0022-1554/90/$3.30 Thejournal of Histochemistry

Copyright

and

Vol. 38, No.

Cytochemistry

by The Histochemical

1990

1

Society,

Original

and Bone

LARRY W. FISHER, ROBEY

BIANCO,”2 PAMELA

Research

Received

for

GEHRON Branch,

National

publication

The messenger droitin/dermatan

institute

)anuary

13,

of Dental

1990

and

MARIAN

Research,

in revised

May

and core pmteins sulfate proteoglycans,

ofthe two small chonbiglycan and decorin, were localized in developing human bone and other tissues by both 35S-labeled RNA probes and antibodies directed against synthetic peptides corresponding to nonhomologous regions of the two core proteins. Biglycan and decorin expression and localization were substantially divergent and sometimes mutually exclusive. In developing bones, spatially restricted patterns of gene expression and/or matrix localization of the two proteoglycans were identified in articular regions, epiphyseal cartilage, vascular canals, subperichondral regions, and periosteum, and indicated the association ofeach molecule with specific developmental events at specific sites. Study of non-skeletal tissues revealed that RNAs

Developing (CS)

human

bone

proteoglycans,

the products tein

of

Mr

tured

bone protein

the same ogous and

PG

(26)

localized in intact 1

2

sulfate

II), which

minor

viewed

as comprising

proteoglycans

to defined Therefore,

1990;

to: Paolo Research, Dip

‘La Sapienza,’

NIH,

Bianco,

Biopatologia

Viale

Bone Research

Regina

Umana,

Elena

324,

to the PG

Human

JOHN

D.

Bethesda,

Maryland

June

14,

1990

TERMINE,

20892.

(0A1883).

Histochem

Cytochem

KEY

WORDS:

Proteoglycans;

lion;

Bone

still

1990)

Biglycan;

development;

be related

Tissue

Decorin;

to the regulation

However,

In situ

hybridiza-

localization.

of assembly

the distribution

and

and

growth

functions

objective

patterns

decorin temporal

of the present

of localization

in developing and spatial

their

provide and

study

and

human patterns

was to define

gene

expression

are

two core

functions.

proteins,

beled RNA probes. of identifying the correlation

For this

and

(b)

in situ

gene

and

aspects of bone for understand-

purpose,

we used

hybridization

(a) antito nonregions of

using

35S-la-

provided two independent in tissues and allowed

This approach two proteoglycans

between

compare

skeletal and non-skeletal tissues. The of expression of the two proteogly-

valuable insights into molecular a necessary frame of reference

biological

and

of biglycan

bodies directed against synthetic peptides corresponding homologous amino acid sequences in the N-terminal the

of colla-

ofbiglycan

unknown.

The the

38:1549-1563,

expression

and

matrix

ways for a

deposition.

period

Branch,

1-00161

of

of decorin fiber growth

functions

106, Bethesda, Sez Anatomia

1990 USA.

decorin was associated with all major type I (and type II) collagen-rich connective tissues. Conversely, biglycan was expressed andlocalized in a range ofspecialized cell types, induding connective tissue (skeletal myofibers, endotheial cells) and epithelial cells (differentiating keratinocytes, renal tubular epithelia). Biglycan core protein was localized at the cell surface ofcertain cell types (e.g., keratinocytes). Whereas the distribution of decorin was consistent with matrix-centered functions, possibly related to regulation of growth ofcollagen fibers, the distribution ofbiglycan pointed to other function(s), perhaps related to cell regulation. (J

ing

connective

of the collagen

its biological

Bldg. 30, Rm.

a subset

accepted

cans should development,

interspe-

correspond

regions

human

homol-

in most

it may

protein of cul-

PG 1/biglycan

The tissue localization ability to inhibit collagen that

are

is quite

significantly

are presently

(20,23,24).

address:

although

of the

sequence

(5). Both

cytochemically

Present

PG I (15) (with

from,

extracellular

ofDental

UniversitI

cartilage

led to the postulate

tissues

to that

protein

sequence

Correspondence

Institute

is identical core

(7-9,15,20,21). and its proven

(3,17,22,25,27),

in vitro

31,

ofHealth,

gen fibers.

of a core pro-

core protein

ubiquitous matrices

(PG

consists

Biglycan

is distinct

decorin

Il/decorin

virtually tissue

ofbovine and

with,

chondroitin

I) and decorin

(5). Biglycan

cell decorin (5,11).

as that

cies variations)

(PG

genes

small

and

size and

fibroblast

two

two CS chains; decorin has a core one CS chain. The core protein sequence

42,510

human

contains

biglycan

of different

ofsimilar

institutes

well

Introduction

Two Small in Developing

F. YOUNG,

National

form

1549-1563, Printedin

Artide

Expression and Localization of the Proteoglycans Biglycan and Decorin Skeletal and Non-skeletal Tissues PAOLO

11, pp.

Inc.

might

Materials

and

Tissues.

Human

National MD 20892. Patologica,

Roma,

Italy.

Methods tissues of gestational

therapeutic

procedures

ately

in Dulbecco’s

placed

2 hr. Tissues

sampled

were

used

for this

minimal included

essential long

bones

age 14-17 study.

The

weeks

medium of the

obtained

and limbs

from

was immedi-

material

dissected (femur,

within tibia,

hu-

‘549

Downloaded from jhc.sagepub.com at UNIV OF MISSISSIPPI on July 8, 2015

1550

BIANCO,

FISHER,

YOUNG,

TERMINE,

GEHRON

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ROBEY

BIGLYCAN

merus,

AND

DECORIN

and ulna),

lung),

skin, feet,

4%

formaldehyde

phate

and

muscle,

placenta.

buffer,

isopentane nary

ribs, calvaria,

skeletal

and

IN

lower jaws, viscera

peripheral

Tissues

(freshly cryostat

assessment

nerves,

were

made

fixed

from

pH 7.2. Additional and

the

of immunostaining

better

buffered

obtained

of morphology.

in

used

samples

were

thesized

study

manufacturer

and

decalcified

in

The

elsewhere

antiserum

LF-15

to amino

11-24

a synthetic

peptide

was

a synthetic

secreted

form

acids

Briefly,

corresponding

bone

PG 1/biglycan.

was generated

LF-30

to keyhole

(4,5).

peptide

of human

to amino

conjugated

used in this study were

to be monospecific

to BSA. Antiserum

corresponding

peptide

antisera

proven against

of the

was conjugated

rabbit

and

was generated

acids

This

“peptide”

two

in detail

5-17 ofhuman limpet

against

PG lI/deco-

hemocyanin.

When

used in Western blot and immunoprecipitation, LF-15 recognized the core protein ofPGI/biglycan, and LF-30 the core protein ofPG Il/decorin, with no crossreactivity

Immunostaining. posed and

were

then

Sections

hydrogen exposed

BSA

x-100

(four

kegaarde

sections

were and

Perry;

Rockville,

peroxide

of ethanol,

cleared with

temperature

except

Chondroitinase recognize

sections

were

cores

0.05

assayed

M calcium before

adopting

slides were

serum

and sense (control)

plasmids

dilution

tamed

using

G-50 (1).

hydrolysis

Because devoid

ABC

Covina, ranging

0.01% 1.25

at room

antibodies

quantita-

glycosaminoglycan lyase

chains,

(protease-free,

from

Pro5ev-

pH

as the

7.2,

for 10 mm

at 37’C

were

Hybridization.

and

Control oflmmunostaining. Control stitution of normal rabbit serum (1:200) tide

with

tests.

These

100 sl of the

were

done

for primary

by incubating

consisted

1 mM

antisera,

1 nanomole of the

sub-

precipitation.

with

Kpn

I. The

reduced

in size

sections

template

by centrifugation

were

by limited

treated

acetic

with

0.2

N

St Louis,

washed in 0.1 M triethanolamine,

anhydride

in PBS, then in 2 x SSC, dehydrated and

formamide,

0.3

Denhardt’s

dextran

alka-

air-dried.

solution,

sulfate.

The

M NaCI,

tRNA

yeast

Final

hybridization

20 mM

probe

Tris HCI, 500

pH

tg/ml,

10

was

2 x

concentration

Aliquots

at 52’C

20 mM

in a humid pH 8.0,

10 mM

DT.1’ at 52’C

at 52C

for 30 mm,

Adventitiously 20 tg/ml

perature,

then

at 4’C oped

slides

for 3-8

corresponding

croscopy.

days

30 mm

x SSC,

The

10 mM

DTT

dehydrated, at room

in the presence fixer,

slides

were

viewed

then at room

in both

15 mm,

washed tem-

2 x SSC

air-dried.

For autoradi-

of Kodak

NTB-2

for 5 hr. then Exposed tap

cleared

with

Tris HCI,

mm

for

DU

sections

were

at 15’C,

in running

in ethanol,

the

10 mM

slides

temperature

ofdesiccant.

washed

temperature.

for 30

and

for 2.5 mm

4 x SSC,

at room

at 52C

in a 1:1 dilution

dipped

M NaCI,

10 mM

by digesting

DTT

squares

0.3

2 x SSC,

mm)

at 37’C.

10 mM

plastic

formamide,

in 0.5 M NaCI,

temperature,

dehydrated The

Ti

were

D-19 developer

in Kodak

for 5

was removed RNAse

to rest vertically

in KOdak

times

the

10 mM dithiothreitoL

in 50%

formamide,

in 2 x SSC,

in 0.1

allowed

in Permount.

for

then

at room the

probe

day,

formamide,

1 x Denhardt’s,

50%

2 x SSC (four

next

in 50%

washed

for 30 mm,

EDTk,

buffer,

10 mm

sion,

1 mM EtifA,

A, 1 tg/ml

1 mM

in RNAse

The

the slides

sequentially

bound RNAse

pH 8.0,

atmosphere.

by immersing

Tris HO,

fixed

absorption.

1 x 10%

were removed

and antigen antibody

ethanol

ofthe mixture sized on the dimensions ofthe sections and the sections were covered with small plastic squares cut out ofheat-sealable “Seal-a-meal” pouches. The slides were hybridized overcpm/pi.

of each

pep-

Both

were applied,

hemalum,

of the

primary

and

of ethanol,

of 50%

and

with

specificity

dilution

in 0.25%

EDTk,

acid,

to check

working

included

by the WI).

after linearization

was purified

Deparaffinized

acetylated

overnight at 4’C, followed by centrifugation at 10,000 x g for 5 mm, and use in immunostaining. Each antibody was also incubated with the nonpeptide

highest

ofimmunostaining

as indicated Madison,

and

the probe

column,

concentrations

mM DT1

for

standard.

were

linearization

1, and

“spin”

The slides were then

CA) before immunostaining. from 0.01 to 2.5 U/mI in 0.1 M

BSA,

U/mI

done

8.0,

night were

below.

the peptide

of attached

Sections

described 35S-Labeled

HCI, digested with proteinase K (from Tritirachium album; Sigma, MO), 1 sg/ml in 10 mM Tris, 2 mM CaC1, for 15 mm at 37’C,

mixture

of an

a

protein-

to that (11).

Promega,

extraction

after

DNAse

line

ography,

absorption

with

on a Sephadex

in PBS,

concentrations

were

described

T3 polymerase

was removed

in ascending

After

All incubations

by phenol

pH 8.0. The sections were then washed

a 1:100

the

(b)

For synthesis of biglycan anti-sense probe, the plasmid was linearized with Kpn I and T3-primed RNA polymerase reaction was used. The sense strand was synthesized using T7-primed RNA polymerase after linearization of the plasmid with XbaI. Decorm anti-sense was synthesized using T7-primed RNA polymerase after linearization with Barn HI; the sense strand was ob-

30 mm Triton

identical

the

and

with

Ruoslahti

were with

biglycan,

were synthesized

the

0.01%

in ascending

followed

probes

in PBS,

in PBS,

(5), and

System;

in PBS,

antiserum

bone

conditions

Gemini

cx-

for

in Permount.

decorin

by Krusius

and

below),

anti-rabbit

dehydrated

chondroitin

acetate,

with

30 miii.

digestion

teus vulgaris; ICN Biochemicals, eral concentrations of ABCase, Tris,

washing goat

washed

incubated

as needed.

Digestion. with

After primary

mounted

the ABCase

protein

(see

IgG antibody (Kirwashing in PBS-Triton with 3,3’-diaminobenzidine and hydrogen

and

hemalum

treated

then goat

in xylene,

counterstained

ofthe

MD)for

the sections were reacted (6), rinsed in distilled water,

x-100,

tively

The

10 mm),

lyase

of normal

dilution

peroxidase-labeled

and

for 30 mm.

a 1:200

for

affinity-purified,

peroxide

ABC

with

for 2 hr. times

on poly-L-lysine-coated

chondroitin

to a 1:5 dilution

incubated

0.1%

mounted

with

BP insert

a 1.6 KB insert

Transcription

(Riboprobe

In Situ

treated

to 0.3%

sections

reactions.

anti-sense

of human

bone

decorin

a 1658

RNA probes (specific activity 2 x 108 cpm/sg) were syn[“SJ-UTP (New England Nuclear; Boston, MA) in T3- or

using

T7-primed

(4,5).

deparaffinized,

ofhuman

used for transcription

containing

(P2) containing

fibroblast

single-stranded

of fixed given the

of the

Antibodies.

The peptide

sequence

The templates (P16)

sequence

plasmid

for human

in aldehyde-fixed,

embedding protocol,

plasmid

protein-encoding

encoding

for a prelimi-

preliminary

complete

Probes.

ofRNA

a pBluescriptSK

pBluescriptSK

in dry ice-cooled

were

in such

Bone

at 4’C

Preparation (a)

in 0.1 M phos-

were snap-frozen sections

liver, hands

EDTe.

described

rin.

whole

Routine paraffin tissue preparation

results

preservation

adrenal,

tendons,

of immunoreactivity

paraffin-embedded vs frozen tissues. material was adopted as the standard identity

(kidney,

eyes,

for 2 hr or overnight

Frozen

loss

1551

paraformaldehyde)

samples

sectioned.

of potential

TISSUES

HUMAN

slides

stopped water,

were

devel-

in 1% acetic counterstained

in xylene,

brightfield

emulexposed

and

and mounted darkfield

mi-

Figure 1 . Sections ofthe whole hand of a 14-week-old human fetus stained for (a) biglycan and (b) decorin core proteins. The articular ends of each bone rudiment show a “cap” of articular cartilage that stains intensely for biglycan and remains unstained for decorin. Deeper to this zone, resting nonarticular cartilage of each epiphysis stains significantly for decorin and weakly for biglycan. Strong staining of unmineralized, newly deposited osteoid is seen in the midshaft of each rudiment. Details ofthefifth metacarpo-phalangealjoint. Note absence ofstaining for decorin (d) in the biglycan-nch articular cartilage(arrowheads), and the waning of biglycan staining(c) in the deep, decorin-rich resting cartilage(r). Also note thatarticular softtissues(astensk), like articular cartilage, stain for biglycan and do not stain for decorin. Bars: a,b - 1 mm; c,d = 50 sm.

Downloaded from jhc.sagepub.com at UNIV OF MISSISSIPPI on July 8, 2015

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hc

ROBEY

BIGLYCAN

AND

DECORIN

IN

HUMAN

TISSUES

‘553

irrespective ofthe ing for biglycan

Results Immunolocalization Core

ofB:glycan

and

cifically

Tissues.

localized

Biglycan

and decorin

to cartilaginous

ing bones.

However,

tems

were

noted

of biglycan

and

joints.

core protein articular cartilage

and

important

sites.

decorin

proteins

core

were speof develop-

components

in the two staining

differences

at particular

Biglycan

prospective

core proteins

bony Figure

1 shows

pat-

for both biglycan ther membranous,

gin. Biglycan,

the localization

in developing

diarthrodial

ular

was localized to an outer “cap” of at each epiphyseal end of bone mdi-

all classical

ticular

capsule,

synovium)

displayed

articular cartilage, rin. This mutually

i.e. , they exclusive

was

same

essentially

the

articular

resting

biglycan

in limb

the

stained

long

same

soft tissues staining

(ar-

profile

as

stained for biglycan but not for decostaining pattern in articular regions

in all long

cartilage

Articular

bones

bones

studied.

However,

somewhat

more

prominently

than

in metacarpal

deconin

for

Because proceeds

endochondral at different

ofdevelopmental

rates

in most

such

begins

in different

bone

stages ofgrowth

ent bone e,camined,

rudiments included long bones oflimbs

portions,

and their metaphyseal

vaded

by marrow

veloping each

displaying

sification.

plates was displayed

inner

part

a centrally

located,

of a central

by concentrically

arranged

and phalangeal

rudiments

mid-diaphysis,

with

center

upper

territorial

and

proliferative

capsules

zone,

of isogenic

periosteal

degrees

matrix staining in lower proliferative,

of the

growth

less intense

Chondrocytes

biglycan

plates

(Figures

2c and

and 2d).

distinct

stained

staining

(Figure

prominently

for decorin

in

was

as dermis,

ci-

tendon,

oflarge

blood veswas instead local-

protein

connective

tissue

on epithelial

na-

myofibers, keratinokidney and skin pro-

examples of the divergence of the localization patterns In skin (Figure 3), decorin cone protein was localized to

Table

1. Distributio,t

and mRNAs

of

b:glycan

in non-skeletal

In limb long was virtually the

(Figures

2e and

in all growth

and

decorin

core

proteins

tissues

Tissue

Biglycan

Kidney

Endothelia Collecting tubules Endocardium Some myocardial fibers “Small” interstitium

Decorin

Interstitium

Subpcricardium “Small” and “large” Penisinusoidal

-

Adrenal

gland

Aorta

Endothelia

-

Intirna Media Adventitia Epidermis Endothelia

Skin

Tendon

zones

Skeletal

corre-

Eye

2f).

connective

tissue

to 2a),

mineralizing

In contrast,

for biglycan

biglycan

interstitium

zones of more immature growth plates (metacarpal, vertebral) showed prominent staining for decorin and but

(such

, adventitia cone

and

was localized for

cells, skeletal Developing

and

was restricted

of chondrocytes

hypertrophic,

ofeithen

Liver

ossifica-

of invasion

to interterritorial

bones, absent

sponding phalangeal,

where

groups

Biglycan

chondrocytes.

showed

matrix (Figure 2b). for either biglycan or decorin

decorin

cytes,

Lung

ofthe underlying hypertrophic cartilage. A feature comto all growth plates was a highly segregated staining pattern

in the

sclera).

cell types

Heart

ossification mon

ofthe

tissues

tis-

ofbiglycan

deconin staining

(e.g.

included endothelial and renal tubular epithelia.

of Os-

hypertrophy

proliferating

variable

inde-

rudiments,

primary

area of cartilage

Metacarpal in their

cartilaginous

forming

surrounded iion

by the differ-

plates were extensively ossifying. In contrast, most

of entirely

few exceptions

of non-skeletal

distribution

and

growth and

consisted

This consisted

times

layer

canalic-

a spectrum

in this study. At the gestational age were fully ossified in their diaphyseal

vasculature

vertebrae

at different rudiments,

walls and

ized to a few specialized connective tissues where deconin was not detected (e.g., endocardium, articular capsules), and to a spectrum

observed.

ossification

lacunar

1). Whereas

or absent

vide suitable

to epi-

(Table

with

cartilage

the perichondrium

proteins

on cornea),

vascular

from

of cion-

to the thin

localized

of a variety

divergent

then minor

around

bones.

A survey

matrices,

physeal

ingrowing

osteocytic

tissue

ture. These

of long

Tissues.

ofspecialized

and phalangeal

was also

lining

a substantially

core

rudiments. A halo ofcartilage matrix that remained unstained for either biglycan or decorin core protein was consistently observed canals

matrix

connective

sels,

non-

at all sites of bone formation, periosteal, or endochondral

but not deconin,

Non-skeletal

staining

weak.

whereas cellular stainwas also true in other

spaces.

ments. Decorin was not detected at these sites but was found in the more deeply located nonarticular resting cartilage, in which was conversely

and decorin perichondral,

ofunmineralized

sues revealed

for biglycan

matrix, This

zones of cartilage, possibly indicating poorer accessibility of the intracellular biglycan core protein as opposed to the intracellular decorin core protein. Newly deposited bone matrix stained intensely

Decorin

Proteins

Skeletal

stainability ofthe was less prominent.

Peripheral Placenta

Media Adventitia Dermal -

muscle

nerve

+

Myofibers

Sclera

Connective

(inner

matrix

part)

tissue

sheaths

Sclera Cornea

Endothelia

Connective

tissue

sheaths

Endothelia

Connective

tissue

core

plates,

of viii

Figure 2. Epiphyseal cartilage of the humerus, upper proliferative zone. Adjacent sections stained for(a) biglycan and (b)decorln. Territorial capsules of isogenic groups of chondrocytes stain for biglycan but not for decorin. lnterterritorial matrix stains for decorin but not for biglycan. (c,d) Same sections as in a and b, growth plates. Note that the cartilage matrix does not stain significantly for either (C) biglycan or (d) decorin. The cartilage cores of developing bone trabeculae also fail to stain, whereas newly deposited osteoid at their surfaces does stain (arrows). #{149},f show, for comparison, metacarpal rudiments, stained for (#{149}) decorin and (I) biglycan. The hypertrophic cartilage in their growth plates (hc), at variance with that of the humeral growth plate, does stain. Note cell staining for decorin in b and d. Bars: a,b - 30 ism; c,d - 60 tm; e,f - 100 tm.

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BIANCO,

1554

FISHER,

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YOUNG,

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structures

tissue,

and

at the

with

possibly

occasionally, ing glomeruli

-

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ROBEY

concentrations

of arteries,

around

whereas

cells and to the tubules. Occasional

mesangial

epithelial exhibited

focal

adventitia

can was localized to endothelial epithelial cells lining collecting glomeruli,

TERMINE,

bigly-

apical surface cells inside

also stained

of the

fon decorin.

cells of the parietal epithelium some staining for biglycan.

Very

of develop-

C,

c:

Expression

ofBiglycan

Developing were

Bones. Although in cartilage and

expressed

were noted

. ..

at specific

canals

(Figure

7c).

dral locations

p

..t “

.

layers

I

-

.‘ .

‘

T

I

-.

‘:

‘-

between -:-

of the

___‘

Figure a Immunolocalization of biglycan and decorin core proteins in fetal skin. (a) Decorin is localized to the collagenous matrix of the dermis, where staining for (b) biglycan is absent. Biglycan core protein is localized to the epidermis, where decorin is not detected. (C) Localization of biglycan core protein at the cell surface of differentiating keratinocytes. Bars: a,b = 20 sm; C - 30 tm.

localized prickle lining

mRNA

arranged

matrix,

to the cell surface

whereas

of differentiating

were

around

vas-

8).

and

decorin

biglycan

was more

decorin).

mRNAs

on bone

Biglycan

edge

high

consistently mRNA

levels of biglycan

biglycan

core protein keratinocytes

was of the

cell layer and, in the dermis, was restricted to the endothelial ofcapillaries. In kidney (Figure 4), deconin was localized

to

fibroblasts ofthe cambial layer.

typical

localization,

mRNA

tial

concentrations

outer

abun-

but was abunlayer. More cell

(i.e. , at the

peniosteum

confirming

connective

epitheial,

rin

and

was not expressed

in contrast, was the periosteum, could be noted

and pre-osteogenic

cells

Tissues. In non-skeletal tissues, the pattern of deconin gene expression coincided with that of core

fibrous

specialized,

in os-

mRNA

ossification

bone formation)

protein

co-

and

at the metaphyseal site of earliest than at the level ofthe midshaft (where

of periosteal

Non-skeletal biglycan and

were

surfaces

ossification is more advanced). Decorin mRNA, expressed at essentially similar levels throughout i.e., no difference in the levels ofdeconin expression

-.-.

collagenous

than

expressed

periosteal

the dermal

(Figure

in osteoblasts

latter,

expressed

leading

__f___

of deconin

significantly in fibroblasts of the outer periosteum dant in pre-osteogenic cells of the inner (cambial)

.

-‘

vertebrae; Figure 6a), cells inside vascular

in chondrocytes

9), biglycan levels

(in the

dantly

4:1

6b) and

(Figure at high

teocytes

;‘-

deconin genes differences

bones. High levels of regions (Figure 5a), in

low levels

chondrocytes

In bone expressed

I

of developing

In contrast,

(Figure

in hypertrophic ‘

,..,

both biglycan and bone cells, important

cular canals ingrowing from the penichondnium to epiphyseal cartilage (Figures 7a and 7b). In growth plates of limb long bones, low levels of biglycan and low or undetectable levels ofdecorin were observed in proliferating cartilage, and high levels ofboth mRNAs .-.-

/

mRNAs

found in articular cartilage (Figure 5b), whereas very high levels were detected in a narrow rim ofcartilage at peripheral subpenichon-

-

(

regions

Decorin

biglycan mRNA were detected in articular forming growth plates (such as in developing and in penichondnium-denived mesenchymal

.4-

-

and

was

the

tissues

and connective

localized

at high

ofgrains

of deconin

of biglycan

tissue

levels

around

expression

and

cell types.

In skin,

in the dermis,

developing

hair

with

lOc and

lOd).

deconin

was seen

over

In kidney the

interstitium

(Figure

1 1), a strong but

not

over

of

deco-

substan-

follicles.

can mRNA, in contrast, was detected in differentiating (Figures lOa and lOb) and in endothelial cells ofdermal (Figures

in all

in a range

Bigly-

keratinocytes capillaries signal tubular

for and

Figure 4. Localization of biglycan and decorin core proteins in developing kidney. (a,b) Low-power views of adjacent sections showing the staining of renal interstitial connective tissue for (b) decorin but notfor (a) biglycan. Arrows in a and b pointto the cross-sections ofthe same small artery. Note the staining of its endothelial lining for biglycan and of its adventitia for decorin. (C) lmmunolocalization of biglycan; detail of inner cortex showing labelling of glomerular (large arrowheads) and extraglomerular (small arrowheads) capillaries, and of the luminal surface of epithelial cells lining cortical collecting tubules (arrows). (d) Immunolocalization of decorin; detail of inner cortex showing staining of interstitial connective tissue, strong staining of the adventitia of an artery (arrows), and lack of staining of glomeruli (g). Bars: a,b - 210 tm; c,d - 60 tm.

Downloaded from jhc.sagepub.com at UNIV OF MISSISSIPPI on July 8, 2015

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BIANCO,

1556

FISHER,

YOUNG,

TERMINE,

GEHRON

ROBEY

Figure 5. Localization ofbiglycan and decorin mRNAs in articular cartilage of the humerus. (a) Superficial chondrocytes show higher levels of biglycan mRNA than of(b) decorin mRNA. mRNA are seen Darkfield images.

glomerular and

detected data

structures.

in epithelial from

in some

cells

Biglycan

mRNA

of collecting

skeletal

immunolocalization

myofibers

was detected

tubules. (Figure

mRNA

12), thus

confirming

of the corresponding

Discussion

in glomeruli

Biglycan

core

High levels of decorin in deeper chondrocytes. Bar = 40 tm.

was

protein.

Using

both

bridization, droitin/dermatan

sequence-specific we have

“peptide”

presented

sulfate

antibodies

evidence

proteoglycans

Figure darkfield

that biglycan

in situ hy-

two small and

6. Developing images

and

the

vertebral

ofautoradiographies

chon-

decorin

dif-

column, of ad-

jacentsections hybridized with (a) biglycan and (b)decorin probes. Strong hybridization signal for biglycan is detected over chondrocyteslying in an ovoid central portion of the vertebral rudiment. This “centrum’ correspends to the forming primary center of ossification, consisting of a central zone of cartilage hypertrophy surrounded by concentrically arranged layers of proliferating cells. Resting cartilage outside of this area is virtually unlabeled, as are the intervertebral discs (id). In contrast, strong hybridization signal for decorin is seen in a peripheral “shell” of resting cartilage, including forming endplates and subperichondral regions. Intervertebral discsalsoshow hybridization signal (in both annulus and nucleus). Significant trophic

Downloaded from jhc.sagepub.com at UNIV OF MISSISSIPPI on July 8, 2015

labeling cartilage.

is also Bars

-

seen in hyper250 tm.

BIGLYCAN

AND

DECORIN

IN

HUMAN

TISSUES

1557

the two genes tal events.

point

to their

For example,

in pre-osteogenic

cells both

cally undifferentiated formation

ofthe

Decorin

expression, was found layers

ation

(epiphyseal)

in contrast,

and

of biglycan

two genes indicates

with

different

not

at the high

of growth whereas

of formation

biglycan

of growth

sion of decorin ical continuity penivascular

in subpenichondral of vascular canals an internal

cartilage

Deconin (e.g.,

sub-

predominates

The

demonstration

chondrocytes to the high

regions. with the

genes

two

growth

plates.

of deconin in penivascular may be functionally equated

cartilage

of the

rudiments.

expression

levels

dif-

of expres-

as in developing

expression

at the sites ofappositional

regions),

site

such

associ-

osteoblast

pattern

of individual

and

specific

with

rudiments,

(13).

vascular

osteogenic

segregated

of the

thought for the

inside

suggests

expression

bone

levels

ofossification

throughout

the highly association

expressed

perichondral

at high

in morphologi-

detected

This

deconin)

in early the

modes

is maximally

center

to be uniform

(but

vertebrae,

developmen-

and

was not

of peniosteum.

f#{232}rentiation.Furthermore, sion ofthe

specific

was expressed

in the periosteum

secondary

non-osteogenic

with

gene

mesenchymal cells in vascular canals, pre-osteogenic cells recruited

to be penichondnium-derived

canals,

association

biglycan

of

in epiphyseal levels of expres-

In fact, the penichondrium

anatommakes

region in epiphyseal

subpetithondral

cartilage. At two sites

in cartilage,

the combined

tion

and immunolocalization tween gene expression and matrix around vascularcanals plates the

oflong

bones,

absence

high

levels

of stainable

explained

with

high rates ular sites.

ofproteoglycan The known

tiguity

with

7. Expression of the biglycan and decorln genes in vascular canals of cartilage. (a) A vascular canal ingrowing from the perichondrium (vc) is surrounded by chondrocytes exhibiting strong hybridization signal for decorin. Chondrocyteswlth comparable high levelsofdecorin mRNAareseen scattered in the surrounding resting cartilage. (b,c)Mjacent sections of epiphyseal vascular canals hybridized with (b)decorin and (C) biglycan probes. Notethe concentratlon ofchondrocytes with high levels ofdecorin mRNA aroundthe canal. Inside the canal, the walls of individual blood vessels (asterisks) and penchondnium-derived mesenchymal cells(m) show strong hybridization signal for biglycan but not for decorin. Bars - 80 tm. Figure

in distribution

removal occurrence

proteins

differed

nificantly

in morphology vs non-articular

articular tnices).

In addition,

the

in regions

the

in developing

human

skeletal

and

immunolocalization ofcartilage

matrix

of the known

two core

to differ

and overall collagen organization regions, territorial vs interternitorial spatial

patterns

of maximal

expression

sig(e.g., maof

contrasted

matrix.

control

with

This

can

or, alternatively,

be

with

from the matrix at those particof degradation of cartilage ma-

vascular canals (12,13) and explanation. Interestingly,

is a common

feature

of areas

in growth close conof unstain-

plates did stain, at variance with growth plates in accordance with the high levels of biglycan

of long bones, and deconin

and tran-

scripts

by in situ data.

The

in growth

car-

in hypertrophic

occurrence

tilage

matrix

pears

a plausible Using

scnibed

free, ence

rates

correlated

with

data

the presence

cartilage

and

plate

but

biglycan-enriched

invasion, in situ

ap-

and

II, Poole

im-

molecule

cartilage.

Our

from data

an outermost matrix studies

articular

cartilage

et al. (17) de-

fetal and non-fetal

of this

Biochemical

in human

our

to DS/PG

reveal

cartilage

cartilage.

which

in bovine

absence

growth

of vascular

plate

be consistent.

ofdecorin the

extent

with

antibody

findings,

ofdecorin

degradation

the

would

a monoclonal

indicated

ofPG

explanation

munolocalization

ular

chondrocytes

ofdifferent

tive articular

non-skeletal tissues. In developing bones,

hybnidiza-

both around vascular canals and in growth plates of In metacarpal, phalangeal, and vertebral growth plates, uninvaded or poorly vasculanized, the matrix of growth

and hypertrophic such previous fer significantly

in the

translational

vasculatune

expression

proteins

tnix components both around plates (14) supports the second able matrix, long bones. which were

ofgene

core

a negative

use of in situ

disclosed a lack of coincidence bematrix deposition. In a rim of cartilage and in hypertrophic cartilage of growth

have

partially

zone

in human

match

of decorin-

fetal

described

(19,20).

artic-

proliferating

prospecthe pres-

However,

these

studies focused on postnatal articular cartilage and are less directly comparable with ours, because sampling for biochemical studies and

direct

of spatial

tissue

immunolocalization

resolution.

Downloaded from jhc.sagepub.com at UNIV OF MISSISSIPPI on July 8, 2015

Our

data

obviously on the distribution

differ in their power of deconin

out-

FISHER,

BIANCO,

1558

YOUNG,

TERMINE,

Figure a nn mRNAs

GEHRON

ROBEY

decolong bone (femur). (a) Low but distinct levels of biglycan mRNAareseenln the proliferative zone (top), and strong signal is seen over hypertrophic chondnocytes (bottom). (b) An area of hypentrophic cartilage hybridized with a biglycan sense (control) probe. (C) Hypertrophic chondrocytes showing strong signal for deconin mRNA. Bars - 50 um.

side

of skeletal

et al. (17).

tissues

In essence,

type

all collagen

I-rich

match we

those

found

previously

deconin

connective

to

tissues.

reported be

highly

Biglycan,

by Poole enriched

in

in contrast,

ap-

pears to be essentially absent from some typical connective tissue matrices, such as dermis, tendon, and cornea, and is expressed in a variety

of unrelated

cell types

not

involved

in the production

of

Detection

the distribution of the closely related a different biological role. The diversity

biglycan ofcell

can suggests a range offunctions expression in skeletal myofibers

specific would

volvement

physiology

of small

junction

PGs

in the

(16). Also, given

(a) the

of biglycan

in the growth

high

types

and

plateofa

strongly indicates expressing bigly-

to each cell. For example, be consistent with the inof the

neuromuscular

homology

of biglycan

core

type I collagen. In addition to some specialized connective tissue cell types, (endothelial cells, skeletal myofibers), these include epithelial cells such as keratinocytes and renal tubular epithelia, in which biglycan (or its core protein, regardless of the type, if any,

protein with glycoprotein lb (5), which in platelets binds Willebrand factor, and (b) the known occurrence of synthesis

ofglycosaminoglycan

ing ofvon

molecule. production

chain)

Although ofsmall

and skeletal muscle identity. Keratinocytes

to be a cell surface-associated

(16), no precise indication existed as to their have been shown to produce a membrane-

associated

large

proteoglycan calization

species of biglycan

to differentiating

appears

there is previous biochemical evidence for the DS/CS proteoglycans by endothelial cells (10)

CS proteoglycan

in intact

tissue

keratinocytes

(prickle

lated

to a differentiative

Whereas the distribution function ofregulation

phase

ofdeconin ofcollagen

and

also

smaller

expression and locells are restricted

cell layer)

tected in the proliferating cells of the basal given the change in cell/matrix interaction requisite for upward dislocation ofepidermal a proliferative

(2),

in culture (18). Both gene core protein in epidermal

and

are not de-

layer. This is of interest that represents the precells as they shift from of their

lifespan.

lends support fiber assembly

to its postuand growth,

storage sion

of von

ofbiglycan

Willebrand

factor

by microvascular

Willebrand

able hypothesis

factor

relevant

in endothelial endothelia

by biglycan

to the regulation

cells,

suggests

expresbind-

potential

in endotheial ofclot

the cells,

formation.

von and

a test-

However,

biglycan is also homologous to gene products regulating morphogenesis in Drosophila (5). It is interesting, therefore, that active morphogenesis and differentiation were a common denominator of all tissues

displaying

biglycan

Whatever the ultimate ences in tissue distribution remarkable

ifmatched

all structure

can

and

teoglycans partial deconin pears

expression

in this

study.

function of biglycan may be, the differof biglycan and decorin are even more to the similarity

they

share

in terms

and core protein sequence. The view that includes decorin in the same subset of ubiquitous interstitial of the

revision seems

extracellular

matrix

on the basis of the data to be a “full-time” matrix

to be a cell surface

or close territorial

of connective presented component, molecule

tissues

of overbiglyproneeds

here. Whereas biglycan ap(perhaps

found

Figure 9. Detection ofbiglycan and deconin mRNAs in peniosteal ossification. (a,b) Metaphysealleading edge ofpeniosteal ossification in the humerus. High levels of biglycan mRNA are detected in osteoblasts and osteocytes in the bony collar (be). Strong hybridization signal is also observed in the inner (osteogenic) penosteum, whereas the outer (fibrous) peniosteum (op) is virtually devoid oflabeling. In contrast, the levels ofdeconin mRNA are comparable throughout the thickness ofthe periosteum, and high in bone cells. (c,d)Subpeniosteal region ofthe mid-diaphysis ofthe humerus. Strong signalfor(c)biglycan mRNA is seen in osteoblasts at the surfaces of bone trabeculae (bt) and in the innermost part of the periosteum. Osteoblasts on trabecular surfaces also show strong signal for decorin mANA, as do fibroblasts in the periosteum (p). Bars - 60 tm.

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and darkfield images showing strong hybridization signal oven differentiating keratinocytes in the epidermis. oven adnexae, and overthe basalcelllayer ofthe epidermis. (c,d) Brlghtfield and darkfield Images of capillaries signal for biglycan mRNA. Bars: a,b - 40 tm; c,d - 20 sm.

Figure 11. Detection of biglycan and deconin mRNAs in developing kidney. (a,b) Brightfield and darkfield images of developing glomeruli in the cortex, showing hybridization signal for biglycan mRNA. (C) Low-power view of a kidney section hybridized with the decorin probe, showing strong signal over the interstitium, whereas tubular structures appear blank. (d,e) Bnightfield and dankfield images of collecting tubules in the renal medulla, showing strong signal for biglycan. Bars: a,b - 100 tm; c - 180 tm; d,e - 140 tm.

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1562

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