Expression and secretion of interleukin-1b, tumour necrosis factor-a and interleukin-10 by hypoxia- and serum-deprivation-stimulated mesenchymal stem cells Implications for their paracrine roles Zongwei Li, Hua Wei, Linzi Deng, Xiangfeng Cong and Xi Chen Research Center for Cardiac Regenerative Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Keywords IL-10; IL-1b; mesenchymal stem cell; paracrine; TNF-a Correspondence X Chen; X Cong, Research Center for Cardiac Regenerative Medicine, The Ministry of Health, Cardiovascular Institute & Fu Wai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, 167 Beilishilu, Beijing 100037, China Fax ⁄ Tel: +86 10 88398584 E-mail: chenxifw@yahoo.com.cn; xiangfeng_cong@yahoo.com.cn (Received 26 April 2010, revised 27 June 2010, accepted 10 July 2010) doi:10.1111/j.1742-4658.2010.07770.x To understand the potential paracrine roles of interleukin-1b (IL-1b), tumour necrosis factor-a (TNF-a) and interleukin-10 (IL-10), the expression and secretion of these factors by rat bone marrow-derived mesenchymal cells stimulated by hypoxia (4% oxygen) and serum deprivation (hypoxia ⁄ SD) were investigated We found that hypoxia ⁄ SD induced nuclear factor kappa Bp65-dependent IL-1b and TNF-a transcription Furthermore, hypoxia ⁄ SD stimulated the translation of pro-IL-1b and its processing to mature IL-1b, although the translation of TNF-a was unchanged Unexpectedly, the release of IL-1b and TNF-a from hypoxia ⁄ SD-stimulated mesenchymal cells was undetectable unless ATP or lipopolysaccharide was present This result suggests that IL-1b and TNF-a are not responsible for the paracrine effects of mesenchymal cells under ischaemic conditions We also found that hypoxia ⁄ SD induced the transcription and secretion of IL-10, which were significantly enhanced by lipopolysaccharide and the proteasomal inhibitor MG132 Moreover, both the conditioned medium from hypoxia ⁄ SD-stimulated mesenchymal cells (MSC-CM) and IL-10 efficiently inhibited cardiac fibroblast proliferation and collagen expression in vitro, suggesting that mesenchymal cell-secreted IL-10 prevents cardiac fibrosis in a paracrine manner under ischaemic conditions Taken together, these findings may improve understanding of the cellular and molecular basis of the anti-inflammatory and paracrine effects of mesenchymal cells Introduction Ischaemic heart disease is a life-threatening condition that may cause sudden cardiac failure and death Many researchers have investigated cell transplantation as an alternative treatment for heart disease Bone marrow-derived mesenchymal stem cells (MSCs) are easily obtainable and expandable, multipotent progeni- tor cells [1] that have emerged as attractive candidates for cellular therapies for heart and other organ-system disorders [2] Although several mechanisms have been proposed for the cardioprotective effects of MSCs, including cardiomyocyte regeneration, spontaneous cell fusion and paracrine action [3], there is a growing Abbreviations BrdU, 5-bromodeoxyuridine; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; hypoxia ⁄ SD, hypoxia and serum deprivation; IL, interleukin; IMDM, Iscove’s modified Dulbecco’s medium; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; NF-jBp65, nuclear factor kappa Bp65; p38, p38 mitogen-activated protein kinase; TNF-a, tumour necrosis factor-a 3688 FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS Z Li et al Paracrine anti-fibrotic effects of MSCs in vitro body of evidence supporting the hypothesis that paracrine mechanisms mediated by MSC-secreted factors play an essential role in the reparative process [4,5] It has been reported that MSC-conditioned medium under normoxic conditions significantly attenuates cardiac fibroblast proliferation and type I and III collagen expression, exerting paracrine anti-fibrotic effects However, researchers did not analyse the active components of the conditioned medium [6] Other researchers have suggested that adrenomedullin and hepatocyte growth factor are paracrine factors secreted by transplanted MSCs, decreasing myocardial fibrosis [7–9] Whether other paracrine factors released by MSCs mediate these cells’ anti-fibrotic effects remains largely unknown Interleukin-1b (IL-1b) and tumour necrosis factor-a (TNF-a) are present in the tissues or systemic circulation in many inflammatory conditions It has also been reported that the expression of IL-1b and TNF-a in MSCs can be augmented by exposure to hypoxia [5] Furthermore, IL-1b can induce cardiomyocyte growth but inhibits cardiac fibroblast proliferation in culture [10] By contrast, MSC transplantation in rat models of myocardial infarction has anti-inflammatory effects, decreasing protein production and gene expression for IL-1b and TNF-a [11] To address these paradoxes of both pro- and anti-inflammatory effects, the secretion of IL-1b and TNF-a from MSCs under ischaemic conditions must be further characterized IL-10 is an anti-inflammatory cytokine that has been reported to be involved in the immunomodulation mediated by transplanted MSCs [12,13] IL-10 is also a potential anti-fibrotic factor in the liver and kidney [14–16] In addition, the protective effect of MSCs against sepsis is dependent on IL-10, which is not directly produced by the injected MSCs but rather by A endogenous macrophages [17] However, it is not known whether MSCs can secrete IL-10 under ischaemic conditions, resulting in a paracrine anti-fibrotic effect in the heart To assess the paracrine effects of IL-1b, TNF-a and IL-10 released by MSCs on cardiac remodelling under ischaemic conditions, conditioned medium from MSCs (MSCs-CM) was collected during hypoxia and serum deprivation (hypoxia ⁄ SD) This medium was used to treat cardiac fibroblasts, enabling observation of the paracrine effects of MSCs The expression and secretion of IL-1b, TNF-a and IL-10 by hypoxia ⁄ SD-stimulated MSCs were also investigated Our data demonstrate that MSCs-CM can inhibit cardiac fibroblast proliferation and collagen synthesis, with < 30 kDa molecules as its major active components MSCs did not secrete IL-1b and TNF-a under hypoxia ⁄ SD conditions, although MSC-secreted IL-10 hindered cardiac fibroblast proliferation and collagen expression These findings suggest that IL-10 may be an important paracrine, anti-fibrotic mediator secreted by MSCs Results MSCs-CM inhibits cardiac fibroblast proliferation and collagen synthesis The effects of MSCs-CM on cardiac fibroblast proliferation and collagen synthesis were detected by [3H]-thymidine and [3H]-proline incorporation As shown in Fig 1A, MSC-CM treatment significantly inhibited [3H]-thymidine and [3H]-proline incorporation under normoxic or hypoxic culture conditions To further clarify the molecular mass range of important active factors in the MSCs-CM, the medium was divided into B Fig MSCs-CM inhibits cardiac fibroblast proliferation and collagen synthesis (A) The effects of MSCs-CM on the incorporation of [3H]-thymidine and [3H]-proline by cardiac fibroblasts under normoxic or hypoxic conditions Each data point represents the mean ± SEM of at least three independent experiments ***P < 0.001 versus normoxic control (Cont) group; ###P < 0.001 and ##P < 0.01 versus hypoxic control (Cont + h) group (B) The effects of the > 30 kDa and < 30 kDa components of MSCs-CM on the incorporation of [3H]-thymidine and [3H]-proline by cardiac fibroblasts under normoxic or hypoxic conditions ***P < 0.001 versus Cont group; ###P < 0.001 versus Cont + h group FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS 3689 Paracrine anti-fibrotic effects of MSCs in vitro Z Li et al > 30 and < 30 kDa components using a 30 kDa molecular mass cut-off ultrafiltration membrane Fractionation revealed that the < 30 kDa components, but not the > 30 kDa components, of the MSCs-CM inhibited cardiac fibroblast proliferation and collagen synthesis (Fig 1B) whereas hypoxia simply augmented this effect (Fig 2B) It has been reported that the nuclear factor-jB (NFjB) signalling pathway plays an important role in regulating IL-1b and TNF-a transcription [18,19] To investigate the role of this pathway in hypoxia ⁄ SDinduced transcription, MSCs were exposed to BAY 11-7082, an NF-jB pathway inhibitor, followed by hypoxia ⁄ SD for h As shown in Fig 2C, the transcription of IL-1b and TNF-a was significantly attenuated by BAY 11-7082 Interestingly, the proteasomal inhibitor MG132 also abrogated hypoxia ⁄ SD-induced IL-1b and TNF-a transcription Next, to clarify the mechanism by which the NF-jB pathway induces IL-1b and TNF-a transcription, the nuclear translocation of NF-jBp65 was assessed by immunocytochemical staining As shown in Fig 2D, NF-jBp65 was mainly distributed in the cytoplasm of control cells By contrast, hypoxia ⁄ SD treatment significantly stimulated the nuclear translocation of Hypoxia ⁄ SD induces NF-jB-dependent IL-1b and TNF-a transcription Because transcription of IL-1b and TNF-a can be augmented in MSCs by hypoxia [5], and because the molecular masss of IL-1b and TNF-a are both 17 kDa (< 30 kDa), changes in IL-1b and TNF-a gene transcription in hypoxia ⁄ SD-stimulated MSCs were examined As shown in Fig 2A, the increased transcription of IL-1b and TNF-a occurred after h of hypoxia ⁄ SD with a gradual increase up to h, after which transcription decreased We also found that transcription of IL-1b and TNF-a was mainly induced by SD, A B C D Fig Hypoxia ⁄ SD induces NF-jB-dependent IL-1b and TNF-a transcription (A) MSCs were incubated under hypoxia ⁄ SD conditions for the indicated number of hours, and the relative mRNA levels of IL-1b and TNF-a were determined by real-time PCR The data are the mean ± SEM of at least three independent experiments *P < 0.05 and **P < 0.01 versus control group (0 h) (B) The relative mRNA levels for IL-1b and TNF-a in MSCs after hypoxia, SD or hypoxia ⁄ SD for h by real-time PCR **P < 0.01 versus Cont group; #P < 0.05 versus SD group (C) MSCs were exposed to BAY 11-7082 or MG132, followed by hypoxia ⁄ SD for h and detection of relative mRNA levels of IL-1b and TNF-a by real-time PCR *P < 0.05 and **P < 0.01 versus hypoxia ⁄ SD treatment group (D) A representative pattern of the nuclear translocation of NF-jBp65, as assessed by immunocytochemical staining of MSCs using anti-(NF-jBp65 primary Ig) (red) and nuclear labelling with 4¢,6-diamidino-2-phenylindone (blue) 3690 FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS Z Li et al Paracrine anti-fibrotic effects of MSCs in vitro NF-jBp65, indicated by strong immunostaining in the nucleus Pretreatment with BAY 11-7082 inhibited hypoxia ⁄ SD-induced NF-jBp65 translocation, with substantial levels of NF-jBp65 staining remaining in the cytoplasm of most cells These results demonstrate that hypoxia ⁄ SD induces IL-1b and TNF-a transcription, which are dependent on activation of the NF-jB pathway Hypoxia ⁄ SD-induced IL-1b and TNF-a transcription depend on the extracellular signal-regulated kinase pathway The extracellular signal-regulated kinase ⁄ (ERK1 ⁄ 2) and p38 mitogen-activated protein kinase (p38) signalling pathways play important roles in hypoxia ⁄ SD-induced apoptosis of MSCs [20,21] and may also affect IL-1b and TNF-a transcriptional regulation [22] To confirm this, 20 lm U0126 (Fig S1A) was used to inhibit the ERK1 ⁄ pathway in MSCs, followed by measurement of IL-1b and TNF-a mRNA levels by real-time PCR As shown in Fig 3A, U0126 completely abolished hypoxia ⁄ SD-induced IL-1b and TNF-a transcriptional upregulation When the MSCs were exposed to 15 lm SB202190 (Fig S1B), a p38-specific inhibitor, hypoxia ⁄ SD-induced IL-1b transcription was inhibited by 60%, although TNF-a transcription was not affected (Fig 3B) Like BAY 11-7082, U0126 could also inhibit NF-jBp65 nuclear translocation (Fig 3C), suggesting that hypoxia ⁄ SD-induced activation of the NF-jB signalling pathway depends on the ERK1 ⁄ signalling pathway Hypoxia ⁄ SD increases the translation of pro-IL-1b but not TNF-a Having demonstrated significant transcriptional upregulation, we next examined protein levels of IL-1b and TNF-a in MSCs-CM Unexpectedly, neither IL-1b nor TNF-a was detectable in MSCs-CM using enzymelinked immunosorbent assay (ELISA) analysis To determine the reason for this lack of IL-1b and TNF-a secretion by MSCs, changes in these factors’ translation in hypoxia ⁄ SD-stimulated MSCs were investigated As shown in Fig 4A, hypoxia ⁄ SD increased pro-IL-1b translation in a time-dependent manner, whereas TNF-a protein expression remained unchanged at each time point Furthermore, MG132, BAY 11-7082 and U0126, all of which abrogated hypoxia ⁄ SD-induced IL-1b and TNF-a transcription, also abolished pro-IL-1b translational upregulation (Fig 4B,C) but failed to affect TNF-a translation A B C Fig IL-1b and TNF-a transcriptional induction depends on the ERK1 ⁄ pathway MSCs were exposed to the ERK1 ⁄ inhibitor U0126 or the p38 inhibitor SB202190, followed by hypoxia ⁄ SD for h (A,B) Relative mRNA levels for IL-1b and TNF-a, as determined by real-time PCR *P < 0.05 versus hypoxia ⁄ SD group (C) A representative pattern of the nuclear translocation of NF-jBp65, as assessed by immunocytochemical staining of MSCs using an anti-(NF-jBp65 primary Ig) (red) and nuclear labelling with 4Â,6-diamidino-2phenylindone (blue) FEBS Journal 277 (2010) 36883698 ê 2010 The Authors Journal compilation ª 2010 FEBS 3691 Paracrine anti-fibrotic effects of MSCs in vitro A Z Li et al B Fig Hypoxia ⁄ SD increases translation of pro-IL-1b but not TNF-a (A) Representative western blots for pro-IL-1b and TNF-a expression in MSCs stimulated by hypoxia ⁄ SD for the indicated number of hours (B) Representative western blots for pro-IL-1b and TNF-a expression in MSCs in the presence and absence of BAY 11-7082 or MG132 *, nonspecific band (C) Representative western blots for pro-IL-1b expression in MSCs in the presence and absence of U0126 *, nonspecific band C (Fig 4B) These results demonstrate that in hypoxia ⁄ SD-stimulated MSCs, IL-1b mRNA can be efficiently translated into pro-IL-1b protein, whereas the translation of TNF-a mRNA is severely repressed A Hypoxia ⁄ SD induces cleavage of pro-IL-1b into mature IL-1b B Given that hypoxia ⁄ SD induced significant translational upregulation of pro-IL-1b and that processing of pro-IL-1b into mature IL-1b requires activating cleavage of pro-caspase [23], the cleavage of both pro-IL-1b and pro-caspase was examined in hypoxia ⁄ SD-stimulated MSCs As shown in Fig 5A, hypoxia ⁄ SD promoted the processing of pro-IL-1b into mature IL-1b, with a stronger induction effect in the presence of the endotoxin LPS Consistent with these data, hypoxia ⁄ SD also induced the cleavage of procaspase 1, with stronger activation in the presence of LPS (Fig 5B) Hypoxia ⁄ SD-stimulated MSCs require a second signal for IL-1b and TNF-a release Although significant cleavage of pro-IL-1b and procaspase occurred intracellularly in hypoxia ⁄ SD-stimulated MSCs, mature IL-1b was undetectable in MSCs-CM (Fig 6A) However, significant release of IL-1b by hypoxia ⁄ SD-stimulated MSCs in the presence of ATP was detected Furthermore, when both LPS and ATP were present, hypoxia ⁄ SD-stimulated MSCs released a larger amount of IL-1b (Fig 6A) We also examined TNF-a expression in hypoxia ⁄ SD-stimulated MSCs in the presence of LPS As shown in Fig 6B, LPS relieved the translational inhibition of TNF-a Moreover, TNF-a release by MSCs was detectable 3692 Fig Hypoxia ⁄ SD induces cleavages of pro-IL-1b and pro-caspase MSCs were stimulated by hypoxia ⁄ SD in the presence or absence of LPS for the indicated number of hours (A) Representative western blots for pro-IL-1b and mature IL-1b in MSCs (B) Representative western blots for pro-caspase and cleaved caspase in MSCs after hypoxia ⁄ SD treatment for h in the presence of LPS (Fig 6C) These findings demonstrate that hypoxia ⁄ SD-stimulated MSCs require a second stimulatory signal in order to secrete IL-1b and TNF-a Hypoxia ⁄ SD induces the transcription and secretion of IL-10 Because of the lack of secretion of the inflammatory cytokines IL-1b and TNF-a from hypoxia ⁄ SD-stimulated MSCs, as well as the significant anti-inflammatory effects of MSCs, expression and secretion of the anti-inflammatory cytokine IL-10 by these cells was investigated As shown in Fig 7A, hypoxia ⁄ SD FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS Z Li et al Paracrine anti-fibrotic effects of MSCs in vitro A Fig Hypoxia ⁄ SD-stimulated MSCs require a second signal for IL-1b and TNF-a release (A) The results of ELISA analysis of supernatants from MSCs after hypoxia ⁄ SD stimulation for 12 h in the presence and absence of ATP and LPS (B) Representative western blots for TNF-a expression in MSCs stimulated by hypoxia ⁄ SD in the presence or absence of LPS for the indicated number of h *, nonspecific band (C) The results of ELISA analysis of supernatants from MSCs after hypoxia ⁄ SD stimulation for 12 h in the presence or absence of LPS C B A Fig Hypoxia ⁄ SD induces expression and secretion of IL-10 (A) Relative IL-10 mRNA levels in MSCs stimulated by hypoxia ⁄ SD for the indicated number of hours Data are the mean ± SEM of at least three independent experiments *P < 0.05 versus control group (0 h) (B) Relative IL-10 mRNA levels in MSCs after hypoxia ⁄ SD treatment for h in the presence and absence of various reagents *P < 0.05 versus control group; ##P < 0.01 versus hypoxia ⁄ SD treatment group (C) The results of ELISA analysis of supernatants from MSCs after hypoxia ⁄ SD stimulation for the indicated number of hours in the presence or absence of LPS *P < 0.05 versus 6-h group; **P < 0.01 versus 12 h group B C induced significant IL-10 transcription after 3, and 12 h Moreover, the transcriptional induction of IL-10 by hypoxia ⁄ SD was abolished by the p38 inhibitor SB202190 but was unexpectedly augmented by the proteasomal inhibitor MG132 and by LPS (Fig 7B) Next, the secretion of IL-10 from hypoxia ⁄ SD-stimulated MSCs was examined by ELISA As shown in Fig 7C, a small amount of IL-10 release from MSCs was detected at the 6-h time point, and this release was elevated at the 12-h time point Furthermore, IL-10 secretion was augmented by the presence of LPS at each time point IL-10 inhibits cardiac fibroblast proliferation and collagen expression The molecular mass of IL-10 is 19 kDa, which is < 30 kDa and thus part of the MSCs-CM fraction that inhibited cardiac fibroblast proliferation and collagen synthesis (Fig 1B) To investigate the potential contribution of IL-10 to the paracrine effects of MSCs, the influence of IL-10 on cardiac fibroblast proliferation was characterized using a 5-bromodeoxyuridine (BrdU) incorporation assay As shown in Fig 8A,B, different IL-10 concentrations significantly inhibited FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS 3693 Paracrine anti-fibrotic effects of MSCs in vitro Z Li et al A B C D Fig MSC-secreted IL-10 is involved in the inhibition of cardiac fibrosis (A) BrdU incorporation in cardiac fibroblasts grown in standard DMEM at 24 h after IL-10 treatment at different concentrations **P < 0.01 and ***P < 0.001 versus Cont group (B) BrdU incorporation in cardiac fibroblasts grown in DMEM with 10% fetal bovine serum at 24 h after IL-10 treatment at different concentrations **P < 0.01 and ***P < 0.001 versus 10% fetal bovine serum treatment group (C) The relative mRNA levels of collagen I, collagen III and a-smooth muscle actin (a-SMA) in cardiac fibroblasts in the presence and absence of IL-10 *P < 0.05 and **P < 0.01 versus Cont group (D) Representative western blots for collagen I and III in the presence and absence of 0.1 lM angiotensin II and IL-10 BrdU incorporation into cardiac fibroblast under normal 10% fetal bovine serum or serum-free culture conditions IL-10 also decreased type I and III collagen and a-smooth muscle actin mRNA levels in cardiac fibroblasts (Fig 8C) Moreover, IL-10 effectively limited angiotensin II-induced type I and III collagen protein expression (Fig 8D) These results indicate that IL-10 can inhibit cardiac fibroblast proliferation and collagen expression, suggesting a paracrine, anti-fibrotic role for this factor Discussion In this study, we focused on the paracrine effects of MSCs on cardiac fibroblast proliferation and collagen expression, as well as the possible paracrine roles of IL-1b, TNF-a and IL-10 in cardiac fibrosis First, our results demonstrate that MSCs-CM have significant anti-fibrotic effects, as indicated by decreased [3H]-thymidine and [3H]-proline incorporation by cardiac fibroblasts Moreover, we found that < 30 kDa components of MSCs-CM play the dominant anti-fibrotic role, suggesting that these anti-fibrotic factors may be soluble small molecules Second, our data show that hypoxia ⁄ SD induces NF-jB-dependent IL-1b and TNFa transcriptional upregulation However, these two factors are not secreted from hypoxia ⁄ SD-stimulated MSCs unless a second signalling stimulus is present This finding suggests that the paracrine roles of TNF-a 3694 and IL-1b after MSC transplantation may be negligible Third, we determined that hypoxia ⁄ SD induces transcription and secretion of IL-10, which significantly inhibits cardiac fibroblast proliferation and collagen expression MSC-secreted IL-10 may thus play a role in the attenuation of cardiac fibrosis under ischaemic conditions NF-jB is a ubiquitous protein transcription factor that induces a variety of genes affecting the inflammatory processes [24,25] Normally, NF-jB is inactive and coupled to IjB protein [26,27] Based on our study, we hypothesize that hypoxia ⁄ SD stimulates the phosphorylation and ubiquitin-related degradation of IjB The active form of NF-jBp65 is then released and translocated into the nucleus to activate the transcription of IL-1b and TNF-a In this report, hypoxia ⁄ SD-induced IL-1b and TNF-a transcription were abolished by the ERK1 ⁄ inhibitor U0126, suggesting that hypoxia ⁄ SD-induced NF-jB activation is dependent on ERK1 ⁄ signalling However, the p38 inhibitor SB202190 only partly inhibited hypoxia ⁄ SDinduced IL-1b transcription and failed to affect the TNF-a mRNA levels Activation of p38 may thus be involved in the regulation of IL-1b mRNA stability by a mechanism independent of NF-jB signalling Pro-IL-1b is synthesized in the cytosol of activated cells without a signal sequence, precluding secretion via the classical endoplasmic reticulum–Golgi route [28] Processing of pro-IL-1b into its active form FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS Z Li et al requires caspase [29], which is itself activated by a molecular scaffold termed the inflammasome [23] It is generally accepted that such IL-1b generation and secretion by monocytes occurs in two steps First, an inflammatory signal, such as the endotoxin LPS, promotes the synthesis and cytoplasmic accumulation of pro-IL-1b A second signal, in the form of exogenous ATP, triggers caspase 1-mediated processing of pro-IL1b and secretion of the mature cytokine [30,31] In our study, hypoxia ⁄ SD enhanced the transcription and translation of pro-IL-1b as well as the cleavage of proIL-1b into mature IL-1b However, IL-1b was not released from hypoxia ⁄ SD-stimulated MSCs unless ATP or LPS was present Interestingly, although hypoxia ⁄ SD induced significant TNF-a transcription, the translation of TNF-a remained unchanged even when TNF-a transcription was inhibited by MG132 or BAY 11-7082 The exact reason for the translational repression of TNF-a is unclear, but there are at least two possibilities: microRNA-mediated TNF-a mRNA translational silencing or TNF-a mRNA AU-rich element-mediated posttranscriptional regulation involving AU-rich elementbinding proteins and processing bodies (P-bodies) [32] Such AU-rich element-mediated translational repression of TNF-a may strongly correlate with IL-10 secretion by MSCs [33] LPS preconditioning enhances the efficacy of MSC transplantation in a rat model of acute myocardial infarction, resulting in superior therapeutic neovascularization and decreased fibrosis [34] Meanwhile, IL-10 has been reported to inhibit fibrosis in the liver [16], kidney [15] and airway [35] In this study, we found that LPS significantly augmented hypoxia ⁄ SD-induced IL-10 transcription and secretion Furthermore, IL-10 effectively inhibited cardiac fibroblast proliferation and collagen expression in vitro, suggesting that IL-10 has the potential to prevent cardiac fibrosis Thus, we hypothesize that the enhanced anti-fibrotic effects of LPS preconditioning may be because of increased IL-10 secretion induced by LPS MG132 also significantly inhibited hypoxia ⁄ SD-induced MSC apoptosis in vitro (data not shown) and enhanced IL-10 expression Therefore, MG132 preconditioning may provide another effective strategy of maximizing the viability, paracrine effects and biological and functional properties of MSCs In conclusion, our work demonstrates that hypoxia ⁄ SD increases the transcription but not the secretion of IL-1b and TNF-a, suggesting that the roles of these factors in the paracrine effects of MSCs are negligible However, hypoxia ⁄ SD also enhances the transcription and secretion of IL-10, which may be an important Paracrine anti-fibrotic effects of MSCs in vitro mediator of the cells’ paracrine anti-fibrotic effects These findings help to improve our understanding of the cellular and molecular basis of MSCs’ anti-inflammatory and paracrine effects Materials and methods Materials Iscove’s modified Dulbecco’s medium (IMDM), Dulbecco’s modified Eagle’s medium (DMEM) and Trizol reagent were purchased from Invitrogen (Carlsbad, CA, USA) M-MLV reverse transcriptase was obtained from Promega (Madison, WI, USA) and Power SYBR Green PCR Master Mix was purchased from Applied Biosystems (Foster City, CA, USA) SB202190, U0126, MG132, BAY 11-7082, LPS and angiotensin II were obtained from Sigma (St Louis, MO, USA) The BrdU cell proliferation assay kit was acquired from Calbiochem (Gibbstown, NJ, USA) ELISA detection kits for IL-1b, TNF-a and IL-10 as well as antibodies against IL-1b and TNF-a were obtained from R&D Systems (Minneapolis, MN, USA), whereas antibodies against ERK, phospho-ERK1 ⁄ 2, p38 and phospho-p38 were purchased from Cell Signalling Technology (Danvers, MA, USA) Antibodies against NF-jBp65, caspase 1, collagen I, collagen III and b-actin and horseradish peroxidise-conjugated secondary antibodies were manufactured by Santa Cruz Biotechnology (Santa Cruz, CA, USA) Cell culture, inhibitor treatment and conditioned medium collection Isolation and expansion of MSCs were conducted as previously reported [20] Briefly, bone marrow was harvested from the tibias and femurs of 80 g rats, plated in IMDM supplemented with 15% heat-inactivated fetal bovine serum and 100 mL)1 penicillin ⁄ streptomycin and incubated at 37 °C in a humidified tissue culture incubator containing 5% CO2 The medium was replaced h after plating and 24 h later to remove nonadherent hematopoietic cells Adherent MSCs were further grown in medium, which was replaced every 48 h The MSCs used in subsequent experiments had been passaged one to three times All procedures were approved by the Animal Care Committee of the Cardiovascular Institute and Fu Wai Hospital (Beijing, China) For inhibitor-based studies, 15 lm SB202190 (p38 inhibitor) [36,37], 20 lm U0126 (ERK1 ⁄ inhibitor) [20], 10 lm MG132 (proteasome inhibitor) or lm BAY 11-7082 (NF-jB inhibitor) was preincubated with MSCs in complete medium for h The cells were subsequently washed in serum-free IMDM and exposed to hypoxia ⁄ SD in the continued presence of inhibitor Hypoxic conditions were generated by incubating the MSCs at 37 °C in a sealed hypoxic GENbox jar fitted with a catalyst to scavenge free oxygen, as described previously [20] FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS 3695 Paracrine anti-fibrotic effects of MSCs in vitro Z Li et al MSC-CM was generated as follows First, 80% confluent cells were administered serum-free DMEM and incubated for h under hypoxic conditions The medium was then collected, clarified by centrifugation and divided into > 30 and < 30 kDa components using 30 kDa molecular mass cut-off ultrafiltration membranes (Millipore, Billerica, MA, USA) if necessary As a control, plates containing medium alone were also subjected to the same conditions Neonatal cardiac fibroblasts were isolated from Sprague– Dawley rats (1–3 days old) and characterized as previously described [38] All experiments were performed on the second or third passage of cardiac fibroblasts after starvation in serum-free DMEM for 24 h The cells were then treated with control medium or MSCs-CM [3H]-Thymidine and [3H]-proline uptake assays Cardiac fibroblasts were transferred to 24-well plates, starved of serum for 24 h and then stimulated with standard medium or MSCs-CM for 24 h [3H]-Thymidine or [3H]-proline (Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China) was added to each well to a final concentration of lCiỈmL)1 during the last h of incubation Stimulation was terminated by rinsing the cardiac fibroblasts three times with NaCl ⁄ Pi and then adding ice-cold 10% trichloroacetic acid for 30 Cell precipitates were washed three times with ice-cold NaCl ⁄ Pi and then solubilized in 1% SDS with 0.1 m sodium hydroxide overnight at room temperature The radioactivity of SDS-soluble protein was determined by liquid scintillation spectrometry (Beckman Model LS6000-SC, Brea, CA, USA) RNA extraction and real-time PCR analysis Total RNA was extracted from MSCs using Trizol reagent according to the manufacturer’s instructions Next, cDNA was generated from lg of total RNA using M-MLV reverse transcriptase and oligo(dT)18 primer Real-time PCR was performed in a total volume of 25 lL containing 0.5 lL RT product, 0.5 lm primers and 12.5 lL Power SYBR Green PCR Master Mix Glyceraldehyde-3-phosphate dehydrogenase mRNA amplified from the same samples served as an internal control The relative expression of each targeted gene was normalized by subtracting the corresponding glyceraldehyde-3-phosphate dehydrogenase threshold cycle (Ct) values using the DDCt comparative method The sequences of all primers used in this work are as follows: IL-1b: 5¢-GCTGTGGCAGCTACCTATGTCTTG-3¢ and 5¢-AGGTCGTCATCATCCCACGAG-3¢; TNF-a: 5¢-AACTCGAGTGACAAGCCCGTAG-3¢ and 5¢-GTAC CACCAGTTGGTTGTCTTTGA-3¢; IL-10: 5¢-CAGACCC ACATGCTCCGAGA-3¢ and 5¢-CAAGGCTTGGCAA CCCAAGTA-3¢; collagen I: TCCTGGCAATCGTGGTT CAA and ACCAGCTGGGCCAACATTTC; collagen III: TGGACAGATGCTGGTGCTGAG and GAAGGCCAG 3696 CTGTACATCAAGGA; alpha smooth muscle actin (a-SMA): AGCCAGTCGCCATCAGGAAC and CCGG AGCCATTGTCACACAC; and glyceraldehyde-3-phosphate dehydrogenase: 5¢-GGCACAGTCAAGGCTGAGAATG-3¢ and 5¢-ATGGTGGTGAAGACGCCAGTA-3¢ Immunocytochemical staining for NF-jBp65 MSCs in IMDM supplemented with 10% fetal bovine serum were plated on six-well glass slides When the cells reached 70–80% confluence, they were preincubated with U0126 or BAY 11-7082 as described above and exposed to hypoxia ⁄ SD for h The cells were then fixed in 2% paraformaldehyde in NaCl ⁄ Pi for 30 min, washed twice with NaCl ⁄ Pi and permeabilized with 0.3% Triton X-100 in NaCl ⁄ Pi for 10 Next, the MSCs were blocked in 2% goat serum for h and incubated with rabbit anti-(NF-jBp65 primary IgG) for 1–2 h The cells were then washed and incubated with rhodamine-labelled goat anti-(rabbit secondary IgG) After three NaCl ⁄ Pi washes and incubation with the nuclear stain 4¢,6-diamidino-2-phenylindone for 20 min, the MSCs were washed in NaCl ⁄ Pi for 10 and mounted in gelvatol for microscopic imaging Protein extraction and western blotting analysis Lysates of stimulated cells were prepared and subjected to SDS ⁄ PAGE as previously described [20] Briefly, stimulated cells were rinsed twice with ice-cold NaCl ⁄ Pi and lysed in ice-cold lysis buffer for 30 Cell lysates were then centrifuged at 13 000 g for 10 at °C and their protein concentrations were determined by the BCA Protein Assay Lysate amounts allowing equal protein loading between lanes were determined and mixed with · SDS sample buffer, boiled for and separated by 10–15% SDS ⁄ PAGE before transferring the proteins onto nitrocellulose membranes by semi-dry transfer After blocking in 5% skim milk for h, the membranes were rinsed and incubated overnight at °C with gentle shaking and with the appropriate diluted primary antibody in 5% BSA, · Tris-buffered saline (TBS) and 0.1% Tween-20 (TBS ⁄ T) Excess antibody was then removed by washing the membranes with TBS ⁄ T and subsequent incubation with horseradish peroxidase-conjugated secondary antibody for h at room temperature After further washes in TBS ⁄ T, the bands were visualized using an enhanced chemiluminescence detection kit and radiographic film exposure ELISA analysis of IL-1b, TNF-a and IL-10 secretion by MSCs The MSCs-CM was concentrated 20 · by ultrafiltration using 10 kDa molecular mass cut-off ultrafiltration membranes (Millipore) following the manufacturer’s instructions Production of IL-1b, TNF-a and IL-10 by MSCs FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS Z Li et al was then determined by ELISA using the commercially available kits mentioned earlier according to the manufacturer’s instructions Absorbance was measured at 450 nm using a microplate reader Results were compared with a standard curve constructed by titrating rat IL-1b, TNF-a and IL-10 BrdU incorporation assay Cardiac fibroblasts were transferred to 96-well plates, starved of serum for 24 h and stimulated with IL-10 for 24 h DNA synthesis at 24 h was measured using a BrdU ELISA kit Briefly, the cells were incubated for h at 37 °C with 20 lLỈwell)1 of BrdU The supernatant was then removed and the cells were fixed in 200 lLỈwell)1 of FixDenat for 30 at room temperature Subsequently, antiBrdU Ig, horseradish peroxidase-conjugated goat anti(mouse IgG) and substrate solution were applied to the wells The absorbance of the samples was measured at 450 nm using a microplate reader Paracrine anti-fibrotic effects of MSCs in vitro Statistical analysis Data are expressed as the mean ± SEM Differences among groups were tested by one-way analysis of variance (ANOVA) Comparisons between two groups were evaluated using Student’s t-test A value of P < 0.05 was considered statistically significant Acknowledgement This study was supported by the 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Lett 580, 4737–4745 Supporting information The following supplementary material is available: Fig S1 Dose–response data for U0126 and SB202190 This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 3688–3698 ª 2010 The Authors Journal compilation ª 2010 FEBS ... (2007) Effect of hypoxia on gene expression of bone marrowderived mesenchymal stem cells and mononuclear cells Stem Cells 25, 1166–1177 Li L, Zhang S, Zhang Y, Yu B, Xu Y & Guan Z (2009) Paracrine. .. the secretion of IL-1b and TNF-a, suggesting that the roles of these factors in the paracrine effects of MSCs are negligible However, hypoxia ⁄ SD also enhances the transcription and secretion of. .. followed by hypoxia ⁄ SD for h and detection of relative mRNA levels of IL-1b and TNF-a by real-time PCR *P < 0.05 and **P < 0.01 versus hypoxia ⁄ SD treatment group (D) A representative pattern of