help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garach-Jehoshua, O.
Right arrow Articles by Koren, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garach-Jehoshua, O.
Right arrow Articles by Koren, R.
Endocrinology Vol. 140, No. 2 713-721
Copyright © 1999 by The Endocrine Society

ARTICLES

1,25-Dihydroxyvitamin D3 Increases the Growth-Promoting Activity of Autocrine Epidermal Growth Factor Receptor Ligands in Keratinocytes1

Osnat Garach-Jehoshua, Amiram Ravid, Uri A. Liberman and Ruth Koren

Basil and Gerald Felsenstein Medical Research Center (A.R., U.A.L., R.K.) and the Department of Physiology and Pharmacology (O.G.-J., U.A.L., R.K.), Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 49100, Israel

Address all correspondence and requests for reprints to: Dr. Ruth Koren, Felsenstein Medical Research Center, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel. E-mail: rkoren{at}post.tau.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Topical treatment of normal skin with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or its synthetic analogs results in enhanced keratinocyte proliferation. Autocrine growth factors belonging to the epidermal growth factor (EGF) family play a major role in controlling keratinocyte proliferation. 1,25-(OH)2D3 enhanced the autonomous proliferation of HaCaT human keratinocytes in the absence of exogenous growth factors. Autonomous and 1,25-(OH)2D3-stimulated proliferations were inhibited by a specific inhibitor of EGF receptor (EGFR) tyrosine kinase, an EGFR-neutralizing antibody, heparin, the heparin antagonist hexadimethrine, and the proteoglycan sulfation inhibitor chlorate. These results indicate the involvement of proteoglycan-dependent EGFR ligands. The initial events in EGFR (i.e. ErbB1) mitogenic signal transduction are dimer formation with another ErbB protein and tyrosine cross-phosphorylation. By immunoprecipitation followed by Western blotting we showed that ErbB1/ErbB3 heterodimers are the major mitogenic signaling entity in 1,25-(OH)2D3-stimulated cells. 1,25-(OH)2D3 did not affect the levels of the proteoglycan-dependent EGFR ligands amphiregulin and heparin-binding EGF nor the synthesis of proteoglycans, as assessed by 35S labeling and ion exchange chromatography. 1,25-(OH)2D3 caused a marked increase in the cellular contents of ErbB1, ErbB2, and ErbB3 proteins. The increase in ErbB proteins that mediates signal transduction by EGFR ligands can account for the stimulatory effect of 1,25-(OH)2D3 on autonomous keratinocyte proliferation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HORMONAL form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], affects the proliferation and differentiation of skin. 1,25-(OH)2D3 is likely to act in an autocrine manner in the epidermis, because keratinocytes produce and respond to it. Many studies were devoted to the antiproliferative effect of 1,25-(OH)2D3 on keratinocytes (1). Recent studies, however, demonstrated a stimulatory effect of 1,25-(OH)2D3 on keratinocyte proliferation (2, 3, 4, 5). It was found that the direction and extent of the effect of 1,25-(OH)2D3 on keratinocyte proliferation are dependent on the specific growth factors present, the cell density, and the concentration of the hormone. The growth-promoting activity of 1,25-(OH)2D3 and its analogs is also manifested in vivo. Topical application of these compounds resulted in epidermal hyperplasia of human and mouse skin (6, 7, 8, 9), enhanced cutaneous wound healing in rats (10), and reversal of glucocorticoid epidermal atrophy in mouse epidermis (11).

Growth factors acting in an autocrine manner play a major role in controlling keratinocyte proliferation (12). These growth factors are produced by keratinocytes in response to paracrine stimuli such as fibroblast growth factor, keratinocyte growth factor, insulin-like growth factor I, interleukin-1, and tumor necrosis factor (13). Of paramount importance among these autocrine factors are members of the epidermal growth factor (EGF) family, which include transforming growth factor-{alpha}, amphiregulin, heparin-binding EGF (HB-EGF), and betacellulin (14, 15, 16). Amphiregulin and HB-EGF bind to membrane proteoglycans, producing a ternary complex with the EGF receptor (EGFR) that is essential for transduction of the mitogenic signal.

In addition to EGFR (also called ErbB1 or HER1), three other transmembrane proteins that belong to the same family, ErbB2, ErbB3, and ErbB4, may cooperate in the signal transduction by EGFR ligands. The members of the ErbB family, except ErbB4, are expressed in keratinocytes (17). Upon binding to a ligand, EGFR forms a homodimer or a heterodimer with another ErbB protein. Dimer formation is followed by cross-phosphorylation of ErbB proteins by the respective receptor tyrosine kinases (18). It is of interest in this context that ErbB3 is almost devoid of tyrosine kinase activity (18). The formation of specific dimer combinations is determined by the relative abundance of the ErbB family members and depends on the specific EGFR ligand available (16, 18, 19). The phosphorylated tyrosine residues on ErbB proteins forming the signaling dimer serve as docking sites for specific SH2-containing proteins. These interactions lead to activation of the Ras-Raf-mitogen-activated protein (MAP) kinase as a main signaling pathway and finally to transcription factor activation and cell mitogenesis. The specific signaling routes are unique to each dimer, depend on cell context, and determine the potency of the mitogenic signal and its regulation (18).

In this work we used HaCaT cells, a line of spontaneously immortalized and nontumorigenic human keratinocytes (20) that can proliferate in culture in the absence of exogenous growth factors. This system provided an appropriate model to study the stimulatory effect of 1,25-(OH)2D3 on the autocrine growth factor network in keratinocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Tissue culture media were obtained from Life Technologies (Grand Island, NY). Tissue culture dishes were purchased from Corning Glass Works (Corning, NY). FCS was obtained from Beit Haemek Industries (Beit Haemek, Israel). 1,25-(OH)2D3 was obtained from Hoffmann-La Roche (Nutley, NJ; a gift from Dr. M. Uskokovic). [Methyl-3H]thymidine was obtained from Rotem Industries Ltd. (Beer Sheva, Israel). EGF was obtained from PeproTech, Inc. (Rocky Hill, NJ). Tyrphostin AG 1478 was a gift from Prof. A. Levitzki, The Hebrew University (Jerusalem, Israel). Porcine mucous heparin, hexadimethrine, sodium chlorate, phenylmethylsulfonylfluoride, aprotinin, leupeptin, and goat antimouse IgG-agarose were obtained from Sigma Chemical Co. (St. Louis, MO). Human albumin was obtained from KAMADA (Kibbutz Beit Kama, Israel). 35S-Labeled sodium sulfate was purchased from DuPont-New England Nuclear, Inc. (Boston, MA). DE-52 cellulose ion exchange was obtained from Whatman International Ltd. (Maidstone, UK). Rabbit polyclonal anti-EGFR and anti-ErbB3 antibodies (Abs) and horseradish peroxidase (HRP)-conjugated donkey antigoat IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Goat antihuman HB-EGF and amphiregulin Abs were from R & D Systems, Inc. (Minneapolis, MN). Rabbit polyclonal antiactive MAP kinase Abs were obtained from Promega Corp. (Madison, WI). HRP-conjugated goat antimouse Fab Abs were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). HRP-conjugated swine antirabbit Igs were obtained from Dako Corp. (Copenhagen, Denmark). Anti-ErbB2 monoclonal Abs (L87 and L96) and anti-ErbB3 monoclonal Abs (no. 252) were gifts from Profs. Yosef Yarden and Michael Sela (21).

Cell culture
The human keratinocyte cell line HaCaT was provided by Prof. N. Fusenig, German Cancer Research Center (Heidelberg, Germany). Cells were maintained in MEM containing 0.075 mM calcium (MEM-75) and 10% FCS, and were used between passages 36–55. Cells were grown in 5-cm petri dishes and subcultured every 3–4 days. At the initiation of proliferation experiments, cells were seeded in 96-well microtiter plates (7000 cells/well) in MEM-75 containing 10% FCS. Four or 24 h after seeding, the medium was replaced with fresh MEM-75 containing human albumin (0.5 mg/ml) without serum and treated with vitamin D metabolites, cytokines, Abs, or pharmacological agents. Ethanol, the vehicle of 1,25-(OH)2D3, was added to control cultures, and its concentration never exceeded 0.06%. Cell number was assessed 5 days later. For proteoglycan determination and immunoblot analysis, cells were seeded in 6-cm petri dishes (450,000 cells/dish) in MEM-75 containing 10% FCS. Twenty-four hours after seeding the medium was replaced with MEM-75 containing albumin. For proteoglycan determination, the medium was replaced again after an additional 24 h with sulfate-free MEM containing albumin. Cells were cultured for an additional 48 h in the presence of 1,25-(OH)2D3 or vehicle.

Crystal violet (CV) staining
HaCaT cell proliferation was assessed by staining with CV as previously described (22). In brief, cells were stained for 30 min with a 0.1% CV solution in 20% ethanol. The dye was rinsed with water and extracted with 70% ethanol, and its absorbency was determined at 550 nm using a microplate reader. In a preliminary experiment we found a linear correlation (r = 0.999) between CV staining and HaCaT cell number over a wide range of cell densities (5,000–40,000 cells/well). Cell proliferation was assessed by subtracting the absorbency of CV absorbed by the cells 24 h after seeding from that obtained at the end of the experiment.

Thymidine incorporation
[3H]Thymidine incorporation into cellular DNA was determined by incubating the cells with 0.5 µCi/well [3H]thymidine for the last 16 h of culture, harvesting with a cell harvester, and quantifying the radioactivity by ß-scintillation counting.

Proteoglycan synthesis
Culture media were replaced with sulfate-free MEM 48 h before harvesting. Cells were exposed to 35S-labeled sodium sulfate for the last 24 h. Conditioned media were collected, and the cells were scraped with a rubber policeman into PBS, centrifuged at 900 x g for 7 min, and resuspended in PBS containing 1% Triton X-100 for 1 h on ice. After centrifugation (16,000 x g for 5 min), supernatants were used for proteoglycan and protein determinations. 35S-Labeled macromolecules from cell extracts and media were subjected to ion exchange chromatography on DE-52 (23). The samples were loaded onto the columns (300-µl gel volume) and washed with 10 bed vol PBS. Columns were eluted with a step NaCl gradient (0.25–1.5 M) in the presence of 0.5% Triton X-100. Fractions (0.6 ml) were counted for radioactivity in a scintillation counter.

Electrophoresis and immunoblot analysis
Cells were removed from the petri dishes by scraping in PBS containing 10 mM EDTA. The cells were centrifuged at 16,000 x g for 1 min and resuspended in 200 µl SDS-containing sample buffer. Samples were sonicated with a Heat System-Ultrasonics, Inc. (Plainview, NY) sonicator (model W-375) at setting 4 for 20 sec under ice, boiled for 3 min, and centrifuged before electrophoresis. Samples were subjected to SDS-PAGE under reducing conditions using 7.5% or 15% polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes by electroblotting, and the membranes were probed with the appropriate Abs. Detection was carried out by HRP-conjugated secondary Abs and enhanced chemiluminescence.

Protein determination
The protein content of cell extracts in sample buffer and Triton X-100 was measured as previously described (24).

Immunoprecipitation
Cells were lysed with a solubilization buffer (1% Triton X-100, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 2 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 150 mM NaCl, and 50 mM HEPES, pH 7.5) for 30 min on ice. The lysates were collected and clarified by centrifugation for 10 min at 16,000 x g. One microgram of anti-ErbB2 (L96) or anti-ErbB3 (no. 252) Ab was incubated for 1 h at 4 C with 20 µl antimouse IgG-agarose in 500 µl PBS followed by incubation for 1 h at 4 C with cell lysates. Immune complexes were collected and washed three times with cold HNTG buffer (0.1% Triton X-100, 10% glycerol, 150 mM NaCl, and 20 mM HEPES, pH 7.5). Bound proteins were released by heating (5 min at 95 C) in sample buffer and subjected to SDS-PAGE and immunoblotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
1,25-(OH)2D3 stimulates autonomous HaCaT cell proliferation
HaCaT cells proliferate in MEM-75 in the absence of serum and exogenous growth factors. The doubling time of this autonomous proliferation is approximately 5 days. 1,25-(OH)2D3 enhanced the autonomous proliferation in a dose-dependent manner at a concentration range of 0.1–100 nM as shown in Fig. 1Go. Although small, the stimulatory effect at 0.1 nM was observed in all experiments performed and was statistically significant (P < 0.02, by paired Student’s t test). The average enhancement of the proliferation rate in cultures treated with 100 nM 1,25-(OH)2D3 was 241 ± 50% (mean ± SEM) of that in control cultures. The effect of 1,25-(OH)2D3 on HaCaT cell proliferation was also assessed by [3H]thymidine incorporation. Seventy-two-hour exposure to the hormone (100 nM) elevated [3H]thymidine incorporation by 82.3% (19,639 ± 2,509 cpm in hormone-treated cultures compared with 10,769 ± 1,651 cpm in vehicle-treated cultures, mean ± SD of five replicate cultures).



View larger version (12K):
[in this window]
[in a new window]
  		Figure 1. The stimulatory effect of 1,25-(OH)2D3 on HaCaT cell autonomous proliferation. HaCaT cells (7 x 103/well) were plated in a 96-well microtiter plate in MEM-75 containing 10% FCS. Twenty-four hours later, the medium was replaced with MEM-75 containing human albumin (0.5 mg/ml). Cells were then treated for 5 days with 1,25-(OH)2D3. Cell number was assessed by CV staining. The extent of proliferation was determined by subtracting CV staining of 24-h parallel cultures. During this period the number of cells in untreated cultures increased by 120 ± 16% (mean ± SEM; n = 11). Data are expressed as the percent enhancement of proliferation compared with that in parallel cultures grown in the absence of the hormone. Data points represent the mean ± SEM of 11 independent experiments.

 
It is noteworthy that 1,25-(OH)2D3 did not stimulate EGF-driven HaCaT cell proliferation (Fig. 2Go). In addition, at 100 nM 1,25-(OH)2D3, the rate of proliferation of 1,25-(OH)2D3-stimulated cells was consistently higher (by 34 ± 9%, mean ± SEM; n = 9 independent experiments) than the rate of proliferation in the presence of a saturating EGF concentration (20 ng/ml). This result indicates that the lack of a stimulatory effect on EGF-driven proliferation is not merely due to the fact that the cells already proliferate at their maximal rate.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. The effect of 1,25-(OH)2D3 on EGF-treated HaCaT cells. Cells were plated and cultured as described in Fig. 1Go. Cells were treated for 5 days with different concentrations of 1,25-(OH)2D3 in the absence or presence of EGF (20 ng/ml). The bars represent five replicate cultures (mean ± SD). Similar results were obtained in three additional independent experiments.

 
Both autonomous and 1,25-(OH)2D3-stimulated HaCaT cell proliferation are mediated by EGFR ligand(s)
Normal human keratinocyte proliferation in culture depends to a large extent on autocrine growth factors that act via the EGFR (12, 14, 25). We assessed the possibility that the autonomous proliferation of HaCaT cells and particularly its stimulation by 1,25-(OH)2D3 are mediated by such EGFR ligands. To this end we used the specific inhibitor of the EGFR tyrosine kinase, tyrphostin AG 1478 (26), and the EGFR-neutralizing monoclonal antibody, Ab-225 (27) (Fig. 3Go). As shown in Fig. 3AGo, AG 1478 at 500 nM inhibited the autonomous, 1,25-(OH)2D3-stimulated, and EGF-driven proliferation. At lower AG 1478 concentrations, 1,25-(OH)2D3-stimulated cells were less sensitive to inhibition by the tyrphostin than untreated HaCaT cells, but were of comparable sensitivity as EGF-treated cells. The relative resistance of EGF-treated cultures is most likely due to the higher occupancy of the EGFR. The reduced sensitivity of 1,25-(OH)2D3-treated cultures to inhibition by AG 1478 may suggest the presence of a higher number of occupied EGFRs in these cultures as well.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. The inhibitory effect of tyrphostin AG 1478 and the anti-EGF receptor monoclonal Ab Ab-225 on HaCaT cell proliferation. HaCaT cells (7 x 103/well) were plated in a 96-well microtiter plate in MEM-75 containing 10% FCS. Twenty-four (A) or 4 (B) h later, the medium was replaced with MEM-75 containing human albumin (0.5 mg/ml). Cells were treated for 5 days with AG 1478 (A) or Ab-225 (B) in the absence or presence of 1,25-(OH)2D3 (100 nM) or EGF (20 ng/ml). Data are expressed as the mean ± SD of five replicate cultures.

 
In a preliminary experiment we found that the proliferation of HaCaT cells cultured in the presence of exogenous EGF (3 ng/ml) was markedly inhibited (mean ± SD, 65 ± 13%; n = 4) in the presence of 0.5 µg/ml Ab-225. As shown in Fig. 3BGo, Ab-225, added 4 h after seeding, at the lowest concentration abolished the autonomous proliferation of untreated HaCaT cells. It inhibited in a dose-dependent manner up to 74% of the proliferation in the presence of 1,25-(OH)2D3. Under the same conditions, an unrelated mouse IgG1 monoclonal antibody directed against human ferritin had no effect on HaCaT cell proliferation, in the absence or presence of 1,25-(OH)2D3, over a concentration range of 0.5–5 µg/ml. It is of interest that Ab-225 added 24 h after seeding, when cell clustering has already occurred, had only a moderate effect on cell proliferation (data not shown). This indicates the probable involvement of juxtacrine stimulation that is not inhibited by the Ab due to steric hindrance or ligand-receptor complex stabilization. Juxtacrine action was indeed shown to contribute significantly to mitogenesis mediated by the autocrine EGFR ligands (28, 29). Light microscope examination revealed increased cell clustering in 1,25-(OH)2D3-treated cultures, a phenomenon that could explain the smaller inhibitory effect of Ab-225 in these cultures. The residual proliferation in 1,25-(OH)2D3-treated cultures at the highest concentration of the antibody could be abolished by the addition of 500 nM AG 1478. Taken together, the results depicted in Fig. 3Go support the idea that autonomous HaCaT cell proliferation and its stimulation by 1,25-(OH)2D3 are both dependent upon ligand(s) of the EGF family.

Both autonomous and 1,25-(OH)2D3-stimulated HaCaT cell proliferations are mediated by proteoglycan-dependent growth factor(s)
The family of the EGFR ligands can be divided into two main groups: proteoglycan-independent ligands (EGF and transforming growth factor-{alpha}) and proteoglycan-binding ligands (amphiregulin, HB-EGF, and betacellulin) (16). We used heparin, hexadimethrine, and sodium chlorate to modulate the interaction of proteoglycans with growth factors and their receptors. Heparin, a soluble glycosaminoglycan, and hexadimethrine, a synthetic cationic polymer, are known to modulate the formation of the ternary complex of EGFR, proteoglycan, and ligand (15, 30). The results in Fig. 4Go show that heparin inhibited dose dependently the autonomous and, to a slightly lesser extent, the 1,25-(OH)2D3-driven proliferation. As expected, heparin did not affect EGF-dependent proliferation. The extent of inhibition by heparin at a concentration of 10 µg/ml varied between experiments and ranged between 59–100%. The residual proliferation in the presence of heparin could be due to the proteoglycan mimetic activity of heparin (31, 32). Hexadimethrine, at nontoxic concentrations (up to 1 µg/ml), inhibited autonomous and 1,25-(OH)2D3-stimulated proliferation and had only a minimal effect on EGF-driven proliferation (Table 1Go). Supporting evidence for the involvement of proteoglycans was also derived from the finding that sodium chlorate, a competitive inhibitor of proteoglycan sulfation, partially inhibited autonomous and 1,25-(OH)2D3-stimulated proliferation (Table 1Go).



View larger version (39K):
[in this window]
[in a new window]
  		Figure 4. The inhibitory effect of heparin on HaCaT cell proliferation. Cells were plated and cultured as described in Fig. 1Go. Cells were treated for 5 days with heparin, 1,25-(OH)2D3 (100 nM), or EGF (20 ng/ml). Data are expressed as the mean ± SD of five replicate cultures. Similar results were obtained in three additional independent experiments.

 

View this table:
[in this window]
[in a new window]
  		Table 1. The inhibitory effect of hexadimethrine and chlorate on HaCaT cell proliferation

 
1,25-(OH)2D3 enhances EGFR-dependent tyrosine phosphorylation and MAP kinase activation
The tyrosine phosphorylation pattern of cell extracts prepared from autonomously proliferating and 1,25-(OH)2D3-stimulated cultures of HaCaT cells was examined by immunoblotting with an antiphosphotyrosine antibody. Treatment with 1,25-(OH)2D3 (100 nM) for 48 h enhanced tyrosine phosphorylation of two closely migrating proteins in the 180–190 kDa range (Fig. 5Go). The relative intensity of tyrosine phosphorylation of these two proteins varied between experiments. The phosphorylation of these proteins was strongly inhibited by treatment with the specific EGFR tyrosine kinase inhibitor AG 1478 (500 nM) for 3 h (Fig. 5BGo) or by treatment with heparin (10 µg/ml) for 48 h (Fig. 5CGo). MAP kinase activation is a major event in EGFR-dependent mitogenic signaling. Indeed, we show here (Fig. 6Go), using a specific antibody against activated ERK1 and ERK2, that treatment with 1,25-(OH)2D3 increased the level of activated MAP kinase in autonomously proliferating HaCaT cells. We also show, using AG 1478, that in both control and hormone-treated cells activation of MAP kinase is completely dependent on EGFR kinase activity.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 5. Effect of 1,25-(OH)2D3 on tyrosine phosphorylation of HaCaT cell proteins. Cells and cell extracts were prepared as described in Materials and Methods. Cells were harvested 72 h after medium replacement. 1,25-(OH)2D3 (100 nM) and heparin (10 µg/ml) were added 48 h before harvesting. AG 1478 (500 nM) was added for the last 3 h of culture. Thirty minutes before harvesting, cells were exposed to sodium orthovanadate (1 mM). Cell extracts were subjected to SDS-PAGE and probed with antiphosphotyrosine monclonal Abs. A, Lanes 1, 3, and 5, Untreated cultures; lanes 2, 4, and 6, 1,25-(OH)2D3-treated cultures. B, Lane 1, 1,25-(OH)2D3-treated culture; lane 2, 1,25-(OH)2D3- and AG 1478-treated culture. C, Lane 1, 1,25-(OH)2D3-treated culture; lane 2, 1,25-(OH)2D3- and heparin-treated culture.

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. Effect of 1,25-(OH)2D3 on the cellular level of activated MAP kinase in HaCaT cells. Cells and cell extracts were prepared as described in Materials and Methods. Cells were harvested 48 h after medium replacement. 1,25-(OH)2D3 (100 nM) and AG 1478 (500 nM) were added at different time points before harvesting. Cell extracts were subjected to SDS-PAGE and probed with antiactivated MAP kinase Abs.

 
Taken together, these results indicate that treatment of HaCaT cells with 1,25-(OH)2D3 enhances proteoglycan-dependent autocrine EGFR-mediated signaling.

1,25-(OH)2D3 does not affect proteoglycan synthesis or sulfation
The stimulation of autonomous HaCaT cell proliferation by 1,25-(OH)2D3 may thus be due to qualitative or quantitative changes in cell-associated or secreted proteoglycans, the levels of members of the ErbB family that mediate EGFR-dependent signaling, or the levels of their ligands. The first alternative was examined by assaying the incorporation of 35S-labeled sulfate into the cell-associated and secreted proteoglycan pools in control and 1,25-(OH)2D3-treated cultures. As shown in Fig. 7Go, 1,25-(OH)2D3 did not affect either the level of 35SO4-2 incorporation or the elution profiles of proteoglycans from the ion exchange columns. These results rule out an effect of the hormone on total proteoglycan synthesis or secretion or a change in the relative abundance of differently charged proteoglycan species. As expected, sodium chlorate at the same concentration as that used in the proliferation assay markedly inhibited the total proteoglycan sulfation and also shifted the elution pattern from the ion exchange columns, increasing the proportion of lesser charged fractions.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 7. The effect of 1,25-(OH)2D3 on the incorporation of 35SO4-2 into cell-associated and secreted proteoglycans. Cells and cell extracts were prepared as described in Materials and Methods. Cells were treated 48 h before harvesting with 1,25-(OH)2D3 (100 nM) or for 24 h with sodium chlorate (20 mM). Cultures were pulsed for the last 24 h before harvesting with 50 µCi/ml 35SO4-2. Cell extracts (A) and culture media (B) were subjected to ion exchange chromatography as described in Materials and Methods.

 
1,25-(OH)2D3 does not affect the level of proteoglycan-dependent EGFR ligands
The major role of the proteoglycan-dependent EGFR ligands, amphiregulin and HB-EGF, in the autonomous proliferation of human keratinocytes is well established (12, 14, 15). Using immunoblot analysis, we show (Fig. 8Go) that both ligands are produced in HaCaT cells, and that their levels are not affected by treatment with 1,25-(OH)2D3. It should be noted that this lack of effect was observed in the same cell extracts where treatment with the hormone increased EGFR-dependent tyrosine phosphorylation.



View larger version (84K):
[in this window]
[in a new window]
  		Figure 8. Effect of 1,25-(OH)2D3 on the cellular level of proteoglycan-dependent EGFR ligands. Cells and cell extracts were prepared as described in Materials and Methods. Cells were harvested 72 h after medium replacement. 1,25-(OH)2D3 (100 nM) was added 48 h before harvesting. Cell extracts were subjected to SDS-PAGE and probed with antiamphiregulin and anti-HB-EGF Abs. Lanes 1, 3, and 5, Control cultures; lanes 2, 4, and 6, 1,25-(OH)2D3-treated cultures.

 
1,25-(OH)2D3 increases the cellular level of ErbB family members
Having ruled out an effect of the hormone on either proteoglycan synthesis or the levels of EGFR proteoglycan-dependent ligands, we have proceeded to examine the effect of the hormone on members of the ErbB family. Our first task was to identify the member(s) of the ErbB family responsible for signaling in 1,25-(OH)2D3-treated HaCaT cells. We have shown in Fig. 5Go that treatment with 1,25-(OH)2D3 enhanced the tyrosine phosphorylation of two closely migrating proteins in the 180–190 kDa range. In Fig. 9AGo, we show that the high molecular mass band of the doublet comigrates in SDS-PAGE with ErbB2 and ErbB3, and both proteins have a higher mol wt than ErbB1 (i.e. EGFR). A similar electrophoretic pattern of the tyrosine-phosphorylated proteins was observed in at least five different experiments, and we never detected a tyrosine-phosphorylated protein with a molecular mass corresponding to that of EGFR. It is thus probable that EGFR-dependent signaling in autonomously proliferating HaCaT cells treated with 1,25-(OH)2D3 is mediated by a heterodimer of EGFR with either ErbB2 or ErbB3. It is noteworthy that the level of ErbB2 protein in HaCaT cells is very low (Fig. 9AGo) and is similar to the level present in normal keratinocytes (33). Moreover, we compared ErbB2 levels in HaCaT cells to those present in MCF-7 breast cancer cells known to express very low level of this receptor (34) and found that the level in HaCaT cells was at least 5-fold lower (data not shown). The composition of the major signaling dimer in 1,25-(OH)2D3-treated autonomously proliferating HaCaT cells was further examined by immunoprecipitation with specific antibodies to ErbB2 or ErbB3 and probing the immunoprecipitates with an antiphosphotyrosine antibody (Fig. 9BGo). It was found that ErbB3, but not ErbB2, is tyrosine phosphorylated in these cultures. Thus, it may be inferred that the heterodimer responsible for the EGFR-dependent tyrosine phosphorylation in this system is ErbB1-ErbB3. This idea was further supported by demonstrating that ErbB1 is coimmunoprecipitated with ErbB3 using an anti-ErbB3 antibody (Fig. 9BGo), indicating physical interaction between these two proteins. This conclusion explains our finding (Fig. 5Go) that ErbB1 is not tyrosine phosphorylated in 1,25-(OH)2D3-treated cultures, in accord with the known lack of tyrosine kinase activity in ErbB3 (18). It is noteworthy that in parallel cultures we established that 30-min incubation of the cells with exogenous EGF (20 ng/ml) caused pronounced tyrosine phosphorylation in a band comigrating with EGFR (data not shown), suggesting that under such conditions the ErbB1 homodimer is the major signaling molecule in HaCaT cells.



View larger version (65K):
[in this window]
[in a new window]
  		Figure 9. Identification of the tyrosine-phosphorylated ErbB protein(s) in 1,25-(OH)2D3-treated HaCaT cells. Cells and cell extracts were prepared as described in Materials and Methods. Cells were harvested 72 h after medium replacement. Cells were treated with 1,25-(OH)2D3 (100 nM) for 48 h before harvesting. A, Cell extracts were subjected to SDS-PAGE and probed with antiphosphotyrosine, ErbB1, ErbB2, or ErbB3 Abs (lanes 1–4, respectively). B, ErbB2 and ErbB3 proteins were immunoprecipitated from 1,25-(OH)2D3-treated cell extracts, subjected to SDS-PAGE, and probed with anti ErbB1, ErbB2, or ErbB3 or with antiphosphotyrosine Abs.

 
We have proceeded to examine the effect of hormone treatment on the cellular level of the proteins belonging to the ErbB family and found that treatment with the hormone caused a time-dependent increase in ErbB1 levels after 16-h incubation (Fig. 10AGo). Forty-eight-hour incubation with 1,25-(OH)2D3 (100 nM) resulted in an average increase of 100 ± 37%, as quantified by laser densitometry (mean ± SEM; P < 0.05; five independent experiments). In parallel experiments we also found a smaller, but consistent, increase in ErbB2 and ErbB3 levels (Fig. 10BGo). The average increase was 53 ± 6% (P < 0.01; n = 5) for ErbB2 and 69 ± 20% (P < 0.05; n = 4) for ErbB3. As the ErbB1-ErbB3 heterodimer seems to be responsible for the signaling induced by the autocrine EGFR ligands, the increase in the levels of these two ErbB family after treatment with 1,25-(OH)2D3 can account for the stimulation of HaCaT cell proliferation.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 10. Effect of 1,25-(OH)2D3 on the cellular level of the ErbB proteins. Cells and cell extracts were prepared as described in Materials and Methods. Cells were harvested 72 h after medium replacement. 1,25-(OH)2D3 (100 nM) was added at different time points (upper panel) or 48 h (lower panel) before harvesting. Cell extracts were subjected to SDS-PAGE and probed with anti-ErbB1, -ErbB2, or -ErbB3 Abs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most salient findings of this study can be summarized as follows. 1) 1,25-(OH)2D3 enhances the proliferation of HaCaT keratinocytes in the absence of exogenous growth factors. 2) Both autonomous and 1,25-(OH)2D3-stimulated proliferation are mediated by the autocrine network of proteoglycan-dependent EGFR ligands. 3) The stimulatory effect of the hormone is probably due to up-regulation of proteins belonging the ErbB family of tyrosine kinase receptors.

The aim of this work was to study the effect of 1,25-(OH)2D3 on the autocrine network of growth factors, which is a major determinant of keratinocyte proliferation in vivo and in vitro (12). The addition of exogenous growth factors and ill defined medium components such as pituitary extract to normal keratinocyte primary cultures is obligatory for their maintenance. This complex mixture includes mediators that may activate major cellular pathways, including protein kinase C and protein kinase A. Such activation may have quantitative and qualitative effects on the mitogenic autocrine network (35, 36) and its regulation by nuclear receptor agonists, including 1,25-(OH)2D3 (37). As the activity of this autocrine network is sufficient to support HaCaT cell proliferation in the absence of any exogenous active mediators, these cells provide an appropriate system to study the regulation of the signaling pathways that mediate the action of the autocrine growth factors. Using this experimental system we found that 1,25-(OH)2D3 enhanced autonomous keratinocyte proliferation. In this system we found no evidence for the inhibitory or biphasic effect of 1,25-(OH)2D3 observed in most studies with normal keratinocytes (2, 3, 4, 5). It is possible that the manifestation of the inhibitory potential of 1,25-(OH)2D3 depends upon the presence of exogenous active mediators in the culture milieu. The growth-promoting effect of 1,25-(OH)2D3 was dose dependent and significant at the physiological concentration of 0.1 nM. As keratinocytes may produce 1,25-(OH)2D3 in vivo, it is probable that local concentrations of the hormone in the skin exceed those in the circulation.

Using pharmacological tools such as a neutralizing EGFR antibody (Ab-225) and a specific EGFR-dependent tyrosine kinase inhibitor (AG 1478), we show that proliferation and mitogenic signaling in both control and 1,25-(OH)2D3-treated cells are mediated by EGFR. Our finding that Ab-225 inhibited proliferation only when added before cell clustering has occurred suggests a major role for juxtacrine signaling via the EGFR (28). Additional support for this idea was gained by the absence of any detectable mitogenic activity in a 10-fold concentrated conditioned medium of 5-day HaCaT cultures treated or untreated with 1,25-(OH)2D3 (data not shown).

The major role of the proteoglycan-dependent subfamily of EGFR ligands in the maintenance of autonomous HaCaT cell proliferation was established by showing that heparin, hexadimethrine, and chlorate (15, 30, 32) markedly inhibited autonomous and 1,25-(OH)2D3-stimulated HaCaT cell proliferation. The finding that even at the highest heparin concentration there was still residual proliferation in the cultures treated with 1,25-(OH)2D3 may be due to the proteoglycan mimetic activity of heparin (31), although a minor contribution of a nonproteoglycan-dependent ligand to the proliferation of hormone-treated cells cannot be ruled out.

As we have found no qualitative differences between the autocrine mitogenic signaling in 1,25-(OH)2D3-treated and untreated cells, the effect of the hormone is probably due to quantitative differences in the cellular levels of proteoglycans, EGFR ligands, or ErbB receptors. Mitogenic signaling by the proteoglycan-dependent growth factors may be affected both by soluble and cell-associated proteoglycans. The possibility that 1,25-(OH)2D3 modulates proteoglycan synthesis or secretion was worth exploring, because these processes are affected by the hormone in chondrocytes (38). We found that 1,25-(OH)2D3 did not affect either in HaCaT cells. In addition, we did not observe any effect on the cellular levels of amphiregulin and HB-EGF, the two main proteoglycan-dependent EGFR ligands known to support normal keratinocyte proliferation in cell and organ culture (12, 14, 15).

The tyrosine phosphorylation pattern of the autonomously proliferating and 1,25-(OH)2D3-treated cells consistently revealed two closely migrating phosphorylated proteins with molecular masses of 180–190 kDa. The phosphorylation of these proteins, which is EGFR and proteoglycan dependent, was enhanced by 1,25-(OH)2D3. There was no apparent phosphorylation at the molecular mass corresponding to the EGFR (170 kDa), although a 170-kDa band was strongly phosphorylated after 30-min exposure to exogenous EGF (data not shown). Comigration and imunoprecipitation experiments demonstrated that the ErbB1-ErbB3 heterodimer is the major signal transducing entity in this system. Such a signaling pathway may also account for the lack of tyrosine phosphorylation of the EGFR, as the tyrosine kinase of ErbB3 is defective. It is commonly accepted that ErbB2 is the preferred heterodimerization partner of all ErbB receptors (19). The absence of detectable tyrosine-phosphorylated ErbB2 in HaCaT cells under our conditions could be due to the very low expression of this protein compared with that of the other family members.

We found that all three ErbB proteins are up-regulated after exposure to 1,25-(OH)2D3. The modest, although significant and reproducible, increase in the cellular level of both constituents of the signaling heterodimer can account for the increased efficiency of mitogenic signaling resulting in enhanced proliferation. As shown previously, the increase in ErbB2 levels could also contribute indirectly to the formation of an ErbB1-ErbB3 heterodimer (19). The increase in ErbB1 levels results in more ternary ligand-proteoglycan-EGFR complexes in 1,25(OH)2D3-treated cells and may thus account for the reduced sensitivity of these cells to the inhibitory effects of heparin, hexadimethrine, chlorate, AG 1478, and Ab-225. One intriguing observation in this study was that the proliferation of 1,25-(OH)2D3-treated HaCaT cells was consistently higher than that of HaCaT cells grown in the presence of a saturating (20 ng/ml) concentration of exogenous EGF. This may be due to the more efficient mitogenic signaling via the ErbB1-ErbB3 heterodimer compared with signaling via the EGFR homodimer (18), the major species in the presence of exogenous EGF. In addition, juxtacrine signaling via the EGFR seems to be more efficient and sustained than signaling mediated by a soluble growth factor (39).

In previous studies 1,25-(OH)2D3 was shown to increase, decrease, or have no effect on EGFR levels in various cellular systems (40, 41, 42). The effect of the hormone may thus strongly depend on the cellular context. It is noteworthy that most previous evidence relies on binding assays with radiolabeled EGF that measure the number of unoccupied receptors and cannot detect the receptors occupied by autocrine and juxtacrine ligands. To our knowledge there are no previous reports about the effect(s) of 1,25-(OH)2D3 on ErbB2 or ErbB3 levels.

Signaling via the EGFR is known to result in the activation of MAP kinase (18). As shown in Fig. 6Go, activated MAP kinase (ERK1 and ERK2) is present in autonomously proliferating HaCaT cells and, as expected, is markedly elevated in 1,25-(OH)2D3-treated cells. Gniadecky (43) has previously shown that exposure of primary keratinocyte cultures to 1,25-(OH)2D3 induced a rapid and transient (lasting 60 min) activation of MAP kinase. We do not know if and how this early event is associated with the long lasting effects reported here. The increase in the levels of activated MAP kinase in our system is time dependent, long lasting (at least 48 h), and totally dependent on the activity of EGFR tyrosine kinase. Prolonged activation of MAP kinase, as described here, is probably required for some events essential for cell cycle entry (44).

In view of the critical role for autocrine signaling of proteoglycan-dependent EGFR ligand in keratinocyte proliferation in general and wound healing in particular (12, 45), our finding that 1,25-(OH)2D3 up-regulates this system may have physiological and clinical implications.


   Acknowledgments
 
We thank Prof. Y. Yarden and A. Yayon of The Weizmann Institute of Science for their assistance and helpful discussions, and Prof. A. Levitzki of The Hebrew University for kindly providing us with AG 1478.


   Footnotes
 
1 This work was performed in partial fulfillment of the requirements for the Ph.D. degree of Osnat Garach-Jehoshua, Sackler Faculty of Medicine, Tel Aviv University (Tel Aviv, Israel). Back

Received May 26, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bikle DD, Pillai S 1993 Vitamin D, calcium and epidermal differentiation. Endocr Rev 14:3–19[CrossRef][Medline]
  2. Itin PH, Pittelkow MR, Kumar R 1994 Effects of vitamin D metabolites on proliferation and differentiation of cultured human epidermal keratinocytes grown in serum-free or defined culture medium. Endocrinology 135:1793–1798[Abstract]
  3. Bollag WB, Ducote J, Harmon CS 1995 Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation. J Cell Physiol 163:248–256[CrossRef][Medline]
  4. Gniadecki R 1996 Stimulation versus inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D3: dependence on cell culture conditions. J Invest Dermatol 106:510–516[CrossRef][Medline]
  5. Svendsen ML, Daneels G, Geysen J, Binderup L, Kragballe K 1997 Proliferation and differentiation of cultured human keratinocytes is modulated by 1,25(OH)2D3 and synthetic vitamin D3 analogues in a cell density-, calcium- and serum-dependent manner. Pharmacol Toxicol 80:49–56[Medline]
  6. Thieroff-Ekerdt R, Gerhard D, Krause A, Wiesinger H 1993 1,25-Dihydroxyvitamin D3 induces epidermal hyperplasia in normal mouse skin. J Invest Dermatol 100:s592
  7. Levy J, Gassmuller J, Schroder G, Audring H, Sonnichsen N 1994 Comparison of the effects of calcipotriol, prednicarbate and clobetasol 17-propionate on normal skin assessed by ultrasound measurement of skin thickness. Skin Pharmacol 7:231–236[Medline]
  8. Lutzow-Holm C, De Angelis P, Clausen OPF 1996 Calcitriol and its analog KH 1060 induce similar changes in keratinocyte cell cycle progression after topical application to mouse skin. A bromodeoxyuridine pulse-chase flow cytometric study. J Invest Dermatol Symp Proc 1:54–59
  9. Gniadecki R, Serup J 1995 Stimulation of epidermal proliferation in mice with 1{alpha},25-dihydroxyvitamin D3 and receptor-active 20-epi analogues of 1{alpha},25-dihydroxyvitamin D3. Biochem Pharmacol 49:621–624[CrossRef][Medline]
  10. Tian XQ, Chen TC, Holick MF 1995 1,25-dihydroxyvitamin D3: a novel agent for enhancing wound healing. J Cell Biochem 59:53–56[CrossRef][Medline]
  11. Gniadecki R 1994 A vitamin D analogue KH 1060 activates the protein kinase C-c-fos signalling pathway to stimulate epidermal proliferation in murine skin. J Endocrinol 143:521–525[Abstract]
  12. Stoll S, Garner W, Elder J 1997 Heparin-binding ligands mediate autocrine epidermal growth factor receptor activation in skin organ culture. J Clin Invest 100:1271–1281[Medline]
  13. Woodworth CD, McMullin E, Iglesias M, Plowman GD 1995 Interleukin 1{alpha} and tumor necrosis factor {alpha} stimulate autocrine amphiregulin expression and proliferation of human papilloma virus-immortalized and carcinoma-derived cervical epithelial cells. Proc Natl Acad Sci USA 92:2840–2844[Abstract/Free Full Text]
  14. Hashimoto K, Higashiyama S, Asada H, Hashimura E, Kobayashi T, Sudo K, Nakagawa T, Damm D, Yoshikawa K, Taniguchi N 1994 Heparin-binding EGF-like growth factor is an autocrine growth factor for human keratinocytes. J Biol Chem 269:20060–20066[Abstract/Free Full Text]
  15. Piepkorn M, Lo C, Plowman GD 1994 Amphiregulin-dependent proliferation of cultured human keratinocytes: autocrine growth, the effects of exogenous recombinant cytokine, and apparent requirement for heparin-like glycosaminoglycans. J Cell Physiol 159:114–120[CrossRef][Medline]
  16. Beerli RR, Hynes NE 1996 Epidermal growth factor related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. J Biol Chem 271:6071–6076[Abstract/Free Full Text]
  17. Marikovsky M, Lavi S, Pinkas-Kramarsky R, Karunagaran D, Liu N, Wen D, Yarden Y 1995 ErbB-3 mediates differential mitogenic effects of NDF/heregulin isoforms on mouse keratinocytes. Oncogene 10:1403–1411[Medline]
  18. Alroy I, Yarden Y 1997 The ErbB signaling network in embryogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 410:83–86[CrossRef][Medline]
  19. Graus-Porta D, Beerli RR, Daly JM, Hynes NE 1997 ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 16:1647–1655[CrossRef][Medline]
  20. Boukamp P, Petrusevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE 1988 Normal keratinization in a spontaneously immortalized aneuploid keratinocyte cell line. J Cell Biol 106:761–771[Abstract/Free Full Text]
  21. Klapper LN, Vaisman N, Hurwitz E, Pinkas-Kramarsky R, Yarden Y, Sela M 1997 A subclass of tumor-inhibitory monoclonal antibodies to ErbB-2/HER2 blocks crosstalk with growth factor receptors. Oncogene 14:2099–2109[CrossRef][Medline]
  22. Saati N, Ravid A, Liberman UA, Koren R 1997 1,25-Dihydroxyvitamin D3 and agents that increase intracellular cAMP synergistically inhibit fibroblast proliferation. In Vitro Cell Dev Biol Anim 33:310–314[Medline]
  23. Svennevig K, Prudz K, Kolset SO 1995 Proteoglycans in polarized epithelial Madin-Darby canine kidney cells. Biochem J 311:881–888
  24. Minamide LS, Bamburg JR 1990 A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents. Anal Biochem 190:66–70[CrossRef][Medline]
  25. Peus D, Hamacher L, Pittelkow MR 1997 EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation. J Invest Dermatol 109:751–756[CrossRef][Medline]
  26. Osherov N, Levitzki A 1994 Epidermal growth factor dependent activation of the src-family kinases. Eur J Biochem 225:1047–1053[Medline]
  27. Sunada H, Magun BE, Mendelsohn J, Macleod CL 1986 Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation. Proc Natl Acad Sci USA 83:3825–3829[Abstract/Free Full Text]
  28. Bosenberg MW, Massague J 1993 Juxtacrine cell signaling molecules. Curr Opin Cell Biol 5:832–838[CrossRef][Medline]
  29. Higashiyama S, Iwamoto R, Goishi K, Raab G, Taniguchi N, Klagsbrun M, Mekada E 1995 The membrane protein CD9/DRAP 27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin binding EGF-like growth factor. J Cell Biol 128:929–938[Abstract/Free Full Text]
  30. Cook PW, Ashton NM, Karkaria CE, Siess DC, Shipley GD 1995 Differential effects of a heparin antagonist (hexadimethrine) or chlorate on amphiregulin, basic fibroblast growth factor, and heparin-binding EGF-like growth factor activity. J Cell Physiol 163:418–429[CrossRef][Medline]
  31. Aviezer D, Yayon A 1994 Heparin dependent binding and autophosphorylation of epidermal growth factor (EGF) receptor by heparin-binding EGF-like growth factor but not by EGF. Proc Natl Acad Sci USA 91:12173–12177[Abstract/Free Full Text]
  32. Besner GE, Whelton D, Crissman-Combs MA, Steffen CL, Kim GY, Brigstock DR 1992 Interaction of heparin-binding EGF-like growth factor with epidermal growth factor receptor: modulation by heparin, heparinase, or synthetic heparin binding HB-EGF fragments. Growth Factors 7:289–296[Medline]
  33. Ahmed NU, Ueda M, Ichihashi M 1997 Increased level of c-erbB-2/neu/HER-2 protein in cutaneous squamous cell carcinoma. Br J Dermatol 136:908–912[CrossRef][Medline]
  34. Wright M, Grim J, Deshane J, Kim M, Strong TV, Siegal GP, Curiel DT 1997 An intracellular anti-erbB-2 single-chain antibody is specifically cytotoxic to human breast carcinoma cells overexpressing erbB-2. Gene Ther 4:317–322[CrossRef][Medline]
  35. Emkey R, Kahn CR 1997 Cross-talk between phorbol-mediated signaling and tyrosine kinase proto-oncogenes. I. Activation of protein kinase C stimulates tyrosine phosphorylation and activation of ErbB2 and ErbB3. J Biol Chem 272:31172–31181[Abstract/Free Full Text]
  36. Tortora G, Damiano V, Bianco C, Baldassarre G, Bianco AR, Lanfrancone L, Pelicci PG, Ciardiello F 1997 The Rialpha subunit of protein kinase A (PKA) binds to Grb2 and allows PKA interaction with the activated EGF-receptor. Oncogene 14:923–928[CrossRef][Medline]
  37. Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG, Juguilon H, Montminy M, Evans RM 1996 Role of CBP/P300 in nuclear receptor signalling. Nature 383:99–103[CrossRef][Medline]
  38. Gerstenfeld LC, Kelly CM, Von Deck M, Lian JB 1990 Effect of 1,25-dihydroxyvitamin D3 on induction of chondrocyte maturation in culture: extracellular matrix gene expression and morphology. Endocrinology 126:1599–1609[Abstract]
  39. Ito Y, Li JS, Takahashi T, Imanishi Y, Okabayashi Y, Kido Y, Kasuga M 1997 Enhancement of the mitogenic effect by artifical juxtacrine stimulation using immobilized EGF. J Biochem (Tokyo) 121:514–520[Abstract/Free Full Text]
  40. Matsumoto K, Hashimoto K, Nishida Y, Hashiro M, Yoshikawa K 1990 Growth inhibitory effects of 1,25-dihydroxyvitamin D3 on normal human keratinocytes cultured in serum-free medium. Biochem Biophys Res Commun 166:916–923[CrossRef][Medline]
  41. Deprez PY, Poujol D, Falette N 1991 1,25-dihydroxyvitamin D3 increases epidermal growth factor receptor gene expression in BT-20 breast carcinoma cells. Biochem Biophys Res Commun 176:1–6[CrossRef][Medline]
  42. Carthy EP, Yamashita W, Hsu A, Ooi BS 1989 1,25-Dihydroxyvitamin D3 and rat vascular smooth muscle cell growth. Hypertension 13:954–959[Abstract/Free Full Text]
  43. Gniadecki R 1996 Activation of Raf-mitogen-activated protein kinase signaling pathway by 1,25-dihydroxyvitamin D3 in normal human keratinocytes. J Invest Dermatol 106:1212–1217[CrossRef][Medline]
  44. Lavoie JN, L’Allemain G, Brunet A, Muller R, Pouyssegur J 1996 Cyclin D1 expression is regulated positively by the p42/44MAPK and negatively by the p38/HOGMAPK pathway. J Biol Chem 271:20608–20616[Abstract/Free Full Text]
  45. Marikovsky M, Breuing K, Liu PY, Eriksson E, Higashiyama S, Farber P, Abraham J, Klagsbrun M 1993 Appearance of a heparin-binding EGF-like growth factor in wound fluid as a response to injury. Proc Natl Acad Sci USA 90:3889–3893[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. M. Casalino-Matsuda, M. E. Monzon, G. E. Conner, M. Salathe, and R. M. Forteza
Role of Hyaluronan and Reactive Oxygen Species in Tissue Kallikrein-mediated Epidermal Growth Factor Receptor Activation in Human Airways
J. Biol. Chem., May 14, 2004; 279(20): 21606 - 21616.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
S. D. Billings, M. D. Southall, T. Li, P. W. Cook, L. Baldridge, W. B. Moores, D. F. Spandau, J. G. Foley, and J. B. Travers
Amphiregulin Overexpression Results in Rapidly Growing Keratinocytic Tumors: An In Vivo Xenograft Model of Keratoacanthoma
Am. J. Pathol., December 1, 2003; 163(6): 2451 - 2458.
[Abstract] [Full Text]

Home page
 Endocr. Rev.Home page
A. Gurlek, M. R. Pittelkow, and R. Kumar
Modulation of Growth Factor/Cytokine Synthesis and Signaling by 1{alpha},25-Dihydroxyvitamin D3: Implications in Cell Growth and Differentiation
Endocr. Rev., December 1, 2002; 23(6): 763 - 786.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garach-Jehoshua, O.
Right arrow Articles by Koren, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garach-Jehoshua, O.
Right arrow Articles by Koren, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.