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Takeshi Iwata - One of the best experts on this subject based on the ideXlab platform.
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HTRA1 high temperature requirement a serine peptidase 1 gene is transcriptionally regulated by insertion deletion nucleotides located at the 3 end of the arms2 age related maculopathy susceptibility 2 gene in patients with age related macular degeneration
Journal of Biological Chemistry, 2015Co-Authors: Daisuke Iejima, Chio Oka, Takeshi Itabashi, Yuich Kawamura, Toru Noda, Shinsuke Yuasa, Keiichi Fukuda, Takeshi IwataAbstract:Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, whereas Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 (age-related maculopathy susceptibility 2) and HTRA1 (high temperature requirement A serine peptidase 1) genes. Transcription regulator activity was localized in mouse retinas using heterozygous HTRA1 knock-out mice in which HTRA1 exon 1 was replaced with β-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein that bound the insertion/deletion sequence, LYRIC (lysine-rich CEACAM1 co-isolated) protein. In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant up-regulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained up-regulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HTRA1 and exhibits characteristics similar to those of wet AMD patients. Background: The biological function of insertion/deletion sequences associated with AMD has not been fully characterized. Results: The HTRA1 regulatory region contains an insertion/deletion sequence that is significantly up-regulated in retinal neuronal cell lines. Conclusion: HTRA1 expression is enhanced by a mutation in the insertion/deletion in the HTRA1 regulatory region. Significance: This is the characterization of the HTRA1 regulatory elements and the effect of insertion/deletion sequences associated with AMD.
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HTRA1 high temperature requirement a serine peptidase 1 gene is transcriptionally regulated by insertion deletion nucleotides located at the 3 end of the arms2 age related maculopathy susceptibility 2 gene in patients with age related macular degeneration
Journal of Biological Chemistry, 2015Co-Authors: Daisuke Iejima, Takeshi Itabashi, Yuich Kawamura, Toru Noda, Shinsuke Yuasa, Keiichi Fukuda, Takeshi IwataAbstract:Abstract Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, while Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 and HTRA1 genes. Transcription regulator activity was localized in mouse retinas using heterozygous HTRA1 knockout mice in which HTRA1 exon 1 was replaced with beta-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines, but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein which bound the insertion/deletion sequence, lysine-rich, CEACAM1 co-isolated protein (LYRIC). In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant upregulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained upregulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HTRA1 and exhibits characteristics similar to those of wet AMD patients.
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overexpression of HTRA1 and exposure to mainstream cigarette smoke leads to choroidal neovascularization and subretinal deposits in aged mice
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Mao Nakayama, Daisuke Iejima, Masakazu Akahori, Junzo Kamei, Asako Goto, Takeshi IwataAbstract:PURPOSE We determined the function of ARMS2 and HTRA1 in the choroid and retina using transgenic (Tg) mice and evaluated the effects of mainstream cigarette smoke on these mice. METHODS The chicken actin promoter (CAG) was used to drive mouse HTRA1, human ARMS2, and ARMS2 (A69S) expression in the entire body of a mouse for one year. Fundus observations were performed with a Spectralis HRA+ optical coherence tomograph (OCT). Eyes were sectioned, stained with hematoxylin and eosin (H&E), and analyzed with immunohistochemistry. Mice were exposed to cigarette smoke for 30 min/d, 5 d/wk for 12 weeks using a mainstream smoking chamber (INH06-CIGR02A, MIPS). After 12 weeks, fundus observations and pathological analyses were performed. RESULTS Approximately 18.2% of 12-month-old HTRA1 Tg mice exhibited choroidal neovascularization (CNV) by OCT and positive immunostaining with anti-CD31 and anti-fibronectin antibodies. Furthermore, elastic van Gieson (EVG) staining showed Bruch's membrane damage in HTRA1 Tg mice. No retinal changes were observed in ARMS2 and ARMS2 (A69S) Tg mice. A total of 12 weeks of exposure to mainstream cigarette smoke led to CNV rates of 7.7% for wild type (Wt) mice and 20% for HTRA1 Tg mice, but had no effect on ARMS2 Tg mice. In addition, abnormal deposits were observed between photoreceptor cells and the RPE in an HTRA1 Tg mouse exposed to mainstream cigarette smoke. CONCLUSIONS The HTRA1 overexpression and mainstream cigarette smoke can independently lead to CNV. The HTRA1 gene is a strong risk factor for wet AMD, but not all of the HTRA1 Tg mice developed CNV, suggesting that CNV development depends on multiple risk factors.
Michael Ehrmann - One of the best experts on this subject based on the ideXlab platform.
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cerebral small vessel disease related protease HTRA1 processes latent tgf β binding protein 1 and facilitates tgf β signaling
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Nathalie Beaufort, Eva Scharrer, Michael Ehrmann, Robert Huber, Elisabeth Kremmer, Henry Houlden, David J Werring, Christof Haffner, Martin DichgansAbstract:High temperature requirement protein A1 (HTRA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor β (TGF-β) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-β signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HTRA1 in TGF-β pathway activation. We identify latent TGF-β binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-β bioavailability, as a novel HTRA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HTRA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-β signaling caused by a lack of HTRA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis.
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detrimental role for human high temperature requirement serine protease a1 HTRA1 in the pathogenesis of intervertebral disc ivd degeneration
Journal of Biological Chemistry, 2012Co-Authors: Andre N Tiaden, Marina Klawitter, Vanda Lux, Ali Mirsaidi, Gregor Bahrenberg, Stephan Glanz, Lilian Quero, Thomas Liebscher, Karin Wuertz, Michael EhrmannAbstract:Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.
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htra proteases regulated proteolysis in protein quality control
Nature Reviews Molecular Cell Biology, 2011Co-Authors: Tim Clausen, Markus Kaiser, Robert Huber, Michael EhrmannAbstract:HTRA proteases perform a variety of protein quality control functions that are of key importance to cell fate. This Review discusses the emerging physiological implications and unique architectural and mechanistic features of bacterial, plant and mammalian HTRAs.
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Substrate-induced remodeling of the active site regulates human HTRA1 activity.
Nature structural & molecular biology, 2011Co-Authors: Linda Truebestein, Markus Kaiser, Tim Clausen, Annette Tennstaedt, Timon Mönig, T. Krojer, Flavia Canellas, Michael EhrmannAbstract:Crystal structures of active and inactive conformations of the human serine protease HTRA1 reveal that substrate binding to the active site is sufficient to stimulate proteolytic activity. HTRA1 attaches to liposomes, digests misfolded proteins into defined fragments and undergoes substrate-mediated oligomer conversion. In contrast to those of other serine proteases, the PDZ domain of HTRA1 is dispensable for activation or lipid attachment, indicative of different underlying mechanistic features.
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serine protease HTRA1 modulates chemotherapy induced cytotoxicity
Journal of Clinical Investigation, 2006Co-Authors: Jeremy Chien, Alfonso Baldi, V Catalano, Pietro Muretto, Gary L Keeney, Kimberly R Kalli, Julie Staub, Giovanni Aletti, Michael Ehrmann, William Arthur ClibyAbstract:Resistance to chemotherapy presents a serious challenge in the successful treatment of various cancers and is mainly responsible for mortality associated with disseminated cancers. Here we show that expression of HTRA1, which is frequently downregulated in ovarian cancer, influences tumor response to chemotherapy by modulating chemotherapy-induced cytotoxicity. Downregulation of HTRA1 attenuated cisplatin- and paclitaxel-induced cytotoxicity, while forced expression of HTRA1 enhanced cisplatin- and paclitaxel-induced cytotoxicity. HTRA1 expression was upregulated by both cisplatin and paclitaxel treatment. This upregulation resulted in limited autoproteolysis and activation of HTRA1. Active HTRA1 induces cell death in a serine protease–dependent manner. The potential role of HTRA1 as a predictive factor of clinical response to chemotherapy was assessed in both ovarian and gastric cancer patients receiving cisplatin-based regimens. Patients with ovarian or gastric tumors expressing higher levels of HTRA1 showed a higher response rate compared with those with lower levels of HTRA1 expression. These findings uncover what we believe to be a novel pathway by which serine protease HTRA1 mediates paclitaxel- and cisplatin-induced cytotoxicity and suggest that loss of HTRA1 in ovarian and gastric cancers may contribute to in vivo chemoresistance.
Chio Oka - One of the best experts on this subject based on the ideXlab platform.
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HTRA1 serine protease expression levels on age related macular degeneration amd patients in yogyakarta
BIO Web of Conferences, 2020Co-Authors: Supanji Supanji, Ayudha Bahana Ilham Perdamaian, Anindita Dianratri, Anditta Syifarahmah, Tri Wahyu Widayanti, Firman Setya Wardhana, Muhammad Bayu Sasongko, Mohammad Eko Prayogo, Angela Nurini Agni, Chio OkaAbstract:This research aims to investigate the HTRA1 serine protease circulating level of Age-related Macular Degeneration (AMD) patients in Yogyakarta, Indonesia. This study was conducted from January to August 2019 which included 38 AMD patients and 16 Non-AMD patients/controls (two groups). Baseline data and blood sample were collected. ELISA assay was used to measure the HTRA1 serine protease circulating level on both groups. SNP genotyping of rs10490924 was using restriction enzyme digestion. This study used The IBM SPSS® version 24 (Chicago, The USA) to determine the relationship between HTRA1 expression level and AMD incidence. AMD patients had higher HTRA1 serine protease level (35.31) than controls (30.08). However, there is no association found between HTRA1 serine protease level and AMD incidence (p-value>0.05, CI 95 %). However, HTRA1 serine protease did not associate positively to AMD incidence in Yogyakarta samples. Further analysis by grouping AMD patient based on the rs10490924 genotype show no statistical correlation between HTRA1 to the incidence of AMD. This result might be due to the lack of samples in the study groups. Future studies with larger number of samples are advised to better see the association between HTRA1 serine protease level and AMD incidence.
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high temperature requirement a1 protease as a rate limiting factor in the development of osteoarthritis
American Journal of Pathology, 2019Co-Authors: Peter H Chen, Tian Tang, Chenlu Liu, Beiyu Wang, Michelle Mian, Chio Oka, Maria BaquerizoAbstract:Preserving the mature articular cartilage of joints is a critical focus in the prevention and treatment of osteoarthritis. We determined whether the genetic inactivation of high-temperature requirement A1 (HTRA1) can significantly attenuate the degradation of articular or condylar cartilage. Two types of mouse models of osteoarthritis were used, a spontaneous mutant mouse model [type XI collagen-haploinsufficient (Col11a1+/-) mice] and two post-traumatic mouse models [destabilization of the medial meniscus (DMM) on the knee and a partial discectomy (PDE) on the temporomandibular joint]. Three different groups of mice were generated: i) HTRA1 was genetically deleted from Col11a1+/- mice (HTRA1-/-;Col11a1+/-), ii) HTRA1-deficient mice (HTRA1-/-) were subjected to DMM, and iii) HTRA1-/- mice were subjected to PDE. Knee and temporomandibular joints from the mice were characterized for evidence of cartilage degeneration. The degradation of articular or condylar cartilage was significantly delayed in HTRA1-/-;Col11a1+/- mice and HTRA1-/- mice after DMM or PDE. The amount of collagen type VI was significantly higher in the articular cartilage in HTRA1-/-;Col11a1+/- mice, compared with that in Col11a1+/- mice. The genetic removal of HTRA1 may delay the degradation of articular or condylar cartilage in mice.
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Loss of HTRA1 serine protease induces synthetic modulation of aortic vascular smooth muscle cells
2018Co-Authors: Muthi Ikawati, Masashi Kawaichi, Chio OkaAbstract:Homozygous mutations of human HTRA1 cause cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). HTRA1-/- mice were examined for arterial abnormalities. Although their cerebral arteries were normal, the thoracic aorta was affected in HTRA1-/- mice. The number of vascular smooth muscle cells (VSMCs) in the aorta was increased in HTRA1-/- mice of 40 weeks or younger, but decreased thereafter. The cross-sectional area of the aorta was increased in HTRA1-/- mice of 40 weeks or older. Aortic VSMCs isolated from HTRA1-/- mice rapidly proliferated and migrated, produced high MMP9 activity, and were prone to oxidative stress-induced cell death. HTRA1-/- VSMCs expressed less smooth muscle α-actin, and more vimentin and osteopontin, and responded to PDGF-BB more strongly than wild type VSMCs, indicating that HTRA1-/- VSMCs were in the synthetic phenotype. The elastic lamina was disrupted, and collagens were decreased in the aortic media. Calponin in the media was decreased, whereas vimentin and osteopontin were increased, suggesting a synthetic shift of VSMCs in vivo. Loss of HTRA1 therefore skews VSMCs toward the synthetic phenotype, induces MMP9 expression, and expedites cell death. We propose that the synthetic modulation is the primary event that leads to the vascular abnormalities caused by HTRA1 deficiency.
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Phenotypic characterization of isolated HTRA1-/- mouse VSMCs.
2018Co-Authors: Muthi Ikawati, Masashi Kawaichi, Chio OkaAbstract:(A) Rapid proliferation of HTRA1-/- VSMCs. HTRA1-/- (KO) and wild type (WT) VSMCs were cultured in medium containing 10%, 5%, or 0% FBS as indicated. Cell proliferation was assayed at the indicated time points (0 h was set at 15 h after plating). Points in the graphs represent means ± SD (n = 3–4). The experiments were carried out independently for three different batches of WT and HTRA1-/- VSMCs at the matching passage. The data shown are a representative result from cells at passage 7. (B-C) Rapid migration of HTRA1-/- VSMCs. (B) Cell migration was analyzed by the wound-healing assay. Cells were cultured in medium containing 10%, 5%, or 0% FBS as indicated. Photographs were taken at the indicated time points. White dotted lines indicate boundaries of the initial wounded area. The experiment was repeated three times and representative results are shown. Cells at passage 13 were used. (C) Cell migration was analyzed by a modified Boyden chamber assay. Cells were cultured in the chamber in medium containing 10% FBS for 24 h. The number of cells that migrated through the membrane was counted after DAPI staining. The bars are means ± SD (n = 3). The experiment was repeated twice times and representative results are shown. Cells at passage 12 were used. (D-E) Production of MMPs by isolated HTRA1-/- mouse VSMCs. (D) Increased MMP9 activity in the culture media of HTRA1-/- VSMCs. WT and HTRA1-/- VSMCs were cultured in medium containing 0.5% FBS. After 24 h, the culture medium was recovered. The cells were also harvested, and cell lysates were analyzed by Western blot for tubulin content. The media, whose volumes were normalized by tubulin, were loaded onto a zymography gel. Three different batches of WT and HTRA1-/- VSMCs at the matching passage (passage 12, P12 or 14, P14) were analyzed. The zymogram was analyzed by densitometer and the results are presented in the bar graph on the right. MMP9 activity of WT VSMCs was set to 1, and the relative MMP9 activity of HTRA1-/- VSMCs is shown as mean ± SD. (E) MMP mRNA expression in WT and HTRA1-/- VSMCs. RNA was extracted from cells that were harvested as described in D. MMP2 and MMP9 mRNA were measured by quantitative RT-PCR and normalized with GAPDH. The relative expression levels in HTRA1-/- VSMCs were calculated using the levels of MMP2 and MMP9 mRNA in WT VSMCs as 1. Values represent mean ± SD (n = 3). Cells at passage 10 were used. Statistical significance in A, C, D, and E was determined by Student’s t-test. *; p
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Effects of PDGF-BB or IGF-1 on proliferation, migration, and MMP activities of wild type and HTRA1-/- mouse VSMCs.
2018Co-Authors: Muthi Ikawati, Masashi Kawaichi, Chio OkaAbstract:(A) Effects of PDGF-BB or IGF-1 on VSMC proliferation. Wild type (WT) and HTRA1-/- (KO) VSMCs were cultured in medium containing 0.5% FBS with or without 20 ng/ml PDGF-BB or 10 ng/ml IGF-1, as indicated. Cell proliferation was assayed at the indicated time points (0 h was set at 15 h after plating). Points represent means ± SD (n = 3–4). The experiments were carried out independently for three different batches of WT and HTRA1-/- VSMCs at the matching passage. The data shown are a representative result from cells at passage 7. Statistical significance was determined by Student’s t-test. *; p
Daisuke Iejima - One of the best experts on this subject based on the ideXlab platform.
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HTRA1 high temperature requirement a serine peptidase 1 gene is transcriptionally regulated by insertion deletion nucleotides located at the 3 end of the arms2 age related maculopathy susceptibility 2 gene in patients with age related macular degeneration
Journal of Biological Chemistry, 2015Co-Authors: Daisuke Iejima, Chio Oka, Takeshi Itabashi, Yuich Kawamura, Toru Noda, Shinsuke Yuasa, Keiichi Fukuda, Takeshi IwataAbstract:Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, whereas Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 (age-related maculopathy susceptibility 2) and HTRA1 (high temperature requirement A serine peptidase 1) genes. Transcription regulator activity was localized in mouse retinas using heterozygous HTRA1 knock-out mice in which HTRA1 exon 1 was replaced with β-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein that bound the insertion/deletion sequence, LYRIC (lysine-rich CEACAM1 co-isolated) protein. In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant up-regulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained up-regulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HTRA1 and exhibits characteristics similar to those of wet AMD patients. Background: The biological function of insertion/deletion sequences associated with AMD has not been fully characterized. Results: The HTRA1 regulatory region contains an insertion/deletion sequence that is significantly up-regulated in retinal neuronal cell lines. Conclusion: HTRA1 expression is enhanced by a mutation in the insertion/deletion in the HTRA1 regulatory region. Significance: This is the characterization of the HTRA1 regulatory elements and the effect of insertion/deletion sequences associated with AMD.
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HTRA1 high temperature requirement a serine peptidase 1 gene is transcriptionally regulated by insertion deletion nucleotides located at the 3 end of the arms2 age related maculopathy susceptibility 2 gene in patients with age related macular degeneration
Journal of Biological Chemistry, 2015Co-Authors: Daisuke Iejima, Takeshi Itabashi, Yuich Kawamura, Toru Noda, Shinsuke Yuasa, Keiichi Fukuda, Takeshi IwataAbstract:Abstract Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, while Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 and HTRA1 genes. Transcription regulator activity was localized in mouse retinas using heterozygous HTRA1 knockout mice in which HTRA1 exon 1 was replaced with beta-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines, but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein which bound the insertion/deletion sequence, lysine-rich, CEACAM1 co-isolated protein (LYRIC). In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant upregulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained upregulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HTRA1 and exhibits characteristics similar to those of wet AMD patients.
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overexpression of HTRA1 and exposure to mainstream cigarette smoke leads to choroidal neovascularization and subretinal deposits in aged mice
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Mao Nakayama, Daisuke Iejima, Masakazu Akahori, Junzo Kamei, Asako Goto, Takeshi IwataAbstract:PURPOSE We determined the function of ARMS2 and HTRA1 in the choroid and retina using transgenic (Tg) mice and evaluated the effects of mainstream cigarette smoke on these mice. METHODS The chicken actin promoter (CAG) was used to drive mouse HTRA1, human ARMS2, and ARMS2 (A69S) expression in the entire body of a mouse for one year. Fundus observations were performed with a Spectralis HRA+ optical coherence tomograph (OCT). Eyes were sectioned, stained with hematoxylin and eosin (H&E), and analyzed with immunohistochemistry. Mice were exposed to cigarette smoke for 30 min/d, 5 d/wk for 12 weeks using a mainstream smoking chamber (INH06-CIGR02A, MIPS). After 12 weeks, fundus observations and pathological analyses were performed. RESULTS Approximately 18.2% of 12-month-old HTRA1 Tg mice exhibited choroidal neovascularization (CNV) by OCT and positive immunostaining with anti-CD31 and anti-fibronectin antibodies. Furthermore, elastic van Gieson (EVG) staining showed Bruch's membrane damage in HTRA1 Tg mice. No retinal changes were observed in ARMS2 and ARMS2 (A69S) Tg mice. A total of 12 weeks of exposure to mainstream cigarette smoke led to CNV rates of 7.7% for wild type (Wt) mice and 20% for HTRA1 Tg mice, but had no effect on ARMS2 Tg mice. In addition, abnormal deposits were observed between photoreceptor cells and the RPE in an HTRA1 Tg mouse exposed to mainstream cigarette smoke. CONCLUSIONS The HTRA1 overexpression and mainstream cigarette smoke can independently lead to CNV. The HTRA1 gene is a strong risk factor for wet AMD, but not all of the HTRA1 Tg mice developed CNV, suggesting that CNV development depends on multiple risk factors.
Emad S Alnemri - One of the best experts on this subject based on the ideXlab platform.
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inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease omi htra2
Journal of Biological Chemistry, 2003Co-Authors: Srinivasa M Srinivasula, Zhijia Zhang, Sanjeev Gupta, Pinaki Datta, Ramesh Hegde, Naeun Cheong, Teresa Fernandesalnemri, Emad S AlnemriAbstract:Abstract The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays demonstrated that Omi/HtrA2 could degrade inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, our data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis.
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structural insights into the pro apoptotic function of mitochondrial serine protease htra2 omi
Nature Structural & Molecular Biology, 2002Co-Authors: Wenyu Li, Jijie Chai, Pingwei Li, Jiawei Wu, Zhijia Zhang, Emad S AlnemriAbstract:HtrA2/Omi, a mitochondrial serine protease in mammals, is important in programmed cell death. However, the underlining mechanism of HtrA2/Omi-mediated apoptosis remains unclear. Analogous to the bacterial homolog HtrA (DegP), the mature HtrA2 protein contains a central serine protease domain and a C-terminal PDZ domain. The 2.0 A crystal structure of HtrA2/Omi reveals the formation of a pyramid-shaped homotrimer mediated exclusively by the serine protease domains. The peptide-binding pocket of the PDZ domain is buried in the intimate interface between the PDZ and the protease domains. Mutational analysis reveals that the monomeric HtrA2/Omi mutants are unable to induce cell death and are deficient in protease activity. The PDZ domain modulates HtrA2/Omi-mediated cell death activity by regulating its serine protease activity. These structural and biochemical observations provide an important framework for deciphering the mechanisms of HtrA2/Omi-mediated apoptosis.