Signaling Invivo Research Articles

4-Hydroxynonenal Promotes Colorectal Cancer Progression Through Regulating Cancer Stem Cell Fate.

Aims: Tumor microenvironment (TME) plays a crucial role in sustaining cancer stem cells (CSCs). 4-hydroxynonenal (4-HNE) is abundantly present in the TME of colorectal cancer (CRC). However, the contribution of 4-HNE to CSCs and cancer progression remains unclear. This study aimed to investigate the impact of 4-HNE on the regulation of CSC fate and tumor progression. Methods: Human CRC cells were exposed to 4-HNE, and CSC signaling was analyzed using quantitative real-time polymerase chain reaction, immunofluorescent staining, fluorescence-activated cell sorting, and bioinformatic analysis. The tumor-promoting role of 4-HNE was confirmed using a xenograft model. Results: Exposure of CRC cells to 4-HNE activated noncanonical hedgehog (HH) signaling and homologous recombination repair (HRR) pathways in LGR5+ CSCs. Furthermore, blocking HH signaling led to a significant increase in the expression of γH2AX, indicating that 4-HNE induces double-stranded DNA breaks (DSBs) and simultaneously activates HH signaling to protect CSCs from 4-HNE-induced damage via the HRR pathway. In addition, 4-HNE treatment increased the population of LGR5+ CSCs and promoted asymmetric division in these cells, leading to enhanced self-renewal and differentiation. Notably, 4-HNE also promoted xenograft tumor growth and activated CSC signaling invivo. Innovation and Conclusion: These findings demonstrate that 4-HNE, as a signaling inducer in the TME, activates the noncanonical HH pathway to shield CSCs from oxidative damage, enhances the proliferation and asymmetric division of LGR5+ CSCs, and thereby facilitates tumor growth. These novel insights shed light on the regulation of CSC fate within the oxidative TME, offering potential implications for understanding and targeting CSCs for CRC therapy.

Neuropeptide inactivation regulates egg-laying behavior to influence reproductive health in Caenorhabditis elegans

Neural communication requires both fast-acting neurotransmitters and neuromodulators that function on slower timescales to communicate. Endogenous bioactive peptides, often called "neuropeptides," comprise the largest and most diverse class of neuromodulators that mediate crosstalk between the brain and peripheral tissues to regulate physiology and behaviors conserved across the animal kingdom. Neuropeptide signaling can be terminated through receptor binding and internalization or degradation by extracellular enzymes called neuropeptidases. Inactivation by neuropeptidases can shape the dynamics of signaling invivo by specifying both the duration of signaling and the anatomic path neuropeptides can travel before they are degraded. For most neuropeptides, the identity of the relevant inactivating peptidase(s) is unknown. Here, we established a screening platform in C.elegans utilizing mass spectrometry-based peptidomics to discover neuropeptidases and simultaneously profile the invivo specificity of these enzymes against each of more than 250 endogenous peptides. We identified NEP-2, a worm ortholog of the mammalian peptidase neprilysin-2, and demonstrated that it regulates specific neuropeptides, including those in the egg-laying circuit. We found that NEP-2 is required in muscle cells to regulate signals from neurons to modulate both behavior and health in the reproductive system. Taken together, our results demonstrate that peptidases, which are an important node of regulation in neuropeptide signaling, affect the dynamics of signaling to impact behavior, physiology, and aging.

Wg/Wnt-signaling-induced nuclear translocation of β-catenin is attenuated by a β-catenin peptide through its interference with the IFT-A complex

Wnt/Wingless (Wg) signaling is critical in development and disease, including cancer. Canonical Wnt signaling is mediated by β-catenin/Armadillo (Arm in Drosophila) transducing signals to the nucleus, with IFT-A/Kinesin 2 complexes promoting nuclear translocation of β-catenin/Arm. Here, we demonstrate that a conserved small N-terminal Arm34-87/β-catenin peptide binds to IFT140, acting as a dominant interference tool to attenuate Wg/Wnt signaling invivo. Arm34-87 expression antagonizes endogenous Wnt/Wg signaling, resulting in the reduction of its target expression. Arm34-87 inhibits Wg/Wnt signaling by interfering with nuclear translocation of endogenous Arm/β-catenin, and this can be modulated by levels of wild-type β-catenin or IFT140, with the Arm34-87 effect being enhanced or suppressed. Importantly, this mechanism is conserved in mammals with the equivalent β-catenin24-79 peptide blocking nuclear translocation and pathway activation, including in cancer cells. Our work indicates that Wnt signaling can be regulated by a defined N-terminal β-catenin peptide and thus might serve as an entry point for therapeutic applications to attenuate Wnt/β-catenin signaling.

IGSF8 is an innate immune checkpoint and cancer immunotherapy target

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival invitro or invivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells invitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and Tcell signaling invivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Discovery of YAP1/TAZ pathway inhibitors through phenotypic screening with potent anti-tumor activity via blockade of Rho-GTPase signaling

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity invivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation invitro. Multi-parameter optimization resulted in BAY-593, an invivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling invivo.

Fish Uses CTLA-4 Immune Checkpoint to Suppress mTORC1-Controlled T-Cell Glycolysis and Immunity.

As an immune checkpoint, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) suppresses the activation, proliferation, and effector function of T cells, thus preventing an overexuberant response and maintaining immune homeostasis. However, whether and how this immune checkpoint functions in early vertebrates remains unknown. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell response by CTLA-4 in bony fish. Tilapia CTLA-4 is constitutively expressed in lymphoid tissues, and its mRNA and protein expression in lymphocytes are upregulated following PHA stimulation or Edwardsiella piscicida infection. Blockade of CTLA-4 signaling enhanced T cell activation and proliferation but inhibited activation-induced T cell apoptosis, indicating that CTLA-4 negatively regulated T cell activation. In addition, blocking CTLA-4 signaling invivo increased the differentiation potential and cytotoxicity of T cells, resulting in an enhanced T cell response during E. piscicida infection. Tilapia CTLA-4 competitively bound the B7.2/CD86 molecule with CD28, thus antagonizing the CD28-mediated costimulatory signal of T cell activation. Furthermore, inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, c-Myc, or glycolysis markedly impaired the CTLA-4 blockade-enhanced T cell response, suggesting that CTLA-4 suppressed the T cell response of tilapia by inhibiting mTORC1/c-Myc axis-controlled glycolysis. Overall, the findings indicate a detailed mechanism by which CTLA-4 suppresses T cell immunity in tilapia; therefore, we propose that early vertebrates have evolved sophisticated mechanisms coupling immune checkpoints and metabolic reprogramming to avoid an overexuberant T cell response.

Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts.

Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip-/- mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip-/- mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip-/- mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the invivo model of lung fibrosis, Tollip-/- mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling invivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip-/- MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip-/- MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip-/- MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip-/- through SMAD2 invitro and invivo. Tollip-/- mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip-/- mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.

Disruption of sirtuin 7 in zebrafish facilitates hypoxia tolerance

SIRT7 is a member of the sirtuin family proteins with nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase activity, which can inhibit the activity of hypoxia-inducible factors independently of its enzymatic activity. However, the role of SIRT7 in affecting hypoxia signaling invivo is still elusive. Here, we find that sirt7-null zebrafish are more resistant to hypoxic conditions, along with an increase of hypoxia-responsive gene expression and erythrocyte numbers, compared with their wildtype siblings. Overexpression of sirt7 suppresses the expression of hypoxia-responsive genes. Further assays indicate that sirt7 interacts with zebrafish hif-1αa, hif-1αb, hif-2αa, and hif-2αb to inhibit their transcriptional activity and mediate their protein degradation. In addition, sirt7 not only binds to the hypoxia responsive element of hypoxia-inducible gene promoters but also causes a reduction of H3K18Ac on these promoters. Sirt7 may regulate hypoxia-responsive gene expression through its enzymatic and nonenzymatic activities. This study provides novel insights into sirt7 function and sheds new light on the regulation of hypoxia signaling by sirt7.

Interleukin-33-activated neuropeptide CGRP-producing memory Th2 cells cooperate with somatosensory neurons to induce conjunctival itch

Allergic conjunctivitis is a chronic inflammatory disease that is characterized by severe itch in the conjunctiva, but how neuro-immune interactions shape the pathogenesis of severe itch remains unclear. We identified a subset of memory-type pathogenic Th2 cells that preferentially expressed Il1rl1-encoding ST2 and Calca-encoding calcitonin-gene-related peptide (CGRP) in the inflammatory conjunctiva using a single-cell analysis. The IL-33-ST2 axis in memory Th2 cells controlled the axonal elongation of the peripheral sensory C-fiber and the induction of severe itch. Pharmacological blockade and genetic deletion of CGRP signaling invivo attenuated scratching behavior. The analysis of giant papillae from patients with severe allergic conjunctivitis revealed ectopic lymphoid structure formation with the accumulation of IL-33-producing epithelial cells and CGRP-producing pathogenic CD4+ Tcells accompanied by peripheral nerve elongation. Thus, the IL-33-ST2-CGRP axis directs severe itch with neuro-reconstruction in the inflammatory conjunctiva and is a potential therapeutic target for severe itch in allergic conjunctivitis.

G protein–biased GPR3 signaling ameliorates amyloid pathology in a preclinical Alzheimer’s disease mouse model

Biased G protein-coupled receptor (GPCR) ligands, which preferentially activate G protein or β-arrestin signaling pathways, are leading to the development of drugs with superior efficacy and reduced side effects in heart disease, pain management, and neuropsychiatric disorders. Although GPCRs are implicated in the pathophysiology of Alzheimer's disease (AD), biased GPCR signaling is a largely unexplored area of investigation in AD. Our previous work demonstrated that GPR3-mediated β-arrestin signaling modulates amyloid-β (Aβ) generation invitro and that Gpr3 deficiency ameliorates Aβ pathology invivo. However, Gpr3-deficient mice display several adverse phenotypes, including elevated anxiety-like behavior, reduced fertility, and memory impairment, which are potentially associated with impaired G protein signaling. Here, we generated a G protein-biased GPR3 mouse model to investigate the physiological and pathophysiological consequences of selective elimination of GPR3-mediated β-arrestin signaling invivo. In contrast to Gpr3-deficient mice, G protein-biased GPR3 mice do not display elevated anxiety levels, reduced fertility, or cognitive impairment. We further determined that G protein-biased signaling reduces soluble Aβ levels and leads to a decrease in the area and compaction of amyloid plaques in the preclinical AppNL-G-F AD mouse model. The changes in amyloid pathology are accompanied by robust microglial and astrocytic hypertrophy, which suggest a protective glial response that may limit amyloid plaque development in G protein-biased GPR3 AD mice. Collectively, these studies indicate that GPR3-mediated G protein and β-arrestin signaling produce discrete and separable effects and provide proof of concept for the development of safer GPCR-targeting therapeutics with more directed pharmacological action for AD.

MP49-02 MODULATION OF SENSORY GLIA GQ-GPCR SIGNALING IN VIVO AFFECTS SPONTANEOUS VOIDING AND BLADDER FUNCTION IN AN ANIMAL MODEL OF CHRONIC PELVIC PAIN

You have accessJournal of UrologyCME1 May 2022MP49-02 MODULATION OF SENSORY GLIA GQ-GPCR SIGNALING IN VIVO AFFECTS SPONTANEOUS VOIDING AND BLADDER FUNCTION IN AN ANIMAL MODEL OF CHRONIC PELVIC PAIN Alison Xiaoqiao Xie, Randall Meacham, and Anna Malykhina Alison Xiaoqiao XieAlison Xiaoqiao Xie More articles by this author , Randall MeachamRandall Meacham More articles by this author , and Anna MalykhinaAnna Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002624.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chronic pelvic pain (CPP) is a bothersome and highly prevalent symptom present in many chronic disorders of the pelvis. Afferent hypersensitivity plays an important role in the generation and maintenance of CPP symptoms including visceral pain, urgency and urinary frequency. We previously showed that activation of Gq-G protein coupled receptors (Gq-GPCR) in satellite glial cells (SGCs) decreases lumbosacral nociceptive responses and alleviates visceral hypersensitivity in a mouse model of CPP. The objective of this study was to further investigate the modulatory capacity of satellite glial activation in regulation of spontaneous voiding and bladder function. METHODS: Intravesical instillation of vascular endothelial growth factor (VEGFA) induces sensory nerve remodeling in the urinary bladder, and increases nociceptive responses in the lumbosacral sensory ganglia associated with visceral hypersensitivity. Adult C57BL/6 mice were subjected to three intravesical instillations of VEGFA within a 2-week period. Selective activation of lumbosacral sensory glia was achieved by expressing Gq-coupled Designer Receptors Exclusively Activated by Designer Drugs (Gq-DREADD) in glial acidic fibrillary protein (GFAP+) expressing glia by using Gfap-Gq-DREADD transgenic mice or targeted adeno-associated viral vector (AAV) delivery. Gq-DREADD agonist, clozapine N-oxide (CNO), was used to selectively activate Gq-GPCR signaling in GFAP+ glia in vivo. Micturition patterns were recorded by performing a voiding spot assay (spontaneous voiding) and cystometry in unanesthetized animals (bladder function). RESULTS: Activation of lumbosacral SGCs significantly altered spontaneous voiding patterns and bladder function in vivo in VEGFA-treated mice. Higher urinary output, shorter intermicturition intervals, and fewer non-voiding contractions were observed in VEGFA-treated animals following activation of Gq-GPCR signaling. The observed glia-mediated neuromodulatory effects were long-lasting and dose-dependent. CONCLUSIONS: Our data demonstrated that activation of sensory glia in the peripheral nervous system significantly regulate bladder functions. Targeting neuron-glia interactions in the sensory ganglia can be used to effectively alleviate visceral pain and regulate voiding function in vivo. Further studies are warranted to test a therapeutic potential of targeting glial cell signaling in treating hypersensitivity-related symptoms in CPP disorders. Source of Funding: Colorado Pilot Program Mentored Award, CCTSI. "Beyond the neurons: the role of peripheral glia in neurogenic bladder dysfunction". 2019-2021R01 DK129260-01. "Activating Peripheral Glia to Relieve Visceral Pain in Animal Models of Urological Chronic Pelvic Pain Syndrome (UCPPS)". 2021-2026 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e853 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Alison Xiaoqiao Xie More articles by this author Randall Meacham More articles by this author Anna Malykhina More articles by this author Expand All Advertisement PDF DownloadLoading ...

Microvilli-derived extracellular vesicles carry Hedgehog morphogenic signals for Drosophila wing imaginal disc development

Morphogens are secreted molecules that regulate and coordinate major developmental processes, such as cell differentiation and tissue morphogenesis. Depending on the mechanisms of secretion and the nature of their carriers, morphogens act at short and long range. We investigated the paradigmatic long-range activity of Hedgehog (Hh), a well-known morphogen, and its contribution to the growth and patterning of the Drosophila wing imaginal disc. Extracellular vesicles (EVs) contribute to Hh long-range activity; however, the nature, the site, and the mechanisms underlying the biogenesis of these vesicular carriers remain unknown. Here, through the analysis of mutants and a series of Drosophila RNAi-depleted wing imaginal discs using fluorescence and live-imaging electron microscopy, including tomography and 3D reconstruction, we demonstrate that microvilli of the wing imaginal disc epithelium are the site of generation of small EVs that transport Hh across the tissue. Further, we show that the Prominin-like (PromL) protein is critical for microvilli integrity. Together with actin cytoskeleton and membrane phospholipids, PromL maintains microvilli architecture that is essential to promote its secretory function. Importantly, the distribution of Hh to microvilli and its release via these EVs contribute to the proper morphogenesis of the wing imaginal disc. Our results demonstrate that microvilli-derived EVs are carriers for Hh long-range signaling invivo. By establishing that members of the Prominin protein family are key determinants of microvilli formation and integrity, our findings support the view that microvilli-derived EVs conveying Hh may provide a means for exchanging signaling cues of high significance in tissue development and cancer.

In vivo endocannabinoid dynamics at the timescale of physiological and pathological neural activity

The brain's endocannabinoid system is a powerful controller of neurotransmitter release, shaping synaptic communication under physiological and pathological conditions. However, our understanding of endocannabinoid signaling invivo is limited by the inability to measure their changes at timescales commensurate with the high lability of lipid signals, leaving fundamental questions of whether, how, and which endocannabinoids fluctuate with neural activity unresolved. Using novel imaging approaches in awake behaving mice, we now demonstrate that the endocannabinoid 2-arachidonoylglycerol, not anandamide, is dynamically coupled to hippocampal neural activity with high spatiotemporal specificity. Furthermore, we show that seizures amplify the physiological endocannabinoid increase by orders of magnitude and drive the downstream synthesis of vasoactive prostaglandins that culminate in a prolonged stroke-like event. These results shed new light on normal and pathological endocannabinoid signaling invivo.

Sinigrin attenuates angiotensin II‑induced kidney injury by inactivating nuclear factor‑κB and extracellular signal‑regulated kinase signaling invivo and invitro.

The present study investigated the function of sinigrin in angiotensin II (Ang II)-induced renal damage. The results demonstrated that systolic blood pressure (SBP) and diastolic blood pressure (DBP) were increased in Ang II-challenged rats, and sinigrin treatment inhibited their increase. The levels of blood urea nitrogen (BUN) and serum creatinine (SCR) were increased by Ang II in the rats, and these were reversed by sinigrin in a dose-dependent manner. In addition, the Ang II-induced elevation of urinary protein levels was inhibited by sinigrin treatment. Glomerular basement membrane thickness and ECM degradation markers, such as collagen I, collagen IV and fibronectin, were suppressed by sinigrin in the Ang II-challenged rats. Moreover, the levels of inflammatory regulators, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), were reduced following sinigrin treatment of the Ang II-challenged rats and in Ang II-exposed proximal tubule epithelial cells. Furthermore, the superoxide dismutase (SOD) and catalase (CAT) levels were downregulated, whereas the malondialdehyde (MDA) levels were upregulated by Ang II; these effects were reversed by sinigrin treatment in vivo and in vitro. Mechanistically, sinigrin inhibited the Ang II-induced phosphorylation of ERK, p65 and IκBα. Thus, sinigrin attenuated Ang II-induced renal injury by inactivating ERK and NF-κB signaling. Sinigrin may thus prove to be a potential candidate for the treatment of hypertension-induced kidney damage.

Design and synthesis of novel Flavone-based histone deacetylase inhibitors antagonizing activation of STAT3 in breast cancer.

Histone deacetylases (HDACs) inhibitors have demonstrated a great clinical achievement in hematological malignancies. However, the efficacy of HDACs inhibitors in treating solid tumors remains limited due to the complicated tumor microenvironment. In this study, we designed and synthesized a class of novel HDACs inhibitors based on the structure of flavones and isoflavones, followed by biological evaluation. To be specific, a lead compound 15a was discovered with strong anti-proliferative effects on a variety of solid tumor cells, especially for breast cancer cells with resistance to SAHA. Studies demonstrated that 15a could significantly inhibit the activity of HDAC 1, 2, 3 (class I) and 6 (class IIB), leading to a dose-dependent accumulation of acetylated histones and α-Tubulin, cell cycle arrest (G1/S phase) and apoptosis in breast cancer cells. Furthermore, the lead compound 15a could also antagonize the activation of STAT3 induced by HDACs inhibition in some breast cancer cells, which further reduced the level of pro-survive proteins in tumor cells and enhanced anti-tumor activity regulated by STAT3 signaling invivo. Overall, our findings demonstrated that the novel compound 15a might be a HDACs inhibitor candidate, which could be used as promising chemotherapeutic agent for breast cancer.

SpiCee: A Genetic Tool for Subcellular and Cell-Specific Calcium Manipulation

Calcium is a second messenger crucial to a myriad of cellular processes ranging from regulation of metabolism and cell survival to vesicle release and motility. Current strategies to directly manipulate endogenous calcium signals lack cellular and subcellular specificity. We introduce SpiCee, a versatile and genetically encoded chelator combining low- and high-affinity sites for calcium. This scavenger enables altering endogenous calcium signaling and functions in single cells invitro and invivo with biochemically controlled subcellular resolution. SpiCee paves the way to investigate local calcium signaling invivo and directly manipulate this second messenger for therapeutic use.

Heat shock protein 90 relieves heat stress damage of myocardial cells by regulating Akt and PKM2 signaling invivo.

Heat shock protein 90 (Hsp90) is associated with resisting heat-stress injury to the heart, particularly in myocardial mitochondria. However, the mechanism underlying this effect remains unclear. The present study was based on the high expression of Hsp90 during heat stress (HS) and involved inducing higher expression of Hsp90 using aspirin in mouse hearts. Higher Hsp90 levels inhibited HS-induced myocardial damage and apoptosis, and mitochondrial dysfunction, by stimulating Akt (protein kinase B) activation and PKM2 (pyruvate kinase M2) signaling, and subsequently increasing mitochondrial Bcl-2 (B-cell lymphoma 2) levels and its phosphorylation. Functional inhibition of Hsp90 using geldanamycin verified that reducing the association of Hsp90 with Akt and PKM2 caused the functional decline of phosphorylated (p)-Akt and PKM2 that initiate Bcl-2 to move into mitochondria, where it is phosphorylated. Protection by Hsp90 was weakened by blocking Akt activation using Triciribine, which could not be recovered by normal initiation of the PKM2 pathway. Furthermore, increased Hsp70 levels induced by Akt activation in myocardial cells may flow into the blood to resist heat stress. The results provided in vivo mechanistic evidence that in myocardial cells, Hsp90 resists heat stress via separate activation of the Akt-Bcl-2 and PKM2-Bcl-2 signaling pathways, which contribute toward preserving cardiac function and mitochondrial homeostasis.

Non-canonical Caspase-1 Signaling Drives RIP2-Dependent and TNF-α-Mediated Inflammation In Vivo

Pro-inflammatory caspase-1 is a key player in innate immunity. Caspase-1 processes interleukin (IL)-1β and IL-18 to their mature forms and triggers pyroptosis. These caspase-1 functions are linked to its enzymatic activity. However, loss-of-function missense mutations in CASP1 do not prevent autoinflammation in patients, despite decreased IL-1β production. Invitro data suggest that enzymatically inactive caspase-1 drives inflammation via enhanced nuclear factor κB (NF-κB) activation, independent of IL-1β processing. Here, we report two mouse models ofenzymatically inactive caspase-1-C284A, demonstrating the relevance of this pathway invivo. In contrast to Casp1-/- mice, caspase-1-C284A mice show pronounced hypothermia and increased levels of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-6 when challenged with lipopolysaccharide (LPS). Caspase-1-C284A signaling isRIP2 dependent and mediated by TNF-α but independent of the NLRP3 inflammasome. LPS-stimulated whole blood from patients carrying loss-of-function missense mutations in CASP1 secretes higher amounts of TNF-α. Taken together, these results reveal non-canonical caspase-1 signaling invivo.

Collagen VI Deficiency Results in Structural Abnormalities in the Mouse Lung

Collagen VI (COL6) is known for its role in a spectrum of congenital muscular dystrophies, which are often accompanied by respiratory dysfunction. However, little is known regarding the function of COL6 in the lung. We confirmed the presence of COL6 throughout the basement membrane region of mouse lung tissue. Lung structure and organization were studied in a previously described Col6a1-/- mouse, which does not produce detectable COL6 in the lung. The Col6a1-/- mouse displayed histopathologic alveolar and airway abnormalities. The airspaces of Col6a1-/- lungs appeared simplified, with larger (29%; P<0.01) and fewer (31%; P<0.001) alveoli. These airspace abnormalities included reduced isolectin B4+ alveolar capillaries and surfactant protein C-positive alveolar epithelial type-II cells. Alterations in lung function consistent with these histopathologic changes were evident. Col6a1-/- mice also displayed multiple airway changes, including increased branching (59%; P<0.001), increased mucosal thickness (34%; P<0.001), and increased epithelial cell density (13%; P<0.001). Comprehensive transcriptome analysis revealed that the loss of COL6 is associated with reductions in integrin-paxillin-phosphatidylinositol 3-kinase signaling invivo. Invitro, COL6 promoted steady-state phosphorylated paxillin levels and reduced cell density (16% to 28%; P<0.05) at confluence. Inhibition of phosphatidylinositol 3-kinase, or its downstream effectors, resulted in increased cell density to a level similar to that seen on matrices lacking COL6.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling invivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of β-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.