ABSTRACT
Rap1-GTPase activates integrins and plays an indispensable role in lymphocyte trafficking, but the importance of Rap1 inactivation in this process remains unknown. Here we identified the Rap1-inactivating proteins Rasa3 and Sipa1 as critical regulators of lymphocyte trafficking. The loss of Rasa3 and Sipa1 in T cells induced spontaneous Rap1 activation and adhesion. As a consequence, T cells deficient in Rasa3 and Sipa1 were trapped in the lung due to firm attachment to capillary beds, while administration of LFA1 antibodies or loss of talin1 or Rap1 rescued lung sequestration. Unexpectedly, mutant T cells exhibited normal extravasation into lymph nodes, fast interstitial migration, even greater chemotactic responses to chemokines and sphingosine-1-phosphate, and entrance into lymphatic sinuses but severely delayed exit: mutant T cells retained high motility in lymphatic sinuses and frequently returned to the lymph node parenchyma, resulting in defective egress. These results reveal the critical trafficking processes that require Rap1 inactivation.
Subject(s)
Integrins , T-Lymphocytes , Cell Adhesion , Integrins/metabolism , GTPase-Activating Proteins/physiology , Lymph Nodes/metabolism , Lung/metabolismABSTRACT
ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.
Subject(s)
GTPase-Activating Proteins/metabolism , ADP-Ribosylation Factors/metabolism , Animals , Cilia/metabolism , Cytoskeletal Proteins/metabolism , Fibroblasts/metabolism , GTPase-Activating Proteins/physiology , Golgi Apparatus/metabolism , Mice , Microtubules/metabolism , Mitochondrial Dynamics , Signal Transduction/geneticsABSTRACT
Here we report on the related TBC/RabGAPs EPI64A and EPI64B and show that they function to organize the apical aspect of epithelial cells. EPI64A binds the scaffolding protein EBP50/NHERF1, which itself binds active ezrin in epithelial cell microvilli. Epithelial cells additionally express EPI64B that also localizes to microvilli. However, EPI64B does not bind EBP50 and both proteins are shown to have a microvillar localization domain that spans the RabGAP domains. CRISPR/Cas9 was used to inactivate expression of each protein individually or both in Jeg-3 and Caco2 cells. In Jeg-3 cells, loss of EPI64B resulted in a reduction of apical microvilli, and a further reduction was seen in the double knockout, mostly likely due to misregulation of Rab8 and Rab35. In addition, apical junctions were partially disrupted in cells lacking EPI64A and accentuated in the double knockout. In Caco2 loss of EPI64B resulted in wavy junctions, whereas loss of both EPI64A and EPI64B had a severe phenotype often resulting in cells with a stellate apical morphology. In the knockout cells, the basal region of the cell remained unchanged, so EPI64A and EPI64B specifically localize to and regulate the morphology of the apical domain of polarized epithelial cells.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Epithelial Cells/metabolism , GTPase-Activating Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Binding Sites , Caco-2 Cells , Cell Line, Tumor , Cell Polarity , Cytoskeletal Proteins , Epithelial Cells/physiology , GTPase-Activating Proteins/physiology , Humans , Microvilli/genetics , Microvilli/metabolism , Phosphoproteins/metabolism , Phosphorylation , Protein Binding/physiologyABSTRACT
The mechanistic target of rapamycin complex 1 (mTORC1) acts as a central regulator of metabolic pathways that drive cellular growth. Abnormal activation of mTORC1 occurs at high frequency in human and mouse hepatocellular carcinoma (HCC). DEP domain-containing protein 5 (DEPDC5), a component of GATOR1 complex, is a repressor of amino acid-sensing branch of the mTORC1 pathway. In the current study, we found that persistent activation of hepatic mTORC1 signaling caused by Depdc5 ablation was sufficient to induce a pathological program of liver damage, inflammation and fibrosis that triggers spontaneous HCC development. Take advantage of the combinatory treatment with a single dose of diethylnitrosamine (DEN) and chronic feeding with high-fat diet (HFD), we demonstrated that hepatic depdc5 deletion did not aggravate DEN&HFD induced liver tumorigenesis, probably due to its protective effects on diet-induced liver steatosis. In addition, we further showed that chronic rapamycin treatment did not have any apparent tumor-suppressing effects on DEN&HFD treated control mice, whereas it dramatically reduced the tumor burden in mice with hepatic Depdc5 ablation. This study provides the novel in vivo evidence for Depdc5 deletion mediated mTORC1 hyperactivation in liver tumorigenesis caused by aging or DEN&HFD treatment. Moreover, our findings also propose that pharmacological inhibition of mTORC1 signaling maybe a promising strategy to treat HCC patients with mutations in DEPDC5 gene.
Subject(s)
Carcinoma, Hepatocellular/pathology , Diet, High-Fat , Diethylnitrosamine/toxicity , Fatty Liver/pathology , GTPase-Activating Proteins/physiology , Liver/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Alkylating Agents/toxicity , Animals , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Proliferation , Disease Models, Animal , Fatty Liver/chemically induced , Fatty Liver/genetics , Fatty Liver/metabolism , Female , Inflammation/immunology , Inflammation/metabolism , Liver/pathology , Liver Neoplasms/chemically induced , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mechanistic Target of Rapamycin Complex 1/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Signal Transduction , Tumor BurdenABSTRACT
Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport.
Subject(s)
Exocytosis/genetics , GTP-Binding Proteins/physiology , GTPase-Activating Proteins/physiology , Membrane Proteins/physiology , Weibel-Palade Bodies/physiology , von Willebrand Factor/physiology , Humans , Protein Transport/geneticsABSTRACT
Acrolein, an electrophilic α,ß-unsaturated aldehyde, is present in foods and beverages, and is a product of incomplete combustion, and thus, reaches high ppm levels in tobacco smoke and structural fires. Exposure to acrolein is linked with cardiopulmonary toxicity and cardiovascular disease risk. The hypothesis of this study is the direct effects of acrolein in isolated murine blood vessels (aorta and superior mesenteric artery, SMA) are transient receptor potential ankyrin-1 (TRPA1) dependent. Using isometric myography, isolated aorta and SMA were exposed to increasing levels of acrolein. Acrolein inhibited phenylephrine (PE)-induced contractions (approximately 90%) in aorta and SMA of male and female mice in a concentration-dependent (0.01-100 µM) manner. The major metabolite of acrolein, 3-hydroxypropylmercapturic acid (3HPMA), also relaxed PE-precontracted SMA. As the SMA was 20× more sensitive to acrolein than aorta (SMA EC50 0.8 ± 0.2 µM; aorta EC50 > 29.4 ± 4.4 µM), the mechanisms of acrolein-induced relaxation were studied in SMA. The potency of acrolein-induced relaxation was inhibited significantly by: 1) mechanically-impaired endothelium; 2) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME); 3) guanylyl cyclase (GC) inhibitor (ODQ); and, 4) a TRPA1 antagonist (A967079). TRPA1 positive immunofluorescence was present in the endothelium. Compared with other known TRPA1 agonists, including allyl isothiocyanate (AITC), cinnamaldehyde, crotonaldehyde, and formaldehyde, acrolein stimulated a more potent TRPA1-dependent relaxation. Acrolein, at high concentration [100 µM], induced tension oscillations (spasms) independent of TRPA1 in precontracted SMA but not in aorta. In conclusion, acrolein is vasorelaxant at low levels (physiological) yet vasotoxic at high levels (toxicological).
Subject(s)
Acetylcysteine/analogs & derivatives , Acrolein/pharmacology , Aorta, Thoracic/drug effects , Mesenteric Artery, Superior/drug effects , TRPA1 Cation Channel/physiology , Acetylcysteine/blood , Acetylcysteine/pharmacology , Acrolein/blood , Animals , Aorta, Thoracic/physiology , Female , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/physiology , Glutathione S-Transferase pi/genetics , Glutathione S-Transferase pi/physiology , Male , Mesenteric Artery, Superior/physiology , Mice, Inbred C57BL , Mice, Knockout , TRPA1 Cation Channel/geneticsABSTRACT
PURPOSE: While chemotherapy remains the standard treatment for triple-negative breast cancer (TNBC), identifying and managing chemoresistant tumors has proven elusive. We sought to discover hallmarks and therapeutically actionable features of refractory TNBC through molecular analysis of primary chemoresistant TNBC specimens. EXPERIMENTAL DESIGN: We performed transcriptional profiling of tumors from a phase II clinical trial of platinum chemotherapy for advanced TNBC (TBCRC-009), revealing a gene expression signature that identified de novo chemorefractory tumors. We then employed pharmacogenomic data mining, proteomic and other molecular studies to define the therapeutic vulnerabilities of these tumors. RESULTS: We reveal the RAS-GTPase-activating protein (RAS-GAP) RASAL2 as an upregulated factor that mediates chemotherapy resistance but also an exquisite collateral sensitivity to combination MAP kinase kinase (MEK1/2) and EGFR inhibitors in TNBC. Mechanistically, RASAL2 GAP activity is required to confer kinase inhibitor sensitivity, as RASAL2-high TNBCs sustain basal RAS activity through suppression of negative feedback regulators SPRY1/2, together with EGFR upregulation. Consequently, RASAL2 expression results in failed feedback compensation upon co-inhibition of MEK1/2 and EGFR that induces synergistic apoptosis in vitro and in vivo. In patients with TNBC, high RASAL2 levels predict clinical chemotherapy response and long-term outcomes, and are associated via direct transcriptional regulation with activated oncogenic Yes-Associated Protein (YAP). Accordingly, chemorefractory patient-derived TNBC models exhibit YAP activation, high RASAL2 expression, and tumor regression in response to MEK/EGFR inhibitor combinations despite well-tolerated intermittent dosing. CONCLUSIONS: These findings identify RASAL2 as a mediator of TNBC chemoresistance that rewires MAPK feedback and cross-talk to confer profound collateral sensitivity to combination MEK1/2 and EGFR inhibitors.
Subject(s)
Drug Resistance, Neoplasm , GTPase-Activating Proteins/physiology , Mitogen-Activated Protein Kinase Kinases/physiology , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Cell Line, Tumor , ErbB Receptors/physiology , Female , HumansABSTRACT
Ultrasonic vocalization (USV) characterization is useful for evaluating communication in mouse models of autism spectrum disorder (ASD). Here, by categorizing USVs into 12 types using a comprehensive classification method, we obtained the qualitative and quantitative characteristics of USV repertoire emitted by ASD-related Dock4 knockout (KO) mice and their wild-type (WT) littermates during social isolation over early postnatal development. Notably, USVs emitted by WT pups exhibited a developmental switch from a pattern with more multiple-note calls, which have more complex acoustic structure, lower pitch and larger volume, into one with more single-note calls, which have simpler acoustic structure, higher pitch and smaller volume. Comparing with WT pups, USVs emitted by Dock4 KO pups had larger volume and consisted of more multiple-note calls with higher pitch in later developmental stage. These findings collectively reveal a developmental pattern of USV in normal mice and identified a set of alterations in Dock4 KO pups.
Subject(s)
Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/physiopathology , Behavior, Animal/physiology , GTPase-Activating Proteins/physiology , Social Isolation , Vocalization, Animal/physiology , Animals , Disease Models, Animal , Female , GTPase-Activating Proteins/genetics , Growth and Development , Male , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
RPA is a critical factor for DNA replication and replication stress response. Surprisingly, we found that chromatin RPA stability is tightly regulated. We report that the GDP/GTP exchange factor DOCK7 acts as a critical replication stress regulator to promote RPA stability on chromatin. DOCK7 is phosphorylated by ATR and then recruited by MDC1 to the chromatin and replication fork during replication stress. DOCK7-mediated Rac1/Cdc42 activation leads to the activation of PAK1, which subsequently phosphorylates RPA1 at S135 and T180 to stabilize chromatin-loaded RPA1 and ensure proper replication stress response. Moreover, DOCK7 is overexpressed in ovarian cancer and depleting DOCK7 sensitizes cancer cells to camptothecin. Taken together, our results highlight a novel role for DOCK7 in regulation of the replication stress response and highlight potential therapeutic targets to overcome chemoresistance in cancer.
Subject(s)
Chromatin/metabolism , DNA Replication , GTPase-Activating Proteins/physiology , Guanine Nucleotide Exchange Factors/physiology , Replication Protein A/metabolism , Animals , Cell Line, Tumor , DNA Repair , Female , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/metabolism , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Humans , Mice , Mice, Nude , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Phosphorylation , Proteolysis , Signal Transduction , Stress, Physiological/genetics , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases/metabolism , rac1 GTP-Binding Protein/metabolismABSTRACT
RASAL2 (RAS protein activator like 2), a RASGTPase activating protein, can catalyze the hydrolysis of RAS-GTP into RAS-GDP to inactivate the RAS pathway in various types of cancer cells. However, the cellular function of RASAL2 remains elusive. Here we showed that RASAL2 can attenuate PRKAA/AMPKα phosphorylation by recruiting phosphatase PPM1B/pp2cß, thus inhibiting the initiation of basal autophagy under normal conditions. In addition, we found that glucose starvation could induce dissociation of PPM1B from RASAL2 and then RASAL2 at S351 be phosphorylated by PRKAA, followed by the binding of phosphorylated-RASAL2 with to PIK3C3/VPS34-ATG14-BECN1/Beclin1 complex to increase PIK3C3 activity and autophagy. Furthermore, RASAL2 S351 phosphorylation facilitated breast tumor growth and correlated to poor clinical outcomes in breast cancer patients. Our study demonstrated that the phosphorylation status of RASAL2 S351 can function as a molecular switch to either suppress or promote AMPK-mediated autophagy. Inhibition of RASAL2 S351 phosphorylation might be a potential therapeutic strategy to overcome the resistance of AMPK-activation agents.Abbreviations: AICAR: aminoimidazole carboxamide ribonucleotide; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ATG14: autophagy related 14; C.C: compound C; CQ: chloroquine; DKO: double-knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PPM1B/pp2cß: protein phosphatase, Mg2+/Mn2+ dependent 1B; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; RASAL2: RAS protein activator like 2; RasGAPs: RasGTPase activating proteins; SQSTM1/p62: sequestosome 1; TNBC: triple-negative breast cancer.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Autophagy , GTPase-Activating Proteins/metabolism , Binding Sites , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Female , GTPase-Activating Proteins/physiology , Glucose/deficiency , Humans , Phosphorylation , Protein Phosphatase 2C/metabolismABSTRACT
The clearance of myelin debris is a critical step in the functional recovery following spinal cord injury (SCI). As phagocytes do, microvascular endothelial cells (MECs) participate in myelin debris clearance at the injury site within one week. Our group has verified that G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) is essential in autophagy and angiogenesis, both of which are tightly related to the uptake and degradation of myelin debris by MECs. Here, we analyzed the performance and mechanism of GIT1 in myelin debris clearance after SCI. The SCI contusion model was established and in vitro MECs were treated with myelin debris. Better recovery from traumatic SCI was observed in the GIT1 WT mice than in the GIT1 KO mice. More importantly, we found that GIT1 prompted MECs to clear myelin debris and further enhanced MECs angiogenesis in vivo and in vitro. Mechanistically, GIT1-mediated autophagy contributed to the clearance of myelin debris by MECs. In this study, we demonstrated that GIT1 may prompt MECs to clear myelin debris via autophagy and further stimulate MECs angiogenesis via upregulating VEGF. Our results indicate that GITI may serve as a promising target for accelerating myelin debris clearance and improving SCI recovery.
Subject(s)
Autophagy , Cell Cycle Proteins/physiology , Endothelial Cells/physiology , GTPase-Activating Proteins/physiology , Myelin Sheath/physiology , Spinal Cord Injuries/pathology , Animals , Cells, Cultured , Mice, Knockout , Microvessels/pathology , Neovascularization, Physiologic , Up-Regulation , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Prostate cancer is the second most commonly diagnosed cancer in men and one of the main leading causes of cancer deaths among men worldwide. Rapid uncontrolled growth and the ability to metastasize to other sites are key hallmarks in cancer development and progression. The Rho family of GTPases and its activators the GTPase-activating proteins (GAPs) are required for regulating cancer cell proliferation and migration. StarD13 is a GAP for Rho GTPases, specifically for RhoA and Cdc42. We have previously shown that StarD13 acts as a tumor suppressor in astrocytoma as well as breast and colorectal cancer. In this study, we performed a functional comparative analysis of StarD13 targets/and or interacting molecules to understand the general role that StarD13 plays in cancers. Our data highlight the importance of StarD13 in modulating several hallmarks of cancer. Findings from database mining and immunohistochemistry revealed that StarD13 is underexpressed in prostate cancers, in addition knocking down Stard13 increased cancer cell proliferation, consistent with its role as a tumor suppressor. Stard13 depletion, however, led to an increase in cell adhesion, which inhibited 2D cell migration. Most interestingly, StarD13 depletion increases invasion and matrix degradation, at least in part, through its regulation of Cdc42. Altogether, the data presented suggest that StarD13 acts as a tumor suppressor inhibiting prostate cancer cell invasion.
Subject(s)
Cell Movement/genetics , GTPase-Activating Proteins/genetics , GTPase-Activating Proteins/physiology , Gene Expression/genetics , Neoplasm Invasiveness/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/physiology , Cell Line, Tumor , Disease Progression , GTPase-Activating Proteins/metabolism , Humans , Male , Tumor Suppressor Proteins/metabolism , cdc42 GTP-Binding Protein , rho GTP-Binding Proteins , rhoA GTP-Binding ProteinABSTRACT
ARHGAP21 is a RhoGAP protein implicated in the modulation of insulin secretion and energy metabolism. ARHGAP21 transient-inhibition increase glucose-stimulated insulin secretion (GSIS) in neonatal islets; however, ARHGAP21 heterozygote mice have a reduced insulin secretion. These discrepancies are not totally understood, and it might be related to functional maturation of beta cells and peripheral sensitivity. Here, we investigated the real ARHGAP21 role in the insulin secretion process using an adult mouse model of acute ARHGAP21 inhibition, induced by antisense. After ARHGAP21 knockdown induction by antisense injection in 60-day old male mice, we investigated glucose and insulin tolerance test, glucose-induced insulin secretion, glucose-induced intracellular calcium dynamics, and gene expression. Our results showed that ARHGAP21 acts negatively in the GSIS of adult islet. This effect seems to be due to the modulation of important points of insulin secretion process, such as the energy metabolism (PGC1α), Ca2+ signalization (SYTVII), granule-extrusion (SNAP25), and cell-cell interaction (CX36). Therefore, based on these finds, ARHGAP21 may be an important target in Diabetes Mellitus (DM) treatment.
Subject(s)
GTPase-Activating Proteins/physiology , Gene Expression Regulation/drug effects , Glucose/pharmacology , Hyperinsulinism/prevention & control , Insulin Secretion , Insulin-Secreting Cells/drug effects , Animals , Homeostasis , Hyperinsulinism/metabolism , Hyperinsulinism/pathology , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Sweetening Agents/pharmacologyABSTRACT
Proteins involved in the spaciotemporal regulation of GLUT4 trafficking represent potential therapeutic targets for the treatment of insulin resistance and type 2 diabetes. A key regulator of insulin- and exercise-stimulated glucose uptake and GLUT4 trafficking is TBC1D1. This study aimed to identify proteins that regulate GLUT4 trafficking and homeostasis via TBC1D1. Using an unbiased quantitative proteomics approach, we identified proteins that interact with TBC1D1 in C2C12 myotubes including VPS13A and VPS13C, the Rab binding proteins EHBP1L1 and MICAL1, and the calcium pump SERCA1. These proteins associate with TBC1D1 via its phosphotyrosine binding (PTB) domains and their interactions with TBC1D1 were unaffected by AMPK activation, distinguishing them from the AMPK regulated interaction between TBC1D1 and AMPKα1 complexes. Depletion of VPS13A or VPS13C caused a post-transcriptional increase in cellular GLUT4 protein and enhanced cell surface GLUT4 levels in response to AMPK activation. The phenomenon was specific to GLUT4 because other recycling proteins were unaffected. Our results provide further support for a role of the TBC1D1 PTB domains as a scaffold for a range of Rab regulators, and also the VPS13 family of proteins which have been previously linked to fasting glycaemic traits and insulin resistance in genome wide association studies.
Subject(s)
GTPase-Activating Proteins/pharmacology , Glucose Transporter Type 4/metabolism , Homeostasis/drug effects , Homeostasis/genetics , Muscle Fibers, Skeletal/metabolism , Proteins/pharmacology , Vesicular Transport Proteins/pharmacology , AMP-Activated Protein Kinases/metabolism , Animals , Cells, Cultured , Diabetes Mellitus, Type 2 , GTPase-Activating Proteins/physiology , HEK293 Cells , Humans , Insulin Resistance , Male , Mice, Transgenic , Proteins/physiology , Vesicular Transport Proteins/physiologyABSTRACT
Despite the best treatment, approximately 10% of fractures still face undesirable repair. Recently, many studies have focused on the importance of macrophages in bone repair; however, the cellular mechanisms by which they work are not yet fully understood. In this study, we explored the functions of macrophage G-protein-coupled receptor interacting protein 1 (GIT1) in healing a tibial monocortical defect model. Using GIT1flox/flox Lyz2-Cre (GIT1 CKO) mice, we observed that a GIT1 deficiency in the macrophages led to an exacerbation of interleukin 1ß (IL1ß) production, more M1-like macrophage infiltration, and impaired intramembranous ossification in vivo. The results of in vitro assays further indicated that the macrophage GIT1 plays a critical role in several cellular processes in response to lipopolysaccharide (LPS), such as anti-oxidation, IL1ß production alleviation, and glycolysis control. Although GIT1 has been recognized as a scaffold protein, our data clarified that GIT1-mediated extracellular-signal-regulated kinase (ERK) phosphorylation could activate nuclear factor (erythroid-derived 2)-like 2 (NRF2) in macrophages after LPS treatment. Moreover, we demonstrated that macrophage GIT1-activated ERK/NRF2 negatively regulates the 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), facilitating the decrease of glycolysis. Our findings uncovered a previously unrecognized role of GIT1 in regulating ERK/NRF2 in macrophages to control the inflammatory response, suggesting that macrophage GIT1 could be a potential target to improve bone regeneration. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..
Subject(s)
Bone Regeneration , Cell Cycle Proteins/physiology , Extracellular Signal-Regulated MAP Kinases , GTPase-Activating Proteins/physiology , Macrophages , NF-E2-Related Factor 2 , Animals , Inflammation , MiceABSTRACT
Cardiac remodeling is a major early event of heart failure, which is regulated by multiple signaling pathways. Here, we demonstrate that TBC1D25 is upregulated during pathological cardiac remodeling. The aim of this study is to determine the role of TBC1D25 in cardiac remodeling and to illustrate the underlying molecular mechanism. Specifically, cardiac remodeling was induced in TBC1D25-KO mice and their wild-type control mice through partial transverse aortic constriction (TAC) of aortic arch. Knockout TBC1D25 exacerbated cardiac hypertrophy, fibrosis and dysfunction. Meanwhile, TBC1D25 overexpression in both H9C2 cells and NRCMs alleviate Angiotensin II-induced cardiomyocyte hypertrophy in vitro. Moreover, TBC1D25 deficiency increases the phosphorylation levels of TAK1 and its downstream molecular (JNK and p38), whereas overexpressed TBC1D25 inhibits phosphorylation of TAK1, JNK and p38. And TAK1 is the key molecule in this process. Furthermore, we demonstrated that TBC1D25 could directly interacts with TAK1 by immunoprecipitation assay and GST pull-down assay, and the interaction needs the amino acids from at least 138 to 226 in the C-terminal region of TBC1D25 and from 1 to 300 in the C-terminal region of TAK1. We conclude that TBC1D25 suppresses pathological cardiac remodeling via regulating TAK1-JNK/p38 signaling pathway, which suggests that TBC1D25 will likely become a promising therapeutic target for heart failure.
Subject(s)
GTPase-Activating Proteins/physiology , Gene Expression Regulation , MAP Kinase Kinase Kinases/metabolism , Signal Transduction , Angiotensin II/metabolism , Animals , Aorta/pathology , Cardiomegaly/metabolism , Echocardiography , Heart Failure , Hypertrophy , MAP Kinase Signaling System , Male , Mice , Mice, Knockout , Myocytes, Cardiac/metabolism , Phosphorylation , Protein Domains , Rats , Rats, Sprague-Dawley , Ventricular Remodeling/physiologyABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a devastating disease with poor prognosis due to limited clinical treatment options. IPF is characterized by the augmented deposition of extracellular matrix driven by myofibroblasts, and the epithelial-mesenchymal transition (EMT) has been known to play an essential role in the mechanism of pulmonary fibrosis. Previous genome-wide association study identified Fam13a as one of genes that showed genetic link with IPF and chronic obstructive pulmonary disease. Here, we analyzed the role of Fam13a in the pathogenesis of pulmonary fibrosis using Fam13a-deficient mice. We found that Fam13a was down-regulated in mouse lungs of bleomycin-induced pulmonary fibrosis model. Of note, genetic deletion of Fam13a exacerbated the lung fibrosis induced by bleomycin in association with enhanced EMT in mice. Moreover, silencing of Fam13a accelerated EMT induced by TGF-ß and TNF-α in alveolar epithelial cells, accompanied by increased active ß-catenin and its nuclear accumulation. Our data revealed a crucial role of Fam13a in the development of pulmonary fibrosis potentially through inhibiting EMT, and thus Fam13a has a therapeutic potential in the treatment of IPF.
Subject(s)
Epithelial-Mesenchymal Transition/physiology , GTPase-Activating Proteins/deficiency , GTPase-Activating Proteins/physiology , Idiopathic Pulmonary Fibrosis/genetics , A549 Cells , Animals , Bleomycin/pharmacology , Cell Nucleus/chemistry , Disease Models, Animal , Down-Regulation , Epithelial-Mesenchymal Transition/drug effects , Extracellular Matrix/physiology , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/genetics , Humans , Idiopathic Pulmonary Fibrosis/chemically induced , Idiopathic Pulmonary Fibrosis/physiopathology , Lung/chemistry , Lung/pathology , Lung/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Myofibroblasts/chemistry , Myofibroblasts/pathology , Transfection , Transforming Growth Factor beta/pharmacology , beta Catenin/analysisABSTRACT
The Rac1-specific guanosine triphosphatase (GTPase)-activating protein Slit-Robo GAP2 (Srgap2) is dramatically upregulated during RANKL-induced osteoclastogenesis. Srgap2 interacts with the cell membrane to locally inhibit activity of Rac1. In this study, we determined the role of Srgap2 in the myeloid lineage on bone homeostasis and the osteoclastic response to TNFα treatment. The bone phenotype of mice specifically lacking Srgap2 in the myeloid lineage (Srgap2 f/f :LysM-Cre; Srgap2 conditional knockout [cKO]) was investigated using histomorphometric analysis, in vitro cultures and Western blot analysis. Similar methods were used to determine the impact of TNFα challenge on osteoclast formation in Srgap2 cKO mice. Bone parameters in male Srgap2 cKO mice were unaffected. However, female cKO mice displayed higher trabecular bone volume due to increased osteoblast surface and bone formation rate, whereas osteoclastic parameters were unaltered. In vitro, cells from Srgap2 cKO had strongly enhanced Rac1 activation, but RANKL-induced osteoclast formation was unaffected. In contrast, conditioned medium from Srgap2 cKO osteoclasts promoted osteoblast differentiation and had increased levels of the bone anabolic clastokine SLIT3, providing a possible mechanism for increased bone formation in vivo. Rac1 is rapidly activated by the inflammatory cytokine TNFα. Supracalvarial injection of TNFα caused an augmented osteoclastic response in Srgap2 cKO mice. In vitro, cells from Srgap2 cKO mice displayed increased osteoclast formation in response to TNFα. We conclude that Srgap2 plays a prominent role in limiting osteoclastogenesis during inflammation through Rac1, and restricts expression of the paracrine clastokine SLIT3, a positive regulator of bone formation. © 2019 American Society for Bone and Mineral Research.
Subject(s)
Bone Resorption , GTPase-Activating Proteins , Osteogenesis , Animals , Bone and Bones , Cell Differentiation , Female , GTPase-Activating Proteins/physiology , Male , Membrane Proteins , Mice , Neuropeptides , Osteoclasts , RANK Ligand , rac1 GTP-Binding ProteinABSTRACT
KEY POINTS: Although the role of TBC1D1 within the heart remains unknown, expression of TBC1D1 increases in the left ventricle following an acute infarction, suggesting a biological importance within this tissue. We investigated the mechanistic role of TBC1D1 within the heart, aiming to establish the consequences of attenuating TBC1D1 signalling in the development of diabetic cardiomyopathy, as well as to determine potential sex differences. TBC1D1 ablation increased plasma membrane fatty acid binding protein content and myocardial palmitate oxidation. Following high-fat feeding, TBC1D1 ablation dramatically increased fibrosis and induced end-diastolic dysfunction in both male and female rats in the absence of changes in mitochondrial bioenergetics. Altogether, independent of sex, ablating TBC1D1 predisposes the left ventricle to pathological remodelling following high-fat feeding, and suggests TBC1D1 protects against diabetic cardiomyopathy. ABSTRACT: TBC1D1, a Rab-GTPase activating protein, is involved in the regulation of glucose handling and substrate metabolism within skeletal muscle, and is essential for maintaining pancreatic ß-cell mass and insulin secretion. However, the function of TBC1D1 within the heart is largely unknown. Therefore, we examined the role of TBC1D1 in the left ventricle and the functional consequence of ablating TBC1D1 on the susceptibility to high-fat diet-induced abnormalities. Since mutations within TBC1D1 (R125W) display stronger associations with clinical parameters in women, we further examined possible sex differences in the predisposition to diabetic cardiomyopathy. In control-fed animals, TBC1D1 ablation did not alter insulin-stimulated glucose uptake, or echocardiogram parameters, but increased accumulation of a plasma membrane fatty acid transporter and the capacity for palmitate oxidation. When challenged with an 8 week high-fat diet, TBC1D1 knockout rats displayed a four-fold increase in fibrosis compared to wild-type animals, and this was associated with diastolic dysfunction, suggesting a predisposition to diet-induced cardiomyopathy. Interestingly, high-fat feeding only induced cardiac hypertrophy in male TBC1D1 knockout animals, implicating a possible sex difference. Mitochondrial respiratory capacity and substrate sensitivity to pyruvate and ADP were not altered by diet or TBC1D1 ablation, nor were markers of oxidative stress, or indices of overt heart failure. Altogether, independent of sex, ablation of TBC1D1 not only increased the susceptibility to high-fat diet-induced diastolic dysfunction and left ventricular fibrosis, independent of sex, but also predisposed male animals to the development of cardiac hypertrophy. These data suggest that TBC1D1 may exert cardioprotective effects in the development of diabetic cardiomyopathy.
Subject(s)
Cardiomyopathies/physiopathology , GTPase-Activating Proteins/physiology , Proteins/physiology , Animals , Cardiomyopathies/genetics , Diet, High-Fat , Female , GTPase-Activating Proteins/genetics , Gene Knockout Techniques , Glucose/metabolism , Heart Ventricles/physiopathology , Insulin , Male , Muscle, Skeletal , Proteins/genetics , Rats , Sex FactorsABSTRACT
Inflammation plays an important role in pathological angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation; however, a comprehensive description of its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4-activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis.