliferation and is suboptimal for transition through early pre-TCR-dependent stages of thymocyte development -LSB- 46,36 -RSB- . Loss of individual Egr family or AP-1 transcription factors does not completely abrogate thymocyte differentiation but this reflects that there is considerable redundancy in AP-1 complexes due to the ability of different family members to pair as dimers -LSB- 47 -- 49 -RSB- . The simultaneous elimination of Egr1 , Egr3 , Ets1 and multiple Fos/Jun family members in CD2 -- C3 DN3s would circumvent the possibility of redundancy -LSB- 50,51 -RSB- . This comprehensive loss of AP-1 activity in pre-T cell lacking RhoA function plus the decreased expression of other transcription factors would rapidly cause global changes in the transcriptional program of a cell . Moreover , the transcription factor defects would be exacerbated by the decreased expression of chromatin modifying enzymes such as SATB1 . In summary , loss of Rho function or failed expression of the pre-TCR complex in Rag2 − / − null mice block T cell development at a common stage . The present data show that the genetic consequences of loss of Rho function versus loss of pre-TCR expression for T cell progenitors are not identical . The present results also show that loss of Rho function in pre-T cells results in downregulation of genes encoding members of the Fos/Jun and Early growth response -LRB- Egr -RRB- family of transcription factors . The collective loss of these transcription factors and the resultant secondary genetic changes explains why loss of RhoA function prevents pre-T cell development .

O B O O O O O O O B O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O