A calpain inhibitor protects against fractalkine production in lipopolysaccharide-treated endothelial cells

( Jaewoong Jang ),( Yoosik Yoon ),( Dong-jin Oh ) 대한신장학회 2017 Kidney Research and Clinical Practice Vol.36 No.3

Background: Fractalkine (CX3CL1) is a chemokine with a unique CX3C motif and is produced by endothelial cells stimulated with lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, interleukin (IL)-1, and interferon-γ. There have been several reports that the caspase/calpain system is activated in endotoxemia, which leads to cellular apoptosis and acute inflammatory processes. We aimed to determine the role of the caspase/calpain system in cell viability and regulation of fractalkine production in LPS-treated endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were stimulated with 0.01-100 μg/mL of LPS to determine cell viability. The changes of CX3CL1 expression were compared in control, LPS (1 μg/mL)-, IL-1α (1 μg/ mL)-, and IL-1β (1 μg/mL)-treated HUVECs. Cell viability and CX3CL1 production were compared with 50 μM of inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. Results: Cell viability was significantly decreased from 1 to 100 μg/mL of LPS. Cell viability was significantly restored with inhibitors of caspase-1, caspase-3, caspase-9, and calpain in LPS-treated HUVECs. The expression of CX3CL1 was highest in IL-1β-treated HUVECs. CX3CL1 production was highly inhibited with a calpain inhibitor and significantly decreased with the individual inhibitors of caspase-1, caspase-3, and caspase-9. Conclusion: The caspase/calpain system is an important modulator of cell viability and CX3CL1 production in LPStreated endothelial cells.

Mutational analysis of <i>caspase 1</i>, <i>4</i>, and <i>5</i> genes in common human cancers

Soung, Young Hwa,Jeong, Eun Goo,Ahn, Chang Hyeok,Kim, Sung Soo,Song, Sang Yong,Yoo, Nam Jin,Lee, Sug Hyung Elsevier 2008 Human pathology Vol.39 No.6

<P><B>Summary</B></P><P>Mounting evidence indicates that deregulation of apoptosis is involved in the mechanisms of cancer development. Mutations of genes encoding caspases, the executioners of apoptosis, have been detected in human cancers, indicating inactivation of apoptosis by the mutations of <I>caspase</I> is an important mechanism in cancer development. The aim of this study was to see whether genes encoding human <I>caspases 1</I>, <I>4</I>, and <I>5</I> are mutated in human cancers. We analyzed the entire coding region and all splice sites of human <I>caspase 1</I>, <I>4</I>, and <I>5</I> genes for the detection of somatic mutations in 337 human cancers, including 103 colorectal, 54 gastric, 60 breast, 60 hepatocellular, and 60 lung carcinomas by a single-strand conformation polymorphism assay. We detected 2 (0.6%) <I>caspase-1,</I> 2 (0.6%) <I>caspase-4,</I> and 15 (4.4%) <I>caspase-5</I> mutations in the 343 cancers. The mutations were detected in 11 gastric carcinomas (2 <I>caspase-1</I> and 9 <I>caspase-5</I> mutations), 6 colorectal carcinomas (2 <I>caspase-4</I> and 4 <I>caspase-5</I> mutations), 1 breast carcinoma (1 <I>caspase-5</I> mutation), and 1 lung carcinoma (1 <I>caspase-5</I> mutation). The mutations consisted of 11 mutations in exons and 8 mutations in noncoding sequences. The 11 mutations in the exons consisted of 3 missense, 1 silent, and 7 frameshift mutation(s). Of note, most (6/9) of the <I>caspase-5</I> mutations in the coding sequences were detected in microsatellite instability (MSI)-positive cancers. These data indicate that somatic mutations of <I>caspase-1</I> and <I>caspase-4</I> genes are rare in common solid cancers. In addition, the data indicate that <I>caspase-5</I> gene is commonly mutated in the MSI-positive cancers, and suggest that inactivation of <I>caspase-5</I> may play a role in the tumorigenesis of MSI-positive cancers.</P>

Cathepsin B Is Implicated in Triglyceride (TG)-Induced Cell Death of Macrophage

정병출,임재원,김성훈,김윤석,Jung, Byung Chul,Lim, Jaewon,Kim, Sung Hoon,Kim, Yoon Suk Korean Society for Clinical Laboratory Science 2020 대한임상검사과학회지(KJCLS) Vol.52 No.3

Macrophage cell death contributes to the formation of plaque, leading to the development of atherosclerosis. The accumulation of triglyceride (TG) is also associated with the pathogenesis of atherosclerosis. A previous study reported that TG induces the cell death of macrophages. This study examined whether the cytoplasmic release of cathepsin B from lysosome is associated with the TG-induced cell death of macrophage. The release of cathepsin B was increased in the TG-treated THP-1 macrophages, but the TG treatment did not affect cathepsin B expression. Furthermore, the inhibition of cathepsin B by its inhibitor, CA-074 Me, partially inhibited the TG-induced cell death of macrophage. TG-triggered macrophage cell death is mediated by the activation of caspase-1, -2, and apoptotic caspases. Therefore, this study investigated whether cathepsin B is implicated in the activation of these caspases. The inhibition of cathepsin B blocked the activation of caspase-7, -8, and -1 but did not affect the activity of caspase-3, -9, and -2. Overall, these results suggest that TG-induced cytoplasmic cathepsin B causes THP-1 macrophage cell death by activating caspase-1, leading to subsequent activation of the extrinsic apoptotic pathway. 대식세포사멸은 죽상판 형성에 영향을 미쳐 죽상동맥경화증 발병에 관여하는 것으로 알려져 있다. 중성지방 역시 죽상동맥경화 발병에 기여한다고 알려져 있는데 최근 본 연구팀에서는 중성지방이 대식세포사멸을 유발한다는 결과를 확인하였다. 본 연구에서는 cathepsin B가 중성지방에 의해 유발되는 대식세포사멸 과정에 관여하는지 확인하고자 연구를 진행하였다. THP-1 대식세포에 중성지방 처리 시 cathepsin B의 발현량에는 변화가 없고 리소좀에 있던 cathepsin B가 세포질로 방출되어 세포질의 cathepsin B가 증가한 것을 확인하였다. 다음으로 cathepsin B 억제제인 CA-074 Me를 처리 시 중성지방에 의해 유도되는 대식세포사멸이 일부 회복되는 것을 확인하였다. 본 연구팀의 이전 연구에서 중성지방에 의한 대식세포사멸이 caspase-1, -2 및 apoptotic caspase 활성화를 매개로 일어남을 확인하였기 때문에 본 연구에서는 이러한 caspase 활성 경로와 cathepsin B와의 연관성에 대해 연구하였다. cathepsin B 억제시 caspase-7, -8 및 -1의 활성은 억제되었으나, caspase-3, -9 및 -2는 활성에는 영향을 미치지 않음을 알 수 있었다. 정리하면, 중성지방에 의해 세포질로 방출된 cathepsin B는 caspase-1 활성화에 기여하고, 활성화된 caspase-1은 외인성 apoptotic caspase 경로를 활성화하여 THP-1 대식세포 사멸을 유발한다는 것을 알 수 있다.

Caspase-8의 양성 피드백 방식을 통한 중성지방-유도 THP-1 대식세포 사멸 증가

정병출 ( Byung Chul Jung ),임재원 ( Jaewon Lim ),김성훈 ( Sung Hoon Kim ),김윤석 ( Yoon Suk Kim ) 대한임상검사과학회 2021 대한임상검사과학회지(KJCLS) Vol.53 No.2

고중성지방혈증은 죽상동맥경화증의 주요한 위험 요인 중 하나이다. 중성지방은 대식세포의 세포 사멸을 유도하여 죽상동맥경화증 발생에 기여하는 것으로 알려져 있다. 본 연구팀은 앞선 연구에서 대식세포의 중성지방-유도 세포 사멸이 pannexin-1 활성화에 의한 세포 외 ATP 농도 증가, caspase-2와 caspase-1 활성화, caspase-8을 포함한 apoptotic caspase 활성화 경로로 일어나는 것을 보고하였다. 한편 다른 연구들에서는 세포 내 다른 여러 기전에서 caspase-8이 caspase-1과 -2의 상위 단백질이라 보고하고 있다. 따라서 본 연구에서는 caspase-8이 중성지방-유도 대식세포 사멸 과정에서 상위단백지로 영향을 미치는지 여부를 조사하기 위해 수행되었다. 본 연구진은 caspase-8이 중성지방-유도 대식세포 사멸 과정에서 caspase-3 활성화 및 PARP 절단을 유도하였다. 다음으로 중성지방이 처리된 대식세포에서 caspase-8 억제 시, caspase-8의 상위 단백질로 보고한 caspase-1 및 -2의 활성이 감소하는 것을 확인하였다. 또한 ATP 처리 시 caspase-8 억제제 처리에 의해 감소된 caspase-2의 활성이 회복되는 것을 확인하였다. 위의 결과를 통해 caspase-8이 중성지방-유도 대식세포 사멸 과정에서 세포 외부 ATP 농도 증가에 관여하는 단백질 또는 그 상위 기전에 양성피드백 방식으로 영향을 미쳐 caspase-1과 -2를 활성화하여 중성지방-유도 대식세포 사멸을 증진시킴을 알 수 있다. Hypertriglyceridemia is the main risk factor for atherosclerosis. It is reported that triglyceride (TG) induces macrophage cell death, and is involved in the formation of plaques and development of atherosclerosis. We previously reported that TG-induced cell death of macrophages is mediated via pannexin-1 activation, which increases the extracellular ATP and subsequent increase in potassium efflux, thereby activating the caspase-2/caspase-1/apoptotic caspases, including the caspase-8 pathway. Contrarily, some studies have reported that caspase-8 is an upstream molecule of caspase-1 and caspase-2 in several cellular processes. Therefore, this study was undertaken to investigate whether caspase-8 influences its upstream molecules in TG-stimulated macrophage cell death. We first confirmed that caspase-8 induces caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage in TG-treated macrophages. Next, we determined that the inhibition of caspase-8 results in reduced caspase-1 and -2 activity, which are upstream molecules of caspase-8 in TG-induced cell death of macrophages. We also found that ATP treatment restores the caspase-8 inhibitor-induced caspase-2 activity, thereby implying that caspase-8 affects the upstream molecules responsible for increasing the extracellular ATP levels in TG-induced macrophage cell death. Taken together, these findings indicate that caspase-8 potentiates the TG-induced macrophage cell death by activating its upstream molecules. Copyright Ⓒ 2021 The Korean Society for Clinical Laboratory Science. All rights reserved.

Caspase-1 Independent Viral Clearance and Adaptive Immunity Against Mucosal Respiratory Syncytial Virus Infection

신예리,이흥규 대한면역학회 2015 Immune Network Vol.15 No.2

Respiratory syncytial virus (RSV) infection is recognized by the innate immune system through Toll like receptors (TLRs) and retinoic acid inducible gene I. These pathways lead to the activation of type I interferons and resistance to infection. In contrast to TLRs, very few studies have examined the role of NOD-like receptors in viral recognition and induction of adaptive immune responses to RSV. Caspase-1 plays an essential role in the immune response via the maturation of the proinflammatory cytokines IL-1β and IL-18. However, the role of caspase-1 in RSV infection in vivo is unknown. We demonstrate that RSV infection induces IL-1β secretion and that caspase-1 deficiency in bone marrow derived dendritic cells leads to defective IL-1β production, while normal RSV viral clearance and T cell responses are observed in caspase- 1 deficient mice following respiratory infection with RSV. The frequencies of IFN-γ producing or RSV specific T cells in lungs from caspase-1 deficient mice are not impaired. In addition, we demonstrate that caspase-1 deficient neonatal or young mice also exhibit normal immune responses. Furthermore, we find that IL-1R deficient mice infected with RSV exhibit normal Th1 and cytotoxic T lymphocytes (CTL) immune responses. Collectively, these results demonstrate that in contrast to TLR pathways, caspase-1 might not play a central role in the induction of Th1 and CTL immune responses to RSV.

Caspase-2 mediates triglyceride (TG)-induced macrophage cell death

( Jaewon Lim ),( Hyun-kyung Kim ),( Sung Hoon Kim ),( Ki-jong Rhee ),( Yoon Suk Kim ) 생화학분자생물학회 2017 BMB Reports Vol.50 No.10

Triglyceride (TG) accumulation causes macrophage cell death, which affects the development of atherosclerosis. Here, we examined whether caspase-2 is implicated in TG-induced macrophage cell death. We found that caspase-2 activity is increased in TG-treated THP-1 macrophages, and that inhibition of caspase-2 activity drastically inhibits TG-induced cell death. We previously reported that TG-induced macrophage cell death is triggered by caspase-1, and thus investigated the relationship between caspase-2 and caspase-1 in TG-induced macrophage cell death. Inhibition of caspase-2 activity decreased caspase-1 activity in TG-treated macrophages. However, caspase-1 inhibition did not affect caspase-2 activity, suggesting that caspase-2 is upstream of caspase-1. Furthermore, we found that TG induces activation of caspase-3, -7, -8, and -9, as well as cleavage of PARP. Inhibition of caspase-2 and -1 decreased TG-induced caspase-3, -7, -8, and -9 activation and PARP cleavage. Taken together, these results suggest that TG-induced macrophage cell death is mediated via the caspase-2/caspase-1/apoptotic caspases/PARP pathways. [BMB Reports 2017; 50(10): 510-515]

Caspase-1 Level in Synovial Fluid Is High in Patients with Spondyloarthropathy but Not in Patients with Gout

손창남,방소영,김지혜,최찬범,김태환,전재범 대한의학회 2013 Journal of Korean medical science Vol.28 No.9

Activation of caspase-1 by NALP3 inflammasomes has been shown to be important in initiating acute gouty arthritis. The objectives of this study were to measure the levels of caspase-1 in synovial fluid in gout and various arthritides, and to elucidate the clinical significance of caspase-1 levels in synovial fluid. Caspase-1, IL-1β, IL-18, and uric acid were measured in synovial fluid from 112 patients with gout and other arthritides, such as rheumatoid arthritis, osteoarthritis, and spondyloarthropathy. Caspase-1 in synovial fluid from patients with crystal-induced arthritis, inflammatory arthritis, osteoarthritis, and spondyloarthropathy was 35.9 ± 86.7, 49.7 ± 107.7, 2.1 ± 7.0, and 152.6 ± 155.7 pg/mL, respectively. The mean level and the frequency of high levels (≥125 pg/mL) of caspase-1 in spondyloarthropathy were significantly higher than those in the other arthritides including gout. Caspase-1 was detectible in the synovial fluid of patients with the various arthritides. Contrary to our hypothesis, the caspase-1 level in the synovial fluid of patients with gout was not higher than in that of other arthritides. High levels of caspase-1 may be helpful in differentiating spondyloarthropathy from other arthritides.

Caspase-1 Independent Viral Clearance and Adaptive Immunity Against Mucosal Respiratory Syncytial Virus Infection

Shim, Ye Ri,Lee, Heung Kyu The Korean Association of Immunobiologists 2015 Immune Network Vol.15 No.2

Respiratory syncytial virus (RSV) infection is recognized by the innate immune system through Toll like receptors (TLRs) and retinoic acid inducible gene I. These pathways lead to the activation of type I interferons and resistance to infection. In contrast to TLRs, very few studies have examined the role of NOD-like receptors in viral recognition and induction of adaptive immune responses to RSV. Caspase-1 plays an essential role in the immune response via the maturation of the proinflammatory cytokines IL-$1{\beta}$ and IL-18. However, the role of caspase-1 in RSV infection in vivo is unknown. We demonstrate that RSV infection induces IL-$1{\beta}$ secretion and that caspase-1 deficiency in bone marrow derived dendritic cells leads to defective IL-$1{\beta}$ production, while normal RSV viral clearance and T cell responses are observed in caspase-1 deficient mice following respiratory infection with RSV. The frequencies of IFN-${\gamma}$ producing or RSV specific T cells in lungs from caspase-1 deficient mice are not impaired. In addition, we demonstrate that caspase-1 deficient neonatal or young mice also exhibit normal immune responses. Furthermore, we find that IL-1R deficient mice infected with RSV exhibit normal Th1 and cytotoxic T lymphocytes (CTL) immune responses. Collectively, these results demonstrate that in contrast to TLR pathways, caspase-1 might not play a central role in the induction of Th1 and CTL immune responses to RSV.

Salmonella Promotes ASC Oligomerization-dependent Caspase-1 Activation

황인화,김은희,유제욱,박상준,홍수정 대한면역학회 2012 Immune Network Vol.12 No.6

Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa)triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-1β) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably,inhibition of potassium efflux failed to block Salmonellapromoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome,intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium. Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa)triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-1β) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably,inhibition of potassium efflux failed to block Salmonellapromoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome,intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium.

Nitric Oxide로 유발된 아포토시스에 대한 Sek 1의 영향

오재민,박래길,소흥섭,문연자,백용아,최민규,박옥규,이호익,정연태 대한해부학회 1999 Anatomy & Cell Biology Vol.32 No.5