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a Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our original observations identifying apo E as an active agent were made through measuring the effect of apo E-enriched high density lipoprotein (HDL) on androstenedione production by rat ovarian theca-interstitial cell preparations [3]. In this cell system, theca cells are the only androgen-producing cells and are therefore the target for the modulatory effects of apo E on steroidogenesis. To use a more defined reagent and not be limited by the amount of apo E available from recombinant or plasma sources, we developed apo E synthetic peptides that retain the biological activities of the native protein [4]. Using these synthetic peptides we determined that for them to be LDL-receptor active and mimic the native apo E modulation of ovarian theca cell androgen production, they needed to contain the LDL receptor-binding domain in a linear, tandem repeat dimer [4–6]. We have recently shown that the selective action of the apo E peptide acetyl-Y(141–155)2C on ovarian theca cell androgen production is due to changes in the rate of transcription of the gene for P450 17
-hydroxylase, C17–20 lyase [7].
Because the apo E peptides that modulate androgen production by ovarian theca cells also bind to the LDL receptor and the LDL receptor-related protein (LRP), we postulated that the peptide effect on the rate of transcription of the gene for P450 17-hydroxylase, C17–20 lyase was mediated by members of the LDL receptor superfamily. This postulate is feasible because all known members of the LDL receptor superfamily are expressed within the rodent ovary [8]. Here, we used a number of experimental approaches to test the hypothesis that the apo E peptides act via members of the LDL receptor superfamily to alter the androgen production of ovarian theca cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whole ovary dispersates containing the theca cells were obtained from immature Sprague-Dawley rats that had been hypophysectomized at 21 days of age [3]. This cell preparation was contaminated with interstitial cells, but only the theca cells are able to produce androstenedione in response to LH stimulation. Animal care was in accordance with the Institutional Animal Care and Use Committee of Northern Arizona University. The cells were cultured at 40 000–50 000 cells per well of a 96-well plate (Costar, Cambridge, MA) in 0.25 ml of serum-free McCoy's 5a medium with penicillin and streptomycin (Life Technologies, Grand Island, NY) in a 95% air, 5% CO2 humidified incubator at 37°C [3]. Ovine LH (NIDDK-oLH-25; 2.3 U/mg, 1 U = activity of 1 mg of NIH-LH-S1) was generously provided by the National Hormone and Pituitary Program of the NIDDK (NIH, Bethesda, MD). The concentration of 1 ng/ml of LH was used in all experiments because it causes maximum theca cell androstenedione production [7]. Purified receptor-associated protein (RAP) was kindly provided by Dr. D. Strickland, American Red Cross, Rockville, MD. The apo E peptides were kindly provided by Dr. L. Curtiss, The Scripps Research Institute, La Jolla, CA.
Cell Number Determination
Changes in cell number due to culture conditions and the experimental treatments were detected using the Quantos cell proliferation assay kit according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). Briefly, the cells were lysed and incubated with the DNA-binding dye for 1 h. The signal was measured using a fluorescence microtiter plate reader and compared to standard curves using both purified DNA and freshly isolated theca-interstitial cells.
Recombinant Apo E and Preparation of the Apo E Peptides
Human recombinant apo E3 at 14 µM in 0.7 M ammonium bicarbonate (PanVera Corporation, Madison, WI) was dialyzed against PBS at room temperature before its addition to the cells.
The apo E peptides were synthesized and purified as previously described: Y(141–155)2, amino acid sequence Y(LRKLRKRLLRDADDLLRKLRKRLLRDADDL); acetyl-Y(141–155)2C, amino acid sequence acetyl-Y(LRKLRKRLLRDADDLLRKLRKRLLRDADDL)C; and apo E (129–162), amino acid sequence (STEELRVRLASHLRKLRKRLLRDADDLQKRLAVY) [6, 9]. The expected molecular weight of each peptide was confirmed by mass spectrometry. The peptide purities and contents of Y(141–155)2, acetyl-Y(141–155)2C, and (129–162) were 93% and 65%, 90% and 52%, and 98% and 60%, respectively. The lyophilized apo E peptides were reconstituted in water at a concentration range of 300–500 µM, dialyzed against water in 1000 molecular weight cut-off tubing to remove contaminating salts, sterile filtered, and stored in the dark at -20°C until they were used.
Treatments Used to Modify Cellular Heparan Sulfate Proteoglycans (HSPG)
Freshly isolated ovarian theca-interstitial cells were treated with heparan sulfate-degrading enzymes for 2 h at 37°C according to published protocol [9–12] before LH and the apo E peptide were added. Heparinase I and heparinase III (Sigma Chemical Co., St. Louis, MO; cat. nos. H 2519 and H 8891, respectively) were used at a final concentration of 3 U/ml, and phosphatidylinositol-specific phospholipase C (Sigma; cat. no. P 5542) was used at a final concentration of 1.0 U/ml. We left the enzymes on the cells, to suppress the cells' restoration of their HSPG, as described by Murano et al. [11]. Heparin (Sigma; cat. no. H 9399) was added to the cells at a final concentration of 100 µg/ml [11].
Analysis of Steroid Production
After 48 h of culture, the supernatants were removed and stored at -20°C until assayed in specific RIAs to measure the content of androstenedione and progesterone [3]. Assay results were calculated by a four-parameter logistic analysis using the software AssayZap (Biosoft, Ferguson, MO). Each point was done in quadruplicate, and the results are representative of at least two individual experiments. Data are shown as the mean ± SEM; the results marked with an asterisk were statistically different (p < 0.05) as determined by Student's t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The apo E peptide-mediated effects on the androstenedione production by theca cells were compared to those of native full-length apo E3. As shown in Figure 1, recombinant apo E3 was similar to the apo E peptide acetyl-Y(141–155)2C in modulating androstenedione production, while progesterone production was unchanged (data not shown). Also, the apo E peptide acetyl-Y(141–155)2C was more potent than the recombinant apo E3: lower concentrations of the peptide were needed for both its stimulation and inhibition of androstenedione production by theca cells. The apo E synthetic peptide acetyl-Y(141–155)2C caused maximum stimulation of androstenedione production at 0.037 µM, while maximum stimulation occurred with the recombinant apo E3 at a 3-fold higher concentration of 0.11 µM. The apo E synthetic peptide acetyl-Y(141–155)2C was even more effective at inhibiting androstenedione production than the recombinant apo E3. Fifty percent inhibition of androstenedione production occurred at 0.33 µM with the apo E synthetic peptide acetyl-Y(141–155)2C, but at the 5-fold greater concentration of 1.8 µM with the recombinant apo E3. Changes in androstenedione levels mediated by apo E were due to its primary effect on the androgen-producing theca cells; but because the cell preparation contains both theca and theca-derived interstitial cells, the theca cell responses have to be considered in the context of the mixed cell population. The apo E peptide acetyl-Y(141–155)2C mimicked the stimulatory and inhibitory effects of full-length, native apo E on androstenedione production; this was consistent with our original report of the activity of apo E in rat HDL [3]. Because the apo E peptide was equivalent in activity to native apo E, and because of practical considerations of cost and ease of working with the peptide relative to the native protein, we used apo E synthetic peptides in the remaining experiments.
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Apo E Peptide Acetyl-Y(141–155)2C-Mediated Stimulation of Androstenedione Production Was Blocked by RAP
As before, the apo E peptide acetyl-Y(141–155)2C had a biphasic effect on androstenedione production; concentrations 
0.10 µM were stimulatory, whereas at concentrations 
0.30 µM, the peptide was inhibitory (Fig. 2). At the apo E peptide concentration of 1.0 µM, androstenedione production was below the level of that seen with only LH stimulation. In contrast, progesterone production was unchanged by any concentration of the apo E peptide (data not shown). We tested the effect of RAP, a protein that blocks native apo E binding to all members of the LDL receptor superfamily [13], on the activities of the apo E peptide. The ovarian theca-interstitial cells were pretreated with RAP (100 nM) for 15 min before the addition of LH and the apo E peptide. RAP pretreatment reduced LH-stimulated androstenedione production of the control cells by 33% (Fig. 2) without altering the amount of progesterone made (data not shown). RAP prevented the apo E peptide-mediated stimulation of androstenedione production. But the apo E peptide-mediated inhibition of androstenedione was not altered: 50% inhibition occurred at the same 1 µM peptide concentration in the absence or presence of RAP (Fig. 2). These results suggested that the apo E peptide stimulated ovarian theca cell androstenedione production via one or more members of the LDL receptor superfamily, but none of these receptors appeared to be involved in mediating the inhibition of androstenedione production.
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Apo E Peptide Stimulation of Androstenedione Production Was Dependent on Its Ability to Bind to the Members of the LDL Receptor Superfamily
We wanted to determine whether the ability of the apo E peptide to stimulate ovarian theca cell androstenedione production was attributable to its ability to bind the LDL receptor. To test this we analyzed the activity of apo E peptide (129–162), which does not bind the LDL receptor. This peptide also serves as a control for peptide specificity because it is approximately the same size as the apo E peptide Y(141–155)2 and contains one repeat of amino acids 141–155, but, as mentioned above, does not bind to the LDL receptor [6]. The apo E peptide Y(141–155)2 at concentrations 0.5 µM stimulated androstenedione production, whereas the apo E peptide (129–162) did not stimulate androstenedione production (Fig. 3). Both apo E peptides inhibited androstenedione production, and the concentration of each peptide that inhibited androstenedione production by 50% was 1.0 µM for apo E peptide Y(141–155)2 and 3.0 µM for apo E peptide (129–162). These results suggested that for an apo E peptide to stimulate ovarian theca cell androstenedione production it must be able to bind to the LDL receptor ligand-binding domain. In contrast, the inhibition of androstenedione production was not dependent on the apo E peptide being LDL receptor active.
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Heparinase Degradation of Ovarian Theca-Interstitial Cell HSPG Prevented Apo E Synthetic Peptide Stimulatory Activity
Another test of the involvement of members of the LDL receptor superfamily in the apo E peptide acetyl-Y(141–155)2C stimulation of androstenedione production would be to define the role of cellular HSPG. Receptors of the LDL receptor superfamily have been shown to be dependent on HSPG for high-affinity binding of apo E [10, 14–16]. To determine whether the stimulatory activity of apo E peptide acetyl-Y(141–155)2C was dependent on intact HSPG, the cells were pretreated with heparinase I, or heparinase III, or heparinase I+heparinase III to degrade glycosaminoglycan chains of cell surface and secreted extracellular HSPG. On the basis of recent work we knew that apo E peptide acetyl-Y(141–155)2C acts in the first 12 h of culture to alter the rate of transcription of the gene for P450 17-hydroxylase, C17–20 lyase [7]. Thus, the degradative enzymes were left in the cultures so the cells could not restore their HSPG during the first 12 h of culture [11]. Because the heparinase pretreatments caused increased androstenedione production in control cells (Fig. 4, bar graphs), we expressed the effect of the apo E peptide on androstenedione production relative to that in the matched control cells. Cells that had been treated with heparinase I, heparinase III, or the combination of the two heparinases for 2 h before the addition of LH and apo E peptide did not have increased androstenedione production in response to the concentrations of the peptide 0.3 µM (Fig. 4). The inhibitory effect of the peptide at the highest concentration of 3.0 µM was slightly blunted in cells treated with heparinase III alone or in combination with heparinase I. Progesterone production was unchanged with all treatments (data not shown). These results suggested that the apo E peptide stimulation of androstenedione production was dependent on the integrity of the theca-interstitial cell HSPG.
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Apo E Peptide Activity Was Not Dependent on Glycosyl-Phosphotidylinositol-Linked HSPG
To prove that the integrity of specific HSPG was required, and that the suppressed apo E peptide stimulatory response in the presence of heparinase was not due to the addition of a degradative enzyme to the cells, we treated the cells with phosphotidylinositol-specific phospholipase C (PI-PLC). This enzyme cleaves the glycosyl-phosphotidylinositol (GPI) linkage that anchors a specific class of cell surface HSPG [17]. PI-PLC pretreatment reduced ovarian theca control cell androstenedione production by 40% (Fig. 5, bar graph) and progesterone production by 20% (data not shown). Because the treatment altered the response of the control cells, the results using the apo E peptide were expressed relative to the matched control cells. The PI-PLC pretreatment of the cells did not alter apo E peptide acetyl-Y(141–155)2C-mediated stimulation or inhibition of androstenedione production (Fig. 5). The removal of GPI-linked HSPG did not alter the effects of the apo E peptide, indicating that specific HSPG were involved in apo E peptide-mediated stimulatory activity.
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Apo E Peptide Bound to Heparin
The LDL receptor-binding domain of apo E is a heparin-binding site, and because of this property, heparin is used to prevent apo E as well as apo B binding to members of the LDL receptor superfamily [18–20]. Heparin was added to determine whether in its presence the effects of the apo E peptide acetyl-Y(141–155)2C on androstenedione production would be prevented. Heparin (100 µg/ml) had a marked stimulatory effect on the response of the control cells, causing androstenedione production to more than double (Fig. 6, bar graph). Heparin completely abolished both apo E peptide-mediated stimulation and inhibition of androstenedione production (Fig. 6). These results indicated that the apo E peptide binds to heparin with a higher affinity than it binds to the theca-interstitial cell HSPG and that when bound to heparin, the apo E peptide cannot interact with androgen-producing theca cells.
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Experimental Treatments Did Not Change Cell Number
A simple explanation for the changes in androstenedione production observed with heparinase, PI-PLC, and heparin treatments was a change in the cell number during the culture period. The cell numbers did not change in control cells with time in culture (data not shown) or with any of the treatments (Fig. 7). These results indicated that the ovarian cells did not divide while in culture and that none of the treatments altered the cell number. Therefore, the observed changes in ovarian theca cell androstenedione production were due to increased or decreased androstenedione production per cell.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The apo E peptide acetyl-Y(141–155)2C induces changes in androstenedione production by altering the rate of transcription of the enzyme, P450 17-hydroxylase, C17–20 lyase, that synthesizes androstenedione from progesterone [7]. The effect of the apo E peptide on theca cell steroid production is selective: the expression of P450 side-chain cleavage is not altered [7]. We reported in our recent paper that apo E peptide-induced changes in androstenedione production always corresponded with the same relative changes in the level of mRNA for P450 17-hydroxylase, C17–20 lyase [7]. Consequently, in the results presented here, i.e., when changes were observed in androstenedione production, we predicted that the mRNA for P450 17-hydroxylase, C17–20 lyase would change in a parallel manner. We conclude that the apo E peptide-stimulated expression of the gene for P450 17-hydroxylase, C17–20 lyase was as a consequence of its interaction with members of the LDL receptor superfamily. This is the first report suggesting that members of the LDL receptor superfamily can mediate a change in the level of expression of the gene required for androgen steroid synthesis. Importantly, exogenous cholesterol did not play a role in this response mediated by the members of the LDL receptor superfamily: the ovarian theca-interstitial cells were cultured in serum-free medium, and the apo E peptide has been shown to be receptor active without added lipids [4, 5, 21].
Native apo E contains 299 amino acids, and the apo E peptide (141–155) contains the amino acid sequence that binds the LDL receptor ligand-binding domain [21]. We have found that for an apo E peptide to bind the LDL receptor, the receptor-binding domain has to be repeated in a tandem, linear dimer [4, 5, 21]. One proof of the requirement for the dimer repeat was that the apo E peptide (129–162) is not LDL receptor active even though it has nested within it one repeat of 141–155 and is approximately the same length as the dimer peptide [5, 6]. Here, androstenedione production was not stimulated by the apo E peptide (129–162). Therefore, for an apo E peptide to stimulate ovarian theca cell androstenedione production, the peptide must be able to bind to the LDL receptor ligand-binding domain.
HSPG are needed for high-affinity binding of apo E to the members of the LDL receptor superfamily [10, 14–16, 18–21]. Our results demonstrate that the integrity of specific HSPG was required for the apo E peptide to stimulate ovarian theca cell androstenedione production. Binding of the apo E peptide to the LDL receptor and the LRP that occurs in human skin fibroblasts is sensitive to heparinase I pretreatment [9]. Heparinase I pretreatment of human skin fibroblasts reduced the amount of apo E peptide/LDL complex cell association by almost 70% [9]. The requirement for HSPG for apo E high-affinity binding to its receptors is also observed in the dominant forms of type III hyperlipoproteinemia. Mutant forms of apo E that interact poorly with HSPG cause a more severe accumulation of remnant lipoprotein in the plasma than was promoted by the mutant form that had very low affinity for the LDL receptor [22]. The apo E peptide binds to heparin with high affinity [23]. A higher affinity of apo E for heparin than for heparan sulfates has been demonstrated by the removal of cell surface HSPG-bound apo E by the addition of heparin [19, 24]. Thus, the prevention of the apo E peptide's activities by heparin suggests that the degree of sulfation, and therefore of negative charges present in the heparan sulfate chain, is an important determinant of apo E peptide binding to HSPG—and supports the conclusion that apo E stimulation of theca cell P450 17-hydroxylase, C17–20 lyase expression and androstenedione production was mediated by members of the LDL receptor superfamily.
Our data indicate that HSPG are integral to ovarian theca cell steroidogenic response to LH stimulation. Androstenedione production was significantly increased by heparinase and heparin treatments, both of which could have affected interaction of locally produced heparin-binding growth factors with these cells. Theca cell-derived growth factors such as basic fibroblast growth factor (bFGF) [25] and hepatocyte growth factor (HGF) [26] inhibit theca cell androstenedione production by binding to their high-affinity signaling receptors [12]. However, prior association of bFGF and HGF with cell surface HSPG appears to be necessary for their binding to these high-affinity receptors [12, 27]. We propose that removal of HSPG from the cell surface by heparinase or competition for inhibitory growth factor binding with exogenous heparin resulted in a reduced inhibitory tone promoted by bFGF and/or HGF, resulting in increased androstenedione production by the control cells. HGF exerts its inhibitory effect at the level of lyase gene expression [26], which is the same action as recently shown by us for the apo E peptide [7]. Basic FGF is also a potent inhibitor of lyase activity [25]. According to this interpretation, if theca cell HSPG are intact, androstenedione production is suppressed by locally produced inhibitory growth factors, such as HGF and bFGF, and the level of these growth factors may therefore control follicle maturation. We propose that the inhibition of androgen synthesis by endogenously produced apo E may be more important later in follicular maturation or atresia when the theca cells luteinize. This hypothesis is in agreement with the previous finding of high levels of apo E mRNA in theca and theca-derived interstitial cells of atretic follicles and increased apo E mRNA levels in these cells upon hCG stimulation [28, 29].
Specific HSPG were required for the apo E peptide to stimulate androstenedione production, as removal of GPI-linked HSPG did not alter either the stimulatory or inhibitory effect of the peptide on theca cell androstenedione production. There are non-HSPG molecules linked to cells via GPI, and their removal by PI-PLC was probably responsible for the inhibition of the control cell response to LH.
It is unknown which members of the LDL receptor superfamily were involved in the apo E peptide stimulatory activity. All of the several members of the LDL receptor superfamily—the LDL receptor, the LRP, glycoprotein (gp) 330 or megalin, the VLDL receptor, the apo E receptor 2, and LR11—are expressed in the ovary, but it is not known which are expressed by ovarian theca cells [8, 30, 31]. All of these members of the LDL receptor superfamily have highly conserved LDL receptor ligand-binding domains; in fact, it is the conservation of this cysteine-rich motif that has enabled the rapid cloning of the additional members of the superfamily. All of the receptors bind RAP, which blocks the binding of apo E [13]. Of these receptors, probably only the LDL receptor can be eliminated as a candidate receptor that mediates the apo E peptide-mediated stimulation of androstenedione production. LDL contains apo B, which only binds to the LDL receptor, and LDL does not selectively alter theca cell androgen production [3]. The members of the LDL receptor superfamily are endocytic, able to internalize the bound apo E via coated pits [32]. The LRP and gp330 have been reported to participate in signal transduction [32–34], and it is probable that the apo E peptide-mediated stimulation of androstenedione production is due to changes in theca cell signal transduction. Apo E peptide stimulation of androstenedione production always occurred, but it varied in magnitude from 20% to 70% greater than the control cell response. The variation in the degree of stimulation may have been a function of the level of expression by the theca cells of the relevant member(s) of the LDL receptor superfamily.
Apo E is made within the rat ovary primarily by theca cells of advanced atretic follicles and by the interstitial cells that are derived from the theca cells of atretic follicles [28, 29]. An early event in follicular atresia is the selective suppression of theca cell androstenedione production as the cells luteinize to become exclusively progesterone-secreting cells [35–38]. During this transition the interstitial cells increase their expression of apo E mRNA, and at the same time the expression of P450 17-hydroxylase, C17–20 lyase is reduced [39]. Because apo E is made within the ovary, we propose that apo E is an autocrine/paracrine agent that modulates the expression of the androgen-synthesizing enzyme, thus regulating the availability of estrogen substrate during follicular maturation and controlling the level of androgens that participate in follicular atresia [40].
Inhibition of ovarian theca cell androstenedione production by apo E peptide was not via its interaction with members of the LDL receptor superfamily or HSPG. Because the apo E peptide has high affinity for lipids [9], inhibition of androstenedione production by theca cells observed at high concentrations of the peptide could be a result of its direct interaction with specific cell membrane lipids [41]. Apo E's action to alter select gene expression may not be restricted to the ovary. Evidence from other physiologic and pathologic situations where apo E is made locally, such as in the brain and within the vessel wall, suggests that apo E may influence lesion development via noncholesterol transport-dependent functions in diseases such as atherosclerosis and Alzheimer's disease [42–45].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Celina V. Zerbinatti, Department of Biological Sciences, South Beaver St., Room 420, Box 5640, Northern Arizona University, Flagstaff, AZ 86011–5640. FAX: 520 523 7500; celina.zerbinatti{at}nau.edu
Accepted: April 14, 1999.
Received: September 21, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-macroglobulin receptor-associated protein. J Biol Chem 1996; 271:31379–31383. in rat and guinea-pig gonads, adrenal glands and brain. J Reprod Fertil 1991; 93:609–622.This article has been cited by other articles:
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N. Argov, U. Moallem, and D. Sklan Lipid Transport in the Developing Bovine Follicle: Messenger RNA Expression Increases for Selective Uptake Receptors and Decreases for Endocytosis Receptors Biol Reprod, August 1, 2004; 71(2): 479 - 485. [Abstract] [Full Text] [PDF]
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T. Fayad, V. Levesque, J. Sirois, D. W. Silversides, and J. G. Lussier Gene Expression Profiling of Differentially Expressed Genes in Granulosa Cells of Bovine Dominant Follicles Using Suppression Subtractive Hybridization Biol Reprod, February 1, 2004; 70(2): 523 - 533. [Abstract] [Full Text] [PDF]
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 B. Sisco, L. J. Hagemann, A. N. Shelling, and P. L. Pfeffer Isolation of Genes Differentially Expressed in Dominant and Subordinate Bovine Follicles Endocrinology, September 1, 2003; 144(9): 3904 - 3913. [Abstract] [Full Text] [PDF]
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 J. Raber, G. Bongers, A. LeFevour, M. Buttini, and L. Mucke Androgens Protect against Apolipoprotein E4-Induced Cognitive Deficits J. Neurosci., June 15, 2002; 22(12): 5204 - 5209. [Abstract] [Full Text] [PDF]
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C. V. Zerbinatti, L. P. Mayer, R. G. Audet, and C. A. Dyer Apolipoprotein E Is a Putative Autocrine Regulator of the Rat Ovarian Theca Cell Compartment Biol Reprod, April 1, 2001; 64(4): 1080 - 1089. [Abstract] [Full Text]
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D. K. Swertfeger and D. Y. Hui Apolipoprotein E Receptor Binding Versus Heparan Sulfate Proteoglycan Binding in Its Regulation of Smooth Muscle Cell Migration and Proliferation J. Biol. Chem., June 29, 2001; 276(27): 25043 - 25048. [Abstract] [Full Text] [PDF]
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