Signaling In Epithelial Cells Research Articles

TRIB3 suppresses milk fatty acids metabolism by inhibiting p‐AKT/PPARG signaling in goat mammary epithelial cells

AbstractTribbles pseudokinase 3 (TRIB3) interacts with a variety of proteins and plays a key role in the regulation of glucose metabolism and glycolysis in nonruminants, but whether it has a specific role in goat mammary lipid metabolism has still been kept unknown. In this study, we observed that TRIB3 is highly expressed in the mammary tissues of lactating dairy goats. Overexpressing TRIB3 in goat mammary epithelial cells (GMECs) suppressed the mRNA expression of GPAM, DGAT1, and PLIN1, which are associated with the formation of triacylglycerol and lipid droplets (p < 0.05). The fatty acid‐sensitive transcription regulator PPARG was also downregulated. Interfering TRIB3 had the opposite effect and decreased Akt phosphorylation. The TRIB3 gene influenced fatty acid composition in GMECs, and its overexpression reduced the total concentration of intracellular triacylglycerol (p < 0.01), this response was verified using BODIPY staining. Overall, these data indicated that TRIB3 suppresses milk fatty acids metabolism through inhibiting p‐AKT/PPARG signaling in GMECs.

Retinoblastoma-Binding Protein 9 Suppresses Intestinal Inflammation and Inflammation-Induced Tumorigenesis in Mice

BACKGROUND & AIMSRetinoblastoma-binding protein 9 (RBBP9) was initially reported as cell cycle regulator via RB/E2F. Accumulating evidence has revealed the importance of RBBP9 in physiological and pathological states including inflammatory disease. However, the functional role of RBBP9 in ulcerative colitis (UC) and colitis-associated cancer (CAC) remains elusive. METHODSHuman samples of UC and CAC were examined by immunohistochemical and bioinformatics analyses. We established dextran sodium sulfate (DSS)-induced colitis, azoxymethane (AOM)/DSS-induced CAC model, and ApcMin/+ sporadic tumor model using wild-type and Rbbp9-/- mice. RNA sequencing was analyzed to identify the phenotype alternation upon Rbbp9 deletion. In addition, genetic and pharmacological inhibition of the Janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) pathway was performed. RESULTSThe expression of RBBP9 was reduced in human UC and CAC samples. The loss of RBBP9 enhanced the activation of interferon (IFN)/JAK/STAT1 signaling, resulting in susceptibility to DSS-induced colitis and AOM/DSS-induced CAC tumors by increasing epithelial cell apoptosis and immune activation. An in vitro kinase assay revealed that RBBP9 directly regulated JAK/STAT1 signaling by suppressing STAT1 phosphorylation. A positive feedback loop involving epithelial cell apoptosis, commensal microbiome invasion, and activation of submucosal immune activity was identified in Rbbp9-/- mouse intestines through enhanced JAK/STAT1 signaling in RBBP9-deficient epithelial cells and macrophages. The genetic inhibition of STAT1 or treatment with the JAK/STAT inhibitor reversed epithelial cell apoptosis and mitigated the enhanced susceptibility to DSS-induced colitis in Rbbp9-/- mice. CONCLUSIONSRBBP9 suppresses the intestinal inflammation by negatively regulating JAK/STAT1 signaling pathway.

Abstract C003: Outer membrane vesicles from Bacteroides fragilis contain coding and non-coding small RNA species that modulate inflammatory signaling in intestinal epithelial cells

Abstract Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbial-derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remains unclear. This paper investigates the role of OMVs and OMV-associated small RNA (sRNA) species from pathogenic and commensal Bacteroides fragilis (ETBF and NTBF respectively) in eliciting different immune responses in intestinal epithelial cells. To distinguish the differences in the sRNA profiles of both strains and their OMVs, small RNA-seq was conducted, and identified enrichment of discrete sRNA species in OMVs that were also differentially expressed between the two strains. To understand the effects of OMVs on pattern recognition receptors, Toll-like receptor (TLR) reporter cells were treated with OMVs, and activation of TLRs 2, 3, 4, and 7 were observed. Treatment of Caco-2 and HT29-MTX cells with OMVs demonstrated increased expression of IL-8. Surprisingly, we discovered that degradation of RNase-accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL-8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV-associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications. Citation Format: Aadil Sheikh, Colin Scano, Julia Xu, Tolulope Ojo, Jessica M. Conforti, Kayla L. Haberman, Bryan King, James Lotter, Alysia Martinez, Harry Ojeas, Michelle Pujol, Juli Watkins, Emily Lin, Bernd Zechmann, Amanda Sevcik, Christie Sayes, Elyssia S Gallagher, Joshua Mell, Garth Ehrlich, Joseph H Taube, K. Leigh Greathouse. Outer membrane vesicles from Bacteroides fragilis contain coding and non-coding small RNA species that modulate inflammatory signaling in intestinal epithelial cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C003.

Abstract A041: Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis

Abstract In the tumor microenvironment (TME) cancer cells engage dynamically with the stromal component in promoting tumor progression and metastasis formation that are accompanied by intratumor fibrosis. Extensive extracellular matrix (ECM) remodeling and stiffness boost fibrosis which co-occurs with poor immune infiltration. Very little is known about the cross-interactions between tumor cells and stroma. A better understanding of this dynamic relationship may advance the development of new therapies. Transforming growth factor (TGF)-β signaling in both cancer epithelial cells and stroma is crucial for the establishment of intratumor fibrosis that enhances tumor progression. We previously discovered that in cancer cells, TGF-β signaling synergizes with RAS signal to regulate classical EMT transcription factors (Snail, Zeb1) and fibrogenic factors (Il11, Pdgf, Ctgfb, Wisp and others). This synergistic program is required for metastasis formation in pancreatic and lung adenocarcinoma (LUAD) models. However, how this dual program is affecting the stromal population in vivo remains unknown. Additionally, the role of each TGF-β dependent fibrogenic factor in regulating the stroma is still unclear. Using metastasis in vivo assays, we tested the metastatic ability of LUAD cells wild-type in comparison to Knock-Out cells targeting each one of the TGF-β dependent fibrogenic factors. We found that these factors are drivers of metastasis. More interestingly, these fibrogenic factors have a preferential effect depending on the metastatic site, suggesting that survival of the cancer cells in a certain tissue is dependent on the stromal niche within the site, which will act in favor or not of their growth. We also deeply characterize the stroma of micro-metastatic lesions by using high-throughput multiplex immunofluorescence to analyze the whole tissue TME at single cell resolution and found that lack of any of these fibrogenic factors compromise the establishment of a robust ECM at the very beginning of the metastatic colonization, to favor immune infiltration and consequently lead to cancer cell clearance. Together, these findings shed light on the potential role of these specific fibrogenic factors in metastasis, how their absence impacts the TME and how targeting these factors could be beneficial in metastatic disease. Citation Format: Elena Spina, Liangji Li, Junho Lee, Joan Massaguè. Deciphering the crosstalk between cancer cells and stroma that drives EMT- coupled fibrosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A041.

Transcriptional regulators ensuring specific gene expression and decision-making at high TGFβ doses.

TGFβ-signaling regulates cancer progression by controlling cell division, migration, and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision-making toward specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing, and morphological assays to quantitatively link signaling, gene expression, and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that most of the TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision-making.

PLNC8 αβ Potently Inhibits the Flavivirus Kunjin and Modulates Inflammatory and Intracellular Signaling Responses of Alveolar Epithelial Cells.

PLNC8 αβ is a cationic antimicrobial peptide that previously has been reported to express both antibacterial and antiviral properties. This study aimed to further elucidate the antiviral effects of PLNC8 αβ and its impact on virus-induced cytotoxicity and inflammatory signaling in human alveolar epithelial cells (A549) infected with the flavivirus Kunjin. Complementary in silico analyses using molecular dynamics (MD) simulation were conducted to investigate the mechanism of action of PLNC8 αβ by studying the interaction of PLNC8 α and β with models of a flavivirus membrane and a eukaryotic plasma membrane, respectively. Our findings demonstrated that PLNC8 αβ significantly reduces both extracellular and intracellular viral loads, as confirmed by plaque reduction assays and RT-PCR. The peptide also mitigated virus-induced cytotoxicity and inflammation. Notably, PLNC8 αβ modulated the virus-induced dysregulation of key signaling and inflammatory genes, such as TLR9, TLR3, NOD2, FOS, JUN, IL6, and CXCL8. MD simulation revealed that PLNC8 αβ exhibits higher binding affinity for a flavivirus membrane model compared to a model of the plasma membrane, likely due to stronger electrostatic interactions with anionic phospholipids. This selective interaction possibly accounts for a potent antiviral activity of PLNC8 αβ combined with a minimal cytotoxicity toward human cells. Overall, PLNC8 αβ shows significant promise as an antiviral agent against flavivirus infections and warrants further exploration for peptide-based antiviral therapies.

Apoptotic extracellular vesicles carrying Mif regulate macrophage recruitment and compensatory proliferation in neighboring epithelial stem cells during tissue maintenance.

Apoptotic cells can signal to neighboring cells to stimulate proliferation and compensate for cell loss to maintain tissue homeostasis. While apoptotic cell-derived extracellular vesicles (AEVs) can transmit instructional cues to mediate communication with neighboring cells, the molecular mechanisms that induce cell division are not well understood. Here, we show that macrophage migration inhibitory factor (Mif)-containing AEVs regulate compensatory proliferation via ERK signaling in epithelial stem cells of larval zebrafish. Time-lapse imaging showed efferocytosis of AEVs from dying epithelial stem cells by healthy neighboring stem cells. Proteomic and ultrastructure analysis of purified AEVs identified Mif localization on the AEV surface. Pharmacological inhibition or genetic mutation of Mif, or its cognate receptor CD74, decreased levels of phosphorylated ERK and compensatory proliferation in the neighboring epithelial stem cells. Disruption of Mif activity also caused decreased numbers of macrophages patrolling near AEVs, while depletion of the macrophage lineage resulted in a reduced proliferative response by the epithelial stem cells. We propose that AEVs carrying Mif directly stimulate epithelial stem cell repopulation and guide macrophages to cell non-autonomously induce localized proliferation to sustain overall cell numbers during tissue maintenance.

Zinc pyrithione ameliorates colitis in mice by interacting on intestinal epithelial TRPA1 and TRPV4 channels

AimsAlthough zinc pyrithione (ZPT) has been studied as topical antimicrobial and cosmetic consumer products, little is known about its pharmacological actions in gastrointestinal (GI) health and inflammation. Our aims were to investigate the effects of ZPT on transient receptor potential (TRP) channels and Ca2+ signaling in intestinal epithelial cells (IECs) and its therapeutic potential for colitis. Main methodsDigital Ca2+ imaging and patch-clamp electrophysiology were performed on human colonic epithelial cells (HCoEpiC) and rat small intestinal epithelial cells (IEC-6). The transcription levels of proinflammatory cytokines such as IL-1β were detected by RTq-PCR. Dextran sulfate sodium (DSS) was used to induce colitis in mice. Key findingsZPT dose-dependently induced Ca2+ signaling and membrane currents in IECs, which were attenuated by selective blockers of transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 4 (TRPV4) channels, respectively. Interestingly, Ca2+ entry via TRPA1 channels inhibited the activity of TRPV4 channels in HCoEpiC, but not vice versa. ZPT significantly promoted migration of IECs by activating TRPA1 and TRPV4 channels. ZPT reversed lipopolysaccharides (LPS)-induced changes in mRNA expression of TRPA1 and TRPV4. Moreover, ZPT decreased mRNA levels of pro-inflammatory factors promoted by LPS in HCoEpiC, which were restored by selective TRPA1 blocker. In whole animal studies in vivo, ZPT significantly ameliorated DSS-induced body weight loss, colon shortening and increases in stool score, serum calprotectin and lactic acid (LD) in mouse model of colitis. SignificanceZPT exerts anti-colitic action likely by anti-inflammation and pro-mucosal healing through TRP channels in IECs. The present study not only expands pharmacology spectrum of ZPT in GI tract, but also repurposes it to a potential drug for colitis therapy.

LMTK2 switches on canonical TGF-β1 signaling in human bronchial epithelial cells.

Transforming growth factor (TGF-β1) is a critical profibrotic mediator in chronic lung disease, and there are no specific strategies to mitigate its adverse effects. Activation of TGF-β1 signaling is a multipart process involving ligands, transmembrane receptors, and transcription factors. In addition, an intricate network of adaptor proteins fine-tunes the signaling strength, duration, and activity. Namely, Smad7 recruits growth arrest and DNA damage (GADD34) protein that then interacts with the catalytic subunit of phosphoprotein phosphatase 1 (PP1c) to inactivate TGF-β receptor (TβR)-I and downregulate TGF-β1 signaling. Little is known about how TGF-β1 releases TβR-I from the GADD34-PP1c inhibition to activate its signaling. Transmembrane lemur tyrosine kinase 2 (LMTK2) is a PP1c inhibitor, and our published data showed that TGF-β1 recruits LMTK2 to the cell surface. Here, we tested the hypothesis that TGF-β1 recruits LMTK2 to inhibit PP1c, allowing activation of TβR-I. First, LMTK2 interacted with the TGF-β1 pathway in the human bronchial epithelium at multiple checkpoints. Second, TGF-β1 inhibited PP1c by an LMTK2-dependent mechanism. Third, TGF-β1 used LMTK2 to activate canonical Smad3-mediated signaling. We propose a model whereby the LMTK2-PP1c and Smad7-GADD34-PP1c complexes serve as on-and-off switches in the TGF-β1 signaling in human bronchial epithelium.NEW & NOTEWORTHY Activation of the transforming growth factor (TGF)-β1 signaling pathway is complex, involving many ligands, transmembrane receptors, transcription factors, and modulating proteins. The mechanisms of TGF-β1 signaling activation/inactivation are not fully understood. We propose for the first time a model by which transmembrane lemur tyrosine kinase 2 (LMTK2) forms a complex with phosphoprotein phosphatase 1 (PP1c) to activate TGF-β1 signaling and Smad7, growth arrest and DNA damage (GADD34), and PP1C form a complex to inactivate TGF-β1 signaling in human bronchial epithelium.

Abstract C012: Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant Pancreatic Cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of only 12%, with limited treatment options. Oncogenic KRAS mutations drive PDAC tumor progression, which occur in the majority of PDAC tumors. KRAS G12D is the most common KRAS mutation, found in 36% of PDAC cases, which is associated with the worst clinical outcomes. Mutant-selective KRAS G12D inhibitors (KRASi G12D), such as MRTX1133, have recently demonstrated initial responses in KRAS G12D-mutant preclinical models. However, mechanisms of adaptive resistance to KRAS G12D inhibition are largely under-investigated and novel therapeutic strategies to overcome drug resistance are urgently needed. Results: Adaptive RAS signaling reactivation was found to dampen KRAS G12D inhibition in a library of KRAS G12D PDAC cell lines, thereby minimizing drug efficacy. Quantitative downstream analyses of drug combination screening using a KRAS G12D cell line library further identified this adaption to KRAS inhibition was cell subtype specific. Fibroblast growth factor receptor (FGFRs) drove adaptive RAS reactivation specifically in mesenchymal PDAC cells, which are aggressive, and intrinsically resistant to KRASi. In contrast, epithelial growth factor receptor (EGFR) reactivated RAS G12D signaling in epithelial cells, which are sensitive to KRASi G12D. Genetic and pharmacological inhibition of FGFR1 or FGFR2 improved drug efficacy by decreasing FGFR-mediated RAS reactivation specifically in mesenchymal PDAC lines. Pharmacological blockade of EGFR enhanced drug efficacy by inhibiting EGFR-mediated RAS reactivation specifically in epithelial PDAC lines. Furthermore, low-concentration of a novel pan- RAS inhibitor, which inhibits both wild-type and mutant RAS, completely abrogated MAPK signaling and significantly improved efficacy, when combined with KRASi G12D, suggesting a pro-survival dependency on wild-type RAS as a compensatory mechanism in response to KRAS G12D inhibition. Conclusion: Upstream RTK-mediated adaptive RAS signaling re-activation in response to KRAS inhibition in PDAC is cell subtype specific. Complete inhibition of RAS signaling by overcoming adaptive RAS rebound improves efficacy of KRASi G12D. Citation Format: Qingxiang (Nick) Lin, Alvin Morales, Haley Barnes, Ryan Bruce Corcoran. Overcoming Cell Subtype-Specific Adaptive Resistance to KRAS G12D Inhibition in KRAS G12D-Mutant Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C012.

Abstract A051: Innate immune cell dynamics and pancreatic tumor progression in alcoholic pancreatitis

Abstract Introduction: Persistent inflammation due to alcoholic chronic pancreatitis (ACP) is associated with pancreatic ductal adenocarcinoma (PDAC). Myeloid cell infiltration within the pancreas, particularly with oncogenic mutant KrasG12D/+ (*Kras), is crucial for driving PDAC carcinogenesis. Our previous work demonstrated that hyperactivation of cyclic AMP response element binding protein (CREB) accelerates ADM/PanIN progression with heightened fibroinflammation. However, the association between CREB in precursor neoplastic lesions and innate myeloid cells remains unclear. Methods: We established an ACP mouse model using an alcohol diet along with prolonged caerulein administration in Ptf1aCreERTM/+; LSL-KrasG12D/+ (KC), and CREB deleted [Crebfl/fl in KC (KCC-/-)] mice. Single-cell RNA sequencing (scRNA-seq) of the mouse pancreas, multiparametric immunophenotyping of tumor-infiltrating myeloid cells and chromatin immunoprecipitation (ChIP-seq) with CREB pulldown were performed. Cytokine arrays and qPCR studies followed co-culture assays using mouse neutrophils (J774M) and macrophages (RAW 264.7) with epithelial cell lines harboring *Kras with in vitro ACP induction were conducted. Results: ACP induction in KC mice led to increased infiltration of neutrophils and alternatively activated macrophages with the pancreas. Conversely, Creb-deleted mice exhibited significantly reduced myeloid cell infiltration despite ACP induction. ChIP-Seq and qPCR identified CREB-regulated chemokines and immunomodulators as mediators of myeloid cell trafficking. Co-culture of mouse neutrophils and macrophages with KC-ACP conditioned media significantly increased IL-1 receptor antagonist (Il-1ra) expression and secretion. Single-cell transcriptomic analysis revealed heightened Il1rn mRNA levels in neutrophils and macrophages in KC-ACP when compared with KC pancreatic tumors. Creb-deleted PDAC models showed strong downregulation of Il1rn in macrophages, confirming CREB’s role in modulating myeloid cell interactions via epithelial cell signaling. Conclusions: Our findings highlight that CREB activation in epithelial cells under ACP conditions facilitates crosstalk with myeloid cells, promoting IL-1ra upregulation in neutrophils and macrophages. This CREB-driven regulation of IL-1ra in myeloid cells contributes to inflammation-associated tumorigenesis. Targeting this axis may offer a novel therapeutic approach to curb pancreatic cancer progression. Citation Format: Siddharth Mehra, Sudhakar Jinka, Varun Krishnamoorthy, Supriya Srinivasan, Anna Bianchi, Andrew Adams, Haleh Amirian, Manan Patel, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. Innate immune cell dynamics and pancreatic tumor progression in alcoholic pancreatitis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A051.

Proteomic Profile of Circulating Extracellular Vesicles in the Brain after Δ9-Tetrahydrocannabinol Inhalation.

Given the increasing use of cannabis in the US, there is an urgent need to better understand the drug's effects on central signaling mechanisms. Extracellular vesicles (EVs) have been identified as intercellular signaling mediators that contain a variety of cargo, including proteins. Here, we examined whether the main psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), alters EV protein signaling dynamics in the brain. We first conducted in vitro studies, which found that THC activates signaling in choroid plexus epithelial cells, resulting in transcriptional upregulation of the cannabinoid 1 receptor and immediate early gene c-fos, in addition to the release of EVs containing RNA cargo. Next, male and female rats were examined for the effects of either acute or chronic exposure to aerosolized ('vaped') THC on circulating brain EVs. Cerebrospinal fluid was extracted from the brain, and EVs were isolated and processed with label-free quantitative proteomic analyses via high-resolution tandem mass spectrometry. Interestingly, circulating EV-localized proteins were differentially expressed based on acute or chronic THC exposure in a sex-specific manner. Taken together, these findings reveal that THC acts in the brain to modulate circulating EV signaling, thereby providing a novel understanding of how exogenous factors can regulate intercellular communication in the brain.

Insoluble HIFa protein aggregates by cadmium disrupt hypoxia-prolyl hydroxylase (PHD)-hypoxia inducible factor (HIFa) signaling in renal epithelial (NRK-52E) and interstitial (FAIK3-5) cells

The kidney is the main organ that senses changes in systemic O2 pressure by hypoxia-PHD-HIFa (HPH) signaling, resulting in adaptive target gene activation, including erythropoietin (EPO). The non-essential transition metal cadmium (Cd) is nephrotoxic and disrupts the renal HPH pathway, which may promote Cd-associated chronic renal disease (CKD). A deeper molecular understanding of Cd interference with renal HPH signaling is missing, and no data with renal cell lines are available. In rat kidney NRK-52E cells, which model the proximal tubule, and murine fibroblastoid atypical interstitial kidney (FAIK3-5) cells, which mimic renal EPO-producing cells, the chemical hypoxia mimetic dimethyloxalylglycine (DMOG; 1 mmol/l) or hypoxia (1% O2) activated HPH signaling. Cd2+ (2.5–20 µmol/l for ≤ 24 h) preferentially induced necrosis (trypan blue uptake) of FAIK3-5 cells at high Cd whereas NRK-52E cells specially developed apoptosis (PARP-1 cleavage) at all Cd concentrations. Cd (12.5 µmol/l) abolished HIFa stabilization and prevented upregulation of target genes (quantitative real-time polymerase chain reaction and immunoblotting) induced by DMOG or hypoxia in both cell lines, which was caused by the formation of insoluble HIFa aggregates. Strikingly, hypoxic preconditioning (1% O2 for 18 h) reduced apoptosis of FAIK3-5 and NRK-52E cells at low Cd concentrations and decreased insoluble HIFa proteins. Hence, drugs mimicking hypoxic preconditioning could reduce CKD induced by chronic low Cd exposure.

HPV18 E7 inhibits LATS1 kinase and activates YAP1 by degrading PTPN14.

High-risk human papillomavirus (HPV) oncoproteins inactivate cellular tumor suppressors to reprogram host cell signaling pathways. HPV E7 proteins bind and degrade the tumor suppressor PTPN14, thereby promoting the nuclear localization of the YAP1 oncoprotein and inhibiting keratinocyte differentiation. YAP1 is a transcriptional coactivator that drives epithelial cell stemness and self-renewal. YAP1 activity is inhibited by the highly conserved Hippo pathway, which is frequently inactivated in human cancers. MST1/2 and LATS1/2 kinases form the core of the Hippo kinase cascade. Active LATS1 kinase is phosphorylated on threonine 1079 and inhibits YAP1 by phosphorylating it on amino acids including serine 127. Here, we tested the effect of high-risk (carcinogenic) HPV18 E7 on Hippo pathway activity. We found that either PTPN14 knockout or PTPN14 degradation by HPV18 E7 decreased the phosphorylation of LATS1 T1079 and YAP1 S127 in human keratinocytes and inhibited keratinocyte differentiation. Conversely, PTPN14-dependent differentiation required LATS kinases and certain PPxY motifs in PTPN14. Neither MST1/2 kinases nor the putative PTPN14 phosphatase active sites were required for PTPN14 to promote differentiation. Together, these data support that PTPN14 inactivation or degradation of PTPN14 by HPV18 E7 reduce LATS1 activity, promoting active YAP1 and inhibiting keratinocyte differentiation.IMPORTANCEThe Hippo kinase cascade inhibits YAP1, an oncoprotein and driver of cell stemness and self-renewal. There is mounting evidence that the Hippo pathway is targeted by tumor viruses including human papillomavirus. The high-risk HPV E7 oncoprotein promotes YAP1 nuclear localization and the carcinogenic activity of high-risk HPV E7 requires YAP1 activity. Blocking HPV E7-dependent YAP1 activation could inhibit HPV-mediated carcinogenesis, but the mechanism by which HPV E7 activates YAP1 has not been elucidated. Here we report that by degrading the tumor suppressor PTPN14, HPV18 E7 inhibits LATS1 kinase, reducing inhibitory phosphorylation on YAP1. These data support that an HPV oncoprotein can inhibit Hippo signaling to activate YAP1 and strengthen the link between PTPN14 and Hippo signaling in human epithelial cells.

Galectin-1 Attenuates PDGF-Mediated AKT Signaling in Retinal Pigment Epithelial Cells.

Galectins have the potential to interact with transmembrane glycoproteins to modulate their functions. Since galectin-1 interacts with PDGF-Rβ, we analyzed the effect of galectin-1 on PDGF-BB-mediated AKT signaling in primary human retinal pigment epithelial (RPE) cells and galectin-1-deficient immortalized human RPE cells (LGALS1-/-/ARPE-19) following incubation with PDGF-BB and galectin-1. Expression and localization of galectin-1, PDGF-Rβ and pAKT were investigated using western blot analysis and immunohistochemical staining. Cell proliferation of RPE cells was analyzed using BrdU ELISA. Following treatment of human RPE cells with human recombinant (hr)-galectin-1 and PDGF-BB, an intense clustering of PDGF-Rβ and colocalization with galectin-1 were detected. By Western blot analysis and immunocytochemistry of human RPE cells, an enhanced PDGF-BB-mediated expression of pAKT was observed, which was substantially reduced by additional incubation with hr-galectin-1. Vice versa, in LGALS1-/-/ARPE-19 cells, the PDGF-BB-induced pAKT signal was enhanced compared to wild-type cells. Furthermore, a decreased expression of PDGF-Rβ in human RPE cells was observed after treatment with PDGF-BB and hr-galectin-1, while in untreated LGALS1-/-/ARPE-19 cells, its constitutive expression was increased. In addition, after treatment of RPE cells with hr-galectin-1, the PDGF-BB-induced proliferation was markedly reduced. In summary, galectin-1 has the distinct potential to reduce PDGF-mediated pAKT signaling and proliferation in human RPE cells-an effect that is most likely facilitated via a decreased expression of PDGF-Rβ.

ASB3 expression aggravates inflammatory bowel disease by targeting TRAF6 protein stability and affecting the intestinal microbiota.

Ubiquitination is a key process that controls protein stability. We determined the ubiquitination of TRAF6 by ASB3 in intestinal epithelial cells during colonic inflammation. Inflammatory bowel disease patients exhibit upregulated ASB3 expression at focal sites, supporting the involvement of degradation of TRAF6, which promotes TLR-Myd88/TRIF-independent NF-κB aberrant activation and intestinal microbiota imbalance. Sustained inflammatory signaling in intestinal epithelial cells and dysregulated protective probiotic immune responses mediated by ASB3 collectively contribute to the exacerbation of inflammatory bowel disease. These findings provide insights into the pathogenesis of inflammatory bowel disease and suggest a novel mechanism by which ASB3 increases the risk of colitis. Our results suggest that future inhibition of ASB3 in intestinal epithelial cells may be a novel clinical strategy.

ISGylation enhances dsRNA-induced interferon response and NFκB signaling in fallopian tube epithelial cells

Heritable mutations in BRCA1 associate with increased risk of high-grade serous tubo-ovarian cancer (HGSTOC). Non-genetic risk factors associated with this cancer, which arises from fallopian tube epithelial (FTE) cells, suggests a role for repetitive ovulation wherein FTE cells are exposed to inflammatory signaling molecules within follicular fluid. We previously reported increased NFκB and EGFR signaling in BRCA1-deficient primary FTE cells, with follicular fluid exposure further increasing abundance of interferon-stimulated gene (ISG) transcripts, including the ubiquitin-like protein ISG15 and other ISGylation pathway members. Both NFκB and type I interferon signaling are upregulated by stimulation of cGAS-STING or MDA5 and RIGI pattern recognition receptors (PRRs). Since some PRRs and their signal transduction pathway members are ISGylated, we tested the impact of ISG15 and ISGylation on IRF3 and NFκB signaling through cGAS-STING or RIGI and MDA5 activation. Expression of ISG15 or UBA7, the E1-like ISG15 activating enzyme, in immortalized FTE cells was disrupted by CRISPR gene editing. Activation of IRF3 by RIGI or MDA5 but not cGAS-STING was attenuated by loss of either ISG15 or UBA7 and this was reflected by a similar effect on NFκB activation and downstream targets. Loss of ISGylation decreased levels of both MDA5 and RIGI, with knock-down of RIGI but not MDA5, decreasing IRF3 and NFκB activation in parental cells. These finding indicate that ISGylation enhances the ability of dsRNA to activate cytokine release and pro-inflammatory signaling. Further work to explore ISGylation as a target for prevention of HGSTOC in BRCA1 mutation carriers is warranted.

Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells

Intracellular pH (pHi) dynamics regulate normal cell function, and dysregulated pHi dynamics is an emerging hallmark of cancer (constitutively increased pHi) and neurodegeneration (constitutively decreased pHi). However, the molecular mechanisms by which pHi dynamics regulate cell biology are poorly understood. Here, we discovered that altering pHi in normal human breast epithelial cells triggers global transcriptional changes. We identified 176 genes differentially regulated by pHi, with pHi-dependent genes clustering in signaling and glycolytic pathways. Using various normal epithelial cell models, we showed pH-dependent Notch homolog 1 protein expression, with increased protein abundance at high pHi. This resulted in pH-dependent downstream signaling, with increased Notch homolog 1 signaling at high pHi. We also found that high pHi increased the expression of glycolytic enzymes and regulators of pyruvate fate, including lactate dehydrogenase and pyruvate dehydrogenase kinase. These transcriptional changes were sufficient to alter lactate production, with high pHi shifting these normal epithelial cells toward a glycolytic metabolism and increasing lactate production. Thus, pHi dynamics transcriptionally regulate signaling and metabolic pathways in normal epithelial cells. Our data reveal new molecular regulators of pHi-dependent biology and a role for increased pHi in driving the acquisition of cancer-associated signaling and metabolic changes in normal human epithelial cells.

Efficacy and Mechanism of Core Traditional Chinese Medicines for Treating Malignant Lymphoma based on Efficacy Studies: A Study Supported by Network Pharmacology and Molecular Docking.

The burden of malignant lymphoma in China is greater than the global equivalent. The randomized controlled trials provide medical evidence that TCM can improve the response and survival in patients with lymphoma. However, the mechanisms underlying remain undefined. Evidence-based data mining for traditional Chinese medicine (TCM) on improving response and survival in malignant lymphoma treatment was performed in this study. In addition, the mechanisms of TCM through network pharmacology and molecular docking were explored. The China national knowledge infrastructure, Wanfang Data, China Science and Technology Journal Database, PubMed, and Web of Science databases were searched to select TCM formulas with response and survival benefits in the treatment of malignant lymphomas. We then analyzed and visualized the tropism of taste, frequency of drug use, dosage, clustering, association rules mining (minimum support threshold as 0.20, the minimum confidence threshold as 0.80 and lift >1), and complex networks for potential core herb compositions using Excel, IBM SPSS Statistics 26, and IBM SPSS Modeler 18. TCM systems pharmacology, GeneCards, Online Mendelian Inheritance in Man, and other databases were used to screen potential core active ingredients and malignant lymphoma-related targets. The intersection targets were used to construct a protein interaction network using Cytoscape to obtain the key targets. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were used to analyze the core target, and molecular docking of key components and targets was performed using CB-Dock2. Twenty-four Chinese herbal formulae were included, encompassing 107 herbs with mainly cold and warm properties and bitter and sweet flavors. They were associated with the yin meridians of the liver, spleen, and lungs. The TCMs underwent association rule analysis, identified 27 association rules, including 12 herb pairs and 13 angle medicine, and clustered into eight classes by clustering analysis. Combined with the results from mining analysis, Pinelliae (Ban-xia), Poria (Fu-ling), Atractylodis macrocephalae (Bai-zhu), Curcumae (E-zhu), and Sparganii (San-leng) were the potential core herbs According to network pharmacology and molecular docking, the main core components of the potential core drugs are hederagenin, cerevisterol, 14- acetyl-12-senecioyl-2E,8E,10E-atractylentriol, 12,13-epoxy-9-hydroxynonadeca-7,10-dienoic acid, cavidine, and baicalein. These core drugs are mainly involved in the pathways of EGFR tyrosine kinase inhibitor resistance, PD-1/L1, natural killer cell-mediated cytotoxicity, NF-κB, epithelial cell signaling in H. pylori infections, and Th17 cell differentiation. They aid in regulating the transmembrane receptor protein tyrosine kinase signaling pathway, ERBB signaling pathway, PI3K signaling pathway, and phosphorylation process. Ten key components and eight key targets, including baicalein and hederagenin, demonstrated strong binding activity. Collectively, some core herbs exerted anti-tumor effects through immune and inflammatory pathway modulation, inhibition of immune escape, and induction of cell apoptosis. These findings support future evidence-based research on malignant lymphoma treatment using TCM.

Inflammatory gene regulation by Cdc42 in airway epithelial cells

Cytokine release from airway epithelial cells is a key immunological process that coordinates an immune response in the lungs. We propose that the Rho GTPase, Cdc42, regulates both transcription and trafficking of cytokines, ultimately affecting the essential process of cytokine release and subsequent inflammation in the lungs. Here, we examined the pro-inflammatory transcriptional profile that occurs in bronchial epithelial cells (BEAS-2B) in response to TNF-α using RNA-Seq and differential gene expression analysis. To interrogate the role of Cdc42 in inflammatory gene expression, we used a pharmacological inhibitor of Cdc42, ML141, and determined changes in the transcriptomic profile induced by Cdc42 inhibition. Our results indicated that Cdc42 inhibition with ML141 resulted in a unique inflammatory phenotype concomitant with increased gene expression of ER stress genes, Golgi membrane and vesicle transport genes. To further interrogate the inflammatory pathways regulated by Cdc42, we made BEAS-2B knockdown strains for the signaling targets TRIB3, DUSP5, SESN2 and BMP4, which showed high differential expression in response to Cdc42 inhibition. Depletion of DUSP5 and TRIB3 reduced the pro-inflammatory response triggered by Cdc42 inhibition as shown by a reduction in cytokine transcript levels. Depletion of SESN2 and BMP4 did not affect cytokine transcript level, however, Golgi fragmentation was reduced. These results provide further evidence that in airway epithelial cells, Cdc42 is part of a signaling network that controls inflammatory gene expression and secretion by regulating Golgi integrity.Summary sentence:We define the Cdc42-regulated gene networks for inflammatory signaling in airway epithelial cells which includes regulation of ER stress response and vesicle trafficking pathways.