Arginase II down regulation recovers Ca /CaMKII-Akt-eNOS signaling and 2+ vascular homeostasis in p32 over expressed. Bon-hyeock Koo, Sungwoo Ryoo Department of Biology, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea BACKGROUND Methods ● Maintaining vascular homeostasis is important physiologically for vascular systems. ● Electron microscopy (EM) ● Intracellular Ca 2+ signaling between mitochondria and cytosol which is regulated by mitochondrial p32 ● Mitochondrial fractionation and their mechanism is important for vascular homeostasis. ●
Immunofluorescence staining and imaging ● Among the various function of p32, our previous research is found that p32 is possible to uptake from cytosol to mitochondria. In this study shown that localization of artificially expressed p32 is placed in ● Preparation of p32-expressing adenovirus mitochondria and ER and also has a Ca 2+ movement function. ● Confocal microscopy and flow cytometry ● p32 overexpression decrease CaMKII/Akt/eNOS signaling and disrupts vascular homeostasis via Ca 2+ ● Measurement of NO and ROS movement from cytosol to mitochondria and ER but arginase II down regulation
can alleviate their signaling and vascular homeostasis ● Aortic vascular tension assay AIM The target organelles of overexpressed p32 and determined whether the p32 involved in the regulation of cytosolic Ca 2+ was associated with Ca 2+ -dependent eNOS activation? RESULTS Cell count Cell count Fig 2. Over-expressed p32 has their function which is Ca 2+ uptake from cytosol to ER and mitochondria. Fig 1. p32 over expression through adenovirus increases p32 level in not only mitochondria but endoplasmic reticulum. Fig 4. Although p32 adenovirus increased p32 expression, arginase II
down-regulation activates CaMKII- Fig 3. Up-regulated p32 induces to decrease NO production and increase ROS generation by inactivating eNOS signaling by reducing p32 CaMKII-Akt-eNOS signaling axis, therefore disrupts vascular homeostasis. level. CONCLUSION REFERENCES ● p32 over expression decrease CaMKII/Akt/eNOS signaling and disrupts vascular Koo BH, et al, Arginase II activity regulates cytosolic Ca 2+ level in a homeostasis via Ca 2+ movement from cytosol to mitochondria and ER p32-dependent manner that contributes to Ca 2+ -dependent vasoconstriction in native low-density
lipoprotein-stimulated vascular smooth muscle cells. Exp Mol Med. 2019 Jun3 ● Arginase II down regulation can alleviate their signaling and vascular homeostasis Koo BH, et al. Arginase II Contributes to the Ca 2+ /CaMKII/eNOS ● Comprehensive relationship of arginase II and p32 will be help to find novel Axis by Regulating Ca 2+ Concentration Between the Cytosol and Mitochondria in a p32-Dependent Manner. J Am Heart Assoc. therapeutics in various vascular diseases. 2018 Sep 18 [H. Cell signaling-1] Arginase II down regulation recovers Ca2+/CaMKII-Akt-eNOS signaling and vascular homeostasis in
p32 over expressed Bon Hyeock Koo¹, Sung Woo Ryoo¹˙* ¹BIT medical-convergence, Kangwon national university, chun cheon 24341, Korea Maintaining vascular homeostasis is important physiologically for vascular systems. Many studies shown lots of methods to alleviates vascular dysfunction and have been studied until now. We focus on intracellular Ca2+ signaling between mitochondria and cytosol which is regulated by mitochondrial p32 and their mechanism is important for vascular homeostasis. p32 is known as HABP1, gC1qR, C1qbp which known to have a variety of functions and is predominantly placed
in mitochondria because p32 has mitochondrial targeting sequence contained in 73N- terminal amino acid. Especially among the p32 functions, our previous research is found that p32 is possible to uptake from cytosol to mitochondria. To find the exactly p32 function, we over expressed the p32 by transfection of p32 cDNA tagged flag in adenovirus and p32 dominantly expressed in not only mitochondria but also endoplasmic reticulum (ER). Over expressed p32 also has a Ca2+ uptake function from cytosol to mitochondria and ER which caused reduction of CaMKII/Akt/eNOS signaling axis and NO production
by decreasing the cytosolic Ca2+ level. However, our study found that arginase II down regulation induces to decrease over expressed p32 levels and then, reduced p32 level is activation of CaMKII/eNOS signaling again. p32-Dependent p38 MAPK Activation by Arginase II Downregulation Contributes to Endothelial Nitric Oxide Synthase Activation in HUVECs Bon-hyeock Koo, Sungwoo Ryoo Department of Biology, College of Natural Sciences, Kangwon National University, Chuncheon 200-701, Republic of Korea BACKGROUND Methods ● p38 MAPK is an enzyme that is activated upon inhibition of arginase II in
endothelial cells and contributes to eNOS activity ● High cholesterol diet 4 weeks ● Our previous studies revealed that p32, is Ca 2+ regulator between mitochondria and cytosol, could be ● Western blotting analysis regulated by arginase II activity. ● Mitochondrial fractionation ● In this study, we confirmed that mitochondrial p32 protein by inhibition of arginase II activity induces an increase in cytosolic Ca 2+ and an increase in NO production by inducing activation of CaMKII-Akt- ● Confocal microscopy and flow cytometry p38MAPK-eNOS signaling. ● Measurement of NO and ROS ● Thus, Repair of
vascular function by arginase II down-regulation may be induced through activation of ● Aortic vascular tension assay p38 MAPK and may occur through CaMKII-Akt-p38 MAPK-eNOS signaling pathway activity. AIM Therefore, arginase II regulated CaMKII-Akt-p38 MAPK-eNOS signaling pathway which This study investigated that arginase II regulated CaMKII-Akt-p38 MAPK-eNOS signaling pathway which is is important enzyme in vascular homeosis. important enzyme cascade to maintaining vascular homeostasis. RESULTS Figure 2. Arginase II down-regulation increased cytosolic Ca 2+ level, which phosphorylated p38
MAPK via activation of CaMKII Figure 4. p38 MAPK activation through arginase II down- regulation was regulated by Akt Figure 5. Arginase II down-regulation increased NO production and decrease ROS generation via eNOs activation, which was Figure 1. Arginase II down-regulation induced to activate eNOS through Figure 3. p32 regulated phosphorylation of p38 MAPK. mediated by p38 MAPK. p38 MAPK phosphorylation. Figure 8. Although ApoE -/- + HCD model could be repaired vascular function by down-regulating arginase II, inhibition of p38 MAPK disrupted endothelial dependent vasorelaxation by
decreasing NO production Figure 6. Arginase II down-regulation increased Ach dependent vasorelaxation and decrease PE-dependent vasoconstriction by activating p38 MAPK Figure 7. ApoE -/- + HCD model decreased activity of eNOS compared with WT and inhibition of p38 MAPK could reduce activation of eNOS. CONCLUSION REFERENCES ● Arginase II down-regulation increased cytosolic Ca 2+ concentration by decreasing activity of p32 which is Ca 2+ regulator between mitochondrial and cytosol. Koo BH et al. Arginase II inhibition prevents interleukin-8 production through regulation of p38 MAPK
phosphorylation activated by loss of mitochondrial membrane potential in nLDL-stimulated hAoSMCs. Exp Mol Med. 2018 Feb 2. ● Increased cytosolic Ca 2+ level induced endothelial dependent vasorelaxation by activating CaMKII-Akt-p38 MAPK-eNOS signaling cascade. Koo BH et al. Arginase II Contributes to the Ca 2+ /CaMKII/eNOS Axis by Regulating Ca 2+ Concentration Between the Cytosol and Mitochondria in a -/- ● Although ApoE + HCD model has reduced activity of CaMKII, Akt, p38 MAPK and eNOS p32-Dependent Manner. J Am Heart Assoc. 2018 Sep 18 compared WT, groups of down regulated arginase II
recovered vascular function. [H. Cell signaling-2] p32-Dependent p38 MAPK Activation by Arginase II Downregulation Contributes to Endothelial Nitric Oxide Synthase Activation in HUVECs Bon Hyeock Koo¹, Sung Woo Ryoo¹˙* ¹BIT medical-convergence, Kangwon national university, chun cheon 24341, Korea Arginase II reciprocally regulates eNOS through a p32-dependent Ca2+ control. We investigated the signaling pathway of arginase II-dependent eNOS phosphorylation. Western blot analysis was applied for examining protein activation and [Ca2+]c was analyzed by microscopic and FACS analyses. NO and ROS
productions were measured using specific fluorescent dyes under microscopy. NO signaling pathway was tested by measuring vascular tension. Following arginase II downregulation by chemical inhibition or gene knock out, increased eNOS phosphorylation at Ser1177 and decreased phosphorylation at Thr495 was depend on p38 MAPK activation, which induced by CaMKII activation through p32-dependent increase in [Ca2+]c. The protein amount of p32 negatively regulated p38 MAPK activation. p38 MAPK contributed to Akt-induced eNOS phosphorylation at Ser1177 that resulted in accelerated NO production and
reduced reactive oxygen species production in aortic endothelia. In vascular tension assay, p38 MAPK inhibitor decreased acetylcholine-induced vasorelaxation responses and increased phenylephrine- dependent vasoconstrictive responses. Here, we demonstrated a novel signaling pathway contributing to understanding of the relationship between arginase II, endothelial dysfunction, and atherogenesis. Hispidin, an extract of Phellinus Linteus, can alleviate endothelial dysfunction by inhibiting arginase Byeongjun Yoon and Sungwoo Ryoo Department of Biology, College of Natural Sciences, Kangwon
National University, Chuncheon 200-701, Republic of Korea ABSTRACTS INTRODUCTIONS ● Hispidin effectively inhibited arginase, which induced phosphorylation CaMKll ● Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO) by 2+ thr 286 and eNOS ser1177 residues in a Ca -dependent manner. using L-arginine as a substrate. ● The activity of eNOS induced by cytosolic Ca 2+ increased NO production, which ● The reduction of NO production due to decrease of eNOS activity is increased the endothelium-dependent relaxation acetylcholine (Ach) response, considered an indicator of almost all
cardiovascular diseases. but the contraction of phenylephrine (PE) was reduced. ● Arginase hydrolyzes L-arginine to L-ornithine, a precursor for spermine, ● In ApoE -/- mice fed HCD, an arteriosclerosis model, eNOS activity was which has two isoforms for arginase, arginase l in the cytosol, and decreased due to a decrease in cytosol Ca 2+ levels, which caused endothelial arginase ll in the mitochondria. dysfunction ● Intracellular Ca level are associated with the activity of Ca /calmodulin- 2+ 2+ -/- ● Treatment of hispidin in ApoE mice fed HCD restored cytosolic Ca 2+ levels and dependent
protein kinase ll (CaMK ll) and eNOS, and Arginase ll can increased vascular reactivity. regulate cytosolic Ca 2+ level. ● In conclusion, arginase inhibition with hispidin can increase eNOS activity in a ● Therefore, we investigated whether arginase inhibition with hispidin can Ca -dependent manner, which can alleviate endothelial dysfunction. alleviate endothelial dysfunction. 2+ RESULTS Materials & methods Hispidin Structure Week 0 1 2 3 4 5 6 7 8 ApoE -/- mice, 39 weeks old, Start experiments fed HCD • Hispidin is a phenol compound in Phellinus Linteus (Sang-hwang mushroom) that is known to
be effective in anti-inflammatory and anti-cancer. • The experiment was conducted after feeding HCD to ApoE -/- mice for 8 weeks Fig. 1. Hispidin inhibited arginase I,II in an uncompetitive Fig. 2. Arginase inhibition with hispidin incuced manner, which induced eNOS and CaMKII activity. an increase cytosolic Ca 2+ level. Fig. 3. Arginase inhibition with hispidin incuced an increase Fig. 4. In arteriosclerosis mice model, Hispidin increases Fig. 5. In arteriosclerosis mice model, the relaxation cytosolic Ca 2+ level. cytosolic Ca 2+ and the production of NO. rate of blood vessels increases with
the treatment of Hispidin. CONCLUSION REFERENCES ● Hispidin can act as an arginase inhibit
