©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Human Keratinocyte Cell Line Produces Two Autocrine Growth Inhibitors, Transforming Growth Factor- and Insulin-like Growth Factor Binding Protein-6, in a Calcium- and Cell Density-dependent Manner (*)

Mitsuyasu Kato , Akira Ishizaki , Ulf Hellman , Christer Wernstedt , Masahisa Kyogoku (1), Kohei Miyazono , Carl-Henrik Heldin , Keiko Funa (§)

From the (1) Ludwig Institute for Cancer Research, Biomedical Center, S-751 24 Uppsala, Sweden Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai, 980 Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Two growth inhibitors were identified in culture medium conditioned by a human keratinocyte cell line, HaCat. TGF- 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.


INTRODUCTION

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 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.

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- 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).

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 concentrations. Culture media of a human skin keratinocyte cell line, HaCat, were used to examine production of growth inhibitors.


MATERIALS AND METHODS

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.

To estimate how much of the growth-inhibitory activity was due to TGF-, 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.


RESULTS

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- Is Secreted in a Cell Density-dependent Manner

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 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- Is Secreted in a Calcium Concentration-dependent Manner

To elucidate the effect of the Ca 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.




DISCUSSION

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 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.

TGF-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).

In spite of the wide distribution of TGF- 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.

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 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.

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- 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Ludwig Institute for Cancer Research, Biomedical Center, Box 595, S-751 24 Uppsala, Sweden. Tel.: 46-18-17-40-05; Fax: 46-18-50-68-67.

The abbreviations used are: IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; TGF, transforming growth factor; FCS, fetal calf serum; DME, Dulbecco's modified Eagle's medium; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. Norbert E. Fusenig for the HaCat cell line and Dr. Stuart A. Aaronson for the Balb/MK cell line.


REFERENCES
  1. Antonell-Orlidge, A., Saunders, K. B., Smith, S. R., and D'Amore, P. A.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4544-4548 [Abstract]
  2. Barreca, A., De Luca, M., Del Monte, P., Bondanza, S., Damonte, G., Cariola, G., Di Marco, E., Giordano, G., Cancedda, R., and Minuto, F.(1992) J. Cell. Physiol. 151, 262-268 [Medline] [Order article via Infotrieve]
  3. Blobel, G., and Dobberstein, B.(1975) J. Cell Biol. 67, 835-851 [Abstract]
  4. Böhmer, F. D., Kraft, R., Otto, A., Wernstedt, C., Hellman, U., Kurtz, A., Müller, T., Rohde, K., Etzold, G., Lehmann, W., Langen, P., Heldin, C.-H., and Grosse, R.(1987) J. Biol. Chem. 262, 15137-15143 [Abstract/Free Full Text]
  5. Choi, Y., and Fuchs, E.(1990) Cell Regul. 1, 791-809 [Medline] [Order article via Infotrieve]
  6. Dennis, P. A., and Rifkin, D. B.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 580-584 [Abstract]
  7. Finch, P. W., Rubin, J. S., Miki, T., Ron, D., and Aaronson, S. A. (1989) Science 245, 752-755 [Medline] [Order article via Infotrieve]
  8. Glick, A. B., Flanders, K. C., Danielpour, D., Yuspa, S. H., and Sporn, M. B.(1989) Cell Regul. 1, 87-97 [Medline] [Order article via Infotrieve]
  9. Glick, A. B., Danielpour, D., Morgan, D., Sporn, M. B., and Yuspa, S. H.(1990) Mol. Endocrinol. 4, 46-52 [Abstract]
  10. Gray, A., Tam, A. W., Dull, T. J., Hayflick, J., Pintar, J., Cavenee, W. K., Koufos, A., and Ullrich, A.(1987) DNA (NY) 6, 283-295 [Medline] [Order article via Infotrieve]
  11. Han, V. K. M., D'Ercole, A. J., and Lund, P. K. (1987a) Science 236, 193-197 [Medline] [Order article via Infotrieve]
  12. Han, V. K. M., Hill, D. J., Strain, A. J., Towle, A. C., Lauder, J. M., Underwood, L. E., and D'Ercole, A. J. (1987b) Pediatr. Res. 22, 245-249 [Abstract]
  13. Han, V. K. M., Lund, P. K., Lee, D. C., and D'Ercole, A. J.(1988) J. Clin. Endocrinol. Metab. 66, 422-429 [Abstract]
  14. Hanks, S. K., Armour, R., Baldwin, J. H., Maldonado, F., Spiess, J., and Holly, R. W.(1988) Proc. Natl. Acad. Sci. U. S. A. 85, 79-82 [Abstract]
  15. Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K., and Yuspa, S. H.(1980) Cell 19, 245-254 [Medline] [Order article via Infotrieve]
  16. Holly, R. W., Böhlen, P., Fava, R., Baldwin, J. H., Kleeman, G., and Armour, R.(1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5989-5992 [Abstract]
  17. Ikeda, T., Lioubin, M. N., and Marquardt, H.(1987) Biochemistry 26, 2406-2410 [Medline] [Order article via Infotrieve]
  18. Kiefer, M. C., Masiarz, F. R., Bauer, D. M., and Zapf, J.(1991) J. Biol. Chem. 266, 9043-9049 [Abstract/Free Full Text]
  19. Kovacina, K. S., Steele-Perkins, G., Purchio, A. F., Lioubin, M., Miyazono, K., Heldin, C.-H., and Roth, R. A.(1989) Biochem. Biophys. Res. Commun. 160, 393-403 [Medline] [Order article via Infotrieve]
  20. Lamson, G., Giudice, L. C., and Rosenfeld, R. G.(1991) Growth Factors 5, 19-28 [Medline] [Order article via Infotrieve]
  21. Lillie, J. H., MacCallum, D. K., and Jepsen, A.(1980) Exp. Cell Res. 125, 153-165 [Medline] [Order article via Infotrieve]
  22. Lyons, K. M., Pelton, R. W., and Hogan, B. L. M.(1989) Genes & Dev. 3, 1657-1688
  23. Martin, J. L., Willetts, K. E., and Baxter, R. C.(1990) J. Biol. Chem. 265, 4124-4130 [Abstract/Free Full Text]
  24. Martin, J. L., Coverley, J. A., and Baxter, R. C.(1994) J. Biol. Chem. 269, 11470-11477 [Abstract/Free Full Text]
  25. Massagué, J.(1990) Annu. Rev. Cell Biol. 6, 597-641 [CrossRef]
  26. Matsumoto, K., Hashimoto, K., Yoshikawa, K., and Nakamura, T.(1991) Exp. Cell Res. 196, 114-120 [Medline] [Order article via Infotrieve]
  27. Miyazono, K., Okabe, T., Urabe, A., Takaku, F., and Heldin, C.-H. (1987) J. Biol. Chem. 262, 4098-4103 [Abstract/Free Full Text]
  28. Miyazono, K., Ichijo, I., and Heldin, C.-H.(1993) Growth Factors 8, 11-22 [Medline] [Order article via Infotrieve]
  29. Morrissey, J. H.(1981) Anal. Biochem. 117, 307-310 [Medline] [Order article via Infotrieve]
  30. Nickoloff, B. J., Misra, P., Morhenn, V. B., Hintz, R. L., and Rosenfeld, R. G.(1988) Dermatologica 177, 265-273 [Medline] [Order article via Infotrieve]
  31. Nielsen, F. C.(1992) Progr. Growth Factor Res. 4, 257-290
  32. Odekon, L. E., Blasi, F., and Rifkin, D. B.(1994) J. Cell. Physiol. 158, 398-407 [Medline] [Order article via Infotrieve]
  33. Oh, Y., Müller, H. L., Lamson, G., and Rosenfeld, R. G.(1993) J. Biol. Chem. 268, 14964-14971 [Abstract/Free Full Text]
  34. Peehl, D. M., and Ham, R. G.(1980) In Vitro 16, 516-525 [Medline] [Order article via Infotrieve]
  35. Rechler, M. M., and Nissley, S. P.(1990) in Peptide Growth Factors and Their Receptors (Sporn, I. M. B., and Roberts, A. B., eds) pp. 263-367, Springer-Verlag, Berlin
  36. Rheinwald, J. G., and Green, H.(1975) Cell 6, 331-344 [Medline] [Order article via Infotrieve]
  37. Roberts, A. B., and Sporn, M. B.(1990) in Peptide Growth Factors and Their Receptors (Sporn, I. M. B., and Roberts, A. B., eds) pp. 419-472, Springer-Verlag, Berlin
  38. Roberts, A. B., Anzano, M. A., Meyers, C. A., Wideman, J., Blacher, R., Pan, Y.-C. E., Stein, S., Lehrman, S. R., Smith, J. M., Lamb, L. C., and Sporn, M. B.(1983) Biochemistry 22, 5692-5698 [Medline] [Order article via Infotrieve]
  39. Roghani, M., Hossenlopp, P., Lepage, P., Balland, A., and Binoux, M. (1989) FEBS Lett. 255, 253-258 [CrossRef][Medline] [Order article via Infotrieve]
  40. Rubin, J. S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S., and Aaronson, S. A.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 802-806 [Abstract]
  41. Sato, Y., and Rifkin, D. B.(1989) J. Cell Biol. 109, 309-315 [Abstract]
  42. Shimasaki, S., and Ling, N.(1991) Progr. Growth Factor Res. 3, 243-266
  43. Shimasaki, S., Gao, L., Shimonaka, M., and Ling, N.(1991) Mol. Endocrinol. 5, 938-948 [Abstract]
  44. Shipley, G. D., Pittelkow, M. R., Wille, J. J., Jr., Scott, R. E., and Moses, H. L.(1986) Cancer Res. 46, 2068-2071 [Abstract]
  45. Wataya-Kaneda, M., Hashimoto, K., Kato, M., Miyazono, K., and Yoshikawa, K.(1994) J. Dermatol. Sci. 8, 38-44 [Medline] [Order article via Infotrieve]
  46. Wille, J. J., Jr., Pittelkow, M. R., Shipley, G. D., and Scott, R. E. (1984) J. Cell. Physiol. 121, 31-44 [Medline] [Order article via Infotrieve]
  47. Zapf, J., Kiefer, M., Merryweather, J., Masiarz, F., Bauer, D., Born, W., Fischer, J. A., and Froesch, E. R.(1990) J. Biol. Chem. 265, 14892-14898 [Abstract/Free Full Text]

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