Gennady Ermak

Calcium and oxidative stress: from cell signaling to cell death

Reactive oxygen and nitrogen species can be used as a messengers in normal cell functions. However, at oxidative stress levels they can disrupt normal physiological pathways and cause cell death. Such a switch is largely mediated through Ca(2+) signaling. Oxidative stress causes Ca(2+) influx into the cytoplasm from the extracellular environment and from the endoplasmic reticulum or sarcoplasmic reticulum (ER/SR) through the cell membrane and the ER/SR channels, respectively. Rising Ca(2+) concentration in the cytoplasm causes Ca(2+) influx into mitochondria and nuclei. In mitochondria Ca(2+) accelerates and disrupts normal metabolism leading to cell death. In nuclei Ca(2+) modulates gene transcription and nucleases that control cell apoptosis. Both in nuclei and cytoplasm Ca(2+) can regulate phosphorylation/dephosphorylation of proteins and can modulate signal transduction pathways as a result. Since oxidative stress is associated with many diseases and the aging process, understanding how oxidants alter Ca(2+) signaling can help to understand process of aging and disease, and may lead to new strategies for their prevention.

PRODUCTION AND RELEASE OF PROOPIOMELANOCORTIN (POMC) DERIVED PEPTIDES BY HUMAN MELANOCYTES AND KERATINOCYTES IN CULTURE : REGULATION BY ULTRAVIOLET B

It is demonstrated that ultraviolet B (UVB) radiation stimulates increased expression of the proopiomelanocortin (POMC) gene which is accompanied by production and release of alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) by both normal and malignant human melanocytes and keratinocytes. The production and release of both peptides are also stimulated by dibutyryl cyclic adenosine monophosphate (dbcAMP) and interleukin 1 alpha (IL-1 alpha) but not by endothelin-1 (ET-1) or tumor necrosis factor-alpha (TNF-alpha). N-acetyl-cysteine (NAC), a precursor of glutathione (GSH), an intracellular free radical scavenger, abolishes the UVB-stimulated POMC peptide production and secretion. Conclusions are as follows: (1) Cultured human cells of cutaneous origin, namely keratinocytes and melanocytes, can produce and express POMC; (2) POMC expression is enhanced by exposure to UVB, possibly through a cyclic AMP-dependent pathway; and (3) The action of UVB on POMC production may involve a cellular response to oxidative stress.

Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease.

The DSCR1(Adapt78) gene was independently discovered as a resident of the “Down syndrome candidate region”and as an “adaptive response”shock or stress gene that is transiently induced during oxidative stress. Recently the DSCR1 (Adapt78) gene product was discovered to be an inhibitor of the serine/threonine phosphatase, calcineurin, and its signaling pathways. We hypothesized thatDSCR1 (Adapt78) might also be involved in the development of Alzheimers disease. To address this question we first studiedDSCR1 (Adapt78) in multiple human tissues and found significant expression in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 (Adapt78) is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When we compared DSCR1 (Adapt78) mRNA expression in post-mortem brain samples from Alzheimers disease patients and individuals who had died with no Alzheimers diagnosis, we found that DSCR1 (Adapt78) mRNA levels were about twice as high in age-matched Alzheimers patients as in controls.DSCR1 (Adapt78) mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimers disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimers symptoms) also exhibited DSCR1 (Adapt78) mRNA levels two to three times higher than controls. Using a cell culture model we discovered that the amyloid β1–42 peptide, which is a major component of senile plaques in Alzheimers, can directly induce increased expression of DSCR1 (Adapt78). Our findings associate DSCR1 (Adapt78) with such major hallmarks of Alzheimers disease as amyloid protein, senile plaques, and neurofibrillary tangles.

Mitochondrial fission and cristae disruption increase the response of cell models of Huntington's disease to apoptotic stimuli

Huntingtons disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, is accompanied by multiple mitochondrial alterations. Here, we show that mitochondrial fragmentation and cristae alterations characterize cellular models of HD and participate in their increased susceptibility to apoptosis. In HD cells, the increased basal activity of the phosphatase calcineurin dephosphorylates the pro‐fission dynamin related protein 1 (Drp1), increasing its mitochondrial translocation and activation, and ultimately leading to fragmentation of the organelle. The fragmented HD mitochondria are characterized by cristae alterations that are aggravated by apoptotic stimulation. A genetic analysis indicates that correction of mitochondrial elongation is not sufficient to rescue the increased cytochrome c release and cell death observed in HD cells. Conversely, the increased apoptosis can be corrected by manoeuvres that prevent fission and cristae remodelling. In conclusion, the cristae remodelling of the fragmented HD mitochondria contributes to their hypersensitivity to apoptosis.

Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin

Evidence is provided that human skin, the largest body organ exposed to multiple stressors, expresses proopiomelanocortin (POMC), corticotropin releasing hormone (CRH) and CRH‐receptor (CRHR) genes in vivo. In vitro studies show that POMC and CRHR mRNAs are transcribed in melanocytes, cells derived from the neural crest, and in keratinocytes, cells derived from the ectoderm. CRH mRNA is transcribed in cultured melanocytes but not in keratinocytes. It is proposed that an equivalent of the ‘hypothalamus‐pituitary axis’ composed of the CRH‐CRHR‐POMC loop is conserved in mammalian skin.

Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin.

Kelvin J. A. Davies,* Gennady Ermak,* Beverley A. Rothermel, Melanie Pritchard, Joseph Heitman, Joohong Ahnn, Flavio Henrique-Silva, Dana Crawford, Silvia Canaider,** Pierluigi Strippoli,** Paolo Carinci,** Kyung-Tai Min, Deborah S. Fox, Kyle W. Cunningham, Rhonda Bassel-Duby, Eric N. Olson, Zhuohua Zhang, R. Sanders Williams, Hans-Peter Gerber,*** Merce Perez-Riba, Hisao Seo, Xia Cao, Claude B. Klee, Juan Miguel Redondo, Lois J. Maltais, Elspeth A. Bruford, Sue Povey, Jeffery D. Molkentin,**** Frank D. McKeon, Elia J. Duh, Gerald R. Crabtree,§§§§ Martha S. Cyert, Susana de la Luna, and Xavier Estivill

HSP70 mediates dissociation and reassociation of the 26S proteasome during adaptation to oxidative stress.

We report an entirely new role for the HSP70 chaperone in dissociating 26S proteasome complexes (into free 20S proteasomes and bound 19S regulators), preserving 19S regulators, and reconstituting 26S proteasomes in the first 1-3h after mild oxidative stress. These responses, coupled with direct 20S proteasome activation by poly(ADP ribose) polymerase in the nucleus and by PA28αβ in the cytoplasm, instantly provide cells with increased capacity to degrade oxidatively damaged proteins and to survive the initial effects of stress exposure. Subsequent adaptive (hormetic) processes (3-24h after stress exposure), mediated by several signal transduction pathways and involving increased transcription/translation of 20S proteasomes, immunoproteasomes, and PA28αβ, abrogate the need for 26S proteasome dissociation. During this adaptive period, HSP70 releases its bound 19S regulators, 26S proteasomes are reconstituted, and ATP-stimulated proteolysis is restored. The 26S proteasome-dependent, and ATP-stimulated, turnover of ubiquitinylated proteins is essential for normal cell metabolism, and its restoration is required for successful stress adaptation.

Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress

Hyperphosphorylated tau proteins accumulate in the paired helical filaments of neurofibrillary tangles seen in such tauopathies as Alzheimers disease. In the present paper we show that tau turnover is dependent on degradation by the proteasome (inhibited by MG132) in HT22 neuronal cells. Recombinant human tau was rapidly degraded by the 20 S proteasome in vitro, but tau phosphorylation by GSK3beta (glycogen synthase kinase 3beta) significantly inhibited proteolysis. Tau phosphorylation was increased in HT22 cells by OA [okadaic acid; which inhibits PP (protein phosphatase) 1 and PP2A] or CsA [cyclosporin A; which inhibits PP2B (calcineurin)], and in PC12 cells by induction of a tet-off dependent RCAN1 transgene (which also inhibits PP2B). Inhibition of PP1/PP2A by OA was the most effective of these treatments, and tau hyperphosphorylation induced by OA almost completely blocked tau degradation in HT22 cells (and in cell lysates to which purified proteasome was added) even though proteasome activity actually increased. Many tauopathies involve both tau hyperphosphorylation and the oxidative stress of chronic inflammation. We tested the effects of both cellular oxidative stress, and direct tau oxidative modification in vitro, on tau proteolysis. In HT22 cells, oxidative stress alone caused no increase in tau phosphorylation, but did subtly change the pattern of tau phosphorylation. Tau was actually less susceptible to direct oxidative modification than most cell proteins, and oxidized tau was degraded no better than untreated tau. The combination of oxidative stress plus OA treatment caused extensive tau phosphorylation and significant inhibition of tau degradation. HT22 cells transfected with tau-CFP (cyan fluorescent protein)/tau-GFP (green fluorescent protein) constructs exhibited significant toxicity following tau hyperphosphorylation and oxidative stress, with loss of fibrillar tau structure throughout the cytoplasm. We suggest that the combination of tau phosphorylation and tau oxidation, which also occurs in tauopathies, may be directly responsible for the accumulation of tau aggregates.

The DSCR1 (Adapt78) isoform 1 protein calcipressin 1 inhibits calcineurin and protects against acute calcium-mediated stress damage, including transient oxidative stress

Although DSCR1 (Adapt78) has been associated with successful adaptation to oxidative stress and calcium stress and with devastating diseases such as Alzheimers and Down syndrome, no rationale for these apparently contradictory findings has been tested. In fact, DSCR1 (Adapt78) has not yet been proved to provide protection against acute oxidative stress or calcium stress. We have addressed this question using cross‐adaptation to H2O2 and the calcium ionophore A23187, stable DSCR1 (Adapt78) transfection and over‐expression in hamster HA‐1 cells, ‘tet‐off’ regulated DSCR1 (Adapt78) isoform 1 transgene expression in human PC‐12 cells, and DSCR1 (Adapt78) antisense oligonucleotides to test the ability of the DSCR1 (Adapt78) protein product calcipressin 1 (a calcineurin inhibitor) to protect against oxidative stress and calcium stress. Under all conditions, resistance to oxidative stress and calcium stress increased as a function of DSCR1 (Adapt78)/calcipressin 1 expression and decreased as gene/protein expression diminished. We conclude that cells may transiently use increased expression of the DSCR1 (Adapt78) gene product calcipressin 1 to provide short‐term protection against acute oxidative stress and other calcium‐mediated stresses, whereas chronic overexpression may be associated with Alzheimer disease progression.—Ermak, G., Harris, C. D., Davies, K. J. A. The DSCR1 (Adapt78) isoform 1 protein calcipressin 1 inhibits calcineurin and protects against acute calcium‐mediated stress damage, including transient oxidative stress. FASEB J. 16, 814–824 (2002)

Metabolism of Serotonin to N-Acetylserotonin, Melatonin, and 5-Methoxytryptamine in Hamster Skin Culture*

Biotransformation of [H]serotonin by cultured hamster skin to H-metabolites corresponding to N-acetylserotonin (NAS), melatonin, and 5-methoxytryptamine (5-MT) was demonstrated. This process was time-dependent, with the highest production of radioactive NAS and melatonin metabolites after 3 and 5 h of incubation followed by a decrease in the rate of metabolite release into the media. Conversely, the formation of radioactive metabolite corresponding to 5-MT increased gradually during skin culture, reaching the highest level after 24 h of incubation. The production of H-metabolites, corresponding to NAS, melatonin, and 5-MT, was stimulated by forskolin with a maximum effect of forskolin at 10 μM concentration. The gas chromatographic/mass spectroscopy analysis of the fraction eluting at the retention time of NAS standard material showed that it contained NAS, further confirming production and release of NAS into the media by hamster skin. Therefore, we conclude that mammalian skin can acetylate serotonin to NAS and postulate that the NAS is further metabolized by the skin to form melatonin which is subsequently transformed to 5-MT.