MICE LACKING MIST1 SHOW INCREASED SENSITIVITY TO CHRONIC ETHANOL EXPOSURE

Rodent models show that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. Mice lacking MIST1 (Misti4\u27), a target of the UPR marker XBP1, show reduced ability to activate the UPR during cell stress. Therefore, I hypothesized that an absence of MIST1 would lead to increased sensitivity to alcohol feeding. The effects of dietary stress on the UPR were examined in pancreatic tissue from 2 to 4 month-old mice placed on a diet containing 36% of kcal from ethanol for 6 weeks. Based on immunofluorescent, histological and immunoblotting assays, MistTAmice showed age related changes in UPR activation. In response to ethanol, they developed periductal accumulations of inflammatory cells, limited induction of autophagy and reduction in the expression of BiP, pelF2a and sXbp1, unlike wild type counterparts that had significantly higher levels of sXbp1 and pelF2a. The UPR was not further activated following initiation of acute pancreatitis. This work suggests that factors affecting MIST1 expression and function in humans may be a predisposing factor for pancreatic disease

Distinct Changes in Placental Ceramide Metabolism Characterize Type 1 and 2 Diabetic Pregnancies with Fetal Macrosomia or Preeclampsia

Disturbances of lipid metabolism are typical in diabetes. Our objective was to characterize and compare placental sphingolipid metabolism in type 1 (T1D) and 2 (T2D) diabetic pregnancies and in non-diabetic controls. Placental samples from T1D, T2D, and control pregnancies were processed for sphingolipid analysis using tandem mass spectrometry. Western blotting, enzyme activity, and immunofluorescence analyses were used to study sphingolipid regulatory enzymes. Placental ceramide levels were lower in T1D and T2D compared to controls, which was associated with an upregulation of the ceramide degrading enzyme acid ceramidase (ASAH1). Increased placental ceramide content was found in T1D complicated by preeclampsia. Similarly, elevated ceramides were observed in T1D and T2D pregnancies with poor glycemic control. The protein levels and activity of sphingosine kinases (SPHK) that produce sphingoid-1-phosphates (S1P) were highest in T2D. Furthermore, SPHK levels were upregulated in T1D and T2D pregnancies with fetal macrosomia. In vitro experiments using trophoblastic JEG3 cells demonstrated increased SPHK expression and activity following glucose and insulin treatments. Specific changes in the placental sphingolipidome characterize T1D and T2D placentae depending on the type of diabetes and feto-maternal complications. Increased exposure to insulin and glucose is a plausible contributor to the upregulation of the SPHK-S1P-axis in diabetic placentae

The Absence of MIST1 Leads to Increased Ethanol Sensitivity and Decreased Activity of the Unfolded Protein Response in Mouse Pancreatic Acinar Cells

Background: Alcohol abuse is a leading cause of pancreatitis in humans. However, rodent models suggest that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. The goal of this study was to determine if an absence of MIST1, a transcription factor required for complete differentiation of pancreatic acinar cells in mice, increased the sensitivity to alcohol. Methods: Two to four month-old mice lacking MIST1 (Mist1 2/2) or congenic C57 Bl6 mice were placed on a Lieber-DeCarli diet (36 % of total kcal from ethanol and fat), a control liquid diet (36 % kcal from fat) or a regular breeding chow diet (22% kcal from fat). After six weeks, pancreatic morphology was assessed. Biochemical and immunofluorescent analysis was used to assess mediators of the unfolded protein response (UPR). Results: Ethanol-fed Mist1 2/2 mice developed periductal accumulations of inflammatory cells that did not appear in wild type or control-fed Mist1 2/2 mice. Wild type mice fed diets high in ethanol or fat showed enhancement of the UPR based on increased accumulation of peIF2a and spliced XBP1. These increases were not observed in Mist1 2/2 pancreatic tissue, which had elevated levels of UPR activity prior to diet exposure. Indeed, exposure to ethanol resulted in a reduction of UPR activity in Mist1 2/2 mice. Conclusions: Our findings suggest that an absence of MIST1 increases the sensitivity to ethanol that correlated wit

The Dynamic Role of Jumonji C Domain Containing Protein 6 in Placental Development and Disease

Perturbations in oxygen sensing are a defining feature of placental-associated pathologies such as preeclampsia, a serious disorder of pregnancy. Preeclamptic placentae have markedly elevated levels of Hypoxia Inducible Factor 1Îą (HIF1A), a master regulator of oxygen homeostasis. Mounting evidence implicates a family of Fe2+ and oxygen-dependent Jumonji C domain containing enzymes (JMJDs) as mediators of the epigenetic code and hypoxic gene expression. While several JMJDs are induced in hypoxia, their role in pregnancy remains unclear. The goal of this study was to characterize JMJD6 function in the placenta in physiological and pathological conditions, and unravel its regulatory relationship with von Hippel Lindau tumour suppressor (VHL), a key executor of the cellular hypoxic response. JMJD6 expression inversely correlated with changes in oxygen tension during placental development, while JMJD6 protein and mRNA were significantly elevated in low oxygen and in early-onset preeclamptic (E-PE) placentae. In vitro demethylation assays revealed that optimal JMJD6-dependent demethylation of its histone targets, H3R2me2s and H4R3me2s, occurred in normoxia, and this was impaired in E-PE placentae due to a hypoxia-iron imbalance. In cytotrophoblast cells, JMJD6 is a positive regulator of VHL gene expression in normoxia. Accordingly, JMJD6 histone targets in E-PE placentae showed marked reductions in their association with VHL promoter regions. Independent of VHL gene regulation, JMJD6 also controlled VHL protein stability through lysyl hydroxylation and promoting its SUMO1-dependent SUMOylation. Importantly, the Jumonji C catalytic domain was found to be indispensable in executing both functions of JMJD6. In summary, these data signify a novel function for JMJD6 as an oxygen sensor in the human placenta, exerting a dual role in regulating VHL gene and protein. Uncovering epigenetic oxygen sensing mechanisms controlling HIF1A is crucial for defining the unique molecular signature of preeclampsia, which may ultimately translate to molecular-based diagnosis and therapy.Ph.D

The absence of MIST1 leads to increased ethanol sensitivity and decreased activity of the unfolded protein response in mouse pancreatic acinar cells

Background: Alcohol abuse is a leading cause of pancreatitis in humans. However, rodent models suggest that alcohol only sensitizes the pancreas to subsequent insult, indicating that additional factors play a role in alcohol-induced pancreatic injury. The goal of this study was to determine if an absence of MIST1, a transcription factor required for complete differentiation of pancreatic acinar cells in mice, increased the sensitivity to alcohol. Methods: Two to four month-old mice lacking MIST1 (Mist1 -/-) or congenic C57 Bl6 mice were placed on a Lieber-DeCarli diet (36% of total kcal from ethanol and fat), a control liquid diet (36% kcal from fat) or a regular breeding chow diet (22% kcal from fat). After six weeks, pancreatic morphology was assessed. Biochemical and immunofluorescent analysis was used to assess mediators of the unfolded protein response (UPR). Results: Ethanol-fed Mist1 -/- mice developed periductal accumulations of inflammatory cells that did not appear in wild type or control-fed Mist1 -/- mice. Wild type mice fed diets high in ethanol or fat showed enhancement of the UPR based on increased accumulation of peIF2α and spliced XBP1. These increases were not observed in Mist1 -/- pancreatic tissue, which had elevated levels of UPR activity prior to diet exposure. Indeed, exposure to ethanol resulted in a reduction of UPR activity in Mist1 -/- mice. Conclusions: Our findings suggest that an absence of MIST1 increases the sensitivity to ethanol that correlated with decreased activity of the UPR. Therefore, events that affect the expression and/or function of MIST1 may be confounding factors in pancreatitis. © 2011 Alahari et al

JMJD6 Dysfunction Due to Iron Deficiency in Preeclampsia Disrupts Fibronectin Homeostasis Resulting in Diminished Trophoblast Migration

The mechanisms contributing to excessive fibronectin in preeclampsia, a pregnancy-related disorder, remain unknown. Herein, we investigated the role of JMJD6, an O2- and Fe2+-dependent enzyme, in mediating placental fibronectin processing and function. MALDI-TOF identified fibronectin as a novel target of JMJD6-mediated lysyl hydroxylation, preceding fibronectin glycosylation, deposition, and degradation. In preeclamptic placentae, fibronectin accumulated primarily in lysosomes of the mesenchyme. Using primary placental mesenchymal cells (pMSCs), we found that fibronectin fibril formation and turnover were markedly impeded in preeclamptic pMSCs, partly due to impaired lysosomal degradation. JMJD6 knockdown in control pMSCs recapitulated the preeclamptic FN phenotype. Importantly, preeclamptic pMSCs had less total and labile Fe2+ and Hinokitiol treatment rescued fibronectin assembly and promoted lysosomal degradation. Time-lapse imaging demonstrated that defective ECM deposition by preeclamptic pMSCs impeded HTR-8/SVneo cell migration, which was rescued upon Hinokitiol exposure. Our findings reveal new Fe2+-dependent mechanisms controlling fibronectin homeostasis/function in the placenta that go awry in preeclampsia

<i>Mist1^−/−</i> pancreatic tissue exhibits increased activation of the UPR.

<p>(<b>A</b>) Western blot analysis of key UPR markers in 2 month old WT and <i>Mist1^−/−</i> whole pancreatic lysates. <i>Mist1^−/−</i> extracts show significantly increased accumulations of BiP/GRP78 (<b>B</b>), GADD34 (<b>C</b>) and sXBP1 (<b>D</b>), but not peIF2α (p = 0.743) or uXBP1 (p = 0.532) relative to WT pancreatic tissue. *P<0.05. n = 3.</p

<i>Mist1^−/−</i> pancreata develop periductal accumulations of inflammatory cells in response to ethanol feeding.

<p>Representative photomicrographs of Gomori's trichrome stained pancreatic sections from WT (<b>A, B</b>) and <i>Mist1^−/−</i> mice (<b>C, D</b>) fed a LDC-HF (<b>A, C</b>) or LDC-E (<b>B, D</b>) diet for 6 weeks. Higher magnification images from the same sections (<b>Ai–Di</b>) were used to highlight specific morphological events. Cellular accumulations (arrowhead) surrounding ducts (*) and adipose accumulations (arrow) were observed only in LDC-E fed <i>Mist1^−/−</i> mice. (<b>E, F</b>) Immunofluorescent analysis for the T-lymphocyte marker CD4 (<b>E</b>) indicated that these accumulations consisted of lymphocytes (arrowhead). Sections were co-stained with DAPI (<b>F</b>) to reveal all cells. Magnification bars = 40 µm.</p

WT and <i>Mist1^−/−</i> mice show no overt response to ethanol feeding.

<p>Caloric intake (<b>A</b>) and weight gain (<b>B</b>) was monitored over six weeks of feeding with a Lieber-DeCarli Ethanol (LDC-E), LDC high fat (LDC-HF) or breeding chow (BC) diet. Letters represent statistically significant differences between groups (see text for details). Groups were compared using Two-way ANOVA and Bonferroni post-hoc test. *P<0.05, **P<0.01, ***P<0.001. n = 7 (BC), n = 11 (LDC-HF), n = 12 (LDC-E). The LDC-HF diet resulted in increased weight gain in both genotypes. At six weeks, mice were killed and serum amylase (<b>C</b>) and pancreatic edema (<b>D</b>) calculated. No significant differences were observed between genotypes or treatments. n = 11 (LDC-HF), n = 12 (LDC-E).</p