Prevention of CCAAT/Enhancer-binding Protein �� DNA Binding by Hypoxia during Adipogenesis

Upon exposure to adipogenesis-inducing hormones, confluent 3T3-L1 preadipocytes express C/EBPβ (CCAAT/enhancer binding protein β). Early induced C/EBPβ is inactive but, after a lag period, acquires its DNA-binding capability by sequential phosphorylation. During this period, preadipocytes pass the G₁/S checkpoint synchronously. Thr¹⁸⁸ of C/EBPβ is phosphorylated initially to prime the factor for subsequent phosphorylation at Ser¹⁸⁴ or Thr¹⁷⁹ by GSK3β, which translocates into the nuclei during the G₁/S transition. Many events take place during the G₁/S transition, including reduction in p27^Kip1 protein levels, retinoblastoma (Rb) phosphorylation, GSK3β nuclear translocation, and C/EBPβ binding to target promoters. During hypoxia, hypoxia-inducible factor-1α (HIF-1α) is stabilized, thus maintaining expression of p27^Kip1, which inhibits Rb phosphorylation. Even under normoxic conditions, constitutive expression of p27^Kip1 blocks the nuclear translocation of GSK3β and DNA binding capability of C/EBPβ. Hypoxia also blocks nuclear translocation of GSK3β and DNA binding capability of C/EBPβ in HIF-1α knockdown 3T3-L1 cells that fail to induce p27^Kip1. Nonetheless, under hypoxia, these cells can block Rb phosphorylation and the G₁/S transition. Altogether, these findings suggest that hypoxia prevents the nuclear translocation of GSK3β and the DNA binding capability of C/EBPβ by blocking the G₁/S transition through HIF-1α-dependent induction of p27^Kip1 and an HIF-1α/p27-independent mechanism.     

Regulation of CCAAT/Enhancer-binding Protein Homologous Protein (CHOP) Expression by Interleukin-1β in Pancreatic β Cells

Apoptosis contributes to immune-mediated pancreatic β cell destruction in type I diabetes. Exposure of β cells to interleukin-1β (IL-1β) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in β cells treated with IL-1β. In addition, prolonged exposure to high glucose further increases IL-1β-triggered CHOP expression. IL-1β also causes increased expression of C/EBP-β and a reduction of MafA, NFATc2, and Pdx-1 expression in β cells. Inhibition of the NF-κB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects β cells from IL-1β-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of β cells.     

Suppressor of Cytokine Signaling 3 Inhibits LPS-induced IL-6 Expression in Osteoblasts by Suppressing CCAAT/Enhancer-binding Protein β Activity

Suppressor of cytokine signaling 3 (SOCS3) is an important intracellular protein that inhibits cytokine signaling in numerous cell types and has been implicated in several inflammatory diseases. However, the expression and function of SOCS3 in osteoblasts are not known. In this study, we demonstrated that SOCS3 expression was transiently induced by LPS in osteoblasts, and apparently contributed to the inhibition of IL-6 induction by LPS treatment. We found that tyrosine 204 of the SOCS box, the SH2 domain, and the N-terminal kinase inhibitory region (KIR) of SOCS3 were all involved in its IL-6 inhibition. Furthermore, we demonstrated that CCAAT/enhancer-binding protein (C/EBP) β was activated by LPS (increased DNA binding activity), and played a key role in LPS-induced IL-6 expression in osteoblasts. We further provided the evidence that SOCS3 functioned as a negative regulator for LPS response in osteoblasts by suppressing C/EBPβ DNA binding activity. In addition, tyrosine 204 of the SOCS box, the SH2 domain, and the N-terminal kinase inhibitory region (KIR) of SOCS3 were all required for its C/EBPβ inhibition. These findings suggest that SOCS3 by interfering with C/EBPβ activation may have an important regulatory role during bone-associated inflammatory responses.     

Src-Mediated Phosphorylation of Dynamin and Cortactin Regulates the “Constitutive” Endocytosis of Transferrin

The mechanisms by which epithelial cells regulate clathrin-mediated endocytosis (CME) of transferrin are poorly defined and generally viewed as a constitutive process that occurs continuously without regulatory constraints. In this study, we demonstrate for the first time that endocytosis of the transferrin receptor is a regulated process that requires activated Src kinase and, subsequently, phosphorylation of two important components of the endocytic machinery, namely, the large GTPase dynamin 2 (Dyn2) and its associated actin-binding protein, cortactin (Cort). To our knowledge these findings are among the first to implicate an Src-mediated endocytic cascade in what was previously presumed to be a nonregulated internalization process.Iron is an essential element for all mammalian organisms that plays essential roles in hemoglobin and myoglobin production (23). Altered iron transport can lead to disease states such as hemochromatosis (23), anemia (5, 23), and neuronal disorders (23). The transferrin receptor (TfR) is an important component of iron regulation in cells. There are two distinct TfRs in humans sharing 45% identity that are homodimeric and bind iron-associated transferrin (Tf) at markedly different affinities (26). While significant attention has been paid toward understanding the basic endocytic machinery that supports the efficient internalization and recycling of the TfR1 and its associated iron-bound ligand, it has been assumed that this transport process is constitutive in nature. This is in direct contrast to the highly regulated internalization pathway used by members of the receptor tyrosine kinase family (RTKs) and the family of G-coupled protein receptors (GPCRs) that utilize phosphorylation and/or ubiquination as signaling modules to regulate internalization.To test if TfR1 internalization might be regulated in a similar fashion, we focused on two essential components of the endocytic machinery: the large GTPase Dyn2 that mediates endocytic vesicle scission (35) and Cort that binds to Dyn2 via an SH3-PRD interaction and has been postulated to regulate actin dynamics to facilitate vesicle invagination and release (36, 40). Both Dyn2 and Cort have shown to be phosphorylated in vivo and in vitro by a variety of kinases (51, 58). Dyn1 interacts with (17) and is phosphorylated by Src in neuronal cells and in other excitable cells in response to activation of GPCRs and epidermal growth factor (EGF) (1, 2). While the Src phosphorylation motifs of dynamin are conserved in the epithelial expressed form of Dyn2, it is unclear if Dyn2 is phosphorylated in response to ligands that induce clathrin-based endocytosis.Cort possesses a series of C-terminal tyrosines that are heavily Src-phosphorylated and implicated in regulating actin remodeling during cell motility (20). In this study, we demonstrate that addition of Tf to cultured epithelial cells results in an internalization of the TfR1 mediated by a Src kinase-dependent phosphoactivation of the Dyn2-Cort-based endocytic machinery. In support of these findings, dominant negative forms of c-Src kinase, when expressed in a hepatocyte-derived cell line (Clone 9), attenuate Tf internalization. Remarkably, cells exposed to Tf showed a 3- to 4-fold increase in Dyn2 and Cort phosphorylation compared to that shown by untreated cells, an increase exceeding that observed in cells treated with EGF. These findings provide new insights into the regulation of what was thought to be a constitutive endocytic process.