2-Alkenylthieno[2,3-b]pyridine-5-carbonitriles: Potent and selective inhibitors of PKCtheta

A series of 2-alkenyl thieno[2,3-b]pyridine inhibitors of PKCθ were synthesized as potential inflammatory modulators. This series led to the discovery of 2-alkenyl amides, which are exceptionally potent and selective inhibitors of PKCθ. Compound 8 has an IC₅₀ of 3.8 nM against PKCθ and shows excellent selectivity over a variety of PKC isoforms.     

The essential role of PKCalpha in the protective effect of heat-shock pretreatment on TNFalpha-induced apoptosis in hepatic epithelial cell line

During sepsis, hepatic apoptosis occurred, which is associated with inactivation of PKCalpha and elevation of tumor necrosis factor-alpha (TNFalpha), an apoptosis trigger. Heat shock, accompanied by the increase of heat-shock protein (Hsp72), has been shown to exhibit a protective role on cell survival. However, Hsp72 was unable to express during sepsis when the apoptosis was markedly increased. We hypothesized that hepatic apoptosis during sepsis may be due to the failure to induce expression of Hsp72, which is activated by PKC-phosphorylated HSF. This study was designed to examine the role of PKCalpha in Hsp72 expression and the anti-apoptotic effect of Hsp72 on hepatic epithelial cells by analyzing a TNFalpha-induced apoptosis system. The following results were observed: (1) Hsp72 was highly expressed at 8 h after heat-shock treatment in a clone 9 hepatic epithelial cell line; (2) the protein expression of PKCalpha in membrane-associated fraction was decreased by TNFalpha treatment; (3) the TNFalpha-induced cell death, especially apoptosis, was diminished by heat-shock pretreatment; (4) in the presence of PKCalpha antisense, which blocks the PKCalpha resynthesis, no protective effect of heat-shock pretreatment was observed, and the protein expression of Hsp72 was significantly suppressed. These results suggest that PKCalpha plays a critical role in the expression of Hsp72, which subsequently protects against TNFalpha-induced hepatic apoptosis.     

Regulation of protein kinase Cδ downregulation by protein kinase Cε and mammalian target of rapamycin complex 2

Phosphorylation and dephosphorylation of PKCs can regulate their activity, stability and function. We have previously shown that downregulation of PKCδ by tumor promoting phorbol esters was compromised when HeLa cells acquired resistance to cisplatin (HeLa/CP). In the present study, we have used these cells to understand the mechanism of PKCδ downregulation. A brief treatment of HeLa cells with phorbol 12,13-dibutyrate (PDBu) induced phosphorylation of PKCδ at the activation loop (Thr505), turn motif (Ser643), hydrophobic motif (Ser662) and Tyr-311 sites to a greater extent in HeLa/CP cells compared to HeLa cells. Prolonged treatment with PDBu led to downregulation of PKCδ in HeLa but not in HeLa/CP cells. The PKC inhibitor Gö 6983 inhibited PDBu-induced downregulation of PKCδ, decreased Thr505 phosphorylation and increased PKCδ tyrosine phosphorylation at Tyr-311 site. However, knockdown of c-Abl, c-Src, Fyn and Lyn had little effect on PKCδ downregulation and Tyr311 phosphorylation. Pretreatment with the phosphatidylinositol 3-kinase inhibitor Ly294002 and mTOR inhibitor rapamycin restored the ability of PDBu to downregulate PKCδ in HeLa/CP cells. Knockdown of mTOR and rictor but not raptor facilitated PKCδ downregulation. Depletion of PKCε also enhanced PKCδ downregulation by PDBu. These results suggest that downregulation of PKCδ is regulated by PKCε and mammalian target of rapamycin complex 2 (mTORC2).     

C-5 Substituted heteroaryl 3-pyridinecarbonitriles as PKCθ inhibitors: Part I

We earlier reported that 3-pyridinecarbonitriiles with a 4-methylindolyl-5-amino group at C-4 and a phenyl group at C-5 were inhibitors of PKCθ. Keeping the group at C-4 of the pyridine core constant, we varied the water solubilizing group on the phenyl ring at C-5 and then replaced the C-5 phenyl ring with several monocyclic heteroaryl rings, including furan, thiophene and pyridine. Analog 6e with a 4-methylindol-5-ylamino group at C-4 and a 5-[(4-methylpiperazin-1-yl)methyl]-2-furyl group C-5 had an IC₅₀ value of 4.5 nM for the inhibition of PKCθ.     

TGF-β1 regulation of dendritic cells

Dendritic cells (DCs) represent antigen-presenting cell (APC) populations in lymphoid and nonlymphoid organs which are considered to play key roles in the initiation of antigen-specific T-cell proliferation. According to current knowledge, the net outcome of T-cell immune responses seems to be significantly influenced by the activation stage of antigen-presenting DCs. Several studies have shown that transforming growth factor-beta 1 (TGF-β1) inhibits in vitro activation and maturation of DCs. TGF-β1 inhibits upregulation of critical T-cell costimulatory molecules on the surface of DCs and reduces the antigen-presenting capacity of DCs. Thus, in addition to direct inhibitory effects of TGF-β1 on effector T lymphocytes, inhibitory effects of TGF-β1 at the level of APCs may critically contribute to previously characterized immunosuppressive effects of TGF-β1. In contrast to these negative regulatory effects of TGF-β1 on function and maturation of lymphoid tissue type DCs, certain subpopulations of immature DCs in nonlymphoid tissues are positively regulated by TGF-β1 signaling. In particular, epithelial-associated DC populations seem to critically require TGF-β1 stimulation for development and function. Recent studies established that TGF-β1 stimulation is absolutely required for the development of epithelial Langerhans cells (LCs) in vitro and in vivo. Furthermore, TGF-β1 seems to enhance antigen processing and costimulatory functions of epithelial LCs.     

The roles of MCP-1 and protein kinase Cδ activation in human eosinophilic leukemia EoL-1 cells

Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-α (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKCδ in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G_i/G_o protein, phospholipase C (PLC), PKCδ, p38 MAPK and NF-κB. MCP-1 activates p38 MAPK via G_i/G_o protein, PLC and PKCδ cascade. MCP-1 also induces NF-κB translocation and the activation is inhibited by PKCδ activation. The increase in the basal expression and activity of PKCδ in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKCδ is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKCδ functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.     

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE₂ was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE₂, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGFβ and tumor necrosis factor-α(TNF). The production of 6-keto-PGF_1α (the stable metabolite of prostacyclin) and of PGF_2α were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGFβ and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE₂ biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6–12 h, and levels had appreciably diminished by 24 h after treatment. Both TGFβ and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE₂ resulting from TNF stimulation was blocked by the addition of an interleukin-1β (IL-1β) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1β production. TGFβ regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines. J. Cell. Biochem. 64:618–631. © 1997 Wiley-Liss, Inc.     

Involvement of Polyamines in the Action of Transforming Growth Factor β and Interleukin-1 on Cultured Chick Embryo Fibroblasts

In this study we have examined the relationship between growth factor-induced proliferation and ODC/polyamine levels. TGFβ promotes cell growth and enhances [³H]-thymidine incorporation in chick embryo fibroblasts maintained in a serum-depleted medium. The action on DNA synthesis declines in the second day of treatment. IL-1 does not affect proliferation or [³H]-thymidine incorporation either when it is added alone or in combination with TGFβ. The response of the cells to TGFβ is associated with a significant stimulation of ODC activity and Put, Spd levels together with an enhancement of the Spd/polyamines ratio. IL-1, which does not act on cell proliferation, fails to activate ODC and to increase polyamine levels, thus indicating that the ODC/polyamine system is most likely to be an important link in the chain of events that leads to growth factor-induced proliferation. © 1997 John Wiley & Sons, Ltd.     

FGF2 suppresses neuronogenesis of a cell line derived from rat olfactory epithelium

Neurogenesis continues throughout adulthood in the mammalian olfactory epithelium (OE), and both neurons as well as nonneuronal cells are reconstituted following experimental injury. Underlying the capacity of the OE to replenish its mature elements is a population of progenitor basal cells. Although the precise lineage relationships among progenitor and mature cell types are incompletely understood, the population of globose basal cells (GBCs) contains immediate precursors to neurons as well as amplifying progenitors, and retroviral lineage analyses suggest that multipotential GBCs are activated following direct injury to the OE. To assess the controls on the process of epithelial regeneration, we have characterized a cell line derived from rat OE and studied the effects of serum and tissue extracts, fibroblast growth factor-2 (FGF2) and transforming growth factor-α (TGFα) on the cells. Using a panel of cell type-specific markers whose patterns of labeling in the OE are well defined, including recently developed markers for GBCs, we characterized the phenotype of the cell line under differing culture conditions. In complete medium, which contains serum and tissue extracts, the cell line displayed characteristics of GBCs that are prominent during regeneration. Serum and extract withdrawal induced the cells to differentiate into neurons. In contrast, FGF2 prevented neuronal differentiation and maintained a GBC phenotype. TGFα had a mitogenic or differentiative effect that was context dependent. Finally, we demonstrate here that FGF2 is contained in mature olfactory neurons and sustentacular cells in vivo, suggesting a physiologic role for this growth factor in OE cell regulation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 411–428, 1997