Rapid subcellular fractionation of the rat liver endocytic compartments involved in transcytosis of polymeric immunoglobulin A and endocytosis of asialofetuin.

The distributions of two endocytosed radiolabelled ligands (polymeric immunoglobulin A and asialofetuin) in rat liver endocytic compartments were investigated by using rapid subcellular fractionation of post-mitochondrial supernatants on vertical density gradients of Ficoll or Nycodenz. Two endocytic compartments were identified, both ligands being initially associated with a light endocytic-vesicle fraction on Ficoll gradients, asialofetuin then accumulating in denser endosomes before transfer to lysosomes for degradation.     

Investigation of endosomal compartments involved in endocytosis and transcytosis of polymeric immunoglobulin A by subcellular fractionation of perfused isolated rat liver.

1. A gamma camera was used to monitor continuously the uptake of radiolabelled polymeric immunoglobulin A (pIgA) into the rat body after intravenous injection. Uptake into liver was fast but, since the peak of liver labelling occurred only after 9-15 min, it was not sufficiently rapid to constitute a pulse dose. A perfused, isolated rat liver system was therefore established which could be given a single pass dose of pIgA; a variety of tests showed such livers remained viable for at least 3 h and could be subsequently fractionated on Ficoll and Nycodenz gradients with normal distributions of marker enzymes. 2. Subcellular fractionation at different times after a single pass dose of pIgA showed that whilst pIgA appeared sequentially in sinusoidal plasma membrane, light endosomes, dense endosomes, very dense endosomes and lysosomes as in vivo, the predominance of pIgA in the light endosome compartment disappeared much earlier than after injection in vivo of pIgA, presumably because this compartment was not being continuously loaded over the first 10-15 min. The time course of appearance of label in bile was unchanged. A large excess of unlabelled asialofetuin did not change these patterns, indicating that the asialoglycoprotein receptor was not involved. 3. Low doses of the microtubule agent colchicine reduced the proportion of pIgA reaching the bile, but subcellular fractionation of treated liver showed that distribution of label amongst liver fractions was little changed, although the overall liver pIgA content had increased. This would suggest that pIgA did not remain in the common compartment which could have supplied bile or lysosomes but rather flowed out of it as rapidly as in untreated liver but towards those compartments supplying the lysosomes. 4. Experiments with nocodazole, which reversibly disrupts microtubules, showed that very little of the pIgA taken into an inhibited liver appeared in the bile after nocodazole was removed 30 min later, even though a second dose of pIgA, given after nocodazole removal, appeared in bile with a normal time course. The first dose of pIgA must therefore have passed beyond the compartments competent to supply the bile before nocodazole was removed. Such compartments were undamaged since the second dose of pIgA appeared in bile normally. We therefore conclude that the bulk of pIgA must be supplied to the bile from light or dense endosomes rather than from very dense endosomes and lysosomes.     

Glucocorticoid regulation of enkephalins in cultured rat adrenal medulla

The effect of dexamethasone on enkephalin-containing (EC) peptide levels and preproenkephalin mRNA levels was determined in adrenal medullary explants (glands) from sham and hypophysectomized (hypox) rats. Culture for 4 days in serum-free medium without dexamethasone resulted in a 13- and 4-fold increase in EC peptide levels in sham and hypox glands, respectively. The addition of dexamethasone (10(-5) M) produced a 20- to 26-fold increase in EC peptides in sham and hypox glands. In serum free medium, hypox glands showed a concentration dependent increase in EC peptides with the ED50 for dexamethasone equal to 5.7 x 10(-7) M. Since the glucocorticoid antagonist RU486 partially blocked the rise in EC peptides in sham glands, it appears that the increase in EC peptides in sham glands in the absence of dexamethasone is a result of a higher concentration of endogenous corticosterone in sham compared to hypox glands. Dexamethasone resulted in a 6-fold increase in preproenkephalin mRNA in hypox glands cultured for 2 days. This increase was approximately proportional to the increase in EC peptides seen at 4 days. In serum free medium progesterone, testosterone, and deoxycorticosterone failed to increase EC peptides in hypox glands. These results indicate that glucocorticoid treatment is required for maximal proenkephalin gene expression and EC peptide biosynthesis in cultured glands.     

Mendelian randomization supports causality between maternal hyperglycemia and epigenetic regulation of leptin gene in newborns

Leptin is an adipokine that acts in the central nervous system and regulates energy balance. Animal models and human observational studies have suggested that leptin surge in the perinatal period has a critical role in programming long-term risk of obesity. In utero exposure to maternal hyperglycemia has been associated with increased risk of obesity later in life. Epigenetic mechanisms are suspected to be involved in fetal programming of long term metabolic diseases. We investigated whether DNA methylation levels near LEP locus mediate the relation between maternal glycemia and neonatal leptin levels using the 2-step epigenetic Mendelian randomization approach. We used data and samples from up to 485 mother-child dyads from Gen3G, a large prospective population-based cohort. First, we built a genetic risk score to capture maternal glycemia based on 10 known glycemic genetic variants (GRS₁₀) and showed it was an adequate instrumental variable (β = 0.046 mmol/L of maternal fasting glucose per additional risk allele; SE = 0.007; P = 7.8 × 10⁻¹¹; N = 467). A higher GRS₁₀ was associated with lower methylation levels at cg12083122 located near LEP (β = −0.072 unit per additional risk allele; SE = 0.04; P = 0.05; N = 166). Direction and effect size of association between the instrumental variable GRS₁₀ and methylation at cg12083122 were consistent with the negative association we observed using measured maternal glycemia. Lower DNA methylation levels at cg12083122 were associated with higher cord blood leptin levels (β = −0.17 log of cord blood leptin per unit; SE = 0.07; P = 0.01; N = 170). Our study supports that maternal glycemia is part of causal pathways influencing offspring leptin epigenetic regulation.     

Induction of HIV-1 resistance: cell susceptibility to infection is an inverse function of globotriaosyl ceramide levels

To examine the role of the glycosphingolipid (GSL), globotriaosylceramide(Gb₃, CD77, p^k blood group antigen) in HIV-1 infection, we havepharmacologically modulated Gb₃ metabolism in an X4 HIV-1 infectablemonocytic cell line (THP-1) that naturally expresses Gb₃ andin a Gb₃-expressing glioblastoma cell line (U87) transfectedto express both CD4 and CCR5 to permit R5 HIV-1 infection. THP-1and U87 cells were treated with either a competitive inhibitorof -galactosidase A, 1-deoxygalactonojirimycin (DGJ) to induceGb₃ accumulation, or a glucosylceramide synthase inhibitor,phenyl-2-palmitylamino-3-pyrrolidino-1-propanol (P4) to depletecells of Gb₃. HIV susceptibility was determined via measurementof p24^gag antigen production by ELISA. In addition, total cellularGb₃ content was determined using thin layer chromatography followedby Verotoxin1 overlay binding. The cell surface expression ofGb₃ was verified by FACS analysis. We found that DGJ significantlydecreased THP-1 and U87 cell susceptibility to HIV-1_IIIB andHIV-1_BaL infection, respectively, at a concentration of approximately100 µM. In contrast, P4 (2 µM) substantiallyincreased cellular susceptibility to HIV-1 infection. Totalcellular GSL analysis verified increased Gb₃ expression in cellstreated with DGJ and considerable reduction of Gb₃ in P4-treatedcells as compared to controls. These results show a reciprocalrelationship between Gb₃ expression and infection with eitherX4 HIV-1_IIIB or R5 HIV-1_Ba-L. These results support previousstudies that Gb₃ provides resistance to HIV infection. VariableGb₃ expression may provide a natural HIV resistance factor inthe general population, and pharmacological manipulation ofGb₃ levels may provide an approach to induction of HIV resistance.     

Protein-tyrosine Phosphatase-�� and Src Functionally Link Focal Adhesions to the Endoplasmic Reticulum to Mediate Interleukin-1-induced Ca2+ Signaling

Calcium (Ca²⁺) signaling by the pro-inflammatory cytokine interleukin-1 (IL-1) is dependent on focal adhesions, which contain diverse structural and signaling proteins including protein phosphatases. We examined here the role of protein-tyrosine phosphatase (PTP) α in regulating IL-1-induced Ca²⁺ signaling in fibroblasts. IL-1 promoted recruitment of PTPα to focal adhesions and endoplasmic reticulum (ER) fractions, as well as tyrosine phosphorylation of the ER Ca²⁺ release channel IP₃R. In response to IL-1, catalytically active PTPα was required for Ca²⁺ release from the ER, Src-dependent phosphorylation of IP₃R1 and accumulation of IP₃R1 in focal adhesions. In pulldown assays and immunoprecipitations PTPα was required for the association of PTPα with IP₃R1 and c-Src, and this association was increased by IL-1. Collectively, these data indicate that PTPα acts as an adaptor to mediate functional links between focal adhesions and the ER that enable IL-1-induced Ca²⁺ signaling.The interleukin-1 (IL-1)³ family of pro-inflammatory cytokines mediates host responses to infection and injury. Impaired control of IL-1 signaling leads to chronic inflammation and destruction of extracellular matrices (1, 2), as seen in pathological conditions such as pulmonary fibrosis (3), rheumatoid arthritis (4, 5), and periodontitis (6). IL-1 elicits multiple signaling programs, some of which trigger Ca²⁺ release from the endoplasmic reticulum (ER) as well as expression of multiple cytokines and inflammatory factors including c-Fos and c-Jun (7, 8), and matrix metalloproteinases (9, 10), which mediate extracellular matrix degradation via mitogen-activated protein kinase-regulated pathways (11).In anchorage-dependent cells including fibroblasts and chondrocytes, focal adhesions (FAs) are required for IL-1-induced Ca²⁺ release from the ER and activation of ERK (12–14). FAs are actin-enriched adhesive domains composed of numerous (>50) scaffolding and signaling proteins (15–17). Many FA proteins are tyrosine-phosphorylated, including paxillin, focal adhesion kinase, and src family kinases, all of which are crucial for the assembly and disassembly of FAs (18–21). Protein-tyrosine phosphorylation plays a central role in regulating many cellular processes including adhesion (22, 23), motility (24), survival (25), and signal transduction (26–29). Phosphorylation of proteins by kinases is balanced by protein-tyrosine phosphatases (PTP), which can enhance or attenuate downstream signaling by dephosphorylation of tyrosine residues (30–32).PTPs can be divided into two main categories: receptor-like and intracellular PTPs (33). Two receptor-like PTPs have been localized to FA (leukocyte common antigen-related molecule and PTPα). Leukocyte common antigen-related molecule can dephosphorylate and mediate degradation of p130^cas, which ultimately leads to cell death (34, 35). PTPα contains a heavily glycosylated extracellular domain, a transmembrane domain, and two intracellular phosphatase domains (33, 36). The amino-terminal domain predominantly mediates catalytic activity, whereas the carboxyl-terminal domain serves a regulatory function (37, 38). PTPα is enriched in FA (23) and is instrumental in regulating FA dynamics (39) via activation of c-Src/Fyn kinases by dephosphorylating the inhibitory carboxyl tyrosine residue, namely Tyr⁵²⁹ (22, 40–42) and facilitation of integrin-dependent assembly of Src-FAK and Fyn-FAK complexes that regulate cell motility (43). Although PTPα has been implicated in formation and remodeling of FAs (44, 45), the role of PTPα in FA-dependent signaling is not defined.Ca²⁺ release from the ER is a critical step in integrin-dependent IL-1 signal transduction and is required for downstream activation of ERK (13, 46). The release of Ca²⁺ from the ER depends on the inositol 1,4,5-triphosphate receptor (IP₃R), which is an IP₃-gated Ca²⁺ channel (47). All of the IP₃R subtypes (subtypes 1–3) have been localized to the ER, as well as other the plasma membrane and other endomembranes (48–50). Further, IP₃R may associate with FAs, enabling the anchorage of the ER to FAs (51, 52). However, the molecule(s) that provide the structural link for this association has not been defined.FA-restricted, IL-1-triggered signal transduction in anchorage-dependent cells may rely on interacting proteins that are enriched in FAs and the ER (53). Here, we examined the possibility that PTPα associates with c-Src and IP₃R to functionally link FAs to the ER, thereby enabling IL-1 signal transduction.     

The Influence of Radiographic Phenotype and Smoking Status on Peripheral Blood Biomarker Patterns in Chronic Obstructive Pulmonary Disease

Background

Chronic obstructive pulmonary disease (COPD) is characterized by both airway remodeling and parenchymal destruction. The identification of unique biomarker patterns associated with airway dominant versus parenchymal dominant patterns would support the existence of unique phenotypes representing independent biologic processes. A cross-sectional study was performed to examine the association of serum biomarkers with radiographic airway and parenchymal phenotypes of COPD.

Methodology/Principal Findings

Serum from 234 subjects enrolled in a CT screening cohort was analyzed for 33 cytokines and growth factors using a multiplex protein array. The association of serum markers with forced expiratory volume in one second percent predicted (FEV1%) and quantitative CT measurements of airway thickening and emphysema was assessed with and without stratification for current smoking status. Significant associations were found with several serum inflammatory proteins and measurements of FEV1%, airway thickening, and parenchymal emphysema independent of smoking status. The association of select analytes with airway thickening and emphysema was independent of FEV1%. Furthermore, the relationship between other inflammatory markers and measurements of physiologic obstruction or airway thickening was dependent on current smoking status.

Conclusions/Significance

Airway and parenchymal phenotypes of COPD are associated with unique systemic serum biomarker profiles. Serum biomarker patterns may provide a more precise classification of the COPD syndrome, provide insights into disease pathogenesis and identify targets for novel patient-specific biological therapies.     

Signaling by FGFR2b controls the regenerative capacity of adult mouse incisors

Rodent incisors regenerate throughout the lifetime of the animal owing to the presence of epithelial and mesenchymal stem cells in the proximal region of the tooth. Enamel, the hardest component of the tooth, is continuously deposited by stem cell-derived ameloblasts exclusively on the labial, or outer, surface of the tooth. The epithelial stem cells that are the ameloblast progenitors reside in structures called cervical loops at the base of the incisors. Previous studies have suggested that FGF10, acting mainly through fibroblast growth factor receptor 2b (FGFR2b), is crucial for development of the epithelial stem cell population in mouse incisors. To explore the role of FGFR2b signaling during development and adult life, we used an rtTA transactivator/tetracycline promoter approach that allows inducible and reversible attenuation of FGFR2b signaling. Downregulation of FGFR2b signaling during embryonic stages led to abnormal development of the labial cervical loop and of the inner enamel epithelial layer. In addition, postnatal attenuation of signaling resulted in impaired incisor growth, characterized by failure of enamel formation and degradation of the incisors. At a cellular level, these changes were accompanied by decreased proliferation of the transit-amplifying cells that are progenitors of the ameloblasts. Upon release of the signaling blockade, the incisors resumed growth and reformed an enamel layer, demonstrating that survival of the stem cells was not compromised by transient postnatal attenuation of FGFR2b signaling. Taken together, our results demonstrate that FGFR2b signaling regulates both the establishment of the incisor stem cell niches in the embryo and the regenerative capacity of incisors in the adult.