Decreased prostaglandin E2 production by inflammatory cytokine and lower expression of EP2 receptor result in increased collagen synthesis in keloid fibroblasts

We investigated the metabolism of arachidonic acid in normal skin-derived fibroblasts (NF) as well as in keloid-derived fibroblasts (KF) in response to macrophage migration inhibitory factor (MIF), a pluripotent cytokine. We found that MIF enhanced cyclooxygenase-2 activity in NF more than in KF. Consistent with this finding, prostaglandin E(2) (PGE(2)), an antifibrogenic molecule, was more significantly increased in NF than in KF by MIF treatment. As regarding E prostanoid receptor 2, the level of expression was significantly lower in KF than in NF. On the other hand, Forskolin, a direct activator of adenylcyclase, decreased collagen synthesis in both NF and KF, which indicates that cAMP plays an important role in regulating collagen synthesis. As PGE(2) induces cAMP production, it is conceivable that increased collagen synthesis in KF might be owing to decreased PGE(2) and cAMP production. These findings may aid in the development of a therapeutic strategy for the regulation of collagen synthesis in keloid fibroblasts.     

cis-Urocanic acid enhances prostaglandin E2 release and apoptotic cell death via reactive oxygen species in human keratinocytes

Urocanic acid (UCA) is a major UVR-absorbing skin molecule that undergoes trans to cis photoisomerization in the epidermis following UVR exposure. Murine studies have established that cis-UCA is an important mediator of UVR-induced immune suppression, but little is known about its signaling pathway. We have previously demonstrated that treatment of normal human epidermal keratinocytes with cis-UCA resulted in increased synthesis of prostaglandin E(2) (PGE(2)) and cell death. Here, using immortalized human keratinocytes, we report that cis-UCA but not trans-UCA generates reactive oxygen species (ROS) in a dose-dependent manner and that the natural antioxidant α-tocopherol can reduce this ROS generation, subsequent PGE(2) release, and apoptotic cell death. Western blot analysis revealed that cis-UCA leads to a transient phosphorylation of EGFR as well as downstream mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and p38. Pharmacological inhibition of their activity attenuated PGE(2) release induced by cis-UCA. After transient activation, cis-UCA downregulated EGFR protein expression that corresponded to activation of caspase-3. In addition, pretreatment with α-tocopherol inhibited EGFR downregulation and caspase-3 activation induced by cis-UCA. These results suggest that cis-UCA exerts its effects on human keratinocytes via intracellular ROS generation that modulates EGFR signaling and subsequently induces PGE(2) synthesis and apoptotic cell death.     

High plasminogen activator inhibitor type 2 expression is a hallmark of scleroderma fibroblasts in vitro

Systemic scleroderma is a chronic disease, which leads to fibrosis of the skin and internal organs. Fibroblasts obtained from patients with this disease demonstrate an activated state in culture. We, in this study, report strong, constitutive overexpression of plasminogen activator inhibitor type-2 (PAI-2) in scleroderma fibroblasts and demonstrate that this induction observed at the mRNA and protein level is dependent on serum addition. Induced PAI-2 protein levels were restricted to the non-glycosylated 47-kDa form, which is located intracellularly. Induction was stable for at least 12 passages. No modulation by fibrogenic cytokines--for example, transforming growth factor-beta1 or connective tissue growth factor--or by antagonizing IL-1 receptors was observed. The data indicate that scleroderma fibroblasts are more sensitive to the induction of PAI-2 expression than control fibroblasts by a presently unknown factor in serum.     

Inhibitors of dipeptidyl peptidase IV-like activity mediate antifibrotic effects in normal and keloid-derived skin fibroblasts

Suppression of collagen and matrix synthesis and inhibition of the fibrogenic cytokine transforming growth factor-beta(1) (TGF-beta(1)) is a major therapeutic goal in the treatment of fibrosis and keloids. Inhibitors of dipeptidyl peptidase IV (DP IV)-like activity affect cell growth and cytokine production and are currently under investigation for the treatment of metabolic, autoimmune and inflammatory diseases. We show here that the inhibitors of DP IV-like activity, Lys[Z(NO(2))]-thiazolidide and Lys[Z(NO(2))]-pyrrolidide, suppress proliferation in human skin fibroblasts and keloid-derived skin fibroblasts in vitro. They significantly decrease TGF-beta(1) expression and secretion of procollagen type I C-terminal peptide in supernatants of both cell types. Furthermore, they abrogate the TGF-beta(1)-induced stimulation of collagen synthesis, matrix deposition, and TGF-beta(1) and fibronectin expression. Both inhibitors lead to dephosphorylation of mitogen-activated protein kinases pp38 and pERK1/2, which are activated upon TGF-beta1 stimulation and have been implicated in fibrogenesis. In a mouse model of dermal fibrosis, induced by repetitive intracutaneous injections of TGF-beta(1), the profibrotic effect of TGF-beta(1) detected by dermal thickening, collagen I, and alpha-smooth muscle actin expression, is significantly suppressed in the presence of inhibitors. Inhibition of DP IV-like enzymatic activity may therefore represent a promising therapeutic approach for the treatment of fibrotic skin disorders and keloids.     

Stimulated expression of decorin and the decorin gene in fibroblasts cultured from patients with localized scleroderma

Decorin mRNA levels, the content of decorin and the synthesis of dermatan sulphate in skin fibroblasts from patients with systemic and localized scleroderma were investigated. Approximately a 2.2-fold increase in decorin mRNA levels, was found by Northern blot analysis in localized scleroderma, but no significant changes were found in systemic scleroderma. Decorin, as measured by an immunoblot assay, was increased 2.6-fold in fibroblast cultures from localized scleroderma but not in those from systemic scleroderma. In contrast, the synthesis of dermatan sulphate was similar in both conditions. These results indicate that altered decorin gene expression causes abnormal proteoglycan metabolism in localized scleroderma.     

Comparison of histamine release and prostaglandin E2 production of human basophils in atopic and normal individuals

Summary The influence of arachidonic acid (AA) metabolism upon histamine release (HR) from human basophils after stimulation with anti-IgE was studied in 23 atopic and 11 normal individuals. HR occurred significantly faster in atopics than in normals; the total amount of HR after a 40 min incubation period was not significantly different between the two groups. Indomethacin and acetylsalicylic acid (ASA) increased the quantity of HR significantly both in atopics and normals without influencing the time course. Addition of exogenous PGE2 decreased HR; here atopics were more affected than normals 5 and 10 min after challenge with anti-IgE. Production of PGE2 after stimulation with anti-IgE was very low in both groups (in the range of 30–50 pg/10⁶ cells) and often below detection limit (10–20 pg/ml). Addition of glutathione (GSH), a coenzyme of PGE2-isomerase, increased PGE2 production 2 to 5-fold during stimulation with anti-IgE. These data support the idea that arachidonic acid metabolites play an important role in modulating the releasability of human basophils. It is suggested that the basophils of atopic individuals may release their histamine faster than normals — perhaps on the basis of a more slowly acting endogenous feedback mechanism by PGE2. Both phenomena support the idea of an altered releasability of basophils from atopics compared to normals.     

Positive correlation of vanilloid receptor subtype1 and prostaglandin E2 expression with pain in leiomyomas

Cutaneous leiomyomas are benign smooth muscle tumors that are occasionally painful. The mechanism of pain related to leiomyoma is not fully understood. To investigate the possible involvement of algoneic factors in pain from cutaneous leiomyomas. We present a case of cutaneous leiomyoma with severe, diffused pain in a large area and collected 10 more specimens of cutaneous leiomyoma with or without pain in patient histories. We immunohistochemiacally examined the expression of algoneic factors: serotonin, histamin, Substance P, PGE2, BDKRB2, VR1 and CGRP. We compared the pain area and expression of algoneic factors to reveal possible correlations. We describe here a patient with a cutaneous leiomyoma 1‐cm in diameter, which caused severe pain diffused throughout an area of 20‐cm around the tumor. The pain completely resolved after surgical excision of the leiomyoma. We observed that the leiomyoma cells expressed CGRP, PGE2 and VR1 in this case. We found a positive correlation between VR1 and PGE2 expression in the leiomyoma cells and areas with pain around the tumors among 11 specimens in total. VR1 and PGE2 might be key algogenic substances in painful leiomyoma.     

Possible involvement of enhanced prostaglandin E2 production in the photosensitivity in xeroderma pigmentosum group A model mice

Xeroderma pigmentosum group A (XPA) gene-deficient mice cannot repair UV-induced DNA damage and easily develop skin cancers by UV irradiation. Therefore, XPA-deficient mice are a useful model of human XP and represent a promising tool for photobiologic studies of the disorder. Exposure to ultraviolet (UV) B (280-320 nm) radiation greatly enhanced inflammation and immunosuppression in these mice. To investigate the molecular mechanisms of enhanced UV inflammation and immunosuppression, we determined the amount of prostaglandin (PG) E2, an inflammatory mediator and immunomodulator, and analysed the expression of cyclooxygenase (COX) mRNA in the ear skin of XPA-deficient mice after UV irradiation. In XPA-deficient mice, the amount of PGE2 significantly increased at 48 and 72 h after UVB irradiation to the level that was 8- and 16-fold higher than those in wild-type mice, respectively. The expression level of COX-2 mRNA increased in a time-dependent manner, although COX-1 mRNA was constantly expressed. Treatment with indomethacin, a potent inhibitor of PG biosynthesis, inhibited UV-induced ear swelling, abrogated local immunosuppression, and decreased the amount of PGE2 in the ear skin of XPA-deficient mice. These results indicate that the excess DNA photoproducts remaining in XPA-deficient cells after UV radiation may induce COX-2 expression. The induced production of PGE2 may be involved in the enhanced inflammation and immunosuppression caused by UV radiation in XPA-deficient mice and XP patients.     

Activation of platelet-activating factor receptor in SZ95 sebocytes results in inflammatory cytokine and prostaglandin E2 production

Platelet-activating factor (PAF) is a group of phosphocholines with various biological effects mediated by the PAF receptor (PAF-R). Activation of the epidermal PAF-R induces the expression of inflammatory mediators, including cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). The upregulation of COX-2 expression has been shown to be involved in sebocyte proliferation, sebaceous gland inflammation and carcinogenesis. The present study was designed to investigate whether PAF-R activation could induce the expression of COX-2 and production of PGE(2), as well as secretion of the inflammatory cytokine, interleukin-8 (IL-8), in the immortalized sebaceous gland cell line SZ95. Using calcium mobilization studies, we first confirmed that PAF can signal through PAF-R in SZ95 sebocytes. We then found that the production of IL-8 was induced following treatment with PAF-R agonist, however blocked by a specific PAF-R antagonist. Induction of COX-2 expression and increased PGE(2) production were observed in SZ95 sebocytes after PAF-R activation. Finally, it was demonstrated that the production of PGE(2), induced by PAF-R activation and mediated by COX-2 expression, was blocked following PAF-R antagonism in SZ95 sebocytes. These studies suggest that SZ95 sebocytes express functional PAF-Rs and PAF-Rs are involved in regulating the expression of inflammatory mediators, including COX-2, PGE(2) and IL-8.     

培养硬皮病皮损成纤维细胞Smad3 DNA结合活性的研究

目的：探讨培养硬皮病成纤维细胞中磷酸化转录因子Smad3的表达和转录因子Smad3的DNA结合活性和表达量。方法：4例硬皮病患者和4例正常人对照进入本研究。采用Western Blot检测了磷酸化Smad3在培养硬皮病成纤维细胞全细胞裂解液中的表达;采用电泳迁移率实验（electrophoretic mobility shift assay,EMSA）检测了培养硬皮病成纤维细胞在核蛋白中Smad3的DNA结合活性,并对其进行了密度扫描和定量分析。结果：磷酸化转录因子Smad3的表达在培养硬皮病成纤维细胞全细胞裂解液中呈高表达,所有正常人对照为阴性。在培养硬皮病成纤维细胞核蛋白中,转录因子Smad3的DNA结合活性是显著增高的;经密度扫描,是正常人对照的21倍;培养正常人成纤维细胞核蛋白中转录因子Smad3的DNA结合活性仅略高于本底颜色。结论：磷酸化转录因子Smad3高表达和Smad3的DNA结合活性显著增高,提示转录因子Smad3在硬皮病纤维化的病理发生过程中具有重要作用。     

Inhibition of collagen expression by azelastine hydrochloride in cultured skin fibroblasts from normal individuals and scleroderma patients

The effects of azelastine hydrochloride on cell proliferation and collagen synthesis in cultured human skin fibroblasts were studied. Azelastine inhibited cell proliferation during proliferating cell phases. Azelastine was found to inhibit collagen synthesis without altering cell proliferation during quiescent phases. It did not alter the ratio of type I to III collagen synthesis. Northern blot analysis of collagen chain mRNAs revealed that the levels of α₁ (I), α₁ (III) and α₁ (VI) mRNAs were reduced by azelastine treatment, whereas the level of α₂ (VI), α₃ (VI) mRNAs were not significantly changed. These results suggest that azelastine modulates collagen synthesis at a pretranslational level. Azelastine inhibited collagen synthesis in fibroblasts from scleroderma patients to the same extent as in normal skin fibroblasts. This drug may be useful in the treatment of fibrotic diseases.     

Bidens pilosa suppresses interleukin-1beta-induced cyclooxygenase-2 expression through the inhibition of mitogen activated protein kinases phosphorylation in normal human dermal fibroblasts

Bidens pilosa (BP) Linn. var. radiata is a plant used in traditional folk medicine. It is clinically effective in various diseases; the pathogenesis of most of these involves cyclooxygenase (COX)-2. To investigate the mechanism on which the clinical effectiveness of BP is based, we examined its effects on COX-2 expression and its major product, prostaglandin (PG)E(2), under conditions of inflammation. We induced inflammation in normal human dermal fibroblasts with interleukin (IL)-1beta and examined the effects of BP on COX-2 expression and PGE(2) production using Western blotting and competitive enzyme immunoassay, respectively. The functional involvements of mitogen activated protein kinases (MAPK) ERK1/2, p38, and JNK in COX-2 expression were also examined by Western blotting. IL-1beta-induced COX-2 expression was regulated by MAPK pathways, especially by p38. BP inhibited the phosphorylation of MAPKs, COX-2 expression, and subsequent PGE(2) production. The physiological activities and clinical effectiveness of BP observed under diverse conditions may be partly attributable to its ability to inhibit MAPK, mainly p38, activity, COX-2 expression, and subsequent PGE(2) production.     

Involvement of PPARgamma in oxidative stress-mediated prostaglandin E(2) production in SZ95 human sebaceous gland cells

Peroxisome proliferator-activated receptor gamma (PPARgamma) is thought to play a role in sebaceous gland cell function. We previously demonstrated in human epidermoid carcinoma KB cells that UVB irradiation activates PPARgamma via the generation of multiple oxidized glycerophosphocholine species with PPARgamma ligand activity. UVB-induced cyclooxygenase 2 (COX-2) expression was also shown to be PPARgamma-dependent. We therefore reasoned that PPARgamma activation and PPARgamma-dependent COX-2 expression may occur as a general consequence of oxidative stress. The present studies were designed to examine the effects of the oxidant tert-butylhydroperoxide (TBH) on PPARgamma activation and COX-2 expression in SZ95 sebocytes. We first verified that functional PPARgamma is expressed and activated by UVB irradiation in these cells. We next demonstrated that TBH increased PPARgamma reporter activity in SZ95 sebocytes. Increased COX-2 protein, mRNA expression, and prostaglandin E(2) (PGE(2)) production was observed after TBH or PPARgamma agonist treatment. The ability of PPARgamma agonists and TBH to induce COX-2 expression and PGE(2) production was blocked by pretreatment with the specific PPARgamma antagonist GW9662. Finally, TBH and PPARgamma agonists failed to elicit a PGE(2) response in SZ95 sebocytes stably expressing a dominant-negative PPARgamma. This study illustrates the importance of the PPARgamma system in regulating cellular responses to oxidative stress.     

Immunohistochemical study of cyclooxygenase-2 and p53 expression in skin tumors

Overexpression of cyclooxygenase-2 (COX-2) has been demonstrated in various cancers, including experimentally promoted tumors, gastrointestinal cancers, breast tumors and skin tumors. The mechanism that controls COX-2 expression is not yet clear. Currently, it is reported that COX-2 expression is frequently associated with mutated p53 genes. The goal of this study was to evaluate the expression patterns of COX-2 and p53 in several skin tumors and their correlation. An immunohistochemical method was used to investigate the expression of COX-2 and p53 proteins on formalin-fixed, paraffin-embedded tissue specimens of squamous cell carcinomas (SCC), basal cell carcinomas (BCC), Bowen's disease (BD), actinic keratosis (AK) and porokeratosis. The expression of COX-2 increased in 50% (5/10) of SCC, 80% (8/10) of BCC, 40% (4/10) of BD, 50% (5/10) of AK, and 20% (2/10) of porokeratosis cases. The expression of p53 increased in 90% (9/10) of SCC, 70% (7/10) of BCC, 70% (7/10) of BD, 50% (5/10) of AK, and 40% (4/10) of porokeratosis cases. COX-2 positivity rates of the p53-positive skin tumors were 56%, 100%, 57%, 80% and 25% in SCC, BCC, BD, AK and porokeratosis, respectively. However, the correlation between p53 and COX-2 expression in skin tumors was not statistically significant ( P  > 0.05). Our results indicate that skin COX-2 and p53 may play roles in skin tumors, but that there is no apparent correlation between the two markers.     

Involvement of EGF receptor activation in the induction of cyclooxygenase-2 in HaCaT keratinocytes after UVB

Because selective inhibition of cyclooxygenase-2 (COX-2) suppressed the induction of skin tumors in mice by UV and as UV has been shown to induce expression of COX-2 in skin and cells, COX-2 may be crucial for photocarcinogenesis of the skin. We studied the mechanism of UVB-induced expression of COX-2 focusing on the signal transduction pathway involved. Hydrogen peroxide (H2O2) treatment of HaCaT cells induced expression of COX-2 and pretreatment with the antioxidant N-acetylcysteine (NAC) partly inhibited the UVB-induced expression of COX-2 protein in HaCaT cells, suggesting that oxidative stress contributes to COX-2 induction. To examine the signaling pathways involved in the UVB-induced expression of COX-2 in HaCaT cells, we analysed the expression of COX-2 protein after treatment with various inhibitors of signaling molecules. Inhibition of EGFR by a specific inhibitor and by a neutralizing antibody suppressed the induction of COX-2 expression by UV. Although a neutralizing antibody to transforming growth factor-alpha (TGF-alpha) suppressed COX-2 expression induced by TGF-alpha, it did not suppress COX-2 expression by UV, indicating that a direct activation of EGFR is involved. Treatment of cells at low temperature (4 degrees C) inhibited UVB-induced JNK activation, but it did not inhibit COX-2 expression by UV. Inhibitors of MEK, p38 MAP kinase and PI3-kinase, suppressed the induction of COX-2 expression by UV. In contrast, an erbB-2 inhibitor augmented the UVB-induced increase of COX-2 protein. These data indicate that oxidative stress in association with activation of EGFR, ERK, p38 MAP kinase, and PI3-kinase plays crucial roles in the UVB induction of expression of COX-2.