Captopril modulates acetylcholinesterase in human keratinocytes

Human keratinocytes synthesize and secrete non-neuronal acetylcholine, which acts as a local cell signaling molecule, regulating functions like proliferation, cell adhesion, motility, desmosomal cell contact, and glandular activity. The keratinocyte acetylcholine axis is composed of the enzymes mediating acetylcholine synthesis (acetyltransferase) and degradation (acetylcholinesterase), and two classes of acetylcholine receptors. In this study we investigated the effect of captopril, an ACE-inhibitor, on acetylcholinesterase and acetylcholine secretion in human keratinocytes. We analyzed the level of acetylcholinesterase in HaCat and NHEK cells by RT-PCR and Western blotting analysis. In addition, the effect of captopril on AChE activity was evaluated. We found that captopril induces a strong AChE up-regulation leading to ACh degradation and reduced secretion. Our results suggest that acantholysis induced by ACE-inhibitors might be linked to altered level of Ach.     

Endogenous survivin modulates survival and proliferation in UVB-treated human keratinocytes

Abstract:  Survivin is a bi-functional member of inhibitor of apoptosis protein family, as it is able to both inhibit apoptosis and to regulate cell cycle. We investigated the role of survivin in human keratinocytes under normal conditions and during UVB irradiation. Survivin siRNA decreases proliferation and induces apoptosis in human keratinocytes, in a mode consistent with the mitotic catastrophe. Low doses UVB increase survivin expression at earlier times, while high doses down-regulate survivin level. Low doses UVB induce cell cycle arrest in G2/M, while high doses UVB cause apoptosis. Moreover, overexpression of survivin protects keratinocytes from UVB-induced apoptosis, and silencing of survivin renders keratinocytes more susceptible to UVB-induced cell death. Finally, survivin siRNA increases UVB-induced reduction of cell proliferation. Taken together, these results indicate that survivin plays a critical role in epidermal homeostasis in normal conditions and during UVB exposure, with possible implication in skin carcinogenesis.     

Regulation of galanin gene expression in human keratinocytes

Morphine produces an exacerbation of DNFB-induced cutaneous inflammation when administered prior to antigen challenge in a rat model of contact hypersensitivity (CHS). Recent evidence indicates that this exacerbation of inflammation is significantly greater in females than that in males. This sex difference in morphine modulation of CHS is due to the activation of central μ-opioid receptor pathways and appears to be corticosterone independent. Furthermore, the presence of female gonadal hormones may account for observed the sex differences, because ovariectomy largely eliminates morphine's enhancement of CHS inflammation without affecting the basal CHS response. Given the involvement of substance P in the pathological exacerbation of CHS, we hypothesized that sex differences in NK1 receptor activation might account for morphine's greater effect on the expression of CHS in females. To that end, male and female CDF rats were treated with the selective NK1 receptor antagonist SR140 333 (1 mg/kg, subcutaneously) 150 min prior to and after DNFB challenge. SR140 333 treatment significantly reduced the magnitude of morphine's enhancement of CHS in female, but not male rats without affecting the baseline CHS response in either sex. Preliminary data suggest that neuropeptide depletion of the skin using capsaicin produces a similar pattern of sex-dependent effects on morphine modulation of CHS. Taken together, these experiments support the hypothesis that greater activity of the peripheral NK1 receptor system in females may in part account for sex differences in the magnitude of CHS following morphine treatment.
This project was supported by NIH grants DA15709 (D.T.L.) and DA016836 (J.C.E). SR140 333 was generously provided by Sanofi-Synthelabo.     

CD24 expression on human keratinocytes

Abstract: CD24 or Nectadrin is a cell surface glycoprotein expressed in pre-B lymphocytes, T lymphocytes, neurons, muscle cells and carcinoma cells. Its function is not completely known, but it has been suggested that it is involved in cell adhesion and signalling. CD24 has recently been identified as the human molecule homologous to the murine heat-stable antigen (HSA). HSA is expressed by murine keratinocytes and delivers costimulatory signals in T-cell activation. Long-term cultures of normal human keratinocytes (HKC) were obtained from skin of human female breast sections and either left untreated or were treated with phorbol-12-myristate-13-acetate (PMA) at 10–100 ng/ml, calcium 0.5–2 mM or IFN-γ 100–1000 U/ml, for 24–48 h. Using RT-PCR and flow cytometry we showed that HKC express low levels of CD24 even under basal conditions, and the treatment with calcium, PMA or IFN-γ increased levels of CD24 mRNA and protein. To the best of our knowledge, this is the first report to measure CD24 expression and production by cultured HKC in basal conditions and after stimulation. Further studies are needed to determine biological and therapeutical relevance of these findings.     

Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro

Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.     

Differentiation-regulated expression of Toll-like receptors 2 and 4 in HaCaT keratinocytes

Toll-like receptors (TLRs) play an important role in the recognition of pathogens in keratinocytes. In this study, we investigated whether the differentiation state of HaCaT keratinocytes correlates with the expression of TLR2 and TLR4 genes. The expression levels of TLR2 and TLR4 in a HaCaT differentiation model system were determined using quantitative real-time RT-PCR (Q-RT-PCR) and flow cytometry. The progression of keratinocyte differentiation was monitored by determining the level of involucrin gene expression using Q-RT-PCR. The expression levels of TLR2 and TLR4 increased with the stage of differentiation and there were strong correlations between the expression level of the involucrin gene and those of the TLR2 gene (r=0.809, P<0.0001) and the TLR4 gene (r=0.568, P<0.02). Increased cell surface expression of TLR2 and TLR4 was also found in differentiated HaCaT keratinocytes by flow cytometric analysis. Our findings suggest that upregulation of TLR expression during differentiation in keratinocytes could be a part of the differentiation process of keratinocytes and could have biological significance in protecting skin against microbes.     

Role of specific membrane receptors in urokinase-dependent migration of human keratinocytes

On the basis of both 125I-labeled plasminogen activator binding analysis and transmission electron microscopy studies of the interaction of a plasminogen activator/gold complex with cell membranes, we have found that human keratinocytes have specific receptors for human urokinase-type plasminogen activator distributed on the cell surface as singlets, or as small or large clusters. The use in binding experiments of the purified A chain of urokinase-plasminogen activator and of anti-A chain monoclonal antibodies has indicated that cell receptors are specific for a sequence present on the A chain, as previously reported for other cells. The interaction of both the native molecule and the purified A chain with such receptors stimulates mobilization of keratinocytes in an in vitro cell model system (Boyden chamber), when present in the lower compartment of the migration apparatus in nanomolar concentrations. Preincubation of chemoattractants with a monoclonal antibody which prevents receptor/ligand interaction also prevents plasminogen activator-induced cell migration. These data suggest that, under the conditions used in this in vitro model system, the plasminogen activator-dependent mobilization of keratinocytes depends on the interaction of the ligand with free receptors on the cell surface, and is independent of plasmin generation.     

Distinct strains of Propionibacterium acnes induce selective human beta-defensin-2 and interleukin-8 expression in human keratinocytes through toll-like receptors

Acne is a chronic inflammatory disease of the pilosebaceous follicle. One of the main pathogenetic factors in acne is the increased proliferation of Propionibacterium acnes (P. acnes) in the pilosebaceous unit. We investigated whether direct interaction of P. acnes with keratinocytes might be involved in the inflammation and ductal hypercornification in acne. The capacities of different P. acnes strains to activate the innate immune response and the growth of cultured keratinocytes were investigated. We have found that two clinical isolates of P. acnes significantly induced human beta-defensin-2 (hBD2) messenger RNA (mRNA) expression; in contrast a third clinical isolate and the reference strain (ATCC11828) had no effect on hBD2 mRNA expression. In contrast, all four strains significantly induced the interleukin-8 (IL-8) mRNA expression. The P. acnes-induced increase in hBD2 and IL-8 gene expression could be inhibited by anti-Toll-like receptor 2 (TLR2) and anti-TLR4 neutralizing antibodies, suggesting that P. acnes-induced secretion of soluble factors in keratinocytes are both TLR2 and TLR4 dependent. In addition, the clinical isolate P. acnes (889) was capable of inducing keratinocyte cell growth in vitro. Our findings suggest that P. acnes modulates the antimicrobial peptide and chemokine expression of keratinocytes and thereby contributes to the recruitment of inflammatory cells to the sites of infections.     

白细胞介素22对人表皮角质形成细胞CC趋化因子配体27表达的影响

目的 研究白细胞介素22（IL-22）对人表皮角质形成细胞CC趋化因子配体27（CCL27）表达的影响及机制。方法 免疫组化法检测10例银屑病患者皮损及5例健康对照组皮肤中CCL27的表达。培养HaCaT细胞,分为8组：对照组用PBS处理,IL-22处理组分别用12.5、25、50、100、200 μg/L IL-22处理,信号通路阻断组先分别加入50 μmol/L AG490（JAK2/STAT3通路抑制剂）或PD98059（MAPK-ERK1/2通路抑制剂）阻断处理,2 h后各加入50 μg/L IL-22,继续于37 ℃、5% CO2孵箱中培养。孵育24 h 后,分别提取 HaCaT 细胞总蛋白并收集上清液,Western印迹、ELISA法分别检测CCL27表达。结果 免疫组化检测示,银屑病患者皮损中CCL27的表达水平明显高于正常人皮肤。Western 印迹显示,HaCaT细胞分别用12.5、25、50、100、200 μg/L IL-22处理后,CCL27相对表达量逐渐升高至0.491 ± 0.013、0.620 ± 0.019、0.623 ± 0.014、0.802 ± 0.052、1.138 ± 0.013,均较对照组（0.413 ± 0.013）升高（P＜0.01）;IL-22 + AG490组及IL-22 + PD98059组CCL27表达量分别为0.411 ± 0.019和0.280 ± 0.012,均低于50 μg/L IL-22组（均P＜0.01）。ELISA法检测显示,各组HaCaT细胞CCL27分泌水平变化趋势与Western印迹法检测结果一致。结论 IL-22可促进HaCaT 细胞CCL27表达,其机制可能与MAPK-ERK1/2和JAK2/STAT3通路有关。