Proteins In Human Keratinocytes Research Articles

Role of the NLRP1 inflammasome in skin cancer and inflammatory skin diseases.

Inflammasomes are cytoplasmic protein complexes that play a crucial role in protecting the host against pathogenic and sterile stressors by initiating inflammation. Upon activation, these complexes directly regulate the proteolytic processing and activation of proinflammatory cytokines interleukin (IL)-1β and IL-18 to induce a potent inflammatory response, and induce a programmed form of cell death called pyroptosis to expose intracellular pathogens to the surveillance of the immune system, thus perpetuating inflammation. There are various types of inflammasome complexes, with the NLRP1 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-1) inflammasome being the first one identified and currently recognized as the predominant inflammasome sensor protein in human keratinocytes. Human NLRP1 exhibits a unique domain structure, containing both an N-terminal pyrin (PYD) domain and an effector C-terminal caspase recruitment domain (CARD). It can be activated by diverse stimuli, such as viruses, ultraviolet B radiation and ribotoxic stress responses. Specific mutations in NLRP1 or related genes have been associated with rare monogenic skin disorders, such as multiple self-healing palmoplantar carcinoma; familial keratosis lichenoides chronica; autoinflammation with arthritis and dyskeratosis; and dipeptidyl peptidase 9 deficiency. Recent research breakthroughs have also highlighted the involvement of dysfunctions in the NLRP1 pathway in a handful of seemingly unrelated dermatological conditions. These range from monogenic autoinflammatory diseases to polygenic autoimmune diseases such as vitiligo, psoriasis, atopic dermatitis and skin cancer, including squamous cell carcinoma, melanoma and Kaposi sarcoma. Additionally, emerging evidence implicates NLRP1 in systemic lupus erythematosus, pemphigus vulgaris, Addison disease, Papillon-Lefèvre syndrome and leprosy. The aim of this review is to shed light on the implications of pathological dysregulation of the NLRP1 inflammasome in skin diseases and investigate the potential rationale for targeting this pathway as a future therapeutic approach.

PDE4 inhibition by difamilast regulates filaggrin and loricrin expression via keratinocyte proline-rich protein in human keratinocytes

BackgroundDifamilast, a topical phosphodiesterase 4 (PDE4) inhibitor, has been shown to be effective for treating atopic dermatitis (AD), but the molecular mechanism involved is unclear. Since skin barrier dysfunction including reduced expression of filaggrin (FLG) and loricrin (LOR) contributes to AD development, difamilast treatment may be able to improve this dysfunction. PDE4 inhibition increases transcriptional activity of cAMP-responsive element binding protein (CREB). Therefore, we hypothesized that difamilast may affect FLG and LOR expression via CREB in human keratinocytes. ObjectiveTo elucidate the mechanism by which difamilast regulates FLG and LOR expression via CREB in human keratinocytes. MethodsWe analyzed normal human epidermal keratinocytes (NHEKs) treated with difamilast. ResultsWe observed increases of intracellular cAMP levels and CREB phosphorylation in difamilast (5 μM)-treated NHEKs. Next, we found that difamilast treatment increased mRNA and protein levels of FLG and LOR in NHEKs. Since reduced expression of keratinocyte proline-rich protein (KPRP) is reported to be involved in skin barrier dysfunction in AD, we examined KPRP expression in difamilast-treated NHEKs. We found that difamilast treatment increased mRNA and protein levels of KPRP in NHEKs. Furthermore, KPRP knockdown using siRNA transfection abolished the upregulation of FLG and LOR in difamilast-treated NHEKs. Finally, CREB knockdown canceled the upregulation of FLG, LOR, and KPRP in difamilast-treated NHEKs, indicating that PDE4 inhibition by difamilast treatment positively regulates FLG and LOR expression via the CREB-KPRP axis in NHEKs. ConclusionThese findings may provide further guidance for therapeutic strategies in the treatment of AD using difamilast.

The Transcriptional Regulation of Genes Involved in the Immune Innate Response of Keratinocytes Co-Cultured with Trichophyton rubrum Reveals Important Roles of Cytokine GM-CSF.

Trichophyton rubrum is the most causative agent of dermatophytosis worldwide. The keratinocytes are the first line of defense during infection, triggering immunomodulatory responses. Previous dual RNA-seq data showed the upregulation of several human genes involved in immune response and epithelial barrier integrity during the co-culture of HaCat cells with T. rubrum. This work evaluates the transcriptional response of this set of genes during the co-culture of HaCat with different stages of T. rubrum conidia development and viability. Our results show that the developmental stage of fungal conidia and their viability interfere with the transcriptional regulation of innate immunity genes. The CSF2 gene encoding the cytokine GM-CSF is the most overexpressed, and we report for the first time that CSF2 expression is contact and conidial-viability-dependent during infection. In contrast, CSF2 transcripts and GM-CSF secretion levels were observed when HaCat cells were challenged with bacterial LPS. Furthermore, the secretion of proinflammatory cytokines was dependent on the conidia developmental stage. Thus, we suggest that the viability and developmental stage of fungal conidia interfere with the transcriptional patterns of genes encoding immunomodulatory proteins in human keratinocytes with regard to important roles of GM-CSF during infection.

Matriptase Cleaves EpCAM and TROP2 in Keratinocytes, Destabilizing Both Proteins and Associated Claudins.

The homologs EpCAM and TROP2, which both interact with claudin-1 and claudin-7, are frequently coexpressed in epithelia including skin. Intestine uniquely expresses high levels of EpCAM but not TROP2. We previously identified EpCAM as a substrate of the membrane-anchored protease matriptase and linked HAI-2, matriptase, EpCAM and claudin-7 in a pathway that is pivotal for intestinal epithelial cells (IEC) homeostasis. Herein, we reveal that TROP2 is also a matriptase substrate. Matriptase cleaved TROP2 when purified recombinant proteins were mixed in vitro. TROP2, like EpCAM, was also cleaved after co-transfection of matriptase in 293T cells. Neither EpCAM nor TROP2 cleavage was promoted by protease-disabled matriptase or matriptase that harbored the ichthyosis-associated G827R mutation. We confirmed that EpCAM and TROP2 are both expressed in skin and detected cleavage of these proteins in human keratinocytes (HaCaT cells) after the physiologic inhibition of matriptase by HAI proteins was relieved by siRNA knockdown. Knockdown of EpCAM or TROP2 individually had only small effects on claudin-1 and claudin-7 levels, whereas elimination of both markedly diminished claudin levels. HAI-1 knockdown promoted EpCAM and TROP2 cleavage accompanied by reductions in claudins, whereas HAI-2 knockdown had little impact. Double knockdown of HAI-1 and HAI-2 induced nearly complete cleavage of EpCAM and TROP2 and drastic reductions of claudins. These effects were eliminated by concurrent matriptase knockdown. Decreases in claudin levels were also diminished by the lysosomal inhibitor chloroquine and cleaved EpCAM/TROP2 fragments accumulated preferentially. We demonstrate that TROP2 and EpCAM exhibit redundancies with regard to regulation of claudin metabolism and that an HAI, matriptase, EpCAM and claudin pathway analogous to what we described in IECs exists in keratinocytes. This study may offer insights into the mechanistic basis for matriptase dysregulation-induced ichthyosis.

Interleukin-38 interacts with destrin/actin-depolymerizing factor in human keratinocytes.

Interleukin (IL)-38 is a member of the IL-1 family of cytokines, which was proposed to exert anti-inflammatory effects. IL-38 is constitutively expressed in the skin, where keratinocytes are the main producing cells. Little information is currently available concerning IL-38 biology. Here, we investigated the subcellular localization and interaction partners of the IL-38 protein in human keratinocytes. IL-38 expression was reduced in primary keratinocytes grown in monolayer (2D) cultures. We thus used IL-38 overexpressing immortalized normal human keratinocytes (NHK/38) to study this cytokine in cell monolayers. In parallel, differentiation of primary human keratinocytes in an in vitro reconstructed human epidermis (RHE) 3D model allowed us to restore endogenous IL-38 expression. In NHK/38 cells and in RHE, IL-38 was mainly cell-associated, rather than released into culture supernatants. Intracellular IL-38 was preferentially, although not exclusively, cytoplasmic. Similarly, in normal human skin sections, IL-38 was predominantly cytoplasmic in the epidermis and essentially excluded from keratinocyte nuclei. A yeast two-hybrid screen identified destrin/actin-depolymerizing factor (DSTN) as a potential IL-38-interacting molecule. Co-immunoprecipitation and proximity ligation assay confirmed this interaction. We further observed partial co-localization of IL-38 and DSTN in NHK/38 cells. Endogenous IL-38 and DSTN were also co-expressed in all epidermal layers in RHE and in normal human skin. Finally, IL-38 partially co-localized with F-actin in NHK/38 cells, in particular along the cortical actin network and in filopodia. In conclusion, IL-38 is found predominantly in the cytoplasm of human keratinocytes, where it interacts with DSTN. The functional relevance of this interaction remains to be investigated.

Sunscreen-induced expression and identification of photosensitive marker proteins in human keratinocytes under UV radiation.

Solar ultraviolet (UV) radiation is the main factor of photocarcinogenesis, photoaging, and photosensitivity; thus protection from biological damaging UV radiation is a concern. Sunscreens containing UV filters are the most preferred means of photoprotection but the safety and efficacy of UV filters are in question. Benzophenone (BP) and its derivatives, namely, benzophenone 1 (BP1), is commonly used in sunscreens as a UV blocker. The aim of this study was to assess the effects of BP and BP1 on the differential expression of proteins in human keratinocytes (HaCaT cells) under exposure to ultraviolet A radiation. Photosensitive proteins were screened from HaCaT cells by two-dimensional (2-D) gel electrophoresis, and identification of these differentially expressed proteins was performed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/TOF mass spectrometry. Protein identification was performed using the search program MASCOT and a database made of SUMO and GhJMJ12 amino acid sequences. Our results showed that the proteins involved directly or indirectly in apoptosis are 70 kDa heat shock protein, long-chain specific acyl-CoA dehydrogenase, serine/threonine-protein kinase, and FAM78A protein, which were upregulated in comparison to control HaCaT cells. The expressions of binding immunoglobulin protein, podocalyxin-like protein, actin, cytoplasmic, and calreticulin precursors were downregulated. The altered protein expression indicated that cell growth arrest and apoptosis were potential mechanisms of cytotoxicity and genotoxicity of BPs. The results of 2-D gel electrophoresis followed by mass spectrometry showed expression of novel proteins involved in promoting or initiating apoptotic pathways. Hence, we conclude that BPs should be avoided as a UV blocker from sunscreens because of its potential to promote apoptotic proteins in human skin keratinocytes.

Effect of S100A8 and S100A9 on expressions of cytokine and skin barrier protein in human keratinocytes.

Atopic dermatitis (AD) is an inflammatory skin disorder caused by immunological dysregulation and genetic factors. Whether the expression levels of cytokine and skin barrier protein were altered by S100 calcium binding protein A8 (S100A8) and S100A9 in human keratinocytic HaCaT cells was examined in the present study. Alterations of cytokine expression were examined by ELISA following treatment with S100A8/9 and various signal protein‑specific inhibitors. Activation of the mitogen activated protein kinase (MAPK) pathway and nuclear factor (NF)‑κB was evaluated by using western blotting and an NF‑κB activity test, respectively. The expression levels of interleukin (IL)‑6, IL‑8 and monocyte chemoattractant protein‑1 increased following treatment with S100A8 and S100A9, and the increase was significantly blocked by specific signaling pathway inhibitors, including toll‑like receptor 4 inhibitor (TLR4i), rottlerin, PD98059, SB203580 and BAY‑11‑7085. Extracellular signal‑regulated kinase (ERK) and p38 MAPK pathways were activated in a time‑dependent manner following treatment with S100A8 and S100A9. Phosphorylation of ERK and p38 MAPK were blocked by TLR4i and rottlerin. S100A8 and S100A9 induced translocation of NF‑κB in a time‑dependent manner, and the activation of NF‑κB was inhibited by TLR4i, rottlerin, PD98059 and SB203580. In addition, S100A8 and S100A9 decreased the expression of skin barrier proteins, filaggrin and loricrin. These results may help to elucidate the pathogenic mechanisms of AD and develop clinical strategies for controlling AD.

Human Papillomavirus 16 E7 Stabilizes APOBEC3A Protein by Inhibiting Cullin 2-Dependent Protein Degradation.

ABSTRACTAPOBEC3 (A3) mutation signatures have been observed in a variety of human cancer genomes, including those of cervical and head and neck cancers caused by human papillomavirus (HPV) infection. However, the driving forces that promote off-target A3 activity remain mostly unclear. Here, we report a mechanism for the dramatic increase of A3A protein levels in HPV-positive keratinocytes. We show that expression of the viral protein E7 from high-risk HPVs, but not E7 from low-risk HPVs, significantly prolongs the cellular half-life of A3A protein in human keratinocytes and HPV-positive cancer cell lines. We have mapped several residues within the cullin 2 (CUL2) binding motif of HPV16 E7 as being important for mediating A3A protein stabilization. Furthermore, we provide direct evidence that both A3A and HPV16 E7 interact with CUL2, suggesting that the E7-CUL2 complex formed during HPV infection may regulate A3A protein levels in the cell. Using an in vitro cytidine deaminase assay, we show that E7-stabilized A3A remains catalytically active. Taken together, our findings suggest that the HPV oncoprotein E7 dysregulates endogenous A3A protein levels and thus provides novel mechanistic insight into cellular triggers of A3 mutations in HPV-positive cancers.IMPORTANCE Human papillomavirus (HPV) is causally associated with over 5% of all human malignancies. Several recent studies have shown that a subset of cancers, including HPV-positive head and neck and cervical cancers, have distinct mutational signatures potentially caused by members of the APOBEC3 cytidine deaminase family. However, the mechanism that induces APOBEC3 activity in cancer cells is poorly understood. Here, we report that the HPV oncoprotein E7 stabilizes the APOBEC3A (A3A) protein in human keratinocytes by inhibiting ubiquitin-dependent protein degradation in a cullin-dependent manner. Interestingly, the HPV E7-stabilized A3A protein maintains its deaminase activity. These findings provide a new insight into cancer mutagenesis enhanced by virus-induced A3A protein stabilization.

The antimicrobial protein S100A7/psoriasin enhances the expression of keratinocyte differentiation markers and strengthens the skin's tight junction barrier.

S100A7/psoriasin is a member of the S100 protein family and is encoded in the epidermal differentiation complex, which contains genes for markers of epidermal differentiation. S100A7/psoriasin is overexpressed in hyperproliferative skin diseases, where it is believed not only to exhibit antimicrobial functions, but also to induce immunomodulatory activities, including chemotaxis and cytokine/chemokine production. To evaluate the effect of S100A7/psoriasin on keratinocyte differentiation and regulation of the tight junction (TJ) barrier. Expression of differentiation markers and TJ proteins in human keratinocytes was determined by real-time polymerase chain reaction and Western blot. The changes in TJ barrier function were assessed by transepithelial electrical resistance and paracellular permeability assays. Glycogen synthase kinase-3 (GSK-3) and mitogen-activated protein kinase (MAPK) activation was analysed by Western blot, whereas β-catenin and E-cadherin activation was evaluated by Western blot and immunofluorescence. S100A7/psoriasin enhanced the expression of several differentiation markers and selectively increased the expression of TJ proteins (e.g. claudins and occludin), which are known to strengthen the TJ barrier. Furthermore, S100A7/psoriasin increased β-catenin and E-cadherin accumulation at cell-cell contact, and enhanced transepithelial electrical resistance while reducing the paracellular permeability of keratinocyte layers. The data suggest that S100A7/psoriasin-mediated regulation of the TJ barrier was via both the GSK-3 and MAPK pathways, as evidenced by the inhibitory effects of inhibitors for GSK-3 and MAPKs. Our finding that S100A7/psoriasin regulates differentiation and strengthens TJ barrier function provides novel evidence that, in addition to antimicrobial and immunoregulatory activities, S100A7/psoriasin is involved in skin innate immunity.

Effect of hyaluronan tetrasaccharides on epidermal differentiation in normal human epidermal keratinocytes

Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation. In addition, HA has been shown to have different biological activities depending on its molecular weight. It has been reported that HA-mediated CD44 activation regulates keratinocyte differentiation. Therefore, the aim of this study was to investigate the influence of HA tetrasaccharides (HA4) on the regulation of keratinocyte differentiation, CD44 gene expression and CD44-phosphorylated protein in human keratinocytes, and compare HA4 with high molecular weight HA. Normal human epidermal keratinocytes (NHEKs) were treated at doses of 1μgmL(-1) HA or HA oligosaccharides (HA4). After treatment, cell viability was checked using an MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each differentiation marker and CD44 mRNA expression was detected by real-time PCR. Each differentiation marker and CD44-phosphorylated protein was assessed by Western blotting. Hyaluronan and HA4 showed no cytotoxicity up to a dose of 1μgmL(-1) . On day 3 after HA4 treatment, each differentiation marker mRNA and K10 protein level was higher than that of the control. On day 9, late differentiation marker mRNA and protein levels were increased with HA and HA4 treatment. In addition, HA4 treatment increased the expression of CD44 mRNA, CD44-phosphorylated protein and intracellular calcium concentrations. HA4 enhanced keratinocyte differentiation and increased CD44-phosphorylated protein levels. HA4 may induce epidermal differentiation through phosphorylation of CD44.

Expression of HPV16 E5 down-modulates the TGFbeta signaling pathway

BackgroundInfection with high-risk human papillomavirus (HR-HPV) genotypes, mainly HPV16 and HPV18, is a major risk factor for cervical cancer and responsible for its progression. While the transforming role of the HPV E6 and E7 proteins is more characterized, the molecular mechanisms of the oncogenic activity of the E5 product are still only partially understood, but appear to involve deregulation of growth factor receptor expression. Since the signaling of the transforming growth factor beta (TGFbeta) is known to play crucial roles in the epithelial carcinogenesis, aim of this study was to investigate if HPV16 E5 would modulate the TGF-BRII expression and TGFbeta/Smad signaling.FindingsThe HPV16 E5 mRNA expression pattern was variable in low-grade squamous intraepithelial lesions (LSIL), while homogeneously reduced in high-grade lesions (HSIL). Parallel analysis of TGFBRII mRNA showed that the receptor transcript levels were also variable in LSILs and inversely related to those of the viral protein. In vitro quantitation of the TGFBRII mRNA and protein in human keratinocytes expressing 16E5 in a dose-dependent and time-dependent manner showed a progressive down-modulation of the receptor. Phosphorylation of Smad2 and nuclear translocation of Smad4 were also decreased in E5-expressing cells stimulated with TGFbeta1.ConclusionsTaken together our results indicate that HPV16 E5 expression is able to attenuate the TGFbeta1/Smad signaling and propose that this loss of signal transduction, leading to destabilization of the epithelial homeostasis at very early stages of viral infection, may represent a crucial mechanism of promotion of the HPV-mediated cervical carcinogenesis.

Dermal fibroblasts influence the expression profile of 14-3-3 proteins in human keratinocytes

We have previously demonstrated that the release of some of the 14-3-3 isoforms from keratinocytes is able to influence the expression of key matrix metalloproteinases (MMPs) in dermal fibroblasts. Conversely, in this study we aimed to investigate whether dermal fibroblasts possess the ability to modulate the expression of 14-3-3 proteins in keratinocytes. In order to address this question, human keratinocytes and dermal fibroblasts were harvested and co-cultured. Intra- and extracellular levels of 14-3-3 proteins (β, η, γ, and σ) were analyzed using western blot analysis, and the gene expression was further assessed by quantitative real-time polymerase chain reaction. Gene analysis revealed an up-regulation of all four 14-3-3 isoforms of interest. In addition, the findings of this study reveal a significant increase in the intracellular levels of 14-3-3 γ and σ in keratinocytes co-cultured with fibroblasts compared to those of the mono-cultured control keratinocytes. Mechanistic investigations also demonstrated the capacity of several mitogen-activated protein kinase-specific inhibitors to markedly reduce induction of 14-3-3 σ in keratinocytes stimulated with fibroblast-conditioned medium. The study concluded that dermal fibroblasts possess the ability to influence the expression of several 14-3-3 isoforms (notably γ and σ) in keratinocytes, suggesting that the two cell types might be capable of bi-directionally influencing the protein expression of one another in vivo.

Functional characterization of T cells differentiated in vitro from bone marrow‐derived CD34+ cells of psoriatic patients with family history

The strong but complex genetic background suggests that inherent and intrinsic rather than exogenous factors have a key role in immunopathogenesis of psoriasis. It is reasonable to speculate that the dysfunctional activity of psoriatic T cells may partly originate from the abnormal haematopoietic cells. To test if T cells originated from haematopoietic progenitor cells in psoriasis patients display functional alternations similar to previously reported abnormalities of circulating T cells. Bone marrow CD34(+) haematopoietic cells were isolated from psoriatic patients with family history and healthy subjects, and differentiated into T cells in vitro in the thymic stromal co-culture system. These cells were further subjected to functional comparisons such as in vitro proliferation, secretion of cytokines such as IL-4, IL-8 and IFN-gamma, and inducing the production of C-myc, Bcl-xL, and Ki67 proteins in human keratinocytes. While bone marrow-derived CD34(+) cells from both patients and healthy volunteers developed into mature T cells within weeks in the thymic environment in vitro, the differentiated T cells from psoriatic patients showed higher proliferation and stronger capacity to secret TH1 cytokines in response to streptococcal superantigen. The differentiated T cells from psoriatic patients, but not from normal controls, induced overexpression of C-myc and Ki67, but not Bcl-XL, in keratinocytes. T cells differentiated from CD34(+) cells of psoriatic patients, but not normal controls, are functionally similar to psoriatic circulating T cells, suggesting that the dysfunctional activity of T cells in psoriatic patients can be traced back to the early development of haematopoietic cells.

Protein kinase C δ and η differently regulate the expression of loricrin and Jun family proteins in human keratinocytes

Barrier function of the epidermis is maintained by precise expression of keratinocyte-specific structural proteins to form the cornified cell envelope (CE). Loricrin, a major component of the CE, is expressed at the late stage of keratinocyte differentiation. In this study, we reveal the isoform-specific function of protein kinase C (PKC) in the regulation of loricrin expression. Both PKCδ and PKCη have been recognized as differentiation-promoting isoforms. However, loricrin expression was inversely controlled by PKCδ and PKCη in cultured keratinocytes and 3D skin culture; i.e. loricrin expression was decreased by PKCδ and increased by PKCη. To clarify the mechanisms that PKCδ and PKCη oppositely regulate the loricrin expression, we examined the expression of activator protein-1 (AP-1) family proteins, which modulate the transcription of loricrin and are downstream molecules of PKC. PKCδ decreased c-Jun expression, whereas PKCη increased JunD, which are positive regulators of loricrin transcription. These findings suggest that inverse effects of PKCδ and PKCη on loricrin expression attributes to the expression of c-Jun and JunD.

The Th17 cytokine IL‐22 induces IL‐20 production in keratinocytes: A novel immunological cascade with potential relevance in psoriasis

Psoriasis is a common chronic skin disease. Recent studies demonstrated that IL-20 and IL-22, cytokines produced by keratinocytes and T cells, respectively, both inhibit keratinocyte terminal differentiation and induce psoriasis-like epidermis alterations. Here, we investigated the relationship between these mediators. Although IL-20 was not able to regulate IL-22 production, IL-22 induced IL-20 mRNA and protein in human keratinocytes. However, IL-22 had only a minimal effect, if any, on IL-19 and IL-26. Cutaneous IL-20 was also elevated in mice following IL-22 application. Accordingly, some of IL-22's effects on differentiation-regulating genes were partially mediated by an endogenous, secreted protein and attenuated by anti-IL-20 Ab. Like IL-22, IL-17A and TNF-alpha induced IL-20 in keratinocytes, whereas IFN-gamma and IL-20 itself did not. Furthermore, IL-17A and TNF-alpha individually strengthened the IL-22-induced IL-20 production. In lesional skin of psoriasis patients, highly elevated IL-20 levels strongly correlated with IL-22, and to a lesser extent, with IL-17A and TNF-alpha. As previously shown for IL-22, IL-20 blood levels correlated with the disease severity, although with a lower significance. This study demonstrates that a T-cell mediator induces a tissue cell mediator with similar effects to its own and therefore suggests the existence of a novel type of pathogenetic cascade.

The role of water channel aquaporin 3 in the mechanism of TNF‐α‐mediated proinflammatory events: Implication in periodontal inflammation

Aquaporin 3 (AQP3) is the predominant water channel protein in human keratinocytes and acts as an inflammatory mediator in some lesions. A chronic, inflammatory process of periodontitis is related with a dramatic change of surrounding fluid homeostasis to plasma extravasation. The exact pattern of aquaporin (AQP) water channel expression and its mechanism in periodontal disease is still unknown. We describe herein an up-regulated AQP3 expression in the epithelial lesion with chronic periodontitis and its functional role. The levels of AQP3 expression in inflamed gingival epithelial tissues were significantly higher than those of healthy subjects. Consistent with these results, AQP3 expression (i.e., levels of mRNA and protein) in cultured rat primary gingival epithelial cells and the human gingival epithelial cell line Ca9-22 were strongly increased in response to TNF-alpha treatment through the 55 kDa TNF-alpha receptor (TNFR I). In this context, small interfering RNA- (siRNA)-mediated "aqp-3 gene silencing," which could reduce AQP3 expression by more than 65%, significantly attenuated selected proinflammatory events of ICAM-1 expression induced by TNF-alpha in Ca9-22. A sixfold increase in leukocyte adherence to TNF-alpha-stimulated epithelial cells was demonstrated by an adherence assay (P < 0.001) and pretreatment with AQP3 siRNA and anti-ICAM-1 antibody reduced leukocyte retention by 85% (P < 0.001). Our study indicates for the first time a novel important mode in the regulation of the inflammatory response through TNF-alpha/TNFR I ligation at the site of epithelial lesions by specialized membrane channel AQP3 and ICAM-1 protein, which is closely implicated in the development of periodontitis mechanisms.

Epidermal Peroxisome Proliferator-activated Receptor γ as a Target for Ultraviolet B Radiation

Ultraviolet B radiation (UVB) is a pro-oxidative stressor with profound effects on skin in part through its ability to stimulate cytokine production. Peroxisome proliferator-activated receptor gamma (PPAR gamma) has been shown to regulate inflammatory processes and cytokine release in various cell types. Since the oxidized glycerophospholipid 1-hexadecyl-2-azelaoyl glycerophosphocholine (azPC) has been shown to be a potent PPAR gamma agonist, this study was designed to assess whether the PPAR gamma system is a target for UVB irradiation and involved in UVB-induced inflammation in epidermal cells. The present studies demonstrated the presence of PPAR gamma mRNA and functional protein in human keratinocytes and epithelial cell lines HaCaT, KB, and A431. The treatment of epidermal cells with the PPAR gamma-specific agonist ciglitazone or azPC augmented cyclooxygenase-2 expression and enzyme activity induced by phorbol 12-myristate-13-acetate or interleukin-1 beta. Lipid extracts from the cell homogenate of UVB-irradiated, but not control, cells contained a PPAR gamma-agonistic activity identified by reporter assay, and this activity up-regulated cyclooxygenase-2 expression induced by phorbol 12-myristate-13-acetate. Subjecting purified 1-hexadecyl-2-arachidonoyl-glycerophosphocholine to UVB irradiation generated a PPAR gamma-agonistic activity, among which the specific PPAR gamma agonist azPC was identified by mass spectrometry. These findings suggested that UVB-generated PPAR gamma-agonistic activity was due to the free radical mediated non-enzymatic cleavage of endogenous glycerophosphocholines. Treatment with the specific PPAR gamma antagonist GW9662 or expression of a dominant-negative PPAR gamma mutant in KB cells inhibited UVB-induced epidermal cell prostaglandin E(2) production. These findings suggested that UVB-generated PPAR gamma activity is necessary for the optimal production of epidermal prostaglandins. These studies demonstrated that epithelial cells contain a functional PPAR gamma system, and this system is a target for UVB through the production of novel oxidatively modified endogenous phospholipids.

Identification of novel genes for secreted and membrane-anchored proteins in human keratinocytes.

Both intercellular and intracellular signals are transduced primarily by interactions of secreted and/or membrane-anchored polypeptides, and they play a pivotal role in regulating proliferation, differentiation and apoptosis of keratinocytes within the epidermis. Despite recent identification of these polypeptides, it is likely that several important molecules remain undisclosed. To identify novel genes encoding secreted or membrane-anchored polypeptides expressed by human keratinocytes. We employed a signal sequence (SS) trap of a 5'-end-enriched cDNA library prepared from primary cultured human keratinocytes. Gene expression analysis was performed using Northern blotting. R Screening of 4018 cDNA clones yielded 82 positive clones (57 independent genes), most of which encoded SSs in their N-termini. Most of the positive clones were known genes registered in the GenBank database. Seven genes were identified in the EST database, four of which encoded novel membrane-anchored polypeptides with features of type I transmembrane proteins; the other three genes encoded novel non-type I transmembrane polypeptides. These EST genes were expressed differentially by keratinocytes subjected to low vs. high calcium concentrations and by basal vs. squamous cell carcinomas. Using the SS trap, we isolated many genes known to be involved in constituting epidermal structures and others that had not previously been associated with keratinocytes. In addition, we identified novel genes (EST genes) that differ in kinetics of gene expression in keratinocyte differentiation. Our results validate the effective use of this SS trap method for identifying secreted and membrane-anchored polypeptides expressed by human keratinocytes. The identification will better illuminate the molecular mechanisms responsible for co-ordinated regulation of epidermal homeostasis.

Power-line frequency electromagnetic fields do not induce changes in phosphorylation, localization, or expression of the 27-kilodalton heat shock protein in human keratinocytes.

The linkage of the exposure to the power-line frequency (50-60 Hz) electromagnetic fields (EMF) with human cancers remains controversial after more than 10 years of study. The in vitro studies on the adverse effects of EMF on human cells have not yielded a clear conclusion. In this study, we investigated whether power-line frequency EMF could act as an environmental insult to invoke stress responses in human keratinocytes using the 27-kDa heat shock protein (HSP27) as a stress marker. After exposure to 1 gauss (100 micro T) EMF from 20 min to 24 hr, the isoform pattern of HSP27 in keratinocytes remained unchanged, suggesting that EMF did not induce the phosphorylation of this stress protein. EMF exposure also failed to induce the translocation of HSP27 from the cytoplasm to the nucleus. Moreover, EMF exposure did not increase the abundance of HSP27 in keratinocytes. In addition, we found no evidence that EMF exposure enhanced the level of the 70-kDa heat shock protein (HSP70) in breast or leukemia cells as reported previously. Therefore, in this study we did not detect any of a number of stress responses in human keratinocytes exposed to power-line frequency EMF.

Ultraviolet irradiation increases FADD protein in apoptotic human keratinocytes

Ultraviolet irradiation (UV) can induce keratinocyte apoptosis by activating death receptors that recruit the intracellular adaptor molecule FADD/MORT1 (Fas-associating death domain protein/mediator of receptor-induced toxicity). We hypothesized that UV could alter FADD expression levels to augment UV-induced keratinocyte apoptosis. In a dose-dependent manner UV B irradiation increased the expression of FADD protein in a human keratinocyte cell line (CCD-1106) with a corresponding increase in caspase-8 cleavage and cellular apoptosis. FADD overexpression induced cell death in 80% of cells compared with 10% spontaneous cell death in controls. Inhibition of FADD protein by adenoviral expression of anti-sense FADD reduced keratinocyte apoptosis. Regulation of FADD expression by UV may serve to enhance death receptor-mediated keratinocyte death.