Thrombopoietic effect of VPAC1 inhibition during megakaryopoiesis.

Megakaryocytes and platelets express the stimulatory G protein (Gs)-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are agonists. The neuropeptide PACAP and VPAC1 were previously found to negatively regulate megakaryopoiesis, and inhibition of their physiological pathway was found to have a thrombopoietic effect in conditions where megakaryopoiesis and thrombopoiesis were impaired, such as chemotherapy-induced thrombocytopenia and congenital thrombocytopenia. The present study explored the thrombopoietic effect of VPAC1 inhibition in a murine model of syngeneic bone marrow transplantation (BMT) and in passive immune thrombocytopenia. Treatment of donor mice with a neutralizing anti-VPAC1 antibody stimulated the initial, most critical recovery of the platelets in irradiated mice. In the passive immune thrombocytopenia model, we observed a thrombopoietic effect, resulting in a less severe platelet drop after induction of their removal in the spleen by an anti-platelet antibody. We concluded that inhibition of the physiological PACAP/VPAC1 pathway could stimulate in vivo megakaryopoiesis. This inhibition can be applied to attenuate thrombocytopenia in conditions where platelets are destroyed as the major pathogenetic mechanism, e.g. immune thrombocytopenia purpura, or need to be produced de novo, e.g. after irradiation and BMT.

Monocytes from type 2 diabetic patients have a pro-inflammatory profile. 1,25-Dihydroxyvitamin D(3) works as anti-inflammatory.

The exact factors contributing to the pathogenesis of type 2 diabetes remain elusive. Lately, it was suggested that inflammation and activation of the innate immune system could be linked to type 2 diabetes pathogenesis and also to the development of common diabetic complications, mainly atherosclerosis. The aim of this study was to investigate the role of monocytes in this sub-clinical inflammatory state and test 1,25-dihydroxyvitamin D(3), the active form of Vitamin D, as an anti-inflammatory agent. For this purpose, monocytes from type 2 diabetic patients were compared to monocytes from healthy controls and type 1 diabetic patients. The expression profile of inflammatory markers in freshly isolated and immune-stimulated monocytes was measured by quantitative real-time RT-PCR. Type 2 diabetic patients showed significantly higher expression levels of TNF-alpha, IL-6, IL-1, IL-8, COX-2, ICAM-1 and B7-1 compared to controls and type 1 diabetic patients. 1,25-Dihydroxyvitamin D(3) was able to down-regulate the expression of TNF-alpha, IL-6, IL-1, and IL-8, confirming its immunomodulatory properties. From these data we concluded that monocytes from type 2 diabetic patients have a pro-inflammatory profile. In addition, 1,25-dihydroxyvitamin D(3) was able to modulate inflammation in these monocytes.

Immune regulation of 25-hydroxyvitamin-D3-1alpha-hydroxylase in human monocytes.

UNLABELLED: Monocytes express 1alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, in response to IFNgamma and CD14/TLR4 activation. Cross-talk between the JAK-STAT, the NF-kappaB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPbeta to its recognition sites in the promoter of the 1alpha-hydroxylase gene is a prerequisite. INTRODUCTION: The activated form of vitamin D3, 1,25(OH)2D3, known for its action in bone and mineral homeostasis, has important immunomodulatory effects. 1,25(OH)2D3 modulates the immune system through specific nuclear receptors, whereas macrophages produce 1,25(OH)2D3. In monocytes, the expression of 1alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, is regulated by immune stimuli. The aim of this study was to elucidate the intracellular pathways through which interferon (IFN)gamma and Toll-like receptor (TLR) modulation regulate expression of 1alpha-hydroxylase in monocytes/macrophages. MATERIALS AND METHODS: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated with IFNgamma (12.5 U/ml) and/or lipopolysaccharide (LPS; 100 ng/ml) for 48 h. The following inhibitors were used: janus kinase (JAK) inhibitor AG490 (50 microM), NF-kappaB inhibitor sulfasalazine (0.25 mM), p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 microM). 1alpha-hydroxylase mRNA expression was monitored by qRT-PCR. Phosphorylation of transcription factors was studied by Western blotting. Transfection of mutated or deletion promoter constructs, cloned in the pGL3-luciferase reporter plasmid, were performed in the RAW264.7 cell line. Cells were stimulated with IFNgamma (100 U/ml) and LPS (100 microg/ml), and promoter activity was studied. Binding of signal transducer and activator of transcription (STAT)1alpha, NF-kappaB, and C/EBPbeta to their respective binding sites in the promoter was analyzed by gel shift assays. RESULTS: 1alpha-hydroxylase mRNA expression in monocytes is synergistically induced by IFNgamma and CD14/TLR4 ligation and paralleled by 1,25(OH)2D3 production. This induction requires the JAK-STAT, NF-kappaB, and p38 MAPK pathways. Each of them is essential, because blocking individual pathways is sufficient to block 1alpha-hydroxylase expression (JAK inhibitor, 60% inhibition, p < 0.01; NF-kappaB inhibitor, 70% inhibition, p < 0.05; p38 MAPK inhibitor, 95% inhibition, p < 0.005). In addition, we show the involvement of the p38 MAPK pathway in phosphorylation of C/EBPbeta. Direct binding of C/EBPbeta to its recognition sites in the 1alpha-hydroxylase promoter is necessary to enable its immune-stimulated upregulation. CONCLUSION: IFNgamma and CD14/TLR4 binding regulate expression of 1alpha-hydroxylase in monocytes in a synergistic way. Combined activation of the JAK-STAT, p38 MAPK, and NF-kappaB pathways is necessary, with C/EBPbeta most probably being the essential transcription factor controlling immune-mediated transcription.

Immune regulation of 25-hydroxyvitamin D-1alpha-hydroxylase in human monocytic THP1 cells: mechanisms of interferon-gamma-mediated induction.

CONTEXT: 25-Hydroxyvitamin D can be activated to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] by the rate-limiting enzyme 1alpha-hydroxylase in cells of the immune system under control of immune stimuli, such as interferon-gamma (IFNgamma). In pathological situations, such as sarcoidosis, this can lead to systemic excess of 1,25(OH)(2)D(3) and hypercalcemia. OBJECTIVE: The aim of this study was to elucidate the intracellular pathways used by the immune system to tightly regulate 1,25(OH)(2)D(3) production in monocytes and macrophages. DESIGN: Human monocytic THP1-cells were differentiated and activated by IFNgamma and a secondary stimulus, such as lipopolysaccharide or phorbol myristate acetate. 1alpha-Hydroxylase mRNA levels were quantified by real-time RT-PCR. The involvement of different signaling pathways in the regulation of this enzyme was investigated using specific pharmacological inhibitors, whereas phosphorylation of signal transducer and activator of transcription 1alpha and CCAAT/enhancer binding protein beta was investigated by Western blotting. RESULTS: In undifferentiated monocytic THP1 cells, IFNgamma needs to be combined with a second stimulus, such as lipopolysaccharide, to induce 1alpha-hydroxylase. In contrast, in phorbol myristate acetate-differentiated THP1 macrophages, IFNgamma alone induces 1alpha-hydroxylase and to much higher levels. Many different signaling pathways need to be activated concurrently to allow immune-mediated 1alpha-hydroxylase up-regulation. We show involvement of the Janus kinase-signal transducer and activator of transcription, MAPK, and nuclear factor-kappaB pathways, with a crucial role for the transcription factor CCAAT/enhancer binding protein beta. Furthermore, histone remodeling involving histone deacetylases and histone acetylase p300 is required. CONCLUSION: The present findings indicate that IFNgamma-mediated 1,25(OH)(2)D(3) production, as observed in granulomatous diseases such as sarcoidosis, will take place only under conditions where the necessary other signaling pathways are also activated.

Regulation of 25-hydroxyvitamin d-1alpha-hydroxylase by IFNgamma in human monocytic THP1 cells.

1,25-DihydroxyVitamin D(3) (1,25(OH)(2)D(3)), a molecule with well-known actions in bone and mineral homeostasis, also plays a role in the immune system. Indeed, the receptor for 1,25(OH)(2)D(3) is found in most immune cells and important immunological effects have been described in vitro, reflected by its capacity to prevent autoimmunity and to prolong graft survival. The aim of this study was to elucidate the intracellular pathways used by the immune system to regulate 1,25(OH)(2)D(3) production. Therefore we studied the regulation of 25-hydroxyvitamin-D-1alpha-hydroxylase (1alpha hydroxylase) in THP1 cells by IFNgamma, demonstrating that its induction is highly dependent on the activation/differentiation by PMA and occurred at a late time point (140-fold at 72 h, P < 0.05). Complete inhibition with actinomycin D indicated that the observed induction was, at least in part, a transcriptional event. Dose-dependent inhibition with cycloheximide demonstrated that the induction was dependent on "de novo" protein synthesis, a finding that correlates with the late time point of up-regulation. The data presented indicate a role for 1,25(OH)(2)D(3), activated by 1alpha hydroxylase, as a late down-tapering signal in the immune cascade.

Monocytic expression behavior of cytokines in diabetic patients upon inflammatory stimulation.

Cytokines are involved in the pathogenesis of type 1 diabetes. The disease is characterized by T cell-mediated beta cell destruction and a biased Th1 cytokine pattern. Type 2 diabetes also presents an inflammatory cytokine imbalance. In this study, mRNA expression of cytokines IL-12, TNF-alpha, IL-1, and IL-6 was studied in monocytes from diabetic patients after in vitro immune stimulation. Whereas IL-12(p40) was highly expressed in type 1 diabetic patients, TNF-alpha, IL-1, and IL-6 transcripts were elevated in type 1 but especially type 2 diabetic patients compared with healthy controls, suggesting an important proinflammatory milieu. We conclude that circulating monocytes from type 1 as well as type 2 diabetic patients have an aberrant cytokine profile when stimulated by an immune stimulus such as IFNgamma. This condition not only is likely to be involved in disease pathogenesis, but may contribute to its later complications.

Regulation of vitamin D homeostasis: implications for the immune system.

Vitamin D homeostasis in the immune system is the focus of this review. The production of both the activating (25- and 1alpha-hydroxylase) and the metabolizing (24-hydroxylase) enzymes by cells of the immune system itself, indicates that 1,25(OH)(2)D(3) can be produced locally in immune reaction sites. Moreover, the strict regulation of these enzymes by immune signals is highly suggestive for an autocrine/paracrine role in the immune system, and opens new treatment possibilities.

The N-terminal coiled coil of the Rhodococcus erythropolis ARC AAA ATPase is neither necessary for oligomerization nor nucleotide hydrolysis.

Deletion mutants of the Rhodococcus erythropolis ARC AAA ATPase were generated and characterized by biochemical analysis and electron microscopy. Based on sequence comparisons the ARC protein was divided into three consecutive regions, the N-terminal coiled coil, the central ARC-specific inter domain and the C-terminal AAA domain. When the ARC AAA domain was expressed separately it formed aggregates of undefined structure. However, when the AAA domain was expressed in conjunction with the preceeding inter domain, but without the N-terminal coiled coil, high-molecular weight-complexes were formed (ARC-DeltaCC) which showed an N-ethylmaleimide-sensitive ATPase activity. In 2D crystallization experiments the ARC-DeltaCC particles yielded crystals nearly identical to those formed by the wild-type ARC complexes. Thus, the N-terminal coiled coil, which was proposed to have a role in the assembly of and/or interaction between the eukaryotic AAA ATPases in the 26S proteasome, is neither essential for assembly nor for ATP hydrolysis of the ARC ATPase. The N-terminal domain of related AAA ATPases mediates the interaction with substrates or co-factors, suggesting a regulatory function for the N-terminal coiled coil of the ARC ATPase. Surprisingly, the mutant ARC protein ARC-DeltaAAA consisting of the N-terminal coiled coil and the central inter domain, but deleted for the C-terminal AAA domain, was shown to form a dodecameric complex with sixfold symmetry. This suggests an important role of the inter domain for the ordered assembly of the ARC ATPase.