One of the unique features of ␤-cells is their relatively low expression of many antioxidant enzymes. This could render ␤-cells susceptible to oxidative damage but may also provide a system that is sensitive to reactive oxygen species as signals. In isolated mouse islets and INS-1(832/13) cells, glucose increases intracellular accumulation of H 2 O 2 . In both models, insulin secretion could be stimulated by provision of either exogenous H 2 O 2 or diethyl maleate, which raises intracellular H 2 O 2 levels. Provision of exogenous H 2 O 2 scavengers, including cell permeable catalase and N-acetyl-Lcysteine, inhibited glucose-stimulated H 2 O 2 accumulation and insulin secretion (GSIS). In contrast, cell permeable superoxide dismutase, which metabolizes superoxide into H 2 O 2 , had no effect on GSIS. Because oxidative stress is an important risk factor for ␤-cell dysfunction in diabetes, the relationship between glucose-induced H 2 O 2 generation and GSIS was investigated under various oxidative stress conditions. Acute exposure of isolated mouse islets or INS-1(832/ 13) cells to oxidative stressors, including arsenite, 4-hydroxynonenal, and methylglyoxal, led to decreased GSIS. This impaired GSIS was associated with increases in a battery of endogenous antioxidant enzymes. Taken together, these findings suggest that H 2 O 2 derived from glucose metabolism is one of the metabolic signals for insulin secretion, whereas oxidative stress may disturb its signaling function. Diabetes

Inorganic arsenic is a well-documented human carcinogen that targets the skin. The induction of oxidative stress, as shown with arsenic, may have a bearing on the carcinogenic mechanism of this metalloid. The transcription factor Nrf2 is a key player in the regulation of genes encoding for many antioxidative response enzymes. Thus, the effect of inorganic arsenic (as sodium arsenite) on Nrf2 expression and localization was studied in HaCaT cells, an immortalized human keratinocyte cell line. We found, for the first time, that arsenic enhanced cellular expression of Nrf2 at the transcriptional and protein levels and activated expression of Nrf2-related genes in these cells. In addition, arsenic exposure caused nuclear accumulation of Nrf2 in association with downstream activation of Nrf2-mediated oxidative response genes. Arsenic simultaneously increased the expression of Keap1, a regulator of Nrf2 activity. The coordinated induction of Keap1 expression and nuclear Nrf2 accumulation induced by arsenic suggests that Keap1 is important to arsenic-induced Nrf2 activation. Furthermore, when cells were pretreated with scavengers of hydrogen peroxide (H(2)O(2)) such as catalase-polyethylene glycol (PEG-CAT) or Tiron, arsenic-induced nuclear Nrf2 accumulation was suppressed, whereas CuDIPSH, a cell-permeable superoxide dismutase (SOD) mimic compound that produces H(2)O(2) from superoxide (*O(2)(-)), enhanced Nrf2 nuclear accumulation. These results indicate that H(2)O(2), rather than *O(2)(-), is the mediator of nuclear Nrf2 accumulation. Additional study showed that arsenic causes increased cellular H(2)O(2) production and that H(2)O(2) itself has the ability to increase Nrf2 expression at both the transcription and protein levels in HaCaT cells. Taken together, these data clearly show that arsenic increases Nrf2 expression and activity at multiple levels and that H(2)O(2) is one of the mediators of this process.

It is widely believed that epithelial stem cells reside in the hair follicle bulge region. We investigated the hematopoietic stem and progenitor cell marker, CD34, as a potential marker of hair follicle bulge keratinocytes. Using a CD34-specific antibody, we identified intense membrane staining on keratinocytes in the bulge region of the mouse hair follicle. CD34 expression colocalized with both slowly cycling (label retaining) cells and keratin 15 expression. Live CD34+ keratinocytes were positively selected using antibodies to CD34 and alpha6 integrin in combination with fluorescent activated cell sorting. Sorted cells were analyzed for DNA content, and a staining profile was generated to confirm these cells as keratinocytes. CD34+ keratinocytes were predominantly in Go/G1, in contrast to CD34- cells, which had well defined G2/M and S phases. In addition, CD34+ keratinocytes were found to express alpha6 integrin more intensely than CD34- cells (p<0.05), identifying this population as an alpha6 integrin bright subset. When seeded at clonal density, CD34+ keratinocytes formed larger colonies than CD34- cells (p<0.05), indicating a higher proliferative potential. All flow-sorted cells were positive for keratin 14 expression, and negative for keratin 1, loricrin, vimentin, and CD31. The majority of CD34+ cells (98%) were positive for keratin 6, establishing this population as basal keratinocytes of follicular origin. CD34 message was detected by reverse transcription polymerase chain reaction predominantly in the CD34+ keratinocytes, confirming specificity of the antibody. This work is the first to demonstrate that CD34 is a specific marker of bulge cell keratinocytes in the cutaneous epithelium. Furthermore, the use of this marker facilitates isolation of live epithelial cells with stem and progenitor cell characteristics, potentially providing a tool for the study of carcinogen target cells, gene therapy, and tissue engineering applications.

Nrf2 is a key transcription factor in the cellular response to oxidative stress. In this study we first identify two phosphorylated forms of endogenous human Nrf2 after chemically-induced oxidative stress and provide evidence that protein kinase CK2-mediated sequential phosphorylation plays potential role in Nrf2 activation and degradation. Human Nrf2 has a predicted molecular mass of 66 kDa. However, immunoblots showed that two bands at 98 and 118 kDa, which are identified as phosphorylated forms, are increased in response to Nrf2 inducers. In addition, human Nrf2 was found to be a substrate for CK2 which mediated two steps of phosphorylation, resulting in two forms of Nrf2 migrating with differing Mr at 98 kDa (Nrf2-98) and 118 kDa (Nrf2-118). Our results support a role in which calmodulin binding regulates CK2 activity, in that cold (25 °C) in Ca 2+ -free media (cold/Ca 2+ -free) decreased both cellular calcium levels and CK2-calmodulin binding and induced Nrf2-118 formation, the latter of which was prevented by CK2 specific inhibitors. Gel-shift assays showed that the Nrf2-118 generated under cold/Ca 2+ -free conditions does not bind to the antioxidant response element, indicating that Nrf2-98 has transcriptional activity. In contrast, Nrf2-118 is more susceptible to degradation. These results provide evidence for phosphorylation by CK2 as a critical controlling factor in Nrf2-mediated cellular antioxidant response.