Interleukin-6 and leukemia inhibitory factor induction of JunB is regulated by distinct cell type-specific cis-acting elements.

Interleukin (IL)-6 plays an important role in a wide range of biological activities, including differentiation of murine M1 myeloid leukemic cells into mature macrophages. At the onset of M1 differentiation, a set of myeloid differentiation primary response (MyD) genes are induced, including the proto-oncogene for JunB. In order to examine the molecular nature of the mechanisms by which IL-6 activates the immediate early expression of MyD genes, JunB was used as a paradigm. A novel IL-6 response element, -65/-52 IL-6RE, to which a 100-kDa protein complex is bound, has been identified on the JunB promoter. Leukemia inhibitory factor (LIF)-induced activation of JunB in M1 cells was also mediated via the -65/-52 IL-6RE. The STAT3 and CRE-like binding sites of the JunB promoter, identified as IL-6-responsive elements in HepG2 liver cells were found, however, to play no role in JunB inducibility by IL-6 in M1 myeloid cells. Conversely, the -65/-52 IL-6RE is shown not to be necessary for JunB inducibility by IL-6 or LIF in liver cells. It appears, therefore, that immediate early activation of JunB is regulated differently in M1 myeloid cells than in HepG2 liver cells. This indicates that distinct cis-acting control elements participate in cell type-specific induction of JunB by members of the IL-6 cytokine superfamily.     

The network of hemopoietic regulatory proteins in myeloid cell differentiation.

There are clones of myeloid leukemic cells that can be induced to undergo terminal cell differentiation to macrophages by normal hemopoietic regulatory proteins. Induction of differentiation in two different clones of myeloid leukemic cells with interleukin 6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in induction of mRNA for the hemopoietic regulatory proteins IL-6, GM-CSF, interleukin 1 alpha and interleukin 1 beta, tumor necrosis factor, and transforming growth factor beta 1. In one of these clones, induction of differentiation with GM-CSF was also associated with induction of mRNA for macrophage colony-stimulating factor (M-CSF) but not for the receptor for M-CSF (c-fms), whereas in the other clone, induction of differentiation with IL-6 was associated with induction of mRNA for both c-fms and M-CSF. The clones also differed in their responsiveness to these regulators. There was no induction of mRNA for granulocyte colony-stimulating factor or interleukin 3 during differentiation of either clone. The results indicate that the genes for a nearly normal network of positive and negative hemopoietic regulatory proteins are induced during differentiation of these myeloid leukemic cells and that there are leukemic clones with specific defects in this network.     

Dissection of the immediate early response of myeloid leukemia cells to terminal differentiation and growth inhibitory stimuli

To better understand the immediate early genetic response of myeloid cells to terminal differentiation and growth inhibitory stimuli, complementary DNA clones of myeloid differentiation primary response (MyD) genes have recently been isolated. In this study, a set of known (junB, c-jun, ICAM-1, H1(0), and H3.3 histone variants) and novel (MyD88, MyD116) MyD genes were used as immediate early molecular markers to further dissect the primary genetic response of myeloid cells to various differentiation and growth inhibitory stimuli. Expression of all of these MyD genes was highly induced in autonomously replicating differentiation inducible M1D+ myeloblasts following induction of terminal differentiation and growth inhibition by interleukin 6. Expression of all MyD genes except MyD88 was induced upon inhibition of M1D+ cell growth and induction of early, but not late, differentiation markers by interleukin 1 and lipopolysaccharide. In sharp contrast, only expression of H1(0) and H3.3 histone variants was increased following inhibition of M1D+ cell growth by interferon beta or gamma, which did not induce any differentiation associated properties. No increase in the expression of any of these MyD genes was seen in a clone of WEHI-3B D- myelomonocytic cells following stimulation with interleukin 6, which neither induced it for differentiation nor inhibited its growth. 12-O-Tetradecanoylphorbol-13-acetate, known to be a potent inducer of jun expression in many cell types, failed to induce high or stable expression of junB and c-jun in M1D+ cells, where it did not induce differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-6- and leukemia inhibitory factor-induced terminal differentiation of myeloid leukemia cells is blocked at an intermediate stage by constitutive c-myc.

Interleukin-6 (IL-6) and leukemia inhibitory factor (LIF), two multifunctional cytokines, recently have been identified as physiological inducers of hematopoietic cell differentiation which also induce terminal differentiation and growth arrest of the myeloblastic leukemic M1 cell line. In this work, it is shown that c-myc exhibited a unique pattern of expression upon induction of M1 terminal differentiation by LIF or IL-6, with an early transient increase followed by a decrease to control levels by 12 h and no detectable c-myc mRNA by 1 day; in contrast, c-myb expression was rapidly suppressed, with no detectable c-myb mRNA by 12 h. Vectors containing the c-myc gene under control of the beta-actin gene promoter were transfected into M1 cells to obtain M1myc cell lines which constitutively synthesized c-myc. Deregulated and continued expression of c-myc blocked terminal differentiation induced by IL-6 or LIF at an intermediate stage in the progression from immature blasts to mature macrophages, precisely at the point in time when c-myc is normally suppressed, leading to intermediate-stage myeloid cells which continued to proliferate in the absence of c-myb expression.     

Synergistic interactions of interleukin 1, interferon-beta, and tumor necrosis factor in terminally differentiating a mouse myeloid leukemic cell line (M1). Evidence that interferon-beta is an autocrine differentiating factor

The effect was investigated of combinations of cytokines known to be cytostatic for some tumor cells, namely interleukin 1 alpha (IL-1 alpha), interferon-beta (IFN-beta), and tumor necrosis factor (TNF), on the growth and differentiation of the mouse myeloid leukemic cell line, M1, cells. IL-1 alpha, IFN-beta, and TNF by themselves are antiproliferative for M1 cells. Treatment of cells with a mixture of any two of the three cytokines resulted in at least additive growth inhibition. None of these cytokines by themselves induced differentiation of M1 cells as assessed by increased expression of Fc receptors (FcR), stimulation of phagocytic activity and by morphologic criteria. However, as little as 1 U/ml IL-1 alpha in conjunction with IFN-beta or TNF increased FcR expression, phagocytic activity and morphologic changes in addition to inhibiting the growth of M1 cells. The combination of IFN-beta and TNF did not induce differentiation, although the growth of the cells was markedly inhibited. Both TNF and lipopolysaccharide (LPS) induced the in vitro production of IFN activity by M1 cells. Furthermore, the induction of differentiation of M1 cells by a combination of IL-1 alpha with either IFN-beta, TNF, or LPS was inhibited by antibody against mouse IFN-beta. Therefore, it appears that IFN-beta provides one of the two required signals for differentiation of M1 cells by these combinations of stimulants, the other being IL-1. Furthermore, the cytostatic effect of TNF by itself on M1 cells was also partly blocked by anti-IFN-beta antibody, suggesting that IFN-beta is also involved in the growth inhibitory effect of TNF for M1 cells. In contrast, the cytostatic effect of IL-1 on M1 cells was not blocked by anti-IFN-beta antibody. In conclusion, both the cytostatic and differentiative effect of TNF appear to be mediated by IFN-beta. Thus, the combination of IL-1 and IFN-beta or inducers of IFN-beta resulted in terminal differentiation of M1 cells. Northern blot analysis using cDNAs for murine IFN-beta1 or human IFN-beta2 showed an increased expression of mRNA for IFN-beta1 but not for IFN-beta2 by stimulation with TNF or LPS, strongly suggesting that IFN-beta 1 rather than IFN-beta 2 is responsible for TNF or LPS effects.     

Both D factor/LIF and IL-6 inhibit the differentiation of mouse teratocarcinoma F9 cells

Differentiation-stimulating factor (D factor)/leukemia inhibitory factor (LIF) and IL-6 are reported to be cytokines having multifaced functions including the induction of differentiation in mouse myeloid leukemia M1 cells. We here report that both D factor/LIF and IL-6 inhibit the differentiation of mouse teratocarcinoma F9 cells induced by retinoic acid alone or combined with dibutyryl cAMP. From the microscopic observation as well as Northern blot analysis using cDNA probes encoding several marker proteins for differentiation of F9 cells, we concluded that D factor/LIF and IL-6 are functionally closely related in the induction of differentiation in M1 cells and in the inhibition of F9 differentiation.     

Interleukin-4 inhibits the differentiation of mouse myeloid leukemia M1 cells induced by dexamethasone, D-factor/leukemia inhibitory factor and interleukin-6, but not by 1 alpha,25-dihydroxyvitamin D3

The effects of interleukin-4(IL-4) on the growth and differentiation of mouse myeloid leukemia M1 cells induced by various differentiation inducers were investigated. IL-4 alone did not have any significant effect on the growth or differentiation of M1 cells, but inhibited their differentiation induced by dexamethasone, D-factor/leukemia inhibitory factor, or interleukin 6. IL-4 also restored the proliferation of M1 cells after growth inhibition during their induction of differentiation by inducers. In contrast, IL-4 enhanced inhibition of growth and induction of differentiation of M1 cells by 1 alpha,25-dihydroxyvitamin D3. These results indicate that modulation of differentiation of M1 cells by IL-4 depends on the differentiation inducer.     

Autocrine beta-related interferon controls c-myc suppression and growth arrest during hematopoietic cell differentiation

Different hematopoietic cells produce minute amounts of beta-related interferon (IFN) following induction of differentiation by chemical or natural inducers. The endogenous IFN binds to type I cell surface receptors and modulates gene expression in the producer cells. We show that self-induction of two members of the IFN-induced gene family differs in the dose response sensitivity and the prolonged kinetics of mRNA accumulation from the response to exogenous IFN-beta 1. Production and response to endogenous IFN are also detected when bone marrow precursor cells differentiate to macrophages after exposure to colony stimulating factor 1. In M1 myeloid cells induced to differentiate by lung-conditioned medium, addition of antibodies against IFN-beta partially abrogates the reduction of c-myc mRNA and the loss in cell proliferative activity, which both occur during differentiation. The endogenous IFN therefore functions as an autocrine growth inhibitor that participates in controlling c-myc suppression and the specific G0/G1 arrest during terminal differentiation of hematopoietic cells.     

Deregulated c-myb disrupts interleukin-6- or leukemia inhibitory factor-induced myeloid differentiation prior to c-myc: role in leukemogenesis.

The c-myb proto-oncogene is abundantly expressed in tissues of hematopoietic origin, and changes in endogenous c-myb genes have been implicated in both human and murine hematopoietic tumors. c-myb encodes a DNA-binding protein capable of trans-activating the c-myc promoter. Suppression of both of these proto-oncogenes was shown to occur upon induction of terminal differentiation but not upon induction of growth inhibition in myeloid leukemia cells. Myeloblastic leukemia M1 cells that can be induced for terminal differentiation with the physiological hematopoietic inducers interleukin-6 and leukemia inhibitory factor were genetically manipulated to constitutively express a c-myb transgene. By using immediate-early to late genetic and morphological markers, it was shown that continuous expression of c-myb disrupts the genetic program of myeloid differentiation at a very early stage, which precedes the block previously shown to be exerted by deregulated c-myc, thereby indicating that the c-myb block is not mediated via deregulation of c-myc. Enforced c-myb expression also prevents the loss in leukemogenicity of M1 cells normally induced by interleukin-6 or leukemia inhibitory factor. Any changes which have taken place, including induction of myeloid differentiation primary response genes, eventually are reversed. Also, it was shown that suppression of c-myb, essential for terminal differentiation, is not intrinsic to growth inhibition. Taken together, these findings show that c-myb plays a key regulatory role in myeloid differentiation and substantiate the notion that deregulated expression of c-myb can play an important role in leukemogenicity.     

Hepatocyte-stimulating factor III shares structural and functional identity with leukemia-inhibitory factor

The coordinate increase in the hepatic production of the acute phase plasma proteins appears to be mediated by several cytokines produced by different cell types. One factor, hepatocyte-stimulating factor III (HSF-III), constitutively produced by human squamous carcinoma (COLO-16) cells, stimulates the synthesis of the same set of acute phase plasma proteins as the structurally distinct IL-6. The physicochemical properties of HSF-III coincide with those of the T cell-derived leukemia-inhibitory factor (LIF). Human rLIF, tested on hepatoma cells, indicated a liver-regulating activity identical to HSF-III. The LIF activity is specifically neutralized by HSF-III antibodies. COLO-16 cells contain an LIF mRNA which is characteristic for lectin-stimulated T cells, suggesting that HSF-III is an epidermal cell-derived form of LIF. This result provides additional evidence for the close relationship between acute phase regulation of the liver and control of proliferation and differentiation of hemopoietic cells by identical cytokines.     

Autoregulation of interleukin 6 and granulocyte-macrophage colony-stimulating factor in the differentiation of myeloid leukemic cells.

Induction of differentiation in one type of clone of mouse myeloid leukemic cells by mouse or human interleukin 6 (IL-6) and in another type of clone by mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to be associated with induction of IL-6 and GM-CSF mRNA and protein. The results indicated that IL-6 and GM-CSF could positively autoregulate their own gene expression during myeloid cell differentiation. It is suggested that this autoregulation may serve to enhance and prolong the signal induced by these proteins in cells transiently exposed to IL-6 or GM-CSF.     

Purification of a murine leukemia inhibitory factor from Krebs ascites cells

A factor capable of inducing terminal differentiation in the murine myeloid leukemia cell line M1 has been purified to apparent homogeneity from the medium conditioned by Krebs II ascites tumor cells. The factor, termed leukemia inhibitory factor (LIF) is a single chain glycoprotein of apparent Mr 58,000 which induces differentiation and inhibits proliferation of the M1 cell line but not the WEHI-3B D+ murine myeloid leukemic cell line and has no detectable proliferative activity on normal myeloid progenitor cells. It was purified using four successive high-efficiency purification steps--anion-exchange chromatography on DEAE-Sepharose; cation-exchange chromatography on CM-Sepharose; affinity chromatography on lentil lectin-Sepharose; and reverse-phase high-performance liquid chromatography on a phenyl-silica matrix--to a specific biological activity of approximately 1.25 X 10(8) units/mg with an overall purification of 12,000-fold and a yield of 73% for the activity failing to bind to DEAE-Sepharose. Sufficient quantities of the factor (12 micrograms, 200 pmol) have been purified to allow structural and functional analysis of the molecule and comparison with other know differentiation inducers.