Cellular Signalling

{beta}-arrestins 1 and 2 are intracellular adaptor proteins that regulate the subcellular localisation and signalling of GPCRs including Parathyroid Hormone Receptor 1 (PTH1R), which produces {beta}-arrestin-dependent signalling that may selectively stimulate bone anabolism. {beta}-arrestins can themselves be regulated by post-translational modifications including phosphorylation, which dictates the translocation and signalling of the few GPCRs studied in this context to date. We aimed to investigate whether phosphorylation of {beta}-arrestins modulates their subcellular translocation in response to PTH1R activation. Translocation of {beta}-arrestins to the plasma membrane and early endosomes was quantified using BRET biosensors and immunocytochemistry with high content analysis. Phosphoknockout or phosphomimetic mutations were introduced to previously demonstrated phosphorylation sites in {beta}-arrestins 1 and 2, initially in bulk to assess overall effects. Plasma membrane translocation was altered only subtly, whereas early endosome translocation was markedly reduced for both mutations but to a greater degree for the phosphomimetic. Contributions of individual or paired sites were assessed by restoration to wildtype from the bulk phosphomimetic mutants. Dual restoration of S13 and T275 in {beta}-arrestin 1, or the analogous sites in {beta}-arrestin 2, restored the greatest degrees of early endosomal translocation, with complete restoration for {beta}-arrestin 1 and partial for {beta}-arrestin 2. Restoration of other sites had minimal or no effect. Dephosphorylation therefore appears to be required for PTH1R-stimulated {beta}-arrestin early endosomal translocation, with different phosphorylation sites implicated than some other GPCRs. These findings raise the possibility of differential {beta}-arrestin regulation by distinct stimuli which may have implications in therapeutic targeting of {beta}-arrestin-dependent pathways.

Myocardin (MYOCD) is the principal trigger-switch for vascular smooth muscle cell (VSMC) growth arrest and the cyto-contractile program of differentiation. While much is known about MYOCD-dependent VSMC differentiation, limited information exists as to its transcriptional regulation. Here, we report moderate mRNA expression of Myeloid Ecotropic viral Integration Site 1 (MEIS1) in both human and mouse VSMCs. MEIS1 mRNA is reduced in parallel with myocardin mRNA and protein under conditions of VSMC de-differentiation. Loss- and gain-of-Meis1 expression studies in cultured human VSMCs and the mouse carotid artery support MEIS1 as an activator of myocardin and VSMC differentiation. CRISPR-mediated knockout of the endogenous Meis1 gene in vitro and elicited lower levels of VSMC contractile markers and an increase in growth rate. Similarly, Femoral artery wire injury, in mice lacking Meis1 showed and increased of neointima formation. Mechanistically, we identified a conserved MEIS1 response element in the proximal promoter of myocardin, that appears to contribute to the promoter activity, suggesting a potential regulatory interaction. These results suggest a previously unrecognized role for the homeodomain protein MEIS1 in the activation of myocardin and the quiescent, VSMC differentiated state.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Background: Pulmonary arterial hypertension (PAH) manifests by pulmonary vascular remodeling, caused by hyper-proliferation and apoptosis resistance of resident PA cells, elevated right ventricular (RV) afterload, and death of RV failure. The goal of this study is to determine the role and mechanisms of action of mitotic spindle-associated microtubule regulator DLG associated protein 5 (DLGAP5) in pulmonary vascular remodeling in PAH. Methods: DLGAP5 expression was assessed in human PAH transcriptomic datasets, PAH patients' lung tissues, pulmonary artery smooth muscle cells (PASMCs), and pulmonary artery adventitial fibroblasts (PAAFs). Loss-of-function, molecular, functional, RNAseq, and network analyses were performed. Smooth muscle (SM)-specific DLGAP5 knockdown using adeno-associated virus serotype 6 (AAV6) in mice with SU5416/hypoxia-induced PH was performed. Results: DLGAP5 was overaccumulated in small muscular PAs, PASMCs, and PAAFs from PAH lungs. siRNA DLGAP5 down-regulated cyclin A2, suppressed hyper-proliferation, increased pro-apoptotic BIM, and induced apoptosis in human PAH PASMCs and PAAFs. DEP domain containing 1 (DEPDC1) was identified and validated as a downstream effector of DLGAP5 in PAH PASMCs responsible for hyper-proliferation and apoptosis resistance of human PAH PASMCs. SM-specific DLGAP5 knockdown with AAV6-enTagln-shDLGAP5 reversed DEPDC1 overaccumulation in small muscular PAs, reduced PA medial thickness, RV systolic pressure, mean PA pressure, and RV hypertrophy in mice with SuHx-induced PH. Conclusions: DLGAP5 supports proliferation and survival of human PAH PASMCs and PAAFs through up-regulation of cyclin A2, DEPDC1, and suppression of BIM, and promotes PA remodeling and PH in mice. DLGAP5 could serve as a potential molecular target to attenuate pulmonary vascular remodeling in PAH.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

Skeletal muscle is a dynamic tissue able to regenerate in response to injury. This regeneration relies primarily on the activation of precursor cells, known as muscle stem cells (MuSC). Upon activation, skeletal MuSC re-enter the cell cycle and proliferate as myoblasts, which subsequently either differentiate and fuse to form new fibers or return to quiescence to maintain long-term regenerative capacity. From primary human myoblasts, we generated reserve cells (RCs), quiescent cells similar to MuSC, and investigated their activation after stimulation with a growth medium containing serum. Here, we show that Orai3 calcium channel is primarily expressed in RC, where it promotes RC activation in a calcium-independent manner. We demonstrate that Orai3 does not contribute to store-operated calcium entry (SOCE) or calcium response induced by serum stimulation in RC cells, supporting the hypothesis that Orai3 plays a unique role in RC, distinct from its calcium channel activity. Using a protein proximity assay (BioID), we identified the large scaffold protein AHNAK2 as a potential partner of Orai3. AHNAK2 is expressed at high level in myotubes, and has a similar effect on RC activation than Orai3. Both Orai3 and AHNAK2 are necessary for proper myoblast differentiation and RC activation, while acting through different signaling pathways. Specifically, we show that Orai3 directly influences the RC fate, whereas AHNAK2 facilitates RC activation via signals derived from myotubes. These findings provide new insights into the molecular mechanisms underlying the activation and quiescence of human MuSC.

Introduction: In both humans and experimental animals, obesity is associated with hypothalamic inflammation and neuronal metabolic dysfunction, which contribute to disrupting body energy homeostasis. Although ciliary neurotrophic factor (CNTF) is a neuroprotective cytokine with recognized anti-obesity effects in peripheral metabolic tissues, its role in obesity-associated hypothalamic dysfunction is still poorly characterized. We investigated whether CNTF exerts any direct effects on mHypoE-N41 (N41) hypothalamic neurons and whether it modulates the effects induced by palmitate (PA), an established model of obesity-associated neuronal metabolic dysfunction. Methods: N41 cells were administered CNTF alone or combined with PA to assess whether CNTF affects PA-induced lipotoxic stress. Gene and protein expression was assessed by qRT-PCR, western blotting, and immunofluorescence analysis. Results: In N41 cells, CNTF activated the STAT3, ERK and AMPK signaling pathways. It also upregulated genes involved in mitochondrial metabolism and fatty acid oxidation, including Ucp2, Cpt1 and Pgc1, as well as the proliferation-associated markers Ki-67 and PCNA. PA treatment induced inflammatory and endoplasmic reticulum stress responses and impaired insulin signaling. In PA-treated N41 cells, CNTF significantly attenuated the expression of pro-inflammatory markers Tnf, Mcp1 and Nfkb, restored insulin-induced AKT phosphorylation, and partially reversed PA-induced changes in the expression of metabolic genes Ucp2 and Cpt1. Conclusions: Collectively, these findings indicate that CNTF exerts direct protective metabolic effects on hypothalamic N41 neurons subjected to lipotoxic stress and supports a role for it in the preservation of neuronal metabolic function under obesogenic conditions.

Background: KCNH2 encodes Kv11.1 channel proteins that conduct the rapidly activating delayed-rectifier K+ current (IKr), which is critical for cardiac repolarization. KCNH2 encodes two functional isoforms, Kv11.1a and Kv11.1b, via alternative transcription start sites. Kv11.1a is the principal determinant of cardiac IKr and ventricular repolarization. The circadian clock, a transcriptional-translational feedback loop that cycles with a period of ~24 hours and drives the circadian expression of many genes, including Kcnh2 in the mouse heart. Because daily body temperature rhythms provide a systemic signal that synchronizes cardiac circadian clocks, we tested whether physiological temperature cycles drive the circadian promoter activity of the cloned human KCNH2 (hKCNH2) promoter. Hypothesis: hKCNH2 is a direct transcriptional target of the circadian clock, with temperature driving its promoter activity through BMAL1:CLOCK acting at a conserved tandem E-box. Methods: We cloned the conserved proximal promoter of KCNH2 (-1631 bp upstream of Kv11.1a exon 1) to generate hKCNH2 promoter luciferase reporter constructs. Constructs were transfected into C2C12 myotubes and synchronized by serum shock (static 37{degrees}C) or temperature cycling (36.5-38.5{degrees}C). Bioluminescence was recorded and assessed for period, phase, and amplitude. BMAL1:CLOCK dependence was tested via dominant-negative CLOCK{Delta}19 co-expression. Results: Temperature cycling did not exhibit the rapid damping characteristic of serum-shock-synchronized oscillations, consistent with continuous entrainment by an external zeitgeber rather than a free-running oscillator. Deletion analysis identified a conserved tandem E-box required for oscillation under both serum shock and temperature cycling, and for BMAL1:CLOCK-dependent transactivation (1.75 {+/-} 0.21 vs. 0.86 {+/-} 0.06 RLU, p = 0.0038). CLOCK{Delta}19 reduced hKCNH2 promoter amplitude under temperature cycling without altering period. Conclusion: The circadian clock regulates KCNH2 promoter activity through daily temperature rhythms.

Peroxisome proliferator-activated receptor (PPAR) is a nuclear receptor that plays a central role in metabolic adaptation by regulating gene expression programs involved in lipid utilization and energy homeostasis. Although the hepatic functions of PPAR and its target genes are well characterized, less is known about its transcriptional activity in extrahepatic tissues. Pemafibrate (K-877), a selective PPAR modulator (SPPARM), is a high-affinity PPAR ligand with improved potency and selectivity compared with conventional fibrates. Here, we investigated pemafibrate-induced tissue-specific transcriptional responses comparing wild type and whole-body PPAR-deficient littermate mice. Twelve-week-old male PPAR+/+ and PPAR-/- mice were treated daily with pemafibrate (0.1 mg/kg) or vehicle by oral gavage for 14 days. We performed genome-wide transcriptomic analyses in the liver, jejunum, kidney, soleus muscle, and BAT. Pemafibrate-induced transcriptional changes were entirely dependent on PPAR, as no differentially expressed genes were detected in PPAR-/- mice. The magnitude and nature of the response were tissue-specific, with the strongest transcriptional effects observed in the liver and jejunum, whereas the kidney, soleus muscle, and BAT showed limited responses. In the liver, pemafibrate primarily induced the expression of genes involved in fatty acid metabolism, mitochondrial function, and energy production. In the jejunum, pemafibrate activated lipid metabolic programs together with tissue-specific pathways related to cellular homeostasis. Collectively, our findings show that pemafibrate is a highly selective PPAR activator with a preferential transcriptional activity in the liver and intestine after oral administration. This study provides a resource for defining tissue-specific PPAR-responsive gene networks and expands our understanding of PPAR functions beyond the liver.

Oxidative phosphorylation (OxPhos) and substrate-level phosphorylation (SLP) are the primary biochemical mechanisms for ATP generation in mammalian cells. Increased dependency on SLP can arise from either transient (e.g., Crabtree effect) or chronic (e.g., Warburg effect) suppression of OxPhos. We evaluated the steady-state bioenergetics of quiescent and proliferating mouse cells matched in genetic background and cell biology, including primary thioglycollate-elicited peritoneal macrophages, malignant VM-M3 glioma with macrophage characteristics, and malignant CT-2A astrocytoma. Data are presented showing: 1) OxPhos insufficiency was the primary bioenergetic hallmark of proliferating malignant cells. 2) Dysregulated proliferation was associated with lower metabolic flexibility under nutrient stress than was growth arrest or regulated proliferation. 3) The Crabtree effect was present in macrophages and involved a glucose- and time-dependent suppression of OxPhos coupled to ATP production through SLP and resistance to mitochondrial inhibitors; lowering glucose concentration or substituting glucose with respiratory fuels increased OxPhos and restored sensitivity to mitochondrial inhibitors. 4) The Warburg effect in tumor cells involved a persistent SLP dependency and resistance to mitochondrial inhibitors under equivalent glucose-replete conditions. 5) OxPhos could sustain the regulated proliferation of non-tumor cells in the absence of a Warburg effect. Collectively, our findings emphasize that comparisons between normal and cancer cell metabolism require long-term experimental endpoints that ensure steady-state ATP sufficiency.

Hepatic stellate cells (HSCs) are the key cellular drivers of liver fibrosis. During liver injury and chronic inflammation HSCs adopt an activated phenotype and secrete fibrotic extracellular matrix (ECM) components such as collagen 1. Mechanical cues derived from the fibrotic ECM drive and support the activation of HSCs, via mechanisms that involve integrins and the mechano-sensitive transcriptional regulator YAP1. It is not yet well understood how external mechanical cues are translated into a molecular response that alters YAP1 nuclear shuttling. There is evidence that suggests the PDZ and LIM domain protein (PDLIM) 5 can regulate YAP1 shuttling in human epithelial cells. We therefore investigated whether PDLIM5 is expressed in HSCs and contributes to YAP1 associated HSC mechano-activation. PDLIM5 protein was detected in HSCs in fibrotic human and mouse liver. PDLIM5 transcript and protein were expressed by primary human and mouse HSCs and by the immortalised HSC LX-2 cell line. PDLIM5 localised with actin stress fibres suggesting a role in HSC adhesion. Co-immunoprecipitation and proximity ligation in LX-2 cells support an association between PDLIM5 and YAP1. We used pharmacological (paclitaxel) and genetic (siRNA and CRISPRi) approaches to inhibit PDLIM5 in HSCs. Inhibiting PDLIM5 reduced YAP1 nuclear localisation and fibrotic gene (COL1A1, ACTA2) expression in LX-2 cells. Overall, these data support a role for PDLIM5 in regulating YAP1 localisation and fibrogenic gene expression in HSCs.

Background: Milrinone, a phosphodiesterase-3 (PDE-3) inhibitor, is widely used to improve cardiac output in pediatric and adult patients. Yet, developmental differences in myocardial responsiveness to milrinone remain incompletely understood. In this study, we examined the impact of postnatal maturation on the acute cardiac effects of milrinone using an intact guinea pig heart model. Methods: Neonatal (0-2 days), juvenile (4-10 days), and adult (> 6 months) guinea pig hearts were excised, Langendorff-perfused, and cardiac metrics were evaluated under basal conditions and in response to acute milrinone treatment (15 minutes, 10 and 100 M sequential concentrations). Pseudo-electrocardiograms were recorded continuously and left ventricular pressure measurements were performed under sinus rhythm and in response to external pacing. Post-rest potentiation was used to assess contractile reserve, and optical action potentials and calcium transients were recorded. Results: Baseline left ventricular developed pressure (LVDP), action potential duration (APD), and calcium transient duration (CaD) increased with postnatal maturation, consistent with developmental cardiomyocyte remodeling and refinement of excitation-contraction coupling. Milrinone increased heart rate in all age groups, with the greatest chronotropic response at 100 M. Milrinone also increased ventricular contractility and relaxation across developmental stages, but the magnitude of the response varied with age and was attenuated during high frequency pacing. Adult hearts had the greatest increase in LVDP during sinus rhythm and robust post-rest potentiation, which were less pronounced in neonatal hearts. APD and CaD were shortened in neonatal and adult hearts, but were minimally affected in juveniles, indicating a non-linear developmental pattern. Conclusions: Milrinone exerts positive chronotropic, inotropic, and lusitropic effects throughout development, but the magnitude and frequency dependence of these responses vary with age. These findings suggest that postnatal maturation influences the cardiac response to PDE3 inhibition. These developmental differences highlight the importance of considering age as a biological factor in pediatric drug selection and dosing.

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Pulmonary arterial hypertension (PAH) is characterized by the hyperproliferative phenotype of pulmonary arterial smooth muscle cells (PAVSMCs) and extensive extracellular matrix (ECM) remodeling. Vacuolar protein sorting 34 (VPS34), a Class III Phosphatidylinositol-3 Kinase (PI3K), regulates cell proliferation, but its specific function in pulmonary vascular remodeling remains poorly understood. We aim to determine the role of VPS34 activation and regulation in PAVSMC proliferation, matrix deposition, and the progression of PAH. First, we found a marked decrease in the inhibitory phosphorylation of VPS34 at Ser164 (P-S164-VPS34), indicative of constitutive VPS34 activation, in remodeled pulmonary arteries (PAs) from human PAH patients and male mice exposed to Sugen/Hypoxia (SuHx). In human PAH PAVSMCs, siRNA-mediated knockdown of VPS34 or pharmacological inhibition via the selective inhibitor SAR405 significantly blunted cell proliferation and reduced the production of fibronectin, a major component of the remodeled ECM. Mechanistically, we found that Akt activation in PAH PAVSMCs promotes VPS34 activation; Protein Phosphatase 1 Catalytic Subunit Alpha is a potential effector in VPS34 activation; and active VPS34 led to the activation of the mTOR/S6 signaling pathway with tuberous sclerosis complex 2 downregulation. Finally, in vivo treatment with SAR405 attenuated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in SuHx mice, while concurrently decreasing vascular fibronectin deposition. Our study demonstrates that VPS34 activation, driven by an Akt-mediated loss of the inhibitory P-S164, is crucial in PAVSMC proliferation and fibrotic vascular remodeling in PAH. Targeting the VPS34 axis represents a promising anti-remodeling therapeutic strategy for the treatment of PAH. Keywords: Pulmonary arterial hypertension, vacuolar protein sorting 34, pulmonary vascular remodeling, pulmonary arterial vascular smooth muscle

Protein misfolding in the endoplasmic reticulum (ER) of podocytes/glomerular epithelial cells (GECs) induces ER stress and contributes to the pathogenesis of glomerular disease. Activation of IRE1 and consequently the unfolded protein response (UPR) and autophagy sustains proteostasis. The present study examines the role of Surf4 and collagen IV-5 (COL4A5) trafficking in podocyte proteostasis. Glomerular Surf4 protein expression increased in IRE1-replete mice with experimental focal segmental glomerulosclerosis (FSGS) compared to podocyte-IRE1 knockout mice with FSGS and control mice. Glomerular Surf4 gene expression was greater in human FSGS and membranous nephropathy compared to healthy controls. In cultured GECs, basal Surf4 expression was greater in IRE1-replete (control) compared to IRE1 knockout GECs, although ER stress (tunicamycin treatment) did not increase Surf4 despite activating the UPR. Tunicamycin enhanced autophagy (LC3-II) in an IRE1-dependent manner, and the amount of Surf4 localized in autophagosomes. In GECs, tunicamycin increased the amount and colocalization of endogenous COL4A5 or ectopically-expressed COL4A5-wild type (WT) and Surf4 in autophagosomes. Compared to COL4A5-WT, the human COL4A5-C1567R mutant (which is misfolded) showed enhanced basal colocalization with Surf4 in autophagosomes; colocalization was similar to COL4A5-WT after tunicamycin treatment. Degradation of COL4A5-C1567R was more rapid compared to COL4A5-WT and was comparable to degradation of COL4A5-WT in the presence of tunicamycin. Thus, during ER stress, subsets of Surf4 and COL4A5 are redirected from the secretory pathway to autophagy. Surf4 may help deliver misfolded ER cargo, such as COL4A5 for autosomal degradation, thereby sustaining proteostasis in podocytes.

Abstract The canonical WNT signaling pathway guides cell growth throughout the human lifespan. In adults, WNT plays an essential role in tissue homeostasis by maintaining tissue stem cells. In the intestine, the WNT transcription factor, TCF4, is necessary for stem cell maintenance. Aberrant activation of WNT signaling is generally accepted as an initiating event in colon cancer, leading to constitutive TCF4 activity. To investigate the role of this key transcription factor in global gene regulation, we abrogated TCF4 expression in colon cancer cells. Loss of TCF4 function resulted in the over-expression of the WNT transcription factor, LEF1. LEF1 was transcriptionally competent and over-compensated for TCF4 (TCF7L2) silencing in a WNT reporter assay. TCF4 enlisted the transcriptional repressor CtBP1, and bound the LEF1 promoter, indicative of direct repression. TCF4 and LEF1 drove different transcriptional programs, with TCF4 favoring MYC and cell cycle progression, while LEF1 favored immune-related gene signatures. TCF4 was the primary regulator of MYC expression, yet also the mediator of LEF1 repression, suggestive of competition centered on TCF4 transcriptional output despite the {beta}-catenin saturated colon cancer nucleus. We conclude that TCF4 has dual activator-repressor activity in colon cancer.

Minor introns are rare but evolutionarily conserved introns in the human genome. Defects in minor spliceosome components have been reported to cause several human diseases. Mutations in ZRSR2 are frequently observed in myelodysplastic syndrome (MDS) patients. However, the precise molecular mechanism by which these mutations cause a loss of ZRSR2 function in minor intron splicing remain elusive. In this study, we characterized the impact of MDS-causing missense mutations in ZRSR2. Most of the mutant ZRSR2 proteins we tested showed different subnuclear localization pattern from that of wild-type ZRSR2 protein. Co-immunoprecipitation (co-IP) analysis revealed that UHM missense mutations disrupted the interaction between ZRSR2 and SF3B complex. We also demonstrated that ZRSR2 preferentially immunoprecipitated minor spliceosome components over major spliceosome components and it directly interacts with SF3B1. Furthermore, co-IP analysis utilizing a variety of ZRSR2 mutants revealed that mutations in the UHM and Zn2 domains significantly attenuated the interaction with SF3B complex. Finally, UHM and Zn2 mutations markedly disrupted the supporting activity of ZRSR2 for minor intron splicing. Our results indicate that disruption of the physical interaction with SF3B complex is one of the hallmarks of MDS-causing mutations of ZRSR2, which results in aberrant splicing of minor introns.

Type I and type III interferons (IFNs) activate the shared Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway but generate distinct inflammatory transcriptional responses in addition to their shared antiviral transcriptional induction. While some mechanisms including cytokine-receptor affinity and receptor abundance have been proposed for regulating type I vs type III IFN specificity, they account for differences in signal amplitude but do not fully explain IFN-specific changes in JAK-STAT activity over time (i.e. the signaling temporal dynamics) which has been shown to be predictive of inflammatory gene activation. Here, we investigated mechanisms that could explain how type I and III IFNs produce distinct temporal dynamics of the IFN-activated transcription factor IFN-stimulated gene factor 3 (ISGF3), leading to IFN-type specific activation of inflammatory genes in lung epithelial cells. We show that IFN-{beta}, a type I IFN, and IFN-{lambda}3, a type III IFN, induced comparable initial ISGF3 activation but subsequently generated distinct signaling trajectories, with IFN-{beta} undergoing potentiation and IFN-{lambda}3 limiting the duration of high ISGF3 activity, preventing robust inflammatory gene induction despite prolonged lower-level activity. Inflammatory gene induction was associated with the magnitude and duration of ISGF3 activity above an activation threshold during the intermediate phase of the signal response. The characteristic IFN-{lambda}3-induced ISGF3 signaling dynamics were maintained at saturating doses of IFN-{lambda}3 indicating the lack of signal activation above the threshold was independent of the dose. Additionally, differences in the abundance of the ISGF3 complex subunits STAT1, STAT2, and interferon regulatory factor 9 (IRF9), or in the induction of known negative regulators did not readily explain the response. Blocking protein synthesis caused rapid loss of IFN-{lambda}3-induced STAT phosphorylation and ISGF3 activity, supporting the presence of a pre-existing negative regulator limiting IFN-{lambda}3-mediated JAK-STAT pathway activation above the threshold required for activating inflammatory genes, while IFN-{beta} and IFN-{lambda}3 co-stimulation localized this regulation upstream of the shared ISGF3 complex. Increasing pathway competence through IFN-{beta} priming enhanced the IFN-{lambda}3-induced ISGF3 response above the inflammatory gene activation threshold leading to induction of inflammatory genes, albeit transiently. Together, these findings support a model in which the magnitude and duration of ISGF3 activity above an inflammatory gene activation threshold, rather than pathway activation alone, contribute to stimulus-specific transcriptional responses and suggest that a negative regulatory mechanism upstream of the ISGF3 complex constrains the temporal dynamics of IFN-{lambda}3 signaling.

Objective Based on the bioinformatics method of multi-database integration, this study systematically analyzes the expression characteristics, clinical pathological correlation, prognostic value, potential molecular mechanisms, and immune infiltration patterns of hepatocyte growth factor receptor (MET) in gastric cancer. Methods The UALCAN and GEPIA databases were employed to examine the differential expression of MET between gastric cancer and normal gastric mucosal tissues, as well as its associations with clinicopathological features. Kaplan-Meier Plotter was utilized to evaluate the impact of MET expression on overall survival (OS) and progression-free survival (PFS). Protein-protein interaction (PPI) network was constructed via LinkedOmics, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of co-expressed genes. Four algorithms, TIMER, CIBERSORT, EPIC, and MCPcounter, were used to cross evaluate the correlation between MET expression and immune cell infiltration; Further validate the cell type specific expression of MET using the gastric cancer single-cell sequencing queues (GSE134520, GSE167297) built into the TISCH database. Results MET expression was significantly elevated in gastric cancer tissues compared with normal gastric mucosa (P < .05), and its expression level was significantly correlated with tumor grade and TNM stage. Patients with high MET expression exhibited significantly poorer OS and PFS than those with low MET expression (P < .05). The PPI network revealed that MET could interact with 20 key proteins, including EGFR, ERBB2, HGF, STAT3, and GRB2 etc. GO enrichment analysis suggests that differentially expressed genes are significantly enriched in functions such as the ERBB signaling pathway, cadherin binding, and DNA repair complexes; KEGG enrichment analysis showed that MET related genes were significantly enriched in pathways such as homologous recombination, nuclear cytoplasmic transport, mismatch repair, and oxidative phosphorylation. Immune infiltration analysis showed that the negative association between MET and B cells infiltration has cross algorithm robustness, while the association with neutrophils, CD8+ T cells, CD4+ T cells, and macrophages exhibits algorithmic heterogeneity or insignificance; There is no significant correlation between MET and common immune checkpoint molecules such as PD-1, PD-L1, CTLA4, etc. Single cell validation further confirmed that MET is mainly enriched in malignant epithelial cells and endothelial cells, and is almost not expressed in immune cells. Conclusions Multidimensional bioinformatic analyses demonstrate that elevated MET expression serves as an independent risk factor for unfavorable prognosis in gastric cancer. MET may mediate dual drug resistance in gastric cancer via crosstalk with multiple signaling molecules (including EGFR, ERBB2, HGF, STAT3 and GRB2) and dysregulation of the homologous recombination repair pathway. Results from multiple-algorithm immune infiltration analysis, single-cell dataset analysis and immune checkpoint correlation analysis indicate that MET exerts only modest direct regulatory effects on the gastric cancer immune microenvironment. This exploratory study offers systematic bioinformatic evidence supporting MET as a candidate prognostic biomarker and potential therapeutic target for gastric cancer. Further functional experiments and prospective cohort studies are required to validate its molecular mechanisms and clinical utility.