L type amino acid transporter 1 (LAT1) is an attractive molecular target for cancer therapy because of its overexpression in many cancer cells. JPH203, a selective LAT1 inhibitor, causes amino acid ...deprivation and suppresses cancer cell proliferation. However, several cancer cells showed resistance to amino acid deprivation. In this study, we aimed to elucidate the molecular mechanism of different sensitivity between 2 breast cancer cells to anti-LAT1 therapy. MDA-MB-231 cells were more resistant to growth suppression effect of JPH203 than T-47D cells (IC50 was 200 ± 12.5 μM for MDA-MB-231, and 5 ± 1.1 μM for T-47D cells; p < 0.05). Transcriptome and biochemical analysis were done in these cells in the presence/absence of JPH203. JPH203 induced intracellular amino acid deprivation stress in both cells, but it upregulated cystathionine γ lyase (CTH), an enzyme for synthesis of antioxidants, only in MDA-MB-231 cells. Moreover, siRNA-mediated CTH knockdown induced oxidative stress in response to JPH203 leading to decreased cell viability in MDA-MB-231 cells. These results suggest that activation of anti-oxidation pathways in response to amino acid deprivation confers resistance to anti-LAT1 therapy.
Protein kinase R-like endoplasmic reticulum kinase (PERK) is one of the endoplasmic reticulum (ER) stress sensors. PERK loss-of-function mutations are known to cause Wolcott-Rallison syndrome. This ...disease is characterized by early-onset diabetes mellitus, skeletal dysplasia, and cardiac valve malformation. To understand the role of PERK in valve formation in vivo, we used an endothelial-specific PERK conditional knockout mice as well as in vitro PERK inhibition assays. We used ProteoStat dyes to visualize the accumulation of misfolded proteins in the endocardial cushion and valve mesenchymal cells (VMCs). Then, VMCs were isolated from E12.5 fetal mice, by fluorescence assisted cell sorting. Proteomic analysis of PERK-deleted VMCs identified the suppression of proteins related to fatty acid oxidation (FAO), especially carnitine palmitoyltransferase II (CPT2). CPT2 is a critical regulator of endocardial-mesenchymal transformation (EndoMT); however how TGF-β downstream signaling controls CPT2 expression remains unclear. Here, we showed that PERK inhibition suppressed, not only EndoMT but also CPT2 protein expression in human umbilical vein endothelial cells (HUVECs) under TGF-β1 stimulation. As a result, PERK inhibition suppressed mitochondrial metabolic activity. Taken together, these results demonstrate that PERK signaling is required for cardiac valve formation via FAO and EndoMT.
Although thousands of long noncoding RNAs (lncRNAs) are localized in the nucleus, only a few dozen have been functionally characterized. Here we show that nuclear enriched abundant transcript 1 ...(NEAT1), an essential lncRNA for the formation of nuclear body paraspeckles, is induced by influenza virus and herpes simplex virus infection as well as by Toll-like receptor3-p38 pathway-triggered poly I:C stimulation, resulting in excess formation of paraspeckles. We found that NEAT1 facilitates the expression of antiviral genes including cytokines such as interleukin-8 (IL8). We found that splicing factor proline/glutamine-rich (SFPQ), a NEAT1-binding paraspeckle protein, is a repressor of IL8 transcription, and that NEAT1 induction relocates SFPQ from the IL8 promoter to the paraspeckles, leading to transcriptional activation of IL8. Together, our data show that NEAT1 plays an important role in the innate immune response through the transcriptional regulation of antiviral genes by the stimulus-responsive cooperative action of NEAT1 and SFPQ.
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•Poly I:C stimulation and virus infection induce NEAT1v2 expression•Poly I:C stimulation and virus infection induces excess formation of paraspeckles•NEAT1 facilitates the expression of antiviral genes including cytokines such as IL8•NEAT1 induction relocates SFPQ from the IL8 promoter to the paraspeckles
Imamura et al. show that NEAT1, a nuclear long noncoding RNA, can cooperatively function with a paraspeckle protein, SFPQ (splicing factor proline/glutamine-rich), to regulate antiviral gene expression.
Peripartum cardiomyopathy (PPCM) is a life-threatening heart failure occurring in the peripartum period. Although mal-angiogenesis, induced by the 16-kDa N-terminal prolactin fragment (16 K PRL), is ...involved in the pathogenesis, the effect of full-length prolactin (23 K PRL) is poorly understood. We transfected neonate rat cardiomyocytes with plasmids containing 23 K PRL or 16 K PRL in vitro and found that 23 K PRL, but not 16 K PRL, upregulated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling, and hypoxia promoted this effect. During the perinatal period, cardiomyocyte-specific PERK homogenous knockout (CM-KO) mice showed PPCM phenotypes after consecutive deliveries. Downregulation of PERK or JAK/STAT signaling and upregulation of apoptosis were observed in CM-KO mouse hearts. Moreover, in bromocriptine-treated CM-KO mice, cardiac function did not improve and cardiomyocyte apoptosis was not suppressed during the peripartum period. These results demonstrate that interaction between 23 K PRL and PERK signaling is cardioprotective during the peripartum term.
Identified as a major downstream effector of the small GTPase RhoA, Rho-associated coiled-coil kinase (ROCK) is a versatile regulator of multiple cellular processes. Angiogenesis, the process of ...generating new capillaries from the pre-existing ones, is required for the development of various diseases such as cancer, diabetes and rheumatoid arthritis. Recently, ROCK has attracted attention for its crucial role in angiogenesis, making it a promising target for new therapeutic approaches. In this review, we summarize recent advances in understanding the role of ROCK signaling in regulating the permeability, migration, proliferation and tubulogenesis of endothelial cells (ECs), as well as its functions in non-ECs which constitute the pro-angiogenic microenvironment. The therapeutic potential of ROCK inhibitors in angiogenesis-related diseases is also discussed.
The efficacy of prior activation of an anti-inflammatory pathway called the cholinergic anti-inflammatory pathway (CAP) through vagus nerve stimulation (VNS) has been reported in renal ...ischemia-reperfusion injury models. However, there have been no reports that have demonstrated the effectiveness of VNS after injury. We investigated the renoprotective effect of VNS in a cisplatin-induced nephropathy model. C57BL/6 mice were injected with cisplatin, and VNS was conducted 24 hours later. Kidney function, histology, and a kidney injury marker (Kim-1) were evaluated 72 hours after cisplatin administration. To further explore the role of the spleen and splenic macrophages, key players in the CAP, splenectomy, and adoptive transfer of macrophages treated with the selective α7 nicotinic acetylcholine receptor agonist GTS-21 were conducted. VNS treatment significantly suppressed cisplatin-induced kidney injury. This effect was abolished by splenectomy, while adoptive transfer of GTS-21-treated macrophages improved renal outcomes. VNS also reduced the expression of cytokines and chemokines, including CCL2, which is a potent chemokine attracting monocytes/macrophages, accompanied by a decline in the number of infiltrating macrophages. Taken together, stimulation of the CAP protected the kidney even after injury in a cisplatin-induced nephropathy model. Considering the feasibility and anti-inflammatory effects of VNS, the findings suggest that VNS may be a promising therapeutic tool for acute kidney injury.
The cholinergic anti-inflammatory pathway (CAP) links the nervous and immune systems and modulates innate and adaptive immunity. Activation of the CAP by vagus nerve stimulation exerts protective ...effects in a wide variety of clinical disorders including rheumatoid arthritis and Crohn’s disease, and in murine models of acute kidney injury including ischemia/reperfusion injury (IRI). The canonical CAP pathway involves activation of splenic alpha7-nicotinic acetylcholine receptor (α7nAChR)-positive macrophages by splenic β2-adrenergic receptor-positive CD4+ T cells. Here we demonstrate that ultrasound or vagus nerve stimulation also activated α7nAChR-positive peritoneal macrophages, and that adoptive transfer of these activated peritoneal macrophages reduced IRI in recipient mice. The protective effect required α7nAChR, and did not occur in splenectomized mice or in mice lacking T and B cells, suggesting a bidirectional interaction between α7nAChR-positive peritoneal macrophages and other immune cells including β2-adrenergic receptor-positive CD4+ T cells. We also found that expression of hairy and enhancer of split-1 (Hes1), a basic helix-loop-helix DNA-binding protein, is induced in peritoneal macrophages by ultrasound or vagus nerve stimulation. Adoptive transfer of Hes1-overexpressing peritoneal macrophages reduced kidney IRI. Our data suggest that Hes1 is downstream of α7nAChR and is important to fully activate the CAP. Taken together, these results suggest that peritoneal macrophages play a previously unrecognized role in mediating the protective effect of CAP activation in kidney injury, and that Hes1 is a new candidate pharmacological target to activate the CAP.
Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying ...vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine.
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