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First published online September 27, 2007
Stem Cells Vol. 25 No. 12 December 2007, pp. 3069 -3080
doi:10.1634/stemcells.2007-0550; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS

Sonic Hedgehog Stimulates Mouse Embryonic Stem Cell Proliferation by Cooperation of Ca2+/Protein Kinase C and Epidermal Growth Factor Receptor As Well as Gli1 Activation

Jung Sun Heo, Min Young Lee, Ho Jae Han

Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

Key Words. Embryonic stem cells • Sonic hedgehog • Gli1 • Ca2+/protein kinase C • Epidermal growth factor receptor

Correspondence: Ho Jae Han, D.V.M., Ph.D., Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea. Telephone: 82-62-530-2831; Fax: 82-62-530-2809; e-mail: hjhan{at}chonnam.ac.kr

Received on July 10, 2007; accepted for publication on September 18, 2007.

First published online in STEM CELLS EXPRESS  September 27, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hedgehog signaling has an essential role in the control of stem cell growth in embryonic tissues. Therefore, this study examined the effect of sonic hedgehog (Shh) on the self-renewal of mouse embryonic stem (ES) cells and its related mechanisms. Shh increased DNA synthesis blocked by the inhibition of the smoothened receptor. Shh required Gli1 activation to induce the increases in Notch/Hes-1 and Wnt/β-catenin. Shh increased the intracellular calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity. We show that the Shh-induced increase in the Gli1 mRNA level requires [Ca2+]i and PKC. Shh increased the phosphorylation of epidermal growth factor receptor (EGFR), which is blocked by the matrix metalloproteinase inhibitor. Subsequently, Shh increased the nuclear factor (NF)-{kappa}B p65 phosphorylation, which was inhibited by blocking PKC and EGFR tyrosine kinase. Shh also increased the level of the cell cycle regulatory proteins in a dose-dependent manner. However, Shh decreased the levels of the cyclin-dependent kinase inhibitory proteins. The effect of Shh on these proteins was inhibited by blocking PKC, EGFR, and NF-{kappa}B as well as transfection of Gli1 small interfering RNA (siRNA). Finally, Shh-induced progression of the G1/S-phase was blocked by the inhibition of PKC and EGFR tyrosine kinase. In conclusion, Shh stimulates mouse ES cell proliferation through Gli1 activation as well as Ca2+/PKC and EGFR.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hedgehog (Hh) signaling is generally lower in adults than in embryos but was recently suggested to play an important role in the control of embryonic and adult stem cell growth [1]. Among the members of the mammalian hedgehog proteins (Sonic, Indian, Desert), sonic hedgehog (Shh) has received the most attention and acts on the target cells to increase the transcription of several genes, including members of Wnt, Notch, and several growth factors such as fibroblast growth factor, transforming growth factor β families, and the receptor Patched (Ptc), which releases the Ptc-mediated repression of Smoothened (Smo) [2–4]. It is known that the end point of the Shh signal pathway, representing the nuclear component of the pathway, is the zinc-finger transcription factor Gli (Ci [Cubitus interruptus] in Drosophila species) [5]. The three Gli proteins, Gli1, 2, and 3, act in a combinational fashion that is context dependent, with Gli1 and -2 being the major positive mediators of Shh signals, and Gli3 having often an antagonistic role [6]. The previous report demonstrated that Hh-Gli signaling regulates the expression of stemness genes in and the self-renewal of glioma cancer stem cells [7].

Smo is a family of serpentine G protein-coupled receptors (GPCRs) that targets the inhibitory G protein (G{alpha}i). However, previous reports showed that Shh did not decrease intracellular cAMP level [8] and, in contrast, increased [Ca2+]i, suggesting the possibility of Smo coupling with G{alpha}q [9]. Since the proliferative effects of Shh may not arise solely from the canonical pathway, we cannot rule out the role of G{alpha}q protein among noncanonical mitogenic mechanisms of Shh. Intracellular calcium and protein kinase C (PKC) are the possible downstream mechanisms of G{alpha}q. Calcium is a ubiquitous second messenger that mediates a wide range of cellular responses such as fluid and electrolyte secretion, exocytosis, gene transcription, and cellular proliferation [10]. Protein kinase C is also an important mediator of signal transduction in response to several cellular signals and is involved in gene expression, cell proliferation, and differentiation. Although these mechanisms are involved in various cellular responses, it is unclear whether they are essential for Shh-induced proliferative signaling in embryonic stem (ES) cells.

The epidermal growth factor receptor (EGFR) pathway appears to be a factor in embryonic stem cell proliferation [11]. EGFR activation leads to the stimulation of many downstream pathways including Ras/ERK and the phosphoinositide 3-kinase (PI3-K) signaling axis, which are responsible for cell growth and death. It was previously reported that EGFR signaling modulates Gli gene expression in epidermal cells [12]. On the other hand, the Shh pathway regulates EGF signaling in epidermal keratinocytes [13], which suggests some crosstalk between Shh and the growth factor signaling pathways. The Shh/Gli pathways can increase the expression of the key regulators of cell cycle progression, which suggests that they promote the transition from a quiescent to a proliferative state [14, 15]. Even though Shh/Gli signaling affects cell proliferation, it is unclear whether this pathway affects ES cells.

Embryonic stem cells are defined as cells with self-renewal capacity and the ability to generate multiple differentiated cell types [16, 17]. It is becoming clearer that both self-renewal and differentiation in stem cells involve numerous interdependent pathways with considerable crosstalk occurring between those pathways. Various extrinsic factors including growth factors and cytokines can target various proteins involved in a particular pathway or interact with proteins in multiple pathways to modulate a specific phenotype [11, 18, 19]. Traditional cell culture techniques facilitate billionfold expansion of embryonic stem cells but result in a gradual loss of their primitive characteristics and self-renewal properties. Therefore, the selection of high-quality input materials, as well as an understanding of their regulatory mechanisms, is essential. In addition, much effort is devoted to the establishment of feeder-free cultures by elucidation of the cytokines and growth factors required for cell propagation. Keeping step with these current demands, this study examined the effect of Shh on embryonic stem (ES) cell expansion and its regulatory mechanisms on self-renewal of mouse ES cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Materials
The mouse ES cells were obtained from the American Type Culture Collection (ES-E14TG2a; Manassas, VA, http://www.atcc.org). The fetal bovine serum was purchased from BioWhittaker (Walkersville, MD, http://www.cambrex.com). The sonic hedgehog, cyclopamine, A 23187, ethylene glycol tetraacetic acid (EGTA), 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), AG 1478, bisindolylmaleimide I, SN 50, BAY11-7082, fluorescence isothiocyanate (FITC)-conjugated goat anti-mouse IgM, and β-actin were acquired from Sigma (St. Louis, http://www.sigmaaldrich.com). The Fluo 3-AM, 5-bromo-2'-deoxyuridine (BrdU), and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Molecular Probes (Eugene, OR, http://probes.invitrogen.com). The [3H] thymidine was supplied by NEN (Boston, http://www.perkinelmer.com). The phospho-EGF receptor, EGF receptor, phospho-nuclear factor (NF)-{kappa}B p65, Notch, Hes-1, Wnt1, β-catenin, cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, http://www.scbt.com). The goat anti-rabbit IgG was supplied by Jackson Immunoresearch Laboratories (West Grove, PA, http://www.jacksonimmuno.com). The Liquiscint was obtained from National Diagnostics (Parsippany, NY, http://nationaldiagnostics.com). All other reagents were of the highest purity commercially available and were used as received.

ES Cell Culture
The mouse ES cells were cultured for 5 days in DMEM (Gibco-BRL, Gaithersburg, MD, Gaithersburg, MD, http://www.invitrogen.com) supplemented with 3.7 g/l sodium bicarbonate, 1% penicillin and streptomycin, 1.7 mM L-glutamine, 0.1 mM β-mercaptoethanol, 5 ng/ml mouse leukemia inhibitory factor, and 15% fetal bovine serum (FBS) without a feeder layer. The cells were passaged with 0.05% trypsin/EDTA onto gelatinized 12-well plates or onto a 60-mm culture dish without a feeder layer and maintained at 37°C in an atmosphere containing air and 5% CO2. After 2–3 days, the cells were washed twice with phosphate-buffered saline (PBS) and maintained in serum-free DMEM with all the supplements. After a 24-hour incubation period, the cells were washed twice with PBS and incubated with fresh serum-free DMEM including all the supplements and the designated agents for the indicated period before the experiments.

Alkaline Phosphatase Staining
Cells were washed twice with PBS and fixed with 4% formaldehyde (in PBS) for approximately 15 minutes at room temperature. The cells were washed with PBS and incubated with an alkaline phosphatase substrate solution (200 µg/ml Naphthol AS-MX phosphate, 2% N,N-dimethylformamide, 0.1 M Tris [pH 8.2], and 1 mg/ml Fast Red TR salt [4-chloro-2-methylbenzenediazonium salt; zinc chloride]) for 10 minutes at room temperature. After washing with PBS, the cells were photographed.

Immunofluorescence Staining with Stage Specific Embryonic Antigen (SSEA-1)
Cells were fixed and treated with mouse anti-SSEA-1 antibody (1:100; Santa Cruz Biotechnology) for 1 hour at room temperature. Subsequently, the FITC-conjugated anti-mouse IgM (1:100) was treated for 1 hour at room temperature. Fluorescence images were obtained using a fluorescence microscope (FluoView 300; Olympus, Tokyo, http://www.olympus-global.com).

[3H] Thymidine Incorporation
The [3H] thymidine incorporation experiments were carried out using the methodology reported by Chen et al. [20]. Zhang et al. [21] reported that most ES cells could be arrested in the G0/G1 phase using a serum deprivation culture. In addition, the synchronized ES cells could successfully re-enter a normal cell cycle after being resupplied with serum. In this study, the cells were cultured in a single well until they reached 50% confluence. They were then washed twice with PBS and maintained in serum-free DMEM including all the supplements. After a 24-hour incubation, the cells were washed twice with PBS and incubated with fresh serum-free DMEM including all the supplements and indicated agents. After the indicated incubation period, 1 µCi of [methyl-3H] thymidine (specific activity: 74 GBq/mmol, 2.0 Ci/mmol; Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) was added to the cultures. The cells were incubated with [3H] thymidine for 1 hour at 37°C. They were then washed twice with PBS, fixed in 10% trichloroacetic acid (TCA) at 23°C for 15 minutes, and washed twice with 5% TCA. The acid-insoluble material was dissolved in 2 N NaOH over a 12-hour period at 23°C. Aliquots were removed and the level of radioactivity was determined using a liquid scintillation counter (LS 6500; Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). The control levels of [3H] thymidine incorporation were determined under the conditions in which the cells had been cultured in serum-free DMEM without Shh. The values were converted from the absolute counts to a percentage of the control in order to allow a comparison between the experimental groups.

Bromodeoxyuridine Incorporation
The level of 5-bromo-2'-deoxyuridine (a thymidine analog) incorporation was measured in order to determine the level of DNA synthesis. The ES cells were serum-starved for 24 hours prior to Shh stimulation. The ES cells were then treated with Shh for 24 hours followed by the addition of 15 µM BrdU. Incubation was continued for an additional 1 hour. After washing several times with PBS, the cells were fixed with methanol (10% [vol/vol] for 10 minutes at 4°C) followed by incubation in 1 N HCl for 30 minutes at room temperature. The cells were then washed and incubated with 0.1 M sodium tetraborate for 15 minutes. The cells were then incubated with Alexa Fluor 488-conjugated mouse anti-BrdU mAb (diluted 1:200; Molecular Probes) in 2% BSA-PBS overnight at 4°C. The cells were then counterstained with DAPI (Molecular Probes). After washing in PBS, the coverslips were mounted onto glass slides with a Dako Fluorescent mounting medium using gelvatol and examined using optical microscopy (FluoView 300). The percentage of BrdU-positive cells was determined by counting the number of BrdU-positive cells per field of vision.

Cell Proliferation Assay
In order to determine the number of cells, the cells were washed twice with PBS and trypsinized from the culture dishes. The cell suspension was mixed with a 0.4% (wt/vol) trypan blue solution, and the number of live cells was determined using a hemocytometer. Cells failing to exclude the dye were considered nonviable.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
The total RNA was extracted from the mouse ES cells using STAT-60, which is a monophasic solution of phenol and guanidine isothiocyanate (Tel-Test, Friendswood, TX, http://www.isotexdiagnostics.com). Reverse transcription was carried out using 3 µg of RNA using a reverse transcription system kit (RT PreMix; AccuPower, Daejeon Korea; http://www.bioneer.com) with the oligo(dT18) primers. Five microliters of the reverse transcription (RT) products was then amplified using a polymerase chain reaction (PCR) kit (PCR PreMix; AccuPower) under the following conditions: denaturation at 94°C for 5 minutes followed by 30 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 30 seconds followed by 5 minutes of extension at 72°C. The primers used were 5'-TGCCAGATATGCTTCAGCCA-3' (sense), 5'-TGTGGCGAA-TAGACAGAGGT-3' (antisense) for Gli1 (291 base pairs [bp]) and 5'-AACCGCGAGAAGATGACCCAGATCATGTTT-3' (sense), 5'-AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3' (antisense) for β-actin (350 bp). PCR for β-actin was also carried out as a control for the quantity of RNA.

Real-Time RT-PCR
The total RNA was extracted from the cells treated with each of the designated agents using STAT-60. The real-time quantification of RNA targets was then performed in the Rotor-Gene 2000 real-time thermal cycling system (Corbett Life Science, Sydney, Australia, http://www.corbettlifescience.com) using a QuantiTect SYBR Green RT-PCR kit (Qiagen, Hilden, Germany, http://www.qiagen.com). The reaction mixture (20 µl) contained 200 ng of the total RNA, 0.5 µM of each primer, the appropriate amounts of enzymes, and fluorescent dyes, as recommended by the supplier. The Rotor-Gene 2000 cycler was programmed as follows: 30 minutes at 50°C for reverse transcription; 15 minutes at 95°C for DNA polymerase activation; 15 seconds at 95°C for denaturing; and 45 cycles of 15 seconds at 94°C, 30 seconds at 55°C, and 30 seconds at 72°C. Data collection was carried out during the extension step (30 seconds at 72°C). The PCR reaction was followed by melting curve analysis to verify the specificity and identity of the RT-PCR products, which can distinguish the specific PCR products from the nonspecific PCR product resulting from primer-dimer formation. The primers used were 5'-CGTGAGACTTTGCAGCCTGA-3' (sense), 5'-GGCGATGTAAGTGATCTGCTG-3' (antisense) for Oct4 (519 bp); 5'-TCTTACATCGCGCTCATCAC-3' (sense), 5'-TCTTGACGAAGCAGTCGTTG-3' (antisense) for FOXD3 (171 bp); and 5'-GTGGAAACTTTTGTCCGAGAC-3' (sense), 5'-TGGAGTGGGAGGAAGAGGTAAC-3' (antisense) for SOX2 (550 bp). The temperature of the PCR products was increased from 65°C to 99°C at a rate of 1°C every 5 seconds, and the resulting data were analyzed using the software provided by the manufacturer.

Measurement of [Ca2+]i
The changes in [Ca2+]i were monitored using Fluo 3-AM dissolved in dimethylsulfoxide. The mouse ES cells in 35-mm culture dishes were rinsed twice with a bath solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, 5.5 mM HEPES, pH 7.4), incubated in bath solution containing 3 µM Fluo 3-AM with 5% CO2-95% O2 at 37°C for 40 minutes, rinsed twice with the bath solution, mounted on a perfusion chamber, and scanned every second using confocal microscopy (x400) (FluoView 300). The fluorescence was excited at 488 nm and the emitted light was read at 515 nm. In order to verify the assay, A 23187 (Ca2+ ionophore) was applied to the cells as positive control. All analyses of [Ca2+]i were processed at a single cell level and are expressed as the relative fluorescence intensity (RFI).

Gli1 Small Interfering Ribonucleic Acid Transfection
The cells were grown to 75% confluence in each dish and transfected for 24 hours with either a SMARTpool of the small interfering RNAs specific to Gli1 (200 pmol/l) or a nontargeting small interfering RNA (as negative control; 200 pmol/l; Dharmacon Inc., Lafayette, CO, http://www.dharmacon.com) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's instructions.

Preparation of Cytosolic and Total Membrane Fractions
The cytosolic and total membrane fractions were prepared using a slight modification of the method reported by Mackman et al. [22]. The medium was removed and replaced with serum-free DMEM that included all the supplements contained in the LIF for 24 hours before the experiments. After removing the medium, the cells were washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF, 10 µg/ml leupeptin, 0.5 mM sodium orthovanadate [pH 7.5]). The resuspended cells were then lysed mechanically on ice by trituration with a 21.1-gauge needle. The cell lysates were initially centrifuged at 1,000g for 10 minutes at 4°C. The supernatants were collected and centrifuged at 100,000g for 1 hour at 4°C to prepare the cytosolic and total particulate fractions. The supernatant (cytosolic fraction) was then precipitated with acetone (5 fold volume of supernatant), incubated on ice for 5 minutes, and then centrifuged at 20,000g for 20 minutes at 4°C. The resulting pellet was resuspended in buffer A containing 1% (vol/vol) Triton X-100. The particulate fractions containing the membrane fraction were washed twice and resuspended in buffer A containing 1% (vol/vol) Triton X-100. The protein in each fraction was quantified using the Bradford procedure [23].

Western Blot Analysis
The cells that had been treated with each of the designated agents after a 24-hour serum starvation were harvested, washed twice with PBS, and lysed with a buffer (20 mM Tris [pH 7.5], 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium orthovanadate) for 30 minutes on ice. The lysates were then cleared by centrifugation (10 minutes at 15,000 rpm, 4°C), and the protein concentration was determined using the Bradford method [23]. Equal amounts of protein (20 µg) were resolved by electrophoresis on 10% SDS-PAGE and transferred to nitrocellulose membranes. The blots on the membrane were washed with Tris-buffered sakine Tween 20 (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20), blocked with 5% skim milk for 1 hour, and incubated with the appropriate primary antibody at the dilutions recommended by the supplier. The membrane was then washed, and the primary antibodies were detected with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase. The bands were visualized by enhanced chemiluminescence (Amersham Biosciences).

Fluorescence-Activated Cell Sorter Analysis
The cells were serum starved for 24 hours and pretreated with either bisindolylmaleimide I or AG 1478 for 30 minutes before incubation with Shh (500 ng/ml) for 24 hours. The cells were then dissociated in trypsin/EDTA, pelleted by centrifugation, and resuspended at approximately 106 cells per milliliter in PBS containing 0.1% BSA. The cells were then fixed in 70% ice-cold ethanol, which was followed by incubation in a freshly prepared nuclei staining buffer (250 µg/ml propidium iodide [PI] and 100 µg/ml RNase) for 30 minutes at 37°C. The cell cycle histograms were generated after analyzing the PI-stained cells by fluorescence-activated cell sorting (FACS) (Beckman Coulter). At least 104 events were recorded for each sample. The samples were analyzed using CXP software (Beckman Coulter).

Statistical Analysis
The results are expressed as the mean ± SE. All the experiments were analyzed by analysis of variance, and some experiments were examined by comparing the treatment means with the control using a Bonferroni-Dunn test. A p value < .05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Effect of Shh on DNA Synthesis and Notch/Hes-1 and Wnt1/β-Catenin
In order to confirm the continued maintenance of pluripotency in the presence of long-term Shh exposure, cells were subcultured up to five passages in the presence of Shh, and then the undifferentiated markers of stem cells were examined. As shown in Figure 1A, alkaline phosphatase enzyme activity and SSEA-1 (carbohydrate epitopes stage-specific embryonic antigen-1) expression were still maintained after five passages. Moreover, it was confirmed that the mRNA expression levels of Oct 4, SOX2, and FOXD3 were increased and the developmental potential still maintained in five passaged cells (supplemental online data). Therefore, these results demonstrate that mouse ES cells conserve an undifferentiated status under even long-term Shh treatment. As shown in Figure 1B, Shh increased the level of Gli1 mRNA expression. However, the repression of Gli1 with the transfection of Gli1 specific siRNA inhibited the Shh-induced increases in Oct 4, SOX2, and FOXD3 mRNA levels (Fig. 1C).


Figure 1
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  		Figure 1. Effect of Shh on the characterization of mouse embryonic stem (ES) cells. The mouse ES cells were subcultured up to five passages in the presence of Shh (500 ng/ml), and then (A) the alkaline phosphatase enzyme activity and immunofluorescence staining with stage specific embryonic antigen-1 were assessed as described in Materials and Methods. The scale bars represent 20 µm. (B): The mouse ES cells were incubated with Shh (500 ng/ml) for 24 hours, and the expression levels of Gli1 (291 base pairs [bp]) and β-actin (350 bp) mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR). Each example shown is representative of five independent experiments. (C): The mouse ES cells were transfected for 24 hours with either a SMARTpool of Gli1 siRNAs (200 pmol/l) or a nontargeting control siRNA (200 pmol/l) using Lipofectamine 2000 before the 24 hour Shh treatment (500 ng/ml). The Oct 4, FOXD3, and SOX2 mRNA levels were determined by real-time RT-PCR. The values are reported as the mean ± SE of five independent experiments with triplicate dishes; *, p < .05 versus control; **, p < .05 versus Shh alone. Abbreviations: AP, alkaline phosphatase; Shh, sonic hedgehog.

 
In further experiments to examine the effect of Shh on cell proliferation, the cells were treated with Shh (0–500 ng/ml) for 24 hours to confirm its effect on DNA synthesis. In this experiment, Shh, at ≥50 ng/ml, significantly increased the level of [3H] thymidine incorporation in a dose-dependent manner during the 24-hour incubation period (Fig. 2A). The number of BrdU-labeled cells was also increased in response to Shh, which is consistent with the observed increase in the level of [3H] thymidine incorporation (Fig. 2B, 2C). Consequently, a significant increase in the number of cells was observed after incubation with Shh (≥50 ng/ml) for 48 hours (Fig. 2D). Therefore, based on these results, we suggest that Shh influences self-renewal rather than differentiation in mouse ES cells under our experimental conditions. Moreover, in order to confirm the involvement of Smo receptor in Shh-induced increase of DNA synthesis, the cells were pretreated with cyclopamine (Smo inhibitor, 10–5 M) before the Shh treatment. The Shh-induced increase in [3H] thymidine incorporation was inhibited by this process (Fig. 2E). Shh also increased the levels of the Notch and Wnt1/β-catenin proteins in a dose-dependent manner (0–500 ng/ml) (Fig. 3A, 3B). However, the Shh-induced increases in the Notch/Hes-1 and Wnt1/β-catenin levels were inhibited when the cells were transfected with the Gli1 specific siRNAs (Fig. 3C, 3D). Consequently, the Shh-induced increase in DNA synthesis was inhibited by a pretreatment with L-685,458 (Notch inhibitor, 10–5 M) (Fig. 3E).


Figure 2
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  		Figure 2. Effect of Shh on cell proliferation. (A): The mouse embryonic stem (ES) cells were incubated for 24 hours with various Shh concentrations (0–500 ng/ml) and pulsed with 1 µCi of [3H] thymidine for 1 hour. (B): The responses of the BrdU-positive cells to different Shh concentrations (0–500 ng/ml) for 24 hours were assessed. The percentage of BrdU-positive cells was determined by counting the number of BrdU-positive cells per field of vision. At least 10 fields of vision per coverslip were counted. (C): The mouse ES cells were incubated with Shh (500 ng/ml) for 24 hours and double-labeled with BrdU and DAPI. The scale bars represent 20 µm. (D): The mouse ES cells were treated with Shh (0–500 ng/ml) for 48 hours, and the number of cells was counted using a hemocytometer. (E): The mouse ES cells were pretreated with cyclopamine for 30 minutes before the 1-hour Shh treatment (500 ng/ml) and then pulsed with 1 µCi of [3H] thymidine for 1 hour. The values are reported as the mean ± SE of four independent experiments with triplicate dishes; *, p < .05 versus control; **, p < .05 versus Shh alone. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; DAPI, 4,6-diamidino-2-phenylindole; hr, hours; Shh, sonic hedgehog.

 


Figure 3
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  		Figure 3. Effect of Shh/Glil on Notch/Hes-1 and Wnt1/β-catenin levels. The mouse embryonic stem (ES) cells were incubated with Shh (0–500 ng/ml). The total protein was extracted and the protein blots were probed with the (A) Notch, (B) Wnt1, or β-catenin specific antibody. (C, D): The mouse ES cells were transfected for 24 hours with either a SMARTpool of Gli1 siRNAs (200 pmol/l) or a nontargeting control siRNA (200 pmol/l) using Lipofectamine 2000 before the 24-hour Shh treatment (100 ng/ml), and the Western blots (Notch, Hes-1, Wnt1, and β-catenin) was assessed. The bands represent 30~50 kDa of Notch, 40~42 kDa of Wnt1, 92 kDa of β-catenin, 20 kDa of Hes-1, and 41 kDa of β-actin, respectively. Each example shown is representative of four independent experiments. (E): The mouse ES cells were pretreated with L-685,458 (Notch inhibitor, 10–5 M) before the Shh treatment (500 ng/ml) for 24 hours and pulsed with 1 µCi of [3H] thymidine for 1 hour. The values are reported as the mean ± SE of four independent experiments with triplicate dishes; *, p < .05 versus control; **, p < .05 versus Shh alone. Abbreviation: Shh, sonic hedgehog.

 
Effect of Shh on Ca2+, PKC, EGF Receptor, and NF-{kappa}B Signaling
The intracellular Ca2+ mobility in response to Shh was examined in order to determine whether the Shh-induced ES cell proliferation involves an increase in [Ca2+]i. As shown in Figure 4A, Shh induced an increase in [Ca2+]i. On the other hand, a pretreatment with cyclopamine inhibited the Shh-induced increase in [Ca2+]i. In order to validate this result, A 23187 (Ca2+ ionophore, 10–6 M), which is a well-known chemical compound that can increase [Ca2+]i, was treated to the cells as positive control. In the next steps, in order to determine whether Shh activates PKC signaling, the translocation of PKC from the cytosolic compartment to the membrane compartment was observed at 1 hour after treatment with 500 ng/ml Shh. Shh enhanced the translocation of PKC {alpha}, {delta}, and {zeta} isoforms from the cytosol to the membrane (Fig. 4B). On the other hand, Shh-induced PKC activation was blocked by cyclopamine (Fig. 4C). The Shh-induced increase in Gli1 gene expression was inhibited by a pretreatment with EGTA (extracellular calcium chelator, 4 x 10–3 M) plus BAPTA-AM (intracellular calcium chelator, 10–5 M) or bisindolylmaleimide I (a PKC inhibitor, 10–7 M) (Fig. 4D). Finally, inhibition of the PKC pathway with bisindolylmaleimide I blocked the Shh-induced increase in [3H] thymidine incorporation (Fig. 4E).


Figure 4
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  		Figure 4. Effect of Shh on [Ca2+]i and PKC activation. (A): The mouse embryonic stem (ES) cells were loaded with 2 µM Fluo 3-AM in serum-free medium for 40 minutes and treated with Shh (500 ng/ml), and mouse ES cells were pretreated with cyclopamine (10–5 M) for 30 minutes and the Shh-induced Ca2+ influx was then measured. In order to validate this assay, A 23187 (Ca2+ ionophore, 10–6 M) was treated to the cells as positive control. The changes in [Ca2+]i were monitored using confocal microscopy and are expressed as the relative fluorescence intensity. (B): The PKC {alpha}, {delta}, and {zeta} isoforms present in either the cytosolic or membrane compartments were detected by Western blotting as described in Materials and Methods. (C): Mouse ES cells were pretreated with cyclopamine for 30 minutes before the 1-hour Shh treatment (500 ng/ml), and the Pan-PKC protein was detected in the membrane compartment. The bands represent 80–90 kDa for Pan-PKC, PKC {alpha}, {delta}, and {zeta} and 41 kDa for β-actin. (D): The mouse ES cells were pretreated with EGTA plus BAPTA-AM or bisindolylmaleimide I for 30 minutes before the 24-hour Shh treatment (500 ng/ml). The Gli1 gene expression levels were then analyzed by real-time reverse transcription-polymerase chain reaction. Each example shown is a representative of five experiments. (E): The mouse ES cells were pretreated with bisindolylmaleimide I (10–6 M) before the Shh treatment (500 ng/ml) for 24 hours and pulsed with 1 µCi of [3H] thymidine for 1 hour. The values are reported as the mean ± SE of four independent experiments with triplicate dishes; *, p < .05 versus control; **, p < .05 versus Shh alone. Abbreviations: BAPTA-AM, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester); PKC, protein kinase C; sec, seconds; Shh, sonic hedgehog.

 
In the experiments to examine whether Shh activates EGF receptor signaling, Shh increased the phosphorylation of the EGF receptor (Fig. 5A). However, a pretreatment with the matrix metalloproteinase (MMP) inhibitor (10–7 M) blocked the effect of Shh on EGF receptor activation. The inhibition of EGF receptor tyrosine kinase with AG 1478 (EGF receptor tyrosine kinase inhibitor, 10–5 M) attenuated the Shh-induced increase in DNA synthesis (Fig. 5B). In the experiment to determine the involvement of NF-{kappa}B activation, Shh increased the level of NF-{kappa}B p65 phosphorylation, which was inhibited by a pretreatment with bisindolylmaleimide I or AG 1478 (Fig. 5C). Finally, Shh-induced DNA synthesis was blocked by inhibiting NF-{kappa}B activation with SN 50 (a NF-{kappa}B inhibitor, 500 ng/ml) or BAY11-7082 (a NF-{kappa}B inhibitor, 2 x 10–5 M) (Fig. 5D).


Figure 5
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  		Figure 5. Effect of Shh on EGFR and NF-{kappa}B signaling. (A): The mouse embryonic stem (ES) cells were pretreated with the MMP inhibitor (10–7 M) before the 1-hour Shh treatment (500 ng/ml). The level of the EGFR was assessed by Western blotting. The bands represent 175 kDa of the phospho-EGFR and EGFR. Each example shown is a representative of four experiments. (B): The mouse ES cells were pretreated with AG 1478 before the 24-hour Shh treatment (500 ng/ml) and pulsed with 1 µCi of [3H] thymidine for 1 hour. (C): The mouse ES cells were pretreated with bisindolylmaleimide I or AG 1478 before the 2-hour Shh treatment (500 ng/ml). The level of phospho-NF-{kappa}B p65 was assessed by Western blotting. The bands represent 65 kDa of phospho-NF-{kappa}B p65 and 41 kDa of β-actin. Each example shown is a representative of four experiments. (D): The mouse ES cells were pretreated with SN 50 (500 ng/ml) or BAY11-7082 (2 x 10–5 M) before a 24-hour Shh treatment and pulsed with 1 µCi of [3H] thymidine for 1 hour. The values represent the mean ± SE of four independent experiments with triplicate dishes; *, p < .05 versus control; **, p < .05 versus Shh alone. Abbreviations: EGFR, epidermal growth factor receptor; MMP, matrix metalloproteinase; Shh, sonic hedgehog.

 
Effect of Shh on Cell Cycle Regulatory Proteins
The cells were treated with Shh (0–500 ng/ml), and the levels of cell cycle regulatory proteins were observed. As shown in Figure 6A, the level of cyclin E was increased in a dose-dependent manner. In contrast, the maximum expressions of CDK 2, cyclin D1, and CDK 4 were observed at 10 ng/ml Shh treatment and then remained stable. When the cells were transfected with a pool of Gli1 specific siRNAs, the Shh-induced increases in the cyclin E/CDK 2 and cyclin D1/CDK 4 protein levels were also inhibited (Fig. 6B). The cells were pretreated with bisindolylmaleimide I or AG 1478 to determine whether Shh-activated PKC and EGF receptor signaling is involved in the Shh-induced increase in the cell cycle regulatory proteins (Fig. 6C). In this experiment, Shh increased the cyclin E/CDK 2 and cyclin D1/CDK 4 levels but decreased the p21cip1/waf1 and p27kip1 levels, which were inhibited by each pretreatment. The inhibition of NF-{kappa}B with SN 50 or BAY11-7082 also decreased the Shh-induced increases in cyclin E/CDK 2 and cyclin D1/CDK 4 protein levels (Fig. 6D). Consequently, Shh significantly increased the percentage of the cell population in the S phase as compared with control. However, a pretreatment with bisindolylmaleimide I or AG 1478 decreased the level of accumulation in the S phase induced by Shh (Fig. 6E, 6F).


Figure 6
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  		Figure 6. Effect of Shh on cell cycle regulatory proteins. (A): The mouse embryonic stem (ES) cells were incubated with Shh (0–500 ng/ml) for 24 hours. The total protein was extracted and the protein blots were probed with the cyclin E, CDK 2, cyclin D1, and CDK 4. (B): The mouse ES cells were transfected for 24 hours with either a SMARTpool of Gli1 siRNAs (200 pmol/l) or a nontargeting control siRNA (200 pmol/l) using Lipofectamine 2000 before a 24-hour Shh treatment. (C, D): The mouse ES cells were pretreated with bisindolylmaleimide I, AG 1478, SN 50, or BAY11-7082 before a 24-hour Shh treatment (500 ng/ml). The levels of cyclin E, CDK 2, cyclin D1, CDK 4, p21cip1/waf1, and p27kip1 were assessed by Western blotting. The levels of cyclin E, CDK 2, cyclin D1, and CDK 4 were assessed by Western blotting. The proteins are expressed in 35–55 kDa, respectively. Each of the examples shown is a representative of three independent experiments. Representative fluorescent-activated cell sorting data (E) and corresponding histograms (F) for the mouse ES cells, which were pretreated with bisindolylmaleimide I or AG 1478 before the 24-hour Shh treatment (500 ng/ml). The cells were washed with phosphate-buffered saline, fixed, stained, and analyzed by flow cytometry. The gates were configured manually to determine the percentage of cells in the G1, S, and G2 phases based on the DNA content. The values represent the mean ± SE of three independent experiments. Abbreviation: Shh, sonic hedgehog.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
This study shows that multiple pathways in parallel are required for Shh-induced proliferation of mouse ES cell (a) canonical Gli1 pathway and (b) noncanonical Ca2+/PKC and EGF receptor activation. The results showed that Shh increased the level of the markers of the S phase including [3H] thymidine and BrdU incorporation, suggesting that Shh stimulates proliferation of mouse ES cells. Moreover, the levels of Oct4, SOX2, and FOXD3 gene expression were increased by Shh, which were reduced by transfection with Gli1 specific siRNA. Therefore, these results suggest that Shh can regulate self-renewal (proliferation plus maintenance of developmental potential) in mouse ES cells. The canonical hedgehog pathway, Gli, was found to play an important role in ES cell proliferation. In this study, a remarkable feature of Gli1 is the regulation of Shh-induced Notch/Hes-1 and Wnt1/β-catenin activation. The RNA interference-mediated inhibition of the Gli1 function is sufficient to inhibit Notch/Hes-1 and Wnt1/β-catenin signaling. The hedgehog, Notch, and Wnt pathways regulate the self-renewal of a variety of stem cells and play an important role in cell proliferation and differentiation in various cell types. Moreover, Hairy and the enhancer of the split homolog (Hes)-1 or β-catenin is an essential downstream factor in each Notch or Wnt-mediated cell proliferation, survival, and migration [24, 25]. The present results also show that these pathways affect the proliferation of mouse ES cells. The inhibition of the Notch pathway decreased the level of Shh-induced DNA synthesis. Although a further study will be needed to determine the proliferative mechanisms of these pathways on ES cells, Notch/Hes-1 and Wnt1/β-catenin signaling can play an important role in the propagation of mouse ES cells.

Among the noncanonical pathways of Shh to induce mitogenic signaling, there is no report of a link between the activation of Ca2+/PKC pathways by the stimulation of Shh and the proliferation of mouse ES cells. A previous report showed that the Smo is involved in the Shh-induced increase in [Ca2+]i to promote DNA synthesis [9]. In this study, Shh stimulated the intracellular [Ca2+]i and induced the translocation of the PKC {alpha} (conventional), {delta} (novel), and {zeta} (atypical) isoforms from the cytosolic to membrane compartment, which was inhibited by repressing the Smo receptor with cyclopamine. It was reported that PKC {delta} is required in hedgehog signaling to stimulate the cell proliferation of the NIH 3T3 cell line [26]. Moreover, in this study, Shh was found to promote Gli1 activation through the Ca2+ and PKC pathways, suggesting these two signaling pathways play a key role in Gli1 regulation and Shh-induced ES cell proliferation. Previous reports also showed that the phorbol ester stimulation of NIH 3T3 cells rapidly induces Gli transcriptional function [26] and that PKC {delta} increases the Gli1 activity in basal cell carcinoma [27]. Although limited data were shown, based upon previous reports and our results, it is hypothesized that the Ca2+/PKC pathways have a regulatory role in Shh signaling in the control of mouse ES cell proliferation.

Crosstalk between heterologous signaling systems of the cells represents a complexity in the molecular communication network that governs a general signaling mechanism to combine and expand signal pathways. Among these mechanisms, recent findings showed that the EGFR is activated by not only its specific ligands but also a variety of physiological and nonphysiological stimuli, which are induced by G protein-coupled receptor, cytokine receptors, cell adhesion, osmotic pressure, and reactive oxygen species (ROS) [28–30]. However, there is no converged understanding about the signaling mediators involved in these networks. Previous studies indicate that the EGFR transactivation is exclusively mediated through intracellular mechanisms, which induce rapid kinetics of the EGFR transactivation, as well as the interaction of the EGFR with MMP-mediated EGF-like ligands [31, 32]. In some cases, the activation of Src can directly phosphorylate the EGFR cytoplasmic tail [33]. Moreover, PKC and Ca2+ activation through G{alpha}q-coupled receptors has been frequently shown to be involved in EGFR transactivation in several cell types [34–39]. In contrast, the MMP-mediated processing of the HB-EGF, and therefore a ligand-dependent mechanism in EGFR transactivation, was first demonstrated by Prenzel and colleagues [40]. In the present study, MMP was suggested as a mediator of Shh-induced EGFR signal transactivation. A few studies showed that MMP-9 was increased in the Shh-expressing cells, which was attenuated by the inhibition of EGF receptor activation or blocking the EGF receptor and ligand interaction [13]. Hedgehog activates EGF receptor signaling by inducing the tissue-specific expression of the Drosophila EGF receptor (DER) ligand [41]. The identity of the MMP and the mechanism still remains to be defined in ES cells. However, previous studies, including ours, that the interaction of Shh and EGFR is involved in various cellular responses [12, 42–44] can suggest that Shh can directly activate EGFR signaling to influence ES cell proliferation. Although the present study does not show the data of EGFR-induced increase in [Ca2+]i, our previous study demonstrated that EGFR activation increased [Ca2+]i in ES cells [11]. On the other hand, based on previous reports that intracellular Ca2+ can activate EGFR signaling [36, 37], the increase in [Ca2+]i through Smo receptor can also trigger EGFR transactivation. Thus, it can be suggested that Ca2+ is both upstream and downstream mediator in Shh-induced transactivation of EGFR. Besides Ca2+ signaling, Shh-induced PKC and EGF receptor activation can converge at the level of a common signal transducer in proliferative signaling. Therefore, this study examined whether or not NF-{kappa}B is activated directly by Shh through PKC and EGF receptor signaling. Calcium dependent signaling pathways such as calcineurin and PKC contribute to NF-{kappa}B activation [45]. Moreover, the EGF receptor signaling mediates the constitutive NF-{kappa}B activity leading to the proliferation and protection from cell death in human pancreatic cancer cells [46]. Previous studies examined the interaction between Shh and NF-{kappa}B and reported that NF-{kappa}B activation induces Shh overexpression, which indicates a contribution to cell proliferation [47, 48]. On the other hand, the present results suggest that Shh is more likely to stimulate NF-{kappa}B activation as a downstream target of PKC and EGF receptor signaling. Therefore, Shh-induced NF-{kappa}B activity influences cell proliferation.

This study also showed that the RNA interference-mediated inhibition of the Gli1 function inhibited Shh-induced increases in cyclin E/CDK 2 and cyclin D1/CDK 4 levels. The Gli binding site in human cyclin D2 promoter, hedgehog/Gli signaling, is likely to induce cyclin D2 transcription directly [15]. Another G1/S-phase regulator, cyclin E, is also controlled by hedgehog/Gli signaling in Drosophila [49], suggesting that the Gli signal pathway is linked to promote the cell position from a quiescent to a proliferative state. Moreover, PKC and EGFR signaling mediated Shh-induced G1/S-phase progression leading to the increases of cyclin E/CDK 2 and cyclin D1/CDK 4 levels. This is consistent with our previous research showing that EGF increased the level of cell cycle regulatory proteins through EGFR-activated PKC and p44/42 mitogen activated protein kinases [11]. In another report to demonstrate involvement of the PKC pathway, TPA stimulation induces cyclin D1 expression and promotes proliferation of mouse embryonic fibroblast C3H 10T1/2 [50]. Interestingly, the blocking of NF-{kappa}B pathway with specific inhibitors attenuated the Shh-induced increases in cell cycle regulatory proteins. Previous experiments in many cell types showed NF-{kappa}B is coordinated with these proteins. The cyclin E/CDK 2 complexes formed at the time of G1/S transition in lymphocytic cells have been shown to associate with NF-{kappa}B complexes [51]. Moreover, NF-{kappa}B acts through increasing the abundance of cyclin D1 and thus the activity of the cyclin D1 kinase [52]. Although these two complexes are important in cell cycle progression, the role of cyclin D/CDK4 in ES cells is still controversial. However, among the functions of cyclin D/CDK4, the inhibitory effect on CDK inhibitors p27Kip1 and p21Cip1 can be suggested to be a functional significance in ES cells [53]. This study also demonstrated decrease of the expression of the p21 and p27 proteins by Shh, indicating important regulators of cell cycle progression in ES cells. A previous report demonstrated that the suppression of hedgehog signaling by cyclopamine increases the level of p21, suggesting suppressed cell proliferation [54]. A disruption of p21 leads to hematopoietic stem cell proliferation and the maintenance of stem cells, which indicates that CDK inhibitors are important regulators of stem cell self-renewal [55]. Although the established pathways in this study have been observed in multiple cell types and under different conditions, these findings led us to propose a hypothesis for a possible new mechanism for how Shh promotes the cell cycle for faster cycling and cell proliferation without affecting differentiation and can affect self-renewal linking to distinct cell signal transduction in ES cells (Fig. 7).


Figure 7
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  		Figure 7. The hypothesized model for the signal pathways involved in Shh-induced embryonic stem (ES) cell proliferation. Shh activates Smo receptors, which increases [Ca2+]i and activates PKC. In another pathway, Shh activates MMP to stimulate EGFR activation. Continuously, PKC and EGFR induce NF-{kappa}B activation to increase cyclinE/CDK2 and cyclin D1/CDK4 protein levels. Activated Smo receptor stimulates Gli, which increases Notch/Hes-1 and Wnt1/β-catenin activation to stimulate ES cell proliferation. The solid line is the proposed pathway and the dashed line is suspected pathway. Abbreviations: CDK, cyclin-dependent kinase; EGFR, epidermal growth factor receptor; MMP, matrix metalloproteinase; NF-{kappa}B, nuclear factor kappa B; PKC, protein kinase C; PTCH, Patched; Shh, sonic hedgehog; Smo, Smoothened.

 
This study presents areas to which defined ES cell culture is expected to provide useful insights as well as provides the necessity of developing defined culture conditions for ES cell expansion without feeder layer coculture. Identifying the complex signaling pathways regulated by Shh that mediate the proliferative responses of embryonic stem cells can indicate how Shh signaling controls stem cell expansion and establishes the stem cell fate. In conclusion, Shh in part stimulates the proliferation of mouse embryonic stem cells, which is mediated not only by Gli1-induced Notch/Hes-1 and Wnt1/β-catenin activation but also by Ca2+/PKC and EGF receptor activation.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This study was supported by a Grant (SC 2210) from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology. The authors acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development through the Brain Korea 21 project, Republic of Korea.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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