Human Keratinocyte Cell Line HaCaT Research Articles

Identification of a cell line producing high levels of TSLP: Advantages for screening of anti-allergic drugs

Thymic stromal lymphopoietin (TSLP) plays critical roles in the induction and exacerbation of allergic diseases. These findings suggest that an inhibitor of TSLP production may be a novel drug for allergic diseases. However, conducting high-throughput screening of such compounds is difficult because there is currently no appropriate in vitro system. In the present study, we demonstrated that the mouse keratinocyte cell line KCMH-1 produced higher amounts of TSLP than the mouse keratinocyte cell line PAM-212, human keratinocyte cell line HaCaT, or bronchial cell line BEAS-2B. A reporter gene assay revealed that transcriptional activity of the TSLP gene was also markedly higher in KCMH-1 than in PAM212 cells. Both dexamethasone and the retinoid X receptor agonist HX600 inhibited the production of TSLP in KCMH-1 cells, which indicated that its production could be pharmacologically regulated. Moreover, the biological activity of TSLP released from KCMH-1 cells in the medium was endorsed by the induction of OX40L expression in bone marrow-derived dendritic cells. These results indicate that KCMH-1 can be utilized in high-throughput screening of inhibitors of TSLP production and also as a source of native TSLP.

Regulatory effect of serum starvation on autophagy in a human keratinocyte cell line HaCaT.

Objective To evaluate the regulatory effect of serum starvation on autophagy in human HaCaT keratinocytes,and to investigate its molecular mechanism.Methods HaCaT cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10％ fetal calf serum (control group) or in DMEM without fetal calf serum (starvation group),for four hours.Then,cell appearance was observed by light microscopy,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell viability,transmission electron microscopy to observe ultrastructural changes,monodansylcadaverine (MDC) staining to identify the formation of autophagosomes,and Western blot to measure the expression of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ,LC3-Ⅰ and autophagy related protein 7 (ATG7).Additionally,the phosphorylation level of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 was semiquantitatively analyzed by Western blot.Results Serum starvation increased the viability of HaCaT cells.Electron microscopy and MDC staining confirmed the formation of autophagosomes in serum-starved HaCaT cells.Compared with the control cells,the serum-starved HaCaT cells showed a significant increase in the transformation of LC3-Ⅰ to LC3-Ⅱ (LC3-Ⅱ/LC3-Ⅰ ratio:3.508 ± 0.415 vs.0.538 ± 0.038,t =13.32,P ＜ 0.01),and expression of ATG7 (ATG7/GAPDH ratio:0.048 ± 0.011 vs.0.021 ± 0.006,t =7.27,P ＜ 0.05).The phosphorylation level of mTOR at ser2448 and ser2481 was significantly lower in the serum-starved HaCaT cells than in the control cells (phosphorylated mTOR (ser2448)/mTOR ratio:0.394 ± 0.048 vs.0.762 ± 0.108,t =7.58,P＜ 0.05; phosphorylated mTOR (ser2481)/mTOR ratio:0.111 ± 0.020 vs.0.263 ± 0.039,t =13.77,P ＜ 0.05).Conclusion Serum starvation can induce autophagy in and increase the viability of HaCaT cells,likely by inhibiting the activation of mTOR. Key words: Keratinocytes; Autophagy; Cells, cultured

Everolimus-induced human keratinocytes toxicity is mediated by STAT3 inhibition

BackgroundMammalian target of rapamycin (mTOR) inhibitors are associated with dermatological adverse events. The chief aim of this study was to examine the relation between the signal transducer and activator of transcription 3 (STAT3) protein and the dermatological adverse events associated with the mTOR inhibitor everolimus.MethodsWe evaluated the effects of STAT3 activity and related signal transduction activities on everolimus-induced cell growth inhibition in the human keratinocyte HaCaT cell line via a WST-8 assay, and on signal transduction mechanisms involved in everolimus treatments via a western blot analysis. Apoptosis was evaluated using an imaging cytometric assay.ResultsThe cell growth inhibitory effects of everolimus were enhanced by stattic or STA-21, which are selective inhibitors of STAT3, treatment in HaCaT cells, although such effects were not observed in Caki-1 and HepG2 cells. Phosphorylation at tyrosine 705 of STAT3 was decreased by treatment with everolimus in a dose-dependent manner in HaCaT cells; in contrast, phosphorylation at serine 727 was not decreased by everolimus, but slightly increased. Furthermore, we found that pretreatment of p38 MAPK inhibitor and transfection with constitutively active form of STAT3 in HaCaT cells resisted the cytostatic activity of everolimus.ConclusionsThese findings suggest that STAT3 activity may be a biomarker of everolimus-induced dermatological toxicity.

Protective effects of Korean red ginseng against radiation-induced apoptosis in human HaCaT keratinocytes

Radiation-induced oral mucositis is a dose-limiting toxic side effect for patients with head and neck cancer. Numerous attempts at improving radiation-induced oral mucositis have not produced a qualified treatment. Ginseng polysaccharide has multiple immunoprotective effects. Our aim was to investigate the effectiveness of Korean red ginseng (KRG) on radiation-induced damage in the human keratinocyte cell line HaCaT and in an in vivo zebrafish model. Radiation inhibited HaCaT cell proliferation and migration in a cell viability assay and wound healing assay, respectively. KRG protected against these effects. KRG attenuated the radiation-induced embryotoxicity in the zebrafish model. Irradiation of HaCaT cells caused apoptosis and changes in mitochondrial membrane potential (MMP). KRG inhibited the radiation-induced apoptosis and intracellular generation of reactive oxygen species (ROS), and stabilized the radiation-induced loss of MMP. Western blots revealed KRG-mediated reduced expression of ataxia telangiectasia mutated protein (ATM), p53, c-Jun N-terminal kinase (JNK), p38 and cleaved caspase-3, compared with their significant increase after radiation treatment. The collective results suggest that KRG protects HaCaT cells by blocking ROS generation, inhibiting changes in MMP, and inhibiting the caspase, ATM, p38 and JNK pathways.

Gardiquimod inhibits the expression of calcium-induced differentiation markers in HaCaT cells

Toll-like receptor 7 (TLR7) is an important member in pattern recognition receptors families. TLR7 signal pathway is involved in the physiological process in many type cells, but the impact of TRL7 on differentiation in the human keratinocytes is still unknown. In this study, we investigated the expression of TLR7 in keratinocytes, and the effect of TLR7 agonist gardiquimod on the expression of calcium (Ca(2+))-induced keratinocytes differentiation markers in HaCaT cells. Immunohistochemistry and western-blotting analysis showed that TLR7 is expressed in basal keratinocytes of normal skin and in the human keratinocyte cell line HaCaT, but not expressed in the keratinocytes of psoriasis lesions. Pretreatment with gardiquimod could down-regulate Ca(2+)-induced differentiation marker expression and activate Raf-MEK-ERK and PI3K-AKT signal pathways in HaCaT cells. However, specific inhibitors studies showed that the down-regulation of the differentiation markers expression by gardiquimod was not dependent on the activation of these two pathways. TLR7 may play an important role in the pathogenesis of psoriasis through regulating the differentiation of the keratinocytes, and will give a new insight into the psoriasis.

Curcumin ameliorates TNF-α-induced ICAM-1 expression and subsequent THP-1 adhesiveness via the induction of heme oxygenase-1 in the HaCaT cells

Adhesion molecules such as ICAM-1 are important in the infiltration of leukocytes into the site of inflammation. In this study, we investigated the inhibitory effects of curcumin on ICAM-1 expression and monocyte adhesiveness as well as its underlying action mechanism in the TNF-α-stimulated keratinocytes. Curcumin induced expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. In addition, curcumin induced Nrf2 activation in dose- and time-dependent manners in the HaCaT cells. Curcumin suppressed TNF-α- induced ICAM-1 expression and subsequent monocyte adhesion, which were reversed by the addition of tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, or HO-1 knockdown using siRNA. Furthermore, Nrf2 knockdown using siRNA reversed the inhibitory effect of curcumin on the TNF-α-induced ICAM-1 expression and adhesion of monocytes to keratinocytes. These results suggest that curcumin may exert its anti-inflammatory activity by suppressing the TNF-α-induced ICAM-1 expression and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes. [BMB Reports 2013;46(8): 410-415]

AnIn VitroHSV-1 Reactivation Model Containing Quiescently Infected PC12 Cells

Advances in the understanding of the infection and reactivation process of herpes simplex type 1 (HSV-1) are generally gained by monolayer cultures or extensive and cost-intensive animal models. So far, no reliable in vitro skin model exists either to investigate the molecular mechanisms involved in controlling latency and virus reactivation or to test pharmaceuticals. Here we demonstrate the first in vitro HSV-1 reactivation model generated by using the human keratinocyte cell line HaCaT grown on a collagen substrate containing primary human fibroblasts. We integrated the unique feature of a quiescently infected neuronal cell line, the rat pheochromocytoma line PC12, within the dermal layer of the three-dimensional skin equivalent. Transmission electron microscopy, a cell-based TCID50 assay, and polymerase chain reaction analysis were used to verify cell latency. Thereby viral DNA could be detected, whereas extracellular as well as intracellular virus activity could not be found. Further, the infected PC12 cells show no spontaneous reactivation within the in vitro skin equivalent. In order to simulate a physiologically comparable HSV-1 infection, we achieved a specific and pointed reactivation of quiescently HSV-1 infected PC12 cells by UVB irradiation at 1000 mJ/cm2.

Non-cytotoxic Dimedone Derivatives: Structure, Antiradical and UV Protective Studies

Dimedone derivatives (L1-L4) with methyl substitution at the ortho, para and meta positions were synthesized and their anti-radical, photoreactive and photostability activities were evaluated. All compounds are characterized by spectroscopic techniques and by single crystal X ray diffraction. UV exposure experiments on pBR322 showed inhibition of plasmid DNA fragmentation by UV radiation in a dose dependent manner. Radical scavenging assays and ESR spectra indicate that these compounds possess antiradical activities and do not photodegrade to form other side products as confirmed by chromatographic analysis. They are non-cytotoxic towards human keratinocyte HaCaT cell line indicates their potentiality in sunscreens.

Illicium verum extract suppresses IFN-γ-induced ICAM-1 expression via blockade of JAK/STAT pathway in HaCaT human keratinocytes

Ethnopharmacological relevanceInterferon-gamma (IFN-γ) signaling in keratinocytes plays an important role in mediating inflammatory conditions such as psoriasis and contact dermatitis. Illicium verum Hook. f. has been used in traditional medicine for treating skin inflammation, rheumatism, asthma, and bronchitis in Asia. Aim of the studyTo investigate the anti-inflammatory effects and regulatory mechanisms of Illicium verum extract (IVE) in the human keratinocyte cell line HaCaT. Materials and MethodsIn this study, we examined the effect of IVE on IFN-γ-induced ICAM-1 expression in HaCaT cells. The levels of IFN-γ receptor α (IFN-γRα), phosphorylated Janus kinase 2 (pJak2), phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and suppressor of cytokine signaling 1 (SOCS1) were analyzed by western blot. Expression of intercellular adhesion molecule-1 (ICAM-1) on the HaCaT cells was determined by reverse transcription-polymerase chain reaction, western blot, and cell-surface enzyme-linked immunosorbent assay. The effects of IVE and its constituents on the adherence of T lymphocytes to IFN-γ-treated HaCaT cells were also investigated. ResultsIVE significantly inhibited IFN-γ-induced mRNA and protein expression of ICAM-1. IVE inhibited IFN-γ-induced IFN-γRα, pJak2 and pSTAT1 expression in HaCaT cells. The expression of SOCS1 was up-regulated by treatment of IVE. In addition, IVE and its constituents (p-anisaldehyde and trans-anethole) effectively suppressed IFN-γ-induced adherence of Jurkat T cells to HaCaT cells and ICAM-1 expression on the cell surface. ConclusionsThese results indicate that the anti-inflammatory effects of IVE may contribute to therapeutic efficacy in IFN-γ-dependent inflammatory skin diseases by modulating the IFN-γ signal pathway.

Enhanced antioxidant effect of trans-resveratrol: potential of binary systems with polyethylene glycol and cyclodextrin

Trans-resveratrol, a polyphenol extracted from Vitis vinifera, has different beneficial effects following its administration on the skin. Here the potential use of binary systems to enhance in vitro and in vivo activity of trans-resveratrol was investigated. Thus the aqueous solubility of trans-resveratrol was investigated in the presence of growing concentrations of polyethylene glycol (PEG) or β-cyclodextrin (βCD) as solubilizing excipients. Then, the solid dispersion of trans-resveratrol with PEG or inclusion complexes trans-resveratrol/βCD were prepared and characterised by different methods. Cytotoxicity and inhibition of reactive oxygen species (ROS) following H2O2 challenge in the presence of trans-resveratrol, alone or associated to the excipients, was evaluated on human keratinocyte HaCaT cell line. Both the trans-resveratrol-containing binary systems induced significant reduction of H2O2-induced ROS production, especially in the case of βCD that was selected for the following phase of the study. Thus, the effect of a cream containing trans-resveratrol, alone or associated to βCD, on different skin parameters such as corneometry, colorimetry and elastometry, was evaluated on human volunteers. All patients showed a visible improvement of clinical conditions with a remarkable decrease of aging signs, but this effect was higher of the hemi face treated with the βCD-containing formulation versus formulation containing trans-resveratrol alone.

Photodynamic effects of 31 different phthalocyanines on a human keratinocyte cell line

Phthalocyanines (Pcs, colored macromolecular compounds with the ability to generate singlet oxygen) represent a promising group of photosensitizers due to their intense absorption in the red and UV portion of the spectrum which leads to their excitation. In order to characterize possible toxic effects associated with eventual practical use and application of these chemicals, we employed an in vitro cell culture model to evaluate cytotoxic effects of 31 different phthalocyanines using neutral red uptake assay. An immortalized human keratinocyte cell line HaCaT was exposed to the tested chemicals for 2 or 24h, either with or without illumination in the last 60min of the exposure period. After 2- or 24-h exposure without illumination, no cytotoxic effects or weak cytotoxic effects were induced by any Pc under the study and EC50 values could not be obtained within the tested concentration ranges (1.25–20mgL−1 or 0.625–10mgL−1). On the other hand, exposure to phthalocyanines under illumination induced a significant cytotoxic effect. The most pronounced cytotoxicity was elicited by Pcs previously shown to have high positive charge densities at peripheral parts of substituent groups, which is most likely the factor responsible for the binding of Pc to negatively charged membranes on the cell surface and thus guaranteeing the tight connection necessary for the singlet oxygen attack on the cell surface.

Abstract 1296: Involvement of RSK2 in human skin cancer.

Abstract Our previous study demonstrated that the total p90 ribosomal S6 kinase 2 (RSK2) protein level was significantly higher in human skin cancer tissues and cancer cells compared with normal skin tissues and premalignant cell lines. However, the role of RSK2 activation and signaling in human skin cancer has not yet been fully elucidated. In this study, we provided evidences that RSK2 plays a key role in human skin cancer. We examined the effect of knocking down RSK2 expression on EGF-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma (MM) cells. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2, but also the nuclear localization and gene expression of RSK2. Moreover, RSK2−/− mouse embryonic fibroblasts (MEFs) showed enhanced sub-G1 accumulation induced by UVB stimulation compared with RSK2+/+ MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with UV. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of MM cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth. Citation Format: Cheol-Jung Lee, Hye Suk Lee, Ji-Young Lee, Yong Yeon Cho. Involvement of RSK2 in human skin cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1296. doi:10.1158/1538-7445.AM2013-1296

Ecto-Nucleoside Triphosphate Diphosphohydrolase 2 Modulates Local ATP-Induced Calcium Signaling in Human HaCaT Keratinocytes

Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP) as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP) accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

Metabolic effects of TiO2 nanoparticles, a common component of sunscreens and cosmetics, on human keratinocytes

The long-term health risks of nanoparticles remain poorly understood, which is a serious concern given their prevalence in the environment from increased industrial and domestic use. The extent to which such compounds contribute to cellular toxicity is unclear, and although it is known that induction of oxidative stress pathways is associated with this process, the proteins and the metabolic pathways involved with nanoparticle-mediated oxidative stress and toxicity are largely unknown. To investigate this problem further, the effect of TiO2 on the HaCaT human keratinocyte cell line was examined. The data show that although TiO2 does not affect cell cycle phase distribution, nor cell death, these nanoparticles have a considerable and rapid effect on mitochondrial function. Metabolic analysis was performed to identify 268 metabolites of the specific pathways involved and 85 biochemical metabolites were found to be significantly altered, many of which are known to be associated with the cellular stress response. Importantly, the uptake of nanoparticles into the cultured cells was restricted to phagosomes, TiO2 nanoparticles did not enter into the nucleus or any other cytoplasmic organelle. No other morphological changes were detected after 24-h exposure consistent with a specific role of mitochondria in this response.

Mitochondrial Translocation of Vitamin D Receptor Is Mediated by the Permeability Transition Pore in Human Keratinocyte Cell Line

BackgroundVitamin D receptor (VDR) is a well known transcriptional regulator, active as heterodimer in association with coactivators and corepressors. In addition it has been described the extranuclear distribution of the receptor and in particular the recently reported mitochondrial localization in platelets and megakaryocytes is intriguing because it appears to be a common feature of steroid receptors. Whereas for other members of the steroid receptor family the mitochondrial function has been explored, up to now nothing is known about a mitochondrial form of VDR in human proliferating cells.Methodology/Principal FindingsIn this study we characterized for the first time the mitochondrial localization of VDR in the human keratinocyte cell line HaCaT. In proliferating HaCaT cells VDR was abundantly expressed in mitochondria in association with its binding partner RXRα and the import was ligand-independent. By immunoprecipitation studies we demonstrated the interaction of VDR with proteins of the permeability transition pore (PTP), VDAC and StAR. We then adopted different pharmacological and silencing approaches with the aim of hampering PTP function, either affecting PTP opening or abating the expression of the complex member StAR. By all means the impairment of pore function led to a reduction of mitochondrial levels of VDR.ConclusionsThe results reported here demonstrate a ligand-independent mitochondrial import of VDR through the permeability transition pore, and open interesting new perspectives on PTP function as transporter and on VDR role in mitochondria.

Nonthermal Plasma Increases Expression of Wound Healing Related Genes in a Keratinocyte Cell Line

The application of physical plasma in medicine has great potential in wound healing. Due to the generation of reactive oxygen and nitrogen species (ROS, RNS), emission of UV radiation and the generated electric fields can be used to stimulate epithelial and immune cells. To understand the processes on a molecular level the human keratinocyte cell line HaCaT was treated with a nonthermal atmospheric pressure argon plasma jet (kinpen). Subsequently, cellular RNA was isolated to conduct a quantitative polymerase chain reaction (qPCR) to monitor the magnitude of translation of genes related to wound healing. Plasma treatment induced an upregulation of vascular endothelial growth factor-a (VEGF-A), heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), granulocyte macrophage colony-stimulating factor (GM-CSF), prostaglandin-endoperoxide synthase 2 (PTGS2) and interleukin-6 (IL-6) at the mRNA levels. This is a very promising result as the corresponding proteins are likely to be secreted and promote the wound healing process. Therefore plasma potentially induces the secretion of certain cytokines and growth factors, and hence, it could be the stimulus which is necessary to induce chronic wounds to heal.

Retinoids regulates the production of matrix metalloproteinases in the human epidermal keratinocyte cell line HaCaT

Retinoids are known to induce the proliferation of fibroblasts, to promote collagen synthesis and to decrease the matrix metalloproteinases (MMPs) expression. Adapalene is a naphthoic acid derivative showing some pharmacological activities similar to the regular retinoids. It is used singly or in combination for treating acne and a few other skin disorders. MMPs are abundantly found in acne lesions and believed to worsen inflammation and scar formation. In this study, we tried to investigate the suppressive effect of adapalene, all-trans retinoic acid and 9-cis retinoic acid on the production of MMP-1, 9, and 13.

Phloroglucinol protects human keratinocytes from ultraviolet B radiation by attenuating oxidative stress

Ultraviolet B generates reactive oxygen species by interacting with water in irradiated skin tissues and cells in turn causes lipid peroxidation, protein modification and further DNA damage. This study examined the cytoprotective effects of phloroglucinol (1,3,5-trihydroxybenzene) on ultraviolet B-irradiated cultured human keratinocytes. The human keratinocyte cell line HaCaT cells were treated with 10 μM of phloroglucinol. After 1 h, the cells were irradiated with ultraviolet B light at 30 mJ/cm(2) and incubated at 37°C. Phloroglucinol scavenged both the superoxide anion and hydroxyl radical in a cell-free system and ultraviolet B-induced intracellular reactive oxygen species. Phloroglucinol reduced ultraviolet B-generated lipid peroxidation, protein modification and DNA strand breaks. The enzymatic effects of phloroglucinol restored cellular glutathione levels, superoxide dismutase and catalase activities, which were impaired by ultraviolet B radiation. Phloroglucinol provides the protective effects in human keratinocyte cell line exposed to ultraviolet B radiation, suggesting that phloroglucinol can be used as a photoprotective agent.

Abstract B55: Involvement of RSK2 in human skin cancer

Abstract Our previous study demonstrated that the total p90 ribosomal S6 kinase 2 (RSK2) protein level was significantly higher in human skin cancer tissues and cancer cells compared with normal skin tissues and premalignant cell lines. We also demonstrated that the N-terminal kinase domain of RSK2 plays an important role in transducing the RSK2 activation signal to its substrates. RSK2 requires activation of its C-terminal domain by its upstream kinases, ERKs, in order to phosphorylate and activate its substrates. For example, a recombinant RSK2 protein purified from E. Coli cannot phosphorylate the RSK2 substrate, NFAT3, but phosphorylation of RSK2 by ERKs at the linker region of RSK2 gives RSK2 the ability to phosphorylate its substrates through its N-terminal kinase domain. This suggests that ERKs/RSK2 signaling plays an important role in RSK2-mediated cell proliferation and cell transformation. Importantly, our study demonstrated that co-expression of RasG12V and RSK2 in NIH3T3 cells increased foci formation compared with RasG12V alone and si-RNA knockdown of RSK2 in RasG12V- or RasG12V/RSK2-co-expressing NIH3T3 cells completely blocked foci formation. Furthermore, we demonstrated that kaempferol, a natural compound abundantly found in dietary foods, inhibited RSK2 N-terminal kinase activity and EGF-induced neoplastic cell transformation. These indicated that the Ras/ERKs/RSK2 signaling pathway plays a key role in cell transformation. However, the role of RSK2 activation and signaling in human skin cancer has not yet been fully elucidated. To elucidate the relationship of RSK2 activity and human skin cancer, we examined the effect of knocking down RSK2 expression on EGF-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma (MM) cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2, but also the nuclear localization and gene expression of RSK2. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of MM cells. Moreover, RSK2−/− mouse embryonic fibroblasts (MEFs) showed enhanced sub-G1 accumulation induced by UVB stimulation compared with RSK2+/+ MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with UV. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth. Citation Format: Cheol-Jung Lee, Mee Hyun Lee, Ke Yao, Hye Suk Lee, Ann M. Bode, Zigang Dong, Yong Yeon Cho. Involvement of RSK2 in human skin cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B55.

Tissue-specific opposing functions of the inflammasome adaptor ASC in the regulation of epithelial skin carcinogenesis.

A chronic inflammatory microenvironment favors tumor progression through molecular mechanisms that are still incompletely defined. In inflammation-induced skin cancers, IL-1 receptor- or caspase-1-deficient mice, or mice specifically deficient for the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) in myeloid cells, had reduced tumor incidence, pointing to a role for IL-1 signaling and inflammasome activation in tumor development. However, mice fully deficient for ASC were not protected, and mice specifically deficient for ASC in keratinocytes developed more tumors than controls, suggesting that, in contrast to its proinflammatory role in myeloid cells, ASC acts as a tumor-suppressor in keratinocytes. Accordingly, ASC protein expression was lost in human cutaneous squamous cell carcinoma, but not in psoriatic skin lesions. Stimulation of primary mouse keratinocytes or the human keratinocyte cell line HaCaT with UVB induced an ASC-dependent phosphorylation of p53 and expression of p53 target genes. In HaCaT cells, ASC interacted with p53 at the endogenous level upon UVB irradiation. Thus, ASC in different tissues may influence tumor growth in opposite directions: it has a proinflammatory role in infiltrating cells that favors tumor development, but it also limits keratinocyte proliferation in response to noxious stimuli, possibly through p53 activation, which helps suppressing tumors.