Growth and differentiation signals by the insulin-like growth factor 1 receptor in hemopoietic cells are mediated through different pathways

The type 1 insulin-like growth factor receptor (IGF-IR) plays an important role in the growth of cells both in vivo and in vitro. The IGF-IR is also capable of inducing differentiation in a number of cell types, raising the question of how the same receptor can send two seemingly contradictory signals, one for growth and one for differentiation. Using 32D cells, which are murine hemopoietic cells, we show that the activated IGF-IR can induce differentiation along the granulocytic pathway in a manner similar to the granulocyte colony-stimulating factor. We find that one of the major substrates of the IGF-IR, the insulin receptor substrate-1 inhibits IGF-I-mediated differentiation of 32D cells. In the absence of insulin receptor substrate-1, functional impairment of another major substrate of the IGF-IR, the Shc proteins, is associated with a decrease in the extent of differentiation. Although the end points of the respective pathways remain to be defined, these results show for the first time that IGF-I-mediated growth or differentiation of hemopoietic cells may depend on a balance between two of its substrates.     

Sterol structure dependence of insulin receptor and insulin-like growth factor 1 receptor activation

The plasma membrane is a dynamic environment with a complex composition of lipids, proteins, and cholesterol. Areas enriched in cholesterol and sphingolipids are believed to form lipid rafts, domains of highly ordered lipids. The unique physical properties of these domains have been proposed to influence many cellular processes. Here, we demonstrate that the activation of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) depends critically on the structures of membrane sterols. IR and IGF1R autophosphorylation in vivo was inhibited by cholesterol depletion, and autophosphorylation was restored by the replacement with exogenous cholesterol. We next screened a variety of sterols for effects on IR activation. The ability of sterols to support IR autophosphorylation was strongly correlated to the propensity of the sterols to form ordered domains. IR autophosphorylation was fully restored by the incorporation of ergosterol, dihydrocholesterol, 7-dehydrocholesterol, lathosterol, desmosterol, and allocholesterol, partially restored by epicholesterol, and not restored by lanosterol, coprostanol, and 4-cholesten-3-one. These data support the hypothesis that the ability to form ordered domains is sufficient for a sterol to support ligand-induced activation of IR and IGF1R in intact mammalian cells.     

GIPC participates in G protein signaling downstream of insulin-like growth factor 1 receptor.

Several recent studies have demonstrated that insulin-like growth factor (IGF)-1-induced mitogen-activated protein kinase (MAP kinase) activation is abolished by pertussis toxin, suggesting that trimeric G proteins of the G(i) class are novel cellular targets of the IGF-1 signaling pathway. We report here that the intracellular domain of the Xenopus IGF-1 receptor is capable of binding to the Xenopus homolog of mammalian GIPC, a PDZ domain-containing protein previously identified as a binding partner of G(i)-specific GAP (RGS-GAIP). Binding of xGIPC to xIGF-1 receptor is independent of the kinase activity of the receptor and appears to require the PDZ domain of xGIPC. Injection of two C-terminal truncation mutants that retained the PDZ domain blocked IGF-1-induced Xenopus MAP kinase activation and oocyte maturation. While full-length xGIPC injection did not significantly alter insulin response, it greatly enhanced human RGS-GAIP in stimulating the insulin response in frog oocytes. This represents the first demonstration that GIPC x RGS-GAIP complex acts positively in IGF-1 receptor signal transduction.     

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by beta1 integrins

We show here that beta1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The beta1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, beta1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, beta1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the beta1 cytodomain plays an important role in mediating beta1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, beta1A associates with IRS-1 and beta1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.     

Role of phospholipase C-gamma1 in insulin-like growth factor I-induced muscle differentiation of H9c2 cardiac myoblasts

Insulin-like growth factor-I (IGF-I) regulates muscle differentiation through phosphatidylinositol 3-kinase (PI 3-kinase). Also it was recently reported that PI 3-kinase is involved in the activation of phospholipase C-gamma1 (PLC-gamma1). We investigated whether PLC-gamma1 therefore plays a role in IGF-I-induced muscle differentiation using H9c2 rat cardiac myoblasts as a model. IGF-I was able to activate PLC-gamma1 via both PI 3-kinase-dependent and tyrosine phosphorylation-dependent mechanisms in this model. However, PI 3-kinase appeared to play a more important role than tyrosine phosphorylation in IGF-I activation of PLC-gamma1. In addition, PLC-gamma1 activation was independent of Akt/protein kinase B (Akt/PKB). Importantly, PLC-gamma1 was involved in IGF-I-induced muscle differentiation in parallel with Akt/PKB. Taken together, these results suggest that IGF-I regulation of muscle differentiation is dependent on the activation of PLC-gamma1 and Akt/PKB, both of which are downstream mediators of PI 3-kinase.     

Proprotein convertases regulate insulin-like growth factor 1-induced membrane-type 1 matrix metalloproteinase in VSMCs via endoproteolytic activation of the insulin-like growth factor-1 receptor

The IGF-1 receptor (IGF-1R) and MT1-MMP are synthesized as larger precursor proproteins, which require endoproteolytic activation by the proprotein convertases (PCs) furin/PC5 to gain full biological activity. The aim of this study was to investigate the contribution of PCs to IGF-1R and/or MT1-MMP activation in vascular smooth muscle cells (VSMCs) as well as VSMC proliferation/migration, which are key elements in vascular remodeling. Furin and PC5 mRNAs and proteins were found in VSMCs. Inhibition of furin-like PCs with the specific pharmacological inhibitor dec-CMK inhibited IGF-1R endoproteolytic activation. Inhibition of IGF-1R maturation abrogated IGF-induced IGF-1R autophosphorylation, PI3-kinase and MAPK induction, as well as VSMC proliferation (p<0.05 vs. controls), whereas it had no effect of PDGF-stimulated signaling pathways or cell growth. Both, IGF-1 and PDGF-BB, induced MT1-MMP expression, but only IGF-1-mediated MT1-MMP induction was inhibited by dec-CMK. Induction of MMP-2 by IGF-1 was inhibited by the PI3-kinase inhibitor wortmannin, but not by the MEK-inhibitor PD98059. Dec-CMK inhibited VSMC chemotaxis comparable to the effects of the MMP-inhibitor GM6001 (both p<0.05 vs. controls), supporting that MMPs are involved. In conclusion, this study demonstrates that targeting furin-like PCs and thus inhibiting IGF-1R activation is a novel target to inhibit IGF-1-mediated signaling and cell functions, such as IGF-1-induced MT1-MMP/MMP-2 in VSMCs.     

Ligand-dependent inhibition of myoblast differentiation by overexpression of the type-1 insulin-like growth factor receptor

The insulin-like growth factors (IGFs) have paradoxical effects on skeletal myoblast differentiation. While low concentrations of IGF stimulate myoblast differentiation, high concentrations of IGF induce a progressive decrease in myoblast differentiation. The mechanism of this inhibition is unknown. Using a retroviral expression vector, we developed a subline of mouse P2 mouse myoblasts (P2-LISN) which expressed 7.5 times higher levels of type-1 IGF receptors than control (P2-LNL6) myoblasts, which were infected with a virus lacking the type-1 IGF receptor sequence. Overexpression of the type-1 IGF receptor caused the IGF dose-response curves of stimulation and progressive inhibition of differentiation to shift to the left. Additionally, at high insulin and IGF-I concentrations, complete inhibition of P2-LISN myoblast differentiation occurred. These results suggest that inhibition of differentiation at high ligand concentrations was not due to the primary involvement of other species of receptors for IGF. Type-1 IGF receptor downregulation as a mechanism for inhibition of differentiation was also ruled out since P2-LISN myoblasts constitutively expressed high levels of type-1 IGF receptors. Additionally, inhibition of differentiation at high concentrations of IGF-I was not correlated with overt stimulation of proliferation or with IGF binding protein (IGF-BP) release into the culture medium. These results indicate that the type-1 IGF receptor mediates two conflicting signal pathways in myogenic cells, differentiation-inducing and differentiation-inhibitory, which predominate at different ligand concentrations. © 1993 Wiley-Liss, Inc.     

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

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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.     

Secretion of Annexin II via activation of insulin receptor and insulin-like growth factor receptor

Annexin II is secreted into the extracellular environment, where, via interactions with specific proteases and extracellular matrix proteins, it participates in plasminogen activation, cell adhesion, and tumor metastasis and invasion. However, mechanisms regulating annexin II transport across the cellular membrane are unknown. In this study, we used coimmunoprecipitation to show that Annexin-II was bound to insulin and insulin-like growth factor-1 (IGF-1) receptors in PC12 cells and NIH-3T3 cells overexpressing insulin (NIH-3T3(IR)) or IGF-1 receptor (NIH-3T3(IGF-1R)). Stimulation of insulin and IGF-1 receptors by insulin caused a temporary dissociation of annexin II from these receptors, which was accompanied by an increased amount of extracellular annexin II detected in the media of PC12, NIH-3T3(IR), and NIH-3T3(IGF-1R) cells but not in that of untransfected NIH-3T3 cells. Activation of a different growth factor receptor, the platelet-derived growth factor receptor, did not produce such results. Tyrphostin AG1024, a tyrosine kinase inhibitor of insulin and IGF-1 receptor, was shown to inhibit annexin II secretion along with reduced receptor phosphorylation. Inhibitors of a few downstream signaling enzymes including phosphatidylinositol 3-kinase, pp60c-Src, and protein kinase C had no effect on insulin-induced annexin II secretion, suggesting a possible direct link between receptor activation and annexin II secretion. Immunocytochemistry revealed that insulin also induced transport of the membrane-bound form of annexin II to the outside layer of the cell membrane and appeared to promote cell aggregation. These results suggest that the insulin receptor and its signaling pathways may participate in molecular mechanisms mediating annexin II secretion.     

Mechanism of STAT3 activation by insulin-like growth factor I receptor

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic analysis of type-1 insulin-like growth factor receptor signaling through insulin receptor substrate-1 and -2 in pancreatic beta cells

Signaling by receptor tyrosine kinases regulates pancreatic β cell function. Inactivation of insulin receptor (InsR), IGF1 receptor (Igf1r), or Irs1 in β cells impairs insulin secretion. Conversely, Irs2 ablation impairs β cell replication. In this study, we examined aspects of the Igf1r regulatory signaling cascade in β cells. To examine genetically the involvement of Irs1 and Irs2 in Igf1r signaling, we generated double mutant mice lacking Igf1r specifically in pancreatic β cells in an Irs1- or Irs2-null background. We show that Igf1r/Irs1 double mutants do not differ phenotypically from Irs1 single mutants and exhibit hyperinsulinemia, while maintaining normal β cell mass and glucose tolerance. In contrast, lack of Igf1r function in β cells aggravates the consequences of Irs2 ablation in double mutants and results in lethal diabetes by 6 weeks of age. This additivity of phenotypic manifestations indicates that Irs2 serves a pathway that is largely independent of Igf1r signaling. Consistent with the view that the latter is the InsR pathway, we show that combined β cell-specific knock-out of both Insr and Igf1r results in a phenocopy of double mutants lacking Igf1r and Irs2. We conclude that Igf1r signals primarily through Irs1 and affects insulin secretion, whereas β cell proliferation is mainly regulated by InsR using Irs2 as a downstream signaling effector. The insulin and IGF pathways appear to control β cell functions independently and selectively.     

Functional properties of an isolated alpha beta heterodimeric human placenta insulin-like growth factor 1 receptor complex

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, resulted in the formation of a functional alpha beta heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native alpha 2 beta 2 heterotetrameric disulfide-linked state. The membrane-bound alpha beta heterodimeric complex displayed similar curvilinear 125I-IGF-1 equilibrium binding compared to the alpha 2 beta 2 heterotetrameric complex. Triton X-100 solubilization of the alkaline pH and DTT-pretreated placenta membranes, followed by Bio-Gel A-1.5m gel filtration chromatography, was found to effectively separate the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric IGF-1 receptor species, 125I-IGF-1 binding to both the isolated alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. Similar to the membrane-bound IGF-1 receptor species, the 125I-IGF-1 binding properties between the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes were not significantly different. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of alpha beta heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent alpha 2 beta 2 heterotetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tuftsin signals through its receptor neuropilin‐1 via the transforming growth factor beta pathway

Tuftsin (Thr‐Lys‐Pro‐Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin‐1 (Nrp1) on the surface of cells. Nrp1 is a single‐pass transmembrane protein, but its intracellular C‐terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti‐inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway.

     

Overexpression of the type-1 insulin-like growth factor receptor increases ligand-dependent proliferation and differentiation in bovine skeletal myogenic cultures

Previous studies demonstrated that overexpression of the type-1 insulin-like growth factor (IGF) receptor (IGF-1R) in skeletal myogenic cell lines increased proliferation and differentiation responses to IGF. However, it was unclear if such manipulations in primary, untransformed skeletal myogenic cells would result in modulation of these responses, which may be more stringently regulated in primary cells than in myogenic cell lines. In this study, low passage untransformed fetal bovine myogenic cultures were infected with a replication-deficient retroviral expression vector (LISN) coding for the human IGF-1R or with a control retroviral vector (LNL6). Bovine myogenic cultures infected with the LISN vector (Bov-LISN) displayed ten times more IGF-1Rs than controls (Bov-LNL6). Bov-LISN myogenic cultures exhibited elevated rates of IGF-I-stimulated proliferation and increased rates of terminal differentiation which were reduced to control levels by the anti-human IGF-1R antibody αIR3. These findings indicate overexpression of the IGF-1R can enhance IGF sensitivity and thereby modify the proliferation and differentiation behavior of untransformed low passage myoblasts. Such manipulations may be useful to increase muscle mass in clinical or agricultural applications. © 1996 Wiley-Liss, Inc.     

Structure and autoregulation of the insulin-like growth factor 1 receptor kinase.

The insulin-like growth factor 1 (IGF1) receptor is closely related to the insulin receptor. However, the unique biological functions of IGF1 receptor make it a target for therapeutic intervention in human cancer. Using its isolated tyrosine kinase domain, we show that the IGF1 receptor is regulated by intermolecular autophosphorylation at three sites within the kinase activation loop. Steady-state kinetic analyses of the isolated phosphorylated forms of the IGF1 receptor kinase (IGF1RK) reveal that each autophosphorylation event increases enzyme turnover number and decreases Km for ATP and peptide. We have determined the 2.1 A-resolution crystal structure of the tris-phosphorylated form of IGF1RK in complex with an ATP analog and a specific peptide substrate. The structure of IGF1RK reveals how the enzyme recognizes peptides containing hydrophobic residues at the P+1 and P+3 positions and how autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Although the nucleotide binding cleft is conserved between IGF1RK and the insulin receptor kinase, sequence differences in the nearby interlobe linker could potentially be exploited for anticancer drug design.     

Fibroblast growth factor receptor 1 regulates the differentiation and activation of osteoclasts through Erk1/2 pathway

To elucidate the direct role and mechanism of FGFR1 signaling in the differentiation and activation of osteoclasts, we conditionally inactivated FGFR1 in bone marrow monocytes and mature osteoclasts of mice. Mice deficient in FGFR1 (Fgfr1^−/−) exhibited misregulated bone remodeling with reduced osteoclast number and impaired osteoclast function. In vitro assay demonstrated that the number of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts derived from bone marrow monocytes of Fgfr1^−/− mice was significantly diminished. The bone resorption activity of mature osteoclasts derived from Fgfr1^−/− mice was also suppressed. Further analysis showed that the osteoclasts with FGFR1 deficiency exhibited downregulated expression of genes related to osteoclastic activity including TRAP and MMP-9. The phosphorylation of Erk1/2 mitogen-activated protein (MAP) kinase was also decreased. Our results suggest that FGFR1 is indispensable for complete differentiation and activation of osteoclasts in mice.