Treatment with siRNAs is commonly associated with GPX4 up-regulation and target knockdown-independent sensitization to ferroptosis.

Small interfering RNAs (siRNAs) are widely used in biomedical research and in clinical trials. Here, we demonstrate that siRNA treatment is commonly associated with significant sensitization to ferroptosis, independently of the target protein knockdown. Genetically targeting mitochondrial antiviral-signaling protein (MAVS) reversed the siRNA-mediated sensitizing effect, but no activation of canonical MAVS signaling, which involves phosphorylation of IkBα and interferon regulatory transcription factor 3 (IRF3), was observed. In contrast, MAVS mediated a noncanonical signal resulting in a prominent increase in mitochondrial ROS levels, and increase in the BACH1/pNRF2 transcription factor ratio and GPX4 up-regulation, which was associated with a 50% decrease in intracellular glutathione levels. We conclude that siRNAs commonly sensitize to ferroptosis and may severely compromise the conclusions drawn from silencing approaches in biomedical research. Finally, as ferroptosis contributes to a variety of pathophysiological processes, we cannot exclude side effects in human siRNA-based therapeutical concepts that should be clinically tested.

vPIF-1 is an insulin-like antiferroptotic viral peptide.

Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin-like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone-releasing hormone antagonist-induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms.

Ubiquitination in T-Cell Activation and Checkpoint Inhibition: New Avenues for Targeted Cancer Immunotherapy.

The advent of T-cell-based immunotherapy has remarkably transformed cancer patient treatment. Despite their success, the currently approved immunotherapeutic protocols still encounter limitations, cause toxicity, and give disparate patient outcomes. Thus, a deeper understanding of the molecular mechanisms of T-cell activation and inhibition is much needed to rationally expand targets and possibilities to improve immunotherapies. Protein ubiquitination downstream of immune signaling pathways is essential to fine-tune virtually all immune responses, in particular, the positive and negative regulation of T-cell activation. Numerous studies have demonstrated that deregulation of ubiquitin-dependent pathways can significantly alter T-cell activation and enhance antitumor responses. Consequently, researchers in academia and industry are actively developing technologies to selectively exploit ubiquitin-related enzymes for cancer therapeutics. In this review, we discuss the molecular and functional roles of ubiquitination in key T-cell activation and checkpoint inhibitory pathways to highlight the vast possibilities that targeting ubiquitination offers for advancing T-cell-based immunotherapies.

Estrogen enhances human osteoblast survival and function via promotion of autophagy.

Estrogen increases bone formation by promoting mineralization and prolonging the lifespan of osteoblasts. To understand the underlying molecular mechanism/s, we identified estrogen-regulated proteins at different stages of human osteoblast differentiation using differential proteomics approach. Among the identified proteins, we observed that estrogen upregulated RAB3GAP1 on day 1 and 5 of differentiation. RAB3GAP1 is critically involved in the process of autophagy, a eukaryotic degradative pathway essential for cell survival. We, therefore, investigated the effect of estrogen on autophagy in differentiating human osteoblasts and their precursors, the mesenchymal stem cells (MSCs). MSCs exhibited high autophagic flux which declined during osteoblast differentiation, resulting in high basal apoptosis in osteoblasts. Estrogen reduced apoptosis in differentiating osteoblasts by promoting autophagy, thus contributing towards their longer lifespan. Further, MSCs were resistant against starvation-induced apoptosis, whereas, differentiating osteoblasts showed significant susceptibility towards it. Estrogen, in addition to promoting mineralization, protected differentiating osteoblasts from starvation-induced apoptosis by increasing autophagic flux. Autophagic flux in RAB3GAP1 knockdown osteoblasts appeared diminished, and showed increased apoptosis even in nutrient-rich conditions, and exhibited significantly impaired mineralization. However, irrespective of the presence of estrogen, starvation further enhanced apoptosis in these cells. Furthermore, estrogen failed to promote mineralization in these osteoblasts. Our study illustrates that autophagy is essential for human osteoblast survival and mineralization, and osteoblasts are susceptible to apoptosis due to reduced autophagy during differentiation. Estrogen, via upregulation of RAB3GAP1, promotes autophagy in osteoblasts during differentiation thereby increasing their survival and mineralization capacity. Our study demonstrates the positive role of autophagy in bone homeostasis.

LYN, a key mediator in estrogen-dependent suppression of osteoclast differentiation, survival, and function.

Estrogen insufficiency at menopause cause accelerated bone loss due to unwarranted differentiation and function of osteoclasts. Unraveling the underlying mechanism/s may identify mediators of estrogen action which can be targeted for improved management of osteoporosis. Towards this, we analyzed the effect of 17ß-estradiol on the proteomes of differentiating human osteoclasts. The major proteomic changes observed included upregulation of LYN by estrogen. We, therefore, investigated the effect of estrogen on osteoclast differentiation, survival, and function in control and LYN knockdown conditions. In control condition, estrogen treatment increased the apoptosis rate and suppressed the calcium signaling by reducing the intracellular Ca2+ levels as well as expression and activation of NFATc1 and c-Src during differentiation, resulting in reduced osteoclastogenesis. These osteoclasts were smaller in size with reduced extent of multinuclearity and produced significantly low levels of bone resorbing enzymes. They also exhibited disrupted sealing zone formation with low podosome density, impaired cell polarization and reduced resorption of dentine slices. Interestingly, in LYN knockdown condition, estrogen failed to induce apoptosis and inhibit activation of NFATc1 and c-Src. Compared to effect of estrogen on osteoclast in control condition, LYN knockdown osteoclasts did not show reduction in production of bone resorbing enzymes and had defined sealing zone formation with high podosome density with no impairment in cell polarization. They resorbed significant area on dentine slices. Thus, the inhibitory action of estrogen on osteoclast was severely restrained in LYN knockdown condition, demonstrating the importance of LYN as a key mediator of the effect of estrogen on osteoclastogenesis.

Estradiol: A Steroid with Multiple Facets.

Seventy-five glorious years have passed since estradiol was discovered by Edward Doisy. From discovery in the ovaries to delineation of diverse physiological effects, research on estrogens has covered a lot of ground. Estrogen receptors that mediate estrogenic effects, have been detected not only in reproductive organs, but also in other body organs. Estrogen receptors function either as conventional transcription factors or as rapid signal transducers. These different modes of action are opted by estrogens to elicit an array of reproductive and non-reproductive functions. It is well established that estrogens promote cell proliferation in various tissues and hence are also linked to carcinogenesis. Anti-estrogens are being used as adjunct therapies for cancers since several years. On the other hand, estrogen-based strategies are used to alleviate adverse effects of menopause. Apart from estrogens synthesized in various organs, exposure to environmental estrogens can also impact physiology. Thus, too much or too less of estrogens can tip the balance and lead to unfavorable consequences. Multiple estrogen receptors with their tissue- or cell type-specific expression eliciting dose-dependent effects make it perplexing to 'unify' estrogenic actions in diverse tissues/organs. This warrants more research on estrogen-mediated effects and their regulation in somatic and reproductive tissues. This review presents physiological and pathological aspects of estrogens thus highlighting the good, bad, and ugly facets of estrogens.

Serum levels of phosphorylated heat shock protein 27 (pHSP27) are associated with bone mineral density in pre- & postmenopausal women: A pilot study.

BACKGROUND & OBJECTIVES: Phosphorylated heat shock protein 27 (pHSP27) has been implicated in the pathogenesis of osteoporosis. oxidative stress and proinflammatory cytokines, which are known to be involved in aetiology of osteoporosis, can trigger HSP27 phosphorylation. Since pHSP27 is present in circulation, it was hypothesized that serum pHSP27 would be elevated in low bone mineral density (BMD) condition and might serve as an indicator of osteoporosis/osteopenia. Hence, the aim of this study was to examine serum levels of pHSP27 in relation with BMD in pre- and postmenopausal women. METHODS: Premenopausal (30 to 40 yr) and postmenopausal (50 to 60 yr) women having either low BMD (osteopenia/osteoporosis) or high BMD were selected (n=80) from a prospective cohort (n=200). Serum levels of pHSP27; along with levels of oestradiol, malondialdehyde, total antioxidant capacity, interleukin (IL)-1, IL-6, tumour necrosis factor - alpha, (TNF-α), c-telopeptide fragments of collagen type I (CTX-1) and osteocalcin were estimated. RESULTS: The serum levels of pHSP27 were significantly elevated in low BMD groups in premenopausal and postmenopausal categories (p<0.05). It also exhibited a significant odds ratio (OR) to differentiate between low and high BMD in both premenopausal (OR=1.734, p=0.013) and postmenopausal (OR=1.463, p=0.042) categories. Additionally, area under the curve to predict low BMD was non-significantly higher for pHSP27 than CTX-1 in premenopausal and postmenopausal categories. INTERPRETATION & CONCLUSIONS: This study highlights a novel relation between serum pHSP27 and BMD in Indian women however, these findings need to be confirmed in larger studies.

Influence of bone mineral density measurement on fracture risk assessment tool® scores in postmenopausal Indian women.

AIM: Fracture risk assessment tool® calculations can be performed with or without addition of bone mineral density; however, the impact of this addition on fracture risk assessment tool® scores has not been studied in Indian women. Given the limited availability and high cost of bone mineral density testing in India, it is important to know the influence of bone mineral density on fracture risk assessment tool® scores in Indian women. Therefore, our aim was to assess the contribution of bone mineral density in fracture risk assessment tool® outcome in Indian women. METHODS: Apparently healthy postmenopausal Indian women (n = 506), aged 40-72 years, without clinical risk factors for bone disease, were retrospectively selected, and their fracture risk assessment tool® scores calculated with and without bone mineral density were compared. RESULTS: Based on WHO criteria, 30% women were osteoporotic, 42.9% were osteopenic and 27.1% had normal bone mineral density. Fracture risk assessment tool® scores for risk of both major osteoporotic fracture and hip fracture significantly increased on including bone mineral density (P < 0.0001). When criteria of National Osteoporosis Foundation, US was applied number of participants eligible for medical therapy increased upon inclusion of bone mineral density, (for major osteoporotic fracture risk number of women eligible without bone mineral density was 0 and with bone mineral density was 1, P > 0.05, whereas, for hip fracture risk number of women eligible without bone mineral density was 2 and with bone mineral density was 17, P < 0.0001). CONCLUSION: Until the establishment of country-specific medication intervention thresholds, bone mineral density should be included while calculating fracture risk assessment tool® scores in Indian women.

Monocyte Proteomics Reveals Involvement of Phosphorylated HSP27 in the Pathogenesis of Osteoporosis.

Peripheral monocytes, precursors of osteoclasts, have emerged as important candidates for identifying proteins relevant to osteoporosis, a condition characterized by low Bone Mineral Density (BMD) and increased susceptibility for fractures. We employed 4-plex iTRAQ (isobaric tags for relative and absolute quantification) coupled with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) to identify differentially expressed monocyte proteins from premenopausal and postmenopausal women with low versus high BMD. Of 1801 proteins identified, 45 were differentially abundant in low versus high BMD, with heat shock protein 27 (HSP27) distinctly upregulated in low BMD condition in both premenopausal and postmenopausal categories. Validation in individual samples (n = 80) using intracellular ELISA confirmed that total HSP27 (tHSP27) as well as phosphorylated HSP27 (pHSP27) was elevated in low BMD condition in both categories (P < 0.05). Further, using transwell assays, pHSP27, when placed in the upper chamber, could increase monocyte migration (P < 0.0001) and this was additive in combination with RANKL (receptor activator of NFkB ligand) placed in the lower chamber (P = 0.05). Effect of pHSP27 in monocyte migration towards bone milieu can result in increased osteoclast formation and thus contribute to pathogenesis of osteoporosis. Overall, this study reveals for the first time a novel link between monocyte HSP27 and BMD.