Decreased osteogenesis, increased cell senescence and elevated Dickkopf-1 secretion in human fracture non union stromal cells

The delicately orchestrated process of bone fracture healing is not always successful and long term non union of fractured bone occurs in 5-20% of all cases. Atrophic fracture non unions have been described as the most difficult to treat and this is thought to arise through a cellular and local failure of osteogenesis. However, little is known about the presence and osteogenic proficiency of cells in the local area of non union tissue. We have examined the growth and differentiation potential of cells isolated from human non union tissues compared with normal human bone marrow mesenchymal stromal cells (BMSC). We report the isolation and culture expansion of a population of non union stromal cells (NUSC) which have a CD profile similar to that of BMSC, i.e. CD34-ve, CD45-ve and CD105+ve. The NUSC demonstrated multipotentiality and differentiated to some extent along chondrogenic, adipogenic and osteogenic lineages. However, and importantly, the NUSC showed significantly reduced osteogenic differentiation and mineralization in vitro compared to BMSC. We also found increased levels of cell senescence in NUSC compared to BMSC based on culture growth kinetics and cell positivity for senescence associated beta galactosidase (SA-beta-Gal) activity. The reduced capacity of NUSC to form osteoblasts was associated with significantly elevated secretion of Dickkopf-1 (Dkk-1) which is an important inhibitor of Wnt signalling during osteogenesis, compared to BMSC. Conversely, treating BMSC with levels of rhDkk-1 that were equivalent to those levels secreted by NUSC inhibited the capacity of BMSC to undergo osteogenesis. Treating BMSC with NUSC conditioned medium also inhibited the capacity of the BMSC to undergo osteogenic differentiation when compared to their treatment with BMSC conditioned medium. Our results suggest that the development of fracture non union is linked with a localised reduced capacity of cells to undergo osteogenesis, which in turn is associated with increased cell senescence and Dkk-1 secretion.

Regulation of mucus secretion by cells isolated from the rat gastric mucosa

     This study was undertaken to further understanding of the mechanisms which regulate mucus secretion by rat stomach cells. Particular objectives were: (i) to develop and use a radiochemical assay to estimate the secretion of mucin by a suspension of gastric mucosal cells in vitro, (ii) to develop and use a solid-phase enzyme immunoassay (EIA) to study the regulation of the release of bulk gastric mucin from the isolated cells and (iii) to compare the results obtained with the two procedures.      Cells were isolated by exposure of gastric mucosa to pronase and EDTA. Cell suspensions were preincubated with D-[6-3H]glucosamine. [3H]-labelled material of high molecular mass released into the incubation medium, was purified by Fast Protein Liquid Chromatography, and appeared to be gastric mucin. Some unidentified [3H]-labelled material of lower molecular mass was also found in the medium. Release of [3H]-labelled high molecular mass material was essentially linearly related to time. Secretin, isoprenaline and carbachol stimulated release of [3H]-labelled high molecular mass material. The half-maximally effective concentrations of secretin and isoprenaline were 2.3nM and 34nM respectively. Histamine, gastrin and epidermal growth factor were without effect.      A rabbit polyclonal antibody was raised by using purified 'native' rat gastric mucin as immunogen. The antibody preparation appeared specific for rat gastric mucin and was used to establish a quantitative solid-phase EIA. Release of bulk mucin was essentially linearly related to time. Phorbol-12-myristate-13-acetate (PMA), forskolin and A23187 dose-dependently stimulated bulk mucin release. Synergistic interactions were observed between PMA and forskolin, and PMA and A23187. Secretin and isoprenaline were confirmed as mucin secretogogues.      In conclusion gastric mucin release was investigated for the first time by using a suspension of gastric mucosal cells. Two different assay procedures were developed. Some pathways and agents responsible for controlling mucin secretion were identified.

Visfatin regulates insulin secretion, insulin receptor signalling and mRNA expression of diabetes-related genes in mouse pancreatic beta-cells

The role of the adipocyte-derived factor visfatin in metabolism remains controversial, although some pancreatic ß-cell-specific effects have been reported. This study investigated the effects of visfatin upon insulin secretion, insulin receptor activation and mRNA expression of key diabetes-related genes in clonal mouse pancreatic ß-cells. ß-TC6 cells were cultured in RPMI 1640 and were subsequently treated with recombinant visfatin. One-hour static insulin secretion was measured by ELISA. Phospho-specific ELISA and western blotting were used to detect insulin receptor activation. Real-time SYBR Green PCR array technology was used to measure the expression of 84 diabetes-related genes in both treatment and control cells. Incubation with visfatin caused significant changes in the mRNA expression of several key diabetes-related genes, including marked up-regulation of insulin (9-fold increase), hepatocyte nuclear factor (HNF)1ß (32-fold increase), HNF4a (16-fold increase) and nuclear factor ?B (40-fold increase). Significant down-regulation was seen in angiotensin-converting enzyme (-3.73-fold) and UCP2 (-1.3-fold). Visfatin also caused a significant 46% increase in insulin secretion compared to control (P<0.003) at low glucose, and this increase was blocked by co-incubation with the specific nicotinamide phosphoribosyltransferase inhibitor FK866. Both visfatin and nicotinamide mononucleotide induced activation of both insulin receptor and extracellular signal-regulated kinase (ERK)1/2, with visfatin-induced insulin receptor/ERK1/2 activation being inhibited by FK866. We conclude that visfatin can significantly regulate insulin secretion, insulin receptor phosphorylation and intracellular signalling and the expression of a number of ß-cell function-associated genes in mouse ß-cells.

Rotational co-culture of clonal β-cells with endothelial cells:effect of PPAR-γ agonism in vitro on insulin and VEGF secretion

Aim: Delayed graft revascularization impedes the success of human islet transplantation. This study utilized rotational co-culture of insulin secreting ß-cells with human umbilical vein endothelial cells (HUVECs) and a peroxisome proliferator-activated receptor gamma (PPAR-?) agonist to promote insulin and vascular endothelial growth factor (VEGF) secretory function. Methods: Clonal BRIN-BD11 (D11) cells were maintained in static culture (SC) and rotational culture (RC) ± HUVEC and ± the TZD (thiazolidinedione) rosiglitazone (10 mmol/l) as a specific PPAR-? agonist. HUVECs were cultured in SC and RC ± D11 and ± TZD. D11 insulin secretion was induced by static incubation with low glucose (1.67 mmol/l), high glucose (16.7 mmol/l) and high glucose with 10 mmol/l theophylline (G+T) and assessed by enzyme-linked immunosorbent assay (ELISA). HUVEC proliferation was determined by ATP luminescence, whereas VEGF secretion was quantified by ELISA. Co-cultured cells were characterized by immunostaining for insulin and CD31. Results: D11 SC and RC showed enhanced insulin secretion in response to 16.7 mmol/l and G+T (p <0.01); without significant alteration by the TZD. Co-culture with HUVEC in SC and RC also increased D11 insulin secretion when challenged with 16.7 mmol/l and G+T (p <0.01), and this was slightly enhanced by the TZD. The presence of HUVEC increased D11 SC and RC insulin secretion in response to high glucose and G+T, respectively (p <0.01). Addition of the TZD increased SC and RC HUVEC ATP content (p <0.01) and VEGF production (p <0.01) in the presence and absence of D11 cells. Conclusions: Rotational co-culture of insulin secreting cells with endothelial cells, and exposure to a PPAR-? agonist may improve the prospects for graft revascularization and function after implantation. © 2011 Blackwell Publishing Ltd.

Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts:the active-state conformation of the enzyme does not affect cell motility but is important for its secretion

Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.

Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly Gq- and to a lesser extent Gs-mediated; p38 activation even had a small Gi-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

Diffuse beta-amyloid (A beta) deposits and neurons: in situ secretion or diffusion of A beta?

The association between diffuse-type beta -amyloid (AP) deposits and neuronal cell bodies in Alzheimer's disease (AD) and Down's syndrome (DS) could result from the secretion of AP from clusters of neurons in situ or the diffusion of A beta from cell processes, glial cells or blood vessels. To decide between these hypotheses, spatial pattern analysis was used to study the relationship between the degree of clustering of neuronal cell bodies and the presence of diffuse deposits in the temporal lobe of patients with DS. Significant clustering of neuronal cell bodies was present in 17/24 (71%) of brain areas studied. in addition, in 23/24 (96%) of brain areas, there was a positive correlation between the presence of diffuse deposits and the density of neurons. Hence, the data support the hypothesis that diffuse deposits develop in situ mainly as a result of the secretion of A beta by local clusters of neurons rather than by significant diffusion. Furthermore, the size of a diffuse deposit is likely to be determined by the number of neurons within a cluster which secrete A beta. The number and density of neurons could also be a factor determining the evolution of a diffuse into a mature amyloid deposit.

Acute and long-term effects of metformin on the function and insulin secretory responsiveness of clonal β-cells

Functional effects of acute and prolonged (48 h) exposure to the biguanide drug metformin were examined in the clonal pancreatic ß-cell line, BRIN-BD11. Effects of metformin on prolonged exposure to excessive increased concentrations of glucose and palmitic acid were also assessed. In acute 20-min incubations, 12.5-50 µm metformin did not alter basal (1.1 mm glucose) or glucose-stimulated (16.7 mm glucose) insulin secretion. However, higher concentrations of metformin (100-1000 µm) increased (1.3-1.5-fold; p

Free radicals and redox signalling in T-cells during chronic inflammation and ageing

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.

Factors important to the secretion and matrix deposition of tissue transglutaminase

Data suggest that for TG2 to be secreted, an intact N-terminal FN binding site (for which TG2 has high affinity) is required, however interaction of TG2 with its high affinity binding partners presents both in the intracellular and extracellular space as well as with specific cell surface receptors may also be involved in this process. Using a site-directed mutagenesis approach, the effects of specific mutations of TG2 on its translocation to the cell surface and secretion into the ECM have been investigated. Mutations include those affecting FN binding (FN1), HSPGs binding (HS1, HS2) GTP/GDP binding site (GTP1, 2) as well as N-terminal and C-terminal domains (TG2 deletion mutants N, and C). By performing transglutaminase activity assays, cell surface protein biotinylation and verifying distribution of TG2 mutants in the ECM we demonstrated that one of the potential heparan sulfate binding site mutants (HS2 mutant) is secreted at the cell surface in a much reduced manner and is less deposited into the ECM than the HS1 mutant. The HS2 mutant showed a low affinity for binding to a heparin sepharose column demonstrating this mutation site may be a potential heparan binding site of TG2. Analogous peptides to this site were shown to have some efficiency in the inhibition of the binding of the FN-TG2 complex to cell surface heparan sulfates in a cell adhesion assay indicating the peptide to be representative of the novel heparin binding site within TG2. The GTP binding site mutants GTP1 and GTP2 exhibited low specific activity however, GTP2 showed more secretion to the cell surface in comparison to GTP1. The FN1 binding mutant did not greatly affect TG2 activity nor did it alter TG2 secretion at the cell surface and deposition into the ECM indicating that fibronectin binding at this site on the enzyme is not an important factor. Interestingly an intact N-terminus (?1-15) appeared to be essential for enzyme externalisation. Removal of the first 15 amino acids (N-terminal mutant) abolished TG2 secretion to the cell surface as well as deposition into the ECM. In addition it reduced the enzymes affinity for binding to heparin. In contrast, deletion of the C-terminal TG2 domain (?594-687) increased enzyme secretion to the cell surface. Consistent with the data presented in this thesis we speculate that TG2 must fulfill two requirements to be successfully secreted from cells. The findings indicate that the closed conformation of the enzyme as well as intact N-terminal tail and a novel HS binding site within the TG2 molecule are key elements for the enzyme’s localisation at the cell surface and its deposition into the extracellular matrix. The importance of understanding the interactions between TG2, heparan sulfates and other TG2 binding partners at the cell surface could have an impact on the design of novel strategies for enzyme inhibition which could be important in the control of extracellular TG2 related diseases.

Preparation, characterisation and entrapment of a non-glycosidic threitol ceramide into liposomes for presentation to invariant natural killer T cells

Dendritic cells (DCs) are able to present glycolipids to invariant natural killer T (iNKT) cells in vivo. Very few compounds have been found to stimulate iNKT cells, and of these, the best characterised is the glycolipid a-galactosylceramide, which stimulates the production of large quantities of interferon-gamma (IFN-?) and interleukin-4 (IL-4). However, aGalCer leads to overstimulation of iNKT cells. It has been demonstrated that the aGalCer analogue, threitol ceramide (ThrCer 2), successfully activates iNKT cells and overcomes the problematic iNKT cell activation-induced anergy. In this study, ThrCer 2 has been inserted into the bilayers of liposomes composed of a neutral lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), or dimethyldioctadecylammonium bromide (DDA), a cationic lipid. Incorporation efficiencies of ThrCer within the liposomes was 96% for DSPC liposomes and 80% for DDA liposomes, with the vesicle size (large multilamellar vs. small unilamellar vesicles) making no significant difference. Langmuir-Blodgett studies suggest that both DSPC and DDA stack within the monolayer co-operatively with the ThrCer molecules with no condensing effect. In terms of cellular responses, IFN-? secretion was higher for cells treated with small DDA liposomes compared with the other liposome formulations, suggesting that ThrCer encapsulation in this liposome formulation resulted in a higher uptake by DCs.

Adiponectin (15-36) stimulates steroidogenic acute regulatory (StAR) protein expression and cortisol production in human adrenocortical cells:role of AMPK and MAPK kinase pathways

Adiponectin is an abundantly circulating adipokine, orchestrating its effects through two 7-transmembrane receptors (AdipoR1 and AdipoR2). Steroidogenesis is regulated by a variety of neuropeptides and adipokines. Earlier studies have reported adipokine mediated steroid production. A key rate-limiting step in steroidogenesis is cholesterol transportation across the mitochondrial membrane by steroidogenic acute regulatory protein (StAR). Several signalling pathways regulate StAR expression. The actions of adiponectin and its role in human adrenocortical steroid biosynthesis are not fully understood. The aim of this study was to investigate the effects of adiponectin on StAR protein expression, steroidogenic genes, and cortisol production and to dissect the signalling cascades involved in the activation of StAR expression. Using qRT-PCR, Western blot analysis and ELISA, we have demonstrated that stimulation of human adrenocortical H295R cells with adiponectin results in increased cortisol secretion. This effect is accompanied by increased expression of key steroidogenic pathway genes including StAR protein expression via ERK1/2 and AMPK-dependent pathways. This has implications for our understanding of adiponectin receptor activation and peripheral steroidogenesis. Finally, our study aims to emphasise the key role of adipokines in the integration of metabolic activity and energy balance partly via the regulation of adrenal steroid production.

Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro:implications for pancreatic islet transplantation

Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p <0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 µg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of ß-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may reduce the need for chronic systemic immunosuppression, thus making islet transplantation a more attractive treatment option for the management of insulin-dependent diabetes.

Polarised secretion of leukaemia inhibitory factor

Leukaemia inhibitory factor (LIF) is a cytokine that is active on a wide variety of cells. Multiple LIF transcripts have been described. The transcripts LIF-D and LIF-M encode different signal peptides, which in mouse have been associated with differential localisation of the mature protein. LIF-D is associated with a freely diffusible protein, whereas the LIF-M is associated with the extracellular matrix. The polarity of LIF secretion has yet to be described and could illuminate the mechanisms of LIF localisation. Here the polarised endogenous secretion of human LIF and IL-6 in Caco-2 cells was characterised under normal culture conditions and following induction with IL-1b. Whether the apical or basolateral membrane was stimulated influenced the pattern of secretion (LIF: Unstimulated, 59% basolateral. Dual stimulation, 68% basolateral. Basolateral stimulation, 79% basolateral. Apical stimulation, 53% basolateral). IL-6 displayed a similar dependence on the site of stimulation but was predominantly secreted at the membrane that was stimulated. To determine the effect of the alternate signal peptides on the polarity of LIF secretion, LIF was epitope tagged with FLAG. Epitope-tagging with FLAG was used to separate endogenous from exogenous protein expression. However, despite the normal biological activity of LIF-FLAG and detection of the FLAG in a western blot, detection of the LIF-FLAG under non-reducing conditions was not observed, and therefore it was unsuitable for secretion studies. Untagged LIF was expressed exogenously in Madin-Darby canine kidney (MDCK) cells under the control of a tetracycline response promoter that allowed a variety of LIF expression levels to be tested. Exogenous murine LIF was secreted predominantly from the apical (60%) membrane of MDCK cells irrespective of the signal peptide expressed.