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At least 1,531 records · Page 85
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DOE OSTI2014

Regulation of MDA-MB-231 cell proliferation by GSK-3β involves epigenetic modifications under high glucose conditions

Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3β at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3β by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3β silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3β activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells. - Highlights: • High glucose induces phosphorylation of histone H3 and dephosphorylation of GSK-3β. • Moreover, hyperglycemia also leads to increased DNA methylation in MDA-MB-231 cells. • Inhibition of GSK-3β prevented histone H3 phosphorylation and reduced DNMT1 levels. • Interplay exists between GSK-3β, histone H3 phosphorylation and DNA methylation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Insight in the multilevel regulation of NER

Nucleotide excision repair (NER) is a key component of the DNA damage response (DDR) and it is essential to safeguard genome integrity against genotoxic insults. The regulation of NER is primarily mediated by protein post-translational modifications (PTMs). The NER machinery removes a wide spectrum of DNA helix distorting lesions, including those induced by solar radiation, through two sub-pathways: global genome nucleotide excision repair (GG-NER) and transcription coupled nucleotide excision repair (TC-NER). Severe clinical consequences associated with inherited NER defects, including premature ageing, neurodegeneration and extreme cancer-susceptibility, underscore the biological relevance of NER. In the last two decades most of the core NER machinery has been elaborately described, shifting attention to molecular mechanisms that either facilitate NER in the context of chromatin or promote the timely and accurate interplay between NER factors and various post-translational modifications. In this review, we summarize and discuss the latest findings in NER. In particular, we focus on emerging factors and novel molecular mechanisms by which NER is regulated.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

The β-catenin signaling pathway induces aggressive potential in breast cancer by up-regulating the chemokine CCL5

β-Catenin signaling plays a pivotal role in the genesis of a variety of malignant tumors, but its role in breast cancer has not been fully elucidated. Here, we examined whether deregulation of β-catenin signaling is related to the aggressive characteristics of certain types of breast cancers. Analysis of cytokine levels in MDA-MB-231 cells overexpressing a constitutively active form of β-catenin (CAβ-catenin) revealed a higher level of CCL5 expression. Cells transfected with CAβ-catenin or stimulated with recombinant CCL5 exhibited increased cell invasion activity and spheroid formation in vitro. Furthermore, CAβ-catenin-transfected MDA-MB-231 cells formed larger tumor masses that contained more Ki-67-positive cells and infiltrating lymphocytes than did the control cells. An inhibitor of CCR5 and a pan-CXCR neutralizing antibody dramatically reduced CAβ-catenin-promoted activities. In addition to CCL5, 6-BIO, a chemical activator of β-catenin, induced cell invasion and spheroid formation in MDA-MB-231 cells. Furthermore, high levels of nuclear β-catenin accumulation were detected in breast cancer in patients with metastasis but not in those without metastasis. Nuclear β-catenin localization is related to increased CCL5 production in breast cancer. These findings suggest that β-catenin expression enhances tumor progression via chemokine production in breast cancers and that β-catenin signaling is a critical regulator of the aggressive traits of breast cancers. - Highlights: •The expression of chemokines, especially CCL5, is positively regulated by β-catenin signaling •The inhibition of CCR5 signaling suppressed β-catenin-induced tumor spreading in vitro and in vivo. • β-catenin was detected in cell nuclei in specimens from carcinoma regions of metastasized breast cancer • β-catenin nuclear translocation and cytokines have implications in leading to metastasis events.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

N-terminal nesprin-2 variants regulate β-catenin signalling

The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragment of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. - Highlights: • N-terminal nesprin-2 variants display cell specific expression patterns. • N-terminal spectrin repeats of nesprin-2 interact with β-catenin. • N-terminal nesprin-2 variants scaffold β-catenin at cell-cell junctions.. • Nesprin-2 variants play multiple roles in β-catenin signalling.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

miR-1207-3p regulates the androgen receptor in prostate cancer via FNDC1/fibronectin

Prostate cancer (PCa) is frequently diagnosed in men, and dysregulation of microRNAs is characteristic of many cancers. MicroRNA-1207-3p is encoded at the non-protein coding gene locus PVT1 on the 8q24 human chromosomal region, an established PCa susceptibility locus. However, the role of microRNA-1207-3p in PCa is unclear. We discovered that microRNA-1207-3p is significantly underexpressed in PCa cell lines in comparison to normal prostate epithelial cells. Increased expression of microRNA-1207-3p in PCa cells significantly inhibits proliferation, migration, and induces apoptosis via direct molecular targeting of FNDC1, a protein which contains a conserved protein domain of fibronectin (FN1). FNDC1, FN1, and the androgen receptor (AR) are significantly overexpressed in PCa cell lines and human PCa, and positively correlate with aggressive PCa. Prostate tumor FN1 expression in patients that experienced PCa-specific death is significantly higher than in patients that remained alive. Furthermore, FNDC1, FN1 and AR are concomitantly overexpressed in metastatic PCa. Consequently, these studies have revealed a novel microRNA-1207-3p/FNDC1/FN1/AR regulatory pathway in PCa. - Graphical abstract: miR-1207-3p/FNDC1/FN1/AR is a novel regulatory pathway in prostate cancer. - Highlights: • Expression of microRNA-1207-3p is significantly lost in prostate cancer (PCa) cells. • MicroRNA-1207-3p regulates proliferation, apoptosis, and migration via direct molecular targeting of the 3′UTR of FNDC1. • MicroRNA-1207-3p regulates proliferation, apoptosis, and migration via direct molecular targeting of the 3′UTR of FNDC1. • FNDC1, FN1, and AR are concurrently overexpressed in metastatic PCa.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

TUG1 promotes lens epithelial cell apoptosis by regulating miR-421/caspase-3 axis in age-related cataract

Background: Age-related cataract is among the most common chronic disorders of ageing and the apoptosis of lens epithelial cells contributes to non-congenital cataract development. We amid to explore the role of TUG1 and miR-421 in the age-related cataract. Methods: The expression level of TUG1, miR-421 and caspase-3 were detected by RT-qPCR. The apoptotic-related protein, caspase-3, Bax and blc-2 were analyzed by western blot. We performed ultraviolet (UV) irradiation to induce SAR01/04 cell apoptosis which was analyzed by flow cytometry. RIP pull-down and luciferase reporter assay were used to verified the combination and regulating among TUG1, miR-421 and caspase-3. Results: Here, we observed that the expression level of TUG1 and caspase-3 in the anterior lens capsules of age-related cataract were significantly higher and miR-421 was significantly lower than that in the normal anterior lens capsules. The apoptosis-related protein, caspase-3, Bax and blc-2 were abnormal expression in the anterior lens capsules of age-related cataract tissue. Our data showed that the expression level of TUG1 and caspase-3 and cell apoptosis rate in SAR01/04 cells treated with UV irradiation was remarkably higher than that in the control. TUG1 negatively regulated miR-421 expression and promoted UV irradiation-induced SAR01/04 cell apoptosis. However, miR-421 inhibitor and pcDNA-caspase-3 could reverse the action of the SRA01/04 cell apoptosis by si-TUG1, which suggested TUG1 promoted UV irradiation-induced apoptosis through downregulating miR-421 expression. Furthermore, this study confirmed TUG1 could been in combination with miR-421, and TUG1 and caspase-3 were both a directly target of miR-421. Conclusion: TUG1 modulated lens epithelial cell apoptosis through miR-421/caspase-3 axis. These findings will offer a novel insight into the pathogenesis of cataract. - Highlights: • TUG1 is increased in age-related cataract and UV irradiation-induced SAR01/04 cell. • Highly expressed TUG1 promotes UV irradiation-induced SAR01/04 cell apoptosis. • TUG1 and caspase-3 are both a direct target of miR-421. • MiR-421 inhibitor increases si-TUG1-inhibited SAR01/04 cells apoptosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

MRTF-A-miR-206-WDR1 form feedback loop to regulate breast cancer cell migration

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. Our previous study has shown that MRTF-A promote the migration of MDA-MB-231 cells and WDR1 promotes breast cancer cell migration. But the exact molecular mechanism on metastasis is still not fully understood, we now report that WDR1 enhanced the effect of MRTF-A induced-MDA-MB-231 cell migration by promoting the expression of the EMT markers and migration markers via RhoA-MRTF-A signaling pathway. Importantly, WDR1 promoted the nuclear importion of MRTF-A by affecting the expression of nuclear transport protein importin. But WDR1 did not affect the expression of MRTF-A. Interestingly, MRTF-A promoted the expression of miR-206 via its promoter CArG box but miR-206 inhibits the migration of breast cancer cells through suppressing the expression of WDR1 and MRTF-A via targeted their 3′UTR. Our data thus provide important and novel insights into MRTF-A-miR-206-WDR1 form feedback loop to regulate breast cancer cell migration. - Highlights: • WDR1 enhanced the effect of MRTF-A induced cell migration via RhoA-MRTF-A signaling pathway. • WDR1 promoted the nuclear importion of MRTF-A. • MRTF-A promoted the expression of miR-206 via its promoter CArG box. • miR-206 inhibits cell migration through suppressing the expression of WDR1 and MRTF-A. • MRTF-A-miR-206-WDR1 form feedback loop to regulate breast cancer cell migration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Msi2 plays a carcinogenic role in esophageal squamous cell carcinoma via regulation of the Wnt/β-catenin and Hedgehog signaling pathways

Highlights: • Msi2 is upregulated in ESCC tissues and cell lines. • Msi2 is associated with tumor progression and poor prognosis of ESCC patients. • Msi2 promotes ESCC cell proliferation, EMT and migration. • Msi2 functions by regulating the Wnt/β-catenin and Hh signaling pathways. Msi2 has been widely reported to be upregulated and strongly associated with fast progress and poor prognosis in many cancers. However, the expression and role of Msi2 in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, we found that Msi2 was upregulated in ESCC clinical samples, and was significantly associated with tumor size, differentiation status, and lymph node metastasis in ESCC patients. Multivariate Cox regression analysis showed that Msi2 was an independent predictor for disease-free survival (DFS) and overall survival (OS). Moreover, knockdown of Msi2 impaired ESCC cell proliferation, epithelial-mesenchymal transition (EMT) and migration, while overexpression of Msi2 promoted ESCC cell proliferation, EMT and migration in vitro. Animal experiments also confirmed that Msi2 promoted ESCC cell proliferation in vivo. Mechanistically, Msi2 promoted ESCC cell proliferation, EMT and migration via regulation of the Wnt/β-catenin and Hedgehog (Hh) signaling pathways. Taken together, our study suggested that Msi2 could serve as a candidate for diagnosis and prognosis and as a potential therapeutic target in ESCC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Knockdown of LncRNA PVT1 inhibits tumorigenesis in non-small-cell lung cancer by regulating miR-497 expression

Highlights: • PVT1 was significantly upregulated and miR-497 was markedly downregulated in NSCLC cell lines. • PVT1 knockdown remarkably inhibited cell viability, invasion and promoted apoptosis in NSCLC cells. • PVT1 could directly interact with miR-497. • Knockdown of PVT1 inhibited tumor growth in vivo and promoted miR-497 expression. • Knockdown of PVT1 inhibited cell viability, invasion and induced apoptosis in NSCLC by regulating miR-497 expression. Plasmacytoma variant translocation1 (PVT1) was reported to be upregulated in non-small-cell lung cancer (NSCLC) tissues, serve as a promising biomarker for diagnosis and prognosis of NSCLC, and promoted NSCLC cell proliferation. However, the detailed molecular mechanism of PVT1 involved in the pathogenesis and development of NSCLC remains largely unknown. In this study, the expression levels of PVT1 and miR-497 in NSCLC cells were determined by qRT-PCR. Cell viability, invasion and apoptosis were detected by MTT assay, cell invasion assay and flow cytometry analysis, respectively. RNA immunoprecipitation (RIP) and luciferase reporter assay were performed to confirm whether PVT1 directly interacts with miR-497. A xenograft mouse model was established to confirm the effect of PVT1 on tumor growth in vivo and the underlying molecular mechanism. Our findings indicated that PVT1 was significantly upregulated and miR-497 was markedly downregulated in NSCLC cell lines. si-PVT1 effectively decreased the expression of PVT1 and increased the expression of miR-497. PVT1 knockdown remarkably inhibited cell viability, invasion and promoted apoptosis in NSCLC cells. RIP and luciferase reporter assay demonstrated that PVT1 could directly interact with miR-497. Moreover, PVT1 overexpression reversed the inhibitory effect of miR-497 on cell viability, invasion and promotion effect on apoptosis of NSCLC cells. Furthermore, in vivo experiment showed that knockdown of PVT1 inhibited tumor growth in vivo and promoted miR-497 expression. In conclusion, knockdown of PVT1 inhibited cell viability, invasion and induced apoptosis in NSCLC by regulating miR-497 expression, elucidating the molecular mechanism of the oncogenic role of PVT1 in NSCLC and providing an lncRNA-directed target for NSCLC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

MicroRNA-30e-5p suppresses non-small cell lung cancer tumorigenesis by regulating USP22-mediated Sirt1/JAK/STAT3 signaling

MicroRNA-30e-5p (miR-30e-5p) is a tumor suppressor that is known to be downregulated in non-small cell lung cancer (NSCLC). However, how miR-30e-5p inhibits NSCLC tumorigenesis is not known. Ubiquitin-specific peptidase 22 (USP22) is upregulated in NSCLC and promotes tumorigenesis via a Sirt1-JAK-STAT3 pathway. In this study, we investigated whether miR-30e-5p inhibits tumor growth by targeting USP22 in NSCLC. Our results reveal that miR-30e-5p expression was correlated negatively with USP22 in NSCLC tissues. Luciferase reporter assays showed that miR-30e-5p negatively regulated USP22 expression by binding to a specific sequence in the 3UTR. MiR-30e-5p overexpression and USP22 knockdown significantly inhibited tumor growth in vivo and induced cell cycle arrest and apoptosis in NSCLC cells in vitro. The effects of miR-30e-5p inhibition were prevented by USP22 knockdown. MiR-30e-5p inhibited SIRT1 expression and increased expression of p53 and the phosphorylated form of STAT3 (pSTAT3). Furthermore, miR-30e-5p prevented USP22-mediated regulation of SIRT1, pSTAT3, and p53 expression. Taken together, these findings suggest that miR-30e-5p suppresses NSCLC tumorigenesis by downregulatingUSP22-mediated Sirt1/JAK/STAT3 signaling. Our study has identified miR-30e-5p as a potential therapeutic target for the treatment of NSCLC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

mmu-miR-1963 negatively regulates the ameloblast differentiation of LS8 cell line by directly targeting Smoc2 3’UTR

Highlights: • mmu-miR-1963 could downregulate the expression of Smoc2 by binding to its 3’UTR. • Smoc2 could promote the amelogenic differentiation of LS8. • mmu-miR-1963 inhibits the ameloblast differentiation of LS8 via Smoc2. RUNX2 is a key regulator of osteogenic differentiation and odontoblastic differentiation. RUNX2 mutations could cause Cleidocranial dysplasia (CCD; OMIM119600), which is featured by abnormal development of bone and teeth. By using microRNA array, we identified a large number of microRNAs that showed different expression between wild-type Runx2 group and mutant groups. The aim of this study is to find out the effect of mmu-miR-1963, which was downregulated in all mutant Runx2 groups, on the ameloblast differentiation of LS8 cells. qPCR and Western Blot results showed the suppressive effect of mmu-miR-1963 on ameloblast differentiation of LS8 cell line. We further confirmed Smoc2 as one direct target of mmu-miR-1963. For the first time, we showed that mmu-miR-1963 could regulate the ameloblast differentiation of LS8 by targeting Smoc2. This study suggests the suppressive role of mmu-miR-1963 on ameloblast differentiation of LS8 via directly targeting the 3’UTR of Smoc2. We also demonstrated that Smoc2 itself could promote the ameloblast differentiation of LS8 for the first time. Our results indicate a novel explanation to the enamel hypoplasia phenotype in part of CCD patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

SPARC is down-regulated by DNA methylation and functions as a tumor suppressor in T-cell lymphoma

Highlights: • SPARC is significantly down-regulated in T-NHL tissues and cell lines. • High levels of SPARC demonstrated a favorable clinical outcome. • SPARC inhibits proliferation, invasion and migration, but promotes apoptosis of T-NHL cells. • 5-Aza-Cdr inhibits proliferation, invasion and migration, but promotes apoptosis of T-NHL cells. The aim of this study was to assess the functional role of SPARC in T-cell non-Hodgkin's lymphoma (T-NHL), as well as the underlying molecular mechanisms. Here, we first identified SPARC expression in T-NHL tissues and cell lines through western blot and real-time PCR (RT-PCR). Overall survival of T-NHL patients with different levels of SPARC was assessed by Kaplan-Meier survival curves. Then cell proliferation, apoptosis, migration and invasion of T-NHL cells with either knockdown or overexpression of SPARC were determined by MTT, flow cytometry, transwell migration and invasion assay, respectively. Finally, the molecular mechanism by which SPARC modulated T-NHL cell progression was assessed. We confirmed that SPARC was significantly down-regulated in T-NHL tissues and cell lines. T-NHL patients with high levels of SPARC demonstrated a favorable clinical outcome. SPARC significantly suppressed cell proliferation, migration and invasion, and EMT process, but facilitated cell apoptosis in T-NHL cells. Further, we found that loss of SPARC expression in T-NHL tissues and cell lines, both in mRNA and protein levels, was associated with the aberrant DNA methylation in SPRAC gene, and the disrupted SPARC expression could be rescued after treatment with the demethylating agent 5-Aza-2′-deoxycitydine (5-Aza-Cdr). Additionally, 5-Aza-Cdr reversed SPARC hypermethylation to restore its biological role as a tumor suppressor in T-NHL cells, including inhibiting cell proliferation, invasion and migration, while promoting cell apoptosis. Our data provided evidence that DNA methylation in SPARC gene may play a role in the progression of T-NHL.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Gastrointestinal factors regulating lipid droplet formation in the intestine

Highlights: • Cytosolic lipid droplets are an important factor for the regulation of chylomicron secretion. • Many gastrointestinal peptides regulate cytosolic lipid droplets and lipid metabolism in liver and adipose tissue. • Chylomicron secretion is an important factor in the development of cardiometabolic diseases. Cytoplasmic lipid droplets (CLD) are considered as neutral lipid reservoirs, which protect cells from lipotoxicity. It became clear that these fascinating dynamic organelles play a role not only in energy storage and metabolism, but also in cellular lipid and protein handling, inter-organelle communication, and signaling among diverse functions. Their dysregulation is associated with multiple disorders, including obesity, liver steatosis and cardiovascular diseases. The central aim of this review is to highlight the link between intra-enterocyte CLD dynamics and the formation of chylomicrons, the main intestinal dietary lipid vehicle, after overviewing the morphology, molecular composition, biogenesis and functions of CLD.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The Growth arrest specific 1 (Gas1) gene is transcriptionally regulated by NeuroD1 via two distal E-boxes

Growth arrest specific 1 (GAS1) is a signaling mediator for the development of the central nervous system that works as a co-receptor for sonic hedgehog (SHH) to induce the amplification of neural progenitors during the patterning of the mammalian neural tube and establishing granular cells in the cerebellum. Recently, we confirmed that Gas1 is also expressed by neural progenitors of the developing cortex and the dentate gyrus of the hippocampus. The presence of GAS1 in progenitor stages indicates that one of its principal roles is the maintenance of these cells during neurogenic events. However, the signals responsible for the expression of Gas1 in progenitor cells are unknown, an aspect that is relevant to understand its functions during neurogenesis. Here, we focused on elucidating the mechanisms of the transcriptional regulation of Gas1 and using comparative genomics methods found two highly conserved E-boxes in the Gas1 promoter which mediate its up-regulation by NeuroD1. Additionally, we found that GAS1 and NeuroD1 co-localize in the neocortex, the dentate gyrus of the hippocampus and the external granular layer of the cerebellum, suggesting a previously unsuspected regulatory relationship. Our data indicate that Gas1 is a direct target of NeuroD1 during the induction of the neurogenic program.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in Drosophila nephrocytes

Highlights: • Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin. • Microtubules, Rho-GTPases and endoplasmic reticulum position the actin cluster. • Nephrocyte diaphragm proteins localize to and regulate actin cluster organization. • Perturbation of any of these inter-dependent components impairs ultrafiltration. Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional macromolecular assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the elements that maintain nephrocyte architecture and the ND are not understood. We show that Drosophila nephrocytes have a unique cytoplasmic cluster of F-actin, which is maintained by the microtubule cytoskeleton and Rho-GTPases. A balance of Rac1 and Cdc42 activity as well as proper microtubule organization and endoplasmic reticulum structure, are required to position the actin cluster. Further, ND proteins Sns and Duf also localize to this cluster and regulate organization of the actin and microtubule cytoskeleton. Perturbation of any of these inter-dependent components impairs nephrocyte ultrafiltration. Thus cytoskeletal components, Rho-GTPases and ND proteins work in concert to maintain the specialized nephrocyte architecture and function.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Connexin 43 regulates the expression of wound healing-related genes in human gingival and skin fibroblasts

Highlights: • Cx43 distinctly forms gap junctions and hemichannels (HCs) in human skin and gingiva. • Compared to human gingival fibroblasts, skin fibroblasts contained few Cx43 HCs. • Cx43 distinctly regulates gene expression in human gingival and skin fibroblasts. Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo-like 3D culture model, we further show that the GJ, HC, and channel-independent functions of Cx43 distinctly regulated wound healing-related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PRMT1 negatively regulates activation-induced cell death in macrophages by arginine methylation of GAPDH

Highlights: • PRMT1 mediates arginine methylation of GAPDH. • PRMT1 inhibits S-nitrosylation of GAPDH in LPS/IFN-γ-activated macrophages. • PRMT1 negatively regulates activation-induced cell death of macrophages through arginine methylation of GAPDH. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in cell death in addition to a role as a glycolytic enzyme. In particular, when cells are exposed to cellular stressors involving nitric oxide (NO) production, GAPDH can undergo NO-induced S-nitrosylation and S-nitrosylated GAPDH has been shown to elicit apoptosis. However, the mechanism underlying the regulation of the pro-apoptotic function of GAPDH remains unclear. Here, we found that protein arginine methyltransferase 1 (PRMT1) mediated arginine methylation of GAPDH in primary bone marrow-derived macrophages in a NO-dependent manner. Moreover, PRMT1 inhibited S-nitrosylation of GAPDH as well as its binding to SIAH1, thereby reducing the nuclear translocation of GAPDH in lipopolysaccharide (LPS)/interferon (IFN)-γ-activated macrophages. Furthermore, depletion of PRMT1 expression by RNA interference potentiated LPS/IFN-γ-induced apoptosis in macrophages. Taken together, our results suggest that PRMT1 has a previously unrecognized function to inhibit activation-induced cell death of macrophages through arginine methylation of GAPDH.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Calcium influx differentially regulates migration velocity and directedness in response to electric field application

Neural precursor cells (NPCs) respond to externally applied direct current electrical fields (DCEFs) by undergoing rapid and directed migration toward the cathode in a process known as galvanotaxis. It is unknown if the underlying mechanisms of galvanotactic migration is common to non-electrosensitive cells and if so, how NPCs and other galvanotactic cells sense and transduce electrical fields into cellular motility. In this study, we show that distinct aspects of NPC galvanotactic migration: motility (quantified through |velocity|) and directedness, are differentially regulated by calcium. We use low-Ca{sup 2+} culture conditions; an intracellular Ca{sup 2+} chelator; and voltage gated calcium channel (VGCC) inhibitors to specific channels expressed on NPCs, to demonstrate the role of Ca{sup 2+} influx in DCEF-induced NPC migration. Consistent with existing literature, we show Ca{sup 2+} is involved in F-actin polymerization that lengthens NPC membrane protrusions necessary for cellular motility. However, inhibiting Ca{sup 2+} results in reduced velocity but has no effect on DCEF-induced directedness. This dissociation between velocity and directedness reveal that these migration parameters can be independently regulated, thus suggesting a parallel process of sensing DCEFs by NPCs.

60 APPLIED LIFE SCIENCES↗