Qiaofang Yi

UVB-induced Senescence in Human Keratinocytes Requires a Functional Insulin-like Growth Factor-1 Receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21(CDKN1A). Furthermore, IGF-1R-dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R-dependent premature senescence.

Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor

Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor-positive and -negative (PAF-R-positive and -negative) cells and PAF-R-deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca2+ flux only in PAF-R-positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R-deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R-expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.

Augmentation of Chemotherapy-Induced Cytokine Production by Expression of the Platelet-Activating Factor Receptor in a Human Epithelial Carcinoma Cell Line

In addition to their known cytotoxic effects, chemotherapeutic agents can trigger cytokine production in tumor cells. Moreover, many chemotherapeutic agents are potent pro-oxidative stressors. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to oxidative stress, and many epidermal carcinomas express PAF receptors (PAF-R) linked to cytokine production, it is not known whether PAF is involved in chemotherapeutic agent-induced cytokine production. These studies examined the role of the PAF system in chemotherapy-mediated cytokine production using a model system created by retroviral-mediated transduction of the PAF-R-negative human epidermal carcinoma cell line KB with the human PAF-R. The presence of the PAF-R in KB cells resulted in augmentation of the production of cytokines IL-8 and TNF-α induced by the chemotherapeutic agents etoposide and mitomycin C. These effects were specific for the PAF-R, as expression of the G protein-coupled receptor for fMLP did not affect chemotherapeutic agent-induced cytokine production. Moreover, ablation of the native PAF-R in the epithelial cell line HaCaT using an inducible antisense PAF-R strategy inhibited etoposide-induced cytokine production. Oxidative stress and the transcription factor NF-κB were found to be involved in this augmentative effect, because it was mimicked by the oxidant tert-butyl-hydroperoxide, which was blocked both by antioxidants and by inhibition of the NFκB pathway using a super-repressor IκBM mutant. These studies provide evidence for a novel pathway by which the epidermal PAF-R can augment chemotherapy-induced cytokine production through an NF-κB-dependent process.

Ultraviolet B Radiation of Human Skin Generates Platelet-activating Factor Receptor Agonists

Ultraviolet B radiation (UVB) is a potent stimulator of epidermal cytokine production. In addition to cytokines, such as tumor necrosis factor‐alpha (TNF‐α), UVB generates bioactive lipids including platelet‐activating factor (PAF). Our previous in vitro studies in keratinocytes or epithelial cell lines have demonstrated that UVB‐mediated production of PAF agonists is due primarily to the pro‐oxidative effects of this stimulant, resulting in the nonenzymatic production of modified phosphocholines (oxidized glycerophosphocholines). The current studies use human skin to assess whether UVB irradiation generates PAF‐receptor agonists, and the role of oxidative stress in their production. These studies demonstrate that UVB irradiation of human skin results in PAF agonists, which are blocked by the antioxidant vitamin C and the epidermal growth factor receptor inhibitor PD168393. Inasmuch as UVB‐generated PAF agonists have been implicated in animal model systems as being involved in photobiologic processes including systemic immunosuppression and cytokine (TNF‐α) production, these studies indicate that this novel activity could be involved in human disease.

Cigarette Smoke Exposure Inhibits Contact Hypersensitivity via the Generation of Platelet-Activating Factor Agonists

Previous studies have established that pro-oxidative stressors suppress host immunity because of their ability to generate oxidized lipids with platelet-activating factor receptor (PAF-R) agonist activity. Although exposure to the pro-oxidative stressor cigarette smoke (CS) is known to exert immunomodulatory effects, little is known regarding the role of PAF in these events. The current studies sought to determine the role of PAF-R signaling in CS-mediated immunomodulatory effects. We demonstrate that CS exposure induces the generation of a transient PAF-R agonistic activity in the blood of mice. CS exposure inhibits contact hypersensitivity in a PAF-R–dependent manner as PAF-R–deficient mice were resistant to these effects. Blocking PAF-R agonist production either by systemic antioxidants or treatment with serum PAF-acetyl hydrolase enzyme blocked both the CS-mediated generation of PAF-R agonists and PAF-R–dependent inhibition of contact hypersensitivity (CHS) reactions, indicating a role for oxidized glycerophosphocholines with PAF-R agonistic activity in this process. In addition, cyclooxygenase-2 inhibition did not block PAF-R agonist production but prevented CS-induced inhibition of CHS. This suggests that cyclooxygenase-2 acts downstream of the PAF-R in mediating CS-induced systemic immunosuppression. Moreover, CS exposure induced a significant increase in the expression of the regulatory T cell reporter gene in Foxp3EGFP mice but not in Foxp3EGFP mice on a PAF-R–deficient background. Finally, regulatory T cell depletion via anti-CD25 Abs blocked CS-mediated inhibition of CHS, indicating the potential involvement of regulatory T cells in CS-mediated systemic immunosuppression. These studies provide the first evidence, to our knowledge, that the pro-oxidative stressor CS can modulate cutaneous immunity via the generation of PAF-R agonists produced through lipid oxidation.

Identification of Staphylococcal Protein A in Infected Atopic Dermatitis Lesions

Staphylococcus aureus (S. aureus) infection is a known trigger for skin inflammation and can modulate immune responses. Atopic dermatitis (AD), a chronic inflammatory pruritic skin disease, affects 10–20% of children and 1–3% of adults (De Benedetto et al., 2009). Due to the loss of skin integrity by scratching, as well as decreased levels of antimicrobial peptides in comparison to normal skin or other inflammatory diseases such as psoriasis (Leung, 2003; Ong et al., 2002), patients with AD are particularly susceptible to staphylococcal skin infections, which can further worsen their skin disease (Bieber, 2008). Studies have suggested several underlying mechanisms for staphylococcus-mediated inflammation, which include production of inflammatory cytokines following either direct infection of keratinocytes or immune cells by the bacteria, or indirectly by bacterial products (Baker, 2006; Leung, 2003; Sasaki et al., 2003; Travers et al., 2001). We have demonstrated previously that lipoteichoic acid (LTA), a gram-positive bacterial lipoprotein, may be an important component of the ability of S. aureus to exacerbate AD lesions (Travers et al., 2010). In the present study, we report that staphylococcal protein A (SPA) could also contribute to this process.

Involvement of P38 MAP kinase in the augmentation of UVB-mediated apoptosis via the epidermal platelet-activating factor receptor

Platelet-activating factor (PAF) is a group of phosphocholines with various biological effects, which are mediated by the PAF receptor (PAF-R). Our previous studies have demonstrated that ultraviolet B radiation (UVB) is a potent stimulus for PAF production, and that the presence of the PAF-R on epithelial cells results in an augmentation of UVB-induced apoptosis. Inasmuch as PAF-R activation results in numerous signal transduction pathways, the present study was designed to characterize the signal transduction pathway responsible for PAF-R-mediated enhanced UVB-induced cytotoxicity. Using a model system of PAF-R-negative and -positive epithelioid KB cells, we demonstrate that inhibitors of p38 MAP kinase block the augmentation of UVB-mediated apoptosis seen in PAF-R-positive KB cells. In contrast, pharmacological and/or molecular inhibition of other pathways linked to PAF-R activation including ERK MAP kinase and NFκB do not affect PAF-R-mediated cytotoxicity. This study demonstrates the important role that p38 MAP kinase plays in PAF-R-mediated augmentation of UVB cytotoxicity.