Persistent Increase In Expression Research Articles

Spinal cord injury-induced neurogenic bowel: A role for host-microbiome interactions in bowel pain and dysfunction

Background and aimsSpinal cord injury (SCI) affects roughly 300,000 Americans with 17,000 new cases added annually. In addition to paralysis, 60% of people with SCI develop neurogenic bowel (NB), a syndrome characterized by slow colonic transit, constipation, and chronic abdominal pain. The knowledge gap surrounding NB mechanisms after SCI means that interventions are primarily symptom-focused and largely ineffective. The goal of the present studies was to identify mechanism(s) that initiate and maintain NB after SCI as a critical first step in the development of evidence-based, novel therapeutic treatment options. MethodsFollowing spinal contusion injury at T9, we observed alterations in bowel structure and function reflecting key clinical features of NB. We then leveraged tissue-specific whole transcriptome analyses (RNAseq) and fecal 16S rRNA amplicon sequencing in combination with histological, molecular, and functional (Ca2+ imaging) approaches to identify potential mechanism(s) underlying the generation of the NB phenotype. ResultsIn agreement with prior reports focused on SCI-induced changes in the skin, we observed a rapid and persistent increase in expression of calcitonin gene-related peptide (CGRP) expression in the colon. This is suggestive of a neurogenic inflammation-like process engaged by antidromic activity of below-level primary afferents following SCI. CGRP has been shown to disrupt colon homeostasis and negatively affect peristalsis and colon function. As predicted, contusion SCI resulted in increased colonic transit time, expansion of lymphatic nodules, colonic structural and genomic damage, and disruption of the inner, sterile intestinal mucus layer corresponding to increased CGRP expression in the colon. Gut microbiome colonization significantly shifted over 28 days leading to the increase in Anaeroplasma, a pathogenic, gram-negative microbe. Moreover, colon specific vagal afferents and enteric neurons were hyperresponsive after SCI to different agonists including fecal supernatants. ConclusionsOur data suggest that SCI results in overexpression of colonic CGRP which could alter colon structure and function. Neurogenic inflammatory-like processes and gut microbiome dysbiosis can also sensitize vagal afferents, providing a mechanism for visceral pain despite the loss of normal sensation post-SCI. These data may shed light on novel therapeutic interventions targeting this process to prevent NB development in patients.

A mosquito lipoxin/lipocalin complex mediates innate immune priming in Anopheles gambiae.

Exposure of Anopheles gambiae mosquitoes to Plasmodium infection enhances the ability of their immune system to respond to subsequent infections. However, the molecular mechanism that allows the insect innate immune system to ‘remember' a previous encounter with a pathogen has not been established. Challenged mosquitoes constitutively release a soluble haemocyte differentiation factor into their haemolymph that, when transferred into Naive mosquitoes, also induces priming. Here we show that this factor consists of a Lipoxin/Lipocalin complex. We demonstrate that innate immune priming in mosquitoes involves a persistent increase in expression of Evokin (a lipid carrier of the lipocalin family), and in their ability to convert arachidonic acid to lipoxins, predominantly Lipoxin A4. Plasmodium ookinete midgut invasion triggers immune priming by inducing the release of a mosquito lipoxin/lipocalin complex.

Radiation-Induced Activation of TGF-β Signaling Pathways in Relation to Vascular Damage in Mouse Kidneys

The purpose of this study was to investigate the long-term effects of radiation-induced alterations in TGF-beta signaling pathways with respect to the development of vascular damage in the irradiated kidney. Total RNA was isolated from mouse kidneys at 1-30 weeks after irradiation, and quantitative real-time PCR analyses were performed for TGF-beta receptors (ALK1, ALK5, endoglin), downstream mediators (Smad7, CTGF), and downstream targets (PAI-1 and Id-1). Expression of endoglin and Smad7 protein as well as nucleo-cytoplasmic distribution of phospho Smad 2/3 and phospho Smad 1/5 was analyzed by immunohistochemistry. Radiation caused a rapid and persistent increase in expression of TGF-beta receptors and mediators from 1-30 weeks after treatment. Expression of Id-1, a downstream target of endothelial cell specific receptor ALK1, was transiently increased (1-10 weeks after irradiation) but returned to control levels at later times. Expression of PAI-1, a downstream target of ALK5, increased progressively from 10-30 weeks after irradiation. These results show that radiation activated TGF-beta signaling pathways in the kidney and shifted the balance in favor of ALK5 signaling, which generally inhibits endothelial cell proliferation and migration. We hypothesize that prolonged activation of ALK5 signaling and relative suppression of ALK1 signaling may provide an explanation for the telangiectatic phenotype observed in irradiated kidneys.

FoxM1 Is Essential for Endothelial Repair following Inflammatory Injury

The Forkhead transcription factor FoxM1 regulates expression of cell cycle genes critical for cellular proliferation. Here, we observed that FoxM1 expression was predominantly induced in endothelial cells in the recovery phase following lipopolysacchride-induced vascular injury. To further investigate the functional role of FoxM1 in the mechanism of endothelial repair, we generated endothelial cell-restricted FoxM1-deficient mice (FoxM1 CKO). Unlike the FoxM1 null mutation mice, these conditional mutant mice are viable and exhibited no overt phenotype. However, in response to the inflammatory mediator lipopolysaccharide, FoxM1 CKO displayed a significantly protracted increase in lung vascular permeability and a marked increase in mortality. BrdU labeling studies demonstrated significantly impaired cellular proliferation in FoxM1 CKO lungs following LPS challenge, which was associated with a persistent increase in expression of the cyclin-dependent kinase inhibitor p27kip1. In addition, endothelial cells isolated from FoxM1 CKO lungs failed to proliferate and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Taken together, these data suggest FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier integrity following vascular injury.

Differential regulation of protease and extracellular matrix protein expression by transforming growth factor-beta 1 in non-small cell lung cancer cells and normal human bronchial epithelial cells

In addition to autoregulating its own expression, transforming growth factor-beta 1 (TGF- β1) also regulates the production of proteases, protease inhibitors and extracellular matrix proteins. To investigate the relationship between plasminogen activator (PA), plasminogen activator inhibitor-1 (PAI-1) and the extracellular matrix in malignant and normal lung epithelial cells and to determine whether malignant lung epithelial cells may be more invasive than normal lung epithelial cells because of differences in expression of these proteins in response to TGF- β, the regulation of PA, PAI-1, fibronectin, laminin and thrombospondin by TGF- β1 in human non-small cell lung cancer (NSCLC) cells was examined and compared with normal human bronchial epithelial (NHBE) cells. TGF- β1 caused a persistent increase in expression of the mRNAs for both PA and PAI-1 in NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. By immunoprecipitation analysis, it was shown that TGF- β1 also induced a corresponding increase in the amount of PAI-1 protein in these NSCLC cells as well. In contrast, while TGF- β1 also increased expression of PAI-1 mRNA in NHBE cells, expression of PA mRNA decreased simultaneously. Treatment of NSCLC cells with TGF- β1 resulted in a persistent increase in expression of the mRNAs for fibronectin, laminin and thrombospondin; expression of fibronectin protein also increased after treatment with TGF- β1 in these cells. When NHBE cells were similarly cultured in the presence of TGF- β1, expression of fibronectin mRNA also increased in a persistent manner; however, only an early transient increase in the level of the mRNAs for laminin and thrombospondin was detected in these cells. These data show that there is differential regulation of the genes for PA and PAI-1 and the extracellular matrix protein fibronectin in response to TGF- β1 not only when NSCLC and NHBE cells are compared, but also when different NSCLC cells are compared with each other.

Effects of Transforming Growth Factor-βl and Phorbol Ester on PALI-1 and PA Genes in Human Lung Cells

Transforming growth factor-β (TGF-β) mediates the production of extracellular matrix proteins, proteases and protease inhibitors in epithelial cells. Both TGF-β and phorbol-12-myristate-13-acetate (PMA) exert both positive and negative effects on mitogenesis in these as well as other cell types. Phorbol esters act through stimulation of protein kinase C (PKC) and are among the most potent tumor promoters known. The present study was conducted to determine whether the effect of TGF-β in human non-small cell lung cancer (NSCLC) and normal human bronchial epithelial (NHBE) cells parallels that of the phorbol esters and whether this effect of TGF-β involves PKC. TGF-β1 and PMA increased expression of TGF-βl mRNA 24 hr after their addition to both NSCLC and NHBE cells. The effects of these agents on expression of the mRNAs for TGF-β2 and TGF-β3 were more complex; while TGF-β2 and TGF-p3β mRNAs increased transiently in response to TGF-p1 in NHBE cells and TGF-β3 mRNA increased transiently in some NSCLC cells, expression of these mRNAs decreased in most of these cells in response to PMA with the exception of the carcinoid NCLH727 where TGF-pZ mRNA increased dramatically. TGF-β1 and PMA both caused a persistent increase in expression of the mRNAs for both plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA) up to 24 hr in most NSCLC cells, with the increase in PAI-1 mRNA beginning several hours before that of PA mRNA. In contmst, while TGF-βl also increased expression of PAI-1 mRNA in NHBE cells, the expression of PA mRNA decreased simultaneously. The effect of PMA on PAI-1 and PA mRNAs was opposite of TGF-β1 in these cells, with expression of PAI-1 mRNA decreasing and PA mRNA increasing after addition of PMA. These data show that there is parallel regulation of the genes for TGF-βl, PAI-1 and PA by TGF-β1 and PMA in NSCLC, but differential regulation of the genes for PAL1 and PA by these agents in NHBE cells. The responses of the mRNAs and proteins of TGF-β1, PAI-1 and PA to TGF-βl and PMA were inhibited by the serind threonine kinase inhibitor H7 in NSCLC cells. Treatment of NSCLC cells with TGF-β1 and PMA resulted in a persistent increase in the expression of fibronectin mRNA and protein. This response was blocked by the addition of H7. Inhibition of these effects by H7 in NSCLC cells suggests that H7 blocks TGF-p responses by inhibiting a protein serindthmnine kinase(s). Because the effects of TGF-p and PMA on the different TGF-p isoforms, PA, PA1 and fibronectin in NHBE and NSCLC cells are complex, our data suggest that there are distinct mechanisms for controlling the different TGF-p isoforms, PA, PA1 and extracellular matrix proteins in normal lung and lung cancer cells.

Altered expression of group I metabotropic glutamate receptors in the hippocampus of amygdala-kindled rats

Kindling is a well documented model of acquired focal epilepsy and synaptic plasticity in the nervous system. Previous biochemical studies have indicated an increase in mGluR-mediated phosphoinositide hydrolysis in the amygdala or hippocampus of fully kindled animals. In this study we have used in situ hybridisation techniques to examine the mRNA expression of group I metabotropic glutamate receptors (mGluR1 and mGluR5 both linked to phosphoinositide hydrolysis) in the hippocampus of amygdala-kindled animals sacrificed 24 h, 7 days or 28 days following the last electrically evoked stage 5 seizure, and in implanted non-stimulated control rats. Results indicate an initial up-regulation in mGluR1 mRNA (expressed as percentage of control) bilaterally in the DG (35–40%) and CA3 (16–48%), and unilaterally in CA4 (12%) in the 24 h post-kindled group. In kindled animals studied 7 days after the last seizure, these changes were either reduced or had returned to control levels. By 28 days mGluR1 mRNA levels had returned to control levels, with only a persistent increase in expression unilaterally in the DG (14%). In contrast, an initial down-regulation in mGluR5 mRNA was observed bilaterally in CA4 (−45 and −25%) and CA1 (−46 and −45%), and unilaterally in DG and CA3 (−27 and −42% respectively) 24 h after the last kindled seizure. In the 7 and 28 day kindled groups significant alterations in expression of mGluR5 mRNA were still apparent. These data show that the mRNAs for mGluR1 and mGluR5 are differentially regulated by kindling, indicating that the expression of each of these receptors is under independent regulatory control. These perturbations in mRNA expression may contribute to kindling epileptogenesis but are unlikely to account for the maintenance of the kindled state.