Hailey-Hailey disease (MIM16960) is a blistering skin disease caused by mutations in the Ca2+ ATPase ATP2C1. We found that the abnormal Ca2+ signaling seen in Hailey-Hailey disease keratinocytes correlates with decreased protein levels of ATP2C1. Human ATP2C1 protein approximated 115 kDa in size. The ATP2C1 is localized to the Golgi apparatus in human keratinocytes, similar to its localization in yeast and Caenorhabditis elegans. To test whether the ATP2C1 controls Golgi Ca2+ stores, we measured intraorganelle Ca2+ concentrations using specifically targeted aequorins. Whereas normal keratinocytes display Golgi Ca2+ levels comparable to other epithelial cells, Hailey-Hailey disease keratinocyte Golgi Ca2+ refill is slower, and the maximum Ca2+ concentration reached is significantly lower. These findings were replicated in vivo, because clinically normal Hailey-Hailey disease epidermis contained lower Ca2+ stores and displayed an abnormal Ca2+ gradient. In this report we localize the ATP2C1, demonstrate its physiologic relevance in mammalian cells, and measure intraorganelle Golgi Ca2+ in keratinocytes.

Live-cell detection of intracellular enzyme activity requires that substrates are cell-permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real-time detection of apoptosis in living cells. We have synthesized a highly cell-permeable caspase-3 substrate, DEVD-NucView488, by linking a fluorogenic DNA-binding dye to the caspase-3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD-NucView488 detected caspase-3 activation within a live-cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD-NucView488 exhibited no toxicity and normal apoptotic progression. DEVD-NucView488 is an ideal substrate for kinetic studies of caspase-3 activation because it detects caspase-3 activity in real-time and also efficiently labels DNA in nuclei of caspase-3-activated cells for real-time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real-time intracellular enzyme activity.

Keratinocyte differentiation, adhesion and motility are directed by extracellular Ca2+ concentration increases, which in turn increase intracellular Ca2+ levels. Normal keratinocytes, in contrast to most non-excitable cells, require Ca2+ release from both Golgi and endoplasmic reticulum Ca2+ stores for efficient Ca2+ signaling. Dysfunction of the Golgi human secretory pathway Ca2+-ATPase hSPCA1, encoded by ATP2C1, abrogates Ca2+ signaling and causes the acantholytic genodermatosis, Hailey-Hailey disease. We have examined the role of the endoplasmic reticulum Ca2+ store, established and maintained by the sarcoplasmic and endoplasmic reticulum Ca2+-ATPase SERCA2 encoded by ATP2A2, in Ca2+ signaling. Although previous studies have shown acute SERCA2 inactivation to abrogate Ca2+ signaling, we find that chronic inactivation of ATP2A2 in keratinocytes from patients with the similar acantholytic genodermatosis, Darier disease, does not impair the response to raised extracellular Ca2+ levels. This normal response is due to a compensatory upregulation of hSPCA1, as inactivating ATP2C1 expression with siRNA blocks the response to raised extracellular Ca2+ concentrations in both normal and Darier keratinocytes. ATP2C1 inactivation also diminishes Darier disease keratinocyte viability, suggesting that compensatory ATP2C1 upregulation maintains viability and partially compensates for defective endoplasmic reticulum Ca2+-ATPase in Darier disease keratinocytes. Keratinocytes thus are unique among mammalian cells in their ability to use the Golgi Ca2+ store to mediate Ca2+ signaling.

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, induces a G 1 cell-cycle arrest of human breast cancer cells, although the direct cellular targets that mediate this process are unknown. Treatment of highly invasive MDA-MB-231 breast cancer cells with I3C shifted the stable accumulation of cyclin E protein from the hyperactive lower-molecular-mass 35-kDa form that is associated with cancer cell proliferation and poor clinical outcomes to the 50-kDa cyclin E form that typically is expressed in normal mammary tissue. An in vitro cyclin E processing assay, in combination with zymography, demonstrated that I3C, but not its natural dimer, 3,3 -diindolylmethane, disrupts proteolytic processing of the 50-kDa cyclin E into the lower-molecular-mass forms by direct inhibition of human neutrophil elastase enzymatic activity. Analysis of elastase enzyme kinetics using either cyclin E or N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanalide as substrates demonstrated that I3C acts as a noncompetitive inhibitor of elastase activity with an inhibitory constant of Ϸ12 M. Finally, siRNA ablation of neutrophil elastase protein production in MDA-MB-231 cells mimicked the I3C-disrupted processing of the 50-kDa cyclin E protein and the indole-induced cell-cycle arrest. Taken together, our results demonstrate that elastase is the first identified specific target protein for I3C and that the direct I3C inhibition of elastase enzymatic activity implicates the potential use of this indole, or related compounds, in targeted therapies of human breast cancers where high elastase levels are correlated with poor prognosis.A critical challenge in controlling breast cancer is the identification of therapeutic agents that can effectively control the growth of both estrogen-responsive and nonresponsive breast cancer cells with reduced side effects, especially during prolonged treatments. Epidemiological studies show that frequent consumption of certain vegetables is associated with a lower incidence of cancers at various sites (1). For example, the consumption of Brassica (cruciferous) vegetables, such as cabbage, broccoli, and Brussels sprouts, is directly associated with decreased risk of reproductive tissue cancers in humans (2, 3) and reduced tumor incidence in experimental animals (4). These studies implicate the existence of specific biologically active phytochemicals that represent a largely untapped source of potent chemotherapeutic agents. One such promising molecule is indole-3-carbinol (I3C), a natural compound derived from glycobrassicin in Brassica vegetables, which has been shown to exhibit potent anticarcinogenic properties in a wide range of cancers such as lung, liver, colon, cervical, endometrial, prostate, and breast cancer (5-7). In addition, out of broad spectrum of analyzed phytochemicals, I3C was 1 of the few that tested positive as a chemopreventative agent in a panel of short-term bioassays relevant to carcinogen-induced DNA damage, tumor initiat...

The phytochemical indole-3-carbinol (I3C), from cruciferous vegetables such as broccoli, has been shown to elicit a potent anti-proliferative response in human breast cancer cell lines. Treatment of the immortalized human mammary epithelial cell line MCF10A with I3C induced a G1 cell cycle arrest, elevated p53 tumor suppressor protein levels and stimulated expression of downstream transcriptional target, p21. I3C treatment also elevated p53 levels in several breast cancer cell lines that express mutant p53. I3C did not arrest MCF10A cells stably transfected with dominant-negative p53, establishing a functional requirement for p53. Cell fractionation and immunolocalization studies revealed a large fraction of stabilized p53 protein in the nucleus of I3C-treated MCF10A cells. With I3C treatment, phosphatidyl-inositol-3-kinase family member ataxia telangiectasia-mutated (ATM) was phosphorylated, as were its substrates p53, CHK2 and BRCA1. Phosphorylation of p53 at the N-terminus has previously been shown to disrupt the interaction between p53 and its ubiquitin ligase, MDM2, and therefore stabilizing p53. Coimmunoprecipitation analysis revealed that I3C reduced by 4-fold the level of MDM2 protein that associated with p53. The p53-MDM2 interaction and absence of p21 production were restored in cells treated with I3C and the ATM inhibitor wortmannin. Significantly, I3C does not increase the number of 53BP1 foci or H2AX phosphorylation, indicating that ATM is activated independent of DNA double-strand breaks. Taken together, our results demonstrate that I3C activates ATM signaling through a novel pathway to stimulate p53 phosphorylation and disruption of the p53-MDM2 interaction, which releases p53 to induce the p21 CDK inhibitor and a G1 cell cycle arrest. ' 2005 Wiley-Liss, Inc.Key words: indole-3-carbinol; breast cancer; p53; MDM2; cell cycle arrest; ATM Indole-3-carbinol (I3C), a phytochemical of Brassica vegetables such as broccoli and cabbage, has been shown to prevent spontaneous and carcinogen-induced mammary tumors.1-3 I3C has a direct anti-proliferative effect in both estrogen-dependent and independent human breast cancer cell lines. 4 In tissue culture, I3C treatment of human adenocarcinoma cells induces a G1 cell cycle arrest that is associated with a decreased expression of cyclindependent kinase 6 (CDK6), and inhibited specific enzymatic activity of CDK2. 4,5 In [ 3 H]I3C-treated MCF-7 cells, the I3C condensation product 3,3 0 -diindolylmethane (DIM) represents 40% of radiolabeled compounds in the nuclear fraction, suggesting that DIM may mediate a subset of the transcriptional responses to I3C. 6 DIM has been shown to decrease CDK2 activity through Sp1-mediated transcriptional upregulation of CDK inhibitor p21. 7In addition to their anti-proliferative effects, treatment with I3C or DIM also causes a p53-independent apoptotic response in cultured breast cancer cells. [8][9][10] I3C was shown to induce apoptosis of the tumorigenic MCF10CA1a cell line, 11 but the mechanism of growth inhibition of the isogen...

We have previously shown that the Na+/H+ antiporter (NHE1) is an essential endogenous pathway responsible for stratum corneum (SC) acidification. Since the epidermis must re-establish its epidermal barrier after acute barrier perturbations, we asked whether the NHE1 was, in turn, regulated by changes in barrier status. We found that in vivo epidermal NHE1 expression was upregulated within hours of barrier disruption. We next asked whether NHE1 was regulated by barrier status per se, or by the SC alkalinization that accompanies barrier perturbation. NHE1 was upregulated by alkalinizing SC pH, whereas this antiporter was downregulated by acidifying SC pH, independent of changes in barrier status. Moreover, acidifying SC pH overrode the effects of barrier break in regulating NHE1 expression, suggesting that SC alkalinization is the major stimulus for increased NHE1 expression. Finally, we confirmed that the keratinocyte NHE1 antiporter is regulated by extracellular pH independent of barrier status, by demonstrating that NHE1 was upregulated in cultured keratinocytes exposed to pH 8.3 medium and downregulated in cultured keratinocytes exposed to pH 6.3 medium. These data suggest that the keratinocyte NHE1 is regulated by extracellular pH. SC barrier break also upregulates NHE1 expression, but this response seems to be mediated by concomitant changes in SC pH.

Human melanoma cells displaying distinct PTEN genotypes were used to assess the cellular role of this important tumor suppressor protein during the anti-proliferative response induced by the chemopreventative agent indole-3-carbinol (I3C), a natural indolecarbinol compound derived from the breakdown of glucobrassicin produced in cruciferous vegetables such as broccoli and Brussels sprouts. I3C induced a G1-phase cell cycle arrest and apoptosis by stabilization of PTEN in human melanoma cells that express wild-type PTEN, but not in cells with mutant or null PTEN genotypes. Importantly, normal human epidermal melanocytes were unaffected by I3C treatment. In wild-type PTEN-expressing melanoma xenografts, formed in athymic mice, I3C inhibited the in vivo tumor growth rate and increased PTEN protein levels in the residual tumors. Mechanistically, I3C disrupted the ubiquitination of PTEN by NEDD4-1 (NEDD4), which prevented the proteasome-mediated degradation of PTEN without altering its transcript levels. RNAi-mediated knockdown of PTEN prevented the I3C induced apoptotic response; whereas, knockdown of NEDD4-1 mimicked the I3C apoptotic response, stabilized PTEN protein levels and down-regulated phosphorylated AKT1 levels. Co-knockdown of PTEN and NEDD4-1 revealed that I3C regulated apoptotic signaling through NEDD4-1 requires the presence of the wild-type PTEN protein. Finally, in silico structural modeling in combination with isothermal titration calorimetry analysis demonstrated that I3C directly interacts with purified NEDD4-1 protein. Implications This study identifies NEDD4-1 as a new I3C target protein, and that the I3C disruption of NEDD4-1 ubiquitination activity triggers the stabilization of the wild-type PTEN tumor suppressor to induce an anti-proliferative response in melanoma.

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