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.

In addition to its central role in cellular stress signaling, the tumor suppressor p53 modulates mitochondrial respiration through its nuclear transcription factor activity and localizes to mitochondria where it enhances apoptosis and suppresses mitochondrial DNA (mtDNA) mutagenesis. Here we demonstrate a new conserved role for p53 in mtDNA copy number maintenance and mitochondrial reactive oxygen species (ROS) homeostasis. In mammals, mtDNA is present in thousands of copies per cell and is essential for normal development and cell function. We show that p53 null mouse and p53 knock-down human primary fibroblasts exhibit mtDNA depletion and decreased mitochondrial mass under normal culture growth conditions. This is accompanied by a reduction of the p53R2 subunit of ribonucleotide reductase mRNA and protein and of mitochondrial transcription factor A (mtTFA) at the protein level only. Finally, p53-depleted cells exhibit significant disruption of cellular ROS homeostasis, characterized by reduced mitochondrial and cellular superoxide levels and increased cellular hydrogen peroxide. Altogether, these results elucidate additional mitochondria-related functions for p53 and implicate mtDNA depletion and ROS alterations as potentially relevant to cellular transformation, cancer cell phenotypes, and the Warburg Effect.