Heart Vessels DOI 10.1007/s00380-013-0427-x
SHORT COMMUNICATION
Reduction of inorganic phosphate-induced human smooth muscle cells calcification by inhibition of protein kinase A and p38 mitogen-activated protein kinase Jeong-Hun Kang • Riki Toita • Daisuke Asai Tetsuji Yamaoka • Masaharu Murata
•
Received: 25 March 2013 / Accepted: 4 October 2013 Ó Springer Japan 2013
Abstract High levels of serum phosphate are associated with calcification of human smooth muscle cells (HSMCs). We investigated whether inhibition of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signals [p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)] can reduce inorganic phosphate (Pi)-induced HSMC calcification. Inhibition of PKA or p38 MAPK by inhibitors or small interfering RNAs (siRNAs) reduced Ca levels and alkaline phosphatase activities in HSMCs treated with high Pi, but inhibition of ERK1/2 and JNK showed no significant changes. Moreover, there were no significant changes in cell viability on adding siRNAs and three inhibitors (PKA, p38, and MEK1/2), but JNK inhibitor slightly reduced cell
Electronic supplementary material The online version of this article (doi:10.1007/s00380-013-0427-x) contains supplementary material, which is available to authorized users. J.-H. Kang (&) T. Yamaoka Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan e-mail: [email protected] R. Toita Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan D. Asai Department of Microbiology, St. Marianna University School of Medicine, Sugao 2-16-1 Miyamae, Kawasaki, Kanagawa 216-8511, Japan M. Murata Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
viability. These results show that PKA and p38 MAPK are involved in the Pi-induced calcification of HSMCs, and may be good targets for reducing vascular calcification. Keywords Smooth muscle cell Calcification Protein kinase A Mitogen-activated protein kinase Alkaline phosphatase
Introduction Vascular calcification, such as coronary and aortic calcification, is markedly observed in patients with atherosclerosis, diabetes, and end-stage renal disease, and increases the risk of cardiovascular disease [1–3]. Intimal calcification occurs mainly in atherosclerotic lesions, and is often activated by inflammatory cells (e.g., macrophages) and cytokines (e.g., tumor necrosis factor-a). Medial calcification occurs in the matrix between smooth muscle cells (SMCs), which form a major component of the medial artery [4, 5]. Damage to SMCs stimulates bone-forming signals and their transformation into osteoblast-like cells. High levels of serum phosphate (P) are associated with increased SMC damage and transformation of SMCs to osteoblastic-like cells, leading to increased medial calcification [5, 6]. After addition of organic phosphate donors such as inorganic phosphate (Pi) or b-glycerophosphate (bGP), SMCs lost their lineage markers such as SM22a and smooth muscle a-actin, and gained osteochondrogenic markers such as alkaline phosphatase (ALP), osteopontin, Runx2, core binding factor a1, and osteocalcin [1, 7–9]. Protein kinase A (PKA) increases ALP activity in SMCs treated with an elevated level of Pi. Activated ALP stimulates the sodium-dependent phosphate cotransporter that can induce the uptake of Pi into SMCs, but reduces the
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level of inorganic pyrophosphate that acts as a vascular calcification inhibitor by directly binding to hydroxyapatite crystals, resulting in the stimulation of Pi-induced calcification in SMCs [10–13]. Similarly to PKA, mitogen-activated protein kinase (MAPK) can have an influence on the Pi-mediated calcification of vascular cells, but there are few data available regarding the effect of MAPK signals [p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)] on the Pi-induced calcification of human SMCs (HSMCs). In the present study, we examined whether inhibition of PKA and MAPK signals can reduce Pi-induced HSMC calcification.
Materials and methods
Systems, Hayward, CA, USA). Ca levels were normalized against total protein concentration. Total protein concentration was analyzed by absorbance at 595 nm using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Assay of ALP activity Intracellular ALP activity in HSMCs was measured using the TRACP and ALP Assay Kit (MK301 colorimetric kit; Takara Bio, Tokyo, Japan). Cells were washed three times with saline and were lysed with 500 ll saline containing 1 % NP-40. The cell lysates were mixed with an assay mixture containing p-nitrophenyl phosphate and incubated at 37 °C for 20 min. The reaction was stopped by the addition of 0.4 M NaOH, and was measured at 405 nm. Protein concentration in the cell lysate was evaluated using
Cell culture and Pi-mediated calcification HSMCs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (High Glucose; Wako, Osaka, Japan). Media were supplemented with smooth muscle growth supplement (SMGS; Cascade Biologics, Tokyo, Japan), 10 % fetal bovine serum, penicillin (100 U/ml), streptomycin (100 lg/ml), and amphotericin B (0.25 lg/ml) (all Gibco/ Invitrogen, Grand Island, NY, USA). The cells were incubated under a humidified atmosphere containing 5 % CO2 at 37 °C. To prepare the in vitro model of HSMC calcification, cells (5 9 104 cells) were incubated in DMEM without SMGS for 24 h in a six-well plate. HSMC calcification was induced by adding Pi (NaH2PO4, 1.5 or 3 mM) for 6 days, and the medium was changed every 2 days. Cell viability Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). HSMCs were incubated for 24 h in a six-well plate. The conditioned medium in each well was replaced with 2 ml of fresh medium containing CCK-8 (200 ll), and the cells were incubated for a further 3 h, before measurement of the absorbance at 450 nm. The cell viability (%) was calculated by normalizing the absorbance of treated cells against untreated control cells. Detection of calcium (Ca) concentration Cells were washed three times with phosphate-buffered saline (Ca2? and Mg2? free) and incubated with 0.6 N HCl for 24 h. The supernatant was used for detecting Ca levels using a QuantiChrom calcium assay kit (BioAssay
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Fig. 1 Time- and dose-dependent change of a Ca level and b cell viability, and c von Kossa staining (9100) after adding inorganic phosphate (Pi) to human smooth muscle cells (n = 6). *P \ 0.05, **P \ 0.005, ***P \ 0.001, one-way analysis of variance
Heart Vessels
the Bio-Rad Protein Assay Dye Reagent, and ALP activity was normalized against total protein concentration.
duplicate for each sample. Statistically significant differences between groups were evaluated by one-way analysis of variance followed by a Tukey–Kramer post hoc test.
RNA interference The small interfering RNAs (siRNAs) (SMARTpool p38 MAPK and PKA) were purchased from Thermo Fisher Scientific (Lafayette, CO, USA) and control siRNA was from Sigma-Aldrich (Tokyo, Japan). Each siRNA (100 nM/ well) was transfected twice in a 72-h interval into HSMCs by using Lipofectamine 2000 according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). HSMC calcification was induced by adding Pi (3 mM) at the start of siRNA transfection. The levels of Ca and ALP activities were detected at 6 days after transfection. Statistical analysis Results are expressed as means, with standard deviation, of six samples. All determinations were carried out in
Results In the present study, the in vitro model of HSMC calcification was prepared by treating with Pi (3 mM) for 6 days. The Ca levels increased time- and dose-dependently, but cell viability decreased after adding Pi to HSMCs. At 6 days, the Pi (3 mM)-treated HSMCs showed seven times higher level of Ca, but lower cell viability (70 %), compared with the control group (Fig. 1). To examine whether inhibition of PKA and MAPK signals (p38, ERK1/2, and JNK) can reduce Pi-induced HSMCs calcification, four inhibitors (H89 for PKA, PD184352 for MEK1/2, SP600125 for JNK, and SB202192 for p38; each 10 lM) were added to the medium containing Pi (3 mM) and HSMCs. Addition of PKA or p38 MAPK
Fig. 2 Change of a, c alkaline phosphatase (ALP) activity and b, c Ca level after adding inhibitors (10 lM) (a, b) or siRNAs (100 nM) (c, d) to the inorganic phosphate-treated human smooth muscle cells (n = 6). *P \ 0.005, **P \ 0.0001, one-way analysis of variance. PD184352 MEK1/2 inhibitor, SP600125 JNK inhibitor, H89 PKA inhibitor, SB202192 p38 MAPK inhibitor
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inhibitor significantly and dose-dependently reduced the levels of Ca and ALP activities in HSMCs treated with Pi, but there was no change after adding ERK1/2 and JNK inhibitors (Figs. 2a, b, S2). On the other hand, inhibitors have no effect on the Ca levels and ALP activities in the absence of Pi (Fig. S3). To confirm the results obtained from the addition of p38 MAPK and PKA inhibitors, p38 MAPK and PKA siRNAs were transfected into HSMCs. Two siRNAs significantly decreased Ca levels and ALP activities (Fig. 2c, d). On the other hand, there were no significant changes in cell viability on adding siRNAs and three inhibitors (PKA, p38, MEK1/2, and JNK) (Fig. S1), meaning that significant reduction in the cell viability is caused mainly by the addition of Pi.
mineralization and ALP expression in calcifying vascular cells treated with Pi and a farnesoid X receptor agonist, 6aethyl chenodeoxycholic acid [20]. In the present study, JNK inhibition had no effect on the ALP activities and Ca levels in HSMCs treated with Pi alone. Thus, JNK inhibition may not reduce the Pi-induce calcification of vascular cells. In conclusion, inhibition of PKA and p38 MAPK by inhibitors and siRNAs efficiently decreased Ca levels and ALP activities in HSMCs treated with Pi. These results suggest that PKA and p38 MAPK are involved in the Piinduced calcification of HSMCs, and their inhibition may be an efficient way to reduce vascular calcification. Acknowledgments This work was financially supported by a grantin-aid for Scientific Research (B) (KAKENHI Grant Number 23310085) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
Discussion In the present study, the effect of PKA and MAPK signals (p38, ERK1/2, and JNK) on Pi-induced HSMC calcification was investigated. Previous studies have reported that inhibition of PKA reduces ALP activity and calcification in high P-treated SMCs [11, 12, 14]. Similarly, our study shows that inhibition of PKA by inhibitor or siRNA significantly reduces Ca levels and ALP activities in HSMCs treated with Pi (3 mM) (Fig. 2). Among MAPK signals, the p38 MAPK can regulate osteogenic differentiation and calcification of calcifying vascular cells [15]. Moreover, the glycation end products induce calcification of human aortic SMCs via p38 signal in the presence of b-GP, but show no stimulation of p38 signal in its absence [16]. These studies may mean that p38 MAPK participates in the calcification of vascular cells, but there are still very few data relating to its effect on the Piinduced HSMC calcification. Our study clearly shows that inhibition of p38 MAPK can reduce ALP activities and Ca levels in Pi-treated HSMCs. ERK can induce osteoblastic differentiation and calcification of vascular cells [16] but, on the contrary, several factors reduce their calcification through the ERK signal pathway, such as taurine [17], ghrelin [18], and apelin [19]. Moreover, ERK inhibitor has no influence on the ALP activity and Ca level in b-GP-induced rat or mouse SMC calcification [17, 18] and in human calcifying vascular SMCs [19]. Our study also shows that addition of ERK1/2 inhibitor has no effect on the Pi-induced HSMC calcification. From these results, directly blocking activation of ERK1/2 by ERK1/2 inhibitors may not inhibit Pi- or b-GPinduced SMC calcification. Furthermore, there are few data relating to the effect of JNK on Pi-induced SMC calcification, but a recent study suggests that JNK inhibition can dose-dependently induce
123
References 1. Jono S, Shioi A, Ikari Y, Nishizawa Y (2006) Vascular calcification in chronic kidney disease. J Bone Miner Metab 24:176–181 2. Ueda H, Harimoto K, Tomoyama S, Tamaru H, Miyawaki M, Mitsusada N, Yasuga Y, Hiraoka H (2012) Relation of cardiovascular risk factors and angina status to obstructive coronary artery disease according to categorical coronary artery calcium score. Heart Vessels 27:128–134 3. Farrag A, Bakhoum S, Salem MA, El-Faramawy A, Gergis E (2013) The association between extracoronary calcification and coronary artery disease in patients with type 2 diabetes mellitus. Heart Vessels 28:12–18 4. Tintut Y, Patel J, Parhami F, Demer LL (2000) Tumor necrosis factor-a promotes in vitro calcification of vascular cells via the cAMP pathway. Circulation 102:2636–2642 5. Amann K (2008) Media calcification and intima calcification are distinct entities in chronic kidney disease. Clin J Am Soc Nephrol 3:1599–1605 6. Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM (2000) Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 87:e10–e17 7. Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM (2001) Smooth muscle cell phenotype transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res 89:1147–1154 8. Giachelli CM, Speer MY, Li X, Rajachar RM, Yang H (2005) Regulation of vascular calcification: roles of phosphate and osteopontin. Circ Res 96:717–722 9. Zarjou A, Jeney V, Arosio P, Poli M, Antal-Szalma´s P, Agarwal A, Balla G, Balla J (2009) Ferritin prevents calcification and osteoblastic differentiation of vascular smooth muscle cells. J Am Soc Nephrol 20:1254–1263 10. Lomashvili KA, Cobbs S, Hennigar RA, Hardcastle KI, O’Neill C (2004) Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol 15:1392–1401 11. Huang MS, Sage AP, Lu J, Demer LL, Tintut Y (2008) Phosphate and pyrophosphate mediated PKA-induced vascular cell calcification. Biochem Biophys Res Commun 374:553–558
Heart Vessels 12. Prosdocimo DA, Wyler SC, Romani AM, O’Neill WC, Dubyak GR (2010) Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia. Am J Physiol Cell Physiol 298:C702–C713 13. Simmons CA, Nikolovski J, Thornton AJ, Matlis S, Mooney DJ (2004) Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells. J Biomech 37:1531–1541 14. Hsu JJ, Lu J, Huang MS, Geng Y, Sage AP, Bradley MN, Tontonoz P, Demer LL, Tintut Y (2009) T0901317, an LXR agonist, augments PKA-induced vascular cell calcification. FEBS Lett 583:1344–1348 15. Tanikawa T, Okada Y, Tanikawa R, Tanaka Y (2009) Advanced glycation end products induce calcification of vascular smooth muscle cells through RAGE/p38 MAPK. J Vasc Res 46:572–580 16. Speer MY, Yang HY, Brabb T, Leaf E, Look A, Lin WL, Frutkin A, Dichek D, Giachelli CM (2009) Smooth muscle cells give rise
17.
18.
19.
20.
to osteochondrogenic precursors and chondrocytes in calcifying arteries. Cir Res 104:733–741 Liao XB, Zhou XM, Li JM, Yang JF, Tan ZP, Hu ZW, Liu W, Lu Y, Yuan LQ (2008) Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway. Amino Acids 34:525–530 Liang QH, Jiang Y, Zhu X, Cui RR, Liu GY, Liu Y, Wu SS, Liao ZB, Xie H, Zhou HD, Wu XP, Yuan LQ, Liao EY (2012) Ghrelin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the ERK pathway. PLoS One 7:e33126 Shan PF, Lu Y, Cui RR, Jiang Y, Yuan LQ, Liao EY (2011) Apelin attenuates the osteoblastic differentiation of vascular smooth muscle cells. PLoS One 6:e17938 Miyazaki-Anzai S, Levi M, Kratzer A, Ting TC, Lewis LB, Miyazaki M (2010) Farnesoid X receptor activation prevents the development of vascular calcification in ApoE-/- mice with chronic kidney disease. Circ Res 106:1807–1817
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SHORT COMMUNICATION
Reduction of inorganic phosphate-induced human smooth muscle cells calcification by inhibition of protein kinase A and p38 mitogen-activated protein kinase Jeong-Hun Kang • Riki Toita • Daisuke Asai Tetsuji Yamaoka • Masaharu Murata
•
Received: 25 March 2013 / Accepted: 4 October 2013 Ó Springer Japan 2013
Abstract High levels of serum phosphate are associated with calcification of human smooth muscle cells (HSMCs). We investigated whether inhibition of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signals [p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)] can reduce inorganic phosphate (Pi)-induced HSMC calcification. Inhibition of PKA or p38 MAPK by inhibitors or small interfering RNAs (siRNAs) reduced Ca levels and alkaline phosphatase activities in HSMCs treated with high Pi, but inhibition of ERK1/2 and JNK showed no significant changes. Moreover, there were no significant changes in cell viability on adding siRNAs and three inhibitors (PKA, p38, and MEK1/2), but JNK inhibitor slightly reduced cell
Electronic supplementary material The online version of this article (doi:10.1007/s00380-013-0427-x) contains supplementary material, which is available to authorized users. J.-H. Kang (&) T. Yamaoka Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan e-mail: [email protected] R. Toita Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan D. Asai Department of Microbiology, St. Marianna University School of Medicine, Sugao 2-16-1 Miyamae, Kawasaki, Kanagawa 216-8511, Japan M. Murata Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
viability. These results show that PKA and p38 MAPK are involved in the Pi-induced calcification of HSMCs, and may be good targets for reducing vascular calcification. Keywords Smooth muscle cell Calcification Protein kinase A Mitogen-activated protein kinase Alkaline phosphatase
Introduction Vascular calcification, such as coronary and aortic calcification, is markedly observed in patients with atherosclerosis, diabetes, and end-stage renal disease, and increases the risk of cardiovascular disease [1–3]. Intimal calcification occurs mainly in atherosclerotic lesions, and is often activated by inflammatory cells (e.g., macrophages) and cytokines (e.g., tumor necrosis factor-a). Medial calcification occurs in the matrix between smooth muscle cells (SMCs), which form a major component of the medial artery [4, 5]. Damage to SMCs stimulates bone-forming signals and their transformation into osteoblast-like cells. High levels of serum phosphate (P) are associated with increased SMC damage and transformation of SMCs to osteoblastic-like cells, leading to increased medial calcification [5, 6]. After addition of organic phosphate donors such as inorganic phosphate (Pi) or b-glycerophosphate (bGP), SMCs lost their lineage markers such as SM22a and smooth muscle a-actin, and gained osteochondrogenic markers such as alkaline phosphatase (ALP), osteopontin, Runx2, core binding factor a1, and osteocalcin [1, 7–9]. Protein kinase A (PKA) increases ALP activity in SMCs treated with an elevated level of Pi. Activated ALP stimulates the sodium-dependent phosphate cotransporter that can induce the uptake of Pi into SMCs, but reduces the
123
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level of inorganic pyrophosphate that acts as a vascular calcification inhibitor by directly binding to hydroxyapatite crystals, resulting in the stimulation of Pi-induced calcification in SMCs [10–13]. Similarly to PKA, mitogen-activated protein kinase (MAPK) can have an influence on the Pi-mediated calcification of vascular cells, but there are few data available regarding the effect of MAPK signals [p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)] on the Pi-induced calcification of human SMCs (HSMCs). In the present study, we examined whether inhibition of PKA and MAPK signals can reduce Pi-induced HSMC calcification.
Materials and methods
Systems, Hayward, CA, USA). Ca levels were normalized against total protein concentration. Total protein concentration was analyzed by absorbance at 595 nm using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Assay of ALP activity Intracellular ALP activity in HSMCs was measured using the TRACP and ALP Assay Kit (MK301 colorimetric kit; Takara Bio, Tokyo, Japan). Cells were washed three times with saline and were lysed with 500 ll saline containing 1 % NP-40. The cell lysates were mixed with an assay mixture containing p-nitrophenyl phosphate and incubated at 37 °C for 20 min. The reaction was stopped by the addition of 0.4 M NaOH, and was measured at 405 nm. Protein concentration in the cell lysate was evaluated using
Cell culture and Pi-mediated calcification HSMCs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (High Glucose; Wako, Osaka, Japan). Media were supplemented with smooth muscle growth supplement (SMGS; Cascade Biologics, Tokyo, Japan), 10 % fetal bovine serum, penicillin (100 U/ml), streptomycin (100 lg/ml), and amphotericin B (0.25 lg/ml) (all Gibco/ Invitrogen, Grand Island, NY, USA). The cells were incubated under a humidified atmosphere containing 5 % CO2 at 37 °C. To prepare the in vitro model of HSMC calcification, cells (5 9 104 cells) were incubated in DMEM without SMGS for 24 h in a six-well plate. HSMC calcification was induced by adding Pi (NaH2PO4, 1.5 or 3 mM) for 6 days, and the medium was changed every 2 days. Cell viability Cell viability was determined using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). HSMCs were incubated for 24 h in a six-well plate. The conditioned medium in each well was replaced with 2 ml of fresh medium containing CCK-8 (200 ll), and the cells were incubated for a further 3 h, before measurement of the absorbance at 450 nm. The cell viability (%) was calculated by normalizing the absorbance of treated cells against untreated control cells. Detection of calcium (Ca) concentration Cells were washed three times with phosphate-buffered saline (Ca2? and Mg2? free) and incubated with 0.6 N HCl for 24 h. The supernatant was used for detecting Ca levels using a QuantiChrom calcium assay kit (BioAssay
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Fig. 1 Time- and dose-dependent change of a Ca level and b cell viability, and c von Kossa staining (9100) after adding inorganic phosphate (Pi) to human smooth muscle cells (n = 6). *P \ 0.05, **P \ 0.005, ***P \ 0.001, one-way analysis of variance
Heart Vessels
the Bio-Rad Protein Assay Dye Reagent, and ALP activity was normalized against total protein concentration.
duplicate for each sample. Statistically significant differences between groups were evaluated by one-way analysis of variance followed by a Tukey–Kramer post hoc test.
RNA interference The small interfering RNAs (siRNAs) (SMARTpool p38 MAPK and PKA) were purchased from Thermo Fisher Scientific (Lafayette, CO, USA) and control siRNA was from Sigma-Aldrich (Tokyo, Japan). Each siRNA (100 nM/ well) was transfected twice in a 72-h interval into HSMCs by using Lipofectamine 2000 according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). HSMC calcification was induced by adding Pi (3 mM) at the start of siRNA transfection. The levels of Ca and ALP activities were detected at 6 days after transfection. Statistical analysis Results are expressed as means, with standard deviation, of six samples. All determinations were carried out in
Results In the present study, the in vitro model of HSMC calcification was prepared by treating with Pi (3 mM) for 6 days. The Ca levels increased time- and dose-dependently, but cell viability decreased after adding Pi to HSMCs. At 6 days, the Pi (3 mM)-treated HSMCs showed seven times higher level of Ca, but lower cell viability (70 %), compared with the control group (Fig. 1). To examine whether inhibition of PKA and MAPK signals (p38, ERK1/2, and JNK) can reduce Pi-induced HSMCs calcification, four inhibitors (H89 for PKA, PD184352 for MEK1/2, SP600125 for JNK, and SB202192 for p38; each 10 lM) were added to the medium containing Pi (3 mM) and HSMCs. Addition of PKA or p38 MAPK
Fig. 2 Change of a, c alkaline phosphatase (ALP) activity and b, c Ca level after adding inhibitors (10 lM) (a, b) or siRNAs (100 nM) (c, d) to the inorganic phosphate-treated human smooth muscle cells (n = 6). *P \ 0.005, **P \ 0.0001, one-way analysis of variance. PD184352 MEK1/2 inhibitor, SP600125 JNK inhibitor, H89 PKA inhibitor, SB202192 p38 MAPK inhibitor
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inhibitor significantly and dose-dependently reduced the levels of Ca and ALP activities in HSMCs treated with Pi, but there was no change after adding ERK1/2 and JNK inhibitors (Figs. 2a, b, S2). On the other hand, inhibitors have no effect on the Ca levels and ALP activities in the absence of Pi (Fig. S3). To confirm the results obtained from the addition of p38 MAPK and PKA inhibitors, p38 MAPK and PKA siRNAs were transfected into HSMCs. Two siRNAs significantly decreased Ca levels and ALP activities (Fig. 2c, d). On the other hand, there were no significant changes in cell viability on adding siRNAs and three inhibitors (PKA, p38, MEK1/2, and JNK) (Fig. S1), meaning that significant reduction in the cell viability is caused mainly by the addition of Pi.
mineralization and ALP expression in calcifying vascular cells treated with Pi and a farnesoid X receptor agonist, 6aethyl chenodeoxycholic acid [20]. In the present study, JNK inhibition had no effect on the ALP activities and Ca levels in HSMCs treated with Pi alone. Thus, JNK inhibition may not reduce the Pi-induce calcification of vascular cells. In conclusion, inhibition of PKA and p38 MAPK by inhibitors and siRNAs efficiently decreased Ca levels and ALP activities in HSMCs treated with Pi. These results suggest that PKA and p38 MAPK are involved in the Piinduced calcification of HSMCs, and their inhibition may be an efficient way to reduce vascular calcification. Acknowledgments This work was financially supported by a grantin-aid for Scientific Research (B) (KAKENHI Grant Number 23310085) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
Discussion In the present study, the effect of PKA and MAPK signals (p38, ERK1/2, and JNK) on Pi-induced HSMC calcification was investigated. Previous studies have reported that inhibition of PKA reduces ALP activity and calcification in high P-treated SMCs [11, 12, 14]. Similarly, our study shows that inhibition of PKA by inhibitor or siRNA significantly reduces Ca levels and ALP activities in HSMCs treated with Pi (3 mM) (Fig. 2). Among MAPK signals, the p38 MAPK can regulate osteogenic differentiation and calcification of calcifying vascular cells [15]. Moreover, the glycation end products induce calcification of human aortic SMCs via p38 signal in the presence of b-GP, but show no stimulation of p38 signal in its absence [16]. These studies may mean that p38 MAPK participates in the calcification of vascular cells, but there are still very few data relating to its effect on the Piinduced HSMC calcification. Our study clearly shows that inhibition of p38 MAPK can reduce ALP activities and Ca levels in Pi-treated HSMCs. ERK can induce osteoblastic differentiation and calcification of vascular cells [16] but, on the contrary, several factors reduce their calcification through the ERK signal pathway, such as taurine [17], ghrelin [18], and apelin [19]. Moreover, ERK inhibitor has no influence on the ALP activity and Ca level in b-GP-induced rat or mouse SMC calcification [17, 18] and in human calcifying vascular SMCs [19]. Our study also shows that addition of ERK1/2 inhibitor has no effect on the Pi-induced HSMC calcification. From these results, directly blocking activation of ERK1/2 by ERK1/2 inhibitors may not inhibit Pi- or b-GPinduced SMC calcification. Furthermore, there are few data relating to the effect of JNK on Pi-induced SMC calcification, but a recent study suggests that JNK inhibition can dose-dependently induce
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References 1. Jono S, Shioi A, Ikari Y, Nishizawa Y (2006) Vascular calcification in chronic kidney disease. J Bone Miner Metab 24:176–181 2. Ueda H, Harimoto K, Tomoyama S, Tamaru H, Miyawaki M, Mitsusada N, Yasuga Y, Hiraoka H (2012) Relation of cardiovascular risk factors and angina status to obstructive coronary artery disease according to categorical coronary artery calcium score. Heart Vessels 27:128–134 3. Farrag A, Bakhoum S, Salem MA, El-Faramawy A, Gergis E (2013) The association between extracoronary calcification and coronary artery disease in patients with type 2 diabetes mellitus. Heart Vessels 28:12–18 4. Tintut Y, Patel J, Parhami F, Demer LL (2000) Tumor necrosis factor-a promotes in vitro calcification of vascular cells via the cAMP pathway. Circulation 102:2636–2642 5. Amann K (2008) Media calcification and intima calcification are distinct entities in chronic kidney disease. Clin J Am Soc Nephrol 3:1599–1605 6. Jono S, McKee MD, Murry CE, Shioi A, Nishizawa Y, Mori K, Morii H, Giachelli CM (2000) Phosphate regulation of vascular smooth muscle cell calcification. Circ Res 87:e10–e17 7. Steitz SA, Speer MY, Curinga G, Yang HY, Haynes P, Aebersold R, Schinke T, Karsenty G, Giachelli CM (2001) Smooth muscle cell phenotype transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res 89:1147–1154 8. Giachelli CM, Speer MY, Li X, Rajachar RM, Yang H (2005) Regulation of vascular calcification: roles of phosphate and osteopontin. Circ Res 96:717–722 9. Zarjou A, Jeney V, Arosio P, Poli M, Antal-Szalma´s P, Agarwal A, Balla G, Balla J (2009) Ferritin prevents calcification and osteoblastic differentiation of vascular smooth muscle cells. J Am Soc Nephrol 20:1254–1263 10. Lomashvili KA, Cobbs S, Hennigar RA, Hardcastle KI, O’Neill C (2004) Phosphate-induced vascular calcification: role of pyrophosphate and osteopontin. J Am Soc Nephrol 15:1392–1401 11. Huang MS, Sage AP, Lu J, Demer LL, Tintut Y (2008) Phosphate and pyrophosphate mediated PKA-induced vascular cell calcification. Biochem Biophys Res Commun 374:553–558
Heart Vessels 12. Prosdocimo DA, Wyler SC, Romani AM, O’Neill WC, Dubyak GR (2010) Regulation of vascular smooth muscle cell calcification by extracellular pyrophosphate homeostasis: synergistic modulation by cyclic AMP and hyperphosphatemia. Am J Physiol Cell Physiol 298:C702–C713 13. Simmons CA, Nikolovski J, Thornton AJ, Matlis S, Mooney DJ (2004) Mechanical stimulation and mitogen-activated protein kinase signaling independently regulate osteogenic differentiation and mineralization by calcifying vascular cells. J Biomech 37:1531–1541 14. Hsu JJ, Lu J, Huang MS, Geng Y, Sage AP, Bradley MN, Tontonoz P, Demer LL, Tintut Y (2009) T0901317, an LXR agonist, augments PKA-induced vascular cell calcification. FEBS Lett 583:1344–1348 15. Tanikawa T, Okada Y, Tanikawa R, Tanaka Y (2009) Advanced glycation end products induce calcification of vascular smooth muscle cells through RAGE/p38 MAPK. J Vasc Res 46:572–580 16. Speer MY, Yang HY, Brabb T, Leaf E, Look A, Lin WL, Frutkin A, Dichek D, Giachelli CM (2009) Smooth muscle cells give rise
17.
18.
19.
20.
to osteochondrogenic precursors and chondrocytes in calcifying arteries. Cir Res 104:733–741 Liao XB, Zhou XM, Li JM, Yang JF, Tan ZP, Hu ZW, Liu W, Lu Y, Yuan LQ (2008) Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway. Amino Acids 34:525–530 Liang QH, Jiang Y, Zhu X, Cui RR, Liu GY, Liu Y, Wu SS, Liao ZB, Xie H, Zhou HD, Wu XP, Yuan LQ, Liao EY (2012) Ghrelin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the ERK pathway. PLoS One 7:e33126 Shan PF, Lu Y, Cui RR, Jiang Y, Yuan LQ, Liao EY (2011) Apelin attenuates the osteoblastic differentiation of vascular smooth muscle cells. PLoS One 6:e17938 Miyazaki-Anzai S, Levi M, Kratzer A, Ting TC, Lewis LB, Miyazaki M (2010) Farnesoid X receptor activation prevents the development of vascular calcification in ApoE-/- mice with chronic kidney disease. Circ Res 106:1807–1817
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