IL-21 Stimulates Human Myeloma Cell Growth through an Autocrine IGF-1 Loop
Emmanuelle MénoretSophie MaïgaGéraldine DescampsCatherine Pellat‐DeceunynckCaroline FraslonMelania CappellanoPhilippe MoreauRégis BatailleMartine Amiot
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Abstract IL-21 is a member of the type I cytokine family related most closely to IL-2 and IL-15. IL-21 is a pleiotropic cytokine, produced by T, NKT, and dendritic cells, which modulates lymphoid and myeloid cell functions. Besides its activities on normal lymphoid cells, it has been shown that IL-21 is a growth factor for myeloma cells. In the present study, we demonstrate that IL-21 generated myeloma colonies from 9 of 24 human myeloma cell lines (HMCL) in a collagen-based assay. Of major interest, the capacity of IL-21 to stimulate clonogenicity was restricted to CD45− HMCL. We found that IL-21 induced tyrosine phosphorylation of STAT-3, STAT-1, and Erk1/2. Interestingly, an Akt activation was observed lately after 30 min to 1 h of IL-21 stimulation, indicating that this Akt phosphorylation could be due to an IGF-1 autocrine loop. This hypothesis was sustained both by the fact that IL-21 treatment induced an IGF-1 mRNA synthesis and that an antagonistic anti-IGF-1 receptor mAb (AVE1642) strongly inhibits the IL-21-induced clonogenicity. Thus, we demonstrated by quantitative PCR that IL-21 induced clonogenicity through an autocrine IGF-1 secretion in HMCL and primary myeloma cells. Because we have previously demonstrated that CD45 phosphatase inhibits the IGF-1 signaling, this inhibitory effect of CD45 explains why the IL-21-induced clonogenicity was restricted to CD45− HMCL. These results support that therapy against IGF-1R, which are presently under investigation in multiple myeloma, could be beneficial, not only to suppress IGF-1-mediated myeloma cell growth, but also IL-21-mediated myeloma cell growth.Platelet-derived growth factor, PDGF, is a potent mitogen for cells of mesenchymal origin such as fibroblasts, smooth muscle cells and glial cells. PDGF is thought to have the potential to act as both a paracrine and an autocrine factor. Studies described here extend these observations to human bone-derived cells. Exogenous PDGF induces biologic activity in two human osteogenic sarcoma cell lines and in one of these, the two PDGF genes, PDGF-1 and PDGF-2/c-sis are expressed. In addition, PDGF stimulates proliferation of normal osteoblastic cells derived from adult human cancellous bone. The expression of the PDGF-1 gene but not the PDGF-2/c-sis gene is demonstrated in normal human adult bone-derived cells by Northern blot analysis and synthesis of PDGF is shown by immunoprecipitation with PDGF antisera. These studies indicate that PDGF has the potential to act as a paracrine or autocrine regulator of bone cells.
Platelet-derived growth factor
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Autocrine growth due to dysregulated growth factor production may have a role in the development of neoplasia. Whether autocrine growth is stimulated by growth factor secretion in an autocrine loop or by intracellular binding of the growth factor to a receptor has been unclear. The carboxyl-terminus coding sequence for murine interleukin-3 (IL-3) was extended with an oligonucleotide encoding a four-amino acid endoplasmic reticulum retention signal. IL-3-dependent hematopoietic cells became growth factor-independent when the modified IL-3 gene was introduced by retroviral gene transfer, despite lack of secretion of the modified IL-3. Hence autocrine growth can occur as a result of the intracellular action of a growth factor and this mechanism may be important in neoplastic and normal cells.
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The reduced growth factor requirements of murine fibroblasts transformed by simian virus 40 (SV 40) have been attributed to insulin-like growth factor (IGF)-I induction by T antigen and consequent activation of IGF-I receptor signaling. The present study shows that the autonomous growth of SV 40-transformed human fibroblasts also requires type-I IGF-I receptor activation but that this is not due to de novo induction of IGF-I gene expression since untransformed human fibroblasts, which fail to proliferate in the absence of serum, also showed IGF-I gene expression under serum-free conditions. DNA synthesis assays confirmed that untransformed cells were responsive to exogenous IGF and indicated that transformed cells were already maximally stimulated. In untransformed fibroblasts, IGF binding was principally to abundant membrane-associated IGFBP-5, whereas in transformed fibroblasts this protein was minimally expressed, and IGF binding was to IGF receptors. Loss of detectable membrane-associated IGFBP-5 in transformed cells was associated with diminished IGFBP-5 gene expression and with loss of IGF-II gene expression. Exogenous IGFBP-5 associated with the membranes of transformed cells and inhibited the autocrine growth of these cells. These findings suggest that loss of IGFBP-5 in SV 40-transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor and increases their sensitivity to autocrine stimulation. The reduced growth factor requirements of murine fibroblasts transformed by simian virus 40 (SV 40) have been attributed to insulin-like growth factor (IGF)-I induction by T antigen and consequent activation of IGF-I receptor signaling. The present study shows that the autonomous growth of SV 40-transformed human fibroblasts also requires type-I IGF-I receptor activation but that this is not due to de novo induction of IGF-I gene expression since untransformed human fibroblasts, which fail to proliferate in the absence of serum, also showed IGF-I gene expression under serum-free conditions. DNA synthesis assays confirmed that untransformed cells were responsive to exogenous IGF and indicated that transformed cells were already maximally stimulated. In untransformed fibroblasts, IGF binding was principally to abundant membrane-associated IGFBP-5, whereas in transformed fibroblasts this protein was minimally expressed, and IGF binding was to IGF receptors. Loss of detectable membrane-associated IGFBP-5 in transformed cells was associated with diminished IGFBP-5 gene expression and with loss of IGF-II gene expression. Exogenous IGFBP-5 associated with the membranes of transformed cells and inhibited the autocrine growth of these cells. These findings suggest that loss of IGFBP-5 in SV 40-transformed fibroblasts facilitates interaction of endogenously produced IGF-I with the IGF-I receptor and increases their sensitivity to autocrine stimulation.
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Autocrine regulation is defined as a mechanism of self-control in growth and differentiation; this mode of regulation among histologically homologous cells is mediated humorally. Autocrine mechanisms involve: 1. Autonomously controlled production and secretion of autocrine mediators. 2. Distribution of autocrine mediators among cells. 3. Expression by cells of functional receptors for autocrine mediators. 4. Transduction and intracellular integration of signals mediated by autocrine mediators. 5. Growth response. 6. Maintenance of autonomous control of growth and/or differentiation state in the progeny Biochemical and biological evidence for most of these steps in various transformed cells makes it possible to analyze autocrine control as a multifaceted process. This process depends on tumor cellularity and histoarchitecture, on time and on external influences on secretion of autocrine mediators (e.g., estrogens in estrogen-dependent breast cancer). We review the quantitative aspects of experimental evidence for autocrine control in tumors and examine the phenomenological and some mechanistic concepts in creating integrative, quantitative, and experimentally verifiable mathematical models of autocrine regulation.
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This thesis describes an investigation into the growth characteristics of a human pre-B cell acute lymphocytic leukaemia cell line, SMS-SB. Most lymphocytic tumours are difficult to adapt to tissue culture and enter a crisis after a few rounds of cell division where the majority of cells die. To sustain proliferation of the remaining cells, addition of exogenous mitogens is usually required. SMS-SB was an unusual leukaemia because the cells did not go through a crisis phase and grew indefinitely, in the absence of exogenous mitogens. This sustained proliferation in tissue culture appears to reflect the synthesis and secretion of an autocrine growth factor (s); the cells are density-dependent for growth, and proliferation can occur in media completely devoid of protein. The original aim of this work was to identify and characterise the autocrine growth factor, termed SB-AF. During investigations to identify cytokines with the ability to substitute for the autocrine growth factor activity, platelet-derived growth factor (PDGF) was shown to stimulate the growth of SMS-SB cells under low cell density conditions; SMS-SB cells are known to secrete PDGF and express PDGF receptors. However, antibody inhibition experiments suggest that PDGF cannot account for all the autocrine activity of SB-AF, thus other cytokine components of SB-AF were sought. CD23 is a 45kDa type-II transmembrane glycoprotein and a member of the C-type lectin superfamily. There is a soluble form of CD23 (sCD23) which is released by cleavage from the surface of cells into the extracellular fluid, and this form has been attributed multiple cytokine activities. It was discovered that sCD23 dramatically promotes thymidine incorporation by SMS-SB cells. The work of this thesis has shown that SMS-SB cells undergo apoptosis when cultured at low cell density; sCD23 is the only cytokine tested with the ability to prevent SMS-SB cell apoptosis. Apoptotic SMS-SB cells have low levels of the proto-oncogene bcl-2 but sCD23 can sustain bcl-2 levels in the cells. The investigations have shown that SMS-SB cells do not express CD23, negating the hypothesis that CD23 is acting in an autocrine fashion. The most interesting discovery made during these investigations was that SMS-SB cells bind CD23-containing liposomes specifically but they do not express the known receptors for CD23, namely CD21, CD11a and CD11b; SMS-SB cells express a novel CD23 receptor. Thus, SMS-SB cells express a novel receptor for CD23 and signalling via this receptor prevent apoptosis of the cells. Preliminary data is presented from CD23 affinity columns used to isolate and characterise the novel CD23 receptor. A protein of 85kDa has been identified as a candidate receptor, but further characterisation is required. SMS-SB cells will provide a good model to examine the role of autocrine growth factors in early B cell development, moreover, the discovery of a novel CD23 receptor on pre-B cells, implies a role for sCD23 in early B cell development. Since sCD23 has previously been shown to promote the growth and maturation of early T cells and myeloid progenitors, it will be interesting to investigate the role of CD23, and the novel receptor, in all aspects of haematopoiesis.
Platelet-derived growth factor
CD23
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Autocrine ligands are important regulators of many normal tissues and have been implicated in a number of disease states, including cancer. However, because by definition autocrine ligands are synthesized, secreted, and bound to cell receptors within an intrinsically self-contained “loop,” standard pharmacological approaches cannot be used to investigate relationships between ligand/receptor binding and consequent cellular responses. We demonstrate here a new approach for measurement of autocrine ligand binding to cells, using a microphysiometer assay originally developed for investigating cell responses to exogenous ligands. This technique permits quantitative measurements of autocrine responses on the time scale of receptor binding and internalization, thus allowing investigation of the role of receptor trafficking and dynamics in cellular responses. We used this technique to investigate autocrine signaling through the epidermal growth factor receptor by transforming growth factor alpha (TGFα) and found that anti-receptor antibodies are far more effective than anti-ligand antibodies in inhibiting autocrine signaling. This result indicates that autocrine-based signals can operate in a spatially restricted, local manner and thus provide cells with information on their local microenvironment.
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Co-receptor
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Significance It is well known that AKT inactivates glycogen synthase kinase 3β (GSK3β) by increasing its phosphorylation. The inactivation of GSK3β leads to aberrant phosphorylation of Tau, which is a hallmark of Alzheimer’s disease. However, we found that if phosphorylated AKT is sulfhydrated, it will be unable to inactivate GSK3β and subsequently increases Tau phosphorylation. The influence of sulfhydrated AKT on GSK3β and Tau phosphorylation was reversed in a transgenic AKT-KI +/+ mouse, where sulfhydration of AKT residue was mutated to alanine. Thus, AKT-sulfhydration represents a unique posttranslational modification of AKT that can be targeted to suppress phosphorylation of GSK3β and subsequently reduce Tau phosphorylation.
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Colony-stimulating factor
Hematopoietic growth factor
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