Vol. 91, No. 3, 1979 December

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 739-746

14. 1979

ELASTIN BIOSYNTHESISBY SMOOTH MUSCLECELLSCULTURED UNDERSCORBUTIC CONDITIONS Timothy Scott-Burden,

Pamela J. Davies and Wieland Gevers

Department of Medical Biochemistry University of Cape Town Medical School Observatory, 7925 South Africa Received

September

18,

1979

SU;MMARY. The proliferation of rat heart smooth muscle cells is unaffected -supplementation of the culture mediumwith ascorbic acid. The presence or absence of the vitamin has a pronounced effect, however, on the amount of elastin which is produced. Scorbutic cultures incorporate significantly more radioactive valine (an amino acid prevalent in elastin) into proteins present in the extracellular matrix than do supplemented controls, while there was no difference between the systems in the incorporation of labelled methionine (absent from elastin). Deficiency of ascorbic acid appears to result in an enhancement of the biosynthesis and extracellular processing of elastin in this culture system. INTRODUCTION. The biosynthesis collagen and elastin, modifications proline

residues (l-3).

lated using cell

amino acids, in particular,

the hydroxylation

of

The importance of ascorbic acid in this regard has

some time and a considerable amount of data has been accumuculture

systems (4-8).

ment for the formation of correctly cules and for their collagen fibre

native connective tissue proteins,

involves a number of important post-translational

of certain

been known for

of the

The absolute ascorbic acid require-

structured

subsequent extracellular

formation,

has accordingly

individual

procollagen

mole-

processing in the process of becomerelatively

well understood

(3). In contrast, of elastin

the role possibly played by ascorbic acid in the biosynthesis

has been poorly documented.

hydroxyproline

in elastin

to the biosynthesis

ascorbic acid (for

may well be of little

of the cross-linked

it is corrmOnpractice

It appears that the presence of

to culture

or no importance in relation

extracellular

protein

smooth muscle cells

the promotion of cell division),

(9,lO).

Since

in the presence of little

work has been done

0006-291X/79/230739-08$Ol.OC/U 739

Copyright All rights

@ I979 by Academic Press. Inc. of reproduction in anyform reserved.

BIOCHEMICAL

Vol. 91, No. 3, 1979

on elastin

AND BIOPHYSICAL

formation by smooth muscle cells

We have been studying the biosynthesis

RESEARCH COMMUNICATIONS

in scorbutic

culture

(11,12).

and degradation of various compon-

ents of extracellular

matrices produced by cultured

rat hearts (13,14).

Because our cultured

cells

smooth muscle cells

divide at the samerate in

the presence or absence of ascorbic acid, and secrete approximately total

amount of extracellular

matrix

proteins under both culture

we decided to use our system to study the biosynthesis of elastin scorbutic

from

the same

conditions, under

conditions.

MATERIALSANDMETHODS Culture of cells and matrix production: The isolation and culture of rat ‘heart smooth muscle cells and the preparation of radioactively labelled extracellular matrix was carried out as previously described (13,14). Compositional analysis: Analysis of matrices was carried out, by sequential enzymatic digestion (14), with the following modifications: After incubation of matrix samples with the requisite enzyme, in phosphate-buffered saline (PBS), pH 7.6, containing 1 mMCaC12, digests were collected and protein determinations carried out according to the method of Lowry -et al. (15). The standards used for the protein determinations were bovine serum albumin in the case of digestion with trypsin (Sigma Chemical Co., type III), elastin (Sigma Chemical Co.) in the case of elastase (Worthington ESFF), and rat tail collagen in that of collagenase (Worthington CLSPA). Cross-linking assay: Assessment of the rate and extent of tropoelastin crosslinking was performed using sequential enzyme digests of matsices laid down by cells which had been pulse-labelled for 4 hrs with L-[2,3,4 q valine. The portion of the radioactivity that was released by elastase alone, after try sin treatment was completed, was taken to represent the amount of elastin t I:at had becomecross-linked (13) (see fig. 3 for details). Incorporation of radioisotopes into cult_ure~~systemcompartments: The simultaneous incorporation of LdHJ valine and Ll4CJ methlonine into different compartments of the rat smooth muscle culture systems was studied in the presence and absence of ascorbic acid. Cultures were incubated in the presence of the labelled compounds(1 l&i/ml culture medium) for 20 hours, after which aliquots of culture media were precipitated by the addition of equal volumes of 20% w/v trichloroacetic acid (TCA) and collected on glass fibre (GFC) filters for the determination of radioactivity. Following the removal of media, culture dishes were washed three times with PBS and the cells lysed with 1% (w/v) sodium dodecyl sulphate (SDS). Lysates were collected and each treated with an equal volume of 2M tris-HCl, pH 8.8, for 1 hr at 370C to hydrolyse aminoacyl-tRNA. Proteins were then precipitated at 10% (w/v) TCA and collected on Matrix which remained on culture dishes was then GFCfilters for counting. washed and dried, prior to dissolution in 2N NaOH(14). Protein determinations were carried out using the method of Determinations: Proline/hydroxyproline ratios were determined using a Lowry et al. (15). BeckmaTil'TVCamino acid analyser.

740

BIOCHEMICAL

Vol. 91, No. 3, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS. Rat heart smooth muscle cells grew with an approximate doubling time of 48 hours, when cultured (Fig.

1).

This result

in the presence or absence of ascorbic acid

confirms a finding

Light microscopy revealed no differences but the matrix material

reported previously

by others (6).

between the two sets of cultures,

produced by scorbutic cultures

had a denser, more

amorphousappearance, both under phase contrast and scanning electron copy (data not shown). protein

14 days in culture,

remaining behind on culture

approximately cells

After

the total

dishes after cell

the same in the case of both cultures.

micros-

amount of matrix

lysis and washing was However, scorbutic

required more time before they began to produce detergent-insoluble

matrix

(Fig.

1).

These findings were reproduced in 3 separate experiments.

Compositional analysis revealed that scorbutic containing

little

cultures

collagen but approximately double the amount of elastin,

when compared with matrices produced by cells cultured ascorbic acid (Table 1). greatly

produced matrices

in the presence of

The ratios of proline to hydroxyproline

increased, from 3,5 to 19;

were also

this was presumably due to the low level

lo-

2

Fig.

1.

O&G

I

10

in culture

I

14

Kinetics of cell division and matrix protein production in rat smooth muscle cell cultures grown in the presence (closed symbols) and absence (open symbols) of ascorbic acid. -Cultures were initiated in 35 tna culture-dished each seeded with.105 cells. Ascorbic acid (50 Wml) was added dailv to the relevant dishes and the medium was chan@d every second day.Cell numbers (0,O) and protein concentrations (A , A) were determined as described in Methods.

741

BIOCHEMICAL

Vol. 91, No. 3, 1979

TABLE 1.

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Compositional analysis of extracellular matrix proteins laid down by rat smooth muscle cells cultured in the presence and absence of ascorbic acid. t Ascorbic

Component (X) Glycoproteins,

noncross-linked elastin elastin

Cross-linked Collagen Proline tlydrowproline

acid

- Ascorbic

acid

22%

22%

37.5% 40% 9.2% 2.6%

70.5% 7% 11.6% 0.6%

The values for protetn composition each represent the means of three separate assays (see Methods). Determinations of proline and hydroxyproline residues were carried out in duplicate (~5% differences) and are the means of the values obtained.

of collagen the

fact

tures

present that

the

treated

with

no hydroxyproline than

compartment than

time

periods

in

of

IfI H -valine,

for

enzymatic

labelled tase

protein pulsed

not

at which

Scorbutic of

solvents

(Fig.

2).

This

harvesting

with

contains

also

the

and cul-

virtually

much less

soluble

cross-linked

dishes were

labelled

preformed Both

the elastin

scorbutic and this

by culturing

digested

from

rate into

and ascorbic was followed

742

with

(13);

scorbutic

by a "cold

over

short presence

dried

trypsin

matrix to remove

subsequent

elas-

elastin.

A similar

cultures

trypsin-insensitive acid-treated

ascorbic

in the

washed

cross-linked

at which the

without

cells

and preparing first

elastin

when measured

elastin

valine

cross-linked

in cultures

vitamin,

was monitored

Matrices

released

in the

was faster

and noncross-linked

r3H]-valine,

which

acid,

Analogous

we have used to dissolve

with

culture

analysis.

3).

(16).

were

accumulated

was used to determine

(Fig.

matrices

matrices

supplemented

to cross-link

elastin

of ascorbic

shown).

those

treatment

secrete

c3H]-valine

glycoproteins

procedure able

(data

in the absence

under-hydroxylated

cr,a'-dipyridyl

of extracellular

acid

produced

was also

in a number

(13)

The rate

elastin

(17,18).

controls

material

in matrices

were form

cultures chase"

period

of the were (de-

BIOCHEMICAL

Vol. 91, No. 3, 1979

AND BIOPHYSICAL

0

38

RESEARCH COMMUNICATIONS

02

6 Time (hr)

10

Fig.

2.

Incorporation of t3H]-valine into the elastase-releasable component of extracellular matrices produced by scorbutic (D--O) and ascorbutic (g--O) cultures. Cells were seeded in 35 tm~ dishes at 105 cells/dish, and were grown for 9 days as described in Methods. On dy 10, the medium was changed and replenished with medium containing [ a-valine (1 uCi/ml). Dishes were then harvested at the times shown. After removal of medium and preparation of matrices as described in Methods, compositional analysis was performed using sequential enzymatic digestion by trypsin, elastase and collagenase (14).

Fig.

3.

Assay of the rate at which elastin becomes cross-linked into trypsinnon-releasable matrix elastin. Cultures were grown as described in Figure 2. On d ten, after a medium change, cultures were labelled for 4 hours with 3H -valine. The medium was remOved, the cells washed three timesv w th PBS, and cold mediua added. Dishes were harvested at the times shown (zero time being the start of the chase period) and matrices prepared and analysed by sequential enzyme digestion as described in Figure 2. Trypsin-non-releasable material (% of total radioactivity released,after trypsin treatment,by elastase) produced by scorbutic culture O--O; trypsin-non-releasable material produced by ascorbutic culture GO; trypsin non-releasable material produced by scorbutic cultures in the presence of S-aminopropionitrile A--A .

tails figure

in Fig.

pulse released

Matrices

and subjected Since

elastase. as well

3).

were

to sequential trypsin

the

initial

by elastase)

more cross-linked,

amount was low

there

harvested

enzymatic

digested

as the noncross-linked period

then

the

digestion

glycoprotein

tropoelastin

3).

was an increase

743

in the

at the

not released

As elastin

shown

by trypsin components

molecules,

of radioactivity (Fig.

at the times

in the

and then of the matrix,

end of the by trypsin

became progressively

percentage

of radioactive

(but

Vol.91,

No.3,

BIOCHEMICAL

1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

PC) Methimine

l PI-i) Valine 60

i

1 ‘bed

LYS

Fig.

material (Fig.

4.

3).

remained

with

(Fig.

3). Since

synthesis

eight

it

in the

an actual

increase

into

acid

we tried

in the

intracellular

intracellular

(19).

results

744

four

that

in cultures hours,

an overall

rate

collagen

did

whether

bioof pro-

decrease

of elastin

cells

com-

cultures

in an accumulation

to establish

than

on lysyl

rate

acid-supplemented

Scorbutic

protein

inhibitor

after

of workers

digestion

trypsin-insensitive

at a faster

medium without

cells,

elastase

of this

was reached

by a number

culture

valine

action

case of ascorbic

of ascorbic

by scorbutic

the

proceeded

and equilibrium

in the

absence

peptides

(+70%)

of elastin

acid,

by use of radiolabelled valine

due to the

has been established

gen production showed

Mat

by subsequent

insignificant

hours

in the

released

of B-aminopropionitrile,

of-ascorbic

pared

but

presence

The cross-linking

deprived

collagen

to trypsin

In the

oxidase.

Med

LYS

Simultaneous incorporation of -valine and f4C]-methionine into muscle cultures. various compartments of rat Cells were grown as described in.Fig. 2. On day 10, the medium was changed and reolenished with radioactive medium for 20 hours. The various compartments of the culture system were then collected as described in Methods, and assayed for radioactivity. Error bars represent the standard errors of the means of six determinations, and results are expressed as dpm/mg protein in each specific compartment. Abbreviations:Lys - lysates; MED - culture medium; MAT - matrix proteins.

insensitive

material

Mat

in collaour

cultures

biosynthesis

incorporated

vitamin-supplemented

much more cultures,

BIOCHEMICAL

Vol. 91, No. 3, 1979

whJlch parallelled matrix tion

proteins,

increased

and into

of methionine,which

partments in the

was the culture

linking

butic

tin)

synthesized

specifically

DISCUSSION. systems

for

subsequent this

correct

requirement

for

in normal

elastin

prominent

part

in the

at the cells.

site

does not

The fact

that suggests

presence

proline

of biosynthesis

of collagen

Our data

show that

absence

of ascorbic

acid,

elastin

at least

elastin,

may be secreted as,

if

not better

is

scorscorbutic

(i.e.

elascontrols.

in culture

molecules

these

and for

alia).

their

However,

residues

play

a

inhibitor, These

in elastin

in connective

hydroxylated

is just

peptide

into

a

sequences

tissue

underhydroxylated

and cross-linked

ACKNOWLEGEMENTS. The support of the South African Atomic tnergy fIoard and National Cancer Association

do not

(9,lO).

susceptible

than,

residues

The biosynthesis

normally

also

proline

hydroxylase

hydroxylation

which

was

under under

required

prolyl

chains

the

of ascorbate

proteins

of elastin.

of

show that

in the case of elastin

that

presence

and cross-

cells

5-8% of the

of the

com-

peptides

by cells

--inter

been shown to proceed that

is

apply

organisation

all

total

incorpora-

acid-supplemented

(3,

only

these

tissue

collagen

acid

into

muscle

acid

fibres

the

absence

ascorbic

of stable

due to the

as well

did

However,

results

synthesis

ascorbic

collagen

in the

suggested

"accident"

in the

into

of labelled

These

smooth

occurred

of secretion

connective

that

structural

of elastin

have even

rat

than

are hydroxylated

has already

coincidental

the

rate

ascorbic

and deposition.

3,3'dipyridyl,

(3).

occurred

words,

into

(20),

paths

in protein

formation

secretion

authors

proteins

We have confirmed the

in the

valine-containing

cross-linking

and secretion

steps

increase

at a faster

medium.

The accumulation

which

In other

which

in elastin

cases.

incorporation

of an overall

of the

present

tissue

RESEARCH COMMUNICATIONS

incorporation

proteins

represents

connective

conditions.

conditions

the

same in both

valine

a result

valine

is not

medium

of the

increased not

the

AND BIOPHYSICAL

secretory in the

insoluble monomers.

Medical Research Council, is gratefully acknowledged.

Vol. 91, No. 3, 1979

BlOCHEMlCAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES

1. Serafini-Fracassini, 2.

2 2 7. 8. 9. 10.

ii: 13. 14. 15. :;: 18. 19. 20.

A., and Smith, J.W. (1974) The Structure and Biochemistry of Cartilage, pp. 6-63, Churchill-Livingstone, London Prockop, D.J., Berg, R.A., Kivirikko, K.X., and Uitto, J. (1976) Biochemistry of Collagen. Ed. Ramachandran, G.N., and Reddi, A.H., pp. 163-317, Plenum Press, NewYork. Fessler, J.H. and Fessler, L.Z. (1978) Ann. Rev. Biochem. 47, 129-162. Peterkofsky, B. (1972) Arch. Biochem. Biophys. 152, 318-328. Peterkofsky, B. (1972) Biochem. Biophys. Res. Commun.49, 1343-1350. Rowe, D.W., Starman, B.J., Fujimoto, W.Y., and Williams, R.H. (1977) In Vitro 13, 824-830. Schwartz, R.I., and Bissell, H.J. (1977) Proc. Natl. Acad. Sci. USA 74, 4453-4457. Blanck, T.J., and Peterkofsky, B. (1975) Arch. Biochem. Biophys. 171, 259-267, Uitto, J., Hoffman, H., and Prockop, D.J. (1976) Arch. Biochem. Biophys. 173, 187-200. Narayanan, K.S., Page, R.C., and Kuzan, F. (1977) Advances in Experimental Medicine and Pathology, Vol. 79: Elastin and Elpstic Tissue, ed. Sandberg, L.B., Gray, W.R., and Franzblau, C., Plenum Press, New York, pp. 491-505. Ross, R. (1971) J. Cell. Biol. 50, 172-186. S.J. (1971) J. Cell. Biol. 50, 159-171. Ross, R., and Kletonoff, Jones, P.A., Scott-Burden, T., and Gevers, W. (1979) Proc. Natl. Acad. Sci, USA 76, 353-357. Jones, P.A., and Scott-Burden, T. (1979) Biochem. Biophys. Res. Commun. 86, 71-77. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Jones, P.A., Personal Conrnunication. Keeley, R.W. (1977) Advances in Experimental Medicine and Pathology, Vol. 79: Elastin and Elastic Tissue, ed. Sandberg, L.B., Gray, W.R. and Franzblau, C. Plenum Press, NewYork, pp. 429-442. Rosenbloom, J ., and Cywinski, A. (1976) FEBS Letters 65, 246-250. Rucker, R.B., Goethlich-Reimann, W., Hobe, J., and Devers, K. (1972) Biophys. Biochem. Acta 279, 213-216. Starcher, B.C., and Galione, M.J. (1976) Anal. Biochem. 74, 441-447.

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