Article
Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process Hyun Sook Hwang 1,2 , Chung Mi Yang 1,2 , Su Jin Park 1,2 and Hyun Ah Kim 1,2, * Received: 7 October 2015; Accepted: 30 November 2015; Published: 8 December 2015 Academic Editor: Ali Mobasheri 1
2
*
Division of rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchon, Anyang, Kyunggi 431-070, Korea; [email protected] (H.S.H.); [email protected] (C.M.Y.); [email protected] (S.J.P.) Institute for Skeletal Aging, Hallym University, Chunchon 200-702, Korea Correspondence: [email protected]; Tel.: +82-31-380-1826; Fax: +82-31-381-8812
Abstract: Monosodium urate (MSU) crystals, which are highly precipitated in the joint cartilage, increase the production of cartilage-degrading enzymes and pro-inflammatory mediators in cartilage, thereby leading to gouty inflammation and joint damage. In this study, we investigated the effect of MSU crystals on the viability of human articular chondrocytes and the mechanism of MSU crystal-induced chondrocyte death. MSU crystals significantly decreased the viability of primary chondrocytes in a time- and dose-dependent manner. DNA fragmentation was observed in a culture medium of MSU crystal-treated chondrocytes, but not in cell lysates. MSU crystals did not activate caspase-3, a marker of apoptosis, compared with actinomycin D and TNF-α-treated cells. MSU crystals did not directly affect the expression of endoplasmic reticulum (ER) stress markers at the mRNA and protein levels. However, MSU crystals significantly increased the LC3-II level in a time-dependent manner, indicating autophagy activation. Moreover, MSU crystal-induced autophagy and subsequent chondrocyte death were significantly inhibited by 3-methyladenine, a blocker of autophagosomes formation. MSU crystals activated autophagy via inhibition of phosporylation of the Akt/mTOR signaling pathway. These results demonstrate that MSU crystals may cause the death of chondrocytes through the activation of the autophagic process rather than apoptosis or ER stress. Keywords: monosodium urate crystals; autophagy; osteoarthritis; chondrocyte; cartilage
1. Introduction The conversion of uric acid, a product of purine degradation, is mediated by uricase, which is lacking in primates, including humans. An increased level of uric acid in serum (>6.8 mg/dL) leads to the monosodium urate (MSU) crystallization and tissue deposition, resulting in acute arthritis and the formation of tophi [1,2]. MSU crystals deposited in the synovium and cartilage activate immune cells such as monocytes, polymorphonuclear cells, and lymphocytes to induce pro-inflammatory cytokines, including interleukin-6, interleukin-1β, and tumor necrosis factor (TNF)-α, leading to cartilage and bone destruction [2–5]. Aside from inflammation, MSU crystals have been reported to induce chondrocyte death in previous studies [6,7]. MSU crystals activate p38 mitogen-activated protein kinase through phosphorylation of proline-rich tyrosine kinase 2, focal adhesion kinase, paxillin, and their adaptor proteins, leading to nitric oxide production and matrix metalloproteinase-3 expression [8]. In addition, MSU crystals bind to toll-like receptor-2 (TLR-2), resulting in the formation of signaling complexes involving the myeloid differentiation primary response gene 88 (MyD88), Ras-related C3 botulinum toxin
Int. J. Mol. Sci. 2015, 16, 29265–29277; doi:10.3390/ijms161226164
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2015, 16, 29265–29277
substrate 1, and phosphoinositide 3-kinase (PI3K), consequently inducing nitric oxide generation within chondrocytes [9]. It has been postulated that MSU crystals drive chondrocyte death by hindering the nutrient Int. the J. Mol. Sci. 2015, 16, page–page supply to chondrocytes and increasing catabolic activity within the cartilage matrix [6]. However, the mechanism to explain such an association between MSU crystal deposition in cartilage and substrate 1, and phosphoinositide 3-kinase (PI3K), consequently inducing nitric oxide generation chondrocyte is not[9].clear. Autophagy is a cellular protective mechanism induced by a within death chondrocytes wide rangeItof such asthat metabolic stress, depletion, endoplasmic reticulum hasstimuli, been postulated MSU crystals driveoxygen chondrocyte death byand hindering the nutrient supply to the chondrocytes and increasing catabolic activity within the cartilage matrix [6]. (ER) stress [10–13]. Mammalian target of rapamycin (mTOR), a negative regulator of autophagy, However, the mechanism to explain such an association between MSU crystal deposition in cartilage can form a complex with Raptor (mTORC1) or Rictor (mTORC2). The function or regulatory and chondrocyte death is not clear. Autophagy is a cellular protective mechanism induced by a wide mechanism of mTOR is dependent upon the formation of a complex containing either Raptor range of stimuli, such as metabolic stress, oxygen depletion, and endoplasmic reticulum (ER) stress or Rictor. mTOR, activated by the PI3K/Akt pathway, inhibits the process of autophagy and [10–13]. Mammalian target of rapamycin (mTOR), a negative regulator of autophagy, can form a activates protein synthesis and ribosome biogenesis through phosphorylation S6 kinaseofand the complex with Raptor (mTORC1) or Rictor (mTORC2). The function or regulatoryof mechanism eIF-4E binding protein [14–16]. Several previous reports have shown that microtubule-associated mTOR is dependent upon the formation of a complex containing either Raptor or Rictor. mTOR, thechains PI3K/Akt pathway,an inhibits the process of autophagy and activates proteinsactivated 1A/1B by light 3 (LC3)-II, autophagy marker, is up-regulated in the protein chondrocytes synthesis and ribosome biogenesis through phosphorylation of S6 kinase and the eIF-4E binding of patients with osteoarthritis (OA) [17,18] and that rapamycin, an inhibitor of mTOR, suppresses protein [14–16]. Several previous reports have shown that microtubule-associated proteins 1A/1B glucocorticoid-stimulated chondrocyte death [19]. These findings indicate a relationship among light chains 3 (LC3)-II, an autophagy marker, is up-regulated in the chondrocytes of patients with autophagy, cartilage degeneration, death.an inhibitor of mTOR, suppresses osteoarthritis (OA) [17,18] and and chondrocyte that rapamycin, In the present study, we demonstrated the type of cell death mechanisms and signaling pathways glucocorticoid-stimulated chondrocyte death [19]. These findings indicate a relationship among autophagy, degeneration, and chondrocyte death. that contribute tocartilage MSU crystal-induced chondrocyte death and investigated whether MSU crystals In the present study, demonstrated thearticular type of cellchondrocytes. death mechanisms and signaling pathways induce autophagy-related cellwe death in human that contribute to MSU crystal-induced chondrocyte death and investigated whether MSU crystals induce autophagy-related cell death in human articular chondrocytes. 2. Results 2. Results 2.1. MSU (Monosodium Urate) Crystals Reduced the Viability of Articular Chondrocytes MSU (Monosodium Urate) Crystals Reducedon the chondrocyte Viability of Articular Chondrocytes To 2.1. investigate the effect of MSU crystals viability, articular chondrocytes were treated withTovarious concentrations (50–200 µg/mL) of MSUviability, crystalsarticular for 24 chondrocytes h or with MSU investigate the effect of MSU crystals on chondrocyte werecrystals treated with various concentrations (50–200 µg/mL) MSU crystals for 24 h or with MSU release crystals assays (200 µg/mL) for various time intervals (0–72 h). ofLactate dehydrogenase (LDH) for various time intervals (0–72 h). dehydrogenase release assays showed(200 thatµg/mL) MSU crystals significantly reduced theLactate viability of primary(LDH) chondrocytes from 50 to showed that MSU crystals significantly reduced the viability of primary chondrocytes from 50 to 200 200 µg/mL (Figure 1A) and negatively affected the viability of chondrocytes in a time-dependent µg/mL (Figure 1A) and negatively affected the viability of chondrocytes in a time-dependent mannermanner (Figure(Figure 1B). 1B).
Figure 1. MSU (monosodium urate) crystal-induced chondrocytes death. (A,B) The effect of MSU
Figure 1. MSU (monosodium urate) crystal-induced chondrocytes death. (A,B) The effect of crystals on the viability of primary chondrocytes. Primary human chondrocytes were treated (A) MSU crystals on the viability ofofprimary chondrocytes. Primary human chondrocytes were with various concentrations MSU crystals (50–200 µg/mL) for 24 h; or (B) with MSU crystals (200 treated (A) withµg/mL) various concentrations of MSU crystals (50–200 µg/mL) for 24 h; or (B) with MSU for 6, 14, 24, 48, and 72 h. Cell viability was measured with an LDH (lactate dehydrogenase)crystals assay. Data the48, mean ± SD experiments more than three (200 µg/mL) for represent 6, 14, 24, and 72 for h. duplicate Cell viability wasfrom measured with andifferent LDH (lactate donors. ** passay. < 0.01, Data *** p
Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process Hyun Sook Hwang 1,2 , Chung Mi Yang 1,2 , Su Jin Park 1,2 and Hyun Ah Kim 1,2, * Received: 7 October 2015; Accepted: 30 November 2015; Published: 8 December 2015 Academic Editor: Ali Mobasheri 1
2
*
Division of rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchon, Anyang, Kyunggi 431-070, Korea; [email protected] (H.S.H.); [email protected] (C.M.Y.); [email protected] (S.J.P.) Institute for Skeletal Aging, Hallym University, Chunchon 200-702, Korea Correspondence: [email protected]; Tel.: +82-31-380-1826; Fax: +82-31-381-8812
Abstract: Monosodium urate (MSU) crystals, which are highly precipitated in the joint cartilage, increase the production of cartilage-degrading enzymes and pro-inflammatory mediators in cartilage, thereby leading to gouty inflammation and joint damage. In this study, we investigated the effect of MSU crystals on the viability of human articular chondrocytes and the mechanism of MSU crystal-induced chondrocyte death. MSU crystals significantly decreased the viability of primary chondrocytes in a time- and dose-dependent manner. DNA fragmentation was observed in a culture medium of MSU crystal-treated chondrocytes, but not in cell lysates. MSU crystals did not activate caspase-3, a marker of apoptosis, compared with actinomycin D and TNF-α-treated cells. MSU crystals did not directly affect the expression of endoplasmic reticulum (ER) stress markers at the mRNA and protein levels. However, MSU crystals significantly increased the LC3-II level in a time-dependent manner, indicating autophagy activation. Moreover, MSU crystal-induced autophagy and subsequent chondrocyte death were significantly inhibited by 3-methyladenine, a blocker of autophagosomes formation. MSU crystals activated autophagy via inhibition of phosporylation of the Akt/mTOR signaling pathway. These results demonstrate that MSU crystals may cause the death of chondrocytes through the activation of the autophagic process rather than apoptosis or ER stress. Keywords: monosodium urate crystals; autophagy; osteoarthritis; chondrocyte; cartilage
1. Introduction The conversion of uric acid, a product of purine degradation, is mediated by uricase, which is lacking in primates, including humans. An increased level of uric acid in serum (>6.8 mg/dL) leads to the monosodium urate (MSU) crystallization and tissue deposition, resulting in acute arthritis and the formation of tophi [1,2]. MSU crystals deposited in the synovium and cartilage activate immune cells such as monocytes, polymorphonuclear cells, and lymphocytes to induce pro-inflammatory cytokines, including interleukin-6, interleukin-1β, and tumor necrosis factor (TNF)-α, leading to cartilage and bone destruction [2–5]. Aside from inflammation, MSU crystals have been reported to induce chondrocyte death in previous studies [6,7]. MSU crystals activate p38 mitogen-activated protein kinase through phosphorylation of proline-rich tyrosine kinase 2, focal adhesion kinase, paxillin, and their adaptor proteins, leading to nitric oxide production and matrix metalloproteinase-3 expression [8]. In addition, MSU crystals bind to toll-like receptor-2 (TLR-2), resulting in the formation of signaling complexes involving the myeloid differentiation primary response gene 88 (MyD88), Ras-related C3 botulinum toxin
Int. J. Mol. Sci. 2015, 16, 29265–29277; doi:10.3390/ijms161226164
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2015, 16, 29265–29277
substrate 1, and phosphoinositide 3-kinase (PI3K), consequently inducing nitric oxide generation within chondrocytes [9]. It has been postulated that MSU crystals drive chondrocyte death by hindering the nutrient Int. the J. Mol. Sci. 2015, 16, page–page supply to chondrocytes and increasing catabolic activity within the cartilage matrix [6]. However, the mechanism to explain such an association between MSU crystal deposition in cartilage and substrate 1, and phosphoinositide 3-kinase (PI3K), consequently inducing nitric oxide generation chondrocyte is not[9].clear. Autophagy is a cellular protective mechanism induced by a within death chondrocytes wide rangeItof such asthat metabolic stress, depletion, endoplasmic reticulum hasstimuli, been postulated MSU crystals driveoxygen chondrocyte death byand hindering the nutrient supply to the chondrocytes and increasing catabolic activity within the cartilage matrix [6]. (ER) stress [10–13]. Mammalian target of rapamycin (mTOR), a negative regulator of autophagy, However, the mechanism to explain such an association between MSU crystal deposition in cartilage can form a complex with Raptor (mTORC1) or Rictor (mTORC2). The function or regulatory and chondrocyte death is not clear. Autophagy is a cellular protective mechanism induced by a wide mechanism of mTOR is dependent upon the formation of a complex containing either Raptor range of stimuli, such as metabolic stress, oxygen depletion, and endoplasmic reticulum (ER) stress or Rictor. mTOR, activated by the PI3K/Akt pathway, inhibits the process of autophagy and [10–13]. Mammalian target of rapamycin (mTOR), a negative regulator of autophagy, can form a activates protein synthesis and ribosome biogenesis through phosphorylation S6 kinaseofand the complex with Raptor (mTORC1) or Rictor (mTORC2). The function or regulatoryof mechanism eIF-4E binding protein [14–16]. Several previous reports have shown that microtubule-associated mTOR is dependent upon the formation of a complex containing either Raptor or Rictor. mTOR, thechains PI3K/Akt pathway,an inhibits the process of autophagy and activates proteinsactivated 1A/1B by light 3 (LC3)-II, autophagy marker, is up-regulated in the protein chondrocytes synthesis and ribosome biogenesis through phosphorylation of S6 kinase and the eIF-4E binding of patients with osteoarthritis (OA) [17,18] and that rapamycin, an inhibitor of mTOR, suppresses protein [14–16]. Several previous reports have shown that microtubule-associated proteins 1A/1B glucocorticoid-stimulated chondrocyte death [19]. These findings indicate a relationship among light chains 3 (LC3)-II, an autophagy marker, is up-regulated in the chondrocytes of patients with autophagy, cartilage degeneration, death.an inhibitor of mTOR, suppresses osteoarthritis (OA) [17,18] and and chondrocyte that rapamycin, In the present study, we demonstrated the type of cell death mechanisms and signaling pathways glucocorticoid-stimulated chondrocyte death [19]. These findings indicate a relationship among autophagy, degeneration, and chondrocyte death. that contribute tocartilage MSU crystal-induced chondrocyte death and investigated whether MSU crystals In the present study, demonstrated thearticular type of cellchondrocytes. death mechanisms and signaling pathways induce autophagy-related cellwe death in human that contribute to MSU crystal-induced chondrocyte death and investigated whether MSU crystals induce autophagy-related cell death in human articular chondrocytes. 2. Results 2. Results 2.1. MSU (Monosodium Urate) Crystals Reduced the Viability of Articular Chondrocytes MSU (Monosodium Urate) Crystals Reducedon the chondrocyte Viability of Articular Chondrocytes To 2.1. investigate the effect of MSU crystals viability, articular chondrocytes were treated withTovarious concentrations (50–200 µg/mL) of MSUviability, crystalsarticular for 24 chondrocytes h or with MSU investigate the effect of MSU crystals on chondrocyte werecrystals treated with various concentrations (50–200 µg/mL) MSU crystals for 24 h or with MSU release crystals assays (200 µg/mL) for various time intervals (0–72 h). ofLactate dehydrogenase (LDH) for various time intervals (0–72 h). dehydrogenase release assays showed(200 thatµg/mL) MSU crystals significantly reduced theLactate viability of primary(LDH) chondrocytes from 50 to showed that MSU crystals significantly reduced the viability of primary chondrocytes from 50 to 200 200 µg/mL (Figure 1A) and negatively affected the viability of chondrocytes in a time-dependent µg/mL (Figure 1A) and negatively affected the viability of chondrocytes in a time-dependent mannermanner (Figure(Figure 1B). 1B).
Figure 1. MSU (monosodium urate) crystal-induced chondrocytes death. (A,B) The effect of MSU
Figure 1. MSU (monosodium urate) crystal-induced chondrocytes death. (A,B) The effect of crystals on the viability of primary chondrocytes. Primary human chondrocytes were treated (A) MSU crystals on the viability ofofprimary chondrocytes. Primary human chondrocytes were with various concentrations MSU crystals (50–200 µg/mL) for 24 h; or (B) with MSU crystals (200 treated (A) withµg/mL) various concentrations of MSU crystals (50–200 µg/mL) for 24 h; or (B) with MSU for 6, 14, 24, 48, and 72 h. Cell viability was measured with an LDH (lactate dehydrogenase)crystals assay. Data the48, mean ± SD experiments more than three (200 µg/mL) for represent 6, 14, 24, and 72 for h. duplicate Cell viability wasfrom measured with andifferent LDH (lactate donors. ** passay. < 0.01, Data *** p
