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Short-type PB-cadherin promotes self-renewal of spermatogonial stem cells via multiple signaling pathways
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Cellular Signalling 20 (2008) 1052 – 1060 www.elsevier.com/locate/cellsig
Short-type PB-cadherin promotes self-renewal of spermatogonial stem cells via multiple signaling pathways Ji Wu a,d,⁎, Yong Zhang a , Geng G. Tian b , Kang Zou a , Clement M. Lee c , Qingsheng Yu a , Zhe Yuan a a
School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China Department of Biological Science, School of Life Science, Nanjin Normal University, Nanjin, Jiangsu 210097, China c Fels. Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao Tong University, Shanghai, 200240, China b
d
Received 2 November 2007; received in revised form 13 January 2008; accepted 14 January 2008 Available online 24 January 2008
Abstract Stem cells represent a unique population of cells with self-renewal capacity. However, the molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Here, we show that short-type PB-cadherin (STPB-C) promoted self-renewal of spermatogonial stem cells (SSCs) via activating Janus kinase/signal transducer and activator of transcription (JAK-STAT) and phosphoinositide-3 kinase (PI3-K)/Akt, and blocking transforming growth factor (TGF)-β1 signaling. These data were obtained with varied approaches, including the use of RNA interference (RNAi), SSC cultures infected by STPB-C retroviral vector, bromodeoxyuridine (BrdU) incorporation assay, and other techniques. These findings have important implications for germ cell biology and create the possibility of using SSCs for biotechnology and medicine. They are also critical in understanding tissue homeostasis, the aging process, tumor formation and degenerative diseases. © 2008 Elsevier Inc. All rights reserved. Keywords: STPB-C; Self-renewal of spermatogonial stem cells; JAK-STAT; PI3-K/Akt; TGF-β1 signaling
1. Introduction Stem cells are characterized by their ability to self-renew and to continuously generate differentiated cells. Uncovering the molecular control of stem cell self-renewal is crucial to the future use of stem cells in regenerative medicine and in understanding tissue homeostasis, the aging process, tumor formation and degenerative diseases [1–3]. To maintain normal spermatogenesis, the processes of self-renewal and differentiation of spermatogonial stem cells (SSCs) must be precisely regulated by intrinsic gene expression in the stem cells and by extrinsic signals, including soluble factors or adhesion molecules from the surrounding microenvironment, the stem cell niche. ⁎ Corresponding author. Department of Biological Science and Biotechnology, School of Life Science and Biotechnology, Shanghai Jiao Tong University, No. 800, Dongchuan Road, Minhang District, Shanghai, 200240, China. Tel.: +86 21 34204933; fax: +86 21 34204051. E-mail address: [email protected] (J. Wu). 0898-6568/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.01.011
A recent study has identified growth factors essential for selfrenewal and expansion of mouse SSCs [4]. Glial cell linederived neurotrophic factor (GDNF) is a key factor in deciding the fate of SSCs [5]. It appears to stimulate self-renewal of mouse SSCs and block their differentiation by acting in a paracrine manner [5,6]. Moreover, it has been found that GDNF-induced cell signaling plays a central role in SSC selfrenewal [4]. However, the molecular mechanism underlying SSC self-renewal remains largely unexplored. Cadherins represent a distinct family of single-transmembrane-domain glycoproteins which are key molecules during development, and serve as specific cell-adhesion molecules acting in a Ca2+-dependent manner [7]. Recent findings have demonstrated that DE-cadherin-mediated cell adhesion is essential for maintaining somatic stem cells in Drosophila ovaries and for the involvement of cadherins in signaling pathways [8]. In our previous work, we identified a novel adhesion molecule in the testis, short-type PB-cadherin (STPB-C), and found that it plays a critical role in promoting survival of gonocytes, the precursor cells of SSCs, in neonatal rats [9]. Furthermore, we observed that
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STPB-C activates the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway [9] by inducing phosphorylation of JAK2 and subsequently STAT3. However, little is known about whether STPB-C regulates proliferation of SSCs, although a relationship exists between STPBC expression and number of gonocytes, suggesting that overexpression of STPB-C may increase survival and/or production of gonocytes in testis of neonatal rats. The JAK-STAT signaling pathway is the principal signaling mechanism for a wide array of cytokines and growth factors, and STAT3 has been shown to have a central role in suppressing apoptosis and in promoting proliferation [10]. Evidence has shown that the JAK-STAT signaling pathway controls stem cell self-renewal in Drosophila spermatogenesis [11,12]. In the present study, we addressed whether STPB-C promoted self-renewal of SSCs, and identified the subcellular mechanisms involved in STPB-C function. We observed that STPB-C had a central role in promoting proliferation of rat SSCs in vivo and in vitro. Moreover, we found that this role of STPB-C was mediated via activation of the JAK-STAT and phosphoinositide-3 kinase (PI3-K)/Akt signaling pathways, and blockade of transforming growth factor (TGF)-β1 signaling function. Taking these data together, our findings have important implications for future gene therapy and for animal mutagenesis. 2. Experimental methods 2.1. Isolation and culture of SSCs Animal work was carried out under the National Institutes of Health guidelines. Testes of 6-day-old Sprague–Dawley rats (Charles River Breeding Labs, Kingston, RI) were isolated and decapsulated under a dissection microscope. SSCs were isolated essentially according to the methods of Nagano et al. (a two-step enzymatic digestion) [13]. In brief, decapsulated testes were incubated in approximately 10 volumes of Hanks' balanced salt solution without calcium or magnesium (HBSS) containing 1 mg/ml collagenase (Type IV, Sigma) at 37 °C with gentle agitation for 15 min. After the dispersion of seminiferous tubules, these tubules were then washed 2 to 4 times in 10 volumes of HBSS, followed by incubation at 37 °C for 5 min in HBSS containing 1 mM EDTA and 0.20% trypsin. When most of the cells were dispersed, the action of trypsin was terminated by adding a 20% volume of fetal bovine serum. The suspension was centrifuged at 1,000 r.p.m./min for 5 min, and the supernatant was carefully removed from the pellet. The cells were suspended, and the clumps of cells were removed by passing the suspension through a 70-µm nylon cell strainer. Testis cells enriched for SSCs were prepared by magnetic-activated cell sorting (MACS) with magnetic microbeads conjugated to anti-Thy-1 antibody (BD Biosciences), following the manufacturer's instructions. Finally, the cells were resuspended in SSC medium (see below) at a final concentration of 1 × 104 cells/ml. The culture system for SSCs consisted of the culture medium and mitotically inactivated STO (ATCC, derived from mouse SIM embryonic fibroblasts, strain SIM) cell feeders (~ 5 × 104 cells/cm2). STO cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high-glucose (Life Technologies, Inc., Gaithersburg, MD), supplemented with 1% nonessential amino acids (Life Technologies), 10% fetal bovine serum (FBS; Life Technologies), 2 mM glutamine (Sigma, St. Louis, MO, USA), 30 mg/l penicillin, and 75 mg/l streptomycin. The cells were treated with 10 µg/ml mitomycin C (Sigma) for 2–3 h. The mitomycin C-treated STO cells were washed in PBS, and plated on 0.2% (w/w) gelatin-coated wells of a 24-well plate. The culture medium for SSCs consisted of Minimum Essential Medium Alpha Medium (MEM-α), 10% FBS, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 2 mM L-Glutamine, 0.1 mM β-mercaptoethanol (Sigma), 10 ng/ml LIF (Santa Cruz Inc.), 20 μg/ml transferring, 5 μg/ml insulin, 60 μM
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putrescine, 10 ng/ml EGF (mouse epidermal growth factor), 30 mg/l penicillin. SSCs were cultured on STO feeders in 35 mm plastic Petri dishes in 3 ml culture medium. All cultures were maintained at 37 °C in a 5% CO2 atmosphere. The medium was changed every 2–3 days.
2.2. Immunoprecipitation and Western blotting Immunoprecipitation and Western blotting methods were as previously described [9]. For Western blotting, membranes were exposed to anti-p-STAT3 (phosphorylated STAT3, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-STAT3 (1:1000; BD Bioscience Clontech, Palo Alto, CA), anti-p-JAK2 (phosphorylated JAK2, 1:100; Santa Cruz Biotechnology), anti-JAK2 (1:200; Santa Cruz Biotechnology), anti-Bog (1:200; BD Bioscience Clontech), antiPCNA (1:1000; BD Bioscience Clontech), anti-TGFβ1 (1:100; Santa Cruz Biotechnology), anti-TβRI (TGF-β receptor I, 1:100; Santa Cruz Biotechnology), anti-TβRII (TGF-β receptor II, 1:100; Santa Cruz Biotechnology), anti-pPI3-K (phosphorylated PI3-K, 1:100; Santa Cruz Biotechnology), anti-PI3-K (1:100; BD Bioscience Clontech), anti-p-Akt (phosphorylated Akt, 1:100; Cell Signaling Technology, Danvers, MA), anti-Akt (1:1000; Cell Signaling Technology), or anti-STPB-C (1:200; Resgen, Huntsville, AL). For immunoprecipitation, protein samples were first pre-cleared in 1% normal rabbit serum for 1 h at 4 °C, followed by addition of protein A–agarose for an additional 1 h, and centrifugation. Supernatant was then incubated with anti-STPB-C for 2 h at 4 °C, followed by addition of protein A–agarose and overnight incubation. Immunoprecipitates were collected by centrifugation at 2500 r.p.m. for 10 min, washed with lysis buffer [14], resuspended in sample buffer, and separated by electrophoresis, as previously described for Western analysis [14]. Membranes were immunodetected with appropriate antibodies to anti-STPB-C (1:200; Resgen; positive control), anti-Bog (1:200; BD Bioscience Clontech), anti-PI3K (1:100; BD Bioscience Clontech), anti-TGFβ1 (1:100; Santa Cruz Biotechnology), anti-TβRI (1:100; Santa Cruz Biotechnology), anti-TβRII (1:100; Santa Cruz Biotechnology), or pre-immune serum (negative control).
2.3. Retroviral infection and TGF-β1 treatment STPB-C obtained from pCMV-STPB-C vector [9] was cloned into the pFBNeo retroviral vector (Stratagene) (pFB-Neo-STPB-C). HEK 293T cells (ATCC), were plated on 60-mm tissue culture plates. One day later, the cells were transfected with 3 µg pVPack-GP (gag–pol-expressing vector), and 3 µg pVpack-VSV-G (env-expressing vector), and 3 µg transfer plasmid, pFB-NeoSTPB-C, empty pFB-Neo vector or pFB-Neo-lacz, using the Virapack Transfection Kit according to the manufacturer's instruction (Stratagene). Retroviral supernatants were collected 48 h post-transfection, and passed through a 0.45-µm filter to remove any contaminating virus-producer cells. Unconcentrated vector stocks were used for transduction and titering immediately after collection. Titers were determined by reacting the cells with X-gal 48 h after transduction. The titer was ~106 c.f.u./ml. One milliliter retroviral supernatant with 10 µg/ml diethylaminoethyl–dextran was added to each 35-mm plastic Petri dish, containing SSCs cultures as described above, the dishes were incubated at 37 °C for 3 h, and then 1 ml culture medium (see above) was added to the dish for 1–2 days. For SSC proliferation assay, SSC cultures were labeled with BrdU after infection for 24 h and fixed for immunofluorescent staining of BrdU. For morphological analysis, STPB-C- or mock-infected SSC cultures were observed with phase-contrast optics after infection for 48 h. SSC cultures infected by STPB-C or mock retroviral vector were treated with TGF-β1 (Transforming growth factor beta 1, 1 ng/ml, R&D Systems, Minneapolis, MN) or vehicle for 24 h. After treatment, proliferation of SSCs was studied using a BrdU incorporation assay, as described below. Immunofluorescent staining for caspase-3 was used to determined apoptosis of SSCs.
2.4. RNA interference (RNAi) Bog specific shRNA expression pRS vector or pRS vector (Origene, Rockville, MD) was transfected into HEK 293T cells (ATCC) with FuGene 6 (Roche), according to the manufacturer's instructions (Origene). The suppressing function of this shRNA vector was determined by Western blotting. After high potency shRNA pRS vector for Bog was selected, this vector (Bog-pRS
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vector) was used as follows. Bog-pRS vector was transfected into Phoenix cells with FuGen 6, and the cells were incubated in a 5% CO2 incubator for 48 h. Retroviral supernatants were collected and passed through a 0.45-µm filter. Titers were determined as described above (~ 107 c.f.u./ml). The viral stock was directly added to SSC cultures for 24 h, followed by infection of STPB-C retroviral vector, BrdU incorporation assays, as described below.
2.5. SSC culture treatment with AG490 or LY294002 SSC cultures were pretreated with 30 µM AG490 (Sigma), 20 µM LY294002 (Cell Signaling) or 30 µM AG490 with viral stock for Bog-pRS vector or DMSO vehicle control for 24h, followed by infection with STPB-C retroviral vector. One day after infection, SSC proliferation was determined by BrdU incorporation assay.
2.6. BrdU labeling BrdU (50 µg/ml; Sigma–Aldrich, St. Louis, MO, USA) was added to SSC cultures, after the treatment described above, for 5 h. The cultures were processed for immunofluorescence as described below.
2.8. Northern blotting Total RNA was isolated from SSC cultures infected with either STPB-C retroviral vector or empty vector, using the Qiagen RNeasy kit according to the manufacturer's instructions. Samples (10 µg RNA/lane) were run on a 1% agarose–formaldehyde gel and transferred by capillary action to a nylon membrane (Amersham Pharmacia Biotech) in 20× saline-sodium citrate (SSC) solution overnight. Prehybridization and hybridization were performed as previously described for Southern blotting [9]. The probe was produced as described by Chow et al. [16].
2.9. Morphological analysis Testes from transgenic (n = 10) and wild-type (n = 12) day 5 rats were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and cleared by routine methods. For morphological analysis, sections were stained with hematoxylin– eosin (H–E) staining. For each animal, 20 sections were examined, each section containing a minimum of five cross-sectioned cords. Thus, the number of SSCs per cord profile was assessed in each mouse.
2.10. Data analysis 2.7. Immunofluorescence SSC cultures were fixed with 4% paraformaldehyde (15 min, room temperature). After fixation, SSC cultures were incubated in blocking solution (10% normal goat serum in PBS, 60 min, room temperature), followed by rinsing and overnight incubation in primary antibody at 4 °C: mouse monoclonal anti-PCNA (1:100 dilution; BD Bioscience Clontech); rabbit monoclonal anticaspase-3 (1:100 dilution; Cell Signaling Technology); or mouse monoclonal anti-BrdU (1:100 dilution; Lab Vision Corporation, Fremont, CA). After extensive rinsing, cultures were incubated in darkness with FITC-conjugated secondary antibody (goat anti-rabbit IgG, 1:200 dilution, or rabbit anti-mouse IgG), then rinsed, mounted in 4',6-diamidino-2-phenylindole (DAPI)-containing medium, and viewed and photographed with a Nikon inverted microscope equipped with a Magna-Fire digital camera.
Quantitative data were expressed as the mean ± SEM from at least three experiments. One-way analysis of variance and Student's t test were used for statistical analysis with Sigmastat (Systat Software, Point Richmond, CA) software. P b 0.05 was considered statistically significant.
3. Results 3.1. STPB-C promotes self-renewal of SSCs in vivo and in vitro To determine if STPB-C promotes proliferation (selfrenewal) of SSCs, we first examined the number of SSCs per
Fig. 1. STPB-C promotes self-renewal of SSCs in vivo. (A) Representative morphology of testes from day 5 control (b) or transgenic (Tg) rats (a), demonstrating that the overall morphology of Tg testicular cords was normal, with many SSCs present. (C) Significantly higher numbers of SSCs per cord were quantified in transgenic (Tg) compared to control (Con) rats (⁎P b 0.01). (B) Representative (four replicates) results of Western blot analysis of PCNA in protein isolated from day 5 testes of transgenic (Tg) or control (Con) rats. Equal loading of protein across lanes was verified by reprobing the blots for β-tubulin.
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cross-sectioned seminiferous cord in day 5 transgenic rats overexpressing STPB-C (Tgs) [9] and controls. We found that the number of SSCs in Tgs was significantly higher than that seen in controls (Fig. 1A, C). Based on our earlier data from protein microarray analysis that proliferating cell nuclear antigen (PCNA) expression is higher in day 4 Tgs than in controls [9], we compared PCNA expression in testes on postnatal day 5 in Tg rats and controls. Using Western blot analysis, we observed that there was a marked increase in PCNA expression in Tgs compared to controls (Fig. 1B). To explore this possibility directly (STPB-C promotes self-renewal of SSCs), we applied a replication-defective retroviral genetransfer system and infected SSC cultures with viral supernatant containing the STPB-C gene (pFB-Neo-STPB-C). After 48 h, it was obvious upon visual inspection of cultures infected with the STPB-C retroviral vector that the number of SSCs increased and many SSC clusters were noted (Fig. 2A–C), while only a few clustered SSCs were seen in vehicle-treated controls (Fig. 2A–C). Bromodeoxyuridine (BrdU) labeling of cultures showed that SSC proliferation was obviously increased in cultures infected with the STPB-C retroviral vector, compared with that in mock-infected cultures (Fig. 2B and C). In STPB-C-
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infected and mock-transfected cultures, the number of PCNApositive SSCs in parallel cultures was similar to the number of BrdU-positive SSCs (Fig. 2D). 3.2. STPB-C activates the PI3-K/Akt signaling pathway Previous data from protein microarray analysis have suggested that STPB-C up-regulates PI3-K expression in neonatal testes [9]. Therefore, we asked whether STPB-C activates the PI3-K/Akt signaling pathway. To test this possibility, we used immunoprecipitation to clarify whether STPB-C physically binds to PI3-K. Proteins isolated from testes of Tgs and controls were immunoprecipitated with STPB-C and immunoblotted with anti-PI3-K. The blots were then stripped and re-probed with anti-STPB-C to confirm the presence of STPB-C. The results indicated that PI3-K was not a STPB-C binding partner (Fig. 3A). Based upon studies showing that phosphorylated JAK activates SH2-containing protein [15,17,18], PI3-K, and the result that STPB-C binds to and activates JAK2 (Fig. 3B) [9], we hypothesized that STPB-C activates the PI3-K/Akt signaling pathway via binding to and activating JAK2. To test this hypothesis, we first determined whether PI3-K/Akt
Fig. 2. STPB-C promotes self-renewal of SSCs in vitro. (A) Representative views of SSC cultures that were infected with either STPB-C retroviral vector (STPB-C; A-b) or empty retroviral vector (Mock; A-a). Arrows indicate clustered SSCs. (B) STPB-C gene transfer induced an increase in BrdU-positive SSCs. Photomicrographs of BrdU immunofluorescence after SSC cultures were infected with STPB-C (B-b) or empty retroviral vector for 24 h (B-a) and BrdU labeling for 5 h. (B-c, d) DAPI immunofluorescence. Arrows indicate BrdU-positive SSCs. (C) Significantly higher numbers of SSCs (No. 1), clustered SSCs (No. 2) or BrdU-positive SSCs (No. 3) were quantified in STPB-C- compared to mock-infected SSC cultures (⁎P b 0.05). (D) The effect of STPB-C infection on expression of PCNA in SSC cultures. (D-a, b) PCNA immunofluorescence. (D-c, d) DAPI immunofluorescence. Note the obvious increase in PCNA-positive SSCs in STPB-C-infected cultures. Arrows indicate PCNA-positive SSCs. Scale bar = 50 µm.
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Fig. 3. STPB-C activates the PI3-K/Akt signaling pathway. (A, B) Protein obtained from testes of transgenic (Tg) or control (Con) rats was immunoprecipitated with STPB-C and then probed with PI3-K (A) or JAK2 (B). Subsequently, blots were stripped and re-probed with anti-STPB-C to confirm the presence of STPB-C in the immunoprecipitate (Ip). (C) Western blots of PI3-K, p-PI3-K (Tyr 508), Akt or p-Akt (Tyr 326) in protein isolated from testes of day 5 transgenic (Tg) or control (Con) rats were quantified by densitometry and the data were expressed as a ratio of PI3-K, p-PI3-K, Atk or p-Akt:β-tubulin. ⁎P b 0.05. (D and E) Western blot analysis of p-PI3-K (D) or p-Akt (E), obtained from STPB-C-infected (STPB-C) or mock-infected (Mock) SSC cultures.
components are overexpressed in day 5 testes of Tgs, compared expression of PI3-K, p-PI3-K, Akt and p-Akt by immunoblotting proteins from control and transgenic rats. With Western blot analysis, we noted an obvious increase in PI3-K or Akt expression in Tgs compared to that in control rats while no significant difference was detected in the expression of p-PI3-K or p-Akt in Tgs and controls (Fig. 3C). Next, we used SSC cultures to examine the changes in expression of p-PI3-K and pAkt following infection with the STPB-C retroviral vector. By 24 h post-infection, expression of p-PI3-K or p-Akt was obviously increased compared with that in mock infection, while no obvious change was detected in the total pool of PI3K or Akt in STPB-C infection (Fig. 3D and E). Taken together, these results provide evidence supporting our hypothesis above.
in SSC cultures infected by STPB-C retroviral vector compared to that in controls (Fig. 4E and F). When SSC cultures infected with the STPB-C retroviral vector or mock-infected cultures were treated with TGF-β1, we found that the number of BrdUpositive SSCs in STPB-C-infected cultures was much higher than that of controls (mock) (Fig. 4C-a–d, D), and this difference was much higher than that in untreated SSCs in parallel cultures (Fig. 4C-e–h, D). However, an obvious decrease in caspase-3-positive SSCs in STPB-C-infected cultures was noted compared with that in controls (Fig. 4B-a–d). In addition, there was no significant difference in mRNA expression of TβRII between STPB-C-infected cultures and controls (Fig. 4G).
3.3. STPB-C blocks TGF-β1 signaling growth inhibition by upregulating Bog expression
Previously, we have identified a role for STPB-C in promoting survival of gonocytes in neonatal rats, and have linked its expression to the JAK-STAT signaling pathway, including that STPB-C transfection of L cells up-regulates JAK-STAT components. To elucidate whether the role of STPB-C in SSC proliferation is also linked to JAK-STAT signaling, we first used Western blot analysis to compare expression of JAK-STAT components in day 5 testes in Tgs and controls. Western blot analysis showed an obviously higher expression of JAK2 and STAT3 in Tgs testes (Fig. 5A). Furthermore, we determined whether overexpression of STPB-C in SSC cultures caused changes in levels of p-JAK2 and/or p-STAT3. With Western blot analysis, we found that both p-JAK2 and p-STAT3 were
We have previously reported that STPB-C up-regulates Bog expression in neonatal testes [9,19]. In addition, a recent study has indicated that overexpression of Bog in cells results in loss of TGF-β1 growth inhibition [20]. We reasoned that STPB-C may lead to loss of TGF-β1 signaling growth inhibition by upregulating Bog expression in SSCs. To test this, we first used immunoprecipitation to elucidate whether STPB-C bound to Bog or TGF-β1. As shown in Fig. 4A, we found that STPB-C bound to Bog, but not to TGF-β1, TβRI or TβRII. Furthermore, we observed that expression of Bog was significantly increased
3.4. STPB-C activates the JAK-STAT signaling pathway
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Fig. 4. STPB-C results in loss of TGF-β1 growth inhibition. (A) Protein obtained from testes of day 5 transgenic rats was immunoprecipitated with STPB-C and then probed with TGF-β1, TβRI, TβRII or Bog. Subsequently, blots were stripped and re-probed with anti-STPB-C to confirm the presence of STPB-C in the immunoprecipitate. (B–D) Overexpression of STPB-C disrupted the growth-inhibitory effect of TGF-β1. SSC cultures were infected with STPB-C or empty (mock) retroviral vector. After 48 h, they were cultured in the presence (C-a–d, D-No.1) or absence (C-e–h, D-No. 2) of TGF-β1 for 24 h and BrdU labeling for 5 h. (C-a, b, e, f) BrdU immunofluorescence. (B-a, b) Caspase-3 immunofluorescence. (C-c, d, g, h, B-c,d) DAPI immunofluorescence. Note obvious increase in BrdU-positive SSCs (C-b, f, D, ⁎P b 0.05, ⁎⁎P b 0.005) and decrease in caspase-3-positive SSCs (B-b) in STPB-C-infected cultures. Arrows indicate BrdU-positive or caspase-3-positive SSCs. (E) Western blot analysis of Bog in samples obtained from mock-infected (Mock) or STPB-C-infected (STPB-C) SSC cultures. Equal loading of protein across lanes was verified by reprobing Western blots for β-tubulin. (F) Western blots from 4 separate experiments were quantified by densitometry and data were expressed as a ratio of Bog: β-tubulin. ⁎P b 0.05. (G) Northern blot analysis of TβRII showing no difference in mRNA expression of TβRII in STPB-C- and mock-infected cultures. The expression of GAPDH was used as an internal standard to control for evenness of loading. Scale bar = 50 µm.
markedly increased in STPB-C-infected cultures compared to that in controls (mock-infected) (Fig. 5B, C). 3.5. STPB-C promotes self-renewal of SSCs by activating JAKSTAT and PI3-K/Akt signaling pathways and blocking TGF-β1 signaling Previous studies have shown that STPB-C activates JAKSTAT signaling by binding to JAK2 [9]. Moreover, our results above indicate that STPB-C also activated PI3-K/Akt signaling by binding to JAK2. To examine the role of the JAK-STAT and PI3-K/Akt pathway in STPB-C-promoted SSC self-renewal, SSC cultures were pretreated with Tyrphostin AG 490 (JAK2 inhibitor) or LY294002 (PI3-kinase inhibitor) before infection with STPB-C retroviral vector. AG 490 or LY294002 markedly blocked STPB-C-promoted SSC proliferation (Fig. 6A-a–f, B). To determine the effects of TGF-β1 signaling on STPB-Cinduced SSC proliferation, SSC cultures were infected with viral stock for Bog-pRS vector (vector for Bog knockdown, see Experimental methods) prior to infection with STPB-C retroviral vector. As shown in Fig. 6A-a,b,g,h, B, C, Bog knock-
down reduced the STPB-C-induced increase in the number of BrdU-positive SSCs. In the end, STPB-C-induced SSC proliferation was completely blocked by Tyrphostin AG 490 treatment and Bog knockdown (Fig. 6A, B). 4. Discussion In adult rodents, continuous availability of differentiating spermatogonia is responsible for lifelong fertility of males and species continuity. The number of these spermatogonia available to enter spermatogenesis is controlled by regulation of SSC proliferation and apoptosis [21]. In this study, we examined a novel cadherin, STPB-C, previously found by us to be highly expressed in neonatal testes [14], and its overexpression in transgenic rats results in abnormally high numbers of gonocytes in neonates [9]. We asked whether it has a role in promoting self-renewal of SSCs, besides supporting gonocyte survival by suppressing apoptosis [9]. First, we found that overexpression of STPB-C led to high numbers of SSCs and high expression of PCNA in rat testes from postnatal day 5. Next, we observed that the number of SSCs and BrdU
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Fig. 5. STPB-C activates JAK-STAT signaling pathway in SSCs. (A) Representative Western blot analysis of JAK2 or STAT3 in protein isolated from testes of day 5 transgenic (Tg) or control (Con) rats. Equal loading of protein across lanes was verified by reprobing the blots for β-tubulin. (B and C) Western blot analysis of p-JAK2 (B) or p-STAT3 (C) in protein samples isolated from STPB-C- or mock-infected SSC cultures.
incorporation obviously increased when SSC cultures were infected by STPB-C retroviral vector. Thus, expression of STPB-C in neonates appears to induce SSC proliferation. GDNF is a member of the TGF-β superfamily. It promotes survival and differentiation of several types of neuron [22,23] and regulates branching of the ureter in the embryonic kidney [24,25]. Meng et al. [5] have reported that gene-targeted mice with one GDNF null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. Moreover, Kubota et al. [4] have found that GDNF is the essential growth factor for SSC self-renewal in most, but not all mammals. For instance, SSCs derived from DBA/2 mouse gonocytes (precursors of SSCs) proliferate in culture using medium containing GDNF, leukocyte inhibitory factor (LIF), epidermal growth factor, basic fibroblast growth factor and 1% FBS [26]. In the same culture conditions [26], however, SSCs from other strains (C57BL/6 or 129/SV) do not proliferate. Therefore, STPB-C is another important factor identified for SSC self-renewal, because we have recently found that STPB-C promotes self-renewal of SSCs from C57BL/6 background mice (Wu, unpublished data), and it plays a central role in inducing self-renewal of rat SSCs. Our results have important implications for future gene therapy and for animal mutagenesis, because SSCs are present in extremely
Fig. 6. STPB-C Promotes self-renewal of SSCs by activating JAK-STAT and PI3-K/Akt signaling pathway and blocking TGF-β1 signaling. (A) SSC cultures were pretreated with AG 490 (A-c), LY294002 (A-e), Bog RNAi (A-g), AG 490 plus Bog RNAi (A-i), or controls (A-a, k), before being infected with STPB-C or mock retroviral vector. (A-a, c, e, g, i, k) BrdU immunofluorescence. (A-b, d, f, h, j, l) DAPI immunofluorescence. (B) The number of BrdU-positive cells per condition was quantified. The values represent the mean ± SE of four independent experiments. For each condition, changes in the number of positive cells, are indicated by a (P b 0.05, relative to mock-infected cultures) or b (P b 0.05, relative to STPB-C-infected cultures). (C) Western blot analysis of Bog in samples obtained from SSC cultures infected with Mock (lane 1), STPB-C (lane 2), STPB-C + AG290 (lane 3), STPB-C + LY294002 (lane 4), STPB-C + Bog RNAi (lane 5), STPB-C + control RNAi (lane 6), STPB-C + AG490 + Bog RNAi (lane 7) or STPB-C + AG490 + control RNAi (lane 8). Equal loading of protein across lanes was verified by reprobing Western blots for β-tubulin.
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low numbers in testes, and it has been difficult to produce transgenic animals by SSCs if SSCs do not expand in vitro. Self-renewal of stem cells requires in the integration of survival signals and proliferation controls with the maintenance of their undifferentiated state. This demands a complex crosstalk between extrinsic signals from the microenvironment and the cell-intrinsic regulators of self-renewal. To establish the mechanism of this process, we have explored the molecular and cellular mechanism of STPB-C in inducing self-renewal of SSCs in this study. In general, there are three different ways in which cadherin expression can influence signaling. Firstly, cadherin-dependent adhesion can induce juxtacrine signaling by bringing the opposing membranes of neighboring cells into close proximity, consequently enabling membrane-linked ligands, or ligands tightly associated with the pericellular matrix, to interact with their receptors on the neighboring cell. Secondly, cadherinmediated adhesion can influence cell signaling by inducing polarization and tight-junction formation. In addition, cadherins can mediate signaling directly. Cytoplasmic or transmembranous signaling molecules may act as co-receptors that become activated by homotypic binding of the cadherins. However, unlike most cadherins, STPB-C lacks an intracellular cateninbinding domain, suggesting that STPB-C may associate with specific cytoplasmic molecules distinct from catenins, and any associated signaling pathway does not involve interaction with cytoskeletal elements. In previous work, we have produced transgenic rats overexpressing STPB-C, in which we have used protein microarrays to seek clues concerning the regulatory pathway for STPB-C. The results suggest that JAK-STAT components are involved in STPB-C action, which has been confirmed by the up-regulation of JAK2, STAT3 and SH2-B in transgenic animals, and down-regulation of these components in cultured cells exposed to anti-STPB-C [9]. Further studies have indicated that STPB-C activates the JAK-STAT pathway by binding to JAK2 and inducing phosphorylation of JAK2, and subsequently STAT3 [9]. In this study, however, we found that STPB-C promoted self-renewal of SSCs by PI3-K/Akt and TGF-β1 signaling, besides JAK-STAT. Firstly, we observed that STPB-C overexpression in SSC cultures induced high levels of p-PI3-k or p-Akt. Secondly, Ferrand et al. [27] have shown that activation of JAK2 is upstream of PI3-K/Akt, and that JAK2 is as an upstream mediator of PI3-K activity. In addition, our previous results have indicated that JAK2 is a STPB-C binding partner [9]. In this study, we found that there was an obvious increase in PI3-K or Akt expression in transgenic rats overexpressing STPB-C, compared to that in controls. Further data have demonstrated that STPB-C overexpression results in increased phosphorylation of PI3-K or Akt in SSC cultures. Moreover, our data show that STPB-C bound to Bog and upregulated its expression. Overexpression of STPB-C or Bog in SSC cultures resulted in the loss of TGF-β1 growth inhibition, and did not significantly affect TGF-β1 receptor II mRNA expression. Bog knockdown partly blocked STPB-C-promoted self-renewal of SSCs. Our results indicated that inhibition of JAK-STAT, PI3-k/Akt and activating TGF-β1 signaling completely blocked STPB-C-induced self-renewal of SSCs.
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Thus, our findings demonstrate that STPB-C promotes selfrenewal of SSCs via the JAK-STAT, PI3-K/Akt and TGF-β1 signaling pathways. In mammals, the JAK-STAT pathway is the principal signaling mechanism for a wide array of cytokines and growth factors. STATs may affect proliferation by regulating the expression of immediate–early genes, such as c-myc and c-fos, as well as cell-cycle regulatory genes, such as the cyclins [28]. Moreover, Simon et al. [29] have reported that the JAK-STAT pathway contributes to PDGF-induced mitogenesis. In addition, STAT3 signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. For instance, STAT3 is constitutively activated in various human gastric cancer cells [30], and blocking STAT3 activation can abolish the antiapoptotic and mitogenic actions of IL-22 in hepatic cells [31]. Woitach et al. [20] have reported that overexpression of Bog in RLE cells results in loss of TGF-β1 signaling growth inhibition. TGF-β1 is a ubiquitous cytokine that is well known for its ability to inhibit epithelial cell proliferation [32]. Furthermore, TGF-β1 exerts potent anti-mitogenic and pro-apoptotic effects on certain cancer cells [33,34]. However, somatic mutations abrogating the TGF-β signaling pathway are found in many gastrointestinal cancers [32]. Recent studies have suggested that PI3-K acts as an immediate downstream molecule of growth factor receptors to mediate mitogenic signaling in cells. The activation of PI3-K is sufficient to drive some types of cells to go through G1/S transition and to proliferate [35]. The most commonly known downstream target of PI3-K is the serine–threonine kinase Akt [36–39]. Activated Akt can phosphorylate multiple proteins implicated in the control of the cell cycle to ultimately stimulate cell growth [40,41]. Inhibition of the PI3-K/Akt pathway by LY294002 significantly reduced the number of newly-formed
Fig. 7. The signaling diagram illustrates that STPB-C promotes self-renewal of SSCs via activating JAK-STAT and PI3-K/Akt, and blocking TGF-β1 signaling.
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neurospheres, which indicates that this is an essential pathway for neural stem cell self-renewal [42]. Therefore, PI3-K/Akt is thought to play a crucial role in the survival and proliferation of various cell types [40,41]. Current understanding of these signaling pathways is consistent with our findings, suggesting a role for JAK-STAT, TGF-β1 or PI3-K/Akt signaling pathways in regulation of growth in SSCs, similar to that in other cell types. In conclusion, with varied approaches, including the use of RNAi, SSC cultures infected by STPB-C retroviral vector, and other techniques, we have defined a new function for STPB-C, promoting SSC self-renewal. Moreover, we found that this function of STPB-C was required to activate the JAK-STAT and PI3-K/Akt, and to block the TGF-β1 signaling pathways (Fig. 7). These data have important implications for germ cell biology and create the possibility of using SSCs for biotechnology and medicine. Acknowledgements This work was supported by a key Program of the National Natural Scientific Foundation of China (No. 30630012, to J.W.), and sponsored by the Shanghai Pujing Program (No. 06PJ14058, to J.W.), Shanghai Leading Academic Discipline Project (No. B205) and the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) in China (No. 20050248010 to J.W.). References [1] [2] [3] [4] [5]
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Cellular Signalling 20 (2008) 1052 – 1060 www.elsevier.com/locate/cellsig
Short-type PB-cadherin promotes self-renewal of spermatogonial stem cells via multiple signaling pathways Ji Wu a,d,⁎, Yong Zhang a , Geng G. Tian b , Kang Zou a , Clement M. Lee c , Qingsheng Yu a , Zhe Yuan a a
School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China Department of Biological Science, School of Life Science, Nanjin Normal University, Nanjin, Jiangsu 210097, China c Fels. Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao Tong University, Shanghai, 200240, China b
d
Received 2 November 2007; received in revised form 13 January 2008; accepted 14 January 2008 Available online 24 January 2008
Abstract Stem cells represent a unique population of cells with self-renewal capacity. However, the molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Here, we show that short-type PB-cadherin (STPB-C) promoted self-renewal of spermatogonial stem cells (SSCs) via activating Janus kinase/signal transducer and activator of transcription (JAK-STAT) and phosphoinositide-3 kinase (PI3-K)/Akt, and blocking transforming growth factor (TGF)-β1 signaling. These data were obtained with varied approaches, including the use of RNA interference (RNAi), SSC cultures infected by STPB-C retroviral vector, bromodeoxyuridine (BrdU) incorporation assay, and other techniques. These findings have important implications for germ cell biology and create the possibility of using SSCs for biotechnology and medicine. They are also critical in understanding tissue homeostasis, the aging process, tumor formation and degenerative diseases. © 2008 Elsevier Inc. All rights reserved. Keywords: STPB-C; Self-renewal of spermatogonial stem cells; JAK-STAT; PI3-K/Akt; TGF-β1 signaling
1. Introduction Stem cells are characterized by their ability to self-renew and to continuously generate differentiated cells. Uncovering the molecular control of stem cell self-renewal is crucial to the future use of stem cells in regenerative medicine and in understanding tissue homeostasis, the aging process, tumor formation and degenerative diseases [1–3]. To maintain normal spermatogenesis, the processes of self-renewal and differentiation of spermatogonial stem cells (SSCs) must be precisely regulated by intrinsic gene expression in the stem cells and by extrinsic signals, including soluble factors or adhesion molecules from the surrounding microenvironment, the stem cell niche. ⁎ Corresponding author. Department of Biological Science and Biotechnology, School of Life Science and Biotechnology, Shanghai Jiao Tong University, No. 800, Dongchuan Road, Minhang District, Shanghai, 200240, China. Tel.: +86 21 34204933; fax: +86 21 34204051. E-mail address: [email protected] (J. Wu). 0898-6568/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.01.011
A recent study has identified growth factors essential for selfrenewal and expansion of mouse SSCs [4]. Glial cell linederived neurotrophic factor (GDNF) is a key factor in deciding the fate of SSCs [5]. It appears to stimulate self-renewal of mouse SSCs and block their differentiation by acting in a paracrine manner [5,6]. Moreover, it has been found that GDNF-induced cell signaling plays a central role in SSC selfrenewal [4]. However, the molecular mechanism underlying SSC self-renewal remains largely unexplored. Cadherins represent a distinct family of single-transmembrane-domain glycoproteins which are key molecules during development, and serve as specific cell-adhesion molecules acting in a Ca2+-dependent manner [7]. Recent findings have demonstrated that DE-cadherin-mediated cell adhesion is essential for maintaining somatic stem cells in Drosophila ovaries and for the involvement of cadherins in signaling pathways [8]. In our previous work, we identified a novel adhesion molecule in the testis, short-type PB-cadherin (STPB-C), and found that it plays a critical role in promoting survival of gonocytes, the precursor cells of SSCs, in neonatal rats [9]. Furthermore, we observed that
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STPB-C activates the Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling pathway [9] by inducing phosphorylation of JAK2 and subsequently STAT3. However, little is known about whether STPB-C regulates proliferation of SSCs, although a relationship exists between STPBC expression and number of gonocytes, suggesting that overexpression of STPB-C may increase survival and/or production of gonocytes in testis of neonatal rats. The JAK-STAT signaling pathway is the principal signaling mechanism for a wide array of cytokines and growth factors, and STAT3 has been shown to have a central role in suppressing apoptosis and in promoting proliferation [10]. Evidence has shown that the JAK-STAT signaling pathway controls stem cell self-renewal in Drosophila spermatogenesis [11,12]. In the present study, we addressed whether STPB-C promoted self-renewal of SSCs, and identified the subcellular mechanisms involved in STPB-C function. We observed that STPB-C had a central role in promoting proliferation of rat SSCs in vivo and in vitro. Moreover, we found that this role of STPB-C was mediated via activation of the JAK-STAT and phosphoinositide-3 kinase (PI3-K)/Akt signaling pathways, and blockade of transforming growth factor (TGF)-β1 signaling function. Taking these data together, our findings have important implications for future gene therapy and for animal mutagenesis. 2. Experimental methods 2.1. Isolation and culture of SSCs Animal work was carried out under the National Institutes of Health guidelines. Testes of 6-day-old Sprague–Dawley rats (Charles River Breeding Labs, Kingston, RI) were isolated and decapsulated under a dissection microscope. SSCs were isolated essentially according to the methods of Nagano et al. (a two-step enzymatic digestion) [13]. In brief, decapsulated testes were incubated in approximately 10 volumes of Hanks' balanced salt solution without calcium or magnesium (HBSS) containing 1 mg/ml collagenase (Type IV, Sigma) at 37 °C with gentle agitation for 15 min. After the dispersion of seminiferous tubules, these tubules were then washed 2 to 4 times in 10 volumes of HBSS, followed by incubation at 37 °C for 5 min in HBSS containing 1 mM EDTA and 0.20% trypsin. When most of the cells were dispersed, the action of trypsin was terminated by adding a 20% volume of fetal bovine serum. The suspension was centrifuged at 1,000 r.p.m./min for 5 min, and the supernatant was carefully removed from the pellet. The cells were suspended, and the clumps of cells were removed by passing the suspension through a 70-µm nylon cell strainer. Testis cells enriched for SSCs were prepared by magnetic-activated cell sorting (MACS) with magnetic microbeads conjugated to anti-Thy-1 antibody (BD Biosciences), following the manufacturer's instructions. Finally, the cells were resuspended in SSC medium (see below) at a final concentration of 1 × 104 cells/ml. The culture system for SSCs consisted of the culture medium and mitotically inactivated STO (ATCC, derived from mouse SIM embryonic fibroblasts, strain SIM) cell feeders (~ 5 × 104 cells/cm2). STO cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high-glucose (Life Technologies, Inc., Gaithersburg, MD), supplemented with 1% nonessential amino acids (Life Technologies), 10% fetal bovine serum (FBS; Life Technologies), 2 mM glutamine (Sigma, St. Louis, MO, USA), 30 mg/l penicillin, and 75 mg/l streptomycin. The cells were treated with 10 µg/ml mitomycin C (Sigma) for 2–3 h. The mitomycin C-treated STO cells were washed in PBS, and plated on 0.2% (w/w) gelatin-coated wells of a 24-well plate. The culture medium for SSCs consisted of Minimum Essential Medium Alpha Medium (MEM-α), 10% FBS, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 2 mM L-Glutamine, 0.1 mM β-mercaptoethanol (Sigma), 10 ng/ml LIF (Santa Cruz Inc.), 20 μg/ml transferring, 5 μg/ml insulin, 60 μM
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putrescine, 10 ng/ml EGF (mouse epidermal growth factor), 30 mg/l penicillin. SSCs were cultured on STO feeders in 35 mm plastic Petri dishes in 3 ml culture medium. All cultures were maintained at 37 °C in a 5% CO2 atmosphere. The medium was changed every 2–3 days.
2.2. Immunoprecipitation and Western blotting Immunoprecipitation and Western blotting methods were as previously described [9]. For Western blotting, membranes were exposed to anti-p-STAT3 (phosphorylated STAT3, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-STAT3 (1:1000; BD Bioscience Clontech, Palo Alto, CA), anti-p-JAK2 (phosphorylated JAK2, 1:100; Santa Cruz Biotechnology), anti-JAK2 (1:200; Santa Cruz Biotechnology), anti-Bog (1:200; BD Bioscience Clontech), antiPCNA (1:1000; BD Bioscience Clontech), anti-TGFβ1 (1:100; Santa Cruz Biotechnology), anti-TβRI (TGF-β receptor I, 1:100; Santa Cruz Biotechnology), anti-TβRII (TGF-β receptor II, 1:100; Santa Cruz Biotechnology), anti-pPI3-K (phosphorylated PI3-K, 1:100; Santa Cruz Biotechnology), anti-PI3-K (1:100; BD Bioscience Clontech), anti-p-Akt (phosphorylated Akt, 1:100; Cell Signaling Technology, Danvers, MA), anti-Akt (1:1000; Cell Signaling Technology), or anti-STPB-C (1:200; Resgen, Huntsville, AL). For immunoprecipitation, protein samples were first pre-cleared in 1% normal rabbit serum for 1 h at 4 °C, followed by addition of protein A–agarose for an additional 1 h, and centrifugation. Supernatant was then incubated with anti-STPB-C for 2 h at 4 °C, followed by addition of protein A–agarose and overnight incubation. Immunoprecipitates were collected by centrifugation at 2500 r.p.m. for 10 min, washed with lysis buffer [14], resuspended in sample buffer, and separated by electrophoresis, as previously described for Western analysis [14]. Membranes were immunodetected with appropriate antibodies to anti-STPB-C (1:200; Resgen; positive control), anti-Bog (1:200; BD Bioscience Clontech), anti-PI3K (1:100; BD Bioscience Clontech), anti-TGFβ1 (1:100; Santa Cruz Biotechnology), anti-TβRI (1:100; Santa Cruz Biotechnology), anti-TβRII (1:100; Santa Cruz Biotechnology), or pre-immune serum (negative control).
2.3. Retroviral infection and TGF-β1 treatment STPB-C obtained from pCMV-STPB-C vector [9] was cloned into the pFBNeo retroviral vector (Stratagene) (pFB-Neo-STPB-C). HEK 293T cells (ATCC), were plated on 60-mm tissue culture plates. One day later, the cells were transfected with 3 µg pVPack-GP (gag–pol-expressing vector), and 3 µg pVpack-VSV-G (env-expressing vector), and 3 µg transfer plasmid, pFB-NeoSTPB-C, empty pFB-Neo vector or pFB-Neo-lacz, using the Virapack Transfection Kit according to the manufacturer's instruction (Stratagene). Retroviral supernatants were collected 48 h post-transfection, and passed through a 0.45-µm filter to remove any contaminating virus-producer cells. Unconcentrated vector stocks were used for transduction and titering immediately after collection. Titers were determined by reacting the cells with X-gal 48 h after transduction. The titer was ~106 c.f.u./ml. One milliliter retroviral supernatant with 10 µg/ml diethylaminoethyl–dextran was added to each 35-mm plastic Petri dish, containing SSCs cultures as described above, the dishes were incubated at 37 °C for 3 h, and then 1 ml culture medium (see above) was added to the dish for 1–2 days. For SSC proliferation assay, SSC cultures were labeled with BrdU after infection for 24 h and fixed for immunofluorescent staining of BrdU. For morphological analysis, STPB-C- or mock-infected SSC cultures were observed with phase-contrast optics after infection for 48 h. SSC cultures infected by STPB-C or mock retroviral vector were treated with TGF-β1 (Transforming growth factor beta 1, 1 ng/ml, R&D Systems, Minneapolis, MN) or vehicle for 24 h. After treatment, proliferation of SSCs was studied using a BrdU incorporation assay, as described below. Immunofluorescent staining for caspase-3 was used to determined apoptosis of SSCs.
2.4. RNA interference (RNAi) Bog specific shRNA expression pRS vector or pRS vector (Origene, Rockville, MD) was transfected into HEK 293T cells (ATCC) with FuGene 6 (Roche), according to the manufacturer's instructions (Origene). The suppressing function of this shRNA vector was determined by Western blotting. After high potency shRNA pRS vector for Bog was selected, this vector (Bog-pRS
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vector) was used as follows. Bog-pRS vector was transfected into Phoenix cells with FuGen 6, and the cells were incubated in a 5% CO2 incubator for 48 h. Retroviral supernatants were collected and passed through a 0.45-µm filter. Titers were determined as described above (~ 107 c.f.u./ml). The viral stock was directly added to SSC cultures for 24 h, followed by infection of STPB-C retroviral vector, BrdU incorporation assays, as described below.
2.5. SSC culture treatment with AG490 or LY294002 SSC cultures were pretreated with 30 µM AG490 (Sigma), 20 µM LY294002 (Cell Signaling) or 30 µM AG490 with viral stock for Bog-pRS vector or DMSO vehicle control for 24h, followed by infection with STPB-C retroviral vector. One day after infection, SSC proliferation was determined by BrdU incorporation assay.
2.6. BrdU labeling BrdU (50 µg/ml; Sigma–Aldrich, St. Louis, MO, USA) was added to SSC cultures, after the treatment described above, for 5 h. The cultures were processed for immunofluorescence as described below.
2.8. Northern blotting Total RNA was isolated from SSC cultures infected with either STPB-C retroviral vector or empty vector, using the Qiagen RNeasy kit according to the manufacturer's instructions. Samples (10 µg RNA/lane) were run on a 1% agarose–formaldehyde gel and transferred by capillary action to a nylon membrane (Amersham Pharmacia Biotech) in 20× saline-sodium citrate (SSC) solution overnight. Prehybridization and hybridization were performed as previously described for Southern blotting [9]. The probe was produced as described by Chow et al. [16].
2.9. Morphological analysis Testes from transgenic (n = 10) and wild-type (n = 12) day 5 rats were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and cleared by routine methods. For morphological analysis, sections were stained with hematoxylin– eosin (H–E) staining. For each animal, 20 sections were examined, each section containing a minimum of five cross-sectioned cords. Thus, the number of SSCs per cord profile was assessed in each mouse.
2.10. Data analysis 2.7. Immunofluorescence SSC cultures were fixed with 4% paraformaldehyde (15 min, room temperature). After fixation, SSC cultures were incubated in blocking solution (10% normal goat serum in PBS, 60 min, room temperature), followed by rinsing and overnight incubation in primary antibody at 4 °C: mouse monoclonal anti-PCNA (1:100 dilution; BD Bioscience Clontech); rabbit monoclonal anticaspase-3 (1:100 dilution; Cell Signaling Technology); or mouse monoclonal anti-BrdU (1:100 dilution; Lab Vision Corporation, Fremont, CA). After extensive rinsing, cultures were incubated in darkness with FITC-conjugated secondary antibody (goat anti-rabbit IgG, 1:200 dilution, or rabbit anti-mouse IgG), then rinsed, mounted in 4',6-diamidino-2-phenylindole (DAPI)-containing medium, and viewed and photographed with a Nikon inverted microscope equipped with a Magna-Fire digital camera.
Quantitative data were expressed as the mean ± SEM from at least three experiments. One-way analysis of variance and Student's t test were used for statistical analysis with Sigmastat (Systat Software, Point Richmond, CA) software. P b 0.05 was considered statistically significant.
3. Results 3.1. STPB-C promotes self-renewal of SSCs in vivo and in vitro To determine if STPB-C promotes proliferation (selfrenewal) of SSCs, we first examined the number of SSCs per
Fig. 1. STPB-C promotes self-renewal of SSCs in vivo. (A) Representative morphology of testes from day 5 control (b) or transgenic (Tg) rats (a), demonstrating that the overall morphology of Tg testicular cords was normal, with many SSCs present. (C) Significantly higher numbers of SSCs per cord were quantified in transgenic (Tg) compared to control (Con) rats (⁎P b 0.01). (B) Representative (four replicates) results of Western blot analysis of PCNA in protein isolated from day 5 testes of transgenic (Tg) or control (Con) rats. Equal loading of protein across lanes was verified by reprobing the blots for β-tubulin.
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cross-sectioned seminiferous cord in day 5 transgenic rats overexpressing STPB-C (Tgs) [9] and controls. We found that the number of SSCs in Tgs was significantly higher than that seen in controls (Fig. 1A, C). Based on our earlier data from protein microarray analysis that proliferating cell nuclear antigen (PCNA) expression is higher in day 4 Tgs than in controls [9], we compared PCNA expression in testes on postnatal day 5 in Tg rats and controls. Using Western blot analysis, we observed that there was a marked increase in PCNA expression in Tgs compared to controls (Fig. 1B). To explore this possibility directly (STPB-C promotes self-renewal of SSCs), we applied a replication-defective retroviral genetransfer system and infected SSC cultures with viral supernatant containing the STPB-C gene (pFB-Neo-STPB-C). After 48 h, it was obvious upon visual inspection of cultures infected with the STPB-C retroviral vector that the number of SSCs increased and many SSC clusters were noted (Fig. 2A–C), while only a few clustered SSCs were seen in vehicle-treated controls (Fig. 2A–C). Bromodeoxyuridine (BrdU) labeling of cultures showed that SSC proliferation was obviously increased in cultures infected with the STPB-C retroviral vector, compared with that in mock-infected cultures (Fig. 2B and C). In STPB-C-
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infected and mock-transfected cultures, the number of PCNApositive SSCs in parallel cultures was similar to the number of BrdU-positive SSCs (Fig. 2D). 3.2. STPB-C activates the PI3-K/Akt signaling pathway Previous data from protein microarray analysis have suggested that STPB-C up-regulates PI3-K expression in neonatal testes [9]. Therefore, we asked whether STPB-C activates the PI3-K/Akt signaling pathway. To test this possibility, we used immunoprecipitation to clarify whether STPB-C physically binds to PI3-K. Proteins isolated from testes of Tgs and controls were immunoprecipitated with STPB-C and immunoblotted with anti-PI3-K. The blots were then stripped and re-probed with anti-STPB-C to confirm the presence of STPB-C. The results indicated that PI3-K was not a STPB-C binding partner (Fig. 3A). Based upon studies showing that phosphorylated JAK activates SH2-containing protein [15,17,18], PI3-K, and the result that STPB-C binds to and activates JAK2 (Fig. 3B) [9], we hypothesized that STPB-C activates the PI3-K/Akt signaling pathway via binding to and activating JAK2. To test this hypothesis, we first determined whether PI3-K/Akt
Fig. 2. STPB-C promotes self-renewal of SSCs in vitro. (A) Representative views of SSC cultures that were infected with either STPB-C retroviral vector (STPB-C; A-b) or empty retroviral vector (Mock; A-a). Arrows indicate clustered SSCs. (B) STPB-C gene transfer induced an increase in BrdU-positive SSCs. Photomicrographs of BrdU immunofluorescence after SSC cultures were infected with STPB-C (B-b) or empty retroviral vector for 24 h (B-a) and BrdU labeling for 5 h. (B-c, d) DAPI immunofluorescence. Arrows indicate BrdU-positive SSCs. (C) Significantly higher numbers of SSCs (No. 1), clustered SSCs (No. 2) or BrdU-positive SSCs (No. 3) were quantified in STPB-C- compared to mock-infected SSC cultures (⁎P b 0.05). (D) The effect of STPB-C infection on expression of PCNA in SSC cultures. (D-a, b) PCNA immunofluorescence. (D-c, d) DAPI immunofluorescence. Note the obvious increase in PCNA-positive SSCs in STPB-C-infected cultures. Arrows indicate PCNA-positive SSCs. Scale bar = 50 µm.
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Fig. 3. STPB-C activates the PI3-K/Akt signaling pathway. (A, B) Protein obtained from testes of transgenic (Tg) or control (Con) rats was immunoprecipitated with STPB-C and then probed with PI3-K (A) or JAK2 (B). Subsequently, blots were stripped and re-probed with anti-STPB-C to confirm the presence of STPB-C in the immunoprecipitate (Ip). (C) Western blots of PI3-K, p-PI3-K (Tyr 508), Akt or p-Akt (Tyr 326) in protein isolated from testes of day 5 transgenic (Tg) or control (Con) rats were quantified by densitometry and the data were expressed as a ratio of PI3-K, p-PI3-K, Atk or p-Akt:β-tubulin. ⁎P b 0.05. (D and E) Western blot analysis of p-PI3-K (D) or p-Akt (E), obtained from STPB-C-infected (STPB-C) or mock-infected (Mock) SSC cultures.
components are overexpressed in day 5 testes of Tgs, compared expression of PI3-K, p-PI3-K, Akt and p-Akt by immunoblotting proteins from control and transgenic rats. With Western blot analysis, we noted an obvious increase in PI3-K or Akt expression in Tgs compared to that in control rats while no significant difference was detected in the expression of p-PI3-K or p-Akt in Tgs and controls (Fig. 3C). Next, we used SSC cultures to examine the changes in expression of p-PI3-K and pAkt following infection with the STPB-C retroviral vector. By 24 h post-infection, expression of p-PI3-K or p-Akt was obviously increased compared with that in mock infection, while no obvious change was detected in the total pool of PI3K or Akt in STPB-C infection (Fig. 3D and E). Taken together, these results provide evidence supporting our hypothesis above.
in SSC cultures infected by STPB-C retroviral vector compared to that in controls (Fig. 4E and F). When SSC cultures infected with the STPB-C retroviral vector or mock-infected cultures were treated with TGF-β1, we found that the number of BrdUpositive SSCs in STPB-C-infected cultures was much higher than that of controls (mock) (Fig. 4C-a–d, D), and this difference was much higher than that in untreated SSCs in parallel cultures (Fig. 4C-e–h, D). However, an obvious decrease in caspase-3-positive SSCs in STPB-C-infected cultures was noted compared with that in controls (Fig. 4B-a–d). In addition, there was no significant difference in mRNA expression of TβRII between STPB-C-infected cultures and controls (Fig. 4G).
3.3. STPB-C blocks TGF-β1 signaling growth inhibition by upregulating Bog expression
Previously, we have identified a role for STPB-C in promoting survival of gonocytes in neonatal rats, and have linked its expression to the JAK-STAT signaling pathway, including that STPB-C transfection of L cells up-regulates JAK-STAT components. To elucidate whether the role of STPB-C in SSC proliferation is also linked to JAK-STAT signaling, we first used Western blot analysis to compare expression of JAK-STAT components in day 5 testes in Tgs and controls. Western blot analysis showed an obviously higher expression of JAK2 and STAT3 in Tgs testes (Fig. 5A). Furthermore, we determined whether overexpression of STPB-C in SSC cultures caused changes in levels of p-JAK2 and/or p-STAT3. With Western blot analysis, we found that both p-JAK2 and p-STAT3 were
We have previously reported that STPB-C up-regulates Bog expression in neonatal testes [9,19]. In addition, a recent study has indicated that overexpression of Bog in cells results in loss of TGF-β1 growth inhibition [20]. We reasoned that STPB-C may lead to loss of TGF-β1 signaling growth inhibition by upregulating Bog expression in SSCs. To test this, we first used immunoprecipitation to elucidate whether STPB-C bound to Bog or TGF-β1. As shown in Fig. 4A, we found that STPB-C bound to Bog, but not to TGF-β1, TβRI or TβRII. Furthermore, we observed that expression of Bog was significantly increased
3.4. STPB-C activates the JAK-STAT signaling pathway
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Fig. 4. STPB-C results in loss of TGF-β1 growth inhibition. (A) Protein obtained from testes of day 5 transgenic rats was immunoprecipitated with STPB-C and then probed with TGF-β1, TβRI, TβRII or Bog. Subsequently, blots were stripped and re-probed with anti-STPB-C to confirm the presence of STPB-C in the immunoprecipitate. (B–D) Overexpression of STPB-C disrupted the growth-inhibitory effect of TGF-β1. SSC cultures were infected with STPB-C or empty (mock) retroviral vector. After 48 h, they were cultured in the presence (C-a–d, D-No.1) or absence (C-e–h, D-No. 2) of TGF-β1 for 24 h and BrdU labeling for 5 h. (C-a, b, e, f) BrdU immunofluorescence. (B-a, b) Caspase-3 immunofluorescence. (C-c, d, g, h, B-c,d) DAPI immunofluorescence. Note obvious increase in BrdU-positive SSCs (C-b, f, D, ⁎P b 0.05, ⁎⁎P b 0.005) and decrease in caspase-3-positive SSCs (B-b) in STPB-C-infected cultures. Arrows indicate BrdU-positive or caspase-3-positive SSCs. (E) Western blot analysis of Bog in samples obtained from mock-infected (Mock) or STPB-C-infected (STPB-C) SSC cultures. Equal loading of protein across lanes was verified by reprobing Western blots for β-tubulin. (F) Western blots from 4 separate experiments were quantified by densitometry and data were expressed as a ratio of Bog: β-tubulin. ⁎P b 0.05. (G) Northern blot analysis of TβRII showing no difference in mRNA expression of TβRII in STPB-C- and mock-infected cultures. The expression of GAPDH was used as an internal standard to control for evenness of loading. Scale bar = 50 µm.
markedly increased in STPB-C-infected cultures compared to that in controls (mock-infected) (Fig. 5B, C). 3.5. STPB-C promotes self-renewal of SSCs by activating JAKSTAT and PI3-K/Akt signaling pathways and blocking TGF-β1 signaling Previous studies have shown that STPB-C activates JAKSTAT signaling by binding to JAK2 [9]. Moreover, our results above indicate that STPB-C also activated PI3-K/Akt signaling by binding to JAK2. To examine the role of the JAK-STAT and PI3-K/Akt pathway in STPB-C-promoted SSC self-renewal, SSC cultures were pretreated with Tyrphostin AG 490 (JAK2 inhibitor) or LY294002 (PI3-kinase inhibitor) before infection with STPB-C retroviral vector. AG 490 or LY294002 markedly blocked STPB-C-promoted SSC proliferation (Fig. 6A-a–f, B). To determine the effects of TGF-β1 signaling on STPB-Cinduced SSC proliferation, SSC cultures were infected with viral stock for Bog-pRS vector (vector for Bog knockdown, see Experimental methods) prior to infection with STPB-C retroviral vector. As shown in Fig. 6A-a,b,g,h, B, C, Bog knock-
down reduced the STPB-C-induced increase in the number of BrdU-positive SSCs. In the end, STPB-C-induced SSC proliferation was completely blocked by Tyrphostin AG 490 treatment and Bog knockdown (Fig. 6A, B). 4. Discussion In adult rodents, continuous availability of differentiating spermatogonia is responsible for lifelong fertility of males and species continuity. The number of these spermatogonia available to enter spermatogenesis is controlled by regulation of SSC proliferation and apoptosis [21]. In this study, we examined a novel cadherin, STPB-C, previously found by us to be highly expressed in neonatal testes [14], and its overexpression in transgenic rats results in abnormally high numbers of gonocytes in neonates [9]. We asked whether it has a role in promoting self-renewal of SSCs, besides supporting gonocyte survival by suppressing apoptosis [9]. First, we found that overexpression of STPB-C led to high numbers of SSCs and high expression of PCNA in rat testes from postnatal day 5. Next, we observed that the number of SSCs and BrdU
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Fig. 5. STPB-C activates JAK-STAT signaling pathway in SSCs. (A) Representative Western blot analysis of JAK2 or STAT3 in protein isolated from testes of day 5 transgenic (Tg) or control (Con) rats. Equal loading of protein across lanes was verified by reprobing the blots for β-tubulin. (B and C) Western blot analysis of p-JAK2 (B) or p-STAT3 (C) in protein samples isolated from STPB-C- or mock-infected SSC cultures.
incorporation obviously increased when SSC cultures were infected by STPB-C retroviral vector. Thus, expression of STPB-C in neonates appears to induce SSC proliferation. GDNF is a member of the TGF-β superfamily. It promotes survival and differentiation of several types of neuron [22,23] and regulates branching of the ureter in the embryonic kidney [24,25]. Meng et al. [5] have reported that gene-targeted mice with one GDNF null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. Moreover, Kubota et al. [4] have found that GDNF is the essential growth factor for SSC self-renewal in most, but not all mammals. For instance, SSCs derived from DBA/2 mouse gonocytes (precursors of SSCs) proliferate in culture using medium containing GDNF, leukocyte inhibitory factor (LIF), epidermal growth factor, basic fibroblast growth factor and 1% FBS [26]. In the same culture conditions [26], however, SSCs from other strains (C57BL/6 or 129/SV) do not proliferate. Therefore, STPB-C is another important factor identified for SSC self-renewal, because we have recently found that STPB-C promotes self-renewal of SSCs from C57BL/6 background mice (Wu, unpublished data), and it plays a central role in inducing self-renewal of rat SSCs. Our results have important implications for future gene therapy and for animal mutagenesis, because SSCs are present in extremely
Fig. 6. STPB-C Promotes self-renewal of SSCs by activating JAK-STAT and PI3-K/Akt signaling pathway and blocking TGF-β1 signaling. (A) SSC cultures were pretreated with AG 490 (A-c), LY294002 (A-e), Bog RNAi (A-g), AG 490 plus Bog RNAi (A-i), or controls (A-a, k), before being infected with STPB-C or mock retroviral vector. (A-a, c, e, g, i, k) BrdU immunofluorescence. (A-b, d, f, h, j, l) DAPI immunofluorescence. (B) The number of BrdU-positive cells per condition was quantified. The values represent the mean ± SE of four independent experiments. For each condition, changes in the number of positive cells, are indicated by a (P b 0.05, relative to mock-infected cultures) or b (P b 0.05, relative to STPB-C-infected cultures). (C) Western blot analysis of Bog in samples obtained from SSC cultures infected with Mock (lane 1), STPB-C (lane 2), STPB-C + AG290 (lane 3), STPB-C + LY294002 (lane 4), STPB-C + Bog RNAi (lane 5), STPB-C + control RNAi (lane 6), STPB-C + AG490 + Bog RNAi (lane 7) or STPB-C + AG490 + control RNAi (lane 8). Equal loading of protein across lanes was verified by reprobing Western blots for β-tubulin.
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low numbers in testes, and it has been difficult to produce transgenic animals by SSCs if SSCs do not expand in vitro. Self-renewal of stem cells requires in the integration of survival signals and proliferation controls with the maintenance of their undifferentiated state. This demands a complex crosstalk between extrinsic signals from the microenvironment and the cell-intrinsic regulators of self-renewal. To establish the mechanism of this process, we have explored the molecular and cellular mechanism of STPB-C in inducing self-renewal of SSCs in this study. In general, there are three different ways in which cadherin expression can influence signaling. Firstly, cadherin-dependent adhesion can induce juxtacrine signaling by bringing the opposing membranes of neighboring cells into close proximity, consequently enabling membrane-linked ligands, or ligands tightly associated with the pericellular matrix, to interact with their receptors on the neighboring cell. Secondly, cadherinmediated adhesion can influence cell signaling by inducing polarization and tight-junction formation. In addition, cadherins can mediate signaling directly. Cytoplasmic or transmembranous signaling molecules may act as co-receptors that become activated by homotypic binding of the cadherins. However, unlike most cadherins, STPB-C lacks an intracellular cateninbinding domain, suggesting that STPB-C may associate with specific cytoplasmic molecules distinct from catenins, and any associated signaling pathway does not involve interaction with cytoskeletal elements. In previous work, we have produced transgenic rats overexpressing STPB-C, in which we have used protein microarrays to seek clues concerning the regulatory pathway for STPB-C. The results suggest that JAK-STAT components are involved in STPB-C action, which has been confirmed by the up-regulation of JAK2, STAT3 and SH2-B in transgenic animals, and down-regulation of these components in cultured cells exposed to anti-STPB-C [9]. Further studies have indicated that STPB-C activates the JAK-STAT pathway by binding to JAK2 and inducing phosphorylation of JAK2, and subsequently STAT3 [9]. In this study, however, we found that STPB-C promoted self-renewal of SSCs by PI3-K/Akt and TGF-β1 signaling, besides JAK-STAT. Firstly, we observed that STPB-C overexpression in SSC cultures induced high levels of p-PI3-k or p-Akt. Secondly, Ferrand et al. [27] have shown that activation of JAK2 is upstream of PI3-K/Akt, and that JAK2 is as an upstream mediator of PI3-K activity. In addition, our previous results have indicated that JAK2 is a STPB-C binding partner [9]. In this study, we found that there was an obvious increase in PI3-K or Akt expression in transgenic rats overexpressing STPB-C, compared to that in controls. Further data have demonstrated that STPB-C overexpression results in increased phosphorylation of PI3-K or Akt in SSC cultures. Moreover, our data show that STPB-C bound to Bog and upregulated its expression. Overexpression of STPB-C or Bog in SSC cultures resulted in the loss of TGF-β1 growth inhibition, and did not significantly affect TGF-β1 receptor II mRNA expression. Bog knockdown partly blocked STPB-C-promoted self-renewal of SSCs. Our results indicated that inhibition of JAK-STAT, PI3-k/Akt and activating TGF-β1 signaling completely blocked STPB-C-induced self-renewal of SSCs.
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Thus, our findings demonstrate that STPB-C promotes selfrenewal of SSCs via the JAK-STAT, PI3-K/Akt and TGF-β1 signaling pathways. In mammals, the JAK-STAT pathway is the principal signaling mechanism for a wide array of cytokines and growth factors. STATs may affect proliferation by regulating the expression of immediate–early genes, such as c-myc and c-fos, as well as cell-cycle regulatory genes, such as the cyclins [28]. Moreover, Simon et al. [29] have reported that the JAK-STAT pathway contributes to PDGF-induced mitogenesis. In addition, STAT3 signaling is constitutively activated in various tumors, and is involved in cell survival and proliferation during oncogenesis. For instance, STAT3 is constitutively activated in various human gastric cancer cells [30], and blocking STAT3 activation can abolish the antiapoptotic and mitogenic actions of IL-22 in hepatic cells [31]. Woitach et al. [20] have reported that overexpression of Bog in RLE cells results in loss of TGF-β1 signaling growth inhibition. TGF-β1 is a ubiquitous cytokine that is well known for its ability to inhibit epithelial cell proliferation [32]. Furthermore, TGF-β1 exerts potent anti-mitogenic and pro-apoptotic effects on certain cancer cells [33,34]. However, somatic mutations abrogating the TGF-β signaling pathway are found in many gastrointestinal cancers [32]. Recent studies have suggested that PI3-K acts as an immediate downstream molecule of growth factor receptors to mediate mitogenic signaling in cells. The activation of PI3-K is sufficient to drive some types of cells to go through G1/S transition and to proliferate [35]. The most commonly known downstream target of PI3-K is the serine–threonine kinase Akt [36–39]. Activated Akt can phosphorylate multiple proteins implicated in the control of the cell cycle to ultimately stimulate cell growth [40,41]. Inhibition of the PI3-K/Akt pathway by LY294002 significantly reduced the number of newly-formed
Fig. 7. The signaling diagram illustrates that STPB-C promotes self-renewal of SSCs via activating JAK-STAT and PI3-K/Akt, and blocking TGF-β1 signaling.
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neurospheres, which indicates that this is an essential pathway for neural stem cell self-renewal [42]. Therefore, PI3-K/Akt is thought to play a crucial role in the survival and proliferation of various cell types [40,41]. Current understanding of these signaling pathways is consistent with our findings, suggesting a role for JAK-STAT, TGF-β1 or PI3-K/Akt signaling pathways in regulation of growth in SSCs, similar to that in other cell types. In conclusion, with varied approaches, including the use of RNAi, SSC cultures infected by STPB-C retroviral vector, and other techniques, we have defined a new function for STPB-C, promoting SSC self-renewal. Moreover, we found that this function of STPB-C was required to activate the JAK-STAT and PI3-K/Akt, and to block the TGF-β1 signaling pathways (Fig. 7). These data have important implications for germ cell biology and create the possibility of using SSCs for biotechnology and medicine. Acknowledgements This work was supported by a key Program of the National Natural Scientific Foundation of China (No. 30630012, to J.W.), and sponsored by the Shanghai Pujing Program (No. 06PJ14058, to J.W.), Shanghai Leading Academic Discipline Project (No. B205) and the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP) in China (No. 20050248010 to J.W.). References [1] [2] [3] [4] [5]
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