Andrea L. Nestor-Kalinoski

A microwave-assisted solution combustion synthesis to produce europium-doped calcium phosphate nanowhiskers for bioimaging applications

Biocompatible nanoparticles possessing fluorescent properties offer attractive possibilities for multifunctional bioimaging and/or drug and gene delivery applications. Many of the limitations with current imaging systems center on the properties of the optical probes in relation to equipment technical capabilities. Here we introduce a novel high aspect ratio and highly crystalline europium-doped calcium phosphate nanowhisker produced using a simple microwave-assisted solution combustion synthesis method for use as a multifunctional bioimaging probe. X-ray diffraction confirmed the material phase as europium-doped hydroxyapatite. Fluorescence emission and excitation spectra and their corresponding peaks were identified using spectrofluorimetry and validated with fluorescence, confocal and multiphoton microscopy. The nanowhiskers were found to exhibit red and far red wavelength fluorescence under ultraviolet excitation with an optimal peak emission of 696 nm achieved with a 350 nm excitation. Relatively narrow emission bands were observed, which may permit their use in multicolor imaging applications. Confocal and multiphoton microscopy confirmed that the nanoparticles provide sufficient intensity to be utilized in imaging applications.

Biliverdin Reductase A Attenuates Hepatic Steatosis by Inhibition of Glycogen Synthase Kinase (GSK) 3β Phosphorylation of Serine 73 of Peroxisome Proliferator-activated Receptor (PPAR) α

Non-alcoholic fatty liver disease is the most rapidly growing form of liver disease and if left untreated can result in non-alcoholic steatohepatitis, ultimately resulting in liver cirrhosis and failure. Biliverdin reductase A (BVRA) is a multifunctioning protein primarily responsible for the reduction of biliverdin to bilirubin. Also, BVRA functions as a kinase and transcription factor, regulating several cellular functions. We report here that liver BVRA protects against hepatic steatosis by inhibiting glycogen synthase kinase 3β (GSK3β) by enhancing serine 9 phosphorylation, which inhibits its activity. We show that GSK3β phosphorylates serine 73 (Ser(P)73) of the peroxisome proliferator-activated receptor α (PPARα), which in turn increased ubiquitination and protein turnover, as well as decreased activity. Interestingly, liver-specific BVRA KO mice had increased GSK3β activity and Ser(P)73 of PPARα, which resulted in decreased PPARα protein and activity. Furthermore, the liver-specific BVRA KO mice exhibited increased plasma glucose and insulin levels and decreased glycogen storage, which may be due to the manifestation of hepatic steatosis observed in the mice. These findings reveal a novel BVRA-GSKβ-PPARα axis that regulates hepatic lipid metabolism and may provide unique targets for the treatment of non-alcoholic fatty liver disease.

An mDia2/ROCK Signaling Axis Regulates Invasive Egress from Epithelial Ovarian Cancer Spheroids

Multi-cellular spheroids are enriched in ascites of epithelial ovarian cancer (OvCa) patients. They represent an invasive and chemoresistant cellular population fundamental to metastatic dissemination. The molecular mechanisms triggering single cell invasive egress from spheroids remain enigmatic. mDia formins are Rho GTPase effectors that are key regulators of F-actin cytoskeletal dynamics. We hypothesized that mDia2-driven F-actin dynamics promote single cell invasive transitions in clinically relevant three-dimensional (3D) OvCa spheroids. The current study is a dissection of the contribution of the F-actin assembly factor mDia2 formin in invasive transitions and using a clinically relevant ovarian cancer spheroid model. We show that RhoA-directed mDia2 activity is required for tight spheroid organization, and enrichment of mDia2 in the invasive cellular protrusions of collagen-embedded OVCA429 spheroids. Depleting mDia2 in ES-2 spheroids enhanced invasive dissemination of single amoeboid-shaped cells. This contrasts with spheroids treated with control siRNA, where a mesenchymal invasion program predominated. Inhibition of another RhoA effector, ROCK, had no impact on ES-2 spheroid formation but dramatically inhibited spheroid invasion through induction of a highly elongated morphology. Concurrent inhibition of ROCK and mDia2 blocked single cell invasion from ES-2 spheroids more effectively than inhibition of either protein alone, indicating that invasive egress of amoeboid cells from mDia2-depleted spheroids is ROCK-dependent. Our findings indicate that multiple GTPase effectors must be suppressed in order to fully block invasive egress from ovarian cancer spheroids. Furthermore, tightly regulated interplay between ROCK and mDia2 signaling pathways dictates the invasive capacities and the type of invasion program utilized by motile spheroid-derived ovarian cancer cells. As loss of the gene encoding mDia2, DRF3, has been linked to cancer progression and metastasis, our results set the stage for understanding molecular mechanisms involved in mDia2-dependent egress of invasive cells from primary epithelial tumors.

Epistatic genetic determinants of blood pressure and mortality in a salt-sensitive hypertension model.

Although genetic determinants protecting against the development of elevated blood pressure (BP) are well investigated, less is known regarding their impact on longevity. We concomitantly assessed genomic regions of rat chromosomes 3 and 7 (RNO3 and RNO7) carrying genetic determinants of BP without known epistasis, for their independent and combinatorial effects on BP and the presence of genetic determinants of survival using Dahl salt-sensitive (S) strains carrying congenic segments from Dahl salt-resistant (R) rats. Although congenic and bicongenic S.R strains carried independent BP quantitative trait loci within the RNO3 and RNO7 congenic regions, only the RNO3 allele(s) independently affected survival. The bicongenic S.R strain showed epistasis between R-rat RNO3 and RNO7 alleles for BP under salt-loading conditions, with less-than-additive effects observed on a 2% NaCl diet and greater-than-additive effects observed after prolonged feeding on a 4% NaCl diet. These RNO3 and RNO7 congenic region alleles had more-than-additive effects on survival. Increased survival of bicongenic compared with RNO3 congenic rats was attributable, in part, to maintaining lower BP despite chronic exposure to an increased dietary salt (4% NaCl) intake, with both strains showing delays in reaching highest BP. R-rat RNO3 alleles were also associated with superior systolic function, with the S.R bicongenic strain showing epistasis between R-rat RNO3 and RNO7 alleles leading to compensatory hypertrophy. Whether these alleles affect survival by additional actions within other BP-regulating tissues/organs remains unexplored. This is the first report of simultaneous detection of independent and epistatic loci dictating, in part, longevity in a hypertensive rat strain.

Tight association between macrophages and adipocytes in obesity: Implications for adipocyte preparation

To determine the cellular architecture of the inflammatory infiltrate in adipose tissue from obese mice, and identify the source of inflammatory cytokines in adipose tissue at a single cell level.

Dia-Interacting Protein (DIP) Imposes Migratory Plasticity in mDia2-Dependent Tumor Cells in Three-Dimensional Matrices

Tumor cells rely upon membrane pliancy to escape primary lesions and invade secondary metastatic sites. This process relies upon localized assembly and disassembly cycles of F-actin that support and underlie the plasma membrane. Dynamic actin generates both spear-like and bleb structures respectively characterizing mesenchymal and amoeboid motility programs utilized by metastatic cells in three-dimensional matrices. The molecular mechanism and physiological trigger(s) driving membrane plasticity are poorly understood. mDia formins are F-actin assembly factors directing membrane pliancy in motile cells. mDia2 is functionally coupled with its binding partner DIP, regulating cortical actin and inducing membrane blebbing in amoeboid cells. Here we show that mDia2 and DIP co-tether to nascent blebs and this linkage is required for bleb formation. DIP controls mesenchymal/amoeboid cell interconvertability, while CXCL12 induces assembly of mDia2:DIP complexes to bleb cortices in 3D matrices. These results demonstrate how DIP-directed mDia2-dependent F-actin dynamics regulate morphological plasticity in motile cancer cells.

Mice with hyperbilirubinemia due to Gilbert’s syndrome polymorphism are resistant to hepatic steatosis by decreased serine 73 phosphorylation of PPARα

Gilberts syndrome in humans is derived from a polymorphism (TA repeat) in the hepatic UGT1A1 gene that results in decreased conjugation and increased levels of unconjugated bilirubin. Recently, we have shown that bilirubin binds directly to the fat-burning nuclear peroxisome proliferator-activated receptor-α (PPARα). Additionally, we have shown that serine 73 phosphorylation [Ser(P)73] of PPARα decreases activity by reducing its protein levels and transcriptional activity. The aim of this study was to determine whether humanized mice with the Gilberts polymorphism (HuUGT*28) have increased PPARα activation and reduced hepatic fat accumulation. To determine whether humanized mice with Gilberts mutation (HuUGT*28) have reduced hepatic lipids, we placed them and C57BL/6J control mice on a high-fat (60%) diet for 36 wk. Body weights, fat and lean mass, and fasting blood glucose and insulin levels were measured every 6 wk throughout the investigation. At the end of the study, hepatic lipid content was measured and PPARα regulated genes as well as immunostaining of Ser(P)73 PPARα from liver sections. The HuUGT*28 mice had increased serum bilirubin, lean body mass, decreased fat mass, and hepatic lipid content as well as lower serum glucose and insulin levels. Also, the HuUGT*28 mice had reduced Ser(P)73 PPARα immunostaining in livers and increased PPARα transcriptional activity compared with controls. A chronic but mild endogenous increase in unconjugated hyperbiliubinemia protects against hepatic steatosis through a reduction in Ser(P)73 PPARα, causing an increase in PPARα transcriptional activity.

Glucocorticoid Receptor β Induces Hepatic Steatosis by Augmenting Inflammation and Inhibition of the Peroxisome Proliferator-activated Receptor (PPAR) α

Glucocorticoids (GCs) regulate energy supply in response to stress by increasing hepatic gluconeogenesis during fasting. Long-term GC treatment induces hepatic steatosis and weight gain. GC signaling is coordinated via the GC receptor (GR) GRα, as the GRβ isoform lacks a ligand-binding domain. The roles of the GR isoforms in the regulation of lipid accumulation is unknown. The purpose of this study was to determine whether GRβ inhibits the actions of GCs in the liver, or enhances hepatic lipid accumulation. We show that GRβ expression is increased in adipose and liver tissues in obese high-fat fed mice. Adenovirus-mediated delivery of hepatic GRβ overexpression (GRβ-Ad) resulted in suppression of gluconeogenic genes and hyperglycemia in mice on a regular diet. Furthermore, GRβ-Ad mice had increased hepatic lipid accumulation and serum triglyceride levels possibly due to the activation of NF-κB signaling and increased tumor necrosis factor α (TNFα) and inducible nitric-oxide synthase expression, indicative of enhanced M1 macrophages and the development of steatosis. Consequently, GRβ-Ad mice had increased glycogen synthase kinase 3β (GSK3β) activity and reduced hepatic PPARα and fibroblast growth factor 21 (FGF21) expression and lower serum FGF21 levels, which are two proteins known to increase during fasting to enhance the burning of fat by activating the β-oxidation pathway. In conclusion, GRβ antagonizes the GC-induced signaling during fasting via GRα and the PPARα-FGF21 axis that reduces fat burning. Furthermore, hepatic GRβ increases inflammation, which leads to hepatic lipid accumulation.

Glucocorticoid receptor beta increases migration of human bladder cancer cells.

Bladder cancer is observed worldwide having been associated with a host of environmental and lifestyle risk factors. Recent investigations on anti-inflammatory glucocorticoid signaling point to a pathway that may impact bladder cancer. Here we show an inverse effect on the glucocorticoid receptor (GR) isoform signaling that may lead to bladder cancer. We found similar GRα expression levels in the transitional uroepithelial cancer cell lines T24 and UMUC-3. However, the T24 cells showed a significant (p < 0.05) increased expression of GRβ compared to UMUC-3, which also correlated with higher migration rates. Knockdown of GRβ in the T24 cells resulted in a decreased migration rate. Mutational analysis of the 3′ untranslated region (UTR) of human GRβ revealed that miR144 might positively regulate expression. Indeed, overexpression of miR144 increased GRβ by 3.8 fold. In addition, miR144 and GRβ were upregulated during migration. We used a peptide nucleic acid conjugated to a cell penetrating-peptide (Sweet-P) to block the binding site for miR144 in the 3′UTR of GRβ. Sweet-P effectively prevented miR144 actions and decreased GRβ expression, as well as the migration of the T24 human bladder cancer cells. Therefore, GRβ may have a significant role in bladder cancer, and possibly serve as a therapeutic target for the disease.

Small molecule agonists of mammalian Diaphanous-related (mDia) formins reveal an effective glioblastoma anti-invasion strategy

Formin agonists impede the most dangerous aspect of glioblastoma—tumor spread into surrounding healthy tissue. Formin activation impairs a novel aspect of the transformed cell and informs the development of antitumor strategies for a cancer needing a more effective therapy.