Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the α subunit of Gq protein (Gαq) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active Gαq (GαqQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of Gαq with shRNA in HaCaT human keratinocytes. Gαq was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase Cδ (PKCδ), and matrix metaloprotease-2 (MMP-2). Moreover, GαqQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that Gαq mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKCδ and MMP-2 in HaCaT cells.     

Prostaglandin E(2) mediates inhibition of insulin secretion by interleukin-1beta.

Interleukin-1beta (IL-1beta) and prostaglandin E(2) (PGE(2)), frequently co-participants in inflammatory states, are two well recognized inhibitors of glucose-induced insulin secretion. Previous reports have concluded that the inhibitory effects of these two autacoids on pancreatic beta cell function are not related because indomethacin, a potent prostaglandin synthesis inhibitor, does not prevent IL-1beta effects. However, indomethacin is not a specific cyclooxygenase inhibitor, and its other pharmacologic effects are likely to inhibit insulin secretion independently. Since we recently observed that IL-1beta induces cyclooxygenase-2 (COX-2) gene expression and PGE(2) synthesis in islet beta cells, we have reassessed the possibility that PGE(2) mediates IL-1beta effects on beta function. By using two cell lines (HIT-T15 and betaHC13) as well as Wistar rat isolated pancreatic islets, we examined the ability of two COX-2-specific antagonists, NS-398 and SC-236, to prevent IL-1beta inhibition of insulin secretion. Both drugs prevented IL-1beta from inducing PGE(2) synthesis and inhibiting insulin secretion; adding back exogenous PGE(2) re-established inhibition of insulin secretion in the presence of IL-1beta. We also found that EP3, the PGE(2) receptor subtype whose post-receptor effect is to decrease adenylyl cyclase activity and, thereby, insulin secretion, is the dominant mRNA subtype expressed. We conclude that endogenous PGE(2) mediates the inhibitory effects of exogenous IL-1beta on beta cell function.     

Human endometrial stromal interleukin-1 beta: autocrine secretion and inhibition by interleukin-1 receptor antagonist

Decidualization of human endometrial stromal cells is suppressed by endometrial IL-1 in an autocrine or paracrine manner, indicating that constant suppression of stromal decidualization by IL-1 requires a neutralizing mechanism for IL-1 action to accept embryo implantation. Since production of IL-1ra in human endometrium is reported to be 10- to 30-fold higher than that of IL-1 alpha/beta, we investigated whether endogenous IL-1 beta secreted from human endometrial stromal cells can be inhibited by IL-1ra by using an in vitro decidualization culture. Human stromal cells were cultured with 8-Br-cAMP to induce decidualization, and concentrations of IL-1 beta, IL-8, and prolactin in the culture supernatants were assayed before and after decidualization. There was no significant difference in mean IL-1 beta concentrations measured before and after decidualization. Addition of IL-1ra to endometrial stromal cell cultures strongly inhibited endogenous IL-8 secretion from the cells. Although IL-1 beta showed a biphasic effect on cell proliferation and a suppressive effect on decidualization of stromal cells, these effects were completely inhibited by IL-1ra. The results imply that a high in vivo concentration of IL-1ra in human endometrial tissues may regulate IL-1 effects on decidualization and cell proliferation of human endometrial stromal cells.     

Influence of barometric pressure on interleukin-1 beta secretion

Monocytes and macrophages are activated by various environmental challenges, including microorganisms, radiation, and pollutants. These cells release cytokines, such as interleukin (IL)-1 beta, that mediate physiological adaptations to stress. This study sought to define further the role of IL-1 beta in general adaptation to environmental stress by testing the hypothesis that high altitude (20,000 ft, 6,096 m) would stimulate IL-1 beta secretion from isolated human blood mononuclear cells. Cells from six young men (aged 22--26 yr) were divided into separate cultures incubated in either standard ambient conditions or in one of three test conditions, hypobaric hypoxia (simulating 20,000 ft), hypobaric normoxia (20,000 ft, O(2) supplemented), and normobaric hypoxia (10% O(2)). This design allowed differentiation between pressure-related vs. oxygen-related effects. Each subject made multiple blood donations in order that cells from all subjects were tested in all conditions. Contrary to the hypothesis, IL-1 beta secretion was not induced at simulated altitude in basal cell cultures. In lipopolysaccharide-stimulated cell cultures, exposure to altitude inhibited IL-1 beta secretion by approximately 40%, and the inhibition was due to the change in pressure (P = 0.039) rather than the change in oxygen. Secretion of other factors (IL-1 receptor antagonist and soluble IL-1 receptor type II) was not inhibited. Although these results are in opposition to the original hypothesis, they provide insight regarding adaptations necessary for hematopoiesis in response to high altitude and also provide a cellular rationale for the mountain sanatoriums of the 19th and early 20th centuries.     

Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages

Several mechanistically distinct models of nonclassical secretion, including exocytosis of secretory lysosomes, shedding of plasma membrane microvesicles, and direct efflux through plasma membrane transporters, have been proposed to explain the rapid export of caspase-1-processed IL-1 beta from monocytes/macrophages in response to activation of P2X7 receptors (P2X7R) by extracellular ATP. We compared the contribution of these mechanisms to P2X7R-stimulated IL-1 beta secretion in primary bone marrow-derived macrophages isolated from wild-type, P2X7R knockout, or apoptosis-associated speck-like protein containing a C-terminal CARD knockout mice. Our experiments revealed the following: 1) a novel correlation between IL-1 beta secretion and the release of the MHC-II membrane protein, which is a marker of plasma membranes, recycling endosomes, multivesicular bodies, and released exosomes; 2) a common and absolute requirement for inflammasome assembly and active caspase-1 in triggering the cotemporal export of IL-1 beta and MHC-II; and 3) mechanistic dissociation of IL-1 beta export from either secretory lysosome exocytosis or plasma membrane microvesicle shedding on the basis of different requirements for extracellular Ca(2+) and differential sensitivity to pharmacological agents that block activation of caspase-1 inflammasomes. Thus, neither secretory lysosome exocytosis nor microvesicle shedding models constitute the major pathways for nonclassical IL-1 beta secretion from ATP-stimulated murine macrophages. Our findings suggest an alternative model of IL-1 beta release that may involve the P2X7R-induced formation of multivesicular bodies that contain exosomes with entrapped IL-1 beta, caspase-1, and other inflammasome components.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

The estrogen-responsive B box protein: a novel enhancer of interleukin-1beta secretion

The estrogen-responsive B box protein (EBBP) and Pyrin belong to a family of structurally related proteins. While mutations in the pyrin gene cause an autoinflammatory disease, the biological function of EBBP is unknown. In this study, we identified the proinflammatory cytokine interleukin-1beta (IL-1beta) as an EBBP-binding partner. Furthermore, caspase-1 and NACHT, LRR and Pyrin domain containing protein (NALP) 1, two components of the recently identified inflammasome, a platform for the activation of caspase-1, also interact with EBBP. These proteins bind to the RFP domain of EBBP, suggesting that this domain of so far unknown function is an important protein-binding domain. EBBP was secreted in a caspase-1-dependent manner from cultured cells, and its secretion was enhanced by IL-1beta. Vice versa, endogenous and overerexpressed EBBP increased IL-1beta secretion. These results provide evidence for a role of EBBP in innate immunity by enhancing the alternative secretion pathway of IL-1beta.     

Potent inhibition of gastric acid secretion by intravenous interleukin-1 beta and -1 alpha in rats.

The influence of human and rat recombinant interleukin-1 (hIL-1 beta and -1 alpha and rIL-1 beta) on acid secretion was investigated in conscious pylorus-ligated rats. Intravenous injection of either hIL-1 beta, hIL-1 alpha or rIL-1 beta dose dependently inhibited gastric acid output with an ED50 of 0.05 microgram, 0.5 microgram and 2.2 micrograms, respectively. The antisecretory action of IL-1 beta was associated with an increase in circulating levels of gastrin. hIL-1 beta-induced inhibition of acid secretion was dose dependently reversed by peripheral injection of the IL-1 receptor antagonist, IL-RA, with a dose ratio of 1:10(3) for complete reversal. The inhibitory effect of hIL-1 beta was blocked by indomethacin and was not modified by IV injections of the CRF receptor antagonist, alpha-helical CRF(9-41), or the monoclonal somatostatin antibody CURE.S6, or by systemic capsaicin pretreatment. These results show that systemic hIL-1 beta-induced inhibition of gastric acid secretion is mediated through IL-1 receptors and prostaglandin pathways, and does not involves CRF receptors, afferent fibers, or changes in circulating gastrin or somatostatin levels.     

Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor

P2X(7) receptors are ATP-gated cation channels; their activation in macrophage also leads to rapid opening of a membrane pore permeable to dyes such as ethidium, and to release of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta). It has not been known what this dye-uptake path is, or whether it is involved in downstream signalling to IL-1beta release. Here, we identify pannexin-1, a recently described mammalian protein that functions as a hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature IL-1beta induced by P2X(7) receptor activation.     

Interleukin-1 beta induces synthesis and secretion of interleukin-6 in human chondrocytes

Increased concentrations of interleukin-6 (IL-6) have been found in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and crystal-related joint diseases. It is therefore of great interest to identify the cells responsible for the production of IL-6, and to investigate whether IL-6 plays a role in the pathogenesis of degenerative or inflammatory joint diseases. Here we show that human interleukin-1 beta (IL-1 beta) induces IL-6 synthesis and secretion in differentiated human chondrocytes. In organ cultures resembling closely the in vivo system 10(6) chondrocytes incubated with 100 units of interleukin-1 beta per ml of medium led to the release of 6 X 10(3) units of IL-6 within 24 h. Chondrocytes cultured in agarose or as monolayers similarly incubated with IL-1 beta produced even higher amounts of IL-6: 70 X 10(3) units per 10(6) cells within 24 h. The induction of IL-6 synthesis by IL-1 beta was also shown at the mRNA level. IL-6 secreted by stimulated chondrocytes showed heterogeneity upon Western blot analysis.     

Thioredoxin-interacting protein mediates NALP3 inflammasome activation in podocytes during diabetic nephropathy

Numerous studies have shown that the NALP3 inflammasome plays an important role in various immune and inflammatory diseases. However, whether the NALP3 inflammasome is involved in the pathogenesis of diabetic nephropathy (DN) is unclear. In our study, we confirmed that high glucose (HG) concentrations induced NALP3 inflammasome activation both in vivo and in vitro. Blocking NALP3 inflammasome activation by NALP3/ASC shRNA and caspase-1 inhibition prevented IL-1β production and eventually attenuated podocyte and glomerular injury under HG conditions. We also found that thioredoxin (TRX)-interacting protein (TXNIP), which is a pro-oxidative stress and pro-inflammatory factor, activated NALP3 inflammasome by interacting with NALP3 in HG-exposed podocytes. Knocking down TXNIP impeded NALP3 inflammasome activation and alleviated podocyte injury caused by HG. In summary, the NALP3 inflammasome mediates podocyte and glomerular injury in DN, moreover, TXNIP participates in the formation and activation of the NALP3 inflammasome in podocytes during DN, which represents a novel mechanism of podocyte and glomerular injury under diabetic conditions.     

The three-dimensional structure of interleukin-1 beta

The three-dimensional structure of human recombinant interleukin-1 beta has been determined at 0.24 nm resolution by X-ray crystallographic techniques. The partially refined model has a crystallographic R-factor of just under 19%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side-chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no significant internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by A. D. McLachlan [(1979) J. Mol. Biol. 133, 557-563] for soybean trypsin inhibitor.     

Inhibited insulin secretion by recombinant human interleukin-1 beta in adrenalectomized rats: involvement of prostaglandin

An involvement of prostaglandin synthesis in reduced insulin secretion by interleukin-1 was investigated in adrenalectomized (ADX) rats. The recombinant human interleukin-1 beta (IL-1) significantly reduced insulin secretion in ADX rats 2 and 4 hr after the injection, although IL-1 stimulated insulin secretion in intact rats. In ADX rat, IL-1 showed dose-dependent inhibition of pancreatic insulin secretion. In addition, insulin response to intravenous glucose loading was also attenuated in ADX rats with pretreatment by IL-1. At 4 hours after injection, ibuprofen (IBP; 0.5-50.0 mg/kg, ip), selective cyclooxygenase blocker, attenuated insulin inhibition by IL-1 in a dose-dependent manner. These data suggest that IL-1 may suppress in vivo insulin release at least in part through the mediation of prostaglandin synthesis in the absence of adrenal glands.     

c-Jun NH2-terminal kinase mediates interleukin-1beta-induced inhibition of lacrimal gland secretion

Sjögren's syndrome, an inflammatory disease affecting the lacrimal and salivary glands, is the leading cause of aqueous tear‐deficient type of dry eye. We previously showed that interleukin‐1β (IL‐1β) protein is up regulated in the lacrimal gland of a murine model of Sjögren's syndrome and that exogenous addition of this cytokine inhibits neurotransmitter release and lacrimal gland protein secretion. In the present study we investigated the role of c‐Jun NH₂‐terminal kinase (JNK) in IL‐1β‐mediated inhibition of lacrimal gland secretion and tear production. In vitro, IL‐1β induced a time‐dependent activation of JNK with a maximum 7.5‐fold at 30 min. SP600125, a JNK inhibitor, inhibited, in a concentration‐dependent manner, IL‐1β‐induced activation of JNK with a maximum of 87% at 10^?4 m . In vivo, IL‐1β stimulated JNK and the expression of the inducible isoform of nitric oxide synthase (iNOS). IL‐1β inhibited high KCl and adrenergic agonist induced protein secretion by 85% and 66%, respectively. SP600125 alleviated the inhibitory effect of IL‐1β on KCl‐ and agonist‐induced protein secretion by 79% and 47%, respectively, and completely blocked the expression of iNOS. Treatment for 7 days with SP600125 increased tear production in a murine model of Sjögren's syndrome dry eye. We conclude that JNK plays a pivotal role in IL‐1β‐mediated inhibition of lacrimal gland secretion and subsequent dry eye.