Property characterization of reconstructed human epidermis equivalents, and performance as a skin irritation model.

In recent years, in vitro skin models combining cell biology and tissue engineering have been developed in order to replace animal models for toxicological studies and to serve as research support to better understand skin biology. This study reports the development and characterization of a epidermal tissue equivalent meant to be used to develop and to evaluate the effect of applied cosmetic ingredients, and for alternative toxicological testing. This epidermis equivalent model was characterized relative to the morphological characteristics of short- and long-term maintained tissues by performing histological studies. We also studied the integrity of the epidermal barrier. Finally, with the goal of validating its use as a skin irritation test, we studied the irritation potential of 20 chemical references listed in OECD Test Guideline N°439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). In 2015, OECD officially published the updated version of the Validated Reference Method (VRM) that uses reconstructed human epidermis models for irritation testing, thus offering the possibility for proposed putative similar test methods to obtain a validation agreement through Performance Standards-based validation. In this study, we observed that the epidermal equivalent we developed showed similarities to human in vivo skin, based on the analyzed parameters. Moreover, its performances as a skin irritation test were similar to the ones described in the OECD Test Guideline N°439.

Modulating the expression of survivin and other basal epidermal proteins protects human skin from UVB damage and oxidative stress.

BACKGROUND: The chromosomal passenger complex (CPC) is an assembly made of four interacting proteins: survivin, borealin, INCENP, and aurora kinase B. CPC is the key regulatory complex responsible for the correct development of cellular mitosis, accompanying each step of the chromosomal segregation. This control of mitosis is particularly important in undifferentiated cells that must renew themselves and also further differentiate and specialize. The epidermis is a self-renewing tissue that needs to continuously generate new cells through proliferation and differentiation of progenitor cells. Both the mitosis supervision by the CPC and a correct extracellular environment are physiologically required for the homeostasis of the adult keratinocyte stem cells (KSCs) of the epidermis. KSCs are mainly found in the basal layer of the epidermis and are responsible for the replenishment and maintenance of the tissue, by compensating for the loss of terminally differentiated cells called corneocytes, especially during aging. AIM: The aim of our study was to investigate the implication of survivin in epidermal renewal and the relationships between survivin expression and UVB-induced DNA damage levels in cultured human keratinocytes and in skin biopsies. In parallel, the effects of a treatment by compound IV08.009 were studied. MATERIAL AND METHODS: Cultured human keratinocytes and skin biopsies were used in this study. KSCs-enriched fractions of keratinocytes were isolated from total keratinocytes by differential attachment to a type IV collagen matrix. Survivin expression levels were assessed by immunoblotting in cultured keratinocytes, and α6-integrin, ß1-integrin, keratin 15, and survivin were observed after immunodetection in skin biopsies cross sections. Comet assay, immunodetection of CPDs and of cleaved-caspase 3, and electron microscopy were used to characterize UVB-induced DNA damage. RESULTS: We demonstrated the ability of compound IV08.009 to efficiently protect ex vivo skin against basal UVB-induced damage. Moreover, comet assay studies demonstrated the efficacy of IV08.009 in protecting DNA damage from UVB stress. We found that IV08.009 protects skin from apoptosis induced by oxidative stress, ex vivo. Electron microscopy confirmed the protective efficiency of IV08.009 on cell ultrastructural damage induced by UVB exposure. CONCLUSION: Compound IV08.009 demonstrated to be effective in regulating survivin expression and in preserving the basal epidermis from stresses such as UVB and H2 O2 . These results suggest a protective activity of IV08.009 on the essential renewing potential of KSCs.

NF-kappaB inhibits T-cell activation-induced, p73-dependent cell death by induction of MDM2.

NF-κB is a key transcription factor involved in the regulation of T-cell activation and proliferation upon engagement of the T-cell receptor (TCR). T cells that lack the IκB kinase (IKKß) are unable to activate NF-κB, and rapidly undergo apoptosis upon activation. NF-κB activation following T-cell receptor engagement induces the expression of Mdm2 through interaction with NF-κB sites in its P1 promoter, and enforced expression of Mdm2 protected T cells deficient for NF-κB activation from activation-induced cell death. In T cells with intact NF-κB signaling, ablation or pharmacologic inhibition of Mdm2 resulted in activation-induced apoptosis. Mdm2 coprecipitates with p73 in activated T cells, and apoptosis induced by inhibition of Mdm2 was p73-dependent. Further, Bim was identified as a p73 target gene required for cell death induced by Mdm2 inhibition, and a p73-responsive element in intron 1 of Bim was characterized. Our results demonstrate a pathway for survival of activated T cells through NF-κB-induced Mdm2, which blocks Bim-dependent apoptosis through binding and inhibition of p73.

Positive feedback regulation of PLCgamma1/Ca(2+) signaling by PKCtheta in restimulated T cells via a Tec kinase-dependent pathway.

PKCtheta plays an essential role in activation of mature T cells. Here, we report that the TCR/CD28-induced tyrosine phosphorylation and activation of PLCgamma1 was significantly impaired in PKCtheta (-/-) primary, restimulated T cells. Consistent with this finding, receptor-induced Ca(2+) mobilization, NF-AT DNA-binding activity and the membrane translocation of PKCalpha, a PLCgamma1-dependent conventional PKC, were also markedly reduced in the same cells. Moreover, a dominant-negative PLCgamma1 mutant blocked the PKCtheta-induced activation of an AP-1 reporter gene in Jurkat and primary cells. Regulation of PLCgamma1 signaling by PKCtheta required the tyrosine kinase Tec since a dominant-negative Tec mutant blocked PKCtheta-induced AP-1 (but not NF-kappaB) activation. In addition, wild-type Tec, but not Itk or Rlk, potently activated AP-1. Furthermore, Tec was found to constitutively associate with PKCtheta, an interaction that like AP-1 activation required the pleckstrin-homology domain of Tec. These findings define a novel PKCtheta-initiated pathway that regulates Ca(2+) signaling and AP-1 activation via Tec and PLCgamma1. Moreover, they identify Tec as a key point downstream of PKCtheta, where TCR- and PKCtheta-induced signaling pathways, leading to AP-1 versus NF-kappaB activation, diverge in T cells.

Blocking NF-kappaB activation in Jurkat leukemic T cells converts the survival agent and tumor promoter PMA into an apoptotic effector.

The transcription factor NF-kappaB promotes cell survival. Using a variant of Jurkat leukemic T cells expressing IkappaB-alphaDeltaN, a super-repressor of NF-kappaB activation we first show that the tumor promoter PMA could prevent Fas-induced apoptosis via activation of NF-kappaB. Moreover, we demonstrate that in the absence of NF-kappaB activation, PMA became a strong inducer of apoptosis through stimulation of the upstream caspases 8 and 9 as well as of the effector caspase 3. A RNase-protection analysis showed that PMA stimulated the expression of several known anti-apoptotic genes (TRAF1, TRAF4, c-IAP-1, c-IAP-2, Bfl-1, Bcl-xl). In the absence of NF-kappaB activation, these survival influences were strongly lowered revealing the apoptotic effect of PMA. These results suggest that NF-kappaB activation could be an important step in the tumor promoting effect of PMA.

Enterohemorrhagic Escherichia coli infection induces interleukin-8 production via activation of mitogen-activated protein kinases and the transcription factors NF-kappaB and AP-1 in T84 cells.

Enterohemorrhagic Escherichia coli (EHEC) infections are associated with hemorrhagic colitis and the hemolytic-uremic syndrome (HUS). In vivo, elevated plasma levels of the proinflammatory cytokine interleukin-8 (IL-8) in EHEC-infected children are correlated with a high risk of developing HUS. As IL-8 gene transcription is regulated by the transcription factors NF-kappaB and AP-1, we analyzed the role of these factors in the regulation of IL-8 production after infection of the epithelial intestinal T84 cell line by EHEC. By 6 h of infection, EHEC had induced significant secretion of IL-8 (35.84 +/- 6.76 ng/ml versus 0.44 +/- 0.04 ng/ml in control cells). EHEC induced AP-1 and NF-kappaB activation by 3 h of infection. Moreover, the three mitogen-activated protein kinases (MAPK) (ERK1/2, p38, and JNK) were phosphorylated in EHEC-infected T84 cells concomitant with induction of AP-1 DNA binding activity, and IkappaB-alpha was phosphorylated and then degraded concomitant with induction of NF-kappaB DNA binding activity. Pretreatment of cells with the highly specific MEK1/2 inhibitor U0126, the p38 inhibitor SB203580, and/or the proteasome inhibitor ALLN led to inhibition of the IL-8 secretion induced in EHEC-infected T84 cells. These findings demonstrate that (i) EHEC can induce in vitro a potent proinflammatory response by secretion of IL-8 and (ii) the secretion of IL-8 is due to the involvement of MAPK, AP-1, and NF-kappaB signaling pathways.