Effect of vitamin D and plasma calcium upon proteoglycan size in chick growth cartilage

Vol. 90, No. 1, 1979 September

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

12, 1979

Pages 65-69

EFFECT OF VITAMIN

D AND PLASMA CALCIUM UPON PROTEOGLYCAN

SIZE IN CHICK GROWTH CARTILAGE Ian R. Dickson, Strangeways

Research

*Shriner's

Hospital

Received

July

Peter

Laboratory,

for

J. Roughley*

Warts'

Crippled

& Egon Kodicek

Causeway,

Children,

Cedar

Cambridge

Avenue,

CBl &RN, U.K.

Montreal,

and

Quebec,

Canada

16,1979 SUMMARY

Proteoglycan subunit isolated from the growth cartilage of rachitic chicks much smaller size than the corresponding preparation from vitamin D-treated The size change can be related to differences in plasma calcium concentration not to the vitamin D status of the chicks.

is

of chicks. and

INTRODUCTION Vitamin skeletally grows

D deficiency immature

in

length,

consequence

rachitic

relatively

large

but

vitamin

D-replete

2B (2). chick

reduction in the

eluting

elute

pore A large in

gel

proportion

(3).

volume

is

and major

D-replete

structural

diet

isolated

diet, isolated

D-deficient

is

of

from

molecules

of a column

the

as shown by

and chromatography

of these

therefore

Collagen

subunits,

proteoglycan

or in the presence It

other

Proteoglycan

electrophoresis

the void

and alkylation buffer

with

The two main

composition the

bone

as a

and proteoglycan.

on a vitamin

compared

formation; wide.

that

(2).

on a vitamin

wide

by which

abnormally

evidence

raised

raised

the process bone

collagen

from normal

size

In the

to be of normal

found

of chicks

of viscosity,

of Sepharose

after

differs

of chicks

are

appears

we have

hydrodynamic

columns

chloride

(1)

becomes

cartilage

cartilage

cartilage

cartilage

measurements

even

of this

as appositional

plate

formation.

of bone

ossification,

as well growth

proteoglycan,

the growth

in a disorder

endochondral

affected

growth type

component,

growth

is

components

constitutional

from

animal

the cartilage

structural of the

results

from

of Sepharose

on the Cl-2B

of 4M guanidinium apparent

that

the

0006-291X/79/170065-05.$01.00/0 65

Copyright @ 1979 by Academic Press. Inc. All rights of reproduction in unvform reserved.

Vol. 90, No. 1, 1979

proteoglycan artefact

monomer resulting

of hyaluronic chick

BIOCHEMICAL

units from

acid.

growth

are

large

in vitro

is

and the results

aggregates

The average

cartilage

AND BIOPHYSICAL

size

smaller

observed

formed

that

are not

in the presence

of proteoglycan

than

RESEARCH COMMUNICATIONS

subunits

an

of traces

from rachitic

of bovine

nasal

cartilage

reduction

in proteoglycan

proteoglycan. We considered size

in

the rachitic

proteolytic the

initially

plate

therefore

in vivo

labelling

chicks

to the whole

studies

being

formed

some factor proteoglycan accompanied stimulated the

size

the

level

showed

in

and then

with

newly

relating molecules

being

by a marked intestinal

molecules state

formed.

calcium

It

and not MATERIALS

size

is

considerably of the

proteoglycan

molecules

were

the

size

There were

seemed more likely

D-deficiency

in the

failure

report

to the vitamin

protein in rachitic

molecules

the

growth

core

(3).

was influencing

This

increased.

of similar

tissue

proteoglycan

due to the

of

of proteoglycan

cartilage

therefore

Vitamin

in chick

life

isotopes

in

large

transport.

formed

the

monomer

of limited

increased

attack,

synthesised

hypocalcaemia

of proteoglycan

that

D-deficiency

calcium

the

radioactive

degraded.

to vitamin

state

of growth

the rachitic

rapidly

a hypothesis

of proteolytic

chains

that

with

probable

of proteoglycan

that

of plasma

large

the rachitic

chance

side

population

was no evidence

in

makes it

the

and the glycosaminoglycan and normal

the

was more consistent

since

cartilage

molecules,and However,

state

degradation

growth

that

of the

D status

of the

chick

is

vitamin

D-

evidence

that

can be related

to

provides

cartilage

size

that

of the

chick.

AND METHODS

One day old Ranger cockerels (Ross Poultry, Lines.) were divided into Two groups were raised on the standard vitamin D-deficient four groups of six. diet (5) containing 1.2% calcium and 0.7% phosphorous: one of these groups was given sg cholecalciferol orally each week. A third group was raised on a similar diet modified to reduce the calcium content to below O.Zj% In the and each chick was given %g cholecalciferol orally each week. fourth group the hypocalcaemia of vitamin D-deficiency was compensated for by feeding the chicks the standard diet supplemented with 2% by weight of calcium as calcium carbonate. At four weeks of age chicks were bled, plasma being separated for analysis for calcium and inorganic phosphate, and then killed. The cartilage growth plate was dissected from the proximal end of the tibiae, minced finely with a scalpel blade and extracted for 48h. at 5' with

66

Vol. 90, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

4M guanidinium chloride (30 ml/g wet weight of tissue) containing proteinase inhibitors (1 mM iodoacetic acid, 1mM phenylmethyl sulphonyl fluoride and 2 mM ethylene diaminetetra-acetic acid). After centrifugation (20,000 r.p.m., 30 min) the specific gravity of the supernatant solution was adjusted with caesium chloride to 1.70 : 0.01 and the solution centrifuged (40,000 r.p.m., 110,000g ‘t8h, lo°C. A stainless steel probe was inserted to the bottom of the tubea&d the contents pumped out and fractions (0.6 ml) collected; those fractions containing the most hexuronic acid (approximately the lower 2530% of the contents of the tube) were pooled, dialysed against water, then saturated sodium chloride and finally, exhaustively against water before lyophilisation. For chromatography approximately l-2 mg of this lyophilised proteoglycan was dissolved in 0.4 ml sodium acetate buffer (0.2M acetate, pH 5.5) and applied to a column of Sepharose Cl-2B (1.6 x 27 cm) and eluted with the same buffer at a flow rate of 9.8 ml/hr. Fractions (1.8 ml) were collected and analysed for hexuronic acid. Growth Cl-23. from

were:

The hexuronic the

figure

cartilage

same group.

1. vitamin

RESULTS AND DISCUSSION proteoglycan was chromatographed

on a column

acid

for

elution

Representative

The corresponding D-treated,

values 10.0

profile

was similar

elution

profiles

of plasma

and 5.9;

vitamin

calcium

for

proteoglycan each group

(mg/dl)

D-deficient,

of Sepharose

for

the

specimens are

shown

chicks

11..6 and 6.6

respectively.

Fractionnumber -Representative for growth cartilage vitamin D-deficient hypocalcaemic (0-O)

hexuronic acid elution profile from a Sepharose Cl-2B column proteoglycan from (a) vitamin D-treated chicks and (b) chicks. Profiles are shown for normocalcaemic (0-a) and chicks.

67

in

Vol. 90, No. 1, 1979

The size

BIOCHEMICAL

of proteoglycan

isolated

replete,

normocalcaemic

chicks

(figure

1) was similar

to that

pooled

normal

and rachitic

concentration were chicks

to correct

tibia1

growth

lb)

raised

to that

on a low

hypocalcaemia plate

from

calcium

from

hypocalcaemic

chicks

chicks

calcium

inorganic

la).

in

in chick

size

may be confined

proteoglycan

from rachitic

resembled Dickson, mammalian

the

smaller

growth

aggregates

in

cartilage

aspirates

only

should

be noted

induced

than that

but

the

from rachitic that

when vitamin

to be related nor

articular

growth

in average

cartilage

cartilage

from other

characteristic

lesions

D-deficiency

is

accompanied

size

and

proteoglycan

of the proteoglycan

growth

this

cartilage

size

of proteoglycan-hyaluronic rat

to

to plasma

In the chick

shown).

larger

of aggregates

the

cartilage

D status

was similar

size

pronounced

appear

chicks

known

Chicks

D-deficient

since

is

of

of the proteoglycan

vitamin

cartilage

the

Little

work).

to be much higher it

than

the

in size

growth

cartilage

not

we

week old

chicks.

showed

to vitamin

(results

and normal

rather

unpublished

growth

to growth

was similar

tibial

from

and neither

concentration

four

normocalcaemia

The size

that

calcium

characteristic

diet

from

of 3.2% calcium

normocalcaemic

D-deficiency.

(figure

phenomenon

tissue

D-replete

resembled

concentration

phosphate

the

and the proximal

as in vitamin

these

this

the

D-

chicks (2)

D-deficient

enlargement

D-replete,

vitamin

weeks,

The differences plasma

from

vitamin but

at four

was widened

isolated

isolated

to a level

Despite the

previously

By raising

in vitamin

of vitamin

hypocalcaemic

studied

diet

showed

cartilage

D-deficient,

D-deficient

plate

RESEARCH COMMUNICATIONS

growth

respectively.

from hatching.

Proteoglycan

rickets. (Figure

tissue

diet

the

of preparations

the hypocalcaemia

fed on the

proximal

from

and vitamin

of the vitamin

able

AND BIOPHYSICAL

(I.

in

acid has been

cartilagenous of rickets

reported tissues

in

the rat

(4) are

by a hypophosphataemia

and normocalcaemia. There biosynthesis

is

evidence of the

that

structural

the

level

of extracellular

components

of cartilage

68

calcium

can affect

matrix.

The type

the of

-

Vol. 90, No. 1, 1979

collagen

synthesised

can be varied proteoglycan calcium

by rabbit

by altering synthesis

(6).

The level

post-translational collagen

the

detailed

in

calcium

the process

of these

is

still

but

it

lysine

is

affected

phenomenon

should

vitamin

residues

D and calcium

normal

is

(7).

yet clear.

an inhibitory

effect

the

only

chick. give

of

whereas

unaffected

when large

D-deficient

and

when the

not

not

vitamin

(5)

the degree

relatively

changes

is

culture

physiologically

influence

does have

ossification

reduced

below

can also

type

proteoglycan

of this between

is

is

of bone collagen

the normocalcaemic

of the relationship bone

of small

investigation

medium

suspension

of the medium

cartilage

constitutional

of endochondral

can do so since formed

culture

significance

in

concentration

vertebral

of plasma

of similar

formation

the process

the

RESEARCH COMMUNICATIONS

chondrocytes

calcium

hydroxylation

The physiological If

the

in

AND BIOPHYSICAL

articular

by chick

concentration

levels

skin

BIOCHEMICAL

factor

upon that

proteoglycan

is

A more

us a clearer

idea

in the physiology

of

formation. ACKNOWLEDGEMENTS

We thank Patricia Maher for technical assistance. supported by grants from the Medical Research Council Organisation of North America.

This research and the Shriners

was

PEFERENCES B. P., Kang, A. H., Trelstad, R. L. and Gross, J. (1972) Biochem. J. 1. Toole, 127, 715-720 I. R. and Roughley, P. J. (1978) Biochem. J. 171, 675-682 Dickson, ;: Roughley, P. J. and Dickson, I. R. (Biochem. J. In Prex 4. Howell, D. S. and Pita, J. C. (1976) Clin. Orthop. u, 208-229 W. G. and Sawyer, B. D. (1976) FEBS Lett. 67, 48-51 5. Desmukh, K., Kline, 6. Shulman, H. J. and Opler, A. (1974) Biochem. Biophys. Res. Commun. 2, 914-919 M. J., Constable, B. J., Morton, L. F. and Kodicek, E. (1973) Biochem. 7. Barnes, Biophys. Acta 328, 373-382

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