Shuchen Gu

Regulation of erythrocyte survival by AMP-activated protein kinase

AMP‐activated protein kinase (AMPK), an energy‐sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca2+ activity ([Ca2+]i) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca2+]i (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence‐activated cell sorting (FACS) analysis. Glucose removal increased [Ca2+]i, decreased cell volume, and increased PS exposure. The AMPK‐inhibitor compound C (20 µM) did not significantly modify eryptosis under glucose‐replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca2+]i by Ca2+ ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR;1 mM). As compared with erythrocytes from wild‐type littermates (ampk+/+), erythrocytes from AMPKctl‐deficient mice (ampk−/−) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk−/− mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes.—Föller, M., Sopjani, M., Koka, S., Gu, S., Mahmud, H., Wang, K., Floride, E., Schleicher, E., Schulz, E., Münzel, T., Lang, F. Regulation of erythrocyte survival by AMP‐activated protein kinase. FASEB J. 23, 1072–1080 (2009)

Janus kinase 3 is expressed in erythrocytes, phosphorylated upon energy depletion and involved in the regulation of suicidal erythrocyte death.

Janus kinase 3, a tyrosine kinase expressed in haematopoetic tissues, plays a decisive role in T-lymphocyte survival. JAK3 deficiency leads to (Severe) Combined Immunodeficiency (SCID) resulting from enhanced lymphocyte apoptosis. JAK3 is activated by phosphorylation. Nothing is known about expression of JAK3 in erythrocytes, which may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure and cell shrinkage. Triggers of eryptosis include energy depletion. The present study utilized immunohistochemistry and confocal microscopy to test for JAK3 expression and phosphorylation, and FACS analysis to determine phosphatidylserine exposure (annexin binding) and cell volume (forward scatter). As a result, JAK3 was expressed in erythrocytes and phosphorylated following 24h and 48h glucose depletion. Forward scatter was slightly but significantly smaller in erythrocytes from JAK3-deficient mice (jak3-/-) than in erythrocytes from wild type mice (jak3+/+). Annexin V binding was similarly low in both genotypes. The JAK3 inhibitors WHI-P131/JANEX-1 (4-(4′-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 156µM) and WHI-P154 (4-[(3′-Bromo-4′-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline, 11.2µM) did not significantly modify annexin V binding or forward scatter. Glucose depletion increased annexin V binding, an effect significantly blunted in jak3-/- erythrocytes and in the presence of the JAK3 inhibitors. The observations disclose a completely novel role of Janus kinase 3, i.e. the triggering of cell membrane scrambling in energy depleted erythrocytes.

Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene.

Loss‐of‐function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apcMin/+) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca2+ activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC‐deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin–luciferase in isolated erythrocytes from apcMin/+ mice and wild‐type mice (apc+/+). Clearance of circulating erythrocytes was estimated by carboxyfluorescein‐diacetate‐succinimidyl‐ester labelling. As a result, apcMin/+ mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Extracellular Ca2+ removal or inhibition of Ca2+ entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca2+‐entry by treatment with Ca2+‐ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apcMin/+ erythrocytes than in apc+/+ erythrocytes. Following retrieval and injection into the circulation of the same mice, apcMin/+ erythrocytes were more rapidly cleared from circulating blood than apc+/+ erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apcMin/+ mice. The observations point to accelerated eryptosis and subsequent clearance of apcMin/+ erythrocytes, which contributes to or even accounts for the enhanced erythrocyte turnover, anaemia and splenomegaly in those mice.

Transcription Factor NF-κB Regulates Expression of Pore-forming Ca2+ Channel Unit, Orai1, and Its Activator, STIM1, to Control Ca2+ Entry and Affect Cellular Functions

The serum and glucocorticoid-inducible kinase SGK1 increases the activity of Orai1, the pore forming unit of store-operated Ca2+ entry, and thus influences Ca2+-dependent cellular functions such as migration. SGK1 further regulates transcription factor nuclear factor κB (NF-κB). This study explored whether SGK1 influences transcription of Orai1 and/or STIM1, the Orai1-activating Ca2+ sensor. Orai1 and STIM1 transcript levels were decreased in mast cells from SGK1 knock-out mice and increased in HEK293 cells transfected with active S422DSGK1 but not with inactive K127NSGK1 or in S422DSGK1-transfected cells treated with the NF-κB inhibitor Wogonin (100 μm). Treatment with the stem cell factor enhanced transcript levels of STIM1 and Orai1 in sgk1+/+ but not in sgk1−/− mast cells and not in sgk1+/+ cells treated with Wogonin. Orai1 and STIM1 transcript levels were further increased in sgk1+/+ and sgk1−/− mast cells by transfection with active NF-κB subunit p65 as well as in HEK293 cells by transfection with NF-κB subunits p65/p50 or p65/p52. They were decreased by silencing of NF-κB subunits p65, p50, or p52 or by NF-κB inhibitor Wogonin (100 μm). Luciferase assay and chromatin immunoprecipitation defined NF-κB-binding sites in promoter regions accounting for NF-κB sensitive genomic regulation of STIM1 and Orai1. Store-operated Ca2+ entry was similarly increased by overexpression of p65/p50 or p65/p52 and decreased by treatment with Wogonin. Transfection of HEK293 cells with p65/p50 or p65/p52 further augmented migration. The present observations reveal powerful genomic regulation of Orai1/STIM1 by SGK1-dependent NF-κB signaling.

Chorein-sensitive polymerization of cortical actin and suicidal cell death in chorea-acanthocytosis

Chorea‐acanthocytosis is an inevitably lethal genetic disease characterized by a progressive hyperkinetic movement disorder and cognitive and behavioral abnormalities as well as acanthocytosis. The disease is caused by loss‐of‐function mutations of the gene encoding vacuolar protein sorting‐associated protein 13A (VPS13A) or chorein, a protein with unknown function expressed in various cell types. How chorein deficiency leads to the pathophysiology of chorea‐acanthocytosis remains enigmatic. Here we show decreased phosphoinositide‐3‐kinase (PI3K)‐p85‐subunit phosphorylation, ras‐related C3 botunlinum toxin substrate 1 (Rac1) activity, and p21 protein‐activated kinase 1 (PAK1) phosphorylation as well as depolymerized cortical actin in erythrocytes from patients with chorea‐acanthocytosis and in K562‐erythrocytic cells following chorein silencing. Pharmacological inhibition of PI3K, Rac1, or PAK1 similarly triggered actin depolymerization. Moreover, in K562 cells, both chorein silencing and PAK1 inhibition with IPA‐3 decreased phosphorylation of Bad, a Bcl2‐associated protein, promoting apoptosis by forming mitochondrial pores, followed by mitochondrial depolarization, DNA fragmentation, and phosphatidylserine exposure at the cell surface, all hallmarks of apoptosis. Our observations reveal chorein as a novel powerful regulator of cytoskeletal architecture and cell survival, thus explaining erythrocyte misshape and possibly neurodegeneration in chorea‐acanthocytosis.—Föller, M., Hermann, A., Gu, S., Alesutan, I., Qadri, S. M., Borst, O., Schmidt, E.‐M., Schiele, F., Müller vom Hagen, J., Saft, C., Schöls, L., Lerche, H., Stournaras, C., Storch, A., Lang, F. Chorein‐sensitive polymerization of cortical actin and suicidal cell death in chorea‐acanthocytosis. FASEB J. 26, 1526–1534 (2012). www.fasebj.org

Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo

BackgroundMembrane androgen receptors (mAR) have been implicated in the regulation of cell growth, motility and apoptosis in prostate and breast cancer. Here we analyzed mAR expression and function in colon cancer.ResultsUsing fluorescent mAR ligands we showed specific membrane staining in colon cell lines and mouse xenograft tumor tissues, while membrane staining was undetectable in healthy mouse colon tissues and non-transformed intestinal cells. Saturation/displacement assays revealed time- and concentration-dependent specific binding for testosterone with a KD of 2.9 nM. Stimulation of colon mAR by testosterone albumin conjugates induced rapid cytoskeleton reorganization and apoptotic responses, even in the presence of anti-androgens. The actin cytoskeleton drug cytochalasin B effectively inhibited the pro-apoptotic responses and caspase-3 activation. Interestingly, in vivo studies revealed that mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors.ConclusionOur results demonstrate for the first time that functional mARs are predominantly expressed in colon tumors and that their activation results in induction of anti-tumor responses in vitro and extensive reduction of tumor incidence in vivo.

Relative resistance of SGK1 knockout mice against chemical carcinogenesis

The serum and glucocorticoid inducible kinase SGK1 was originally cloned from mammary tumor cells. SGK1 was found to be up‐regulated in a variety of tumors, but down‐regulated in several distinct tumors. Thus, evidence for a role of SGK1 in tumor growth remained conflicting. According to in vitro observations, SGK1 is up‐regulated by the oncogene β‐catenin and negatively regulates the proapoptotic transcription factor FOXO3a, which in turn stimulates transcription of the Bcl2‐interacting mediator BIM. This study aimed to define the role of SGK1 in colon carcinoma in vivo. SGK1 knockout mice (sgk1−/−) and their wild type littermates (sgk1+/+) were subjected to chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2‐dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium for 7 days). Moreover, SGK1 was silenced in HEK293 cells. FOXO3a and BIM protein abundance was determined by Western blotting and immunohistochemistry. Following chemical cancerogenesis, sgk1−/−mice developed significantly less colonic tumors than sgk1+/+mice. According to Western blotting and immunohistochemistry, SGK1 deficiency enhanced the expression of FOXO3a and BIM both, in vitro and in vivo. SGK1 deficiency counteracts the development of colonic tumors, an effect at least in part due to up‐regulation of FOXO3a and BIM.

Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2

The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.

Sgk1 sensitivity of Na+/H+ exchanger activity and cardiac remodeling following pressure overload

Sustained increase of cardiac workload is known to trigger cardiac remodeling with eventual development of cardiac failure. Compelling evidence points to a critical role of enhanced cardiac Na+/H+ exchanger (NHE1) activity in the underlying pathophysiology. The signaling triggering up-regulation of NHE1 remained, however, ill defined. The present study explored the involvement of the serum- and glucocorticoid-inducible kinase Sgk1 in cardiac remodeling due to transverse aortic constriction (TAC). To this end, experiments were performed in gene targeted mice lacking functional Sgk1 (sgk1−/−) and their wild-type controls (sgk1+/+). Transcript levels have been determined by RT-PCR, cytosolic pH (pHi) utilizing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, Na+/H+ exchanger activity by the Na+-dependent realkalinization after an ammonium pulse, ejection fraction (%) utilizing cardiac cine magnetic resonance imaging and cardiac glucose uptake by PET imaging. As a result, TAC increased the mRNA expression of Sgk1 in sgk1+/+ mice, paralleled by an increase in Nhe1 transcript levels as well as Na+/H+ exchanger activity, all effects virtually abrogated in sgk1−/− mice. In sgk1+/+ mice, TAC induced a decrease in Pgc1a mRNA expression, while Spp1 mRNA expression was increased, both effects diminished in the sgk1−/− mice. TAC was followed by a significant increase of heart and lung weight in sgk1+/+ mice, an effect significantly blunted in sgk1−/− mice. TAC increased the transcript levels of Anp and Bnp, effects again significantly blunted in sgk1−/− mice. TAC increased transcript levels of Collagen I and III as well as Ctgf mRNA and CTGF protein abundance, effects significantly blunted in sgk1−/− mice. TAC further decreased the ejection fraction in sgk1+/+ mice, an effect again attenuated in sgk1−/− mice. Also, cardiac FDG-glucose uptake was increased to a larger extent in sgk1+/+ mice than in sgk1−/− mice after TAC. These observations point to an important role for SGK1 in cardiac remodeling and development of heart failure following an excessive work load.

SGK1-dependent intestinal tumor growth in APC-deficient mice.

Adenomatous polyposis coli (APC) is inactivated in familial adenomatous polyposis and sporadic colorectal cancer. Mice carrying defective APC (apc^Min/+) spontaneously develop gastrointestinal tumors. APC binds GSK3β, which phosphorylates β-catenin thus fostering its degradation. β-catenin upregulates the serum- and glucocorticoid-inducible kinase Sgk1, which inhibits GSK3β. The present study explored the role of SGK1 in tumor growth of apc^Min/+mice. apc^Min/+mice were crossed with SGK1-knockout mice (sgk1^-/-) and their wild type littermates (sgk1^+/+) generating apc^Min/+/sgk1^-/-mice and apc^Min/+/sgk1^+/+mice. β-catenin abundance was determined by Western blotting and confocal microscopy. As a result apc^Min/+/sgk1^+/+mice developed significantly more intestinal tumors than apc^Min/+/sgk1^-/-mice. Following chemical cancerogenesis, colonic β-catenin protein abundance was significantly higher in sgk1^+/+mice than in sgk1^-/-mice. β-catenin expression was significantly increased in HEK293 cells treated with dexamethasone for upregulation of Sgk1. In conclusion, SGK1 expression favors the development of intestinal tumors in APC-deficient mice, an effect at least partially due to enhanced β-catenin protein abundance.