scholarly journals Expression of a Constitutively Active Form of Hck in Chondrocytes Activates Wnt and Hedgehog Signaling Pathways, and Induces Chondrocyte Proliferation in Mice

2020 ◽  
Vol 21 (8) ◽  
pp. 2682
Author(s):  
Viviane K. S. Kawata Matsuura ◽  
Carolina Andrea Yoshida ◽  
Hisato Komori ◽  
Chiharu Sakane ◽  
Kei Yamana ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Signaling Pathways ◽  
Hedgehog Signaling ◽  
Hind Limb ◽  
Active Form ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Chondrocyte Proliferation ◽  
Src Tyrosine Kinase ◽  
Chondrocyte Maturation ◽  
Constitutively Active

Runx2 is required for chondrocyte proliferation and maturation. In the search of Runx2 target genes in chondrocytes, we found that Runx2 up-regulated the expression of hematopoietic cell kinase (Hck), which is a member of the Src tyrosine kinase family, in chondrocytes, that Hck expression was high in cartilaginous limb skeletons of wild-type mice but low in those of Runx2–/– mice, and that Runx2 bound the promoter region of Hck. To investigate the functions of Hck in chondrocytes, transgenic mice expressing a constitutively active form of Hck (HckCA) were generated using the Col2a1 promoter/enhancer. The hind limb skeletons were fused, the tibia became a large, round mass, and the growth plate was markedly disorganized. Chondrocyte maturation was delayed until E16.5 but accelerated thereafter. BrdU-labeled, but not terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive, chondrocytes were increased. Furthermore, Hck knock-down reduced the proliferation of primary chondrocytes. In microarray and real-time RT-PCR analyses using hind limb RNA from HckCA transgenic mice, the expression of Wnt (Wnt10b, Tcf7, Lef1, Dkk1) and hedgehog (Ihh, Ptch1, and Gli1) signaling pathway genes was upregulated. These findings indicated that Hck, whose expression is regulated by Runx2, is highly expressed in chondrocytes, and that HckCA activates Wnt and hedgehog signaling pathways, and promotes chondrocyte proliferation without increasing apoptosis.

Evidence for NFAT1 as a Tumor Suppressor in T-ALL

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3804-3804
Author(s):  
Martin R Mueller ◽  
Bruno K Robbs ◽  
Andre LS Cruz ◽  
Yoshiteru Sasaki ◽  
Irena Stevanovic ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Tumor Suppressor ◽  
Transgenic Mice ◽  
Active Form ◽  
Signalling Pathway ◽  
Resting Cells ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Constitutively Active

Abstract NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT translocates to the nucleus, where it orchestrates developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least three different kinases: CK1, GSK-3 and DYRK. NFAT signalling is further implicated in lymphocyte homeostasis and its deregulation has been suggested to be involved in the pathogenesis of different malignancies. Previous studies have primarily concentrated on NFAT2, which was found to be overexpressed and constitutively activated in a majority of biopsies from Diffuse Large B-Cell lymphomas and Burkitt’s lymphomas, presumably reflecting activation of the Ca/NFAT signalling pathway as part of their pathogenesis. Other recent studies have demonstrated that NFAT2 activation can lead to increased expression of different cell survival factors (CD154, BLyS) in several NHL subtypes. Here, we analyzed the role of NFAT1 in malignant transformation and in the pathogenesis of T-ALL using a transgenic mouse model for the disease. We show that sustained activity of NFAT1 and NFAT2 induces opposite phenotypes in NIH 3T3 cells. While NIH 3T3 cells infected with empty retrovirus showed normal viability and stopped growing upon reaching confluence, cells infected with a constitutively active version of NFAT2 overgrew the monolayer and continued to proliferate beyond confluence. NIH 3T3 cells expressing a constitutively active form of NFAT1 on the contrary exhibited a remarkable reduction in cell proliferation and never reached confluence, suggesting opposite roles for NFAT1 and NFAT2 in the control of cell growth and proliferation. To directly test whether NFAT1 has the capability to suppress tumor growth and to subvert a transformed cell type, we generated NIH 3T3 cell clones harboring the oncogene H-rasV12, which showed a typical transformed phenotype. Expression of constitutively active NFAT1 in the transformed NIH 3T3-HrasV12 cells leads to a significant reduction in cell proliferation which was accomanied by a large decrease in DNA replication and an accumulation in the G1 phase of the cell cycle. To test if NFAT1 has tumor suppressor characteristics in vivo, we generated transgenic mice conditionally expressing a hyperactivable form of NFAT1 from the ROSA26 locus. These mice were subsequently bred to Tel-Jak2 transgenic mice, which develop T-ALL at 8–20 wks of age. Mice that expressed hyperactivable NFAT1 in their T cells in addition to the disease inducing Tel-Jak2 transgene exhibited a significantly attenuated phenotype of the disease. Infiltration of vital organs such as lung, liver and bone marrow was significantly delayed and overall survival was almost twice as long in the animals that expressed hyperactivable NFAT1 in their T cell compartment (88 days vs. 159 days). To summarize, our data identify NFAT1 as a novel tumor suppressor gene in T-ALL and emphasize the importance of the Ca/NFAT signalling pathway in the pathogenesis of hematologic malignancies.

scholarly journals Elucidating Role of Ezh2 in Tolerogenic Function of NOD Bone Marrow-Derived Dendritic Cells Expressing Constitutively Active Stat5b.

2019 ◽  
Author(s):  
Echarki Zerif ◽  
Denis Gris ◽  
Gilles Dupuis ◽  
Abdelaziz Amrani
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Active Form ◽  
Nod Mice ◽  
Autoreactive T Cell ◽  
Tolerogenic Function ◽  
Constitutively Active

Tolerogenic dendritic cells are crucial to control development of autoreactive T cell responses and prevention of autoimmunity. We have reported that NOD.CD11cStat5b-CA transgenic mice expressing a constitutively active form of Stat5b under the control of CD11c promoter are protected from diabetes and that Stat5b-CA-expressing DCs are tolerogenic and halt ongoing diabetes in NOD mice. However, the molecular mechanisms by which Stat5b-CA modulates DC tolerogenic function is not fully understood. Here, we used bone marrow-derived DCs from NOD.CD11cStat5b-CA transgenic mice (Stat5b-CA.BMDC) and found that Stat5b-CA.BMDC displayed high levels of MHC class II, CD80, CD86, PD-L1 and PD-L2 and produced elevated amounts of TGFβ but low amounts of TNF and IL-23. Stat5b-CA.BMDCs upregulated Irf4 and downregulated Irf8 genes and protein expression and promoted CD11c+CD11b+ DC2 subset differentiation. Interestingly, we found that the histone methyltransferase Ezh2 interacted with Stat5b-CA complex that bound GAS sequences in the Irf8 enhancer whereas Ezh2 did not interact with GAS sequences in the case of the Irf4 promoter. Injection of Stat5b-CA.BMDCs to prediabetic NOD mice halted progression of islet inflammation and protected against diabetes. Importantly, inhibition of Ezh2 in tolerogenic Stat5b-CA.BMDCs reduced their ability to prevent diabetes development in NOD recipient mice. Taken together, our data suggest that the active form of Stat5b induces tolerogenic DC function by modulating IRF4 and IRF8 expression through recruitment of Ezh2 and highlight the fundamental role of Ezh2 in Stat5b-mediated induction of tolerogenic DCs function.

Enhanced calcium cycling and contractile function in transgenic hearts expressing constitutively active Gαo* protein

2008 ◽  
Vol 294 (3) ◽  
pp. H1335-H1347 ◽  
Author(s):  
Ming Zhu ◽  
Agnieszka A. Gach ◽  
GongXin Liu ◽  
Xiaomei Xu ◽  
Chee Chew Lim ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Ryanodine Receptor ◽  
Protein Phosphatase ◽  
Contractile Function ◽  
Active Form ◽  
Protein Phosphatase 1 ◽  
Wild Type ◽  
Specific Expression ◽  
Constitutively Active

In contrast to the other heterotrimeric GTP-binding proteins (G proteins) Gs and Gi, the functional role of Go is still poorly defined. To investigate the role of Gαo in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of Gαo1* (Gαo*), the predominant Gαo isoform in the heart. Gαo expression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in Gαo* transgenic mice in vivo, along with increased L-type Ca2+ channel current density, calcium transients, and cell shortening in ventricular Gαo*-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca2+ handling proteins were largely unchanged, except for a modest reduction in Na+/Ca2+ exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in Gαo* ventricles. Density and affinity of β-adrenoceptors, cAMP levels, and PKA activity were comparable in Gαo* and wild-type myocytes, but protein phosphatase 1 activity was reduced upon Gαo* expression, particularly in the vicinity of the ryanodine receptor. We conclude that Gαo* exerts a positive effect on Ca2+ cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca2+ handling proteins in hearts with constitutive Gαo activation.

scholarly journals Sensorimotor Gating Deficits in Transgenic Mice Expressing a Constitutively Active Form of Gsα

2003 ◽  
Vol 29 (3) ◽  
pp. 494-501 ◽  
Author(s):  
Thomas J Gould ◽  
Scott P Bizily ◽  
Jan Tokarczyk ◽  
Michele P Kelly ◽  
Steven J Siegel ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Active Form ◽  
Sensorimotor Gating ◽  
Constitutively Active

scholarly journals Transcriptomic and Metabolic Network Analysis of Metabolic Reprogramming and IGF-1 Modulation in SCA3 Transgenic Mice

2021 ◽  
Vol 22 (15) ◽  
pp. 7974
Author(s):  
Yu-Te Lin ◽  
Yong-Shiou Lin ◽  
Wen-Ling Cheng ◽  
Jui-Chih Chang ◽  
Yi-Chun Chao ◽  
...  
Keyword(s):  
Network Analysis ◽  
Transgenic Mice ◽  
Signaling Pathways ◽  
Metabolic Network ◽  
Metabolic Reprogramming ◽  
Spinocerebellar Ataxia Type ◽  
Positive Effects ◽  
Metabolic Network Analysis ◽  
Potential Biomarker ◽  
Metabolic Remodeling

Spinocerebellar ataxia type 3 (SCA3) is a genetic neurodegenerative disease for which a cure is still needed. Growth hormone (GH) therapy has shown positive effects on the exercise behavior of mice with cerebellar atrophy, retains more Purkinje cells, and exhibits less DNA damage after GH intervention. Insulin-like growth factor 1 (IGF-1) is the downstream mediator of GH that participates in signaling and metabolic regulation for cell growth and modulation pathways, including SCA3-affected pathways. However, the underlying therapeutic mechanisms of GH or IGF-1 in SCA3 are not fully understood. In the present study, tissue-specific genome-scale metabolic network models for SCA3 transgenic mice were proposed based on RNA-seq. An integrative transcriptomic and metabolic network analysis of a SCA3 transgenic mouse model revealed that metabolic signaling pathways were activated to compensate for the metabolic remodeling caused by SCA3 genetic modifications. The effect of IGF-1 intervention on the pathology and balance of SCA3 disease was also explored. IGF-1 has been shown to invoke signaling pathways and improve mitochondrial function and glycolysis pathways to restore cellular functions. As one of the downregulated factors in SCA3 transgenic mice, IGF-1 could be a potential biomarker and therapeutic target.

Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs

Biomaterials ◽  
2012 ◽  
Vol 33 (7) ◽  
pp. 2097-2108 ◽  
Author(s):  
Duohong Zou ◽  
Zhiyuan Zhang ◽  
Jiacai He ◽  
Kai Zhang ◽  
Dongxia Ye ◽  
...  
Keyword(s):  
Blood Vessel ◽  
Active Form ◽  
Blood Vessel Formation ◽  
Vessel Formation ◽  
Constitutively Active

scholarly journals Trafficking to the primary cilium membrane

2017 ◽  
Vol 28 (2) ◽  
pp. 233-239 ◽  
Author(s):  
Saikat Mukhopadhyay ◽  
Hemant B. Badgandi ◽  
Sun-hee Hwang ◽  
Bandarigoda Somatilaka ◽  
Issei S. Shimada ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Signaling Pathways ◽  
Primary Cilium ◽  
Hedgehog Signaling ◽  
Diffusion Barriers ◽  
Disease Pathogenesis ◽  
Ciliary Membrane ◽  
Multiple Organs ◽  
Sensory Reception

The primary cilium has been found to be associated with a number of cellular signaling pathways, such as vertebrate hedgehog signaling, and implicated in the pathogenesis of diseases affecting multiple organs, including the neural tube, kidney, and brain. The primary cilium is the site where a subset of the cell's membrane proteins is enriched. However, pathways that target and concentrate membrane proteins in cilia are not well understood. Processes determining the level of proteins in the ciliary membrane include entry into the compartment, removal, and retention by diffusion barriers such as the transition zone. Proteins that are concentrated in the ciliary membrane are also localized to other cellular sites. Thus it is critical to determine the particular role for ciliary compartmentalization in sensory reception and signaling pathways. Here we provide a brief overview of our current understanding of compartmentalization of proteins in the ciliary membrane and the dynamics of trafficking into and out of the cilium. We also discuss major unanswered questions regarding the role that defects in ciliary compartmentalization might play in disease pathogenesis. Understanding the trafficking mechanisms that underlie the role of ciliary compartmentalization in signaling might provide unique approaches for intervention in progressive ciliopathies.

Abstract 037: Transient Cardiac Expression Of Constitutively Active G alpha Q Activates Renin-angiotensin System, Leading To Progressive Heart Failure And Ventricular Arrhythmias In Transgenic Mice

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Naoko Matsushita ◽  
Masamichi hirose ◽  
Yasuchika Taeishi ◽  
Satoshi Suzuki ◽  
Toshihide Kashihara ◽  
...  
Keyword(s):  
Heart Failure ◽  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Ventricular Arrhythmias ◽  
Renin Angiotensin System ◽  
Gene Expressions ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Progressive Heart Failure ◽  
Constitutively Active

Introduction: Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) caused progressive heart failure and ventricular arrhythmias after the initiating stimulus becomes undetectable. However, the mechanisms are still unknown. Renin-angiotensin system plays a critical role in the development of cardiac hypertrophy and heart failure. We examined the effects of chronic administration of olmesartan on ventricular function, the number of premature ventricular contractions (PVC), and ventricular remodeling in Gαq-TG mice. Methods and Results: Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan treatment prevented the severe reduction of left ventricular fractional shortening and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan -treated Gαq-TG. The QT interval was significantly shorter in olmesartan-treated Gαq-TG than vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased in vehicle-treated Gαq-TG. Olmesartan significantly decreased these gene expressions in Gαq-TG. Moreover, protein expressions of canonical transient receptor potential (TRPC) channels 3 and 6 increased in vehicle-treated Gαq-TG hearts. Olmesartan significantly decreased TRPC6 expressions in Gαq-TG. Angiotensin converting enzyme (ACE) 1 and 2 gene expressions were also increased in vehicle-treated Gαq-TG and was not decreased to the control level in olmesartan-treated Gαq-TG. Conclusions: These findings suggest that renin-angiotensin system has an important role in the development of cardiac hypertrophy and heart failure even if the initiating stimulus is different from the activation of renin-angiotensin system.

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