Vitamin D3 activates the autolysosomal degradation function against Helicobacter pylori through the PDIA3 receptor in gastric epithelial cells.

Helicobacter pylori (H. pylori) is a common human pathogenic bacterium. Once infected, it is difficult for the host to clear this organism using the innate immune system. Increased antibiotic resistance further makes it challenging for effective eradication. However, the mechanisms of immune evasion still remain obscure, and novel strategies should be developed to efficiently eliminate H. pylori infection in stomachs. Here we uncovered desirable anti-H. pylori effect of vitamin D3 both in vitro and in vivo, even against antibiotic-resistant strains. We showed that H. pylori can invade into the gastric epithelium where they became sequestered and survived in autophagosomes with impaired lysosomal acidification. Vitamin D3 treatment caused a restored lysosomal degradation function by activating the PDIA3 receptor, thereby promoting the nuclear translocation of PDIA3-STAT3 protein complex and the subsequent upregulation of MCOLN3 channels, resulting in an enhanced Ca2+ release from lysosomes and normalized lysosomal acidification. The recovered lysosomal degradation function drives H. pylori to be eliminated through the autolysosomal pathway. These findings provide a novel pathogenic mechanism on how H. pylori can survive in the gastric epithelium, and a unique pathway for vitamin D3 to reactivate the autolysosomal degradation function, which is critical for the antibacterial action of vitamin D3 both in cells and in animals, and perhaps further in humans. Abbreviations: 1,25D3: 1α, 25-dihydroxyvitamin D3; ATG5: autophagy related 5; Baf A1: bafilomycin A1; BECN1: beclin 1; CagA: cytotoxin-associated gene A; CFU: colony-forming unit; ChIP-PCR: chromatin immunoprecipitation-polymerase chain reaction; Con A: concanamycin A; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; CTSD: cathepsin D; GPN: Gly-Phe-ß-naphthylamide; H. pylori: Helicobacter pylori; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; MCOLN3: mucolipin 3; MCU: mitochondrial calcium uniporter; MOI: multiplicity of infection; NAGLU: N-acetyl-alpha-glucosaminidase; PDIA3: protein disulfide isomerase family A member 3; PMA: phorbol 12-myristate 13-acetate; PRKC: protein kinase C; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; SS1: Sydney Strain 1; TRP: transient receptor potential; VacA: vacuolating cytotoxin; VD3: vitamin D3; VDR: vitamin D receptor.

CCR1-mediated STAT3 tyrosine phosphorylation and CXCL8 expression in THP-1 macrophage-like cells involve pertussis toxin-insensitive Gα(14/16) signaling and IL-6 release.

Agonists of CCR1 contribute to hypersensitivity reactions and atherosclerotic lesions, possibly via the regulation of the transcription factor STAT3. CCR1 was demonstrated to use pertussis toxin-insensitive Gα(14/16) to stimulate phospholipase Cß and NF-κB, whereas both Gα(14) and Gα(16) are also capable of activating STAT3. The coexpression of CCR1 and Gα(14/16) in human THP-1 macrophage-like cells suggests that CCR1 may use Gα(14/16) to induce STAT3 activation. In this study, we demonstrated that a CCR1 agonist, leukotactin-1 (CCL15), could indeed stimulate STAT3 Tyr(705) and Ser(727) phosphorylation via pertussis toxin-insensitive G proteins in PMA-differentiated THP-1 cells, human erythroleukemia cells, and HEK293 cells overexpressing CCR1 and Gα(14/16). The STAT3 Tyr(705) and Ser(727) phosphorylations were independent of each other and temporally distinct. Subcellular fractionation and confocal microscopy illustrated that Tyr(705)-phosphorylated STAT3 translocated to the nucleus, whereas Ser(727)-phosphorylated STAT3 was retained in the cytosol after CCR1/Gα(14) activation. CCL15 was capable of inducing IL-6 and IL-8 (CXCL8) production in both THP-1 macrophage-like cells and HEK293 cells overexpressing CCR1 and Gα(14/16). Neutralizing Ab to IL-6 inhibited CCL15-mediated STAT3 Tyr(705) phosphorylation, whereas inhibition of STAT3 activity abolished CCL15-activated CXCL8 release. The ability of CCR1 to signal through Gα(14/16) provides a linkage for CCL15 to regulate IL-6/STAT3-signaling cascades, leading to expression of CXCL8, a cytokine that is involved in inflammation and the rupture of atherosclerotic plaque.

Prostacyclin receptor-dependent inhibition of human erythroleukemia cell differentiation is STAT3-dependent.

We have previously demonstrated that activation of prostacyclin (IP) receptors in human erythroleukemia (HEL) cells phosphorylates the signal transducer and activator of transcription 3 (STAT3) via Gα(s) and Gα(16) hybrid signalling. This current study was designed to determine if functional responses to cicaprost in HEL cells were dependent on STAT3 phosphorylation. Cicaprost significantly enhanced the rapid change in HEL cell morphology induced by phorbol-12-myristate-13-acetate (PMA), and this effect was inhibited by the IP receptor antagonist RO1138452 and a STAT3 inhibitory peptide. Other indicators of PMA-induced HEL cell differentiation, such as increased expression of CD41/CD61 and an increase in cell complexity/granularity, were inhibited by cicaprost in an IP receptor-dependent and STAT3-dependent manner. Although thrombopoietic cytokines promote megakaryocytic differentiation and platelet production via activation of STAT3, the predominant STAT3-dependent effects of cicaprost in HEL cells were inhibitory towards the process of PMA-induced megakaryocytopoeisis.

Effects of cannabinoid receptor agonists on immunologically induced histamine release from rat peritoneal mast cells.

Immunologic activation of mast cells through the cross-linking of high affinity IgE receptors results in the release of inflammatory mediators which are important in the pathogenesis of allergic reactions. Early studies investigating the effects of palmitoylethanolamide on animal models of inflammation and on rat mast cells led to the hypothesis that endogenous cannabinoids might act as local autacoids which suppressed inflammation by reducing the activation of mast cells. However, more recent studies produced contradicting results. In order to evaluate if cannabinoid receptors are present in mast cells, we studied the effects of endocannabinoids (anandamide and palmitoylethanolamide) and synthetic cannabimimetics (CP 55,940, WIN 55,212-2 and HU-210) on histamine release from rat peritoneal mast cells. When incubated with mast cells alone, only anandamide could induce significant level of histamine release at concentrations higher than 10(-6) M. When mast cells were activated with anti-IgE, the histamine release induced was not affected by anandamide, palmitoylethanolamide and CP 55,940. In contrast, both WIN 55,212-2 and HU-210 enhanced anti-IgE-induced histamine release at 10(-5) M and preincubation did not increase the potency. The histamine releasing action of anandamide and the enhancing effects of WIN 55,212-2 and HU-210 on anti-IgE-induced histamine release were not reduced by the cannabinoid receptor antagonists, AM 281 and AM 630. In conclusion, the present study does not support the hypothesis that cannabinoids suppress mast cell activation. Instead, some of the cannabinoid receptor-directed ligands tested enhanced mast cell activation. However, the high concentrations required and the failure of cannabinoid receptor antagonists to reverse such effects also question the existence of functional cannabinoid receptors in mast cells.