Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-␥ (IFN-␥), lipopolysaccharide (LPS), or tumor necrosis factor-␣ can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-␤1 (TGF-␤1) can decrease proinflammatory activation. The molecular mediators regulating the balance of these opposing effectors remain incompletely understood. Herein, we identify Kruppel-like factor 4 (KLF4) as being markedly induced in response to IFN-␥, LPS, or tumor necrosis factor-␣ and decreased by TGF-␤1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-␤1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-␥, LPS, or IFN-␥ plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-␤1 and Smad3 signaling. The KLF4 induction of the iNOS promoter is mediated by two KLF DNA-binding sites at ؊95 and ؊212 bp, and mutation of these sites diminished induction by IFN-␥ and LPS. We further provide evidence that KLF4 interacts with the NF-B family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-␤1/ Smad3 induction of the PAI-1 promoter independent of KLF4 DNA binding through a novel antagonistic competition with Smad3 for the C terminus of the coactivator p300/CBP. These findings support an important role for KLF4 as a regulator of key signaling pathways that control macrophage activation.Macrophage activation is an integral process in the development of atherosclerosis as well as a number of other chronic inflammatory diseases such as emphysema, inflammatory bowel disease, psoriasis, arthritis, and pancreatitis. Once within the site of inflammation, macrophages elaborate a broad range of cytokines, growth factors, and proteolytic enzymes that may participate in the damage and repair that ensues. Identification of mechanisms that may regulate macrophage activation is, thus, of considerable interest.One of the key events in macrophage response to proinflammatory stimuli is the expression of inducible nitric-oxide synthase (iNOS) 2 and the formation of nitric oxide, an important mediator involved in many host defense actions in macrophages. However, increased amounts of leukocyte-derived nitric oxide can be detrimental by promoting tissue damage in a variety of inflammatory disease states (1, 2). Given the importance of iNOS in a variety of pathophysiological conditions, control of its expression has been the subject of considerable investigation (1). Indeed, several studies have shown that induction or inhibition of iNOS in macrophages by pro-or anti-inflammatory stimuli, respectively, can occur at the level of transcription. For example, proinflammatory stimuli such as LPS or IFN-␥ involve activation of NF-B or interferon-responsive element...

Monocyte differentiation involves the participation of lineage-restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppellike family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte-restricted and stage-specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL-60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester-induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1À/À fetal liver cells along the monocytic lineage and can activate the monocytic-specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation.

The stochasticity of gene expression presents significant challenges to the modeling of genetic networks. A two-state model describing promoter switching, transcription, and messenger RNA (mRNA) decay is the standard model of stochastic mRNA dynamics in eukaryotic cells. Here, we extend this model to include mRNA maturation, cell division, gene replication, dosage compensation, and growth-dependent transcription. We derive expressions for the time-dependent distributions of nascent mRNA and mature mRNA numbers, provided two assumptions hold: 1) nascent mRNA dynamics are much faster than those of mature mRNA; and 2) gene-inactivation events occur far more frequently than gene-activation events. We confirm that thousands of eukaryotic genes satisfy these assumptions by using data from yeast, mouse, and human cells. We use the expressions to perform a sensitivity analysis of the coefficient of variation of mRNA fluctuations averaged over the cell cycle, for a large number of genes in mouse embryonic stem cells, identifying degradation and gene-activation rates as the most sensitive parameters. Furthermore, it is shown that, despite the model’s complexity, the time-dependent distributions predicted by our model are generally well approximated by the negative binomial distribution. Finally, we extend our model to include translation, protein decay, and auto-regulatory feedback, and derive expressions for the approximate time-dependent protein-number distributions, assuming slow protein decay. Our expressions enable us to study how complex biological processes contribute to the fluctuations of gene products in eukaryotic cells, as well as allowing a detailed quantitative comparison with experimental data via maximum-likelihood methods.

The presence of protein–DNA binding reactions often leads to analytically intractable models of stochastic gene expression. Here we present the linear-mapping approximation that maps systems with protein–promoter interactions onto approximately equivalent systems with no binding reactions. This is achieved by the marriage of conditional mean-field approximation and the Magnus expansion, leading to analytic or semi-analytic expressions for the approximate time-dependent and steady-state protein number distributions. Stochastic simulations verify the method’s accuracy in capturing the changes in the protein number distributions with time for a wide variety of networks displaying auto- and mutual-regulation of gene expression and independently of the ratios of the timescales governing the dynamics. The method is also used to study the first-passage time distribution of promoter switching, the sensitivity of the size of protein number fluctuations to parameter perturbation and the stochastic bifurcation diagram characterizing the onset of multimodality in protein number distributions.

CD4؉ CD25

IntroductionLeukocyte development requires the coordination of stage-specific transcription factors to help orchestrate the processes by which a progenitor cell emerges as a functional leukocyte. Indeed, aberrant expression or function of many of these transcription factors has been associated with several disease conditions, such as leukemia, lymphoma, autoimmunity, and chronic inflammation. Moreover, recent studies have indicated that Krüppel-like factors (KLFs) may be among those key trans-acting factors contributing to the orchestration of several aspects of leukocyte biology, including cell lineage commitment, differentiation, and function.The original Krüppel factor was characterized in Drosophila melanogaster as a "gap" segmentation gene, homozygous mutation of which resulted in the absence of thorax and anterior abdomen in embryos. [1][2][3][4] Thus, the German researchers named this gene Krüppel (English "cripple"). A conserved family of nuclear proteins encoded by Drosophila Krüppel were identified in 1986 and exhibited a striking structural similarity to the DNA-binding "finger motif " of transcription factor IIIA. 5 The first mammalian gene with homology to Krüppel was identified in 1993, and its encoded protein was named erythroid Krüppel-like factor (EKLF) in accordance with its erythroid cell-specific expression. 6 The function of EKLF was demonstrated by the fact that EKLF bound to human and murine adult ␤-globin CACCC elements via its DNA-binding domain, whereas the non-DNA-binding domain mediated transcriptional activation. 7 The importance of EKLF in differentiation and development was later demonstrated by loss-offunction studies showing that homozygous EKLF Ϫ/Ϫ mice developed a fatal ␤-thalassemia during fetal liver erythropoiesis. 8,9 To date, members of the mammalian KLF family number 17. 10 Identified by various experimental approaches, KLF1 (EKLF) through KLF17 have been termed according to their chronologic order of identification ( Figure 1). Each family member is a zinc finger transcription factor. The distinguishing feature of KLFs compared with other zinc finger-containing proteins, therefore, is the presence of a highly conserved DNA-binding domain composed of 3 C 2 H 2 zinc fingers at or near the C-terminus. [11][12][13] As such, most KLFs are able to bind the CACCC element and GC box consensus sequences. Furthermore, the KLFs share a highly conserved 7-residue sequence, TGEKP(Y/F)X, between zinc fingers. 14 The non-DNA-binding regions of each, however, are highly divergent and can function as trans-activation or trans-repression domains. Collectively, these features distinguish the KLFs from the larger family of zinc-finger transcription factors ( Figure 1A).By regulating gene transcription, KLFs are involved in many physiologic and pathologic processes, such as cell differentiation, proliferation, cell growth, and apoptosis during normal development or under different disease conditions ( Figure 1B). 13,15,16 This review focuses on the transcriptional control of leukocyte cell b...

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