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Abstract-Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. Recently, we have found that microRNA (miRNA) miR-145 is the most abundant miRNA in normal vascular walls and in freshly isolated VSMCs; however, the role of miR-145 in VSMC phenotypic modulation and vascular diseases is currently unknown. Here we find that miR-145 is selectively expressed in VSMCs of the vascular wall and its expression is significantly downregulated in the vascular walls with neointimal lesion formation and in cultured dedifferentiated VSMCs.

We report a mechanism by which the adapter protein Gene 33 (also called RALT and MIG6) regulates epidermal growth factor receptor (EGFR) signaling. We find that Gene 33 inhibits EGFR autophosphorylation and specifically blunts epidermal growth factor (

mediates pathogenic inflammatory responses to intestinal ischemia-reperfusion injury. Am J Physiol Gastrointest Liver Physiol 299: G833-G843, 2010. First published August 5, 2010 doi:10.1152/ajpgi.00065.2010.-Acute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) are major causes of death in trauma patients. Gut inflammation and loss of gut barrier function as a consequence of splanchnic ischemia-reperfusion (I/R) have been implicated as the initial triggering events that contribute to the development of the systemic inflammatory response, ALI, and MODS. Since hypoxia-inducible factor (HIF-1) is a key regulator of the physiological and pathophysiological response to hypoxia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Utilizing partially HIF-1␣-deficient mice in a global trauma hemorrhagic shock (T/HS) model, we found that HIF-1 activation was necessary for the development of gut injury and that the prevention of gut injury was associated with an abrogation of lung injury. Specifically, in vivo studies demonstrated that partial HIF-1␣ deficiency ameliorated T/HS-induced increases in intestinal permeability, bacterial translocation, and caspase-3 activation. Lastly, partial HIF-1␣ deficiency reduced TNF-␣, IL-1␤, cyclooxygenase-2, and inducible nitric oxide synthase levels in the ileal mucosa after T/HS whereas IL-1␤ mRNA levels were reduced in the lung after T/HS. This study indicates that prolonged intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. Consequently, these results provide unique information on the initiating events in trauma-hemorrhagic shock-induced ALI and MODS as well as potential therapeutic insights. hemorrhagic shock; inflammation; multiple organ dysfunction syndrome; acute lung injury IN PATIENTS SUSTAINING major trauma, the development of the systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction (MODS) is a major clinical problem resulting in 50 -80% of all deaths in surgical intensive care units. Since the pathophysiology of this syndrome remains incompletely understood and therapy remains largely supportive (16), studies focusing on the basic biology of traumainduced SIRS, organ injury/dysfunction, and MODS have been major areas of investigation. These mechanistic studies have generated several working hypotheses, one of which is the gut hypothesis of MODS. A key element in the gut hypothesis of MODS is that a splanchnic ischemia-reperfusion (I/R) insult leading to gut inflammation and loss of barrier function is the initial triggering event that turns the gut into the "motor" of MODS (19). However, the exact mechanisms by which gut I/R leads to intestinal injury and how an intestinal ischemic insult is transduced into a systemic inflammatory response remains incomplete. To date, the majority of the molecular and cellular studies investigating shock-induced gut injury and gut-induced MODS have focused pr...

The Ras 3 Raf 3 MEK1͞2 3 extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf͞Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a lossof-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin. extracellular signal-regulated kinase ͉ mitogen-activated protein kinase͞merlin M itogen-activated protein kinase (MAPK) kinase kinase (MAP3K) 3 MAPK͞extracellular signal-related kinase (ERK) kinase (MEK) 3 MAPK modules are evolutionarily conserved signaling networks that regulate numerous processes crucial to cell proliferation, survival, inflammation, and metabolism (1). The regulation of MAP3Ks is still poorly understood, especially with regard to how proximal elements that recruit different MAPKs integrate these pathways to produce an appropriate response. Mixed-lineage kinase 3 (MLK3) is one of a family of MAP3Ks, the MLKs, originally identified as specific regulators of the c-Jun N-terminal kinase (JNK) group of MAPKs (1, 2). MLK3 has been linked to TNF activation of JNK (3, 4), and, indeed, targeted disruption of mlk3 in mice impairs TNF recruitment of JNK (4). Other MAPK pathways are unaffected in mlk3 Ϫ/Ϫ mice (4). In contrast, we found that RNA interference (RNAi) silencing of mlk3 revealed, in multiple, diverse cell types, a broader function for MLK3. Thus, our results indicate that MLK3 is required for activation of ERK, JNK, and p38 MAPKs by mitogenic and proinflammatory stimuli (3). The reasons for the discrepancy between our findings and those for the mlk3 Ϫ/Ϫ mice are unclear and may reflect a compensatory redundancy among the different MLKs expressed in these mice, a phenomenon that presumably did not occur in our cultured cell models. Nevertheless, our findings point to a signal-integrating role for MLK3.Of particular interest, we uncovered a potential non-MAP3K role for MLK3 (3). B-Raf and the related Raf-1 are major MAP3Ks coupling Ras to ERKs 1 and 2 (ERK1͞2) and MAPK͞ ERK kinases 1 and 2 (MEK1͞2) (5). We observed that MLK3 was necessary for the signal-dependent phosphorylation of B-Raf...

Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3 ؊/؊ murine embryonic fibroblasts contained an enhanced level of HIF-1␣ under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1␣ under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1␣ in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer halflife than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1␣ stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.

. The female intestine is more resistant than the male intestine to gut injury and inflammation when subjected to conditions associated with shock states. Am J Physiol Gastrointest Liver Physiol 288: G466 -G472, 2005. First published October 21, 2004; doi:10.1152/ajpgi.00036.2004.-Having documented that proestrus female rats are more resistant to shock-induced acute gut and hence lung injury than male rats, we tested the hypothesis that the female gut is more resistant to injury and produces less of an inflammatory response than the male gut when exposed to conditions associated with shock states (hypoxia and acidosis) utilizing the ex vivo Ussing chamber system. Ileal mucosal membranes harvested from normal male and female rats mounted in Ussing chamber systems were exposed to normoxia or 40 min of hypoxia at a normal pH (pH 7.3) or acidosis (pH 6.8). Cytokine and nitric oxide levels in the serosal compartment of the Ussing chamber were measured at the end of the 3-h experimental period to assess the immunoinflammatory response, whereas FITC-dextran (mol wt 4,300) was employed to assess barrier function. Histomorphological changes were used to quantitate gut mucosal injury. Hypoxia, acidosis, or hypoxia plus acidosis was associated with a significant increase in proinflammatory cytokine production [interleukin (IL)-6, tumor necrosis factor, and macrophage inflammatory protein (MIP)-2] by the male compared with the female intestinal segments. In contrast, the female gut manifested a higher anti-inflammatory response (nitric oxide and IL-10) and improved intestinal barrier function as well as less evidence of mucosal injury than the male intestinal segments. Administration of estradiol or the testosterone receptor antagonist, flutamide, to male rats abrogated the increase in gut injury and the increased IL-6 and MIP-2 response observed after hypoxia plus acidosis. These results suggest that gender differences in the ex vivo intestinal response to stresses, such as hypoxia and acidosis, exist and that the administration of estradiol or blockade of the testosterone receptor to male rats mitigates these gender differences.Ussing chamber; gender; ileal membrane; hypoxia/low pH; nitric oxide ALTHOUGH MULTIPLE ORGAN DYSFUNCTION syndrome (MODS) is the leading cause of death in intensive care units today (14), effective therapies have been slow to be developed, at least in part, because of an incomplete understanding of its basic biology (14). Consequently, studies directed at elucidating the pathophysiology of MODS have assumed major clinical importance. Over the years, we and others have focused on the role of gut ischemia/injury as a factor in the subsequent development of MODS (7, 13). Most recently, these studies have documented that, after trauma-hemorrhagic shock (T/ HS), the gut becomes a cytokine-generating organ (17) and that factors exiting the gut via the mesenteric lymphatics contribute to lung injury, endothelial cell dysfunction, and neutrophil activation (2, 14 -16, 19, 41). Although these studies illust...

Cell matrix adhesion is required for cell proliferation and survival. Here we report that mutation by gene targeting of the cytoplasmic tail of β1 integrin leads to defective proliferation and survival both in vivo and in vitro. Primary murine embryonic fibroblasts (MEFs) derived from mutant homozygotes display defective cell cycle coupled to impaired activation of the FAK-PI3K-Akt and Rac-JNK signaling pathways. Expression in homozygous MEFs of a constitutively active form of Rac is able to rescue proliferation, survival, and JNK activation. Moreover, although showing normal Erk phosphorylation, mutant cells fail to display Erk nuclear translocation upon fibronectin adhesion. However, expression of the constitutively activated form of Rac restores Erk nuclear localization, suggesting that adhesion-dependent Rac activation is necessary to integrate signals directed to promote MAPK activity. Altogether, our data provide the evidence for an epistatic interaction between the β1 integrin cytoplasmic domain and Rac, and indicate that this anchorage-dependent signaling pathway is crucial for cell growth control.