IGF-1 protects against angiotensin II-induced cardiac fibrosis by targeting αSMA

The insulin-like growth factor 1 receptor (IGF-1R) signaling in cardiomyocytes is implicated in physiological hypertrophy and myocardial aging. Although fibroblasts account for a small amount of the heart, they are activated when the heart is damaged to promote cardiac remodeling. However, the role of IGF-1R signaling in cardiac fibroblasts is still unknown. In this study, we investigated the roles of IGF-1 signaling during agonist-induced cardiac fibrosis and evaluated the molecular mechanisms in cultured cardiac fibroblasts. Using an experimental model of cardiac fibrosis with angiotensin II/phenylephrine (AngII/PE) infusion, we found severe interstitial fibrosis in the AngII/PE infused myofibroblast-specific IGF-1R knockout mice compared to the wild-type mice. In contrast, low-dose IGF-1 infusion markedly attenuated AngII-induced cardiac fibrosis by inhibiting fibroblast proliferation and differentiation. Mechanistically, we demonstrated that IGF-1-attenuated AngII-induced cardiac fibrosis through the Akt pathway and through suppression of rho-associated coiled-coil containing kinases (ROCK)2-mediated α-smooth muscle actin (αSMA) expression. Our study highlights a novel function of the IGF-1/IGF-1R signaling in agonist-induced cardiac fibrosis. We propose that low-dose IGF-1 may be an efficacious therapeutic avenue against cardiac fibrosis.Subject terms: Heart failure, Heart failure     

A novel functional crosstalk between DDR1 and the IGF axis and its relevance for breast cancer

ABSTRACT

In the last decades increasing importance has been attributed to the Insulin/Insulin-like Growth Factor signaling (IIGFs) in cancer development, progression and resistance to therapy. In fact, IIGFs is often deregulated in cancer. In particular, the mitogenic insulin receptor isoform A (IR-A) and the insulin-like growth factor receptor (IGF-1R) are frequently overexpressed in cancer together with their cognate ligands IGF-1 and IGF-2. Recently, we identified discoidin domain receptor 1 (DDR1) as a new IR-A interacting protein. DDR1, a non-integrin collagen tyrosine kinase receptor, is overexpressed in several malignancies and plays a role in cancer progression and metastasis.

Herein, we review recent findings indicating that DDR1 is as a novel modulator of IR and IGF-1R expression and function. DDR1 functionally interacts with IR and IGF-1R and enhances the biological actions of insulin, IGF-1 and IGF-2. Conversely, DDR1 is upregulated by IGF-1, IGF-2 and insulin through the PI3K/AKT/miR-199a-5p circuit. Furthermore, we discuss the role of the non-canonical estrogen receptor GPER1 in the DDR1-IIGFs crosstalk. These data suggest a wider role of DDR1 as a regulator of cell response to hormones, growth factors, and signals coming from the extracellular matrix.     

Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.     

Mediation of the actions of insulin and insulin-like growth factor-1 on preimplantation mouse embryos in vitro.

Previous studies showed that both insulin and insulin-like growth factor-1 (IGF-1) stimulate metabolism and growth of preimplantation embryos. Because the effects of insulin occur with very low doses, it was suggested that its effects were mediated by its own receptors. However, the effects of IGF-1 occurred at higher doses, suggestive of cross reaction with the insulin receptor but still in the range for mediation via its own receptor. The aim of this study was to investigate the mediation of the metabolic and growth effects of insulin and IGF-1 using a specific insulin receptor antagonist. The antagonistic B-10 Fab fragment (B-10f) completely blocked stimulation of protein synthesis by both insulin and IGF-1, indicating that the insulin receptor mediates this action of both hormones. Alternately, only insulin's stimulation of inner cell mass mitogenesis and morphological development was inhibited by the B-10 Fab fragment. This showed that growth stimulation by insulin and IGF-1 was mediated via different receptors, insulin through its own receptor and IGF-1 through some other receptor. However, mediation via the IGF-2 receptor is not excluded since IGF-1 stimulates compaction when there is evidence for only the presence of the IGF-2 receptor. In summary, insulin or IGF-1 at physiological concentrations stimulates preimplantation mouse embryos, suggesting an important role for both these growth factors in early development.     

Insulin and Insulin-like Growth Factor-1 Receptors Act as Ligand-specific Amplitude Modulators of a Common Pathway Regulating Gene Transcription

Insulin and insulin-like growth factor-1 (IGF-1) act on highly homologous receptors, yet in vivo elicit distinct effects on metabolism and growth. To investigate how the insulin and IGF-1 receptors exert specificity in their biological responses, we assessed their role in the regulation of gene expression using three experimental paradigms: 1) preadipocytes before and after differentiation into adipocytes that express both receptors, but at different ratios; 2) insulin receptor (IR) or IGF1R knock-out preadipocytes that only express the complimentary receptor; and 3) IR/IGF1R double knock-out (DKO) cells reconstituted with the IR, IGF1R, or both. In wild-type preadipocytes, which express predominantly IGF1R, microarray analysis revealed ∼500 IGF-1 regulated genes (p < 0.05). The largest of these were confirmed by quantitative PCR, which also revealed that insulin produced a similar effect, but with a smaller magnitude of response. After differentiation, when IR levels increase and IGF1R decrease, insulin became the dominant regulator of each of these genes. Measurement of the 50 most highly regulated genes by quantitative PCR did not reveal a single gene regulated uniquely via the IR or IGF1R using cells expressing exclusively IGF-1 or insulin receptors. Insulin and IGF-1 dose responses from 1 to 100 nm in WT, IRKO, IGFRKO, and DKO cells re-expressing IR, IGF1R, or both showed that insulin and IGF-1 produced effects in proportion to the concentration of ligand and the specific receptor on which they act. Thus, IR and IGF1R act as identical portals to the regulation of gene expression, with differences between insulin and IGF-1 effects due to a modulation of the amplitude of the signal created by the specific ligand-receptor interaction.     

Essential role of insulin and insulin-like growth factor 1 receptor signaling in cardiac development and function

Cardiovascular disease is the leading cause of death in people with type 2 diabetes and is linked to insulin resistance even in the absence of diabetes. Here we show that mice with combined deficiency of the insulin receptor and insulin-like growth factor 1 (IGF-1) receptor in cardiac and skeletal muscle develop early-onset dilated cardiomyopathy and die from heart failure within the first month of life despite having a normal glucose homeostasis. Mice lacking the insulin receptor show impaired cardiac performance at 6 months, and mice lacking the insulin receptor plus one Igf1r allele have slightly increased mortality. By contrast, mice lacking the IGF-1 receptor or the IGF-1 receptor plus one Ir allele appear normal. Morphological characterization and oligonucleotide array analysis of gene expression demonstrate that prior to development of these physiological defects, mice with combined deficiency of both insulin and IGF-1 receptors have a coordinated down-regulation of genes encoding components of the electron transport chain and mitochondrial fatty acid beta-oxidation pathways and altered expression of contractile proteins. Thus, while neither the insulin receptor nor IGF-1 receptor in muscle is critical for glucose homeostasis during the first month of life, signaling from these receptors, particularly the insulin receptor, is required for normal cardiac metabolism and function.     

Insulin and insulin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors.

Chimeric insulin/insulin-like growth factor-1 receptors and insulin receptor alpha-subunit point mutants were characterized with respect to their binding properties for insulin and insulin-like growth factor-1 (IGF-1) and their ability to translate ligand interaction into tyrosine kinase activation in intact cells. We found that replacement of the amino-terminal 137 amino acids of the insulin receptor (IR) with the corresponding 131 amino acids of the IGF-1 receptor (IGF-1R) resulted in loss of affinity for both ligands. Further replacement of the adjacent cysteine region with IGF-1R sequences fully reconstituted affinity for IGF-1, but only marginally for insulin. Unexpectedly, replacement of the IR cysteine-rich domain alone by IGF-1R sequences created a high affinity receptor for both insulin and IGF-1. The binding characteristics of all receptor chimeras reflected the potential of both ligands to regulate the receptor tyrosine kinase activity in intact cells. Our chimeric receptor data, in conjunction with IR amino-terminal domain point mutants, strongly suggest major contributions of structural determinants in both amino- and carboxyl-terminal IR alpha-subunit regions for the formation of the insulin-binding pocket, whereas, surprisingly, the residues defining IGF-1 binding are present predominantly in the cysteine-rich domain of the IGF-1R.     

Insulin receptor structure and its implications for the IGF-1 receptor

The insulin receptor (isoforms IR-A and IR-B) and the type-I insulin-like growth factor receptor (IGF-1R) are homologous, multi-domain tyrosine kinases that bind insulin and IGF-1 with differing specificity. IR is involved in metabolic regulation and IGF-1R in normal growth and development. IR-A also binds IGF-2 with an affinity comparable to IGF-1R and, like the latter, is implicated in a range of cancers. The recent structure of the IR ectodomain dimer explains many features of ligand-receptor binding and provides insight into the structure of the intact ligand-binding site in both receptors. The structures of the L1-CR-L2 fragments of IR and IGF-1R reveal major differences in the regions that govern ligand specificity. The IR ectodomain X-ray structure raises doubts about that obtained by STEM reconstruction.     

Insulin-like growth factor 1 receptor signaling regulates skin development and inhibits skin keratinocyte differentiation

The insulin-like growth factor 1 receptor (IGF-1R) is a multifunctional receptor that mediates signals for cell proliferation, differentiation, and survival. Genetic experiments showed that IGF-1R inactivation in skin results in a disrupted epidermis. However, because IGF-1R-null mice die at birth, it is difficult to study the effects of IGF-1R on skin. By using a combined approach of conditional gene ablation and a three-dimensional organotypic model, we demonstrate that IGF-1R-deficient skin cocultures show abnormal maturation and differentiation patterns. Furthermore, IGF-1R-null keratinocytes exhibit accelerated differentiation and decreased proliferation. Investigating the signaling pathway downstream of IGF-1R reveals that insulin receptor substrate 2 (IRS-2) overexpression compensates for the lack of IGF-1R, whereas IRS-1 overexpression does not. We also demonstrate that phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1 and 2 are involved in the regulation of skin keratinocyte differentiation and take some part in mediating the inhibitory signal of IGF-1R on differentiation. In addition, we show that mammalian target of rapamycin plays a specific role in mediating IGF-1R impedance of action on keratinocyte differentiation. In conclusion, these results reveal that IGF-1R plays an inhibitory role in the regulation of skin development and differentiation.     

Impact of reduced insulin-like growth factor-1/insulin signaling on aging in mammals: novel findings

Growth hormone deficiency or resistance resulting from spontaneous or experimentally produced mutations in laboratory mice delay aging and increase lifespan. Alterations in insulin-like growth factor-1 (IGF-1) and insulin signaling emerged as likely mechanisms linking growth hormone and aging, and increased longevity was reported in mice with selective deletion of IGF-1 receptor in all tissues or insulin receptor in fat. Recent studies in mice with reduced IGF-1 levels or deletion of pregnancy-associated plasma protein-A, a protease that cleaves one of the IGF-1 binding proteins, strongly support the role of IGF-1 in the control of longevity. Reports of increased lifespan in mice with deletion of insulin receptor substrate (IRS) 1, reduced expression of IRS2, or selective deletion of IRS2 in the brain specifically implicate the IRS-PI3K-Akt-Foxo signaling pathway (which is shared by IGF-1 and insulin) in the control of aging. These important novel findings also strengthen the evidence for evolutionary conservation of mechanisms regulating lifespan in worms, insects and mammals.     

Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors.

BALB/c3T3 cells are exquisitely growth regulated and require both platelet-derived growth factor and insulin-like growth factor-1 (IGF-1) for optimal proliferation. BALB/c3T3 cells that constitutively express IGF-1 and elevated levels of IGF-1 receptor (IGF-1R) are capable of growth in serum-free medium without the addition of any exogenous growth factors. BALB/c3T3 cells overexpressing only the IGF-1R plasmid required IGF-1 or insulin for serum-free growth. Antisense oligodeoxynucleotides complementary to IGF-1R mRNA inhibited IGF-1-mediated cell growth. Under these conditions, neither the epidermal growth factor receptor nor phospholipase C gamma 1 was autophosphorylated. These findings indicate that constitutive expression of IGF-1 and IGF-1R allows 3T3 cells to grow in serum-free medium without addition of those exogenous growth factors that are required by the parent cell line.     

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II

Background

Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2.

Methodology/Principal Findings

In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation.

Conclusions/Significance

This study provides novel insight into the mechanism of receptor binding and activation by IGF-II, which may be important for the future development of inhibitors of its action for the treatment of cancer.     

RACK1 recruits STAT3 specifically to insulin and insulin-like growth factor 1 receptors for activation, which is important for regulating anchorage-independent growth

Current understanding of the activation of STATs is through binding between the SH2 domain of STATs and phosphotyrosine of tyrosine kinases. Here we demonstrate a novel role of RACK1 as an adaptor for insulin and insulin-like growth factor 1 receptor (IGF-1R)-mediated STAT3 activation specifically. Intracellular association of RACK1 via its N-terminal WD domains 1 to 4 (WD1-4) with insulin receptor (IR)/IGF-1R is augmented upon respective ligand stimulation, whereas association with STAT3 is constitutive. Purified RACK1 or RACK1 WD1-4 associates directly with purified IR, IGF-1R, and STAT3 in vitro. Insulin induces multiprotein complex formation of RACK1, IR, and STAT3. Overexpression or downregulation of RACK1 greatly enhances or decreases, respectively, IR/IGF-1R-mediated activation of STAT3 and its target gene expression. Site-specific mutants of IR and IGF-1R impaired in RACK1 binding are ineffective in mediating recruitment and activation of STAT3 as well as in insulin- or IGF-1-induced protection of cells from anoikis. RACK1-mediated STAT3 activation is important for insulin and IGF-1-induced anchorage-independent growth in certain ovarian cancer cells. We conclude that RACK1 mediates recruitment of STAT3 to IR and IGF-1R specifically for activation, suggesting a general paradigm for the need of an adaptor in mediating activation of STATs by receptor protein tyrosine kinases.     

Leukocyte antigen-related deficiency enhances insulin-like growth factor-1 signaling in vascular smooth muscle cells and promotes neointima formation in response to vascular injury

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.     

Insulin and aging

In invertebrates, signaling pathways homologous to mammalian insulin and insulin-like growth factor (IGF-1) signal transduction have a major role in the control of longevity. There are numerous indications that these pathways also influence aging in mammals, but separating the role of insulin from the effects of IGF-1 and growth hormone (GH) is difficult. In mice, selective disruption of the insulin receptor in the adipose tissue extends longevity. Increases in lifespan were also reported in mice with deletion of insulin receptor substrate 1 (IRS1) in whole body or IRS2 only in the brain. GH deficiency or resistance in mutant mice leads to hypoinsulinemia and enhanced insulin sensitivity along with remarkably extended longevity. These characteristics resemble animals subjected to calorie restriction. Studies of physiological characteristics and polymorphisms of insulin-related genes in exceptionally long-lived people suggest a role of insulin signaling in the control of human aging.     

The differential effects of pp120 (Ceacam 1) on the mitogenic action of insulin and insulin-like growth factor 1 are regulated by the nonconserved tyrosine 1316 in the insulin receptor

pp120 (Ceacam 1) undergoes ligand-stimulated phosphorylation by the insulin receptor, but not by the insulin-like growth factor 1 receptor (IGF-1R). This differential phosphorylation is regulated by the C terminus of the beta-subunit of the insulin receptor, the least conserved domain of the two receptors. In the present studies, deletion and site-directed mutagenesis in stably transfected hepatocytes derived from insulin receptor knockout mice (IR(-/-)) revealed that Tyr(1316), which is replaced by the nonphosphorylatable phenylalanine in IGF-1R, regulated the differential phosphorylation of pp120 by the insulin receptor. Similarly, the nonconserved Tyr(1316) residue also regulated the differential effect of pp120 on IGF-1 and insulin mitogenesis, with pp120 downregulating the growth-promoting action of insulin, but not that of IGF-1. Thus, it appears that pp120 phosphorylation by the insulin receptor is required and sufficient to mediate its downregulatory effect on the mitogenic action of insulin. Furthermore, the current studies revealed that the C terminus of the beta-subunit of the insulin receptor contains elements that suppress the mitogenic action of insulin. Because IR(-/-) hepatocytes are derived from liver, an insulin-targeted tissue, our observations have finally resolved the controversy about the role of the least-conserved domain of insulin and IGF-1Rs in mediating the difference in the mitogenic action of their ligands, with IGF-1 being more mitogenic than insulin.     

The luteinizing hormone-releasing hormone inhibits the anti-apoptotic activity of insulin-like growth factor-1 in pituitary alphaT3 cells by protein kinase Calpha-mediated negative regulation of Akt

The luteinizing hormone-releasing hormone (LHRH) receptor is a G protein-coupled receptor involved in the synthesis and release of pituitary gonadotropins and in the proliferation and apoptosis of pituitary cells. Insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor that has a mitogenic effect on pituitary cells. In this study, we used the alphaT3 gonadotrope cell line as a model to characterize the IGF-1R signaling pathways and to investigate whether this receptor interacts with the LHRH cascade. We found that IGF-1 activated the IGF-1R, insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase, and Akt in a time-dependent manner in alphaT3 cells. The MAPK (ERK1/2, p38, and JNK) pathways were only weakly activated by IGF-1. In contrast, LHRH strongly stimulated the MAPK pathways but had no effect on Akt activation. Cotreatment with IGF-1 and LHRH had various effects on these signaling pathways. 1) It strongly increased IGF-1-induced tyrosine phosphorylation of IRS-1 and IRS-1-associated phosphatidylinositol 3-kinase through activation of the epidermal growth factor receptor. 2) It had an additive effect on ERK1/2 activation without modifying the phosphorylation of p38 and JNK1/2. 3) It strongly reduced IGF-1 activation of Akt. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and cell cycle analysis revealed that, in addition to having an additive effect on ERK1/2 activation, cotreatment with IGF-1 and LHRH also had an additive effect on cell proliferation. The LHRH-induced inhibition of Akt stimulated by IGF-1 was completely blocked by Safingol, a protein kinase C (PKC) alpha-specific inhibitor, and by a dominant negative form of PKCalpha. Finally, we showed that the inhibitory effect of LHRH on IGF-1-induced PKCalpha-mediated Akt activation was associated with a marked reduction in Bad phosphorylation and a substantial decrease in the ability of IGF-1 to rescue alphaT3 cells from apoptosis induced by serum starvation. Our results demonstrate for the first time that several interactions take place between IGF-1 and LHRH receptors in gonadotrope cells.