Quantitative phosphoproteomic analysis reveals involvement of PD-1 in multiple T cell functions.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.

Multivariate Modulation of the Zr MOF UiO-66 for Defect-Controlled Combination Anticancer Drug Delivery.

Metal-organic frameworks (MOFs) are emerging as leading candidates for nanoscale drug delivery, as a consequence of their high drug capacities, ease of functionality, and the ability to carefully engineer key physical properties. Despite many anticancer treatment regimens consisting of a cocktail of different drugs, examples of delivery of multiple drugs from one MOF are rare, potentially hampered by difficulties in postsynthetic loading of more than one cargo molecule. Herein, we report a new strategy, multivariate modulation, which allows incorporation of up to three drugs in the Zr MOF UiO-66 by defect-loading. The drugs are added to one-pot solvothermal synthesis and are distributed throughout the MOF at defect sites by coordination to the metal clusters. This tight binding comes with retention of crystallinity and porosity, allowing a fourth drug to be postsynthetically loaded into the MOFs to yield nanoparticles loaded with cocktails of drugs that show enhancements in selective anticancer cytotoxicity against MCF-7 breast cancer cells in vitro. We believe that multivariate modulation is a significant advance in the application of MOFs in biomedicine, and anticipate the protocol will also be adopted in other areas of MOF chemistry, to easily produce defective MOFs with arrays of highly functionalised pores for potential application in gas separations and catalysis.

Layered rare-earth hydroxides as multi-modal medical imaging probes: particle size optimisation and compositional exploration.

Recently, layered rare-earth hydroxides (LRHs) have received growing attention in the field of theranostics. We have previously reported the hydrothermal synthesis of layered terbium hydroxide (LTbH), which exhibited high biocompatibility, reversible uptake of a range of model drugs, and release-sensitive phosphorescence. Despite these favourable properties, LTbH particles produced by the reported method suffered from poor size-uniformity (670 ± 564 nm), and are thus not suitable for therapeutic applications. To ameliorate this issue, we first derive an optimised hydrothermal synthesis method to generate LTbH particles with a high degree of homogeneity and reproducibility, within a size range appropriate for in vivo applications (152 ± 59 nm, n = 6). Subsequently, we apply this optimised method to synthesise a selected range of LRH materials (R = Pr, Nd, Gd, Dy, Er, Yb), four of which produced particles with an average size under 200 nm (Pr, Nd, Gd, and Dy) without the need for further optimisation. Finally, we incorporate Gd and Tb into LRHs in varying molar ratios (1 : 3, 1 : 1, and 3 : 1) and assess the combined magnetic relaxivity and phosphorescence properties of the resultant LRH materials. The lead formulation, LGd1.41Tb0.59H, was demonstrated to significantly shorten the T2 relaxation time of water (r2 = 52.06 mM-1 s-1), in addition to exhibiting a strong phosphorescence signal (over twice that of the other LRH formulations, including previously reported LTbH), therefore holding great promise as a potential multi-modal medical imaging probe.

Magnetically driven preparation of 1-D nano-necklaces capable of MRI relaxation enhancement.

We report a novel magnetically-facilitated approach to produce 1-D 'nano-necklace' arrays composed of 0-D magnetic nanoparticles, which are assembled and coated with an oxide layer to produce semi-flexible core@shell type structures. These 'nano-necklaces' demonstrate good MRI relaxation properties despite their coating and permanent alignment, with low field enhancement due to structural and magnetocrystalline anisotropy.

Gender-specific regulation of tyrosine hydroxylase in thymocyte differentiation antigen-1 knockout mice.

Thymocyte differentiation antigen-1 (Thy-1) is a cell surface glycoprotein found on T cells and neurons and is involved in cell-to-cell interactions. In addition, Thy-1 knockouts (KO) are a potential mouse model of restless legs syndrome (RLS) based on clinical observations and the role of dopamine in the disease. In this study, we analyzed the activity and quantity of tyrosine hydroxylase (TH; the rate-limiting enzyme in dopamine production) and determined phosphorylation levels for the enzyme phosphoserine-40 (pSer-40). There was no significant difference in the total TH activity and pSer-40 TH levels between Thy-1 KO and control groups; however, TH specific activity was significantly lower (by 26%) in Thy-1 KO mice. This difference is due in part to increased TH protein levels in this group (increased by 29%). When analyzed by gender, Thy-1 KO female mouse striata contained less TH specific activity compared with control females (decreased by 41%) and male control or Thy-1 KO animals (decreased by 30%). TH specific activity and pSer-40 TH levels in male Thy-1 KO and control displayed no differences. However, pSer-40 TH was significantly higher in control females (38%) compared with control or Thy-1 KO males. The Thy-1 KO females exhibited significantly lower (28%) pSer-40 TH (normalized to GAPDH or TH) than control females. Indeed, the Thy-1 KO females had 50% of the pSer-40 TH found in controls. Our results suggest a gender effect on TH specific activity, TH protein levels, and serine-40 phosphorylation of TH in Thy-1 KO female mice.

Thermo-responsive nano-in-micro particles for MRI-guided chemotherapy.

In this work, we develop nano-in-micro thermo-responsive microspheres as theranostic systems for anti-cancer hyperthermia. Firstly, layered double hydroxide (LDH) nanoparticles were synthesized and subsequently loaded with the chemotherapeutic agents methotrexate (MTX) or 5-fluorouracil (5FU). The drug-loaded LDH particles were then co-encapsulated with superparamagnetic iron oxide nanoparticles (SPIONs) into poly(acrylamide-co-acrylonitrile) microparticles via spray drying. The SPIONs are able to act as MRI contrast agents, thus resulting in potential theranostic formulations. Concave microparticles were observed by electron microscopy, and elemental mapping results suggest the LDH and SPION particles were homogeneously distributed inside the microparticles. In vitro dissolution tests showed that the drug was released over a prolonged period of time with the microspheres having distinct release curves at 37 and 43 °C. The relaxivity (r2) profiles were also found to be different over the temperature range 35 to 46 °C. Mathematical relationships between r2, release and temperature data were established, demonstrating that the microparticles have the potential for use in MRI-guided therapy. In vitro cell experiments revealed that the formulations permit synergistic hyperthermia-aided chemotherapy in cultured Caco-2 and A549 cells. Thus, the microparticles prepared in this work have potential as smart stimuli-responsive theranostics for hyperthermia-aided chemotherapy.

Effect of HFE variants on sphingolipid expression by SH-SY5Y human neuroblastoma cells.

C282Y and H63D are two common variants of the hemochromatosis protein HFE. SH-SY5Y human neuroblastoma cells stably transfected to express either wild type HFE (WT-HFE), or the C282Y or H63D allele were analyzed for effect of expression of the mutant proteins on transcription of 14 enzymes involved in sphingolipid metabolism. Cells expressing the C282Y variant showed significant increases (>2-fold) in transcription of five genes and decreases in two compared to that seen for cells expressing WT-HFE, while cells expressing the H63D variant showed an elevation in transcription of one gene and a decrease in two. These changes were seen as alterations in ganglioside composition, cell surface binding by the binding subunit of cholera toxin, expression of sphingosine-kinase-1 and synthesis of sphingosine-1-phosphate. These changes may explain why C282Y-HFE is a risk factor for colon and breast cancer and possibly protective against Alzheimer's disease while H63D-HFE is a risk factor for neurodegenerative diseases.

Hypoxia-inducible factor pathway activation in restless legs syndrome patients.

BACKGROUND AND PURPOSE: These studies tested the hypothesis that hypoxia inducible factor-1α (HIF-1α) pathway activation occurs in substantia nigra neurons and brain microvasculature in patients with restless legs syndrome. METHODS: Immunohistochemical analyses of substantia nigra tissue from six RLS and six control subjects were analyzed for HIF-1α, neuronal nitric oxide synthase (nNOS) and nitrotyrosine immunoreactivity. Microvessel lysates were obtained from cortex tissue from four RLS and four control subjects and the lysates were quantified for HIF-2α and vascular endothelial growth factor (VEGF) expression using immunoblot analyses. HIF-1α activation of peripheral blood monocyte cells (PBMCs) (14 RLS and 9 control) was determined through immunoblot analysis of PBMC lysates for EPO. RESULTS: HIF-1α immunoreactivity in substantia nigra neurons was significantly increased in five of six RLS patients as compared with controls. In addition, nNOS and nitrotyrosine expression are up-regulated in the substantia nigra of four of six RLS patients as compared with controls. HIF-2α and VEGF expression are significantly up-regulated in the microvasculature lysates from four RLS cortical brain tissue as compared with controls. Erythropoietin levels are significantly increased in RLS PBMCs. CONCLUSIONS: These results demonstrate that the hypoxia pathway is activated in multiple cell types in individuals with RLS. Increased nNOS and nitrotyrosine suggests that nitric oxide is involved in the activation. Activation of the hypoxia pathway can result from or contribute to cellular iron deficiency. These observations suggest a novel direction to explore in RLS that is tied to the iron deficiency model but better explains the findings in postmortem studies.

The effect of formulation morphology on stimuli-triggered co-delivery of chemotherapeutic and MRI contrast agents.

Most conventional chemotherapeutics have narrow therapeutic windows, and thus their delivery remains challenging and often raises safety and efficacy concerns. Theranostic platforms, with simultaneous encapsulation of therapeutic and diagnostic agents, have been proposed as next-generation formulations which can overcome this issue. In this work, we used electrohydrodynamic approaches to fabricate core@shell formulations comprising a pH responsive Eudragit L100 shell embedded with superparamagnetic iron oxide nanoparticles (SPIONs), and a thermo-responsive poly(N-isopropylacrylamide) (PNIPAM)/ethyl cellulose core loaded with the model drug carmofur. By varying the weight ratio of core polymer to shell polymer, the morphology of PNIPAM/ethyl cellulose@Eudragit L100 microparticles could be changed from concave to spherical. Smooth cylindrical fibres could also be generated. All the formulations exist as amorphous solid dispersions of drug-in-polymer, with distinct core@shell architectures. The fibres have clear thermo-responsive drug release profiles, while no thermo-responsive properties can be seen with the particles. All the formulations can protect SPIONs from degradation in gastric fluids (pH âˆ¼ 1.5), and around the physiological pH range the materials offer effective and pH-responsive relaxivity. The r2 values also display clear linear relationships with drug release data, suggesting the potential of using MRI signals to track drug release in vivo. Mathematical equations were established to track drug release in vitro, with very similar experimental and predicted release profiles obtained.

Genetically Defined, Syngeneic Organoid Platform for Developing Combination Therapies for Ovarian Cancer.

The paucity of genetically informed, immunocompetent tumor models impedes evaluation of conventional, targeted, and immune therapies. By engineering mouse fallopian tube epithelial organoids using lentiviral gene transduction and/or CRISPR/Cas9 mutagenesis, we generated multiple high-grade serous tubo-ovarian cancer (HGSC) models exhibiting mutational combinations seen in patients with HGSC. Detailed analysis of homologous recombination (HR)-proficient (Trp53-/-;Ccne1OE;Akt2OE;KrasOE ), HR-deficient (Trp53-/-;Brca1-/-;MycOE ), and unclassified (Trp53-/-;Pten-/-;Nf1-/- ) organoids revealed differences in in vitro properties (proliferation, differentiation, and "secretome"), copy-number aberrations, and tumorigenicity. Tumorigenic organoids had variable sensitivity to HGSC chemotherapeutics, and evoked distinct immune microenvironments that could be modulated by neutralizing organoid-produced chemokines/cytokines. These findings enabled development of a chemotherapy/immunotherapy regimen that yielded durable, T cell-dependent responses in Trp53-/-;Ccne1OE;Akt2OE;Kras HGSC; in contrast, Trp53-/-;Pten-/-;Nf1-/- tumors failed to respond. Mouse and human HGSC models showed genotype-dependent similarities in chemosensitivity, secretome, and immune microenvironment. Genotype-informed, syngeneic organoid models could provide a platform for the rapid evaluation of tumor biology and therapeutics. SIGNIFICANCE: The lack of genetically informed, diverse, immunocompetent models poses a major barrier to therapeutic development for many malignancies. Using engineered fallopian tube organoids to study the cell-autonomous and cell-nonautonomous effects of specific combinations of mutations found in HGSC, we suggest an effective combination treatment for the currently intractable CCNE1-amplified subgroup.This article is highlighted in the In This Issue feature, p. 211.

SHP2 inhibition diminishes KRASG12C cycling and promotes tumor microenvironment remodeling.

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site-specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.

pH-Responsive nanocomposite fibres allowing MRI monitoring of drug release.

Magnetic resonance imaging (MRI) is one of the most widely-used non-invasive clinical imaging tools, producing detailed anatomical images whilst avoiding side effects such as trauma or X-ray radiation exposure. In this article, a new approach to non-invasive monitoring of drug release from a delivery vehicle via MRI was developed, using pH-responsive Eudragit L100 and S100 fibres encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) and carmofur (a drug used in the treatment of colon cancer). Fibres were prepared by electrospinning, and found to be smooth and cylindrical with diameters of 645 ± 225 nm for L100 and 454 ± 133 nm for S100. The fibres exhibited pH responsive dissolution behaviour. Around the physiological pH range, clear pH-responsive proton relaxation rate changes due to matrix swelling/dissolution can be observed: r2 values of L100 fibres increase from 29.3 ± 8.3 to 69.8 ± 2.5 mM-1 s-1 over 3 h immersion in a pH 7.4 medium, and from 13.5 ± 2.0 mM-1 s-1 to 42.1 ± 3.0 mM-1 s-1 at pH 6.5. The r2 values of S100 fibres grow from 30.4 ± 4.4 to 64.7 ± 1.0 mM-1 s-1 at pH 7.4, but at pH 6.5, where the S100 fibres are not soluble, r2 remains very low (< 4 mM-1 s-1). These dramatic changes in relaxivity demonstrate that pH-responsive dissolution results in SPION release. In vitro drug release studies showed the formulations gave rapid release of carmofur at physiological pH values (pH 6.5 and 7.4), and acid stability studies revealed that they can protect the SPIONs from digestion in acid environments, giving the fibres potential for oral administration. Exploration of the relationship between relaxivity and carmofur release suggests a linear correlation (R2 > 0.94) between the two. Mathematical equations were developed to predict carmofur release in vitro, with very similar experimental and predicted release profiles obtained. Therefore, the formulations developed herein have the potential to be used for non-invasive monitoring of drug release in vivo, and could ultimately result in dramatic reductions to off-target side effects from interventions such as chemotherapy.

Protective effects of a cathepsin K inhibitor, SB-553484, in the canine partial medial meniscectomy model of osteoarthritis.

OBJECTIVE: Cathepsin K (cat K), a cysteine protease expressed in osteoclasts, chondrocytes and synovial fibroblasts, degrades several bone and cartilage matrix components suggesting its potential role in osteoarthritis (OA). We investigated the effects of SB-553484, an inhibitor of cat K, on lesion severity and biomarkers of collagen degradation in the canine partial medial meniscectomy model. METHODS: A partial medial meniscectomy was performed in mature female beagle dogs. Animals were dosed orally with vehicle or SB-553484 at 50mg/kg BID for 28 days. The femorotibial joints were evaluated for gross and microscopic histological changes. Biomarkers of collagen degradation were also analyzed. RESULTS: In dogs treated with SB-553484, subjective gross and calculated degeneration scores decreased significantly by 29% and 46%, respectively. Histopathologic evaluation demonstrated that the summed tibial degeneration score decreased significantly by 21%. Inhibition of tibial cartilage degeneration was significant in zone 1 (32%) and the depth ratio of any tibial matrix change was decreased significantly by 28%. Urinary biomarkers of bone and cartilage degradation were also significantly reduced. CONCLUSION: Treatment with SB-553484 resulted in mild to moderate beneficial effects on gross and histopathological parameters. Reduction of biomarkers of collagen type I and II degradation indicated a direct effect of the compound on bone and cartilage. These data suggest that the prevention of cartilage degradation by cat K inhibition may represent a valid strategy for pharmacological intervention in OA and that monitoring collagen degradation biomarkers may provide an indication of the protective effects of inhibition of bone and cartilage degradation.

Environmental influences on serotonin and cyclic nucleotides in rat cerebral cortex.

The response to different environmental conditions and negative air ions was investigated on cerebral cortical serotonin and cyclic nucleotides. The results indicated that negative air ions alter the weight of the cerebral cortex and that concentrations of serotonin and cyclic nucleotides can be altered both by different environments and by negative air ions. The data stress the importance of the role of the environment when studying the structure and chemistry of the cerebral cortex.

Cerebrospinal Fluid Ceruloplasmin, Haptoglobin, and Vascular Endothelial Growth Factor Are Associated with Neurocognitive Impairment in Adults with HIV Infection.

Dysregulated iron transport and a compromised blood-brain barrier are implicated in HIV-associated neurocognitive disorders (HAND). We quantified the levels of proteins involved in iron transport and/or angiogenesis-ceruloplasmin, haptoglobin, and vascular endothelial growth factor (VEGF)-as well as biomarkers of neuroinflammation, in cerebrospinal fluid (CSF) from 405 individuals with HIV infection and comprehensive neuropsychiatric assessments. Associations with HAND [defined by a Global Deficit Score (GDS) ≥ 0.5, GDS as a continuous measure (cGDS), or by Frascati criteria] were evaluated for the highest versus lowest tertile of each biomarker, adjusting for potential confounders. Higher CSF VEGF was associated with GDS-defined impairment [odds ratio (OR) 2.17, p = 0.006] and cGDS in unadjusted analyses and remained associated with GDS impairment after adjustment (p = 0.018). GDS impairment was also associated with higher CSF ceruloplasmin (p = 0.047) and with higher ceruloplasmin and haptoglobin in persons with minimal comorbidities (ORs 2.37 and 2.13, respectively; both p = 0.043). In persons with minimal comorbidities, higher ceruloplasmin and haptoglobin were associated with HAND by Frascati criteria (both p < 0.05), and higher ceruloplasmin predicted worse impairment (higher cGDS values, p < 0.01). In the subgroup with undetectable viral load and minimal comorbidity, CSF ceruloplasmin and haptoglobin were strongly associated with GDS impairment (ORs 5.57 and 2.96, respectively; both p < 0.01) and HAND (both p < 0.01). Concurrently measured CSF IL-6 and TNF-α were only weakly correlated to these three biomarkers. Higher CSF ceruloplasmin, haptoglobin, and VEGF are associated with a significantly greater likelihood of HAND, suggesting that interventions aimed at disordered iron transport and angiogenesis may be beneficial in this disorder.

Eltrombopag, a thrombopoietin mimetic, crosses the blood-brain barrier and impairs iron-dependent hippocampal neuron dendrite development.

Essentials Potential neurodevelopmental side effects of thrombopoietin mimetics need to be considered. The effects of eltrombopag (ELT) on neuronal iron status and dendrite development were assessed. ELT crosses the blood-brain barrier and causes iron deficiency in developing neurons. ELT blunts dendrite maturation, indicating a need for more safety studies before neonatal use. SUMMARY: Background Thrombocytopenia is common in sick neonates. Thrombopoietin mimetics (e.g. eltrombopag [ELT]) might provide an alternative therapy for selected neonates with severe and prolonged thrombocytopenia, and for infants and young children with different varieties of thrombocytopenia. However, ELT chelates intracellular iron, which may adversely affect developing organs with high metabolic requirements. Iron deficiency (ID) is particularly deleterious during brain development, impairing neuronal myelination, dopamine signaling and dendritic maturation and ultimately impairing long-term neurological function (e.g. hippocampal-dependent learning and memory). Objective To determine whether ELT crosses the blood-brain barrier (BBB), causes neuronal ID and impairs hippocampal neuron dendrite maturation. Methods ELT transport across the BBB was assessed using primary bovine brain microvascular endothelial cells. Embryonic mouse primary hippocampal neuron cultures were treated with ELT or deferoxamine (DFO, an iron chelator) from 7 days in vitro (DIV) through 14 DIV and assessed for gene expression and neuronal dendrite complexity. Results ELT crossed the BBB in a time-dependent manner. 2 and 6 µm ELT increased Tfr1 and Slc11a2 (iron-responsive genes involved in neuronal iron uptake) mRNA levels, indicating neuronal ID. 6 µm ELT, but not 2 µm ELT, decreased BdnfVI, Camk2a and Vamp1 mRNA levels, suggesting impaired neuronal development and synaptic function. Dendrite branch number and length were reduced in 6 µm ELT-treated neurons, resulting in blunted dendritic arbor complexity that was similar to DFO-treated neurons. Conclusions Eltrombopag treatment during development may impair neuronal structure as a result of neuronal ID. Preclinical in vivo studies are warranted to assess ELT safety during periods of rapid brain development.

Acute and chronic effects of developmental iron deficiency on mRNA expression patterns in the brain.

Because of the multiple biochemical pathways that require iron, iron deficiency can impact brain metabolism in many ways. The goal of this study was to identify a molecular footprint associated with ongoing versus long term consequences of iron deficiency using microarray analysis. Rats were born to iron-deficient mothers, and were analyzed at two different ages: 21 days, while weaning and iron-deficient; and six months, after a five month iron-sufficient recovery period. Overall, the data indicate that ongoing iron deficiency impacts multiple pathways, whereas the long term consequences of iron deficiency on gene expression are more limited. These data suggest that the gene array profiles obtained at postnatal day 21 reflect a brain under development in a metabolically compromised setting that given appropriate intervention is mostly correctable. There are, however, long term consequences to the developmental iron deficiency that could underlie the neurological deficits reported for iron deficiency.

Identification of iron responsive genes by screening cDNA libraries from suppression subtractive hybridization with antisense probes from three iron conditions.

The goal of the present study is to identify genes that respond to iron availability. Suppression subtraction hybridization (SSH) was used to generate cDNA libraries from iron loaded and control human astrocytoma cells (SW1088). The cDNA libraries were screened with antisense cDNA probes obtained from mRNA isolated from astrocytoma cells exposed to three conditions: (i) normal media (control), (ii) deferox-amine treated (iron deficient) or (iii) iron loaded. The screening of the cDNA libraries with antisense probes from the three conditions enhanced the screening efficiency and decreased the number of false positives. Positive clones were identified and sequenced. The genes of interest were further analyzed by determining changes in hybridization signal on northern blots from astrocytoma cells exposed to iron or deferoxamine over different time intervals. Our analysis identified cDNAs corresponding to known iron responsive genes such as L-chain ferritin, but also revealed a number of mRNAs with novel sequences and mRNAs previously not known to be responsive to iron such as one of the ABC transporters and Thy-1 glycoprotein. Thus our results suggest that the expression of a number of genes may be influenced by changes in iron availability.

High molecular weight proteoglycans biosynthesized in culture by pigeon aortas.

The properties of aortic proteoglycans synthesized in vitro were examined to demonstrate synthesis of intact proteoglycans by aortic tissue in culture and to compare labeling and synthetic rates of two different populations of proteoglycan. Following 3, 6, or 9 h of incubation in medium containing [35S]sodium sulfate and [3H]serine, the tissue was extracted with 4.0 M guanidine hydrochloride containing protease inhibitors. Extracts were chromatographed on Sepharose CL-4B and subjected to buoyant density centrifugation under dissociative conditions. Radioactive precursors were incorporated into two major populations of aortic proteoglycan, one of high molecular weight eluting near the void volume of Sepharose CL-4B (Proteoglycan I) and one of lower molecular weight (Proteoglycan II) having a Kav of 0.40-0.44. The radioactively labeled proteoglycans were localized at densities 1.50-1.56 g/ml (Preparation 1) and 1.43-1.49 g/ml (Preparation 2) following CsCl buoyant density centrifugation. Both proteoglycan populations had increased incorporation of 35S and 3H over time. At all times the lower molecular weight proteoglycan had a higher specific activity (dpm 35S and 3H/micrograms hexuronic acid). At 3, 6, and 9 h, the specific activity of Proteoglycan II was 8.2-, 6.7- and 3.0-fold higher than Proteoglycan I using 35S and 13.0-, 8.1- and 2.7-fold higher using 3H, suggesting different synthetic rates for the two proteoglycans. The results illustrate synthesis of intact proteoglycans during short-term artery culture. The proteoglycan types have size and buoyant density characteristics as described for artery, but based upon changes in specific activity ratios, the two proteoglycan populations differ in rates of synthesis.

Interleukin-1beta increases binding of the iron regulatory protein and the synthesis of ferritin by increasing the labile iron pool.

This study was undertaken to begin to elucidate the mechanisms by which cytokines influence intracellular iron homeostasis. Intracellular iron homeostasis is maintained by the coordinated regulation of ferritin and transferrin receptor synthesis. The synthesis of these proteins is coordinated by cytoplasmic iron regulatory proteins (IRP), which bind to iron responsive elements (IRE) on their mRNAs. We evaluated the effects of interleukin-1beta (IL-1beta) on iron metabolism in human astrocytoma cells (SW1088). Exposure to IL-1beta for 16 h increased binding of the IRPs to the IRE and also increased ferritin synthesis. Using the iron sensitive dye calcein, we determined that the intracellular labile iron pool increased within 4 h of IL-1beta exposure and continued to increase for 8 h, returning to normal by 16 h. We propose that the cytokine induced increase in the labile iron pool stimulates ferritin synthesis resulting in a subsequent decrease in the labile iron pool. The decrease in the labile iron pool is consistent with the increase in IRE/IRP interaction measured at 16 h. These results indicate that cytokines can influence the labile iron pool and the post-transcriptional regulatory mechanism for maintaining iron homeostasis. These results contribute to understanding the response of ferritin to inflammation by suggesting ferritin synthesis may reflect changes in the labile iron pool. The approach used in this study may provide a model system for studying relations between the labile iron pool and proteins responsible for maintaining intracellular homeostasis