Embryonic stem (ES) cell pluripotency is regulated by a combination of extrinsic and intrinsic factors. Previously we have demonstrated that phosphoinositide 3-kinase (PI3K)-dependent signaling is required for efficient self-renewal of murine ES cells. In the study presented here, we have investigated the downstream molecular mechanisms that contribute to the ability of PI3Ks to regulate pluripotency. We show that inhibition of PI3K activity with either pharmacological or genetic tools results in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels. Inhibition of glycogen synthase kinase 3 (GSK-3) activity by PI3Ks plays a key role in regulation of Nanog expression, because blockade of GSK-3 activity effectively reversed the effects of PI3K inhibition on Nanog RNA, and protein expression and self-renewal under these circumstances were restored. Furthermore, GSK-3 mutants mimicked the effects of PI3K or GSK-3 inhibition on Nanog expression. Importantly, expression of an inducible form of Nanog prevented the loss of self-renewal observed upon inhibition of PI3Ks, supporting a functional relationship between PI3Ks and Nanog expression. In addition, expression of a number of putative Nanog target genes was sensitive to PI3K inhibition. Thus, the new evidence provided in this study shows that PI3K-dependent regulation of ES cell self-renewal is mediated, at least in part, by the ability of PI3K signaling to maintain Nanog expression. Regulation of GSK-3 activity by PI3Ks appears to play a key role in this process.Embryonic stem cell pluripotency underpins their potential utility as a source of differentiated progeny for use in regenerative medicine. Leukemia inhibitory factor (LIF) 2 plays an important role in maintaining self-renewal of murine ES (mES) cells (1, 2) via activation of STAT3 (3-6) and induction of c-Myc (7). LIF also activates additional signals including the Ras/ERK kinase pathway (8, 9), ribosomal S 6 kinases (10), phosphoinositide 3-kinases (11), and Src kinases (12). However, whereas LIF-induced STAT3 activation promotes self-renewal, LIF-induced ERK activation appears to promote differentiation (8), leading to the proposal that the balance between STAT3 and ERK signals contributes to the determination of mES cell fate (13).Other extrinsic factors that also play a role in maintenance of mES cell self-renewal include bone morphogenic protein 4 (BMP4), which acts in synergy with LIF to maintain self-renewal via Smad-mediated induction of Id transcriptional repressor expression (14). A further report suggests BMP4 inhibition of p38 mitogen-activated protein kinase (MAPK) may also contribute to maintenance of self-renewal (15). Wnt signaling has been implicated in regulation of pluripotency of both mES and human ES (hES) cells, work stemming largely from use of the GSK-3 inhibitor 5-bromoindirubin-3-oxime (BIO) (16,17).A number of intrinsic regulators in the form of transcription factors have been identified that play important roles in regulatio...

Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine mainly acting on monocytes and T cells that elicits its biological effects by interacting with the seven-transmembrane helix receptor CCR2B. The vaccinia virus strain Lister and many other poxviruses express soluble proteins (vCCI) that bind MCP-1 and other CC chemokines and inhibit their function. In order to define the interaction site of MCP-1 with vCCI from vaccinia, surface exposed residues of MCP-1 were identified and mutated to alanine. The MCP-1 variants were expressed, purified, and their interaction with vCCI was characterized. The site on MCP-1 for vCCI binding is dominated by arginine 18 with important additional contributions from tyrosine 13 and arginine 24. These residues define a binding site that largely overlaps with the CCR2B receptor interaction site. The viral chemokine-binding protein vCCI thus inhibits the biological function of MCP-1 by directly masking its CCR2B receptor-binding site.Chemokines are small (8 -14 kDa) structurally related proteins that regulate cell trafficking of various leukocyte subtypes through interaction with a set of G protein-coupled receptors. The two major subfamilies are the CXC chemokines which act on neutrophils and non-hemopoietic cells and the CC chemokines which bind to receptors mainly expressed on monocytes, T cells, eosinophils, and basophils. Additional members of the chemokine family are the C chemokine lymphotactin and the CX3C chemokine fraktalkine. Chemokines are important for the development, homeostasis, and function of the immune system and play a pivotal role in host defense (for reviews, see Refs. 1-3).Given the importance of chemokines for defense against pathogens it is no surprise that viruses themselves developed mechanisms to neutralize the function of chemokines to further their own propagation or evade host defense (4). Poxviruses express secreted chemokine neutralizing proteins termed virus-encoded chemokine-binding proteins (vCKBP).1 Two families of vCKBPs have thus far been identified that use distinct mechanisms of chemokine neutralization (5). The vCKBP-1 family binds C, CC, and CXC chemokines with low affinity, possibly by interaction with the proteoglycan-binding site on chemokines, thereby interfering with the proper localization and presentation of chemokines in inflamed tissues (6, 7). The vCKBP-2 proteins, however, bind preferably CC chemokines with high affinity and inhibit the interaction of chemokines with their cellular receptors (8 -10). vCKBP-2 proteins have been identified in strains of vaccinia (11), cowpox (12), rabbitpox (13), myxomavirus (14), and variola virus (15). Prototypic members of this protein family are the M-T1 gene product from myxoma virus and the rabbitpox virus major secreted 35-kDa protein (10). In rabbits infected with a rabbitpox virus which had the gene for the secreted 35-kDa protein deleted, an increased leukocyte influx into virus-infected tissues was observed, confirming the role of this protein in inhibition of chemokine-medi...