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Logo of jbcThe Journal of Biological Chemistry
J Biol Chem. 2012 February 17; 287(8): 5898–5909.
Published online 2011 December 27. doi:  10.1074/jbc.M111.325282
PMCID: PMC3285358

This article has been retractedRetraction in: J Biol Chem. 2016 July 1; 291(27): 14389    See also: PMC Retraction Policy

Glypican-1 Stimulates Skp2 Autoinduction Loop and G1/S Transition in Endothelial CellsAn external file that holds a picture, illustration, etc.
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The heparan sulfate proteoglycan glypican-1 (GPC1) is involved in tumorigenesis and angiogenesis and is overexpressed frequently in tumor and endothelial cells (ECs) in human gliomas. We demonstrated previously that in brain EC, GPC1 regulates mitotic cyclins and securin as well as mitosis and that GPC1 is required for progression through the cell cycle. To characterize the molecular mechanism underlying cell cycle regulation by GPC1, we systematically investigated its effects on key G1/S checkpoint regulators and on major signaling pathways reportedly activated by Dally (Division abnormally delayed) the Drosophila GPC1 homologue. We found that elevated GPC1 affected a wide range of G1/S checkpoint regulators, leading to inactivation of the G1/S checkpoint and increased S phase entry, apparently by activating the mitogen-independent Skp2 autoinduction loop. Specifically, GPC1 suppressed CDK inhibitors (CKIs), including p21, p27, p16, and p19, and the D cyclins, and induced CDK2 and Skp2. GPC1 may trigger the Skp2 autoinduction loop at least partially by suppressing p21 transcription as knockdown of p21 by RNAi can mimic the effect of GPC1 on the cell cycle regulators related to the loop. Moreover, multiple mitogenic signaling pathways, including ERK MAPK, Wnt and BMP signaling, were significantly stimulated by GPC1 as has been reported for Dally in Drosophila. Notably, the c-Myc oncoprotein, which is frequently up-regulated by both ERK and Wnt signaling and functions as a potent transcription repressor for CKIs as well as D cyclins, was also significantly induced by GPC1. These findings provide mechanistic insights into how GPC1 regulates the cell cycle and proliferation.

Keywords: Cell Cycle, Cyclins, Endothelial Cell, Mitosis, Proteoglycan


Glypicans (GPCs)2 are heparan sulfate proteoglycans (HSPGs) that are attached to the extracellular surface of the plasma membrane by a glycosylphosphatidylinositol anchor. In contrast to the syndecan family of HSPGs, which are characterized by a transmembrane core protein with a linear conformation, the protein cores of GPCs putatively possess a globular conformation that is stabilized by disulfide bonds formed between 14 highly conserved cysteine residues. In GPCs, the heparan sulfate (HS) are situated adjacent to the cell surface, whereas in syndecans, the HS attachment sites are located near the N terminus. These structural features endow GPCs with several distinctive functional properties that distinguish them from other HSPGs such as the syndecans. In GPCs, the juxtamembrane position of HS chains may increase the accessibility of HS to HS-dependent ligand-receptor interactions. The glycosylphosphatidylinositol anchor facilitates active shedding by phospholipases, a preferential localization in cholesterol-rich lipid rafts and caveolae, and endocytosis and recycling. Studies in Drosophila have demonstrated that shed GPCs take part in the transport of Wnts, hedgehog (Hh), and bone morphogenic proteins (BMPs) and established tissue gradients of these morphogens. Endocytosis and recycling of GPCs, in particular of GPC1, play an important role in glypican HS remodeling, transcellular molecule transport, and polyamine and basic peptide uptake by the cells (13).

The expression of GPCs is regulated temporally and spatially during normal development, and accumulating evidence suggests their involvement in the development and morphogenesis (4). Of six mammalian glypican family members (GPC1–6), GPC1 is highly expressed in developing brain and is most ubiquitously expressed in adult tissues, including several types of tumors from different tissue origins (59). Studies in a GPC1 knock-out mouse model suggest that in the developing brain, GPC1 may control brain size by activating FGF17, whereas the expression of GPC1 in tumor cells and host ECs appears to contribute both to tumor growth, angiogenesis, and metastasis (10, 11). Notably, GPC1 is expressed highly in both tumor and endothelial cells in human gliomas (7, 8). As GPC1 can act as co-receptor or promoter of many angiogenic growth factors identified in gliomas, including VEGF, FGFs, PDGF, heparin-binding EGF (HB-EGF), HGF, and IGF-1 (1), the presence of abundant GPC1 in glioma ECs may contribute significantly to EC proliferation and angiogenesis in this highly angiogenic malignancy.

In vitro studies in cultured mouse brain ECs have shown a significant effect of GPC1 on EC proliferation and cell cycle progression (12). Knockdown of GPC1 in mouse ECs can dramatically inhibit cell growth by inducing tetra- and polyploidy, whereas overexpression of GPC1 in these cells either promotes cell proliferation or disrupts cell cycle progression by inducing aneuploidy dependent on the expression level of GPC1. Cell cycle progression and ultimately cell proliferation are regulated via different cell cycle checkpoints, principally including the G1/S checkpoint (R-point), G2/M checkpoint, and spindle assembly checkpoint within the M phase. Each cell cycle checkpoint is governed by different cell cycle regulators, which are regulated transcriptionally and/or post-translationally in a cell cycle stage-specific fashion and frequently interact with each other in the form of a network (13). Our previous work in cultured mouse brain ECs pointed toward an effect of GPC1 on anaphase-promoting complex/cyclosome (APC/C)-mediated protein degradation and cell cycle progression in the G2 and/or M phase (12).

Given the critical importance of maintaining the integrity of cell cycle regulation throughout the cell cycle and the decisive role for the G1/S checkpoint in cell proliferation, we chose to further investigate the potential roles of GPC1 at this phase of the cell cycle. Here, we report that GPC1 affects a wide range of G1/S checkpoint regulators, leading to increased G1-S cell cycle progression apparently by activating the mitogen-independent Skp2 autoinduction loop. These findings may provide important insights into the signaling mechanisms by which GPC1 promotes EC proliferation.


Reagents and Plasmids

Antibodies to cyclins D1, D2, D3, A, and B1, p16, p18, p19, p21, p27, p57, CDK4, CDK6, CDK2, Skp2 and Id1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to p15, p-ERK, p-Smad-1, -5, and -8, c-Myc, and pRb were from Cell Signaling Technology (Danvers, MA). An antibody to BrdU was from BD Biosciences, and the MEK1 inhibitor PD98059 was purchased from Calbiochem-Novabiochem (San Diego, CA). The MEK1/2 inhibitor U0126, the pGL4.70 luciferase assay system, and Dual-Luciferase reporter assay system were from Promega (Madison, WI). pCMV/β-Gal was purchased from Applied Biosystems (Carlsbad, CA). The E2F transactivation luciferase reporter construct pGL2(E2F)2 was from Dr. J. Lees (Massachusetts Institute of Technology Center for Cancer Research, Cambridge, MA). TCF-luciferase reporter TOP-FLASH and its mutant FOP-FLASH derivative were obtained from Millipore (Billerica, MA). An axin expression construct pCS2+MT-Axin was from Dr. F. Costantini (Columbia University Medical Center, New York, NY). The dominant-negative TCF4 cDNA construct pcDNA/Myc DeltaN TCF4 and the c-Myc promoter-luciferase reporter Myc-del2-luc were gifts from Dr. B. Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MD). pMKO.1.puro.GFP.shRNA and pRS-mouse p21 shRNA were from Dr. W. Hahn (Dana Farber Cancer Institute, Boston, MA) and Dr. R. Bernards (Netherlands Cancer Institute, Amsterdam, The Netherlands), respectively. pRS-control shRNA and pRS-Myc shRNA were from Dr. J. Manley (Columbia University, New York, NY), and BMP-responsive element (BRE)-luciferase reporter BRE-luc was from Dr. M. Hoffmann (University of Wisconsin, Madison, WI).

Cell Culture and Transfection

Mouse microvascular endothelial cells derived from brains of H-2Kb-tsA58 ImmortoMice (14) were provided by Dr. Isaiah J. Fidler (University of Texas, MD Anderson Cancer Center, Houston, TX) and grown on gelatin-coated tissue culture surfaces in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2-mm l-glutamine, 100 units/ml of penicillin/streptomycin, sodium pyruvate, nonessential amino acids, and a vitamin solution (Invitrogen). The cells were maintained at a lower permissive temperature (32 °C) and transferred to 37 °C 2 days prior to setting up cultures for treatment. At 32 °C, the temperature-sensitive SV40 large T antigen is stable while it is rapidly degraded at 37 °C. For reporter assays, the cells were transiently transfected with Lipofectamine 2000 (Invitrogen) in the presence of 10% FBS according to the manufacturer's instructions. In p21 knockdown immunoblot analysis, the cells were transiently transfected by nucleofection with Amaxa Nucleofector (Amaxa, Koeln, Germany) using program T-13 following the manufacturer's instructions. For p21 knockdown BrdU labeling experiments, the cells were co-transfected with GFP cDNA and p21 or luciferase shRNA constructs in a 1:5 DNA ratio and after 24 h, sorted for GFP-positive cells on a SORP BD FACSAria sorter. The GFP-positive cells were cultured in growth medium for 36 h and then in serum-free starvation medium for 12 h and pulse-labeled with 20 μm BrdU (Sigma) for 30 min before harvest. A chemiluminescence-based BrdU assay was conducted as described previously (7). Human umbilical vein endothelial cells were grown on gelatin-coated tissue culture dishes in EGM-2 endothelial cell growth medium (Cambrex Corp.).

Flow Cytometry

For DNA profile analysis, cells were stained with propidium iodide (Sigma-Aldrich). To examine DNA replication activity, the treated cells were pulse-labeled with 20 μm BrdU for 30 min before harvest. The cells were immunofluorescently stained with mouse anti-BrdU monoclonal antibody (dilution 1:3.5) and Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes) (dilution 1:50) as well as propidium iodide by a method of simultaneous nucleus extraction and acid hydrolysis as described previously (12). All of the samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences), and the data were processed using FlowJo 9 software (FlowJo, Ashland, OR).

Reporter Assays

2 × 104 of mouse brain ECs were seeded in 24-well tissue culture plates overnight. The cells were transiently transfected for 6 h to overnight with Lipofectamine 2000 with different firefly luciferase reporters (125–500 ng) plus pGL4.70 or pCMV/β-Gal as an internal control (25 ng) and additional DNA constructs (250–500 ng) where applicable. The cells were further cultured or transduced with control or GPC1 adenovirus for 48 h and harvested for reporter assays. Firefly luciferase and dual luciferase assays were performed with Promega's Luciferase assay system and Dual-Luciferase reporter assay system, respectively. A standard ortho-nitrophenyl-β-d-galactopyranoside (ONPG) β-galactosidase assay was performed when pCMV/β-Gal was used as the internal control. The resulting firefly luciferase activities in different wells were normalized to the internal control for experimental variations.

Immunoblot Analysis

For analysis of HSPGs, total HSPGs were extracted from cells, digested with heparitinase and chondroitinase ABC, and analyzed by immunoblotting with anti-pan-HSPG antibody 3G10 (Cape Cod, Inc., East Falmouth, MA) as described previously (8). For analysis of total cyclin D1, D2, D3, A, and B1, pRb, CDK4, CDK6, CDK2, p16, p15, p18, p19, p21, p27, p57, Skp2, Emi1, c-Myc, and Id1 as well as p-ERK and p-Smad-1, -5, and -8, cells were lysed in a phosphoprotein lysis buffer and 50 μg of total protein were used for immunoblot analysis as described previously (12). β-Actin was used as the loading control.

Quantitative RT-PCR

Total RNAs were isolated using RNeasy Mini kit (Qiagen, Valencia, CA), and first-strand cDNAs were synthesized using the Omniscript reverse transcription kit (Qiagen) with random hexamers as the primers according to the manufacturer's instructions. To confirm the absence of genomic DNA contamination in the RNA samples, reverse transcription (RT) reactions excluding reverse transcriptase were also conducted. The RT reactions were diluted in water to 10 ng of total RNA/μl, and 5 μl of the diluted RT reactions were applied to each quantitative PCR reaction. For 18 S rRNA amplification, the RT reactions were further diluted 100 times. The quantitative PCR was performed on an iCycler using iQ SYBR Green Supermix (Bio-Rad) and gene-specific PCR primers at 200 nm/each (Table 1). The relative mRNA levels between experimental and control samples (R) were determined using the formula R = (1 + E)−ΔCt, where E is PCR efficiency, Ct is the threshold cycle number, and ΔCt is equal to ((Ct of experimental sample) − (Ct of control sample)). The PCR efficiency of each primer pair was determined by serial dilution of the RT reactions. The PCR result was normalized to 18 S rRNA.

PCR primers used in quantitative RT-PCR

Statistical Analysis

A paired Student's t test was performed to determine the statistical significance of the results in quantitative RT-PCR and reporter assays.


GPC1 Stimulates S Phase Entry

Our previous results suggested that GPC1 can negatively regulate APC/C-mediated protein degradation, affecting the cell cycle progression during the G2 and M phases (12). However, the cell cycle regulatory machinery is highly integrated, e.g. APC/C itself and Emi1, the principle inhibitor for both APC/CCdh1 and APC/CCdc20, can be directly or indirectly regulated by CDKs and E2F during the G1/S phase (15, 16). Therefore, GPC1 may exert its regulatory effect on the cell cycle primarily by affecting the cell cycle regulatory machinery in G1/S phase, i.e. the G1/S check point (or R-point). To test this hypothesis and gain new insights into the signaling mechanisms by which GPC1 might affect cell cycle progression, a systematic investigation into the effect of GPC1 on the G1/S checkpoint was conducted. BrdU pulse-labeling of the cells demonstrated a significant increase in DNA synthesis activity following GPC1 transduction at 5 and 10 MOI (Fig. 1, A–C), indicating stimulation of S phase entry by GPC1. A decline in BrdU incorporation was observed when increasing doses of GPC1 adenovirus were applied (Fig. 1C). This could be attributed to the fact that at the time of BrdU pulse-labeling, and at high levels of GPC1 overexpression, less numbers of G1 cells remained to enter S phase (Fig. 1B).

GPC1 inactivates the G1/S checkpoint and stimulates S phase entry. A, immunoblot analysis with anti-pan-HSPG antibody 3G10. Mouse brain ECs were treated with increasing doses of control or GPC1 adenovirus (Ad) as indicated. B and C, mouse brain ECs were ...

S phase entry from G1 is dominantly controlled by E2F transcription factor family members, which activate genes involved in cell cycle progression and DNA replication (17). A reporter assay showed that the transactivation activity of E2F was significantly stimulated by GPC1 in a dose-dependent manner (Fig. 1D). Consistent with this observation, the transcript level of several direct E2F target genes critical for G1/S transition or S phase progression, including cyclin E, cyclin A, Skp2 and Emi1, were up-regulated by GPC1 as shown by quantitative RT-PCR following GPC1 transduction at 10 MOI (Fig. 1E). E2F activity is dominantly regulated by the pRb protein. pRb is hypophosphorylated in quiescent cells and becomes hyperphosphorylated via different CDKs during cell cycle progression. Hypophosphorylated pRb binds to and inhibits E2F, whereas hyperphosphorylated pRb disassociates itself from E2F, leading to E2F activation (18). Because of the central importance of this regulatory mechanism in cell cycle regulation, the phosphorylation status of pRb was examined. As shown in Fig. 1F, overexpression of GPC1 resulted in a considerable increase in pRb phosphorylation. The total pRb protein level was not affected significantly at GPC1 adenovirus doses under 50 MOI. These results, taken together, indicate that introduction of GPC1 into the cells effectively inactivates the G1/S checkpoint by phosphorylating pRb, producing increased S phase entry.

GPC1 Impairs Cyclin D-CDK4/6 Kinases

As a key step for cell cycle G1 phase progression, cyclin D members, including D1, D2, and D3, are synthesized in response to mitogenic stimuli and form active kinase complexes with CDK4/6 to initiate pRb phosphorylation (19). To determine whether GPC1 may induce phosphorylation of pRb through cyclin D-CDK4/6, the expression of these molecules was examined. Unexpectedly, upon GPC1 transduction, even at a relatively low MOI (such as 5), the protein level of all three cyclin D isoforms was dramatically down-regulated (Fig. 2A). The protein level of both CDK4 and CDK6 was not affected. As cyclin D is essential for the kinase activity of CDK4/6, the near absence of all three cyclin D isoforms following GPC1 overexpression would lead to considerable inactivation of the kinases. mRNA analysis by quantitative RT-PCR showed that the transcript level of all three cyclin D isoforms, particularly cyclin D1 and D2, was dramatically down-regulated by GPC1 (Fig. 2B), suggesting that the depletion of cyclin D proteins following GPC1 overexpression may be attributed, at least partially, to the reduction in their transcripts.

GPC1 impairs cyclin D-CDK4/6 kinases. A, mouse brain ECs were transduced with different doses of control or GPC1 adenovirus (Ad) as indicated for 48 h, and immunoblot analysis was performed for cyclin D1–3, CDK4, and CDK6, with β-actin ...

In addition to the described positive regulation via D cyclins, the kinase activity of CDK4/6 is negatively regulated by Ink4 CKIs, including p16, p15, p18, and p19, by forming ternary complexes (20). The level of Ink4 family members is regulated in response to extra- and intracellular signaling during the cell cycle as well (21). As shown in Fig. 3A, the protein level of p16 and p19 was significantly suppressed by GPC1. The mRNA level of all Ink4 members, particularly p15, was down-regulated by GPC1, despite the fact that the protein level of both p15 and p18 was unchanged (Fig. 3). Collectively, these results revealed that GPC1 inactivates the G1/S checkpoint utilizing a cyclin D-CDK4/6-independent mechanism and that GPC1 suppresses the expression of both D cyclins and members of the Ink4 family.

GPC1 down-regulates Ink4 CKIs. A, mouse brain ECs were transduced with different doses of control or GPC1 adenovirus as indicated for 48 h, and the protein levels of p16, p15, p18, and p19 were determined by immunoblot analysis, with β-actin as ...

GPC1 Stimulates CDK2 and CDK1 Kinases and Skp2 Autoinduction Loop

During the G1-S cell cycle progression, phosphorylation of pRb is normally initiated by cyclin D-CDK4/6 in response to mitogens, followed by cyclin E-CDK2 activity to reach a hyperphosphoryled state. The continued activity of cyclin A-CDK2 and -CDK1 and cyclin B1-CDK1 maintains this pRb hyperphosphorylation throughout S, G2, and M phases of the cell cycle (13). Our previous study has shown a robust induction of cyclin A and B1 by GPC1 with unaltered CDK1 protein levels (12). As both cyclin A and B1 can activate CDK1, this suggests stimulation of CDK1 kinase by GPC1. The protein level of cyclin E, which together with cyclin A activates CDK2, was not affected by GPC1 (12). However, a further analysis of the effect of GPC1 on CDK2 and the Cip/Kip family of CKIs, which includes p21, p27, and p57, demonstrated that the protein level of CDK2 was up-regulated significantly by GPC1 (Fig. 4A), whereas both p21 and p27 were down-regulated significantly (Fig. 4B). The inhibitory effect of GPC1 on p21 was dramatic. Quantitative RT-PCR showed a robust reduction of p21 mRNA by GPC1 as well (Fig. 4C), suggesting that p21 may be down-regulated at the transcriptional level. The mRNA level of both CDK2 and p27 was not significantly altered, whereas that of p57 was induced by GPC1 (Fig. 4C), although its protein level was unaltered (Fig. 4B). Given the prominent role for both p21 and p27 in regulating the kinase activity of CDK2 and CDK1 and consequently the cell cycle (22), the prominent down-regulation of p21 and p27, in combination with up-regulated CDK2 and cyclins A and B1, would be expected to result in a significant activation of both cyclin-dependent kinases. To examine the generality of our findings, we extended our observations to primary human umbilical vein endothelial cells. Flow cytometry showed that overexpression of GPC1 induced an even more dramatic shift from diploidy to tetra- and aneuploidy than in the mouse brain ECs (supplemental Fig. S1A). As in the mouse brain ECs, both cyclin D1 and p21 protein level were markedly down-regulated by GPC1, whereas CDK2 was dramatically up-regulated (supplemental Fig. S1B). Overexpression of GPC1 in human umbilical vein endothelial cells led to significant down-regulation of pRb protein (supplemental Fig. S1B). This is different from the mouse brain ECs where pRb hyperphosphorylation was induced by GPC1 (Fig. 1F). However, the ultimate cellular effect of both types of pRb changes (loss of pRB or hyperphosphorylation) on the G1/S checkpoint would be essentially the same, i.e. both would lead to E2F activation and S phase entry. These results indicate that the observed cell cycle effect of GPC1 is not cell line specific nor can it be attributed to the immortalization of the mouse brain ECs.

GPC1 up-regulates CDK2 and down-regulates p21Cip1 and p27Kip1. A and B, mouse brain ECs were transduced with different doses of control or GPC1 adenovirus for 48 h, and the protein levels of CDK2, p21, and p27 as well as p57 were determined by immunoblot ...

The dysfunction of cyclin D-CDK4/6 and the coordinate alterations in cyclins, CDKs, and CKIs, which all point toward CDK2 activation, strongly suggest that GPC1 may inactivate pRb by a cyclin D-CDK4/6-independent mechanism, namely the Skp2 autoinduction loop (23). It represents a positive feedback loop within the pRb-E2F regulation network, comprised of pRb-E2F, Skp2, p27/p21, and cyclin E/A-CDK2. In the normal cell cycle context, this loop amplifies cyclin D-CDK4/6-mediated mitogen-stimulated E2F activation and ultimately renders subsequent cell cycle progression mitogen-independent. However, direct activation of this loop by cell manipulations, e.g. by ectopic expression of Skp2, which targets p27 and p21 for degradation, rather then by the mitogen-stimulated activation of cyclin D-CDK4/6, could allow for mitogen-independent entry into S phase (24). We examined whether GPC1 stimulates the expression of Skp2, in addition to the observed alterations in other loop components, which all support an activated Skp2 autoinduction loop. In concert with the activation of its transcription activator E2F (Fig. 1B) and the down-regulation of its ubiquitination targets p21 and p27 (Fig. 4B), both mRNA and protein levels of Skp2 were significantly increased upon GPC1 overexpression (Figs. 1E and and55A). However, the effect of GPC1 on Skp2 might be secondary to E2F activation as activated E2F could not only induce Skp2 gene transcription but also reduce APC/CCdh1 mediated Skp2 protein degradation because of the increasing levels of Emi1 and CDK activities, both of which act to inhibit APC/CCdh1 activity.

Knockdown of p21 mimics GPC1 effect on the Skp2 autoinduction loop. A, mouse brain ECs were treated with different doses of control or GPC1 adenovirus (Ad) for 48 h, and the protein level of Skp2 was determined by immunoblot analysis, with β-actin ...

Among the Skp2 autoinduction loop mediators, the transcript level of p21 was down-regulated dramatically by GPC1 (Fig. 4C). Diminished transcription may contribute significantly to the observed decrease in p21 protein level, and this appears to be mediated by signals originating outside of the Skp2 autoinduction loop. Moreover, it would not be surprising if down-regulation of Cip/Kip CKIs could enhance ongoing E2F activation signaling and subsequently S phase entry through the Skp2 autoinduction loop, even if it alone might not be able to trigger cell cycle progression de novo. To examine whether down-regulation of p21 could stimulate the Skp2 autoinduction loop and E2F activation as well as S phase entry, p21 expression in the cells was transiently knocked down using a p21 shRNA construct. As shown in Fig. 5B, similar to GPC1 overexpression, knockdown of p21 induced hyperphosphorylation of pRb and increased the protein levels of cyclin A and B1, Emi1, and Skp2. A moderate reduction in p27 protein was seen, whereas the levels of cyclin D1 and CDK2 were not detectably altered, although cyclin D1 also is reportedly targeted by Skp2 for degradation (25). The luciferase reporter assay showed a dose-dependent up-regulation of E2F transactivation activity by the p21 shRNA construct (Fig. 5C). The BrdU incorporation assay showed increased DNA replication activity following expression of p21 shRNA in the cells (Fig. 5D). All of these results put together suggest that the Skp2 autoinduction loop was stimulated by GPC1 and that GPC1-induced down-regulation of the p21 transcript may contribute significantly to this stimulation.

Multiple Mitogenic Signaling Pathways Are Stimulated by GPC1

Our data have demonstrated significant effects of GPC1 on the cell cycle and on a wide variety of cell cycle regulators, indicating the significance and complexity of the action of GPC1 on cells. GPC1, similar to other HSPGs, can act as co-receptor for many heparan sulfate-binding growth factors, leading to sustained activation of their cognate signaling receptors (1). If growth factor co-receptor activity was the predominant mechanism of GPC1-induced cell cycle changes, one would predict that the GPC1 effect would be more pronounced in the presence of serum, which contains many heparan sulfate-binding growth factors. However, surprisingly, the effect of GPC1 overexpression on both DNA profile and cell cycle regulators was independent of serum stimulation (supplemental Fig. S2). Moreover, ectopic expression of GPC1 reduced low serum-induced apoptosis (supplemental Fig. S2A). This observation suggests a more specific or possibly growth factor-independent mechanism of action. However, the involvement of mitogenic signaling pathways stimulated by either ligand-free activation of receptor tyrosine kinases or by mitogens not present in serum cannot be excluded entirely.

In addition to their general HSPG functions, glypicans have shown specific or preferential participation in the signaling pathways of certain mitogens and morphogens, specificities that have been attributed to their core protein structure. For example, the Drosophila homologue of GPC1, Dally (division abnormally delayed), plays an essential role in Wnt and Dpp signaling and in the development of Drosophila (26, 27). To dissect the signaling mechanisms by which GPC1 affects G1-S cell cycle progression, we examined the effect of GPC1 on these signaling pathways and their contribution to GPC1-induced E2F activation.

GPC1 triggers a robust stimulation of ERK MAPKs. As shown in Fig. 6A, a GPC1 dose-dependent induction of phospho-ERK by GPC1 was observed in the presence of 10% FBS. The MEK/ERK-specific inhibitors PD98059 and U0126 disrupted the ERK signal and suppressed both basal and GPC1-stimulated E2F transactivation in a dose-dependent manner (Fig. 6, B–E). Similarly, canonical Wnt signaling also was significantly and dose-dependently stimulated by GPC1 as shown by the TCF reporter assay (Fig. 7A). No significant effect of GPC1 on the TCF reporter was observed when its TCF binding sites were mutated (Fig. 7B). Ectopic expression of Axin, the scaffolding protein for the β-catenin degradation complex, or of dominant-negative TCF4, effectively inhibited Wnt signaling (Fig. 7, C and E) and suppressed both basal and GPC1-stimulated E2F activities (Fig. 7, D and F). These observations suggest a significant engagement of both ERK and Wnt signaling in the E2F activation in these cells, although the magnitude of their involvement in GPC1-induced E2F activation and the exact signaling mechanisms remain unclear.

GPC1 activates ERK MAPK signaling. A, mouse brain ECs were treated with different doses of control or GPC1 adenovirus in the presence of 10% FBS for 48 h, and the protein level of p-ERK-1/2 was determined by immunoblot analysis, with β-actin as ...
GPC1 stimulates canonical Wnt signaling. A and B, mouse brain ECs were co-transfected with TOP-FLASH (A) or FOP-FLASH (B) and pGL4.70 and transduced subsequently with different doses of control or GPC1 adenovirus for 48 h. Dual luciferase assay was performed, ...

The c-Myc oncogene is induced frequently by the ERK and Wnt pathways both transcriptionally and post-translationally and has been implicated as potent transcriptional repressor of CKIs, including p21 (28). With this in mind, we examined the expression of c-Myc in the cells. Consistent with ERK and Wnt pathway activation, both c-Myc promoter and protein were significantly induced by GPC1 (Fig. 8, A and B). In addition, co-transfection of the E2F transactivation reporter with a well documented c-Myc shRNA construct led to a decrease in E2F activity, which was more pronounced during GPC1 overexpression (Fig. 8C).

GPC1 induces c-Myc protein and activates its promoter. A, mouse brain ECs were transduced with different doses of control or GPC1 adenovirus for 48 h, and the protein level of c-Myc was determined by immunoblot analysis, with β-actin as loading ...

Signaling of BMPs, the vertebrate homologues of Dpp, is modulated by glypicans during activation of their cognate receptor (29). BMP signaling can promote cell cycle progression through inhibition of TGF-β signaling by competing for Smad4, which is shared by both signaling cascades (30), or through induction of the Id proteins, which can either directly dissociate pRb from E2F or suppress E-protein-mediated transcription of p21, p15, and p16 genes (31). TGF-β signaling normally inhibits cell cycle progression by synergistically suppressing c-Myc and inducing p15 and p21 through TGF-β-specific R-Smads, which complex with Smad4 and the Sp1 transcription factor (32). Here, we found that the activity of BMP signaling was significantly stimulated by GPC1 in a dose-dependent manner, as shown by immunoblot analysis for phospho-Smad-1, -5, and -8 (Fig. 9A), the BMP-specific R-Smads, and by reporter assay with a BRE-luciferase reporter (Fig. 9B). Interestingly, the expression of Ids, the prominent mitogenic transcriptional targets of BMP signaling, appeared to be suppressed rather than induced by GPC1 (Fig. 9, C and D), indicating again the complexity of GPC1 signaling while ruling out the participation of Ids in any potential BMP-mediated cell cycle promoting effect.

GPC1 activates BMP signaling without inducing Id proteins. A and C, mouse brain ECs were treated with different doses of control or GPC1 adenovirus for 48 h, and the protein level of p-Smad-1, -5, and -8 (A) and Id1 (C) was determined by immunoblot analysis ...


Malignant gliomas (glioblastoma multiforme) are generally highly aggressive and angiogenic compared with tumors arising at many other organ sites. Growing glioma tumors co-opt and destabilize the normal brain vasculature, leading to vessel regression and reduced perfusion in addition to a breakdown of the blood-brain barrier. These vascular changes usually cause marked hypoxia and necrosis within the tumor and consequently active EC proliferation and tumor angiogenesis. The resulting tumor blood vessels are structurally and functionally abnormal leading to high interstitial fluid pressure and hypoxia, thus producing a tumor microenvironment that selects for the survival of more aggressive and malignant tumor cells. Interference with tumor angiogenesis including EC proliferation shows promise for the management and control of this malignancy. The current consensus on brain tumor angiogenesis is that hypoxia and genomic changes in the tumor cells result in the secretion of a variety of angiogenic growth factors, which stimulate EC proliferation and migration, key steps in the formation of new blood vessels. With this understanding, anti-angiogenic therapeutics targeting GF signaling, primarily VEGF signaling, have been used in combination with conventional cytotoxic therapies in clinical trials for glioblastoma, although the outcome has been mixed (33, 34). Our findings here show that GPC1, which is frequently overexpressed in glioma blood vessel ECs and in the glioma cells, can inactivate the G1/S cell cycle checkpoint and thus stimulate EC proliferation, apparently by activating the mitogen-independent Skp2 autoinduction loop. The Skp2 autoinduction loop, comprised of pRb-E2F, Skp2, p27/p21, and cyclin E/A-CDK2, functions as a positive feedback loop to amplify ongoing E2F activation signaling or trigger G1-S cell cycle progression independently of mitogens (23) (Fig. 10). Thus, our results implicate a novel molecular mechanism involving GPC1, which may play a significant role in the mediation of EC proliferation during glioma angiogenesis. It is possible that the expression of GPC1 in glioma ECs is induced by the highly hypoxic and poorly vascularized brain tumor microenvironment, providing the ECs with a complementary mechanism to adapt for survival and growth. It is not yet clear whether GPC1 alone is sufficient to trigger a cell cycle progression de novo; however, in addition to the ability of GPC1 to activate heparan sulfate-binding growth factor signaling as a co-receptor, its stimulation of the Skp2 autoinduction loop would be expected to enhance and accelerate ongoing cell cycle progression toward mitosis and make EC proliferation less dependent on mitogens. Transient removal of GPC1 from cultured mouse brain ECs can significantly down-regulate mitotic cyclins, up-regulate CKIs, and impede the cell cycle progression and cell proliferation (12). More importantly, this is not a cell line-specific effect, as similar observations can be made in different EC lines derived from both mouse and human (12, supplemental Fig. S1). These results imply that GPC1 signaling is engaged in the cell cycle progression in ECs and that GPC1 might serve as an anti-angiogenic therapeutic target in tumors such as gliomas.

Schematic of GPC1 stimulation of G1-S cell cycle progression by activating the Skp2 autoinduction loop. The Skp2 autoinduction loop, a positive feedback mechanism during E2F activation, is comprised of pRb-E2F, Skp2, p27/p21, and cyclin E/A-CDK2. Cellular ...

The molecular effects induced by GPC1 overexpression as observed in this study are relatively consistent across a range of GPC1 expression levels and thus appear biologically relevant. Ectopic expression of GPC1 in ECs significantly down-regulates CKIs, including p16, p19, p21, and p27, and up-regulates CDK2, Skp2, Emi1, and mitotic cyclins, including cyclin A and cyclin B1 (12) (Figs. 225). Except for p16 and p19, whose change may not affect the cell cycle due to the near absence of D cyclins upon GPC1 overexpression (Fig. 2), all of these molecular alterations can lead to elevated CDK2 and CDK1 activities and consequently hyperphosphorylation of pRb, activation of E2F and cell cycle progression toward mitosis, as observed in this study and well documented in different experimental contexts by others (13). This coordinated induction of such broad cell cycle stimulatory alterations by GPC1 could impose a dramatic effect on cell proliferation in addition to reflecting the significance and complexity of GPC1 signaling.

It has to be noted that many of these molecular alterations induced by GPC1 might be more or less secondary to E2F activation, as cyclin A, Emi1, and Skp2 are direct transcriptional targets of E2F, whereas APC/CCdh1, which targets mitotic cyclins and Skp2 for degradation, is suppressed by Emi1, CDKs, and SCFSkp2 (35, 36). In addition, CDK2 can be targeted by the SCF complex for degradation, and SCFSkp2 and CDK2-mediated ubiquitin-proteasomal degradation plays a prominent role in the regulation of both p27 and p21 (37, 38). As shown in our study (Fig. 4), GPC1 can significantly down-regulate the protein level of p27 while up-regulating that of CDK2 without affecting their transcript levels. However, among these molecules, which are either within or closely related to the Skp2 autoinduction loop, the p21 transcript is dramatically down-regulated by GPC1 (Fig. 4C). p21 transcriptional suppression, which likely contributes to the reduction in p21 protein level, appears to be regulated by signals from beyond the autoinduction loop. Several distinct transcription factors, principally Miz-1, Sp1/Sp3, p53, and E-box-binding proteins (such as E-proteins), have been identified to mediate the gene transcription of p21 (39). It has been well documented that increasing levels of p21 and/or p27 in response to DNA damage and cytotoxic or differentiation-inducing stimuli can suppress CDK2 and/or CDK1 and arrest the cell cycle (40). In contrast, knockdown of p21 alone or plus p27 could restore CDK2 and/or CDK1 activity and consequently the cell proliferation attenuated by growth inhibitors (41, 42). Consistent with this model, our experiments show that knockdown of p21 is sufficient to induce the Skp2 autoinduction loop and E2F transactivation as well as S phase entry (Fig. 5, B–D), suggesting that GPC1 may, at least partially, promote EC G1-S cell cycle progression by suppressing p21 transcription (Fig. 10). The effect produced by p21 RNAi appears less pronounced than that induced by GPC1 (Fig. 5B), which may be attributed to either incomplete silencing of p21 expression or the involvement of additional signaling mechanisms.

It is intriguing that D cyclins are dramatically down-regulated along with multiple CKIs when the cell cycle is stimulated by GPC1. Apart from indicating that GPC1 inactivates the G1/S checkpoint by a mechanism independent of cyclin D-CDK4/6, this contradictory effect of GPC1 on cell cycle regulators may hint at underlying molecular mechanisms. Indeed, the transcript levels for all three cyclin D isoforms, p15 and p21 were dramatically down-regulated by GPC1 (Figs. 2B, ,33B, and and44C). Promoter analyses show that the promoters of these genes share common key DNA regulatory motifs, including an initiator element (Inr, recognized by Wiz-1) and multiple Sp1/Sp3 binding sites. Studies have demonstrated that both Inr and Sp1/Sp3 binding sites are critical for these promoters as deletion of either of them can significantly abolish the basal activity of these promoters (39, 4345). It would be plausible to suggest that GPC1 may disrupt these transcription factors either by direct interaction or through an intermediate, which is induced by GPC1 and interferes with Wiz-1 and/or Sp1. The c-Myc oncoprotein, a basic helix-loop-helix protein transcription factor, has emerged as a potent transcription repressor for CKIs, including p21, p15, and p16, by interacting with both Wiz-1 and Sp1 (28). The significance of this c-Myc function is well demonstrated by the fact that TGF-β induction of p15 and p21 for arresting the cell cycle requires concurrent suppression of c-Myc expression by TGF-β signaling (46). Although D cyclins are reportedly the direct transcriptional targets of c-Myc, it has been noted that constitutive expression of c-Myc could dramatically repress cyclin D1 transcription initiation as well (47). These observations suggest c-Myc as a probable candidate for an intermediate of GPC1-induced transcription repression of D cyclins and CKIs (Fig. 10). This notion is supported by our findings that both c-Myc promoter and protein were significantly induced by GPC1, and that introduction of c-Myc-specific shRNA into the cells led to a decrease in E2F activity, more significantly when GPC1 was overexpressed (Fig. 9). However, further work is needed to specify the molecular interactions of c-Myc with these promoters.

As part of our effort to identify the signaling mechanisms of GPC1 upstream of the cell cycle regulators, we have found that multiple mitogenic signaling pathways, including ERK MAPK, Wnt, and BMP signaling, are significantly stimulated by GPC1 as is Dally in Drosophila (1). Notably, both ERK and Wnt signalings can enhance c-Myc by inducing c-Myc transcription and protein stabilization (48, 49) (Fig. 10).

This study has shown broad effects of GPC1 on the cell cycle. Ectopic expression of GPC1 apparently inactivates the G1/S checkpoint and stimulates S phase entry and DNA replication, likely by activating the mitogen-independent Skp2 autoinduction loop (Figs. 115 and supplemental Fig. S2). We previously have shown that high GPC1 levels can also affect mitosis, inducing multipolar mitotic spindles and endoreduplication (12). These events during mitosis and likely DNA re-replication in S phase could occur concurrently in the GPC1-transduced cell population, resulting in a DNA profile with an expanded S phase-like subpopulation and aneuploidy at the expense of diploid cells (Fig. 1B) (12). Such multifaceted cell cycle effects of GPC1 are well supported by our findings at the molecular level as discussed above and in our previous study (12). Principally, boosting CDK2 and CDK1 kinases by up-regulating CDK2 and mitotic cyclins and by down-regulating CKIs can drive the cell cycle to progress through G1, S, and G2 phases. However, depletion of CDK1 kinase activity by APC/C-mediated ubiquitin-proteosomal degradation of mitotic cyclins before or at metaphase is the prerequisite for the cells to exit mitosis, including both chromatid segregation and cytokinesis (16). Mitotic cyclins and securin, particularly cyclin B1, were stabilized markedly by overexpression of GPC1 (12). This would at least temporarily delay the progression of the cell cycle through mitosis even if the cells could normally progress into M phase. In fact, deregulated CDK2 activity frequently can induce DNA re-replication and centrosome overproduction during S phase as demonstrated in GPC1-overexpressing cells (12, 50). Nevertheless, as the cell cycle effect of GPC1 is overall dose-dependent, a physiologically controlled GPC1 overexpression is expected to be functionally mitogenic, employing similar molecular mechanisms but producing less cell cycle abnormalities in comparison with the in vitro experimental system used in this study.

In summary, these findings provide novel insights into the molecular effects of GPC1 on the cell cycle and cell signaling in ECs.


We thank Dr. M. Hoffmann, Dr. J. Manley, Dr. R. Bernards, Dr. W. Hahn, Dr. B. Vogelstein, Dr. F. Costantini, and Dr. J. Lees for providing DNA constructs.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Figs. S1 and S2.

2The abbreviations used are:

bone morphogenic protein
anaphase promoting complex/cyclosome
BMP-responsive element
cyclin-dependent kinase
cyclin-dependent kinase inhibitor
endothelial cell
multiplicity of infection
heparan sulfate proteoglycan
Skp1-Cullin-F-box protein complex
division abnormaly delayed
retinoblastoma tumor suppressor
T-cell factor.


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