Mitochondrial stress activates YAP/TAZ through RhoA oxidation to promote liver injury.

Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1; also known as TAZ) are the main effectors of the Hippo pathway and their dysregulation contributes to diseases in tissues including the liver. Although mitochondria are capable of transmitting signals to change transcriptomic landscape of diseased hepatocytes, such retrograde signaling and the related nuclear machinery are largely unknown. Here, we show that increased YAP activity is associated with mitochondrial stress during liver injury; and this is required for secondary inflammation, promoting hepatocyte death. Mitochondrial stress inducers robustly promoted YAP/TAZ dephosphorylation, nuclear accumulation, and target gene transcription. RNA sequencing revealed that the majority of mitochondrial stress transcripts required YAP/TAZ. Mechanistically, direct oxidation of RhoA by mitochondrial superoxide was responsible for PP2A-mediated YAP/TAZ dephosphorylation providing a novel physiological input for the Hippo pathway. Hepatocyte-specific Yap/Taz ablation suppressed acetaminophen-induced liver injury and blunted transcriptomic changes associated with the pathology. Our observations uncover unappreciated pathway of mitochondrial stress signaling and reveal YAP/TAZ activation as the mechanistic basis for liver injury progression.

Hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation.

BACKGROUND: Interferon Regulatory Factor 3 (IRF3) is a transcription factor that plays a crucial role in the innate immune response by recognizing and responding to foreign antigens. Recently, its roles in sterile conditions are being studied, as in metabolic and fibrotic diseases. However, the search on the upstream regulator for efficient pharmacological targeting is yet to be fully explored. Here, we show that G protein-coupled receptors (GPCRs) can regulate IRF3 phosphorylation through of GPCR-Gα protein interaction. RESULTS: IRF3 and target genes were strongly associated with fibrosis markers in liver fibrosis patients and models. Conditioned media from MIHA hepatocytes overexpressing IRF3 induced fibrogenic activation of LX-2 hepatic stellate cells (HSCs). In an overexpression library screening using active mutant Gα subunits and Phos-tag immunoblotting, Gαs was found out to strongly phosphorylate IRF3. Stimulation of Gαs by glucagon or epinephrine or by Gαs-specific designed GPCR phosphorylated IRF3. Protein kinase A (PKA) signaling was primarily responsible for IRF3 phosphorylation and Interleukin 33 (IL-33) expression downstream of Gαs. PKA phosphorylated IRF3 on a previously unrecognized residue and did not require reported upstream kinases such as TANK-binding kinase 1 (TBK1). Activation of Gαs signaling by glucagon induced IL-33 production in hepatocytes. Conditioned media from the hepatocytes activated HSCs, as indicated by α-SMA and COL1A1 expression, and this was reversed by pre-treatment of the media with IL-33 neutralizing antibody. CONCLUSIONS: Gαs-coupled GPCR signaling increases IRF3 phosphorylation through cAMP-mediated activation of PKA. This leads to an increase of IL-33 expression, which further contributes to HSC activation. Our findings that hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation provides an insight for understanding the complex intercellular communication during liver fibrosis progression and suggests therapeutic opportunities for the disease. Video Abstract.

Identification of the primary ciliary proteins IFT38 and IFT144 to enhance serum-mediated YAP activation and cell proliferation.

Primary cilia are essential cellular antennae that transmit external signals into intracellular responses. These sensory organelles perform crucial tasks in triggering intracellular signaling pathways, including those initiated by G protein-coupled receptors (GPCRs). Given the involvement of GPCRs in serum-induced signaling, we investigated the contribution of ciliary proteins in mitogen perception and cell proliferation. We found that depletion of cilia via IFT88 silencing impaired cell growth and repressed YAP activation against serum and its mitogenic constituents, namely lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). To identify the key player of serum mitogen signaling, a mutant cell line library with 30 ablated individual ciliary proteins was established and screened based on YAP dephosphorylation and target gene induction. While 9 of them had altered signaling, ablation of IFT38 or IFT144 led to a particularly robust repression of YAP activation upon LPA and S1P. The deficiency of IFT38 and IFT144 attenuated cell proliferation, as corroborated in either 2-dimensional cultures or tumor spheroids. In subcutaneous skin melanoma patients, expression of IFT38 and IFT144 was associated with unfavorable outcomes in overall survival. In conclusion, our study demonstrates the involvement of ciliary proteins in mitogen signaling and identifies the regulatory roles of IFT38 and IFT144 in serum-mediated Hippo pathway signaling and cellular growth.

Identification of Small GTPases That Phosphorylate IRF3 through TBK1 Activation Using an Active Mutant Library Screen.

Interferon regulatory factor 3 (IRF3) integrates both immunological and non-immunological inputs to control cell survival and death. Small GTPases are versatile functional switches that lie on the very upstream in signal transduction pathways, of which duration of activation is very transient. The large number of homologous proteins and the requirement for site-directed mutagenesis have hindered attempts to investigate the link between small GTPases and IRF3. Here, we constructed a constitutively active mutant expression library for small GTPase expression using Gibson assembly cloning. Small-scale screening identified multiple GTPases capable of promoting IRF3 phosphorylation. Intriguingly, 27 of 152 GTPases, including ARF1, RHEB, RHEBL1, and RAN, were found to increase IRF3 phosphorylation. Unbiased screening enabled us to investigate the sequence-activity relationship between the GTPases and IRF3. We found that the regulation of IRF3 by small GTPases was dependent on TBK1. Our work reveals the significant contribution of GTPases in IRF3 signaling and the potential role of IRF3 in GTPase function, providing a novel therapeutic approach against diseases with GTPase overexpression or active mutations, such as cancer.

Shikonin Exerts Cytotoxic Effects in Human Colon Cancers by Inducing Apoptotic Cell Death via the Endoplasmic Reticulum and Mitochondria-Mediated Pathways

The apoptotic effects of shikonin (5,8-dihydroxy-2-[(1R)-1-hydroxy-4-methylpent-3-enyl]naphthalene-1,4-dione) on the human colon cancer cell line SNU-407 were investigated in this study. Shikonin showed dose-dependent cytotoxic activity against SNU-407 cells, with an estimated IC50 value of 3 µM after 48 h of treatment. Shikonin induced apoptosis, as evidenced by apoptotic body formation, sub-G1 phase cells, and DNA fragmentation. Shikonin induced apoptotic cell death by activating mitogen-activated protein kinase family members, and the apoptotic process was mediated by the activation of endoplasmic reticulum (ER) stress, leading to activation of the PERK/elF2α/CHOP apoptotic pathway, and mitochondrial Ca2+ accumulation. Shikonin increased mitochondrial membrane depolarization and altered the levels of apoptosis-related proteins, with a decrease in B cell lymphoma (Bcl)-2 and an increase in Bcl-2-associated X protein, and subsequently, increased expression of cleaved forms of caspase-9 and -3. Taken together, we suggest that these mechanisms, including MAPK signaling and the ER-and mitochondria-mediated pathways, may underlie shikonin-induced apoptosis related to its anticancer effect.

7,8-Dihydroxyflavone Protects High Glucose-Damaged Neuronal Cells against Oxidative Stress

Oxidative stress is considered a major contributor in the pathogenesis of diabetic neuropathy and in diabetes complications, such as nephropathy and cardiovascular diseases. Diabetic neuropathy, which is the most frequent complications of diabetes, affect sensory, motor, and autonomic nerves. This study aimed to investigate whether 7,8-dihydroxyflavone (7,8-DHF) protects SH-SY5Y neuronal cells against high glucose-induced toxicity. In the current study, we found that diabetic patients exhibited higher lipid peroxidation caused by oxidative stress than healthy subjects. 7,8-DHF exhibits superoxide anion and hydroxyl radical scavenging activities. High glucose-induced toxicity severely damaged SH-SY5Y neuronal cells, causing mitochondrial depolarization; however, 7,8-DHF recovered mitochondrial polarization. Furthermore, 7,8-DHF effectively modulated the expression of pro-apoptotic protein (Bax) and anti-apoptotic protein (Bcl-2) under high glucose, thus inhibiting the activation of caspase signaling pathways. These results indicate that 7,8-DHF has antioxidant effects and protects cells from apoptotic cell death induced by high glucose. Thus, 7,8-DHF may be developed into a promising candidate for the treatment of diabetic neuropathy.

Purpurogallin Protects Keratinocytes from Damage and Apoptosis Induced by Ultraviolet B Radiation and Particulate Matter 2.5

Purpurogallin, a natural phenol obtained from oak nutgalls, has been shown to possess antioxidant, anticancer, and anti-inflammatory effects. Recently, in addition to ultraviolet B (UVB) radiation that induces cell apoptosis via oxidative stress, particulate matter 2.5 (PM(2.5)) was shown to trigger excessive production of reactive oxygen species. In this study, we observed that UVB radiation and PM(2.5) severely damaged human HaCaT keratinocytes, disrupting cellular DNA, lipids, and proteins and causing mitochondrial depolarization. Purpurogallin protected HaCaT cells from apoptosis induced by UVB radiation and/or PM(2.5). Furthermore, purpurogallin effectively modulates the pro-apoptotic and anti-apoptotic proteins under UVB irradiation via caspase signaling pathways. Additionally, purpurogallin reduced apoptosis via MAPK signaling pathways, as demonstrated using MAPK-p38, ERK, and JNK inhibitors. These results indicate that purpurogallin possesses antioxidant effects and protects cells from damage and apoptosis induced by UVB radiation and PM(2.5).

A Benzylideneacetophenone Derivative Induces Apoptosis of Radiation-Resistant Human Breast Cancer Cells via Oxidative Stress

Benzylideneacetophenone derivative (1E)-1-(4-hydroxy-3-methoxyphenyl) hept-1-en-3-one (JC3) elicited cytotoxic effects on MDA-MB 231 human breast cancer cells-radiation resistant cells (MDA-MB 231-RR), in a dose-dependent manner, with an IC₅₀ value of 6 μM JC3. JC3-mediated apoptosis was confirmed by increase in sub-G1 cell population. JC3 disrupted the mitochondrial membrane potential, and reduced expression of anti-apoptotic B cell lymphoma-2 protein, whereas it increased expression of pro-apoptotic Bcl-2-associated X protein, leading to the cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase. In addition, JC3 activated mitogen-activated protein kinases, and specific inhibitors of these kinases abrogated the JC3-induced increase in apoptotic bodies. JC3 increased the level of intracellular reactive oxygen species and enhanced oxidative macromolecular damage via lipid peroxidation, protein carbonylation, and DNA strand breakage. Considering these findings, JC3 is an effective therapy against radiation-resistant human breast cancer cells.

Transcatheter closure of a residual shunt after surgical repair of traumatic ventricular septal defect.

We report a patient with a significant residual left-to-right shunt who underwent surgical repair for traumatic VSD. This 13-year-old boy was stabbed with a pair of scissors through his heart. After initial life-saving surgery with lacerated left ventricular wall repair and VSD closure, residual VSD was noted. Six months later, we performed a successful transcatheter closure of the residual VSD with an Amplatzer muscular VSD occluder.