Gautam Adhikary

Role of oxidative stress in intermittent hypoxia‐induced immediate early gene activation in rat PC12 cells

Intermittent hypoxia (IH) occurs in many pathophysiological conditions. The molecular mechanisms associated with IH, however, have received little attention. Previous studies have reported that the c‐fos gene via formation of activator protein‐1 (AP‐1) transcription factor contributes to adaptive responses to continuous hypoxia. In the present study, using a cell culture model we examined whether IH activates c‐fos and AP‐1 and if so, by what mechanisms. Experiments were performed on rat phaeochromocytoma cells exposed to 21% O2 (normoxia) or 60 and 120 cycles of IH, each cycle consisting 15 s of hypoxia followed by 4 min of normoxia. IH resulted in a significant elevation of c‐fos mRNA as well as transcriptional activation. IH was more potent and induced a longer lasting activation of c‐fos than comparable cumulative duration of continuous hypoxia. IH increased AP‐1 activity and tyrosine hydroxylase (TH) mRNA, an AP‐1‐regulated downstream gene, and these effects were prevented by antisense c‐fos. Superoxide dismutase mimetic, a potent scavenger of superoxide anions, prevented IH‐induced c‐fos, AP‐1 and TH activations. IH increased superoxide anion levels in mitochondria as evidenced by decreased aconitase enzyme activity and increased levels of hydrogen peroxide, a stable dismutated product of superoxide anions. Complex I of the mitochondrial electron transport chain was markedly inhibited in IH exposed cells. Pharmacological inhibitors of complex I mimicked the effects of IH during normoxia and occluded the effects of IH on c‐fos activation, suggesting the involvement of the mitochondrial electron transport chain in the generation of superoxide anions during IH. These results suggest IH‐induced c‐fos‐mediated transcriptional activation involves oxidative stress.

The Bmi-1 polycomb protein antagonizes the (−)-epigallocatechin-3-gallate-dependent suppression of skin cancer cell survival

The polycomb group (PcG) proteins are epigenetic regulators of gene expression that enhance cell survival. This regulation is achieved via action of two multiprotein PcG complexes--PRC2 (EED) and PRC1 [B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1)]. These complexes modulate gene expression by increasing histone methylation and reducing acetylation--leading to a closed chromatin conformation. Activity of these proteins is associated with increased cell proliferation and survival. We show increased expression of key PcG proteins in immortalized keratinocytes and skin cancer cell lines. We examine the role of two key PcG proteins, Bmi-1 and enhancer of zeste homolog 2 (Ezh2), and the impact of the active agent in green tea, (-)-epigallocatechin-3-gallate (EGCG), on the function of these regulators. EGCG treatment of SCC-13 cells reduces Bmi-1 and Ezh2 level and this is associated with reduced cell survival. The reduction in survival is associated with a global reduction in histone H3 lysine 27 trimethylation, a hallmark of PRC2 complex action. This change in PcG protein expression is associated with reduced expression of key proteins that enhance progression through the cell cycle [cyclin-dependent kinase (cdk)1, cdk2, cdk4, cyclin D1, cyclin E, cyclin A and cyclin B1] and increased expression of proteins that inhibit cell cycle progression (p21 and p27). Apoptosis is also enhanced, as evidenced by increased caspase 9, 8 and 3 cleavage and increased poly(adenosine diphosphate ribose) polymerase cleavage. EGCG treatment also increases Bax and suppresses Bcl-xL expression. Vector-mediated enhanced Bmi-1 expression reverses these EGCG-dependent changes. These findings suggest that green tea polyphenols reduce skin tumor cell survival by influencing PcG-mediated epigenetic regulatory mechanisms.

Toll-like receptors at the ocular surface.

The Toll-like receptor (TLR) family of pathogen recognition molecules has an important role in recognizing microbial pathogens and microbial breakdown products. Activation of TLRs in the corneal epithelium induces CXC chemokine production and recruitment of neutrophils to the corneal stroma. Although essential for pathogen killing, neutrophils can cause extensive tissue damage, leading to visual impairment and blindness. In this review, we examine the role of TLRs in microbial keratitis and in noninfectious corneal inflammation, most commonly associated with contact lens wear. we present recent findings on TLR signaling pathways in the cornea, including MyD88- and TRIF-dependent responses and discuss the role of resident macrophages and dendritic cells. Finally, we examine the potential for targeting the TLR pathway as a potential therapeutic intervention for microbial keratitis and contact lens-associated corneal inflammation.

Intracellular pathways linking hypoxia to activation of c-fos and AP-1.

Organisms respond to hypoxia through detection of blood oxygen levels by sensors at peripheral chemoreceptors and by receptors in certain key cells of the body. The pathways over which peripheral chemoreceptor signals are transmitted to respiratory muscles are well established. However, the intracellular pathways that transmit hypoxic stimulus to gene activation are just being identified. Using anti-sense c-fos strategy, we have shown that c-fos is essential for the activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of downstream genes such as tyrosine hydroxylase (TH; Mishra et al. 1998). The purpose of the present study was to identify intracellular pathways that link hypoxia to activation of c-fos. The results of the present study show that hypoxia causes Ca2+ influx through L-type voltage gated Ca2+ channels and that hypoxia-induced c-fos gene expression is Ca2+/calmodulin dependent. We also demonstrate that hypoxia activates the extracellular-regulated kinase (ERK) and p38, but not JNK. Further, phosphorylation of ERK is essential for c-fos activation via SRE cis-element. Further characterization of nuclear signalling pathways provides evidence for the involvement of Src, a non receptor protein tyrosine kinase, and Ras, a small G protein, in the hypoxia-induced c-fos gene expression. These results suggest a possible role for non-receptor protein tyrosine kinases in propagating signals from G-protein coupled receptors to the activation of immediate early genes such as c-fos during hypoxia.

Role of c-fos in hypoxia-induced AP-1 cis-element activity and tyrosine hydroxylase gene expression

Previous studies have demonstrated that hypoxia stimulates expression of the c-fos gene in intact animals and isolated cells. The purpose of the present study was to assess the functional significance of c-fos activation during hypoxia. Using antisense c-fos strategy, we tested the hypothesis that c-fos is essential for activation of activator protein-1 transcription factor complex (AP-1) and subsequent stimulation of down stream genes such as tyrosine hydroxylase (TH) gene during hypoxia. Experiments were performed on rat pheochromocytoma 12 (PC12) cells. AP-1 activity was determined by a reporter gene assay using a luciferase expression vector driven by two copies of an AP-1 cis-element (AP-1-Luc). Cells transfected with AP-1-Luc construct were exposed to normoxia (21% O2) or to varying intensities and/or durations of hypoxia. AP-1 activity increased in response to hypoxia. The magnitude of the response depended on the intensity and duration of the hypoxic stimulus. Increases in AP-1 activity could not be elicited in neuroblastoma cells, indicating that hypoxia-induced increase in AP-1 activity is a cell selective phenomenon. Antisense c-fos abolished hypoxia-induced AP-1 activation in PC12 cells. Hypoxia increased tyrosine hydroxylase-chloramphenicol acetyl transferase activity (TH-CAT), and antisense c-fos and mutations at AP-1 binding sites in TH promoter abolished this effect. These results provide direct evidence that c-fos is essential for functional activation of AP-1 and subsequent activation of delayed response genes such as TH in PC12 cells.

PKC-δ and -η, MEKK-1, MEK-6, MEK-3, and p38-δ Are Essential Mediators of the Response of Normal Human Epidermal Keratinocytes to Differentiating Agents

Previous studies suggest that the novel protein kinase C (PKC) isoforms initiate a mitogen-activated protein kinase (MAPK) signaling cascade that regulates keratinocyte differentiation. However, assigning these functions has relied on treatment with pharmacologic inhibitors and/or manipulating kinase function using overexpression of wild-type or dominant-negative kinases. As these methods are not highly specific, an obligatory regulatory role for individual kinases has not been assigned. In this study, we use small interfering RNA knockdown to study the role of individual PKC isoforms as regulators of keratinocyte differentiation induced by the potent differentiating stimulus, 12-O-tetradecanoylphorbol-13-acetate (TPA). PKC-delta knockdown reduces TPA-activated involucrin promoter activity, nuclear activator protein-1 factor accumulation and binding to DNA, and cell morphology change. Knockdown of PKC downstream targets, including MEKK-1, MEK-6, MEK-3, or p38-delta, indicates that these kinases are required for these responses. Additional studies indicate that knockdown of PKC-eta inhibits TPA-dependent involucrin promoter activation. In contrast, knockdown of PKC-alpha (a classical PKC isoform) or PKC-epsilon (a novel isoform) does not inhibit these TPA-dependent responses. Further studies indicate that PKC-delta is required for calcium and green tea polyphenol-dependent regulation of end responses. These findings are informative as they suggest an essential role for selected PKC and MAPK cascade enzymes in mediating a range of end responses to a range of differentiation stimuli in keratinocytes.

Inhibition of corneal inflammation by liposomal delivery of short-chain, C-6 ceramide

Ceramide is recognized as an antiproliferative and proapoptotic sphingolipid metabolite; however, the role of ceramide in inflammation is not well understood. To determine the role of C6‐ceramide in regulating inflammatory responses, human corneal epithelial cells were treated with C6‐ceramide in 80 nm diameter nanoliposome bilayer formulation (Lip‐C6) prior to stimulation with UV‐killed Staphylococcus aureus. Lip‐C6 (5 μM) inhibited the phosphorylation of proinflammatory and proapoptotic MAP kinases JNK and p38 and production of neutrophil chemotactic cytokines CXCL1, CXCL5, and CXCL8. Lip‐C6 also blocked CXC chemokine production by human and murine neutrophils. To determine the effect of Lip‐C6 in vivo, a murine model of corneal inflammation was used in which LPS or S. aureus added to the abraded corneal surface induces neutrophil infiltration to the corneal stroma, resulting in increased corneal haze. Mice were treated topically with 2 nMoles (811 ng) Lip‐C6 or with control liposomes prior to, or following, LPS or S. aureus stimulation. We found that corneal inflammation was significantly inhibited by Lip‐C6 but not control liposomes given prior to, or following, activation by LPS or S. aureus. Furthermore, Lip‐C6 did not induce apoptosis of corneal epithelial cells in vitro or in vivo, nor did it inhibit corneal wound healing. Together, these findings demonstrate a novel, anti‐inflammatory, nontoxic, therapeutic role for liposomally delivered short‐chain ceramide.

Polycomb Group Proteins Are Key Regulators of Keratinocyte Function

The Polycomb group (PcG) proteins are epigenetic suppressors of gene expression that function through modification of histones to change chromatin structure and modulate gene expression and cell behavior. Recent studies show that PcG proteins are expressed in epidermis, that their levels change during differentiation and in disease states, and that PcG expression is regulated by agents that influence cell proliferation and survival. The results indicate that PcG proteins regulate keratinocyte cell-cycle progression, apoptosis, senescence, and differentiation. These proteins are expressed in progenitor cells, in the basal layer, and in suprabasal keratinocytes, and the level, timing, and distribution of expression suggest that the PcG proteins have a central role in maintaining the balance between cell survival and death in multiple epidermal compartments. Additional studies indicate an important role in skin cancer progression.

C-Jun NH2 terminal kinase (JNK) is an essential mediator of Toll-like receptor 2-induced corneal inflammation.

TLRs play an important role in the host inflammatory response to bacteria and bacterial products by activating a cascade of intracellular events leading to production of proinflammatory and chemotactic cytokines. To determine the role of MAPKs in TLR‐ induced corneal inflammation, we stimulated human corneal epithelial (HCE) cells with TLR2 ligands, tripalmitoyl‐S‐glycero‐Cys‐(Lys)4 (Pam3Cys) or inactivated Staphylococcus aureus, and examined the time course of expression of MAPKs and the effect of MAPK inhibition on IkBα degradation and CXC chemokine production. We found that S. aureus and Pam3Cys stimulate phosphorylation of JNK, p38 MAPK, and ERK within 4 h and that blockade of JNK, but not p38 or ERK phosphorylation, had an inhibitory effect on IkBα degradation and CXC chemokine production. To determine if JNK is also important in TLR2‐induced corneal inflammation in vivo, we examined JNK1−/− mice and pharmacological inhibitors in a murine model of TLR2‐induced corneal inflammation which is characterized by neutrophil recruitment to the corneal stroma and development of corneal haze. We found that corneal inflammation was significantly impaired in JNK1−/− mice compared with control mice, and in mice treated with the JNK inhibitor compared with vehicle control. Taken together with results from HCE cells, these findings demonstrate that JNK has an essential role in TLR2‐induced corneal inflammation.

Protein Kinase C (PKC) δ Suppresses Keratinocyte Proliferation by Increasing p21Cip1 Level by a KLF4 Transcription Factor-dependent Mechanism

PKCδ increases keratinocyte differentiation and suppresses keratinocyte proliferation and survival. However, the mechanism of proliferation suppression is not well understood. The present studies show that PKCδ overexpression increases p21Cip1 mRNA and protein level and promoter activity and that treatment with dominant-negative PKCδ, PKCδ-siRNA, or rottlerin inhibits promoter activation. Analysis of the p21Cip1 promoter upstream regulatory region reveals three DNA segments that mediate PKCδ-dependent promoter activation. The PKCδ response element most proximal to the transcription start site encodes six GC-rich DNA elements. Mutation of these sites results in a loss of PKCδ-dependent promoter activation. Gel mobility supershift and chromatin immunoprecipitation reveal that these DNA elements bind the Kruppel-like transcription factor KLF4. PKCδ increases KLF4 mRNA and protein level and KLF4 binding to the GC-rich elements in the p21Cip1 proximal promoter. In addition, KLF4-siRNA inhibits PKCδ-dependent p21Cip1 promoter activity. PKCδ increases KLF4 expression leading to enhanced KLF4 interaction with the GC-rich elements in the p21Cip1 promoter to activate transcription.