Tissue specificity and developmental expression of rat osteopontin

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages I129-I136

November 13, 1987

Tissue specificity and developmental expression of rat osteopontin Kyonggeun Yoon, Robert Buenaga, and Gideon A. Rodan Department of Bone Biology and Osteoporosis Research, Merck Sharp and Dohme Research Laboratories, West Point, PA 19486 Received September 15, 1987

Summary.

Osteopontin

is a 44 kd phosphoprotein abundant in bone m a t r i x .

We i s o l a t e d

a partial

examine

its

tissue

and i t s

hormonal

length

cDNA f o r

specificity,

regulation.

its

rat

osteopontin

expression

during

Estimates

by

the

of

osteotropic

osteopontin

i s turned on r e l a t i v e l y

Osteopontin

is

used

hormones

mRNA l e v e l s

a recently

to

in bone

Osteopontin mRNA is

dexamethasone

indicate

l a t e in c a l v a r i a l

it

bone development

Osteopontin mRNA is most abundant

but is also found in c o n s i d e r a b l e amounts in kidney. regulated

and

that

and

1,25(OH)2D 3.

the o s t e o p o n t i n

development,

gene

o 1987AcademicP r . . . .

Inc.

discovered non-collagenous bone matrix

protein, which contains the Arg Gly Asp Ser amino acid sequence and was proposed to play a role in bone cell attachment (1,2). 44 kd acidic, glutamic for

sialated,

acid/glutamine

about 50% of

phosphorylated glycoprotein,

in which serine,

and aspartic

residues account

but

its

proteins,

not

been established.

function,

demonstrated the presence of fibroblast-like

acid/asparagine

amino a c i d s (3,4).

hydroxyapatite has

cells,

Osteopontin is a

this

Osteopontin binds firmly

like

that

of

other

in the bone matrix

which may be preosteoblasts,

expression of this protein may be an early manifestation differentiation (5). to

explore

the

non-collagenous

Immunolocalization studies

protein

to

have

and in

suggesting that of osteoblastic

The object of the p i l o t studies presented here was

potential

usefulness of

osteoblastic differentiation.

osteopontin

Using a partial

as

a marker of

length osteopontin cDNA,

we examined the distribution of osteopontin mRNA among adult rat tissues 0006-291X/87 $1.50 1129

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and the changes in mRNA l e v e l s during bone development by comparison to other

osteoblast

products:

type

osteonectin and osteocalcin.

1,25(OH)2D 3

hormones,

I

collagen,

alkaline

phosphatase,

We also examined the e f f e c t of osteotropic

and

glucocorticoid

on

osteopontin

mRNA in

o s t e o b l a s t i c c e i l s (ROS 17/2.8). Mat_,eELal~_._~n#_Methods I s o ] a t i g n _ o f ostegpontin cDNA. rat

osteosarcoma

ROS 17/2.8

A cDNA l i b r a r y constructed from mRNA of

cells

was screened with a o l i g o n u c l e o t i d e ,

36-mer (GTCICCGTCGICAICGICGTCGTCAICAICGTCCAI), derived from the published sequence

of

rat

screened

with

osteopontin

the

(3).

Approximately 3.0xlO 5 plaques

oligonucleotide

kinased

by

32p

yAIP.

were

The f i l t e r s

were hybridized f o r 18 h at 37°C in 6xSSC, 0.1% SDS and washed at 50°C in IxSSC, 0.1% SDS.

Several

p o s i t i v e clones were i s o l a t e d a f t e r m u l t i p l e

rounds of plaque p u r i f i c a t i o n .

The phage DNA was prepared by lamda sorb

(obtained

from

The

insert

containing

1300 bp was subcloned into pBS/M13+ (from Stratagene,

Inc.).

Restriction

and

Promega, Inc.) partial

and

digested

sequence analysis

with

EcoRl.

confirmed

the

cDNA as

an

authentic r a t osteopontin cDNA. Preparati_ o_n__oL.RNAa_ndNorthe_rn_ana!ysis. from

adult

various

Sprague-Dawley

stages

of

described previously (6), L-glutamine, and I . I and 5% f e t a l

rats

and

gestation.

fetal

Fresh

tissue was dissected

calvaria

ROS 17/2.8

cells

were were

dissected

at

maintained

as

in F-I2 medium containing 28 mM HEPES, 2.5 mM

mM CaCI2 and supplemented with I00 ~g/ml

bovine serum.

by CsCI c e n t r i f u g a t i o n

kanamycin

Total RNA from tissues or c e l l s was isolated

as described

by Chirgwin e t a l .

(7).

Briefly,

t i s s u e (0.5 g) was dropped into I0 ml of 5M guanidium isothiocyanate and immediately homogenized using polytron bursts

at

the maximum speed.

homogenates were pH7.5). rotor

loaded

RNA p e l l e t

at 35,000

gel

and transferred

several 20 second

a

CsCI cushion after

(5.7M

CsCI in

12 h c e n t r i f u g a t i o n

RNA was isolated from confluent lhe t o t a l

cDNA i n s e r t s .

of

EDTA

liTO.l

150 cm plates by

out

Inserts

(Hybond N) by e l e c t r o b l o t t i n g for

of

12--18 h with 5-10xlO 6 c.p.m. cDNA ~I

(I)

collagen,

phosphatase and osteopontin were prepared by r e s t r i c t i o n and f r a c t i o n a t e d

O.IM

and

RNA was f r a c t i o n a t e d on a 0.8 % agarose

to nylon f i l t e r

The h y b r i d i z a t i o n was carried labeled

for

Debris was removed by c e n t r i f u g a t i o n

was collected

rpm.

s i m i l a r method (7).

on

(Brinkman)

on I% Sea Plaque agarose gel

1130

(8). 32p

alkaline

enzyme digest

(FMC Co. Rockland,

ME).

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The cDNA probes for rat collagen : l

(1) and for bovine osteonectin were

kindly provided by Drs. Rowe, Kream (9) and Termine (lO). phosphatase has been cloned in this laboratory ( l l ) .

Rat alkaline

Each fragment was

labeled with 32p dCIP using random primers and klenow fragment according to

the

method described

oligonucleotide,

by

Feinberg and

Vogelstein

(12).

Kinased

42-mer (GIAGGCGICCIGGAAGCCAATGTGGICCGCIAGCICGTCACA)

was

used to probe osteocalcin mRNA.

Re_~su).ts__an_dpiscus___sion To

address the

question

of

tissue

specificity

for

osteopontin

synthesis, Northern blot analysis was carried out on t o t a l RNA prepared from 12 d i f f e r e n t rat tissues.

As shown in Figure l ,

osteopontin cDNA

hybridizes to a single band of 1.5 kd mRNA in total RNA from calvaria and kidney.

A much lower level of osteopontin

mRNA was also observed in

brain and lung seen in overexposed autoradiographs and none was detected under these conditions

in

l i v e r , spleen and testes. was found

in

the

skin,

muscle, cartilage,

heart,

intestine,

The most abundant level of osteopontin

osteoblastic

osteosarcoma cells

A

ROS 17/2.8,

mRNA which

B q~

y 28S

18S

Fig. I.

Northern blot analysis of total RNA from various rat tissues. Panel A: Northern blot of 20 ~g total RNAs from each tissue were separated by electrophoresis and hybridized to 32p-labeled osteopontin cDNA. The size of osteopontin mRNAhad previously been determined to

be approximately 1.5 kb (3).

RNA size

markers are the ribosomal 18S and 28S RNAs. Panel B: Northern blot of 20 ~g total RNAs from rat calvaria, kidney and ROS 17/2.8 cells, exposed for a shorter time.

1131

Vol. 148, No. 3, 1 9 8 7

exhibited

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a 5-fold

higher level,

than adult calvaria or kidney. the

presence

of

estimated

(5)

scanning,

Immunohistochemical data has documented

osteopontin

osteoprogenitor cells

by densitometric

as well

in

osteoblasts,

osteocytes

as in kidney where its

and

localization

appeared to be lysosomal and where i t was thought to be degraded (13). lhe mRNA findings

are consistent with most of

suggest considerable s p e c i f i c i t y , synthesis by osteogenic

cells,

but not

these observations and

exclusivity,

for

osteopontin

the kidney being potentially

other organ synthesizing this protein.

the major

The role of this protein in the

two major organs which participate in calcium and phosphate metabolism, have the highest levels of alkaline phosphatase and are the major target organs for parathyroid hormone, is certainly of interest. We next examined the relative abundance of osteopontin mRNA at five time points during the development of' calvaria and compared i t to that of type

I

collagen,

Equal amounts of gestation,

alkaline

phosphatase, osteonectin

RNA extracted

immediately after birth

1

from calvaria and adult

2345

1

~28S

at

and

lO,

2345

COL

~ 28S

•.18S

,18S

OP

B 1

1 2345

2345 28S

-28S ON

-18S

•..18S

OC

D

C 1132

18 day of

(250-300 g) rats

AP

A

15,

osteocalcin.

(Fig.

2)

Vol. 148, No. 3, 1987

B I O C H E M I C AAND L BIOPHYSICAL RESEARCH COMMUNICATIONS

were hybridized with estimated

from

the

five

probes.

autoradiography scans

steady-state levels f o r

The

is

relative

shown in

amount of

Fig.

3.

mRNA

The mRNA

these f i v e osteoblast products were highest in

the RNA of newborns and were lower in a d u l t c a l v a r i a .

Interestingly,

the pattern of expression of these f i v e genes during development was not identical.

Type I collagen, osteonectin and a l k a l i n e phosphatase reached

a high level of expression at an e a r l y embryonic age and maintained i t throughout gestation, whereas osteocalcin and osteopontin rose l a t e r and peaked in

the

newborn.

It

has

been postulated that

a small

set

of

transacting d i f f e r e n t i a t o r s may regulate several genes during development (14).

The

temporal

profiles

suggesting a c e r t a i n

extent of

similarities

exist

seem to

appear

to

be

unique

for

independent r e g u l a t i o n .

between collagen,

e a c h gene,

However, some

osteonectin and a l k a l i n e

phosphatase on one hand and osteocalcin and osteopontin on the hand,

consistent

similarity

Fig. 2.

with

extends

coordinate

to

the

Northern blots

for

regulation

effects

of

of

these

1,25(OH)2D3

osteoblastic

genes.

which

"markers" during

other This

stimulates

calvaria

development. Total RNAs isolated from rat calvaria at different ages were loaded in each lane: lanes l , and 18 days of gestation. and adult. in

2, 3 represent lO, 15

Lanes 4 and 5 correspond to newborn

Equal amounts of RNA (lO ~g) were electrophoresed

agarose/formaldehyde gels,

blotted

to

nylon f i l t e r s

and

hybridized with cDNA probes for A) AP, rat alkaline phosphatase and OP, rat osteopontin; B) COL, rat procollagen eI ( I ) ; and C) ON, bovine osteonectin and OC, rat osteocalcin probes. and COL probes were hybridized formaldehyde,

5 x

SSC, 5 x

in

solution

AP, OP

containing

50%

Denhart's (0.1% Ficoll,

0.1%

polyvinylpyrrolidone and 0.1% bovine serum albumin) at

42°C

overnight and washed in O.l x SSC at 65°C.

OC and ON probes

were hybridized and washed at lower stringency, by hybridization with the same solution at room temperature and washed in l x SSC at 55°C. Each probe hybridized to the mRNA species of correct size.

Two bands observed in collagen eI

(I)

represent mRNAs

which u t i l i z e two different polyadenylation sites

(19).

The

total amounts of RNA loaded are shown in panel D by ethidium bromide staining of 28S and 18S rRNAs in each lane.

1133

Vol. 148, No. 3, 1987

B I O C H E M I C AAND L BIOPHYSICAL RESEARCH COMMUNICATIONS

lO0

80 -,:_

60

n-

40

20

12

,4

"

16

,8

20

NB

Embryonic Age, deys

Fig. 3.

Changes in the relative level of during ddevelopment. The extent of in Fig. 2 was quantitated by integration of each peak using

osteoblastic marker mRNAs hybridization for each probe densitometric scanning and an Image Technology image

analyzer. Each point is the average from three independent measurements. Relative intensity of hybridization for each probe was normalized to the highest level of mRNA observed (newborn). Each curve represents the quantitation of mRNAs of alkaline phosphatase (a), collagen : l (I)(o), osteonectin (A), osteopontin (X) and osteocalcin (o).

osteocalcin production and dexamathasone which i n h i b i t s iL (15,16). seen in Fig. 4, 1,25(OH)2D3 treatment

As

increased the steady-state levels

of osteopontin mRNA lO-fold, whereas dexamethasone treatment reduced i t to

undetectable

levels.

Osteopontin secretion

was also

increase in response to 1,25(OH)2D3 (17) in this cell two fold,

reported

to

line a l b e i t only

suggesting additional post-transcriptional regulation.

It

is

of interest that hormonal responses of alkaline phosphatase and collagen also d i f f e r from those of osteocalcin and osteopontin Glucocorticoids

stimulate

1,25(OH)2D 3 i n h i b i t s physiological

the

alkaline

phosphatase expression

expression of

significance

of

type

I

collagen

t h e s e observations

established. 1134

in these cells ( 1 8 ) and

(19,20).

remains

to

The be

Vol. 148, No. 3, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

2

3

4

5

28S'.

18S

Fig. 4.

Hormonal regulation of osteopontin mRNA. The ROS 17/2.8 cells were plated at 20,000 cells/cm2 in 5% serum. Seventy-two hours later, 2 nM of 1,25(OH)2D3 or 30 nM, and l nM dexamethasone were added.

Three days later,

RNA was extracted as described in

Methods. Each lane was loaded with 20 ~g total RNA isolated from: l ,

2 nM 1,25(OH)2D3; 2, control; 3, control; 4, 30 nM

dexamethasone; and 5,

l

nM dexamethasone-treated ROS 17/2.8

cells. The Northern blots of the dexamethasone treated cells were exposed for a longer time (compare control lanes 2 and 3).

In conclusion, osteopontin mRNA is most abundant in bone, although significant

steady-state

levels

are

also

developmental pattern of mRNA enrichment

found

in

kidney.

The

indicates that the osteopontin

gene is turned on at a r e l a t i v e l y l a t e r stage of c a l v a r i a l development than alkaline phosphatase and collagen, osteocalcin.

and resembles in that

respect

Dexamethasone reduces and 1,25(OH)2D3 increases the levels

of osteopontin mRNA, s i m i l a r to the effects of these osteotropic hormones on osteocalcin (15,16,21).

Osteopontin thus deserves consideration as an

interesting marker of osteoblastic a c t i v i t y .

Ac knowl edgments We thank Dr. Mark A. Thiede for the ROS 17/2.8 l i b r a r y and Dr. Masaki Noda f o r the p a r t i a l and Dr.

sequencing of cDNA.

We also thank Dr. David Rowe

Barbara Kream f o r the rat procollagen cDNA clone and Dr.

Termine f o r bovine osteonectin cDNA clone. f o r sharing with us prepublished information. 1135

3ohn

We thank Dr. William Butler

Vol. 148, No. 3, 1 9 8 7

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

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