From the Department of Pathology, Anatomy and Cell
Biology, Jefferson Medical College, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107, the ¶ Center for Extracellular
Matrix Biology, Institute of Biosciences and Technology, Texas A & M
University, Houston, Texas 77030, and the
Kimmel Cancer Center,
Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Several independent lines of evidence have implicated decorin, a small leucine-rich proteoglycan, in the inhibition of cell proliferation. However, the mechanism by which decorin mediates its effect on cell proliferation is unclear. Here we report, for the first time, decorin-mediated increases in intracellular Ca2+ levels of single A431 cells. The effects of decorin persisted in the absence of extracellular Ca2+ but were blocked by AG1478, an epidermal growth factor (EGF)-specific tyrosine kinase inhibitor, and by down-regulation of the EGF receptor. The effects of decorin were not mimicked by the structurally homologous protein, biglycan. Our results indicate a novel action of decorin on the EGF receptor, which results in mobilization of intracellular Ca2+ providing a possible mechanism by which decorin causes growth suppression.
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INTRODUCTION |
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Stimulation of phospholipase C
(PLC)1 by cell surface
receptors results in the generation of the second messenger inositol 1,4,5-trisphosphate and consequent activation of Ca2+
channels located within the membrane of intracellular Ca2+
stores (1). Growth factors, such as epidermal growth factor (EGF)
activate receptor tyrosine kinases, which dimerize, autophosphorylate, and generate inositol 1,4,5-trisphosphate via phosphorylation and
consequent stimulation of the PLC- isoform (2). The ensuing increases in the cytosolic Ca2+ concentration
([Ca2+]i), which are often both temporally and
spatially complex (3), are implicated in a wide range of cellular
processes.
Decorin is a key regulator of extracellular matrix assembly and is a member of an expanding family of proteoglycans characterized by a central domain with tandem leucine-rich repeats (4). Molecular modeling has revealed an arch-shaped molecule with a concave surface capable of interacting with various other proteins (5). Targeted disruption of the decorin gene leads to mice with a skin fragility phenotype and altered collagen fibril morphology (6), thereby providing genetic evidence that decorin is a "modifier" of collagen fibrillogenesis during development.
Increasing evidence suggests an inhibitory role for this secreted proteoglycan in cell proliferation (7). In a variety of cell types, overexpression of decorin inhibits cell growth (8, 9). Furthermore, levels of decorin are markedly increased in quiescent cells and reduced in transformed cells (10, 11). Decorin may also play an important role in tumorigenesis, because not only is decorin expression decreased in various tumor cell types, but secretion of decorin by stromal cells surrounding tumors is increased (12). This may represent a response by the host to reduce the rate of carcinoma spread.
Although decorin overexpression is associated with an up-regulation of p21 (13), an inhibitor of cyclin-dependent kinases required for progression through the cell cycle (14), upstream targets for decorin, and the exact mechanism by which decorin inhibits cellular growth are unknown. Recently, we have shown that decorin stimulates phosphorylation of the EGF receptor and activation of the mitogen-activated protein (MAP) kinase pathway (15). Because EGF receptors can couple to PLC, decorin may also elevate [Ca2+]i, implicating a role for Ca2+ in the inhibitory effects of decorin on cell proliferation. Here, we report for the first time that activation of the EGF receptor by decorin results in elevation of [Ca2+]i in single A431 squamous carcinoma cells. Therefore, Ca2+ signals may be important in mediating decorin-induced growth inhibition, which ultimately may retard tumor progression in vivo.
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EXPERIMENTAL PROCEDURES |
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Materials-- Human squamous carcinoma A431 cells were obtained from ATCC (Rockville, MD). Highly purified human recombinant decorin and biglycan synthesized by human fibrosarcoma HT1080 cells using the vaccinia virus/T7 phage expression system were purified as described previously (16, 17). Fura-2 and Pluronic F-127 acid were from Teflabs, ionomycin was from Calbiochem, and human recombinant EGF was from Life Technologies, Inc. All other reagents were from Sigma or Fisher.
Cell Culture-- A431 cells were cultured on glass coverslips coated with poly-D-lysine (5 µg/cm3) and maintained at 37 °C in Dulbecco's modified Eagle's medium containing 5% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 50 µg/ml streptomycin in an atmosphere of CO2/air (5%:95%). At 70% confluence, the cells were cultured for a further 48 h in serum-free medium prior to imaging experiments.
Single Cell Ca2+ Imaging-- Imaging experiments were performed at room temperature in a medium composed of 121 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 10 mM glucose, 2 mM CaCl2, 0.25% (w/v) bovine serum albumin, and 20 mM HEPES (pH 7.4 at room temperature). [Ca2+]i was determined by loading the cells with fura-2 by incubation with fura-2/acetoxymethyl ester (5 µM) in the presence of Pluronic F-127 acid (0.02% w/v) for 20 min. Coverslips were then mounted on an inverted epifluorescence microscope, and fura-2 was excited at 340 and 380 nm and emission (420-600 nm) collected using a charged coupled device camera. Autofluorescence was determined following quench of fura-2 by Mn2+ in Ca2+-free medium supplemented with 1 mM MnCl2 and 2 µM ionomycin. The ratio of the fluorescence at 340 and 380 nm was converted to [Ca2+] according to Grynkiewicz et al. (18).
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RESULTS AND DISCUSSION |
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Decorin Increases [Ca2+]i in Single A431
Cells--
We have previously shown that decorin-mediated growth
inhibition in A431 cells is associated with phosphorylation of the EGF receptor (15). Generally, EGF stimulates cell growth and proliferation, but the proliferation of certain cells is paradoxically inhibited by
this growth factor (19). The prototype of such cells is the A431 human
squamous carcinoma cell, which expresses a high number of EGF receptors
(~2 × 106/cell) (20). Because activation of EGF
receptors results in autophosphorylation and can potentially stimulate
PLC-, we examined the effects of decorin on
[Ca2+]i in individual fura-2-loaded A431 cells.
Fig. 1 demonstrates typical
[Ca2+]i increases in response to stimulation of
A431 cells with decorin. From five separate cell passages, decorin
increased [Ca2+]i in 49 ± 12% of cells. In
the 498 responding cells, [Ca2+]i increased from
a resting level of 30 ± 1 nM to a peak of 123 ± 3 nM. Similar responses were observed after removal of Ca2+ from the extracellular medium (Fig. 1C).
These results indicate that decorin mobilizes Ca2+
primarily from intracellular stores as opposed to directly stimulating Ca2+ entry across the plasma membrane.
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Decorin-induced Responses are Prevented by AG1478, a Specific
Inhibitor of EGF Receptor Tyrosine Kinase--
To establish whether
decorin-mediated increases in [Ca2+]i were via
activation of the EGF receptor, we examined the effects of the
tyrphostin AG1478, an EGF-selective tyrosine kinase inhibitor on
responses to EGF (21) and decorin (15). From nine separate cell
passages, EGF elevated [Ca2+]i in 90 ± 4%
of cells from a resting level of 28 ± 1 nM to a peak
level of 191 ± 3 nM (1369 responding cells out of 1492 cells, Fig. 3A).
Pretreatment of the cells with AG1478 (2 µM) reduced the
number of cells responding to EGF (Fig. 3B) by 97 ± 2% (27 responding cells out of 533 cells from five separate cell
passages). AG1478 completely abolished the
[Ca2+]i responses to decorin (0 responding cells
out of 499 cells from two separate cell passages; Fig. 3C).
Notably, we used a concentration of AG1478 that is about of
that required to inhibit c-src kinase activity (21) but that effectively blocks activation of the EGF receptor kinase and MAP kinase
caused by either EGF or exogenous decorin (9). Moreover, we found that
MAP kinase activation by platelet-derived growth factor was unaffected
by the same concentrations of AG1478 (9). In addition, AG1478 did not
affect [Ca2+]i responses to ATP. In five separate
cell passages, ATP (200 µM) elevated
[Ca2+]i by 776 ± 44 nM (907 cells) and 870 ± 14 nM (670 cells) in the absence and
the presence of AG1478, respectively. The inhibitory effects of AG1478
on EGF responses were reversed if AG1478 was washed out 45 min prior to
stimulation (not shown).
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Decorin Responses Are Inhibited by EGF Receptor Down-regulation-- We next examined the effects of EGF and decorin on [Ca2+]i in A431 cells in which EGF receptors had been down-regulated. Prolonged exposure of cells to EGF has been shown to reduce the number of EGF receptors at the cell surface (22). Under these conditions, the number of cells responding to EGF (Fig. 4A) and decorin (Fig. 4B) was reduced by 96 ± 3% (4 responding cells out of 255 cells from three separate cell passages) and 99% (3 responding cells out of 424 cells from two separate cell passages), respectively. Down-regulation of EGF receptors reduced [Ca2+]i increases in response to ATP by 58 ± 9%.
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ACKNOWLEDGEMENTS |
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We thank D. Moscatello, A. Morgan, and L. D. Robb-Gaspers for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants RO1 DK38422 (to A. P. T.), RO1 CA39481 (to R. V. I.), and RO1 CA47282 (to R. V. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a Wellcome Prize Traveling Research Fellowship (UK).
** To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Jefferson Alumni Hall, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. Fax: 215-923-2218; E-mail: Iozzo{at}lac.jci.tju.edu or Thomasap{at}umdnj.edu.
1 The abbreviations used are: PLC, phospholipase C; EGF, epidermal growth factor; MAP, mitogen-activated protein.
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REFERENCES |
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