Seung-Yoon Park

The Induction of MicroRNA Targeting IRS-1 Is Involved in the Development of Insulin Resistance under Conditions of Mitochondrial Dysfunction in Hepatocytes

Background Mitochondrial dysfunction induces insulin resistance in myocytes via a reduction of insulin receptor substrate-1 (IRS-1) expression. However, the effect of mitochondrial dysfunction on insulin sensitivity is not understood well in hepatocytes. Although research has implicated the translational repression of target genes by endogenous non-coding microRNAs (miRNA) in the pathogenesis of various diseases, the identity and role of the miRNAs that are involved in the development of insulin resistance also remain largely unknown. Methodology To determine whether mitochondrial dysfunction induced by genetic or metabolic inhibition causes insulin resistance in hepatocytes, we analyzed the expression and insulin-stimulated phosphorylation of insulin signaling intermediates in SK-Hep1 hepatocytes. We used qRT-PCR to measure cellular levels of selected miRNAs that are thought to target IRS-1 3′ untranslated regions (3′UTR). Using overexpression of miR-126, we determined whether IRS-1-targeting miRNA causes insulin resistance in hepatocytes. Principal Findings Mitochondrial dysfunction resulting from genetic (mitochondrial DNA depletion) or metabolic inhibition (Rotenone or Antimycin A) induced insulin resistance in hepatocytes via a reduction in the expression of IRS-1 protein. In addition, we observed a significant up-regulation of several miRNAs presumed to target IRS-1 3′UTR in hepatocytes with mitochondrial dysfunction. Using reporter gene assay we confirmed that miR-126 directly targeted to IRS-1 3′UTR. Furthermore, the overexpression of miR-126 in hepatocytes caused a substantial reduction in IRS-1 protein expression, and a consequent impairment in insulin signaling. Conclusions/Significance We demonstrated that miR-126 was actively involved in the development of insulin resistance induced by mitochondrial dysfunction. These data provide novel insights into the molecular basis of insulin resistance, and implicate miRNA in the development of metabolic disease.

Stabilin-1 mediates phosphatidylserine-dependent clearance of cell corpses in alternatively activated macrophages

Stabilin-1 is specifically expressed in alternatively activated macrophages. These macrophages participate in anti-inflammatory and healing processes, and display a high phagocytic capacity. In this study, we provide evidence that stabilin-1 is a membrane receptor that performs a crucial function in the clearance of cell corpses. Stabilin-1 is expressed on the cell surface of alternatively activated macrophages and is recruited to the sites of recognition and engulfment of apoptotic bodies, as well as to early phagosomes. Blocking stabilin-1 in macrophages results in defective engulfment of aged red blood cells. Ectopic expression of stabilin-1 induces the binding and engulfment of aged cells in mouse fibroblast L cells. The binding and phagocytosis are dependent on phosphatidylserine (PS), which is well known as an engulfing ligand. Furthermore, using PS-coated beads, we demonstrate that PS directly interacts with stabilin-1 and is sufficient for stabilin-1-mediated phagocytosis. EGF-like domain repeat in stabilin-1 is responsible for PS recognition and binding. Thus, our results demonstrate that stabilin-1, found on alternatively activated macrophages, is a phagocytic receptor mediating the clearance of apoptotic cells in a PS-dependent manner. Therefore, this protein might play an important role in the maintenance of tissue homeostasis and prevention of autoimmunity.

Mechanism for phosphatidylserine-dependent erythrophagocytosis in mouse liver.

Aged or damaged RBCs are effectively removed from the blood circulation by Kupffer cells in the liver, but little is known regarding the mechanism of the clearance process. Here we show that stabilin-1 and stabilin-2 in hepatic sinusoidal endothelial cells (HSECs) are critical in effectively clearing damaged RBCs in mouse liver. Damaged RBCs and phosphatidylserine (PS)-coated beads were effectively sequestered in the hepatic sinusoid regardless of the presence of Kupffer cells, suggesting a role for HSECs in PS-dependent sequestration of PS-exposed RBCs in the liver. HSECs mediate tethering of damaged RBCs in a PS-dependent manner via stabilin-1 and stabilin-2. In a sinusoid-mimicked coculture system consisting of macrophages layered over HSECs, there was significant enhancement of the phagocytic capacity of macrophages, and this was mediated by stabilin-1 and stabilin-2 in HSECs. Liver-specific knockdown of stabilin-1 and stabilin-2 inhibited the sequestration of damaged RBCs in the hepatic sinusoid and delayed the elimination of damaged cells in an in vivo animal model. Thus, the roles of stabilin-1 and stabilin-2 in hepatic sequestration of PS-exposed RBCs may represent a potential mechanism for the clearance of damaged RBCs by Kupffer cells and for the control of some pathologic conditions such as hemolytic anemia.

Epidermal Growth Factor-Like Domain Repeat of Stabilin-2 Recognizes Phosphatidylserine during Cell Corpse Clearance

ABSTRACT Exposure of phosphatidylserine (PS) on the cell surface occurs early during apoptosis and serves as a recognition signal for phagocytes. Clearance of apoptotic cells by a membrane PS receptor is one of the critical anti-inflammatory functions of macrophages. However, the PS binding receptors and their recognition mechanisms have not been fully investigated. Recently, we reported that stabilin-2 is a PS receptor that mediates the clearance of apoptotic cells, thus releasing the anti-inflammatory cytokine, transforming growth factor β. In this study, we showed that epidermal growth factor (EGF)-like domain repeats (EGFrp) in stabilin-2 can directly and specifically recognize PS. The EGFrps also competitively impaired apoptotic cell uptake by macrophages in in vivo models. We also showed that calcium ions are required for stabilin-2 to mediate phagocytosis via EGFrp. Interestingly, at least four tandem repeats of EGF-like domains were required to recognize PS, and the second atypical EGF-like domain in EGFrp was critical for calcium-dependent PS recognition. Considering that PS itself is an important target molecule for both apoptotic cells and nonapoptotic cells during various cellular processes, our results should help elucidate the molecular mechanism by which apoptotic cell clearance in the human body occurs and also have implications for targeting PS externalization of nonapoptotic cells.

Cross Talk between Engulfment Receptors Stabilin-2 and Integrin αvβ5 Orchestrates Engulfment of Phosphatidylserine-Exposed Erythrocytes

ABSTRACT Efficient cell corpse clearance is critical for health in organisms. Apoptotic cells displaying phosphatidylserine (PS) are recognized by engulfment receptors and ingested through two conserved pathways. In one pathway, engulfment receptor brain-specific angiogenesis inhibitor 1 (BAI-1) or integrin functions upstream of ELMO/DOCK180 and activate the small GTPase Rac1. In the other pathway, engulfment receptor CED-1 or stabilin-2 acts in concert with the adaptor protein GULP to activate Rac1. Stabilin-2, a PS receptor, facilitates phagocytosis of apoptotic cells and mediates the production of anti-inflammatory cytokines. Here, we propose that the stabilin-2 extracellular domain consisting of integrin-binding fasciclin 1 (FAS1) domains coordinates the activities of the two phagocytic pathways via direct interactions with integrin. Interactions between stabilin-2 and integrin were determined using biochemical assays, including coimmunoprecipitation and fluorescence resonance energy transfer (FRET). These interactions appear to have functional relevance, since knockdown of endogenous αvβ5 expression or treatment with a function-blocking αvβ5 antibody significantly decreased stabilin-2-mediated phagocytosis in the absence of soluble factors. Our data collectively suggest that the engulfment receptors of the two phagocytic pathways communicate with each other to orchestrate engulfment of damaged erythrocytes. Coordinated phagocytic signaling would be advantageous for physiological and pathological circumstances that require rapid clearance of abnormal (apoptotic or aged) cells.

C1q tumor necrosis factor alpha-related protein isoform 5 is increased in mitochondrial DNA-depleted myocytes and activates AMP-activated protein kinase.

Depletion of mtDNA in myocytes causes insulin resistance and alters nuclear gene expression that may be involved in rescuing processes against cellular stress. Here we show that the expression of C1q tumor necrosis factor α-related protein isoform 5 (C1QTNF5) is drastically increased following depletion of mtDNA in myocytes. C1QTNF5 is homologous to adiponectin in respect to domain structure, and its expression and secretion from myocytes correlated negatively with the cellular mtDNA content. Similar to adiponectin, C1QTNF5 induced the phosphorylation of AMP-activated protein kinase (AMPK), leading to increased cell surface recruitment of GLUT4 and increased glucose uptake. Treatment of cells with purified recombinant C1QTNF5 increased the phosphorylation of acetyl-CoA carboxylase and stimulated fatty acid oxidation. C1QTNF5-mediated phosphorylation of AMPK or acetyl-CoA carboxylase was unaffected by depletion of adiponectin receptors such as AdipoR1 or AdipoR2, which indicated that adiponectin receptors do not participate in C1QTNF5-induced activation of AMPK. Serum C1QTNF5 levels were significantly higher in obese/diabetic animals (OLETF rats, ob/ob mice, and db/db mice). These results highlight C1QTNF5 as a putative biomarker for mitochondrial dysfunction and a potent activator of AMPK.

Chondrocyte-specific ablation of Osterix leads to impaired endochondral ossification.

Osterix (Osx) is an essential transcription factor required for osteoblast differentiation during both intramembranous and endochondral ossification. Endochondral ossification, a process in which bone formation initiates from a cartilage intermediate, is crucial for skeletal development and growth. Osx is expressed in differentiating chondrocytes as well as osteoblasts during mouse development, but its role in chondrocytes has not been well studied. Here, the in vivo function of Osx in chondrocytes was examined in a chondrocyte-specific Osx conditional knockout model using Col2a1-Cre. Chondrocyte-specific Osx deficiency resulted in a weak and bent skeleton which was evident in newborn by radiographic analysis and skeletal preparation. To further understand the skeletal deformity of the chondrocyte-specific Osx conditional knockout, histological analysis was performed on developing long bones during embryogenesis. Hypertrophic chondrocytes were expanded, the formation of bone trabeculae and marrow cavities was remarkably delayed, and subsequent skeletal growth was reduced. The expression of several chondrocyte differentiation markers was reduced, indicating the impairment of chondrocyte differentiation and endochondral ossification in the chondrocyte-specific Osx conditional knockout. Taken together, Osx regulates chondrocyte differentiation and bone growth in growth plate chondrocytes, suggesting an autonomous function of Osx in chondrocytes during endochondral ossification.

Depletion of mitochondrial DNA up-regulates the expression of MDR1 gene via an increase in mRNA stability.

The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the carcinogenesis. However, whether the depletion of mtDNA induces multidrug resistance in cancer cells has not been fully investigated. To elucidate the association of cellular mtDNA content and drug resistance, we generated HCT-8 colon cancer cells which revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. The mtDNA-depleted cells showed a decreased sensitivity and accumulation of anti-cancer drugs, suggesting that mtDNA depletion could develop multidrug resistance (MDR) phenotype in HCT-8 cells. We found that the expression level of MDR1 mRNA and its translated product P-glycoprotein was increased in the mtDNA-depleted cells, indicating that the decrease of sensitivity and accumulation of anti-cancer drug in the mtDNA-depleted cells might be due to a substantial increase in the expression of P-glycoprotein. Furthermore, increased expression of MDR1 mRNA and P-glycoprotein was due to an increase of mRNA stability rather than transcriptional activation. Taken together, these results indicate that mtDNA depletion can induce an increased P-glycoprotein expression via an increase of mRNA stability and suggest that the mtDNA depletion in cancer cells plays an important role in the induction of MDR phenotype.

Stabilin-2 is involved in lymphocyte adhesion to the hepatic sinusoidal endothelium via the interaction with αMβ2 integrin

Although lymphocyte recirculation to the endothelium plays a critical role in the movement of immune cells from the blood into tissues and sites of inflammation, the mechanisms involved in lymphocyte trafficking via the hepatic circulation have yet to be elucidated fully. In this study, we investigated the role of stabilin‐2, which is expressed specifically in the sinusoidal endothelium, in the adhesion of lymphocytes to the hepatic endothelium. Stabilin‐2‐expressing cells mediate the adhesion of PBLs. This interaction was attributed specifically to the interaction of stabilin‐2 with αMβ2 integrin. Using mutant stabilin‐2 molecules with deletions in the extracellular domain, we mapped the binding site for αMβ2 integrin to the fasciclin 1 (FAS1) domains of stabilin‐2. The specificity of the interaction between αMβ2 integrin and the FAS1 domain was confirmed further by binding assays using neutralizing antibodies. More physiologically, we showed that the down‐regulation of stabilin‐2 results in the defective binding of lymphocytes to hepatic sinusoidal endothelial cells under conditions of static and physiological flow. Together, these data show that stabilin‐2 can reconstitute the lymphocyte–endothelial adhesion cascade under physiological shear stress. We propose a critical role for stabilin‐2 in lymphocyte adhesion to specialized endothelia, such as that of the hepatic sinusoid.

NF-κB activates fibronectin gene expression in rat hepatocytes

Abstract Fibronectin (FN) plays a role in various biological processes such as fibrosis and tumor metastasis. In this study, we investigated the regulation of FN gene expression by NF-κB transcription factor. Transient expression of NF-κB p65 increased FN promoter activity in rat hepatocytes. Deletion analysis of FN promoter localized the NF-κB-responsive region at the position between −1214 and −1126. Mutations in a putative NF-κB element (5′-GAGAATTTCC-3′) at −1180 blocked most of the p65-induced promoter activity. Electromobility shift assays showed that the expression of p65 induced the binding of the p65/p65 homodimer to the NF-κB site at −1180. Stably p65-expressing cells showed increase of promoter activity, FN protein, and its mRNA levels over control cells. Furthermore, treatment of cells with interleukin-1β, a NF-κB-stimulating cytokine, also increased promoter activity, FN production, and mRNA levels. These results show that NF-κB activates FN gene expression by binding to the responsive element at −1180 as the p65/p65 homodimer in rat hepatocytes.