PKR is an interferon‐induced serine‐threonine protein kinase that plays an important role in the mediation of the antiviral and antiproliferative actions of interferons. PKR is present at low basal levels in cells and its expression is induced at the transcriptional level by interferons. PKR's kinase activity stays latent until it binds to its activator. In the case of virally infected cells, double‐stranded (ds) RNA serves as PKR's activator. The dsRNA binds to PKR via two copies of an evolutionarily conserved motif, thus inducing a conformational change, unmasking the ATP‐binding site and leading to autophosphorylation of PKR. Activated PKR then phosphorylates the α‐subunit of the protein synthesis initiation factor 2 (eIF2α) thereby inducing a general block in the initiation of protein synthesis. In addition to dsRNA, polyanionic agents such as heparin can also activate PKR. In contrast to dsRNA‐induced activation of PKR, heparin‐dependent PKR activation has so far remained uncharacterized. In order to understand the mechanism of heparin‐induced PKR activation, we have mapped the heparin‐binding domains of PKR. Our results indicate that PKR has two heparin‐binding domains that are nonoverlapping with its dsRNA‐binding domains. Although both these domains can function independently of each other, they function cooperatively when present together. Point mutations created within these domains rendered PKR defective in heparin‐binding, thereby confirming their essential role. In addition, these mutants were defective in kinase activity as determined by both in vitro and in vivo assays.

Human innate immunity plays a pivotal role in host defense against various microbial challenges. Mediated by a family of Toll-like-receptors (TLR) and associated intracellular downstream signaling molecules, human innate immunity can specifically recognize diverse microbial products and many other non-microbial environmental cues. Beyond its role of providing first line of defense, activation of innate immunity signaling can lead to expression of diverse pro- and anti- inflammatory mediators, which are critical for regulating various cell and tissue metabolism. Alteration in innate immunity signaling may therefore lead to infection and inflammatory diseases such as atherosclerosis, diabetes, and cancer. TLR receptors as well as intracellular signaling proteins can serve as therapeutic targets for treating various inflammatory diseases. Several synthetic ligands of TLR receptors such as lipid A analogs, poly(I:C), loxoribine, oligodeoxynucleotides have been shown to be effective in regulating innate immune response. This review discusses the potential, challenge, and recent progress of developing synthetic as well as naturally occurring TLR ligands in regulating innate immunity and treating inflammatory diseases.

Uncontrolled proliferation of vascular smooth muscle cells (VSMCs) contribute to intimal hyperplasia during atherosclerosis and restenosis. Heparin is an antiproliferative agent for VSMCs and has been shown to block VSMC proliferation both in tissue culture systems and in animals. Despite the well documented antiproliferative actions of heparin, its cellular targets largely remain unknown. In an effort to characterize the mechanism of the antiproliferative property of heparin, we have analyzed the effect of heparin on cell cycle in VSMC. Our results indicate that the heparin-induced block in G 1 to S phase transition is imposed by p27 kip1 -mediated inhibition of cyclin-dependent kinase 2 activity. Further analysis of p27 kip1 mRNA levels showed that the increase in p27 kip1 protein levels in heparin-treated VSMC occurs at posttranscriptional levels. We present evidence that heparin causes stabilization of p27 kip1 protein during G 1 phase and thereby prevents activation of cyclin-dependent kinase 2.The proliferation of vascular smooth muscle cells (VSMCs) 1 is a key event in the development of atherosclerotic lesions and postangioplasty restenosis (1). In a normal artery, the VSMCs are in a non-proliferative quiescent state and show a well differentiated contractile phenotype. After the vascular injury, there is a loss of differentiated phenotype and a shift to a synthetic phenotype, which is also accompanied by entry into the cell cycle and proliferation (2). Several cytokines, growth factors, vasoregulatory molecules, and extracellular matrix components exert their effects on the proliferation of VSMC. The development of an atherosclerotic lesion can be blocked significantly by effective inhibition of VSMC proliferation (3). Natural glycosaminoglycans such as heparin are also known inhibit VSMC proliferation in vitro in tissue culture (4 -6) and in vivo in animal models (7,8). Despite the well documented antiproliferative effect of heparin on VSMC, the molecular mechanisms responsible for inhibition of cell cycle remain uncharacterized.Cellular proliferation is regulated primarily by regulation of the cell cycle (9), which consists of four distinct sequential phases (G 0 /G 1 , S, G 2 , and M). This tightly regulated temporal order is controlled by the sequential activation of certain serine/threonine protein kinases known as cyclin-dependent kinases (Cdks) that phosphorylate the Rb protein (10). In quiescent cells, Rb exists in its hypophosphorylated state and is thus able to bind and sequester the members of E2F family of transcription factors (11). Phosphorylation of Rb at multiple sites by Cdks causes the release of E2F because hyperphosphorylated Rb cannot bind and sequester E2F factors, thus enabling them to activate transcription of genes whose products are absolutely essential for further cell cycle progression (12). The activity of Cdks is further regulated negatively by a number of Cdk inhibitors, which are grouped into two classes (13) (14), and the members of Cip family (p21 cip1 , p27 kip1 , and ...

PACT is a stress-modulated, cellular activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) and is an important regulator of PKR-dependent signaling pathways. The research presented here is aimed at understanding the regulation of PACT expression in mammalian cells. PACT is expressed ubiquitously in different cell types at varying abundance. We have characterized the sequence elements in PACT promoter region that are required for its expression. Using deletion analysis of the promoter we have identified the minimal basal promoter of PACT to be within 101 nucleotides upstream of its transcription start site. Further mutational analyses within this region, followed by electrophoretic mobility shift analyses (EMSAs) and chromatin immunoprecipitation (ChiP) analysis have shown that Specificity protein 1 (Sp1) is the major transcription factor responsible for PACT promoter activity.