From the
Two growth inhibitors were identified in culture medium
conditioned by a human keratinocyte cell line, HaCat. TGF-
Normal keratinocytes grow in culture only on a feeder layer of
fibroblasts (Rheinwald and Green, 1975) or in a medium with low calcium
concentration (Hennings et al., 1980; Peehl and Ham, 1980).
When keratinocytes are cultured at physiological Ca
Over the past decade, several
growth inhibitors for epithelial cells have been successfully purified
from different sources, including normal tissues (Roberts et
al., 1983; Böhmer et al., 1987) and culture media
conditioned by epithelial cells (Holly et al., 1980; Ikeda
et al., 1987). TGF-
In the present study, we
attempted to isolate and characterize factors secreted by keratinocytes
which might be responsible for the growth arrest at high cell density
and physiological Ca
To estimate
how much of the growth-inhibitory activity was due to TGF-
In this paper we have attempted to identify the growth
inhibitors which are secreted by a human keratinocyte cell line, HaCat.
Growth and differentiation of cultured keratinocytes are considerably
affected by the Ca
TGF-
In spite of the wide
distribution of TGF-
The other growth inhibitor in confluent HaCat
conditioned media was identified as IGFBP-6. IGFBP-6 was purified from
various sources (Roghani et al., 1989; Zapf et al.,
1990; Martin et al., 1990), however, the physiological role of
the molecule is not completely understood (for reviews, see Lamson
et al.(1991) and Shimasaki and Ling(1991)). IGFs and insulin
stimulate the proliferation of many types of cells including
keratinocytes (for reviews, see Rechler and Nissley(1990) and
Nielsen(1992)). Keratinocytes have receptors for IGFs (Nickoloff et
al., 1988), and fibroblast-derived IGF-II stimulates keratinocyte
growth in a paracrine fashion (Barreca et al., 1992).
Insulin/IGFs are the only factors absolutely required to support colony
formation of normal keratinocytes (Wille et al., 1984). IGF-II
was mainly detected in dermal fibroblasts and at a lower level in
keratinocytes of fetal skin tissue (Han et al., 1987a, 1987b).
IGF-II mRNA was 200 times more abundant than IGF-I mRNA in human fetal
skin tissue (Han et al., 1988), and only IGF-II mRNA was
detected in adult skin tissue (Gray et al., 1987). Elevated
levels of IGF-II have been observed in various tumors including
squamous cell carcinoma (Gray et al., 1987). These
observations suggest that IGF-II is an important growth factor for
keratinocytes.
IGFBP-6 is a unique IGFBP which predominantly binds
IGF-II (Roghani et al., 1989; Martin et al., 1990)
and inhibits the function of IGF-II by blocking its binding to the
receptors. We found that IGFBP-6 was secreted only at confluency
(Fig. 2) and at a physiological Ca
In light of the recent
report showing that IGFBP-3 has a direct growth-inhibitory effect on
cells through putative surface receptors (Oh et al., 1993), we
asked whether IGFBP-6 might also have such an effect. However, this
appears less likely since the growth-inhibitory activity of purified
IGFBP-6 was abolished when an excess amount of insulin was supplemented
to the assay culture (Fig. 7). Insulin activates the type I IGF
receptor which mediates growth-stimulatory effects of insulin/IGFs, but
does not bind the type II IGF receptor or IGFBP-6. However, we cannot
exclude the possibility that a strong signal provoked by insulin via
the type I receptor could overcome a suppressive effect of IGFBP-6
acting directly on the cells. The most likely mechanism for the
growth-inhibitory effect of IGFBP-6 involves binding of IGF-II, which
thereby is prevented from binding and activating its receptors. IGF-II
might either be produced by Mv1Lu cells and keratinocytes in an
autocrine manner or be present in 1% of FCS in the culture media. Thus,
modulation of IGF-II or IGFBP-6 secretion might be of critical
importance in growth regulation of keratinocytes. Another intriguing
possibility is that the interaction of IGFBP-6 with IGF-II might affect
the availability of the mannose 6-phosphate/IGF-II receptor which has
shown to be involved in the proteolytic activation of TGF-
In
summary, we showed a possible autocrine growth-inhibitory system to
operate in keratinocytes. Activation of TGF-
We thank Dr. Norbert E. Fusenig for the HaCat cell
line and Dr. Stuart A. Aaronson for the Balb/MK cell line.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
was
detected in media conditioned by growing or confluent HaCat cells, as
well as in media conditioned at physiological (1 mM) or low
(0.03 mM) Ca
concentrations. However, a
considerable part of transforming growth factor
(TGF-
) in
media conditioned at a physiological Ca
concentration
was in active form, whereas most TGF-
in media conditioned at a
low Ca
concentration was latent. The other
growth-inhibitory activity, which was detected only in media
conditioned by confluent cells at a physiological Ca
concentration, was purified to homogeneity by a four-step
procedure. The N-terminal amino acid sequence of the 33-kDa protein was
identical with that of insulin-like growth factor binding protein-6
(IGFBP-6). Purified IGFBP-6 inhibited the growth of HaCat and Balb/MK
keratinocyte cell lines, as well as Mv1Lu cells. The growth activity
was also demonstrated by human recombinant IGFBP-6. In summary, HaCat
cells secrete at least two possible autocrine growth inhibitors:
TGF-
which is secreted constitutively, but activated in a
Ca
-dependent manner, and IGFBP-6 which is secreted in
a cell density- and Ca
-dependent manner.
concentrations on irradiated 3T3 cells (Rheinwald and Green,
1975) or floating collagen gels (Lillie et al., 1980), they
proliferate and gradually stratify to form differentiated squamous cell
layers. In low calcium medium, however, keratinocytes grow as a
monolayer and do not start the terminal differentiation process
(Hennings et al., 1980; Peehl and Ham, 1980). Keratinocyte
growth factor (Rubin et al., 1989; Finch et al.,
1989), insulin-like growth factor-II (IGF-II)
(
)
(Barreca et al., 1992), and hepatocyte growth
factor/scatter factor (Matsumoto et al., 1991), have been
identified as paracrine growth factors for keratinocytes secreted by
fibroblasts. However, it is not known why keratinocytes continue to
grow and do not initiate the terminal differentiation process at low
Ca
concentrations.
is one of the best characterized
autocrine growth inhibitors for keratinocytes; TGF-
s act on
proliferating basal cells to retard their growth (Choi and Fuchs,
1990). TGF-
2 has been shown to be expressed in the stratifying
keratinocytes in developing mouse epidermis (Lyons et al.,
1989) and by keratinocytes during the terminal differentiation process
(Glick et al., 1989, 1990).
concentrations. Culture media of
a human skin keratinocyte cell line, HaCat, were used to examine
production of growth inhibitors.
Cell Culture
A spontaneously immortalized human
keratinocyte cell line, HaCat, was generously provided by Dr. Norbert
E. Fusenig (DKFZ, Heidelberg). It was maintained in Eagle's
minimal essential medium supplemented with 10% fetal calf serum (FCS,
Life Technologies, Inc.), penicillin (100 IU/ml), and streptomycin (100
µg/ml). Mv1Lu cells (CCl-64 cells; American Type Culture
Collection, ATCC, Rockville, MD) were maintained in the same medium for
HaCat cells. Balb/MK mouse keratinocyte cell line was a generous gift
of Dr. Stuart A. Aaronson (NCI, Bethesda, MD). Subclones of HaCat and
Balb/MK cells, being adapted to MCDB 153 medium (Sigma) at 0.03
mM Ca supplemented with 5% dialyzed FCS and
antibiotics, were established by gradual replacement of the media
(HaCat
and Balb/MK
, respectively). For the
culture of Balb/MK
cells, the medium was also
supplemented with epidermal growth factor (5 ng/ml), ethanolamine (0.1
mM), phosphoryl ethanolamine (0.1 mM), and
hydrocortisone (1.4 µM).
Conditioned Medium
HaCat cells were cultured in
75- or 175-cm culture flasks or 850-cm
roller
bottles using Eagle's minimal essential medium supplemented with
10% FCS and antibiotics at a seeding density of 3
10
cells/cm
. On the 2nd day after confluency was
reached, cells were washed twice with phosphate-buffered saline, and
the medium was changed to 0.2 ml/cm
Dulbecco's
modified Eagle's (DME) medium supplemented with 20 mM
Hepes. The serum-free conditioned media were collected after 1 or 2
days of incubation, and the cells were trypsinized and counted. The
conditioned media of HaCat
cells were collected in MCDB
153 medium with either 0.03 or 1 mM CaCl
. The
collected conditioned media were centrifuged, and supernatants were
stored at -20 °C until use.
Assay for Growth-inhibitory
Activity
Growth-inhibitory activity in HaCat conditioned media
was assayed by cell counting and a [H]thymidine
incorporation assay with Mv1Lu cells as responder cells. Mv1Lu cells
were seeded in duplicate or triplicate into 24-well tissue culture
plates (Costar) at a cell density of 1
10
cells/cm
in MCDB 107 medium (Kyokuto Pharmaceutical
Industries, Tokyo) containing 1% FCS. At 24 h after seeding, the cell
test samples were added so that the final concentration of FCS was
always 1%. Following an additional 16 to 20 h of incubation, 0.3
µCi of [
H]thymidine (5.0 Ci/mmol, Amersham)
was added, and the incubation proceeded for another 1 h. The
incorporated
H radioactivity was counted as described
previously (Miyazono et al., 1987). All experiments were
performed more than twice. Growth-inhibitory activities of purified
IGFBP-6, as well as human recombinant IGFBP-6 (Austral Biologicals, San
Ramon, CA), were also measured by the same procedure.
,
TGF-
neutralizing antibody (JO69; R& Systems, Minneapolis,
MN) was incubated with the test samples 1 h prior to application to the
assay cells. The final concentration of the antibody was 10 µg/ml
IgG in the assay culture.
Purification of a Growth Inhibitor for Mv1Lu and HaCat
Cells
The HaCat conditioned medium, 3 liters at a time, was
centrifuged to remove cell debris, filtered through a 0.2-µm
nitrocellulose membrane, and diluted to 9 liters by distilled water.
The material was loaded on a HiLoad Q-Sepharose HP column (HR 26/10,
Pharmacia Biotech, Uppsala, Sweden), and the sample was eluted with a
linear gradient of NaCl (0-500 mM) in 10 mM
Tris-HCl buffer, pH 7.4. Fractions eluting between 200 and 300
mM NaCl contained growth-inhibitory activity which was
nonsuppressible by TGF- antibodies; these fractions were combined,
diluted three times by distilled water, and subjected to chromatography
on a Mono Q column (HR 5/5, Pharmacia), using a NaCl gradient between
50 and 200 mM in 10 mM Tris-HCl, pH 7.4. The
fractions containing high concentrations of growth-inhibitory activity
(13 ml) were concentrated to 400 µl using vacuum centrifugation and
applied on a Superose 12 column (HR 10/30, Pharmacia). The column was
equilibrated with 150 mM NaCl, 10 mM Tris-HCl, pH
7.4, and eluted at a flow rate of 0.5 ml/min.
Separation by Reversed-phase HPLC
The narrow-bore
HPLC equipment used for reversed-phase separations was a Beckman Gold,
Model 126 connected to a Waters 990 + Diode Array Detector. The
column used was a Brownlee Aquapore C4, 2.1 30 mm column. The
samples were eluted by a linear gradient of 1-propanol in 0.13%
trifluoroacetic acid, at a flow rate of 100 µl/min. The eluate was
monitored at 215 nm, and the fractions were collected manually into
1.5-ml Eppendorf tubes.
SDS-Gel Electrophoresis
SDS-gel electrophoresis
was performed according to the method of Blobel and Dobberstein(1975).
Samples were heated in the absence of dithiothreitol at 95 °C for 3
min and applied to a gradient polyacrylamide gel (7-18%). The gel
was fixed with glutaraldehyde after electrophoresis and silver-stained
(Morrissey, 1981). The following markers of molecular masses were used:
bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase
(30 kDa), and -lactalbumin (14.4 kDa).
Amino Acid Sequence Analysis
N-terminal amino acid
sequence was determined by an Applied Biosystems Model 470A
Protein/Peptide Sequencer equipped with an on-line detection system,
Applied Biosystems Model 120A phenylthiohydantoin amino acid analyzer.
The cysteine residues were identified as phenylthiohydantoin
derivatives of pyridylethyl-L-cysteine after reduction with
mercaptoethanol and alkylation with 4-vinylpyridine.
Effect of Confluent HaCat Conditioned Medium on Mv1Lu
Cell Growth
At first we investigated whether growth inhibitors
were produced by confluent cultures of a keratinocyte cell line at a
physiological Ca concentration. The density of the
HaCat cells was very stable and remained about 3.5
10
cells/cm
at confluency. Under serum-free conditions,
the confluent cells remained in a healthy state for at least 5 days if
the culture media were changed every second day. DME medium was
conditioned for 48 h by the confluent cultures, and its effect on Mv1Lu
cell proliferation was measured in the absence and presence of the
conditioned medium. In the presence of 50% HaCat conditioned medium,
the growth of Mv1Lu cells was totally suppressed (Fig. 1).
Figure 1:
Mv1Lu cell growth-inhibitory activity
in confluent HaCat conditioned medium. Mv1Lu cells were seeded at 1
10
cells/dish (35 mm in diameter) and cultured for
1 day in DME supplemented with 1% FCS. One day after seeding, the
culture medium was changed to DME with 1% FCS supplemented with
(
--
) or without (
--
) 50%
(v/v) of HaCat conditioned medium. Culture medium was changed every 2nd
day, and cell numbers were counted. Each value represents the mean
± S.D. of triplicate wells in a representative experiment
performed more than twice.
A Growth Inhibitor Different from TGF-
In order to study whether
cell density influenced the secretion of growth-inhibitory activity,
HaCat conditioned media were collected daily after 24 h of incubation
at different growth phases. HaCat cells at a seeding density of 3
Is Secreted
in a Cell Density-dependent Manner
10
cells/cm
reached confluency on day
4. The medium collected on day 3, which was conditioned by
exponentially growing HaCat cells, showed no growth-modulating activity
on Mv1Lu cells. However, the medium conditioned by confluent HaCat
cells showed clear growth-inhibitory activity (Fig. 2). By the
addition of TGF-
neutralizing antibodies, a growth-stimulatory
effect was revealed in the conditioned medium from growing cells,
suggesting that these cells secrete TGF-
as well as
growth-stimulatory factors. In contrast, the TGF-
antibody
neutralized only a small part of the growth-inhibitory activity in the
conditioned media from confluent HaCat cells (Fig. 2). Under our
experimental conditions, about 0.1 nM (2.5 ng/ml) TGF-
1
gave maximal growth inhibition on Mv1Lu cells, and the TGF-
neutralizing antibody at the concentration used in this study totally
neutralized more than 0.3 nM TGF-
1 or TGF-
2
activity. The amount of TGF-
growth-inhibitory activity as
estimated by neutralization by the antibody was similar in the media
conditioned by growing and confluent cells, in contrast to the
non-TGF-
growth-inhibitory activity which was secreted only by
confluent cells.
Figure 2:
Effect of the cell density on the
secretion of TGF- and other growth inhibitor(s). HaCat cells were
seeded at 8
10
cells/flask (25 cm
in
area) and cultured in Eagle's minimal essential medium
supplemented with 10% FCS. The medium was changed to DME without serum
and after HaCat conditioned medium was collected at 24 h, and cells
were counted (
--
). Mv1Lu growth-inhibitory
activity in the conditioned media was measured by
[
H]thymidine incorporation assay with
(
--
) or without (
--
)
TGF-
neutralizing antibody. Each value represents the mean
± S.D. of triplicate wells in a representative experiment
performed more than twice.
A Growth Inhibitor Different from TGF-
To elucidate the
effect of the Ca Is Secreted
in a Calcium Concentration-dependent Manner
concentration on the secretion of
TGF-
and non-TGF-
growth inhibitor(s), a subcloned cell line,
HaCat
, was established. The culture media conditioned by
confluent HaCat
cells were collected at different
Ca
concentrations. The medium conditioned at 0.03
mM Ca
did not show any growth-inhibitory
activity for Mv1Lu cells (Fig. 3A). However, after
heating of the media, which activates latent TGF-
, a clear
growth-inhibitory activity which was totally neutralized by TGF-
antibodies, was seen (Fig. 3B). Thus, cells maintained
at low Ca
concentration secreted TGF-
in a
latent form. On the other hand, the medium conditioned at a 1
mM Ca
concentration showed a clear
growth-inhibitory activity without prior heating, and less than half of
the activity was neutralized by TGF-
antibodies
(Fig. 3A). Heat treatment of the conditioned medium
enhanced the activity. The amount of growth-inhibitory activity
remaining after neutralization of TGF-
in the heated media was
similar to the amount remaining in the media without prior heating
(Fig. 3, A and B). These results suggest that
TGF-
is secreted both at low and high Ca
concentrations, but partially activated only at high
Ca
concentration, and, moreover, that a heat-stable
growth inhibitor different from TGF-
is secreted only by confluent
HaCat
cells maintained in a medium with a high
Ca
concentration.
Figure 3:
Effect of calcium concentration on the
secretion of TGF- and other growth inhibitor(s). A subclone of the
HaCat cell line (HaCat
) was cultured in MCDB 153 medium
with 0.03 mM calcium supplemented with 5% dialyzed FCS until
confluency was reached. Then cells were washed twice with
phosphate-buffered saline, and the medium was conditioned for 24 h at
different calcium concentrations. Mv1Lu cell growth-inhibitory activity
in the conditioned media was assayed by a
[
H]thymidine incorporation assay before
(A) and after (B) heat treatment (85 °C, 10 min)
and in the presence (
--
) or absence
(
--
) of a TGF-
neutralizing antibody.
Calcium concentration of the conditioned media was adjusted to 1
mM before assay. Each value represents the mean ± S.D.
of triplicate wells in a representative experiment performed more than
twice.
Separation of Two Growth Inhibitors in HaCat Conditioned
Medium by Gel Chromatography on a Superose 12 Column
Dialysis
and gel permeation chromatography of HaCat conditioned medium were done
for rough estimation of molecular size of the growth-inhibitory
activity different from TGF-. Thirty ml of a confluent HaCat
conditioned medium was dialyzed against 20 mM Tris-HCl buffer
(pH 7.4), lyophilized, and then reconstituted in 0.3 ml of water. The
concentrated conditioned medium was separated by gel chromatography on
a Superose 12 column (Fig. 4A). When the
growth-inhibitory activity of each fraction was examined without prior
heating, a single peak of growth-inhibitory activity was observed in
fractions 26-30 corresponding to a size of about 80 kDa. By heat
treatment of each fraction, another distinct peak was revealed in
fractions 17-21 close to the void volume of the column. TGF-
antibodies totally neutralized the growth-inhibitory activity in
fraction 18 (Fig. 4B), but had only a slight effect on
the growth-inhibitory activity in fraction 28 (Fig. 4C).
Thus, the high molecular mass activity corresponds to a latent
TGF-
complex, whereas that of lower molecular mass is distinct
from TGF-
.
Figure 4:
Gel
chromatography of keratinocyte-derived growth inhibitors on a Superose
12 column. A, gel chromatography of a dialyzed and 100
concentrated HaCat conditioned medium on a Superose 12 column. The
elution positions of markers of molecular mass are indicated by
arrows (V, void volume; B, bovine serum
albumin, 67 kDa; O, ovalbumin, 45 kDa; C,
chymotrypsinogen A, 25 kDa; R, ribonuclease, 14 kDa).
Ten-µl aliquots of the fractions (2% v/v) were added to the cells
for assay before (
--
) or after
(
--
) prior heating (85 °C, 10 min);
means in duplicate wells. B and C, analysis of the
effects on [
H]thymidine incorporation in Mv1Lu
cells of fractions 18 (B) and 28 (C) from the
Superose 12 chromatography without treatment
(
--
), after heat activation
(
--
), after incubation with TGF-
antibodies (
--
), and after heat activation and
incubation with TGF-
antibodies (
--
);
means ± S.D., triplicate wells.
Purification and Structural Characterization of a Growth
Inhibitor Different from TGF-
For the purification of the
growth inhibitor, confluent HaCat conditioned medium was subjected to
ion exchange chromatography on a HiLoad Q-Sepharose HP column
(Fig. 5A). The active fractions were subjected to a
second ion exchange chromatography using a Mono Q column
(Fig. 5B). The active fractions were concentrated and
further separated by chromatography on a Superose 12 column
(Fig. 5C). A growth-inhibitory activity which was not
neutralized by TGF- antibodies eluted at a position corresponding
to a size of about 80 kDa. Final purification was obtained by HPLC
using a C4 reversed-phased column (Fig. 5D). The yield
was approximately 100 µg from 3 liters of conditioned medium.
Analysis by SDS-gel electrophoresis and silver staining of the active
fraction from the HPLC revealed a single component of about 33 kDa
under nonreducing condition (Fig. 5E).
Figure 5:
Purification of a growth inhibitor
different from TGF-. Growth-inhibitory activity of HaCat
conditioned medium was subjected to consecutive chromatographies on a
HiLoad Q Sepharose HP column (A), on a Mono Q column
(B), on a Superose 12 column (C), and on a C4
reverse-phase HPLC column (D). For a
[
H]thymidine incorporation assay of Mv1Lu cells,
aliquots from each fraction were analyzed without prior activation of
TGF-
by heating (10 µl/well). The fractions indicated by
closed circles in each chromatography were pooled and
subjected to the next purification step. The high optic density at late
fractions from a HiLoad Q-Sepharose HP and a Mono Q chromatography
corresponds to the absorbance by phenol red. E, analysis by
SDS-gel electrophoresis under nonreduced conditions followed by silver
staining of an active fraction (fraction 5) from the reverse-phase
HPLC.
N-terminal
amino acid sequencing of the purified protein yielded the sequence
R(L,A)C(A)PGCGQGVQAGCPGGCVEEEDGGXPAEGC. Leucine and alanine
were detected as minor components in the first cycle and alanine as a
minor component in the second. A homology search revealed that the
sequence was identical with that of insulin-like growth factor binding
protein-6 (IGFBP-6), the amino acid sequence of which has been deduced
from a human cDNA sequence obtained from a human placental cDNA library
(Shimasaki et al., 1991) and from a human osteosarcoma cell
line (Kiefer et al., 1991). The purified IGFBP-6 was applied
again on a Superose 12 gel permeation column. It gave a single peak
between fractions 27 and 29 corresponding to a size of about 80 kDa.
Since the size estimated by SDS-gel electrophoresis under nonreducing
conditions was smaller (33 kDa), we explored the possibility that
IGFBP-6 occurred as a dimer. Analysis by SDS-gel electrophoresis and
silver staining after cross-linking of purified IGFBP-6 using
3,3`-bis(sulfosuccinimido)suberate, yielded a component of
approximately 70 kDa, supporting the notion that IGFBP-6 occurs as a
noncovalent dimer (data not shown).
Purified IGFBP-6 Has Growth-inhibitory Activity on HaCat,
Balb/MK, and Mv1Lu Cells
Purified IGFBP-6 exerted a
dose-dependent growth-inhibitory activity on HaCat and Balb/MK
keratinocyte cell lines as well as Mv1Lu cells. Half-maximal effects on
each of these cell lines were similar and obtained at concentrations of
0.3-1 µg/ml (Fig. 6). Human recombinant IGFBP-6 also
inhibited growth of Mv1Lu cells. The effect was, however, less potent,
and the half-maximal effect was exerted by 1-3 µg/ml. Thus,
IGFBP-6 has autocrine growth-inhibitory activity at least for the HaCat
keratinocyte cell line.
Figure 6:
Growth-inhibitory activity of purified
IGFBP-6 on Mv1Lu, HaCat, and Balb/MK cells and of recombinant IGFBP-6
on MV1Lu cells. [H]Thymidine incorporation assays
were used to analyze the dose dependence of the growth-inhibitory
activity of purified IGFBP-6 on Mv1Lu (
--
), HaCat
(
--
), and Balb/MK (
--
)
cells. Recombinant IGFBP-6 inhibited the growth of Mv1Lu
(
--
) but with lower potency. Each value
represents the mean of duplicate wells in a representative experiment
performed more than twice.
Growth-inhibitory Activity of IGFBP-6 Is Abolished by an
Excess Amount of Insulin
To see if growth-inhibitory activity of
IGFBP-6 is due to binding to IGF-II or IGF-I and thereby preventing
activation of type I IGF receptor which mediates the growth-stimulatory
effect of IGFs, the growth inhibition assay was done in the presence of
10 mg/ml of insulin. At this high concentration insulin binds to the
type I IGF receptor and stimulates cell growth, whereas IGFBP-6 can not
bind insulin. Indeed, IGFBP-6 did not show any growth-inhibitory effect
on Mv1Lu cells in the presence of insulin (Fig. 7). This result
suggests that the growth-inhibitory effect of IGFBP-6 is mediated by
the decreased ligand binding to the type I IGF receptor and not by a
direct effect of IGFBP-6 on cells.
Figure 7:
Growth-inhibitory activity of purified
IGFBP-6 in the presence of insulin. Growth-inhibitory activity of
purified IGFBP-6 on Mv1Lu cells was analyzed by
[H]thymidine incorporation assays in the absence
(
--
) or presence (
--
) of
10 mg/ml insulin. Each value represents the mean of duplicate wells in
a representative experiment performed more than
twice.
concentrations in culture media.
At low Ca
concentrations (<0.1 mM),
keratinocytes grow well and remain undifferentiated. In contrast, at
physiological Ca
concentrations (1.0-1.5
mM), keratinocytes start to stratify and undergo the terminal
differentiation process (Hennings et al., 1980; Peehl and Ham,
1980). The growth-inhibitory activity being neutralized by the
TGF-
antibody was detected in media conditioned by growing or
confluent HaCat cells, both at physiological (1 mM) or low
(0.03 mM) Ca
concentrations (
Fig. 2
and Fig. 3). However, a considerable part of
TGF-
in media conditioned at a physiological Ca
concentration was in active form, whereas only latent TGF-
was detected in media conditioned at a low Ca
concentration (Fig. 3). Thus, the increased growth rate and
the blockade of differentiation of keratinocytes at low Ca
concentrations may at least in part be due to the loss of two
autocrine growth inhibitor pathways, i.e. impaired TGF-
activation and decreased IGFBP-6 secretion.
s are produced
by many different cell types and affect growth and differentiation of
many cell types (for reviews, see Roberts and Sporn(1990),
Massagué(1990), and Miyazono et al. (1993)). TGF-
2
was purified as an autocrine growth inhibitor of BSC-1, monkey kidney
epithelial cells (Holly et al., 1980; Hanks et al.,
1988), and as a Mv1Lu cell growth inhibitor in conditioned media from
PC-3 cells, a human prostatic cancer cell line (Ikeda et al.,
1987). It also has growth-inhibitory activity on keratinocytes (Shipley
et al., 1986) and is secreted by keratinocytes as an autocrine
growth inhibitor during the terminal differentiation process (Glick
et al., 1989, 1990). In normal skin tissue, a strong
immunoreactivity for TGF-
2 was found in intercellular spaces of
keratinocytes in the suprabasal and the upper layers, whereas a weak
cytoplasmic staining was seen in the proliferating basal layers
(Wataya-Kaneda et al., 1994). This staining pattern suggests
that the secretion of TGF-
2 is also regulated in the skin tissue.
In our in vitro system, TGF-
activities did not differ in
media conditioned by growing or confluent HaCat cells at a
physiological calcium concentration. However, the TGF-
activities
in conditioned media at different Ca
concentrations
suggested that physiological concentrations of Ca
are
required for the activation of TGF-
(Fig. 3). Most of
TGF-
was present in a latent form in medium conditioned at a low
Ca
concentration, whereas a considerable part
(20-30%) of TGF-
in medium conditioned at a high
Ca
concentration was recovered in active form,
similar to TGF-
in conditioned medium of keratinocytes treated by
retinoic acid (Glick et al., 1989).
and its receptors, the action of TGF-
appears to be carefully regulated by the activation process. There are
several cell culture systems which have been reported to secrete active
TGF-
or activate exogenous latent TGF-
(for review, see
Miyazono et al.(1993)). In co-culture of endothelial cells and
smooth muscle cells, plasmin has been shown to activate latent
TGF-
(Antonell-Orlidge et al., 1989; Sato and Rifkin,
1989). Binding of latent TGF-
to the mannose 6-phosphate/IGF-II
receptor facilitated the proteolytic activation of TGF-
, and
excess mannose 6-phosphate blocked generation of activated TGF-
(Kovacina et al., 1989; Dennis and Rifkin, 1991; Odekon et
al., 1994). Whether Ca
-dependent TGF-
activation by keratinocytes might involve these mechanisms remains to
be elucidated.
concentration (Fig. 3). The proliferating HaCat cells were
also suppressed by the purified IGFBP-6 (Fig. 6), which suggested
that this molecule might work as an autocrine growth inhibitor for
keratinocytes at physiological Ca
concentrations.
Furthermore, the growth-inhibitory activity of human recombinant
IGFBP-6 was confirmed on Mv1Lu cells, although with much lower
efficiencies. The difference in the efficiency of IGF-II binding
between recombinant and purified IGFBP-6 is reported (Martin et
al., 1994). It might be due to the structural difference since
recombinant IGFBP-6 is not glycosylated and occurs as a monomer as
compared to native IGFBP-6 which seems to exist mostly in a dimeric
form as we could show by gel filtration.
.
and secretion of
IGFBP-6 seem to occur in a cell density- and Ca
concentration-dependent manner. It remains to be elucidated
whether these molecules act not only in the growth inhibition of
keratinocytes, but also in their terminal differentiation which can be
induced by the physiological concentration of calcium.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.