Studying Metabolic Abnormalities in the Costello Syndrome HRAS G12V Mouse Model: Isolation of Mouse Embryonic Fibroblasts and Their In Vitro Adipocyte Differentiation

2021 ◽  
pp. 397-409
Author(s):  
Miray Fidan ◽  
Saravanakkumar Chennappan ◽  
Ion Cristian Cirstea
Keyword(s):  
Mouse Model ◽  
Adipocyte Differentiation ◽  
Costello Syndrome ◽  
Metabolic Abnormalities ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals PAPA-1 Is a Nuclear Binding Partner of IGFBP-2 and Modulates Its Growth-Promoting Actions

2009 ◽  
Vol 23 (2) ◽  
pp. 169-175 ◽  
Author(s):  
Kenichi Miyako ◽  
Laura J. Cobb ◽  
Malik Francis ◽  
Alden Huang ◽  
Bonnie Peng ◽  
...  
Keyword(s):  
Glutathione S Transferase ◽  
Mouse Embryonic Fibroblasts ◽  
Promoting Effect ◽  
Embryonic Fibroblasts ◽  
Igf Binding Proteins ◽  
Cellular Effects ◽  
Igf Binding ◽  
Growth Promoting ◽  
Interfering Rna

Abstract IGF-binding proteins (IGFBPs) have multiple cellular effects, which occur by both IGF-dependent and -independent mechanisms. IGFBP-2 is involved in the regulation of both normal and carcinogenic cell growth. To further understand the actions of IGFBP-2, we carried out a yeast two-hybrid screen to search for intracellular partner proteins using a human prostate cDNA library. We isolated Pim-1-associated protein-1 (PAP-1)-associated protein-1 (PAPA-1) as an IGFBP-2-binding protein, whose expression and subcellular localization is regulated by both IGFBP-2 and androgens. Coimmunoprecipitation and glutathione S-transferase pull-down assay confirmed the interaction in vitro, and confocal microscopy showed the colocalization of IGFBP-2 and PAPA-1 in the nucleus. Suppression of PAPA-1 by small interfering RNA treatment enhanced the growth-promoting effect of IGFBP-2. Conversely, IGFBP-2-promoted bromodeoxyuridine incorporation into LNCaP cells was abrogated by the simultaneous overexpression of myc-hPAPA-1. Mouse embryonic fibroblasts from IGFBP-2 knockout mouse showed diminished growth activity compared with wild type, and expression of FLAG-mPAPA-1 decreased cell proliferation in IGFBP-2 knockout, but not control mouse embryonic fibroblasts. These studies suggest that the growth-promoting role of IGFBP-2 in prostate cancer is inhibited by its intracellular interaction with PAPA-1.

scholarly journals Tm7sf2 gene promotes adipocyte differentiation of mouse embryonic fibroblasts and improves insulin sensitivity

2021 ◽  
Vol 1868 (1) ◽  
pp. 118897
Author(s):  
Leonardo Gatticchi ◽  
Maya Petricciuolo ◽  
Paolo Scarpelli ◽  
Lara Macchioni ◽  
Lanfranco Corazzi ◽  
...  
Keyword(s):  
Insulin Sensitivity ◽  
Adipocyte Differentiation ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals MEK-inhibitor-mediated rescue of skeletal myopathy caused by activating Hras mutation in a Costello syndrome mouse model

2021 ◽  
Author(s):  
William E. Tidyman ◽  
Alice F. Goodwin ◽  
Yoshiko Maeda ◽  
Ophir D. Klein ◽  
Katherine A. Rauen
Keyword(s):  
Mouse Model ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Costello Syndrome ◽  
Skeletal Myopathy ◽  
Mitogen Activated Protein

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes due to mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due in part to an inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction of myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction of p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS.

Skp2 promotes adipocyte differentiation via a p27Kip1-independent mechanism in primary mouse embryonic fibroblasts

2009 ◽  
Vol 379 (2) ◽  
pp. 249-254 ◽  
Author(s):  
Mitsuru Okada ◽  
Tamon Sakai ◽  
Takehiro Nakamura ◽  
Mimi Tamamori-Adachi ◽  
Shigetaka Kitajima ◽  
...  
Keyword(s):  
Adipocyte Differentiation ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Primary Mouse ◽  
Independent Mechanism

scholarly journals Cdk2 and Cdk4 Activities Are Dispensable for Tumorigenesis Caused by the Loss of p53

2009 ◽  
Vol 29 (10) ◽  
pp. 2582-2593 ◽  
Author(s):  
V. C. Padmakumar ◽  
Eiman Aleem ◽  
Cyril Berthet ◽  
Mary Beth Hilton ◽  
Philipp Kaldis
Keyword(s):  
Cyclin D ◽  
P53 Mutations ◽  
Spontaneous Tumors ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Oncogenic Potential ◽  
Cyclin Dependent Kinases ◽  
Cell Cycle Inhibitors

ABSTRACT The loss of p53 induces spontaneous tumors in mice, and p53 mutations are found in approximately 50% of human tumors. These tumors are generally caused by a number of events, including genomic instability, checkpoint defects, mitotic defects, deregulation of transcriptional targets, impaired apoptosis, and G1 deregulation or a combination of these effects. In order to determine the role of proteins involved in G1 control in tumorigenesis, we focused on Cdk2 and Cdk4, two cyclin-dependent kinases that in association with cyclin E and cyclin D promote the G1/S phase transition. We analyzed the consequence of loss of Cdk2 in p53-null animals by generating Cdk2 − / − p53 − / − mice. These mice are viable and developed spontaneous tumors, predominantly lymphoblastic lymphomas, similar to p53 − / − mice. In contrast, the genotypes Cdk4 − / − p53 − / − were mostly lethal, with few exceptions, and Cdk2 − / − Cdk4 − / − p53 − / − mice die during embryogenesis at embryonic day 13.5. To study the oncogenic potential, we generated mouse embryonic fibroblasts (MEFs) and found that p53 − / −, Cdk2 − / − p53 − / −, Cdk4 − / − p53 − / −, and Cdk2 − / − Cdk4 − / − p53 − / − MEFs grew at similar rates without entering senescence. Ras-transformed MEFs of these genotypes were able to form colonies in vitro and induce tumors in nude mice. Our results suggest that tumorigenicity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates considering the use of broad-spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy.

scholarly journals Anthraquinones as Inhibitors of SOS RAS-GEF Activity

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1128
Author(s):  
Alberto Fernández-Medarde ◽  
Rocío Fuentes-Mateos ◽  
Rósula García-Navas ◽  
Andrea Juan ◽  
José María Sánchez-López ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Chemical Structures ◽  
Ras Genes ◽  
Ras Activation ◽  
Marine Compounds ◽  
Ras Isoforms

Recent breakthroughs have reignited interest in RAS GEFs as direct therapeutic targets. To search for new inhibitors of SOS GEF activity, a repository of known/approved compounds (NIH-NACTS) and a library of new marine compounds (Biomar Microbial Technologies) were screened by means of in vitro RAS-GEF assays using purified, bacterially expressed SOS and RAS constructs. Interestingly, all inhibitors identified in our screenings (two per library) shared related chemical structures belonging to the anthraquinone family of compounds. All our anthraquinone SOS inhibitors were active against the three canonical RAS isoforms when tested in our SOS GEF assays, inhibited RAS activation in mouse embryonic fibroblasts, and were also able to inhibit the growth of different cancer cell lines harboring WT or mutant RAS genes. In contrast to the commercially available anthraquinone inhibitors, our new marine anthraquinone inhibitors did not show in vivo cardiotoxicity, thus providing a lead for future discovery of stronger, clinically useful anthraquinone SOS GEF blockers.

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