Organic Anion Transporting Polypeptide (OATP) 1B3 is a Significant Transporter for Hepatic Uptake of Conjugated Bile Acids in Humans.

BACKGROUND & AIMS: OATP1B3/SLCO1B3 is a human liver-specific transporter for the clearance of endogenous compounds (eg, bile acid [BA]) and xenobiotics. The functional role of OATP1B3 in humans has not been characterized, as SLCO1B3 is poorly conserved among species without mouse orthologs. METHODS: Slc10a1-knockout (Slc10a1-/-), Slc10a1hSLCO1B3 (endogenous mouse Slc10a1 promoter-driven human-SLCO1B3 expression in Slc10a1-/- mice), and human SLCO1B3 liver-specific transgenic (hSLCO1B3-LTG) mice were generated and challenged with 0.1% ursodeoxycholic-acid (UDCA), 1% cholic-acid (CA) diet, or bile duct ligation (BDL) for functional studies. Primary hepatocytes and hepatoma-PLC/RPF/5 cells were used for mechanistic studies. RESULTS: Serum BA levels in Slc10a1-/- mice were substantially increased with or without 0.1% UDCA feeding compared with wild-type (WT) mice. This increase was attenuated in Slc10a1hSLCO1B3-mice, indicating that OATP1B3 functions as a significant hepatic BA uptake transporter. In vitro assay using primary hepatocytes from WT, Slc10a1-/-, and Slc10a1hSLCO1B3-mice indicated that OATP1B3 has a similar capacity in taking up taurocholate/TCA as Ntcp. Furthermore, TCA-induced bile flow was significantly impaired in Slc10a1-/- mice but partially recovered in Slc10a1hSLC01B3-mice, indicating that OATP1B3 can partially compensate the NTCP function in vivo. Liver-specific overexpression of OATP1B3 markedly increased the level of hepatic conjugated BA and cholestatic liver injury in 1% CA-fed and BDL mice. Mechanistic studies revealed that conjugated BAs stimulated Ccl2 and Cxcl2 in hepatocytes to increase hepatic neutrophil infiltration and proinflammatory cytokine production (eg, IL-6), which activated STAT3 to repress OATP1B3 expression by binding to its promoter. CONCLUSIONS: Human OATP1B3 is a significant BA uptake transporter and can partially compensate Ntcp for conjugated BA uptake in mice. Its downregulation in cholestasis is an adaptive protective response.

Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling.

BACKGROUND AND AIMS: Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. APPROACH AND RESULTS: Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5- ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1 -P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2 , and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid-induced CCL2 and CXCL2 in hepatocytes. CONCLUSIONS: This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.

Hepatic TNFRSF12A promotes bile acid-induced hepatocyte pyroptosis through NFκB/Caspase-1/GSDMD signaling in cholestasis.

Tumor necrosis factor receptor superfamily member-12A (TNFRSF12A) plays a critical role in inflammation and cell death. It is expressed in multiple tissues yet extremely low in normal liver. To date, little is known about its role in cholestasis. Therefore, we sought to delineate the role of TNFRSF12A in cholestasis and its underlying mechanisms. Human liver tissues were collected from patients with obstructive cholestasis (OC) or primary biliary cholangitis (PBC). Tnfrsf12a knockout (KO) mice were generated. Cholestasis was induced by bile-duct ligation (BDL) or 0.1% 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-feeding. Human hepatoma PLC/PRF/5-ASBT and THP1 cell lines or primary mouse hepatocytes were used for mechanistic studies. Hepatic TNFRSF12A expression was markedly increased in OC or PBC patients. Genetic ablation of Tnfrsf12a in BDL- and 0.1%DDC-induced cholestatic mice significantly attenuated cholestatic liver injury with remarkable reduction of hepatocyte pyroptosis but without changing scores of necroptosis and apoptosis. Morphological features of hepatocyte pyroptosis and increased levels of pyroptosis-related proteins, NLRP3, cleaved-Caspase-1, and cleaved-GSDMD in OC patients and BDL-mice confirmed this observation. Further mechanistic studies revealed that bile acids (BAs) induced TNFRSF12A expression by enhancing the transcription factor c-JUN binding to the TNFRSF12A promoter and subsequently initiated hepatocyte pyroptosis by the NFκB/Caspase-1/GSDMD signaling. Interestingly, TWEAK, a typical ligand of TNFRSF12A, secreted by infiltrated macrophages in cholestatic livers, enhanced TNFRSF12A-induced hepatocyte pyroptosis. Taken together, we report, for the first time, that hepatic TNFRSF12A is dramatically increased in human cholestasis. Deletion of TNFRSF12A inhibits BAs-induced hepatocyte pyroptosis through the NFκB/Caspase-1/GSDMD signaling and thereby ameliorates cholestatic liver injury. As such, targeting TNFRSF12A could be a promising approach to treating cholestasis.

Ferulic acid regulates miR-17/PTEN axis to inhibit LPS-induced pulmonary microvascular endothelial cells apoptosis through activation of PI3K/Akt pathway.

Acute lung injury (ALI) is mainly mediated by the damage of pulmonary microvascular endothelial cells (PMVECs). LPS is one of the pathogenic factors leading to microcirculatory abnormalities of ALI. Ferulic acid (FA) exhibits therapeutic effects against various diseases. During lipopolysaccharide-induced acute respiratory distress syndrome, FA, when given beforehand, could depress inflammation and oxidative stress. However, the concrete role and underlying mechanism of FA in ALI have not been well characterized. Ten µg/mL Lipopolysaccharide (LPS) was used to treat rat PMVECs for 24 hr. qRT-PCR was used to detect the level of miR-17 and phosphatase and tensin homolog deleted on chromosome ten (PTEN). Western blot was used to analyze the associated proteins in the PI3K/Akt pathway, and the apoptosis-related proteins. Flow cytometric analysis was performed to detect the apoptosis of PMVECs. MTT assay was constructed to detect the cell viability. Luciferase assay was conducted to detect the target gene of miR-17 and PTEN. A cell model for in vitro studying the role of FA in ALI was established using PMVECs. Our data demonstrate that FA up-regulates miR-17 and declines apoptosis induced by LPS. FA inhibits apoptosis mediated by up-regulating miR-17. Furthermore, we found miR-17 targeted PTEN negatively. FA inhibits cleaved caspase-3 and Bax expression through the PI3K/Akt pathway mediated by up-regulating miR-17. Over-expression of PTEN could contribute to the similar expression trend of the PI3K/Akt signal pathway protein compared to miR-17 inhibitor transfected cells. FA inhibits PMVECs apoptosis induced by LPS via miR-17/PTEN to further regulate the activation of the PI3K/Akt pathway in ALI. We anticipate that our data will provoke additional studies for ALI clinical therapy.

Robust Least-SquareLocalization Based on Relative Angular Matrix in Wireless Sensor Networks.

Accurate position information plays an important role in wireless sensor networks (WSN), and cooperative positioning based on cooperation among agents is a promising methodology of providing such information. Conventional cooperative positioning algorithms, such as least squares (LS), rely on approximate position estimates obtained from prior measurements. This paper explores the fundamental mechanism underlying the least squares algorithm's sensitivity to the initial position selection and approaches to dealing with such sensitivity. This topic plays an essential role in cooperative positioning, as it determines whether a cooperative positioning algorithm can be implemented ubiquitously. In particular, a sufficient and unnecessary condition for the least squares cost function to be convex is found and proven. We then propose a robust algorithm for wireless sensor network positioning that transforms the cost function into a globally convex function by detecting the null space of the relative angle matrix when all the targets are located inside the convex polygon formed by its neighboring nodes. Furthermore, we advance one step further and improve the algorithm to apply it in both the time of arrival (TOA) and angle of arrival/time of arrival (AOA/TOA) scenarios. Finally, the performance of the proposed approach is quantified via simulations, and the results show that the proposed method has a high positioning accuracy and is robust in both line-of-sight (LOS) and non-line-of-sight (NLOS) positioning environments.