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Background/Aims. As autophagy is linked to several pathological conditions, like cancer and neurodegenerative diseases, it is crucial to understand its regulatory signaling network. In this study, we investigated the role of the serum- and glucocorticoid-induced protein kinase 1 (SGK1) in the control of autophagy. Methods. To measure autophagic activity in vivo, we quantified the abundance of the autophagy conjugates LC3-PE (phosphatidylethanolamine) and ATG12-ATG5 in tissue extracts of SGK1 wild-type (Sgk1+/+) and knockout (Sgk1−/−) mice that were either fed or starved for 24 h prior sacrifice. In vitro, we targeted SGK1 by RNAi using GFP-WIPI1 expressing U-2 OS cells to quantify the numbers of cells displaying newly formed autophagosomes. In parallel, these cells were also assessed with regard to LC3 and ULK1 by quantitative Western blotting. Results. The abundance of both LC3-PE (LC3-II) and ATG12-ATG5 was significantly increased in red muscle tissues of SGK1 knockout mice. This was found in particular in fed conditions, suggesting that SGK1 may keep basal autophagy under control in red muscle in vivo. Under starved conditions, significant differences were observed in SGK1-deficient white muscle tissue and, under fed conditions, also in the liver. In vitro, we found that SGK1 silencing provoked a significant increase of cells displaying WIPI1-positive autophagosomes and autophagosomal LC3 (LC3-II). Moreover, autophagic flux assessments revealed that autophagic degradation significantly increased in the absence of SGK1, strongly suggesting that SGK1 inhibits both autophagosome formation and autophagic degradation in vitro. In addition, more ULK1 protein lacking the inhibitory, TORC1-specific phosphorylation at serine 758 was detected in the absence of SGK1. Conclusions. Combined, our data strongly support the idea that SGK1 inhibits the process of autophagy. Mechanistically, our data suggest that SGK1 should act upstream of ULK1 in regulating autophagy, and we hypothesize that SGK1 contributes to the regulation of ULK1 gene expression.

Ewing sarcoma (ES) is a malignant tumor that occurs mostly in children. However, the underlying mechanisms of ES are still unknown. Analyzing the results of two previous miRNA array reports, we found that miR-146b-5p might be an onco-miRNA in ES progression. To test this hypothesis, we detected the expression levels of miR-146b-5p by real-time PCR and observed the effects of miR-146b-5p on the progression of ES cells by CCK8 and transwell assays. Bioinformatics and luciferase assays were used to identify the target genes of miR-146b-5p. It showed that the expression levels of miR-146b-5p were upregulated in ES cell lines compared with human mesenchymal stem cells (MSCs). Up- or downregulation of miR-146b-5p in ES cell lines could effectively promote or block the proliferation, migration, and invasion of ES cells, respectively. Furthermore, we demonstrated that BTG2 was one of the target genes and mediated the effects of miR-146b-5p in ES cells. Interestingly, we also found that miR-146b-5p was partly involved in the anticancer effects of pemetrexed in ES cells. Our study revealed that miR-146b-5p affected the progression of ES by suppressing BTG2, which might shed light on anticancer drug development and ES treatment in the future.

There is substantial evidence that mitochondrial dysfunction plays a significant role in the pathogenesis of Parkinson disease (PD). This contribution probably encompasses defects of oxidative phosphorylation, mitochondrial turnover (mitophagy), mitochondrial derived oxidative stress and apoptotic signalling. Human cytomegalovirus immediate-early protein pUL37 × 1 induces Bax mitochondrial translocation and inactivation to prevent apoptosis. Over-expressing pUL37 × 1 in neuronal cells protects against staurosporin and 6-hydroxydopamine induced apoptosis and cell death. Protection is not enhanced by bax silencing in pUL37 × 1 over-expressing cells, suggesting a bax-dependent mechanism of action. pUL37 × 1 increases glycolysis and induces mitochondrial hyperpolarization, a bax independent anti-apoptotic action. pUL37 × 1 increases glycolysis through activation of phosphofructokinase by a calcium-dependent pathway. The dual anti-apoptotic mechanism of pUL37 × 1 may be considered a novel neuroprotective strategy in diseases where mitochondrial dysfunction and apoptotic pathways are involved.

Connective tissue growth factor (CTGF) is a 38-kd protein involved in several human fibrotic disorders including atherosclerosis and skin and renal fibrosis. Although it has been shown that human and experimental liver fibrosis is associated with CTGF expression through up-regulation of CTGF mRNA by hepatic stellate cells (HSC), the role of CTGF in the liver has not yet been determined. The aim of the present study was to assess the effects of CTGF on rat primary HSC and its regulation in a well-established model of in vitro liver fibrogenesis. Incubation of primary HSC with recombinant CTGF induced a significant migratory (2.3-fold, 50 ng/ml CTGF) and proliferative effect (1.8-fold, 100 ng/ml CTGF). Type I collagen mRNA expression, as assessed by a real-time RT-PCR procedure, was also increased when cells were incubated in the presence of CTGF (2-fold, 50 ng/ml). Transforming growth factor-β1 (TGF-β1) strongly stimulated CTGF mRNA expression, a direct mechanism observed in the absence of any intermediate protein synthesis. Furthermore, spontaneous activation of HSC plated on plastic and stimulation by vascular endothelial growth factor, lipid peroxidation products (HNE, MDA), acetaldehyde, and platelet-derived growth factor (PDGF)-BB significantly up-regulated CTGF mRNA expression in HSC. PDGF-induced CTGF stimulation might be related in part to TGF-β1 secretion because CTGF mRNA up-regulation observed after PDGF-BB stimulation was abrogated in the presence of neutralizing TGF-β1 antibody. In conclusion, this study extends the role of CTGF in HSC activation and suggests that CTGF up-regulation might be a central pathway during HSC activation.

The mitogen-activated protein kinase (MAPK) superfamily comprises classical MAPK (also called ERK), c-Jun amino-terminal or stress-activated protein kinase (JNK or SAPK), and p38. Although MAPK is essential for meiotic processes in Xenopus oocytes and the spindle assembly checkpoint in Xenopus egg extracts, the role of members of the MAPK superfamily in M phase or the spindle assembly checkpoint during somatic cell cycles has not been elucidated. The kinase p38, but not MAPK or JNK, was activated in mammalian cultured cells when the cells were arrested in M phase by disruption of the spindle with nocodazole. Addition of activated recombinant p38 to Xenopus cell-free extracts caused arrest of the extracts in M phase, and injection of activated p38 into cleaving embryos induced mitotic arrest. Treatment of NIH 3T3 cells with a specific inhibitor of p38 suppressed activation of the checkpoint by nocodazole. Thus, p38 functions as a component of the spindle assembly checkpoint in somatic cell cycles.

Connective Tissue Growth Factor and IGF-I Are Produced by Human Renal Fibroblasts and Cooperate in the Induction of Collagen Production by High Glucose Suzanne Lam 1 , Reinier N. van der Geest 1 , Nicole A.M. Verhagen 1 , Frans A. van Nieuwenhoven 2 , Ingrid E. Blom 2 , Jan Aten 3 , Roel Goldschmeding 2 , Mohamed R. Daha 1 and Cees van Kooten 1 1 Department of Nephrology, Leiden University Medical Center, Leiden, the Netherlands 2 Department of Pathology, University Medical Center Utrecht, Utrecht, the Netherlands 3 Department of Pathology, Academic Medical Center, Amsterdam, the Netherlands Address correspondence and reprint requests to Dr. Cees van Kooten, Leiden University Medical Center, Department of Nephrology, C3P, Albinusdreef 2, 2333 ZA Leiden, Netherlands. E-mail: Kooten{at}lumc.nl Abstract Tubulointerstitial fibrosis is an important component in the development of diabetic nephropathy. Various renal cell types, including fibroblasts, contribute to the excessive matrix deposition in the kidney. Although transforming growth factor-β (TGF-β) has been thought to play a major role during fibrosis, other growth factors are also involved. Here we examined the effects of connective tissue growth factor (CTGF) and IGF-I on collagen type I and III production by human renal fibroblasts and their involvement in glucose-induced matrix accumulation. We have demonstrated that both CTGF and IGF-I expressions were increased in renal fibroblasts under hyperglycemic conditions, also in the absence of TGF-β signaling. Although CTGF alone had no effect on collagen secretion, combined stimulation with IGF-I enhanced collagen accumulation. Furthermore, IGF-I also had a synergistic effect with glucose on the induction of collagens. Moreover, we observed a partial inhibition in glucose-induced collagen secretion with neutralizing anti-CTGF antibodies, thereby demonstrating for the first time the involvement of endogenous CTGF in glucose-induced effects in human renal fibroblasts. Therefore, the cooperation between CTGF and IGF-I might be involved in glucose-induced matrix accumulation in tubulointerstitial fibrosis and might contribute to the pathogenesis of diabetic nephropathy. DMEM, Dulbecco’s modified Eagle’s medium ECM, extracellular matrix ELISA, enzyme-linked immunosorbent assay IGFBP, IGF-binding protein mAb, monoclonal antibody OD, optical density TBP, TATA-box binding protein TGF-β, transforming growth factor-β Footnotes Accepted September 21, 2003. Received March 21, 2003. DIABETES

Transforming growth factor (TGF)-β and connective tissue growth factor may be implicated in extracellular matrix protein deposition in asthma. We have recently reported that TGF-β increased connective tissue growth factor expression in airway smooth muscle cells isolated from patients with asthma. In this study, we examined fibronectin and collagen production and signal transduction pathways after stimulation with TGF-β and connective tissue growth factor. In both asthmatic and nonasthmatic airway smooth muscle cells, TGF-β and connective tissue growth factor led to the production of fibronectin and collagen I. Fibronectin and collagen expression was extracellular regulated kinase–dependent in both cell types but phosphoinositide-3 kinase–dependent only in asthmatic airway smooth muscle cells. p38 was implicated in fibronectin but not collagen expression in both cell types. TGF-β induction of fibronectin and collagen was in part mediated by an autocrine action of connective tissue growth factor. Phosphorylation of SMAD-2 may represent an additional pathway because this was increased in asthmatic cells. Our results suggest that these two cytokines may be important in the deposition of extracellular matrix proteins and that the signal transduction pathways may be different in asthmatic and nonasthmatic cells.

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate-early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin αvβ3and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an αvβ3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

The efficient isolation and ex vivo expansion of antigen-specific T cells are crucial for successful adoptive immunotherapy against uncontrollable infections and cancers. Several methods have been reported for this purpose, for example, employing MHC-multimeric complexes, interferon-gamma secretion, and antibodies specific for molecules expressed on T-cell surfaces, including CD25, CD69, CD107a, CD137, and CD154. Of the latter, CD137 has been shown to be one of the most promising targets since it is only expressed on CD8 + T cells early after encountering antigen, while being almost undetectable on resting cells. However, detailed comparisons between CD137-based and other methods have not yet been conducted. In this study, we therefore compared three approaches (with CD137, CD107a, and tetramers) using HLA-A24-restricted CMV pp65 and EBV BRLF1 epitopes as model antigens. We found that the CD137-based isolation of antigen-stimulated CD8 + T cells was comparable to tetramer-based sorting in terms of purity and superior to the other two methods in terms of subsequent cell expansion. The method was less applicable to CD4 + T cells since their CD137 upregulation is not sufficiently high. Collectively, this approach is most likely to be optimal among the methods tested for the isolation and expansion of antigen-specific CD8 + cells.

Functional Characteristics of Connective Tissue Growth Factor on Vitreoretinal Cells Takeshi Kita , Yasuaki Hata , Muneki Miura , Shuhei Kawahara , Shintaro Nakao and Tatsuro Ishibashi From the Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Address correspondence and reprint requests to Yasuaki Hata, MD, PhD, Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan. E-mail: hatachan{at}med.kyushu-u.ac.jp Abstract Connective tissue growth factor (CTGF) level is elevated in eyes with proliferative vitreoretinal diseases, such as proliferative diabetic retinopathy and proliferative vitreoretinopathy (PVR), as we previously reported, but its functional characteristics on vitreoretinal cells are yet to be clarified. In this study, we demonstrated a growth-promoting effect of CTGF on cultured hyalocytes and bovine retinal pigment epithelial cells (BRPEs) with the induction of p44/p42 mitogen-activated protein kinase phosphorylation and [ 3 H]thymidine incorporation. CTGF also stimulated the synthesis of fibronectin by hyalocytes and BRPEs without significant effect on collagen gel contraction by these cells. On the other hand, CTGF had no direct effects on the proliferation, migration, or in vitro tube formation by vascular endothelial cells. Nevertheless, CTGF promoted vascular endothelial growth factor (VEGF) gene expression by hyalocytes and BRPEs. Although the concentrations of both CTGF and VEGF in the human vitreous samples with proliferative vitreoretinal diseases were elevated, there was no significant correlation between these concentrations. These findings indicate that CTGF appears to be involved in the formation of proliferative membranes without direct regulation of their cicatricial contraction in the pathogenesis of proliferative vitreoretinal diseases. Whereas CTGF might have no direct effects or minimal effects, if any, on retinal neovascularization, it is possible that CTGF has indirect effects by modulating the expression of VEGF. BAEC, bovine aortic endothelial cell BREC, bovine retinal capillary endothelial cell BRPE, bovine retinal pigment epithelial cell CTGF, connective tissue growth factor DMEM, Dulbecco's modified Eagle's medium FBS, fetal bovine serum HUVEC, human umbilical vein endothelial cell MAPK, mitogen-activated protein kinase PDR, proliferative diabetic retinopathy PVR, proliferative vitreoretinopathy REC, retinal capillary endothelial cell RPE, retinal pigment epithelial cell RRD, rhegmatogenous retinal detachment TGF-β, transforming growth factor-β VEGF, vascular endothelial growth factor Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 15 February 2007. DOI: 10.2337/db06-1644. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted February 5, 2007. Received November 23, 2006. DIABETES