Proteolysis-inducing factor differentially influences transcriptional regulation in endothelial subtypes.

Proteolysis-inducing factor (PIF) is a novel sulfated glycoprotein initially identified as a protein capable of triggering muscle proteolysis during the process of cancer cachexia. Only skeletal muscle and liver exhibit substantial binding of PIF in adult tissue. Here, we demonstrate that PIF induces transcriptional regulation in both the liver endothelial cell line SK-HEP-1 and in human umbilical vein endothelial cells (HUVECs) but not in pulmonary artery endothelial cells. PIF differentially induces activation of nuclear factor-kappaB, resulting in the induction of proinflammatory cytokines [interleukin (IL)-8 and IL-6] and increased expression of the cell surface proteins intercellular adhesion molecule-1 and vascular cell adhesion molecule in SK-HEP-1 and HUVECs only. In addition, PIF induces the shedding of syndecans from the cell surface. Syndecans are involved in wound repair, metastasis of cancers, and embryonic development. These results suggest that PIF may play additional roles in the proinflammatory response observed in cancer cachexia but may also have a role without the cachectic process.

Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factor-kappaB.

The airway epithelium is injured by oxidants inhaled as atmospheric pollutants or produced during inflammatory responses. We studied the effect of modulating the antioxidant intracellular glutathione, both using thiol compounds and by the adaptive effect of hyperoxia, on oxidant-induced injury and activation of the nuclear factor-kappaB (NF-kappaB) in two cell lines: the human bronchial (16HBE) and type II alveolar epithelial cells (A549). The thiol antioxidants glutathione (GSH) and glutathione monoethyl ester (GSH-MEE) [2 mM] increased GSH levels (nmol/mg protein) in A549 cells (GSH 383 +/- 26 and GSH-MEE 336 +/- 23 vs control 171 +/- 13, P < 0.001) and in 16HBE cells (GSH 405 +/- 33, GSH-MEE 362 +/- 37 vs control 198 +/- 12, P < 0.001, N = 3). Treatment of hyperoxia (95% oxygen) also increased GSH levels between 4 and 24 hr exposure compared with control (P < 0.01). Hydrogen peroxide (H(2)O(2)) (0.01 mM) induced NF-kappaB activation, whereas hyperoxia exposure did not affect NF-kappaB activation in either cell line. Pretreatment with dl-buthionine (SR)-sulfoximine, which decreased intracellular glutathione, increased NF-kappaB binding induced by H(2)O(2) and increased lactate dehydrogenase (LDH) release (P < 0.001). Pretreatment with the thiol compounds and hyperoxia totally inhibited H(2)O(2)-induced NF-kappaB binding and cell injury as measured by LDH release. These data indicate the importance of intracellular glutathione and inhibition of NF-kappaB in both protection/tolerance against oxidant-induced epithelial cell injury, and NF-kappaB activation in response to oxidative stress which may be important in lung inflammation. Thus, increasing intracellular glutathione may be of therapeutic relevance if able to modulate NF-kappaB activation and hence attenuate inflammation.

Proteolysis-inducing factor regulates hepatic gene expression via the transcription factors NF-(kappa)B and STAT3.

A novel protein, proteolysis-inducing factor (PIF), has been isolated from the urine of patients with pancreatic cancer and is capable of inducing muscle proteolysis in vitro. Only adult skeletal muscle and liver exhibit substantial binding of PIF. We have investigated the effect of PIF on hepatic gene expression. Primary cultures of human hepatocytes and the human cell line HepG2 were incubated in the presence of PIF to assess its effects on hepatic transcription factors, proinflammatory cytokine production, and acute phase proteins. PIF activates both the transcription factors NF-kB and STAT3, which result in the increased production of IL-8, IL-6, and C-reactive protein and the decreased production of transferrin. The function of PIF, beyond muscle degradation, is unknown but here we show that it is involved in hepatic gene expression, and is thus likely to be involved in the proinflammatory response observed in cachexia. These results may also suggest a potential role for PIF during embryonic development. The expression of PIF peaks during the embryonic period E8 to E9, a stage that is crucial in the development of skeletal muscle and liver and during which both NF-kB and STAT3 activation can also be observed.

Hydrogen peroxide-induced epithelial injury: the protective role of intracellular nonprotein thiols (NPSH).

Injury to the alveolar region is a hallmark of the adult respiratory distress syndrome (ARDS) whereas injury to the epithelium of the conducting airways is a characteristic of asthma. Reactive oxygen species have been implicated as mediators of lung injury in both of these conditions. We have investigated the relationship between intracellular nonprotein thiols (NPSH), and the release of the cytosolic enzyme lactate dehydrogenase (LDH), as an index of cell injury, following treatment of the human alveolar type II-like epithelial cell line (A549 cells) or the human bronchial epithelial cell line (16HBE140-) with hydrogen peroxide (H2O2). We have also assessed the protective effects of pre-incubation of both of these cells lines with H2O2 or enhancement of intracellular NPSH against H2O2-induced cell injury. Exposure of A549 and 16HBE140- cells to H2O2 (0.1 mM and 1 mM respectively for 16 h) produced the release of 40% of the total cellular LDH. H2O2 exposure produced an initial dose-dependent decrease in NPSH in A549 cells, with a subsequent increase to above control values. 16HBE140- cells also showed a dose-dependent decrease in NPSH following exposure to H2O2. Pretreatment of A549 cells with 0.1 mM H2O2 followed by subsequent exposure to H2O2 did not protect against H2O2-induced LDH release in this epithelial cell line. Pre-incubation with 2 mM N-acetylcysteine (NAC) increased NPSH but not intracellular reduced glutathione and resulted in total inhibition of H2O2-induced LDH release in both cell types. Pretreatment with reduced glutathione protected both cell types against the injurious effects of H2O2, whereas glutathione monethyl ester (GSHMEE) only partially protected A549 cells and had no effect in 16HBE140- cells. Intracellular cysteine levels were increased in both cell lines following NAC exposure but not sufficiently to account for the increase in NPSH levels. These observations raise the possibility that a critical concentration of nonprotein thiols may be necessary to protect pulmonary epithelial cells against hydrogen peroxide-induced injury.