145

Biochimica et Biophysica A cta, 607 ( 1 9 8 0 ) 1 4 5 - - 1 6 0

E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 9 6 1 5

COLLAGEN BIOSYNTHESIS BY HUMAN SKIN FIBROBLASTS

I. OPTIMIZATION OF THE CULTURE CONDITIONS FOR SYNTHESIS OF
TYPE I AND TYPE III PROCOLLAGENS *

B A R B A R A A. BOOTH, K A T H Y L. P O L A K and J O U N I U I T T O

Division of Dermatology, Department of Medicine, Washington University School of

Medicine, St. Louis, MO 63110 (U.S.A.)
( R e c e i v e d A u g u s t 1 6 t h , 1979)

Key words: Collagen synthesis; Procollagen synthesis; (Human skin fibroblast)

Summary

Skin fibroblasts in culture can provide a convenient means to study aberrations of collagen metabolism in a variety of clinical conditions. In the present
study, the culture conditions for the synthesis of procollagen by cultured
human skin fihroblasts were optimized by independently varying parameters in
the cell culture environment. To study the synthesis of procollagen the cell
cultures were labeled with [3H]proline and the collagenous polypeptides were
determined either by measuring the synthesis of hydroxy[3H]proline or by
assaying the 3H-labeled proteins digested into dialyzable 3H-labeled peptides by
bacterial collagenase. On the basis of the experimental results, the following
culture conditions are suggested for optimal synthesis of procollagen: (a) cell
culture medium should be supplemented with ascorbic acid (25--50 #g/ml) and
fetal calf serum (20%); (b) the pH of the culture medium should be kept above
7.2 and preferably in the pH range 7.5--7.8; (c) the cell cultures should be used
one to two days after reaching visual confluency. Under these conditions
the synthesis and secretion of [3H]procollagen was found to be linear through
a 24 h incubation period, and procollagen was demonstrated to be a major
gene product of the fibroblasts. The relative synthesis of type I and type III
procoUagens was also monitored by isolating these genetically distinct procollagens by DEAE-cellulose chromatography or by measuring type I and III
collagens by sodium dodecyl sulfate polyacrylamide gel electrophoresis after
* A preliminary report of part of this work has b e e n presented at the 36th Annual Meeting of the
American Federation of Clinical Research, Washington, DC, May 1971 [1].
Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2.ethanesulfonie acid; Bes, 2-[bis(2-hydroxyethyl)amino]ethanesulfonie acid; Trieine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glyeine; Tris, 2-amino2-hydroxymet~hyl-l,3-propanediol;SDS0 sodium dodeeyl sulfate.

146

limited pepsin proteolysis. No marked changes were observed in type I/III

procollagen ratios in situations where the total formation of hydroxy[3H] proline was significantly affected. The average coefficient of variance for procollagen synthesis between replicate cultures was found to be relatively small
(14%), and the optimization of the culture conditions for the control cells has,
therefore, created a reliable and reproducible basis for employing human skin
fibroblasts to study collagen metabolism in acquired and inherited diseases.

Introduction
Collagen is the most abundant structural protein of the connective tissues;
for example in human skin, collagen represents well over 70% of the dry weigh~
o f the tissue [2,3]. Collagen is actually a family of genetically distinct proteins
[4]. In human skin, the two major interstitial collagens are t y p e I and type III
comprising a b o u t 80 and 15% of the dermal collagen, respectively [4,5]. The
remaining 5% consists of less abundant forms of collagen, such as t y p e I trimer,
A-B collagens, and basement membrane or type IV collagen [6--8]. Each of the
genetically distinct types of collagen is synthesized as a precursor molecule,
procollagen, and its biosynthesis involves several unique reactions which are
required for fibrillogenesis of collagen in normal connective tissues [2,9]. A
defect at any one of these reactions can lead to abnormal fiber for/nation and
functionally defective collagen. Biochemical studies during the past few years
have demonstrated defects in the structure and metabolism of collagen in
several connective tissue disorders [3,10,11]. A large part of these studies
e m p l o y e d cultured human skin fibroblasts. In most studies, however, culture
conditions were n o t rigorously controlled and some conflicting results have
been obtained using cell cultures.
In the present study, we have examined the synthesis of procollagen by
human skin fibroblasts in culture. Various parameters of the culture conditions
were varied in order to study their effects on procollagen synthesis and then to
optimize the c o n d i t i o n s . f o r synthesis of procollagen. Also, the newly synthesized type I and III procollagens were isolated by chromatographic procedures, and possible variations in their relative synthesis were monitored under
experimental conditions.
Materials and Methods
H u m a n skin fibroblast cultures. Primary cultures of human skin fibroblasts
were initiated either from 3-ram skin punch biopsies or from skin obtained in
connection with various surgical procedures after obtaining informed consent.
Cells were subcultivated in Dulbecco's modified Eagle's medium + glutamine
(KC Biologicals) with 30 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (Hepes) buffer, pH 7.55 at 22C, and containing 200 units/ml of penicillin
and 200 gg/ml of streptomycin. Secondary cultures were initiated from a pool
of cells obtained b y trypsinization, and the cells were plated on 75-cm 2 flasks
(Falcon Plastics) at a density of a b o u t 5 l 0 s cells per flask, or onto 850-cm 2
roller bottles at the density of 6 106 cells per bottle. The medium, containing
20% fetal calf serum, was changed at 2~lay intervals until the desired growth

147
stage was reached. For studies on procollagen biosynthesis, cells were labeled at
different stages of growth and under variable experimental conditions (see
Results) with ~4C-or 3H-labeled proline. In most experiments, ascorbic acid, 25
or 50 ~g/ml, was added to the culture medium 24 or 4 h prior to labeling. The
labeling was initiated by changing the medium to Dulbecco's modified Eagle's
medium containing the same amount of ascorbic acid, 20 ~g/ml ~-aminopropionitrile, 200 units/ml penicillin, 50 /~g/ml streptomycin, 30 mM Hepes
buffer (pH 7.55 at 22C), variable amounts of fetal calf or human serum and
radioactive proline. In most experiments the serum included in the labeling
medium was dialyzed against 0.15 M NaC1, 30 mM Hepes, pH 7.5, at 4C. The
cultures were incubated for the time periods indicated in Results by slow
shaking in a tissue culture incubator at 37C. At the end of the labeling period
the medium was removed, rapidly cooled on an ice bath and stock solutions
containing protease inhibitors were added to give final concentrations of 20
mM disodium EDTA, 10 mM N-ethylmaleimide, and 0.3 mM phenylmethylsulfonylfluoride. The cell layer was rinsed three times with 3 ml of 0.4 M NaC1,
0.01 M Tris-HC1, pH 7.5, containing 20 mM disodium EDTA, 10 mM N-ethylmaleimide, and 0.3 mM phenylmethylsulfonylfluoride, and the cells were
scraped into 5 ml of the same solution using a rubber policeman; the cells were
sonicated at 60 Hz for 30 s in an ice bath. Aliquots of the medium and cell
homogenate were prepared for assay of collagenous proteins or for DEAEcellulose chromatography, as described below.
For the experiments on environmental pH, media were made containing
varying concentrations of bicarbonate as discussed in Results. Organic buffers,
Bes (PKa 6.88), Hepes (pK a 7.31) and Tricine (pKa 7.79), were used at 30 mM
to provide buffering capacity at the desired pH range. The media was equilibrated to 37C and the pH was adjusted. The pH of media in the cultures was
checked by removing the cultures from the 37C incubator and immediately
measuring the medium pH using a regular pH meter (Radiometer) and electrode
(Sensorex).
Chromatography on DEAE-cellulose. To prepare the newly synthesized procollagen for DEAE-cellulose chromatography, the radioactive collagenous
proteins from the medium were isolated by precipitation with 20% (NH4)2SO4
(114 mg/ml). The precipitate was dissolved in approx. 5 ml of starting buffer
consisting of 2 M urea and 1 mM disodium EDTA in 0.025 M Tris-HC1, pH 7.5,
at 4C. The sample was dialyzed against 500 ml of the same buffer for 2 h,
changing the dialysis buffer three times. The sample was then chromatographed
on a 2.5 10.0 cm column of microgranular (preswollen) DEAE-cellulose
(DE52; Whatman Biochemicals, Ltd.). The sample was eluted with a linear
gradient prepared with 300 ml of starting buffer containing 2 M urea and 1 mM
disodium EDTA in 0.025 M Tris-HC1, pH 7.5, and 300 ml of limit buffer consisting of 0.22 M NaC1, 2 M urea and 1 mM disodium EDTA in 0.025 M TrisHC1, pH 7.5, at 8C [12,13]. Chromatography was performed at a flow rate of
180 ml/h; 8-ml fractions were collected and 0.4 ml aliquots were counted in
3 ml ACS counting solution (Amersham). The peaks of radioactive protein
were pooled, aliquots were dialyzed against distilled water, and radioactive
hydroxyproline and proline were determined after hydrolysis in 6 M HC1, as
described below.

148

SDS-polyacrylamide gel electrophoresis. To estimate the relative proportions of newly synthesized triple-helical type I and type III [3H]procollagens,
the 3H-labeled protein recovered from the cell culture media by (NH4)2SO4
precipitation (see above) was subjected to limited proteolytic digestion by
pepsin. For pepsin digestion, the precipitates were dissolved in 0.5 M acetic
acid and then dialyzed against the same buffer. 100 /~g/ml pepsin {Worthington, twice crystallized) was added and the samples were incubated for 3 h at
4C. At the end of the incubation, the pH of the digests was brought to 8.5 to
inactivate the pepsin. The samples were then heated for 5 min at 100C in the
presence of 2% SDS, 5 mM iodoacetamide, 20 mM disodium EDTA, 10 mM
N-ethylmaleimide, and 0.3 mM phenylmethylsulfonylfluoride.The samples were
electrophoresed on 6% polyacrylamide slab gels, as described previously [13].
To separate al(III)-chains from type I collagen a-chains, the electrophoresis
run was started without reduction; however, after 1 h the electrophoresis was
interrupted and 50 #1 of 2-mercaptoethanol was added to each sample slot. The
electrophoresis was continued an additional 2.5--3 h, until the dye front
reached the lower end of the gel. The 3H-labeled polypeptides were visualized
and quantitated by scanning fluorographs, as described previously [13].
Other assays. To measure the synthesis of radioactive hydroxyproline and to
determine the incorporation of total radioactivity into proteins, aliquots of the
medium and cell homogenates were dialyzed against tap water and then hydrolyzed in 6 M HC1 at l l 0 C for 24 h. The radioactive hydroxyproline in the
hydrolyzate was separated from proline on a 10 0.75 cm column of Beckman
type W2 polystyrene resin, eluted with 0.02 M sodium citrate buffer, pH 3.24.
Fractions of 0.1 ml were collected and 0.4 ml aliquots were counted in 3 ml of
ACS counting solution using a Beckman LS 3155 P liquid scintillation counter.
Alternatively, the radioactive hydroxyproline and the total radioactivity were
assayed by a specific radiochemical method, as described previously [14].
As an independent measure of the synthesis of collagenous proteins the
samples were incubated with purified bacterial collagenase, and the release of
dialyzable radioactive peptides was taken as a measure of collagenous proteins.
For collagenase digestion the samples were extensively dialyzed against 0.15 M
NaC1 and 5 mM CaC12 in 0.05 M Tris-HC1 buffer, pH 7.6, at 4C. Purified
bacterial collagenase (Advanced Biofactors, New York, NY) {final concentration 50 /~g/ml), 2.5 mM N-ethylmaleimide, and 30 /~M phenylmethylsulfonylfluoride were added and the samples were incubated for 2 h at 25C [15]. The
incubation was stopped by adding one-tenth vol. of 0.2 M disodium EDTA,
and the samples were dialyzed against 0.15 M NaC1 and 1 mM disodium EDTA
in 0.05 M Tris-HC1, pH 7.6, at 4C. The release of radioactive peptides by
dialysis was taken as a measure of collagenous proteins in the sample. In some
experiments the small peptides released by the collagenase digestion were
collected by dialyzing the digest first against 10 vols. of 0.15 M NaC1 Tris-HC1,
pH 7.6. The peptides recovered outside the dialysis bag were hydrolyzed in
6 M HC1, and radioactive hydroxyproline and proline were assayed, as
described above.
For assay of DNA and total cell protein, aliquots of the cell homogenates
were dialyzed against 0.15 M NaC1, 0.001 M Tris-HC1, pH 7.5. DNA was
measured by a diphenylamine method, as described by Burton [16], and the
protein was assayed according to Lowry et al. [17].

149
Results

Synthesis and secretion of type I and type III procollagens

To examine the synthesis of collagenous proteins by the cultured skin
fibroblasts, the cells were incubated under various experimental conditions
with radioactive proline and the formation of radioactive hydroxyproline in the
non~lialyzable protein was used as an index of collagen formation; the values
of radioactivity were related to DNA or cell protein. In initial experiments, the
cells in monolayer cultures, 2 days after having reached visual confluency, were
incubated for varying time periods with [aH]proline in the presence of 50
#g/ml ascorbic acid and 20% fetal calf serum. The cells rapidly incorporated
[aH]proline into non~lialyzable protein and a significant amount of hydroxy[aH]proline was detected after a 10 rain labeling period (Fig. 1). The incorporation of [aH]proline as well as the synthesis of hydroxy[aH]proline were linear
through 24 h of incubation. Examination of the medium hydroxy[aH]proline
separately from the cell fraction demonstrated that there was a considerable
delay in the secretion of procollagen by the fibroblasts: over 60 min was
required before any hydroxyproline could be detected in the medium (Fig. 1).
The secretion of the hydroxy[3H]proline-containing macromolecules appeared
to proceed linearly from 4 to 24 h of incubation.
Reproducibility of the procollagen synthesis was checked between replicate
cultures, using several different cell lines. The average coefficient of variance
for dpm non~tialyzable hydroxy[aH]proline/mg protein for all groups of
replicates (n = 49) in 12 consecutive experiments was 14 *- 8% (S.D.).
In order to examine separately the synthesis of genetically distinct procollagens, the radioactive proteins recovered in the medium were chromatographed on DEAE-cellulose under conditions which separate human type I and
type III procollagens [12,18]. In a typical chromatogram two major peaks of
radioactivity could be observed (Fig. 2). The first peak eluting symmetrically
as a relatively sharp peak in fractions 41--47 consisted of a collagenous 3Hlabeled protein since approximately 40% of the [3H]prolyl residues in the
newly synthesized polypeptides were converted to hydroxy[aH]proline. Also,
about 90% of the radioactivity in this peak was converted to dialyzable peptides by digestion with highly purified bacterial collagenase. On the basis of
cyanogen bromide peptide mapping and s-chain composition, as estimated by
CM~ellulose chromatography and polyacrylamide gel electrophoresis in SDS,
the collagenous protein in the first peak has been identified as type I [aH]procollagen (Uitto, J., Booth, B.A. and Polak, K.L., unpublished results). The
second major area of radioactivity (fractions 52--64 in Fig. 2) in most case
appeared to represent a complex of 3H-labeled proteins; it was usually relatively
broad and it frequently displayed several distinct peaks of radioactivity
(Fig. 2). This complex, however, clearly contained collagenous proteins since
variably from 25 to 43% of the [aH]prolyl residues in the newly synthesized
protein were converted to hydroxy[3H]proline. Further purification of the
hydroxy[aH]proline~ontaining protein has allowed the identification of this
macromolecule as type III [aH]procollagen (Uitto, J., Booth, B.A. and Polak,
K.L., unpublished results). Since type III [aH]procollagen in the chromatogram was not consistently separated from other [aH]proline-labeled, non-

150
I

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Cl[s + Medium

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I

Procollagen

20

04

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12
16
20
TIME OF INCUBATION(h)

24

,-'~'~-,,pea

10

20

30

40

50

60

70

80

FRACTION NUMBER

F i g . 1 . S y n t h e s i s and s e c r e t i o n o f p r o c o l l a g e n b y h u m a n skin fibroblasts. Cells in early c o n f l u e n t cultures w e r e i n c u b a t e d in m o d i f i e d D u l b e c c o ' s m o d i f i e d Eagle's m e d i u m c o n t a i n i n g 20% d i a l y z e d fetal calf
s e r u m a n d 2 5 # g / m l ascorbic acid. A f t e r a 4 h p r e i n c u b a t i o n , the cells w e r e l a b e l e d w i t h [ 3 H ] p r o l i n e . A t
t h e t i m e p o i n t s i n d i c a t e d the cell and m e d i u m f r a c t i o n s w e r e r e m o v e d and a s s a y e d for n o n - d i a l y z a b l e
h y d r o x y [ 3 H ] p r o l i n e and cellula D N A . . . . . . .
, c e l l s ; . . . . . .
o, m e d i u m ;
, cells + medium.
F i g . 2 . D E A E - c e l l u l o s e c h r o m a t o g r a p h y o f Partially purified m e d i u m 3 H . l a b e l e d p r o t e i n s . Skin fibroblasts
in early c o n f l u e n t c u l t u r e s w e r e labeled w i t h [ 3 H ] p r o l i n e , as d e s c r i b e d in the t e x t and in the l e g e n d t o
Fig. 1. A f t e r a 2 0 h
labeling p e r i o d , t h e m e d i u m 3 H - l a b e l e d p r o t e i n was p r e c i p i t a t e d w i t h 2 0 %
( N H 4 ) 2 S O 4 a f t e r a d d i t i o n o f p r o t e a s e inhibitors. The p r e c i p i t a t e was d i s s o l v e d in D E A E - c h r o m a t o g r a p h y
starting b u f f e r , d i a l y z e d , and c h r o m a t o g r a p h e d o n D E A E - c e l l u l o s e as d e s c r i b e d in Materials and M e t h o d s .
T h e s e p a r a t e d t y p e I and t y p e III p r o c o l l a g e n s w e r e assayed, a f t e r p o o l i n g the f r a c t i o n s i n d i c a t e d b y the
h o r i z o n t a l bars, by d e t e r m i n i n g their h y d r o x y [ 3 H ] p r o l i n e c o n t e n t .

collagenous proteins, the newly synthesized type I and type III procollagens,
separated by DEAE-cellulose chromatography, were quantitated by assaying
the radioactive hydroxyproline in the pooled areas, as indicated in Fig. 2.
Examination of the medium 3H-labeled protein at 6, 12, and 20-h labeling
points by DEAE-ceUulose chromatography revealed essentially identical
patterns. In order to obtain a m a x i m u m amount of radioactivity on the linear
portion of the synthesis curve, of both type I and type III procollagens, a 20 h
incubation time was used in the subsequent experiments.

Effect of ascorbic acid

When varying concentrations of ascorbic acid were added to the culture
medium 4 h prior to and at the time of labeling, a marked stimulation of
hydroxy[3H]proline synthesis in the non-dialyzable fraction with as little as
1 ~g/ml (5.6 ~M) of ascorbic acid was noted, and maximal effects were
obtained with 5--50 pg/ml (Fig. 3). In the presence of 100 #g/ml of ascorbic
acid the synthesis of radioactive hydroxyproline was slightly less than was
observed with 50 pg/ml. It should be noted that the maximal stimulation by
ascorbic acid varied with different cell lines, the radioactive hydroxyproline
in ascorbic acid-containing cultures being from 140 to 290% of the controls.
Examination of non-dialyzable hydroxy[3H]proline separately in the cell and
the medium fractions indicated that when the cells were incubated with no
added ascorbic acid the small amount of hydroxy[3H]proline synthesized was

151

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7'5

ASCORBIC ACID ( p g / m [ )

by human skin fibroblasts in the

Fig. 3. S y n t h e s i s o f n o n - d l a l y z a b l e c o l l a g e n h y d r o x y [ 3 H ] p r o l i n e
p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f a s c o r b i c a c i d . Cells i n e a r l y c o n f l u e n t c u l t u r e s w e r e p r e i n c u b a t e d
for 4 h in the presence of 0--100/~g/ml (0--0.56 raM) ascorbic acid and then labeled for 20 h with [3H]p r o l l n e . N o n - d l a l y z a b l e h y d r o x y [ 3 H ] p r o l i n e i n t h e cell a n d m e d i u m f r a c t i o n s , as well as c e l l u l a r p r o t e i n
and DNA were determined, o ......
o, h y d r o x y [ 3 H ] p r o l l n e ( h y p r o ) , d p m ( X 1 0 - S ) / m g p r o t e i n ;
~,
hydroxy[BH]prollne (hypro), dpm (X10"4)fl~g DNA.

predominantly in the cells (Table I). When ascorbic acid was added, the synthesis of hydroxy[SH]proline increased, and at the same time the relative
proportion of the hydroxy[SH]proline found in the cells decreased. The total
amount of hydroxy[SH]proline in the cell fraction, however, was maximal at
1 ~g/ml ascorbic acid, probably because the procoUagen formed at this concentration was still somewhat underhydroxylated and was not secreted at an
optimal rate (see Discussion). Examination of type I and type III procollagens
by DEAE-ceUulose chromatography in the media from cultures incubated with
0 or 25 ~g/ml of ascorbic acid demonstrated that these procollagens were
present in the same ratio.
TABLE I
THE EFFECT OF ASCORBIC ACID ON THE SYNTHESIS AND SECRETION OF PROCOLLAGEN
FIBROBLASTS

BY

S k i n f i b r o b l a s t c u l t t t r e s in e a r l y c o n f l u e n c y w e r e i n c u b a t e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s m e d i u m c o n t a l n i n g 2 0 % f e t a l c a l f s e r u m a n d v a r y i n g c o n c e n t r a t i o n s o f a s c o r b i c a c i d . A f t e r 4 h p r c i n c u b a t i o n , t h e cult u r e s w e r e l a b e l e d w i t h 3 0 /~Ci [ S H ] p r o l l n e . A f t e r 2 0 h l a b e l i n g t h e cell a n d m e d i u m f r a c t i o n s w e r e

a s s a y e d f o r n o n - d l a l y z a b l e [ 3 H ] h y d r o x y p r o l l n e a n d c e l l u l a r p r o t e i n . T h e values f o r h y d r o x y [ 3 H ] p r o l l n e
are e x p r e s s e d as d p m (X 1 0 - 4 ) / r a g cell p r o t e i n ; m e a n + S.D. o f t h r e e p a r a l l e l d e t e r m i n a t i o n s . T h e v a l u e s
f o r p e r c e n t o f t o t a l are e x p r e s s e d as t h e m e a n d p m h y d r o x y [ 3 H ] p r o l l n e in i n d i v i d u a l s a m p l e s as p e r c e n t
o f t o t a l d p m h y d r o x y [ 3 H ] p r o l i n e in cells + m e d i u m ( m e a n + S.D.).
Concentration of
ascorbic acid (~g/ml)

0
1
5
25
50
100

Medium

Cells

Hydroxy[ 3H ]Proline

% of total

Hydroxy[ 3 H ] proline

% of total

0.90.5
11.06.1
19.75.6
22.17.8
21.58.3
17.73.5

431
59e4
809
866
884
874

1.20.5
7.33.4
4.81.8
3.30.8
3.12.1
2.50.5

571
414
209
146
124
134

152

Previously, it has been shown that ascorbic acid concentration in cell culture
medium decreases rapidly [19]. It was of interest, therefore, to study the effect
o f varying preincubation times on hydroxy[3H]proline synthesis to determine
h o w long the stimulatory effect of ascorbic acid lasts. 50 #g/ml ascorbic acid
was added to confluent cultures at varying times before labeling; in order not
to change the ascorbic acid concentration, all cells were fed only at the 48 h
point. Two sets of controls were included: one set of cells had no added
ascorbic acid at any time, and the other received 50 /~g/ml of ascorbic acid in
fresh medium at 4 h prior to and at the time of labeling. The cells were then
labeled for 20 h with [3H]proline. The results, shown in Table II, indicate that
when no ascorbic acid was added to the culture, the hydroxy[3H]proline per
cell protein or D N A was lower than in any culture receiving ascorbic acid and
the percent hydroxy[3H]proline relative to total 3H radioactivity was also
decreased. In some experiments (see Expt. 2 in Table II), the synthesis of
hydroxy[3H]proline and the percent of hydroxy[3H]proline appeared to be
somewhat lower when ascorbic acid was added to the cultures at the time of
labeling when compared to cells preincubated with ascorbic acid at least for
4 h. When ascorbic acid was added 4, 8, 24, or 48 h prior to labeling, values for
hydroxy[3H]proline were not significantly different either from each other, or
from the controls receiving ascorbic acid 4 h prior to as well as at the time of
labeling.

Effect o f serum
To study the effect of varying serum concentrations on the synthesis of

T A B L E II
THE RATE OF PROCOLLAGEN
SYNTHESIS
BY FIBROBLASTS
ASCORBIC ACID AT VARIOUS TIMES PRIOR TO LABELING

AFTER

THE

ADDITION

OF

Skin f i b r o h l a s t c u l t u r e s in early c o n f l u e n c y w e r e i n c u b a t e d in D u l b e c c o ' s m o d i f i e d Eagle's m e d i u m c o n taining 10% d i a l y z e d f e t a l calf s e r u m , as d e s c r i b e d in Materials and M e t h o d s . T h e m e d i u m w a s c h a n g e d

4 8 h b e f o r e labeling, and 5 0 D g ] m l a s c o r b i e acid w a s a d d e d at 0 - - 4 8 h prior t o labeling; t h e c o n t r o l cultures did n o t r e c e i v e a n y a s c o r b i c acid. T h e c u l t u r e s w e r e t h e n l a b e l e d w i t h 3 0 # C i o f [ 3 H ] p r o l i n e , and
a f t e r 2 0 h labeling t h e y w e r e a s s a y e d for n o n - d i a l y z a b l e 3 H , n o n - d i a l y z a b l e h y d r o x y [ 3 H ] p r o l i n e , cellular
p r o t e i n and D N A . T h e values are m e a n S . D o f 3 - - 4 parallel d e t e r m i n a t i o n s .
A s c o r b i c acid a d d e d
prior to labeling ( h )

Exp. 1
24
8
4
0

Control
Exp. 2
48
24
4
0

Control

H y d r o x y [ 3 H ] proline

H y d r o x y [ 3 H ] proline
(dpm(X 10-3)/20 h)

( 1 0 0 ) / ( t o t a l

Per m g p r o t e i n

Per/~g D N A

230
240
260
230
130

+
+
+

24
41
10
42
18

4.1
4.2
4.5
4.1
2.5

+
+

0.5
0.7
0.3
0.9
0.5

11.1
11.5
11.3
9.6
7.1

+
+

0.8
0.6
1.1
1.3
0.4

250
300
270
220
200

49
70
36
30
29

5.8
4.6
4.4
3.5
3.3

0,8
1.2
0.6
0.5
0.4

11.9
11.0
11.1
9.2
8.1

0.2
1.1
0.5
1.6
0.2

3H)

153

non<lialyzable hydroxy[3H]proline, the cells were incubated with [3H]proline

in media containing dialyzed fetal calf serum. Medium, containing 0--60% fetal
calf serum and 50 ~g/ml ascorbic acid, was added to the cultures 4 h prior to
labeling and again when the cultures were labeled. Both cell protein and D N A
content of the cultures increased as the serum concentration increased
(Fig. 4B). At the same time, the hydroxy[3H]proline synthesis per flask
increased with increasing serum concentration up to 3 0 % (Fig. 4A). W h e n the
synthesis of hydroxy[3H]proline was related to cellularprotein or D N A , the
range of 10--30% serum concentration appeared to produce a stimulation and
on the whole, depending on the reference parameter used, the 2 0 % serum conA

~'~"

~3.0

?---:

[3H] Hydrxypr!'ine
.

i~.

Z
w

uJ

1.0 ~"

Z__

"##/~
/

""C~...k

EQ.

Per Protein

,i-i-

1.0

g.5 ~

e~
C3
>"T"

1'0 ~0 3b 4b 5b

"1-

CONCENTRATION OF SERUM(%)

30

B ....

VI

o / ""~/'~---DNA

q 20

40 ~_

u.l

lb

7o g

4b 5b 6b o

CONCENTRATION OF SERUM (%)

Fig. 4 , S y n t h e s i s o f [ 3 H ] p r o c o l l a g e n in Lhe p r e s e n c e o l varying c o n c e n t r a t i o n s o f fetal c a l l s e r u m . Ceils in
early c o n f l u e n t c u l t u r e s w e r e first p r e l n c u b a t e d for 1 2 h in m e d i u m w i t h o u t s e r u m a n d t h e n f o r 4 h in
m e d i u m contA|nqng 0 - - 6 0 % d i a l y z e d f e t a l c a l f s e r u m and 5 0 # g / m ] a s e o r b i e acid. Cultures w e r e l a b e l e d
f o r 2 0 h w i t h [ 3 H ] p r o l / n e and n o n - d i a l y z a b l e h y d r o x y [ 3 H ] p r o U n e in the c e l l and m e d i u m fractions, as
w e l l as cellular p r o t e i n a n d D N A w e r e d e t e r m i n e d . ( A ) . . . . .
.A synthesis of hydr0xy[3H]proline, dpm
(X10-5)/flask;
~, d p m ( X 1 0 - 4 ) / # g D N A ; o . . . . . .
o, d p m ( X 1 0 - S ) / m g p r o t e i n . ( B ) C o n t e n t o f
cellular P r o t e i n ( m g / f l a s k ; o . . . . . .
o ) , and D N A (Mglflask;
-~).

154
centration appeared optimal. The synthesis of h y d r o x y [3H]proline per flask in
the presence of 60% fetal calf serum, although higher than in cultures with no
added serum, was less than with 20% serum (Fig. 4A). No change in type I/
t y p e III procollagen ratio was noted when the media from cultures incubated
with 0, 20, or 60% of fetal calf serum was examined by DEAE-cellulose
chromatography. Further experiments employing 20% heat-inactivated adult
h u m a n serum gave results similar to those obtained with 20% fetal calf serum.
Since the cultures used in above experiments had been initially grown in
m e d i u m containing 20% fetal calf serum, a control experiment was conducted
in order to determine if the decreased hydroxy[3H]proline synthesis at concentrations less than 20% was due to a change in serum concentration just prior
to labeling. Two different cell lines were used. One line was grown from passage
in medium containing 5% or 20% serum; the other line was grown in the
presence of 10% serum. One to two days after having reached visual confluency, each of these cell lines, grown in different serum concentrations, was
labeled in media containing 5, 10, or 20% serum. The results, shown in Table
III, indicate t h a t cells synthesized the most hydroxy[3H]proline when labeled
in medium containing 20% serum irrespective of whether initially grown in 5,
10, or 20% serum-containing medium. In addition, cells grown in the presence
o f 20% fetal calf serum synthesized more hydroxy[3H]proline than the same
cell line initially grown in medium containing 5% serum (Table III).

Effect of environmental pH
Our standard culture medium, Dulbecco's modified Eagle's medium, contains 44 mM bicarbonate. Cultures fed with this medium, and adjusted with
various organic buffers (see Materials and Methods) to various pH values
between 7.0 and 8.0, were all in the pH range 7.5--7.8 after 3 h incubation
under 5% CO2, indicating t h a t the bicarbonate buffer under standard culture
conditions efficiently stabilized the pH of the cultures. During the subsequent
TABLE III
T H E R A T E O F P R O C O L L A G E N S Y N T H E S I S BY F I B R O B L A S T S G R O W N A N D L A B E L E D I N M E D I A
CONTAINING VARYING CONCENTRATIONS OF FETAL CALF SERUM
S k i n f i b r o b l a s t s a t P a s s a g e w e r e p l a t e d in m e d i a c o n t a i n i n g 5, 1 0 , o r 2 0 % n o n - d i a l y z e d f e t a l c a l f s e r u m .
O n e d a y a f t e r t h e c u l t u r e s h a d r e a c h e d v i s u a l c o n f l u e n c y , t h e c e i l s w e r e l a b e l e d f o r 20 h w i t h [ 3 H ] p r o i l n e
in m e d i u m c o n t a i n i n g 5, 1 0 , o r 2 0 % d i a l y z e d f e t a l c a l f s e r u m a n d 25 ~ g / m l a s c o r b i c a c i d . A t t h e e n d o f
the labeling period, the cultures were assayed for non-dialyzable 3H, non-dialyzable h y d r o x y [ 3 H ] p r o l i n e
and cellular protein. The values for h y d r o x y [ 3 H ] p r o l i n e ( [ 3 H ] h y p r o )
are e x p r e s s e d as d p m ( X 1 0 - 3 ) / 2 0 h
p e r m g cell p r o t e i n . T h e v a l u e s f o r p e r c e n t h y p r o a r e e x p r e s s e d as 1 0 0 X h y d r o x y [ 3 H ] p r o l i n e / t o t a l 3H.
Cells g r o w n in 5% a n d 20% s e r u m w e r e t h e s a m e line, w h i l e ceils g r o w n i n 1 0 % s e r u m w e r e a d i f f e r e n t
line.
Concentration of
f e t a l c a l f s e r u m in
l a b e l i n g m e d i u m (%)

5
10
20

C o n c e n t r a t i o n o f f e t a l c a l f s e r u m in g r o w t h m e d i u m (%)
5

10

20

[3H]Hypro

% hypro

[3H]Hypro

% hypro

[3H]Hypro

% hypro

2 4 3 + 55
3 0 5 41
3 4 7 + 50

12 + 1
11 + 1
12 1

2 1 4 + 13
2 2 7 41
2 4 5 38

12 1
11 + 1
11 + 2

2 8 8 + 28
3 7 0 85
443 *

15+ 2
15 3
14 *

* T h e v a l u e s a r e m e a n + S.D. o f t h r e e p a r a l l e l d e t e r m i n a t i o n s , e x c e p t f o r t h e p o i n t i n d i c a t e d (n = 1).

155
2 days, the pH of all cultures slowly decreased due to metabolic acidification
(see Ref. 20). In order to be able to maintain cultures at varying pH values,
different concentrations of bicarbonate were tested. In the presence of 2 mM
bicarbonate all cultures rapidly acidified. When the bicarbonate concentration
was adjusted to 20 mM, the pH ranges, adjusted by organic buffers, were maintained reasonably close to the initial starting pH for 2 days. To test the effect
of pH on procollagen synthesis, cells were incubated for one day and then
labeled for an additional 20 h at low (6.8--7.2), intermediate (7.2--7.5), and
high (7.7--8.2) pH. The synthesis of non-dialyzable hydroxy[3H]proline was
decreased in cultures labeled at low pH when compared to cells exposed to
intermediate or high pH (Table IV). The percentage of hydroxy[3H]proline was
also slightly increased at high pH. SDS gel electrophoresis showed no change in
type I/type III collagen ratios when the media from cultures incubated at
different pH values were examined after limited pepsin proteolysis.

Synthesis of procollagen at different growth stages of the cell cultures

To examine the synthesis of [3H]procollagen at various stages of growth
of the cultured flbroblasts, identical subcultures of cells were plated on plastic
flasks, and the synthesis of coUagenous proteins was followed at 2-day intervals
by incubating cells with [3H]proline. Two independent methods were used to
assay the synthesis of collagenous 3H-labeled proteins: (a) the synthesis of
hydroxy[3H]proline in the non<lialyzable fraction of cells and medium, and
(b) release of dialyzable 3H-labeled peptides from the newly synthesized
protein by digestion with specific bacterial collagenase. In order to account for
the increased cell population as growth progressed, the radioactivity was
expressed relative to DNA and cell protein. Examination of the synthesis of
coUagenous 3H-labeled protein, measured as collagenase-released peptides, indicated that their formation was highest at the log phase of growth and decreased
thereafter (Fig. 5). Examination of the hydroxy[3H]proline in cell and medium
T A B L E IV
THE RATE OF PROCOLLAGEN
ED TO VARIOUS pH RANGES

S Y N T H E S I S BY F I B R O B L A S T S

I N C U B A T E D IN M E D I A A D J U S T -

S k i n f i b r o b l a s t s i n e a r l y c o n f l u e n t c u l t u r e s w e r e i n c u b a t e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s m e d i u m
containing 10% dialyzed fetal calf serum and adjusted to varying pH ranges using organic buffers for
2 4 h P r i o r t o l a b e l i n g . The organic buffers (30 m M ) u s e d w e r e Bes ( p H 6 . 8 - - 7 . 2 ) , H e p e s ( p H 7 . 2 - - 7 . 5 ) and
T r i c i n e ( p H 7 . 7 - - 8 . 2 ) . A f t e r 2 0 h l a b e l i n g in the presence o f [ 3 H ] p r o l i n e a n d 2 5 / J g / m l a s c o r b i c acid, t h e
cultures were assayed for total non-dialyzable 3H, non-dialyzable hydroxy[3H]proline, cellular protein
a n d D N A . T h e v a l u e s a r e e x p r e s s e d as m e a n s S.D. o f t h r e e p a r a l l e l d e t e r m i n a t i o n s .
Hydroxy [ 3H ]proline
(dpm(X 10-3)/20 h)

Low (6.8--7.2)
Intermediate (7.2--7.5)
High (7.7--8.2)

Hydroxy [ 3 H ]proline
(X 1 0 0 ) / t o t a l 3 H

Per mg protein

P e r ~g D N A

5 3 2 57 *
811 54
933 158

7.9 1 . 4 *
10.5 1.8
10.7 + 1.0

12.3 0.7
11.0 0.6
1 4 . 8 1 . 0 **

* T h e v a l u e s a r e s t a t i s t i c a l l y d i f f e r e n t f r o m t h e c o r r e s p o n d i n g values o b t a i n e d i n i n t e r m e d i a t e a n d h i g h
p H r a n g e s (P ~ 0 . 0 5 ) .
** T h e v a l u e is s t a t i s t i c a l l y d i f f e r e n t f r o m t h e c o r r e s p o n d i n g values o b t a i n e d i n l o w a n d i n t e r m e d i a t e p H
ranges (P ~ 0 . 0 5 ) .

156

9
6
06~"

Confluency
2~
C~

Z
r~

[3H] Hyp,o
"-I0

x
E
cL
q:3

0,4 'o
x
-c3

O~05
02 ~_

>Collagenase

"0

I
2

I
4

t
6
DAYS

t
8
AFTER

L
10

I
12

lt4

PASSAGE

Fig. 5. S y n t h e s i s o f [ 3 H ] p r o c o l l a g e n a t d i f f e r e n t s t a g e s o f cell g r o w t h . Cells w e r e p l a t e d a t l o w d e n s i t y

a n d i n c u b a t e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s m e d i u m c o n t a i n i n g 2 0 % f e t a l c a l f s e r u m . A f t e r v a r y i n g t i m e p e r i o d s , t h e cells w e r e l a b e l e d f o r 2 0 h w i t h [ 3 H ] p r o l i n e in t h e p r e s e n c e o f 5 0 /~g/ml a s c o r b i e
acid. The cultures were assayed for collagenous proteins by measuring the non-dialyzable hydroxy[3H]proHne (hypro) or by assaying collagenase-released 3Holabeled peptides.
e, [3H]hypro,
dpm (X10 -3)/#g DNA; o ......
o, c o l l a g e n a s e - r e l e a s e d 3 H - l a b e l e d p e p t i d e s , d p m ( X 1 0 "4)fl~g D N A .

fractions indicated, on the other hand, that procollagen synthesis, measured as

formation of radioactive hydroxyproline, was highest at 1 day after the cells
had reached visual confluency (Fig. 5). It appeared, therefore, that at the early
stage of log phase of growth the cells contained a pool of collagenous polypeptides which were not efficiently hydroxylated, even in the presence of 50
pg/ml of ascorbic acid.
In further experiments, the relative ratios of type I and type III procollagens
in the media from cultures at different growth stages were examined by DEAEcellulose chromatography. No difference in the ratio of these procollagens was
observed in cultures which, on the basis of their DNA contents, were 63 and
330% confluent as compared to cultures just having reached normal confluency.
Discussion
Human skin fibroblasts in culture have been extensively employed in studies
on connective tissue metabolism in various clinical conditions and these studies
have provided useful information on the molecular defects in collagen in a
variety of acquired and inherited diseases [2,3,10,11]. It is evident, however,
that in several of these studies the fibroblast culture conditions have not been
rigorously controlled, and there have been conflicting results in studies of
collagen synthesis using cell culture experiments to study conditions like
scleroderma and some forms of the Ehlers-Danlos syndrome (see Refs. 21 and
22). In the present study, therefore, attempts were made to optimize fibroblast
culture conditions in order to yield reproducible and reliable results in ongoing
studies on procollagen synthesis in diseases.
In order to optimize the fibroblast culture conditions, several parameters
of the culture milieu were independently varied, and on the basis of these
experiments the following culture conditions were found to be optimal for
synthesis and secretion of procollagen by human skin fibroblasts: (a) Cells,

157
when incubated in Dulbecco's modified Eagle's medium, should be supplemented 4--24 h in advance of labeling with 25--50 Izg/ml ascorbic acid and
20% fetal calf serum; (b) the pH of the culture should be maintained above pH
7.2, preferably in the pH range 7.5--7.8; (c) the optimal synthesis of procollagen polypeptides and their subsequent hydroxylation occurs in cultures 1--2
days after they have reached visual confluency. Employing these conditions the
rate of procollagen synthesis, measured as formation of non-dialyzable radioactively labeled hydroxyproline, was found to be linear through a 24 h incubation period. During the 20 h labeling period procollagen was found to be a
major gene product of the fibroblasts in that about 20--25% of the newly synthesized 3H-labeled material in the total culture was procollagen. In the
medium fraction about 40--50% of the newly synthesized polypeptides were
collagenous. It has been demonstrated that a fraction of newly synthesized
procollagen polypeptides is rapidly degraded intracellularly [23,24], and
some of the degradation products are dialyzable thus being unaccounted
for in the total procollagen synthesis. Our results (Booth, B.A. and Uitto, J.,
unpublished observations) indicated that under optimal conditions the degradation of newly synthesized intracellular procollagen polypeptides represents a
minor pathway in the total processing of the collagenous polypeptides, while
most of the pro~-chains clearly attain the triple-helical conformation. Also, the
fraction degraded intracellularly is highly variable, perhaps depending on the
critical control of the incubation temperature below the the Tm of the collagen
triple helix. On this basis it appears that assay of hydroxy[3H]proline or
collagenase<legradable 3H-peptides gives a relatively accurate estimate of the
rate of proco]lsgen synthesis by human skin fibroblasts in culture.
Ascorbic acid, even in concentrations as low as 1 #g/ml, clearly increased
the synthesis of radioactive hydroxyproline. The exact concentration of
ascorbic acid required for maximal stimulation was somewhat variable depending on the cell line employed, but in most experiments ascorbic acid in concentrations from 5 to 50 ~g/ml appeared to be optimal. These observations are in
agreement with previous studies indicating that ascorbic acid is required for
optimal synthesis of collagen hydroxyproline by human skin fibroblasts [25-27]. It should be noted that the addition of ascorbic acid in concentrations of
50 ~g/ml into the culture medium from 4 to 48 h prior to labeling resulted
in equally effective stimulation of hydroxy[3H]proline formation. This observation indicates that even though the concentration of ascorbic acid is probably
decreasing in the culture medium during the preincubation [19], the concentration at the end of the 48 h preincubation period is still sufficient to allow
maximal hydroxylation of prolyl residues over the next 20 h. In contrast, however, addition of 50 #g/ml ascorbic acid at the time of the labeling resulted in
slightly lower synthesis of hydroxy[3H]proline. This observation can be probably e x p l ~ e d by previous observations [27--30] indicating that ascorbic acid,
in addition to participating as a cofactor in the prolyl hydroxylation reaction,
may be required for activation of prolyl hyclroxylase. This activation may
require up to 4 h, reflected as a lag in the initiation of prolyl hydroxylation. In
parallel with the increased synthesis of hyclroxyproline the secretion of procollagen was enhanced by the addition of ascorbic acid. This observation is
probably explained by previous demonstrations that a critical amount of

158
hydroxyproline is required for the procollagen polypeptides to attain the triplehelical conformation and that the triple helix, in turn, is a prerequisite for the
secretion of procollagen at an optimal rate [2,9]. The omission of ascorbic acid
from the culture medium, therefore, appears to lead to synthesis of underhydroxylated procollagen polypeptides which then accumulate intracellularly.
The presence of serum was found to be necessary for maintaining the fibroblast cultures and for the synthesis o f procollagen. In most experiments fetal
calf serum was added to the culture medium although heat-inactivated human
serum yielded similar results. The serum used during the labeling of the cultures
with radioactive proline was dialyzed prior to use to minimize dilution of the
radioactive proline by unlabeled amino acid. The optimum concentration of
fetal calf serum appeared to be 20% although the range from 10 to 30% was
about equally effective. While this manuscript was in preparation a preliminary
report on efforts to optimize the fibroblast culture conditions for collagen synthesis was also given by another group [31]. Their results, for the most part,
were in good agreement with the observations presented here, but a difference
was noted in that 30% fetal calf serum in their system proved to be inhibitory
for the synthesis of procollagen type I (Grove, D. and Pinnell, S., personal
communication). This discrepancy may be explained by the fact that the fetal
calf sera used in these two studies were obtained from different commercial
sources, and various batches of fetal calf serum may, therefore, have different
properties affecting the cell growth and procollagen synthesis. Nevertheless,
these observations underline the necessity for each investigator to optimize the
culture conditions with components used in his or her laboratory.
An additional parameter tested was the pH of the culture medium. As noted
in Results, the standard culture medium containing 44 mM bicarbonate in the
presence of 5% CO: atmosphere was efficiently buffered and attempts to
modify the pH by non-toxic concentrations of organic buffers for a few days
were not successful. In order to be able to adjust and maintain the pH at low
(6.8--7.2), intermediate (7.2--7.5), and high (7.7--8.2) ranges the bicarbonate
concentration was lowered to 20 mM and the pH was controlled by various
buffers. In this experimental system, the collagen production was affected by
the environmental pH; in the low pH range the synthesis of hydroxy[3H] proline was clearly less than in the intermediate or high pH range. These results
are in accordance with a previous report studying the effect of environmental
pH on collagen production by cultured fibroblasts [32]. It was concluded,
therefore, that the pH of culture medium for optimal collagen synthesis should
probably be kept above pH 7.2 and preferably in the pH range 7.5--7.8.
In further studies collagen production by fibroblasts at various stages of cell
growth was studied by labeling the cells at 2-day intervals after passage. The
results indicated that the synthesis of radioactive hydroxyproline, in relation to
DNA or cellular protein, was highest one or two days after the cell cultures had
reached visual confluency. When collagen production was assayed by means of
measuring the radioactive 3H-labeled polypeptides which were digested into
dialyzable peptides by bacterial collagenase the results suggested that synthesis
of collagen was higher during the early log phase of cell growth. These observations, therefore, suggest that the hydroxylation of prolyl residues at the early
stages of cell culture is not optimal. This suggestion is further supported by

159

previous reports indicating that the activity of prolyl hydroxylase in the early
log phase of cell culture is very low and that the activity markedly increases
when the cultures reach confluency [33,34].
In the studies reported here the relative synthesis of genetically distinct
procollagens of type I and type III were also monitored under varying experimental conditions. The procollagens of type I and t y p e HI were isolated by
DE AE-cellulose chromatography and then quantitated by hydroxy[3H]proline
assay of the separated molecules. The identity of these procollagens has been
confirmed on the basis of CNBr-peptide mapping and a-chain composition
[ 18] (Uitto, J., Booth, B.A. and Polak, K.L., unpublished results). Alternatively,
in some experiments the genetic types of collagen were measured using polyacrylamide gel electrophoresis after limited pepsin proteolysis [13]. The results
demonstrated that the relative ratio of type I/III procollagens was unaltered
under various experimental conditions when the total synthesis of hydroxy[3H]proline changed. These experimental conditions included varying concentrations of ascorbic acid and fetal calf serum, changes in the environmental pH,
and also the variations in the stage of cell growth. The latter observation is in
disagreement with a recent report [35] suggesting that human skin or guinea
pig fibroblasts, when incubated in high~lensity cultures, synthesize relatively
less type I than type III procollagen. On the other hand, in a previous study
[36] employing rabbit fibroblasts no differences in the synthesis of type I and
type III procollagens were noted at different stages of cell growth. The results
of the latter study as well as of ours would, therefore, indicate that the synthesis of type I and type III procollagens is under a relatively rigid control, and
changes in their relative synthesis can be seen only in some inherited disorders
of connective tissue or when the genetic material is chemical perturbed [37-4O].
On the basis of the experiments performed here the culture conditions were
optimized for maximal synthesis of procollagen, as indicated above. Under the
optimized conditions the synthesis of procollagen by fibroblast cultures was
reasonably reproducible; the average coefficient of variance for replicate cultures was 14 -* 8%. On this basis, an experimental situation affecting the rate
of procollagen synthesis has to cause, in general, approximately a 30% increase
or decrease in the synthesis of hydroxyproline for the changes to be of
statistical significance. The optimization of the control cultures has created a
reliable basis for us to study aberrations in the metabolism of collagen employing fibroblasts in both acquired and inherited diseases affecting the skin and
other connective tissues.

Acknowledgements
The authors are grateful to Mr. Gary Kinder for his excellent technical
assistance. This study was supported in part by United States Public Health
Service, National Institutes of Health grants AM 12129 and TO AM 07284, and
by a grant National Foundation-March of Dimes. J.U. is a recipient of Research
Career Development Award 5-KO4-AM-00455 from National Institutes of
Health.

160

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