Abstract NO is a crucial mediator of the inflammatory response, but its in vivo role as a determinant of lung inflammation remains unclear. We addressed the in vivo role of NO in regulating the activation of NF-κB and expression of inflammatory proteins using an in vivo mouse model of sepsis induced by i.p. injection of Escherichia coli. We observed time-dependent degradation of IκB and activation of NF-κB accompanied by increases in inducible NOS, macrophage inflammatory protein-2 (MIP-2), and ICAM-1 expression after E. coli challenge, which paralleled the ability of lung tissue to produce high-output NO. To determine the role of NO in this process, mice were pretreated with the NO synthase (NOS) inhibitor NG-methyl-l-arginine. Despite having relatively modest effects on NF-κB activation and ICAM-1 or inducible NOS expression, the NOS inhibitor almost completely inhibited expression of MIP-2 in response to E. coli challenge. These responses were associated with the inhibition of migration of neutrophils in lung tissue and increased permeability induced by E. coli. In mice pretreated with NG-methyl-l-arginine, coadministration of E. coli with the NO donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate substantially restored MIP-2 expression but decreased ICAM-1 expression. The results suggest that NO generated after administration of E. coli serves as an important proinflammatory signal to up-regulate MIP-2 expression in vivo. Thus, NO production in high quantities may be important in the mechanism of amplification of the lung inflammatory response associated with sepsis.
In addition to exerting a potent anti-elastase function, α-1 antitrypsin (A1AT) maintains the structural integrity of the lung by inhibiting endothelial inflammation and apoptosis. A main serpin secreted in circulation by hepatocytes, A1AT requires uptake by the endothelium to achieve vasculoprotective effects. This active uptake mechanism, which is inhibited by cigarette smoking (CS), involves primarily clathrin- but also caveola-mediated endocytosis and may require active binding to a receptor. Because circulating A1AT binds to high-density lipoprotein (HDL), we hypothesized that scavenging receptors are candidates for endothelial uptake of the serpin. Although the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) internalizes only elastase-bound A1AT, the scavenger receptor B type I (SR-BI), which binds and internalizes HDL and is modulated by CS, may be involved in A1AT uptake. Transmission electron microscopy imaging of colloidal gold-labeled A1AT confirmed A1AT endocytosis in both clathrin-coated vesicles and caveolae in endothelial cells. SR-BI immunoprecipitation identified binding to A1AT at the plasma membrane. Pretreatment of human lung microvascular endothelial cells with SR-B ligands (HDL or LDL), knockdown of SCARB1 expression, or neutralizing SR-BI antibodies significantly reduced A1AT uptake by 30–50%. Scarb1 null mice exhibited decreased A1AT lung content following systemic A1AT administration and reduced lung anti-inflammatory effects of A1AT supplementation during short-term CS exposure. In turn, A1AT supplementation increased lung SR-BI expression and modulated circulating lipoprotein levels in wild-type animals. These studies indicate that SR-BI is an important mediator of A1AT endocytosis in pulmonary endothelium and suggest a cross talk between A1AT and lipoprotein regulation of vascular functions.
Recently, we demonstrated in cultured endothelial cells (ECs) and in vivo that intersectin-1s (ITSN-1s) deficiency impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e., enlarged endocytic structures, membranous rings/tubules), underrepresented under normal conditions. We show now that these endocytic structures internalize Alk5, a broadly expressed transforming growth factor beta receptor I (TGFβ–RI), leading to its ubiquitination and degradation. Moreover, the apoptotic/activated vascular cells of the ITSN-1s knockdown mice (KD-ITSN) release in the systemic circulation Alk5-bearing microparticles (MPs) able to interact and transfer the Alk5/TGFβ–RI to endocytic-deficient ECs; the interaction results in lung ECs survival and phenotypic alteration toward proliferation in a process involving Erk1/2 activation. We also show that non-productive assembly of Alk5/Smad/SARA (Smad Anchor for Receptor Activation) signaling complex and preferential formation of Alk5/mSos/Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary ECs. Altogether, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 via MPs and ECs survival/proliferation and define a novel molecular mechanism for TGFβ/Alk5-dependent Erk1/2MAPK signaling significant for proliferative signaling and abnormal growth.
Intersectin-1s (ITSN-1s), a multidomain adaptor protein, plays a vital role in endocytosis, cytoskeleton rearrangement and cell signaling. Recent studies have demonstrated that deficiency of ITSN-1s is a crucial early event in pulmonary pathogenesis. In lung cancer, ITSN-1s deficiency impairs Eps8 ubiquitination and favors Eps8-mSos1 interaction which activates Rac1 leading to enhanced lung cancer cell proliferation, migration and metastasis. Restoring ITSN-1s deficiency in lung cancer cells facilitates cytoskeleton changes favoring mesenchymal to epithelial transformation and impairs lung cancer progression. ITSN-1s deficiency in acute lung injury leads to impaired endocytosis which leads to ubiquitination and degradation of growth factor receptors such as Alk5. This deficiency is counterbalanced by microparticles which, via paracrine effects, transfer Alk5/TGFβRII complex to non-apoptotic cells. In the presence of ITSN-1s deficiency, Alk5-restored cells signal via Erk1/2 MAPK pathway leading to restoration and repair of lung architecture. In inflammatory conditions such as pulmonary artery hypertension, ITSN-1s full length protein is cleaved by granzyme B into EHITSN and SH3A-EITSN fragments. The EHITSN fragment leads to pulmonary cell proliferation via activation of p38 MAPK and Elk-1/c-Fos signaling. In vivo, ITSN-1s deficient mice transduced with EHITSN plasmid develop pulmonary vascular obliteration and plexiform lesions consistent with pathological findings seen in severe pulmonary arterial hypertension. These novel findings have significantly contributed to understanding the mechanisms and pathogenesis involved in pulmonary pathology. As demonstrated in these studies, genetically modified ITSN-1s expression mouse models will be a valuable tool to further advance our understanding of pulmonary pathology and lead to novel targets for treating these conditions.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe syndromes resulting from the diffuse damage of the pulmonary parenchyma. ALI and ARDS are induced by a plethora of local or systemic insults, leading to the activation of multiple pathways responsible for injury, resolution, and repair or scarring of the lungs. Despite the large efforts aimed at exploring the roles of different pathways in humans and animal models and the great strides made in understanding the pathogenesis of ALI/ARDS, the only viable treatment options are still dependent on ventilator and cardiovascular support. Investigation of the pathophysiological mechanisms responsible for initiation and resolution or advancement toward lung scarring in ALI/ARDS animal models led to a better understanding of the disease's complexity and helped in elucidating the links between ALI and systemic multiorgan failure. Although animal models of ALI/ARDS have pointed out a variety of new ideas for study, there are still limited data regarding the initiating factors, the critical steps in the progression of the disease, and the central mechanisms dictating its resolution or progression to lung scarring. Recent studies link deficiency of intersectin‐1s (ITSN‐1s), a prosurvival protein of lung endothelial cells, to endothelial barrier dysfunction and pulmonary edema as well as to the repair/recovery from ALI. This review discusses the effects of ITSN‐1s deficiency on pulmonary endothelium and its significance in the pathology of ALI/ARDS.
Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78.
