Loss Of Cell Function Research Articles

Metformin Delays Satellite Cell Activation and Maintains Quiescence.

The regeneration of the muscle tissue relies on the capacity of the satellite stem cell (SC) population to exit quiescence, divide asymmetrically, proliferate, and differentiate. In age-related muscle atrophy (sarcopenia) and several dystrophies, regeneration cannot compensate for the loss of muscle tissue. These disorders are associated with the depletion of the satellite cell pool or with the loss of satellite cell functionality. Recently, the establishment and maintenance of quiescence in satellite cells have been linked to their metabolic state. In this work, we aimed to modulate metabolism in order to preserve the satellite cell pool. We made use of metformin, a calorie restriction mimicking drug, to ask whether metformin has an effect on quiescence, proliferation, and differentiation of satellite cells. We report that satellite cells, when treated with metformin in vitro, ex vivo, or in vivo, delay activation, Pax7 downregulation, and terminal myogenic differentiation. We correlate the metformin-induced delay in satellite cell activation with the inhibition of the ribosome protein RPS6, one of the downstream effectors of the mTOR pathway. Moreover, in vivo administration of metformin induces a belated regeneration of cardiotoxin- (CTX-) damaged skeletal muscle. Interestingly, satellite cells treated with metformin immediately after isolation are smaller in size and exhibit reduced pyronin Y levels, which suggests that metformin-treated satellite cells are transcriptionally less active. Thus, our study suggests that metformin delays satellite cell activation and differentiation by favoring a quiescent, low metabolic state.

Endogenous DNA damage as a driver of senescence and aging

Aging is defined as a loss of tissue homeostasis and resilience in the face of stress and is attributed to stochastic damage that occurs over time. However, in measuring the various types of cellular damage it is impossible to determine what damage caused aging and what is a consequence of it. Genetic depletion of the DNA repair enzyme in mice or cells leads to the accelerated accumulation of spontaneous endogenous DNA damage, which cannot be repaired. With this as an initiating event, we found that deletion of ERCC1‐XPF in primary fibroblasts causes a rapid acceleration of senescence. In vivo, depletion of ERCC1‐XPF also causes accelerated accumulation of senescent cells in numerous tissues including lung, heart, pancreas, liver, spleen, skin and brain. There is accelerated onset of age‐related pathology and spontaneous onset of multiple age‐related diseases including cerebral atrophy, chronic kidney disease, loss of vision and hearing, osteoporosis and disc degeneration. We also discovered increased production of reactive oxygen species and evidence of oxidative damage to multiple types of macromolecules in cells. Furthermore, in ERCC1‐XPF deficient mice we see loss of stem cell function, mitochondrial dysfunction, epigenetic changes, alterations in autophagy and stress responses. In all cases, changes seen in ERCC1‐XPF‐deficient mice were recapitulated in aged wild‐type mice. Finally, tissue‐specific deletion of ERCC1‐XPF is enough to cause rapid onset of age‐related disease such as cardiomyopathy, diabetes and dementia. These data establish endogenous DNA damage as a driver of aging. Furthermore, the response to DNA damage impacts all pillars of aging.Support or Funding InformationNIA P01 AG043376, NIA U19 AG056278, The Glenn FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Oxidized low-density lipoprotein promotes vascular endothelial cell dysfunction by stimulating miR-496 expression and inhibiting the Hippo pathway effector YAP.

Oxidized low‐density lipoprotein (ox‐LDL) can damage vascular endothelial cells and cause atherosclerosis, but its epigenetic regulatory mechanism has not been fully elucidated. We show that ox‐LDL induced significant apoptosis and loss of function in human umbilical vascular endothelial cells (HUVECs). At the same time, ox‐LDL significantly decreased the expression of Hippo–YAP/ZAP (Yes‐associated protein/YLP motif–containing 1) pathway proteins as compared to that of the control. The luciferase reporter system confirmed that microRNA (miR)‐496 silenced YAP gene expression by binding to its 3′ untranslated region (3′ UTR). Ox‐LDL–treated miR‐496 overexpression HUVECs had a higher apoptosis rate and more severe dysfunction compared to the control cells. This in‐depth study shows that ox‐LDL inhibits YAP protein expression by inducing miR‐496 expression, leading to its inability to enter the nucleus, thereby losing its function as a transcriptional cofactor for activating the downstream genes. Our findings reveal that, through epigenetic modification, ox‐LDL can inhibit the normal expression of Hippo–YAP/ZAP pathway proteins via miR‐496 expression and induce vascular endothelial cell dysfunction.

Nonlinear dynamics with Hopf bifurcations by targeted mutation in the system of rock-paper-scissors metaphor

The role of mutation, which is an error process in gene evolution, in systems of cyclically competing species has been studied from various perspectives, and it is regarded as one of the key factors for promoting coexistence of all species. In addition to naturally occurring mutations, many experiments in genetic engineering have involved targeted mutation techniques such as recombination between DNA and somatic cell sequences and have studied genetic modifications through loss or augmentation of cell functions. In this paper, we investigate nonlinear dynamics with targeted mutation in cyclically competing species. In different ways to classic approaches of mutation in cyclic games, we assume that mutation may occur in targeted individuals who have been removed from intraspecific competition. By investigating each scenario depending on the number of objects for targeted mutation analytically and numerically, we found that targeted mutation can lead to persistent coexistence of all species. In addition, under the specific condition of targeted mutation, we found that targeted mutation can lead to emergences of bistable states for species survival. Through the linear stability analysis of rate equations, we found that those phenomena are accompanied by Hopf bifurcation which is supercritical. Our findings may provide more global perspectives on understanding underlying mechanisms to control biodiversity in ecological/biological sciences, and evidences with mathematical foundations to resolve social dilemmas such as a turnover of group members by resigning with intragroup conflicts in social sciences.

Liraglutide protects renal mesangial cells against hyperglycemia‑mediated mitochondrial apoptosis by activating the ERK‑Yap signaling pathway and upregulating Sirt3 expression.

Diabetic nephropathy results from hyperglycemia‑mediated renal glomerular cell death via mitochondrial apoptosis. There is an emerging requirement for novel approaches with mitochondrial protective effects that alleviate the hyperglycemia‑induced loss of functional cells during diabetic renal damage. Liraglutide, a type of glucagon‑like peptide‑1 agonist, has been suggested to inhibit the progression of obesity and hyperglycemia. However, the contributions and mechanism of action of liraglutide on hyperglycemia‑mediated cell mitochondrial apoptosis in diabetic kidneys have not been illustrated. The present study demonstrated that liraglutide may protect human renal mesangial cells (HRMCs) against hyperglycemia‑induced cell death by inhibiting mitochondrial apoptosis. Liraglutide administration also maintained HRMC viability and promoted HRMC proliferation within a high glucose stress environment. Functional studies demonstrated that hyperglycemia triggered mitochondrial dysfunction, including mitochondrial potential reduction, mitochondrial permeability transition pore opening, reactive oxygen species overproduction and the activation of the mitochondrial apoptotic pathway. However, liraglutide treatment preserved mitochondrial function and prevented activation of mitochondrial apoptosis by upregulating sirtuin 3 (Sirt3) expression. Deletion of Sirt3 abrogated the protective effects of liraglutide on mitochondrial homeostasis following high glucose challenge. In addition, molecular analysis confirmed that liraglutide upregulated Sirt3 via activating the extracellular signal‑regulated kinase‑Yes‑associated protein (ERK‑Yap) signaling pathway. Inhibition of the ERK‑Yap axis negated the action of liraglutide on Sirt3 activation, leading to mitochondrial injury and HRMC apoptosis. Taken together, the present study illustrated that liraglutide protected renal mesangial cells from hyperglycemia‑mediated mitochondrial apoptosis by upregulating Sirt3 expression and activation of the ERK‑Yap signaling pathway.

Abstract B172: Molecular mechanisms of dual-specificity phosphatase 22 (DUSP22) in suppression of tumorigenesis: The potential involvement of the EGFR/PD-L1 axis

Abstract DUSP22, belonging to members of the dual-specificity phosphatases (DUSPs) family, can control the activity of various protein kinases and transcription factors through the dephosphorylation process. Many DUSP22-targeted proteins are involved in signaling pathways that are crucial for tumorigenesis, and inflammation. Genetic alteration of DUSP22 or DUSP22 downregulation was observed in some cancers. However, little is known about the roles of DUSP22 during tumor suppression. Here, utilizing TGCA databases, our analysis showed low DUSP22 expression is associated with shorter disease-free survival of lung and prostate cancer patients. The IL-6/STAT3 and EGFR/ERK1/2 axes are generally recognized as an important oncogenic and mitogenic pathway in prostate cancer. We found that DUSP22 expression markedly reduced p-STAT3 (Tyr705), and p-EGFR (Tyr1068)/p-ERK1/2 expression levels after IL-6 and EGF stimulation, respectively, in prostate cancer cells. Similarly, DUSP22 can inhibit the EGFR/ERK1/2 axis in lung cancer cells that harbor EGFR exon 19 deletion mutation, supporting a repressive role for DUSP22 in regulating growth receptor signaling pathways. In addition, we found the exogenous expression of DUSP22 significantly reduced the cell growth of prostate and lung cancer cells. More importantly, DUSP22 can serve as a negative regulator of androgen receptor in prostate cancer cells through reducing EGF-induced AR phosphorylation and inhibiting DHT-mediated nuclear AR translocation. Taken together, our current data indicate that loss of DUSP22 function or DUSP22 downregulation may accelerate lung and prostate tumorigenesis through EGFR-dependent pathways. Importantly, it has been reported that increased PD-L1 expression by EGFR activation mediates the immune escape in EGFR-driven lung cancer (NSCLC). We found that the inhibition of the EGFR signaling by DUSP22 can reduce PD-L1 expression in both lung and prostate cancer cells, suggesting that loss of DUSP22 function in cancer cells may not only enhance cell growth, but also potentially repress antitumor immunity through upregulation of PD-L1. Our findings implicate that anti-PD1/PD-L1 therapy could be a potential option if lung or prostate cancer patients are found with low DUSP22 expression. Currently, we are using multiple approaches to elucidate molecular mechanisms of DUSP22 ablation for accelerating lung and prostate tumorigenesis in vivo. Citation Format: Wen-Jye Lin, Cheng-Wei Chang, Hsiu-Ping Lin, Hui-Min Ho. Molecular mechanisms of dual-specificity phosphatase 22 (DUSP22) in suppression of tumorigenesis: The potential involvement of the EGFR/PD-L1 axis [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B172.

LAMP2 expression dictates azacytidine response and prognosis in MDS/AML.

Chaperone-mediated autophagy (CMA) is a highly selective form of autophagy. During CMA, the HSC70 chaperone carries target proteins endowed with a KFERQ-like motif to the lysosomal receptor LAMP2A, which then translocate them into lysosomes for degradation. In the present study, we scrutinized the mechanisms underlying the response and resistance to Azacytidine (Aza) in MDS/AML cell lines and bone marrow CD34+ blasts from MDS/AML patients. In engineered Aza-resistant MDS cell lines and some AML cell lines, we identified a profound defect in CMA linked to the absence of LAMP2A. LAMP2 deficiency was responsible for Aza resistance and hypersensitivity to lysosome and autophagy inhibitors. Accordingly, gain of function of LAMP2 in deficient cells or loss of function in LAMP2-expressing cells rendered them sensitive or resistant to Aza, respectively. A strict correlation was observed between the absence of LAMP2, resistance to Aza and sensitivity to lysosome inhibitors. Low levels of LAMP2 expression in CD34+ blasts from MDS/AML patients correlated with lack of sensitivity to Aza and were predictive of poor overall survival. We propose that CD34+/LAMP2Low patients at diagnosis or who become CD34+/LAMP2Low during the course of treatment with Aza might benefit from a lysosome inhibitor already used in the clinic.

The impact of melatonin on antioxidant defense mechanisms in Alzheimer’s disease

During aging, the increased risk of neurodegenerative disease development is observed. One of the most common and most serious disorders is Alzheimer’s disease manifested by the loss of nerve cell functions. In the course of the disease, extracellular senile plaques consisting of beta-amyloid and intracellular neurofibrillary tangles of hyper-phosphorylated tau protein are formed. As a result of aging, the repair potential of damaged nerve cells is reduced. Free radicals formed in excess generate augmented oxidative stress, which contributes to the damaging of biomolecules and the development of pathological changes. This mechanism is favored by a decrease in the efficiency of antioxidant enzymes and the deficiency of antioxidants, observed in aging organism. With age, the secretion of the pineal hormone melatonin, functioning as a biochemical biological clock, is significantly reduced. This compound has been proved to be a very effective antioxidant that plays a key role in protecting cells against excessive damage, especially in nervous tissue. Studies have shown that supplementation with exogenous melatonin can prevent oxidative stress-induced degeneration of neurons. Considering the action of melatonin and the pathogenesis of Alzheimer’s disease, the idea of using the hormone supplementation in the prevention and alleviation of the effects of the disease seems to be extremely interesting.

Induced in vivo knockdown of the Brca1 gene in skeletal muscle results in skeletal muscle weakness.

Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function. BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle. Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity. Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations. These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. Recent evidence suggests that the breast cancer 1 early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The present study aimed to determine whether induced-loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in the skeletal muscle of adult mice to generate skeletal muscle-specific Brca1 homozygote knockout (Brca1KOsmi ) mice. Brca1KOsmi exhibited kyphosis and decreased maximal isometric force in limb muscles compared to age-matched wild-type mice. Brca1KOsmi skeletal muscle shifted toward an oxidative muscle fibre type and, in parallel, increased myofibre size and reduced capillary numbers. Unexpectedly, myofibre bundle mitochondrial respiration was reduced, whereas contraction-induced lactate production was elevated in Brca1KOsmi muscle. Brca1KOsmi mice accumulated mitochondrial DNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and these were more susceptible to swelling. In summary, skeletal muscle-specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health.

Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome.

Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3-5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960-1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.

Homology-independent multiallelic disruption via CRISPR/Cas9-based knock-in yields distinct functional outcomes in human cells

BackgroundCultured human cells are pivotal models to study human gene functions, but introducing complete loss of function in diploid or aneuploid cells has been a challenge. The recently developed CRISPR/Cas9-mediated homology-independent knock-in approach permits targeted insertion of large DNA at high efficiency, providing a tool for insertional disruption of a selected gene. Pioneer studies have showed promising results, but the current methodology is still suboptimal and functional outcomes have not been well examined. Taking advantage of the promoterless fluorescence reporter systems established in our previous study, here, we further investigated potentials of this new insertional gene disruption approach and examined its functional outcomes.ResultsExemplified by using hyperploid LO2 cells, we demonstrated that simultaneous knock-in of dual fluorescence reporters through CRISPR/Cas9-induced homology-independent DNA repair permitted one-step generation of cells carrying complete disruption of target genes at multiple alleles. Through knocking-in at coding exons, we generated stable single-cell clones carrying complete disruption of ULK1 gene at all four alleles, lacking intact FAT10 in all three alleles, or devoid of intact CtIP at both alleles. We have confirmed the depletion of ULK1 and FAT10 transcripts as well as corresponding proteins in the obtained cell clones. Moreover, consistent with previous reports, we observed impaired mitophagy in ULK1−/− cells and attenuated cytokine-induced cell death in FAT10−/− clones. However, our analysis showed that single-cell clones carrying complete disruption of CtIP gene at both alleles preserved in-frame aberrant CtIP transcripts and produced proteins. Strikingly, the CtIP-disrupted clones raised through another two distinct targeting strategies also produced varied but in-frame aberrant CtIP transcripts. Sequencing analysis suggested that diverse DNA processing and alternative RNA splicing were involved in generating these in-frame aberrant CtIP transcripts, and some infrequent events were biasedly enriched among the CtIP-disrupted cell clones.ConclusionMultiallelic gene disruption could be readily introduced through CRISPR/Cas9-induced homology-independent knock-in of dual fluorescence reporters followed by direct tracing and cell isolation. Robust cellular mechanisms exist to spare essential genes from loss-of-function modifications, by generating partially functional transcripts through diverse DNA and RNA processing mechanisms.

Ras-Driven B-Cell Transformation Targets Developmental Rewiring of Cytokine to Pre-B Cell Receptor Signaling

Ras-pathway lesions are oncogenic drivers in ~45% of B-cell acute lymphoblastic leukemia (B-ALL) cases. Activating mutations of NRAS and KRAS are oncogenic drivers in B-ALL while the BRAFV600E mutation occurs in almost all cases of B-cell hairy cell leukemia. Less frequent lesions resulting in increased ERK-signaling in B-cell malignancies include activating mutations of RAF1, MAP2K1 and the PTPN11 phosphatase as well as deleterious mutations of the Ras-GTPase activator NF1. Interestingly, increased immunoglobulin light chain gene expression was observed in B-ALL cases with RAS-pathway lesions (COG P9906), reflecting engagement of pre-B cell receptor (pre-BCR) downstream signaling. Here we tested the hypothesis that oncogenic RAS-signaling in B-ALL mimics pre-BCR-induced developmental rewiring of signal transduction at the pro-B to pre-B cell transition and identified PTPN6 and BCL6 as therapeutic targets in RAS-driven B-ALL.During early B-cell development, pro-B cells transition from cytokine- to pre-B cell receptor (pre-BCR)-dependent survival and proliferation signals. Inducible activation of immunoglobulin (Ig) µ heavy chain (µHC) expression induced developmental progression and surface expression of Ig κ light chains. Notably, inducible activation of oncogenic NRASG12D had the same effect and resulted in increased surface expression of Ig κ light chains. Furthermore, studying genetic models for this transition revealed that both pre-BCR signaling and RAS-oncogenes suppressed cytokine receptor/STAT5-signaling and induced massive de novo expression of the proto-oncogene and transcriptional repressor BCL6. Our genetic studies revealed that the SH2-domain containing protein tyrosine phosphatase PTPN6 was activated by oncogenic RAS-signaling and essential for the switch from STAT5 to BCL6-activation. Given that oncogenic RAS activated PTPN6, we tested the role of PTPN6 in RAS-driven leukemogenesis. To this end, ablation of Ptpn6 in NRASG12D-driven B-ALL resulted in depletion of cells from cell culture in competitive-growth assays and reduced the number of colonies formed in semi-solid methylcellulose. Collectively, these findings suggest that PTPN6 represents a potential therapeutic intervention point in RAS-driven B-ALL.In addition to PTPN6, we investigated the role of BCL6 in RAS-driven B-ALL. Aberrant activation of oncogenic RAS results in oncogene-induced senescence (OIS) characterized by induction of ARF/p53 and irreversible cell cycle arrest in the G1 phase. For oncogenic Ras-signaling, BCL6 was required to oppose ERK-mediated activation of p21, p27 and p53 checkpoint molecules in B-ALL. Here we tested the hypothesis that BCL6 bypasses the RAS-mediated OIS program to facilitate transformation. To this end, increases in number of colonies formed in semi-solid methylcellulose were observed upon inducible activation of Bcl6 in NRASG12D B-ALL cells. Furthermore, loss of Bcl6 function in NRASG12D B-ALL cells resulted in depletion of cells from cell culture in competitive growth assays and reduced colony forming ability. Importantly, expression of NRASG12D in Bcl6+/+ pre-B cells resulted in transformation and fatal leukemia in transplant recipient mice. In striking contrast, Bcl6-/- pre-B cells transduced with NRASG12D failed to initiate fatal disease in vivo. Furthermore, pharmacological inhibition of BCL6 restored sensitivity to chemotherapy in patient-derived KRASG12V B-ALL cells.In conclusion, we identified oncogenic RAS-signaling as functional mimics of pre-BCR signaling. Oncogenic RAS induced expression of BCL6 at the expense of cytokine receptor/STAT5-signaling. Our genetic studies identified PTPN6 as a critical effector molecule of the switch from cytokine receptor to pre-BCR signaling. Importantly, we identified PTPN6 and BCL6 as potential therapeutic intervention points in RAS-driven B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Notchless defines a stage-specific requirement for ribosome biogenesis during lineage progression in adult skeletal myogenesis.

Cell fate decisions occur through the action of multiple factors, including signalling molecules and transcription factors. Recently, the regulation of translation has emerged as an important step for modulating cellular function and fate, as exemplified by ribosomes that play distinct roles in regulating cell behaviour. Notchless (Nle) is a conserved nuclear protein that is involved in a crucial step in ribosome biogenesis, and is required for the maintenance of adult haematopoietic and intestinal stem/progenitor cells. Here, we show that activated skeletal muscle satellite cells in conditional Nle mutant mice are arrested in proliferation; however, deletion of Nle in myofibres does not impair myogenesis. Furthermore, conditional deletion of Nle in satellite cells during homeostasis did not impact on their fate for up to 3 months. In contrast, loss of Nle function in primary myogenic cells blocked proliferation because of major defects in ribosome formation. Taken together, we show that muscle stem cells undergo a stage-specific regulation of ribosome biogenesis, thereby underscoring the importance of differential modulation of mRNA translation for controlling cell fate decisions.

Insulin-like growth factor receptor signaling in breast tumor epithelium protects cells from endoplasmic reticulum stress and regulates the tumor microenvironment

BackgroundEarly analyses of human breast cancer identified high expression of the insulin-like growth factor type 1 receptor (IGF-1R) correlated with hormone receptor positive breast cancer and associated with a favorable prognosis, whereas low expression of IGF-1R correlated with triple negative breast cancer (TNBC). We previously demonstrated that the IGF-1R acts as a tumor and metastasis suppressor in the Wnt1 mouse model of TNBC. The mechanisms for how reduced IGF-1R contributes to TNBC phenotypes is unknown.MethodsWe analyzed the METABRIC dataset to further stratify IGF-1R expression with patient survival and specific parameters of TNBC. To investigate molecular events associated with the loss of IGF-1R function in breast tumor cells, we inhibited IGF-1R in human cell lines using an IGF-1R blocking antibody and analyzed MMTV-Wnt1-mediated mouse tumors with reduced IGF-1R function through expression of a dominant-negative transgene.ResultsOur analysis of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset revealed association between low IGF-1R and reduced overall patient survival. IGF-1R expression was inversely correlated with patient survival even within hormone receptor-positive breast cancers, indicating reduced overall patient survival with low IGF-1R was not due simply to low IGF-1R expression within TNBCs. Inhibiting IGF-1R in either mouse or human tumor epithelial cells increased reactive oxygen species (ROS) production and activation of the endoplasmic reticulum stress response. IGF-1R inhibition in tumor epithelial cells elevated interleukin (IL)-6 and C-C motif chemokine ligand 2 (CCL2) expression, which was reversed by ROS scavenging. Moreover, the Wnt1/dnIGF-1R primary tumors displayed a tumor-promoting immune phenotype. The increased CCL2 promoted an influx of CD11b+ monocytes into the primary tumor that also had increased matrix metalloproteinase (MMP)-2, MMP-3, and MMP-9 expression. Increased MMP activity in the tumor stroma was associated with enhanced matrix remodeling and collagen deposition. Further analysis of the METABRIC dataset revealed an increase in IL-6, CCL2, and MMP-9 expression in patients with low IGF-1R, consistent with our mouse tumor model and data in human breast cancer cell lines.ConclusionsOur data support the hypothesis that reduction of IGF-1R function increases cellular stress and cytokine production to promote an aggressive tumor microenvironment through infiltration of immune cells and matrix remodeling.

SBF-1 preferentially inhibits growth of highly malignant human liposarcoma cells

Frequent local recurrence and metastasis are generally involved in human liposarcoma, but the management is a challenge. There is an urgent need for improved effective therapy. In the present study, we reported that SBF-1, a steroidal glycoside, inhibited the growth of cultured highly malignant human liposarcoma SW872-S cells in vitro and in vivo. SBF-1 down-regulated the phosphorylation of protein kinase B (AKT) and thus reduced cell adhesion to fibronectin and laminin. Then we found that SBF-1 inhibited the expression of oxysterol binding protein (OSBP) in SW872-S cells, indicating that OSBP may be involved in malignant liposarcoma cell survival. Cancer cell growth and AKT phosphorylation were inhibited significantly upon knockdown of OSBP in SW872-S cells in vitro. Taken together, these results suggest that SBF-1 causes an apparent loss of OSBP function in SW872-S cells, resulting in growth inhibition. Based on our findings, OSBP serves as a potential therapeutic target for human liposarcoma.

RNA-binding protein, human antigen R regulates hypoxia-induced autophagy by targeting ATG7/ATG16L1 expressions and autophagosome formation.

Autophagy, a prosurvival mechanism offers a protective role during acute kidney injury. We show novel findings on the functional role of RNA binding protein, HuR during hypoxia-induced autophagy in renal proximal tubular cells-2 (HK-2). HK-2 cells showed upregulated expressions of HuR and autophagy-related proteins such as autophagy related 7 (ATG7), autophagy related 16 like 1 (ATG16L1), and LC3II under hypoxia. Increased autophagosome formation was visualized as LC3 puncta in hypoxic cells. Further, short hairpin-RNA-mediated loss of HuR function in HK-2 cells significantly decreased ATG7 and ATG16L1 protein expressions. Bioinformatics prediction revealed HuR motif binding on the coding region of ATG7 and AU-rich element at 3'UTR ATG16L1 messnger RNA (mRNA). The RNA immunoprecipitation study showed that HuR was predominantly associated with ATG7 and ATG16L1 mRNAs under hypoxia. In addition, HuR enhanced autophagosome formation by regulating LC3II expressions. These results show that HuR regulates ATG7 and ATG16L1 expressions and thereby mediate autophagy in HK-2 cells. Importantly, HuR knockdown cells underwent apoptosis during hypoxia as observed through the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Collectively, these findings show the crucial role of HuR under hypoxia by regulating autophagy and suppressing apoptosis in renal tubular cells.

Zinc deficiency drives Th17 polarization and promotes loss of Treg cell function

A high number of illnesses and disorders are connected to zinc deficiency. Equally, T cell polarization and a balance between different T helper (Th) cell subsets are essential. Therefore, in this study, the influence of zinc deficiency on T cell polarization and on respective signaling pathways was investigated. We uncovered a significantly increased number of regulatory T cells (Treg) and Th17 cells in expanded T cells during zinc deficiency after 3 days of combined treatment with IL-2 and TGF-β1 (Treg) or IL-6 and TGF-β1 (Th17). No difference in Th1 and Th2 cell polarization between zinc-deficient and zinc-adequate status was prominent. On the molecular level, Smad signaling was significantly enhanced by stimulation with TGF-β1/IL-6 during zinc deficiency compared to adequate zinc condition. This represents an explanation for the elevated Th17 cell numbers associated with autoimmune disease especially during zinc deficiency. Moreover, Treg cell numbers are increased during zinc deficiency as well. However, those cells might be nonfunctional since a lower expression of miR-146a was uncovered compared to normal zinc concentrations. In summary, an adequate zinc homeostasis is fundamental to slow down or probably stop the progression of autoimmune diseases and infections. Therefore, supplementing zinc might be a therapeutic approach to dampen autoimmune diseases connected to Th17 cells.

The use of hydrogels for cell-based treatment of chronic kidney disease.

Chronic kidney disease (CKD) is a major and growing public health concern with increasing incidence and prevalence worldwide. The therapeutic potential of stem cell therapy, including mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) holds great promise for treatment of CKD. However, there are significant bottlenecks in the clinical translation due to the reduced number of transplanted cells and the duration of their presence at the site of tissue damage. Bioengineered hydrogels may provide a route of cell delivery to enhance treatment efficacy and optimise the targeting effectiveness while minimising any loss of cell function. In this review, we highlight the advances in stem cell therapy targeting kidney disease and discuss the emerging role of hydrogel delivery systems to fully realise the potential of adult stem cells as a regenerative therapy for CKD in humans. MSCs and EPCs mediate kidney repair through distinct paracrine effects. As a delivery system, hydrogels can prolong these paracrine effects by improving retention at the site of injury and protecting the transplanted cells from the harsh inflammatory microenvironment. We also discuss the features of a hydrogel, which may be tuned to optimise the therapeutic potential of encapsulated stem cells, including cell-adhesive epitopes, material stiffness, nanotopography, modes of gelation and degradation and the inclusion of bioactive molecules. This review concludes with a discussion of the challenges to be met for the widespread clinical use of hydrogel delivery system of stem cell therapy for CKD.

PLG72 induces superoxide radicals via interaction and aggregation with SOD1

G72 has been characterised as a susceptibility gene that can have wide-ranging effects in a number of neurodegenerative diseases, including schizophrenia and major depression. Indeed, its product, pLG72, is a potential serum biomarker for schizophrenia. Previous transcriptomic and biochemical studies have indicated that pLG72 may induce the production of mitochondrial reactive oxygen species (ROS), resulting in cell damage. Here, we investigated the mechanism of pLG72 by transfecting a human U87 glioblastoma cell line with a G72 construct. By employing ROS-specific scavengers, we discovered that superoxide radicals were specifically induced in the pLG72-expressing cells. We also found that pLG72 interacted and co-localised with superoxide dismutase 1 (SOD1), resulting in aggregation of SOD1 with a concomitant 23% or 74% reduction of total SOD activity, depending on the amount of G72 transfection plasmid. Finally, we found that transfection of U87 cells with the G72 construct caused a 29% decrease in cell proliferation. The observed loss of SOD1 function in pLG72-expressing cells may explain the elevated ROS levels and inhibition of U87 cell proliferation and has implications for understanding the onset of neurodegenerative diseases in humans.

Tmc2 expression partially restores auditory function in a mouse model of DFNB7/B11 deafness caused by loss of Tmc1 function

Mouse Tmc1 and Tmc2 are required for sensory transduction in cochlear and vestibular hair cells. Homozygous Tmc1∆/∆ mice are deaf, Tmc2∆/∆ mice have normal hearing, and double homozygous Tmc1∆/∆; Tmc2∆/∆ mice have deafness and profound vestibular dysfunction. These phenotypes are consistent with their different spatiotemporal expression patterns. Tmc1 expression is persistent in cochlear and vestibular hair cells, whereas Tmc2 expression is transient in cochlear hair cells but persistent in vestibular hair cells. On the basis of these findings, we hypothesized that persistent Tmc2 expression in mature cochlear hair cells could restore auditory function in Tmc1∆/∆ mice. To express Tmc2 in mature cochlear hair cells, we generated a transgenic mouse line, Tg[PTmc1::Tmc2], in which Tmc2 cDNA is expressed under the control of the Tmc1 promoter. The Tg[PTmc1::Tmc2] transgene slightly but significantly restored hearing in young Tmc1∆/∆ mice, though hearing thresholds were elevated with age. The elevation of hearing thresholds was associated with deterioration of sensory transduction in inner hair cells and loss of outer hair cell function. Although sensory transduction was retained in outer hair cells, their stereocilia eventually degenerated. These results indicate distinct roles and requirements for Tmc1 and Tmc2 in mature cochlear hair cells.