Eupatolide, isolated from Liriodendron tulipifera, sensitizes TNF-mediated dual modes of apoptosis and necroptosis by disrupting RIPK1 ubiquitination.

Ubiquitination of RIPK1 plays an essential role in the recruitment of the IKK complex, an upstream component of pro-survival NF-κB. It also limits TNF-induced programmed cell death by inhibiting the spatial transition from TNFR1-associated complex-I to RIPK1-dependent death-inducing complex-II or necrosome. Thus, the targeted disruption of RIPK1 ubiquitination, which induces RIPK1-dependent cell death, has proven to be a useful strategy for improving the therapeutic efficacy of TNF. In this study, we found that eupatolide, isolated from Liriodendron tulipifera, is a potent activator of the cytotoxic potential of RIPK1 by disrupting the ubiquitination of RIPK1 upon TNFR1 ligation. Analysis of events upstream of NF-κB signaling revealed that eupatolide inhibited IKKß-mediated NF-κB activation while having no effect on IKKα-mediated non-canonical NF-κB activation. Pretreatment with eupatolide drastically interfered with RIPK1 recruitment to the TNFR1 complex-I by disrupting RIPK1 ubiquitination. Moreover, eupatolide was sufficient to upregulate the activation of RIPK1, facilitating the TNF-mediated dual modes of apoptosis and necroptosis. Thus, we propose a novel mechanism by which eupatolide activates the cytotoxic potential of RIPK1 at the TNFR1 level and provides a promising anti-cancer therapeutic approach to overcome TNF resistance.

All-in-one IQ toggle switches with high versatilities for fine-tuning of transgene expression in mammalian cells and tissues.

The transgene toggling device is recognized as a powerful tool for gene- and cell-based biological research and precision medicine. However, many of these devices often operate in binary mode, exhibit unacceptable leakiness, suffer from transgene silencing, show cytotoxicity, and have low potency. Here, we present a novel transgene switch, SIQ, wherein all the elements for gene toggling are packed into a single vector. SIQ has superior potency in inducing transgene expression in response to tebufenozide compared with the Gal4/UAS system, while completely avoiding transgene leakiness. Additionally, the ease and versatility of SIQ make it possible with a single construct to perform transient transfection, establish stable cell lines by targeting a predetermined genomic locus, and simultaneously produce adenovirus for transduction into cells and mammalian tissues. Furthermore, we integrated a cumate switch into SIQ, called SIQmate, to operate a Boolean AND logic gate, enabling swift toggling-off of the transgene after the removal of chemical inducers, tebufenozide and cumate. Both SIQ and SIQmate offer precise transgene toggling, making them adjustable for various researches, including synthetic biology, genome engineering, and therapeutics.

Rubiarbonol B induces RIPK1-dependent necroptosis via NOX1-derived ROS production.

The activation of receptor-interacting protein kinase 1 (RIPK1) by death-inducing signaling complex (DISC) formation is essential for triggering the necroptotic mode of cell death under apoptosis-deficient conditions. Thus, targeting the induction of necroptosis by modulating RIPK1 activity could be an effective strategy to bypass apoptosis resistance in certain types of cancer. In this study, we screened a series of arborinane triterpenoids purified from Rubia philippinesis and identified rubiarbonol B (Ru-B) as a potent caspase-8 activator that induces DISC-mediated apoptosis in multiple types of cancer cells. However, in RIPK3-expressing human colorectal cancer (CRC) cells, the pharmacological or genetic inhibition of caspase-8 shifted the mode of cell death by Ru-B from apoptosis to necroptosis though upregulation of RIPK1 phosphorylation. Conversely, Ru-B-induced cell death was almost completely abrogated by RIPK1 deficiency. The enhanced RIPK1 phosphorylation and necroptosis triggered by Ru-B treatment occurred independently of tumor necrosis factor receptor signaling and was mediated by the production of reactive oxygen species via NADPH oxidase 1 in CRC cells. Thus, we propose Ru-B as a novel anticancer agent that activates RIPK1-dependent cell death via ROS production, and suggest its potential as a novel necroptosis-targeting compound in apoptosis-resistant CRC.

Octyl syringate is preferentially cytotoxic to cancer cells via lysosomal membrane permeabilization and autophagic flux inhibition.

The autophagy-mediated lysosomal pathway plays an important role in conferring stress tolerance to tumor cells during cellular stress such as increased metabolic demands. Thus, targeted disruption of this function and inducing lysosomal cell death have been proved to be a useful cancer therapeutic approach. In this study, we reported that octyl syringate (OS), a novel phenolic derivate, was preferentially cytotoxic to various cancer cells but was significantly less cytotoxic to non-transformed cells. Treatment with OS resulted in non-apoptotic cell death in a caspase-independent manner. Notably, OS not only enhanced accumulation of autophagic substrates, including lapidated LC3 and sequestosome-1, but also inhibited their degradation via an autophagic flux. In addition, OS destabilized the lysosomal function, followed by the intracellular accumulation of the non-digestive autophagic substrates such as bovine serum albumin and stress granules. Furthermore, OS triggered the release of lysosomal enzymes into the cytoplasm that contributed to OS-induced non-apoptotic cell death. Finally, we demonstrated that OS was well tolerated and reduced tumor growth in mouse xenograft models. Taken together, our study identifies OS as a novel anticancer agent that induces lysosomal destabilization and subsequently inhibits autophagic flux and further supports development of OS as a lysosome-targeting compound in cancer therapy. • Octyl syringate, a phenolic derivate, is preferentially cytotoxic to various cancer cells. • Octyl syringate destabilizes the lysosomal function. • Octyl syringate blocks the autophagic flux. • Octyl syringate is a potential candidate compound for cancer therapy.

Chemical modulation of SQSTM1/p62-mediated xenophagy that targets a broad range of pathogenic bacteria.

The N-degron pathway is a proteolytic system in which the N-terminal degrons (N-degrons) of proteins, such as arginine (Nt-Arg), induce the degradation of proteins and subcellular organelles via the ubiquitin-proteasome system (UPS) or macroautophagy/autophagy-lysosome system (hereafter autophagy). Here, we developed the chemical mimics of the N-degron Nt-Arg as a pharmaceutical means to induce targeted degradation of intracellular bacteria via autophagy, such as Salmonella enterica serovar Typhimurium (S. Typhimurium), Escherichia coli, and Streptococcus pyogenes as well as Mycobacterium tuberculosis (Mtb). Upon binding the ZZ domain of the autophagic cargo receptor SQSTM1/p62 (sequestosome 1), these chemicals induced the biogenesis and recruitment of autophagic membranes to intracellular bacteria via SQSTM1, leading to lysosomal degradation. The antimicrobial efficacy was independent of rapamycin-modulated core autophagic pathways and synergistic with the reduced production of inflammatory cytokines. In mice, these drugs exhibited antimicrobial efficacy for S. Typhimurium, Bacillus Calmette-Guérin (BCG), and Mtb as well as multidrug-resistant Mtb and inhibited the production of inflammatory cytokines. This dual mode of action in xenophagy and inflammation significantly protected mice from inflammatory lesions in the lungs and other tissues caused by all the tested bacterial strains. Our results suggest that the N-degron pathway provides a therapeutic target in host-directed therapeutics for a broad range of drug-resistant intracellular pathogens.Abbreviations: ATG: autophagy-related gene; BCG: Bacillus Calmette-Guérin; BMDMs: bone marrow-derived macrophages; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CFUs: colony-forming units; CXCL: C-X-C motif chemokine ligand; EGFP: enhanced green fluorescent protein; IL1B/IL-1ß: interleukin 1 beta; IL6: interleukin 6; LIR: MAP1LC3/LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Mtb: Mycobacterium tuberculosis; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PB1: Phox and Bem1; SQSTM1/p62: sequestosome 1; S. Typhimurium: Salmonella enterica serovar Typhimurium; TAX1BP1: Tax1 binding protein 1; TNF: tumor necrosis factor; UBA: ubiquitin-associated.

IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish.

Though various transgene expression switches have been adopted in a wide variety of organisms for basic and biomedical research, intrinsic obstacles of those existing systems, including toxicity and silencing, have been limiting their use in vertebrate transgenesis. Here we demonstrate a novel QF-based binary transgene switch (IQ-Switch) that is relatively free of driver toxicity and transgene silencing, and exhibits potent and highly tunable transgene activation by the chemical inducer tebufenozide, a non-toxic lipophilic molecule to developing zebrafish with negligible background. The interchangeable IQ-Switch makes it possible to elicit ubiquitous and tissue specific transgene expression in a spatiotemporal manner. We generated a RASopathy disease model using IQ-Switch and demonstrated that the RASopathy symptoms were ameliorated by the specific BRAF(V600E) inhibitor vemurafenib, validating the therapeutic use of the gene switch. The orthogonal IQ-Switch provides a state-of-the-art platform for flexible regulation of transgene expression in zebrafish, potentially applicable in cell-based systems and other model organisms.

8-Geranylumbelliferone isolated from Paramignya trimera triggers RIPK1/RIPK3-dependent programmed cell death upon TNFR1 ligation.

In tumor necrosis factor (TNF) signaling, IκB kinase (IKK) complex-mediated activation of NF-κB is a well-known protective mechanism against cell death via transcriptional induction of pro-survival genes occurring as a late checkpoint. However, recent belief holds that IKK functions as an early cell death checkpoint to suppress the death-inducing signaling complex by regulating receptor interacting protein kinase1 (RIPK1) phosphorylation. In this study, we propose that two major gernaylated 7-hydroxy coumarins, 6-geranyl-7-hydroxycoumarin (ostruthin) and 8-geranyl-7-hydroxycoumarin (8-geranylumbelliferone, 8-GU) isolated from Paramignya timera, facilitate RIPK1-dependent dual modes of apoptosis and necroptosis by targeting IKKß upon TNF receptor1 (TNFR1) ligation. Analysis of events upstream of NF-κB revealed that 8-GU and ostruthin drastically inhibited TNF-induced IKK phosphorylation, while having no effect on TAK1 phosphorylation and TNFR1 complex-I formation. Interestingly, 8-GU did not affect the cell death induced by Fas ligand or TNF-related apoptosis-inducing ligand or that induced by DNA-damaging agents, indicating that 8-GU sensitizes TNF-induced cell death exclusively. Moreover, 8-GU accelerated TNF-driven necroptosis by up-regulating necrosome formation in FADD deficient cancer cells harboring RIPK3. Thus, the present study provides new insights into the molecular mechanism underlying geranylated 7-hydroxy coumarin-mediated control of the RIPK1-dependent early cell death checkpoint and suggests that 8-GU is a potential anti-cancer therapeutic via an alternative apoptosis-independent strategy to overcome TNF resistance.

3-O-acetylrubianol C (3AR-C) induces RIPK1-dependent programmed cell death by selective inhibition of IKKß.

In tumor necrosis factor (TNF) signaling, phosphorylation and activation of receptor interacting protein kinase 1 (RIPK1) by upstream kinases is an essential checkpoint in the suppression of TNF-induced cell death. Thus, discovery of pharmacological agents targeting RIPK1 may provide new strategies for improving the therapeutic efficacy of TNF. In this study, we found that 3-O-acetylrubianol C (3AR-C), an arborinane triterpenoid isolated from Rubia philippinesis, promoted TNF-induced apoptotic and necroptotic cell death. To identify the molecular mechanism, we found that in mouse embryonic fibroblasts, 3AR-C drastically upregulated RIPK1 kinase activity by selectively inhibiting IKKß. Notably, 3AR-C did not interfere with IKKα or affect the formation of the TNF receptor1 (TNFR1) complex-I. Moreover, in human cancer cells, 3AR-C was only sufficient to sensitize TNF-induced cell death when c-FLIPL expression was downregulated to facilitate the formation of TNFR1 complex-II and necrosome. Taken together, our study identified a novel arborinane triterpenoid 3AR-C as a potent activator of TNF-induced cell death via inhibition of IKKß phosphorylation and promotion of the cytotoxic potential of RIPK1, thus providing a rationale for further development of 3AR-C as a selective IKKß inhibitor to overcome TNF resistance in cancer therpay.

Tropomyosin-receptor kinase fused gene (TFG) regulates lipid production in human sebocytes.

The endoplasmic reticulum (ER) is an organelle in which important cellular events such as protein synthesis and lipid production occur. Although many lipid molecules are produced in the ER, the effect of ER-organizing proteins on lipid synthesis in sebocytes has not been completely elucidated. Tropomyosin-receptor kinase fused gene (TFG) is located in ER exit sites and participates in COPII-coated vesicle formation along with many scaffold proteins, such as Sec. 13 and Sec. 16. In this study, we investigated the putative role of TFG in lipid production in sebocytes using an immortalized human sebocyte line. During IGF-1-induced lipogenesis, the level of the TFG protein was increased in a time- and dose-dependent manner. When TFG was over-expressed using recombinant adenovirus, lipid production in sebocytes was increased along with an up-regulation of the expression of lipogenic regulators, such as PPAR-γ, SREBP-1 and SCD. Conversely, down-regulation of TFG using a microRNA (miR) decreased lipid production and the expression of lipogenic regulators. Based on these data, TFG is a novel regulator of lipid synthesis in sebocytes.

Induction of alopecia areata in C3H/HeJ mice using polyinosinic-polycytidylic acid (poly[I:C]) and interferon-gamma.

Alopecia areata (AA) is a chronic, relapsing hair-loss disorder that is considered to be a T-cell-mediated autoimmune disease. Several animal models for AA have been created to investigate the pathophysiology and screen for effective therapeutic targets. As C3H/HeJ mice develop AA spontaneously in a low frequency, a novel animal model is needed to establish an AA-like condition faster and more conveniently. In this study, we present a novel non-invasive AA rodent model that avoids skin or lymph-node cell transfer. We simply injected C3H/HeJ mice subcutaneously with interferon-gamma (IFNγ) along with polyinosinic:polycytidylic acid (poly[I:C]), a synthetic dsRNA, to initiate innate immunity via inflammasome activation. Approximately 80% of the IFNγ and poly(I:C) co-injected mice showed patchy AA lesions after 8 weeks. None of the mice displayed hair loss in the IFNγ or poly(I:C) solely injection group. Immunohistochemical staining of the AA lesions revealed increased infiltration of CD4+ and CD8+ cells infiltration around the hair follicles. IFNγ and poly(I:C) increased the expression of NLRP3, IL-1ß, CXCL9, CXCL10, and CXCL11 in mouse skin. Taken together, these findings indicate a shorter and more convenient means of AA animal model induction and demonstrate that inflammasome-activated innate immunity is important in AA pathogenesis.

Possible Role of Single Stranded DNA Binding Protein 3 on Skin Hydration by Regulating Epidermal Differentiation.

BACKGROUND: Skin hydration is a common problem both in elderly and young people as dry skin may cause irritation, dermatological disorders, and wrinkles. While both genetic and environmental factors seem to influence skin hydration, thorough genetic studies on skin hydration have not yet been conducted. OBJECTIVE: We used a genome-wide association study (GWAS) to explore the genetic elements underlying skin hydration by regulating epidermal differentiation and skin barrier function. METHODS: A GWAS was conducted to investigate the genetic factors influencing skin hydration in 100 Korean females along with molecular studies of genes in human epidermal keratinocytes for functional study in vitro. RESULTS: Among several single nucleotide polymorphisms identified in GWAS, we focused on Single Stranded DNA Binding Protein 3 (SSBP3) which is associated with DNA replication and DNA damage repair. To better understand the role of SSBP3 in skin cells, we introduced a calcium-induced differentiation keratinocyte culture system model and found that SSBP3 was upregulated in keratinocytes in a differentiation dependent manner. When SSBP3 was overexpressed using a recombinant adenovirus, the expression of differentiation-related genes such as loricrin and involucrin was markedly increased. CONCLUSION: Taken together, our results suggest that genetic variants in the intronic region of SSBP3 could be determinants in skin hydration of Korean females. SSBP3 represents a new candidate gene to evaluate the molecular basis of the hydration ability in individuals.

Erratum: Possible Role of Single Stranded DNA Binding Protein 3 on Skin Hydration by Regulating Epidermal Differentiation.

[This corrects the article on p. 432 in vol. 30, PMID: 30065583.].

Correction: Adiponectin Signaling Regulates Lipid Production in Human Sebocytes.

[This corrects the article DOI: 10.1371/journal.pone.0169824.].

Effects of Brn2 overexpression on eccrine sweat gland development in the mouse paw.

Eccrine sweat glands regulate body temperature by secreting water and electrolytes. In humans, eccrine sweat glands are ubiquitous in the skin, except in the lips and external genitalia. In mice, eccrine sweat glands are present only in the paw pad. Brn2 is a protein belonging to a large family of transcription factors. A few studies have examined Brn2 in melanoma cells and epidermal keratinocytes. This study investigated changes in the skin in the K5-Brn2 transgenic mouse, which overexpresses Brn2 and contains the keratin 5 promotor. Interestingly, the volume of eccrine sweat glands was reduced markedly in the K5-Brn2 transgenic mouse compared with the wild-type, while the expression of aquaporin 5, important molecule in sweat secretion, was increased in each sweat gland cell, probably to compensate for the reduction in gland development. However, sweating response to a pilocarpine injection in the hind paw was significantly decreased in the K5-Brn2 transgenic mouse compared with the wild-type. The paw epidermis was thicker in the K5-Brn2 transgenic mouse compared with the wild-type. Taken together, eccrine sweat gland development and sweat secretion were suppressed markedly in the K5-Brn2 transgenic mouse. These results may be associated with dominant development of the epidermis by Brn2 overexpression in the paw skin.

UCHL1 Regulates Melanogenesis through Controlling MITF Stability in Human Melanocytes.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is involved in many signaling pathways via the ubiquitin-proteasome system. UCHL1 is expressed in the human skin and serves as a neuronal marker; however, its functions in melanogenesis remain unknown. Here, we investigated the role of UCHL1 in melanogenesis and elucidated the underlying mechanism using human melanocytes. UCHL1 downregulation by small interfering RNA resulted in upregulation of microphthalmia-associated transcription factor (MITF), tyrosinase, dopachrome tautomerase, tyrosinase-related protein-1, and melanin. In contrast, overexpression of UCHL1 in melanocytes via adenovirus transfection led to downregulation of tyrosinase, dopachrome tautomerase, and tyrosinase-related protein-1 and decreased melanin contents. Furthermore, UCHL1 reduced the protein, but not mRNA, levels of MITF, the upstream regulator of tyrosinase, dopachrome tautomerase, and tyrosinase-related protein-1. Inhibition of de novo protein synthesis and treatment of normal human primary epidermal melanocytes with proteasome inhibitor MG132 revealed that UCHL1 negatively regulates the stability of MITF by binding to the ubiquitinated protein. Finally, overexpression of MITF via an adenovirus restored the level of melanogenesis reduced by UCHL1. Collectively, our findings indicate a role of UCHL1 in regulating skin pigmentation. Suppression of MITF activity by UCHL1 via protein degradation might aid in the development of new therapeutic approaches for melanoma or dyspigmentation disorders.

Double-stranded RNA induces inflammation via the NF-κB pathway and inflammasome activation in the outer root sheath cells of hair follicles.

Alopecia areata (AA), a chronic, relapsing, hair-loss disorder, is considered to be a T cell-mediated autoimmune disease. It affects approximately 1.7% of the population, but its precise pathogenesis remains to be elucidated. Despite the recent attention focused on the roles of inflammasomes in the pathogenesis of autoinflammatory diseases, little is known about inflammasome activation in AA. Thus, in this study, we investigated the pattern of NLRP3 inflammasome activation in the outer root sheath (ORS) cells of hair follicles. We found that interleukin (IL)-1ß and caspase-1 expression was increased in hair follicle remnants and inflammatory cells of AA tissue specimens. After stimulation of ORS cells with the double-stranded (ds)RNA mimic polyinosinic:polycytidylic acid (poly[I:C]), the activation of caspase-1 and secretion of IL-1ß were enhanced. Moreover, NLRP3 knockdown decreased this poly(I:C)-induced IL-1ß production. Finally, we found that high-mobility group box 1 (HMGB1) translocated from the nucleus to the cytosol and was secreted into the extracellular space by inflammasome activation. Taken together, these findings suggest that ORS cells are important immunocompetent cells that induce NLRP3 inflammasomes. In addition, dsRNA-induced IL-1ß and HMGB1 secretion from ORS cells may contribute to clarifying the pathogenesis and therapeutic targets of AA.

Targeted deletion of Crif1 in mouse epidermis impairs skin homeostasis and hair morphogenesis.

The epidermis, which consists mainly of keratinocytes, acts as a physical barrier to infections by regulating keratinocyte proliferation and differentiation. Hair follicles undergo continuous cycling to produce new one. Therefore, optimum supply of energy from the mitochondria is essential for maintaining skin homeostasis and hair growth. CRIF1 is a mitochondrial protein that regulates mitoribosome-mediated synthesis and insertion of mitochondrial oxidative phosphorylation polypeptides into the mitochondrial membrane in mammals. Recent studies reveal that conditional knockout (cKO) of Crif1 in specific tissues of mice induced mitochondrial dysfunction. To determine whether the mitochondrial function of keratinocytes affects skin homeostasis and hair morphogenesis, we generated epidermis-specific Crif1 cKO mice. Deletion of Crif1 in epidermis resulted in impaired mitochondrial function and Crif1 cKO mice died within a week. Keratinocyte proliferation and differentiation were markedly inhibited in Crif1 cKO mice. Furthermore, hair follicle morphogenesis of Crif1 cKO mice was disrupted by down-regulation of Wnt/ß-catenin signaling. These results demonstrate that mitochondrial function in keratinocytes is essential for maintaining epidermal homeostasis and hair follicle morphogenesis.