No abstract

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.

After interferon‐gamma (IFN‐gamma) treatment of cells the appearance of tyrosine phosphorylated Stat1 in the nucleus was maximal within 20–30 min, remained for 2–2.5 h and activated molecules disappeared by 4 h. In the absence of continued signaling from the receptor (imposed by staurosporine treatment) previously activated Stat1 disappeared completely within 60 min, implying continuous generation and removal of active molecules during extended IFN‐gamma treatment. Proteasome inhibitors prolonged the time of activation of Stat1 by prolonging signaling from the receptor but not by blocking removal of already activated Stat1 molecules. By analyzing with 35S labeling the distribution of total Stat1 and activated Stat1, we concluded that the Stat1 molecules promptly cycle into the nucleus as tyrosine phosphorylated molecules and later return quantitatively to the cytoplasm as non‐phosphorylated molecules. Therefore, the removal of the activated Stat1 molecules from the nucleus appears not to be proteolytic but must depend on a protein tyrosine phosphatase(s).

Obesity is an important public health problem associated with a number of disease states such as diabetes and arteriosclerosis. As such, an understanding of the mechanisms governing adipose tissue differentiation and function is of considerable importance. We recently reported that the Krü ppel-like zinc finger transcription factor KLF15 can induce adipocyte maturation and GLUT4 expression. In this study, we identify that a second family member, KLF2/Lung Krü ppel-like factor (LKLF), as a negative regulator of adipocyte differentiation. KLF2 is highly expressed in adipose tissue, and studies in cell lines and primary cells demonstrate that KLF2 is expressed in preadipocytes but not mature adipocytes. Constitutive overexpression of KLF2 but not KLF15 potently inhibits peroxisome proliferator-activated receptor-␥ (PPAR␥) expression with no effect on the upstream regulators C/EBP␤ and C/EBP␦. However, the expression of C/EBP␣ and SREBP1c/ADD1 (adipocyte determination and differentiation factor-1/sterol regulatory element-binding protein-1), two factors that feedback in a positive manner to enhance PPAR␥ function, was also markedly reduced. In addition, transient transfection studies show that KLF2 directly inhibits PPAR␥2 promoter activity (70% inhibition; p < 0.001). Using a combination of promoter mutational analysis and gel mobility shift assays, we have identified a binding site within the PPAR␥2 promoter, which mediates this inhibitory effect. These data identify a novel role for KLF2 as a negative regulator of adipogenesis.

Resistance to the stimulatory effects of insulin on glucose utilization is a key feature of type 2 diabetes, obesity, and the metabolic syndrome. Recent studies suggest that insulin resistance is primarily caused by a defect in glucose transport. GLUT4 is the main insulinresponsive glucose transporter and is expressed predominantly in muscle and adipose tissues. Whereas GLUT4 has been shown to play a critical role in maintaining systemic glucose homeostasis, the mechanisms regulating its expression are incompletely understood. We have cloned the murine homologue of KLF15, a member of the Krü ppel-like family of transcription factors. KLF15 is highly expressed in adipocytes and myocytes in vivo and is induced when 3T3-L1 preadipocytes are differentiated into adipocytes. Overexpression of KLF15 in adipose and muscle cell lines potently induces GLUT4 expression. This effect is specific to KLF15 as overexpression of two other Krü ppel-like factors, KLF2/LKLF and KLF4/GKLF, did not induce GLUT4 expression. Both basal (3.3-fold, p < 0.001) and insulin-stimulated (2.4-fold, p < 0.00001) glucose uptake are increased in KLF15-overexpressing adipocytes. In co-transfection assays, KLF15 and MEF2A, a known activator of GLUT4, synergistically activates the GLUT4 promoter. Promoter deletion and mutational analyses provide evidence that this activity requires an intact KLF15-binding site proximal to the MEF2A site. Finally, co-immunoprecipitation assays show that KLF15 specifically interacts with MEF2A. These studies indicate that KLF15 is an important regulator of GLUT4 in both adipose and muscle tissues.Glucose uptake into cells is regulated by two families of cellular transporters, the sodium-linked glucose transporters (kidney, intestine) and the facilitated glucose transporters (GLUTs). With respect to the latter, GLUT4 is the main effector of insulin-stimulated glucose transport and is located primarily in muscle and adipose tissues (1).Clinical and experimental observations suggest that insulinstimulated glucose transport via GLUT4 is critical in maintaining systemic glucose homeostasis. For example, heterozygous mice deficient in GLUT4 exhibit muscle insulin resistance and develop diabetes (2). Tissue-specific disruption of GLUT4 in adipose tissue and skeletal muscle results in the development of insulin resistance and glucose intolerance (3, 4). In human type 2 diabetic patients, impairment of insulin-stimulated glucose transport is responsible for resistance to insulin-stimulated glycogen synthesis in muscle (5-7).Studies in adipose and muscle tissues reveal that expression of the GLUT4 glucose transporter is controlled at the level of transcription (8, 9). In vitro and in vivo promoter studies support a role for members of the MADS-box family of transcription factors termed MEF2 proteins in the regulation of the GLUT4 promoter. However, these studies suggest that MEF2 binding alone is not sufficient to fully support GLUT4 expression (10 -12).The Krü ppel-like family of transcription factors are important regulators ...