Previous animal studies have identified a role for activation of innate immunity in the pathogenesis of ventilator-associated lung injury. These studies have used large tidal volume ventilation to study the effect of alveolar overdistension on induction of inflammatory pathways. We hypothesized an alternative mechanism for the pathogenesis of lung injury in which moderate tidal volume ventilation does not independently cause clinical inflammation but rather interacts with innate immune activation by bacterial products, resulting in an enhanced inflammatory response. We measured cytokine expression and lung injury in normal and lipopolysaccharide (LPS)-treated anesthetized rabbits randomized to either spontaneous respiration or mechanical ventilation. Outcome parameters were analyzed by two-way factorial analysis of variance to identify synergism between ventilation and systemic LPS. Mechanical ventilation alone resulted in minimal cytokine expression in the lung but did enhance LPS-induced expression of tumor necrosis factor-alpha, the CXC chemokines interleukin-8 and growth-related protein-alpha, and the CC chemokine monocyte chemoattractant protein-1. Increased mRNA expression and activation of the transcription factors nuclear factor-kappaB and activator protein-1 accompanied the cytokine responses. We conclude that moderate volume ventilation strategies augment the innate immune response to bacterial products in the lung and may play a role in the development of acute lung injury in patients with sepsis.

The interaction of the multimodular heterogeneous nuclear ribonucleoprotein (hnRNP) K protein with many of its protein and nucleic acid partners is regulated by extracellular signals. Acting as a docking platform, K protein could link signal-transduction pathways to DNA- and RNA-directed processes such as transcription, mRNA processing, transport, and translation. Treatment of hepatocyte culture with insulin increased K protein tyrosine phosphorylation. Insulin altered K protein interaction with RNA and DNA in vitro. Administration of insulin into mice had similar effects on K protein in liver. Coimmunoprecipitations of RNA with K protein revealed preferential in vivo K protein binding of a subset of transcripts, including the insulin-inducible c-fos mRNA. These results suggest a class of insulin pathways that signal nucleic acid-directed processes that involve K protein

The heterogeneous nuclear ribonucleoprotein K protein is an RNA- and DNA-binding protein implicated in the regulation of multiple processes that comprise gene expression. We used chromatin immunoprecipitation (ChIP) assays to explore K protein interactions with serum-inducible, constitutively expressed and untranscribed gene loci in vivo. In the rat HTC-IR hepatoma cell line, serum treatment induced transient increases in the mRNA levels of two immediate-early genes, egr-1 and c-myc. ChIP analysis showed that the induction of egr-1 and c-myc genes was associated with a transient recruitment of K protein to multiple sites within each of these loci, including the promoter and transcribed regions. In contrast, recruitment of K protein to the constitutively transcribed beta-actin locus and to randomly chosen non-transcribed loci was far weaker. In rat mesangial cells, c-myc was constitutively expressed while egr-1 remained serum responsive. In these cells, ChIP analysis showed serum-induced recruitment to the inducible egr-1 but not to the c-myc locus. Pre-treatment with the transcription inhibitor actinomycin D blocked the inducible but not the constitutive binding of K protein to these loci. Taken together, the results of this study suggest that the transient recruitment of K protein to serum-responsive loci depends on the inducible transcription of these immediate-early genes.

Mesangial cells in glomeruli are located under a fenestrated capillary endothelium and are exposed to hydrostatic pressure necessary to sustain normal filtration (1-3). Progressive renal diseases, such as diabetic nephropathy, remnant kidney, and hypertensive nephropathy, lead to prolonged glomerular hypertension, which is involved in the mesangial cell proliferation that is considered to be the most important factor mediating glomerular sclerosis (4 -11). However, the mechanism of these changes under glomerular hypertension remains largely unknown. Hishikawa et al. (12) have reported that, in vascular smooth muscle cells, pressure promotes DNA synthesis and cell growth probably via protein kinase C (PKC), 1 although the detailed mechanism between pressure as an extracellular stimulus and proliferation is unknown.Various growth factors and mitogenic stimuli are known to induce the activation of mitogen-activated protein kinase (MAPK), a serine/threonine kinase (13,14). This kinase activity is up-regulated through phosphorylation on tyrosine and threonine residues by MAPK/extracellular signal-regulated kinase kinases (MEKs) (15, 16). MEKs are substrates for Raf-1 (17, 18), which has been reported to be activated either through receptors involved in Ras or a PKC-dependent pathway (19,20). These MAPK activators cause the translocation of MAPK from the cytosol to the nucleus (21-23), where transcription factors such as Elk-1 (24) and c-Ets (25, 26) are substrates for MAPK. This indicates that MAPK serves as an important regulator of transcriptional activity related to proliferation. Recently, Lavoie et al. (27) reported that cyclin D1 expression, which is one of the earliest cell cycle-related events to occur during the G 0 /G 1 to S phase transition, is regulated positively by MAPK. Therefore, increasing interest has been paid to the role of MAPK in the cell cycle (28 -30). We recently showed that pressure enhances G 1 /S progression and promotes the rate of DNA synthesis in mesangial cells (31). However, MAPK activation and its physiological effects in glomerular hypertension are presently unknown. We investigated MAPK activation and cell proliferation in mesangial cells using a pressure-loading apparatus. We show here that applied pressure is a novel activator of MAPK. Furthermore, we demonstrate that MAPK activation plays a role in pressure-induced proliferation, probably via cyclin D1 expression. , anti-phosphoElk-1 (Ser-383) antibody, Elk-1 fusion protein, and PD98059 (MEK EXPERIMENTAL PROCEDURES Materials