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Received Date : 25-Dec-2013 Accepted Date : 11-Jun-2014 Article type
: Original Scientific Article
Sphingosine-1-Phosphate mediates AKT/ERK maintenance of dental pulp homeostasis
H.Y. Pan, H. Yang, M.Y. Shao, J. Xu, P. Zhang, R. Cheng*, T. Hu*
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu，Sichuan，China
* T. Hu and R. Cheng are both corresponding authors
Running Title: pulp homeostasis maintenance
Keywords: S1P, ERK/AKT, ROCK, apoptosis, proliferation, microinflammation
Corresponding authors: Tao Hu, Professor and Ran Cheng, Associate professor Department of Operative Dentistry and Endodontics Department of Preventive Dentistry State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University , 14# ,3rd section, Renmin South Road, Chengdu，Sichuan，610041 E-mail: [email protected] ；[email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/iej.12335 This article is protected by copyright. All rights reserved.
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Abstract Aim To investigate the cell status of dental pulp cells (DPCs) in a S1P-induced microinflammation environment and the possible mechanisms of cell homeostasis maintenance by S1P.
Methodology S1P receptor (S1PR) expression was examined in DPCs within a local S1P-induced microinflammation model established using 1ｕM S1P. U0126 (ERK inhibitor), LY294002 (AKT inhibitor), and Y27632 (ROCK inhibitor) were used to inhibit corresponding signaling pathways of DPCs. CCK8 and cell cycle analysis tested cell proliferation. Immunofluorescence staining JC-1 detected changes of mitochondrial membrane potential (ΔΨm). Tests for apoptosis and the apoptosis-related proteins Bax and Bcl-2 were assessed by flow cytometry and western blot analysis, respectively. Expressions of ERK and AKT were evaluated by western blot analysis. The results were analyzed using the Student’s t-test with SPSS and the significance level set at P < 0.05.
Results Expressions of S1PR1, S1PR2 and S1PR3 in DPCs differed among individuals. DPCs maintained self-homeostasis in response to S1P-induced microinflammation via S1PRs. During this repair process, ERK, AKT, and ROCK had a short-term complementary interaction at 60min, but then AKT and ERK gradually played decisive roles after 24h in proliferation enhancement and apoptosis inhibition, respectively (p>0.05).
Conclusions The AKT–ERK balance may determine whether DPC homeostasis in S1P-induced microinflammation is maintained by synergistic regulation of cell growth and apoptosis.
Introduction Blood vessels in the dental pulp are involved in both acute and chronic inflammation. During acute inflammation, various inflammatory mediators and leukocytes may cause plasma leakage and high pressure in the pulp space (Dahlén et al. 1981, Cooper et al. 2014). Dental pulp tissue can react to moderate external stimuli and induce reparative dentinogenesis. The pulps in teeth that have been stimulated by occlusal pressure or caries for long periods may result in vascular reactivity and local microinflammation, thereby switching on reparative dentinogenesis or dental tissue repair (Howard et al. 2010). Thus, there must be a mechanism to maintain homeostasis to avoid harmful stimulation and severe inflammation. Certain blood vessel-derived growth factors might be involved in this process, e.g. vascular endothelial growth factor (Mullane et al. 2008, Botero et al. 2010) and tumor necrosis factor-alpha (Paula-Silva et al. 2009).
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Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and S1P are plasma components and potent immune-modulating factors. S1P mediates broad biological effects, including cell growth and survival, cell differentiation, cell migration, angiogenesis, and immune regulation (Young & Van Brocklyn, 2006, Howard et al. 2010). When encountering inflammatory stimuli, S1P is released locally from platelets plasma into tissue injury sites so as to exert homeostatic and/or inflammatory functions depending on the S1P receptors (English et al. 2000, Obinata & Hla, 2012). The plasma concentration of S1P is 0.1–1 μM (Murata et al. 2000). Thus, the local congregated rise of S1P may represent a very early stage of an inflammation microenvironment.
Microinflammation means the form of low-grade and asymptomatic inflammation in the early stages of the inflammation responses (Wang et al. 2012). It induces systematic homeostasis dysbiosis and contributes to the inflammatory progress in various chronic disease, such as systemic bone mass, renal disease, uremia and acidosis, oxidative stress, bowel disease and dental diseases (Hamidi et al. 2014, Schindler 2004). However, little attention has been paid to the microenvironment in pulp diseases so far as well as to homeostatic regulation of dental pulp cells (DPCs) in this environment.
This study aimed to investigate S1P receptor expression on DPCs and cell homeostasis status in a S1P-induced microinflammation environment. Possible signaling pathways in proliferative and apoptotic balance were analyzed as an initial exploration.
Materials and Methods Cell Culture Five human impacted third molars or premolars were collected from adults (aged between 18 and 26 years) with informed consent according to the institutional standards. Individual cell samples were numbered as 1, 2, 3, 4, 5. The isolation, culture and identity of DPCs were confirmed as reported previously (Pan et al. 2013). The cells were used between three and seven passages. S1P is regarded as an immune-modulating lipid that is secreted by activated platelets in response to various physiological or pathological stimuli. High plasma concentrations of S1P were used to simulate the local microinflammatory state. DPCs were washed twice with phosphate-buffered saline and serum starved for 24 h before use. The cells were treated with 10 μM Y27632 (Rho-associated kinase (ROCK) inhibitor, Sigma-Aldrich), 50 μM LY294002 (AKT inhibitor, Sigma-Aldrich), and 10 μM U0126 (extracellular signal-regulated kinase (ERK) inhibitor, Sigma-Aldrich) for 40 min, and then stimulated with 1 μM S1P (Sigma-Aldrich) for specific times. The dosage of 1 μM S1P, 10 μM Y27632, 10 μM U0126 and 50 μM LY294002 on DPCs are known to be safe and without additional side effects (Wang et al. 2006, Spagnuolo et al. 2008, Cheng et al. 2010, 2011, Pan et al. 2013).
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Cell Proliferation Assay 1×103 cells were grown in 96-well culture plates and were utilized for experimentation at 60-70% confluence. Cells were starved for 24 h and treated as required for 24 h. Cell proliferation after stimulation of 24 hours was tested using Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) according to the protocol and previous studies (Chen et al. 2008, Meng et al. 2014). The optical density was measured at 450 nm on a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). Samples 1-5 were used and each sample repeated three times.
To investigate the cell cycle and proliferation index (PI) of DPCs in the microinflammatory environment for 24 hours, a cell cycle detection kit (Nanjing KeyGEN Biotech. Co., Ltd, Nanjing, P. R. China) was used according to the manufacturer (Yim et al. 2012). Cells were seeded in 50 mL culture bottles overnight in 5 mL culture medium. Cells were starved for 24 h and treated as required for 24 h. About 1×106 cells were harvested by trypsinization and fixed in 70% ethanol overnight at –20°C. After washing twice with PBS, the cells were suspended in 100 μL of RNase solution for 30 min at 37°C, followed by 400 μL of propidium iodide solution for 30 min at 4°C while protected from the light. The stained cells were collected using a flow cytometer (Beckman FC500; Beckman Coulter, Miami, IL, USA). The PI was calculated as follows: PI= ( S+ G2 /M ) / ( G0 /G1 + S+ G2 /M )(Cheng et al. 2012) . Samples 1-5 were used and each sample was repeated at least twice.
Mitochondrial Membrane Potential (ΔΨm) Reduced mitochondrial membrane potential (ΔΨ) is a hallmark for apoptosis. JC-1 is a recommended dye used to measure the ΔΨ changes, thereby distinguishing dead cells from living cells. Healthy cells emitted red-orange fluorescence in JC-1 aggregates. In dead cells, JC-1 monomers present in the cytoplasm emitted green fluorescence. However, JC-1 alone scarcely discriminates intact/early apoptotic cells against necrotic/late apoptotic cells. ΔΨ was analyzed using a JC-1 kit (Beyotime Biotech, Nantong, China). Cells were seeded in 6-well plates and incubated overnight with 2 mL of culture medium. Cells were starved for 24 h and treated as required for 24 h. After changing the cell culture media, 1 mL JC-1 dye and 1 mL culture medium were added and plates were incubated at 37°C for 20 min. The cells were then washed twice and visualized using a fluorescence microscope (Carl Zeiss, Göttingen, Germany). Three individual samples (samples 1,3,5) were used and each sample was repeated at least five times.
Cell Apoptosis Assay To determine the apoptotic index of DPCs after 24 hour stimulation during microinflammation, a KeyGEN apoptosis detection kit (Nanjing KeyGEN Biotech. Co., Ltd, Nanjing, P. R. China) were used as reported previously (Yim et al. 2012, Cheng et al. 2012, Pan et al. 2013). The stained cells were collected using a flow cytometer (Beckman FC500; Beckman Coulter, Miami, USA). The apoptosis
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rate included early apoptosis and late apoptosis but excluded necrotic cells. Samples 1-5 were used in apoptosis analysis and each sample was repeated at least three times.
Western Blotting To measure the cellular protein levels, cell extracts were subjected to standard western blotting (Cheng et al. 2010, 2011). The antibodies used were against S1P1, S1P2 (Proteintech Group Inc., Chicago, IL, USA), S1P3, phosphorylated AKT (p-AKT, Ser473), phosphorylated extracellular signal-regulated kinase (p-ERK, Thr202/Tyr204), AKT, ERK, Bcl-2, Bax, and GAPDH (Epitomics, Inc., Burlingame, CA, USA). In the preliminary experiment, phosphorylation events of ERK and AKT in S1P-dependent microinflammation for 5min, 10min, 30min, 1h, 2h, 12h and 24h were explored. Then, the three key time points at 30min, 60min and 12h were selected. Samples 1-5 were used to test S1P receptor expression and the test was repeated at least three times per sample. Samples 2,3,4 were tested in AKT, ERK and ROCK diversifications, and samples 2,3,5 were used in Bax and Bcl-2 investigation. Each sample was repeated at least twice. The quantification of phosphorylation was normalized by the corresponding native protein.
Statistical Analysis The data from two groups were compared using Student’s t test. In all cases, P < .05 indicated a significant difference. Data are expressed as the mean ± SEM.
Results DPCs Expressed S1P Receptors S1P receptors S1PR1, S1PR2, and S1PR3 were detected using western blot analysis. The DPCs expressed S1PR1, S1PR2, and S1PR3. However, the level of these three receptors differed between donors (Fig. 1). The result indicated that S1P could affect DPCs through S1PR1, S1PR2, and S1PR3.
S1P Promoted the Proliferation of Dental Pulp Cells To investigate the possible signaling pathway involved in cell proliferation in microinflammation, DPCs were stimulated using S1P, U0126 (an ERK inhibitor), LY294002 (an AKT inhibitor), or Y27632 (a ROCK inhibitor) for 24 h. Cells in control were only treated with serum-free medium with or without inhibitors alone for the same time.
S1P treatment increased cell growth by 20.6% compared with the control. In the microinflammatory state, cell viability decreased by 21.1%, 26.0%, and 43.3% after U0126 (ERK inhibitor), Y27632 (ROCK inhibitor), and LY294002 (AKT inhibitor) treatment, respectively (Fig. 2A).
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S1P treatment increased the PI by 13.5%. By contrast, in the S1P-induced microinflammation environment U0126, Y27632 and LY294002 decreased the PI by 16.6%, 4.5% and 23.3%, respectively (Fig. 2B).
In the normal state AKT inhibition significantly reduced cell proliferation by both cell counting numbers and cell cycle influence. ERK and ROCK had little effect (Fig.2). However, in the S1P-induced microinflammation environment DPCs proliferated obviously on ERK/AKT/ROCK-dependent pathways. The pro-proliferative effect of AKT was stronger than ERK and ROCK(Fig.2) indicating that cell proliferation mechanisms, as a stress response to S1P-induced microinflammation, differed from cells in the normal environment. DPCs may enhance self-protection by functional proliferation behavior.
S1P Induced Complementary Effects in ERK, AKT, and ROCK The results described above suggested that ERK, AKT, and ROCK are involved in the S1P-induced cell proliferation and cell cycle. The interactions between ERK, AKT, and ROCK were then examined. DPCs were pretreated with U0126, LY294002, or Y27632 for 40 min and then stimulated with S1P for 60 min. S1P stimulation activated ERK and AKT within 30 min and 60 min, respectively. Interestingly, there was a complementary effect between ERK, AKT, and ROCK. U0126 inhibited ERK phosphorylation but activated AKT. LY294002 inhibited AKT but increased p-ERK compared with S1P alone. Y27632 increased p-ERK and p-AKT content markedly (Fig. 3A). However, the complementary effect disappeared at 24 h (Fig. 3B).
Inhibition of the ERK Pathway Induced DPC Apoptosis To obtain an overall view of cell vitality, flow cytometry was used to measure the apoptotic rate. In the microinflammation state S1P decreased cell apoptosis by 58.6% compared with the control. U0126 (ERK inhibitor), Y27632 (ROCK inhibitor), and LY294002 (AKT inhibitor) blocked S1P-induced survival, as shown by 501%, 108%, and 96% increases in cell apoptosis, respectively. In the quiescent state, ERK inhibition was associated with a significant rise in cell apoptosis by 144% (Fig. 4B). In the normal state, ERK inhibition significantly increased cell apoptosis, but AKT and ROCK had little effect. By contrast, cell apoptosis decreased in the S1P-influenced microinflammation state, thus increasing cell survival ability. ERK, AKT, and ROCK all had an anti-apoptotic effect, although this was much stronger for ERK than for AKT and ROCK (Fig. 4B). In accordance to the proliferation phenomenon above, the mediation of apoptosis in the microinflammation state was considerably distinct compared to the normal state.
To examine further the effects of S1P on the ERK, AKT, and ROCK pathways during cell apoptosis, the ΔΨm and the expression of Bcl-2 and Bax were measured after 24 h. Bax and Bcl-2 are important Bcl-2 family proteins that act as pro- and antiapoptotic modulators during cell
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apoptosis, respectively. ERK inhibition increased Bax expression and decreased the ΔΨm compared with the S1P group, indicating an apoptotic tendency (Fig. 4A and 4C).
The other two inhibitors also affected the ΔΨm according to the yellow/orange cells expression. Yellow staining appeared to come from an overlay of red and green staining suggesting that all cells may be losing mitochondrial polarization but are at different stages of the dying process(Fig. 4A). However, the expressions of green immunofluorescence in AKT inhibition and ROCK inhibition were significantly lower than that in ERK block, which confirmed the ERK-related decisive role in cell apoptosis regulation in the flow cytometry test (Fig. 4B). Furthermore, AKT and ROCK had different effects on Bax and Bcl-2 expression: S1P and AKT inhibition had little effect on Bax and Bcl-2 expression compared with the S1P group. ROCK inhibition increased both Bcl-2 and Bax expression in microinflammation. (Fig. 4C).
Discussion LPLs are generated by various cells including activated platelets, epithelial cells, macrophages, and some cancer cells (Goetzl & An 1998). Two major LPLs, LPA and S1P, activate G protein-coupled receptors in the endothelial differentiation gene family and regulate similar cellular function signaling pathways (Takuwa et al. 2002). Five specific G protein-coupled receptors, S1P1–S1P5 (Young & Van Brocklyn 2006), mediate the cellular processes regulated by S1P. S1P1, S1P2, and S1P3 receptors are expressed widely: S1P4 is expressed mainly in the immune system, but S1P5 receptors are limited to the nervous system (Means & Brown 2009, Schuchardt et al. 2011). The current study revealed that DPCs express receptors S1P1–S1P3 but that the expression of these receptors differs between individuals. Despite the interindividual differences, the cells from different donors responded similarly in the experiments. This established the foundation of S1P effects on DPCs. Previous studies have shown that differences in S1P receptor expression are involved in physiological processes, such as cell growth and apoptosis, and pathological processes, such as the inflammatory response (Payne et al. 2002, Qu et al. 2012). It can be hypothesized that different receptors have complementary effects with each other.
DPCs homeostasis plays a critical role in postnatal tooth formation, dental repair and pulp regeneration as a response to physiological and pathological stimuli in terms of balancing cell proliferation and cell death (Rangiani et al. 2012). S1P is widely researched on broad biological functions via several signaling pathways including ERK, p38 mitogen-activated protein kinase (MAPK), C-Jun N-terminal kinase, phospholipases C and D, phosphoinositide 3 (PI3)-kinase/AKT, and Rho GTPase (Xu et al. 2004, Schuchardt et al. 2011). However, whether S1P could mediate DPCs homeostasis and be involved in dental pulp repair and what the related mechanism is unknown.
The present data demonstrated that pulp repair occurred by way of S1P-maintained DPCs homeostasis against microinflammation. More importantly, DPCs homeostasis maintenance differed
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in the S1P-dependent microinflammation environment from that in the normal environment (Fig2 , Fig4). In the normal state, AKT determined cell proliferation while ERK decided cell survival. The balance of AKT and ERK may make a contribution to cell quiescence living. In contrast, as a response to microinflammtory stimuli, ERK, AKT and ROCK all participated in cell viability regulation to participate in pulp repair. Besides, ERK and AKT played a much more decisive role than ROCK.
Among various S1P downstream pathways, MAPK/ERK and PI3K/AKT are known to mediate S1P-induced mitogenic effects in many cell types, which subsequently modulate cell proliferation and cell death (An et al. 2000, Van Brocklyn et al. 2002). Glioma cells exhibit mitogenic effects via ERK/MAPK and PI3K activation after S1P stimulation (Van Brocklyn et al. 2002). An increase in proliferation of rat primary chondrocytes and ERK induced by S1P but not by p38 is involved in this process (Kim et al. 2006). PI3K/AKT is one of the best predictors of LPL-enhanced cell survival ability via coupling to the Gi pathways (Radeff-Huang et al. 2004). In the current study, DPC proliferation increased when cells were exposed to S1P, and AKT had a stronger effect on proliferation compared with ERK and ROCK.
MAPK/ERK and PI3K/AKT are also known to influence cell apoptosis, thereby mediating cell growth and death (Yim et al. 2012, Nakahara et al. 2012，Pan et al. 2013). Some reports demonstrated that ERK contributed to cell apoptosis in pathological injury (Zhuang & Schnellmann 2006). S1P also seems to protect cells from radiation and H2O2-induced apoptosis via the AKT pathway (Waeber et al. 2004, Bonnaud et al. 2010，Nakahara et al. 2012). However, in a previous study of ischemic damage on DPCs, another LPL, LPA, was used to protect DPCs from serious inflammatory injury. ERK played a key role in the anti-apoptotic effect but AKT had little effect (Pan et al. 2013). In the present study, DPCs survival in the microinflammation state depended on a combined effect of ERK, AKT and ROCK. However, ERK dominated cell survival to a large extent compared with AKT and ROCK. This indicates the cell survival ability enhanced by ERK pathway is essential for various types of pulp repair process. The differences of the effect of AKT on DPCs LPL-induced apoptosis inhibition may vary depending on the extent or seriousness of environmental inflammation.
Another interesting phenomenon in this study is that ERK and AKT could compensate for each other both in short-term stimuli and long-term influence (Fig 3). ERK inhibition significantly activated AKT while AKT inhibition obviously enhanced ERK activation. ROCK transiently interfered with interactions of ERK and AKT. ERK and AKT are likely to have collaborated together to maintain cell homeostasis in case of excessive or uncontrolled proliferation or apoptosis.
Rho/ROCK pathway, another important S1P downstream signaling, is a key mediator in DPCs migration, adhesion and related to S1P chemotactic response (Wang et al. 2008, Howard et al. 2010). S1P induces vigorous migration of DPCs to injury sites to become involved in dental pulp repair processes (Howard et al. 2010, Cheng et al. 2010, 2011). Recently, the ROCK pathway was
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shown to interfere with S1P-induced cell proliferation and apoptosis in various cell types with a prominent effect or little function. (Hurst et al. 2008, Zhang et al. 2011, Wu et al. 2012, Hsu et al. 2012). Thus, this study focused on how ROCK functioned with DPC homeostasis in microinflammation. However, it was noted that ROCK had little effect on DPC cell proliferation and apoptosis in the 24h microinflammatory environment. On the one hand, this may be caused by cell-specific diversity of ROCK influence on cell homeostasis. One the other hand, the results revealed that ROCK inhibition increased the levels of Bax and Bcl-2, thereby balancing the pro- and anti-apoptotic effects of Bcl-2 family members at the same time. This neutralized effect may balance ROCK mediations in cell proliferation and cell death. Moreover, the data confirmed that ROCK produced a complementary effect with ERK and AKT within 1 h, but this effect disappeared at 24 h. This short and immediate reaction suggested ROCK might participate in dental repair by a transiently indirect regulation in ERK/AKT-dependent way. It needs much further and thorough investigations on whether this phenomenon transiently switches on DPCs homing to injury sites at early stage of dental pulp repair and then subsequently induces ERK/AKT balance in late stage of homeostasis.
In this study, the focus was on DPCs homeostasis modulation in pulp repair to S1P-induced microinflammation and possible signaling mechanisms. The complex but vital mechanisms on how S1P modulates DPCs in the normal state and how DPCs switch cell viability regulation in response to different environments needs further research.
Conclusion DPCs expressed S1P1–S1P3 receptors but that the expression levels differed between individuals. The AKT, ERK and ROCK pathways were involved in S1P-induced cell proliferation and antiapoptosis. Interestingly, the balance between the AKT and ERK pathways mediated homeostasis of cell growth and death in the local microinflammatory state.
Acknowledgments The authors deny any conflicts of interest. This study was supported by grants from the National Natural Science Foundation of China (Numbers 81371134, 81000429, 30973322 and 31170894) and the Research Fund for the Doctoral Program of Higher Education (No. 20100181120066). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
References An S, Zheng Y, Bleu T (2000) Sphingosine 1-phosphate-induced cell proliferation, survival, and related signaling events mediated by G protein-coupled receptors Edg3 and Edg5. The Journal of Biological Chemistry 275,288–96.
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Bonnaud S, Niaudet C, Legoux F, et al. (2010) Sphingosine-1-phosphate activates the AKT pathway to protect small intestines from radiation-induced endothelial apoptosis. Cancer Research 70,9905–15. Botero TM, Son JS, Vodopyanov D, Hasegawa M, Shelburne CE, Nör JE (2010) MAPK signaling is required for LPS-induced VEGF in pulp stem cells. Journal of Dental Research 89,264–9. Chen Y, Takita J, Choi YL, et al.(2008) Oncogenic mutations of ALK kinase in neuroblastoma. Nature 455,971-4. Cheng MR, Li Q, Wan T, et al.(2012) Galactosylated chitosan/5-fluorouracil nanoparticles inhibit mouse hepatic cancer growth and its side effects. World Journal of Gastroenterology18,6076-87. Cheng R, Cheng L, Shao MY, et al. (2010) Roles of lysophosphatidic acid and the Rho-associated kinase pathway in the migration of dental pulp cells. Experimental Cell Research 316,1019–27. Cheng R, Shao MY, Yang H, et al. (2011) The effect of lysophosphatidic acid and Rho-associated kinase patterning on adhesion of dental pulp cells. International Endodontic Journal 44,2–8. Cooper PR, Holder MJ, Smith AJ (2014) Inflammation and regeneration in the dentin-pulp complex: a double-edged sword. Journal of Endodontics 40 Suppl 4, S46-51. Dahlén SE, Björk J, Hedqvist P, et al. (1981) Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory response. Proceedings of the National Academy of Sciences USA 78,3887-91. English D, Welch Z, Kovala AT, et al.(2000) Sphingosine 1-phosphate released from platelets during clotting accounts for the potent endothelial cell chemotactic activity of blood serum and provides a novel link between hemostasis and angiogenesis. The FASEB Journal 14,2255-65. Goetzl EJ, An S (1998) Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate. The FASEB Journal 12,1589–98. Hamidi S, Schäfer-Korting M, Weindl G.(2014) TLR2/1 and sphingosine 1-phosphate modulate inflammation, myofibroblast differentiation and cell migration in fibroblasts. Biochimica et Biophysica Acta 1841,484-94 Howard C, Murray PE, Namerow KN (2010) Dental pulp stem cell migration. Journal of Endodontics 36,1963–6. Hsu A, Zhang W, Lee JF, et al. (2012) Sphingosine-1-phosphate receptor-3 signaling up-regulates epidermal growth factor receptor and enhances epidermal growth factor receptor-mediated carcinogenic activities in cultured lung adenocarcinoma cells. International Journal of Oncology 40, 1619-162. Hurst JH, Mumaw J, Machacek DW, et al. (2008) Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology. BMC Neuroscience 9,118. Kim MK, Lee HY, Kwak JY, Park JI, Yun J, Bae YS (2006) Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation. Biochemistry and Biophysical Research Communications 345,67–73.
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Means CK, Brown JH (2009) Sphingosine-1-phosphate receptor signalling in the heart. Cardiovascular Research 82,193–200. Meng W, Wu Y, He X, et al.(2014) A Systems Biology Approach Identifies Effective Tumor-Stroma Common Targets for Oral Squamous Cell Carcinoma. Cancer Res 74,2306-15. Mullane EM, Dong Z, Sedgley CM, et al. (2008) Effects of VEGF and FGF2 on the revascularization of severed human dental pulps. Journal of Dental Research 87,1144–8. Murata N, Sato K, Kon J, et al.（2000）Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions. Biochemical Journal 352,809–15. Nakahara T, Iwase A, Nakamura T, et al. (2012) Sphingosine-1-phosphate inhibits H2O2-induced granulosa cell apoptosis via the PI3K/Akt signaling pathway. Fertility and Sterility 98,1001–8.e1. Obinata H, Hla T.(2012) Sphingosine 1-phosphate in coagulation and inflammation. Seminars in Immunopathology 34,73-91. Pan HY, Cheng L, Yang H, et al. (2013) Lysophosphatidic acid rescues human dental pulp cells from ischemia-induced apoptosis. Journal of Endodontics 40,217-22 Paula-Silva FW, Ghosh A, Silva LA, Kapila YL (2009) TNF-α promotes an odontoblastic phenotype in dental pulp cells. Journal of Dental Research 88,339–44. Payne SG, Milstien S, Spiegel S (2002) Sphingosine-1-phosphate: dual messenger functions. FEBS Letters 531,54–7. Qu Z, Cheuk BL, Cheng SW (2012) Differential expression of sphingosine-1-phosphate receptors in abdominal aortic aneurysms. Mediators of Inflammation 2012,643609. Radeff-Huang J, Seasholtz TM, Matteo RG, Brown JH (2004) G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival. Journal of Cellular Biochemistry 92,949–66. Rangiani A, Cao ZG, Liu Y, et al.(2012) Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation. International Journal of Oral Science 4,189-95. Schindler R.(2004) Causes and therapy of microinflammation in renal failure. Nephrol Dial Transplant. 19 Suppl 5,V34-40. Schuchardt M, Tölle M, Prüfer J, van der Giet M (2011) Pharmacological relevance and potential of sphingosine 1-phosphate in the vascular system. British Journal of Pharmacology 163,1140–62. Spagnuolo G, D'Antò V, Valletta R, et al.(2008) Effect of 2-hydroxyethyl methacrylate on human pulp cell survival pathways ERK and AKT. Journal of Endodontics 34,684-8.
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Takuwa Y, Takuwa N, Sugimoto N (2002) The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities. The Journal of Biochemistry 131,767–71. Van Brocklyn J, Letterle C, Snyder P, Prior T (2002) Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: role of ERK MAP kinase and phosphatidylinositol 3-kinase β. Cancer Letters 181,195–204. Waeber C, Blondeau N, Salomone S (2004) Vascular sphingosine-1-phosphate S1P1 and S1P3 receptors. Drug News&Perspectives 17,365–82. Wang FM, Hu T, Tan H et al. (2006) p38 Mitogen-activated protein kinase affects Transforming growth factor-β/Smad signaling in human dental pulp cells. Molecular and Cellular Biochemistry 291, 49–54. Wang F, Zhang P, Jiang H, et al.(2012) Gut bacterial translocation contributes to microinflammation in experimental uremia. Digestive Diseases and Sciences 57,2856-62. Wang L, Lee JF, Lin CY, Lee MJ (2008) Rho GTPases mediated integrin αvβ3 activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells. Histochemistry and Cell Biology 129,579–88. Wu J, Li J, Hu H, Liu P, Fang Y, Wu D (2012) Rho-kinase inhibitor, fasudil, prevents neuronal apoptosis via the Akt activation and PTEN inactivation in the ischemic penumbra of rat brain. Cellular and Molecular Neurobiology 32,1187–97. Xu CB, Hansen-Schwartz J, Edvinsson L (2004) Sphingosine signaling and atherogenesis. Acta Pharmacologica Sinica 25,849–54. Yim NH, Cho WK, Lee JH, Jung YP, Yang HJ, Ma JY (2012) HRT, herbal formula, induces G(2)/M cell cycle arrest and apoptosis via suppressing Akt signaling pathway in human colon cancer cells. Evidence-based Complementary and Alternative Medicine 2012,2871893. Young N, Van Brocklyn JR (2006) Signal transduction of sphingosine-1-phosphate G protein-coupled receptors. The Scientific World Journal 6,946–6. Zhang L, Valdez JM, Zhang B, et al. (2011) ROCK inhibitor Y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency. PLOS One 6,e18271. Zhuang S, Schnellmann RG (2006) Death-promoting role for extracellular signal-regulated kinase. Journal of Pharmacology and Experimental Therapeutics 319,991–7.
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Figure legends Figure 1. Expression of S1P receptors S1P1, S1P2, and S1P3 in DPCs. Samples 1–5 were derived from different donors. S1P1–S1P3 expression levels differed between donors. Ctrl, positive control, mesenchymal stem cells from rat bone marrow; GAP, GAPDH.
Figure 2. S1P-induced cell proliferation mainly via AKT and less via ERK and ROCK. (A) Proliferation measured using CCK8. Results were expressed as OD value (Mean ± SE). N=5. (B) Flow cytometric detection of the cell cycle.. Results were expressed as Proliferation index (PI). N=5. N, numbers of individual cell sample. *P < .05 compared with control. **P< .05 compared with S1P treatment.
Figure 3. The signaling pathways of S1P, ERK, AKT, and ROCK exhibited short-term complemental effects. (A) A complementary effect between ERK, AKT, and ROCK at 60min activated by S1P. N=3. (B) The effects of S1P on p-ERK and p-AKT after 24 h. The interactions between ERK, AKT, and ROCK disappeared. N=3. GAP, GAPDH; p, phosphorylated; Ctrl, control. N, numbers of individual cell sample.
Figure 4. The role of ERK in cell apoptosis was greater than that of AKT and ROCK. (A) DPCs were stained with JC-1 to quantify the ΔΨm. U0126 produced more apoptotic cells than the other inhibitors. N=3. Magnification, 100×. (B) Apoptosis measured using flow cytometry. Results were expressed as apoptosis index. N=5. (C) The effects of S1P and inhibitors on expression of apoptosis-associated proteins Bcl-2 and Bax. S1P and AKT inhibition had little effect on Bcl-2 and Bax, whereas Y27632 increased the expression of Bcl-2 and Bax after 24 h. ERK increased the Bax level compared with the control. N=3. GAP, GAPDH; p, phosphorylated; Ctrl, control. N, numbers of individual cell sample. *P < .05 compared with control. **P < .05 compared with S1P-treated cells.
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Received Date : 25-Dec-2013 Accepted Date : 11-Jun-2014 Article type
: Original Scientific Article
Sphingosine-1-Phosphate mediates AKT/ERK maintenance of dental pulp homeostasis
H.Y. Pan, H. Yang, M.Y. Shao, J. Xu, P. Zhang, R. Cheng*, T. Hu*
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu，Sichuan，China
* T. Hu and R. Cheng are both corresponding authors
Running Title: pulp homeostasis maintenance
Keywords: S1P, ERK/AKT, ROCK, apoptosis, proliferation, microinflammation
Corresponding authors: Tao Hu, Professor and Ran Cheng, Associate professor Department of Operative Dentistry and Endodontics Department of Preventive Dentistry State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University , 14# ,3rd section, Renmin South Road, Chengdu，Sichuan，610041 E-mail: [email protected] ；[email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/iej.12335 This article is protected by copyright. All rights reserved.
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Abstract Aim To investigate the cell status of dental pulp cells (DPCs) in a S1P-induced microinflammation environment and the possible mechanisms of cell homeostasis maintenance by S1P.
Methodology S1P receptor (S1PR) expression was examined in DPCs within a local S1P-induced microinflammation model established using 1ｕM S1P. U0126 (ERK inhibitor), LY294002 (AKT inhibitor), and Y27632 (ROCK inhibitor) were used to inhibit corresponding signaling pathways of DPCs. CCK8 and cell cycle analysis tested cell proliferation. Immunofluorescence staining JC-1 detected changes of mitochondrial membrane potential (ΔΨm). Tests for apoptosis and the apoptosis-related proteins Bax and Bcl-2 were assessed by flow cytometry and western blot analysis, respectively. Expressions of ERK and AKT were evaluated by western blot analysis. The results were analyzed using the Student’s t-test with SPSS and the significance level set at P < 0.05.
Results Expressions of S1PR1, S1PR2 and S1PR3 in DPCs differed among individuals. DPCs maintained self-homeostasis in response to S1P-induced microinflammation via S1PRs. During this repair process, ERK, AKT, and ROCK had a short-term complementary interaction at 60min, but then AKT and ERK gradually played decisive roles after 24h in proliferation enhancement and apoptosis inhibition, respectively (p>0.05).
Conclusions The AKT–ERK balance may determine whether DPC homeostasis in S1P-induced microinflammation is maintained by synergistic regulation of cell growth and apoptosis.
Introduction Blood vessels in the dental pulp are involved in both acute and chronic inflammation. During acute inflammation, various inflammatory mediators and leukocytes may cause plasma leakage and high pressure in the pulp space (Dahlén et al. 1981, Cooper et al. 2014). Dental pulp tissue can react to moderate external stimuli and induce reparative dentinogenesis. The pulps in teeth that have been stimulated by occlusal pressure or caries for long periods may result in vascular reactivity and local microinflammation, thereby switching on reparative dentinogenesis or dental tissue repair (Howard et al. 2010). Thus, there must be a mechanism to maintain homeostasis to avoid harmful stimulation and severe inflammation. Certain blood vessel-derived growth factors might be involved in this process, e.g. vascular endothelial growth factor (Mullane et al. 2008, Botero et al. 2010) and tumor necrosis factor-alpha (Paula-Silva et al. 2009).
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Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and S1P are plasma components and potent immune-modulating factors. S1P mediates broad biological effects, including cell growth and survival, cell differentiation, cell migration, angiogenesis, and immune regulation (Young & Van Brocklyn, 2006, Howard et al. 2010). When encountering inflammatory stimuli, S1P is released locally from platelets plasma into tissue injury sites so as to exert homeostatic and/or inflammatory functions depending on the S1P receptors (English et al. 2000, Obinata & Hla, 2012). The plasma concentration of S1P is 0.1–1 μM (Murata et al. 2000). Thus, the local congregated rise of S1P may represent a very early stage of an inflammation microenvironment.
Microinflammation means the form of low-grade and asymptomatic inflammation in the early stages of the inflammation responses (Wang et al. 2012). It induces systematic homeostasis dysbiosis and contributes to the inflammatory progress in various chronic disease, such as systemic bone mass, renal disease, uremia and acidosis, oxidative stress, bowel disease and dental diseases (Hamidi et al. 2014, Schindler 2004). However, little attention has been paid to the microenvironment in pulp diseases so far as well as to homeostatic regulation of dental pulp cells (DPCs) in this environment.
This study aimed to investigate S1P receptor expression on DPCs and cell homeostasis status in a S1P-induced microinflammation environment. Possible signaling pathways in proliferative and apoptotic balance were analyzed as an initial exploration.
Materials and Methods Cell Culture Five human impacted third molars or premolars were collected from adults (aged between 18 and 26 years) with informed consent according to the institutional standards. Individual cell samples were numbered as 1, 2, 3, 4, 5. The isolation, culture and identity of DPCs were confirmed as reported previously (Pan et al. 2013). The cells were used between three and seven passages. S1P is regarded as an immune-modulating lipid that is secreted by activated platelets in response to various physiological or pathological stimuli. High plasma concentrations of S1P were used to simulate the local microinflammatory state. DPCs were washed twice with phosphate-buffered saline and serum starved for 24 h before use. The cells were treated with 10 μM Y27632 (Rho-associated kinase (ROCK) inhibitor, Sigma-Aldrich), 50 μM LY294002 (AKT inhibitor, Sigma-Aldrich), and 10 μM U0126 (extracellular signal-regulated kinase (ERK) inhibitor, Sigma-Aldrich) for 40 min, and then stimulated with 1 μM S1P (Sigma-Aldrich) for specific times. The dosage of 1 μM S1P, 10 μM Y27632, 10 μM U0126 and 50 μM LY294002 on DPCs are known to be safe and without additional side effects (Wang et al. 2006, Spagnuolo et al. 2008, Cheng et al. 2010, 2011, Pan et al. 2013).
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Cell Proliferation Assay 1×103 cells were grown in 96-well culture plates and were utilized for experimentation at 60-70% confluence. Cells were starved for 24 h and treated as required for 24 h. Cell proliferation after stimulation of 24 hours was tested using Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) according to the protocol and previous studies (Chen et al. 2008, Meng et al. 2014). The optical density was measured at 450 nm on a microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). Samples 1-5 were used and each sample repeated three times.
To investigate the cell cycle and proliferation index (PI) of DPCs in the microinflammatory environment for 24 hours, a cell cycle detection kit (Nanjing KeyGEN Biotech. Co., Ltd, Nanjing, P. R. China) was used according to the manufacturer (Yim et al. 2012). Cells were seeded in 50 mL culture bottles overnight in 5 mL culture medium. Cells were starved for 24 h and treated as required for 24 h. About 1×106 cells were harvested by trypsinization and fixed in 70% ethanol overnight at –20°C. After washing twice with PBS, the cells were suspended in 100 μL of RNase solution for 30 min at 37°C, followed by 400 μL of propidium iodide solution for 30 min at 4°C while protected from the light. The stained cells were collected using a flow cytometer (Beckman FC500; Beckman Coulter, Miami, IL, USA). The PI was calculated as follows: PI= ( S+ G2 /M ) / ( G0 /G1 + S+ G2 /M )(Cheng et al. 2012) . Samples 1-5 were used and each sample was repeated at least twice.
Mitochondrial Membrane Potential (ΔΨm) Reduced mitochondrial membrane potential (ΔΨ) is a hallmark for apoptosis. JC-1 is a recommended dye used to measure the ΔΨ changes, thereby distinguishing dead cells from living cells. Healthy cells emitted red-orange fluorescence in JC-1 aggregates. In dead cells, JC-1 monomers present in the cytoplasm emitted green fluorescence. However, JC-1 alone scarcely discriminates intact/early apoptotic cells against necrotic/late apoptotic cells. ΔΨ was analyzed using a JC-1 kit (Beyotime Biotech, Nantong, China). Cells were seeded in 6-well plates and incubated overnight with 2 mL of culture medium. Cells were starved for 24 h and treated as required for 24 h. After changing the cell culture media, 1 mL JC-1 dye and 1 mL culture medium were added and plates were incubated at 37°C for 20 min. The cells were then washed twice and visualized using a fluorescence microscope (Carl Zeiss, Göttingen, Germany). Three individual samples (samples 1,3,5) were used and each sample was repeated at least five times.
Cell Apoptosis Assay To determine the apoptotic index of DPCs after 24 hour stimulation during microinflammation, a KeyGEN apoptosis detection kit (Nanjing KeyGEN Biotech. Co., Ltd, Nanjing, P. R. China) were used as reported previously (Yim et al. 2012, Cheng et al. 2012, Pan et al. 2013). The stained cells were collected using a flow cytometer (Beckman FC500; Beckman Coulter, Miami, USA). The apoptosis
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rate included early apoptosis and late apoptosis but excluded necrotic cells. Samples 1-5 were used in apoptosis analysis and each sample was repeated at least three times.
Western Blotting To measure the cellular protein levels, cell extracts were subjected to standard western blotting (Cheng et al. 2010, 2011). The antibodies used were against S1P1, S1P2 (Proteintech Group Inc., Chicago, IL, USA), S1P3, phosphorylated AKT (p-AKT, Ser473), phosphorylated extracellular signal-regulated kinase (p-ERK, Thr202/Tyr204), AKT, ERK, Bcl-2, Bax, and GAPDH (Epitomics, Inc., Burlingame, CA, USA). In the preliminary experiment, phosphorylation events of ERK and AKT in S1P-dependent microinflammation for 5min, 10min, 30min, 1h, 2h, 12h and 24h were explored. Then, the three key time points at 30min, 60min and 12h were selected. Samples 1-5 were used to test S1P receptor expression and the test was repeated at least three times per sample. Samples 2,3,4 were tested in AKT, ERK and ROCK diversifications, and samples 2,3,5 were used in Bax and Bcl-2 investigation. Each sample was repeated at least twice. The quantification of phosphorylation was normalized by the corresponding native protein.
Statistical Analysis The data from two groups were compared using Student’s t test. In all cases, P < .05 indicated a significant difference. Data are expressed as the mean ± SEM.
Results DPCs Expressed S1P Receptors S1P receptors S1PR1, S1PR2, and S1PR3 were detected using western blot analysis. The DPCs expressed S1PR1, S1PR2, and S1PR3. However, the level of these three receptors differed between donors (Fig. 1). The result indicated that S1P could affect DPCs through S1PR1, S1PR2, and S1PR3.
S1P Promoted the Proliferation of Dental Pulp Cells To investigate the possible signaling pathway involved in cell proliferation in microinflammation, DPCs were stimulated using S1P, U0126 (an ERK inhibitor), LY294002 (an AKT inhibitor), or Y27632 (a ROCK inhibitor) for 24 h. Cells in control were only treated with serum-free medium with or without inhibitors alone for the same time.
S1P treatment increased cell growth by 20.6% compared with the control. In the microinflammatory state, cell viability decreased by 21.1%, 26.0%, and 43.3% after U0126 (ERK inhibitor), Y27632 (ROCK inhibitor), and LY294002 (AKT inhibitor) treatment, respectively (Fig. 2A).
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S1P treatment increased the PI by 13.5%. By contrast, in the S1P-induced microinflammation environment U0126, Y27632 and LY294002 decreased the PI by 16.6%, 4.5% and 23.3%, respectively (Fig. 2B).
In the normal state AKT inhibition significantly reduced cell proliferation by both cell counting numbers and cell cycle influence. ERK and ROCK had little effect (Fig.2). However, in the S1P-induced microinflammation environment DPCs proliferated obviously on ERK/AKT/ROCK-dependent pathways. The pro-proliferative effect of AKT was stronger than ERK and ROCK(Fig.2) indicating that cell proliferation mechanisms, as a stress response to S1P-induced microinflammation, differed from cells in the normal environment. DPCs may enhance self-protection by functional proliferation behavior.
S1P Induced Complementary Effects in ERK, AKT, and ROCK The results described above suggested that ERK, AKT, and ROCK are involved in the S1P-induced cell proliferation and cell cycle. The interactions between ERK, AKT, and ROCK were then examined. DPCs were pretreated with U0126, LY294002, or Y27632 for 40 min and then stimulated with S1P for 60 min. S1P stimulation activated ERK and AKT within 30 min and 60 min, respectively. Interestingly, there was a complementary effect between ERK, AKT, and ROCK. U0126 inhibited ERK phosphorylation but activated AKT. LY294002 inhibited AKT but increased p-ERK compared with S1P alone. Y27632 increased p-ERK and p-AKT content markedly (Fig. 3A). However, the complementary effect disappeared at 24 h (Fig. 3B).
Inhibition of the ERK Pathway Induced DPC Apoptosis To obtain an overall view of cell vitality, flow cytometry was used to measure the apoptotic rate. In the microinflammation state S1P decreased cell apoptosis by 58.6% compared with the control. U0126 (ERK inhibitor), Y27632 (ROCK inhibitor), and LY294002 (AKT inhibitor) blocked S1P-induced survival, as shown by 501%, 108%, and 96% increases in cell apoptosis, respectively. In the quiescent state, ERK inhibition was associated with a significant rise in cell apoptosis by 144% (Fig. 4B). In the normal state, ERK inhibition significantly increased cell apoptosis, but AKT and ROCK had little effect. By contrast, cell apoptosis decreased in the S1P-influenced microinflammation state, thus increasing cell survival ability. ERK, AKT, and ROCK all had an anti-apoptotic effect, although this was much stronger for ERK than for AKT and ROCK (Fig. 4B). In accordance to the proliferation phenomenon above, the mediation of apoptosis in the microinflammation state was considerably distinct compared to the normal state.
To examine further the effects of S1P on the ERK, AKT, and ROCK pathways during cell apoptosis, the ΔΨm and the expression of Bcl-2 and Bax were measured after 24 h. Bax and Bcl-2 are important Bcl-2 family proteins that act as pro- and antiapoptotic modulators during cell
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apoptosis, respectively. ERK inhibition increased Bax expression and decreased the ΔΨm compared with the S1P group, indicating an apoptotic tendency (Fig. 4A and 4C).
The other two inhibitors also affected the ΔΨm according to the yellow/orange cells expression. Yellow staining appeared to come from an overlay of red and green staining suggesting that all cells may be losing mitochondrial polarization but are at different stages of the dying process(Fig. 4A). However, the expressions of green immunofluorescence in AKT inhibition and ROCK inhibition were significantly lower than that in ERK block, which confirmed the ERK-related decisive role in cell apoptosis regulation in the flow cytometry test (Fig. 4B). Furthermore, AKT and ROCK had different effects on Bax and Bcl-2 expression: S1P and AKT inhibition had little effect on Bax and Bcl-2 expression compared with the S1P group. ROCK inhibition increased both Bcl-2 and Bax expression in microinflammation. (Fig. 4C).
Discussion LPLs are generated by various cells including activated platelets, epithelial cells, macrophages, and some cancer cells (Goetzl & An 1998). Two major LPLs, LPA and S1P, activate G protein-coupled receptors in the endothelial differentiation gene family and regulate similar cellular function signaling pathways (Takuwa et al. 2002). Five specific G protein-coupled receptors, S1P1–S1P5 (Young & Van Brocklyn 2006), mediate the cellular processes regulated by S1P. S1P1, S1P2, and S1P3 receptors are expressed widely: S1P4 is expressed mainly in the immune system, but S1P5 receptors are limited to the nervous system (Means & Brown 2009, Schuchardt et al. 2011). The current study revealed that DPCs express receptors S1P1–S1P3 but that the expression of these receptors differs between individuals. Despite the interindividual differences, the cells from different donors responded similarly in the experiments. This established the foundation of S1P effects on DPCs. Previous studies have shown that differences in S1P receptor expression are involved in physiological processes, such as cell growth and apoptosis, and pathological processes, such as the inflammatory response (Payne et al. 2002, Qu et al. 2012). It can be hypothesized that different receptors have complementary effects with each other.
DPCs homeostasis plays a critical role in postnatal tooth formation, dental repair and pulp regeneration as a response to physiological and pathological stimuli in terms of balancing cell proliferation and cell death (Rangiani et al. 2012). S1P is widely researched on broad biological functions via several signaling pathways including ERK, p38 mitogen-activated protein kinase (MAPK), C-Jun N-terminal kinase, phospholipases C and D, phosphoinositide 3 (PI3)-kinase/AKT, and Rho GTPase (Xu et al. 2004, Schuchardt et al. 2011). However, whether S1P could mediate DPCs homeostasis and be involved in dental pulp repair and what the related mechanism is unknown.
The present data demonstrated that pulp repair occurred by way of S1P-maintained DPCs homeostasis against microinflammation. More importantly, DPCs homeostasis maintenance differed
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in the S1P-dependent microinflammation environment from that in the normal environment (Fig2 , Fig4). In the normal state, AKT determined cell proliferation while ERK decided cell survival. The balance of AKT and ERK may make a contribution to cell quiescence living. In contrast, as a response to microinflammtory stimuli, ERK, AKT and ROCK all participated in cell viability regulation to participate in pulp repair. Besides, ERK and AKT played a much more decisive role than ROCK.
Among various S1P downstream pathways, MAPK/ERK and PI3K/AKT are known to mediate S1P-induced mitogenic effects in many cell types, which subsequently modulate cell proliferation and cell death (An et al. 2000, Van Brocklyn et al. 2002). Glioma cells exhibit mitogenic effects via ERK/MAPK and PI3K activation after S1P stimulation (Van Brocklyn et al. 2002). An increase in proliferation of rat primary chondrocytes and ERK induced by S1P but not by p38 is involved in this process (Kim et al. 2006). PI3K/AKT is one of the best predictors of LPL-enhanced cell survival ability via coupling to the Gi pathways (Radeff-Huang et al. 2004). In the current study, DPC proliferation increased when cells were exposed to S1P, and AKT had a stronger effect on proliferation compared with ERK and ROCK.
MAPK/ERK and PI3K/AKT are also known to influence cell apoptosis, thereby mediating cell growth and death (Yim et al. 2012, Nakahara et al. 2012，Pan et al. 2013). Some reports demonstrated that ERK contributed to cell apoptosis in pathological injury (Zhuang & Schnellmann 2006). S1P also seems to protect cells from radiation and H2O2-induced apoptosis via the AKT pathway (Waeber et al. 2004, Bonnaud et al. 2010，Nakahara et al. 2012). However, in a previous study of ischemic damage on DPCs, another LPL, LPA, was used to protect DPCs from serious inflammatory injury. ERK played a key role in the anti-apoptotic effect but AKT had little effect (Pan et al. 2013). In the present study, DPCs survival in the microinflammation state depended on a combined effect of ERK, AKT and ROCK. However, ERK dominated cell survival to a large extent compared with AKT and ROCK. This indicates the cell survival ability enhanced by ERK pathway is essential for various types of pulp repair process. The differences of the effect of AKT on DPCs LPL-induced apoptosis inhibition may vary depending on the extent or seriousness of environmental inflammation.
Another interesting phenomenon in this study is that ERK and AKT could compensate for each other both in short-term stimuli and long-term influence (Fig 3). ERK inhibition significantly activated AKT while AKT inhibition obviously enhanced ERK activation. ROCK transiently interfered with interactions of ERK and AKT. ERK and AKT are likely to have collaborated together to maintain cell homeostasis in case of excessive or uncontrolled proliferation or apoptosis.
Rho/ROCK pathway, another important S1P downstream signaling, is a key mediator in DPCs migration, adhesion and related to S1P chemotactic response (Wang et al. 2008, Howard et al. 2010). S1P induces vigorous migration of DPCs to injury sites to become involved in dental pulp repair processes (Howard et al. 2010, Cheng et al. 2010, 2011). Recently, the ROCK pathway was
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shown to interfere with S1P-induced cell proliferation and apoptosis in various cell types with a prominent effect or little function. (Hurst et al. 2008, Zhang et al. 2011, Wu et al. 2012, Hsu et al. 2012). Thus, this study focused on how ROCK functioned with DPC homeostasis in microinflammation. However, it was noted that ROCK had little effect on DPC cell proliferation and apoptosis in the 24h microinflammatory environment. On the one hand, this may be caused by cell-specific diversity of ROCK influence on cell homeostasis. One the other hand, the results revealed that ROCK inhibition increased the levels of Bax and Bcl-2, thereby balancing the pro- and anti-apoptotic effects of Bcl-2 family members at the same time. This neutralized effect may balance ROCK mediations in cell proliferation and cell death. Moreover, the data confirmed that ROCK produced a complementary effect with ERK and AKT within 1 h, but this effect disappeared at 24 h. This short and immediate reaction suggested ROCK might participate in dental repair by a transiently indirect regulation in ERK/AKT-dependent way. It needs much further and thorough investigations on whether this phenomenon transiently switches on DPCs homing to injury sites at early stage of dental pulp repair and then subsequently induces ERK/AKT balance in late stage of homeostasis.
In this study, the focus was on DPCs homeostasis modulation in pulp repair to S1P-induced microinflammation and possible signaling mechanisms. The complex but vital mechanisms on how S1P modulates DPCs in the normal state and how DPCs switch cell viability regulation in response to different environments needs further research.
Conclusion DPCs expressed S1P1–S1P3 receptors but that the expression levels differed between individuals. The AKT, ERK and ROCK pathways were involved in S1P-induced cell proliferation and antiapoptosis. Interestingly, the balance between the AKT and ERK pathways mediated homeostasis of cell growth and death in the local microinflammatory state.
Acknowledgments The authors deny any conflicts of interest. This study was supported by grants from the National Natural Science Foundation of China (Numbers 81371134, 81000429, 30973322 and 31170894) and the Research Fund for the Doctoral Program of Higher Education (No. 20100181120066). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
References An S, Zheng Y, Bleu T (2000) Sphingosine 1-phosphate-induced cell proliferation, survival, and related signaling events mediated by G protein-coupled receptors Edg3 and Edg5. The Journal of Biological Chemistry 275,288–96.
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Bonnaud S, Niaudet C, Legoux F, et al. (2010) Sphingosine-1-phosphate activates the AKT pathway to protect small intestines from radiation-induced endothelial apoptosis. Cancer Research 70,9905–15. Botero TM, Son JS, Vodopyanov D, Hasegawa M, Shelburne CE, Nör JE (2010) MAPK signaling is required for LPS-induced VEGF in pulp stem cells. Journal of Dental Research 89,264–9. Chen Y, Takita J, Choi YL, et al.(2008) Oncogenic mutations of ALK kinase in neuroblastoma. Nature 455,971-4. Cheng MR, Li Q, Wan T, et al.(2012) Galactosylated chitosan/5-fluorouracil nanoparticles inhibit mouse hepatic cancer growth and its side effects. World Journal of Gastroenterology18,6076-87. Cheng R, Cheng L, Shao MY, et al. (2010) Roles of lysophosphatidic acid and the Rho-associated kinase pathway in the migration of dental pulp cells. Experimental Cell Research 316,1019–27. Cheng R, Shao MY, Yang H, et al. (2011) The effect of lysophosphatidic acid and Rho-associated kinase patterning on adhesion of dental pulp cells. International Endodontic Journal 44,2–8. Cooper PR, Holder MJ, Smith AJ (2014) Inflammation and regeneration in the dentin-pulp complex: a double-edged sword. Journal of Endodontics 40 Suppl 4, S46-51. Dahlén SE, Björk J, Hedqvist P, et al. (1981) Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory response. Proceedings of the National Academy of Sciences USA 78,3887-91. English D, Welch Z, Kovala AT, et al.(2000) Sphingosine 1-phosphate released from platelets during clotting accounts for the potent endothelial cell chemotactic activity of blood serum and provides a novel link between hemostasis and angiogenesis. The FASEB Journal 14,2255-65. Goetzl EJ, An S (1998) Diversity of cellular receptors and functions for the lysophospholipid growth factors lysophosphatidic acid and sphingosine 1-phosphate. The FASEB Journal 12,1589–98. Hamidi S, Schäfer-Korting M, Weindl G.(2014) TLR2/1 and sphingosine 1-phosphate modulate inflammation, myofibroblast differentiation and cell migration in fibroblasts. Biochimica et Biophysica Acta 1841,484-94 Howard C, Murray PE, Namerow KN (2010) Dental pulp stem cell migration. Journal of Endodontics 36,1963–6. Hsu A, Zhang W, Lee JF, et al. (2012) Sphingosine-1-phosphate receptor-3 signaling up-regulates epidermal growth factor receptor and enhances epidermal growth factor receptor-mediated carcinogenic activities in cultured lung adenocarcinoma cells. International Journal of Oncology 40, 1619-162. Hurst JH, Mumaw J, Machacek DW, et al. (2008) Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology. BMC Neuroscience 9,118. Kim MK, Lee HY, Kwak JY, Park JI, Yun J, Bae YS (2006) Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation. Biochemistry and Biophysical Research Communications 345,67–73.
This article is protected by copyright. All rights reserved.
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Means CK, Brown JH (2009) Sphingosine-1-phosphate receptor signalling in the heart. Cardiovascular Research 82,193–200. Meng W, Wu Y, He X, et al.(2014) A Systems Biology Approach Identifies Effective Tumor-Stroma Common Targets for Oral Squamous Cell Carcinoma. Cancer Res 74,2306-15. Mullane EM, Dong Z, Sedgley CM, et al. (2008) Effects of VEGF and FGF2 on the revascularization of severed human dental pulps. Journal of Dental Research 87,1144–8. Murata N, Sato K, Kon J, et al.（2000）Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions. Biochemical Journal 352,809–15. Nakahara T, Iwase A, Nakamura T, et al. (2012) Sphingosine-1-phosphate inhibits H2O2-induced granulosa cell apoptosis via the PI3K/Akt signaling pathway. Fertility and Sterility 98,1001–8.e1. Obinata H, Hla T.(2012) Sphingosine 1-phosphate in coagulation and inflammation. Seminars in Immunopathology 34,73-91. Pan HY, Cheng L, Yang H, et al. (2013) Lysophosphatidic acid rescues human dental pulp cells from ischemia-induced apoptosis. Journal of Endodontics 40,217-22 Paula-Silva FW, Ghosh A, Silva LA, Kapila YL (2009) TNF-α promotes an odontoblastic phenotype in dental pulp cells. Journal of Dental Research 88,339–44. Payne SG, Milstien S, Spiegel S (2002) Sphingosine-1-phosphate: dual messenger functions. FEBS Letters 531,54–7. Qu Z, Cheuk BL, Cheng SW (2012) Differential expression of sphingosine-1-phosphate receptors in abdominal aortic aneurysms. Mediators of Inflammation 2012,643609. Radeff-Huang J, Seasholtz TM, Matteo RG, Brown JH (2004) G protein mediated signaling pathways in lysophospholipid induced cell proliferation and survival. Journal of Cellular Biochemistry 92,949–66. Rangiani A, Cao ZG, Liu Y, et al.(2012) Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation. International Journal of Oral Science 4,189-95. Schindler R.(2004) Causes and therapy of microinflammation in renal failure. Nephrol Dial Transplant. 19 Suppl 5,V34-40. Schuchardt M, Tölle M, Prüfer J, van der Giet M (2011) Pharmacological relevance and potential of sphingosine 1-phosphate in the vascular system. British Journal of Pharmacology 163,1140–62. Spagnuolo G, D'Antò V, Valletta R, et al.(2008) Effect of 2-hydroxyethyl methacrylate on human pulp cell survival pathways ERK and AKT. Journal of Endodontics 34,684-8.
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Takuwa Y, Takuwa N, Sugimoto N (2002) The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities. The Journal of Biochemistry 131,767–71. Van Brocklyn J, Letterle C, Snyder P, Prior T (2002) Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: role of ERK MAP kinase and phosphatidylinositol 3-kinase β. Cancer Letters 181,195–204. Waeber C, Blondeau N, Salomone S (2004) Vascular sphingosine-1-phosphate S1P1 and S1P3 receptors. Drug News&Perspectives 17,365–82. Wang FM, Hu T, Tan H et al. (2006) p38 Mitogen-activated protein kinase affects Transforming growth factor-β/Smad signaling in human dental pulp cells. Molecular and Cellular Biochemistry 291, 49–54. Wang F, Zhang P, Jiang H, et al.(2012) Gut bacterial translocation contributes to microinflammation in experimental uremia. Digestive Diseases and Sciences 57,2856-62. Wang L, Lee JF, Lin CY, Lee MJ (2008) Rho GTPases mediated integrin αvβ3 activation in sphingosine-1-phosphate stimulated chemotaxis of endothelial cells. Histochemistry and Cell Biology 129,579–88. Wu J, Li J, Hu H, Liu P, Fang Y, Wu D (2012) Rho-kinase inhibitor, fasudil, prevents neuronal apoptosis via the Akt activation and PTEN inactivation in the ischemic penumbra of rat brain. Cellular and Molecular Neurobiology 32,1187–97. Xu CB, Hansen-Schwartz J, Edvinsson L (2004) Sphingosine signaling and atherogenesis. Acta Pharmacologica Sinica 25,849–54. Yim NH, Cho WK, Lee JH, Jung YP, Yang HJ, Ma JY (2012) HRT, herbal formula, induces G(2)/M cell cycle arrest and apoptosis via suppressing Akt signaling pathway in human colon cancer cells. Evidence-based Complementary and Alternative Medicine 2012,2871893. Young N, Van Brocklyn JR (2006) Signal transduction of sphingosine-1-phosphate G protein-coupled receptors. The Scientific World Journal 6,946–6. Zhang L, Valdez JM, Zhang B, et al. (2011) ROCK inhibitor Y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency. PLOS One 6,e18271. Zhuang S, Schnellmann RG (2006) Death-promoting role for extracellular signal-regulated kinase. Journal of Pharmacology and Experimental Therapeutics 319,991–7.
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Figure legends Figure 1. Expression of S1P receptors S1P1, S1P2, and S1P3 in DPCs. Samples 1–5 were derived from different donors. S1P1–S1P3 expression levels differed between donors. Ctrl, positive control, mesenchymal stem cells from rat bone marrow; GAP, GAPDH.
Figure 2. S1P-induced cell proliferation mainly via AKT and less via ERK and ROCK. (A) Proliferation measured using CCK8. Results were expressed as OD value (Mean ± SE). N=5. (B) Flow cytometric detection of the cell cycle.. Results were expressed as Proliferation index (PI). N=5. N, numbers of individual cell sample. *P < .05 compared with control. **P< .05 compared with S1P treatment.
Figure 3. The signaling pathways of S1P, ERK, AKT, and ROCK exhibited short-term complemental effects. (A) A complementary effect between ERK, AKT, and ROCK at 60min activated by S1P. N=3. (B) The effects of S1P on p-ERK and p-AKT after 24 h. The interactions between ERK, AKT, and ROCK disappeared. N=3. GAP, GAPDH; p, phosphorylated; Ctrl, control. N, numbers of individual cell sample.
Figure 4. The role of ERK in cell apoptosis was greater than that of AKT and ROCK. (A) DPCs were stained with JC-1 to quantify the ΔΨm. U0126 produced more apoptotic cells than the other inhibitors. N=3. Magnification, 100×. (B) Apoptosis measured using flow cytometry. Results were expressed as apoptosis index. N=5. (C) The effects of S1P and inhibitors on expression of apoptosis-associated proteins Bcl-2 and Bax. S1P and AKT inhibition had little effect on Bcl-2 and Bax, whereas Y27632 increased the expression of Bcl-2 and Bax after 24 h. ERK increased the Bax level compared with the control. N=3. GAP, GAPDH; p, phosphorylated; Ctrl, control. N, numbers of individual cell sample. *P < .05 compared with control. **P < .05 compared with S1P-treated cells.
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Accepted Article This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
