Gene expression in response to cyclic mechanical stretch in primary human dermal fibroblasts.

The human dermal skin is permanently exposed to mechanical stress, for instance during facial expression, which might cause wrinkles with age. Cyclic mechanical stretching of cells results in cellular and cytoskeleton alignment perpendicular to the stretch direction regulating cellular response. With gene expression profiling it was aimed to identify the differentially expressed genes associated with the regulation of the cytoskeleton to investigate the stretch-induced cell alignment mechanism. Here, the transcription activity of the genome in response to cyclic mechanical stress was measured using DNA microarray technology with Agilent SurePrint G3 Human GE 8x60k Microarrays, based on the overall measurement of the mRNA. Gene expression was measured at the beginning of the alignment process showing first reoriented cells after 5 h stretching and at the end after 24 h, where nearly all cells are aligned. Gene expression data of control vs. stretched primary human dermal fibroblasts after 5 h and 24 h demonstrated the regulation of differentially expressed genes associated with metabolism, differentiation and morphology and were deposited at http://www.ncbi.nlm.nih.gov/geo with the accession number GSE58389.

Role of HuR and p38MAPK in ultraviolet B-induced post-transcriptional regulation of COX-2 expression in the human keratinocyte cell line HaCaT.

COX-2 (cyclooxygenase-2) is a pivotal player in inflammatory processes, and ultraviolet radiation is a known stimulus for COX-2 expression in skin cells. Here, an induction of COX-2 expression in HaCaT human keratinocytes was observed only upon exposure of cells to UVB (280-320 nm) but not to UVA radiation (320-400 nm), as demonstrated by reverse transcription-PCR and Western blotting. Prostaglandin E(2) levels were elevated in cell culture supernatants of HaCaT cells exposed to UVB. COX-2 mRNA stability was dramatically increased by UVB irradiation. Both the stabilization of COX-2 mRNA and the enhancement of COX-2 steady-state mRNA and protein levels caused by UVB were prevented both by inhibition and small interfering RNA-induced depletion of p38(MAPK), a kinase strongly activated upon exposure to UVB, suggesting p38(MAPK)-dependent mRNA stabilization as a mechanism of UVB-induced COX-2 expression. A dramatic decrease in COX-2 expression induced by UVB was elicited by small interfering RNA-based depletion of a stress-responsive mRNA stabilizing protein regulated by p38(MAPK), i.e. HuR; UVB-induced elevation of COX-2 mRNA and protein levels coincided with an accumulation of HuR in the cytoplasm and was attenuated in cells depleted of HuR. Moreover, UVB-induced generation of prostaglandin E(2) by HaCaT cells was blunted by HuR depletion, suggesting that stress kinases (such as p38(MAPK)) as well as HuR are excellent targets for approaches aiming at interfering with induction of COX-2 expression by UVB.

Nickel and copper ion-induced stress signaling in human hepatoma cells: analysis of phosphoinositide 3'-kinase/Akt signaling.

Nickel compounds may act as carcinogens, affecting both initiation and promotion stages of carcinogenesis due, in large parts, to their capability of inducing DNA damage and of modulating cellular signaling cascades known to affect cellular proliferation, respectively. We have previously demonstrated that the phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is stimulated in cells exposed to copper ions, resulting in phosphorylation and nuclear exclusion of FoxO transcription factors. Here, human hepatoma cells were exposed to nickel or copper ions, followed by comparative analysis of PI3K/Akt-dependent signaling. Exposure of hepatoma cells to copper ions resulted in extensive oxidation of cellular glutathione, while no such effect was detected with nickel ions. Similarly, copper ions were more than 100-fold more toxic to cells than nickel, as deduced from analyses of colony forming abilities. Despite this lack of oxidative and cytotoxic action, exposure of hepatoma cells to Ni(2+) resulted in a significant activation of Akt that was abrogated by inhibitors of PI3K. Interestingly, activation of Akt--although coincident with a phosphorylation of Akt substrates, such as glycogen synthase kinase-3--did not result in significant nuclear exclusion of FoxO1a. In line with this finding, no significant modulation of the activity of a FoxO-responsive promoter construct was observed in cells exposed to nickel ions. In summary, exposure of HepG2 human hepatoma cells to nickel ions results in stimulation of the Ser/Thr kinase Akt in a PI3K-dependent fashion, activation most likely being independent of oxidative processes. In sharp contrast to copper ions, nickel-induced Akt activation is not propagated further downstream to FoxO-dependent signaling beyond the phosphorylation of FoxO1a and 3a.

Thalidomide resistance is based on the capacity of the glutathione-dependent antioxidant defense.

Thalidomide as an effective treatment for multiple myeloma and leprosy has also caused birth defects in thousands of children five decades ago particularly in Europe. Thus its use in humans remains limited. The rapid and fatal approval of thalidomide at that time ultimately was a consequence of the sole use of thalidomide-insensitive species in animal toxicity tests. Here, we aimed at elucidating the molecular basis for the resistance of mice to thalidomide teratogenicity. By using hydroethidine staining we demonstrate that thalidomide induces the formation of superoxide in embryonic fibroblasts of thalidomide-sensitive species but not in those of mice. As determined by trypan blue staining, scavenging of superoxide prevents thalidomide-induced apoptosis, a marker for thalidomide teratogenicity. Mouse embryonic fibroblasts are found to have higher glutathione levels than those of sensitive species and can be sensitized for thalidomide by glutathione depletion with diethyl maleate or diamide. Accordingly, experimental increase of glutathione levels in human embryonic fibroblasts by adding N-acetyl cysteine or glutathione ethyl ester to the culture medium counteracts thalidomide-induced apoptosis. Finally, we show that thalidomide-induced molecular pathology downstream of superoxide is essentially identical in human and sensitized mouse embryonic fibroblasts. In conclusion, thalidomide-resistance is based on the capacity of the glutathione-dependent antioxidant defense. We provide a basis to pharmacologically overcome the limitations of thalidomide use at humans and describe substantial differences between human and mouse embryonic cells regarding the protection against oxidative stress.