Glucocorticoid-induced Apoptosis Research Articles

A New Indole Derivative, LWX-473, Overcomes Glucocorticoid Resistance in Jurkat Cells by Activating Mediators of Apoptosis.

Glucocorticoids (GCs) are commonly used as the primary chemotherapy for lymphoid malignancies, including acute lymphoblastic leukemia (ALL). However, the development of GC resistance limits their prolonged use. In this study, we investigated the potential of a newly synthesized indole derivative called LWX-473, in combination with the classic GC Dexamethasone (DEX), to enhance the responsiveness of Jurkat cells to GC treatment. Our findings demonstrate that LWX-473 alone or in combination with DEX significantly improves GC-induced cell apoptosis and arrests the cell cycle in the G1 phase. Notably, the combination of LWX-473 and DEX exhibits superior efficacy in killing Jurkat cells compared to LWX-473 alone. Importantly, this compound demonstrates reduced toxicity towards normal cells. Our study reveals that LWX-473 has the ability to restore the sensitivity of Jurkat cells to DEX by modulating the mitochondrial membrane potential, activating the expression of DEX-liganded glucocorticoid receptor (GR), and inhibiting key molecules in the JAK/STAT signaling pathway. These findings suggest that LWX-473 could be a potential therapeutic agent for overcoming GC resistance in lymphoid malignancies.

TCF1-LEF1 co-expression identifies a multipotent progenitor cell (TH2-MPP) across human allergic diseases.

Repetitive exposure to antigen in chronic infection and cancer drives T cell exhaustion, limiting adaptive immunity. In contrast, aberrant, sustained T cell responses can persist over decades in human allergic disease. To understand these divergent outcomes, we employed bioinformatic, immunophenotyping and functional approaches with human diseased tissues, identifying an abundant population of type 2 helper T (TH2) cells with co-expression of TCF7 and LEF1, and features of chronic activation. These cells, which we termed TH2-multipotent progenitors (TH2-MPP) could self-renew and differentiate into cytokine-producing effector cells, regulatory T (Treg) cells and follicular helper T (TFH) cells. Single-cell T-cell-receptor lineage tracing confirmed lineage relationships between TH2-MPP, TH2 effectors, Treg cells and TFH cells. TH2-MPP persisted despite in vivo IL-4 receptor blockade, while thymic stromal lymphopoietin (TSLP) drove selective expansion of progenitor cells and rendered them insensitive to glucocorticoid-induced apoptosis in vitro. Together, our data identify TH2-MPP as an aberrant T cell population with the potential to sustain type 2 inflammation and support the paradigm that chronic T cell responses can be coordinated over time by progenitor cells.

Cortistatin prevents glucocorticoid-associated osteonecrosis of the femoral head via the GHSR1a/Akt pathway

Long-term use of glucocorticoids (GCs) is known to be a predominant cause of osteonecrosis of the femoral head (ONFH). Moreover, GCs can mediate apoptosis of various cell types by exaggerating oxidative stress. We have previously found that Cortistatin (CST) antagonizes oxidative stress and improves cell apoptosis in several conditions. In this study, we detected that the CST expression levels were diminished in patients with ONFH compared with femoral neck fracture (FNF). In addition, a GC-induced rat ONFH model was established, which impaired bone quality in the femoral head. Then, administration of CST attenuated these ONFH phenotypes. Furthermore, osteoblast and endothelial cells were cultured and stimulated with dexamethasone (Dex) in the presence or absence of recombinant CST. As a result, Dex induced impaired anabolic metabolism of osteoblasts and suppressed tube formation in endothelial cells, while additional treatment with CST reversed this damage to the cells. Moreover, blocking GHSR1a, a well-accepted receptor of CST, or blocking the AKT signaling pathway largely abolished the protective function of CST in Dex-induced disorder of the cells. Taken together, we indicate that CST has the capability to prevent GC-induced apoptosis and metabolic disorder of osteoblasts in the pathogenesis of ONFH via the GHSR1a/AKT signaling pathway.

Ortho-silicic Acid Prevents Glucocorticoid-Induced Femoral Head Necrosis by Promoting Akt Phosphorylation to Inhibit Endoplasmic Reticulum Stress-Mediated Apoptosis and Enhance Angiogenesis and Osteogenesis.

Glucocorticoid-induced osteonecrosis of the femoral head (SONFH) is the most prevalent form of secondary osteonecrosis affecting the femoral head. Glucocorticoids can cause damage to both vascular endothelial cells and osteoblasts. Previous studies have demonstrated that silicon can improve the resistance of vascular endothelial cells to oxidative stress and positively impact bone health. However, the impact of silicon on SONFH has yet to be investigated. We examined the influence of ortho-silicic acid (OSA, Si(OH)4) on the apoptosis and proliferation of vascular endothelial cells after glucocorticoid induction. Additionally, we evaluated the expression of apoptosis-related genes such as cleaved-caspase-3, Bcl-2 and Bax. The impact of glucocorticoids and OSA on the function of vascular endothelial cells was evaluated through wound healing, transwell and angiogenesis assays. Osteogenic function was subsequently evaluated through alizarin red staining, alkaline phosphatase staining and expression levels of osteogenic genes like RUNX2 and ALP. Moreover, we investigated the potential role of OSA in vivo using the SONFH animal model. At concentrations below 100μM, OSA exhibits no toxicity on vascular endothelial cells and effectively reverses glucocorticoid-induced apoptosis in these cells. OSA increases the resilience of vascular endothelial cells against oxidative stress and enhances osteoblast differentiation. Our study revealed that glucocorticoids activate endoplasmic reticulum stress, a process that mediates the apoptosis of vascular endothelial cells. OSA ameliorated the endoplasmic reticulum stress associated with glucocorticoids through the increased expression of p-Akt levels. In vivo, OSA treatment effectively improved SONFH by enhancing vascular endothelial cell function and promoting osteogenic differentiation. OSA counteracted the adverse effects of glucocorticoids both in vitro and in vivo, demonstrating a beneficial therapeutic effect on SONFH.

Human Mesenchymal Stem Cells-Derived Exosome Mimetic Vesicles Regulation of the MAPK Pathway and ROS Levels Inhibits Glucocorticoid-Induced Apoptosis in Osteoblasts.

Long-term extensive use of glucocorticoids will lead to hormonal necrosis of the femoral head, and osteoblasts play an important role in the prevention of osteonecrosis. However, there is no complete cure for necrosis of the femoral head. Mesenchymal stem cell- (MSCs-) derived exosomes are widely used for the repair of various tissue lesions. Therefore, the aim of this study was to investigate the mechanism of dexamethasone- (DEX-) induced osteoblast apoptosis and the therapeutic effect of human umbilical cord MSC- (hucMSC-) derived exosome mimetic vesicles (EMVs) on osteoblast-induced apoptosis by DEX. The viability and apoptosis of primary MC3T3-E1 cells were determined by the Cell Counting Kit-8 (CCK-8), FITC-Annexin V/PI staining and immunoblot. The intracellular levels of reactive oxygen species (ROS) after DEX treatment were measured by 2', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA) staining. In this study, hucMSC-EMVs and N-acetyl-l-cysteine (NAC) were used as therapeutic measures. The expression of B-cell lymphoma 2-associated X, Bcl 2, HO-1, and nuclear factor erythroid-derived 2-like 2 and MAPK- signaling pathway in osteogenic cell MC3T3-E1 cells treated with Dex was analyzed by the immunoblotting. DEX significantly induced osteoblasts MC3T3-E1 apoptosis and ROS accumulation. MAPK-signaling pathway was activated in MC3T3-E1 after DEX treatment. hucMSC-EMVs intervention significantly downregulated DEX-induced MAPK-signaling pathway activation and ROS accumulation. In addition, hucMSC-EMVs can reduce the apoptosis levels in osteoblast MC3T3-E1 cells induced by DEX. Our study confirmed that hucMSC-EMVs regulates MAPK-signaling pathway and ROS levels to inhibit DEX-induced osteoblast apoptosis.

Butein inhibits corticosterone-induced apoptosis of Neuro2A cells by maintaining MEK-ERK signaling.

Stress-induced overactivation of glucocorticoid signaling may contribute to mental illness by inducing neuronal death and dysfunction. We previously reported that pretreatment with the plant flavonoid butein inhibits corticosterone (CORT)-induced apoptosis of Neuro2A (N2A) cells. In the current study, we examined whether MEK-ERK and PI3K-AKT signaling pathways are involved in neuroprotection by butein. N2A cells were pre-incubated with serum-free DMEM containing 0.5μM butein for 30min, and then incubated with serum-free DMEM containing 0.5µM butein, 50µM CORT, 50µM LY294002, or 50µM PD98059 as indicated for 24h. We subsequently performed the MTT assay and the western blot analysis. As expected, CORT considerably reduced N2A cell viability and increased relative expression of the apoptosis effector cleaved caspase-3, whereas pretreatment with butein blocked these cytotoxic effects. Treatment with CORT alone also decreased both AKT and ERK protein phosphorylation. Butein pretreatment had no effect on AKT phosphorylation, and only partially reversed the reduction in phosphorylated ERK. However, cotreatment with butein and the PI3K inhibitor LY294002 during CORT exposure enhanced ERK phosphorylation, whereas cotreatment with butein and the ERK phosphorylation/activation inhibitor PD98059 enhanced AKT phosphorylation, suggesting that MEK-ERK negatively regulates AKT phosphorylation. Moreover, the protective efficacy of butein was blocked by PD98059 cotreatment but not LY294002 cotreatment. These findings suggest that butein protects neurons against glucocorticoid-induced apoptosis by sustaining ERK phosphorylation and downstream signaling.

FAR591 promotes the pathogenesis and progression of SONFH by regulating Fos expression to mediate the apoptosis of bone microvascular endothelial cells

The specific pathogenesis of steroid-induced osteonecrosis of the femoral head (SONFH) is still not fully understood, and there is currently no effective early cure. Understanding the role and mechanism of long noncoding RNAs (lncRNAs) in the pathogenesis of SONFH will help reveal the pathogenesis of SONFH and provide new targets for its early prevention and treatment. In this study, we first confirmed that glucocorticoid (GC)-induced apoptosis of bone microvascular endothelial cells (BMECs) is a pre-event in the pathogenesis and progression of SONFH. Then, we identified a new lncRNA in BMECs via lncRNA/mRNA microarray, termed Fos-associated lincRNA ENSRNOT00000088059.1 (FAR591). FAR591 is highly expressed during GC-induced BMEC apoptosis and femoral head necrosis. Knockout of FAR591 effectively blocked the GC-induced apoptosis of BMECs, which then alleviated the damage of GCs to the femoral head microcirculation and inhibited the pathogenesis and progression of SONFH. In contrast, overexpression of FAR591 significantly promoted the GC-induced apoptosis of BMECs, which then aggravated the damage of GCs to the femoral head microcirculation and promoted the pathogenesis and progression of SONFH. Mechanistically, GCs activate the glucocorticoid receptor, which translocates to the nucleus and directly acts on the FAR591 gene promoter to induce FAR591 gene overexpression. Subsequently, FAR591 binds to the Fos gene promoter (–245∼–51) to form a stable RNA:DNA triplet structure and then recruits TATA-box binding protein associated factor 15 and RNA polymerase II to promote Fos expression through transcriptional activation. Fos activates the mitochondrial apoptotic pathway by regulating the expression of Bcl-2 interacting mediator of cell death (Bim) and P53 upregulated modulator of apoptosis (Puma) to mediate GC-induced apoptosis of BMECs, which leads to femoral head microcirculation dysfunction and femoral head necrosis. In conclusion, these results confirm the mechanistic link between lncRNAs and the pathogenesis of SONFH, which helps reveal the pathogenesis of SONFH and provides a new target for the early prevention and treatment of SONFH.

Rspo1 inhibited apoptosis of glucocorticoid-induced osteoblasts via Wnt/β-catenin pathway in Legg-Calve-Perthes disease.

The pathogenesis of Legg-Calve-Perthes disease (LCPD), a juvenile form of avascular necrosis of the femoral head (ANFH), is not fully understood. The purpose of this work was to study the regulatory effect of R-spondin 1 (Rspo1) on osteoblastic apoptosis and evaluate the pre-clinical efficacy of recombinant human protein Rspo1 (rhRspo1) in treatment of LCPD. This is an experimental study. In vivo rabbit ANFH model was established. Human osteoblast cell line hFOB1.19 (hFOB) was used to overexpress and silence Rspo1 in vitro. Additionally, hFOB cells were induced with glucocorticoid (GC) and methylprednisolone (MP), and treated with rhRspo1. The expressions of Rspo1, β-catenin, Dkk-1, Bcl-2, and caspase-3, and the apoptosis rate of hFOB cells were examined. The expressions of Rspo1 and β-catenin were lower in ANFH rabbits. The expression of Rspo1 was decreased in GC-induced hFOB cells. Compared to the control group, after 1 μM MP induction for 72 h, the expressions of β-catenin and Bcl-2 were higher, while Dkk-1, caspase-3 and cleaved caspase-3 expressions were lower in Rspo1 overexpression and rhRspo1-treated groups. The apoptosis rate of GC-induced hFOB cells was decreased in Rspo1 overexpression and rhRspo1-treated groups compared to the control group. R-spondin 1 inhibited GC-induced osteoblast apoptosis via Wnt/β-catenin pathway, which might be associated with the development of ANFH. Moreover, rhRspo1 had a potential pre-clinical therapeutic effect on LCPD.

Geniposide ameliorates glucocorticoid-induced osteoblast apoptosis by activating autophagy

Long-term exposure to glucocorticoid (GC) contributes to the development of osteoporosis (OP), which is correlated with the risk of fracture. Pathologically, GC-induced bone loss is associated with osteoblast apoptosis. Geniposide (GEN), a natural occurring compound derived from Eucommia ulmoides, has been reported to ameliorate dexamethasone (DEX)-induced OP. Our previous study shows that GEN exhibits protective activity against DEX-induced OP by attenuating endoplasmic reticulum stress and decreasing apoptosis in osteoblasts. However, the molecular mechanisms of GEN in inhibiting DEX-induced osteoblast apoptosis still need further elucidation. In this article, a molecular target network of GEN against OP was screened. It was found that GEN might interact with OP by mediating PI3K/AKT pathway, which is the upstream factor in regulating autophagy. GEN exhibited protective activity against DEX-induced apoptosis by activating autophagy in vivo and in vitro. Blockage of autophagy, activation of PI3K/AKT/mTOR pathway, or inhibition of GLP-1R activity could eliminate the protective effects of GEN against DEX-induced apoptosis. Collectively, GEN ameliorated DEX-induced osteoblast apoptosis by activating autophagy through GLP-1R/PI3K/AKT/mTOR pathway.

Potential Mechanisms by which Glucocorticoids Induce Breast Carcinogenesis through Nrf2 Inhibition.

Breast cancer is the most common malignancy among women worldwide. Several studies indicate that, in addition to established risk factors for breast cancer, other factors such as cortisol release related to psychological stress and drug treatment with high levels of glucocorticoids may also contribute significantly to the initiation of breast cancer. There are several possible mechanisms by which glucocorticoids might promote neoplastic transformation of breast tissue. Among these, the least known and studied is the inhibition of the nuclear erythroid factor 2-related (Nrf2)-antioxidant/electrophile response element (ARE/EpRE) pathway by high levels of glucocorticoids. Specifically, Nrf2 is a potent transcriptional activator that plays a central role in the basal and inducible expression of many cytoprotective genes that effectively protect mammalian cells from various forms of stress and reduce the propensity of tissues and organisms to develop disease or malignancy including breast cancer. Consequently, a loss of Nrf2 in response to high levels of gluco-corticoids may lead to a decrease in cellular defense against oxidative stress, which plays an important role in the initiation of human mammary carcinogenesis. In the present review, we provide a comprehensive overview of the current state of knowledge of the cellular mechanisms by which both glucocorticoid pharmacotherapy and endogenous GCs (cortisol in humans and corticosterone in rodents) may contribute to breast cancer development through inhibition of the Nrf2-ARE/EpRE pathway and the protective role of melatonin against glucocorticoid-induced apoptosis in the immune system.

Vitamin B2 Prevents Glucocorticoid-Caused Damage of Blood Vessels in Osteonecrosis of the Femoral Head.

Osteonecrosis of the femoral head (ONFH) is a disorder that can cause collapse of the femoral head. The damage and dysfunction of femoral head microvascular endothelial cells are related to the pathogenesis of glucocorticoid-induced ONFH. Reports suggest that vitamin B2 can promote osteoblast differentiation and prevent low bone mineral density and prevent reperfusion oxidative injury. To explore the effect and possible molecular mechanism of vitamin B2 on the ONFH and Human Umbilical Vein Endothelial Cells (HUVECs), we performed a rat model of ONFH by dexamethasone. The rats were randomly divided into four groups: control group, vitamin B2 group, dexamethasone group, and dexamethasone combined with vitamin B2 treatment group. HUVECs were used to further prove the role and mechanism of vitamin B2 in vitro. In patients, according to immunohistochemical and qRT-PCR of the femoral head, the angiogenic capacity of the ONFH femoral head is compromised. In vivo, it showed that vitamin B2 could inhibit glucocorticoid-induced ONFH-like changes in rats by suppressing cell apoptosis, promoting the regeneration of blood vessels, and increasing bone mass. According to in vitro results, vitamin B2 could induce the migration of HUVECs, enhance the expression of angiogenesis-related factors, and inhibit glucocorticoid-induced apoptosis. The underlying mechanism may be that vitamin B2 activates the PI3K signaling pathway. Vitamin B2 alleviated dexamethasone-induced ONFH, and vitamin B2 could promote the proliferation and migration of HUVECs and inhibit their apoptosis by activating the PI3K/Akt signaling pathway. Vitamin B2 may be a potentially effective treatment for ONFH.

Targeting signaling pathways vulnerabilities for the treatment of IKZF1-deleted ph-negative B lymphoblastic leukemia.

7033 Background: Approximately 20% of children diagnosed with B lymphoblastic leukemia (B-ALL) will suffer a relapse, and most adults with B-ALL have a poor prognosis. Genome-wide association studies of B-ALL patients have identified frequent deletions of the gene IKZF1, encoding the master regulator of lymphoid development, IKAROS. These deletions are associated with therapy resistance, increased risk of relapse, and inferior survival. Currently, how loss of IKAROS function contributes to therapy resistance and increased risk of relapse is not fully understood. We used CRISPR-Cas9 genome editing to develop human B-ALL cell lines with various IKZF1 deletions that genetically and phenotypically recapitulate those occurring in patients. Using these isogenic cell lines, we have previously shown IKZF1 deletion results in cell-intrinsic chemoresistance and increased activation of the JAK/STAT signaling pathway (Rogers, Gupta et al. 2021). Methods: Given JAK/STAT is often dysregulated in poor-prognosis leukemia, we investigated the potential mechanisms of aberrant JAK/STAT activation and the therapeutic potential of targeting JAK/STAT in our engineered cell lines. We treated our cells with SH-4-54 (STAT3/5 inhibitor) or tofacitinib (JAK1 inhibitor) alone and in combination with dexamethasone. To elucidate how loss of IKAROS mediates an increase in JAK-STAT activity, we also performed RNAseq of known pathway regulators, comparing IKZF1 wild-type with IKZF1 knockout. Results: The JAK/STAT negative regulator Suppressor of Cytokine Signaling 2 (SOCS2) was significantly downregulated with IKZF1 deletion, validated by RTqPCR and immunoblotting. We further analyzed publicly-available RNAseq data from > 650 pediatric B-ALL samples, finding that SOCS2 expression is significantly lower in patients with low IKZF1expression (likely corresponding to IKZF1 deletion) compared to those with high IKZF1 expression. When we treated our engineered cell lines with tofacitinib or SH-4-54, IKZF1 wild-type cells were sensitive to each compound, suggesting JAK/STAT signaling plays a vital role in cell survival. In contrast, the IKZF1-deleted cells were relatively resistant to JAK/STAT inhibitors alone. However, in combination with dexamethasone, treatment of cells with sub-IC50 levels of SH-4-54 or tofacitinib resulted in re-sensitization to glucocorticoid-induced apoptosis. Conclusions: Our findings support that IKZF1 deletion leads to a targetable upregulation of the JAK/STAT pathway that, when inhibited, results in relative re-sensitization to dexamethasone. JAK/STAT pathway upregulation in IKZF1 deleted cells may be mediated by decreased expression of SOCS2. These results provide initial promise for targeting these vulnerabilities for the treatment of this poor-prognosis disease.

Anti-inflammatory and Tendon-Protective Effects of Adipose Stem Cell-Derived Exosomes with Concomitant Use of Glucocorticoids.

Glucocorticoid (GC) injections are commonly used in clinical practice to relieve pain and improve function in patients with multiple shoulder disabilities but cause detrimental effects on rotator cuff tendons. Adipose stem cell-derived exosomes (ASC-Exos) reportedly recover impaired tendon matrix metabolism by maintaining tissue homeostasis. However, it is unclear whether additional treatment with ASC-Exos overrides the detrimental effects of GCs without interfering with their anti-inflammatory effects. Thus, we aimed to investigate the anti-inflammatory effect of ASC-Exos with GCs and protective effect of ASC-Exos against GC-induced detriments. The present study comprised in vitro and in vivo studies. In vitro inflammatory analysis revealed that ASC-Exos exerted a synergic anti-inflammatory effect with GCs by significantly decreasing secretion of proinflammatory cytokines by RAW cells and increasing secretion of anti-inflammatory cytokines. In vitro cytoprotective analysis showed that ASC-Exos overrode GC-induced detrimental effects on tenocytes by significantly improving GC-suppressed cellular proliferation, migration, and transcription of tenocytic matrix molecules and degradative enzyme inhibitors and significantly decreasing GC-induced cell senescence, apoptosis, and transcription of ROS and tenocytic degradative enzymes. In vivo studies revealed that additional ASC-Exo injection restored impairments in histological and biomechanical properties owing to GC administration. Collectively, these results suggest that ASC-Exos exert a stronger anti-inflammatory effect in combination with GCs, overriding their detrimental effects on rotator cuff tendons.

MTOR inhibition downregulates glucose-6-phosphate dehydrogenase and induces ROS-dependent death in T-cell acute lymphoblastic leukemia cells

mTOR activation is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL) and is associated with resistance to glucocorticoid (GC)-based chemotherapy. We previously showed that altering redox homeostasis primes T-ALL cells to GC-induced apoptosis. Here we investigated the connection between the mTOR pathway and redox homeostasis using pharmacological inhibitors and gene silencing.In vitro studies performed on T-ALL cell lines and CG-resistant patient-derived T-ALL xenograft (PDX) cells showed that the mTOR inhibitor everolimus increased reactive oxygen species (ROS) levels, augmented lipid peroxidation, and activated the ROS-controlled transcription factor NRF2. These effects were accompanied by a decrease in the levels of NADPH and of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), which is a major source of cytosolic NADPH needed for maintaining the cellular ROS-scavenging capacity. The mTOR inhibitor everolimus induced mitochondrial inner membrane depolarization and dose-dependent apoptosis of T-ALL cells, but did not kill normal T-cells. Importantly, the combination of everolimus and the GC dexamethasone had a synergistic effect on killing T-ALL cells. The effects of mTOR inhibition were blunted by ROS scavengers and phenocopied by siRNA-mediated G6PD silencing. In vivo studies of NOD/SCID mice inoculated with refractory T-ALL PDX demonstrated that everolimus overcame dexamethasone resistance in conditions of high tumor burden that mimicked the clinical setting of acute leukemia.These findings provide insight into the crosstalk between mTOR and ROS homeostasis in T-ALL cells and furnish mechanistic evidence to support the combination of glucocorticoids with mTOR inhibitors as a therapeutic avenue for treating refractory T-ALL.

MicroRNA expression signature and target prediction in familial and sporadic primary macronodular adrenal hyperplasia (PMAH)

BackgroundPrimary macronodular adrenal hyperplasia (PMAH), previously termed ACTH-independent macronodular adrenal hyperplasia (AIMAH), is a rare cause of Cushing’s syndrome usually characterized by functioning adrenal macronodules and increased cortisol production.MethodsTo screen and analyse the microRNA (miRNA) profile of PMAH in order to elucidate its possible pathogenesis, a miRNA microarray was used to test tissue samples from patients with familial PMAH, patients with sporadic PMAH and normal control samples of other nontumour adrenocortical tissues and identify characteristic microRNA expression signatures. Randomly selected miRNAs were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the key signalling pathways and miRNAs involved in PMAH pathogenesis were determined by gene ontology and pathway analysis.ResultsCharacteristic microRNA expression signatures were identified for patients with familial PMAH (16 differentially expressed microRNAs) and patients with sporadic PMAH (8 differentially expressed microRNAs). The expression of the selected miRNAs was confirmed by qRT-PCR, suggesting the high reliability of the miRNA array analysis results. Pathway analysis showed that the most enriched pathway was the renal cell carcinoma pathway. Overexpression of miR-17, miR-20a and miR-130b may inhibit glucocorticoid-induced apoptosis in PMAH pathogenesis.ConclusionWe identified the miRNA signatures in patients with familial and sporadic PMAH. The differentially expressed miRNAs may be involved in the mechanisms of PMAH pathogenesis. Specific miRNAs, such as miR-17, miR-20a and miR-130b, may be new targets for further functional studies of PMAH.

Anti-PD-1 Checkpoint Therapy Can Promote the Function and Survival of Regulatory T Cells.

We have previously shown in a model of claudin-low breast cancer that regulatory T cells (Tregs) are increased in the tumor microenvironment (TME) and express high levels of PD-1. In mouse models and patients with triple-negative breast cancer, it is postulated that one cause for the lack of activity of anti-PD-1 therapy is the activation of PD-1-expressing Tregs in the TME. We hypothesized that the expression of PD-1 on Tregs would lead to enhanced suppressive function of Tregs and worsen antitumor immunity during PD-1 blockade. To evaluate this, we isolated Tregs from claudin-low tumors and functionally evaluated them ex vivo. We compared transcriptional profiles of Tregs isolated from tumor-bearing mice with or without anti-PD-1 therapy using RNA sequencing. We found several genes associated with survival and proliferation pathways; for example, Jun, Fos, and Bcl2 were significantly upregulated in Tregs exposed to anti-PD-1 treatment. Based on these data, we hypothesized that anti-PD-1 treatment on Tregs results in a prosurvival phenotype. Indeed, Tregs exposed to PD-1 blockade had significantly higher levels of Bcl-2 expression, and this led to increased protection from glucocorticoid-induced apoptosis. In addition, we found in vitro and in vivo that Tregs in the presence of anti-PD-1 proliferated more than control Tregs PD-1 blockade significantly increased the suppressive activity of Tregs at biologically relevant Treg/Tnaive cell ratios. Altogether, we show that this immunotherapy blockade increases proliferation, protection from apoptosis, and suppressive capabilities of Tregs, thus leading to enhanced immunosuppression in the TME.

Artemisinin Reverses Glucocorticoid-Induced Injury in Bone Marrow-Derived Mesenchymal Stem Cells through Regulation of ERK1/2-CREB Signaling Pathway.

Glucocorticoids are the most common cause of secondary osteoporosis, which affects both women (pre- and postmenopausal) and men. In cases of prolonged treatment, glucocorticoids promote the loss and inactivation of the differentiational function of bone marrow mesenchymal stromal cells (BMSCs), risking the development of skeletal system diseases such as osteoporosis. This study reports for the first time the protective effect of the antimalarial artemisinin against glucocorticoid-induced insults on primary cultured rat BMSCs. At relatively low concentrations, artemisinin treatment improved BMSC survival by promoting a decline of reactive oxygen species (ROS) production that correlated with the decrease of caspase-3 activation, LDH release, mitochondrial membrane potential (Δψm) loss, and apoptosis induced by dexamethasone (DEXA). In addition, artemisinin improved the osteogenic differentiation of DEXA-damaged cells. DEXA inhibited extracellular-signal-regulated kinase 1/2 (ERK1/2) and cAMP response element binding protein (CREB) phosphorylation, and artemisinin treatment promoted their activation in a concentration-dependent manner. PD98059, the specific inhibitor of the ERK1/2 pathway, blocked ERK1/2 phosphorylation and artemisinin protection. Similarly, siCREB attenuated the protective effect of artemisinin, strongly suggesting the involvement of the ERK1/2-CREB pathway in the protective action of artemisinin against DEXA-induced damage in BMSCs. In addition, we found that the expression of antiapoptotic protein B-cell lymphoma 2 protein (BCL-2) was also upregulated by artemisinin. These studies demonstrate the therapeutic potential of artemisinin in the survival improvement of BMSCs exposed to glucocorticoid-induced apoptosis and suggest that artemisinin-mediated protection may occur via the activation of ERK1/2-CREB signaling pathway.

Repression of the Orphan Nuclear Receptor Esrrb Results in Glucocorticoid Resistance in ALL

▪Acute lymphoblastic leukemia (ALL) is an aggressive hematological malignancy, in which 20-30% of pediatric patients relapse or undergo induction failure (Vrooman, 2009). ALL is treated with a combination of chemotherapeutic agents, including glucocorticoids (GCs). Pediatric ALL patients who exhibit resistance to GCs at diagnosis or relapse have a poor prognosis(Dördelmann, 1999). We performed an unbiased whole genome survival-based shRNA screen in primary mouse T-ALL cells to identify GC resistance genes. This shRNA screen identified known mediators of GC resistance as well as novel genes involved in chromatin remodeling, cell differentiation and metabolism.One of the top hits in the GC resistance screen were shRNAs targeting the orphan nuclear receptor ESRRB. ESRRB is essential for the maintenance of pluripotency in mouse embryonic stem cells and decreases in ESRRB expression stimulate differentiation (Zhang, 2008). We observe no effects of ESRRB deficiency on leukemic cell growth or differentiation. However, silencing of Esrrb prevents GC-induced apoptosis in mouse and human T-ALL cell lines in vitro . Although the screen was done in mouse T-ALL cells, we find that ESRRB-deficiency also results in GC resistance in human B-ALL cells. As many published GC resistance genes fail to translate in vivo, we transplanted mice with T-ALL cells transduced with shRNAs targeting Esrrb or empty vector. Dexamethasone treatment prevented disease progression in mice transplanted with control T-ALL cells however, dexamethasone treatment had no significant effect on disease development in mice transplanted with ESRRB deficient T-ALL cells, indicating that ESRRB deficiency confers GC resistance in vivo.As ESRRB has no established role in T-cells or the GC response, we performed RNA-sequencing on normal and ESRRB-deficient T-ALL cells in the absence and presence of GCs to uncover the mechanism of GC resistance. ESRRB deficient T-ALL cells fail to upregulate 65% of GC-induced genes, indicating that decreases in ESRRB expression prevents optimal GC-induced gene expression and apoptosis. Furthermore, we identified Esrrb as a GC-induced gene, supporting its role in the GC response. Due to the global repression of GC-induced gene expression in ESRRB-deficient leukemic cells, we examined GC receptor (GR) localization, but found that ESRRB had no detectable effects on GR localization.Collectively these data suggest that ESRRB may function as a novel co-activator of GR mediated transcription. Consistently, we found ESRRB expression decreased in a subset of relapse ALL patients, suggesting that ESRRB may contribute to GC resistance in patients. These findings identify ESRRB as a novel GR co-activator, whose expression is suppressed in a subset of relapse ALL patients. We demonstrate that treatment with the synthetic ESRRB agonist GSK4716 stimulates GC-induced apoptosis and gene expression in human ALL cell lines. These data raise the intriguing possibility that ESRRB agonists may re-sensitize GC resistant leukemia patients to GC therapy. DisclosuresNo relevant conflicts of interest to declare.

Protection of Antigen-Primed Effector T Cells From Glucocorticoid-Induced Apoptosis in Cell Culture and in a Mouse Model of Multiple Sclerosis.

Induction of T cell apoptosis constitutes a major mechanism by which therapeutically administered glucocorticoids (GCs) suppress inflammation and associated clinical symptoms, for instance in multiple sclerosis (MS) patients suffering from an acute relapse. The sensitivity of T cells to GC action depends on their maturation and activation status, but the precise effect of antigen-priming in a pathological setting has not been explored. Here we used transgenic and congenic mouse models to compare GC-induced apoptosis between naïve and antigen-specific effector T cells from mice immunized with a myelin peptide. Antigen-primed effector T cells were protected from the pro-apoptotic activity of the synthetic GC dexamethasone in a dose-dependent manner, which resulted in their accumulation relative to naïve T cells in vitro and in vivo. Notably, the differential sensitivity of T cells to GC-induced apoptosis correlated with their expression level of the anti-apoptotic proteins Bcl-2 and Bcl-XL and a loss of the mitochondrial membrane potential. Moreover, accumulation of antigen-primed effector T cells following GC treatment in vitro resulted in an aggravated disease course in an adoptive transfer mouse model of MS in vivo, highlighting the clinical relevance of the observed phenomenon. Collectively, our data indicate that antigen-priming influences the T cells’ sensitivity to therapeutically applied GCs in the context of inflammatory diseases.

Hyper-activated platelet lysates prevent glucocorticoid-associated femoral head necrosis by regulating autophagy

Platelet Rich Plasma (PRP) can activate angiogenic and osteogenic pathways, making it a highly promising therapeutic agent for bone growth. Super active platelet lysate (sPL) is derived from platelet-rich plasma (PRP) through ultra-low temperature freeze-thawing. The aim of this study was to evaluate the potential therapeutic effect of sPL on glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). sPL increased the proliferation of GC-treated osteoblasts and endothelial cells, and inhibited apoptosis in vitro. Furthermore, sPL promoted healing of necrotic bone tissues in a rat ONFH model by restraining GC-induced apoptosis and increase autophagy of the osteoblasts. Overall, the results of this study provide a theoretical basis for the clinical application of sPL in ONFH.