[Show abstract][Hide abstract] ABSTRACT: Insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine by a number of cytokine receptors and is implicated in the activation of phosphatidylinositol 3'-kinase (PI3-kinase). Here, we demonstrate that induction of granulocytic differentiation of human promyeloid HL-60 cells leads to an increase in the amount of IRS-2 that is phosphorylated in response to insulin-like growth factor (IGF)-I. Although PI3-kinase is often activated following interaction with IRS-1, we could not detect IRS-1 protein, IRS-1 mRNA, or IRS-1-precipitable PI3-kinase enzymatic activity. However, PI3-kinase activity that was coimmunoprecipitated with either anti-phosphotyrosine or anti-IRS-2 following IGF-I stimulation was increased 100-fold. Heightened tyrosine phosphorylation of IRS-2 during granulocytic differentiation was not caused by an increase in expression of the tyrosine kinase IGF-I receptor, as measured by the amount of both the alpha- and beta-subunits. Instead, immunoblotting experiments with an Ab to IRS-2 revealed that induction of granulocytic differentiation caused a large increase in IRS-2, and this occurred in the absence of detectable IRS-1 protein. These IRS-2-positive cells could not differentiate into more mature myeloid cells in serum-free medium unless IGF-I was added. These data are consistent with a model of granulocytic differentiation that requires at least two signals, the first of which leads to an increase in the cytoplasmic pool of IRS-2 protein and a second molecule that acts to tyrosine phosphorylate IRS-2 and enhance granulocytic differentiation.
The Journal of Immunology 02/2000; 164(1):113-20. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyclin-dependent kinase inhibitors such as p27(KIP1) have recently been shown to lead to cellular differentiation by causing cell cycle arrest, but it is unknown whether similar events occur in differentiating promyeloid cells. Hematopoietic progenitor cells undergo lineage-restricted differentiation, which is accompanied by expression of distinct maturation markers. Here we show that the classical growth factor insulin-like growth factor I (IGF-I) potently promotes vitamin D(3)-induced macrophage differentiation of promyeloid cells, as assessed by measurement of a coordinate increase in expression of the integrin alpha subunit CD11b, the CD14 lipopolysaccharide receptor, and the macrophage-specific esterase, alpha-naphthyl acetate esterase, as early as 24 h following initiation of terminal differentiation. Addition of IGF-I to cells undergoing vitamin D(3)-induced differentiation also leads to an early increase in expression of cyclin E, phosphorylation of the retinoblastoma tumor suppressor protein, and a doubling of the cell number. Early expression of CD11b (24 h) is simultaneously accompanied by inhibition in the expression of p27(KIP1). Cell cycle analysis with propidium iodide revealed that CD11b expression at 24 h following initiation of differentiation occurs at all phases of the cell cycle instead of only those cells arrested in G(0)/G(1). Similarly, development of a novel double-labeling intra- and extracellular flow-cytometric technique demonstrated that single cells expressing the mature leukocyte differentiation antigen CD11b can also incorporate the thymidine analog bromodeoxyuridine. Likewise, expression of the intracellular DNA polymerase delta cofactor/proliferating-cell nuclear antigen at 24 h is also simultaneously expressed with the surface marker CD11b, indicating that these cells continue to proliferate early in their differentiation program. Finally, at 24 h following induction of differentiation, IGF-I promoted a fourfold increase in the uptake of [(3)H]thymidine by purified populations of CD11b-expressing cells. Taken together, these data demonstrate that the initial steps associated with terminal macrophage differentiation occur concomitantly with progression through the cell cycle and that these very early differentiation events do not require the accumulation of p27(KIP1).
Molecular and Cellular Biology 10/1999; 19(9):6229-39. · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heightened expression of both a proinflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), and a survival peptide, insulin-like growth factor I (IGF-I), occurs in diverse diseases of the central nervous system, including Alzheimer's disease, multiple sclerosis, the AIDS-dementia complex, and cerebral ischemia. Conventional roles for these two proteins are neuroprotection by IGF-I and neurotoxicity by TNF-alpha. Although the mechanisms of action for IGF-I and TNF-alpha in the central nervous system originally were established as disparate and unrelated, we hypothesized that the signaling pathways of these two cytokines may interact during neurodegeneration. Here we show that concentrations of TNF-alpha as low as 10 pg/ml markedly reduce the capacity of IGF-I to promote survival of primary murine cerebellar granule neurons. TNF-alpha suppresses IGF-I-induced tyrosine phosphorylation of insulin receptor substrate 2 (IRS-2) and inhibits IRS-2-precipitable phosphatidylinositol 3'-kinase activity. These experiments indicate that TNF-alpha promotes IGF-I receptor resistance in neurons and inhibits the ability of the IGF-I receptor to tyrosine-phosphorylate the IRS-2 docking molecule and to subsequently activate the critical downstream enzyme phosphatidylinositol 3'-kinase. This intracellular crosstalk between discrete cytokine receptors reveals a novel pathway that leads to neuronal degeneration whereby a proinflammatory cytokine inhibits receptor signaling by a survival peptide.
Proceedings of the National Academy of Sciences 09/1999; 96(17):9879-84. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Why a primary lymphoid organ such as the thymus involutes during aging remains a fundamental question in immunology. Aging is associated with a decrease in plasma growth hormone (somatotropin) and IGF-I, and this somatopause of aging suggests a connection between the neuroendocrine and immune systems. Several investigators have demonstrated that treatment with either growth hormone or IGF-I restores architecture of the involuted thymus gland by reversing the loss of immature cortical thymocytes and preventing the decline in thymulin synthesis that occurs in old or GH-deficient animals and humans. The proliferation, differentiation and functions of other components of the immune system, including T and B cells, macrophages and neutrophils, also demonstrate age-associated decrements that can be restored by IGF-I. Knowledge of the mechanism by which cytokines and hormones influence hematopoietic cells is critical to improving the health of aged individuals. Our laboratory has recently demonstrated that IGF-I prevents apoptosis in promyeloid cells, which subsequently permits these cells to differentiate into neutrophils. We also demonstrated that IL-4 acts much like IGF-I to promote survival of promyeloid cells and to activate the enzyme phosphatidylinositol 3'-kinase (PI 3-kinase). However, the receptors for IGF-I and IL-4 are completely different, with the intracellular beta chains of the IGF receptor possessing intrinsic tyrosine kinase activity and the alpha and gammac subunit of the heterodimeric IL-4 receptor utilizing the Janus kinase family of nonreceptor protein kinases to tyrosine phosphorylate downstream targets. Both receptors share many of the components of the PI 3-kinase signal transduction pathway, converging at the level of insulin receptor substrate-1 or insulin receptor subtrate-2 (formally known as 4PS, or IL-4 Phosphorylated Substrate). Our investigations with IGF-I and IL-4 suggest that PI 3-kinase inhibits apoptosis by maintaining high levels of the anti-apoptotic protein Bcl-2. The sharing of common activation molecules, despite vastly different protein structures of their receptors, forms a molecular explanation for the possibility of cross talk between IL-4 and IGF-I in regulating many of the events associated with hematopoietic differentiation, proliferation and survival.
[Show abstract][Hide abstract] ABSTRACT: A decline in plasma concentrations of both growth hormone and IGF-I occurs during aging of humans and rodents, and this is accompanied by involution of the thymus gland. Exogenous growth hormone induces the synthesis of IGF-I, which acts on bone marrow-derived hematopoietic progenitors of the myeloid and lymphoid lineages to promote their replication and survival. The increase in survival of these cells is caused by the ability of IGF-I to inhibit their apoptotic death. In contrast to the multipotential colony-stimulating-factor IL-3, inhibition of apoptosis by IGF-I requires the activation of the critical intracellular effector PI 3-kinase. These data establish that hematopoietic progenitors can use more than one intracellular signaling pathway in order to maintain their survival. The data also extend the original hypothesis that IGF-I shares with the colony-stimulating factors the properties of promoting DNA synthesis and inhibiting programmed cell death. Collectively, these data establish that hematopoietic progenitor cells are important targets for IGF-I, and this is likely to be important in understanding thymic aging.
Annals of the New York Academy of Sciences 06/1998; 840:518-24. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositides that are phosphorylated at the D3 position have been reported to activate an atypical, Ca2-independent protein kinase C (PKC) isoform designated PKC-zeta, and overexpression of this enzyme leads to monocytic differentiation. In this study, we cultured human HL-60 promyeloid cells with vitamin D3 and insulin-like growth factor-I (IGF-I), a 70-amino-acid peptide that activates phosphatidylinositol 3'-kinase (PI 3-kinase) in murine promyeloid cells. Two days later, the proportion of cells differentiating into macrophages in serum-free medium, as assessed by expression of the alpha-subunit of the beta2 integrin CD11b, increased from 5 +/- 1% to 25 +/- 3%. Addition of IGF-I increased the proportion of cells differentiating into CD11b-positive macrophages to 78 +/- 5%. In the absence of vitamin D3, IGF-I did not induce expression of CD11b (6 +/- 1%). The IGF-I-promoted macrophage differentiation was blocked specifically by preincubation of HL-60 cells with a mAb (alphaIR3) directed against the IGF type I receptor. Similarly, pretreatment of cells with either alphaIR3 or an IGF-binding protein, IGFBP-3, led to a 75% inhibition of CD11b expression when cells were cultured with vitamin D3 in serum-containing medium. IGF-I, but not vitamin D3, caused a sevenfold increase in the enzymatic activity of both PI 3-kinase and atypical PKC-zeta. Inhibition of IGF-I-inducible PI 3-kinase with either wortmannin or LY294002 abrogated the IGF-I-induced activation of PKC-zeta and totally blocked the enhancement in macrophage differentiation caused by IGF-I. These data establish that PKC-zeta is a putative downstream target of PI 3-kinase that is activated during IGF-I-promoted macrophage differentiation.
The Journal of Immunology 03/1998; 160(3):1393-401. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor-I (IGF-I) promotes cell division and prevents programmed cell death in hemopoietic progenitors. Human HL-60 promyeloid cells differentiate toward the granulocytic lineage when stimulated with retinoic acid (RA) in serum-containing medium. When deprived of serum, however, we found that these cells differentiate poorly in the presence of RA, as assessed by expression of the alpha subunit of the beta2 integrin heterodimer, CD11b/CD18. However, when IGF-I is added to RA-treated cells, the proportion of CD11b-positive cells increases to a level similar to that in RA-treated cells cultured in serum-containing medium. Cells treated with RA alone not only differentiate poorly but also undergo apoptosis, as assessed by flow cytometry using propidium iodide and HO33342. In serum-free medium, one-third of RA-treated cells become apoptotic compared with only 5% apoptotic cells in the absence of RA. However, addition of IGF-I to RA-treated cells prevents the appearance of this apoptotic population and increases phosphatidylinositol 3'-kinase (PI 3-kinase) activity by fivefold. Wortmannin, a PI 3-kinase inhibitor, potently decreases this IGF-I-induced lipid kinase activity, blocks the ability of IGF-I to prevent apoptosis, and inhibits IGF-I-enhanced CD11b expression. These data demonstrate that IGF-I acts on RA-treated progenitors to promote their differentiation along the granulocytic lineage. IGF-I acts by rescuing these cells from apoptotic cell death via a downstream pathway that is dependent upon PI 3-kinase.
The Journal of Immunology 08/1997; 159(2):829-37. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flow cytometry was used to examine the expression of type I insulin-like growth factor receptors (IGF-IR) on three types of human hematopoietic cells that represent different stages of myeloid lineage development. Both HL-60 (promyeloid) and U-937 (monocytic) cells express abundant IGF-IR protein (> 79% cells positive for the IGF-IR), whereas KG-1 myeloblasts express negligible levels of IGF-IR (< 1% IGF-IR-positive cells). Exogenous IGF-I, IGF-II, and an IGF-I analog that binds poorly to IGF-binding protein-3 (des-IGF-I) increased DNA synthesis of HL-60 and U-937 cells in a dose-dependent (1-25 ng/ml) fashion by 2- to 4-fold in serum-free medium, whereas KG-1 cells did not respond to any of these growth factors. The IGF-induced increase in proliferation of HL-60 promyeloid cells was inhibited by soluble IGF-binding protein-3 (500 ng/ml) when these cells were stimulated with 10 ng/ml of either IGF-I (53 +/- 8%) or IGF-II (59 +/- 8%), but not with des-IGF-I (3 +/- 1%). In contrast, the anti-IGF-IR monoclonal antibody (mAb; alpha IR-3) inhibited the DNA synthesis caused by 10 ng/ml exogenous IGF-I (67 +/- 6%), IGF-II (72 +/- 8%), and des-IGF-1 (82 +/- 9%). Proliferation of KG-1 myeloblasts, however, was neither stimulated by the IGFs nor inhibited by the anti-IGF-IR mAb. In the absence of exogenous IGF-I, the mAb directed against the IGF-IR significantly suppressed basal DNA synthesis of HL-60 promyeloid (72 +/- 5%) and U-937 monocytic (39 +/- 7%) cells, but did not affect DNA synthesis of KG-1 myeloblasts (8 +/- 1%) compared to an isotype-matched control mAb. Similarly, the alpha IR-3 mAb abrogated vitamin D3-induced differentiation of the HL-60 cells into macrophages in serum-free medium, as assessed by expression of the leucam surface protein, CD11b. As the alpha IR-3 mAb inhibits DNA synthesis in the presence and absence of exogenous IGF-I on receptor-bearing cells, but not IGF-IR-negative cells, these data demonstrate that both endocrine and autocrine IGF-I are potent growth factors in human myeloid cells where expression of the surface receptor, rather than the ligand, is the critical control element. More importantly, these data support the hypothesis that autocrine IGF-I may play a significant role in the differentiation of promyeloid cells into macrophages.
[Show abstract][Hide abstract] ABSTRACT: Primary (thymus) and secondary (spleen) murine lymphoid tissues express a 25-kDa protein that binds IGF-I. To determine the cellular source of this insulin-like growth factor binding protein (IGFBP), 11 murine or human cell lines representing T, B, and myeloid cells at various stages of differentiation were characterized by IGF-I affinity cross-linkage and Western ligand blotting. Mature myeloid cells, but not T or B cells, secrete a 25-kDa protein that is capable of binding IGF-I. CSF-1-derived bone marrow macrophages also synthesize this 25-kDa IGFBP. Thymic macrophages, which were estimated to secrete 2 ng of binding protein/10(6) cells-h, were used in conjunction with [125I] IGF-I affinity cross-linking to develop a protein binding immunomobility-shift assay to identify which IGFBP is produced by these cells. An anti-IGFBP-4Ab, but not an anti-IGFBP-2 Ab or normal rabbit serum, shifted the [125I] IGF-IGFBP complex to a higher m.w. position, indicating that the single 25-kDa IGFBP is IGFBP-4. Northern blotting confirmed that transcripts for IGFBP-4 as well as IGF-I are expressed in thymic macrophages. A putative 278-bp IGFBP-4 cDNA fragment (residues 341-618) of rat) that contains two unique cysteine residues found only in IGFBP-4 was cloned and sequenced from thymic macrophages. These clones differed from the rat sequence at only six residues (97% homology), and the deduced amino acid sequence from the murine cDNA was identical with that of the rat sequence. Subsequent studies revealed that IGF-I stimulates DNA synthesis in thymic macrophages. However, two different IGF-I analogues differing in the amino-terminus that bind equally well to the IGF-I receptor but poorly to IGFBPs are as effective as IGF-I at 100-fold lower concentrations. These data demonstrate that murine macrophages are a source of a single 25-kDa secreted protein that binds IGF-, that the molecular identity of this protein is IGFBP-4, and that this binding protein may antagonize the extracellular effects of IGF-I.
The Journal of Immunology 02/1996; 156(1):64-72. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hypocellularity of primary lymphoid organs is a distinctive and reproducible characteristic of aged humans and animals. Similar changes have been reported in both hypophysectomized and dwarf rodents. In the bone marrow of these animals, there is an associated reduction in the number of erythroid, lymphoid and myeloid elements. Implantation of growth hormone (GH)-secreting GH3 pituitary cells or infusion of growth hormone into aged rodents dramatically improves cellularity of both the thymus gland and bone marrow. At present it is unknown whether these effects are due to direct effects of growth hormone on hematopoietic cells or if they are caused by the induction of insulin-like growth factor-1 (IGF-1) synthesis. We recently discovered that colony-stimulating factor-1 (CSF-1) and interleukin-3 (IL-3) induce expression and synthesis of the IGF-1 peptide in murine bone marrow cells. Transcripts for IGF-1 increase approximately 50-fold during differentiation over the negligible levels that are expressed in freshly isolated bone marrow cells. Two potential functions of macrophage-derived IGF-1 are to: (a) increase the proliferation of early or committed bone marrow progenitors and (b) reduce their rate of cell death. In support of the first possibility, IGF binding protein-3 significantly inhibits the proliferation of CSF-1-treated bone marrow cells and this inhibition can be reversed by addition of exogenous IGF-1. In support of the second possibility, we have induced apoptosis of both nonadherent bone marrow cells and a myeloid progenitor cell line by depriving these cells of CSFs. Preliminary results indicate that addition of IGF-1 to these cells reduces apoptotic cell death by 50%. These data establish that two different CSFs, CSF-1 and IL-3, induce abundant expression of IGF-1 as these cells differentiate into more mature hematopoietic cells. This model offers a novel approach for investigating the developmental expression of IGF-1 during defined differentiation pathways of hematopoietic cells. If IGF-1 is indeed proven to act as a survival factor for hematopoietic progenitors, these data would support the idea that the hypocellularity of primary lymphoid tissues in aged animals is related to the limited availability to these cells of either growth hormone or IGF-1.
Hormone Research 02/1996; 45(1-2):38-45. · 2.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This article presents a detailed overview of the conceptual and technical considerations involved in the measurement of insulin-like growth factor-I (IGF-I) mRNAs in leukocytes. Two different quantitative techniques that take advantage of the in vitro synthesis of antisense and sense synthetic IGF-I RNA, respectively, are described: the ribonuclease protection assay (commonly referred to as solution hybridization) and competitive RT-PCR. We have improved the ribonuclease protection assay by constructing tandem, cassette riboprobes to generate multigene antisense RNAs of varying sizes. This approach permits the simultaneous quantitation of two or more mRNAs in a single RNA sample, one of which can serve as an internal standard for comparison of IGF-I transcripts among various treatments. The second approach of competitive RT-PCR represents an improvement in previous technologies by cloning a competing IGF-I sequence into an RNA expression vector. The resulting synthetic sense competitor IGF-I RNA (1.1 kb) serves as an internal standard during both the reverse transcription and amplification steps. We have used both the ribonuclease protection assay and competitive RT-PCR to define the macrophage as the major cellular source of leukocyte-derived IGF-I and to characterize these macrophage-derived mRNAs as being derived almost exclusively from exon 1. In addition, these techniques have allowed us to study the ontogeny of IGF-I expression in differentiating bone marrow macrophages and show that hematopoietic progenitors are induced to express abundant IGF-I transcripts as they differentiate into macrophages in the presence of CSF-1. These techniques can be readily adapted for measuring steady-state transcripts for a variety of leukocyte-derived hormones.
[Show abstract][Hide abstract] ABSTRACT: Leukocytes synthesize a variety of hormones that were once thought to be unique products of endocrine tissues. Understanding the regulation of leukocyte-derived hormone synthesis requires an accurate means for measuring steady-state expression of specific mRNA transcripts. Here we describe a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) technique to accurately quantitate macrophage-derived insulin-like growth factor-I (IGF-I) mRNA, and demonstrate the utility of this approach for measuring expression of leukocyte-derived hormone transcripts. A riboprobe was constructed to generate approximately 1 kb of synthetic competitor IGF-I RNA (exons 1 and 3-6) that differed from cellular IGF-I RNA by insertion of 122 bp of beta-actin RNA. One set of oligonucleotide primers could thus be used to simultaneously reverse transcribe and amplify both 144 bp of cellular (exons 3 and 4) and 266 bp of competitor IGF-I RNA. Densitometric scanning of the PAGE-separated PCR products revealed that the ratio of competitor to cellular amplified DNA bore a linear relationship (r2 > or = 0.98) to the amount of competitor RNA for both rat liver and splenocytes. However, rat liver contained 104 x 10(6) IGF-I molecules per microgram of total cellular RNA compared to only 2 x 10(6) IGF-I molecules for splenocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: Prolactin has been shown to have an immunoregulatory role in the rodent immune response. A prolactin-like molecule has also been found in mouse splenocytes and a human B-lymphoblastoid cell line. We have evaluated whether human peripheral blood mononuclear cells (PBMCs) synthesize and/or secrete prolactin. We used the polymerase chain reaction (PCR) to generate a 276-base-pair prolactin product from human PBMCs, and Southern blot analysis confirmed that it was related to prolactin. Western blotting using a polyclonal antibody to prolactin indicated that cell extracts prepared from human PBMCs contained a high molecular mass (60-kDa) immunoreactive prolactin. To determine whether this PBMC prolactin was being secreted, we developed a highly sensitive and specific hormonal enzyme-linked immunoplaque assay. With this assay, we were able to detect human prolactin secretion from concanavalin A (Con A)- or phytohemagglutinin-stimulated PBMCs but not from unstimulated PBMCs. We next sought to determine whether this secreted prolactin could function as an autocrine growth factor in lymphoproliferation. We observed that anti-human prolactin antiserum significantly inhibited human PBMC proliferation in response to Con A or phytohemagglutinin. We conclude that a prolactin-like molecule is synthesized and secreted by human PBMCs and that it functions in an autocrine manner as a growth factor for lymphoproliferation.
Proceedings of the National Academy of Sciences 09/1992; 89(16):7713-6. · 9.81 Impact Factor