Eliglustat exerts anti-fibrotic effects by activating SREBP2 in TGF-ß1-treated myofibroblasts derived from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic lung disease. Myofibroblasts play a critical role in fibrosis. These cells produce the extracellular matrix (ECM), which contributes to tissue regeneration; however, excess ECM production can cause fibrosis. Transforming growth factor-ß (TGF-ß)/Smad signaling induces ECM production by myofibroblasts; therefore, the inhibition of TGF-ß/Smad signaling may be an effective strategy for IPF treatment. We recently reported that miglustat, an inhibitor of glucosylceramide synthase (GCS), ameliorates pulmonary fibrosis by inhibiting the nuclear translocation of Smad2/3. In the present study, we examined the anti-fibrotic effects of another GCS inhibitor, eliglustat, a clinically approved drug for treating Gaucher disease type 1, in myofibroblasts derived from patient with IPF (IPF-MyoFs). We found that eliglustat exerted anti-fibrotic effects independent of GCS inhibition, and inhibited TGF-ß1-induced expression of α-smooth muscle actin, a marker of fibrosis, without suppressing the phosphorylation and nuclear translocation of Smad2/3. RNA sequencing analysis of eliglustat-treated human lung fibroblasts identified sterol regulatory element-binding protein 2 (SREBP2) activation. Transient overexpression of SREBP2 attenuated the TGF-ß1-induced increase in the expression of Smad target genes in IPF-MyoFs, and SREBP2 knockdown nullified the inhibitory effect of eliglustat on TGF-ß1-induced expression of α-SMA. These results suggested that eliglustat exerts its anti-fibrotic effects through SREBP2 activation. The findings of this study may contribute to the development of novel therapeutic strategies for IPF treatment.

Functional Roles of CD26/DPP4 in Bleomycin-Induced Pulmonary Hypertension Associated with Interstitial Lung Disease.

Pulmonary hypertension (PH) with interstitial lung diseases (ILDs) often causes intractable conditions. CD26/Dipeptidyl peptidase-4 (DPP4) is expressed in lung constituent cells and may be related to the pathogenesis of various respiratory diseases. We aimed to clarify the functional roles of CD26/DPP4 in PH-ILD, paying particular attention to vascular smooth muscle cells (SMCs). Dpp4 knockout (Dpp4KO) and wild type (WT) mice were administered bleomycin (BLM) intraperitoneally to establish a PH-ILD model. The BLM-induced increase in the right ventricular systolic pressure and the right ventricular hypertrophy observed in WT mice were attenuated in Dpp4KO mice. The BLM-induced vascular muscularization in small pulmonary vessels in Dpp4KO mice was milder than that in WT mice. The viability of TGFß-stimulated human pulmonary artery SMCs (hPASMCs) was lowered due to the DPP4 knockdown with small interfering RNA. According to the results of the transcriptome analysis, upregulated genes in hPASMCs with TGFß treatment were related to pulmonary vascular SMC proliferation via the Notch, PI3K-Akt, and NFκB signaling pathways. Additionally, DPP4 knockdown in hPASMCs inhibited the pathways upregulated by TGFß treatment. These results suggest that genetic deficiency of Dpp4 protects against BLM-induced PH-ILD by alleviating vascular remodeling, potentially through the exertion of an antiproliferative effect via inhibition of the TGFß-related pathways in PASMCs.

Functional roles of CD26/DPP4 in bleomycin-induced pulmonary fibrosis.

The pathogenesis of pulmonary fibrosis involves complex interplay between cell types and signaling pathways. Recurrent alveolar epithelial injury can occur during pulmonary inflammation, causing dysregulation of epithelial repair. Dysregulated repair interacts with mesenchymal, inflammatory, and endothelial cells to trigger fibroblast-to-myofibroblast activation. CD26/dipeptidyl peptidase-4 (DPP4) is a type II membrane protein mediating pleiotropic effect. However, the mechanistic role of CD26/DPP4 in pulmonary fibrosis remains unclear. In this study, we aimed to characterize Dpp4 deficiency in a mouse bleomycin (BLM)-induced pulmonary fibrosis model and in cell culture systems of human lung fibroblasts (HLFs). Dpp4 knockout (Dpp4 KO) mouse lungs exhibited lower Ashcroft scale indices, collagen content, and numbers of fibroblasts and myofibroblasts compared with those in C57BL/6 wild-type (WT) mice. Upregulation of Tgfb1 and Tgfb2 mRNA levels in the lungs after BLM treatment was lower in Dpp4 KO mice compared with those in WT mice. Although TGF-ß-driven endothelial-to-mesenchymal transition (EndMT) has been implicated as one of the mechanisms of pulmonary fibrosis, a number of partial EndMT cells in lungs did not differ between Dpp4 KO mice and WT mice. The proliferation capacity and mRNA levels of COL1A1, a collagen deposition-related gene, in cultured HLFs were suppressed in DPP4 small interfering RNA-treated cells. This study indicates that the genetic deficiency of DPP4 has protective effects against BLM-induced pulmonary fibrosis, partly through the reduction in TGF-ß expression and inhibition of fibroblast activation in the lung. Our study suggests that CD26/DPP4 inhibition is a potential therapeutic strategy for pulmonary fibrosis.

N-butyldeoxynojirimycin (miglustat) ameliorates pulmonary fibrosis through inhibition of nuclear translocation of Smad2/3.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease. The disease involves excessive accumulation of fibroblasts and myofibroblasts, and myofibroblasts differentiated by pro-fibrotic factors promote the deposition of extracellular matrix proteins such as collagen and fibronectin. Transforming growth factor-ß1 is a pro-fibrotic factor that promotes fibroblast-to-myofibroblast differentiation (FMD). Therefore, inhibition of FMD may be an effective strategy for IPF treatment. In this study, we screened the anti-FMD effects of various iminosugars and showed that some compounds, including N-butyldeoxynojirimycin (NB-DNJ, miglustat, an inhibitor of glucosylceramide synthase (GCS)), a clinically approved drug for treating Niemann-Pick disease type C and Gaucher disease type 1, inhibited TGF-ß1-induced FMD by inhibiting the nuclear translocation of Smad2/3. N-butyldeoxygalactonojirimycin having GCS inhibitory effect did not attenuate the TGF-ß1-induced FMD, suggesting that NB-DNJ exerts the anti-FMD effects by GCS inhibitory effect independent manner. N-butyldeoxynojirimycin did not inhibit TGF-ß1-induced Smad2/3 phosphorylation. In a mouse model of bleomycin (BLM)-induced pulmonary fibrosis, intratracheal or oral administration of NB-DNJ at an early fibrotic stage markedly ameliorated lung injury and deterioration of respiratory functions, such as specific airway resistance, tidal volume, and peak expiratory flow. Furthermore, the anti-fibrotic effects of NB-DNJ in the BLM-induced lung injury model were similar to those of pirfenidone and nintedanib, which are clinically approved drugs for the treatment of IPF. These results suggest that NB-DNJ may be effective for IPF treatment.

Regulation of neural stem cell proliferation and survival by protein arginine methyltransferase 1.

Protein arginine methyltransferase 1 (PRMT1), a major type I arginine methyltransferase in mammals, methylates histone and non-histone proteins to regulate various cellular functions, such as transcription, DNA damage response, and signal transduction. PRMT1 is highly expressed in neural stem cells (NSCs) and embryonic brains, suggesting that PRMT1 is essential for early brain development. Although our previous reports have shown that PRMT1 positively regulates oligodendrocyte development, it has not been studied whether PRMT1 regulates NSC proliferation and its survival during development. To examine the role of PRMT1 in NSC activity, we cultured NSCs prepared from embryonic mouse forebrains deficient in PRMT1 specific for NSCs and performed neurosphere assays. We found that the primary neurospheres of PRMT1-deficient NSCs were small and the number of spheres was decreased, compared to those of control NSCs. Primary neurospheres deficient in PRMT1 expressed an increased level of cleaved caspase-3, suggesting that PRMT1 deficiency-induced apoptosis. Furthermore, p53 protein was significantly accumulated in PRMT1-deficient NSCs. In parallel, p53-responsive pro-apoptotic genes including Pmaip1 and Perp were upregulated in PRMT1-deficient NSCs. p53-target p21 mRNA and its protein levels were shown to be upregulated in PRMT1-deficient NSCs. Moreover, the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay showed that the loss of PRMT1 led to cell cycle defects in the embryonic NSCs. In contrast to the above in vitro observations, NSCs normally proliferated and survived in the fetal brains of NSC-specific PRMT1-deficient mice. We also found that Lama1, which encodes the laminin subunit α1, was significantly upregulated in the embryonic brains of PRMT1-deficient mice. These data implicate that extracellular factors provided by neighboring cells in the microenvironment gave a trophic support to NSCs in the PRMT1-deficient brain and recovered NSC activity to maintain brain homeostasis. Our study implies that PRMT1 plays a cell-autonomous role in the survival and proliferation of embryonic NSCs.

Multi­omics analysis of right ventricles in rat models of pulmonary arterial hypertension: Consideration of mitochondrial biogenesis by chrysin.

In pulmonary arterial hypertension (PAH), right ventricular failure is accompanied by metabolic alterations in cardiomyocytes, which may be due to mitochondrial dysfunction and decreased energy production. Chrysin (CH) is a phytochemical with pharmacological activity that is involved in the regulation of mitochondrial biogenesis. The present study investigated the role of CH in the right ventricle (RV) by analyzing the cardiac transcriptome and metabolome of a SU5416(a vascular endothelial growth factor receptor blocker, /hypoxia (Su/Hx) rat model of PAH. RNA­sequencing of the RV transcriptome between Su/Hx, Su/Hx with CH (Su/Hx + CH) and control groups, extracellular matrix (ECM) organization and ECM­receptor interaction­associated genes were upregulated in the RV of Su/Hx but not Su/Hx + CH rats. Furthermore, expression of mitochondrial function­, energy production­, oxidative phosphorylation­ and tricarboxylic acid (TCA) cycle­associated genes was decreased in the RV of Su/Hx rats; this was reverse by CH. Metabolomic profiling analysis of Su/Hx and Su/Hx + CH rats showed no significant changes in glycolysis, TCA cycle, glutathione, NADH or NADPH. By contrast, in the RV of Su/Hx rats, decreased adenylate energy charge was partially reversed by CH administration, suggesting that CH was involved in the improvement of mitochondrial biogenesis. Reverse transcription­quantitative PCR analysis revealed that expression of peroxisome proliferator­activated receptor γ, a master regulator of fatty acid metabolism and mitochondrial biogenesis, was increased in the RV of Su/Hx + CH rats. CH ameliorated cardiac abnormality, including cardiac fibrosis, RV hypertrophy and PH. The present study suggested that CH altered patterns of gene expression and levels of mitochondrial metabolites in cardiomyocytes, thus improving RV dysfunction in a Su/Hx PAH rat model.

Pathophysiological Roles of Stress-Activated Protein Kinases in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is one of the most symptomatic progressive fibrotic lung diseases, in which patients have an extremely poor prognosis. Therefore, understanding the precise molecular mechanisms underlying pulmonary fibrosis is necessary for the development of new therapeutic options. Stress-activated protein kinases (SAPKs), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38) are ubiquitously expressed in various types of cells and activated in response to cellular environmental stresses, including inflammatory and apoptotic stimuli. Type II alveolar epithelial cells, fibroblasts, and macrophages are known to participate in the progression of pulmonary fibrosis. SAPKs can control fibrogenesis by regulating the cellular processes and molecular functions in various types of lung cells (including cells of the epithelium, interstitial connective tissue, blood vessels, and hematopoietic and lymphoid tissue), all aspects of which remain to be elucidated. We recently reported that the stepwise elevation of intrinsic p38 signaling in the lungs is correlated with a worsening severity of bleomycin-induced fibrosis, indicating an importance of this pathway in the progression of pulmonary fibrosis. In addition, a transcriptome analysis of RNA-sequencing data from this unique model demonstrated that several lines of mechanisms are involved in the pathogenesis of pulmonary fibrosis, which provides a basis for further studies. Here, we review the accumulating evidence for the spatial and temporal roles of SAPKs in pulmonary fibrosis.

Transcriptomic Evaluation of Pulmonary Fibrosis-Related Genes: Utilization of Transgenic Mice with Modifying p38 Signal in the Lungs.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing lung disease that is caused by the dysregulation of alveolar epithelial type II cells (AEC II). The mechanisms involved in the progression of IPF remain incompletely understood, although the immune response accompanied by p38 mitogen-activated protein kinase (MAPK) activation may contribute to some of them. This study aimed to examine the association of p38 activity in the lungs with bleomycin (BLM)-induced pulmonary fibrosis and its transcriptomic profiling. Accordingly, we evaluated BLM-induced pulmonary fibrosis during an active fibrosis phase in three genotypes of mice carrying stepwise variations in intrinsic p38 activity in the AEC II and performed RNA sequencing of their lungs. Stepwise elevation of p38 signaling in the lungs of the three genotypes was correlated with increased severity of BLM-induced pulmonary fibrosis exhibiting reduced static compliance and higher collagen content. Transcriptome analysis of these lung samples also showed that the enhanced p38 signaling in the lungs was associated with increased transcription of the genes driving the p38 MAPK pathway and differentially expressed genes elicited by BLM, including those related to fibrosis as well as the immune system. Our findings underscore the significance of p38 MAPK in the progression of pulmonary fibrosis.

Inhibition of the SET8 Pathway Ameliorates Lung Fibrosis Even Through Fibroblast Dedifferentiation.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown etiopathogenesis. The activation of extracellular matrix (ECM)-producing myofibroblasts plays a key role in fibrotic tissue remodeling. The dedifferentiation of myofibroblasts has attracted considerable attention as a promising target for the development of effective therapeutic interventions against IPF. Here, we screened a small library of epigenetics-related inhibitors using dedifferentiation assay of lung myofibroblasts prepared from a patient at the terminal stages of IPF and chose UNC0379. The inhibition of SET8, a histone H4 lysine 20 (H4K20) monomethyltransferase, by UNC0379 markedly suppressed the expression of α-smooth muscle actin (SMA) and ED-A-fibronectin in myofibroblasts. In IPF myofibroblasts, SET8 expression and H4K20 monomethylation (H4K20me1) levels, which were significantly higher than those in normal human lung fibroblasts, were reduced upon treatment with UNC0379. Hence, the changes in the expression of the two fibrotic markers clearly correlated with those in SET8 expression and H4K20me1 level. Furthermore, in a mouse model of bleomycin (BLM)-induced lung fibrosis, the intratracheal administration of UNC0379 at an early fibrotic stage markedly ameliorated the histopathological changes associated with collagen deposition in the lungs. However, treatment with UNC0379 did not significantly affect the number of proinflammatory cells or cytokine production in the bronchoalveolar lavage fluids from mice treated with BLM. In the BLM-injured lung, SET8 was predominantly localized to the nuclei of α-SMA-positive cells, which colocalized with H4K20me1. Taken together, our results indicate that the inhibition of SET8 resulting in myofibroblast dedifferentiation may partly mitigate lung fibrosis without affecting the inflammatory responses.

Transcriptomic changes involved in the dedifferentiation of myofibroblasts derived from the lung of a patient with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown etiology. Under pathological conditions in lungs with IPF, myofibroblasts serve a key role in fibrogenesis via the accumulation of an excessive amount of extracellular matrix. To develop effective therapeutic interventions against IPF, studies have recently focused on how to dedifferentiate established myofibroblasts. The present study revealed that JQ1, an inhibitor of bromodomain and extra­terminal proteins, markedly suppressed the expression levels of α­smooth muscle actin and ED­A­fibronectin in myofibroblasts prepared from the lung of a patient with end­stage IPF. Furthermore, these findings were supported by transcriptome analysis using RNA sequencing, in which differentially expressed genes (DEGs) downregulated by JQ1 treatment were significantly enriched in the fibrosis­related signaling pathway. On the other hand, the upregulated DEGs in response to JQ1 treatment were significantly enriched in glutathione metabolism, which may affect the cell status of fibroblast/myofibroblast. To the best of our knowledge, this was the first study to comprehensively analyze transcriptome profiles associated with dedifferentiation of IPF myofibroblasts.

Pharmacological properties of ß-adrenoceptors mediating rat superior mesenteric artery relaxation and the effects of chemical sympathetic denervation.

AIMS: ß-Adrenoceptors (ß-ADRs) mediating the relaxation of rat superior mesenteric arteries (SMAs) were pharmacologically identified, and the effects of chemical sympathetic denervation on ß-ADR-mediated relaxation were examined. MAIN METHODS: The tension changes of endothelium-denuded SMAs were isometrically recorded and the mRNA of endothelium-denuded SMA ß-ADR was detected using RT-PCR. KEY FINDINGS: In endothelium-denuded SMAs contracted with ≥10-7 M phenylephrine (an α1-ADR agonist), isoprenaline (a ß-ADR agonist)-induced relaxation was competitively inhibited by 3 × 10-9-10-8 M propranolol (a ß1,2-ADR antagonist), but not further affected by ≥10-8 M propranolol. Although isoprenaline-induced relaxation was not affected by ICI-118,551 (10-9-10-8 M; a ß2-ADR antagonist), it was competitively inhibited by atenolol (10-7-3 × 10-7 M; a ß1-ADR antagonist) in the presence of ICI-118,551. In the presence of 10-7 M propranolol, isoprenaline- and CGP-12177A (a ß3-ADR partial agonist)-induced relaxation was competitively inhibited by high concentrations of bupranolol (a ß1,2,3-ADR antagonist), with pA2 values of 6.49 and 5.76, respectively. We detected the mRNA of ß1- and ß3-ADRs in endothelium-denuded SMAs. Treatment with 6-hydroxydopamine (a catecholaminergic neurotoxin) reduced maximal isoprenaline-induced relaxation in the presence and absence of 10-7 M propranolol, but not CGP-12177A-induced relaxation. SIGNIFICANCE: Isoprenaline-induced relaxation of rat SMAs is mediated by ß1- and ß3-ADRs. ß-ADR-mediated relaxation of rat SMAs is shown to be attenuated by chemical sympathetic denervation. The differences in the effects of bupranolol and chemical sympathetic denervation on the responses to isoprenaline and CGP-12177A in rat SMAs might be explained by the possible presence of multiple ß3-ADRs with different pharmacological properties.

Cooperative action of APJ and α1A-adrenergic receptor in vascular smooth muscle cells induces vasoconstriction.

The apelin receptor (APJ), a receptor for apelin and elabela/apela, induces vasodilation and vasoconstriction in blood vessels. However, the prolonged effects of increased APJ-mediated signalling, involving vasoconstriction, in smooth muscle cells have not been fully characterized. Here, we investigated the vasoactive effects of APJ gain of function under the control of the smooth muscle actin (SMA) gene promoter in mice. Transgenic overexpression of APJ (SMA-APJ) conferred sensitivity to blood pressure and vascular contraction induced by apelin administration in vivo. Interestingly, ex vivo experiments showed that apelin markedly increased the vasoconstriction of isolated aorta induced by noradrenaline (NA), an agonist for α- and ß-adrenergic receptors, or phenylephrine, a specific agonist for α1-adrenergic receptor (α1-AR). In addition, intracellular calcium influx was augmented by apelin with NA in HEK293T cells expressing APJ and α1A-AR. To examine the cooperative action of APJ and α1A-AR in the regulation of vasoconstriction, we developed α1A-AR deficient mice using a genome-editing technique, and then established SMA-APJ/α1A-AR-KO mice. In the latter mouse line, aortic vasoconstriction induced by a specific agonist for α1A-AR, A-61603, were significantly less than in SMA-APJ mice. These results suggest that the APJ-enhanced response requires α1A-AR to contract vessels coordinately.

Stress-Activated Protein Kinases in Spinal Cord Injury: Focus on Roles of p38.

Spinal cord injury (SCI) consists of three phases-acute, secondary, and chronic damages-and limiting the development of secondary damage possibly improves functional recovery after SCI. A major component of the secondary phase of SCI is regarded as inflammation-triggered events: induction of cytokines, edema, microglial activation, apoptosis of cells including oligodendrocytes and neurons, demyelination, formation of the astrocytic scar, and so on. Two major stress-activated protein kinases (SAPKs)-c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK)-are activated in various types of cells in response to cellular stresses such as apoptotic stimuli and inflammatory waves. In animal models of SCI, inhibition of either JNK or p38 has been shown to promote neuroprotection-associated functional recovery. Here, we provide an overview on the roles of SAPKs in SCI and, in particular, the pathological role of p38 will be discussed as a promising target for therapeutic intervention in SCI.

Antifibrotic effects of cyclosporine A on TGF-ß1-treated lung fibroblasts and lungs from bleomycin-treated mice: role of hypoxia-inducible factor-1α.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder that is characterized by aberrant tissue remodeling and the formation of fibroblastic foci that are composed of fibrogenic myofibroblasts. TGF-ß1 is one of the factors that are responsible for fibrosis as it promotes fibroblast to myofibroblast differentiation (FMD) and is associated with up-regulation of α-smooth muscle actin. Therefore, inhibition of FMD may represent an effective strategy for the treatment of IPF. Here, we describe the treatment of human lung fibroblasts (WI-38 and HFL-1 cells) with cyclosporine A (CsA), which reduces TGF-ß1-induced FMD via degradation of hypoxia-inducible factor-1α (HIF-1α). In addition, in primary myofibroblast-like cells that were obtained from a patient with pulmonary fibrosis, treatment with CsA and an HIF-1α inhibitor (HIFi) decreased the expression levels of α-smooth muscle actin and fibronectin, which indicated that CsA and HIFi promote dedifferentiation of myofibroblasts. In mice intratracheally administered CsA or HIFi at an early fibrotic stage [7, 8, and 9 d postinstillation (dpi) of bleomycin], marked alleviation of lung fibrosis was observed at 14 dpi. These results suggest that CsA exhibits antifibrotic effects by degrading HIF-1α and that the CsA-HIF-1α axis provides new insights into therapeutic options for the treatment of IPF.-Yamazaki, R., Kasuya, Y., Fujita, T., Umezawa, H., Yanagihara, M., Nakamura, H., Yoshino, I., Tatsumi, K., Murayama, T. Antifibrotic effects of cyclosporine A on TGF-ß1-treated lung fibroblasts and lungs from bleomycin-treated mice: role of hypoxia-inducible factor-1α.

Genetic and Pharmacological Inhibition of p38α Improves Locomotor Recovery after Spinal Cord Injury.

One of the mitogen-activated protein kinases, p38α plays a crucial role in various inflammatory diseases and apoptosis of various types of cells. In this study, we investigated the pathophysiological roles of p38α in spinal cord injury (SCI), using a mouse model. Lateral hemisection at T9 of the SC was performed in wild type (WT) and p38α+/- mice (p38α-/- showed embryonic lethality). p38α+/- mice showed a better functional recovery from SCI-associated paralyzed hindlimbs compared to WT mice at 7 days post-injury (dpi), which remained until 28 dpi (an end time point of monitoring the behavior). In histopathological analysis at 28 dpi, there was more axonal regeneration with remyelination on the caudal side of the lesion epicenter in p38α+/- mice than in WT mice. At 7 dpi, infiltration of inflammatory cells into the lesion and expression of cytokines in the lesion were reduced in p38α+/- mice compared with WT mice. At the same time point, the number of apoptotic oligodendrocytes in the white matter at the caudal boarder of the lesion of p38α+/- mice was lower than that of WT mice. At 14 dpi, more neural and oligodendrocyte precursor cells in the gray matter and white matter, respectively, were observed around the lesion epicenter of p38α+/- mice compared with the case of WT mice. At the same time point, astrocytic scar formation was less apparent in p38α+/- than in WT mice, while compaction of inflammatory immune cells associated with the wound contraction was more apparent in p38α+/- than in WT mice. Furthermore, we verified the effectiveness of oral administration of SB239063, a p38α inhibitor on the hindlimb locomotor recovery after SCI. These results suggest that p38α deeply contributes to the pathogenesis of SCI and that inhibition of p38α is a beneficial strategy to recovery from SCI.

Role of CD69 in the pathogenesis of elastase-induced pulmonary inflammation and emphysema.

Cluster of differentiation 69 (CD69), known as an early activation marker of lymphocytes, has been demonstrated to regulate inflammatory events in various disease models. Although the increased number of CD69-expressed T lymphocytes in the lungs of patients with chronic obstructive pulmonary disease (COPD) has been reported, a functional role of CD69 in the pathogenesis of COPD remains unknown. To address to this question, CD69-deficient (CD69KO) mice and wild-type (WT) mice were subjected to a mouse model of porcine pancreatic elastase (PPE)-induced pulmonary inflammation and emphysema. In the two genotypes, PPE increased counts of macrophages, neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) and induced emphysematous changes in the lung, whereas those two pathological signs were significantly enhanced in CD69KO mice compared to WT mice. Moreover, the PPE-induced levels of IL-17 and IL-6 in BALF were significantly higher in CD69KO mice than in WT mice at the acute inflammatory phase. Immunofluorescent studies showed that IL-17 and IL-6 were predominantly expressed in CD4+ and γδ T cells and macrophages, respectively. Concomitant administration of IL-17- and IL-6-neutralizing antibodies significantly attenuated the PPE-induced emphysematous changes in the two genotypes. These findings suggest that CD69 negatively regulates the development of PPE-induced emphysema in part at least through modulating function of IL-17-producing T cells.

Severe Hypomyelination and Developmental Defects Are Caused in Mice Lacking Protein Arginine Methyltransferase 1 (PRMT1) in the Central Nervous System.

Protein arginine methyltransferase 1 (PRMT1) is involved in cell proliferation, DNA damage response, and transcriptional regulation. Although PRMT1 is extensively expressed in the CNS at embryonic and perinatal stages, the physiological role of PRMT1 has been poorly understood. Here, to investigate the primary function of PRMT1 in the CNS, we generated CNS-specific PRMT1 knock-out mice by the Cre-loxP system. These mice exhibited postnatal growth retardation with tremors, and most of them died within 2 weeks after birth. Brain histological analyses revealed prominent cell reduction in the white matter tracts of the mutant mice. Furthermore, ultrastructural analysis demonstrated that myelin sheath was almost completely ablated in the CNS of these animals. In agreement with hypomyelination, we also observed that most major myelin proteins including myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin-associated glycoprotein (MAG) were dramatically decreased, although neuronal and astrocytic markers were preserved in the brain of CNS-specific PRMT1 knock-out mice. These animals had a reduced number of OLIG2(+) oligodendrocyte lineage cells in the white matter. We found that expressions of transcription factors essential for oligodendrocyte specification and further maturation were significantly suppressed in the brain of the mutant mice. Our findings provide evidence that PRMT1 is required for CNS development, especially for oligodendrocyte maturation processes.

PRMT8 as a phospholipase regulates Purkinje cell dendritic arborization and motor coordination.

The development of vertebrate neurons requires a change in membrane phosphatidylcholine (PC) metabolism. Although PC hydrolysis is essential for enhanced axonal outgrowth mediated by phospholipase D (PLD), less is known about the determinants of PC metabolism on dendritic arborization. We show that protein arginine methyltransferase 8 (PRMT8) acts as a phospholipase that directly hydrolyzes PC, generating choline and phosphatidic acid. We found that PRMT8 knockout mice (prmt8 (-/-)) displayed abnormal motor behaviors, including hindlimb clasping and hyperactivity. Moreover, prmt8 (-/-) mice and TALEN-induced zebrafish prmt8 mutants and morphants showed abnormal phenotypes, including the development of dendritic trees in Purkinje cells and altered cerebellar structure. Choline and acetylcholine levels were significantly decreased, whereas PC levels were increased, in the cerebellum of prmt8 (-/-) mice. Our findings suggest that PRMT8 acts both as an arginine methyltransferase and as a PC-hydrolyzing PLD that is essential for proper neurological functions.

Bidirectional role of IL-6 signal in pathogenesis of lung fibrosis.

BACKGROUND: Various signals are known to participate in the pathogenesis of lung fibrosis. Our aim was to determine which signal is predominantly mobilized in the early inflammatory phase and thereafter modulates the development of lung fibrosis. METHODS: Mice received a single dose of 3 mg/kg body weight of bleomycin (BLM) and were sacrificed at designated days post-instillation (dpi). Lung homogenates and sections from mice in the early inflammatory phase were subjected to phospho-protein array analysis and immunofluorescence studies, respectively. Bronchoalveolar lavage fluid (BALF) from mice was subjected to an enzyme-linked immunosorbent assay (EIA) for interleukin (IL)-6 and evaluation of infiltrated cell populations. The effects of endogenous and exogenous IL-6 on the BLM-induced apoptotic signal in A549 cells and type 2 pneumocytes were elucidated. In addition, the effect of IL-6-neutralizing antibody on BLM-induced lung injury was evaluated. RESULTS: Phospho-protein array revealed that BLM induced phosphorylation of molecules downstream of the IL-6 receptor such as Stat3 and Akt in the lung at 3 dpi. At 3 dpi, immunofluorescence studies showed that signals of phospho-Stat3 and -Akt were localized in type 2 pneumocytes, and that BLM-induced IL-6-like immunoreactivity was predominantly observed in type 2 pneumocytes. Activation of caspases in BLM-treated A549 cells and type 2 pneumocytes was augmented by application of IL-6-neutralizing antibody, a PI3K inhibitor or a Stat3 inhibitor. EIA revealed that BLM-induced IL-6 in BALF was biphasic, with the first increase from 0.5 to 3 dpi followed by the second increase from 8 to 10 dpi. Blockade of the first increase of IL-6 by IL-6-neutralizing antibody enhanced apoptosis of type 2 pneumocytes and neutrophilic infiltration and markedly accelerated fibrosis in the lung. In contrast, blockade of the second increase of IL-6 by IL-6-neutralizing antibody ameliorated lung fibrosis. CONCLUSIONS: The present study demonstrated that IL-6 could play a bidirectional role in the pathogenesis of lung fibrosis. In particular, upregulation of IL-6 at the early inflammatory stage of BLM-injured lung has antifibrotic activity through regulating the cell fate of type 2 pneumocytes in an autocrine/paracrine manner.

p38α controls self-renewal and fate decision of neurosphere-forming cells in adult hippocampus.

Neural stem cells (NSC) from the adult hippocampus easily lose their activity in vitro. Efficient in vitro expansion of adult hippocampus-derived NSC is important for generation of tools for research and cell therapy. Here, we show that a single copy disruption or pharmacological inhibition of p38α enables successful long-term neurosphere culture of adult mouse hippocampal cells. Expanded neurospheres with high proliferative activity differentiated into the three neuronal lineages under differentiating conditions. Thus, inhibition of p38α can maintain adult hippocampal NSC activity in vitro.