Decreased IGF Type 1 Receptor Signaling in Mammary Epithelium during Pregnancy Leads to Reduced Proliferation, Alveolar Differentiation, and Expression of Insulin Receptor Substrate (IRS)-1 and IRS-2

The IGFs and the IGF type 1 receptor (IGF-1R) are essential mediators of normal mammary gland development in mice. IGF-I and the IGF-1R have demonstrated functions in formation and proliferation of terminal end buds and in ductal outgrowth and branching during puberty. To study the functions of IGF-1R during pregnancy and lactation, we established transgenic mouse lines expressing a human dominant-negative kinase dead IGF-1R (dnhIGF-1R) under the control of the whey acidic protein promoter. We provide evidence that the IGF-1R pathway is necessary for normal epithelial proliferation and alveolar formation during pregnancy. Furthermore, we demonstrate that the whey acidic protein-dnhIGF-1R transgene causes a delay in alveolar differentiation including lipid droplet formation, lumen expansion, and β-casein protein expression. Analysis of IGF-1R signaling pathways showed a decrease in P-IGF-1R and P-Akt resulting from expression of the dnhIGF-1R. We further demonstrate that disruption of the IGF-1R decreases mammary epithelial cell expression of the signaling intermediates insulin receptor substrate (IRS)-1 and IRS-2. No alterations were observed in downstream signaling targets of prolactin and progesterone, suggesting that activation of the IGF-1R may directly regulate expression of IRS-1/2 during alveolar development and differentiation. These data show that IGF-1R signaling is necessary for normal alveolar proliferation and differentiation, in part, through induction of signaling intermediates that mediate alveolar development.

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Type 1 Insulin-Like Growth Factor Receptor/Insulin Receptor Substrate 1 Signaling Confers Pathogenic Activity on Breast Tumor Cells Lacking REST

Molecular and Cellular Biology ◽

10.1128/mcb.01149-14 ◽

2015 ◽

Vol 35 (17) ◽

pp. 2991-3004 ◽

Cited By ~ 6

Author(s):

Kassondra Meyer ◽

Brittany Albaugh ◽

Barry Schoenike ◽

Avtar Roopra

Keyword(s):

Growth Factor ◽

Insulin Receptor ◽

Tumor Cells ◽

Breast Tumor ◽

Growth Factor Receptor ◽

Insulin Receptor Substrate ◽

Insulin Like Growth Factor ◽

Insulin Receptor Substrate 1 ◽

Breast Tumor Cells

Loss of repressor element 1 silencing transcription factor (REST) occurs in 20% of breast cancers and correlates with a poor patient prognosis. However, the molecular basis for enhanced malignancy in tumors lacking REST (RESTless) is only partially understood. We used multiplatform array data from the Cancer Genome Atlas to identify consistent changes in key signaling pathways. Of the proteins screened in the reverse-phase protein array, we found that insulin receptor substrate 1 (IRS1) is the most highly upregulated protein in RESTless breast tumors. Analysis of breast tumor cell lines showed that REST directly represses IRS1, and cells lacking REST have increased levels of IRS1 mRNA and protein. We find that the upregulation of IRS1 function is both necessary and sufficient for enhanced signaling and growth in breast cancer cells lacking REST. IRS1 overexpression is sufficient to phenocopy the enhanced activation of the signaling hubs AKT and mitogen-activated protein kinase (MAPK) of MCF7 cells lacking REST. Loss of REST renders MCF7 and MDA-MB-231 breast tumor cells dependent on IRS1 activity for colony formation in soft agar. Inhibition of the type 1 insulin-like growth factor receptor (IGF1R) reduces the enhanced signaling, growth, and migration in breast tumor cells that occur upon REST loss. We show that loss of REST induces a pathogenic program that works through the IGF1R/IRS1 pathway.

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The Pleckstrin Homology (PH) Domain-Interacting Protein Couples the Insulin Receptor Substrate 1 PH Domain to Insulin Signaling Pathways Leading to Mitogenesis and GLUT4 Translocation

Molecular and Cellular Biology ◽

10.1128/mcb.22.20.7325-7336.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 7325-7336 ◽

Cited By ~ 26

Author(s):

Janet Farhang-Fallah ◽

Varinder K. Randhawa ◽

Anjaruwee Nimnual ◽

Amira Klip ◽

Dafna Bar-Sagi ◽

...

Keyword(s):

Signal Transduction ◽

Insulin Receptor ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Insulin Receptor Substrate ◽

Glut4 Translocation ◽

Ph Domain ◽

Insulin Receptor Substrate 1 ◽

Interacting Protein ◽

Negative Mutant

ABSTRACT Receptor-mediated tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) is required for the propagation of many of insulin's biological effects. The amino-terminal pleckstrin homology (PH) domain of IRS-1 plays a pivotal role in promoting insulin receptor (IR)-IRS-1 protein interactions. We have recently reported the isolation of a PH domain-interacting protein, PHIP, which selectively binds to the IRS-1 PH domain and is stably associated with IRS-1 in mammalian cells. Here we demonstrate that overexpression of PHIP in fibroblasts enhances insulin-induced transcriptional responses in a mitogen-activated protein kinase-dependent manner. In contrast, a dominant-negative mutant of PHIP (DN-PHIP) was shown to specifically block transcriptional and mitogenic signals elicited by insulin and not serum. In order to examine whether PHIP/IRS-1 complexes participate in the signal transduction pathway linking the IR to GLUT4 traffic in muscle cells, L6 myoblasts stably expressing a myc-tagged GLUT4 construct (L6GLUT4myc) were transfected with either wild-type or dominant-interfering forms of PHIP. Whereas insulin-dependent GLUT4myc membrane translocation was not affected by overexpression of PHIP, DN-PHIP caused a nearly complete inhibition of GLUT4 translocation, in a manner identical to that observed with a dominant-negative mutant of the p85 subunit of phosphatidylinositol 3-kinase (Δp85). Furthermore, DN-PHIP markedly inhibited insulin-stimulated actin cytoskeletal reorganization, a process required for the productive incorporation of GLUT4 vesicles at the cell surface in L6 cells. Our results are consistent with the hypothesis that PHIP represents a physiological protein ligand of the IRS-1 PH domain, which plays an important role in insulin receptor-mediated mitogenic and metabolic signal transduction.

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Insulin receptor substrate-1/2 mediates IL-4-induced migration of human airway epithelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.90453.2008 ◽

2009 ◽

Vol 297 (1) ◽

pp. L164-L173 ◽

Cited By ~ 14

Author(s):

Steven R. White ◽

Linda D. Martin ◽

Mark K. Abe ◽

Bertha A. Marroquin ◽

Randi Stern ◽

...

Keyword(s):

Epithelial Cells ◽

Insulin Receptor ◽

Airway Epithelial Cells ◽

Dominant Negative ◽

Insulin Receptor Substrate ◽

Airway Epithelial ◽

Hairpin Rna ◽

Concurrent Treatment ◽

Receptor Associated Protein ◽

Air Liquid Interface

Migration of airway epithelial cells (AEC) is an integral component of airway mucosal repair after injury. The inflammatory cytokine IL-4, abundant in chronic inflammatory airways diseases such as asthma, stimulates overproduction of mucins and secretion of chemokines from AEC; these actions enhance persistent airway inflammation. The effect of IL-4 on AEC migration and repair after injury, however, is not known. We examined migration in primary human AEC differentiated in air-liquid interface culture for 3 wk. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Concurrent treatment with IL-4 up to 10 ng/ml accelerated migration significantly in fully differentiated AEC. As expected, IL-4 treatment induced phosphorylation of the IL-4 receptor-associated protein STAT (signal transducer and activator of transcription)6, a transcription factor known to mediate several IL-4-induced AEC responses. Expressing a dominant negative STAT6 cDNA delivered by lentivirus infection, however, failed to block IL-4-stimulated migration. In contrast, decreasing expression of either insulin receptor substrate (IRS)-1 or IRS-2 using a silencing hairpin RNA blocked IL-4-stimulated AEC migration completely. These data demonstrate that IL-4 can accelerate migration of differentiated AEC after injury. This reparative response does not require STAT6 activation, but rather requires IRS-1 and/or IRS-2.

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Nuclear Translocation of Insulin Receptor Substrate-1 by the Simian Virus 40 T Antigen and the Activated Type 1 Insulin-like Growth Factor Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m204658200 ◽

2002 ◽

Vol 277 (35) ◽

pp. 32078-32085 ◽

Cited By ~ 52

Author(s):

Marco Prisco ◽

Francesca Santini ◽

Raffaele Baffa ◽

Mingli Liu ◽

Robert Drakas ◽

...

Keyword(s):

Growth Factor ◽

Insulin Receptor ◽

Nuclear Translocation ◽

Growth Factor Receptor ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Insulin Receptor Substrate ◽

Insulin Receptor Substrate 1

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Insulin down-regulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway

Journal of Endocrinology ◽

10.1677/joe.0.1790253 ◽

2003 ◽

Vol 179 (2) ◽

pp. 253-266 ◽

Cited By ~ 62

Author(s):

Y Hirashima ◽

K Tsuruzoe ◽

S Kodama ◽

M Igata ◽

T Toyonaga ◽

...

Keyword(s):

Insulin Receptor ◽

Kinase Inhibitor ◽

Insulin Response ◽

Dominant Negative ◽

Akt Pathway ◽

Insulin Receptor Substrate ◽

Dependent Manner ◽

Gene Promoters ◽

Gene Assay

Insulin receptor substrate (IRS)-1 and IRS-2 are the major substrates that mediate insulin action. Insulin itself regulates the expression of the IRS protein in the liver, but the underlying mechanisms of IRS-1 and IRS-2 regulation are not fully understood. Here we report that insulin suppressed the expression of both IRS-1 and IRS-2 proteins in Fao hepatoma cells. The decrease in IRS-1 protein occurred via proteasomal degradation without any change in IRS-1 mRNA, whereas the insulin-induced suppression of IRS-2 protein was associated with a parallel decrease in IRS-2 mRNA without changing IRS-2 mRNA half-life. The insulin-induced suppression of IRS-2 mRNA and protein was blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, but not by the MAP kinase-ERK kinase (MEK) inhibitor, PD098059. Inhibition of Akt by overexpression of dominant-negative Akt also caused complete attenuation of the insulin-induced decrease in IRS-2 protein and partial attenuation of its mRNA down-regulation. Some nuclear proteins bound to the insulin response element (IRE) sequence on the IRS-2 gene in an insulin-dependent manner in vitro, and the binding was also blocked by the PI 3-kinase inhibitor. Reporter gene assay showed that insulin suppressed the activity of both human and rat IRS-2 gene promoters through the IRE in a PI 3-kinase-dependent manner. Our results indicate that insulin regulates IRS-1 and IRS-2 through different mechanisms and that insulin represses IRS-2 gene expression via a PI 3-kinase/Akt pathway.

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