Dopamine deficiency contributes to early visual dysfunction in a rodent model of type 1 diabetes.

Dopamine (DA) functions as an essential neuromodulator in the brain and retina such that disruptions in the dopaminergic system are associated with common neurologic disorders such as Parkinson's disease. Although a reduction in DA content has been observed in diabetes, its effects in the development of diabetes-induced neuropathy remains unknown. Because the retina is rich in DA and has a well known diabetes-induced pathology (diabetic retinopathy or DR), this study was designed to examine the role of retinal DA deficiency in early visual defects in DR. Using rodent models of type 1 diabetes mellitus, we investigated whether diabetes caused a reduction in retinal DA content in both rats and mice and determined whether restoring DA levels or activating specific DA receptor pathways could improve visual function (evaluated with optokinetic tracking response) of diabetic mice, potentially via improvement of retinal function (assessed with electroretinography). We found that diabetes significantly reduced DA levels by 4 weeks in rats and by 5 weeks in mice, coincident with the initial detection of visual deficits. Treatment with l-DOPA, a DA precursor, improved overall retinal and visual functions in diabetic mice and acute treatment with DA D1 or D4 receptor agonists improved spatial frequency threshold or contrast sensitivity, respectively. Together, our results indicate that retinal DA deficiency is an underlying mechanism for early, diabetes-induced visual dysfunction and suggest that therapies targeting the retinal dopaminergic system may be beneficial in early-stage DR.

Overexpression of Ste20-related proline/alanine-rich kinase exacerbates experimental colitis in mice.

Inflammatory bowel disease, mainly Crohn's disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in inflammatory bowel disease.

Adenosine 2B receptor expression is post-transcriptionally regulated by microRNA.

We have reported that epithelial adenosine 2B receptor (A(2B)AR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor alpha (TNF-alpha). Here, we examined the mechanism that governs A(2B)AR expression during colitis. A 1.4-kb sequence of the A(2B)AR promoter was cloned into the pFRL7 luciferase vector. Anti-microRNA (miRNA) was custom-synthesized based on specific miRNA binding sites. The binding of miRNA to the 3'-untranslated region (UTR) of A(2B)AR mRNA was examined by cloning this 3'-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-alpha induced a 35-fold increase in A(2B)AR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A(2B)AR mRNA following TNF-alpha treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3'-UTR of the A(2B)AR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-alpha reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A(2B)AR levels by >60%. Blockade of miR27b increased A(2B)AR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A(2B)AR mRNA. Collectively, these data demonstrate that TNF-alpha-induced A(2B)AR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A(2B)AR expression in murine colitis.

MicroRNA-92b regulates expression of the oligopeptide transporter PepT1 in intestinal epithelial cells.

MicroRNAs (miRNAs), which are noncoding RNAs that posttranscriptionally inhibit expression of target genes, have recently emerged as important regulators of many cellular functions such as cell differentiation. The epithelial di/tripeptide membrane transporter PepT1 is expressed in highly differentiated cells (the villous tip) but not in undifferentiated cells (the crypt) of the small intestine. Here, we investigated the regulation of PepT1 expression by miRNAs and its functional consequences. We observed a reverse correlation between the expression levels of PepT1 and mature miRNA-92b (miR-92b) during the differentiation of intestinal epithelial Caco2-BBE cells, suggesting a miR-92b-mediated regulation of PepT1 expression. We demonstrate that miR-92b suppressed PepT1 expression at both mRNA and protein levels, with subsequent reduced PepT1 transport activity, in Caco2-BBE cells by directly targeting the PepT1 3'-untranslated region. In addition, miR-92b suppresses bacterial peptide-induced proinflammatory responses in intestinal epithelial cells by inhibiting PepT1 expression. Altogether, our study provides for the first time evidence for the regulation of PepT1 expression at a posttranscriptional level by miRNAs in intestinal epithelial cells during pathophysiological states.

Prohibitin is a novel regulator of antioxidant response that attenuates colonic inflammation in mice.

BACKGROUND & AIMS: Increased free radicals and/or impaired antioxidant defenses have been shown to play a pathogenetic role in human and animal models of inflammatory bowel disease. Our previous studies showed that prohibitin (PHB) levels are decreased during colitis and that cultured intestinal epithelial cells overexpressing PHB are protected from oxidative stress. This study investigated the effect of intestinal epithelial cell-specific PHB overexpression on oxidative stress associated with experimental colitis and the potential mechanism by which PHB functions as an antioxidant using PHB transgenic mice. METHODS: Colitis was induced using 2 established mouse models (Salmonella typhimurium and dextran sodium sulfate) in PHB transgenic mice and wild-type littermates. Oxidative stress was determined by measuring glutathione and protein carbonyl levels in the cecum or colon. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcriptional regulator of oxidant responses, expression, and activation, was assessed in colon mucosa and cultured intestinal epithelial cells overexpressing PHB. RESULTS: Cells overexpressing PHB showed sustained Nrf2 nuclear accumulation and DNA binding during oxidant stress. PHB transgenic mice exhibited decreased oxidative stress and colitis and increased Nrf2 messenger RNA expression, nuclear protein translocation, and DNA binding compared with wild-type littermates during colitis. CONCLUSIONS: These results show that PHB is a regulator of Nrf2 expression in intestinal epithelial cells during oxidative conditions and prevents inflammation-associated oxidative stress and injury through sustained activation of Nrf2. Our data show that PHB is a novel regulator of antioxidants and suggest that restoration of PHB levels represents a potential therapeutic approach in inflammatory bowel disease.

Nuclear factor-kappaB is a critical mediator of Ste20-like proline-/alanine-rich kinase regulation in intestinal inflammation.

Inflammatory bowel disease (IBD) is thought to result from commensal flora, aberrant cellular stress, and genetic factors. Here we show that the expression of colonic Ste20-like proline-/alanine-rich kinase (SPAK) that lacks a PAPA box and an F-alpha helix loop is increased in patients with IBD. The same effects were observed in a mouse model of dextran sodium sulfate-induced colitis and in Caco2-BBE cells treated with the pro-inflammatory cytokine tumor necrosis factor (TNF)-alpha. The 5'-flanking region of the SPAK gene contains two transcriptional start sites, three transcription factor Sp1-binding sites, and one transcription factor nuclear factor (NF)-kappaB-binding site, but no TATA elements. The NF-kappaB-binding site was essential for stimulated SPAK promoter activity by TNF-alpha, whereas the Sp1-binding sites were important for basal promoter activity. siRNA-induced knockdown of NF-kappaB, but not of Sp1, reduced TNF-alpha-induced SPAK expression. Nuclear run-on and mRNA decay assays demonstrated that TNF-alpha directly increased SPAK mRNA transcription without affecting SPAK mRNA stability. Furthermore, up-regulation of NF-kappaB expression and demethylation of the CpG islands induced by TNF-alpha also played roles in the up-regulation of SPAK expression. In conclusion, our data indicate that during inflammatory conditions, TNF-alpha is a key regulator of SPAK expression. The development of compounds that can either modulate or disrupt the activity of SPAK-mediated pathways is therefore important for the control and attenuation of downstream pathological responses, particularly in IBD.

Nanoparticle-based therapeutic delivery of prohibitin to the colonic epithelial cells ameliorates acute murine colitis.

BACKGROUND: Intestinal epithelial expression of antioxidants and nuclear factor kappa B (NF-κB) contribute to mucosal barrier integrity and epithelial homeostasis, two key events in the pathogenesis of inflammatory bowel disease (IBD). Genetic restoration of intestinal epithelial prohibitin 1 (PHB) levels during experimental colitis reduces the severity of disease through sustained epithelial antioxidant expression and reduced NF-κB activation. To determine the therapeutic potential of restoring epithelial PHB during experimental colitis in mice, we assessed two methods of PHB colonic mucosal delivery: adenovirus-directed administration by enema and poly(lactic acid) nanoparticle (NPs) delivery by gavage. METHODS: As a proof-of-principle to demonstrate the therapeutic efficacy of PHB, we utilized adenovirus-directed administration by enema. Second, we used NPs-based colonic delivery of biologically active PHB to demonstrate therapeutic use for human IBD. Colitis was induced by oral administration of dextran sodium sulfate (DSS) in water for 6-7 days. Wildtype mice receiving normal tap water served as controls. RESULTS: Both methods of delivery resulted in increased levels of PHB in the surface epithelial cells of the colon and reduced severity of DSS-induced colitis in mice as measured by body weight loss, clinical score, myeloperoxidase activity, proinflammatory cytokine expression, histological score, and protein carbonyl content. CONCLUSIONS: This is the first study to show oral delivery of a biologically active protein by NPs encapsulated in hydrogel to the colon. Here we show that therapeutic delivery of PHB to the colon reduces the severity of DSS-induced colitis in mice. PHB may represent a novel therapeutic target in IBD.

Ste20-related proline/alanine-rich kinase (SPAK) regulated transcriptionally by hyperosmolarity is involved in intestinal barrier function.

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-kappaB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-kappaB and Sp1 to bind to their binding sites. Knock-down of either NF-kappaB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-kappaB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function.

Ecto-phosphorylation of CD98 regulates cell-cell interactions.

Ecto-phosphorylation plays an important role in many cellular functions. The transmembrane glycoprotein CD98 contains potential phosphorylation sites in its extracellular C-terminal tail. We hypothesized that extracellular signaling through ecto-protein kinases (ePKs) might lead to ecto-phosphorylation of CD98 and influence its multiple functions, including its role in cell-cell interactions. Our results show that recombinant CD98 was phosphorylated in vitro by ePKs from Jurkat cells and by the commercial casein kinase 2 (CK2). Alanine substitutions at serines-305/307/309 or serines-426/430 attenuated CK2-mediated CD98 phosphorylation, suggesting that these residues are the dominant phosphorylation sites for CK2. Furthermore, CD98 expressed in the basolateral membranes of intestinal epithelial Caco2-BBE cells was ecto-phosphorylated by Jurkat cell-derived ePKs and ecto-CK2 was involved in this process. Importantly, cell attachment studies showed that the ecto-phosphorylation of CD98 enhanced heterotypic cell-cell interactions and that the extracellular domain of CD98, which possesses the serine phosphorylation sites, was crucial for this effect. In addition, phosphorylation of recombinant CD98 increased its interactions with Jurkat and Caco2-BBE cells, and promoted cell attachment and spreading. In conclusion, here we demonstrated the ecto-phosphorylation of CD98 by ePKs and its functional importance in cell-cell interactions. Our findings reveal a novel mechanism involved in regulating the multiple functions of CD98 and raise CD98 as a promising target for therapeutic modulations of cell-cell interactions.

Interleukin-6 transcriptionally regulates prohibitin expression in intestinal epithelial cells.

Prohibitin (PHB) is a highly conserved protein that has multiple functions in the cell. We recently demonstrated that PHB plays an important role in combating oxidative stress and its expression is down-regulated in human and animal models of inflammatory bowel disease. Little is known regarding the regulation of PHB expression in intestine or other tissues. In this study we examined the regulation of PHB expression in intestinal epithelial cells using the model cell line Caco2-BBE. We successfully cloned the 1192-bp human PHB promoter region and identified the transcription start site 1594 bp upstream from the translation start site due to an intervening intron. We show that the acute phase cytokine interleukin-6 (IL-6) increases PHB protein and mRNA abundance and induces PHB promoter activation. The IL-6 response element site in the PHB promoter is required for maximal basal promoter activity and responsiveness to IL-6. IL-6 also increases binding of nuclear proteins to the IL-6 response element in the PHB promoter that are supershifted by a STAT3 antibody. Both basal promoter activity and IL-6 responsiveness are attenuated by signal transducer and activator of transcription 3 short interference RNA, suggesting that signal transducer and activator of transcription 3 mediates PHB activity by IL-6. Confirming these in vitro results, IL-6(-/-) mice exhibit reduced PHB expression in the colon compared with wild-type mice. These results suggest that IL-6 modulates PHB expression in cultured intestinal epithelial cells and in the intestine in vivo.