ER Calcium Homeostasis Research Articles

Wolfram Syndrome 1: A Neuropsychiatric Perspective on a Rare Disease.

Wolfram syndrome 1 (WS1) is an uncommon autosomal recessive neurological disorder that is characterized by diabetes insipidus, early-onset non-autoimmune diabetes mellitus, optic atrophy, and deafness (DIDMOAD). Other clinical manifestations are neuropsychiatric symptoms, urinary tract alterations, and endocrinological disorders. The rapid clinical course of WS1 results in death by the age of 30. Severe brain atrophy leads to central respiratory failure, which is the main cause of death in WS1 patients. Mutations in the WFS1 gene, located on chromosome 4p16, account for approximately 90% of WS1 cases. The gene produces wolframin, a transmembrane glycoprotein widely distributed and highly expressed in retinal, neural, and muscular tissues. Wolframin plays a crucial role in the regulation of apoptosis, insulin signaling, and ER calcium homeostasis, as well as the ER stress response. WS1 has been designated as a neurodegenerative and neurodevelopmental disorder due to the numerous abnormalities in the ER stress-mediated system. WS1 is a devastating neurodegenerative disease that affects patients and their families. Early diagnosis and recognition of the initial clinical signs may slow the disease's progression and improve symptomatology. Moreover, genetic counseling should be provided to the patient's relatives to extend multidisciplinary care to their first-degree family members. Regrettably, there are currently no specific drugs for the therapy of this fatal disease. A better understanding of the etiology of WS1 will make possible the development of new therapeutic approaches that may enhance the life expectancy of patients. This review will examine the pathogenetic mechanisms, development, and progression of neuropsychiatric symptoms commonly associated with WS1. A thorough understanding of WS1's neurophysiopathology is critical for achieving the goal of improving patients' quality of life and life expectancy.

The A-kinase anchoring protein Yotiao decrease the ER calcium content by inhibiting the store operated calcium entry

The meticulous regulation of ER calcium (Ca2+) homeostasis is indispensable for the proper functioning of numerous cellular processes. Disrupted ER Ca2+ balance is implicated in diverse diseases, underscoring the need for a systematic exploration of its regulatory factors in cells. Our recent genomic-scale screen identified a scaffolding protein A-kinase anchoring protein 9 (AKAP9) as a regulator of ER Ca2+ levels, but the underlying molecular mechanisms remain elusive. Here, we reveal that Yotiao, the smallest splicing variant of AKAP9 decreased ER Ca2+ content in animal cells. Additional testing using a combination of Yotiao truncations, knock-out cells and pharmacological tools revealed that, Yotiao does not require most of its interactors, including type 1 inositol 1,4,5-trisphosphate receptors (IP3R1), protein kinase A (PKA), protein phosphatase 1 (PP1), adenylyl cyclase type 2 (AC2) and so on, to reduce ER Ca2+ levels. However, adenylyl cyclase type 9 (AC9), which is known to increases its cAMP generation upon interaction with Yotiao for the modulation of potassium channels, plays an essential role for Yotiao's ER-Ca2+-lowering effect. Mechanistically, Yotiao may work through AC9 to act on Orai1-C terminus and suppress store operated Ca2+ entry, resulting in reduced ER Ca2+ levels. These findings not only enhance our comprehension of the interplay between Yotiao and AC9 but also contribute to a more intricate understanding of the finely tuned mechanisms governing ER Ca2+ homeostasis.

NRAS Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth.

Our findings suggest that targeting the AHCYL1-IP3R axis presents a novel therapeutic approach for NRAS-mutated melanomas, with potential applicability to all cancers harboring RAS mutations, such as KRAS-mutated human colorectal cancers.

Abstract 283: AHCYL1 is crucial for NRAS mutant-expressing melanoma tumor growth by governing ER calcium homeostasis

Abstract Cancer cells apply metabolic adaptations for proliferation and survival such as the Warburg Effect and the maintenance of redox balance, which are common for different cancer types. However, it remains elusive whether different cancer oncogenic backgrounds require oncogene-specific metabolic adaptations, which could represent unique cancer vulnerabilities for novel therapy development. Herein we report that, using a shRNA library designed to target a subset of genes related to cell metabolism, we identified adenosylhomocysteinase like 1 (AHCYL1) as a “synthetic lethal” partner of oncogenic NRAS mutations, which is selectively important for the proliferation and survival of NRAS mutant-expressing melanoma cells, but not for cells harboring BRAF mutations. AHCYL1 is an inhibitory binding partner of endoplasmic reticulum (ER) calcium channel protein inositol trisphosphate receptor (IP3R) by competing off its agonist inositol triphosphate (IP3) and consequently blocking ER calcium release. In consonance with our finding, AHCYL1 expression levels correlate with mutational status of NRAS but not with BRAF in diverse human melanoma cell lines. In addition, exogenous expression of mutant NRAS but not BRAF in immortal melanocytes MEL-ST cells results in upregulated AHCYL1 transcription and protein expression. Notably, these findings are consistent with the results of Cancer Genome Atlas (TCGA) database analysis, which reveal elevated AHCYL1 transcription in melanoma patients harboring NRAS mutations but not BRAF V600E mutation. Further transcription factor screening studies suggested that the transcription of AHCYL1 is regulated by activating transcription factor 2 (ATF2), which is supported by TCGA database analysis results showing that ATF2 transcription level is selectively higher in NRAS-mutant melanoma patients. Furthermore, AHCYL1-deficiency by shRNA-mediated knockdown and CRISPR/Cas9-mediated knockout significantly reduces in vitro cell proliferation and in vivo tumor growth potential of NRAS mutant-expressing melanoma cells, but not for control melanoma cells expressing BRAF V600E mutant. Mechanistically, AHCYL1 deficiency in NRAS mutant-expressing melanoma cells triggers calcium leakage from the ER, leading to sustained activation of the ER unfolded protein response (UPR) that subsequently attenuates cell proliferation and initiates apoptosis. Altogether, these findings suggest a positive feedback mechanism by which mutant NRAS enhances AHCYL1 expression that contributes to NRAS mutation-dependent melanoma transformation and tumor growth. The AHCYL1-IP3R axis may represent a novel therapeutic target for treatment of NRAS mutant-expressing tumors including melanoma. Citation Format: Chufan Cai, Jiayi Tu, Jeronimo Najarro, Rukang Zhang, Xue Gao, Hao Fan, Freya Zhang, Jiacheng Li, Michele Ciboddo, Shannon Elf, Rong Wu, Jing Chen. AHCYL1 is crucial for NRAS mutant-expressing melanoma tumor growth by governing ER calcium homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 283.

DNAJB12 and Hsp70 Mediate Triage of Misfolded Membrane Proteins for Proteasomal versus Lysosomal Degradation.

The endoplasmic reticulum (ER) fills the cell with a continuous network of sealed membrane tubules and sheets. The ER is subdivided into microdomains mediating one-third of total protein biosynthesis, oxidative protein folding, secretion, protein quality control, calcium signaling, marcoautophagy/autophagy, stress sensing, and apoptosis. Defects in ER-calcium homeostasis underlie several diseases. Damage to the ER by misfolded membrane proteins is suppressed by specific HSPA/Hsp70 and DNAJ/Hsp40 chaperone pairs that select intermediates for ubiquitination and ER-associated degradation (ERAD) via the proteasome. The ER-transmembrane Hsp40 chaperone DNAJB12 and HSPA/Hsp70 also target toxic intermediates of misfolded membrane proteins for ER-associated autophagy (ERAA). DNAJB12-HSPA/Hsp70 maintain membrane protein degradation intermediates in detergent-soluble and degradation-competent states. DNAJB12-HSPA/Hsp70 also interact with the autophagy initiation kinase ULK1 on ER tubules containing ERAD-resistant misfolded membrane proteins (ERAD-RMPs). Omegasomes are ER microdomains where the autophagosome precursor or phagophore (PG) forms. ER tubules loaded with ERAD-RMPs enter omegasomes where they are converted into ER-connected PG (ER-PG). The Atg8 (autophagy related 8)-family member GABARAP (GABA type A receptor-associated protein) facilitates transfer of ERAD-RMPs from ER-PGs to autolysosomes (AL) that dock transiently with omegasomes. This article describes a model for DNAJB12-HSPA/Hsp70 action during the conformation-dependent triage in the ER of misfolded membrane proteins for folding versus proteasomal or AL degradation.

CLIC4 localizes to mitochondrial-associated membranes and mediates cardioprotection.

Mitochondrial-associated membranes (MAMs) are known to modulate organellar and cellular functions and can subsequently affect pathophysiology including myocardial ischemia-reperfusion (IR) injury. Thus, identifying molecular targets in MAMs that regulate the outcome of IR injury will hold a key to efficient therapeutics. Here, we found chloride intracellular channel protein (CLIC4) presence in MAMs of cardiomyocytes and demonstrate its role in modulating ER and mitochondrial calcium homeostasis under physiological and pathological conditions. In a murine model, loss of CLIC4 increased myocardial infarction and substantially reduced cardiac function after IR injury. CLIC4 null cardiomyocytes showed increased apoptosis and mitochondrial dysfunction upon hypoxia-reoxygenation injury in comparison to wild-type cardiomyocytes. Overall, our results indicate that MAM-CLIC4 is a key mediator of cellular response to IR injury and therefore may have a potential implication on other pathophysiological processes.

Wolfram Syndrome 1: From Genetics to Therapy.

Wolfram syndrome 1 (WS1) is a rare neurodegenerative disease transmitted in an autosomal recessive mode. It is characterized by diabetes insipidus (DI), diabetes mellitus (DM), optic atrophy (OA), and sensorineural hearing loss (D) (DIDMOAD). The clinical picture may be complicated by other symptoms, such as urinary tract, endocrinological, psychiatric, and neurological abnormalities. WS1 is caused by mutations in the WFS1 gene located on chromosome 4p16 that encodes a transmembrane protein named wolframin. Many studies have shown that wolframin regulates some mechanisms of ER calcium homeostasis and therefore plays a role in cellular apoptosis. More than 200 mutations are responsible for WS1. However, abnormal phenotypes of WS with or without DM, inherited in an autosomal dominant mode and associated with one or more WFS1 mutations, have been found. Furthermore, recessive Wolfram-like disease without DM has been described. The prognosis of WS1 is poor, and the death occurs prematurely. Although there are no therapies that can slow or stop WS1, a careful clinical monitoring can help patients during the rapid progression of the disease, thus improving their quality of life. In this review, we describe natural history and etiology of WS1 and suggest criteria for a most pertinent approach to the diagnosis and clinical follow up. We also describe the hallmarks of new therapies for WS1.

Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3

Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analog misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.

Endoplasmic reticulum maintains ion homeostasis required for plasma membrane repair

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.

Intersecting roles of ER stress, mitochondrial dysfunction, autophagy, and calcium homeostasis in HIV-associated neurocognitive disorder.

HIV-associated neurocognitive disorder (HAND) is a collective term describing the spectrum of neurocognitive deficits that arise from HIV infection. Although the introduction to highly active antiretroviral therapy (HAART) has prolonged the lifespan of HIV patients, neurocognitive impairments remain prevalent, as patients are left perpetually with HIV. Currently, physicians face a challenge in treating HAND patients, so a greater understanding of the mechanisms underlying HAND pathology has been a growing focus in HIV research. Recent research has revealed the role disrupted calcium homeostasis in HIV-mediated neurotoxicity. Calcium plays a well-established role in the crosstalk between the mitochondrion and ER as well as in regulating autophagy, and ER stress, mitochondrial dysfunction, and impaired autophagic activity are considered hallmarks in several neurodegenerative and neurocognitive disorders. Therefore, it is paramount that the intricate inter-organelle signaling in relation to calcium homeostasis during HIV infection and the development of HAND is elucidated. This review consolidates current knowledge regarding the neuropathology of neurocognitive disorders and HIV infection with a focus on the underlying role of calcium during ER stress, mitochondrial dysfunction, and autophagy associated with the progression of HAND. The details of this intricate crosstalk during HAND remain relatively unknown; further research in this field can potentially aid in the development of improved therapy for patients suffering from HAND.

Nrf2 signaling links ER oxidative protein folding and calcium homeostasis in health and disease.

We report a signaling pathway linking two fundamental functions of the ER, oxidative protein folding, and intracellular calcium regulation. Cells sense ER oxidative protein folding through H2O2, which induces Nrf2 nuclear translocation. Nrf2 regulates the expression of GPx8, an ER glutathione peroxidase that modulates ER calcium levels. Because ER protein folding is dependent on calcium, this pathway functions as rheostat of ER calcium levels. Protein misfolding and calcium dysregulation contribute to the pathophysiology of many diseases, including amyotrophic lateral sclerosis, in which astrocytic calcium dysregulation participates in causing motor neuron death. In human-derived astrocytes harboring mutant SOD1 causative of familial amyotrophic lateral sclerosis, we show that impaired ER redox signaling decreases Nrf2 nuclear translocation, resulting in ER calcium overload and increased calcium-dependent cell secretion, leading to motor neuron death. Nrf2 activation in SOD1 mutant astrocytes with dimethyl fumarate restores calcium homeostasis and ameliorates motor neuron death. These results highlight a regulatory mechanism of intracellular calcium homeostasis by ER redox signaling and suggest that this mechanism could be a therapeutic target in SOD1 mutant astrocytes.

Correction: Genetic and clinical aspects of Wolfram syndrome 1, a severe neurodegenerative disease.

The original version of this Article erroneously cropped part of the abstract. The abstract has now been corrected to read 'Wolfram syndrome 1 (WS1) is a rare autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness, and other abnormalities. WS1 usually results in death before the age of 50 years. The pathogenesis of WS1 is ascribed to mutations of human WFS1 gene on chromosome 4p encoding a transmembrane protein called wolframin, which has physiological functions in membrane trafficking, secretion, processing, and/or regulation of ER calcium homeostasis. Different types of WFS1 mutations have been identified, and some of these have been associated with a dominant, severe type of WS. Mutations of CISD2 gene cause autosomal recessive Wolfram syndrome 2 (WS2) characterized by the absence of diabetes insipidus and psychiatric disorders, and by bleeding upper intestinal ulcer and defective platelet aggregation. Other WFS1-related disorders such as DFNA6/14/38 nonsyndromic low-frequency sensorineural hearing loss and Wolfram syndrome-like disease with autosomal dominant transmission have been described. WS1 is a devastating disease for the patients and their families. Thus, early diagnosis is imperative to enable proper prognostication, prevent complications, and reduce the transmission to further progeny. Although there is currently no effective therapy, potential new drugs have been introduced, attempting to improve the progression of this fatal disease." in both the PDF and HTML versions of the Article.

Seipin regulates lipid homeostasis by ensuring calcium-dependent mitochondrial metabolism.

Seipin, the gene that causes Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), is important for adipocyte differentiation and lipid homeostasis. Previous studies in Drosophila revealed that Seipin promotes ER calcium homeostasis through the Ca2+-ATPase SERCA, but little is known about the events downstream of perturbed ER calcium homeostasis that lead to decreased lipid storage in Drosophila dSeipin mutants. Here, we show that glycolytic metabolites accumulate and the downstream mitochondrial TCA cycle is impaired in dSeipin mutants. The impaired TCA cycle further leads to a decreased level of citrate, a critical component of lipogenesis. Mechanistically, Seipin/SERCA-mediated ER calcium homeostasis is important for maintaining mitochondrial calcium homeostasis. Reduced mitochondrial calcium in dSeipin mutants affects the TCA cycle and mitochondrial function. The lipid storage defects in dSeipin mutant fat cells can be rescued by replenishing mitochondrial calcium or by restoring the level of citrate through genetic manipulations or supplementation with exogenous metabolites. Together, our results reveal that Seipin promotes adipose tissue lipid storage via calcium-dependent mitochondrial metabolism.

Genetic and clinical aspects of Wolfram syndrome 1, a severe neurodegenerative disease.

Wolfram syndrome 1 (WS1) is a rare autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness, and other abnormalities. WS1 usually results in death before the age of 50 years. The pathogenesis of WS1 is ascribed to mutations of human WFS1 gene on chromosome 4p encoding a transmembrane protein called wolframin, which has physiological functions in membrane trafficking, secretion, processing, and/or regulation of ER calcium homeostasis. Different types of WFS1 mutations have been identified, and some of these have been associated with a dominant, severe type of WS. Mutations of CISD2 gene cause autosomal recessive Wolfram syndrome 2 (WS2) characterized by the absence of diabetes insipidus and psychiatric disorders, and by bleeding upper intestinal ulcer and defective platelet aggregation. Other WFS1-related disorders such as DFNA6/14/38 nonsyndromic low-frequency sensorineural hearing loss and Wolfram syndrome-like disease with autosomal dominant transmission have been described. WS1 is a devastating disease for the patients and their families. Thus, early diagnosis is imperative to enable proper prognostication, prevent complications, and reduce the transmission to further progeny. Although there is currently no effective therapy, potential new drugs have been introduced, attempting to improve the progression of this fatal disease.

The sigma-1 receptor mediates the beneficial effects of pridopidine in a mouse model of Huntington disease

The tri-nucleotide repeat expansion underlying Huntington disease (HD) results in corticostriatal synaptic dysfunction and subsequent neurodegeneration of striatal medium spiny neurons (MSNs). HD is a devastating autosomal dominant disease with no disease-modifying treatments. Pridopidine, a postulated “dopamine stabilizer”, has been shown to improve motor symptoms in clinical trials of HD. However, the target(s) and mechanism of action of pridopidine remain to be fully elucidated. As binding studies identified sigma-1 receptor (S1R) as a high-affinity receptor for pridopidine, we evaluated the relevance of S1R as a therapeutic target of pridopidine in HD. S1R is an endoplasmic reticulum - (ER) resident transmembrane protein and is regulated by ER calcium homeostasis, which is perturbed in HD. Consistent with ER calcium dysregulation, we observed striatal upregulation of S1R in aged YAC128 transgenic HD mice and HD patients. We previously demonstrated that dendritic MSN spines are lost in aged corticostriatal co-cultures from YAC128 mice. We report here that pridopidine and the chemically similar S1R agonist 3-PPP prevent MSN spine loss in aging YAC128 co-cultures. Spine protection was blocked by neuronal deletion of S1R. Pridopidine treatment suppressed supranormal ER Ca2+ release, restored ER calcium levels and reduced excessive store-operated calcium (SOC) entry in spines, which may account for its synaptoprotective effects. Normalization of ER Ca2+ levels by pridopidine was prevented by S1R deletion. To evaluate long-term effects of pridopidine, we analyzed expression profiles of calcium signaling genes. Pridopidine elevated striatal expression of calbindin and homer1a, whereas their striatal expression was reduced in aged Q175KI and YAC128 HD mouse models compared to WT. Pridopidine and 3-PPP are proposed to prevent calcium dysregulation and synaptic loss in a YAC128 corticostriatal co-culture model of HD. The actions of pridopidine were mediated by S1R and led to normalization of ER Ca2+ release, ER Ca2+ levels and spine SOC entry in YAC128 MSNs. This is a new potential mechanism of action for pridopidine, highlighting S1R as a potential target for HD therapy. Upregulation of striatal proteins that regulate calcium, including calbindin and homer1a, upon chronic therapy with pridopidine, may further contribute to long-term beneficial effects of pridopidine in HD.

Mephebrindole, a synthetic indole analog coordinates the crosstalk between p38MAPK and eIF2α/ATF4/CHOP signalling pathways for induction of apoptosis in human breast carcinoma cells.

The efficacy of cancer chemotherapeutics is limited by side effects resulting from narrow therapeutic windows between the anticancer activity of a drug and its cytotoxicity. Thus identification of small molecules that can selectively target cancer cells has gained major interest. Cancer cells under stress utilize the Unfolded protein response (UPR) as an effective cell adaptation mechanism. The purpose of the UPR is to balance the ER folding environment and calcium homeostasis under stress. If ER stress is prolonged, tumor cells undergo apoptosis. In the present study we demonstrated an 3,3'-(Arylmethylene)-bis-1H-indole (AMBI) derivative 3,3'-[(4-Methoxyphenyl) methylene]-bis-(5-bromo-1H-indole), named as Mephebrindole (MPB) as an effective anti-cancer agent in breast cancer cells. MPB disrupted calcium homeostasis in MCF7 cells which triggered ER stress development. Detailed evaluations revealed that mephebrindole by activating p38MAPK also regulated GRP78 and eIF2α/ATF4 downstream to promote apoptosis. Studies extended to in vivo allograft mice models revalidated its anti-carcinogenic property thus highlighting the role of MPB as an improved chemotherapeutic option.

Deficiency of osteoblastic Arl6ip5 impaired osteoblast differentiation and enhanced osteoclastogenesis via disturbance of ER calcium homeostasis and induction of ER stress-mediated apoptosis.

ADP-ribosylation-like factor 6 interacting protein 5 (Arl6ip5), which belongs to the prenylated rab-acceptor-family, has an important role in exocytic protein trafficking, glutathione metabolism and involves in cancer progression. However, its expression pattern and functional role in bone are unknown. Here we demonstrate that Arl6ip5 knock-out mice (Arl6ip5 Δ2/Δ2) show marked decrease of bone mineral density, trabecular bone volume and trabecular thickness. Histomorphometric studies reveal that bone formation parameters are decreased but bone resorption parameters and mRNA level of osteoclast-specific markers are increased in Arl6ip5Δ2/Δ2 mice. In osteoblast, we demonstrate that Arl6ip5 abundantly expresses in osteoblastic cells and is regulated by bone metabolism-related hormones and growth factors. In vitro analysis reveals that osteoblast proliferation and differentiation are impaired in Arl6ip5 knocked-down and deficient primary osteoblast. Arl6ip5 is also found to function as an ER calcium regulator and control calmodulin signaling for osteoblast proliferation. Moreover, Arl6ip5 insufficiency in osteoblast induces ER stress and enhances ER stress-mediated apoptosis. CCAAT/enhancer-binding protein homologous protein (Chop) is involved in the regulation of apoptosis and differentiation in Arl6ip5 knocked-down osteoblasts. For osteoclastogenesis, Arl6ip5 insufficiency in osteoclast precursors has no effect on osteoclast formation. However, knocked-down osteoblastic Arl6ip5 induces receptor activator of nuclear factor-κB ligand (RANKL) expression and enhances osteoclastogenesis. In addition, ER stress and Chop are involved in the RANKL expression in Arl6ip5 knocked-down osteoblasts. In conclusion, we demonstrate that Arl6ip5 is a novel regulator of bone formation in osteoblasts.

Use of microarray analysis to unveil transcription factor and gene networks contributing to Beta cell dysfunction and apoptosis.

The beta cell fate following immune-mediated damage depends on an intricate pattern of dozens of genes up- or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat beta cells exposed to the proapoptotic cytokines, IL-1beta and/or IFN-gamma. The picture emerging from these experiments is that beta cells are not passive bystanders of their own destruction. On the contrary, beta cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine-exposed beta cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF-kappaB. NF-kappaB blocking prevents cytokine-induced beta cell death, and characterization of NF-kappaB-dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of beta cell differentiated status, cytosolic and ER calcium homeostasis, attraction of mononuclear cells, and apoptosis. Identification of this and additional "transcription factor networks" is being pursued by cluster analysis of gene expression in insulin-producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the beta cell "decision" to undergo or not apoptosis in early T1DM.

Changes in interferon-regulatory factor-1 mRNA levels after transient ischemia in rat brain.

To evaluate whether the interferon system in the brain is activated by a severe form of metabolic stress, and to elucidate the possible mechanism underlying this activation, changes in the interferon regulatory factor-1 (irf-1) mRNA levels were evaluated after transient cerebral ischemia, and after exposure of primary neuronal cells to experimental conditions resulting in a depletion of ER calcium stores. Following 30 min ischemia and 2 h recovery, irf-1 mRNA levels rose significantly and stayed high for up to 24 h of recovery. Irf-1 mRNA levels were also significantly increased in neurons in vitro after exposing cells to conditions resulting in ER calcium store depletion with or without a parallel increase in cytoplasmic calcium activity. It is concluded that transient cerebral ischemia induces activation of the interferon system and that disturbances of ER calcium homeostasis may play a role in this process.