scholarly journals Interleukin-2 Receptor β Thr-450 Phosphorylation Is a Positive Regulator for Receptor Complex Stability and Activation of Signaling Molecules

2015 ◽  
Vol 290 (34) ◽  
pp. 20972-20983 ◽  
Author(s):  
Blanca E. Ruiz-Medina ◽  
Jeremy A. Ross ◽  
Robert A. Kirken
Keyword(s):  
Tyrosine Phosphorylation ◽  
Human Lymphocytes ◽  
Functional Characterization ◽  
Phosphorylation Site ◽  
Interleukin 2 ◽  
Cell Types ◽  
Effector Proteins ◽  
Positive Regulator ◽  
Multiple Cell

T, B, and natural killer cells are required for normal immune response and are regulated by cytokines such as IL-2. These cell signals are propagated following receptor-ligand engagement, controlling recruitment and activation of effector proteins. The IL-2 receptor β subunit (IL-2Rβ) serves in this capacity and is known to be phosphorylated. Tyrosine phosphorylation of the β chain has been studied extensively. However, the identification and putative regulatory roles for serine and threonine phosphorylation sites have yet to be fully characterized. Using LC-MS/MS and phosphospecific antibodies, a novel IL-2/IL-15 inducible IL-2Rβ phosphorylation site (Thr-450) was identified. IL-2 phosphokinetic analysis revealed that phosphorylation of IL-2Rβ Thr-450 is rapid (2.5 min), transient (peaks at 15 min), and protracted compared with receptor tyrosine phosphorylation and occurs in multiple cell types, including primary human lymphocytes. Pharmacological and siRNA-mediated inhibition of various serine/threonine kinases revealed ERK1/2 as a positive regulator, whereas purified protein phosphatase 1 (PP1), dephosphorylated Thr-450 in vitro. Reconstitution assays demonstrated that Thr-450 is important for regulating IL-2R complex formation, recruitment of JAK3, and activation of AKT and ERK1/2 and a transcriptionally active STAT5. These results provide the first evidence of the identification and functional characterization for threonine phosphorylation of an interleukin receptor.

scholarly journals MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 630
Author(s):  
Huili Lyu ◽  
Cody M. Elkins ◽  
Jessica L. Pierce ◽  
C. Henrique Serezani ◽  
Daniel S. Perrien
Keyword(s):  
Heterotopic Ossification ◽  
Muscle Injury ◽  
Cell Types ◽  
Fibrodysplasia Ossificans Progressiva ◽  
Inflammatory Signaling ◽  
Conditional Deletion ◽  
Pathway Crosstalk ◽  
Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

scholarly journals Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 66
Author(s):  
Rashmita Pradhan ◽  
Phuong A. Ngo ◽  
Luz d. C. Martínez-Sánchez ◽  
Markus F. Neurath ◽  
Rocío López-Posadas
Keyword(s):  
Intestinal Mucosa ◽  
Rho Gtpases ◽  
Current Knowledge ◽  
Cell Types ◽  
Effector Proteins ◽  
Special Focus ◽  
Specific Cell ◽  
Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

scholarly journals Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2745-2754 ◽  
Author(s):  
Alistair J. Standish ◽  
Jonathan J. Whittall ◽  
Renato Morona
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Capsular Polysaccharide ◽  
Regulatory Mechanism ◽  
Tyrosine Phosphatase ◽  
Phosphorylation Site ◽  
Regulatory Proteins ◽  
Amidase Activity ◽  
The Past

Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the Streptococcus pneumoniae capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that in vitro pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function in vitro with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal lytA of S. pneumoniae, autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide.

TMOD-21. A NOVEL IN-VITRO METHOD TO MODEL MACROPHAGES IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi220-vi220
Author(s):  
Hasan Alrefai ◽  
Andee Beierle ◽  
Lauren Nassour ◽  
Nicholas Eustace ◽  
Zeel Patel ◽  
...  
Keyword(s):  
Cell Types ◽  
In Vitro Models ◽  
Tumor Initiating Cells ◽  
Serum Free ◽  
Brain Tumor Initiating Cells ◽  
Multiple Cell ◽  
Anti Inflammatory ◽  
Free Media ◽  
Inflammatory Macrophages

Abstract BACKGROUND The GBM tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemoresistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Others have addressed this problem by employing 3D bioprinting to incorporate astrocytes and macrophages in an extracellular matrix; however, they used serum-containing media and classically polarized anti-inflammatory macrophages. Serum has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, highlighting the importance of excluding it from these models. Additionally, tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. METHODS THP-1 monocytes and normal human astrocytes (NHAs) were transitioned into serum-free HL-1 and neurobasal-based media, respectively. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft(PDX) lines, using a transwell insert. CD206 expression was used to validate polarization and a cytokine array was used to characterize the cells. RESULTS There was no difference in proliferation rates at 72 hours for THP-1 monocytes grown in serum-free HL-1 media compared to serum-containing RPMI 1640 (p > 0.95). Macrophages polarized via transwell inserts expressed the lymphocyte chemoattractant protein, CCL2, whereas resting(M0), pro-inflammatory(M1), and anti-inflammatory(M2) macrophages did not. Additionally, these macrophages expressed more CXCL1 and IL-1ß relative to M1 macrophages. We have also demonstrated a method to maintain a tri-culture model of GBM PDX cells, NHAs, and TAMs in a serum-free media that supports the growth/maintenance of all cell types. CONCLUSIONS We have demonstrated a novel method by which we can polarize macrophages towards a tumor-supportive phenotype that differs in cytokine expression from traditionally polarized macrophages. This higher-fidelity method of modeling TAMs in GBM can aid in the development of targeted therapeutics that may one day enter the clinic in hopes of improving outcomes in GBM.

scholarly journals Maturation of acute T-lymphoblastic leukemia cells after CD2 ligation and subsequent treatment with interleukin-2

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1182-1192 ◽  
Author(s):  
F Mentz ◽  
F Ouaaz ◽  
A Michel ◽  
C Blanc ◽  
P Herve ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Lymphoblastic Leukemia ◽  
Interleukin 1 ◽  
Interleukin 2 ◽  
Subsequent Treatment ◽  
Leukemic Cells ◽  
Cell Phenotype ◽  
Cell Functions

Abstract In this study, we have investigated the ability of various cytokines to induce the maturation of acute lymphoblastic leukemia (T-ALL) cells with early T-cell phenotype. Leukemic blasts from 17 untreated T-ALL patients were assayed for their ability to acquire mature T-cell markers, CD3/T-cell receptor (TCR) in particular, after incubation with one or a combination of recombinant human interleukin-1 (IL-1), IL-2, IL-4, IL-7, and CD2-specific monoclonal antibody (MoAb). IL-7 or IL-2 induced the proliferation of some leukemic cells, whereas sequential cell treatment with CD2-MoAb and then IL-2 promoted CD3/TCR expression on nearly all CD2+ cells (15 of 16), except for 1 T-ALL that developed into CD3-CD16+CD56+ cells. Differentiation of T-ALL cells was also evidenced through the downregulation of CD34 precursor cell antigen, the generation of CD4+ and CD8+ cells from CD4+ CD8+ precursors, and the acquisition of mature T-cell functions. CD2 ligation induced a progressive increase of surface expression of IL-2 receptor alpha (IL- 2R alpha) and IL-2R beta and an accelerated in vitro death of leukemic cells. The ligation of IL-2R by IL-2 rescued T-ALL cells from death and promoted their progression toward more mature cells expressing extracellular CD3/TCR alpha beta complexes. Intracellular analysis indicates that TCR alpha transcription and membrane translocation of both TCR alpha and TCR beta were promoted in these conditions. Analysis of intracellular signals transduced during T-ALL differentiation indicated that CD2-ligation induced Ca2+ influx and that the ligation of CD2 and IL-2R induced distinct tyrosine phosphorylation patterns. The addition of inhibitors of tyrosine phosphorylation abolished T-ALL cell differentiation, which suggests the involvement of tyrosine kinases in this phenomenon. Together, we showed the constant maturation of leukemic early T cells after stimulation of surface CD2 and the high- affinity IL-2R.

scholarly journals The proliferative response of B cell chronic lymphocytic leukemia to interleukin 2: functional characterization of the interleukin 2 membrane receptors

Blood ◽  
1987 ◽  
Vol 69 (6) ◽  
pp. 1667-1673 ◽  
Author(s):  
I Touw ◽  
L Dorssers ◽  
B Lowenberg
Keyword(s):  
Monoclonal Antibodies ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Functional Characterization ◽  
Interleukin 2 ◽  
Lymphocytic Leukemia ◽  
Membrane Receptors ◽  
Thymidine Uptake ◽  
Cell Chronic Lymphocytic Leukemia

Abstract To determine the growth properties of B cell chronic lymphocytic leukemia (B CLL) and to identify possible abnormalities thereof, we examined the in vitro action of interleukin 2 (IL2) in four patients. Using radiolabeled IL2 and monoclonal antibodies reactive with IL2 membrane receptors we show that CLL cells, after their activation in vitro, express IL2 receptors of a high- as well as a low-affinity type, exactly as has been reported for normal T and B blasts. In three of the four reported cases, CLL proliferation (measured with 3H-thymidine incorporation) depended on the addition of phytohemagglutinin (PHA) to activate the cells and IL2 (optimal concentration, 10 to 100 U IL2/mL). In contrast, the cells of the fourth case of CLL (CLL-4) proliferated in an autonomous fashion, ie, without a need for PHA and IL2 in culture. Specific blocking of the IL2-binding sites with anti-IL2 receptor monoclonal antibodies almost completely inhibited the proliferation of these cells, which indicated that functional IL2 receptors were required for the autonomous proliferation. The demonstration of low concentrations of IL2 activity in the culture medium conditioned by the cells suggests that endogenous IL2 had been responsible for the spontaneous 3H-thymidine uptake by the CLL cells of patient 4. However, we were unable to extract IL2 mRNA from the cells (neither fresh nor after various in vitro incubations) in quantities detectable by Northern blot analysis that would prove that the CLL cells of patient 4 were actively synthesizing IL2 during culture. Thus, individual cases of B CLL are subject to variable growth regulation involving functional IL2 receptors on the cell surface: after activation with PHA the cells respond to exogenous IL2 in a fashion similar to normal B lymphocytes, or the cells are stimulated by endogenous IL2 (or an IL2-like activity) and do not require activation with PHA.

scholarly journals Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1306
Author(s):  
Ann-Kristin Afflerbach ◽  
Mark D. Kiri ◽  
Tahir Detinis ◽  
Ben M. Maoz
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Types ◽  
In Vitro Models ◽  
Cell Source ◽  
Fundamental Research ◽  
Multiple Cell ◽  
Vitro Model

The human-relevance of an in vitro model is dependent on two main factors—(i) an appropriate human cell source and (ii) a modeling platform that recapitulates human in vivo conditions. Recent years have brought substantial advancements in both these aspects. In particular, mesenchymal stem cells (MSCs) have emerged as a promising cell source, as these cells can differentiate into multiple cell types, yet do not raise the ethical and practical concerns associated with other types of stem cells. In turn, advanced bioengineered in vitro models such as microfluidics, Organs-on-a-Chip, scaffolds, bioprinting and organoids are bringing researchers ever closer to mimicking complex in vivo environments, thereby overcoming some of the limitations of traditional 2D cell cultures. This review covers each of these advancements separately and discusses how the integration of MSCs into novel in vitro platforms may contribute enormously to clinical and fundamental research.

scholarly journals Three-dimensional testicular organoids as novel in vitro models of testicular biology and toxicology

2019 ◽  
Vol 5 (3) ◽  
Author(s):  
Sadman Sakib ◽  
Anna Voigt ◽  
Taylor Goldsmith ◽  
Ina Dobrinski
Keyword(s):  
Reproductive Biology ◽  
Monolayer Culture ◽  
Three Dimensional ◽  
Cell Types ◽  
In Vitro Models ◽  
Toxicity Screening ◽  
Current State ◽  
Potential Applications ◽  
Multiple Cell

Abstract Organoids are three dimensional structures consisting of multiple cell types that recapitulate the cellular architecture and functionality of native organs. Over the last decade, the advent of organoid research has opened up many avenues for basic and translational studies. Following suit of other disciplines, research groups working in the field of male reproductive biology have started establishing and characterizing testicular organoids. The three-dimensional architectural and functional similarities of organoids to their tissue of origin facilitate study of complex cell interactions, tissue development and establishment of representative, scalable models for drug and toxicity screening. In this review, we discuss the current state of testicular organoid research, their advantages over conventional monolayer culture and their potential applications in the field of reproductive biology and toxicology.

Sign in / Sign up

Export Citation Format

Share Document